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A r q u i v o s b r a s i l e i r o s d e

78 02publicação oficial do conselho brasileiro de oftalmologia

março/abril 2015

issn 0004-2749versão impressa

indexada nas bases de dados

MEDLINE | EMBASE | ISI | SciELO

Impact of wildfire smoke on ocular

surface

Intrastromal ring with and without

crosslinking for keratoconus

Automatic behavioural analysis for

IOL decision

Intravitreal adalimumab in rabbits

Frequency of publication: Bimonthly Arq Bras Oftalmol. São Paulo, v. 78, issue 2, pages 67-140, Mar/Apr. 2015

Continuous publication since 1938

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Literatura Latino-americana em Ciências da Saúde

Contents

OFFICIAL PUBLICATION OF THE BRAZILIAN COUNCIL OF OPHTHALMOLOGY (CBO) ISSN 0004-2749(Printed version)


Frequency of publication: Bimonthly Arq Bras Oftalmol. São Paulo, v. 78, issue 2, pages 67-140, Mar/Apr. 2015

PUBLICAÇÃO OFICIAL DOCONSELHO BRASILEIRO

DE OFTALMOLOGIA

EditorialV Publish (negative results too) or perish Publique (resultados negativos também) ou pereça Eduardo Melani Rocha, Mario Luiz Ribeiro Monteiro

Original Articles67

without affecting iNOS or MMP-9 Colírio de cetorolaco reduz inflamação e atrasa a epitelização em resposta a queimadura por álcali em coelhos, sem afetar iNOS ou MMP-9 Tiago Barbalho Lima, Alexandre Pinto Ribeiro, Luciano Fernandes da Conceição, Marcio Bandarra, Wilson Gomez Manrique, José Luiz Laus

73 Results of pars plana vitrectomy after complicated phacoemulsification surgery Resultados pós-vitrectomia via pars plana em pacientes submetidos à cirurgia de facoemulsificação com complicação intraoperatória Alexandre Grandinetti, Diogo Suenaga, Fernanda M. Oliveira, Krissis Saliba Uliana Cruz, Laurinda Meneguette, Luciane Bugman Moreira

76 Femtosecond-assisted intrastromal corneal ring implantation for keratoconus treatment: a comparison with crosslinking combination

Implante de anel intraestromal com laser de femtosegundo no tratamento de ceratocone: comparação da combinação com o crosslinking Peter Alexander von Harbach Ferenczy, Maiara Dalcegio, Marcela Koehler, Thiago Silveira Pereira, Hamilton Moreira, Luciane Bugmann Moreira

82 Blood gas analyzer utility in evaluating oxygen kinetics of the aqueous humor Utilidade de um analisador sanguíneo de gás para a avaliação cinética do oxigênio no humor aquoso Ismail Ersan, Sedat Arikan, Huseyin Toman, Selcuk Kara, Baran Gencer, Mesut Erbas, Hasan Ali Tufan, Metehan Uzun

85 Association of high-density lipoprotein and apolipoprotein E genetic variants with age-related macular degeneration Associação de lipoproteína de alta densidade e variantes genéticas da apolipoproteína E com degeneração macular relacionada à idade Sabrina Mayara Cezario, Maria Clara Jéssica Calastri, Camila Ive Ferreira Oliveira, Tayanne Silva Carmo, Marcela Augusta de Souza Pinhel, Moacir Fernandes de Godoy,

Rodrigo Jorge, Carina Costa Cotrim, Dorotéia Rossi Silva Souza, Rubens Camargo Siqueira

89 Cytotoxicity and genotoxicity of intravitreal adalimumab administration in rabbit retinal cells Citotoxicidade e genotoxicidade da administração de adalimumabe intravítreo nas células da retina de coelhos Álcio Coutinho de Paula, Marcos Pereira de Ávila, David Leonardo Cruvinal Isaac, Rodrigo Salustiano, Aliny Pereira de Lima, Francyelli Mariana Mello,

Flávia de Castro Pereira, Pedro Henrique de Paula Silva, Elisângela de Paula Silveira Lacerda

94 Preoperative automatic visual behavioural analysis as a tool for intraocular lens choice in cataract surgery Análise comportamental visual automática pré-operatória como ferramenta para escolha de lente intraocular em cirurgia de catarata Heloisa Neumann Nogueira, Mônica Alves, Paulo Schor

100 Quercetin protects the retina by reducing apoptosis due to ischemia-reperfusion injury in a rat model Quercetina protege a retina reduzindo a apoptose consequente à lesão por isquemia e reperfusão em um modelo de rato Sedat Arikan, Ismail Ersan, Turan Karaca, Selcuk Kara, Baran Gencer, Ihsan Karaboga, Hasan Ali Tufan

105 Optical coherence tomography and multifocal electroretinography of patients with advanced neovascular age-related macular degeneration before, during, and after treatment with ranibizumab

Tomografia de coerência óptica e eletrorretinografia multifocal de pacientes com degeneração macular relacionada à idade, neovascular avançada, antes, durante e após o tratamento com ranibizumabe

Izabela Negrão Frota de Almeida, Luciana Negrão Frota de Almeida, Edmundo Frota de Almeida Sobrinho, Bruno Duarte Gomes, Givago da Silva Souza, Alexandre Antonio Marques Rosa, Luiz Carlos L. Silveira

110 Impact of wildfire smoke in Buenos Aires, Argentina, on ocular surface Efeitos da fumaça de incêndios na superfície ocular em Buenos Aires, Argentina Martin Berra, Gustavo Galperín, Laura Dawidowski, Julia Tau, Isabel Márquez, Alejandro Berra

Case Reports115 Bilateral acute iris transillumination (BAIT) initially misdiagnosed as acute iridocyclitis Transiluminação de íris aguda bilateral (BAIT) inicialmente diagnosticada como iridociclite aguda Saban Gonul, Banu Bozkurt

118 Acute retinal necrosis following intravitreal dexamethasone (Ozurdex®) implant Necrose aguda de retina após implante de dexametasona intravítrea (Ozurdex®) Murat Kucukevcilioglu, Mustafa Eren, Umit Yolcu, Gungor Sobaci

120 Distrofia macular associada à síndrome de Kjellin: um relato de caso Vinícius Monteiro de Castro, André Meirelles, Rafael Saran Arcieri, Katharina Messias, André Messias

123 Oculo-peritoneal shunt: draining aqueous humor to the peritoneum Derivação óculo-peritoneal: drenagem do humor aquoso para o peritônio Ana Maldonado-Junyent, Arturo Maldonado-Bas, Andrea Gonzalez, Francisco Pueyrredón, María Maldonado-Junyent, Arturo Maldonado-Junyent,

Diego Rodriguez, Mariano Bulacio

Review Articles126 Impression cytology in the evaluation of ocular surface tumors: review article Citologia de impressão na avaliação de tumores da superfície ocular: artigo de revisão Jeison de Nadai Barros, Simone Ribeiro Araújo de Almeida, Marcia Serva Lowen, Marcelo Carvalho da Cunha, José Álvaro Pereira Gomes

Letters to the Editor133 A calipers-free intravitreal anti-VEGF injection technique Técnica de injeção intravítrea de anti-VEGF sem compasso Zafer Oztas, Cezmi Akkin, Filiz Afrashi, Sedat Selim

135 Dengue and chiasmal compression Compressão de quiasma e dengue Viroj Wiwanitkit

137 Instructions to Authors

V

Editorial

http://dx.doi.org/10.5935/0004-2749.20150018

Publish (negative results too) or perish

Publique (resultados negativos também) ou pereça

Eduardo MElani rocha1, Mario luiz ribEiro MontEiro2

Over the past decades, the quotation “publish or perish” has been echoing in the minds of everyone with an academic carrier. The good will behind these three words is that science is a public task, science does not have frontiers, and science grows better with multiple and interdisciplinary collaboration. Meanwhile, not ensuring a certain amount of hype about the product of a scientific task is the same as condemning it to death. Furthermore, publication of research results is considered an obligation that the researcher has to the society, the university to which he/she belongs to, and the research agency supporting the studies.

Everything is perfect until someone gets “negative results,” when the above-mentioned rationale is dis-rupted. Let us define negative results before continuing further. Negative results are data collected from a well-designed project, well-performed assays, and from analyses that do not confirm the hypothesis of the study. This leads the researcher to accept the “null hypothesis” (H0), indicating that there is no relationship between the two measured phenomena, or from a statistical point of view, showing that the probability of the phenomena occurs by chance and not because the intervention is above the adopted significance level (p, usually 5%). A simple example in ophthalmic research is when the benefit of treatment with a placebo is similar or superior to that treatment with the drug being tested, with a comparison of the mean outcome measurements resulting in a p-value of ≥0.05 or 5%. In this example, the study accepted the null hypothesis when concluded that the drug being tested was not more efficient than the placebo. The scientific community would label this study a “negative result” study.

In the scientific world, a negative result leads to disappointment in the research group involved in the study, followed by displeasure of the agency or company involved in the financial support, and worst of all, skepticism and disinterest of reviewers and editors of scientific journals. The final result of the process is hidden, buried data, thus, ignored by the scientific community(1). Interestingly and ironically, in journalism, there is a general interest in bad (or negative) news, whereas good news is usually only briefly (or not) reported.

However, in science negative results should not be treated in a negative way. Although we may understand the journalistic interest for bad news to gain public attention, we should not accept that only positive research results are worthy because they tend to gain attention from medical journals (and their reviewers). Negative results in science may represent the result of significant time and money spent to obtain them. The cost of not announcing them properly may lead to greater expenditure when other scientific groups repeat the work. Scientific community will ignore the previous effort because they had never been published. The efforts of authors who had obtained negative results must be credited and not labeled as failure or bad planning. Moreo-ver, negative results give a balance for comparing potential therapeutic approaches in systematic reviews and meta-analysis. Hidden negative results only nourish the unacceptable interests of those who benefit from these results being hidden. Most importantly, negative results may imply that a new (and usually expensive) drug or technology does not add significant advantage over well-established ones. This information may save time and money for patients and the society, not to mention avoiding previously unknown side effects that usually are discovered only after new drugs are released. In some situations, negative results may in fact be the good results, and reporting them may avoid unnecessary treatments, testing, or surgeries.

In their most recent book, “Think like a freak,” Steven D. Levitt and Stephen J. Dubner quote the former New York mayor Michael Bloomberg: “In medicine and science, if one follows a path that reveals a dead end, it will have taken a relevant contribution, because we know that it will not be accurate to travel the same path”(2). Efforts to revert the practice of aversion to negative results are being made by the biomedical community. Among those efforts are the clinical trials registers(3). Their objective is to announce every clinical trial prior to initiating data collection from patients. Data will be known to the registers, despite the results being unfavorable for the sponsors supporting the studies. Another effort is made by scientific journals, whose name and mission reinforce the commitment to judge and publicize negative results; such journals include Journal of Negative Results in Biomedicine, the Journal of Negative Results-Ecology and Evolutionary Biology, and the Journal of Articles in Support of the Null Hypothesis(4).

Submitted for publication: February 7, 2015 Accepted for publication: February 7, 20151 Department of Ophthalmology, Otorhinolaryngology and Head and Neck Surgery, Ribeirão Preto

Medical School (FMRP), University of São Paulo (USP), Ribeirão Preto, SP, Brazil.2 Department of Ophthalmology and Otorhinolaryngology, School of Medicine, University of São Paulo

(USP), São Paulo, SP, Brazil.

Funding: No specific financial support was available for this study.

Disclosure of potential conflicts of interest: None of the authors have any potential conflict of interest to disclose.

Corresponding author: Eduardo M. Rocha. Av. Bandeirantes, 3.900 - FMRP-USP - 14049-900 - Ri beirão Preto - SP - Brazil - E-mail: [email protected]

Publish (negative results too) or perish

VI

In the midst of the pressure to publish study data and the low priority given by major journals to articles with negative results, academic researchers are faced with another nightmare, the so-called “predator journals.” As pointed out by Jeffrey Beall, predator journals proceed with a list of misconducts that jeopardize the reputation of their authors and the patience of their readers and scientific community. For example, they do not follow the ethical principles of open access journals, do not use ISSN or DOI numbers, and worse, falsely claim to have an impact factor or use some informal measures (e.g., estimated factor or view factor). These journals frequently use spam messages, alleging to have a motto to serve the scientific community while focusing on receiving author fees for publication with an average charge of approximately US$ 2000(5,6). It is easy to predict that au-thors with negative results are frequent victims of such journals, but they must be aware that this practice may compromise their carriers instead of pushing them up. Recent scandals noticed in the press revealed that some of these journals clearly do not have peer reviews or any reviews at all. Manuscripts generated by a robotic word processor or made up by one single phrase repeated throughout the entire text were published as scientific papers in two of these journals(7).

Instead of giving in to the temptation of publishing in predator journals or not publishing their results, re-searchers with negative results should instead learn how to emphasize the importance of their studies both in the rationale and in the discussion of their papers and present it to peer-reviewed journals with a clear editorial commitment to ethics and honest science. ABO - Arquivos Brasileiros de Oftalmologia reinforces the editorial line to publish original papers in the field of ophthalmology with good scientific quality, whether the results confirm or disprove the study hypothesis, because both types of results are useful and required to have a good understanding of scientific questions in order to guide readers in their clinical practice and future research. Publication of negative results is necessary to prevent the credibility of the scientific community from perishing.

REFERENCES 1. Lexchin J, Bero LA, Djulbegovic B, Clark O. Pharmaceutical industry sponsorship and

research outcome and quality: systematic review. BMJ. 2003;326(7400):1167-70. 2. Levitt SD, Dubner SJ. Think like a freak. New York: HarperCollins Publishers; 2014. 3. National Institute of Health. Clinical trials register. Washington, DC; NIH. [cited 2015

Jan 29]. Available from: https://clinicaltrials.gov/ 4. O’Hara B. Negative results are published. Nature. 2011;471(7339):448-9. Comment on:

Nature. 2011;470(7335):437.

5. Beall J. Criteria for determining predatory open-access publishers. Scholary Open Access: critical analysis of scholarly open-access publishing. [cited 2015 Jan 29]. Avai lable from: http://scholarlyoa.com/2012/08/04/criteria-for-determining-predatory-open-access- publishers/

6. Butler D. Investigating journals: The dark side of publishing. Nature. 2013;495(7442): 433-5.

7. Schwartsman H. Brincando com a ciência. Folha de São Paulo [Internet]. 2015 Jan 03. [cited 2015 Jan 29] Disponível em: http://www1.folha.uol.com.br/colunas/heliosch wartsman/2015/01/1569975-brincando-com-a-ciencia.shtml

Original Article

67Arq Bras Oftalmol. 2015;78(2):67-72http://dx.doi.org/10.5935/0004-2749.20150019

INTRODUCTIONNon-steroidal anti-inflammatory drugs (NSAIDs) used in ulcera ti ve

keratitis may delay healing of epithelial lesions and enhance corneal stroma degradation(1,2). An elevated expression of different metal lo-proteinases (MMPs) in the cornea locally treated with sodium diclo-fenac or ketorolac tromethamine has been demonstrated, even without preservatives(2,3).

Nitric oxide (NO) is a simple gaseous molecule found in small quantities in atmospheric air. It is synthesized with the help of nitric

oxide synthase (NOS)(4). Three distinct forms of NOS have been recognized: two constitutive isoforms [nNOS (NOS-1) in the ner-vous system and eNOS (NOS-3) in endothelial cells] and the iNOS in ducible isoform (NOS-2)(5), which is expressed in different cells following transcriptional activation by cytokines or endotoxins(6). In ocular tissues, NO is involved in different events(7). In vitro, it has been demonstrated that stimulation of endothelial and bovine keratocyte cells by lipopolysaccharides and cytokines induces iNOS expression, releasing a large amount of NO(8). iNOS expression in corneal lesions

Ketorolac eye drops reduce inflammation and delay re-epithelization in response

to corneal alkali burn in rabbits, without affecting iNOS or MMP-9

Colírio de cetorolaco reduz inflamação e atrasa a epitelização em resposta a queimadura por álcali em coelhos, sem afetar iNOS ou MMP-9

tiago barbalho liMa1, alExandrE Pinto ribEiro2, luciano FErnandEs da concEição1, Marcio bandarra3, Wilson goMEz ManriquE3, José luiz laus1

Submitted for publication: 24, 2014 Accepted for publication: 1, 20141 Department of Medicine and Surgery, College of Agricultural and Veterinarian Sciences, State

University of São Paulo (UNESP), Jaboticabal, SP, Brazil.2 Department of Veterinary Clinical Medicine, College of Veterinary and Agricultural Sciences, Federal

University of Mato Grosso (UFMT), Cuiabá, MT, Brazil.3 Department of Veterinary Pathology, College of Agricultural and Veterinarian Sciences State University

of São Paulo (UNESP), Jaboticabal, SP, Brazil.

Funding: FAPESP process number 2011/07848-0 and CNPq process number 300833/2010-5 Disclosure of potential interest conflicts: None of the authors have any potential conflict of interest

to disclose. Corresponding author: José Luiz Laus. Departamento de Clínica e Cirurgia Veterinária - FCAV-UNESP -

Via de Acesso Prof. Paulo Donato Castellane, s/n - Jaboticabal, SP - 14884-900 - Brazil E-mail: [email protected]

Approved by the following Research Ethics Committee on Animal Use: Comissão de Ética do Uso de Animais (CEUA) - UNESP (protocol number 007616/11).

ABSTRACTPurposes: To assess the effects of 0.5% ketorolac tromethamine without preser-vatives on the expression of iNOS and MMP-9 in alkali burn ulcers. Methods: Twelve eyes of 120-day-old male rabbits were treated (TG) every 6 h with 0.5% ketorolac tromethamine and 12 other eyes were treated with saline solution (CG), immediately after the occurrence of ulcers by 1 M sodium hydroxide (NaOH). Re-epithelialization was monitored using fluorescein every 6 h. After 24 h, six corneas (n=6) of each group were collected (M1). The others (n=6) were col-lected after reepithelialization (M2). At both moments, the inflammatory infiltrate and the conditions of the newly formed epithelium were histologically analyzed. iNOS and MMP-9 were evaluated by immunohistochemistry. Results: Mean epithelialization time in TG was 55 ± 0.84 h. In CG, it was 44 ± 1.06 h (p=0.001). At M1, corneas of TG had lower inflammatory exudation compared with (p <0.001). At M2, TG revealed discrete inflammatory exudation (p>0.05) and lower numbers of epithelial layers compared with CG. The mean iNOS in stromal cells did not differ in TG over both moments compared with CG (p>0.05) At M2, the central corneal region expressed more iNOS in both groups compared with the peripheral region. No significant differences were observed in iNOS scores of epithelial immunostaining between the groups and across M1 and M2 (p=0.69). Epithelial immunostaining scores for MMP-9 did not differ in TG compared with CG (p=0.69). The average immunostaining score of MMP-9 in stromal cells showed no differences between groups or moments. There was no correlation between immunostaining of iNOS and MMP-9 or between the amount of inflammatory cells and immunostaining of iNOS. Conclusions: Use of 0.5% keratolac tromethamine reduced inflammation and delayed reepithelialization in a cornea alkali burn model without impacting the expression of iNOS or MMP-9.

Keywords: Corneal ulcer/chemically induced; Ketrolac tromethamine/adminis-tration; iNOS, MMP-9

RESUMOObjetivos: Avaliarem-se os efeitos do cetorolaco de trometamina 0,5%, sem con-servante, sobre a expressão da iNOS e da MMP-9, em córneas com úlceras químicas. Métodos: Doze olhos de coelhos machos, 120 dias de idade, foram tratados (GT ), a cada 6 horas, com o cetorolaco de trometamina 0,5% e outros 12 com solução salina (GC), imediatamente à ocorrência de úlceras por hidróxido de sódio (NaOH) 1 mol/L. A reepitelização foi monitorada por fluresceína a cada seis horas. Decorridas 24 horas, seis córneas (n=6) de cada grupo foram colhidas (primeiro momento). As demais (n=6) o foram após a sua reepitelização (segundo momento). Em ambos os momentos, avaliaram-se o infiltrado inflamatório e as condições do epitélio neoformado (HE). Por imuno-histoquímica, avaliou-se a imunomarcação de iNOS e de MMP-9. Resultados: A média do tempo de epitelização no GT foi de 55 ± 0,84 horas. No GC, ela foi de 44 ± 1,06 horas (p=0,001). Às 24 horas, as córneas do GT apresentaram menor exsudação inflamatória (p<0,01). No segundo momento, o GT mostrou discreta exsu-dação inflamatória (p>0,05) e menor número de camadas epiteliais comparativamente ao GC. A média de imunomarcação de iNOS em células do estroma não diferiu do GT, em ambos os momentos (p>0,05). No segundo momento, a região central da córnea expressou mais iNOS, comparativamente à periférica, em ambos os grupos. Não se observaram diferenças significativas nos escores de imunomarcação epitelial de iNOS entre os grupos e os momentos (p=0,69). Os escores de imunomarcação epitelial para MMP-9 não diferiram entre os grupos (p=0,69). A média de imunomarcação da MMP-9 em células do estroma não exibiram diferenças entre os grupos e momentos da avaliação (p=0,32). Não houve correlação entre a imunomarcação de iNOS e de MMP-9, assim como quanto ao quantitativo de células inflamatórias e à imunomarcação de iNOS. Conclusões: Cetorolaco 0,5% reduziu a inflamação e atrasou a epitelização na quei-madura corneal por álcali sem alterar a expressão de iNOS ou MMP-9

Descritores: Úlcera da córnea/induzida quimicamente; Cetorolaco de trometamina/administração; iNOS; MMP-9

Ketorolac eye drops reduce inflammation and delay re-epithelization in response to corneal alkali burn in rabbits, without affecting iNOS or MMP-9

68 Arq Bras Oftalmol. 2015;78(2):67-72

induced by ultraviolet radiation has been verified(9). In vivo, high levels of NO can be involved in corneal inflammatory diseases(8). iNOS has been expressed in chemically ulcerated murine corneas, inhibiting neovascularization(10). Peroxynitrite and NO reduce the levels of the tissue inhibitor of metalloproteinase-1 (TIMP-1), increasing the gela-tinolytic activity of MMPs in corneal fibroblasts cultivated in vitro(11).

MMP-9 is produced by epithelial cells and neutrophils and has a role in remodeling the corneal stroma after keratectomy(12). MMP-9 is involved in corneal stroma degradation, facilitating the migration and proliferation of neo-vessels in ulcers(12). iNOS expression has been studied in corneal ulcers caused by alkali(10); however, the effect of NSAIDs on its activity is unknown.

Ketorolac tromethamine is used in ocular pain control, despite being toxic to the epithelium(3), and is capable of raising MMPs ex-pression(2). In addition, its activity can be increased by iNOS, as de-monstrated in vitro(11).

The present study aimed to evaluate the effect of 0.5% ketorolac tromethamine without preservative on iNOS and MMP-9 expression in rabbit corneas with ulcers chemically induced by sodium hydro-xide (NaOH).

METHODSMale rabbits (average weight, 3.4 kg; age, 120 days) of the

White New Zealand breed were selected [Oryctolagus cuniculus (Linnaeus, 1758)]. Animals were evaluated prior to the experiment using biomicroscopy with a slit lamp (XL-1 Slit lamp®; Shin-Nippon, Japan), applanation tonometry (Tono Pen XL®; Medtronic, Jackson-ville, U.S.A.), indirect binocular ophthalmoscopy (Indirect Binocular ophthalmoscope FOH®; Eyetec S.A.), and fluorescein dye (Fluorescein 5 strips®-Ophthalmos Ltda., São Paulo/SP, Brazil). Healthy animals were individually housed in a ventilated environment, in appropriate, clean, and sanitized cages with a diet based on commercial food and drinking water ad libitum.

After induction of anesthesia by intramuscular injection of 15 mg/kg of ketamine [(S)-(+)-Ketamine®; Cristália, São Carlos/SP, Brazil] with 0.5 mg/kg midazolam (Dormire®; Cristália, São Carlos/SP, Brazil), periocular trichotomy was performed. A drop of proximetacaine was instilled (Anestalcon®; Alcon, São Paulo/SP, Brazil), and antiseptic treatment of the cornea, conjunctival sac, and conjunctivae were performed with iodine-polyvinylpyrrolidone (Laboriodine; Glicola-bor Ind. Farmacêutica Ltda, Ribeirão Preto/SP, Brazil) diluted in saline (Sodium Chloride Solution 0.9%; JP Indústria Farmacêutica, Ribeirão Preto/SP, Brazil; 1:50). General anesthesia was achieved using masks with isoflurane (Forane®; Cristália, São Carlos/SP, Brazil), diluted in 100% oxygen in an open circuit.

After routine preparation of the operation field, a disk of filter paper (Whatman No. 40; F. Maia Ltda, Cotia/SP, Brazil), 6.0 mm in diameter, soaked in a solution of 1 M sodium hydroxide (NaOH) was gently maintained over the paracentral region of the cornea for 1 min. Immediately, careful washing of the entire corneal surface with saline solution 0.9% was performed.

Immediately after the lesions have been inflicted, 12 eyes were treated with 30 μL of 0.5% ketorolac tromethamine (Acular®; Aller-gan, Guarulhos/SP, Brazil), without preservatives (the treated group, TG), and 12 other eyes received, under similar regimen, 0.9% saline solution (control group, CG), both at regular intervals of 6 h, until the collection of corneas.

With lesions lasting for 24 h (M1, first moment), six corneas (n=6) from each group were collected. Remaining corneas (n=6) were col-lected after re-epithelialization (M2, second moment). Immediately, the preparation of samples for histology and immunohistochemistry was performed.

For monitoring re-epithelialization, clinical observations was performed using fluorescein dye tests and biomicroscopy with slit light and cobalt blue filter (Fluorescein in stripes®; Ophthalmos, São Paulo/SP, Brazil) every 6 h.

HISTOLOGY

Corneas were processed and analyzed at the Laboratory of Immunohistochemistry at the Department of Veterinary Pathology, College of Agricultural and Veterinary Sciences, State University of São Paulo, Jaboticabal, SP, Brazil. The specimens were maintained in 10% buffered formalin for 24 h and then in 70% alcohol for 3 days. Next, samples were embedded in paraffin and cut sagittally in 5-µm sections. Hematoxylin-eosin (HE) was used for staining.

Using 40× light microscopy magnification (Olympus BX51; Olym pus Optical Brazil, Ltda., São Paulo/SP, Brazil), the epithelial and conditions of the newly formed stroma were analyzed with regard to cellularity, disposition of collagen fibers, and inflammatory infil-tration. Three random fields in the center of the corneas (adjacent to the lesion area) and three fields in the periphery (adjacent to the limbus) were analyzed.

IMMUNOHISTOCHEMISTRY

Five-micrometer-thick sections were mounted on electrically char-ged slides and processed, employing the streptavidin-biotin complex technique, for labeling of iNOS and MMP-9. Sections were deparaffi-nized and hydrated in decreasing xylene batteries, followed by al-cohol dehydration and rinsing in distilled water. Endogenous protein blocking was performed (Protein Block-DAKO® X0909). Retrieval of antigens was performed under heat and pressure (Pot Pascal-DAKO® S2800) in 10 mM buffered solution of sodium citrate with pH=6.0. Next, polyclonal primary antibody against iNOS (1:600) and MMP-9 (1:200) were added. The material was incubated in a humid, dark chamber at a temperature of 23°C for 14 h. As secondary antibody, a polymer bound with peroxidase (ADVANCE-DAKO® K4069 HRP Rabbit/Mouse) was used, followed by incubation in a humid, dark chamber for 1 h. Visualization with diaminobenzidine chromogen (DAB; EnVision + System-HRP-DAKO®) was eventually performed. Counterstaining was performed using Harris’s hematoxylin. For the negative control, only the primary antibody was excluded from the reaction. Slides were washed three times for 5 min using PBS (pH=7.4).

For quantification, average values of three random fields were calculated during evaluation of epithelial immunostaining, using a semiquantitative index(13). For stromal immunostaining, three ran-dom fields in the central region (adjacent to the lesion area) and three fields in the peripheral region (adjacent to the limbus) were counted.

STATISTICAL ANALYSIS

Data were evaluated for normality employing Kolmogorov- Smirnov’s test. Variance analysis was used for repeated measure-ments, and further analyses were conducted using Bonferroni’s and Tukey’s tests. Correlations between immunostaining scores of iNOS and MMP-9 and between iNOS and the amount of inflammatory cells were analyzed using Pearson’s correlation test. A minimum sig-nificance level of p<0.05 was used. It was considered that variables showed weak correlation when p<0.05, moderate correlation when p<0.01, and strong correlation when p<0.001. Results were expressed as means and standard error of the mean.

RESULTSAverage epithelialization time was 55 ± 0.84 h (second time) in

TG and 44 ± 1.06 h in CG (p=0.001).

HISTOLOGY

Sections stained with Hematoxylin-Eosin staining exhibited areas denuded of epithelium; this was more extensive in corneas treated with 0.5% ketorolac tromethamine without preservative than those of CG after 24 h. The newly formed epithelium in TG was largely cha-racterized by a single layer. Control corneas showed newly formed epithelium, with alternating areas of single and two or more layers.

Lima TB, et al.

69Arq Bras Oftalmol. 2015;78(2):67-72

At M2, the treated corneas proved to be epithelialized, someti-mes exhibiting areas of epithelium in double layers still not adhering in the center of the lesion. In the same period, control corneas revea-led a higher stratification.

Regarding stromal conditions, in both groups and at both mo-ments, low cellularity, collagen fiber disorganization, and edema were observed.

Regarding inflammatory cellularity, control corneas exhibited margination and diapedesis of polymorphonuclear cells, with a pre-dominance of neutrophils and with mononuclear cells being rare. After 55 h, a mild inflammatory exudate was noticed in the treated corneas, with rare polymorphonuclear and inflammatory mononu-clear cells compared with controls. The amount of inflammatory cells in the corneal periphery at M1, was significantly lower in the TG (395.5 ± 40.97) compared with the CG (1644 ± 246.6; p<0.001). Values were also smaller in the central region of TG (159.7 ± 25.06) at 24 h compared with the CG (474.8 ± 104.3; p>0.05). Moreover, at M1, the amount of inflammatory cells in the periphery of control corneas (1644 ± 246.6) was significantly higher compared with that in the central area (474.8 ± 104.3 cells; p<0.001; Figure 1). At M2, the amount of inflammatory cells in the periphery was lower in TG (430.5 ± 71.41) compared with CG (960.3 ± 170.3). In the central region, similar results were found in TG (276.3 ± 30.11) and CG (277.8 ± 40.53; Figure 1). The number of cells in the corneal periphery of CG significantly decreased between M1 and M2 (p<0.01; Figure 1).

IMMUNOHISTOCHEMISTRY

iNOS immunostaining of the epithelium was conspicuously more intense in the margins of the lesions. Regarding the stroma, immu-nostaining of the cytoplasm and of the nuclei of polymorphonuclear and mononuclear cells and keratocytes was observed to be more intense in the region adjacent to the lesion (Figure 2).

At M1, iNOS expression in the corneal epithelium was similar between the two groups. At M2, a non-significant lower expression was observed in TG (1.5 ± 0.22) compared with CG (2 ± 0.44; p=0.69; Figure 3).

When the periphery and the stroma center were analyzed at M1, no difference in iNOS expression in the treated corneas was observed. At M2, a significantly higher expression was found in the central re-gion (48.83 ± 9.93) of treated corneas compared with the peripheral region (14.5 ± 2.88) in the same group (p<0.01). iNOS expression in

the central region (45.5 ± 7.43) in control corneas was significantly higher compared with the peripheral region (12.67 ± 5.07) at M2 (p<0.05; Figure 3). At M1, there was no difference between the re-gions in the two groups.

Regarding MMP-9, immunostaining was predominantly evident in the cytoplasm of epithelial cells (Figure 4). MMP-9 expression was observed in smaller quantity in keratocytes and in polymorphonuclear and in mononuclear cells.

When assessing immunostaining scores in the epithelium of trea-ted corneas at M1, a lower MMP-9 expression was observed (1.16 ± 0.16) compared with CG (1.5 ± 0.34). At M2, MMP-9 expression in the epithelium of treated corneas was lower (1.5 ± 0.3) compared with the expression observed in CG (1.5 ± 0.22); however, this difference was not statistically significant (p=0.69).

Regarding average quantity of immunostained cells at M1, MMP-9 expression was lower in the periphery of treated corneas (7 ± 1.98) compared with control corneas (23.5 ± 7.34; p<0.001). In the central region, there were negligible differences between TG (24 ± 9.45) and CG (26.5 ± 8.92). At M2, the mean quantity of immunostained cells in the periphery of treated (9.16 ± 4.24) and control (9 ± 3.97) corneas did not differ. In the central area, there was a higher expression in TG (22.83 ± 13.41) than in CG (15.5 ± 6.52). No significant difference was found when comparing the values (p=0.32).

No correlation was observed between iNOS and MMP-9 in the epithelium of TG (r=0.30 and p=0.34) and in the stroma of treated (r=0.20 and p=0.54) and CG (r=0.41 and p=0.18). Moderate correla-tion (r=0.70 and p=0.01) was observed between iNOS and MMP-9 expression in the epithelium of CG.

No correlation was observed between iNOS expression and the amount of inflammatory cells in the stroma in TG (r=0.28 and p=0.38) or CG (r=0.56 and p=0.06).

DISCUSSIONRepeated instillation of ketorolac tromethamine influences the

development of corneal lesions(14) as also evidenced by the results found in this study. The commercial formulation, i.e., without preser-vatives, was chosen because it may be toxic to the epithelium(15,16). Control corneas epithelialized faster, and this observation supports the fact that the used drug was toxic and the effect could have resul-ted from changes in the cytoskeleton of epithelial cells(17). Similarly, desquamation and decrease in microvilli in corneas treated with ke-torolac tromethamine without preservatives have been observed(3).

The effect of ketorolac tromethamine and its preservatives on the epithelialization of debrided corneas with 95% ethyl alcohol has been studied in rabbits(14). The authors found that eyes treated with NSAIDs were still ulcerated 60 h after abrasion. In the present study, treated eyes had epithelialized by 55 h following abrasion.

The present investigation showed significantly lower inflammatory exudation in treated eyes. Similar findings were presented after using ketorolac tromethamine with preservatives in chemical ulcers in rab-bits(14). Infiltration by polymorphonuclear leukocytes, mainly neutrophils, is necessary for epithelialization. The inflammatory infiltrate influences resident cells by producing cytokines that modulate tissue repair(18).

The number of inflammatory cells in the periphery was higher in control corneas. The amount of cells in the central region did not sig-nificantly differ between the groups. In a sterile inflammation model, central infiltrates consist mainly of neutrophils and monocytes(10); this in agreement with the findings of this study.

It has been demonstrated that iNOS is expressed in rat corneas during the inflammatory course following chemical abrasion by alkali(10). The expression reached a maximum between 4 and 7 days fol lowing cauterization in the central corneal area. These data were consistent the findings of this study, in which the greatest number of positive iNOS cells occurred in M2 of the evaluation; in the central re-gion, despite a higher number of inflammatory cells in the periphery.

Figure 1. Mean and standard error* of numbers of inflammatory cells in the periphery (P) and center (C) of male adult rabbit corneas ulcerated with 1 M NaOH and treated locally with 0.5% ketorolac tromethamine without preservative (gray) or with 0.9% saline solution (white) at first moment (M1, 24 hours after chemical burn) and second moment (M2, after reepithelialization). *Bonferroni’s Test.



A

C

B

D

Figure 2. Photomicrographs of iNOS immunostaining of male adult rabbit corneas ulcerated with 1 M NaOH and locally treated with 0.5% trome-thamine ketorolac (A and B) without preservative or (C and D) with 0.9% saline solution. A) Immunostained cells (arrows) in the corneal stroma treated at first moment (M1, 24 hours after chemical burn). B) Immunostained cells (arrows) in the corneal stroma treated at second moment (M2, after reepithelialization). C) Immunostained cells (arrows) in the control corneal stroma treated at M1. D) Immunostained cells (arrows) in the control corneal stroma at M2. Diaminobenzidine. (Scale bar=50 µm).

Figure 3. Mean and standard error* of quantitative iNOS immunostaining of stromal cells in the periphery (P) and center (C) of male adults rabbit corneas ulcerated with 1 M NaOH and treated locally with 0.5% ketorolac tromethamine without preservative (gray) or with 0.9% saline solution (white) at first moment (M1, 24 hours after chemical burn) and second moment (M2, after reepithelialization). *Bonferroni’s Test.

Immunostaining of iNOS were similar to that observed for neu-trophils(10). However, few cells showed positive labeling for macro-

phages. These observations suggest that neutrophils and monocytes may be the primary source of NO in corneas. Using the same model of chemical abrasion and considering the type of inflammatory infiltrate found in the corneas in this study, the same cells are speculated to be involved in iNOS expression. However, no correlation between iNOS expression and the amount of inflammatory cells was found. Langerhans cells have been found in the central epithelium after thermal cauterization(19) and they may act in NO production.

Immunostaining of iNOS in keratocytes was observed in areas adjacent to the lesions. In vitro, iNOS may be expressed by keratocytes upon stimulation by cytokines(8). The possibility that these cells have contributed to in vitro production of NO could not be excluded. Reac-tions that occur in different stages of corneal repair, however, are complex compared with those processed in vitro(10).

iNOS expression was higher when compared with controls but the difference was nonsignificant. Contrary to these results, a proan-giogenic role of NO in vitro has been reported(20,21). Such differences may be related with the expression of distinct isoforms of NOS de-pendent on the experimental model employed. The mechanism by which NO inhibits angiogenesis in the corneal cauterization model is not completely understood. However, it is clear that inhibition of angiogenesis by iNOS decreases the amount of inflammatory cells by inducing apoptosis(22). When inhibiting inflammatory exudation, NSAIDs decrease neovascularization, with iNOS playing an important role. Involvement of other NOS isoforms, except iNOS, in vasculature induction has been demonstrated(9). These findings justify the expres-

Lima TB, et al.


A

C

B

D

Figure 4. Photomicrographs of MMP immunostaining in male adult rabbit corneas ulcerated with 1 M NaOH and locally treated with 0.5% ketorolac tromethamine (A and B) without preservative or (C and D) with 0.9% saline solution. A. Immunostaining of the corneal epithelium (arrows) treated at first moment (M1, 24 hours after chemical burn). B. Immunostaining of the corneal epithelium (arrows) treated at second moment (M2, after reepithelialization). C. Immunostaining (arrows) of the control cornea at M1. D. Immunostaining of the epithelium (arrows) of the control cornea at M2. Diaminobenzidine (Bar=50 µm).

sion of iNOS in TG and the lack of correlation between an event and the amount of inflammatory cells.

Kim et al. studied iNOS immunostaining after laser-assisted la-mellar keratoplasty(22). Chen et al. observed iNOS immunostaining in corneal epithelium upon stimulation by ultraviolet radiation(9). In the present study, iNOS expression was similar in both groups, despite the study drug being toxic to the epithelium.

Regarding MMP-9, we observed pronounced immunostaining in the epithelial area adjacent to the lesion. Authors have reported that basal epithelial cells synthesized MMP-9 in corneas that under-went lamellar keratectomy(23); in addition, inflammatory cells and keratocytes were positive by immunostaining for MMP-9.MMP-9 is involved in the early stages of corneal epithelium repair(23,24). In the present study, MMP-9 expression was observed in the epithelium at both M1 and M2. When stromal cells were evaluated, a higher expres-sion in the corneal central region was observed at M1. The highest activity of MMP-9 was observed at M1, which was also observed in cases of keratotomy(23).

Changes in MMPs due to the local use of NSAIDs may delay cor-neal epithelium repair(25). Collagenases may impair cicatricial healing, breaking the basal membrane interactions of epithelial cells(25). MMP production was found in the intact corneal epithelium after NSAIDs instillation(2). Such findings indicate their involvement in the worse-ning of corneal ulcers(2,25).

Delayed corneal repair associated with the use of local NSAIDs and expression of MMP-1, MMP-8, MMP-2 at significantly high levels, and low levels of MMP-9 in corneas treated with ketorolac trometha-

mine have been reported(2). In this study, lower expression of MMP-9 was observed in the epithelium of TG compared with CG, but without significant differences. Other authors found no difference in the ex-pression of MMPs; however, differences in the amount of MMP-9 and MMP-2 were determined by zymography(26).

In inflammatory processes, it has been demonstrated that NO and peroxynitrite can alter levels of MMPs and tissue inhibitors of MMPs (TIMPs)(27). However, no correlation between MMP-9 expression and iNOS was found in this study. Data are available on the involvement of oxidative stress in inflammatory processes, in which macrophages, polymorphonuclear cells, and other inflammatory cells are involved in the activation of lytic enzymes(11). High concentrations of peroxy-nitrite in vitro reduced TIMP-1 and hence the inhibition of MMP-9(28). Similar results were found by Brown et al.(11) in 2004, showing a cellular response to oxidative stress induced by peroxynitrite, suggesting a relation between elements of oxidative stress with activity of degra-dative enzymes. Other MMPs may be activated and involved in the reactions, as would be with regard to the higher activity of TIMPs.

Based on the obtained results, 0.5% ketorolac tromethamine without preservative may induce less inflammatory exudation and delay corneal epithelialization, eliciting non-significant changes in the expression of iNOS and MMP-9.

ACKNOWLEDGEMENTSThe authors are thankful to the Fundação de Amparo à Pesquisa

of the State of São Paulo-FAPESP (proc. 2011/07848-0) and to the



Conselho Nacional de Desenvolvimento Científico e tecnológico - CNPq (proc. 300833/2010-5) for financial support.

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Original Article


INTRODUCTIONWith the increasing popularity of phacoemulsification as a me-

thod of choice for cataract surgery, the incidence of complications such as inadvertent posterior capsule tear, nuclear fragments, and intraocular lens (IOL) loss into the vitreous cavity has increased greatly(1-6).

These complications are directly related to the surgeon’s ex-pertise and tend to increase in specific cases that represent major challenges. These cases include inadequate zonular support (pseu-doexfoliation, trauma, and previous vitrectomy), mature and hyper-mature cataracts, high axial myopia, insuficient mydriasis, patient movements during the perioperative period, among others(2,6,7-12).

Nuclear and IOL fragmentation in the vitreous may trigger serious consequences, including permanent visual loss, if not treated pro-perly. These complications induce a severe inflammatory response

that is proportional to the excessive intraocular manipulation trauma and the nuclear fragment size or IOL model and material. These pa-tients may also develop chronic uveitis, secondary glaucoma, corneal edema, and retinal detachment. One study showed a 52% incidence of glaucoma in such a condition(3).

To determine the best clinical or surgical treatment strategy for such conditions, ophthalmologists mainly consider fragment size and the presence or absence of corneal edema and glaucoma(13-20).

Studies on the removal time of vitrectomy nuclear fragments indicated that it is not necessary for this procedure to be performed on the same day and that these fragments can be removed up till 1-2 weeks after surgery(3,6,13-20).

