corneal nerves in refractive surgery and dry eye
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Department of Ophthalmology
University of Helsinki
Helsinki, Finland
CORNEAL NERVES IN REFRACTIVE
SURGERY AND DRY EYE
ILPO S. TUISKU
Academic Dissertation
To be publicly discussed,
with the permission of the Medical Faculty of the University of Helsinki,
in Lecture Hall 2 of Biomedicum, Haartmaninkatu 8,
Helsinki, on February 22nd, 2008, at 12 noon.
Helsinki 2008
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Supervised by:
Professor Timo Tervo
Department of Ophthalmology
University of Helsinki
FINLAND
Reviewed by:
Professor Hannu Uusitalo
Department of Ophthalmology
University of Tampere
FINLAND
and
Professor MarjaLiisa Vuori
Department of Ophthalmology
University of Turku
FINLAND
Opponent:
Professor Friedrich Kruse
Department of Ophthalmology
University of ErlangenNürnberg
GERMANY
© Ilpo S. Tuisku
ISBN 9789529233076 (paperback)
ISBN 9789521045004 (PDF)
Yliopistopaino
Helsinki 2008
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To Johanna, Tom, and Matias
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CONTENTS
LIST OF ORIGINAL PUBLICATIONS 6
ABBREVIATIONS 7
ABSTRACT 9
INTRODUCTION 10
REVIEW OF THE LITERATURE 11
1. Corneal structure 11
2. Corneal innervation and sensitivity 13
3. Neurosecretorial regulation of tearing 15
4. Tear fluid cytokines 16
5. Corneal wound healing 17
6. Antigenpresenting cells 20
7. Excimer laser 20
8. Photorefractive keratectomy – PRK 218.1 Nerve regeneration after PRK 228.2 Corneal sensitivity after PRK 23
9. Laser in situ keratomileusis – LASIK 239.1 Nerve regeneration after LASIK 249.2 Corneal sensitivity after LASIK 25
10. Dry eye 27
11. Dry eye after refractive surgery 29
12. Sjögren’s syndrome 3012.1 Neurological manifestations in Sjögren’s syndrome 31
AIMS OF THE STUDY 32
SUBJECTS AND METHODS 33
1. Subjects 331.1 PRK patients 331.2 LASIK patients and controls 331.3 Primary Sjögren’s syndrome patients and controls 34
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2. Methods 352.1 Clinical examination 352.2 Tear fluid analysis 362.3 In vivo confocal microscopy – IVCM 382.4 Noncontact esthesiometry 392.5 PRK 402.6 LASIK 402.7 Statistical analyses 41
RESULTS 42
1. Tear fluid cytokines, haze, and nerve regeneration after PRK 421.1 Tear fluid cytokines after PRK 421.2 IVCM three months after PRK 421.3 Correlations between IVCM data and cytokines 44
2. Dry eye and corneal sensitivity after high myopic LASIK 442.1 Subjective symptoms 442.2 Objective dry eye tests 442.3 Corneal sensitivity 45
3. Corneal morphology and sensitivity in primary Sjögren’s syndrome 453.1 Subjective symptoms 453.2 Objective dry eye tests 453.3 Corneal sensitivity 463.4 IVCM in primary Sjögren’s syndrome 46
DISCUSSION 48
1. Tear fluid cytokines and haze after PRK 48
2. Nerve regeneration after PRK 48
3. Corneal sensitivity after high myopic LASIK 50
4. Corneal sensitivity in primary Sjögren’s syndrome 52
5. Corneal nerves in primary Sjögren’s syndrome 53
SUMMARY AND CONCLUSIONS 57
ACKNOWLEDGMENTS 58
REFERENCES 60
ORIGINAL PUBLICATIONS 74
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LIST OF ORIGINAL PUBLICATIONS
This dissertation is based on the following original publications, which are referred to in the text
by Roman numerals I – IV:
I Ilpo S. J. Tuominen, Timo M. T. Tervo, AnnaMaija Teppo, Tuuli U. Valle, Carola
GrönhagenRiska and Minna H. Vesaluoma. Human tear fluid PDGFBB, TNF
and TGF 1 vs corneal haze and regeneration of corneal epithelium and subbbasal
nerve plexus after PRK. Exp. Eye Res. 2001;72:631–641.
