Effects of riboflavin/UVA corneal cross-linking on keratocytes and collagen fibres in human cornea

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<ul><li><p>Original Article</p><p>Effects of riboavin/UVA corneal cross-linking onkeratocytes and collagen bres in human cornea_ 49..56Rita Mencucci MD,1 Mirca Marini PhD MD,2 Iacopo Paladini MD,1 Erica Sarchielli PhD,2 Eleonora SgambatiPhD,3 Ugo Menchini MD1 and Gabriella B Vannelli PhD MD21Department of Oto-Neuro-Ophthalmological Surgical Sciences, Eye Clinic, 2Department of Anatomy Histology and Forensic Medicine University of Florence, Policlinic of Careggi, Florence, and 3Department of Health Sciences, University of Molise, Campobasso, Italy</p><p>ABSTRACTPurpose: To evaluate the effects of corneal cross-linking on keratocytes and collagen fibres in humancorneas.</p><p>Methods: Fifteen corneal buttons were examined. Tenwere from patients with keratoconus submitted topenetrating keratoplasty and five of them weretreated with cross-linking 6 months before penetrat-ing keratoplasty. Five normal corneal buttons fromhealthy donors were used as controls. All sampleswere prepared for TUNEL assay and Western blotanalysis for the detection of keratocyte apoptosis andimmunohistochemical analysis for the morpholo-gical evaluation of keratocytes and collagen fibrediameter.</p><p>Results: Normal corneas exhibited no TUNEL-positivekeratocytes and keratoconic and cross-linkedcorneas showed moderate apoptotic cells mainly inthe anterior part of the stroma. This apoptotic trendwas confirmed by the cleavage of poly (ADP-ribose)polymerase assessed using Western blot. The Ki-67staining showed a significant increase in the kerato-cyte proliferation in cross-linked corneas comparedwith normal and keratoconus. In cross-linkedcorneas CD34-positive keratocytes were regularlydistributed throughout the whole corneal stroma asin the control, and keratoconus was associated withpatchy loss of immunoreactivity. The immunohis-tochemical analysis of collagen type I showed a sig-nificant increase in fibre diameter of cross-linkedcorneas compared with control and keratoconus.</p><p>Conclusion: Corneal cross-linking leads to keratocytedamage; after 6 months a repopulation by prolifer-ating cells, a distribution of CD34-positive kerato-cytes as in control and an increase in collagen fibrediameter were observed. These modifications are themorphological correlate of the process leading to anincrease in biomechanical stability.</p><p>Key words: collagen bre, human cornea, keratocyte,riboavin/UVA corneal cross-linking.</p><p>INTRODUCTION</p><p>Keratoconus is a non-inflammatory usually bilateralprogressive corneal pathology with frequency of 1 in2.000 in the general population. It is a pathologycharacterized by a progressive thinning and ectasiaof the stroma that results in a cone-shaped corneaand severe visual impairment, making corneal trans-plantation necessary in approximately 20% ofpatients.1 This pathology is characterized by axialthinning, fragmentation of the epithelial basementmembrane, breaks and scarring at the level of Bow-mans membrane, keratocytes alteration and, ulti-mately, stromal scarring.</p><p>Keratocytes, the principal cells of the stroma, playan important role in the preservation of cornealtransparency and mechanical stability. They areresponsible for the synthesis and maintenance of thecollagen component and the extracellular matrix.2</p><p>Some studies on keratoconic corneas revealedchanges in keratocyte morphology, decrease in kera-tocyte density and apoptosis increase.3 The causes ofcorneal thinning in keratoconus are still not clear.Takahashi et al. noted that the number of lamellae in</p><p> Correspondence: Dr Rita Mencucci, Department of Oto-Neuro-Ophthalmological Surgical Sciences Eye Clinic, Viale GB Morgagni 85, 50134</p><p>Florence, Italy. Email: rita.mencucci@uni.it</p><p>Received 27 July 2009; accepted 3 November 2009.