This study aims to identify the causes and results of pars plana vitrectomy (PPV) in patients who underwent phacoemulsification with intraoperative complications and to analyze whether the surgical

Results of pars plana vitrectomy after complicated phacoemulsification surgery

Resultados pós-vitrectomia via pars plana em pacientes submetidos à cirurgia de facoemulsificação com complicação intraoperatória

alExandrE grandinEtti1, diogo suEnaga2, FErnanda M. olivEira3, Krissis saliba uliana cruz2, laurinda MEnEguEttE2, lucianE bugMan MorEira4

Submitted for publication: June 9, 2014 Accepted for publication: December 11, 20141 Department of Surgery, Federal University of Parana (UFPR), Curitiba, PR, Brazil.2 Anterior Segment Sector, Paraná Eye Hospital, Curitiba, PR, Brazil.3 Retina and Vitreous Sector, Paraná Eye Hospital, Curitiba, PR, Brazil.4 Department of Ophthalmology, Medical School, Positivo University, Curitiba, PR, Brazil.



Corresponding author: Krissis Saliba Uliana Cruz. Rua Frei Caneca, 3.159 - Guarapuava, PR - 85015-220 - Brazil - E-mail: [email protected]

Approved by the following research ethics committee: Positivo University (CAAE 27185914. 6.0000.0093).

ABSTRACTPurpose: To identify the causes and outcomes of pars plana vitrectomy (PPV) in patients undergoing phacoemulsification with intraoperative complication and to analyze whether the interval between phacoemulsification and PPV interferes with best-corrected final visual acuity. Methods: This descriptive and retrospective analytical study was conducted in Paraná Eye Hospital in 2013. Data were collected from medical records of 38 patients who underwent complicated phacoemulsification and also required PPV. Results: The most frequent complication as a result of phacoemulsification was posterior capsule rupture, observed in 35 patients (92.10%), followed by capsular bag detachment, in three patients (7.89%). Twenty-eight patients (73.68%) had cortical fragments that were removed during PPV. Twelve patients (31.57%) had their intraocular lens repositioned. PPV was performed on the same day of pha-coemulsification in one patient (2.63%), within 1 week in 15 patients (39.47%), between 1 week and 1 month in 13 patients (34.21%), and 1 month after phacoe-mulsification in 9 patients (23.68%). Conclusion: This study is in agreement with worldwide literature, asserting that major complications of phacoemulsification are posterior capsule rupture and capsular bag detachment, and in addition, there is an improvement in the final visual acuity in almost half the cases, even when there are complications during modern cataract surgery, when complementary appropriate treatment is provided.

Keywords: Phacoemulsification/complications; Vitrectomy/methods; Cataract ex-traction

RESUMOObjetivos: Identificar as causas e os resultados da vitrectomia via pars plana (VPP) em pacientes submetidos à cirurgia de facoemulsificação com complicação intrao-peratória, analisando se o tempo cirúrgico entre a facoemulsificação e a VPP interfere na melhor acuidade visual corrigida final. Métodos: Estudo analítico descritivo e retrospectivo realizado no Hospital de Olhos do Paraná em 2013. Os dados foram coletados de prontuários de 38 pacientes que foram submetidos à cirurgia de facoemulsificação complicada e que também preci-saram de VPP. Resultados: A complicação intraoperatória mais frequente na cirurgia de fa-coemulsificação, nos pacientes estudados, foi à ruptura de cápsula posterior, que ocorreu em 35 pacientes (92,10%), seguido de desinserção zonular em 3 pacientes (7,89%). Em 28 pacientes (73,68%) foram encontrados restos corticais, que foram removidos durante a VPP. Em 12 pacientes (31,57%) foi realizado o reposicionamento da lente intraocular. A cirurgia de VPP foi realizada no mesmo dia da facoemul-sificação em 1 paciente (2,63%), dentro de 7 dias em 15 pacientes (39,47%), entre 1 semana e 1 mês em 13 pacientes (34,21%) e após 1 mês da facoemulsificação em 9 pacientes (23,68%). Conclusão: O presente estudo encontrou dados semelhantes aos descritos na li teratura mundial, que afirmam que as principais complicações da facoemulsificação são a ruptura de cápsula posterior e desinserção zonular; e que a acuidade visual final melhora, em aproximadamente metade dos casos, mesmo após ocorrer complica-ções na cirurgia de catarata moderna, quando instituído tratamento complementar adequado.

Descritores: Facoemulsificação/complicações; Vitrectomia/métodos; Extração de ca tarata

Results of pars plana vitrectomy after complicated phacoemulsification surgery


interval between phacoemulsification and PPV interferes with the best-corrected final visual acuity (VA).

METHODSA descriptive and retrospective analytical study was conducted

at Paraná Eye Hospital by reviewing 38 patient records.The inclusion criteria were as follows: patients who underwent

cataract surgery by phacoemulsification with intraoperative compli-cations and required postoperative PPV immediately after cataract surgery. Considered surgeries were performed between January and December 2013.

The exclusion criteria were as follows: patients with incomplete follow-up during the study period or those with incomplete medical records.

The following data were collected for this study: gender, age, ori-gin, best-corrected visual acuity (BCVA) prior to phacoemulsification and a month after PPV, and complications or procedures required during the last surgery.

The ophthalmologic examination included the following pro-cedures: average VA with best correction according to the Snellen chart, biomicroscopy with Zeiss slit lamp, intraocular pressure measu-rement by Perkins tonometer, and retinal mapping by EyeTech indi-rect ophthalmoscopy. Legacy 20000™ (Alcon) and Infiniti® (Alcon) were used for phacoemulsification, and Stellaris (Bausch & Lomb) was used for vitrectomy.

Third-year ophthalmology residents and fellows of the cataract and anterior segment department of Paraná Eye Hospital performed the phacoemulsification surgeries. Experienced physicians expertise in retinal procedures performed the PPV procedures.

The study design was submitted to and approved by the Ethics Committee of Positivo University. Patient identities were not collec-ted, ensuring patient anonymity. Because the aim of the study was the collection of medical appointment data, there were no interven-tions in the physician conduct, which occurred independent of the study.

RESULTSBetween January and December 2013, 42 patients underwent

complicated cataract surgery followed by PPV. Four of these patients were excluded from the study because of incomplete records or follow-up. Of the 38 remaining patients, 12 were male (31.5%) and 26 female (68.4%). The average age was 69.42 ± 13.89 years (minimum of 5 years, maximum of 84 years). Half of the patients were from Curitiba, Paraná and the other half were from other locations.

Prior to phacoemulsification, eye comorbidities (besides cataract) were found in six patients (15.78%): glaucoma (five patients) and age- related macular degeneration (one patient). Of the 38 patients, 13 (34.21%) had diabetes mellitus type II and 11 (28.94%) had systemic arterial hypertension without fundoscopic changes.

Table 1 shows the general view of the BCVA values prior to pha-coemulsification (BCVA pre-surgery) and 30 days after vitrectomy (BCVA post-surgery).

When analyzed individually, 18 (47.36%) patients presented an improvement in the final BCVA after PPV compared with BCVA prior to phacoemulsification, whereas 11 (28.94%) had worsening of BCVA. BCVA remained unchanged in nine (23.68%) patients. Of the 18 pa-tients with improvement in the final BCVA, 15 (83.33%) underwent PPV within the first month after phacoemulsification.

Eighteen (47.37%) phacoemulsification procedures were perfor-med within the first half of 2013 and 20 (52.43%) in the second half. Vitrectomy (PPV) was performed on the same day as phacoemulsifi-cation in one patient (2.63%), within 7 days in 15 patients (39.47%), between 1 week and 1 month in 13 patients (34.21%), and 1 month after phacoemulsification in nine patients (23.68%)(Table 2).

Posterior capsule tear was the most frequent intraoperative com-plication (35 patients, 92.10%), followed by detachment of the cap-sular bag in three patients (7.89%). Cortical fragments were found in 28 patients (73.68%) and were removed during PPV. Twelve patients (31.57%) required intraocular lens repositioning in the ciliary sulcus.

DISCUSSIONCortical fragments were observed and surgically removed in

73.68% of the patients who underwent PPV. The remaining patients underwent PPV for removal of the nucleus from the vitreous. Gilliland showed a similar occurrence of cortical fragments in patients who underwent PPV(3).

Lavinski et al. revealed that the surgical interval between cataract surgery and PPV was more than 15 days for most patients. In the present study, this surgical interval ranged from up to 15 days (16 patients; 42.10%) to more than 15 days (22 patients; 57.89%)(21). On the other hand, the authors suggested that a shorter surgical interval between the two procedures results in an improvement of the final VA. This finding is consistent with that of the present study, given that of the 18 patients that showed improvement in the final BCVA after PPV, 13 (72.22%) had undergone this procedure within the first month after phacoemulsification(21,22).

The average patient age in the present study was 69.42 years, similar to that obtained by Santacruz I in his RCP and final VA study,

Table 1. Best-corrected visual acuity (BCVA) prior to phacoemulsifica-tion (Pre-Phaco) and after pars plana vitrectomy (Post-PPV), n=38

BCVA

Pre-Phaco Post-PPV

N % N %

20/20 to 20/30 00 0 06 015.78

20/40 to 20/60 14 036.8 09 023.68

20/70 to 20/200 09 023.6 08 021.05

20/300 to 20/400 01 02.63 03 007.89

Worse than 20/400 14 036.8 12 031.57

Total 38 100.0 38 100.00

BCVA= best-corrected visual acuity.

Table 2. Best-corrected visual acuity (BCVA) prior to phacoemulsification (Pre-Phaco) and post pars plana vitrectomy (Post-PPV), according to the interval between surgeries, n=38

Surgical interval between phaco and PPV

Pre-Phaco Post-PPV

<20/400 ≥20/400Average of the eyes

≥20/400 <20/400 ≥20/400Average of the eyes

≥20/400

Up to 7 days 7 09 20/70 5 11 20/50

Between 8 and 30 days 3 10 20/60 2 11 20/60

After 30 days 4 05 20/70 5 04 20/40

Grandinetti A, et al.


and by Falavarjani et al. in their study of PPV for the removal of the nucleus from the vitreous (23).

Tavares et al. observed ocular comorbidities in 35% of patients, with glaucoma being the most frequent condition. These findings are consistent with those of in the present study(24).

It is important to emphasize that RCP is a complication that can occur with any surgeon and its proper management requires expe-rience. Its management includes vitrectomy in order to minimize final VA losses(25).

As demonstrated, the results obtained in the present study are consistent with existing worldwide literature statistics.

It is crucial to point out the importance of an integrated and readily available team of retina specialists in order to manage such complications, provide the best prognosis, and consequently, increa-se quality of life.

CONCLUSIONIn the present study, the most frequent intraoperative compli-

cation during phacoemulsification was the posterior capsule tear, followed by capsular bag detachment.

Almost half of the patients presented improvement in the final BCVA after PPV compared with BCVA prior to phacoemulsification.

Most of the patients who presented improvement in the final BCVA underwent PPV within the first month after phacoemulsifica-tion, suggesting that a shorter surgical interval between phacoemul-sification with complication and PPV improves the final BCVA.

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Original Article

76 Arq Bras Oftalmol. 2015;78(2):76-81 http://dx.doi.org/10.5935/0004-2749.20150021

INTRODUCTIONKeratoconus is a non-inflammatory ectatic corneal disorder. It is

characterized by paracentral corneal thinning and increased corneal curvature leading to irregular astigmatism, myopia, and protrusion(1). Initial treatment includes using spectacles and rigid contact lenses. Several surgical procedures, such as corneal transplantation, epike-ratophakia, and photorefractive keratectomy, have been developed for more advanced cases; however, some of these have yielded di-sappointing results(2-4).

Intrastromal corneal ring segments (ICRS) are an interesting al-ternative for keratoconus treatment in patients with clear corneas and contact lens intolerance(5-7). There are several different models with varying sizes and arch thicknesses. These segments induce an arch shortening effect in the lamella leading to central flattening of the cornea. The main advantages of this procedure are its safety, reversibility, and stability as well as the fact that segments do not affect the corneal visual axis(8-13). The intrastromal tunnel for ring implantation was initially manually constructed; however, complica-

Femtosecond-assisted intrastromal corneal ring implantation for

keratoconus treatment: a comparison with crosslinking combination

Implante de anel intraestromal com laser de femtosegundo no tratamento de ceratocone:

comparação da combinação com o crosslinking

PEtEr alExandEr von harbach FErEnczy1, Maiara dalcEgio1, MarcEla KoEhlEr2, thiago silvEira PErEira2, haMilton MorEira3, lucianE bugMann MorEira4

Submitted for publication: September 11, 2014 Accepted for publication: January 19, 20151 Anterior Segment Sector, Hospital de Olhos do Paraná, Curitiba, PR, Brazil.2 Refractive Surgery Sector, Hospital de Olhos do Paraná, Curitiba, PR, Brazil.3 Hospital de Olhos do Paraná, Curitiba, PR, Brazil.4 Contact Lens Sector, Hospital de Olhos do Paraná, Curitiba, PR, Brazil.



Corresponding author: Peter Alexander von Harbach Ferenczy. Alameda Presidente Taunay, 483 - Curitiba, PR - 80420-180 - Brazil - E-mail: [email protected]

Approved by the following research ethics committee: Universidade Positivo: number 465.316; date: November 22, 2013.

ABSTRACT Purpose: To compare visual outcomes, corneal astigmatism, and keratometric readings in patients with keratoconus who underwent intrastromal corneal ring implantation (ICRSI) alone with those who underwent ICRSI combined with ultra-violet A riboflavin-mediated corneal collagen crosslinking (CXL).Methods: Pre- and post-operative best-corrected distance visual acuity (BCDVA), spherical error, cylindrical error, and mean keratometry were retrospectively compa-red over a period of 2 years in patients with keratoconus who underwent only ICRSI (group 1) versus those in patients who underwent combined ICRSI-CXL (group 2). Results: Thirty-two eyes of 31 patients were evaluated. CXL was performed in 10 cases (31%), and there were no complications or need for ring repositioning. BCDVA improved from 0.54 to 0.18 in the group 1 and from 0.56 to 0.17 in the group 2. Spherical and cylindrical errors and mean keratometry values significan-tly decreased in both groups. No patient postoperatively had visual acuity (VA) of less than 20/60 on refraction, and 78% exhibited VA better than or equal to 20/40 with spectacles (72% of group 1 and 90% of group 2). Improvement in the spherical equivalent (SE) value was observed in the group 1 (from -5.89 ± 3.37 preoperatively to -2.65 ± 2.65 postoperatively; p<0.05) and group 2 (from -6.91 ± 1.93 preoperatively to -2.11 ± 3.01 postoperatively; p<0.05). Conclusion: Both techniques can be considered safe and effective in improving VA and refractive SE values, in decreasing the curvature of the cone apex in the topographical analysis, and in decreasing corrected diopters postoperatively in patients with keratoconus.

Keywords: Cornea; Corneal stroma; Keratoconus; Prosthesis implantation; Cross-lin king reagents; Riboflavin/therapeutic use; Ultraviolet rays; Visual acuity

RESUMOObjetivo: Comparar os resultados visuais, astigmatismo corneano e ceratometria em pacientes com ceratocone submetidos a implante de anel corneano intraestromal (ICRSI) e quando em combinação com radiação ultravioleta associado ao crosslinking do colágeno corneano mediada pela riboflavina (CXL). Métodos: Comparou-se retrospectivamente pacientes com ceratocone submetidos somente a implante de anel corneano intraestromal (grupo 1) versus o mesmo pro-cedimento associado ao crosslinking em um período de 2 anos. Avaliou-se acuidade visual com correção, equivalente esférico, ápice do cone na topografia e adaptação com lentes de contato pré e pós operatórios. Resultados: O estudo avaliou 32 olhos de 31 pacientes. Em 10 casos (31%) foi reali-zado crosslinking corneano, não havendo complicações ou necessidade de reposi-cionamento do anel. Acuidade visual corrigida pré e pós-operatória, componentes esférico e cilíndrico da refração e valores de ceratometria media diminuíram signi-ficativamente em ambos os grupos. Após o implante, nenhum paciente apresentou acuidade visual pior que 20/60 e 78% apresentaram acuidade corrigida melhor ou igual a 20/40 (72% do grupo 1 e 90% do grupo 2). Observou-se diminuição no valor do equivalente esférico no grupo 1 (de -5,89 ± 3,37 pré-operatório para -2,65 ± 2,65 pós-operatório; p<0,05) e no grupo 2 (de -6,91 ± 1,93 pré-operatório para -2,11 ± 3,01 pós-operatório; p<0,05). Conclusão: Ambas as técnicas podem ser consideradas seguras e eficazes na melhora da acuidade visual e equivalente esférico, diminuição do ápice de curvatura do cone na análise topográfica e na redução de dioptrias a serem corrigidas no pós-operatório de pacientes com ceratocone.

Descritores: Córnea; Substância própria; Ceratocone; Implante de prótese; Reagentes para licações cruzadas; Riboflavina/uso terapêutico; Raios ultravioleta; Acuidade visual

Ferenczy PAH, et al.


tions, such as epithelial defects, depth asymmetry, and perforation, were reported(5,6,13).

Femtosecond laser has recently been used to create the tunnel for ring implantation. This technique reportedly creates a tunnel with precise depth, width, and location leading to minimal haze and ede ma as well as minimal surgical complications. The laser acts via photodisruption and can be programed to create tunnels for seg-ment placement at predetermined depths. Studies have shown that tunnel creation using this technique is easier, more precise, and more predictable than the technique involving a conventional mechanical microkeratome(7).

We studied the outcomes of patients with keratoconus who underwent intrastromal corneal ring implantation (ICRSI) using fem-tosecond laser (Ziemer LDV) at the Hospital de Olhos do Paraná. Our aim was to evaluate the visual improvement in these patients, the safety of this technique, and the differences between ICRSI alone and its combination with postoperative ultraviolet A riboflavin-mediated corneal collagen crosslinking (CXL) procedure.

METHODSThis was an observational study approved by the Research Ethics

Committee of Positivo University on November 22, 2013 under the protocol number 465.316.

The medical records of patients with keratoconus seen at Hospital de Olhos do Paraná who underwent ICRSI using femtosecond laser or who had ICRSI combined with CXL were retrospectively analyzed. The medical records from January 2011 to December 2012 were reviewed, and the time of the follow-up that was considered for the analysis was from 6-15 months.

The inclusion criteria were as follows: (1) eyes with keratoconus in the topographical analysis confirmed by two experienced ophthal-mologists; (2) presence of the clear central cornea; (3) corrected visual acuity (VA) of less than 20/40 according to the Snellen chart; (4) into-lerance to contact lenses or no improvement in VA with contact len-ses; (5) a minimum central corneal thickness of 380 μ and a minimum corneal thickness of 400 μ at the site of the incision and construction of the corneal tunnel for ring implants; and (6) a minimum central corneal thickness of 400 μ for the CXL procedure.

The exclusion criteria were as follows: (1) incomplete clinical data in the medical records; (2) loss to follow-up of patients 3 months pos-toperatively; (3) history of eye diseases such as glaucoma, cataract, diabetic retinopathy, and age-related macular disease; (4) preopera-tive plano spherical equivalent (SE); (5) history of eye surgery; and (6) collagen disease or pregnancy.

In this study, ICRSI (CornealRing, Visiontech®, Belo Horizonte, Brazil) was performed by two experienced surgeons using the femto-second laser (Ziemer LDV, Ziemer Ophthalmic Systems, Switzerland) to create the stromal tunnel. The channel’s inner diameter was set to 4.8 mm and the outer diameter was set between 6.02 to 6.26 mm; the entry cut thickness was set to 1.3 mm and 10 degrees in length (at the steepest topographical axis), the velocity for was set for 3.2 mm/s, 1.0 mm/s and 5.2 mm/s for the stroma, vertical incision and insertion, respectively. The power was set at 100% for the stroma and at 150% for the vertical incision. A vacuum setting at 750 mbar on controlled mode with automatic release mode was applied. The nomogram is available online at www.cornealring.com.

The selected clinical and topographical data were assessed pre- and postoperatively. The clinical examination data included the best spectacle-corrected VA, spherical and cylindrical diopter values, and SE. The topographical data included the maximum, minimum, and mean keratometric values (Kmax, Kmin, and Kmed, respectively) as well as the corneal apex value in diopters (D). In addition, the infor-mation on the transoperative and postoperative complications and on the performance of the corneal crosslinking was considered.

The differences in VA and topographical data between pre- and post-ICRSI period as well as the perioperative and postoperative com-plications were assessed during the study period.

CXL was performed on eyes that showed evidence of ectasia and that still had low VA 3 months after ICRSI. Ectasia was considered if the topography showed at least 0.5 D of progression in the corneal curvature index over a 6-month period.

After central corneal abrasion in the operating room and under appropriate sterile conditions, 0.5% proparacaine hydrochloride drops were used as local anesthesia. The central corneal epithelium was removed (9 mm) with a blunt spatula. A photosynthetic ribofla-vin 0.1% solution (10 mg riboflavin-5-phosphate in 10 mL of dextran T-500 20%), was applied to the cornea every 5 min for 30 min before the ultraviolet A (UVA) irradiation.

The cornea was then exposed to UVA irradiation with a solid state device: the X-Link (Opto Electronics, San Carlos, Brazil), which emitted light at a wavelength of 370 ± 5 nm at an irradiance of 3 mW/cm2 or 5.4 J/cm2. The corneal exposure lasted 30 min, while the riboflavin solution was applied every 5 min.

The data were described as the mean, median, minimum and maximum values, and standard deviations. The preoperative compa-risons were performed using Student’s t-test for independent sam ples. The postoperative comparisons and differences between pre- and postoperative values were based on the model analysis of covariance (ANCOVA), including the preoperative evaluation as a co-variate. To compare the pre- and post-ratings within each treatment, we used Student’s t-test for paired samples; for variable VA logMAR comparisons, we used the nonparametric Mann-Whitney test. Com-parisons between pre- and postoperative values within each group were made utilizing the non-parametric Wilcoxon test. P values of <0.05 were considered statistically significant. The normal variables were evaluated by the Kolmogorov-Smirnov test. All analyses were performed using IBM SPSS Statistics v.20.

RESULTSThirty-one patients with keratoconus who underwent ICRSI using

femtosecond laser (32 eyes) were studied. The group 1 consisted of 14 (63%) men and eight (37%) women with ages between 16 and 52 years (mean 28.9 ± 8.2 years). The group 2 consisted of seven (70%) men and three (30%) women with ages between 19 and 37 years (mean, 27 ± 6.4).

Except for the mean K (p=0.02), all other preoperative variables were similar between the two groups (age: p=0.53; gender: p=1; spherical refractive error: p=0.67; astigmatism: p=0.09; corneal apex: p=0.49; and VA in logMAR: p=0.76), as presented in table 1.

Table 1. Evaluation of associated factors with conus progression after ICRS

Variable

Group

P value*ICRSI ICRSI + CXL

Age (years) 28.90 ± 8.20 27.00 ± 6.40 0.529

Male 63.6% 70.0% 1

Spheric pré -3.99 ± 3.08 -4.45 ± 2.18 0.674

Cilindric pré -3.80 ± 1.96 -4.93 ± 1.03 0.098

SE pré -5.89 ± 3.37 -6.91 ± 1.93 0.379

K-average pré 50.90 ± 2.75 47.06 ± 2.24 0.018

Apex pré 58.19 ± 2.99 56.56 ± 4.75 0.493

VA logMAR 0.54 ± 0.24 0.56 ± 0.34 0.764

*Student’s t-test or exact Fisher test, p<0.05

Femtosecond-assisted intrastromal corneal ring implantation for keratoconus treatment: a comparison with crosslinking combination


Table 2. Differences in best-corrected visual acuity between groups

Treatment n Mean Median Minimum Maximum Standard deviation P value

Preoperative VA ICRSI 22 0.54 0.47 -0.30 1.30 0.24

logMAR ICRSI + CXL 10 0.56 0.47 -0.30 1.30 0.34 0.764

Postoperative VA ICRSI 22 0.18 0.09 -0.00 0.47 0.17

logMAR ICRSI + CXL 10 0.17 0.17 -0.09 0.47 0.11 0.646

Difference pre- and post-operative ICRSI 22 0.36 0.38 -0.17 0.83 0.22

ICRSI + CXL 10 0.40 0.34 -0.13 0.83 0.25 0.857

*= nonparametric Mann-Whitney test, p<0.05.VA= visual acuity; ICRSI= intrastromal corneal ring implantation; CXL= crosslinking.

Figure 1. Pre- and postoperative VA data per groups (logMAR).

CXL was performed in 10 eyes (31%) at a mean time of 5.8 ± 2.04 months after the ring insertion. In all cases in which CXL was per-formed, there were no complications or need for ring repositioning.

VA of patients preoperatively ranged from 20/40 to 20/400 (Snellen chart) in both groups. No patient postoperatively presented with VA worse than 20/60, and 78% exhibited VA better than or equal to 20/40 with spectacles in both groups. Table 2 and figure 1 show differences in the best-corrected VAs before and after the procedure.

In the group 1, the spherical refraction ranged from -9.50 D to plano preoperatively, with a mean cylindrical value of -3.80 D. In the group 2, the spherical refraction ranged from -7.00 to -0.25 D preo-peratively, with a mean cylindrical value of -4.93 D.

Postoperatively, the spherical refraction in the first group ranged from -7.00 to +1.50 D; the mean cylindrical value was -2.22 D. In the group 2, the spherical refraction ranged from -7.00 to +4.75 D, and the mean cylindrical value was -2.38 D.

The SE improved in both groups; SE improved from -5.89 ± 3.37 to -2.65 ± 2.65 (p<0.001) in the group 1 and from -6.91 ± 1.93 to -2.11 ± 3.01 postoperatively (p<0.001) in the group 2.

Regarding the keratometric values in the pre- and postoperative topographical analyses, only one implant did not demonstrate a decreased corneal apex value. However, all other patients had an improvement in VA and SE (group 1, p=0.001; group 2, p=0.01). Table 3 and figure 2 show the pre- and postoperative keratometric data.

The mean K value of the corneal apex improved after the pro-cedure (group 1, p=0.001; group 2, p=0.007), although there was no significant statistical difference between the groups (Table 4 and Figure 3).

In the group 2, when comparing the data 3 months after ICRSI, we observed statistical differences in the spherical refractions (p=0.007), astigmatism (p=0.01), SEs (p=0.002), corneal apices (p=0.006), and VAs (p=0.007) (Table 5).

DISCUSSIONThe correction of irregular astigmatism caused by primary corneal

ectasia is a challenging process. With advancing topographical chan-ges, optical correction becomes ineffective, and the main treatment consists of rigid contact lenses. These provide a uniform surface that neutralizes the myopia and irregular astigmatism associated with keratoconus. When these patients become intolerant to contact lenses, even in the absence of a lesion at the ectasia apex, corneal transplantation is recommended(14).

Several surgical procedures have been proposed as an alternative to penetrating keratoplasty for the treatment of keratoconus, such as photorefractive keratectomy, epikeratoplasty, sectorial keratectomy, and lamellar keratoplasty; however, some of these have yielded di sap-pointing results(2-4). The deep lamellar keratoplasty and penetrating ke-ratoplasty procedures have considerably improved in recent years(15,16). The best spectacle-corrected VA, refractive results, and com plication rates have been similar for both techniques, although the nature of complications varied depending on the technique. Deep lamellar kera-toplasty is technically more challenging than penetrating keratoplasty; however, it prevents endothelial rejection and may reduce the risk for late endothelial failure(15). Despite its complications, such as the side effects of corticosteroids and allogeneic reac tions, the success rate of penetrating keratoplasty is 93%-96%(17,18).

In 2000, Colin et al.(19) described their preliminary results for kera-toconus management using ICRSI. Since then, several studies have shown that this was a safe procedure for the correction of corneal ectasias and astigmatism using the manual tunnel dissection tech-nique(19-22). However, several complications related to the manual dissection technique have been observed: epithelial defects, anterior and posterior corneal perforation, superficial placement and displa-cement of the segment, stromal thinning, extension of the incision to the center of the cornea or close to the limbus, infectious keratitis due to the introduction of epithelial cells in the tunnel, extrusion of the segment, and stromal edema around the tunnel(23, 24). Rabinowitz et al.(25) observed epithelial defects in 50% of patients when using the manual tunnel dissection technique.

A small amount of long-term data is available for ICRS placement, and any effect of ICRSI on disease progression remains uncertain. In a case series of Intacs ICRSI with 3-year follow-up in 13 eyes, significant increases in the average K values were observed between 6 months and 3 years, indicating that disease stabilization was not achieved by ICRSI alone(8).

Intacs alone may not stop progressive keratoconus. Alió et al.(8) found a 1.67 D rise in mean K values between 6 months and 36 months in a series of 13 eyes after ICRSI. This was expected, particularly in



Table 3. Differences in mean keratometric data between groups


Mean K pre ICRSI 13 50.90 50.89 47.34 55.74 2.75

ICRSI + CXL 07 47.06 47.15 42.61 49.72 2.24 0.018**

Mean K post ICRSI 13 47.12 47.43 43.33 51.76 2.56

ICRSI + CXL 07 44.50 45.61 42.03 46.05 1.78 0.768**

Difference pre and post ICRSI 13 03.78 03.99 -2.29 06.27 2.10

ICRSI + CXL 07 02.56 02.45 0.41 05.16 1.85 0.768**

*= Student’s t-test for independent samples, p<0.05; **= ANCOVA. VA= visual acuity; ICRSI = intrastromal corneal ring implantation; CXL= crosslinking.

Table 4. Differences in corneal apex between groups


Apex pre ICRSI 13 58.19 58.18 53.03 63.40 2.99

ICRSI + CXL 07 56.65 57.72 48.06 62.43 4.75 0.493**

Apex post ICRSI 13 53.57 52.02 47.13 64.77 4.27

ICRSI + CXL 07 51.06 51.49 47.53 55.11 2.62 0.309**

Difference pre and post ICRSI 13 04.63 05.46 -6.59 08.35 3.66

ICRSI + CXL 07 05.59 06.23 -1.38 10.43 3.67 0.309**

*= Student’s t-test for independent samples, p<0.05; **= ANCOVA. VA= visual acuity; ICRSI= intrastromal corneal ring implantation; CXL= crosslinking.

se= standard error; sd= standard deviation.

Figure 2. Pre and postoperative mean K data per groups (diopters).

cases of rapidly-progressing keratoconus, because the ring insertion does not treat the underlying structural issue of the weakened colla-gen. Therefore, combining CXL with ICRS insertion in patients with progressive keratoconus is an attempt to ensure stability. It has been proven that CXL, unlike ring insertion, increased the biomechanical rigidity by 4.5-fold. In addition to the crosslinking of the collagen lamellae, the collagen fibril diameter also increases(26).

The literature has not shown significant differences between refractive outcomes among different models with same shapes and sizes for ICRS. Haddad et al. compared Intacs SK ICRS and Keraring SI6 ICRS; both ICRS models significantly improved the visual function

in patients with keratoconus with comparable postoperative profiles and no major complications(27).

The first clinical result on the use of femtosecond laser for tun-nel creation was reported in 2003 by Ratkay-Traub et al.(28). They evaluated a limited series of 16 eyes and obtained refractive results similar to those observed in patients who had the tunnel construc-ted manually. Others have also reported a significant improvement in corrected and uncorrected VA and in keratometric values after ICRSI using femtosecond laser(25,29,30). In a study by Coskunseven et al. in 2008(30), 68% of 50 eyes exhibited improved corrected VA. The mean keratometry decreased from 50.6 D to 47.5 D, and the mean SE

Femtosecond-assisted intrastromal corneal ring implantation for keratoconus treatment: a comparison with crosslinking combination


Table 5. Follow-up of the ICRS + crosslinking group after 3 months

Variable Pré Pós 3 m P value*

VA CC 20.00 ± 0.00 20.00 ± 0 .00 -

VA logMAR pré 0.56 ± 0.34 0.21 ± 0.12 0.007

Spheric pré -4.45 ± 2.18 -2.33 ± 3.13 0.007

Cilindric pré -4.93 ± 1.03 -2.30 ± 1.88 0.010

SE pré -6.91 ± 1.93 -3.38 ± 3.42 0.002

K-average pré 47.20 ± 2.10 44.10 ± 4.00 0.088

Apex pré 56.90 ± 4.40 52.10 ± 3.00 0.006

*= paired Student’s t-test, p<0.05.

se= standard error; sd= standard deviation.

Figure 3. Pre and postoperative mean apex data per groups (diopters).

decreased from -5.6 D to -2.4 D over a year. In our study, we observed an improvement in the corrected VA in 93.7% eyes. The mean kerato-metry decreased from 50.90 D to 47.12 D, and the mean SE decreased from -5.89 D to -2.65 D over a year in the group that also had ICRSI. When associated to CXL, the mean keratometry decreased from 47.06 D to 44.50 D, and the mean SE decreased from -6.91 D to -2.11 D during the same period. We also found two recipients in the group 1 that did not gain any lines of vision (in one case, the SE changed from -5.00 D to -8.00 D; and in the other, the SE improved from -2.25 to -0.5 D with no gain in VA).

Kubaloglu et al.(29) compared the manual technique with the fem-tosecond laser and reported a significantly higher rate of epithelial defects when using the manual technique (44% vs. 14%). In this study, authors obtained similar refractive SE and VA results with both tunnel construction techniques and observed that the incidence of complications was significantly lower when using the femtosecond laser technique than that with the manual technique. Moreover, it has been confirmed that using laser for tunnel construction made the procedure easier, quicker, and more comfortable for the patient and surgeon and allowed a more precise corneal dissection at a predetermined depth(25,27). In 2011, Coskunseven et al. described the occurrence of intraoperative complications during ring implantation in 850 eyes using femtosecond laser, including incomplete tunnel formation (2.6%), galvanometer lag error due to system malfunction (0.6%), endothelial perforation (0.6%), incorrect entry into the tunnel (0.2%), and loss of vacuum (0.1%) as well as postoperative compli-

cations, such as ring migration (0.8%), corneal melting (0.2%), and infection (0.1%)(28). In our study, no intraoperative or postoperative complications were observed, including in the group that also un-derwent CXL.

CONCLUSIONIn summary, we observed a significant improvement in VA and

refractive SE values, decreased curvature of the cone apex in the topographical analysis, and decreased corrected diopters postope-ratively. ICRS implants do not ensure control of the ectasia and CXL does not affect visual outcomes in patients who underwent the ICRSI procedure. Furthermore, no postoperative complications were observed with the femtosecond laser and CXL procedures. Both techniques were safe and effective in reducing irregular astigmatism and myopia in patients with keratoconus.

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thinning disorders. Surv Ophthalmol. 1984;28(4):293-322. 2. McDonald MB, Kaufman HE, Durrie DS, Keates RH, Sanders DR. Epikeratophakia for

keratoconus. The nationwide study. Arch Ophthalmol. 1986;104(9):1294-300. 3. Koch DD. Refractive surgery for keratoconus: a new approach. J Cataract Refract Surg.

2000;26(8):1099-100. 4. Sekundo W, Stevens JD. Surgical treatment of keratoconus at the turn of the 20th

cen tury. J Refract Surg. 2001;17(1):69-73. 5. Kanellopoulos AJ, Pe LH, Perry HD, Donnenfeld ED. Modified intracorneal ring

segment implantations (INTACS) for the management of moderate to advanced keratoconus: efficacy and complications. Cornea. 2006;25(1):29-33.

6. Bourcier T, Borderie V, Laroche L. Late bacterial keratitis after implantation of intrastro-mal corneal ring segments. J Cataract Refract Surg. 2003;29(2):407-9.

7. Ertan A, Bahadir M. Topography-guided vertical implantation of Intacs using a femtosecond laser for the treatment of keratoconus. J Cataract Refract Surg. 2007; 33(1):148-51.

8. Alió JL, Shabayek MH, Artola A. Intracorneal ring segments for keratoconus correc-tion: long-term follow-up. J Cataract Refract Surg. 2006;32(6):978-85.

9. Burris TE, Ayer CT, Evensen DA, Davenport JM. Effects of intrastromal corneal ring size and thickness on corneal flattening in human eyes. Refract Corneal Surg. 1991; 7(1):46-50.

10. Alió JL, Artola A, Hassanein A, Haroun H, Galal A. One or 2 Intacs segments for the correction of keratoconus. J Cataract Refract Surg. 2005;31(5):943-53.

11. Tunc Z, Deveci N, Sener B, Bahcecioglu H. [Corneal ring segments (INTACS) for the treatment of asymmetrical astigmatism of the keratoconus. Follow-up after 2 years]. J Fr Ophthalmol. 2003;26(8):824-30. French.

12. Boxer Wachler BS, Christie JP, Chandra NS, Chou B, Korn T, Nepomuceno R. Intacs for keratoconus. Ophthalmology. 2003;110(5):1031-40.

13. Ruckhofer J, Stoiber J, Alzner E, Grabner G; Multicenter European Corneal Correction Assessment Study Group. One-year results of European multicenter study of intras-tromal corneal ring segments. Part 2: complications, visual symptoms, and patient satisfaction. J Cataract Refract Surg. 2001;27(2):287-96.

14. Zadok D, Schwarts S, Marcovich A, Barkana Y, Morad Y, Eting E, et al. Penetrating keratoplasty for keratoconus: long-term results. Cornea. 2005;24(8):959-61.

15. Watson SL, Ramsay A, Dart JK, Bunce C, Craig E. Comparison of deep lamellar kera-toplasty and penetrating keratoplasty in patients with keratoconus. Ophthalmology. 2004;111(9):1676-82.

16. Buzard KA, Fundingsland BR. Corneal transplant for keratoconus: results in early and late disease. J Cataract Refract Surg. 1997;23(3):398-406.

17. Sharif KW, Casey TA. Penetrating keratoplasty for keratoconus: complication and long- term success. Br J Ophthalmology. 1991;75(3):142-6.

18. Tuft SJ, Gregory WM, Davison CR. Bilateral penetrating keratoplasty for keratoconus. Ophthalmology. 1995;102(3):462-8.

19. Colin J, Cochener B, Savary G, Malet F. Correcting keratoconus with intracorneal rings. J Cataract Refract Surg. 2000;26(8):1117-22. Comment in: J Cataract Refract Surg. 2000;26(8):1099-100; J Cataract Refract Surg. 2001;27(3):341.

20. Lovisolo CF, Fleming JF. Intracorneal ring segment for iatrogenic keratectasia after laser in situ keratomileusis or photorefractive keratectomy. J Cataract Refract Surg. 2002;18(5):535-41.

21. Colin J, Cochener B, Savary G, Malet F, Holmes-Higgin D. INTACS inserts for treating keratoconus: one-year results. Ophthalmology. 2001;108(8):1409-14.

22. Ruckhofer J, Stoiber J, Alzner E, Grabner G; Multicenter European Corneal Correction Assessment Study Group. One year results of European multicenter study of intra-corneal ring segments. Part 1: refractive outcomes. J Cataract Refract Surg. 2001; 27(2):277-86.



23. Bourcier T, Borderie V, Laroche L. Late bacterial keratitis after implantation of intras-tromal corneal ring segments. J Cataract Refract Surg. 2003;29(2):407-9.

24. Siganos CS, Kymionis GD, Kartakis N, Theodoraids MA, Theodorakis MA, Astyrakakis N, et al. Management of keratoconus with Intacs. Am J Ophthalmology. 2003;135(1):64-70.

25. Rabinowitz YS, Li X, Ignacio TS, Maguen E. INTACS inserts using the femtosecond laser compared to the mechanical spreader in the treatment of keratoconus. Cataract Refract Surg. 2008;22(8):764-71. Comment in: J Refract Surg. 2007;23(3):221-2; author reply 222.

26. Ratkay-Traub I, Ferincz IE, Juhasz T, Kurtz RM, Krueger RR. First clinical results with the femtosecond neodynium-glass laser in refractive surgery. J Refract Surg. 2003; 19(2):94-103.

27. Kubaloglu A, Sari SE, Cinar Y, Cingu K, Koytak A, Coskun E, et al. Comparison of mecha-

nical and femtosecond laser tunnel creation for intrastromal corneal ring segment implantation in keratoconus: prospective randomized clinical trial. J Cataract Refract Surg. 2010;36(9):1556-61.

28. Coskunseven E, Kymionis GD, Tsiklis NS, Atun S, Arslan E, Siganos CS, et al. Com-plications of intrastromal corneal ring segment implantation using a femtosecond laser for channel creation: a survey of 850 eyes with keratoconus. Acta Ophthalmol. 2011;89(1):54-7.

29. Ertan A, Colin J. Intracorneal rings for keratoconus and keratectasia. J Cataract Refract Surg. 2007;33(7):1303-14.

30. Haddad W, Fadlallah A, Dirani A, El Rami H, Fahd D, Khanafer D, et al. Comparison of 2 types of intrastromal corneal ring segments for keratoconus. J Cataract Refract Surg. 2012;38(7):1214-21.

40o Congresso da Sociedade Brasileira de Retina e Vítreo

18 a 20 de abril de 2015Costão do Santinho

Florianópolis - SC

Informações: Site: www.retina2015.com.br

E-mail: [email protected]

Original Article


INTRODUCTION The aqueous humor (AH) is critical to the maintenance of avas-

cular eye structures, such as the cornea and the lens. It provides nu trients and removes the metabolites from these avascular structu-res(1). Oxygen is supplied to the AH via a transcorneal passage from the atmosphere, the limbal circulation, the arterioles of the ciliary body, and the iridial vessels(2,3).

Contact lenses, especially those with low oxygen permeability, dramatically hinder the passage of atmospheric oxygen to the cor-nea, causing the oxygen concentration in the corneal stroma and in the AH to decrease(4-6). This hypoxia may give a rise to oxygen flux from the AH to the cornea, resulting in a decrease of AH oxygen tension(5).

A high incidence of eye surgery has been reported to be related to corneal complications, such as corneal edema. These issues result from various conditions, e.g., the alteration of endothelial function in diabetes(7), Fuchs’ corneal dystrophy(8), endothelial injury from previous inflammation, surgery, or trauma(9), and chronic obstructive pulmonary disease(10). Changes in corneal endothelial cell density and morphology appear to make the cornea more sensitive to hypo-xia(11). Postoperative complications, such as corneal edema, caused by a decreased supply of ocular oxygen, can be interpreted as a result of inadequate ventilation to the eye structures in patients under local or general anesthesia. As corneal endothelial cells are not counted

before prescribing contact lens or before most intraocular surgeries, clinicians must maintain an adequate oxygen supply to the anterior chamber via the trancorneal passage and systemic circulation to avoid causing additional alterations in corneal endothelial pump function.

Most data regarding oxygen kinetics in the anterior chamber of the eye originate from indirect fluid measurements, including the application of polarographic electrode analysis inside the eye(2,4,5,12,13) or on the corneal surface(14), ocular scanning fluorometry(15), or optical oxygen sensors(16) and from direct measurements of the AH, using a blood-gas analyzer, obtained via anterior chamber paracentesis(6,17).

To the best of our knowledge, no data of simultaneous evalua-tions of the PO2, PCO2, and pH of AH and arterial blood samples exist. In the present study, we aimed to simultaneously measure the PO2, PCO2, and pH of blood and AH samples from rabbits.

METHODSAdult male New Zealand rabbits, weighing between 2000 g and

3500 g, were obtained for this study and were treated during all experiments in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and with the approval of the Ethics Committee of Canakkale Onsekiz Mart University School of

Blood gas analyzer utility in evaluating oxygen kinetics of the aqueous humor

Utilidade de um analisador sanguíneo de gás para a avaliação cinética do oxigênio no humor aquoso

isMail Ersan1, sEdat ariKan1, husEyin toMan2, sElcuK Kara1, baran gEncEr1, MEsut Erbas2, hasan ali tuFan1, MEtEhan uzun3

Submitted for publication: January 12, 2015 Accepted for publication: February 6, 20151 Department of Ophthalmology, Canakkale Onsekiz Mart University School of Medicine, Canakkale,

Turkey.2 Department of Anesthesia and Reanimation, Canakkale Onsekiz Mart University School of Medicine,

Canakkale, Turkey.3 Department of Physiology, Canakkale Onsekiz Mart University School of Medicine, Canakkale,

Turkey.