II Ilpo S. Tuisku, Nina Lindbohm, Steven E. Wilson, Timo M. Tervo. Dry eye and
corneal sensitivity after high myopic LASIK. J. Refract. Surg. 2007;23:338342.
III Ilpo S. J. Tuominen, Yrjö T. Konttinen, Minna H. Vesaluoma, Jukka A.O.
Moilanen, Maaret Helintö, Timo M. T. Tervo. Corneal innervation and morphology
in primary Sjögren’s syndrome. Invest. Ophthalmol. Vis. Sci. 2003;44:25452549.
IV Ilpo S. Tuisku, Yrjö T. Konttinen, Liisa M. Konttinen, Timo M. Tervo. Alterations
in corneal sensitivity and nerve morphology in patients with primary Sjögren’s
syndrome. Submitted 2007.
These publications have been reprinted with the kind permission of their copyright holders. In
addition, some unpublished material is presented.
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ABBREVIATIONS
APC Antigenpresenting cellArF Argon fluorideARVO Association for Research in Vision and OphthalmologyBSCVA Best spectaclecorrected visual acuityBM Basal membraneBUT Tear breakup timeCCK CholecystokininCGRP Calsitonin generelated peptideCMTF Confocal microscopy through focusingD DiopterDC Dendritic cellDLK Diffuse lamellar keratitisECM Extracellular matrixEGF Epidermal growth factorExcimer Excited dimerFas Fas (Apo95) receptorFasL FasligandFML FluorometholoneGAL GalaninHGF Hepatinocyte growth factorIGF Insulinlike growth factorIL InterleukinIVCM In vivo confocal microscopykDa kiloDaltonKGF Keratinocyte growth factorLASIK Laser assisted in situ keratomileusisLINE LASIKinduced neurotrophic epitheliopathyMENK MethionineenkephalinNCE Noncontact esthesiometerNFB Nerve fiber bundleNGF Nerve growth factorNPY Neuropeptide YOSDI Ocular surface disease indexPACAP Pituitary adenylate cyclaseactivating polypeptidePDGF Plateletderived growth factorPRK Photorefractive keratectomypSS Primary Sjögren’s syndromeSP Substance PSS Sjögren’s syndromesSS Secondary Sjögren’s syndromeTGF Transforming growth factor TNF Tumor necrosis factor – Trk Tyrosine kinaseTSCM Tandem scanning confocal microscopyUCVA Uncorrected visual acuityVAS Visual analog scaleVIP Vasoactive intestinal polypeptide
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ABSTRACT
This study aimed to investigate the morphology and function of corneal sensory nerves in 1)patients after corneal refractive surgery and 2) patients with dry eye due to Sjögren’s syndrome.A third aim was to explore the possible correlation between cytokines detected in tears anddevelopment of postPRK subepithelial haze. The main methods used were tear fluid ELISAanalysis, corneal in vivo confocal microscopy, and noncontact esthesiometry.
The results revealed that after PRK a positive correlation exists between the regeneration ofsubbasal nerves and the thickness of regenerated epithelium. Pre or postoperative levels of thetear fluid cytokines TGF 1, TNF , or PDGFBB did not correlate with the development ofcorneal haze objectively estimated by in vivo confocal microscopy 3 months after PRK. Afterhigh myopic LASIK, a discrepancy between subjective dry eye symptoms and objective signsof dry eye was observed. The majority of patients reported ongoing dry eye symptoms even 5years after LASIK, although no objective clinical signs of dry eye were apparent. In addition,no difference in corneal sensitivity was observed between these patients and controls. PrimarySjögren’s syndrome patients presented with corneal hypersensitivity, although their cornealsubbasal nerve density was normal. However, alterations in corneal nerve morphology (nervesprouting and thickened stromal nerves) and an increased number of antigenpresenting cellsamong subbasal nerves were observed, implicating the presence of an ongoing inflammation.