</p><p>Clinical and Experimental Ophthalmology 2010; 38: 4956 doi: 10.1111/j.1442-9071.2010.02207.x</p><p> 2010 The AuthorsJournal compilation 2010 Royal Australian and New Zealand College of Ophthalmologists</p></li><li><p>the keratoconic cornea is significantly lower than inthe normal cornea but the thickness of lamellae isunaltered.4 Moreover, some studies showed no dif-ference in interfibrillar spacing between keratoconicand control corneas, demonstrating that thinning ofthe corneal stroma in keratoconus is not the result ofcloser packing of the fibrils, but it is due to theprogressive loss of lamellae from the stroma.46 It wasalso reported that the orientation of collagen fibrilswithin the lamellae is altered in keratoconus,suggesting that the loss of structural integrity couldplay an important role in the pathogenesis of thedisease.7</p><p>Recently, a new method was developed forthe treatment of progressive keratoconus: cornealcollagen cross-linking, that increases the stiffness ofthe cornea using UVA and the photosensitizerriboflavin.8,9 Several studies reported that collagencross-linking can delay or stop keratoconus progres-sion treating some underlying pathophysiologicalmechanisms, improving corneal shape and produc-ing a better quality of vision.10 Extensive experimen-tal studies on rabbit and porcine eyes showed asignificant increase in corneal rigidity of approxi-mately 70% in treated versus untreated corneas afterriboflavin/UVA collagen cross-linking.1114 It wasdemonstrated that this procedure can strengthen theweak corneal structure by increasing collagen cross-links, which have the role to anchor the collagenfibres preventing the cornea from bulging out andbecoming irregular, as in the keratoconus.14 It hasbeen suggested that a process of keratocytes-inducedapoptosis could be essential for replacement of cellsand new well-structured collagen as confirmed by invivo confocal examinations.15 Wollensak et al. in 2004showed the induction of keratocytes apoptosis in therabbit cornea 24 h after cross-linking.16</p><p>Some studies hypothesized that riboflavin/UVAcross-linking increases corneal stroma strength andretards the progression of keratoconus by cross-linking of collagen molecules with mechanisms thatare still largely unknown.17,18 However, recently itwas described in rabbits and spiny dogfish sharksthat the increase in cross-linking of collagen mol-ecules is caused by reactions that require productionof singlet oxygen, whose half-life is extended in thepresence deuterium oxide.19</p><p>A previous study reported a statistically signifi-cant increase in corneal collagen fibre diameter afterriboflavin/UVA cross-linking in rabbit corneas, espe-cially in the anterior stroma that underlies theincreased biomechanical stiffness of the cornea afterthe treatment.20 The aggregation of collagen exposedto UVA in the presence of riboflavin was also impu-tated to tyrosine modification.21 A recent study dem-onstrated the presence of high-molecular-weightcollagen type I polymers in the porcine cross-linked</p><p>corneas that correlates with an increased collagenfibre diameter after the treatment.22</p><p>The clinical importance of a keratocyte loss is notquite clear; at present the effects of cross-linkingtreatment on stromal keratocyte population are notcompletely understood.17,18</p><p>The aim of our study was to evaluate the effect ofriboflavin/UVA corneal cross-linking treatment onhuman keratocytes and stromal fibres.