Corresponding author: Ismail Ersan. Canakkale Onsekiz Mart University, School of Medicine. Depart-ment of Ophthalmology - Canakkale Onsekiz Mart Universitesi Tip Fakultesi, Goz - Hastalıkları AD - Canakkale - Turkey - E-mail: [email protected]

Approved by the following research ethics: Animal Studies Committee of the Canakkale Onsekiz Mart University School of Medicine (2013/09-02 and 2013/09-03).

ABSTRACTPurpose: To measure the partial pressure of oxygen (PO2) and carbon dioxide (PCO2) and the pH of aqueous humor (AH) and arterial blood samples from rabbits using a blood gas analyzer. Methods: Twenty New Zealand rabbits were anesthetized intramuscularly with ketamine and xylazine and were then allowed to breathe room air. Using a gas blood analyzer, arterial blood and AH samples were analyzed for PO2, PCO2, and pH. Results: The mean arterial blood pressure was 87.14 ± 15.0 mmHg. The mean blood and AH PO2 were 95.18 ± 11.76 mmHg and 88.83 ± 9.92 mmHg, the mean blood and AH PCO2 were 25.86 ± 5.46 mmHg and 29.50 ± 5.36 mmHg, and the mean blood and AH pH were 7.38 ± 0.06 and 7.33 ± 0.09, respectively. Conclusions: The blood gas analyzer was easily employed to evaluate the aqueous humor in rabbits. When comparing the results of studies evaluating aqueous PO2, care should be taken to determine the methods used in these studies.

Keywords: Aqueous humor; Blood gas analysis; Oxygen/physiology; Carbon dio-xi de/physiology; Rabbits

RESUMOObjetivo: Medir a pressão parcial de oxigênio (PO2) e dióxido de carbono (PCO2), e o pH de humor aquoso (AH) e de amostras de sangue arterial de coelhos. Método: Vinte coelhos New Zealand foram anestesiados por via intramuscular com cetamina e xilazina, em seguida, foram liberados a respirar o ar ambiente. Utilizando um analisador sanguíneo de gás, amostras de sangue arterial e AH foram analisadas para PO2, PCO2, e pH.Resultados: A pressão arterial média foi de 87,14 ± 15,0 mmHg. A PO2 média do sangue e AH foi 95,18 ± 11,76 mmHg e 88,83 ± 9,92 mmHg; a PCO2 média do sangue e AH foi de 25,86 ± 5,46 mmHg e 29,50 ± 5,36 mmHg; o pH médio do sangue e AH foi 7,38 ± 0,06 e 7,33 ± 0,09, respectivamente. Conclusões: O analisador de gases no sangue foi facilmente empregadas para ava-liar o humor aquoso em coelhos. Quando se comparam os resultados de estudos que avaliaram PO2 do humor aquoso, deve ser tomado cuidado para determinar os métodos utilizados nestes estudos.

Descritores: Humor aquoso; Gasometria; Oxigênio/fisiologia; Dióxido de carbono/fi siologia; Coelhos

Ersan I, et al.


Medicine. The rabbits were evaluated for ocular and systemic problems, and only healthy rabbits were included in the study. Throughout the experimental period, the animals were fed with standard rabbit food and had access to water. The experiments were conducted between 09:00 and 16:00. All measurements were conducted at sea level. All the rabbits were made to fast overnight (approximately 8 h) before undergoing the experimental procedures.

On the day of the experiment, each rabbit was carried into the operating room. The left main auricular artery was catheterized for arterial blood sampling, and the rabbit was monitored by electrocar-diography. The rabbit was anesthetized intramuscularly with 35 mg/kg ketamine (Ketasol, Richter Pharma AG) and 5 mg/kg xylazine (Rompun®, Bayer Healthcare LLC).

One milliliter of arterial blood was obtained from the rabbit’s ear artery; susequently, using a heparinized tuberculin syringe with a 24-G needle, 0.2 ml AH was obtained from the right eye of the animal by anterior chamber paracentesis while it breathed room air. If an air bubble was visually observed in the syringe, the blood sample was discarded, and the rabbit was excluded from the study. All included samples were transferred immediately to the blood gas analyzer (Gas-tat 600 Series Blood Gas Analyzer, Techno Medica Co. Ltd., Yokohama, Japan) that was actively running in the operating room and were analyzed for PO2, PCO2, and pH.

All data were analyzed using SPSS for Windows (version 16.0, SPSS Inc., Chicago, IL, USA). Measurements were presented as mean ± stan-dard deviation. The Shapiro-Wilk test was used to identify the nor-mality of distribution. The Spearman rank correlation test was used to determine the relationship between measures of arterial and AH samples. A p value <0.05 was considered to be statistically significant.

RESULTSBlood and AH samples in the present study were obtained from

20 rabbits under general anesthesia, and both samples were analyzed for PO2, PCO2, and pH. Mean arterial blood pressure was 87.14 ± 15.0 mmHg. Mean blood and AH PO2 were 95.18 ± 11.76 mmHg and 88.83 ± 9.92 mmHg, respectively. Table 1 summarizes the results of the blood and AH sample analyses.

The arterial blood PO2 and PCO2 values were not correlated with values of AH samples (Spearman ρ=-0.068 and 0.292, p=0.777 and 0.212, respectively). The arterial blood pH was correlated with AH pH (Spearman ρ=0.457, p=0.043).

DISCUSSIONCorneal homeostasis depends to a great extent on the amount

of energy obtained by the aerobic Krebbs cycle and the anaerobic Embden-Meyerhof pathway(18). In case of low oxygen levels during contact lens wear, the cornea shifts the anaerobic Embden-Meyerhof pathway, resulting in stromal lactic acid accumulation and an acidic shift in the stromal pH. The increase in stromal lactic acid creates an osmotic load and decreases the pumping function of the endothe-lium from prolonged corneal hypoxia; both of these changes affect the increase in stromal edema(19). Besides corneal stromal changes, endothelial blebs, endothelial polymegatism, and pleomorphism are also potentially related to contact lens wear(19).

Because of the dynamic interaction between the cornea and AH, its biochemical analysis has long received much attention. It must be stressed that the biochemical analysis of aqueous fluid has not been validated using different types of analyzers; as such, AH analysis has no “gold standard.” In fact, there is poor agreement regarding AH oxygen tension in reports using different analytical methods(2,4-6,12-17). Using polarographic oxygen electrodes, Stefansson et al.(5) reported a mean aqueous PO2 of 23 ± 2 mmHg in rabbits, whereas Fitch et al.(13) reported a mean aqueous PO2 of 63 ± 9 mmHg in rats breathing room air. Using ocular scanning fluorometry, mean aqueous PO2 in rabbits was 23 ± 3 mmHg (range, 20-29 mmHg)(15). Shui et al.(16), using an optical oxygen sensor (optode) in the center of the anterior chamber, reported an aqueous PO2 of 27 ± 2 mmHg in rabbits breathing 20% oxygen and >100 mmHg when the animals were ventilated with 60% oxygen; they did not measure oxygen levels over 100 mmHg, as the sensor could not reliably measure higher oxygen values.

Jampol et al. reported mean aqueous PO2 levels in rabbits kept under normobaric conditions breathing room air as 63.5 ± 12.3 mmHg using a blood gas analyzer(17). Sharifipour et al. used a blood gas ana-lyzer in human patients breathing 21% oxygen undergoing cataract surgery under local anesthesia, and recorded blood and AH PO2 at 85.7 ± 7.9 mmHg and 112.3 ± 6.2 mmHg, respectively(6).

The key aspect of polarographic oxygen electrodes and optodes is their ability to measure PO2 in different regions of the eye. In reports using these electrodes, lower PO2 measurements may be attributed to the oxygen consumption property of the electrodes(16,20).

In the present study, the mean aqueous PO2 levels were in par-tial accord with studies using polarographic oxygen electrodes in rabbits(2) and rats(13) and with studies using blood gas analyzers in rabbits(17) and humans(6); however, PO2 levels in this study were higher than those in studies using polarographic oxygen electrodes in rabbits(5) and rats(13). PO2 levels were also higher in the present study than in studies using ocular scanning fluorometry(15) and optodes(16) in rabbits.

The differences between the studies may have been due to air con tamination during paracentesis when using a blood gas analyzer. To avoid this complicating factor, we discarded samples if air con-tamination occurred. A second possible explanation for the higher results in studies using a blood gas analyzer may be delayed analysis. Using plastic syringes, which do not have a good oxygen barrier, may lead to overestimated PO2 levels in aqueous samples. To avoid this complicating factor, all samples in the present study were analyzed by a blood gas analyzer, which was ready for use and in the operating room, within a minute of taking the sample.

The higher values reported in some earlier studies compared with those reported in the present study may be related to the very large corneal surface of the subject’s eye in relation to eye volume(13).

With regard to the Shui et al. study, in which the breathing con-ditions of rabbits had been increased from 20% to 60% oxygen, using an optode reportedly caused the PO2 to increase by factors of appro-ximately 5 to over 12 times in different parts of the eye(16). Disparities in AH PO2 levels between the different studies may be the result of the lack of standardized ventilation during sampling. To prevent these inconsistencies, the blood and AH samples must be analyzed simultaneously and arterial pressures must be measured. In addition, the altitude at which the study was conducted can affect the partial pressure of the inspired oxygen, and may contribute to these diffe-rences. The present study was conducted at sea level.

The intent of the present study was to measure the mean PO2, PCO2, and pH values of the AH simultaneously with those of the blood; due to the limitations of the blood gas analyzer, the evaluation of O2 and CO2 kinetics were beyond the scope of the study.

The significance of these AH measurements lies in the importan-ce of oxidative (e.g., cataract and glaucoma) and hypoxic (e.g., ru-beosis iridis) events that are a characteristic of numerous anterior seg ment pathologies. Measuring AH PO2, PCO2, and pH levels with an

Table 1. Arterial blood and aqueous humor gas measurements in rabbits

SamplesPO2 (mmHg) mean ± SD

PCO2 (mmHg) mean ± SD

pH mean ± SD

Arterial blood samples 95.18 ± 11.76 25.86 ± 5.46 7.38 ± 0.06

Aqueous humor samples 88.83 ± 09.92 29.50 ± 5.36 7.33 ± 0.09

Blood gas analyzer utility in evaluating oxygen kinetics of the aqueous humor


easily accessible single device, i.e., the blood gas analyzer, gives inves-ti gators an opportunity to examine anterior segment pathologies.

In conclusion, when comparing the results of studies measuring PO2 of the AH with the same or different devices, respiratory and he-modynamic parameters, which affect blood and tissue oxygenation during the study, must be taken into account. The present study simultaneously measured the PO2, PCO2, and pH of the AH as well as that of arterial blood samples in rabbits under ketamine/xylasine anesthesia. The use of the blood gas analyzer in the operating room ensured that the study rapidly and easily measured the PO2, PCO2, and pH of the AH.

REFERENCES 1. To C, Kong C, Chan C, Shahidullah M, Do C. The mechanism of aqueous humor for-

mation. Clin Exp Optom. 2002;85(6):335-49. 2. Kwan M, Niinikoski J, Hunt TK. In vivo measurements of oxygen tension in the cornea,

aqueous humor, and anterior lens of the open eye. Invest Ophthalmol. 1972;11(2):108-14. 3. Bill A. Some aspects of the ocular circulation. Friedenwald lecture. Invest Ophthalmol Vis

Sci. 1985;26(4):410-24. 4. Stefansson E, Wolbarsht ML, Landers MB. The corneal contact lens and aqueous

humor hypoxia in cats. Invest Ophthalmol Vis Sci. 1983;24(8):1052-4. 5. Stefánsson E, Foulks GN, Hamilton RC. The effect of corneal contact lenses on the

oxygen tension in the anterior chamber of the rabbit eye. Invest Ophthalmol Vis Sci. 1987;28(10):1716-9.

6. Sharifipour F, Idani E, Zamani M, Helmi T, Cheraghian B. Oxygen Tension in the aqueous humor of human eyes under different oxygenation conditions. J Ophthalmic Vis Res. 2013;8(2):119-25.

7. Keoleian GM, Pach JM, Hodge DO, Trocme SD, Bourne WM. Structural and functional

studies of the corneal endothelium in diabetes mellitus. Am J Ophthalmol. 1992; 113(1):64-70.

8. Seitzman GD. Cataract surgery in Fuchs’ dystrophy. Curr Opin Ophthalmol. 2005;16(4): 241-5.

9. Yasukawa T, Suga K, Yokoo N, Asada S. Cataract surgery in a patient with severe chronic iritis and corneal endothelial damage. J Cataract Refract Surg. 1998;24(7):885-8.

10. Soler N, García-Heredia A, Marsillach J, Mackness B, Mackness M, Joven J, et al. Pa-raoxonase-1 is associated with corneal endothelial cell alterations in patients with chronic obstructive pulmonary disease. Invest Ophthalmol Vis Sci. 2013;54(8):5852-8.

11. Ziadi M, Moiroux P, d’Athis P, Bron A, Brun J-M, Creuzot-Garcher C. Assessment of in-duced corneal hypoxia in diabetic patients. Cornea. 2002;21(5):453-7.

12. Heald K, Langham ME. Permeability of the cornea and the blood-aqueous barrier to oxygen. Br J Ophthalmol. 1956;40(12):705-20.

13. Fitch CL, Swedberg SH, Livesey JC. Measurement and manipulation of the partial pressure of oxygen in the rat anterior chamber. Curr Eye Res. 2000;20(2):121-6.

14. Kleinstein RN, Kwan M, Fatt I, Weissman BA. In vivo aqueous humor oxygen tension-as estimated from measurements on bare stroma. Invest Ophthalmol Vis Sci. 1981;21(3): 415-21.

15. McLaren JW, Dinslage S, Dillon JP, Roberts JE, Brubaker RF. Measuring oxygen tension in the anterior chamber of rabbits. Invest Ophthalmol Vis Sci. 1998;39(10):1899-909.

16. Shui YB, Fu JJ, Garcia C, Dattilo LK, Rajagopal R, McMillan S, et al. Oxygen distribution in the rabbit eye and oxygen consumption by the lens. Invest Ophthalmol Vis Sci. 2006; 47(4):1571-80.

17. Jampol LM, Orlin C, Cohen SB, Zanetti C, Lehman E, Goldberg MF. Hyperbaric and transcorneal delivery of oxygen to the rabbit and monkey anterior segment. Arch Oph thalmol. 1988;106(6):825-9.

18. Leung BK, Bonanno JA, Radke CJ. Oxygen-deficient metabolism and corneal edema. Prog Retin Eye Res. 2011;30(6):471-92.

19. Liesegang TJ. Physiologic changes of the cornea with contact lens wear. CLAO J. 2002; 28(1):12-27. Review.

20. Park Y-H, Shui Y-B, Beebe DC. Comparison of two probe designs for determining in-traocular oxygen distribution. Br J Ophthalmol. 2011;95(1):118-22.

XX Congresso do Conselho Latino-Americano de Estrabismo - CLADE

29 de abril a 2 de maio de 2015Punta Cana - República Dominicana

Informações: Site: clade2015rd.com

Original Article


INTRODUCTIONAge-related macular degeneration (AMD) is an irreversible, pro-

gressive retinal disease as well as one of the major causes of blindness in individuals aged >55 years(1). It can be classified as early, in termediate, or late, according to the presence of anomalies of the retinal pigmen-ted epithelium (RPE), size of drusen (yellow pigments comprising cholesterol and apolipoproteins)(2), area of atrophy of the RPE (geo-graphic atrophy), and presence or absence of choroidal neovascula-rization (exudative and atrophic forms)(2-4).

In addition to genetic predisposition, which accounts for 70% of the risk of disease development(5), advanced age is also considered

a risk factor, which results in the deposition of lipid particles and for mation of drusen in the retina (Bruch’s membrane, BM), thereby affecting retinal function(6).

Apolipoprotein E (APOE) acts in the metabolism of triglyceride-rich lipoproteins (TG)(7) and is also present in RPE and BM(4). This gly co-protein is encoded by a gene represented by three alleles, apoE2, apoE3, and apoE4; apoE2 and apoE4 are associated with increased and decreased risk of AMD, respectively(7).

The carotenoids lutein and zeaxanthin are absorbed together with intestinal fat and transported to the retina by low- and high-density lipoproteins (LDL and HDL, respectively)(8). These carotenoids act as

Association of high-density lipoprotein and apolipoprotein E genetic variants

with age-related macular degeneration

Associação de lipoproteína de alta densidade e variantes genéticas da apolipoproteína E com degeneração macular relacionada à idade

sabrina Mayara cEzario1, Maria clara Jéssica calastri1, caMila ivE FErrEira olivEira1, tayannE silva carMo1, MarcEla augusta dE souza PinhEl2, Moacir FErnandEs dE godoy 1, rodrigo JorgE3, carina costa cotriM3, dorotéia rossi silva souza1, rubEns caMargo siquEira1,3

Submitted for publication: January 14, 2015 Accepted for publication: February 6, 20151 Medical School of São José do Rio Preto (FAMERP), São José do Rio Preto, SP, Brazil.2 Medical School of Ribeirão Preto (FMRP-USP), Ribeirão Preto, SP, Brazil.3 Department of Ophthalmology, Medical School of Ribeirão Preto (FMRP-USP), Ribeirão Preto, SP,

Brazil.

Funding: This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo/FAPESP and Faculdade de Medicina de São José do Rio Preto/FAMERP.


Corresponding author: Sabrina Mayara Cezario. Faculdade de Medicina de São José do Rio Preto (FAMERP) - Pós-Graduação em Ciências da Saúde. Avenida Brigadeiro Faria Lima, 5.416 - São José do Rio Preto, SP - 15090-000 - Brazil - E-mail: [email protected]

This study has been approved by the following research ethics committee: Comitê de Ética em Pesquisa FAMERP (CAAE: 02711112.0.0000.5415).

ABSTRACT Purpose: This study aimed to evaluate the association of age-related macular degeneration (AMD) with apolipoprotein E (APOE) variants and serum lipid pro-files, including levels and fractions of total serum cholesterol (TC), low-density lipoprotein cholesterol (LDLc), and high-density lipoprotein cholesterol (HDLc), and triglycerides (TG). Methods: Genotyping of APOE-HhaI was performed in 134 patients (study group, SG) and 164 individuals without AMD (control group, CG), aged 50-89 years. Lipid profiles were analyzed in a subgroup of 30 subjects of both groups, matched according to age and sex. The significance level was set at P<0.05. Results: APOE E3/E3 was more prevalent (SG=74.6%; CG=77.4%), with no diffe-rence between both groups (P=0.667). The same result was observed for risk genotypes (APOE E -/2: SG=7.4%; CG=10.3%, P=0.624). Serum levels of TC, LDLc, and TG revealed similar median values between SG (193.5, 116, and 155 mg/dL, respectively) and CG (207.5, 120, and 123.5 mg/dL, respectively; P >0.05). For HDLc, a higher median value was observed in SG (53.3 mg/dL) versus CG (42.5 mg/dL; P=0.016). Logistic regression analysis showed the same value, and the HDLc/TC ratio was -11.423 (P=0.014), as also confirmed by an increase in HDLc in SG. The association between lipid profiles and apolipoprotein E genotypes was similar in both groups (P>0.05). Conclusions: APOE-HhaI is not associated with AMD. However, an increase in serum HDLc level appears to exert a protective effect against the disease, irrespective of the genetic variants of apoE.

Keywords: Polymorphism; genetic; Apolipoprotein E; Triglycerides; Lipids; Cho-lesterol; Macular degeneration; Cholesterol, HDL; LDL-receptor related proteins

RESUMO Objetivo: Este estudo teve como objetivo avaliar a associação de degeneração ma-cular relacionada à idade (DMRI) com variantes de alipoproteína E (APOE) e perfil lipídico sérico, incluindo níveis séricos de colesterol total ( TC) e frações de proteínas relacionadas a receptor de LDL (LDLc) e HDL colesterol (HDLc), e triglicérides ( TG). Métodos: Realizou-se genotipagem de APOE-HhaI em 134 pacientes (grupo de es-tudo - SG) e 164 indivíduos sem a doença (grupo controle - CG), na faixa etária entre 50-89 anos. O perfil lipídico sérico foi analisado em um subgrupo de 30 indivíduos de ambos os grupos, pareados por idade e sexo. Admitiu-se nível de significância para valor-P<0,05. Resultados: APOE E3/E3 prevaleceu (SG=74,6%; CG=77,4%), sem diferença entre os grupos (P=0,667), o mesmo ocorreu para genótipos de risco (APOE -/E2: SG=7,4%; CG=10,3%, P=0,624). Níveis séricos de TC, LDLc e TG mostraram medianas semelhan -tes entre SG (193,5; 116; 155 mg/dL, respectivamente) e CG (207,5; 120; 123,5 mg/dL respectivamente; P>0,05). Para HDLc notou-se valor de mediana elevado em SG (53,3 mg/dL) versus CG (42,5 mg/dL; P=0,016), constatado também na análise de regressão logística, cuja razão HDLc/TC mostrou coeficiente -11,423 (P=0,014), con-firmando acréscimo de HDLc em SG. A relação entre perfil lipídico sérico e genótipos de APOE mostrou semelhança entre os grupos (P>0,05). Conclusão: APOE-HhaI não se associa a DMRI, no entanto, o acréscimo no nível sérico de HDLc parece ter efeito protetor contra a doença, independente de variantes genéticas da apoE.

Descritores: Polimorfismo genético; Apolipoproteins E; Lipídeos; Triglicerídeos; Coleste-rol; Degeneração macular; HDL-colesterol; Proteínas relacionadas a receptor de LDL

Association of high-density lipoprotein and apolipoprotein E genetic variants with age-related macular degeneration


antioxidants, limiting oxidative damage to the retina cells, exerting a protective effect against AMD(9). Elucidating the association between serum lipid profiles and genetic variants of apoE may establish the underlying molecular mechanisms for the development and pre-vention of this disease. The present study aimed to evaluate the asso ciation of APOE-HhaI polymorphism with lipid profiles in patients with AMD.

METHODSA total of 298 subjects were studied, including 134 patients (age,

51-89 years; mean, 72 ± 9.3 years; sex, 50% male) with AMD (study group, SG), and 164 subjects (age, 50-81 years; mean, 59 ± 7.2 years; sex, 57% male) under ophthalmological treatment for complaints other than AMD (control group, CG). All subjects were examined and followed up by an ophthalmologist, and a questionnaire with perso-nal data and medical history was provided to them. Exclusion criteria included age of <50 years and/or diagnosis of diabetes mellitus.

DNA was extracted from whole blood collected in edetic acid (EDTA) using the salting-out method(10). APOE polymorphic frag-ments were amplified by polymerase chain reaction (PCR) (Mas-tercycler, Eppendorf ) using 0.5 µL of each deoxynucleotide (0.8 mM), 2.5 µL 10´ PCR buffer, 2.5 µL 10% dimethylsulfoxide, 2.5 µL of each primer (2.5 µM), 0.2 µL Taq polymerase (5 U/µL), 11 µL Milli Q water, and 2 µL diluted genomic DNA (0.2 µg). APOE (rs429358 and rs7412) polymorphisms were analyzed using the following primers: P1: 5’-ACAGAATTCGCCCCGGCCTGGTACAC-3’ and P2: 5’-TAAGCTTGGCA CGGCTGTCCAAGGA-3’(11). Genomic DNA (50 ng) amplification (ESCO Thermocycler) comprised 30 cycles (94ºC for 5 min, 94ºC for 30 s, 65ºC for 30 s, and 72º for 30 s), followed by a cycle of 10 min at 72°C for the final chain extension. Moreover, for the analysis of restriction frag-ment length polymorphism, the amplification product was digested with HhaI, followed by 5.0% agarose gel electrophoresis at 110 V for 2 h and 30 min, staining with GelRed® (Uniscience), and visualization of the DNA fragments under ultraviolet light(12).

For serum lipid profile analysis [total cholesterol (TC), high-densi-ty lipoprotein cholesterol (HDLc), low-density lipoprotein cholesterol (LDLc), and triglycerides (TG)], electronic medical records of the pa tients were assessed according to the standards of the V Brazilian Guidelines for Dyslipidemia and Prevention of Atherosclerosis(13).

Statistical analysis was performed using the Minitab and Gra-phPad statistical software. A descriptive analysis with mean, standard deviation, median, and percentage was used. Qualitative variables were tested with Fisher’s exact or chi-square test. Genotype distri-bution analysis was performed with Hardy-Weinberg equilibrium testing. Further, for quantitative variables with Gaussian distributions, analysis of variance (ANOVA) was applied to compare three or more groups. For quantitative variables without Gaussian distribution, the Mann-Whitney test was used to compare the two groups. Logistic regression analysis was applied to estimate the probability of AMD occurrence considering the analyzed variables. The alpha error was set at 5% with a significance level of P<0.05.

This study was approved by the Ethics Research Committee of the Medical School of Sao Jose do Rio Preto (FAMERP). All subjects were informed about the study and confirmed their willingness to participate by signing an informed consent.

RESULTSThe distributions of genotypes and allele frequencies of APOE-HhaI

are presented in table 1. The homozygous wild-type E3E3 ge notype and allele E3 was prevalent in SG (74.6% and 0.87, respectively) and CG (77.4% and 0.88, respectively), with no significant difference between both groups (P=0.667 and 0.654, respectively). The same result was observed for genotypes with at least one E4 allele (E -/4; P=0.328) and one E2 (E -/2; P=0.624).

Table 2 shows similar results for serum median levels of TC, LDL cholesterol (LDLc), and TG between SG (193.5, 116, and 155 mg/dL, res-pectively) and CG (207.5, 120, and 123.5 mg/dL, respectively, P>0.05). For HDL cholesterol (HDLc), a higher median value was observed in SG (53.3 mg/dL) than in CG (42.5 mg/dL; P=0.016). This finding was confirmed by logistic regression analysis, providing an HDLc/TC ratio of -11.423 (P=0.014). The lipid profile revealed no association with the genotypes of APOE-HhaI (P>0.05; Table 3).

Table 1. Distribution of genotype and allele frequencies of APOE-HhaI polymorphism in individuals with age-related macular degeneration (SG, Study Group) and in controls (CG)

APOE-HhaI genotype

SG (N=134) CG (N=164)

P valueN % N %

E2E2 000 - 000 - -

E2E3 010 7.50 016 009.80 0.623

E2E4 000 - 001 000.60 -

E3E3 100 74.60 127 077.40 0.667

E3E4 023 17.20 020 012.20 0.294

E4E4 001 0.70 000 - -

Total 134 100.00 164 100.00

E -/4 024 17.90 021 012.80 0.328

E -/2 010 7.40 017 010.30 0.624

Allele N Abs. freq. N Abs. freq.

E2 010 0.40 017 000.50 0.694

E3 233 0.87 290 000.88 0.654

E4 025 0.19 021 000.60 0.239

Total 268 1.00 328 001.00

Fisher’s or chi-squared tests; N= total number of individuals; Abs. freq.= absolute frequency.

Table 2. Median, minimum, and maximum values for lipid profile in patients with age-related macular degeneration (SG, Study Group) and in controls (CG)

Lipid profile SG CG

P value(mg/dL) (N=30) (N=30)

TC

Median 193.5 207.5 0.662

Minimum 144.0 107.0

Maximum 327.0 313.0

HDLc

Median 42.5 53.3 0.016*

Minimum 24.0 22.0

Maximum 70.0 80.0

LDLc

Median 115.0 120.0 0.882

Minimum 70.0 51.0

Maximum 255.0 237.0

TG

Median 151.0 123.5 0.813

Minimum 046.0 054.0

Maximum 561.0 333.0

Mann-Whitney Test; TC= total cholesterol; HDLc= high-density lipoprotein cholesterol; LDLc= low-density lipoprotein cholesterol; TG= triglycerides; SD= standard deviation; N= number of individuals. *= statistically significant.

Cezario SM, et al.


DISCUSSIONIn this study, similar to other studies(14,15), an association of APOE-HhaI

polymorphism with AMD could not be confirmed, which is in con-trast to another study(16). The E3E3 genotype and E3 allele were pre valent in patients and controls, as observed in another study(7). It has been demonstrated a higher frequency of allele E3, particularly in individuals without the disease(17). In this study, the sample size played an important role, as did the diverse ethnic background of the different populations. The Brazilian population is largely composed of individuals of Amerindian, African, and European descent(18).

Meta-analysis have clearly shown the protective effect of the E4 allele, even in heterozygotes(19), which were not observed in this study. Although the E2 allele is considered a risk allele(16), no differen-ce in risk was observed between the two groups. This finding is in accordance with that of other studies(14,15).

The protective nature of apoE4 can be explained by the lack of cysteine residues at positions 112 and 158, in contrast to apoE2 and apoE3. This lack prevents the formation of disulfide bridges with other proteins, as found in the binding between apoE3 and apoA-II in HDL(20). The inability of apoE4 to form dimers with other proteins found in HDL facilitates the transport of lipoproteins in MB, conside-ring the small particle size, preventing the formation of drusen(20,21). This prevention could be maximized by the concomitant presence of apoE4 and high levels of HDL. Although our study has not confirmed an association between genetic variants of APOE, it has shown higher levels of HDL, particularly in CG, consistent with another study(22), thereby suggesting that increased HDLc is a protective factor against AMD. However, Chakravarthy et al.(23) did not observe this association, and other reports indicate higher risks of diseases accompanying high serum levels of HDLc(24).

HDL particles are responsible for the transport of the carotenoids lutein and zeaxanthin, which are present in dark green leafy vege-tables, maize, squash, broccoli, peas, and egg yolk(25). In addition to their antioxidant activity, these carotenoids hold great potential for improving macular pigment density(26) and, therefore, help filtering blue light. Thus, HDL is studied for its possible key role in protecting against AMD(27).

Changes in the metabolism of cholesterol, particularly of HDL, may influence drusen accumulation and consequently promote AMD development. This suggests that increasing levels of HDLc associated with a carotenoid-diet rich could interfere with disease progression, particularly in patients currently under treatment, thereby improving its prognosis. Thus, reduced levels of HDLc can increase the disease risk, as found in this study.

Cholesterol can be obtained from systemic sources or recycled in the retina, through circulation of blood lipids. Thus, the retinal rod cells are capable of using both lipids from liver and those recycled from RPE(28). This dual capability can explain the association between increased serum cholesterol level and AMD, which was not observed in this study. Some authors agree with this finding(3,23); however, other authors disagree(22,29). The sample size, age, sex, and patients’ lifestyle should be considered, while evaluating published results.

The present study assessed the association of lipid profile with APOE-HhaI polymorphism. ApoE plays a role in the metabolism of TG-rich lipoproteins (chylomicrons and very LDL) as well as in the association of this metabolism with HDL(3,30). Importantly, apoE3 and particularly apoE4 reveal affinity with the membrane receptor apoB/E, whereas apoE2 is does not, which influences serum levels of TC, LDLc, and TG(19). Thus, APOE-HhaI genotypes could contribute to changes in the levels of serum lipids and consequently cause greater deposition in RPE and form drusen(28).

Patients and controls showed similar distributions of CT, LDLc, HDLc, and TG values for all genotypes of APOE-HhaI. However, the small sample size is a limitation of this study. The study aimed to match both groups (patients and controls) according to sex and age in order to minimize confounding factors in the statistical analysis of the association of lipid profiles with genetic polymorphism. The findings should be confirmed in further studies with larger sample sizes and varied populations.

CONCLUSIONAPOE-HhaI has no association with AMD. However, increased serum

levels of HDLc appear to offer protection against the disease, irres-

Table 3. Median, minimum and maximum values for lipid profile in patients with age-related macular degeneration (AMD) and without (controls) considering APOE-HhaI polymorphism

Lipid profile (mg/dL)

AMD (N=30) Controls (N=30)

P-value

E-/2 (a) E3E3 (b) E -/4 (c) E-/2 (d) E3E3 (e) E-/4 (f)

N=5 N=22 N=3 N=3 N=24 N=3

TC

Median 207.0 193.00 219.0 233.0 212.0 152.0 0.121

Minimum 175.0 144.00 172.0 191.0 123.0 107.0

Maximum 210.0 327.00 227.0 278.0 313.0 157.0

HDLc

Median 050.0 042.50 042.0 054.0 054.0 044.0 0.191

Minimum 030.0 024.00 036.0 051.0 022.0 040.0

Maximum 068.0 063.00 070.0 055.0 080.0 053.0

LDLc

Median 114.0 117.50 140.0 126.0 123.0 083.0 0.234

Minimum 106.0 070.00 107.0 098.6 055.0 051.0

Maximum 138.0 255.00 142.0 206.0 237.0 093.0

TG

Median 164.0 152.65 145.0 192.0 120.0 080.0 0.544

Minimum 095.0 046.00 075.0 085.0 055.0 054.0

Maximum 295.0 561.0 184.0 227.0 333.0 127.0

One-way ANOVA test; TC= total cholesterol; HDLc= high density lipoprotein cholesterol; LDLc= low density lipoprotein cholesterol; TG= triglycerides; SD= standard deviation; N= number of individuals.

Association of high-density lipoprotein and apolipoprotein E genetic variants with age-related macular degeneration


pective of the genetic variants of apoE. This observation should be confirmed in larger sample sizes and patient subgroups in different populations.

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26. Trieschmann M, Beatty S, Nolan JM, Hense HW, Heimes B, Austermann U, et al. Chan-ges in macular pigment optical density and serum concentrations of its constituent carotenoids following supplemental lutein and zeaxanthin: the LUNA study. Exp Eye Res. 2007;84(4):718-28.

27. Kijlstra A, Tian Y, Kelly ER, Berendschot TT. Lutein: more than just a filter for blue light. Prog Retin Eye Res. 2012;31(4):303-15.

28. Curcio CA, Johnson M, Rudolf M, Huang JD. The oil spill in ageing Bruch membrane. Br J Ophthalmol. 2011;95(12):1638-45.

29. Ulas F, Balbaba M, Ozmen S, Celebi S, Dogan U. Association of dehydroepiandroste-rone sulfate, serum lipids, C-reactive protein and body mass index with age-related macular degeneration. Int Ophthalmol. 2013;33(5):485-91.

30. Rohn TT, Day RJ, Catlin LW, Brown RJ, Rajic AJ, Poon WW. Immunolocalization of an amino-terminal fragment of apolipoprotein E in the Pick’s disease brain. PLoS One. 2013;8(12):e80180.

Original Article


INTRODUCTIONTumor necrosis factor alpha (TNF-α) is an inflammatory cytokine

derived from T cells and macrophages, involved in the regulation of cellular apoptotic activity(1,2). Despite having a critical role in con-trolling inflammatory processes in the human body, raised levels of TNF-α have been associated with cellular and vascular malformations, edemas, and neurovascular degeneration(1,3,4). For instance, patients with non-infectious uveitis demonstrate increased levels of TNF-α, suggesting that TNF-α is a key mediator of uveal inflammation(5-7). Therefore, suppression of TNF-α activity through antagonist drug admi nistration offers a promising treatment for patients with ocular inflammatory diseases(2,8-10).

ABSTRACT Purpose: To assess the cytotoxicity and genotoxicity of intravitreal adalimumab treatment in an animal experimental model using cytological and molecular te chniques.Methods: Eighteen rabbits were randomly assigned to three groups: control, adalimumab treatment, and placebo. Cytotoxicity on retinal cells was evaluated using flow cytometry assays to determine the level of apoptosis and necrosis. Ge-notoxicity was evaluated by comet assays to assess DNA damage, and quantitative real-time polymerase chain reaction (qPCR) was used to evaluate expression of apoptosis-inducing caspases (8 and 3). Results: No cytotoxicity or genotoxicity was observed in any of the two treatment groups (adalimumab and placebo) following intravitreal administration compared with the control group. Flow cytometry analysis revealed that more than 90% of the cells were viable, and only a low proportion of retinal cells presented apoptotic (~10%) or necrotic (<1%) activity across all groups. Molecular damage was also low with a maximum of 6.4% DNA degradation observed in the comet assays. In addi-tion, no increase in gene expression of apoptosis-inducing caspases was observed on retinal cells by qPCR in both the adalimumab and placebo groups compared with the control group. Conclusion: The use of adalimumab resulted in no detectable cytotoxicity or ge notoxicity on retinal cells for up to 60 days upon administration. These results therefore indicate that adalimumab may be a safe option for intravitreal application to treat ocular inflammatory diseases in which TNF-α is involved.

Keywords: Intravitreal injections; Retina; Antibodies, monoclonal, humanized/to xicity; Apoptosis; Tumor necrosis factor alpha; Ani mals; Rabbits

RESUMOObjetivo: Acessar a citotoxicidade e genotoxicidade do tratamento intravítreo de adalimumabe em um modelo experimental animal utilizando técnicas citológicas e moleculares. Métodos: Dezoito coelhos foram aleatoriamente selecionados em três grupos: con trole, tratamento intravítreo com adalimumabe e placebo. Os efeitos tóxicos nas células da retina foram avaliados através de ensaios de citometria de fluxo, para a de -terminação de atividade apoptótica e necrótica. A genotoxidade foi avaliada através de ensaios cometa para determinar danos ao DNA e através de PCR em tempo real para avaliar a expressão genética de caspases (8 e 3) promotoras de apoptose celular. Resultados: Não foram detectadas citotoxicidade e genotoxidade nos dois grupos de tratamento, adalimumabe e placebo, em comparação com o controle. A citometria de fluxo determinou que mais de 90% das células eram viáveis após o tratamento, e uma pequena quantidade de células da retina apresentaram apoptose (~10%) ou necrose (<1%) em todos os grupos. O dano molecular também foi baixo com uma degradação no DNA de no máximo 6,4% detectados nos ensaios cometa. Adicionalmente, não foram observados aumentos na expressão genética das caspases que induzem a apoptose através dos ensaios de PCR em tempo real. Conclusão: O tratamento intravítreo com adalimumabe não promoveu nenhuma citotoxicidade e genotoxicidade detectável em células da retina por até sessenta dias. Estes resultados, portanto, indicam que o adalimumabe pode ser uma opção segura para o tratamento de doenças oculares inflamatórias em que o TNF-α está envolvido.

Descritores: Injecções intravítreas; Retina; Anticorpos monoclonais humanizados/toxicidade; Apoptose; Fator de necrose tumoral alfa; Animais; Coelhos

Adalimumab is a recombinant human monoclonal antibody spe-cifically inhibiting TNF-α activity. Systemic use of TNF-α inhibitors is widely recognized as a treatment for autoimmune(11) and rheumatic diseases(12). Subcutaneous adalimumab administration shows pro-mising clinical improvement in patients with refractive uveitis(13) and macular edema secondary to non-infectious uveitis(14). However, systemic administration of adalimumab and other anti-TNF agents has produced several side effects(15). Thus, the use of intravitreal in-jection is the preferred method for treating ocular diseases because it provides higher drug concentrations with fewer injections of low dosage, preventing the risk of systemic exposure. However, few studies on the safety of intravitreal use of adalimumab have been

Cytotoxicity and genotoxicity of intravitreal adalimumab administration

in rabbit retinal cells

Citotoxicidade e genotoxicidade da administração de adalimumabe intravítreo nas células

da retina de coelhos

Álcio coutinho dE Paula1, Marcos PErEira dE Ávila1, david lEonardo cruvinal isaac1, rodrigo salustiano1, aliny PErEira dE liMa2, FrancyElli Mariana MEllo2, FlÁvia dE castro PErEira2, PEdro hEnriquE dE Paula silva3, ElisângEla dE Paula silvEira lacErda2

Submitted for publication: November 24, 2014 Accepted for publication: February 6, 20151 Ophthalmic Center Reference (CEROF), Federal University of Goiás (UFGO), Goiânia, GO, Brazil.2 Molecular Genetics and Cytogenetics, Institute of Biological Science, Federal University of Goiás

(UFGO), Goiânia, GO, Brazil.3 Instituto de Ciências Farmacêuticas, Estudos e Pesquisa (ICF) Goiânia, GO, Brazil.



Corresponding author: Álcio Coutinho de Paula. CEROF/UFG. 1a Avenida, s/n - Setor Leste Uni-versitário - Goiânia, GO - 74605-020 - Brazil - E-mail: [email protected]

Approved by the following research ethics committee: Animal Experimentation, Federal University of Goiás (009/11).

Cytotoxicity and genotoxicity of intravitreal adalimumab administration in rabbit retinal cells


published; for these, electroretinography and histological techniques have been used(14,16-18). These techniques cannot detect toxicity at the submicroscopic level and therefore we tested new methods to address intravitreal adalimumab toxicity at the cellular and molecular level in this study.

The purpose of this study was to evaluate, for the first time, the toxicity of adalimumab intravitreal administration in an animal mo del using cytological and molecular techniques. We administered adali-mumab intravitreously in rabbits to obtain live retina samples, with a view to determine apoptotic activity at the cellular level using flow cytometry. At the molecular level, we assed DNA damage using co met assays and expression of apoptosis-inducing caspases using quantitative real-time polymerase chain reaction (qPCR).

METHODSMEDICAL PROCEDURES AND EXPERIMENTAL DESIGN

Eighteen New Zealand albino male rabbits weighing 2.5-3.0 kg were used to analyze cytotoxic and genotoxic effects of intravitreal adalimumab injections. The experimental procedures followed the ARVO statement for the Use of Animals in Ophthalmic and Vision Re-search as well as our institutional guidelines. Animals were anestheti-zed before performing any medical procedures. They were subjected to dilated fundus examination to determine overall ocular health at the beginning of the experiment and after 30 and 60 days using eye drops of 10% phenylephrine (Allergan® Pharmaceuticals, San Diego, CA, USA) and 1% tropicamide (Mydriacyl®, Alcon Laboratories, Fort Worth, TX, USA). We used an indirect binocular ophthalmoscope (Welch Allyn, Skaneateles Falls, NY, USA) with 20 diopter lenses (Volk Optical, Mentor, OH, USA) to examine the vitreous cavity, retina, and opacity of the medium. Only animals with healthy eyes were selected for the study.

Animals were randomly allocated to three groups: group 1 was the control (no injections, n=2), group 2 received adalimumab in jec-tions (n=8), and group 3 placebo injections (n=8). Animals in group 2 were administered 0.1 mL (0.5 mg) of adalimumab (Humira®, Abbott, Abbott Park, IL, USA) via the right eye. The procedure was performed by inserting a 30-gauge needle via pars plana, 1-mm distant from the limbus. The treatment was reapplied after 30 days. Animals in group 3 were administered the same volume (0.1 mL) of a balanced saline solution (BSS®, Alcon, Fort Worth, TX, USA) using the same method, which was also reapplied after 30 days.

RETINA DISSECTION AND ANALYSIS

To perform the biological assays, the retinas of all study animals were dissected. Immediately after eye enucleation, an ocular dissection was performed and retina samples were obtained by the dislocation method. Retinal cell samples were analyzed by flow cytometry, co -met assays, and qPCR at the Molecular Genetics and Cytogenetic Laboratory at the Federal University of Goiás, Goiânia, Brazil.