Based on these results, the relationship between nerve regeneration and epithelial thickness 3months after PRK appears to reflect the trophic effect of corneal nerves on epithelium. Inaddition, measurement of tear fluid cytokines may not be suitable for screening patients for riskof scar (haze) formation after PRK. Presumably, at least part of the symptoms of “LASIKassociated dry eye” are derived from aberrantly regenerated and abnormally functioning cornealnerves. Thus, they may represent a form of corneal neuropathy or “phantom pain” rather thanconventional dry eye. Corneal nerve alterations and inflammatory findings in Sjögren’ssyndrome offer an explanation for the corneal hypersensitivity or even chronic pain orhyperalgesia often observed in these patients. In severe cases of disabling chronic pain inpatients with dry eye or after LASIK, when conventional therapeutic possibilities fail to offerrelief, consultation of a physician specialized in pain treatment is recommended.
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INTRODUCTION
The cornea is an avascular and delicate optically transparent tissue. Its transparency ismaintained by metabolically active pump systems located mainly in the endothelium. Cornealsensory nerves also have a crucial role in maintaining corneal architecture and transparency.The cornea is one of the most densely innervated peripheral tissues in humans. Corneal nervesinterfere with epithelial mitotic activity and proliferation, and presumably also with theregulation of keratocyte function. Secretion of tear fluid is also regulated by the corneal nervesforming the afferent loop of the cornealacrimal gland reflex arc. Several systemic and cornealdisorders as well as ocular surgery may damage the corneal nerves and impair their function.Consequently, the regulation of epithelial cell and keratocyte metabolism and also tearformation and corneal sensitivity are disturbed until innervation is restored.
Corneal refractive surgery (PRK and LASIK) severs corneal sensory nerves, impairing cornealsensitivity and function. Corneal nerve regeneration begins shortly, but the original fine nervearchitecture may never be completely restored. Patients experience varying degrees of dry eyesymptoms for a 1 to 6month period following refractive surgery. Occasionally, extensive dryeye symptoms or even ocular pain may persist for years, with or without dry eye signs. On theother hand, patients may have clinical signs compatible with dry eye, but present with minimalor no symptoms.
Dry eye is a common external eye disease that arises from a wide variety of etiologies.Sjögren’s syndrome (SS) is a systemic autoinflammatory disorder of unknown etiology, and dryeye is one of its major manifestations. Primary Sjögren’s syndrome (pSS) occurs without anyassociation with other rheumatological diseases. Consequently, it represents a relativelyhomogeneous group of patients.
Patients presenting with symptoms compatible with dry eye, but with minimal or no dry eyesigns found on ocular examination are relatively frequently seen after refractive surgery. Thegoal of this thesis was to explore the reasons behind this clinical discrepancy by investigatingcorneal nerves and sensitivity in patients after refractive surgery and in patients with SS.
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REVIEW OF THE LITERATURE
1. Corneal structure
The cornea is an avascular tissue accounting for most of the refractive power of the human eye.Due to its transparency, it allows light to enter the eye and to be projected to the retina.According to a recent metaanalysis, the average thickness of the central cornea is 534 µmcentrally, although values are affected by measurement techniques: for slitlampbased opticalpachymetry, the mean central corneal thickness is 530 m, and for ultrasonic pachymetry 544
m (Doughty and Zaman 2000). The normal cornea is thinnest in the center and thickestperipherally, measuring approximately 650 m.
Anatomically, the cornea can be divided into five sublayers: 1. epithelium, 2. Bowman’s layer,3. stroma or substantia propria, 4. Descemet’s membrane, and 5. endothelium.
The epithelium consists of 57 layers of nonkeratinized squamous epithelial cells, measuring50–60 m in thickness. Three morphological cell types are present in human cornealepithelium: superficial epithelial cells, intermediate wing cells, and the innermost basalepithelial cells (Ehlers 1970). The corneal epithelium is a dynamic tissue in which cells areconstantly renewed and lost; nevertheless, the total mass is kept steady by mechanisms not yetfully elucidated. A strong body of evidence suggests that corneal epithelial cells arise fromlimbal stem cells (reviewed by Tseng 1989 and Dua et al. 2000). Once inside the cornea, cellsslowly move towards the apex in a centripetal fashion; this is supported by the XYZ theory byThoft and Friend (1983) as well as by various clinical observations (Bron 1973, Kaye 1980,Lemp and Mathers 1989). Evidence for the XYZ theory has also been gained from in vivoconfocal microscopy studies (Auran et al. 1995) and experimental in vivo studies on transgenicmice (Nagasaki and Zhao 2003).