</p><p>METHODS</p><p>Human corneal samples were collected and dividedin three study groups: (i) five cadaver normal corneasfrom Eye-Bank of Lucca as control; (ii) five cornealbuttons with keratoconus without subepithelialscarring removed after penetrating keratoplasty(PKP); and (iii) five corneal buttons with keratoco-nus treated with cross-linking 6 months before,according to Wollensak technique,8 and thenremoved to perform a PKP because of the poorvision. All keratoconus donors gave permission touse their tissue for research. Tissue procurement anduse was carried out in accordance with declaration ofHelsinki and local regulations. In the groups 2 and 3all keratoconus were in Krumeich stage 3.23 Thepatients were informed about the severe stage oftheir keratoconus and gave their permission toperform corneal cross-linking before PKP. In theclinical history of the three groups there was no traceof any other corneal surgical treatment or any ste-roids therapy. Antibiotics drops were used 5 daysafter cross-linking until the new epithelium wasformed completely. The age range of the subjects was3040 years and was similar in the three groups.</p><p>In all groups we analysed central corneal buttonsof similar diameter. Immediately after removal, thecornea buttons were cut and small pieces were pro-cessed for light microscopy and Western blotanalysis.</p><p>TUNEL (TdT-mediated dUTP Nick-EndLabeling) assay</p><p>Apoptotic cells were identified with Klenow FragelTM DNA Fragmentation Detection Kit (OncogeneResearch Products, San Diego, CA, USA). Briefly,paraffin embedded tissues were deparaffinized,and proteins were digested by applying proteinaseK (20 mg/mL) to specimens for 15 min at roomtemperature. After washing in distilled water,endogenous peroxidase was quenched in 3% hydro-gen peroxide in methanol for 5 min. As described bythe manufacturer, DNA strand breaks were labelledby attaching them to biotin-labelled and unlabelleddeoxynucleotides. Biotinylated nucleotides were</p><p>50 Mencucci et al.</p><p> 2010 The AuthorsJournal compilation 2010 Royal Australian and New Zealand College of Ophthalmologists</p></li><li><p>detected using a streptavidin-horse radish peroxi-dase conjugate. The immunoreaction product wasvisualized using 3,3-diaminobenzidine tetrahydro-chloride as chromogen. For counterstaining ofnuclei, 0.3% (w/v) methyl green was used. Theslides were evaluated and photographed using aNikon Microphot-FXA microscope (Nikon, Tokyo,Japan). The number of apoptotic cells was countedin 15 separate fields for each slide.</p><p>Western blot analysis</p><p>Cornea specimens were incubated with collagenase(0.5 mg/mL) for 20 h at 37C and centrifuged for15 min at 10 000 g. The pellet was washed inphosphate-buffered saline, homogenized in ice-coldlysis buffer (50 mM TrisHCl, pH 7.5, 150 mM NaCl,1 mM EDTA, 1% Triton, 0.25% SDS) supplementedwith a protease inhibitor cocktail (Sigma-Aldrich,St. Louis, MO, USA) and centrifuged for 15 min at4C at 10 000 g. The supernatant was collected, andthe protein concentration was measured using a Coo-massie Bio-Rad protein assay kit. Protein aliquots(30 mg) were diluted in 4 reducing Laemmlissample buffer (250 mM TrisHCl, pH 6.8, 20% glyc-erol, 8% SDS, 20% 2-mercaptoethanol, 0.008% bro-mophenol blue) and loaded onto 10% SDS-PAGE.After SDS-PAGE, proteins were transferred on poly-vinylidene difluoride membranes. The equal proteinloading in each lane was verified with Ponceau Ssolution staining. Then, membranes were blocked1 h at room temperature in 5% BSA-Tween Tris-buffered saline (TTBS) buffer (0.1% Tween-20,20 mM TrisHCl, 150 mM NaCl, pH 7.5), washed inTTBS and incubated at 4C overnight with PARP(poly (ADP-ribose) polymerase) primary policlonalantibody (1 : 1000, H-250, Santa Cruz, CA, USA)diluted in TTBS, followed by peroxidase conjugatedsecondary IgG. The reacted proteins were revealedby the enhanced chemiluminescence system (ECLplus; Amersham Bioscience GE Healthcare, Milan,Italy). Image acquisition and densitometric analysiswere performed with Quantity One software on aChemiDoc XRS instrument (BIO-RAD Laboratories,Hercules, CA, USA).