FLOW CYTOMETRY

Death of retinal cells was determined using an Annexin V-FITC Apoptosis Detection Kit (Sigma-Aldrich, St. Louis, Mo, USA), according to the manufacturer’s instructions. Detection of apoptosis was based on the binding properties of phosphatidylserine annexin to the cell membrane (Annexin V+ to early and Annexin V- to late apoptotic cells), whereas the detection of necrosis was based on the binding properties of propidium iodide to cell DNA. Isolated cells (100 µL) were suspended in 400 µL of buffer solution and subsequently 5 µL of Annexin and 1 µL of propidium iodide were added. Flow cytometry analyses were performed using a FACSCalibur™ (BD Biosciences, San Jose, CA, USA) flow cytometer in conjunction with the software Diva ModFit®.

COMET ASSAY DNA damage was assessed by comet assays, a single-cell gel elec-

trophoresis method, as previously described(19). For the comet assays, retinal samples were placed in Falcon® tubes with 500 µL of 100% trypsin solution (Cultilab, Campinas, SP, Brazil) for 5 min. Subsequen-tly, 500 µL of culture medium supplemented with fetal bovine serum (10%) was added. A 20-µL cell suspension was homogenized with 100 µL of low-melting-point agarose (0.5%) spread onto microscope slides pre-coated with normal-melting-point agarose (1.5%). After 10 min at 4°C, slides were immersed in a cold lysis solution (2.4 M NaCl, 100 mM EDTA, 10 mM Tris, 10% DMSO, and 1% Triton-X, pH 10) for 24 h. After lysis, slides were placed in an electrophoresis chamber and covered with electrophoresis buffer (300 mM NaOH/1 mM EDTA, pH >13) for 20 min to allow for DNA unwinding.

The electrophoresis proceeded for 20 min (25 V and 300 mA). Subsequently, slides were submerged for 15 min in a neutralization buffer (0.4M Tris-HCl, pH 7.5), dried at room temperature, and fixed in 100% ethanol for 5 min. Slide staining was performed immedia-tely before analysis with ethidium bromide (20 µg/mL). Slides were prepared in duplicates and 100 cells were screened per sample (50 cells from each slide) using a fluorescence microscope (Leica, Wetzlar, Germany) interfaced with a computer.

Nucleus analysis was performed using the image software Comet Score ver. 15 (TriTek Corp., Sumerduck, VA, USA), according to the migration of DNA fragments as follows: class 0 (no damage), class 1 (little damage with a short tail length smaller than the diameter of the nucleus), class 2 (medium damage with a tail length one or two times the diameter of the nucleus), class 3 (significant damage with a tail length between two and a half to three times the diameter of the nucleus), and class 4 (significant damage with a long tail of damage greater than three times the diameter of the nucleus)(20). A damage index (DI) value was assigned to each comet assay according to for-mula (1), where n=number of cells in each class analyzed. DI=(0 x n0) + (1 x n1) + (2 x n2) + (3 x n3) + (4 x n4) (1) DI may range from 0 (completely undamaged; 100 cells x 0) to

400 (maximum damage; 100 cells x 4)(21).

QPCRThe molecular markers used to evaluate the apoptotic activity

included the expression of the messenger RNA (mRNA) of caspase 8 (apoptosis initiator) and caspase 3 (apoptosis effector). Retinal cell mRNAs were extracted using TRIzol reagent (Sigma-Aldrich, St. Louis, MO, USA), according to the manufacturer’s instructions. Total mRNA (2 µg) was then used to produce complementary DNA (cDNA) using a random initiator (Applied Biosystems, Foster City, CA, USA) with a total reaction mixture of 20 μL. The primer sequences used were 5’-ACGAAACCTCCGTGGACGCAA-3’ and 5’-AGACCGGGACGACATTC CAGTG-3’ (185 bp) for caspase 3 and 5’-TGGCAGCAGATGATGACAA TGGTG-3’ and 5’-TGGAAGCACTGTCAGAAACAGCAC-3’ (135 bp) for caspase 8. The qPCR was performed using a Line-gene K Real-time PCR Detection System (Bioer Technology, Binjiang, China) in total volumes of 20 μL reaction mixture: 2 μL of cDNA, 10 μL of SYBR Green PCR Master Mix (LGC Biotecnology, Teddington, Middlesex, UK), and 2 μL of each primer (400 nM). The qPCR was initiated at 95°C for 40 thermal cycles of 15 min each, followed by 15 s at 55°C and 30 s at 72°C. All qPCR analyses were performed in triplicates.


Data was presented as mean ± SE (standard error). The cytotoxi-city of adalimumab in the flow cytometry experiment was analyzed using a multivariate permutational analyses of variance (PERMA-NOVA). Data was first reassembled in a Bray-Curtis similarity matrix, after which unrestricted permutations of raw data were applied. PERMANOVA were conducted using Primer-e 6 PERMANOVA+1.0 software (Ver. 6.1.14). The genotoxicity of adalimumab measured by

Paula AC, et al.


the comet assays (comet tail length and damage index) was analyzed using one-way fixed effect analyses of variances (ANOVA–SYSTAT Ver. 12). For the ANOVA, assumptions of homogeneity of variance and normality were assessed by scatter plots and normal curves of residuals, respectively(22). Finally, the expression of caspase 8 and 3 as measured by qPCR was also analyzed using PERMANOVA, with the same operational inputs used for the abovementioned cytotoxicity analyses above.

RESULTSCELLULAR APOPTOSIS AND NECROSIS ANALYSIS

The parameters of cell viability, apoptosis, late apoptosis, and ne-crosis were determined by flow cytometry analysis. There were no significant differences in any of these parameters between the healthy (control), adalimumab injection, and the placebo injection groups (PERMANOVA, F4,29=0.859, p>0.05). The viability of retinal cells was similar for all groups, with over 90% viable cells detected by flow cytometry (Figure 1 A). The mean percentage of early and late cell apoptosis (Figures 1 B and 1 C respectively) was lower for the control group (0.51 ± 0.50 and 1.37 ± 1.16, respectively) than for both the ada-limumab (4.05 ± 1.23 and 3.58 ± 0.52, respectively) and placebo (3.52 ± 0.58 and 2.65 ± 0.51, respectively) groups. However, no significant differences were detected. The proportion of cell necrosis detected by flow cytometry was very low, with values of 0.15 ± 0.12% for the controls, 0.26 ± 0.08% for the adalimumab group, and 0.30 ± 0.15 % for the placebo group (Figure 1 D).

DNA DAMAGE ANALYSIS

Low DNA damage was observed after the intravitreal injections with adalimumab and placebo. The comet assays showed that there was no significant difference in DNA damage (tail length) between the control, adalimumab, and placebo groups (ANOVA, F2,18=2.437, p>0.05, Figure 2). The percentage of tail length was similar at 5.0 ± 0.42 for the control group, 6.4 ± 0.28 for the adalimumab group, and 5.0 ± 0.36 for the placebo group (Figure 2). The DI was also low for all groups (<40), with no significant differences (ANOVA, F2,16=3.312, p>0.05) in DI between the controls (30.6 ± 2.3), the adalimumab group (38.6 ± 3.0), and the placebo group (28.5 ± 1.8; Figure 3).

APOPTOSIS-INDUCING CASPASES 8 AND 3 EXPRESSION ANALYSIS

To determine the values of mRNA expression for both caspases (8 and 3) in retinal cells, we used healthy animals (controls) as a calibra-tor group with an absolute genetic expression value of 1, with values over and below representing higher and lower gene expression, res-pectively. There were no significant differences between the control, adalimumab, and placebo groups (PERMANOVA F2,18=0.8482 and p>0.05). Gene expression in the adalimumab group was 1.26 and 0.95 for caspase 8 and 3, respectively (Figure 4). For the placebo group, the values were 1.19 for caspase 8 and 0.48 for caspase 3 (Figure 4).

DISCUSSIONIn this study, we detected no significant cytological or molecular

toxicity to the retinal cells; therefore, these results provide strong evi-dence that adalimumab is safe for intravitreal treatment of non-infec-tious ocular inflammatory diseases. Our data show that ada limumab treatment results in only slight cytotoxic and genotoxic effects on rabbit retinal cells for up to 60 days, comparable with the effects ob-served in the control and placebo groups. Cellular apop to tic activity was very low with >90% viable retinal cells obser ved by flow cytome-try. Furthermore, the comet assay and qPCR de mons trated that DNA damage was minimal and expression of the apop to sis-inducing caspases 8 and 3 was low.

The use of intravitreal adalimumab has been evaluated previously in animal models by dose dependence studies. The administration of doses up to 5 mg of adalimumab produced no functional or structu-ral ocular toxicity(16-18). These studies evaluating adalimumab toxicity have, however, used electroretinography and histological methods, which cannot detect at sub-microscopic levels. Therefore, comple-mentary studies using cellular and molecular techniques should be used for early detection of cytotoxic and genotoxic effects such as the onset of cellular apoptosis or expression of caspases. Furthermore, we used a longer time span of 60 days compared with the span of 14 or 42-days previously used. This allows a longer contact between the substance and the retina and more closely resembles the clinical situation.

Based on the systemic adalimumab administration(18), a study suggested that 0.5 mg may be the therapeutically appropriate dose for intravitreal treatment. Using flow cytometry analysis, we confir-

Figure 1. Flow cytometry analysis showing the percentage of viable (A), apoptotic (B), late apoptotic (C), and necrotic (D) cells. Error bars (±standard error). Note the change of y-axis scale.

A B

DC

Cytotoxicity and genotoxicity of intravitreal adalimumab administration in rabbit retinal cells


Molecular assays have been increasingly used to determine ge-notoxicity of novel chemicals and pharmaceuticals(26). The comet assay is a microgel electrophoresis technique that measures DNA da mage at single-cell level(19). Single-cell gel electrophoresis (comet assay) has proven to be a highly sensitive method to assess DNA damage induced by several agents(27). For example, TNF-α-induced experiments in pancreatic cells resulted in a 50% increase in comet tail length(28). The use of etanercept, another TNF-α antagonist drug, has also resulted in DNA damage as observed by comet assay(29). In our study, we observed only small increases in comet tail length in the adalimumab and placebo groups compared with the control group. Large molecules, such as monoclonal antibodies, have limited access to the genetic material via cellular membranes and generally promo-te low molecular damage(26). Therefore, the minimal DNA damage observed here shows that adalimumab confers no genotoxicity on retinal cells for up to 60 days.

Evaluation of the molecular mechanisms that induce apoptosis is also an effective strategy for drug genotoxicity evaluation. The process of apoptotic cell death involves the activation of caspase pro-teases. Caspase 8 is an initiator of cellular death signaling pathways, whereas caspase 3 is an effector caspase linked to the process of cellular destruction that accompanies apoptotic signals(24). The use of adalimumab and other anti-TNF agents such as infliximab may increase the expression of apoptosis-inducing caspases in response to drug administration in in vitro studies of human monocytes and in vivo animal studies(24,30). In this study, however, there were no sig nificant increases in the expression of these apoptosis-inducing caspases in retinal cells in any of the treatment groups (adalimumab and placebo) compared with the control group. These contrasting re-sults may reflect differences in drug administration type and suggest that intravitreal adalimumab treatment is a less toxic administration method than subcutaneous injections.

CONCLUSIONIn conclusion, we have demonstrated that the use of adali-

mumab results in no detectable cytotoxicity and genotoxicity in rabbit retinal cells for up to 60 days after intravitreal administration, in dica ting that this treatment is a safe option applicable to human cli nical studies that aim to determine the efficacy of adalimumab for various ocular inflammatory diseases where TNF is implicated. Finally, the use of cytological and molecular techniques provides early and more reliable detection of drug toxicity, offering new approaches in ophtomological research.

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Figure 3. Damage index of retinal cells as determined by comet assay. Error bars (± stan-dard error).

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XV Congresso da Sociedade Caipira de Oftalmologia

XIV Simpósio da Sociedade de Enfermagem em Oftalmologia

7 a 9 de maio de 2015Ipê Park Hotel

São José do Rio Preto - SP

Informações: Site: www.cenacon.com.br/eventos/2015/caipira/

Original Article


INTRODUCTIONQuality of vision is an important part of the quality of life(1); im-

paired vision therefore has a negative impact on the quality of life by causing difficulties in daily activities and emotional issues(2-4). The progressive increase in life expectancy worldwide is a major public health challenge, placing cataract as one of the important issues. Kara-José et al.(5) estimated the prevalence of cataract in Brazil to be 6.8% in the agre group of 50-59 years and as high as 68.3% in indivi-duals aged above 80 years, indicating a rise prevalence with age.

According to the World Health Organization, 39 million people worldwide are blind, the main cause being cataract(6,7). Daily difficul-ties due to ocular conditions were reported by 82.7% of patients in a Brazilian study, in which the most frequently reported difficulties of patients with cataract were walking (72.5%), doing housework (64.8%), and watching television (64.8%)(8). Thus, cataract indeed greatly impacts the quality of life.

Cataract surgery followed by intraocular lens (IOL) implant can considerably improve vision(2,9). IOL is used after surgery to replace the natural lenses that were removed(10). New materials and different IOL models have been developed and improved to promote better acuity and visual function without the need for glasses(11,12). The monofocal IOL is most commonly implanted in patients undergoing cataract surgery, and enables correction of distance visual acuity; ho-wever, glasses are required for near-sighted activities(13). The multifo-cal IOL corrects distance and near-sighted acuities(14) and reduces the need to wear glasses. There are also strategies such as “monovision,” which corrects one eye for far sight and the other eye for near sight, reducing the need for glasses with great success(15,16).

However, despite great improvements in the new generation of lenses, they present side effects, such as intense brightness, forma-tion of halos, and inconsistent intermediate sight(14,15,17), affecting the daily activities and the quality of life of the users(17,18). Therefore, it is

Preoperative automatic visual behavioural analysis as a tool for intraocular lens

choice in cataract surgery

Análise comportamental visual automática pré-operatória como ferramenta para escolha

de lente intraocular em cirurgia de catarata

hEloisa nEuMann noguEira1, Mônica alvEs1, Paulo schor2

Submitted for publication: October 3, 2014 Accepted for publication: December 25, 20141 Pontifícia Universidade Católica de Campinas (PUCCAMP), Campinas, SP, Brazil.2 Department of Ophthalmology and Visual Sciences, Paulista School of Medicine (EPM), Federal

University of São Paulo (UNIFESP), São Paulo, SP, Brazil.



Corresponding author: Heloisa Neumann Nogueira. Departmento de Oftalmologia e Ciências Visuais. Universidade Federal de São Paulo. Rua Botucatu, 821 - São Paulo, SP - 04023-062 - Brazil - E-mail: [email protected]

ABSTRACTPurpose: Cataract is the main cause of blindness, affecting 18 million people worldwide, with the highest incidence in the population above 50 years of age. Low visual acuity caused by cataract may have a negative impact on patient quality of life. The current treatment is surgery in order to replace the natural lens with an artificial intraocular lens (IOL), which can be mono- or multifocal. However, due to potential side effects, IOLs must be carefully chosen to ensure higher patient satisfaction. Thus, studies on the visual behavior of these patients may be an im-portant tool to determine the best type of IOL implantation. This study proposed an anamnestic add-on for optimizing the choice of IOL. Methods: We used a camera that automatically takes pictures, documenting the patient’s visual routine in order to obtain additional information about the fre quency of distant, intermediate, and near sights. Results: The results indicated an estimated frequency percentage, suggesting that visual analysis of routine photographic records of a patient with cataract may be useful for understanding behavioural gaze and for choosing visual management strategy after cataract surgery, simultaneously stimulating interest for customized IOL manufacturing according to individual needs.

Keywords: Cataract extraction; Cataract/psychology; Lenses, intraocular; Quality of life; Vision, ocular; Visual acuity; Sickness impact profile

RESUMOObjetivo: A catarata é a principal causa de cegueira e acomete 18 milhões de pessoas no mundo, com maior incidência na população acima de 50 anos. A baixa acuidade visual causada pela catarata gera um impacto negativo na qualidade de vida de pa-cientes. O tratamento atual é feito por meio de cirurgia com a substituição do cristalino opacificado por uma lente intraocular (LIO) que pode ser monofocal ou multifocal. No entanto, a escolha da lente intraocular deve ser cuidadosamente realizada para garantir maior satisfação dos pacientes Assim, o estudo do comportamento visual desses pacientes pode ser uma ferramenta importante para definir qual o melhor tipo de lente intraocular a ser implantada. O presente estudo propôs a avaliação de uma ferramenta adicional à anamnese na a escolha da lente intraocular. Método: Com o uso de uma câmera programada para realizar o registro automático de fotos, foi documentanda a rotina visual do paciente, a fim de se obterem maiores informações sobre a frequência com que o mesmo utiliza a visão para longe, meia distância ou para perto. Resultados: Os resultados indicaram uma estimativa em porcentagem dessa frequên-cia, sugerindo que a análise dos registros fotográficos da rotina visual de um paciente portador de catarata pode ser de grande ajuda no entendimento do seu comportamento visual e para a escolha da estratégia de reabilitação visual após a cirurgia de catarata e, inclusive, despertar o interesse pela confecção de lentes intraoculares personalizadas de acordo com as necessidades de cada paciente.

Descritores: Extração de catarata; Catarata/psicologia; Lentes intraoculares; Quali-dade e vida; Visão ocular; Fotografias; Acuidade visual; Perfil de impacto da doença

Nogueira HN, et al.


necessary to carefully select patients to maximize the success of this technology and patient satisfaction(17,19). This selection is made ac-cording to the level of patient awareness, and the patient should be made aware of the advantages and limitations of multifocal IOL(20). It is critical to discuss and determine patient expectations because the individual approach and proper patient need assessment are most important when choosing the type of IOL(13).

Nowadays, we are increasingly multitasking that demand rapid alternation far and near sights such as reading and handling tablets or cell phones while watching television or using a computer and following a GPS while driving. Hence, the ability to quickly focus on objects within variable distances is a common and important activi-ty(17). Analysis of visual behavior can generate relevant information regarding patients with various disorders, particularly in the case of cataract, and be used to inform individual approaches.

Several commercially available devices are used to perform eye tracking to analyze visual behavior under various circumstances. Eye tracking can be used to gain information on how people acquire and process information when reading, browsing the internet, shopping, interpreting image exams, or managing and executing other tasks requiring visual skills to discern the surrounding environment(21). This technology can also be used in the medical field to assess patients with Parkinson’s disease (22) or schizophrenia(23), to improve refractive surgery(24,25), and to understand the role of literacy in health(26), among other approaches. Examples of technologies developed for this purpose include EyeLink 1000 and EyeLink II (SR Research), Optical Head Tracking Module and Eye Tracking Glasses (SMI), Tobii X2 30 Eye Tracker, Tobii Glasses 2 Eye Tracker and other models of the Tobii Company, and GP3 Eye Tracker (Gazepoint).

Based on the above, it can be concluded that the visual behavior of patients with cataract is extremely important for deciding the visual strategy to be adopted after phacoemulsification and, consequently, for patient satisfaction and quality of life. This choice is currently ba-sed on anamnestic and questionnaire data, obtaining guidelines in relation to visual demands.

New strategies and technological advances to treat cataract-as-sociated visual impairment are in constant evolution and have been rapidly incorporated. Thus, a better understanding of patient beha-vioural gaze has gained relevance. No tools have been described in the literature that can aid the physician or patient in the choice of these prostheses, and judgment errors may result in discontent and even repeated surgery to remove the lenses. This study proposes a new technology as an add-on to the anamnesis used today, introdu-cing the use of a camera that automatically takes pictures and allows a daily assessment of the visual behavior of patients with cataract.

METHODSWe used a Narrative Clip camera (http://getnarrative.com/), with

the following features: dimensions, 36 × 36 × 9 mm; weight, 20 g; capacity, 6000 photographs; image-capture frequency, 30 sec; reso-lution, 5 megapixels; battery life, 2 days (Figure 1).

In this case series, we selected functionally independent, men-tally unimpaired patients with senile cataract. All included patients had already undergone cataract surgery in at least one eye and under similar conditions (phacoemulsification and IOL implant). Also, we collected patient information and a brief report on their daily acti-vities, as a database to be analyzed together with the photographic records.

PATIENT 1Identification: Male, 66 years old, driver.Report on Daily Activities: After waking up at 6 a.m., the patient

drives his car for 20 to 30 min before reaching work. He works from 7 a.m. to 5 p.m., providing internal transport for company staff, driving a van. At night, he has dinner and watches television at an approxi-

mate distance of 2 m. He reads the newspaper on a daily basis for approximately 40 min. During his day off and spare time, he watches television, visits family, and does not go out much.

PATIENT 2Identification: Female, 64 years old, teacher.Report of Daily Activities: After waking up at 6 a.m., the patient

has breakfast at home, subsequently spending 10 min of walking to reach her place of work. She works from 7 a.m. to 4 p.m., performing computer tasks and activities that require reading. After work, she walks back home, where she spends most of her time watching television. She usually does not spend much time doing household chores. She reads books on a daily basis for approximately 1 h, and does embroidery two or three times a week.

PATIENT 3Identification: Female, 54 years old, nurse.Report of Daily Activities: After waking up at 6 a.m. the patient

has breakfast at home. She drives or carpools to work; twice a week she goes by bus. She works from 8 a.m. to 5 p.m. in a storeroom of health products purchase and distribution department, which mainly involves using a computer, and also works three night shifts per week in a hospital. She attends Pilates classes three times a week; and on weekends, she visits family, goes to the movies, and watches television. She also attends a post-graduate class once a month.

PATIENT 4Identification: Female, 61 years old, retired lawyer.Report of Daily Activities: After waking up at 7 a.m., the patient

has breakfast at home. She does her household chores, likes to cook, and does hydrogymnastics twice a week. She attends an arts-and- crafts course twice a week and likes to sew. She frequently visits other cities and spends quite a few hours driving every day. She goes to the mall and watches movies and television approximately 1 h a day.

To compare the references obtained from the reports of daily activities with the photographic records, we classified the activities reported by the patients as follows: Driving: Mainly far and intermediate sight and less frequently near

sight (checking car dashboard, speedometer, radio, GPS, etc.) Watching television: Intermediate sight Reading, using the computer and cell phone, doing craftwork,

cooking, eating: Near sight Talking to other people: Intermediate and near sight Walking on the street: Far and intermediate sight

Figure 1. Narrative Clip camera. The image shows the Narrative Clip camera held by an adult hand for size reference. Features: dimensions, 36 × 36 × 9 mm; weight, 20 g; capacity, 6000 photographs; image-capture frequency, 30 s; resolution, 5 megapixels; battery life, 2 days.

Preoperative automatic visual behavioural analysis as a tool for intraocular lens choice in cataract surgery


Patients were instructed to use a shirt with a firm collar reaching the level of the sternal notch, so that the camera could be attached using the clip located on its back side. They were instructed to use the camera throughout the day, immediately on waking up, and to remove it only when its use was considered inappropriate. They were also instructed to point the camera towards the object being obser-ved. The camera was delivered to the patient’s home on the previous afternoon, and collected on the day after it was used.

Every 10 records were selected from the photographs recorded by patients using the camera over a period of one day, i.e., at 5-min inter-vals. The photographs were assessed and classified in terms of distance by our team, as near sight (0-1 m), intermediate sight (1-4 m), or far sight (>4 m). We then evaluated the photographs with each patient, so that they could confirm the distance classification and inform us about the true importance of events recorded. Next, we established the propor-tion (%) of time that each patient used near, intermediate, and far sight, according to the number of photographs placed within each of the three classifications versus the total number of photographs selected.

RESULTS This study separately assessed the visual behavior of each patient.

The results for each patient are enumerated. Because the camera was around the neck and not at the eye level, in certain situations it was not clear what object the patient was looking at. Table 1 shows the proportions (%) of near, intermediate, and far sight for each patient.

PATIENT 1Patient satisfaction regarding IOL: The patient has a monofocal

lens. He is satisfied with his vision and says that he only needs his glasses for activities that require near sight, such as reading.

According to the report on daily activities provided by the pa-tient, we judged that he used mainly far and intermediate sight. The camera was used during his day off. The photographic records matched the activities mentioned by the patient, performed du-ring days off and weekends, where the main activity was watching television. This fact is proven by graph 1 (Figure 2), which shows an in termediate sight proportion of 87.5%, whereas near and far sight proportions were 8.4% and 4.1%, respectively. In an overall evaluation of his day off, we infer that the patient mainly uses intermediate sight for his daily activities. Figure 2 shows examples of photographs taken by this patient.


lens in only one eye, and she needs glasses to perform daily activities, because most of her activities require near sight. She has difficulties reading and suffers from eye fatigue.

According to the report of daily activities provided by the patient, we assessed that she used mainly near and intermediate sight. The camera was used during a normal day at work. According to the re-sults shown in graph 2 (Figure 3), the photographic records matched the report of daily activities, with proportions of 49.8% and 48.2% for near and intermediate sight, respectively. During photograph eva-luation, the patient confirmed all our classifications. Figure 3 shows examples of photographs taken by this patient.


lens and is satisfied with its performance when using far and interme-diate sight. She only needs to use glasses for delicate tasks requiring near sight.

According to the report on daily activities provided by the pa-tient, we judged that she used mainly near and intermediate sight. The camera was used during a vacation day. The photographic records partially matched the classification of the report on daily activities, indicating proportions of 39.1% and 41.9% for near and intermediate sight, respectively, as shown in graph 3 (Figure 4). During the photograph evaluation, the patient confirmed all our classifications. Figure 4 shows examples of photographs taken by this patient.

Table 1. Proportions (%) of far, intermediate, and near sight

Patient Patient 1 Patient 2 Patient 3 Patient 4

Far 04.10 02.00 19.00 18.00

Intermediate 87.50 48.20 41.90 35.20

Near 08.40 49.80 39.10 46.80

A B

Figure 2. Examples of photographic records obtained by Patient 1 while using the Narrative Clip camera for one day. In image A, the patient was watching television (photograph with modified content due to privacy issues); intermediate sight. Image B illustrates the patient using the telephone and reading a book; near sight. Graph 1. Proportion (%) of time spent by Patient 1 using near, intermediate, and far sight.

Nogueira HN, et al.


A B

Figure 3. Examples of photographic records obtained by Patient 2 while using the Narrative Clip camera for one day. In image A, the patient was at work; near and intermediate sight. In image B, the patient was walking down the street; intermediate and far sight. Graph 2: Proportion (%) of time spent by Patient 2 using near, intermediate, and far sight.

BA

Figure 4. Examples of photographic records obtained by Patient 3 while using the Narrative Clip camera for one day. In image A, the patient was performing household chores; near sight. In image B, the patient was traveling by bus; intermediate and far sight. Graph 3: Proportion (%) of time spent by Patient 3 using near, intermediate, and far sight.

PATIENT 4Patient satisfaction regarding IOL: The patient has multifocal len-

ses in both eyes. She is very satisfied with her near and intermediate sight, but has difficulties with far sight; she says it is too bright and blurry. She also complains of eye pain.

According to the report of daily activities provided by the patient, we considered that she used mainly near and intermediate sight. The camera was used during a normal day of daily activities that, according to the patient, do not vary on weekends because she is retired. The photo-graphic records matched the classification of the report of daily activities, resulting in proportions of 46.8% and 35.2% for near and intermediate sight, respectively, as shown in graph 4 (Figure 5). During photograph evaluation, the patient only made few changes to our classifications. Figure 5 shows examples of photographs taken by this patient.

DISCUSSION AND FUTURE PERSPECTIVESThe choice of IOL after cataract surgery is currently based only on

the medical history of patients, so the surgeon can suggest types and

brands of available lenses to be implanted. Several studies have been performed to evaluate the best multifocal IOL diopter addition(20). However, knowing the distance at which the patient executes most of his/her daily tasks, i.e., the patient’s visual behavior, is critical for choosing an optimal IOL(27-29). Through this unprecedented study, besides achieving a detailed medical history, we were able to add information regarding the type of patient activities performed throughout an entire day; distance estimates of patient sight; and estimates of the proportions of near, intermediate, and far sight. We were therefore able to collect important behavioural gaze data that can lead to developing a better approach toward the patients, inclu-ding determination of the best type of IOL to be selected for each individual patient and even the more appropriated near vision lens power in multifocal IOLs.

As mentioned earlier, the most commonly used IOL at present is the monofocal lens, which, when implanted in both eyes, requires the use of near-sighted glasses(13). However, particularly for patients who mostly use near sight in their daily professional or leisure activities, it is necessary to consider alternatives, such as monovision, where one

Preoperative automatic visual behavioural analysis as a tool for intraocular lens choice in cataract surgery


BA

Figure 5. Examples of photographic records obtained by Patient 4 while using the Narrative Clip camera for one day. In image A, the patient was driving; mainly far and intermediate sight but also near sight. In image B, the patient was using the computer; near sight. Graph 4: Proportion (%) of time spent by Patient 4 using near, intermediate, and far sight.

eye is corrected for far sight and the other for the distance most often used by the patient(15,16). Multifocal lenses are also an option, with the second eye focus being adjusted to the patient’s needs.

The selected patients had already undergone cataract surgery at least in one eye; hence, it was possible to perform a general assess-ment of the strategy used for each patient, taking into account the performance of the lenses and the satisfaction of the patients. In the case of Patient 1, who was classified as having mainly far sight, the monofocal lens appeared to have been a good choice. Patient 2 was classified as having mainly near and intermediate sight and was not satisfied with monofocal lenses when carrying out activities requiring near sight. A possible strategy for this case would be monovision with the implant of a lens that corrected the vision of the other eye for near sight, so that the patient would be able to make the most of her vision during daily activities. Another alternative would be to discuss the possibility of implanting a monofocal IOL, which would correct near sight and be compatible with the patient’s main needs. Patient 3 was classified as having mainly near and intermediate sight. She had monofocal lenses implanted in both eyes, and she was satisfied with her intermediate and far sight; however, she needed glasses for near sight activities, particularly those related to her work. Therefore, monovision would have been a good strategy for this pa-tient. Patient 4 showed a more even distribution of all three types of sight compared with the other patients, but was classified as having mainly near sight, followed by intermediate sight. She had multifocal lenses implanted in both eyes and reported being very satisfied with her visual performance. The only exception was at night, when she complained about seeing intense brightness and the need for extra light to be able to read. As mentioned earlier, the problems stated by Patient 4 are quite common for patients implanted with this type of IOL, and she appeared to be well informed about these issues, which is a determining factor for allocating patients to receiving multifocal lens implants. This leads us to conclude that the strategy used was correct for this patient.

The patients were asked to use the camera for an entire working day. Patients 1 and 3 were on vacation, so their records were conse-quently compatible with the activity report of leisure days. However, these do not correspond to the tasks they would have performed on a usual working day (professional activities). To obtain a more accu-rate analysis that would give a full classification of the vision of these patients, it would be necessary to document their activities during a working day. Patient 1 spends most of his time driving, which requires

intermediate and far sight more frequently than near sight. As for Pa-tient 2, according to her report of daily activities, the expected result would be a higher proportion of near sight, rather than the result of intermediate sight that was obtained.

All patients showed interest and willingness not only to use the camera to capture the photographs but also to discuss the results, although knowing that they would not directly benefit from this study. We had some difficulty evaluating the photographs with re gard to the three types of sight for certain activities - for example, driving involves all three sights - when calculating the proportion of activities involving far, intermediate, and near sights. Despite the fact that the camera was not positioned at eye level, and despite the difficulty in terms of standardizing its height at the level of the sternal notch, the expected final results were not affected. However, it became clear that patients selected for this type of study require good cognitive function to be able to follow the guidance provided by the research team. In addition, a device shaped as a pair of glasses that can determine the distance between the observer and the object could give more accurate photo documentation of the true visual behavior. Furthermore, it would be interesting to have records of data on pupil diameter corresponding to each visual scenario, given the importance of this parameter in the choice of IOL for certain types of bifocal lenses.

With regard to visual behavior, it is important to highlight that this is an issue that involves the individuality of the patient and that has gained importance concerning the improvement of strategies to rehabilitate visual impairment in cataract patients. This is peculiar, since in the medical field diseases are usually analyzed in a general way for a given population. The data obtained were analyzed separa-tely for each patient because only this would allow for an appropriate choice of IOL that would correct their near, intermediate, or far sight, or a combination thereof, according to the needs of each individual patient. We therefore opted to perform an analysis that would esti-mate the proportion, as a percentage, with which each patient used near, intermediate, or far sight, in order to highlight the importance of the concept of individuality and, in the not so distant future, stimulate interest in the manufacture of personalized IOLs according to the needs of each patient.

This study brings to light a novel method to improve current understanding of behavioural gaze. Our results open up new perspec tives on cataract visual impairment rehabilitation but can even be broadly extrapolated as a feasible tool to provide individual evaluation of ’ visual needs.

Nogueira HN, et al.


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Original Article


INTRODUCTIONIschemia-reperfusion (I/R) injury occurs because of temporary dis-

ruption and subsequent improvement of the blood flow. This type of injury may become serious because significant amounts of reactive oxygen species (ROS), which are very toxic to cells, are produced as a result of tissue reoxygenation. Both direct and indirect mechanisms may be involved in ROS-induced cellular toxicity. In the direct me-

RESUMOObjetivo: O objetivo deste estudo é investigar o efeito da quercetina, contra a morte celular por apoptose induzida por lesão consequente à isquemia-reperfusão (I/R) na retina de ratos. Método: Vinte e quatro ratos foram divididos em quatro grupos iguais: controle, is-quêmico, solvente e quercetina. O modelo lesão por I/R foi realizado por meio da elevação da pressão intraocular acima da pressão de perfusão, em todos os grupos. Injecções intraperitoneais de 20 mg/kg de quercetina ou sulfóxido de dimetilo (DMSO) foram realizadas nos grupos quercetina e solvente, respectivamente, imediatamente antes da lesão por I/R, permitindo que as retinas fossem reperfundidas. Quarenta e oito horas após a lesão, as espessuras de camada de células ganglionares da retina (RGCL), camada nuclear interna (INL), camada plexiforme interna (IPL), camada ple-xiforme externa (OPL), e a camada nuclear externa (ONL) foram medidas em todos os grupos. Além disso, o número de células TUNEL (+) e caspase-3 (+) tanto na camada nuclear interna quanto na camada nuclear externa foi avaliada em todos os grupos. Resultados: A administração de quercetina diminuiu o afinamento de todas as camadas da retina em comparação com o grupo isquêmico. A espessura média da camada nuclear interna nos grupos quercetina e isquêmico foi de 21 ± 5,6 µm e 16 ± 6,4 µm, respectivamente (p<0,05). A espessura média da camada nuclear externa no grupo quercetina e isquêmico foi 50 ± 12,8 µm e 40 ± 8,7 µm, respectivamente (p<0,05). O efeito anti-apoptótico de quercetina em termos de redução do número de células TUNEL (+) e caspase-3 (+) foi significativa na INL. O número médio de células TUNEL (+) da camada nuclear interna no grupo isquêmico e quercetina foi 476,8 ± 45,6/mm2 e 238,72 ± 251/mm², respectivamente (p<0,005). O médio número de células de cas-pase-3 (+) na INL do grupo isquêmico e quercetina foi 633,6 ± 38,7/mm² e 342,4 ± 36,1/mm², respectivamente (p<0,001). Conclusão: A utilização de quercetina pode ser benéfica no tratamento de lesão da re tina por I/R devido ao seu efeito anti-apoptótico nas camadas da retina, particu-larmente na camada nuclear interna.

Descritores: Quercetina; Retina/efeitos de drogas; Traumatismo por reperfusão; Apop to se; Isquemia; Modelos animais de doenças; Ratos

chanism, because both mitocondria and DNA are surrounded by membranes, ROS may lead to disruption of mitocondrial function and alterations in the structure of DNA, as a result of peroxidation of polyunsaturated fatty acids present in these membranes(1). In the indirect mechanism, ROS induce hypersecrection of excitatory amino acids such as glutamate and aspartate(2). Hypersecrection of excitatory amino acids leads to an influx of excessive free calcium by

Quercetin protects the retina by reducing apoptosis due to ischemia-reperfusion

injury in a rat model

Quercetina protege a retina reduzindo a apoptose consequente à lesão por isquemia e reperfusão em um modelo de rato

sEdat ariKan1, isMail Ersan1, turan Karaca2, sElcuK Kara1, baran gEncEr1, ihsan Karaboga2, hasan ali tuFan1

Submitted for publicattion: October 28, 2014 Accepted for publicattion: February 9, 20151 Department of Ophthalmology, Canakkale Onsekiz Mart University School of Medicine, Canakkale,

Turkey.2 Department of Histology and Embriyology, Trakya University School of Medicine, Edirne, Turkey.



Corresponding author: Sedat Arikan. Canakkale Onsekiz Mart University, School of Medicine - De-partment of Ophthalmology - Canakkale Onsekiz Mart Universitesi Tip Fakultesi - Goz Hastalıkları AD - Canakkale - Turkey - E- mail: [email protected]

Approved by the following research ethics committee: Canakkale Onsekiz Mart University Ethics Committee of Experimental Animals (2011/09-11).

ABSTRACTPurpose: This study aimed to investigate the effect of quercetin on apoptotic cell death induced by ischemia-reperfusion (I/R) injury in the rat retina. Methods: Twenty-four rats were divided into four equal groups: control, ischemic, solvent, and quercetin. I/R injury was achieved by elevating the intraocular pressure above the perfusion pressure. Intraperitoneal injections of 20 mg/kg of quercetin and dimethyl sulfoxide (DMSO) were performed in the quercetin and solvent groups, respectively, immediately prior to I/R injury, and the researchers allowed for the retinas to be reperfused. Forty-eight hours after injury, the thicknesses of the retinal ganglion cell layer (RGCL), inner nuclear layer (INL), inner plexiform layer (IPL), outer plexiform layer (OPL), and outer nuclear layer (ONL) were measured in all groups. Moreover, the numbers of terminal deoxynucleotidyl transferase dUTP nick-end-labeled [TUNEL (+)] cells and caspase-3 (+) cells in both INL and ONL were evaluated in all groups. Results: The administration of quercetin was found to reduce the thinning of all retinal layers. The mean thickness of INL in the quercetin and ischemic groups was 21 ± 5.6 µm and 16 ± 6.4 µm, respectively (P<0.05). Similarly, the mean thickness of ONL in the quercetin and ischemic groups was 50 ± 12.8 µm and 40 ± 8.7 µm, respectively (P<0.05). The antiapoptotic effect of quercetin in terms of reducing the numbers of both TUNEL (+) cells and caspase-3 (+) cells was significant in INL. The mean number of TUNEL (+) cells in INL in the ischemic and quercetin groups was 476.8 ± 45.6/mm2 and 238.72 ± 251/mm², respectively (P<0.005). The mean number of caspase-3 (+) cells in INL of ischemic and quercetin groups was 633.6 ± 38.7/mm2 and 342.4 ± 36.1/mm2, respectively (P<0.001). Conclusion: The use of quercetin may be beneficial in the treatment of retinal I/R injury because of its antiapoptotic effect on the retinal layers, particularly in INL.

Keywords: Quercetin; Retina/drug effects; Reperfusion injury; Apoptosis; Ischemia; Disease models; Rats

Arikan S, et al.


stimulation of the N-methyl-D-aspartate (NMDA) receptor-operated channels, and increased levels of intracellular free calcium may subsequently trigger apoptosis of neuronal cells by a process called excitotoxicity(2,3). Neurotrophin, deprivation, glial activation, ischemia, glutamate excitotoxicity, and oxidative stress lead to induction of apoptotic cell death of retinal ganglion cells(3).

Retinal cell death is mostly due to apoptosis rather than necro-sis, and this provides an important advantage to the retina because apop tosis does not result in an extreme inflammatory response; thus, limiting cell loss(4). However, in the event of excessive or deficient apop tosis, such as in I/R injury, increased loss of retinal neurons may lead to visual impairment if the macula is affected(5,6). I/R injury is the main pathogenetic mechanism underlying several clinically impor-tant di seases such as myocardial infarction, stroke, spinal injury, and shock, as well as some sight-threatening disorders such as diabetic retinopathy, retinal vascular occlusion, and glaucoma(7). Thus, there may be a potential benefit from the use of medications, vitamins, and subs tances possessing both antioxidative and antiapoptotic effect as adjuvants in the treatment of eye diseases associated with I/R injury.

Flavonoids are plant-derived substances possessing several an-tioxidative properties. Among the flavonoids, quercetin is known as a powerful antioxidant and free radical-scavenging substance(8). Fur-thermore, quercetin also posseses antiapoptotic effects(9). Some stu-dies have evaluated the antiapoptotic effect of quercetin; however, little data is available regarding the antiapoptotic effect of quercetin on retinal I/R injury(10,11).

In the present study, we aimed to evaluate whether quercetin decreases apoptotic cell loss in a rat retinal I/R injury model. Because the intensity of apoptosis occuring in the different layers of the retina may differ because of differences in the susceptibility of the retinal layers to I/R injury, we separately evaluated the antiapoptotic effect of quercetin on both the inner and outer nuclear layers of the retina.

METHODSTwenty-four male Winstar Albino rats weighing 250-300 g, which

were obtained from Saki Yenilli Research Animal Laboratory, were in-cluded in the study. After obtaining approval from Canakkale Onsekiz Mart University Ethics Committee of Experimental Animals, the rats were housed in cages with free access to standard food and drinking water, and they were maintained under controlled conditions, inclu-ding a 12-h light/dark cycle (08:00-20:00, light; 20:00-08:00, dark), an ambient temperature of 23-25°C, and a humidity in the range of 55%-60% in the Canakkale Onsekiz Mart University Laboratory of Animal Research Center.

All operations were performed under aseptic conditions. For anes-thetizing the rats, a single dose of 80 mg/kg ketamine (Ketasol, Richter Pharma Ag, Wels, Austria) and 5 mg/kg xylazine (Rompun, Bayer, İs-tan bul) was intraperitonally injected. To maintain body temperature within the physiological range, the rats were moved to special con-tainers to ensure sufficient covering of their bodies. After instilling one or two drops of 0.5% proparakain HCl (Alcaine; Alcon, USA) for corneal analgesia, 10% povidone iodine was administered into the conjonctival sac to ensure desinfection of the ocular surface.

Twenty-four rats were divided into four groups of six according to weight and were used as control, ischemic, solvent, and quercetin treatment groups, respectively. In the control group, the right an te-rior chamber of each animal was penetrated without drug adminis-tration.

In the ischemic group, the I/R injury model was developed using the method previously described by Buchi et al.(12). According to this method, to establish retinal ischemia, the right anterior chamber was cannulated with a 30-gauge needle attached to an infusion set, and its reservoir was placed approximately 200 cm above the animal. After opening the reservoir of the infusion set containing physiological saline, the intraocular pressure (IOP) was raised above 50 mmHg, until

whitening of the iris and loss of the red reflex of the fundus were ob-served, which are signs of retinal ischemia. All procedures and clinical examinations in the present study were performed using a portable biomicroscope (Shin Nippon, Japan). After 60 min of retinal ischemia, IOP was reverted to normal pressure by removing the infusion needle from the anterior chamber to allow the retinas to be reperfused. In the solvent group, after inducing retinal ischemia in the right eyes, di-methyl sulfoxide (DMSO) was intraperitoneally injected immediately before allowing the retinas to be reperfused. In the quercetin group, after inducing retinal ischemia in the right eyes, a dose of 20 mg/kg quercetin (Sigma-Aldrich Chemical Co., United Kingdom) dissolved in DMSO was intraperitoneally injected immediately before allowing the retinas to be reperfused. After 48 h, the right eye of each animal was enucleated to perform histopathological and immunohistoche-mical evaluations of the retina, and the experimental animals were sacrified.