Superficial epithelial cells, which form a barrier against foreign substances, are connected toeach other by tight junctions and desmosomes. They have numerous microvilli and microplicaeon their surfaces that increase the adherence of tear fluid mucins. The life span of surfaceepithelial cells is only a few days, after which they are shed in tear fluid. The intermediate wingcells form 2–3 layers beneath the surface epithelium and have winglike extensions. The basalepithelial cells form a monolayer of cells anchored tightly to the basement membrane with theaid of hemidesmosomes and different anchoring fibrils (Gipson et al. 1989). Basal epithelialcells have limited capacity to divide before terminal differentiation, thus being partly
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responsible for the excellent capability of the epithelium to regenerate (Hanna and O’Brien1960, Kruse et al. 1990, reviewed by Li et al. 2007).
Bowman’s layer was first described by Sir William Bowman at the London OphthalmicHospital (Moorfields) in 1847. Bowman’s layer separates the epithelium from the stroma,acting as an anterior limiting membrane and giving tensile strength to the cornea. In Bowman’slayer, individual collagen fibrils are interwoven densely to form a feltlike sheet. The thicknessof this acellular layer is 8 12 m (Komai and Ushiki 1991).
The stroma, or substantia propria, consists of keratocytes, extracellular matrix, and nerve fibers.The stroma constitutes the largest portion of the cornea; its thickness is approximately 470 m.Three hundred to five hundred collagen lamellae running from limbus to limbus are oriented atprecise angles with respect to adjacent lamellae, contributing to corneal transparency andstrength (Komai and Ushiki 1991, reviewed by Ihanamäki et al. 2004). Stromal collagenlamellae (consisting of types I, III, V, XII, and XIII) are surrounded by several proteoglycansresponsible for proper spacing of collagen and stromal hydration (reviewed by Ihanamäki et al.2004).
Descemet’s membrane dividing the stroma from the endothelium acts as a basement membranefor the endothelium. The thickness of Descemet’s membrane gradually increases from birth (3µm) to adulthood (810 m) (Johnson et al. 1982). The endothelium is a monolayer of 5 mthick cells and is the innermost layer of the cornea, functioning in fluid pumping and regulationof corneal hydration. Endothelial cells are not known to be capable of dividing, thus lackingmitotic activity (Nishida 2005).
Figure 1. Schematic drawing of cornea: 1) epithelium, 2) subbasal nerve plexus and Bowman’s layer, 3)stromal nerves, 4) stroma, and 5) Descemet’s membrane and endothelium.
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2. Corneal innervation and sensitivity
Corneal afferent sensory neurons are derived from the Gasserian ganglion and enter the eye ballvia long ciliary nerves, which are branches of the nasociliary portion of the ophthalmic divisionof the trigeminal nerve. In some cases, the inferior cornea receives additional innervation fromthe maxillary branch of the trigeminal nerve (Zander and Weddel 1951, Ruskell 1974, Rozsaand Beuerman 1982). Long ciliary nerves are myelinated until they penetrate the limbus andform nerve bundles surrounded only by the Schwann’s cells (Zander and Weddel 1951). Theabsence of myelin on central corneal axons is essential for maintaining corneal transparency. Inthe human cornea, thick nerve trunks move from the periphery below the anterior third of thestroma due to organization of collagen lamellae (Muller et al. 2001, Radner and Mallinger2002). Nerve fibers run forward in a radial fashion towards the center of the cornea andpenetrate Bowman’s layer, then turning abruptly and continuing parallelly to the cornealsurface, simultaneously losing their Schwann’s cell ensheathment (Schimmelpfennig 1982,Muller et al. 1996). Nerve fibers form a network by branching both vertically and horizontallybetween Bowman’s layer and basal epithelial cells; this network is called the subbasal nerveplexus (Muller et al. 1997). Nerve terminals are then sent between epithelial cells (Rozsa andBeuerman 1982), electromicroscopic studies have shown evidence that nerve terminalsinvaginate both basal epithelial cells and wing cells (Muller et al. 1996). Beaded nerve fibers,with a diameter of approximately 2 µm, contain many mitochondria and glycogen, indicatingactive metabolism, and are thought to also contain neuropeptides (Muller et al. 1996). Most ofthe corneal neurons are classified as Ctype, with a conducting velocity less than 2 m/s, and therest are myelinated A type axons, typically with conduction velocities between 2 and 15 m/s(Belmonte and Tervo 2005).