</p><p>Immunohistochemical studies</p><p>Immunohistochemical studies were performed ondeparaffinized and rehydrated sections, as previ-ously described.24 The slides were exposed to hydro-gen peroxide 3% solution to quench endogenousperoxidase activity. Slides were rinsed in tap water,immersed in ethylenediaminetetraacetic acid (pH 8)and microwaved for 20 min at 350 W to enhanceantigen exposure. The monoclonal anti-Ki-67 (1 : 50</p><p>dilution, 7B11; Zymed laboratories San Francisco,CA, USA), anti-CD-34 (1 : 100 dilution, Dakopatts,Carpinteria, CA, USA), anti-collagen type I (1 : 500dilution; COL-1, Sigma-Aldrich), anti-a smoothmuscle actin (1 : 100 diluition; 1A4, Sigma-Aldrich)and polyclonal anti-desmin (1 : 1000; Santa Cruz,CA, USA) antibodies were added to the slides andincubated overnight at 4C. Sections were rinsedin phosphate-buffered saline, incubated with bio-tinylated secondary antibody and then withstreptavidin-biotin peroxidase complex (Ultravisionlarge volume detection system anti-polyvalent,Laboratory-Vision, Fremont, CA, USA). The reac-tion product was developed with the 3,3-diaminobenzidine tetrahydrochloride as chromogen(Sigma-Aldrich). Slides were washed in running tapwater followed by dehydration and coverslipmounting. Controls were performed by processingslides lacking the primary antibody or stained withthe corresponding non-immune serum and counter-stained with haematoxylin. The slides were evalu-ated and photographed using a Nikon Microphot-FXA microscope (Nikon). In the anterior stroma, thecollagen fibres diameter was measured with a com-puterized image analyser programme (Image J1.38) on 15 fields for slide. Computer-assistedquantification of CD34 staining has been made usingAdobe Photoshop 6.0 software (Adobe System Incor-porated, San Jose, CA, USA).25</p><p>Statistical analysis</p><p>Data are expressed as the mean SD. A one-wayANOVA followed by post-hoc test (Bonferroni correc-tion for multiple comparisons) was performed. Thelevel of P &lt; 0.05 was accepted as statistically signifi-cant. Comparisons of percentages were analysedstatistically after conversion through arcsine transfor-mation from the binomial to the normal distribution.</p><p>RESULTS</p><p>The anterior stroma was identified considering 250300 mm from basal membrane of the epithelium.</p><p>Keratocytes</p><p>The apoptosis was evaluated with TUNEL assay andWestern blotting analysis. The light microscopy inthe TUNEL-stained sections showed apoptotic cellsin the anterior stroma of cross-linked and kerato-conic corneas (Fig. 1a). The number of apoptoticcells both in keratoconus (4.75 1.28; n = 4) and incross-linking (4.51 1.52; n = 4) was statisticallysignificantly increased in comparison with con-trol (1.25 0.91; n = 5; P &lt; 0.01 and P &lt; 0.05,</p><p>Effects of cross-linking on human cornea 51</p><p> 2010 The AuthorsJournal compilation 2010 Royal Australian and New Zealand College of Ophthalmologists</p></li><li><p>respectively) (Fig. 1b). Western blotting analysiswas performed using PARP antibody. PARP is aprotein involved in the cell protection from apop-totic stimuli, and its cleavage is considered one ofthe classical characteristics of apoptosis. In fact,during the apoptotic process the full-length activeform (116 kDa) is cleaved to an inactive form(85 kDa) that is not able to protect cells from pro-grammed cell death. As shown in Figure 2a, bothisoforms were present and more expressed in cross-linked and keratoconic specimens compared withcontrol. In particular, the densitometric analysis ofthe bands showed a PARP expression increase of,respectively, 338.8% (438.8% 81.2) in keratoco-nus and 326.4% (426.4% 83) in cross-linkingcompared with control (100% 21; P &lt; 0.01) takenas 100 (Fig. 2b).</p><p>The keratocyte proliferation was evaluated withKi-67 staining. Almost no Ki-67-positive cells weredetected in the control corneas and only ra...</p></li></ul>