HISTOPATHOLOGICAL AND IMMUNOHISTOCHEMICAL EVALUATIONS

Eye tissue samples, fixed in 10% buffered formaldehyde fixative so lution and embedded in paraffin 48 h after acute retinal ischemia, were examined with hematoxylin and eosin (H&E) to evaluate his-topathology. Sections that were 5 µm thick were obtained using a microtome (Leica, RM2245) and stained with H&E. Retinal thickness was calculated in three separate regions: peripheral (100-150 µm from the ora serrata), central (100-150 µm from the optic nerve), and midperipheral (halfway between the central and peripheral). Two representative sections were randomly selected from the same three positions for each eye, and their mean values were calculated(13). The sections were also incubated with polyclonal anti-Caspase-3 antibody (Abcam, ab4051; dilution 1:100) at room temperature for 90 min. Sections were washed three times with PBS and incubated with biotinylated secondary antibody (Ultra Vision Detection System-HRP kit, Thermo, Fremont, USA), and subsequently streptavidin peroxidase (Ultra Vision Detection System-HRP kit, Lab Vision, Fremont, USA) was added and sections were maintained at room temperature for 20 min.

TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE DUTP NICK-END LABELING (TUNEL) ASSAY

Enucleated eyes were immersed in 10% formaldehyde fixative solution for 48 h at room temperature and embedded in paraffin; 5-µm sections were obtained, and the slides were air dried. Apopto-tic cells were identified using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL; Calbiochem, San Diego, CA, USA), as previously reported(14). Nuclear counterstaining was performed using 3,3’-diaminobenzidine (DAB), and sections were counterstained with hematoxylin.


Data were expressed as mean ± standard deviation (SE). The Bartlett test was used to determine whether the data were heterogeneous or homogeneous. Bonferroni multiple comparison test was applied to identify differences between means. Differences were considered statistically significant at probability (P) levels of <0.05.

RESULTSThe mean thicknesses of RGCL, IPL, OPL, INL, and ONL were

19 ± 2.7 µm, 60 ± 12.1 µm, 11 ± 3.0 µm, 27 ± 9.9 µm, and 55 ± 13.3 µm, respectively, in the control group; 8 ± 1.4 µm; 22 ± 6.5 µm, 7 ± 1.9 µm, 16 ± 6.4 µm, and 40 ± 8.7 µm in the ischemic group; 9 ± 0.9 µm, 22 ± 7.4 µm, 7.4 ± 1.6 µm, 17 ± 5.2, and 41 ± 10.2 µm in the solvent group; and 10 ± 3.8 µm, 25 ± 8.2 µm, 9 ± 2.4 µm, 21 ± 5.6 µm, and 50 ± 12.8 µm, respectively, in the quercetin group (Figures 1 and 2).

Values indicating the mean thicknesses of RGCL, IPL, and OPL measured in the quercetin group were statistically significantly grea-ter those measured in the ischemic and the solvent group (P<0.05 and P<0.05 respectively). However, among all of the retinal layers,

Quercetin protects the retina by reducing apoptosis due to ischemia-reperfusion injury in a rat model


the reduction in the thicknesses of both ONL and INL was statistically significant, particularly in the quercetin group; ONL mean thickness in the quercetin and ischemic groups were 50 ± 12.8 µm and 40 ± 8.7 µm, respectively (P<0.05), and INL mean thickness in the quercetin and is-chemic groups were 21 ± 5.6 µm and 16 ± 6.4 µm, respectively (P<0.05).

THE EFFECT OF QUERCETIN ON THE NUMBER OF TUNEL (+) CELLS IN INL AND ONL

The mean number of TUNEL (+) cells counted in INL was 10.88 ± 3.5/mm², 476.8 ± 45.6/mm², 336 ± 34.9/mm², and 238.72 ± 251/mm² in the control, ischemic, solvent, and quercetin groups, respectively. The mean number of TUNEL (+) cells in INL was significantly lower in the quercetin group than in the ischemic group (P<0.005). Moreover, the mean number of TUNEL (+) cells counted in ONL was 197.76 ± 18.4/mm², 176 ± 16.8/mm², and 71.68 ± 11.8/mm2 in the ischemic, solvent, and quercetin groups, respectively. The mean number of TUNEL (+) cells in ONL was also significantly lower in the quercetin group than in the ischemic group (P<0.05; Figures 3 and 4).

CHANGES IN THE NUMBER OF CASPASE-3 (+) CELLS IN INL AND ONL The mean number of caspase-3 (+) cells detected in INL was 62.56

± 0.2/mm², 633.6 ± 38.7/mm², 550 ± 34.2/mm², and 342.4 ± 36.1/mm² in the control, ischemic, solvent, and quercetin groups, respectively. Treatment with quercetin was found to significantly decrease the number of caspase-3 (+) cells in INL by a ratio of nearly 46% when compared with the ischemic group (P<0.05). Moreover, the mean number of caspase-3 (+) cells detected in ONL was 43.6 ± 9.4/mm², 41.2 ± 6.7/mm², and 29,7 ± 8.4/mm² in the ischemic, solvent, and quercetin groups, respectively. Quercetin treatment was found to significantly decrease the number of caspase-3 (+) cells in ONL by a ratio of nearly 32% when compared with the ischemic group (P<0.05; Figures 5 and 6).

DISCUSSIONThe antioxidant, free radical-scavenging, and neuroprotective

effects of quercetin is well known(15-17). Besides these important bene-fits of quercetin, it also has a protective effect against apoptotic cell damage. Some experimental studies have revealed the antiapoptotic effect of quercetin on I/R injury in various organs(18,19); however, we have not been able to find any experimental studies evaluating the benefit of quercetin in treatment of I/R injury of the retina.

Because apoptosis is the mechanism predominantly involved in retinal cell death, antiapoptotic drugs or substances are considered

* P<0.05 compared with the control group. ¥ P<0.05 compared with the control, ischemic, and solvent groups.Figure 1. The thicknesses of the retinal layers (µm) as measured in each group.

A

C

B

D

Figure 2. Representative photographs showing retinal thickness detected in the rat retina. A) Normal appearance of the retina (control group). B and C) Retinal thickness in the ischemic group and solvent group, respectively; in both of B and C, the thicknesses of the retina was found to be significantly reduced after ischemia. D) Retinal thickness in the quercetin group. Total retinal thickness in the quercetin group was significantly less reduced compared with the ischemic group at end of the experiment. H&E staining (×400).

Figure 4. Representative photographs of TUNEL staining of rat retinas in control (A), ischemic (B), solvent (C), and quercetin (D) groups. The number of cells positive by TUNEL staining was increased in ischemic rats. However, treatment with quercetin markedly reduced retinal cell apoptosis. Arrows: TUNEL (+) cells. (×400).

A

C

B

D

a= P<0.001 compared with the control group.b= P<0.005 compared with the control and ischemic groups.c= P<0.05 compared with the control, ischemic, and solvent groups.d= P<0.05 compared with the control group.Figure 3. Number of TUNEL (+) cells detected in the inner nuclear layer (INL) and outer nuclear layer (ONL) in each group.

Arikan S, et al.


particularly beneficial in the treatment and prevention of retinal di-seases that are associated with the formation of excessive apoptosis, such as I/R injury.

The exact mechanism of cell death due to retinal ischemia is incompletely known; however, it was previously demonstrated that in conditions of oxidative stress, retinal ganglion cells are damaged as a result of increased intracellular ROS and calcium influx(20). During I/R, overproduction of ROS leads to excessive apoptosis by inducing the secretion of glutamate and calcium influx(21-23). Apart from indu-cing apoptosis, increased intracellular calcium levels may also cause cellular damage by hindering mitocondrial functions, decreasing the level of intracellular adenosine triphosphate (ATP), inducing ROS production, and stimulating cellular proteases and nitric oxide synthase(24). Therefore, it is conceivable that the maintainance of intracellular free calcium levels at an optimal concentration may be critical for treating I/R injury.

Flavonoids, such as quercetin, contain a flavonol nucleus that is surrounded by functional groups that have the ability to maintain the intracellular free calcium concentration at low levels by influencing glutathione metabolism, particularly in conditions of excessive ROS

production(25,26). Therefore, it may be reasonable to use quercetin in the treatment of I/R injury because of its beneficial biochemical properties.

A supressor effect of quercetin against the activities of caspase-3 and calpain, which are the representitive proteases in apoptotic and necrotic pathways, was demonstrated in purified primary rat retinal ganglion cell cultures and in human retinal pigment epithelium cell cultures(27,28). Moreover, an antiapoptotic effect of quercetin in terms of reducing caspase-3 activity has also been demonstrated in a myocardial I/R model(29). Because quercetin was found to induce cardioprotection in the myocardial I/R model, we thought that it may also be beneficial for retinal protection in the ocular I/R model; hence, we aimed to investigate this relationship in the present study.

To develop the ocular I/R model, we applied a widely used me-thod, which involves increasing the intraocular pressure above the ocular perfusion pressure for nearly 60 min(5). In the histological exa-mination of the retina, this procedure was found to cause retinal da mage through different types of cell death mechanisms such as apoptosis, necrosis, and nonlysosomal vesiculate(5). Hughes demons-trated increased apoptosis and INL thinning in a tissue model of acute ischemic injury(30). In addition, a number of investigations have shown that a peak in apoptosis occurs 6 to 18 h after I/R, returning to control levels after 48 h(31,32). Therefore, in the present study, we administered quercetin immediately after the induction of ischemia but before the begining of reperfusion, i.e., before the peak of apoptosis. Forty-eight hours later, we found that the thickness of the retinal layers in the quercetin group was significantly less decreased than that in the is-chemic and solvent groups.

Moreover, a significant decrease in the number of TUNEL (+) and caspase-3 (+) cells was detected in both INL and ONL in the quercetin group in comparison with the ischemic group. Because TUNEL (+)and caspase-3 (+) cells are important markers in the determination of apoptosis, obtaining a reduction in the number of such markers and a decrease in the thinning of retinal layers clearly demonstrates the antiapoptotic effect of quercetin on I/R-induced retinal injury. Although the decrease in the thinning of INL and ONL was found to be almost similar, the antiapoptotic effect of quercetin appeared much more evident in INL than in ONL. We believe that this disparity may arise from a difference in susceptibility of INL and ONL to ische-mic damage. In a previous study, the intensity of apoptotic cell loss was found to be more significant in INL than in ONL in the earlier phases of I/R injury(33). Consistent with this finding, we also observed that the number of both TUNEL (+) and caspase-3 (+) cells was much higher in INL than in ONL.

In the present study, the reason for the significantly increased degree of apoptosis in INL may result from excitotoxicity, which is coupled to hypersecretion of excitatory amino acids such as gluta-mate. This idea is supported by the findings of both Massey et al. and Brandstätter et al. who showed that expression of glutamate recep-tors is confined to neurons of the ganglion cell layer and INL(34,35). This supports the hypothesis that quercetin may achieve its antiapoptotic effect depending on the intensity of the apoptosis, and it may be beneficial, particulary in the presense of increased apoptosis. On the other hand, because the number of both TUNEL (+)and caspase-3 cells was much lower in ONL in all the groups, it is speculated that the antiapoptotic effect of quercetin may not be prominent in ONL because of decereased induction of apoptosis. However, in an animal model of I/R injury, an increased rate of apoptosis in ONL was repor-ted to be associated with extended postischemic survival time(36). In our study, upon evaluation of the early phases of I/R injury, we speculate that a significant decrease in the thickness of ONL in the ischemic group may also be due to a different cell death mechanism such as necrosis in addition to apoptosis. Therefore, the assessment of the intensity of apoptosis in ONL should be also elucidated in the later phases of the I/R injury model, to gain additional insight into the antiapoptotic effects of quercetin in all the layers of the retina.

a= P<0.0001 compared with the control groupb= P<0.001 compared with the control and ischemic groupsc= P<0.005 compared with the control and quercetin groupsd= P<0.05 compared with the control, ischemic, and solvent groupsFigure 5. The number of caspase-3 (+) cells in the inner nuclear layer (INL) and outer nucler layer (ONL) in each group.

A

C

B

D

Figure 6. Representative photographs of caspase-3 (+) cells. A) Control group; B) Is-chemic group; C) Solvent group, and D) Quercetin group. The number of caspase-3 (+) cells observed among the quercetin-treated retinal cells was significantly lower than that among the ischemic retinal cells. Arrows: Caspase-3 (+) cells. Immunoperoxidase, hematoxylin counterstain (×400).

Quercetin protects the retina by reducing apoptosis due to ischemia-reperfusion injury in a rat model


Caspase-3 is an important marker of apoptosis, and it is capable of decreasing the thickness of retinal layers as a result of activating the proteolytic cascade(37). Hence in the current study, the possible mechanism of the effect of quercetin on change in retinal thickness over a relatively short time may arise from its inhibitory effect on cas-pase-3 cells. In a previous study, a dose of 20 mg/kg i.p. quercetin was found to be sufficient for achieving cerebral protection in a cerebral I/R injury model; we also used quercetin at a dose of 20 mg/kg i.p. in our retinal I/R injury model(38). Although, we could not assess the penetrance of quercetin into the retinal layers, we believe that when systemically used, quercetin may accumulate in the retinal layers at adequate concentrations during ischemic injury because of the possibly broken blood-retinal barrier. On the other hand, an antia-poptotic effect of quercetin was also previously demonstrated in the retinal layers of the streptozotocin-induced diabetic rats even after oral administration(39). This result may indicate that quercetin has a good oral bioavailability; therefore, if the therapeutic dose is adjusted for humans in future studies, oral use of quercetin may be beneficial in the treatment of various retinal diseases associated with I/R injury.

Toxicity studies of quercetin and electrophysiological tests of the retina could not be performed in the present study because of inadequate laboratory equipment; we view this as a major limitation of this study. Because we histologically examined the retinal tissues 48 h after I/R injury, the antiapoptotic effect of quercetin in the later periods of the injury could not be properly evaluated, and this is ano-ther major limitation of this study. However, we used an additional number of rats to create a control group. However, we believe that if we had used the left eyes of either the ischemic group or the solvent group as a control group, we would not have been able to perform accurate comparisons between the control group and the other experimental groups because of the influence of possibly increased inflammatory cytokines produced in the eye with I/R injury, as in the case of sympathetic ophthalmia.

CONCLUSIONTo the best of our knowledge, this study is the first to demonstrate

the neuroprotective effect of quercetin on apoptotic cell injury in a retinal I/R model. Because a significant antiapoptotic effect of quer-cetin was obtained when it was used even after the begining of is-chemia, the present study could be valuable with respect to revealing the therapeutic effects of quercetin on retinal diseases, for which the pathophysiological mechanism involves I/R injury.

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28. Kook D, Wolf AH, Yu AL, Neubauer AS, Priglinger SG, Kampik A, et al. The protective effect of quercetin against oxidative stress in the human RPE in vitro. Invest Ophthalmol Vis Sci. 2008;49(4):1712-20.

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Original Article


INTRODUCTIONAccording to the World Health Organization, age-related macular

degeneration (AMD) is the third leading cause of visual impairment worldwide resulting in blindness with a prevalence of 8.7%. AMD is also the leading cause of visual impairment among elderly in in-dus trialized countries. It is likely that AMD prevalence increases in proportion to the increase of the elderly population. Epidemiological

data from recent years indicate that the AMD incidence increased from 5% to 27%(1).

In neovascular AMD, there is a subretinal neovascular membrane and exudation results in intraretinal edema. After treatment with rani-bizumab, retinal edema decreases, leading to retinal thinning. Recent studies have suggested that other assessment methods should be used in neovascular AMD, including optical coherence tomography

Optical coherence tomography and multifocal electroretinography of patients with

advanced neovascular age-related macular degeneration before, during, and after

treatment with ranibizumab

Tomografia de coerência óptica e eletrorretinografia multifocal de pacientes com degeneração macular relacionada à idade, neovascular avançada, antes, durante e após o tratamento com ranibizumabe

izabEla nEgrão Frota dE alMEida1, luciana nEgrão Frota dE alMEida2, EdMundo Frota dE alMEida sobrinho3,4, bruno duartE goMEs2, givago da silva souza2,5, alExandrE antonio MarquEs rosa3,4, luiz carlos l. silvEira2,5,6

Submitted for publication: November 5, 2014 Accepted for publication: February 6, 20151 Complexo Hospitalar Padre Bento de Guarulhos, Guarulhos, São Paulo, SP, Brazil. 2 Instituto de Ciências Biológicas, Federal University of Pará, Belém, PA, Brazil. 3 Instituto de Ciências da Saúde, Federal University of Pará, Belém, PA, Brazil. 4 Hospital Universitário Bettina Ferro de Souza, Federal University of Pará, Belém, PA, Brazil. 5 Núcleo de Medicina Tropical, Federal University of Pará, Belém, PA, Brazil.6 Ceuma University, São Luís, MA, Brazil.

Funding: This study was supported by FINEP IBN Net; CNPq-PRONEX/FAPESPA #2268 and #316799/2009; CNPq #620037/2008-3, #476744/2009-1, and #475860/2010-1; and CAPES-PROCAD #182/2007.


Corresponding author: Izabela Negrão Frota de Almeida. Rua Dr. Sérgio Meira, 230, 94/2 - São Paulo, SP - 01153-010 - Brazil - E-mail: [email protected]

ABSTRACTPurpose: To evaluate retinal morphology and function of patients with advanced neovascular age-related macular degeneration (AMD) before, during, and after treatment with ranibizumab. Methods: Twenty-one eyes diagnosed with advanced AMD were studied with optical coherence tomography (OCT) and multifocal electroretinography (mfERG). Three intravitreal injections of ranibizumab were administered at 1-month intervals. Evaluations were performed before the first injection (D0) and at 30 (D30), 60 (D60), and 90 days (D90) after the first injection and compared to an age-matched control group (n=21 eyes). Results: The thickness of macular retinal layers increased before treatment due to the presence of intraretinal fluid. A thick retinal pigment epithelium-choriocapillaris complex (RPE-CC) suggested the presence of choroidal neovascular membrane. Intraretinal edema decreased after treatment (P<0.01), but persisting RPE-CC thick-ness resulted in a subretinal scar. Three different annular retinal areas were studied with mfERG (from center to periphery: rings R1, R2, and R3). The amplitude of the first negative component (N1) decreased in R1, R2, and R3 at D30, D60, and D90 when compared with that in controls (P<0.05); the N1 implicit time was delayed in R3 at D30 (P<0.05). The amplitude of the first positive component (P1) was reduced in R1 and R2 at D30, D60, and D90 when compared with that in controls (P<0.01); the P1 implicit time was delayed in R1 at D0 and D60 (P<0.05), in R2 at D0, D30, and D90 (P<0.01), and in R3 at D30 and D60 (P<0.05). Conclusion: Ranibizumab reduces intraretinal edema, even in advanced cases. Central macular activity appeared to increase after the initiation of treatment, improving over time.

Keywords: Macular degeneration/drug therapy; Antibodies; monoclonal, huma ni-zed/therapeutic use; Tomography; optical coherence; Electroretinography; In travitreal injections

RESUMOObjetivo: Avaliar a morfologia e função da retina em pacientes com doença ma-cular relacionada à idade (DMRI), neovascular avançada, antes, durante e após o tratamento com ranibizumabe. Métodos: Vinte e um olhos com diagnóstico de DMRI avançada foram avaliados pela tomografia de coerência óptica (OCT ) e eletrorretinografia multifocal (mfERG). Três injeções intravítreas de ranibizumabe foram administradas em intervalos de 1 mês. As avaliações foram realizadas antes da primeira injeção (D0) e aos 30 (D30), 60 (D60), e 90 dias (D90) após a primeira injeção e comparados com um grupo controle (n=21 olhos). Resultados: A espessura macular estava aumentada antes do tratamento devi-do à presença de fluido intrarretiniano, e o aumento da espessura do complexo EPR-CC foi compatível com a presença de membrana neovascular coroidal. O edema intrarretiniano diminuiu após o tratamento (P<0,01). Três diferentes áreas retinianas anulares (do centro para a periferia: anéis R1, R2 e R3) foram consideradas no mfERG. A amplitude do componente N1 diminuiu nos anéis R1, R2 e R3 em D30, D60 e D90 comparados com o grupo controle (P<0,05); e o tempo implícito de N1 aumentou no anel R3 em D30 (P<0,05). A amplitude do componente P1 diminuiu em R1 e R2 nos dias D30, D60 e D90 comparados com os controles (P<0,01); o tempo implícito de N1 aumentou no anel R1 em D0 e D60 (P<0,05), no anel R2 em D0, D30 e D90 (P<0,01) e no anel R3 em D30 e D60 (P<0,05). Conclusão: O ranibizumabe reduziu o edema intrarretiniano, mesmo em casos avan-çados. A atividade central macular parece aumentar após o início do tratamento e melhorar ao longo do tempo.

Descritores: Degeneração macular/quimioterapia; Anticorpos monoclonais hu ma-nizado/uso terapêutico; Tomografia de coerência óptica; Eletrorretinografia; In jeções intravítreas

Optical coherence tomography and multifocal electroretinography of patients with advanced neovascular age-related macular degeneration before, during, and after treatment with ranibizumab


(OCT) for qualitative and quantitative image analysis and accuracy improvement by characterizing the retinal morphology impairment. The major OCT limitation is the difficulty to perform the tomography of the very same retinal region before and after treatment in order to make an appropriate comparison(2).

Multifocal electroretinography (mfERG) provides a topographic map of retinal function using parameters that ensure a proper evalua-tion of the photopic response. The objective measurements provided by mfERG enable quantification of responses to light from photo-receptors, second order neurons, and inner retinal neurons. mfERG enables simultaneous measurements of responses originating from localized areas in the central and peripheral retina. In this way, mfERG differs from full-field electroretinography (ffERG), which records the global response of the whole retina. Information provided by mfERG for specific retinal areas may assist in explaining functional losses observed by psychophysical methods and complement the structu-ral evaluation provided by ophthalmological observations and OCT imaging. Furthermore, mfERG may reveal the occurrence of retinal dysfunction even when ophthalmological observation, OCT imaging, and psychophysical evaluation suggest little or no impairment of the visual system(3).

One of the few studies using mfERG to evaluate neuroretinal function after the treatment of AMD with ranibizumab described sta-bilization or improvement of visual acuity and reduction of macular thickness after treatment, but also a reduction in mfERG amplitude in patients compared with that in controls(4). The authors suggested that edema reduction could lead to visual acuity improvement, but the drug may also cause tissue hypoxia leading to deterioration of re tinal function(4).

Most studies on the use of ranibizumab for the treatment of neo-vascular AMD evaluated patients with relatively good vision (20/30-20/150)(5-8). The objective of the present study was to use mfERG to study patients with advanced neovascular AMD (visual acuity 20/100 or worse) in order to determine if retinal function improves after ranibizumab treatment.

A short communication of the results of this work was presented in the Annual Meeting of the Association for Research in Vision and Ophthalmology (ARVO)(9).

METHODSThis interventional case series was performed in the Núcleo de

Me dicina Tropical and Hospital Universitário Bettina Ferro de Souza, both in Universidade Federal do Pará. We evaluated 17 patients (23 eyes) diagnosed with advanced neovascular AMD; two eyes were excluded because they had a visual acuity better than 20/100. There-fore, a total of 21 eyes with a visual acuity ≤20/100 (advanced neo-vascular AMD) were evaluated. We excluded patients with cataract opacity because this condition prevents reliable OCT imaging and mfERG recording.

Three intravitreal injections of anti-angiogenic ranibizumab were administered at 1-month intervals. OCT and mfERG (103 hexagons) were performed before the first injection (D0) and at 30, 60, and 90 days after injection (D30, D60, and D90, respectively). Macular OCT (fast macular thickness map, 6 mm) was performed at 10-µm axial resolution with Stratus OCT 3.0 (Carl Zeiss Meditec, Jena, Thuringia, Germany)(10). Total macular volume measurements were made using the default software provided by the device manufacturer.

The mfERG was performed with the Veris System 6.010 (Elec-tro-Diagnostic Imaging, EDI, Redwood City, CA, U.S.A.), which was used for stimulation, recording, and data extraction. We used a high spatial resolution (1280 × 1024 pixels) and temporal resolution (60 Hz) FMS III microdisplay (EDI) to present a black and white stimulus for the patient’s eye. The stimulus was composed by an array of 103 hexagons covering 45° squared visual angle. The hexagons were sca-led considering the changes in retinal cone density with eccentricity

in order to elicit a uniform mfERG across retinal areas at different re tinal eccentricities. The luminance modulation of each hexagon was driven by a complete cycle of an m-sequence of 214-1 elements. The same m-sequence was used to control luminance modulation of all hexagons, but the starting point of the m-sequence reading for each hexagon differed. One m-state controlled hexagon lumi-nance at 200 cd/m2 (flash period), while another m-state switched off hexagon luminance to a residual screen luminance of 0.01 cd/m2 (non-flash period). The base period of the m-sequence reading was 16.6 ms. A red cross (1° of visual angle) was used in the center of the hexagon array as a fixation mark. An infrared camera (EDI) was used for eye movements monitoring. The recording duration lasted for approximately 4 min and 33 sec. The retinal activity was recorded by a corneal Dawson-Trick-Litzkow (DTL) electrode (active electrode) and surface electrodes that were used as reference (placed at the temporal canthus of the tested eye) and ground (placed at the center of the forehead) electrodes. The analog signal was amplified × 50,000, sampled at 960 Hz, and on-line filtered between 10 Hz and 300 Hz with a differential amplifier Model 15LT (Grass, Quincy, MA, U.S.A.). The analog signal was digitized using an acquisition board (PCIE series, National Instruments, Austin, TX, U.S.A.). The peak-to-baseline amplitude and implicit time of the first negative component (N1) and first positive component (P1) components were measured. The analysis was made in three different annular retinal areas: R1, which was composed by hexagons corresponding to the central 8° of the retina; R2, which was composed by hexagons located between 8° and 24° of eccentricity; and R3, which was composed by hexagons located between 24° and 45° of eccentricity. The average of the data from each region was obtained. All electrophysiological procedures were performed according to the guidelines of the International Society for Clinical Electrophysiology of Vision (ISCEV)(11).

A sex- and age-matched control group of 21 eyes was also eva-luated using OCT and mfERG. We compared the results from OCT and mfERG patients with the control group using one-way ANOVA followed by Tukey’s post-hoc test (α=0.05).

All patients were studied according to the tenets of Declaration of Helsinki. Institutional approval was granted by the Committee for Research Ethics of the Núcleo de Medicina Tropical (Protocol #027/2010). Informed consent was obtained from all patients.

RESULTSPatients included 12 women and 5 men, 70.76 ± 2.24 years old.

Their visual acuities (logMAR ± standard error) at D0 and D90 were 1.75 ± 0.13 and 1.91 ± 0.14, respectively (P=0.28).

OCT performed prior to the treatment revealed an increased macular thickness probably due to intraretinal edema. We speculated that the increased thickness of the retinal pigment epithelium-cho -riocapillaris complex (RPE-CC) was compatible with the choroidal neovascular membrane. The temporal evolution of the retinal thickness was as follows: D0=8.44 ± 0.35 mm, D30=7.13 ± 0.22 mm, D60=7.16 ± 0.24 mm, and D90=7.23 ± 0.28 mm. Subjects without AMD had 6.52 ± 0.09 mm. Follow-up OCT examination showed a sig-nificant reduction in macular volume when D0 was compared with follow-up examination (P<0.01 one-way ANOVA, Tukey’s post-hoc test), but persistence of the increased retinal thickness compared with controls (Figures 1-2).

The results were grouped in three annular retinal areas for mfERG analysis: R1 (central area, 0°-8°), R2 (parafoveal area, 8°-24°), and R3 (peripheral area, 24°-45°). In each of these rings, we analyzed N1 and P1.

Prior to treatment (D0), N1 amplitudes in R1 did not differ among patients and controls, but after the first injection (D30), the ampli-tude was significantly reduced (P<0.01). This difference decreased 2 months and 3 months after the first injection (D60 P<0.01, D90 P<0.05, one-way ANOVA, Tukey’s post-hoc test; Figure 3 A). In R2, the N1 amplitude was significantly reduced in patients compared

Almeida INF, et al.


with that in controls over the entire testing period (P<0.01, one-way ANOVA, Tukey’s post-hoc test; Figure 3 A). In R3, the N1 amplitude was significantly reduced at D30, D60, and D90 compared with that in controls (P<0.01, one-way ANOVA, Tukey’s post-hoc test; Figure 3A).

In R1 and R2, N1 implicit time was not significantly different in patients and controls (Figure 3 B). In R3, N1 amplitude was delayed at D30 compared with that in controls (P<0.05, one-way ANOVA, Tukey’s post-hoc test; Figure 3 B).

Prior to treatment (D0), P1 amplitudes in R1 and R2 did not differ between patients and controls, but the amplitude was significantly reduced in patients at D30, D60, and D90 compared with that in con-trols (P<0.01, one-way ANOVA, Tukey’s post-hoc test; Figure 4 A). P1 amplitude in R3 did not differ between the two groups (Figure 4 A).

In R1, P1 implicit time at D0 and D60 was significantly delayed in patients compared with that in the control group (P<0.05, one-way ANOVA, Tukey’s post-hoc test; Figure 4 B). In R2, P1 implicit time was delayed at D30, D60, and D90 in patients compared with that in controls (P<0.01, one-way ANOVA, Tukey’s post-hoc test; Figure 4 B). In R3, P1 implicit time at D30 and D60 was significantly delayed in patients compared with that in controls (P<0.05, one-way ANOVA, Tukey’s post-hoc test; Figure 4 B).

Tables 1 and 2 show the mean values of the amplitude of mfERG components along the therapy.

DISCUSSIONIn a multicenter study (the MARINA Study), Boyer and colleagues

found improved visual acuity in the ranibizumab-treated group(12), a result corroborated by another multicenter study(13). Most multi-center studies evaluated only patients with good visual acuity, and a few studies evaluated the response of anti-angiogenic therapy in patients with advanced AMD. Despite subjective reports of improved perception in the central scotoma, visual acuity remained altered, with no significant difference before and after treatment. However, a significant reduction in retinal thickness was observed by measuring total macular volume before and after treatment. The results of this work showed a stabilization of visual acuity in patients before and after treatment, but the patients’ visual acuity was different from that observed in previous studies.

There was a significant reduction in retinal thickness after the first month of treatment compared with other days of examination, which is in agreement with other studies(14,15). Keane et al. used OCT to study 95 patients with neovascular AMD after intravitreal injections of ranibizumab(14). The total subretinal fluid (SRF) volume reached its lowest level by 1 month after the injection (P<0.001). Karagiannis et al. evaluated 34 patients treated with monthly injections of intravitreal bevacizumab for six months followed by monthly injections of rani-bizumab for 12 months(15). In these patients, OCT showed a decrease in retinal thickness (P=0.033) after bevacizumab, and this reduction improved after changing to ranibizumab (P=0.09).

Some authors suggested that patient history and age should be considered before analyzing mfERG since these factors may alter the retinal response. Importantly, mfERG responses became smaller and delayed with advancing age(16). The responses mediated by re-tinal cones occurring before retinal damage became observable by ophthalmoscopy(17). Some studies suggested that the implicit time of the photopic mfERG responses was more sensitive for detecting areas with retinal dysfunction than response density(18).

We estimated the amplitude and implicit time mean values for N1 and P1 mfERG components as a function of the time after rani-bizumab injections. We focused on the temporal evolution of these parameters along the therapy. In this study, constant factors that could change these parameters, such as the hexagon areas, were not considered when estimating amplitude density. Previous studies revealed a delay in N1, P1, and the second negative component (N2)

Figure 1. Optical coherence tomography (OCT) of macular volume of controls (dashed line) and patients (D0, D30, D60, and D90).

A

C

B

D

Figure 2. Optical coherence tomography (OCT) of patients at (A) D0, (B) D30, (C) D60, and (D) D90. An increased thickness of the inner layers of the macula area was seen at D0 due to intraretinal fluid (white arrow) as well as increased thickness of the retinal pigment epithelium-choriocapillaris complex (RPE-CC) (red arrow). Intraretinal edema was reduced at D30, D60, and D90 (P<0.01, ANOVA, Tukey’s test), but the RPE-CC (red arrow) resulted in a subretinal scar.

Optical coherence tomography and multifocal electroretinography of patients with advanced neovascular age-related macular degeneration before, during, and after treatment with ranibizumab


Table 1. mfERG amplitude and implicit time of the N1 component for three retinal areas at different days of therapy. Averaged values for controls are shown for comparison

R1 amplitude (µv)

R2 amplitude (µv)

R3 amplitude (µv)

GT-D0 -11.43 ± 0.99** -6.24 ± 0.43* -5.84 ± 0.51*

GT-D30 -8.09 ± 0.61* -4.41 ± 0.37* -4.71 ± 0.50*

GT-D60 0-8.54 ± 0.79** -4.41 ± 0.37* -4.49 ± 0.43*

GT-D90 0-9.88 ± 0.99** -5.18 ± 0.48* -4.67 ± 0.46*

GC -13.87 ± 0.95 0 -8.46 ± 0.58* -7.26 ± 0.50*

R1 implicit time (ms)



GT-D0 17.79 ± 0.69 16.80 ± 0.54 15.47 ± 0.21*

GT-D30 15.23 ± 1.19 17.76 ± 0.91 16.46 ± 0.34*

GT-D60 14.02 ± 0.92 16.18 ± 0.93 16.04 ± 0.41*

GT-D90 17.06 ± 0.46 16.81 ± 0.61 15.83 ± 0.23*

GC 15.74 ± 0.48 14.94 ± 0.23 15.16 ± 0.21*

GT= group of patients receiving therapy; GC= group of control subjects; D0= day of the first injection; D30= 30th day after therapy initiation; D60= 60th day after therapy initiation; D90= 90th day after therapy initiation; *= P<0.01; **= P<0.05.

Table 2. mfERG amplitude and implicit time of the P1 component for three retinal areas at different days of therapy. Averaged values for controls were also shown for comparison

R1 amplitude (µv)

R2 amplitude (µv)

R3 amplitude (µv)

GT-D0 14.24 ± 1.61* 10.26 ± 1.06* 11.10 ± 1.27

GT-D30 11.57 ± 1.33* 07.89 ± 0.93* 08.29 ± 1.08

GT-D60 11.21 ± 0.95* 07.64 ± 0.79* 08.50 ± 0.90

GT-D90 12.39 ± 1.10* 08.39 ± 0.90* 09.14 ± 1.15

GC 18.57 ± 1.25* 13.26 ± 0.89* 12.79 ± 0.94




GT-D0 35.36 ± 0.76** 34.73 ± 0.71* 33.38 ± 0.51*

GT-D30 32.94 ± 0.98** 35.65 ± 0.97* 34.19 ± 0.79*

GT-D60 35.67 ± 1.14** 34.21 ± 0.54* 32.62 ± 0.33*

GT-D90 34.31 ± 1.07** 34.91 ± 0.68* 33.55 ± 0.63*

GC 31.44 ± 0.32** 31.61 ± 0.22* 31.20 ± 0.15*

GT= group of patients receiving therapy; GC= group of control subjects; D0= day of the first injection; D30= 30th day after therapy initiation; D60= 60th day after therapy initiation; D90= 90th day after therapy initiation; *= P<0.01; **= P<0.05.

Figure 3. (A) Amplitude and (B) implicit time of the N1 component of the multifocal electroretinogram (mfERG).

A

B

A

B

Figure 4. (A) Amplitude and (B) implicit time of the P1 component of the multifocal electroretinogram (mfERG).

in 16%, 23%, and 31%, respectively, but response density decreased by only 4.2% with the stimulated area(19). A study evaluating only two patients with exudative AMD demonstrated a decrease in mfERG res-ponses but no correlation between lesion size and response density or visual acuity(20). Another study that evaluated nine patients with AMD found no changes in ffERG after injecting bevacizumab (Avas-tin), but some improvement was detected in the mfERG responses, suggesting that the drug improved macular function as indicated by a better mfERG response and was not toxic to the retina once there were no ffERG changes(21). Feigl et al.(4) evaluated the mfERG response in three patients before, during, and after ranibizumab treatment. They divided the stimulus in two concentric rings (R1 and R2) and found no significant differences in N1 and P1 amplitudes for R1 or R2 prior to treatment in patients compared with those in controls. After

Almeida INF, et al.


the third treatment, N1 and P1 amplitudes in R1 and R2 significantly decreased compared with those in controls. The implicit time of N1 and P1 in R1 and R2 did not change after treatment, but it was sig-nificantly delayed in two out of three patients when compared with those in controls. The authors argued that the mfERG represented AMD neovascularization (mfERG evaluated retinal function in up to 25° of the visual angle and thus represents the area affected by the neovascularization membrane), whereas visual acuity reflected the function of the foveal area, a region <1° of the visual angle. They sugges ted that the adverse effects of anti-VEGF in affected retinal areas were larger than the area clinically affected by AMD(4).

The role of VEGF in choroidal neovascularization is well establi-shed(22). Inflammation and focal retinal ischemia induce VEGF pro-duction, leading to the proliferation of neovascular membranes from the choriocapillaris(23). Ranibizumab was demonstrated as acting as anti-ocular VEGF; it neutralizes VEGF but whether it has an adverse effect on the healthy peripheral retina is not clear(24). Some studies suggest that VEGF inhibition may cause retinal ischemia(25).

The present work suggests a stabilization of visual acuity and reduction of edema after treatment, which is in agreement with other studies(26). Costa and colleagues suggested that the edema reduction alone could result in an improvement and/or stabilization of visual acuity(27). However, conflicting mfERG data were reported in other studies and suggested that AMD interrupted delicate intraretinal mechanisms. The progression of the disease associated or not with the adverse effects of anti-VEGF could interfere with neuroretinal functions(28).

We observed a reduction in the electrical activity of the central macular region (R1) after the initiation of treatment in patients under-going anti-angiogenic therapy with ranibizumab. As AMD affected mainly the central region, changes were expected in R1, which had the largest concentration of retinal neurons affected by the disease and was the place where the drug’s anti-angiogenic effect was expec-tedly higher(4). Some experimental studies suggested a possible toxic effect of high-dose intravitreal VEGF inhibitors on retinal neurons(29,30). The lower retinal electrical activity observed immediately after treat-ment raised the possibility of a deleterious effect of the medication on macular function, which fortunately recovered over time. Perhaps the preferential occurrence of macular lesions in advanced AMD faci-litated a very large increase in the levels of anti-angiogenic factors in the central retina, temporarily causing this deleterious effect.

The present study showed a reduction of retinal thickness due to a reduction of edema and tissue fibrosis. The mfERG findings showed that the amplitude of mfERG components decreased and that the implicit time of these components increased, suggesting a worse-ning of retinal activity. The non-significant reduction in visual acuity indicated that retinal activity impairment did not affect it. Along the therapy, we found an improvement of the mfERG response. More controlled studies will be necessary for more conclusive results of the effects of ranibizumab on AMD.

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Original Article


INTRODUCTIONThe Metropolitan Area of Buenos Aires (MABA) is comprises Buenos

Aires city and 24 districts, with an estimated population of 14 million, being the 10th largest megacity in the world and the 3rd in Latin America. By its 200 km2, Buenos Aires is the largest city in Argentina, with a population of approximately 3 million people.

In the Delta Islands of the Paraná River, approximately 112 miles a way from the city of Buenos Aires, pasture burning for grazing li-vestock is a fairly common practice. Between April 16th and April 24th 2008, there was a series of wildfires that affected over 70,000 hectares. The extent of the fire, the drought, and the wind direction generated a thick cloud of smoke that caused the most severe episode of acute air pollution in the MABA ever recorded. The northern and northeas-

ABSTRACTPurpose: To evaluate the acute impact of the wildfire smoke episode in 2008 on the ocular surface of subjects living in the Metropolitan Area of Buenos Aires (MABA).Methods: A total of 86 subjects were evaluated: Group 1 comprised patients from a public ophthalmology hospital (N=35) and Group 2 comprised healthy volunteers (N=51). All subjects answered a questionnaire on ocular symptoms and underwent ophthalmologic examination [bulbar conjunctival hyperemia, corneal fluorescein staining, rose bengal vital staining, tear break-up time (TBUT), Schirmer I test, tear lysozyme, and impression cytology] during and after the acute episode. Concentrations of carbon monoxide (CO), nitrogen dioxide (NO2), and particulate matter (PM) were measured before, during, and after the acute episode. Results: Both groups showed a statically significant increase in ocular symptoms and bulbar conjunctival hyperemia and a statically significant decrease in tear break-up time during the acute episode. Group 1 showed more severe symptoms and a statistically significant increase in fluorescein and rose bengal staining intensities during the acute episode. We found a significant negative correlation between ocular symptoms and tear break-up time. During the episode, the levels of CO, NO2, and particulate matter in MABA were four times higher than the usual average levels for the same period in 2007 and 2009. Conclusions: Increased air pollution from the burning of biomass is associated with a decrease in the stability of the tear film (TBUT), generating areas of ocular surface exposure that may be the cause of the increased feeling of irritation. Group 1 was more affected by not having a healthy ocular surface, and thus consulted an ophthalmologist. Cytological changes in the conjunctiva were not observed, which could be due to the short duration of the episode.

Keywords: Urban fires; Conjunctiva/cytology; Cytodiagnosis; Air pollution; Tears/physiology; Fluorescein/diagnostic use; Rose bengal/diagnostic use; Hyperemia; Argentina

RESUMOObjetivo: Avaliar os efeitos agudos da fumaça do episódio de incêndio violento ocor-rido em 2008, sobre a superfície ocular de sujeitos que vivem na Região Metropolitana de Buenos Aires (MABA). Métodos: Um total de 86 indivíduos foram avaliados: Grupo 1: pacientes de um hospital público de oftalmologia (N=35) e Grupo 2: voluntários saudáveis (N=51). Todos os participantes responderam a um questionário sobre os sintomas oculares e foram submetidos a exame oftalmológico (hiperemia conjuntival bulbar, teste de fluoresceína, corante rosa bengala, tempo de ruptura do filme lacrimal (TBUT ), teste de Schirmer I, lisozima lacrimal e citologia de impressão) durante e após o episódio agudo. As concentrações de monóxido de carbono, dióxido de nitrogênio e partículas (PM) foram medidas antes, durante e após o episódio agudo.Resultados: Ambos os grupos apresentaram aumento estatisticamente significativo dos sintomas oculares, hiperemia conjuntival bulbar, e diminuição estatisticamente significativa no tempo de ruptura do filme lacrimal durante o episódio agudo. Grupo 1 apresentou maior intensidade dos sintomas e aumento estatisticamente significativo no teste de fluoresceína e rosa bengala durante o episódio agudo. Encontramos uma correlação negativa significativa entre os sintomas oculares e tempo de ruptura do filme lacrimal. Durante o episódio agudo de 2008, os níveis de CO, NO2 e PM na Região Metropolitana de Buenos Aires foram 4 vezes maiores do que os níveis médios habituais para o mesmo período de 2007 e 2009. Conclusões: O aumento da poluição do ar a partir da queima de biomassa está associado a uma diminuição da estabilidade do filme lacrimal (TBUT ) gerando zonas da exposição da superfície ocular, que podem ser a causa do aumento da sensação de irritação. Grupo 1 foi mais afetado por não ter superfície ocular saudável e, portanto, consultaram um oftalmologista. Mudanças citológicas da conjuntiva não foram observadas e isso poderia ser devido à curta duração do episódio.