Figure 2. After the stromal nerves have penetrated Bowman’s layer, they run parallel to the cornealsurface between the basal epithelium and Bowman’s layer, forming a neural network called the subbasalplexus. 1) Wing cells 2) basal epithelial cells, and 3) Bowman’s layer.
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Several neural transmitters and neuropeptides have been demonstrated in the corneal nerves.Substance P (SP) (Tervo et al. 1982), calcitonin generelated peptide (CGRP) (Stone et al.1986), and galanin (GAL) (Jones and Marfurt 1998) are of sensory origin. Catecholamines(Toivanen et al. 1987) and neuropeptide Y (NPY) (Jones and Marfurt 1998) are of sympatheticorigin, and galanin (GAL) (Jones and Marfurt 1998), cholecystokinin (CCK) (Stone et al.1984), vasoactive intestinal peptide (VIP) (Jones and Marfurt 1998), and methionineenkephalin(MENK) (Jones and Marfurt 1998) are of parasympathetic origin.
Development of modern corneal imaging techniques, such as in vivo confocal microscopy(IVCM), has enabled corneal nerves in living corneas to be analyzed. IVCM allowsvisualization of the subbasal nerve plexus, and is also used in imaging normal corneas(OliveiraSoto and Efron 2001, Grupcheva et al. 2002 and Patel et al. 2005), patients after PRK(Heinz et al. 1996, Kauffmann et al. 1996, Linna and Tervo 1997, Bohnke et al. 1998, Frueh etal. 1998, Moilanen et al. 2003, Erie et al. 2005b), patients after LASIK (Kauffmann et al. 1996,Linna et al. 2000, Lee et al. 2002, Calvillo et al. 2004, Bragheeth and Dua 2005, Erie et al.2005b), patients with herpetic keratitis (Rosenberg et al. 2002), patients with keratoconus (Pateland McGhee 2006), patients with diabetes mellitus (Rosenberg et al. 2000b), and patients withvarious corneal dystrophies, e.g. epithelial basement membrane dystrophy (Rosenberg et al2000a), lattice dystrophy type II (Rosenberg et al. 2001), cornea plana (Vesaluoma et al. 2000),and Fuchs’ dystrophy (Mustonen et al. 1998). Patel et al. (2005) have developed a noveltechnique to elucidate the overall distribution of subbasal nerves in the human cornea by laserscanning in vivo confocal microscopy. Multiple images are obtained from several locations inthe cornea, and overlapping images are later reconstructed to confluent montages. With the aidof this novel technique, they observed that subbasal nerves were settled in a whorllike pattern,similar to that seen in the epithelium in corneal verticillata. On the basis of their observations,they hypothesized that epithelial cells and nerves would migrate centripetally in tandem (Patelet al. 2005).
Normal corneal sensitivity is higher in the center of the cornea than at the periphery (Millodotand Larson 1969). Accordingly, the central cornea is 56 times more densely innervated thanthe peripheral cornea (Muller et al. 1997). The cornea is one of the most densely innervatedperipheral tissues in humans; the nerve density is estimated to be 300–400 times higher than in,for example, the human finger (Rozsa and Beurman 1982, reviewed by Muller et al. 2003).Based on electrophysiological studies, different functional types of sensory nerve fibers exist inthe cornea. The majority of corneal nerves are polymodal nociceptors (70%), which areactivated by nearnoxious mechanical energy, heat, chemical irritants, and a large variety ofendogenous chemical mediators. Mechanonociceptors, accounting for 1520% of cornealperipheral axons, respond only to coarse mechanical forces in the order of magnitude close tothat required to damage corneal epithelial cells. Coldsensitive thermal axons accounting for the
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remaining 1015% of corneal peripheral axons, respond by increasing their firing rate when thecorneal temperature falls below normal levels (reviewed by Belmonte et al. 2004, Belmonte andTervo 2005). Several corneal diseases, corneal dystrophies, and ocular surgery may cause adecline in corneal sensitivity (reviewed by Muller et al. 2003). In addition, corneal sensitivityhas been reported to diminish with increasing age (Millodot 1977), although the density andorientation of the subbasal nerves seem to be unaffected (Erie et al. 2005a).