Descritores: Incêndios urbanos; Conjuntiva/citologia; Citodiagnóstico; Poluição do ar; Lágrimas/fisiologia; Fluoresceína/uso diagnóstico; Rosa bengala/uso diagnóstico; Hiperemia; Argentina

tern areas of the province of Buenos Aires, including MABA, had been swathed in thick smoke (Figure 1 A, B). It is estimated that the wildfires of the Delta of the Paraná River affected approximately 20 million people. The smoke was so dense in the city of Buenos Aires that on April 18th, the airports and highways were closed, and classes in all schools were suspended (Figure 1 B, C). The levels of carbon mo noxide (CO), nitrogen dioxide (NO2), and particulate matter (PM) registered during the episode were the highest ever registered in the region of the River de la Plata.

Most of the previous studies on the impact of wildfires were con-ducted focusing on short-term outcomes related to hospital admittan-ces or specific cohorts, particularly susceptible to respiratory distress, such as patients with chronic obstructive pulmonary disease or asthma or those who are directly exposed to the fires, such as firemen(1-3).

Impact of wildfire smoke in Buenos Aires, Argentina, on ocular surface

Efeitos da fumaça de incêndios na superfície ocular em Buenos Aires, Argentina

Martin bErra1,2, gustavo galPErín1,2, laura daWidoWsKi3, Julia tau2, isabEl MÁrquEz4, alEJandro bErra2,4

Submitted for publication: September 8, 2014 Accepted for publication: January 26, 20151 Hospital de Oftalmología Pedro Lagleyze, Buenos Aires, Argentina.2 Laboratorio de Investigaciones Oculares, Departamento de Patología, Facultad de Medicina, Uni-

versidad de Buenos Aires, Buenos Aires, Argentina.3 Grupo de Monitoreo Ambiental, Comisión Nacional de Energía Atómica, Buenos Aires, Argentina. 4 Laboratorio BioFundus, Buenos Aires, Argentina.



Corresponding author: Martín Berra. Laboratorio de Investigaciones Oculares. Departamento de Patología, Facultad de Medicina. Universidad de Buenos Aires - J.E Uriburu 950 - Postal Code 1114, Ciudad Autónoma de Buenos Aires, Buenos Aires - Argentina - E-mail: [email protected]

Berra M, et al.


The ocular surface is daily in contact with air, and epidemiolo-gical studies have indicated increasing incidence of dry eye disease (DED) worldwide(4-5). This common ocular condition has multiple causes that are not entirely understood. The emerging awareness that environmental factors can contribute to DED is supported by recent studies(6-8). Moreover, impact of the environment on the pathophysiology of DED has been studied and confirmed in animal models of DED(8-11).

There are few studies that show the ocular impact of wildfires(12-13). The wildfire episode in the northern area of the province of Buenos Aires was a unique opportunity to study the ocular impact of the exposure to the smoke produced by pasture burning on patients and healthy volunteers in a megacity.

METHODSPOPULATION OF STUDY

The study population consisted of two groups, each with two sub groups (Table 1).

Group 1 (G1) comprised 35 patients who visited the Emergency Unit of Hospital Oftalmologico Pedro Lagleyze on April 19th due to ocular complaints; Hospital Oftalmologico Pedro Lagleyze is the largest Ophthalmology Hospital in Argentina and located in the study area.

Group 2 (G2) included 51 healthy volunteers who accompanied the patients to the hospital on April 19th. Both groups were evaluated on a second appointment on May 22nd, when the air pollutant levels had recovered to normal values in the city of Buenos Aires (Table 1).

We included 35/67 patients and 51/72 healthy volunteers becau-se only 35 patients and 51 healthy volunteers visited the hospital on both April 19th and May 22nd.

To be included in G1 and G2, subjects had to give their informed consent before being enrolled in the study, had to be between 18 and 65 years old, and had to have lived and worked in MABA between March and May 2008, far from areas close to stationary sources of significant emissions, such as factories. The exclusion criteria for G2 included having a previous history of ocular surface disease or using contact lenses or any systemic or topical ocular drugs.

This study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethical Committee of the Hospital Oftalmologico Pedro Lagleyze, Buenos Aires, Argentina.

OCULAR SYMPTOMS QUESTIONNAIRE

All subjects answered a symptoms questionnaire composed by ques tions that assessed the presence of ocular burning, dryness, foreign body sensation, irritation, and itching. Subjects were asked to grade the intensity of the symptoms on a scale of 1 to 3 (1 = minor, 2 = mild, and 3 = severe). We also investigated if any factor worsened the symptoms, such as the time of the day of exposure, staying in- or outdoors, if it was the first time they experienced the symptoms, and if they were using any eye drops to improve the ocular symptoms.

OPHTHALMOLOGIC EXAMINATION The usual ocular surface tests available in the hospital and our

standard of care in our institution were used to evaluate the subjects.

Bulbar conjunctival hyperemia

Bulbar conjunctival hyperemia was recorded using five levels of severity from grade 0 (normal) to grade 4 (severe)(14) with a slit lamp (SL-8Z; Topcon Corp., Tokyo, Japan). The inter-subject variation in gra-ding was determined to be 1.0 scale units. Interpretation of grading levels was as follows: 0 = Normal, 1 = Trace, 2 = Mild, 3 = Moderate, and 4 = Severe.

Corneal fluorescein staining

Fluorescein staining with cobalt blue light was used to assess the presence or absence of corneal keratitis. Corneal fluorescein staining was evaluated with fluorescein strips (Diagnóstico Ocular, Buenos Aires, Argentina), which were wetted with 0.9% sodium chloride and gently applied to the inferior fornix. The cornea was divided into five regions (central, superior, inferior, nasal, and temporal), and each re-gion was graded by a scale, with values ranging from grade 0 (normal) to grade 4 (severe)(14). Grades of each area were added to produce a final score.

Conjunctival rose bengal vital staining

Vital staining with rose bengal was performed by instilling one drop of 1% rose bengal solution (Farmacia Magister, Buenos Aires, Argen-tina) into the inferior fornix. The staining pattern was evaluated and graded according to the Van Bijsterveld scoring system on a scale that ranges from 0 to 9. Scores higher than 4 were considered abnormal(15).

Tear break-up time

Tear break-up time (TBUT) was measured by instilling one drop of 1% fluorescein solution (Farmacia Magister, Buenos Aires, Argentina) into the inferior fornix. The subject was asked to blink several times

Figure 1. A) Satellite image (MODIS/Aqua) of the wildfires in the Delta of the Parana River on April 18th, 2008. Red boxes indicate wildfire areas. Scale: 1 px=250 m (NASA. Fires and smoke over Argentina, seen from MODIS on the Aqua satellite at 2008/18/04 at 17:50 UTC. Scale: 1 px=250 m) http://rapidfire.sci.gsfc.nasa.gov/gallery/?20081090418/Argentina.A2008109.1750.250 m.jpg, 2008). B and C) Images of the same location in the city of Buenos Aires at two different moments: (B) April 18th, 2008, during the wildfires in the Delta of the Paraná River (http://nidodecaranchos.blogspot.com/2008_04_01_archive.html). (C) May 20th, 2008 (http://cliobuenosaires.blogspot.com/2010/05/el-obelisco-un- simbolo-que-define.html).

A

B C

Table 1. Study population

Group Subgroup Subjects NTime of examination

relative to the acute episode

G1 G1A Patients 35 During (19th April)

G1B The same as G1A 35 After (22nd May)

G2 G2A Healthy volunteers 51 During (19th April)

G2B The same as G2A 51 After (22nd May)



and then stop, at which point TBUT was assessed by monitoring the time (in seconds) elapsing to the appearance of the initial black spot on the cornea under slit lamp examination with a cobalt blue filter(16). Values of 10 seconds or below were considered abnormal(17).

Schirmer’s I test

Subjects were evaluated by Schirmer’s I test without topical anes-thesia. One graded sterile Schirmer strip (Opthalmos™, Brazil) was pla-ced in the lateral canthus of the inferior lid margin of both eyes, and the subjects were instructed to keep their eyes closed during the test. After 5 minutes, the length of wetting was measured in millimeters, and values of 10 mm or less were considered abnormal(17).

Tear lysozyme concentration

Tears were collected by gently applying a 5-mm diameter filter paper disc in the inferior conjunctival cul-de-sac of both eyes for one minute with eyes closed. Samples were kept at -20°C until processed. To determine tear lysozyme concentration, we used the Micrococcus lysodeikticus (ATCC 4698, M3770; Sigma-Aldrich, St. Louis, MO) agar diffusion assay(18) in Mueller Hinton agar plates (Bio Merieux, Marcy l’Etoile, France). Each disc was placed in the plate with the M. lyso-deik ticus (2 × 106 CFU/mL) suspension gel, and the inhibition halo was measured after 24 hours. To calculate the lysozyme concentration, a standard curve was obtained using identical discs wetted with 10,000, 1,000, 100, and 10 µg/mL of lysozyme (ATCC 4698, L6876; Sigma-Aldrich) diluted in phosphate-buffered saline (Invitrogen Corp., Carlsbad, CA). Values of 1000 µg/mL or below were considered abnormal(19-20).

Impression cytology

Semicircular filters, approximately 15 mm diameter, (Polyvinylide-ne Fluoride -PVDF-, 22-μm pore size; Millipore Corp., Bedford, MA, USA) were applied to the inferior tarsal and bulbar conjunctiva after instillation of one drop of topical anesthetic (tetracaine) in each eye, and any excess fluid was wiped away. The paper fragments were gently pressured with the blunt end of the forceps, and the fragments were peeled off and immediately immersed in tubes containing absolute ethanol. After fixation, specimens were rehydrated in 70% ethyl alcohol, then placed successively in periodic acid-Schiff reagent, sodium metabisulfite, Gill’s hematoxylin, and Scott’s tap water. Speci-mens were then rinsed with 95% alcohol and absolute alcohol. Xylene was used to make the filter paper transparent. Slides were examined under a conventional light microscope, using 400× magnification. Mor phometric analysis was performed using a point-counting techni-que, PAS positive areas were counted across 10 ran dom microscopic high-power fields (HPF) on a 100-point, and 50 lines grid on a video system coupled to the microscope. The result for each subject was calcu-lated as the average of the 10 HPF counts. A single investigator perfor-med all the observations and was blinded to the origin of the samples(18).

Ocular damage was based on the scale of Nelson, comprising degrees of severity. This scale is based on the density of goblet cells and appearance of epithelial cells (grade squamous metaplasia). Level 0 is considered normal; 1, slightly altered; 2, moderately altered; and 3, severely altered(21).

Assessment of air pollutants during and after the acute episode

Average CO and NO2 concentrations over cumulative periods of three minutes were measured. The calibration of the equipment was made with high quality certified synthetic air. Nondispersive infrared absorptiometry was used to measure CO with an HORIBA monitor, model APMA-360, using a gas standard of 12 ppm to fix the span scale calibration. The HORIBA monitor APNA-360 was used for measuring NO2 on the basis of the chemiluminescence method. The equipment was calibrated with a NO gas standard of 99.7 ppm diluted with a 1:1000 ratio by means of an HORIBA device, SGGU.514. These measures were performed before, during, and after the acute episode. Data from particulate matter (PM) were obtained from the ambient agency of the city of Buenos Aires.


Results are expressed as the mean ± standard deviation. When a test was performed in both eyes, the mean of the two measurements was used in the statistical analysis. Statistical analysis was performed using the T-test for independent samples to compare data between groups and the T-test for paired samples to compare data within groups. Linear regression was used to analyze the association between symptom levels and tear break-up time values. All tests were per-formed with a significance level of 0.05 using SPSS Statistics 17.0. (Copyright© 2007 Sun Microsystems, Inc., 4150 Network Circle, Santa Clara, California 95054, USA).

RESULTSOCULAR SYMPTOMS QUESTIONNAIRE

The individuals of group G1 were patients who went sponta-neously to the Emergency Unit of Hospital Oftalmologico Pedro Lagleyze with ocular complaints on April 19th, 2008. The 35 patients in G1 included 25 women and 10 men, with an average age of 42.5 ± 10.1 years. A total of 31/35 patients had a history of ocular surface pathology: Eighteen suffered from dry eyes (seven had Sjögren’s syndrome), 10 from allergic conjunctivitis, and three suffered from ocular pemphigoid. Four patients had no history of ocular surface pathology but were included in this group because their ocular surfa-ce complaints were of a severity sufficient to attend hospital. During the acute episode, all patients (G1A) experienced at least three of the symptoms evaluated (burning, dryness, foreign body sensation, itchi-ness, and irritation). Burning, foreign body sensation, and irritation were the most frequent and severe symptoms (100%) referred during the acute episode. A total of 83% experienced the symptoms during the whole day; 66% reported worsening of the symptoms when outdoors; and only 11% experienced these symptoms for the first time. There was a significant difference in the frequency and severity of the symptoms when the patients were compared over time, i.e., during vs. after the acute episode (G1A vs. G1B, p<0.01; Figure 2).

Figure 2. Severity of symptoms during and after the acute episode. The figure depicts the mean grade of the reported symptoms ± the standard deviation of the mean for each of the two groups at two different time points [during (19th of April) and after the episode (22nd of May)]. G1A = patient data during the episode; G1B = patient data after the episode; G2A = healthy volunteers data during the episode; G2B = healthy volunteers data after the episode.

Berra M, et al.


Table 2. Test results expressed as means ± standard deviation obtained during and after the acute episode for each group

GroupsBulbar conjuctival

hyperemiaCornea fluorescein

stainingRose bengal vital staining TBUT (seconds)

Schirmer’s I test (mm/5 min)

Tear lyzozyme (ug/ml)

Impression cytology (10 HPF)

G1A 1.8 ± 1.0 3.7 ± 1.2 6.4 ± 2.1 4.4 ± 1.1 11.3 ± 9.5 1533 ± 987 202 ± 145

G1B 1.1 ± 1.0 1.9 ± 0.7 3.7 ± 1.8 6.2 ± 1.3 10.1 ± 8.2 1612 ± 1072 186 ± 120

G2A 0.8 ± 0.3 0.7 ± 0.3 1.9 ± 1.2 4.9 ± 1.2 17.6 ± 9.7 2489 ± 412 298 ± 132

G2B 0.3 ± 0.1 0.6 ± 0.2 1.7 ± 0.8 7.8 ± 1.2 15.3 ± 7.5 2570 ± 426 315 ± 151

*p<0.05 among groups (G1A vs. G1B and G2A vs. G2B). All the differences between groups (G1A vs. G2A and G1B vs. G2B) were statistically significant.

* * * *

**

Figure 3. Correlation between severity of symptoms and tear break-up time (TBUT). There was a statistically significant correlation between the reported severity of ocular burning, dry eye, foreign body sensation, itching and irritation, and mean TBUT values. Values are expressed as means ± 0.95 confidence intervals.

A total of 51 healthy volunteers were evaluated, including 23 women and 28 men, with an average age of 39.1 ± 11.9 years. During the acute episode, 79% of healthy volunteers (G2A) experienced at least three of the symptoms evaluated. The most frequent and severe symptom was ocular irritation (80%). Most people reported sustaining ocular irritation during the whole day (55%) or at the end of the day (41%). In addition, 65% reported worsening of the symptoms when outdoors and 57% experienced these symptoms for the first time. There was a significant difference in the frequency and severity of the symptoms when the healthy volunteers were compared over time, i.e., during vs. after the acute episode (G2A vs. G2B, p<0.01; Figure 2).

OPHTHALMOLOGIC EXAMINATION

Table 2 shows the values obtained from G1A-B (N=35) and G2A-B (N=51) for all tests performed.

The patients (G1) showed a statistically significant increase in bulbar conjunctival hyperemia (G1A vs. G1B, p=0.0061), corneal fluorescein staining (G1A vs. G1B, p<0.0001), and rose bengal vital staining (G1A vs. G1B, p<0.0001) during the acute episode.

The healthy volunteers (G2) only showed a statistically significant increase in bulbar conjunctival hyperemia (G2A vs. G2B, p=0.0001). No statistically significant differences were found in corneal fluorescein staining (G2A vs. G2B, p>0.05) or rose bengal vital staining (G2A vs. G2B, p>0.05) during the acute episode.

No statistically significant differences within the groups were found when analyzing the values obtained by Schirmer’s I test, tear lysozyme, and impression cytology.

The healthy volunteers showed statistically significant differences when compared with patients for the entire test performed during and after the acute episode (G1A vs. G2A and G1B vs. G2B, p<0.05).

Both patients (G1) and healthy volunteers (G2) showed a statisti-cally significant (p<0.0001) decrease in TBUT values during the acute episode. We also observed a negative correlation between severity of symptoms and TBUT values (Figure 3), significant (p<0.05) for the symptoms dryness and irritation (Table 3).

POLLUTANT LEVELS

During April 16th to April 20th, the measured mean levels of CO, NO2, and PM concentrations were four times higher than the usual average levels for this time of the year. Figure 4 depicts the concen-trations of these substances before, during, and after the episode. Hourly measured values of CO reached 17 ppm on April 17th, which is 8.5 times as much as the average value (approximately 2 ppm) registered in the area. Moreover, the 8 hours average value was grea-ter than the standard value recommended by the WHO Air Quality Guidelines (AQG) established by the World Health Organization(22)

in 29 opportunities. For NO2 and PM, average levels are usually not exceeding 50% of the maximum levels recommended by the WHO, but during the acute episode, the concentrations were twice as high as those recommended in the WHO guidelines(23-24).

DISCUSSIONIn this study, we assessed the impact of acute exposure to high

levels of pollutants produced by pasture burning on the ocular sur-face of subjects living in an affected megacity.

Previous studies have demonstrated that ocular symptoms are a frequent finding in people exposed to the smoke generated by fires, including wildfires(12-13,24).

During the acute episode, the patients who attended the hos-pital emergency room and the healthy volunteers experienced ocu lar symptoms, an increase in bulbar conjunctival hyperemia, and a reduc-tion in TBUT values.

These results indicate that the patients showed more severe symp-toms and a greater reduction in TBUT values. Meanwhile, Schirmer´s I test, tear lysozyme, and impression cytology showed no diffe rences.

Low TBUT values and high concentrations of air pollutants (PM10, NO2, and CO) measured during the acute episode indicated an in-fluence of high concentrations of air pollutants on tear film stability; this result is similar to a previous study(25).

The smoke generated by pasture burning is a complex mixture of potentially hazardous substances, including particles composed of polycyclic aromatic hydrocarbons, carbon monoxide, aldehydes, or-ganic acids, organic compounds, nitrogen oxides, and sulfur(26). These substances are directly in contact with the tear film and apparen tly cause tear film instability. The reported symptoms of burning, foreign body sensation, itching, and irritation are possibly caused by direct



Table 3. Data obtained from linear regression analysis. Dependent variable = TBUT

Constant variables

Unstandardized coefficients

Standardized coefficients

B Std. error Beta t pBurning -0.195 0.132 -0.126 -1.478 <0.141

Dryness -0.388 0.128 -0.261 -3.029 <0.003

Foreign body sensation

-0.108 0.102 -0.064 -1.061 <0.290

Irritation -0.524 0.113 -0.370 -4.622 <0.001

Itching -0.072 0.096 -0.052 -0.745 <0.457

toxicity or as a consequence of ocular dryness caused by tear film ins tability.

CONCLUSION In this study, we demonstrated that acute exposure to high levels

of air pollution causes tear film instability without remodulation of the ocular surface. Furthermore, the perceptions of symptoms of nor mal subjects were not severe enough to trigger a visit to the emer -gency unit. In contrast, patients who suffered from dry eyes, allergic conjunctivitis, Sjögren’s syndrome, and ocular pemphigoid experien-ced these symptoms with a higher degree of severity, trigge ring a

visit to the emergency unit. This indicates that this last group of pa-tients is more susceptible to developing and/or suffering from these ocular symptoms.

REFERENCES 1. Lipsett M, Waller K, Shusterman D, Thollaug S, Brunner W. The respiratory health

impact of a large urban fire. Am J Public Health. 1994;84(3):434-8. 2. Mott JA, Meyer P, Mannino D, Redd SC, Smith EM, Gotway-Crawford C, et al. Wildland

forest fire smoke: health effects and intervention evaluation, Hoopa, California, 1999. West J Med. 2002;176(3):157-62. Comment in: West J Med. 2002;176(3):162-3.

3. Vedal S, Dutton SJ. Wildfire air pollution and daily mortality in a large urban area. Environ Res. 2006;102(1):29-35. Comment in: Environ Res. 2008;106(3):423-4; discussion 425.

4. Moss SE, Klein R, Klein BE. Prevalence of and risk factors for dry eye syndrome. Arch Ophthalmol. 2000;118(9):1264-8.

5. Moss SE, Klein R, Klein BE. Incidence of dry eye in an older population. Arch Ophthal-mol. 2004;122(3):369-73.

6. Iyer JV, Lee SY, Tong L. The dry eye disease activity log study. Scientific World J. 2012; 2012:589875.

7. Wolkoff P, Skov P, Franck C, Petersen LN. Eye irritation and environmental factors in the office environment-hypotheses, causes and a physiological model. Scand J Work Environ Health. 2003;29(6):411-30. Comment in: Scand J Work Environ Health. 2003; 29(6):407-9.

8. Alves M, Novaes P, Morraye Mde A, Reinach PS, Rocha EM. Is dry eye an environmental disease? Arq Bras Oftalmol. 2014;77(3):193-200.

9. Barabino S, Dana MR. Animal models of dry eye: a critical assessment of opportunities and limitations. Invest Ophthalmol Vis Sci. 2004;45(6):1641-6.

10. Nakamura S, Shibuya M, Nakashima H, Hisamura R, Masuda N, Imagawa T, et al. Invol-vement of oxidative stress on corneal epithelial alterations in a blink-suppressed dry eye. Invest Ophthalmol Vis Sci. 2007;48(4):1552-8.

11. Stern ME, Schaumburg CS, Siemasko KF, Gao J, Wheeler LA, Grupe DA, et al. Autoan-tibodies contribute to the immunopathogenesis of experimental dry eye disease. Invest Ophthalmol Vis Sci. 2012;53(4):2062-75.

12. Kunzli N, Avol E, Wu J, Gauderman WJ, Rappaport E, Millstein J, et al. Health effects of the 2003 Southern California wildfires on children. Am J Respir Crit Care Med. 2006;174(11):1221-8. Comment in: Am J Respir Crit Care Med. 2007;175(6):629; author reply 629; Am J Respir Crit Care Med. 2006;174(11):1168-9.

13. Viswanathan S, Eria L, Diunugala N, Johnson J, McClean C. An analysis of effects of San Diego wildfire on ambient air quality. J Air Waste Manag Assoc. 2006;56(1):56-67.

14. Efron N. Grading scales for contact lens complications. Ophthalmic Physiol Opt. 1998; 18(2):182-6.

15. Lemp MA. Report of the National Eye Institute/Industry workshop on Clinical Trials in Dry Eyes. CLAO J. 1995;21(4):221-32.

16. van Bijsterveld OP. Diagnostic tests in the Sicca syndrome. Arch Ophthalmol. 1969; 82(1):10-4.

17. The definition and classification of dry eye disease: report of the Definition and Classi fication Subcommittee of the International Dry Eye WorkShop (2007). Ocul Surf. 2007;5(2):75-92.

18. Novaes P, do Nascimento Saldiva PH, Kara-Jose N, Macchione M, Matsuda M, et al. Ambient levels of air pollution induce goblet-cell hyperplasia in human conjunctival epithelium. Environ Health Perspect. 2007;115(12):1753-6.

19. Gonzalez-Garcia MJ, Gonzalez-Saiz A, de la Fuente B, Morilla-Grasa A, Mayo-Iscar A, San-José J, et al. Exposure to a controlled adverse environment impairs the ocular surface of subjects with minimally symptomatic dry eye. Invest Ophthalmol Vis Sci. 2007;48(9):4026-32.

20. Jones LT. The lacrimal secretory system and its treatment. Am J Ophthalmol. 1966; 62(1):47-60.

21. Nelson JD. Impression cytology. Cornea. 1988;7(1):71-81. 22. World Health Organization. Carbon Monoxide. 2nd ed. Geneva: WHO; 1999. (Environ-

mental Health Criteria 213) 23. World health Organization. WHO Air quality guidelines for particulate matter, ozone,

nitrogen, dioxide and sulfur dioxide. Global update 2005. Summary of risk assessment [Internet]. Geneva: WHO; 2005. (WHO/SDE/PHE/OEH/06.02.) [cited 2008 Jul 27]. Avai-lable from: http://whqlibdoc.who.int/hq/2006/WHO_SDE_PHE_OEH_06.02_eng.pdf

24. Morgan O, Verlander NQ, Kennedy F, Moore M, Birch S, Kearney J, et al. Exposures and reported symptoms associated with occupational deployment to the Buncefield fuel depot fire, England 2005. Occup Environ Med. 2008;65(6):404-11.

25. Novaes P, Saldiva PH, Matsuda M, Macchione M, Rangel MP, Kara-Jose N, et al. The effects of chronic exposure to traffic derived air pollution on the ocular surface. En-viron Res. 2010;110(4):372-4.

26. Zelikoff JT, Chen LC, Cohen MD, Schlesinger RB. The toxicology of inhaled woodsmoke. J Toxicol Environ Health B Crit Rev. 2002;5(3):269-82.

Figure 4. A) Carbon Monoxide (CO) and nitrogen dioxide (NO2) hourly measured levels before, during, and after the acute episode. B) Levels of total particulate matter (PM) before, during, and after the acute episode. Data were obtained from the Envi-ronmental Protection Agency of Buenos Aires.

A

B

Case Report


INTRODUCTIONBilateral acute iris transillumination (BAIT) is a very rare condition

characterized by the bilateral acute loss of iris pigment epithelium, iris transillumination, pigment showering, persistent mydriasis, and occasional increased intraocular pressure (IOP)(1,2). Patients with BAIT generally present with acute ocular pain, photophobia, and red eyes(1), which are also observed in patients with iridocyclitis. Further-more, the circulating pigment particles in the anterior chamber may be confused with the inflammatory cells seen in patients with iridocyclitis. Therefore, the presenting symptoms and findings of BAIT may result in a misdiagnosis of acute iridocyclitis. We report a case of BAIT in a patient who was initially misdiagnosed with iridocyclitis and treated with corticosteroid therapy.

CASE REPORTA 30-year-old male was referred to our clinic to seek another

opi nion for his diagnosed iridocyclitis, which was unresponsive to treatment. Two months previously, he had been admitted to another clinic with acute bilateral ocular pain, severe photophobia, and red eyes. He was diagnosed with iridocyclitis and treated with topical and systemic corticosteroids. However, his signs and symptoms did not improve. The patient also had a history of upper respiratory tract

infection and use of the systemic cefazolin 3 months previously. On admission, his best-corrected visual acuity was 20/20 in both eyes. Slit-lamp examination revealed bilateral conjunctival hyperemia, 1+ circulating pigment in the anterior chamber, diffuse iris transillumina-tion, pigment dusting on the anterior lens capsule, and mydriatic and distorted pupils (Figures 1 A, B and 2 A, B). There were no inflamma-tory keratic precipitates on the corneal endothelium or inflammatory cells in the anterior vitreous of either eye. Corneal sensation was intact in both eyes. Gonioscopy showed heavy pigment deposition in the trabecular meshwork bilaterally (Figure 2 C, D). There was no evidence of posterior iris bowing or peripheral anterior synechiae on the gonioscopy. IOP was 20 mmHg in the right eye and 18 mmHg in the right eye. The fundus examination was normal in both eyes. The patient had undergone a complete laboratory evaluation for uveitis in the other clinic. The results of those tests, including erythrocyte se-dimentation rate, complete blood cell count, biochemistry, urinalysis, venereal disease research laboratory test, intradermal purified protein derivative test, and computed tomography of the chest, were normal. Human leukocyte antigen-B51 (HLA-B51) and HLA-B27 were negative.

On admission to our clinic, the patient was using topical predni-solone acetate 1% 8 times a day, topical cyclopentolate 1% twice a day, and a fixed carbonic anhydrase inhibitor/beta blocker combina-tion. The cyclopentolate was discontinued, and the prednisolone was

Bilateral acute iris transillumination (BAIT) initially misdiagnosed as

acute iridocyclitis

Transiluminação de íris aguda bilateral (BAIT) inicialmente diagnosticada como iridociclite aguda

saban gonul1, banu bozKurt1

Submitted for publication: May 6, 2014 Accepted for publication: June 6, 20141 Department of Ophthalmology, Faculty of Medicine, Selcuk University, Konya, Turkey.



Corresponding author: Saban Gonul. Selcuk University Faculty of Medicine - Department of Ophthal-mology - Konya - Turkey - E-mail: [email protected]

ABSTRACTBilateral acute iris transillumination (BAIT) is a relatively new clinical entity cha-racterized by bilateral acute loss of iris pigment epithelium, iris transillumination, pigment dispersion in the anterior chamber, and sphincter paralysis. We report the case of a 30-year-old male who was initially diagnosed with acute iridocyclitis in a different clinic and treated with topical and systemic corticosteroids. He was referred to our clinic to seek another opinion because his symptoms did not improve. An ocular examination revealed bilateral pigment dispersion into the anterior chamber, diffuse iris transillumination, pigment dusting on the anterior lens capsule, atonic and distorted pupils, and increased intraocular pressure, sugges ting a diagnosis of BAIT rather than iridocyclitis. Clinicians should be aware of the differential diagnosis of syndromes associated with pigment dispersion from iridocyclitis to avoid aggressive anti-inflammatory therapy and detailed investigation for uveitis.

Keywords: Iris diseases/diagnosis; Pigment epithelium of eye/pathology; Transillu-mination; Iridocyclitis/diagnosis; Diagnosis, differential; Case reports

RESUMOA transiluminação de íris aguda bilateral (do inglês, bilateral acute iris transillumi-nation, BAIT ) é uma entidade clínica relativamente nova, caracterizada pela perda aguda bilateral do epitélio pigmentado da íris, transiluminação iriana, dispersão de pigmentos na câmara anterior, e paralisia do esfíncter pupilar. Nós relatamos o caso de um homem de 30 anos que foi diagnosticado com iridociclite aguda e tratado com corticosteroides tópicos e sistêmicos. Ele foi encaminhado ao nosso serviço para outra opinião, porque seus sintomas não melhoram com a terapia. Um exame oftalmológico revelou dispersão bilateral de pigmentos para a câmara anterior, transiluminação difusa de íris, pigmento difusa na cápsula anterior do cristalino, pupilas atônicas e distorcidas, e um aumento da pressão intraocular, o que sugere um diagnóstico de BAIT em vez de iridociclite. Os médicos devem estar cientes do diagnóstico diferencial das síndromes associadas à dispersão de pigmento com a iridociclite para evitar a terapia antiinflamatória agressiva e investigação detalhada para uveíte.

Descritores: Doenças da íris/diagnóstico; Epitélio pigmentado ocular/patologia; Transiluminação; Iridociclite/diagnóstico; Diagnóstico diferencial; Relatos de casos

Bilateral acute iris transillumination (BAIT) initially misdiagnosed as acute iridocyclitis


tapered and discontinued after a month. The patient remained stable for the rest of his 3-month follow-up, without the need for steroid treatment. During follow-up examinations, IOP did not increase, the pigment dispersion in the anterior chamber decreased, and the iris transillumination defects remained stable.

DISCUSSIONIris atrophy with and without transillumination may be caused

by several diseases, such as viral iridocyclitis, pigment dispersion syn-drome (PDS), pseudoexfoliation syndrome, Fuchs uveitis syndrome (FUS), Vogt-Koyanagi-Harada disease, acute angle-closure glaucoma, ocular trauma, bilateral acute depigmentation of the iris (BADI), and BAIT(1-5).

The most common differential diagnosis in pigment dispersion and iris transillumination is PDS, which typically occurs in young myo-pic adults. It is usually asymptomatic and characterized by posterior bowing of the iris, Krukenberg’s spindle, and slit-like, radial, midpe-ripheral transillumination defects(6), none of which were detected in our patient.

Herpes simplex and cytomegalovirus may cause unilateral irido-cyclitis with sectorial/diffuse iris atrophy and transillumination. Although an aqueous tap for polymerase chain reaction (PCR) analy sis is requi-

red for a definite diagnosis, negative results do not exclude a viral etiology(7). We were unable to obtain aqueous humor for viral analysis, because the patient did not consent to the procedure. However, symmetrical and bilateral involvement, lack of keratic precipitates, and atonic pupils with compromised reaction to light helped us to differentiate BAIT from viral uveitis.

FUS is a chronic, asymptomatic, low-grade unilateral uveitis cha-racterized by diffuse, stellate, and medium-sized keratic precipitates and iris stromal atrophy, with or without heterochromia, and without posterior synechiae(8), which were also not detected in the present case.

Our patient displayed the clinical findings of BAIT, including acute onset of severe photophobia and red eyes, bilateral severe transillu-mination of the iris, pigment showering in the anterior chamber, a mydriatic and distorted pupils, and increased IOP. This condition should also be differentiated from BADI, which has a more benign course and shorter duration of pigment discharge, a lower incidence of elevated IOP, which is transient, and reversibility of iris changes in some patients(5). Both conditions have a common etiology but the pigment discharge seems to be only from the iris pigment epithelium in BAIT and from the iris stroma in BADI. The features that differentiate BADI from BAIT are depigmentation of the iris without transillumina-tion defect and unaffected pupils(1).

BAIT may also masquerade as iridocyclitis, as in the case of our patient, who was referred for evaluation of bilateral acute iridocyclitis. On our initial examination, he had no inflammatory keratic precipita-tes on the corneal endothelium but had all of the clinical findings of BAIT. Therefore, we ruled out acute iridocyclitis.

Although the exact etiopathogenesis of BAIT remains unclear, several publications have reported a relationship between BAIT and systemic use of moxifloxacin(2) and clarithromycin(9), upper respira-tory tract infections(1), and a toxic effect following fumigation(10). Our patient had a history of upper respiratory tract infection and use of systemic cefazolin 3 weeks before the onset of his symptoms. It is difficult to establish which of those factors was the cause of BAIT. Tugal-Tutkun et al.(1) reported 26 patients with BAIT, 19 of whom had a history of upper respiratory tract infection and use of systemic an-tibiotics, including moxifloxacin, ampicillin/sulbactam, amoxicillin/clavulanate, trimethoprim/sulfamethoxazole, cefixime, and penicillin V. However, they concluded that the relationship between BAIT and systemic antibiotic use was coincidental, because there are no repor-ted ocular adverse effects related to the topical use of these antibio-tics, in particular moxifloxacin, which has efficient ocular penetration. We use moxifloxacin in the anterior chamber for prophylaxis of endo-phthalmitis after cataract surgery, and we have not encountered any cases of BAIT associated with its use. Therefore, it appears that BAIT in the present case was triggered by the virus that caused the upper respiratory tract infection.

In conclusion, BAIT should be differentiated from other diseases causing pigment dispersion into the anterior chamber and from iridocyclitis. It is important to make a correct differential diagnosis of BAIT from anterior uveitis to avoid the unnecessary use of corticoste-roids and detailed investigation for uveitis. Early diagnosis of this con-dition is also important because of the risk of marked increases in IOP.

REFERENCES 1. Tugal-Tutkun I, Onal S, Garip A, Taskapili M, Kazokoglu H, Kadayifcilar S, et al. Bilateral

acute iris transillumination. Arch Ophthalmol. 2011;129(10):1312-19. 2. Morshedi RG, Bettis DI, Moshirfar M, Vitale AT. Bilateral acute iris transillumination

following systemic moxifloxacin for respiratory illness: report of two cases and review of the literature. Ocul Immunol Inflamm. 2012;20(4):266-72.

3. Maestrini HA, Maestrini AA, Machado Dde O, Santos DV, Almeida HG. Bilateral acute depigmentation of the iris (BADI): first reported case in Brazil. Arq Bras Oftalmol. 2013; 76(1):42-4.

4. Tugal-Tutkun I, Urgancioglu M. Bilateral. Acute depigmentation of the iris. Graefes Arch Clin Exp Ophthalmol. 2006;244(6):742-6.

5. Tugal-Tutkun I, Araz B, Taskapili M, Akova YA, Yalniz-Akkaya Z, Berker N, et al. Bilateral

A

C

B

D

Figure 2. Slit-lamp photographs of right (A) and left (B) eyes showing mydriatic, distor-ted pupils with poor response to light, and a small amount of pigment dusting on the anterior lens capsule. Gonioscopy of right (C) and left (D) inferior angles showing heavy inferior trabecular meshwork pigmentation.

Figure 1. Slit-lamp photographs of right (A) and left (B) eyes showing diffuse iris tran-sillumination defects using the retroillumination technique. The photographs were taken without pharmacologic dilation of the pupils.

A B

Gonul S, Bozkurt B


acute depigmentation of the iris: report of 26 new cases and four-year follow-up of two patients. Ophthalmology. 2009;116(8):1552-7.

6. Niyadurupola N, Broadway DC. Pigment dispersion syndrome and pigmentary glau-coma: a major review. Clin Experiment Ophthalmol. 2008;36(9):868-82.

7. De Groot-Mijnes JD, Rothova A, van Loon AM, Schuller M, Ten Dam-Van Loon NH, De Boer JH, et al. Polymerase chain reaction and Goldmann-Witmer coefficient analysis are complimentary for the diagnosis of infectious uveitis. Am J Ophthalmol. 2006; 141(2):313-8.

8. Mohamed Q, Zamir E. Update on Fuchs’ uveitis syndrome. Curr Opin Ophthalmol. 2005; 16(6):356-63.

9. Tranos P, Nasr MB, Asteriades S, Vakalis A, Georgalas I. iris Bilateral difusas atrofia após o uso de claritromicina oral. Cutan Ocul Toxicol. 2014;33(1):79-81.

10. Gonul S, Bozkurt B, Okudan S, Tugal-Tutkun I. Bilateral acute iris transillumination following a fumigation therapy: a village-based traditional method for the treatment of ophthalmomyiasis. Cutan Ocul Toxicol. 2014. [Epub ahead of print]. doi number: 10.3109/15569527.2014.886589.

XVI Simpósio Internacional da Sociedade Brasileira de Glaucoma

14 a 16 de maio de 2015Goiânia-GO

Informações: E-mail: [email protected]

Site: www.sbglaucoma.com

Case Report


INTRODUCTIONThe dexamethasone implant (Ozurdex®; Allergan Inc., Irvine, CA,

USA) is a novel treatment modality mainly used to treat macular ede-ma associated with retinal vein occlusions and to treat noninfectious posterior uveitis(1). However, there are quite satisfactory data about its efficacy in multiple clinical situations associated with refractory macular edema, including diabetic macular edema, macular edema asso ciated with uveitis or Irvine-Gass syndrome, and retinitis pigmen-tosa(2,3). Major concerns with intravitreal dexamethasone (IV-DEX) are increased intraocular pressure and cataract progression(1,2). Here we report an unusual case of IV-DEX-related acute retinal necrosis (ARN) in a rheumatoid arthritis patient with refractory posterior uveitis.

CASE REPORTA 52-year-old woman presented with floaters and decreased

vision in the left eye. She had a 7-year history of rheumatoid arthritis. For the year prior to presentation, she had been receiving azathiopri-ne 150 mg daily to treat posterior uveitis in the left eye. One month prior to presentation, intravitreal dexamethasone (IV-DEX) implanta-tion was performed for resistant macular edema in the left eye.

On examination, visual acuity was 20/20 in the right eye and 20/40 in the left eye. There were no pathological findings in the right eye. Slit-lamp examination of the left eye revealed 3+ anterior cham-ber cells. Fundus examination revealed vitreous haze and debris, disc edema, vessel attenuation, scattered retinal hemorrhages, exudation, and peripheral confluent areas of retinal whitening. The implant was

ABSTRACTA 52-year-old woman undergoing azathioprine treatment for rheumatoid arthritis developed acute retinal necrosis a month after intravitreal dexamethasone (Ozur-dex®) implantation for posterior uveitis in the left eye. Varicella zoster virus (VZV) DNA was detected in the anterior chamber and vitreous samples on polymerase chain reaction (PCR) analysis. Retinal detachment occurred despite systemic and intravitreal antiviral therapy. Favorable structural and functional outcomes were achieved after retinal surgery with silicone oil. To the authors’ knowledge, this is the first reported case of acute retinal necrosis following placement of an Ozurdex® implant. Physicians practicing Ozurdex® implantations should be aware of this unusual but devastating complication. Extra caution and frequent follow-up are required in all immunocompromised patients receiving Ozurdex® implantation.

Keywords: Retinal necrosis syndrome, acute; Immunosuppression; Intravitreal in -jections/methods; Dexamethasone/administration & dosage; Case reports

RESUMOUma mulher de idade de 52 anos em tratamento azatioprina para a artrite reumatóide desenvolveu necrose aguda de retina um mês após implantação Ozurdex® para uveíte posterior do olho esquerdo. DNA de varicela zoster (VZV) foi detectado em amostras de câmara anterior e vítreo por análise de PCR. Apesar da terapia antiviral sistêmica e intravítrea, o paciente apresentou descolamento de retina. Desfecho favorável estrutural e funcional foi obtida após a cirurgia retiniana com óleo de silicone. Pelo conhecimento dos autores, este é o primeiro caso relatado de necrose aguda de retina após a colocação de um implante Ozurdex®. Os médicos que implantam Ozurdex® devem estar cientes desta complicação incomum, mas devastadora. É necessário cuidado extra e acompanhamento frequente dos pacientes que recebam o implante Ozurdex® e apresentem qualquer condição imunocomprometedora.

Descritores: Síndrome de necrose retiniana aguda; Imunossupressão; Injeções in tra-vítreas/métodos; Dexametasona/administração & dosagem; Relatos de casos

easily observed in the inferior vitreous (Figure 1). Fluorescein an-giography demonstrated staining of the optic disc and vessels, and clear-cut zones of retinal ischemia (Figure 2). Immune suppression caused by the azathioprine treatment was ruled out by a normal white cell count.

The presumed diagnosis was acute retinal necrosis. Anterior chamber paracentesis and vitreous tap samples were analyzed using viral polymerase chain reaction (PCR). Varicella zoster virus (VZV) DNA was identified, but no herpes simplex virus, cytomegalovirus, or Epstein-Barr virus DNA was detected. Systemic and laboratory inves-tigations for syphilis, AIDS, and hepatitis were also negative, and there was no evidence of any systemic azathioprine-related adverse effect.

The patient was commenced on intravenous acyclovir (3 × 750 mg/day) treatment for 10 days to manage ARN, in addition to to pical prednisolone acetate 1% drops every hour and cyclopen-tolate 1% drops thrice a day. The patient was followed as inpatient. However, this treatment regimen did not resolve the areas of retinal whitening or the anterior chamber inflammation. Therefore, she received 4 intravitreal ganciclovir injections a week apart, and oral valacyclovir 1 g thrice daily. One month after the first ganciclovir in-jection, the patient developed retinal detachment from the necrotic retinal area in the left eye, and visual acuity decreased to counting fingers at 1 m. She underwent repair of the retinal detachment with vitrectomy, laser retinopexy, and silicone oil. She was discharged after surgery and maintained on oral valacyclovir therapy. Four months after surgery, her visual acuity was 20/60 in the left eye (Figure 3). The right eye re mains unaffected.

Acute retinal necrosis following intravitreal dexamethasone (Ozurdex®) implant

Necrose aguda de retina após implante de dexametasona intravítrea (Ozurdex®)

Murat KucuKEvcilioglu1, MustaFa ErEn1, uMit yolcu2, gungor sobaci3

Submitted for publication: April 22, 2014 Accepted for publication: August 29, 20141 Department of Ophthalmology, Gulhane Military Academy of Medicine, Ankara, Turkey.2 Department of Ophthalmology, Siirt Military Hospital, Siirt, Turkey.3 Department of Ophthalmology, Hacettepe University Faculty of Medicine, Ankara, Turkey.



Corresponding author: Murat Kucukevcilioglu. GATA Goz Klinigi - Etlik/Ankara - Turkey E-mail:[email protected]

Kucukevcilioglu M, et al.


DISCUSSIONThe clinical presentation and laboratory workup of the presented

case lead to a diagnosis of ARN. To the best of our knowledge, there is no previous report of ARN after an IV-DEX implantation (Pubmed and Medline search). However, ARN has been described following intravitreal injections of triamcinolone(4,5). Recently, Ramaiya et al. reported a case of ARN 1 year after fluocinolone acetonide (Retisert®; Bausch and Lomb, Irvine, CA, USA) implantation in a young patient with intractable uveitis(6). Prior to implantation, the authors had tried different immunotherapeutic agents, including azathioprine with high dose oral steroid, to control the inflammation. Shah et al. retrospectively analyzed viral retinitis after triamcinolone injections and found increased number of injections or long duration of drug activity as a risk factor(5). Systemic immunosuppression is another risk factor, and its incidence was found to be 2 times higher in the immu-nocompromised subset than in the rest of the subjects(7). The current

Figure 1. Fundus photography of the left eye at presentation showing mild optic disc edema, macular striations, occlusive vasculitis (white arrow heads), scattered retinal hemorrhages, clear cut whitening of peripheral retina (white arrows), and the IV-DEX implant in the inferior vitreous (black arrow).

Figure 2. Fluorescein angiography images showing annular (A) retinal ischemia in the periphery and (B) optic disc leakage.

A B

Figure 3. Fundus photography of the left eye 4 months after surgery showing attached retina under silicone oil tamponade, mild obscuration of disc edges, and some retinal scarring.

patient was on azathioprine for rheumatoid arthritis, and despite the normal white cell count, the combined effect of local and systemic immunosuppression could have played a role in the development of ARN. However, further studies are needed to confirm this possibility.

In the literature, early vitrectomy with laser demarcation is recommended for severe ARN(8,9). In this case, we initiated medical treatment because the macula was unaffected and visual acuity was good at presentation. After progression to retinal detachment, vitrectomy with laser retinopexy and silicone oil tamponade limited the disease progression and provided a favorable visual outcome in the short term. However, long-term follow up is needed to determine whether this unusual gain in vision is sustained.

In conclusion, physicians practicing IV-DEX implantations should be aware of this unusual but devastating complication. Extra caution and frequent follow-up is required in immunocompromised patients receiving IV-DEX implantation.

REFERENCES 1. Haller JA, Bandello F, Belfort R Jr, Blumenkranz MS, Gillies M, Heier J, et al. Randomized,

sham-controlled trial of dexamethasone intravitreal implant in patients with macular edema due to retinal vein occlusion. Ophthalmology. 2010;117(6):1134-1146.e3.

2. Dutra Medeiros M, Postorino M, Navarro R, Garcia-Arumí J, Mateo C, Corcóstegui B. Dexamethasone intravitreal implant for treatment of patients with persistent diabetic macular edema. Ophthalmologica. 2014;231(3):141-6.

3. Srour M, Querques G, Leveziel N, Zerbib J, Tilleul J, Boulanger-Scemama E, et al. Intra-vitreal dexamethasone implant (Ozurdex) for macular edema secondary to retinitis pigmentosa. Graefes Arch Clin Exp Ophthalmol. 2013;251(6):1501-6.

4. Han JM, Ahn J, Park KH, Woo SJ. Presumed necrotizing viral retinitis after intravitreal triamcinolone injection: case report. Korean J Ophthalmol 2011;25(6):451-4.

5. Shah AM, Oster SF, Freeman WR. Viral retinitis after intravitreal triamcinolone injection in patients with predisposing medical comorbidities. Am J Ophthalmol. 2010;149(3): 433-40 e1.

6. Ramaiya KJ, Rao PK. Herpetic necrotizing retinitis following flucinolone acetonide in travitreal implant. Ocul Immunol Inflamm. 2011;19(1):72-4.

7. Haller JA, Bandello F, Belfort R Jr, Blumenkranz MS, Gillies M, Heier J, et al. Dexametha-sone intravitreal implant in patients with macular edema related to branch or central retinal vein occlusion twelve-month study results. Ophthalmology. 2011;118(12): 2453-60

8. Hillenkamp J, Nolle B, Bruns C, Rautenberg P, Fickenscher H, Roider J. Acute retinal necrosis: clinical features, early vitrectomy, and outcomes. Ophthalmology. 2009;116(10): 1971-5 e2.

9. Lau CH, Missotten T, Salzmann J, Lightman SL. Acute retinal necrosis features, mana-gement, and outcomes. Ophthalmology. 2007;114(4):756-62.

Case Report


INTRODUCTIONHereditary spastic paraplegia (HSP) is a clinically and genetically

heterogeneous group of diseases involving weakness and spasticity of the lower extremities combined with additional neurological or non-neurological manifestations(1). There are almost 48 subtypes of HSP; however, only the 2 subtypes involving mutations of SPG11 and SPG15 are associated with Kjellin’s syndrome(1).

The inheritance of Kjellin’s syndrome is autosomal recessive, and the syndrome is characterized by spastic paraplegia, mental retar-dation, amyotrophy, thin corpus callosum, and macular dystrophy(2). Individuals with this syndrome present with macular changes, most often described as fundus flavimaculatus or Stargardt disease-like, particularly on the basis of fluorescein angiography findings(3).

Here we describe ophthalmological findings in a patient with Kjellin’s syndrome, extending previous reports by demonstrating reti-nal functional and multimodal retinal imaging studies.

CASE REPORTA 34-year-old white male was admitted to São Paulo University

Hospital in Ribeirão Preto, for investigation of spastic paraparesis. At 20 years of age, he had developed muscle weakness in the lower limbs with difficulty in walking. The symptoms progressed slowly; however, 2 years later he had lost the ability to walk unaided.

Since childhood, he had experienced learning difficulties and mild cognitive impairment combined with hypoacusis. His mother repor-ted that he had recently displayed aggressive behavior. There was no

long-term history of visual acuity impairment; however, he reported progressive worsening of vision over the past 2 years Prior to admis-sion, there was no significant family history and the only medication was fluoxetine 20 mg per day.

Neurological examination revealed dysarthria, frontal release signs, preserved perception of touch and pain, spasticity of the lower limbs with a scissors gait, and loss of strength and muscle atrophy in the lower limbs and interosseous muscles of the hands. Laboratory studies revealed negative HIV, VDRL, FTA-abs, HTLV I/II, and hepatitis B and C serology. Vitamin B12 and folic acid levels were within normal limits.

Brain magnetic resonance imaging showed significant volume-tric loss and corpus callosum atrophy. Electroneuromyography showed severe denervation of segmental cranial, cervical, thoracic, and lum-bosacral muscles, suggesting diffuse lower motor neuron disease. Audiometry detected profound sensorineural hearing loss.

OPHTHALMOLOGICAL EVALUATION

Only mild visual acuity loss was detected, with best-corrected vi-sual acuity of 20/25 in the right eye and 20/30 in the left eye. Pupillary direct and consensual light reflexes were normal and ocular motility was preserved. Intraocular pressure was within the normal range: 14 mmHg in the right eye and 15 mmHg in the left eye (Goldmann). Slit lamp biomicroscopy revealed transparent cornea and lenses with no noteworthy structural changes. The fundus presented multiple round yellowish flecks at the level of the retinal pigment epithelium scattered at the posterior pole. Some of these flecks were elongated

Macular dystrophy associated with Kjellin’s syndrome: a case report

Distrofia macular associada à síndrome de Kjellin: um relato de caso

vinícius MontEiro dE castro1, andré MEirEllEs1, raFaEl saran arciEri1, Katharina MEssias1, andré MEssias1

Submitted for publication: April 22, 2014 Accepted for publication: July 9, 20141 Department of Ophthalmology, Otorhinolaryngology and Head & Neck Surgery, Medical School of

Ribeirão Preto, Federal University of São Paulo, Ribeirão Preto, SP, Brazil.



Corresponding author: André Messias. Departamento de Oftalmologia, Otorrinolaringologia e Cirurgia da Cabeça e Pescoço - FMRP - USP. Av. Bandeirantes, 3.900 - Ribeirão Preto, SP - 14049-900 - Brazil - E-mail: [email protected]

ABSTRACTHereditary spastic paraplegia (HSP) is characterized by weakness and spasticity of the lower extremities. Kjellin’s syndrome is a rare syndrome associated with HSP. The syndrome is characterized by the presence of bilateral retinal flecks, similar to the findings in Stargardt disease and fundus flavimaculatus. We report the case of a 34-year-old male who presented with complete features of Kjellin’s syndrome, with typical retinal findings observed on multimodal imaging (spectral domain optical coherence tomography [SD-OCT], near-infrared reflectance and autofluorescence imaging). The ophthalmological changes at early stages of the disease may not impair visual acuity. Therefore, the detection of central retinal degeneration requires thorough fundus examination.

Keywords: Macular degeneration; Spastic paraplegia, hereditary/diagnosis; Diag-nostic techniques, ophthalmological; Case reports

RESUMOA paralisia espástica hereditária (HSP) é caracterizada por fraqueza e espasticidade das extremidades inferiores. A síndrome de Kjellin é uma rara associação de HSP com a presença de flecks retinianos similares aos encontrados em pacientes com doença de Stargardt ou fundus flavimaculatus. Descrevemos os achados em imagens mul-timodais da retina (tomografia de coerência óptica de domínio espectral [SD-OCT ], reflectância próxima ao infravermelho e autofluorescência) em um paciente de 34 anos que apresenta conjunto completo de sinais e sintomas da síndrome de Kjellin. As alterações retinianas em estágios iniciais da doença podem aparecer, mesmo sem redução da acuidade visual, e por isso, para detecção da degeneração central da retina, é necessário exame minucioso do fundo de olho.

Descritores: Degeneração macular; Paraplegia espástica hereditária/diagnóstico; Técnicas de diagnóstico oftalmológico; Relatos de casos

Castro VM, et al.


and showed partial confluence with neighboring flecks (Figure 1). Imaging was performed with a Spectralis HRA + OCT (Heidelberg Engineering, Heidelberg, Germany). Areas of increased fundus auto-fluorescence (FA) were noted, with surrounding borders of decreased FA intensity, corresponding to the visible yellowish fundus lesions (Figure 2). Spectral domain optical coherence tomography (SD-OCT) showed elevated lesions along the retinal pigment epithelium (RPE)/Bruch complex, preserving the external limiting membrane (Figure 3).

Full field and multifocal electroretinography (ERG and mfERG, respectively) were recorded and stored for offline analysis using an Espion E2 system (Diagnosys LLC, Littleton, MA, USA) in accordance with the International Society for Clinical Electrophysiology of Vision (ISCEV) guidelines. The ERG amplitude and implicit time were within normal limits; however, mfERG (Diagnosys LLC) revealed areas of amplitude reduction. Microperimetry (MAIA; CenterVue, Padova, Italy) showed test points with reduction of sensitivity but fixation was relatively stable.

DISCUSSIONThe presented patient shows typical phenotypic traits of Kjellin’s

syndrome characterized by absence of the corpus callosum and ma-cular changes. These traits are suggestive of HSP subtypes 11 and 15, known to be associated with deformity of the corpus callosum(1,4-6).

The natural history of macular dystrophy in Kjellin’s syndrome is such that patients rarely complain of low visual acuity unless it is as-sociated with cataract or optic neuropathy(1,3). In this context, micro-perimetry could be more sensitive to detect functional impairment in Kjellin’s syndrome, because preserved visual acuity is normally found if the fovea is not involved. However, microperimetry findings show a clear distinction in comparison with eccentric and unstable fixation and the loss of sensitivity found in Stargardt disease(7).

As expected, no changes were recorded on ERG, presumably because of the mild and focal retinal impairment typical of Kjellin’s syndrome(8). However, there were obvious changes on mfERG, with areas of reduced amplitude bilaterally(8).

Interestingly, some characteristics of the yellowish-white flecks interspersed with diffuse pigmented lesions found in Kjellin’s syn-drome also differ from those found in Stargardt disease and fundus flavimaculatus. In Kjellin’s syndrome, the areas of increased FA are surrounded by halos of decreased FA(9,10).

On SD-OCT, the presence of elevated lesions along the RPE/Bruch complex, corresponding to areas of increased FA without atrophy of the RPE/Bruch complex and the inner and outer segment (IS/OS) layer, also differ from the findings in Stargardt disease and fundus flavimaculatus(9-11).

The diagnosis of macular changes in HSP is important for the ge-netic evaluation because macular changes indicate association with subtypes 11 and 15. The mutations occur in KIAA 1840 (SPG11) and ZFYVE 26 (SPG15) that encode the proteins spatacsin and spastizin, respectively(1). These proteins are part of the AP5 membrane-trafficking complex and have been identified in animal models of photorecep-tor cells(12). This suggests that the retinal changes in Kjellin’s syndrome might be the result of a metabolic defect, causing accumulation of products related to lipofuscin.

In summary, this case represents Kjellin’s syndrome with a complete SPG11 and SPG15 phenotype with macular changes. The ophthalmological changes, particularly at early stages of the disease, may not impair visual acuity but can be detected with microperime-try, careful fundus examination, and autofluorescence and/or SDOCT.

Figure 1. Color fundus photograph showing symmetric multiple uni-form round yellowish lesions at the posterior pole.

Figure 2. Fundus autofluorescence showing high levels of autofluores-cence in the center of the biomicroscopically visible flecks and sur-rounding halos with decreased autofluorescence signal.

Figure 3. Spectral domain optical coherence tomography [SD-OCT] showing elevated lesions located along the retinal pigment epithelium (RPE)/Bruch complex and preserving the external limiting membrane.

Macular dystrophy associated with Kjellin’s syndrome: a case report


ACKNOWLEDGMENTWe thank Mr. and Mrs. Balit for revising the manuscript.

REFERENCES 1. Finsterer J, Loscher W, Quasthoff S, Wanschitz J, Auer-Grumbach M, Stevanin G. Here-

ditary spastic paraplegias with autosomal dominant, recessive, X-linked, or maternal trait of inheritance. J Neurol Sci. 2012;15(1-2):1-18.

2. Webb S, Patterson V, Hutchinson M. Two families with autosomal recessive spastic paraplegia, pigmented maculopathy, and dementia. J Neurol Neurosurg Psychiatry. 1997;63(5):628-32.

3. Puech B, Lacour A, Stevanin G, Sautiere BG, Devos D, Depienne C, et al. Kjellin syndro-me: long-term neuro-ophthalmologic follow-up and novel mutations in the SPG11 gene. Ophthalmology. 2011;118(3):564-73.

4. Stevanin G, Azzedine H, Denora P, Boukhris A, Tazir M, Lossos A, et al. Mutations in SPG11 are frequent in autosomal recessive spastic paraplegia with thin corpus callosum, cognitive decline and lower motor neuron degeneration. Brain. 2008;131(Pt 3):772-84.

5. Orlen H, Melberg A, Raininko R, Kumlien E, Entesarian M, Söderberg P, et al. SPG11 mutations cause Kjellin syndrome, a hereditary spastic paraplegia with thin corpus callosum and central retinal degeneration. Am J Med Genet B Neuropsychiatr Genet. 2009;5B(7):984-92.

6. Hanein S, Martin E, Boukhris A, Byrne P, Goizet C, Hamri A, et al. Identification of the SPG15 gene, encoding spastizin, as a frequent cause of complicated autosomal-re -cessive spastic paraplegia, including Kjellin syndrome. Am J Hum Genet. 2008;82(4): 992-1002.

7. Reinhard J, Messias A, Dietz K, Mackeben M, Lakmann R, Scholl HP, et al. Quantifying fixation in patients with Stargardt disease. Vision Res. 2007;47(15):2076-85.

8. Frisch IB, Haag P, Steffen H, Weber BH, Holz FG. Kjellin’s syndrome: fundus autofluo-rescence, angiographic, and electrophysiologic findings. Ophthalmology. 2002;109(8): 1484-91.

9. Querques G, Prato R, Coscas G, Soubrane G, Souied EH. In vivo visualization of pho toreceptor layer and lipofuscin accumulation in stargardt’s disease and fundus fl avimaculatus by high resolution spectral-domain optical coherence tomography. Clin Ophthalmol. 2009;3:693-9.

10. Voigt M, Querques G, Atmani K, Leveziel N, Massamba N, Puche N, et al. Analysis of retinal flecks in fundus flavimaculatus using high-definition spectral-domain optical coherence tomography. Am J Ophthalmol. 2010;150(3):330-7.

11. Gomes NL, Greenstein VC, Carlson JN, Tsang SH, Smith RT, Carr RE, et al. A comparison of fundus autofluorescence and retinal structure in patients with Stargardt disease. Invest Ophthalmol Vis Sci. 2009;50(8):3953-9.

12. Murmu RP, Martin E, Rastetter A, Esteves T, Muriel MP, El Hachimi KH, et al. Cellular distribution and subcellular localization of spatacsin and spastizin, two proteins involved in hereditary spastic paraplegia. Mol Cell Neurosci. 2011;47(3):191-202.

6a Jornada Paulista de Oftalmologia (UNICAMP, UNESP e USP - Ribeirão Preto)

23 a 25 de abril de 2015Vitória Hotel

Campinas - SP

Informações: Site: www.jdeeventos.com.br

Case Report


INTRODUCTIONGlaucoma has a pathophysiology that is very similar to that of

hydrocephalus. Both diseases are characterized by a disturbance of the production and absorption of liquids. The function of the cere-brospinal fluid (CSF) and the aqueous humor and their secretion and absorption are remarkably similar.

Hydrocephalus is defined as a disorder of the physiology of CSF(1). The prevalence of hydrocephalus in the population is 1-2%(2-3). Under normal conditions, CSF is produced by the choroid plexus(1) at a rate of 500 ml/day. It is reabsorbed gradually by a passive process into the venous system via the arachnoid villi(4) (arachnoid granulation).

In total, 70,000 cases of hydrocephalus are annually admitted to hospitals in the United States; of these, between 18 and 33,000 will be given CSF shunt devices(3).

Glaucoma is the leading cause of irreversible blindness world-wide(5-6), with a prevalence of up to 4.74%(7). It is characterized by damage to the optic nerve fiber layer, resulting in progressive visual field disturbance. Intraocular pressure (IOP) is the most important risk factor for glaucoma; and currently, decreasing IOP is the only treatment option. Aqueous humor is produced by the ciliary body at a rate of 2.8-3.6 ml per day(8), circulating from the posterior chamber to the anterior chamber, and it is absorbed into the venous system through the trabecular meshwork.

ABSTRACT In 2010, there were estimated to be approximately 60.5 million people with glau-coma. This number is expected to increase to 79.6 million by 2020. In 2010, there were 8.4 million people with bilateral blindness caused by glaucoma, and this number is expected in increase to 11.2 million by 2020. Filtering implants are special devices that have been developed to reduce intraocular pressure in patients with refractory glaucoma. The success rate of these implants is relatively low, and they continue to fail over time. To avoid failure caused by the formation of scar tissue around the implants, attempts have been made to drain the aqueous humor to various sites, including the venous system, lacrimal sac, sinuses, and conjunctival fornix. Recently, a system to shunt aqueous humor from the anterior chamber to the peritoneum has been developed. The surgical technique involved in this system is a modification of the technique currently used by neurosurgeons for the treatment of hydrocephalus. We present the first case operated using this technique.

Keywords: Hydrocephalus; Glaucoma; Glaucoma drainage implants; Aqueous humor; Filtering surgery; Humans; Case reports

RESUMOCalcula-se que em 2010 havia provavelmente 60,5 milhões de pessoas com glaucoma, com aumento previsto para 79,6 milhões em 2020; a cegueira bilateral por glaucoma era detectada em 8,4 milhões de pessoas, em 2010, e com aumento estimado para 11,2 milhões em 2020. Dispositivos especiais foram desenvolvidos para reduzir a pressão intraocular em pacientes com glaucoma refratário, chamados implantes de filtragem. A taxa de sucesso destes implantes é relativamente baixa, e sabe-se que elas continuam a diminuir ao longo do tempo. Para evitar as falhas produzidas pela cicatrização de tecido em torno dos implantes, foram feitas tentativas de drenagem do humor aquoso para diferentes locais, tais como o sistema venoso, saco lacrimal, os seios paranasais e fórnice conjuntival. Revendo o tratamento atual da hidrocefalia, uma técnica de derivação a partir da câmara anterior para o peritônio foi desenvolvida. A técnica cirúrgica desenvolvida é uma modificação da técnica atualmente utilizada por neurocirurgiões para o tratamento de hidrocefalia. Apresentamos o primeiro caso operado com esta técnica.

Descritores: Hidrocefalia; Glaucoma; Implantes para drenagem de glaucoma; Humor aquoso; Cirurgia filtrante; Humanos; Relatos de casos

The latest development in the treatment of hydrocephalus has been the introduction of programmable valves(9). These enable the desired pressure levels for each patient to be regulated in the doctor’s office, avoiding the complications of under- or over-drainage of CSF, without the need for further surgery to change the devices(10). Reviewing the current management of hydrocephalus, a system to shunt aqueous humor from the anterior chamber to the peritoneum has been developed. The surgical technique is a modification of the technique currently used by neurosurgeons to treat hydrocephalus, with the ventricular catheter being replaced by an ocular catheter.

CASE REPORT The patient presented in this paper is enrolled in an ongoing re-

sear ch protocol at the Clínica de Ojos Maldonado Bas. The protocol has been approved by Oulton-Romagosa Institutional Health Research Ethics Committee, and respects the norms and standards enshrined in the Nuremberg Code, the Declaration of Helsinki, CIOMS/WHO Guidelines, the Universal Declaration on Bioethics and Human Rights (UNESCO), and other international and national instruments gover-ning biomedical research.

A 40-year-old male patient with glaucoma presented to the cli-nic. He had a history of bilateral keratoplasty for keratoconus during

Oculo-peritoneal shunt: draining aqueous humor to the peritoneum

Derivação óculo-peritoneal: drenagem do humor aquoso para o peritônio

ana Maldonado-JunyEnt1, arturo Maldonado-bas2, andrEa gonzalEz3, Francisco PuEyrrEdón4, María Maldonado-JunyEnt1, arturo Maldonado-JunyEnt1, diEgo rodriguEz3, Mariano bulacio5

Submitted for publication: August 29, 2014 Accepted for publication: August 29, 20141 Anterior Segment Department, Clínica de Ojos Maldonado-Bas, Córdoba, Argentina.2 Clínica de Ojos Maldonado-Bas, Córdoba, Argentina.3 General Surgery Department, Hospital Municipal de Urgencias of the city of Córdoba, Córdoba,

Argentina.4 Department of Neurosurgery, Sanatorio Allende, Córdoba, Argentina.5 General Surgery Department, Hospital Misericordia Nuevo Siglo, Córdoba, Argentina.


Disclosure of potential conflicts of interest: None of the authors have any potential conflicts of interest to disclose.

Corresponding author: Ana Maldonado-Junyent. Clínica de Ojos Maldonado-Bas - Achával Rodriguez 544 - Córdoba 5014 - Argentina - E-mail: [email protected]

Approved by the following research ethics committee: Approval No:013, CIEIS:OULTON-ROMAGOSA

Oculo-peritoneal shunt: draining aqueous humor to the peritoneum


adolescence. After the corneal surgery he had developed bilateral glaucoma. He had undergone cataract surgery and 2 filtering surge-ries in the right eye (OD) and filtering surgery in the left eye (OS). The visual acuity was no light perception in the OD and 4/10 in the OS. Slit-lamp examination of the OD showed keratoplasty with epithelial edema, a non-reactive dilated pupil with sector iridectomy, capsular pseudoexfoliation, pseudophakia, upper scleral thinning, and 2 non- functioning filtering blebs. IOP was 50 mmHg. Slit-lamp exami nation of the OS showed keratoplasty, permeable iridectomy, a trans parent lens, and a functioning filtering bleb. IOP was 11 mmHg.

An aqueous humor shunt to the peritoneum was performed in the OD. A hydrocephalus valve (Medtronic PS Medical® Strata® NSC) was used, regulated at level 2.5 (to operate at pressures between 14 and 16 mmHg).

SURGICAL TECHNIQUE

The surgical procedure begins with the formation of a fornix-ba-sed conjunctival flap. The conjunctiva is lifted and its rear face is inci-sed, making an opening of sufficient size to allow a surgical guidewire to pass with a silicone tube mounted on it. An incision is made in the retroauricular region of the scalp (Figure 1A), and the ocular catheter mounted on the surgical guidewire is passed from the opening of the rear face of the conjunctiva to the scalp incision (Figure 1B). A tunneler is passed from the scalp incision to the periumbilical region (Figure 1C), and an opening is made in the skin and the peritoneum

in this region. The hydrocephalus valve is positioned and the ocular and peritoneal catheters are assembled with the hydrocephalus valve (Figure 2A). Next, puncture of the anterior chamber of the eye is performed, and the ocular catheter is inserted. A chamber main-tainer is placed (Figure 2B) and connected to a bottle of balanced saline solution to test the permeability of the system (Figure 2C). The peritoneal catheter is then introduced. All surgical wounds are closed.

RESULTSFor the first twenty days postoperatively, the IOP was constant

at 15 mmHg. The anterior chamber had good depth at all follow-up examinations over this period. The keratoplasty became edematous during the first 72 hours, but this resolved with topical medication (prednisolone acetate).

To assess the precision with which these implants regulate pressure, during follow-up at week 4 the valve was adjusted to level 2.0 (to operate at pressures between 10 and 12 mmHg) and IOP im-mediately decreased to 11 mmHg (Figures 3 A-C). During the third postoperative month, the valve was regulated back to level 2.5. IOP increased to 15 mmHg over the following 24 hours.

On slit-lamp examination, and in the tomographic and radiologi-cal investigations in the fourth week after surgery, the catheters and valve were confirmed to be well located. (Figures 4 A-C).

Although the follow-up period of this patient is not yet signifi-cant to assess the efficacy of the surgery, this is the first report in the

A B C

Figure 3. A) Checking the performance level of the device. B) Programming the device to level 2.0. C) Medtronic tools to adjust pressures.

Figure 1. A) Scalp incision. B) The ocular catheter is passed into the scalp incision. C) The tunneler is passed from the scalp incision to the periumbilical region.

A B C

A B C

Figure 2. A) The ocular and peritoneal catheters are assembled with the hydrocephalus valve. B) Placing a chamber maintainer. C) Testing the permeability of the system.

Maldonado-Junyent A, et al.


A B C

Figure 4. A) Slit-lamp examination showing the ocular catheter in position. B) Tomographic view showing the route of the ocular catheter (black arrows). C) Radiological view showing the peritoneal catheter in position (black arrows).

literature of this type of shunt. Filtering aqueous humor outside the orbital cavity, thus avoiding the main reason for failure of filtering sur-gery, and regulating IOP in the clinic during the postoperative period without patient re-intervention, are the main focus of this research.

More studies are needed in the future to evaluate the effective-ness of this type of shunt, and to continue adapting the remarkable technological advances in implants for hydrocephalus for use in oph-thalmology. A multidisciplinary approach has enabled us to successfully develop this oculo-peritoneal shunt technique.

ACKNOWLEDGEMENTSWe wish to thank Moisés Dib MD, Enrique Bepmale, Alberto Siri

MD, Fernando Nolé MD, Sandra Aban, Julio Carranza, Diego Maldona-do-Junyent, Ana Junyent, Joss Heywood, and Héctor Tori.

REFERENCES 1. Garton HJL, Piatt JH Jr. Hydrocephalus. Pediatr Clin North Am. 2004;51(2):305-25.

2. Greenberg M. Handbook of Neurosurgery. 6th ed. New York: Thieme Medical; 2006. 3. Bondurant CP, Jimenez DF. Epidemiology of cerebrospinal fluid shunting. Pediatr

Neurosurg. 1995;23(5):254-8; discussion 259. 4. Albeck MJ, Borgesen SE, Gjerris F, Schmidt JF, Sorensen PS. Intracranial pressure

and cerebrospinal fluid outflow conductance in healthy subjects. J Neurosurg. 1991; 74(4):597-600.

5. Resnikoff S, Pascolini D, Etya’ale D, Kocur I, Pararajasegaram R, Pokharel GP, et al. Global data on visual impairment in the year 2002. Bull World Health Organ. 2004;82(11):844-51.

6. Quigley HA, Broman AT. The number of people with glaucoma worldwide in 2010 and 2020. Br J Ophthalmol. 2006;90(3):262-7. Comment in: Br J Ophthalmol. 2006; 90(3):253-4.

7. Varma R, Ying-Lai M, Francis BA, Nquyen BB, Deneen J, Wilson MR, Azen SP; Los An-geles Latino Eye Study. Prevalence of open-angle glaucoma and ocular hypertension in Latinos: the Los Angeles Latino Eye Study. Ophthalmology. 2004;111(8):1439-48. Comment in: Ophthalmology. 2005;112(4):733; author reply 733-4.

8. Virno M, Taverniti L, De Gregorio F, Sedran L, Longo F. Increase in aqueous humor production following D1 receptors activation by means of ibopamine. Int Ophthal-mol. 1997; 20(1-3):141-6.

9. Lollis SS, Mamourian AC, Vaccaro TJ, Duhaime AC. Programmable CSF shunt valves: radiographic identification and interpretation. AJNR Am J Neuroradiol. 2010;31(7):1343-6.

10. Watt S, Agerlin N, Romner B. Initial experience with the Codman Certas adjustable valve in the management of patients with hydrocephalus. Fluids Barriers CNS. 2012;9(1):21.

Review Article


INTRODUCTIONOcular surface tumors encompass malignant, premalignant, and

benign lesions arising from the conjunctiva, limbus, or cornea. These neoplasms may originate mainly from squamous epithelia, melano-cytes, or lymphocyte cells(1).

Clinical examination of the tumors based on slit-lamp biomi-croscopy by a trained professional frequently yields a correct diagnosis, if the clinician is familiar with the clinical characteristics. However, in some instances, only a broad differential diagnosis is possible, and slit-lamp biomicroscopy cannot reliably exclude uncommon diag nosis such as amelanotic malignant melanoma, highlighting the importance of acquiring a clinical diagnosis before administering a treatment. The gold standard is obtaining a biopsy, either incisional or excisional, for histopathology. The main risk of clinical misdiagnosis of an excised benign lesion is exposing the pa-

tient to unnecessary surgery; to prevent this, adjunctive diagnostic tests can be performed(2).

Diagnosis may be improved by cytological examination, high-resolution anterior segment ultrasound (UBM), in vivo confocal microscopy, and optical coherence tomography. Cytological sam-pling is a relatively noninvasive method and is thereby preferable when treatment with nonsurgical techniques such as administration of a topical chemotherapeutic agent with an antineoplastic drug is considered(3). It may also assist in evaluating lesions in cases where surgery may not be appropriate, including patients not medically fit for surgical biopsy(4). In 1954, Larmande and Timsit were the first to use cytodiagnosis in ophthalmology to assist in the evaluation of tumors of the sclerocorneal limbus(5).

Ocular surface cytology can be performed by several methods, including spatula scraping, brush cytology, and impression cytology

Impression cytology in the evaluation of ocular surface tumors: review article

Citologia de impressão na avaliação de tumores da superfície ocular: artigo de revisão

JEison dE nadai barros1,2, siMonE ribEiro araúJo dE alMEida1, Marcia sErva loWEn3, MarcElo carvalho da cunha2, José Álvaro PErEira goMEs1

Submitted for publication: September 17, 2014 Accepted for publication: December 4, 20141 Department of Ophthalmology & Visual Sciences, Universidade Federal de São Paulo, São Paulo,

SP, Brazil.2 Clínica de Olhos Dr. Moacir Cunha, São Paulo, SP, Brazil.3 Department of Pathology, Universidade Federal de São Paulo, São Paulo, SP, Brazil.



Coresponding author: Jeison de Nadai Barros. Al. Gabriel Monteiro da Silva, 1.000 - São Paulo, SP - 01442-000 - Brazil

E-mail: [email protected]

ABSTRACTImpression cytology (IC) has been widely used as a method for evaluating the ocular surface and superficial cells layers in the diagnosis and follow-up after treatment of several ocular surface tumors of both epithelial and melanocytic origin. Infor-mation regarding this can be found in the English-language literature since 1992. Using either cellulose acetate or Biopore membranes for specimen collection, a high correlation has been found between IC and tissue histology. Compared with exfoliative cytology with spatula, IC is less traumatic to the patient’s eye, provides a precise location of the area being studied, and allows accurate observation of the cells the way they exist in vivo. The additional advantage of IC is the preservation of limbal stem cells responsible for continuous corneal epithelium renewal; these can be affected after incisional or excisional biopsy at the corneoscleral limbus, which is the most frequent site of appearance of tumors in the stratified epithelium. Treatment for ocular surface squamous neoplasia has historically included surgery, but nonsurgical interventions have also been adopted. Hence, in certain cases, ophthalmologists may prefer interventions less invasive than surgical biopsy such as of impression cytology for both initial diagnosis and therapeutic monitoring of treatment for ocular surface lesions. Nevertheless, it should be considered that IC may be less helpful if the results conflict with the clinical picture or if the clinical diagnosis is uncertain and results are negative. In such cases, surgical biopsy is required for accurate diagnosis. The purpose of this review is to examine the published literature on the utilization of IC for the diagnosis and management of ocular surface tumors and to discuss the requirement for further investigation on the subject.

Keywords: Conjunctiva; Cornea; Limbus corneae; Conjunctival neoplasms/diag-nosis; Eye neoplasms; Cytodiagnosis; Cytological techniques; Diagnostic techniques, oph thalmological; Review

RESUMOA citologia de impressão (CI) tem sido amplamente utilizada como um método de avaliação da superfície ocular e das camadas de células superficiais no diagnóstico e no seguimento após tratamento de vários tumores da superfície ocular de origem epitelial ou melanocítica. As informações podem sem encontradas na literatura em língua inglesa desde 1992. Utilizando-se de membranas de acetato de celulose ou Biopore na coleta dos espécimes, uma alta correlação tem sido encontrada entre a CI e a histologia do tecido. Comparando-se com a citologia esfoliativa, a citologia de impressão é menos traumática para o olho do paciente, fornece uma localização precisa da área estudada e permite ver as células da forma como elas organizam-se in vivo. A vantagem adicional da citologia de impressão é a preservação das célu-las-tronco germinativas responsáveis pela renovação contínua do epitélio da córnea. Elas podem ser afetadas após biópsia cirúrgica na região do limbo que é o sítio mais frequentemente acometido pelos tumores do epitélio estratificado. O tratamento para a neoplasia escamosa da superfície ocular tem sido historicamente a cirurgia, mas intervenções não cirúrgicas também foram adotadas. Por esta razão, em certos casos, oftalmologistas podem recorrer a formas menos invasivas que a biópsia cirúr-gica (como a citologia de impressão) tanto para o diagnóstico inicial quanto para o monitoramento terapêutico das lesões da superfície ocular. No entanto, deve-se ter em mente que a citologia de impressão deixa de ser útil quando seu resultado não coincide com o quadro clínico ou quando o diagnóstico clínico é incerto e o resultado da citologia de impressão negativo. Nesses casos, a biópsia cirúrgica deve ser realizada para o diagnóstico. O objetivo desta revisão é examinar a literatura sobre a utilização da citologia de impressão no diagnóstico e tratamento dos tumores da superfície ocular bem como discutir a necessidade de uma investigação mais aprofundada sobre o assunto.

Descritores: Túnica conjuntiva; Córnea; Limbo da córnea; Neoplasias da túnica con-juntiva/diagnóstico; Neoplasias oculares; Citodiagnóstico; Técnicas citológicas; Técnicas de diagnóstico oftalmológico; Revisão

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(IC). IC is a well-established technique for collecting superficial epi-thelial layers by applying collecting devices (either cellulose acetate filter papers or Biopore membrane device), so that cells adhere to their surface and are removed from the eye to be processed further for analysis by various appropriate methods. IC was first developed to diagnose dry-eye status, and it is now used to diagnose various ocular surface disorders, including neoplasia. It represents a non- or minimally invasive biopsy technique applicable to the conjunctiva, cornea, and limbal area for both diagnosis and follow-up after treat-ment of tumors(6). Because repeated surgical biopsies of suspicious ocular surface lesions may cause complications such as scarring, lid deformity, limbal stem cell deficiency (LSCD), and great discomfort to the patient, IC can assist in the evaluation(4).

The present review examines and updates the published literature on the utilization of IC for the diagnosis and management of ocular surface tumors and discusses the requirement for further investiga-tion on the subject.

IC TECHNIQUE After a complete ophthalmological examination, including slit-

lamp biomicroscopy, IC can be performed according to methods previously described(4). A drop of topical anesthesia is consistently used. Then, the collection of the superficial cell layers of the ocular surface is performed by forceps-assisted application of a membrane with submicroscopic pores, such as MF-Millipore, onto the patient’s lesion. Membranes are often precut in different shapes and sizes for orientation purposes during processing. Most authors agree to using membranes with pore sizes ranging 0.025-0.45 µm. It is essential to consider the pore size because it affects the consistency of cell collection (the larger the pore size, greater the cellularity) and the re -

solution of the details under the microscope (morphology was better preserved in the smaller pore size papers). The membrane is firmly pressed against the area to be sampled with the aid of a swab or a solid rod for some seconds and then peeled off using the forceps. Whenever needed, more samples can be collected. They are imme-diately transferred to be fixed in a solution containing glacial acetic acid, 37% formaldehyde, and ethyl alcohol in a 1:1:20 volume ratio, taking care to completely immerse the membranes. After samples have been fixed, different staining techniques can be performed laboratory analysis. The most used stains include periodic acid-Schiff (PAS), hematoxylin-eosin, Gill’s hematoxylin, and Papanicolaou. The cells can be mounted on a slide after fixation and staining ready for interpretation. PAS is used to stain goblet cells and their secretions and hematoxylin as a counterstain to stain epithelial cells. Papani-colaou helps to better interpret the epithelial changes of squamous metaplasia and the distinct nuclear patterns. These stains have also been used together. Although over the last decade several techni-ques have used IC samples, light microscopy remains the most used method. To evaluate IC specimens by light microscopy, several featu-res are universally evaluated: the morphology of the epithelial cells, the degree of squamous metaplasia, the nuclear to cytoplasmic (N/C) ratio; the density, shape, and PAS intensity of goblet cells present; and the presence of nonepithelial cells, including inflammatory cells, melanocytic cells, and microorganisms. Atypical cells are identified by the presence of nuclear enlargement, hyperchromasia, irregular nuclear outline, and coarse nuclear chromatin and eventually by the presence of prominent nucleoli, under magnifications of 100×, 200×, and 400×. If different types of atypical cells are observed in the same specimen, more severe stage is considered.

APPLICATION OF IC IN THE EVALUATION OF LESIONS OF MELANOCYTIC ORIGIN

Lesions of melanocytic origin are as common as epithelial tumors and include conjunctival racial melanosis, primary acquired mela-nosis (PAM), secondary melanosis, nevus, and melanoma. Although majority of the melanocytic lesions are benign, some can be malig-

nant; therefore, distinguishing various conjunctival lesions is crucial(7). The first IC study of pigmented lesions from the conjunctiva

was published in 1992(8). A 73% correlation between IC and histo-pathology was observed in the diagnosis of 24 tumors, of which three were nevi, nine were melanomas, 10 were cases of PAM, and two were cases of secondary melanosis; examples are shown in fi-gure 1. An increased nuclear-to-cytoplasmic (NC) ratio, an irregular nuclear chromatin pattern, the presence of large nucleoli, and the observation of mitosis and anisokaryosis were regarded as cytolo-gical features of malignancy in cells containing melanin. When the relative proportion of atypical melanocytes was low, lesions were cytologically diagnosed as premalignant melanosis equivalent to the histological diagnosis PAM with atypia. If cancerous cells were abundant, the diagnosis was suggestive of melanoma. The authors reported that repeated examinations may increase the sensitivity of the cytological technique. Authors stated that although a diagnostic biopsy may remain necessary for determination of the origin and extent of those lesions, recurrent tumors or suspicious areas may be biopsied less frequently using IC, thus reducing the risk of side effects and patient discomfort(8).

In 2007, a study revealed 68 melanocytic conjunctival lesions, of which 31 were nevi, nine were melanoma, and 28 were PAM. The authors compared the Biopore membrane IC (referred to as “Biopore”) with exfoliative cytology (EC) in these lesions. Twenty-three of the 26 samples analyzed by Biopore and 20 of the 24 samples analyzed by EC correlated with the corresponding histology. Biopore accurately pre-dicted the outcome in 88% and EC in 83% of the lesions. The authors concluded that Biopore could be used in cytology of melanocytic lesions and was easier and faster to interpret than EC. If difficult with Biopore, sampling of the fornix, caruncula, and ocular material in children could be performed by EC. Because some melanocytic lesions will be covered with one or more layers of normal epithelium, cytology could only provide a realistic picture of a lesion when it was able to sample deeper than the most superficial layer of epithelial cells. Biopore, however, may sample only the first layer of cells on the conjunctiva, unless it is repeated several times to acquire cells of deeper layers(9). Similarly, IC with cellulose acetate filters was able to sample deeper layers when performed repeatedly(10).

A case of a patient with an irregular pigmented lesion of the lower eyelid margin simulating malignant tumor, which was treated based on the results of IC and diagnosed with secondary melanosis by histology, was presented in 2009. The importance of IC was em-phasized as an effective and safe method circumventing unnecessary and extensive procedures(11).

A few melanocytic lesions, including four nevi and one melano-ma, were examined in another study, and for such cases, results of both IC and histopathological features correlated(5).

IC features of 35 conjunctival nevi from children and adults re-ferred to as more noticeable were described in 2009. Approximately 26% were amelanotic but could be identified as localized areas of hyperemia. Using criteria derived from histology, IC was reported for conjunctival nevi when nests or clusters of nevus cells were observed within the epithelium layer containing or not containing mucous- secreting goblet cells. Epithelial cell layers demonstrated normal mor-phology, or, when the lesion was elevated, showed signs of squamous metaplasia (SM). IC confirmed the clinical diagnosis by demonstrating typical histopathological features of the superficial layers of conjunc-tival nevi in 91.4% of the cases. For amelanotic nevi, IC also allows differential diagnosis from other non-pigmented lesions(12).

Recently, a case of an amelanotic corneally displaced malignant conjunctival melanoma was described. The authors showed that IC performed prior to the treatment provided the first clue for the diagnosis later confirmed by histopathology. IC samples revealed abundant clusters of pleomorphic atypical tumor-dissociated cells with different sizes and anisokaryosis characterized by large and irre-gular nuclei with occasionally prominent nucleoli in a cytomorpho-



logy not resembling epithelial cells. Some of the atypical cells were spindle-shaped. Melanin pigment was absent. A few nonneoplastic squamous epithelial cells were also observed. Clinical diagnosis of amelanotic melanoma is challenging, and IC can assist in supporting the initial diagnosis when interpreted by a trained cytologist or un-der guidance of an ocular pathologist. For amelanotic melanoma, IC enables differential diagnosis from other nonpigmented lesions(13). In addition, incisional biopsy of melanoma should be avoided because of the risk of local tumor dissemination(14).

APPLICATION OF IC IN THE EVALUATION OF LESIONS OF EPITHELIAL ORIGIN

Ocular surface squamous neoplasia (OSSN) is the most common tumor of the ocular surface. The spectrum of OSSN ranges from mild to severe dysplasia, through full-thickness epithelial involvement, to invasive squamous cell carcinoma (SCC). Although the clinical appearance of a lesion can be suggestive of OSSN, tissue biopsy is necessary to confirm the diagnosis because the different stages of OSSN are extremely difficult to distinguish by slit-lamp biomicrosco-py, with an accuracy of clinical diagnosis by experienced clinicians of approximately 40%(3).

It has been reported that IC immunostained with cytokeratin antibodies and HMB-45 was useful to differentiate a pigmented con-junctival seborrheic keratosis masquerading as malignant melano-ma. IC disclosed basaloid cells intermixing with squamoid cells, and these cells demonstrated positive immunoreactivity to cytokeratin

and no reactivity to HMB-45 and therefore were proven to represent an epithelium-derived tumor despite of being pigmented. This re-port illustrated that IC combined with immunocytochemical staining may be a valuable diagnostic aid in the differentiation of pigmented conjunctival tumors prior to treatment(15).

The published correlation rate with IC for predicting the subse-quent histological findings ranged between 77% (55/71) and 80% (20/25), and both cellulose acetate(16) and Biopore membranes(17) have been successfully used. The difficulty in interpreting these IC specimens caused by the paucity of published criteria was overcome with the publication by Nolan et al., who described in detail the cyto-morphology of OSSN based on a high number of cases. The following cytological criteria were used to diagnose intraepithelial OSSN: nuclear enlargement (more than two times the dimensions of the nucleus of normal conjunctival cells), presence of irregular nuclear contour, coarsely clumped chromatin, nuclear pleomorphism, binucleation or multinucleation, and evident nucleoli. When nuclear enlargement was less than twice the dimensions in normal conjunctival cells or when it was limited to only few squamous cells, the specimen was ca-tegorized as having atypical squamous cells indefinite for dysplasia. If none of the abovementioned characteristics was observed, the spe-cimen was regarded negative. The finding of syncytia-like groupings, intraepithelial infiltration of inflammatory cells, and macronucleoli may be suggestive of SCC in some samples(18). Nevertheless, at pre-sent, no unique specific cytological feature to differentiate SCC from intraepithelial lesions in IC specimens has been identified. According

Figure 1. Examples of IC in the evaluation of lesions of melanocytic origin: A) Anterior segment slit-lamp photograph demonstrating a conjuncti-val nevus. B) IC obtained from the same lesion demonstrating a cluster of nevus cells (arrow) among epithelial cells (Hematoxylin-Eosin staining; original magnification, 400×). C) Anterior segment slit-lamp photograph of malignant melanoma. D) IC obtained from the sample depicted in (C) demonstrated clusters of pleomorphic atypical tumor-dissociated cells with different sizes and anisokaryosis characterized by large and irregular nuclei in a cytomorphology not resembling epithelial cells. Brown melanin granules can be seen inside the cytoplasm of the malignant melanocytes (Hematoxylin-Eosin staining; original magnification, 200×).

A

C

B

D

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to these reports, there were no false-positives identified by IC(16-19). Examples are shown in figure 2.

Notably, the cytology of subclinical intraepithelial OSSN has al ready been described. The cytological pattern for OSSN with no clinically visible abnormality differed from that observed in the eyes with clinically detectable disease; there were often a few dysplastic cells lying within sheets of normal epithelium(18).

In 2002, Chan et al. showed that IC obtained from surface cells overlying a pterygium was abnormal, typically exhibiting SM with increased goblet cell density. Altered cytology could also be de-monstrated in the inferior bulbar conjunctiva and interpalpebral conjunctiva, without clinical evidence of pterygium. This suggested a graded series of changes occurring throughout the bulbar conjunc-tiva, with the most advanced occurring directly over the pterygium, confirming that it was indeed an ocular surface disorder(20).

A case of conjunctiva-cornea intraepithelial neoplasia (CCIN) treated with topical mitomycin-C (MMC) and interferon alfa-2b in cycles was described in 2003. The patient was referred for LCSD and epithelial defect but IC specimens were suggestive of CCIN. After differentiation from LCSD by dye staining and IC, the patient was successfully treated(21).

Another study found that IC had a positive and negative predic-tive value of 97.4% and 53.9%, respectively, when compared with histology(5).

In 2009, Barros et al. described an index score modified from the

Bethesda system for reporting cervical cytologic diagnoses to diffe-rentiate SCC from pre-invasive ocular surface lesions by IC (n=39). They revealed a predictive index score of ≥4.25 representing the best cut-off point for SCC with a sensitivity of 95%, specificity of 93%, positive predictive value of 95%, and negative predictive value of 93%(4). Four of seven parameters included in their regression model (nuclear enlargement >three-fold, syncytial-like groupings, increased NC ratio, and indistinct cytoplasm border) were visible using clinical confocal microscopy (CCM). One parameter (prominent nucleoli) is currently undetectable by CCM. The last two parameters (cellular hyperchromasia and eosinophilic cytoplasm) would require specific stains unavailable in vivo. The introduction of in vivo stains or bio-markers to better visualize these cellular details would be useful to improve image quality and to obtain more detailed information. A novel CCM specific index score to differentiate SCC from preinvasive ocular surface lesions is still necessary(22).

IC may be less sensitive for cases with keratotic lesions because an abundance of surface keratin can make sampling inaccurate(5). To minimize this problem, authors have recommended collecting at least two samples over the same area from a suspicious lesion(4,23). For diagnosing OSSN, adding a second and a third evaluation of IC provided significantly more sensitivity than including only one(23). Nevertheless, it should considered that IC is very helpful, unless the result conflicts with the clinical scenario or when the actual clinical diagnosis is uncertain and the result is negative. In these cases, surgi-

A

C

B

DFigure 2. Example of IC in the evaluation of lesions of epithelial origin (ocular surface squamous neoplasia): A) Anterior segment slit-lamp photograph demonstrating conjunctival intraepithelial neoplasia. B) IC obtained from this lesion demonstrating atypical epithelial cells with mild nuclear enlar-gement, anisokaryosis, and remarkable hyperchromasia (Hematoxylin-Eosin staining; original magnification, 400×). C) Anterior segment slit-lamp photograph of invasive squamous cell carcinoma of the conjunctiva. D) IC demonstrating atypical epithelial cells showing nuclear enlargement, marked increase in the nuclear-to-cytoplasmic ratio, anisokaryosis, hyperchromasia, and a syncytial-like arrangement with absence of well-defined cytoplasmic borders (Hematoxylin-Eosin staining; original magnification, 400×).



cal biopsy needs to be performed for accurate diagnosis(5,23).In the study by Ballalai et al., 0.02% topical MMC was used to

treat patients with OSSN. Before the treatment, cytology showed the presence of neoplastic cells in patients with primary tumors, avoiding surgical biopsy and treatment delay(24).

A great advantage of using IC is the preservation of limbal stem cells, responsible for renewal of corneal epithelium throughout life. In most OSSN cases, the lesions affect predominantly the limbus and have a ten-dency to recur. IC offers a safer tool for diagnosis than repeated biopsy(4). Moreover, IC can be used during post-surgery fol low-up to identify any recurrence of the disease as well as the effects of topical treatment such as chemotherapy with antineoplastic drugs like MMC(25).

APPLICATION OF IC FOLLOWING TUMOR TREATMENT Treatment for OSSN has historically been surgery but nonsurgical

interventions have also been adopted. Adjunctive therapies allowed the treatment of subclinical disease at a site different from that of the clinically evident tumor. Nevertheless, topical chemotherapeutic drugs can be potentially toxic to the ocular surface(26). In 2001, IC was used to study the effects of topical MMC in the treatment of OSSN; 0.04% MMC induced cell death mainly by apoptosis or rarely by necrosis and changes induced in the ocular surface persisted for at least 8 months. MMC induced cytomegaly, cytoplasmic vacuolation, nucleomegaly with nuclear wrinkling, and binucleation or multinucleation. The N/C ratio in these enlarged cells was normal. These changes mimicked those observed following radiation therapy in uterine cervical cancer. Nuclear and cell size increased along with increasing N/C ratio in some dysplastic cells(25). Yamamoto et al. used IC during diagnosis and follow-up, resulting in successful treatment with 5-fluorouracil of an intraepithelial OSSN with LSCD that was refractive to topical MMC(27).

Dogru et al. evaluated the tear function and ocular surface alte-rations in patients with primary intraepithelial OSSN before and after treatment with 0.04% topical MMC. Initial IC specimens showed loss of goblet cells, higher grades of SM, and areas of isolated keratinized, bi-nucleated, and actively mitotic disfigured epithelial cells in all patients. The mean goblet cell density and SM grade were observed to having significantly improved at the last visit of the patients. IC proved useful in attaining the diagnosis of OSSN, evaluating the effect of treatment and showing MMC-related long-term changes on the ocular surface(28).

In 2005, Prabhasawat et al. reported complete tumor regression observed clinically and by IC, demonstrating the efficacy of 0.002% topical MMC as an adjunctive and alternative treatment in primary and recurrent OSSN; IC exhibited tumor-free specimens with cellular elongation as a result of chemotherapy(29).

Notably, cytological changes mimicking malignancy have been reported in conjunctiva up to 6 weeks following topical MMC thera-py. Nevertheless, there are features which help to differentiate these changes: epithelial cells affected by the drug show a proportionate increase in both cytoplasm and nucleus, preserving a normal NC ratio (cytomegaly), unlike the case of increased NC ratio (cariomegaly) in OSSN. The distinction of MMC-related changes from OSSN cells in IC specimens can be performed when the cell border is clearly visible and the N/C ratio can be estimated. Differentiation becomes difficult in cells with large hyperchromatic nuclei where the cell outline is not clearly defined because of overlapping cells or attenuation of the vacuolated cytoplasm. Therefore, studying such cells for which cell size can be clearly assessed is crucial(25).

Westekemper et al. examined ocular surface integrity of ten pa-tients with large and diffuse conjunctival melanoma who underwent proton beam radiation. The IC revealed conjunctival SM in nine cases, indicating a radiogenic, persisting disturbance in the differentiation of the conjunctival epithelial cells. The tear film instability correlated with goblet cell loss and meibomian gland dysfunction(30).

The use of topical MMC has been described by some authors not only for OSSN but also for melanocytic lesions such as PAM with aty-pia. However, its prolonged use may be associated with a high inci-

dence of complications like LSCD. IC diagnoses ocular surface lesions and also evaluates possible local side effects following treatment. Five cases of proven LSCD by IC resulting as a complication of topical treatment with MMC for PAM with atypia have been reported(31).

Rodríguez Feijoo et al. reported that making an accurate diffe-rential diagnosis between keratoacanthoma and SCC by histology as well as carrying out close monitoring after surgery due to the possibility of relapse and conversion to SCC is important. Therefore, they proposed the use of IC as a method for monitoring such pa-tients. After the treatment, IC exhibited large altered epithelial cells with intracellular union changes and an NC ratio of 1:20. A second series of IC tests performed 3 months after the first series showed the same results(32).

Recently, Faramarzi and Feizi evaluated the efficacy of perilesio-nal/subconjunctival injections of an antivascular endothelial growth factor, bevacizumab, for treatment of a group of 10 eyes with primary OSSN. Based on clinical presentation and IC results, they showed that the treatment was effective in terms of decreasing the size of con-junctival OSSN when the lesion was limited to the conjunctiva. Howe-ver, this therapy had no effect on corneal extensions of the OSSN(33).

APPLICATION OF IC IN THE EVALUATION OF TUMORS OF SEBACEOUS ORIGIN

In 2003, Sawada et al. demonstrated that IC detected conjunc-tival intraepithelial invasion from sebaceous cell carcinoma of the eyelid in four patients with severe unilateral blepharoconjunctivitis. IC showed numerous inflammatory cells and abnormal tumor cells with atypia and characteristic cytoplasmic vacuoles, consistent with dissolved sebaceous contents(34-35). They represented areas where lipid was contained before it was dissolved by alcohol; an example is shown in figure 3. The diagnosis was confirmed by histology from full-thickness wedge resection of the eyelids. When pagetoid spread in advanced cases of sebaceous cell carcinoma results in a superficial or full-thickness replacement of the normal conjunctival epithelium with tumor cells, the superficial abnormal cells can be detected by IC. However, areas on the conjunctiva with pagetoid spread may exist without full-thickness epithelial disease. In such cases, IC may sam-ple only the superficial normal epithelial cells and may fail to detect the tumor cells concealed in the deeper layers. Because sebaceous carcinoma can masquerade as several benign conditions such as blepharitis, the investigations should include IC and biopsy in cases that are not responsive to medication. If cellular atypia is present, a full-thickness lid biopsy should be performed(34).

APPLICATION OF IC IN THE EVALUATION OF ULCERATIVE EYELID MALIGNANCY

Thirty-two histopathologically proven malignant eyelid lesions diagnosed over a 2-year period, including 13 basal cell carcinomas, 11 sebaceous carcinomas, four SCC, two malignant melanomas, and two poorly differentiated carcinomas, formed the study group described very recently. The results of IC were compared with those of obtained by histopathological analysis in the study group and with an age- and sex-matched group of benign cases as controls. The sensitivity of IC was 84% (27/32) for the diagnosis of malignancy and 28% (9/32) for categorization of the type of malignancy. Because of its low sensiti-vity in terms of cytological categorization of the type of malignancy, IC cannot be recommended in the primary diagnosis of eyelid ma-lignancies. Nevertheless, with experience and improvement in the technique, it may prove to be a useful tool in deciding future mana-gement, particularly in recurrences of histopathologically confirmed eyelid malignancies, where biopsies may be avoided(36).

IC, IMAGING, AND HISTOPATHOLOGY Clinical examination is subjective, is unable to assess cellular

morphology, and may not detect subclinical microscopic diseases. A surgical biopsy to confirm the resolution of an OSSN could miss small

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residual lesions. Thus, an incisional biopsy may miss lesions that were not included in the excised tissue. The biopsy is based on clinically vi-sible disease and may produce false-negative results. The false clinical impression of tumor resolution can result in premature termination of topical treatment and an increased risk of recurrence. These lesions can spread along the basal conjunctival layers far beyond the clinical lesion, and thus may be missed clinically. Excisional biopsy, despite being the most traditional and accurate means, may induce conjunc-tival scarring, LSCD, and visually disturbing corneal scarring. Due to the multifocal nature of OSSN, surgical excision results in extensive collateral damage to adjacent areas of normal epithelium(37).

In addition to IC, newer diagnostic techniques including CCM(22), toluidine blue(38), and ultra-high resolution anterior segment optical coherence tomography (UHR-OCT)(39) have been reported to aid in the diagnosis of OSSN. All these techniques have limitations and require skilled professionals to perform the tests and interpret the results. IC assesses only superficial layers of cells, which are not always representative of deeper layers, whereas CCM does not provide cross- sectional views hence not being useful for determining the vertical and horizontal extent of the lesion. Therefore, ensuring that the exact same area of the ocular surface is analyzed by CMM at follow-up exa-minations can be challenging. Regarding UHR-OCT, lesions, which are thickly pigmented lesions or show leukoplakia, tend to impede the penetration of light to deeper tissues, impairing the determina-tion of the posterior limit of the lesion. Optical information at the time of the study was not sufficient to study signs of cellular atypia and was not able to rule out microinvasion(37). Similar to UHR-OCT, IC may not distinguish in situ from minimally invasive disease(6).

Each imaging modality has the advantage of being noninvasive, and each has been is useful in the detection of OSSN. However, both UBM and some confocal microscopy devices require contact with the ocular surface, increasing both the length of time and technical expertise required for their performance. Furthermore, although confocal microscopy has the advantage of detailing individual cell morphology, which is currently outside of the capability of UHR-OCT, it targets a very limited area. OCT has the advantage of higher-reso-lution images, but shadowing may occur in thick lesions or those with leukoplakia. UBM has greater depth of penetration but lower resolution and cannot evaluate the epithelial versus subepithelial nature of a lesion. No data are available regarding inter- and intra-observer variability for the assessment of ocular surface pathology using the UHR-OCT(40). Despite UHR-OCT having the advantage over IC of providing relatively deeper scans of the entire epithelium and the underlying tissue, it cannot reliably detect invasion(37). In addition,

UHR-OCT machines are largely limited to academic institutions(39).IC may be an inexpensive tool that can be used in the outpatient

clinic setting to help provide an objective evaluation of suspicious lesions that enables patients to make better informed decisions regarding the treatment requirements. Results of IC may also help the ophthalmologist decide whether incisional or excisional biopsy should be performed and whether any other associated procedures, such as freeze-thaw cryotherapy of the sclera/limbus and/or ethanol application to the cornea, are required. IC provides a flat mount of an area as large as the size of the applied filter paper with well-preserved morphology. In comparison, conjunctival smears destroy much of the morphological information and conjunctival biopsies provide infor-mation from a relatively small sample of the surface epithelium, both because of the difficulty of preparing flat mounts and because of their small sizes. Therefore, IC is ideal for sampling the corneal epithelium(40).

FINAL COMMENTSOSSN masquerades as scar tissue or pannus; in addition, it can

appear in association with pterygia(3). Thus, the question of using IC for the detection of OSSN in the setting of concomitant ocular surface disease requires further studies. Recently, Barros et al. reported that IC demonstrated high agreement with the results of the histopatholo-gical analysis for detecting atypical epithelial cells from unsuspected OSSN in cases of pterygia from Brazil, showing unsuspected and associated OSSN cells in 13 specimens (40%)(41).

IC presents great advantages: (1) it provides a source of intact and well-preserved epithelial cells from the ocular surface in any type of ocular surface pathology; (2) it is a nonsurgical, easy-to-perform, quick, and inexpensive technique that can always be performed on an outpatient basis; (3) only topical anesthesia is required, and no side effects or contraindications have ever been noted and thus it can be applied to children; (4) repeated IC sampling in the same patient over time is an excellent way to demonstrate changes due to a certain event, to monitor the progress of a disease, or to follow the effect of a therapeutic intervention; (5) IC maintains cell-to-cell contacts, preventing the problems of EC or brush cytology, which may destroy much of the cell morphology, cause overlapping of cells, and ham-per clear visualization of the in vivo arrangement of the cells; (6) IC samples can be processed using any type of microscopy in addition to polymerase chain reaction (PCR), immunoblotting analyses, and/or flow cytometry. Based on all these advantages, IC has become the technique of choice for sampling ocular surface epithelium for being a very useful research tool in both basic and clinical aspects(41-42).

A B

Figure 3. A) Anterior segment slit-lamp photograph demonstrating conjunctival intraepithelial invasion from a sebaceous cell carcinoma of the eyelid. B) IC showing inflammatory cells and a tumor cell with atypia, abnormal prominent nucleoli, and characteristic cytoplasmic vacuoles (arrow) consistent with intracellular dissolved sebaceous contents. These cells were PAS-negative, suggestive of non-goblet cell origin. No evident goblet cells were observed in the epithelium (PAS and Hematoxylin-Eosin staining; original magnification, 400×).



Although IC cannot replace histology, it has an important role in the diagnosis and management of patients with OSSN in a less in-vasive manner. A tool such as IC that aids the diagnosis of OSSN is of particular relevance to Brazilian patients, who live in a country closer to the equator line, with a climate and an ultraviolet-B light index that may contribute to the appearance and development of such tumors in its population. The correlation between sun exposure and OSSN has been well established(43). The importance of IC lies in its capacity to detect both the presence and extent of OSSN when the clinical diagnosis is difficult, to detect subclinical disease and follow up on previously diagnosed disease(4,18). Expertise in IC is acquired by continuing experience including close reviews, correlation with all possible subsequent histology specimens, and clinicopathological correlation. This enables the cytologist to gain familiarity and become aware of the eventual difficult areas such as keratinizing lesions(17).

Because IC has not presented sensitivity and specificity of 100%, the prospective use of the Barros score for predicting SCC needs to be further evaluated using a large number of patients(4). The develop-ment of a novel immunohistochemical analysis with a proliferative index such as that for Ki-67 could aid in IC specimens becoming a diagnostic marker for OSSN and in obtaining prognostic information regarding the risk of recurrence in a manner similar to the current use of histology(44). This combination of IC and immunocytochemistry was first described by Krenzer and Freddo in normal human conjunc-tiva in 1997, enabling the simultaneous evaluation of IC specimens for immunoreactivity to cytokeratin and morphological details(45). Nevertheless, as indicated in this review, there was only a single case(15) using this combined technique in the evaluation of an ocular surface tumor. Thus, the sensitivity and reliability of IC combined with immunocytochemical staining in the differentiation of ocular surface tumors need further evaluation in large-scale studies.

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8. Paridaens AD, McCartney AC, Curling OM, Lyons CJ, Hungerford JL. Impression cyto-logy of conjunctival melanosis and melanoma. Br J Ophthalmol. 1992;76(4):198-201.

9. Keijser S, Missotten GS, De Wolff-Rouendaal D, Verbeke SL, Van Luijk CM, Veselic-Char vat M, et al. Impression cytology of melanocytic conjunctival tumours using the Biopore membrane. Eur J Ophthalmol. 2007;17(4):501-6.

10. Singh R, Joseph A, Umapathy T, Tint NL, Dua HS. Impression cytology of the ocular surface. Br J Ophthalmol. 2005;89(12):1655-9. Comment in: Br J Ophthalmol. 2008; 92(1):157-8.

11. Oliveira CS, Barros J de N, Souza S de B, Cvintal T, Schellini SA. [Melanosis in eyelid margin with malignancy suspect: case report]. Arq Bras Oftalmol. 2009;72(5):706-9. Portuguese.

12. Barros J de N, Lowen MS, Mascaro VLD, Andrade TP, Martins MC. Impression cytology features of conjunctival nevi referred as more noticeable. Arq Bras Oftalmol. 2009; 72(2):205-10.

13. Barros J de N, Motono M, Costa FD, Cunha MC, Chojniak MM. Amelanotic corneally displaced malignant conjunctival melanoma: a case report evaluated with impres-sion cytology. Arq Bras Oftalmol. 2014;77(1):57-9.

14. Lim L, Madigan MC, Conway RM. Conjunctival melanoma: a review of conceptual and treatment advances. Clin Ophthalmol. 2013;6:521-31.

15. Tseng SH, Chen YT, Huang FC, Jin YT. Seborreic keratosis of conjunctiva simulating a malignant melanoma: an immunocytochemical study with impression cytology. Ophthalmology. 1999;106(8):1516-20.

16. Nolan GR, Hirst, LW, Wright RG, Bancroft BJ. Application of impression cytology to the diagnosis of conjunctival neoplasms. Diagn Cytopathol. 1994;11(3):246-9.

17. Tole DM, McKelvie PA, Daniell M. Reliability of impression cytology for the diagnosis

of ocular surface squamous neoplasia employing the biopore membrane. Br J Oph-thalmol. 2001;85(2):154-8. Comment in: Br J Ophthalmol. 2001;85(7):888.

18. Nolan GR, Hirst, LW, Bancroft BJ. The citomorphology of ocular surface squamous neoplasia by using impression cytology. Cancer. 2001;93(1):60-7.

19. McKelvie PA, Daniell M, McNab A, Loughnan M, Santamaria JB. Squamous cell car-cinoma of the conjunctiva: a series of 26 cases. Br J Ophthalmol. 2002;86(2):168-73. Comment in: Br J Ophthalmol. 2002;86(12):1462.

20. Chan CM, Liu YP, Tan DT. Ocular surface changes in pterygium. Cornea. 2002;21(1):38-42. 21. Di Pascuale MA, Espana EM, Tseng SC. A case of conjunctiva-cornea intraepithelial

neoplasia successfully treated with topical mitomycin C and interferon alfa-2b in cycles. Cornea. 2004;23(1):89-92.

22. Parrozzani R, Lazzarini D, Dario A, Midena E. In vivo confocal microscopy of ocular surface squamous neoplasia. Eye (Lond). 2011;25(4):455-60.

23. Kheirkhah A, Mahbod M, Farzbod F, Zavareh MK, Behrouz MJ, Hashemi H. Repeated applications of impression cytology to increase sensitivity for diagnosis of conjunc-tival intraepithelial neoplasia. Br J Ophthalmol. 2012;96(2):229-33.

24. Ballalai PL, Erwenne CM, Martins MC, Lowen MS, Barros JN. Long-term results of topi-cal mitomycin C 0.02% for primary and recurrent conjunctival-corneal intraepithelial neoplasia. Ophthal Plast Reconstr Surg. 2009;25(4):296-9.

25. McKelvie P, Daniell M. Impression cytology following mitomycin C therapy for ocular surface squamous neoplasia. Br J Ophthalmol. 2001;85(9):1115-9.

26. Adler E, Turner JR, Stone DU. Ocular surface squamous neoplasia: a survey of changes in the standard of care from 2003 to 2012. Cornea. 2013;32(12):1558-61.

27. Yamamoto N, Ohmura T, Sizuki H, Shirasawa H. Successful treatment with 5-fluorouracil of conjunctival intraepithelial neoplasia refractive to mitomycin C. Ophthalmology. 2002;109(2):249-52. Comment in: Ophthalmology. 2003; 110(4):625-6; author reply 626; Ophthalmology. 2003; 110(7):1289; Ophthalmology. 2003;110(6):1262-3; author reply 1263.

28. Dogru M, Erturk H, Shimazaki J, Tsubota K, Gul M. Tear function and ocular surface changes with topical mitomycin C treatment for primary corneal intraepithelial neoplasia. Cornea. 2003;22(7):627-39.

29. Prabhasawat P, Tarinvorakup P, Tesavibul N, Uiprasertkul M, Kosrirukvongs P, Boorana-pong W, et al. Topical 0.002% mitomycin C for the treatment of conjunctival-corneal intraepithelial neoplasia and squamous cell carcinoma. Cornea. 2005; 24(4):443-8.

30. Westekemper H, Anastassiou G, Sauerwein W, Chauvel P, Bornfeld N, Steuhl KP, et al. [Analysis of ocular surface alterations following proton beam radiation in eyes with conjunctival malignant melanoma]. Ophthalmologe. 2006;103(7):588-95. German.

31. Lichtinger A, Pe’er J, Frucht-Pery J, Solomon A. Limbal stem cell deficiency after topi-cal mitomycin C therapy for primary acquired melanosis with atipia. Ophthalmology. 2010;117(3):431-7.

32. Rodríguez Feijoo D, Romero Moreno I, López Gutierrez C, Usabiaga Usandizaga M, Cisneros Carpio M, Amias Gorostiza A, et al. [Conjunctival keratoacanthoma: diagno-sis, treatment and monitoring by conjunctival impression cytology]. Arch Soc Esp Oftalmol. 2012;87(3):82-5. Spanish.

33. Faramarzi A, Feizi S. Subconjunctival bevacizumab injection for ocular surface squamous neoplasia. Cornea. 2013;32(7):998-1001.

34. Sawada Y, Fischer JL, Verm AM, Harrison AR, Yuan C, Huang AJ. Detection by impres-sion cytologic analysis of conjunctival intraepithelial invasion from eyelid sebaceous cell carcinoma. Ophthalmology. 2003;110(10):2045-50.

35. Santo RM, Bordon PB, Barros J de N, Schellini SA, Erwenne CM. Tumores da superfície ocular. In: Gomes JA, Alves MR, editores. Superfície ocular córnea limbo conjuntiva filme lacrimal. 2nd ed. Rio de Janeiro: Cultura Médica; 2011. p. 141-70.

36. Sen S, Lyngdoh AD, Pushker N, Meel R, Bajaj MS, Chawla B. Impression cytology diagnosis of ulcerative eyelid malignancy. Cytopathology. Forthcoming 2014.

37. Shousha MA, Karp CL, Canto AP, Hodson K, Oellers P, Kao AA, et al. Diagnosis of ocular surface lesions using ultra-high-resolution optical coherence tomography. Ophthalmology. 2013;120(5):883-91.

38. Romero IL, Barros J de N, Martins MC, Ballalai PL. The use of 1% toluidine blue eye drops in the diagnosis of ocular surface squamous neoplasia. Cornea. 2013;32(1):36-9.

39. Thomas BJ, Galor A, Nanji AA, El Sayyad F, Wang J, Dubovy SR, et al. Ultra high-re-solution anterior segment optical coherence tomography in the diagnosis and ma nagement of ocular surface squamous neoplasia. Ocul Surf. 2014;12(1):46-58.

40. Dart J. Impression cytology of the ocular surface - research tool or routine clinical investigation? Br J Ophthalmol. 1997;81(11):930.

41. Barros J de N, Lowen MS, Moraes-Filho MN, Martins MC. Impression cytology for detection of unsuspected ocular surface squamous neoplasia cells in pterygia. Arq Bras Oftalmol. 2014;77(5):305-9.

42. Calonge M, Diebold Y, Sáez V, Enriquez de Salamanca A, García-Vásquez C, Corrales RM, et al. Impression cytology of the ocular surface: a review. Exp Eye Res. 2004; 78(3):457-72.

43. Newton R, Ferlay J, Reeves G, Beral V, Parkin DM. Effect of ambient solar ultravio-let radiation on incidence of squamous-cell carcinoma of the eye. Lancet. 1996; 347(9013):1450-1.

44. Ohara M, Sotozono C, Tsuchihashi Y, Kinoshita S. Ki-67 labeling index as a marker of malignancy in ocular surface neoplasms. Jpn J Ophthalmol. 2004;48(6):524-9.

45. Krenzer KL, Freddo TF. Cytokeratin expression in normal human bulbar conjunctiva obtained by impression cytology. Invest Ophthalmol Vis Sci. 1997;38(1):142-52.

Letters to the Editor


A calipers-free intravitreal anti-VEGF

injection technique

Técnica de injeção intravítrea de anti-VEGF sem compasso

Dear Editor,Intravitreal injection of drugs such as anti-vascular endothelial

growth factor (anti-VEGF) agents and corticosteroids has shown en-couraging results in the treatment of many ocular diseases. In deve-loped countries, this type of injection is probably the most frequently performed vitreoretinal procedure. However, it has the potential for serious complications, such as endophthalmitis, lens injury, and retinal detachment(1). To avoid these unfortunate complications, it is important to assess the safety and cost-effectiveness of intravitreal injection. A safe, cost-effective, calipers-free intravitreal injection techni-que is presented here.

The patient is placed in the supine position in the operating room. The skin, lids, and lashes are sterilized using 10% povidone iodine, and

then, several drops of proparacaine 0.5% and povidone iodine 5% are instilled in the conjunctival cul-de-sac. A speculum is inserted 2 min after the first instillation of 5% povidone iodine drops. An oral ins-truction is given during the injection to position the eye to either the upper-right or upper-left side on the basis of laterality. The injection site is marked from the limbus by using a sterile polypropylene needle (30G x 13 mm, BD Microlance 1 mL; Becton Dickinson, USA) cap until a circular track appears in the sclera (Figures 1 A, B). Subsequently, the needle cap is removed, and a needle is inserted through the point of intersection of the circle and a tangent parallel to the limbus. The point of intersection is 3.5 mm from the limbus (Figures 2 A, B). To avoid lens damage in phakic patients, the needle can be inserted just behind the marked area or 3.5-4 mm from the limbus. Then, the drug is gently administered. We applied tamponade for a few seconds after the procedure by using a sterile cotton-tip applicator. In the first 100 consecutive intravitreal injections, the injection site was checked by using calipers to ensure a distance of 3.5 mm from the limbus. No lens damage, retinal breaks, retinal detachment, or endophthalmitis due to the procedure were detected.

The recommended injection site for intravitreal anti-VEGF injection is 3.5-4 mm posterior to the limbus in the inferotempo-ral quadrant of the globe for pseudophakic and phakic eyes(2,3). Ophthalmic surgi cal calipers are useful for determining the in-

Figure 1. Marking the injection site by using the sterile needle cap; (A) marked area in the sclera (B).

A B

Figure 2. The point of intersection of the circle and a tangent parallel to the limbus (A) is 3.5 mm from the limbus, and the needle is inserted towards the center of the eye from that point (B).

A B

Cartas ao Editor | Letters to the editor


jection site. However, this technique is expensive when multiple intravitreal anti-VEGF in jections are performed because individually sterilized sets must be used for each patient. Our technique using a sterile needle cap that covers and protects the needle from de-bris or contamination does not require the use of surgical calipers. Therefore, our technique is cost-effecti ve. Although Wilson and Scott(4) mentioned the use of the hub of a sterile tuberculin syringe for marking the injection site, they did not describe the technique. In addition, the proposed technique ensures adequate anesthesia before administering the injection. Our technique can be used for both superior and inferior temporal intravitreal injections. However, to avoid iatrogenic inferior retinal breakage or detachment, we prefer superior intravitreal injections.

In our study, we used Becton Dickinson BD Microlance 1-mL 30G x 13-mm caps, which are found in various international markets. Using different needle caps may lead to different distances from the limbus. Therefore, when using needles of different types, we recom mend that the distance should be measured first with surgical calipers be-fore the needle cap is used.

In conclusion, our calipers-free technique enables safe and cost-effec -tive intravitreal injections.

Zafer Oztas1, Cezmi Akkin1,

Filiz Afrashi1, Sedat Selim2

Submitted for publication: December 4, 2014 Accepted for publication: December 11, 20141 Department of Ophthalmology, Ege University Faculty of Medicine, Izmir, Turkey.2 Department of Ophthalmology, Izzet Baysal Training and Research Hospital, Bolu, Turkey.



Corresponding author: Zafer Oztas. Ege Universitesi Tip Fakultesi - Goz Hastaliklari AD - Bornova, Izmir - 35040 - Turkey - Email: [email protected]

REFERENCES 1. Fung AE, Rosenfeld PJ, Reichel E. The International Intravitreal Bevacizumab Safety Survey:

using the internet to assess drug safety worldwide. Br J Ophthalmol. 2006; 90(11):1344-9. Comment in: Br J Ophthalmol. 2006;90(11):1333-4; Br J Ophthalmol. 2006; 90(11):1440-1.

2. Aiello LP, Brucker AJ, Chang S, Cunningham ET Jr, D’ Amico DJ, Flynn LR, et al. Evolving guidelines for intravitreous injections. Retina. 2004;24(5 Suppl):S3-19. Comment in: Retina. 2005;25(7):949-50; author reply 950.

3. Doshi RR, Bakri SJ, Fung AE. Intravitreal injection technique. Semin Ophthalmol. 2011; 26(3):104-13.

4. Wilson ME, Scott AW. How to Give Intravirteal Injections? EyeNet Magazine April 2013: 45-7. http://www.aao.org/publications/eyenet/201304/pearls.cfm

Letters to the Editor

135Arq Bras Oftalmol. 2015;78(2):135http://dx.doi.org/10.5935/0004-2749.20150035

Dengue and chiasmal compression

Compressão de quiasma e dengue

Dear Editor,The recent report on “dengue and chiasmal compression” is

very interesting(1). Suzuki et al. reported a case “presenting with dengue fever and bilateral acute visual loss caused by chiasmal compression due to Rathke’s cleft cyst apoplexy” and concluded that “apoplexy of sellar and suprasellar tumors should be conside-red in the differential diagnosis of patients with acute visual loss and dengue fever(1)”. The hemorrhagic complications of severe dengue infection are not uncommon and may present with ophthalmolo-gical symptoms. In fact, tumor apoplexy may be a complication to dengue and can sometimes be observed in cases without any previous tumor history. Focusing on the specific reported case of Rathke’s cleft cyst apoplexy, Kim noted that “the occurrence of symptomatic pituitary hemorrhage into a Rathke’s cleft cyst (RCC) is extremely rare(2)”. Indeed, there are also other conditions relating to bilateral visual loss in dengue that should be included in differential diagnoses(3), e.g., dengue maculopathy, which may

result in subacute bilateral visual loss(4); retinal hemorrhage, optic neuropathy, and angle closure glaucoma(5-6).

Viroj Wiwanitkit

Submitted for publication: December 16, 2014 Accepted for publication: January 15, 2015

Surin Rajabhat University, Surin, Thailand.



Corresponding author: Viroj Wiwanitkit. E-mail: [email protected]

REFERENCES 1. Suzuki AC, Araújo RB, Souza EC, Monteiro ML. Bilateral acute visual loss from

Rathke’s cleft cyst apoplexy in a patient with dengue fever. Arq Bras Oftalmol. 2014; 77(5):330-3.

2. Kim E. A Rathke’s Cleft Cyst presenting with apoplexy. J Korean Neurosurg Soc. 2012; 52(4):404-6.

3. Schmitt ER, Kohnen T, von Jagow B. [Acute bilateral loss of visual acuity following dengue fever]. Klin Monbl Augenheilkd. 2013;230(11):1142-3. [in German].

4. Braithwaite T, Nabarro L, Tufail A. Subacute bilateral vision loss resulting from dengue maculopathy. BMJ Case Rep. 2013;2013. pii: bcr2013200542.

5. Kumar V, Kataria R, Mehta VS. Dengue hemorrhagic fever: a rare cause of pituitary tumor hemorrhage and reversible vision loss. Indian J Ophthalmol. 2011;59(4):311-2.

6. Pierre Filho Pde T, Carvalho Filho JP, Pierre ET. Bilateral acute angle closure glaucoma in a patient with dengue fever: case report. Arq Bras Oftalmol. 2008;71(2):265-8.


Instructions Authors

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9. Abbreviations and Acronyms. Abbreviations and acronyms should be preceded by the spelled-out abbreviation on first mention and in the legends of tables and figures (even if they have been pre-viously mentioned in the text). Titles and abstracts should not contain abbreviations and acronyms.

10. Units of Measurement: Values of physical quantities should be used in accordance with the standards of the International System of Units.

11. Language. Texts should be clear to be considered appropriate for publication in a scientific journal. Use short sentences, written in a direct and active voice. Foreign words should be in italics. Thera-peutic agents should be mentioned by their generic names with the following information in parentheses: trade name, manufacturer’s name, city, state and country of origin. All instruments or apparatus should be mentioned including their trade name, manufacturer’s name, city, state and country of origin. The superscript symbol of trademark ® or™ should be used in all names of instruments or trade names of drugs. Whenever there are doubts about style, terminology, units of measurement and related issues, refer to the AMA Manual of Style 10th edition.

12. Original Documents. Corresponding authors should keep the original documents and the letter of approval from the Research Ethics Committee for studies involving humans or animals, the con-sent form signed by all patients involved, the statement of agreement with the full content of the study signed by all authors and the state-ment of conflict of interest of all authors, as well as the records of the data collected for the study results.

13. Corrections and Retractions. Errors may be noted in published manuscripts that require the publication of a correction. However, some errors pointed out by any reader may invalidate the results or the authorship of a manuscript. If substantial doubt arises about the honesty or integrity of a submitted manuscript, it is the editor’s responsibility to exclude the possibility of fraud. In these situations, the editor will inform the institutions involved and the funding agen-cies about the suspicion and wait for their final decision. If there is confirmation of a fraudulent publication in ABO, the editor will act in compliance with the protocols suggested by the International Com-mittee of Medical Journal Editors (ICMJE) and by the Committee on Publication Ethics (COPE).

CHECKLISTBefore submitting their manuscript, authors should make sure

that all the following items are available: □Manuscript prepared in accordance with the instructions to

authors. □Maximum number of words, tables, figures, and references

according to the type of manuscript. □ Title page including the clinical trial registration number is not

included in the main document □ No figures and tables are included in the main document of

the manuscript. □ All figures and tables were uploaded separately as supple-

mentary documents. □ Author Contribution Statement completed and saved as a

digital file to be sent as a supplementary document. □ Form for Disclosure of Potential Conflicts of Interest of all

authors completed and saved as digital files to be sent as supplementary documents.

□ Digital version of the report provided by the Institutional Re-view Board containing the approval of the project to be sent as a supplementary document.


LIST OF WEBSITESAuthorship Principles according to the ICMJEhttp://www.icmje.org/recommendations/browse/roles-and-responsibilities/defining-the-role-of-authors-and-contributors.html

http://www.cbo.com.br/site/files/Formulario Contribuicao dos Autores.pdf

CONSORT (Consolidated Standards of Reporting Trials) http://www.consort-statement.org/consort-statement/

STARD (Standards for the Reporting of Diagnostic accuracy studies) http://www.stard-statement.org/

PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) http://www.prisma-statement.org/index.htm

STROBE (Strengthening the Reporting of Observational studies in Epidemiology) http://www.strobe-statement.org/

Online interface for submission of manuscripts to ABOhttp://www.scielo.br/ABO

International Committee of Medical Journal Editors (ICMJE)http://www.icmje.org/

Uniform requirements for manuscripts submitted to biomedical journalshttp://www.nlm.nih.gov/bsd/uniform_requirements.html

http://www.wma.net/en/30publications/10policies/b3/index.html

Principles of the Association for Research in Vision and Ophthal-mology (ARVO)http://www.arvo.org/About_ARVO/Policies/Statement_for_the_Use_of_Animals_in_Ophthalmic_and_Visual_Research/

reviewed medical journalshttp://www.wame.org/about/wame-editorial-on-coi

http://www.icmje.org/coi_disclosure.pdf

http://www.clinicaltrials.gov

http://www.anzctr.org.au

International Standard Randomised Controlled Trial Number - ISRCTNhttp://isrctn.org/

Registry - UMIN CTRhttp://www.umin.ac.jp/ctr/index.htm

Nederlands Trial Registerhttp://www.trialregister.nl/trialreg/index.asp

Registros Brasileiros de Ensaios Clínicoshttp://www.ensaiosclinicos.gov.br/

http://www.ncbi.nlm.nih.gov/sites/entrez?db=mesh&term=

http://decs.bvs.br/

Format suggested by the International Committee of Medical Journal Editors (ICMJE)http://www.nlm.nih.gov/bsd/uniform_requirements.html

List of Journal Indexed in Index Medicushttp://www.ncbi.nlm.nih.gov/journals

AMA Manual of Style 10th editionhttp://www.amamanualofstyle.com/

Protocols of the International Committee of Medical Journal Editors (ICMJE)http://www.icmje.org/recommendations/browse/publishing-and-editorial-issues/scientific-misconduct-expressions-of-concern-and-retraction.html

Protocols of the Committee on Publication Ethics (COPE)http://publicationethics.org/resources/flowcharts

Chief Executive Officer: Fernando Steven Ullmann;Commercial Director: Helen Suzana Perlmann; Art Director: Elza Rudolf;

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Edited byIPSIS GRÁFICA E EDITORA S.A.

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