Corneal sensitivity can be tested in the clinical setting by gently touching the ocular surfacewith a wisp of cotton and observing the blink reflex or by comparing the subjective sensationwith that evoked from the other eye. This information is easily obtained in clinical settings.However, the results are not quantified and represent only an approximation of the functionalstatus of corneal nerves. The CochetBonnet esthesiometer represents a more quantitativeapproach and uses a calibrated nylon hair of variable length. It measures mainly coarsemechanical sensation and cannot discriminate between mechanical, thermal and chemicalsensations. Moreover, it has a limited ability to accurately measure corneal sensitivity at lowstimulus thresholds compared with modern noncontact gas esthesiometers (Murphy et al. 1998).The noncontact gas esthesiometer uses an air jet of adjustable flow and temperature that maycontain CO2 in a variable concentration to reduce local pH. This allows mechanical, thermal, orchemical stimulation of a specific limited area of the cornea (Belmonte et al. 1999). Thenoncontact gas esthesiometer is more accurate and has better repeatability than the CochetBonnet esthesiometer (Murphy et al. 1998).
3. Neurosecretorial regulation of tearing
Main lacrimal glands are innervated by parasympathetic and sympathetic nerves (Botelho et al.1966, Sibony et al. 1988). In addition, scarce sensory nerves have been identified in lacrimalglands (Botelho et al. 1966). Nerves are located in close proximity to acinar, ductal, andmyoepithelial cells as well as to blood vessels (Botelho et al. 1966, Sibony et al. 1988).
Stimulation of the lacrimal gland and secretion occur via the cornea – trigeminal nerve –brainstem – facial nerve – lacrimal gland reflex arc. Afferent sensory nerves of the cornea andconjunctiva are activated by stimuli to the ocular surface. Efferent parasympathetic andsympathetic nerves are then activated to stimulate secretion from acinary and tubular cells(Botelho 1964). Neurotransmitters and neuropeptides released by lacrimal gland nerves includeacetylcholine (Botelho 1964), vasoactive intestinal peptide (VIP) (Uddman et al. 1980),neuropeptide Y (NPY) (Tsukahara and Jacobowitz 1987), substance P (Nikkinen et al. 1984),and calcitonin generelated peptide (CGRP) (Tsukahara and Jacobowitz 1987).
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While regulation of tearing is under tight neural control, a loss of innervation in the inflamedlacrimal gland has been suggested to explain associated decreased secretory function in SS dryeye (Hakala and Niemelä 2000). Accordingly, lymphocytic infiltration of lacrimal glands wasassociated with decreased reflex tearing (Schirmer’s test II with nasal stimulation) in dry eye(Tsubota et al. 1996). However, several studies have shown that viable nerves do exist innonfibrotic areas of lacrimal glands, while they are absent in fibrotic areas (Zoukhri et al. 1998aand Zoukhri et al. 1998b). Hence, the lack of lacrimal gland secretion in SS cannot be explainedby a loss of neural support. Zoukhri and Kublin (2001) found that remaining nerves were unableto release their neurotransmitters and this also correlated with the lack of lacrimal gland proteinsecretion. In conclusion, autonomic nerves in the lacrimal gland are not lost in SS dry eye, butthe inability to release their neurotransmitters seems to play an important role in impairedlacrimal gland secretion.
4. Tear fluid cytokines
Corneal cells are known to express different cytokines and/or their receptors potentiallymodulating wound healing (Wilson et al. 1992, 1994a, 1994b, 1996a, Li and Tseng 1996).
The transforming growth factor (TGF) – family, consisting of TGFβ1, TGFβ2, and TGFβ3,are polypeptides of approximately 25 kDa (Kokawa et al. 1996). TGF is generally thought toinhibit epithelial, endothelial, and leukocyte cell growth and to stimulate proliferation offibroblasts (Song et al. 2002). TGFβ2 seems to be the major isoform present in all types ofcorneal cells (Nishida et al. 1995), but TGFβ1 has also been detected in small amounts in allcorneal layers (Wilson et al. 1992, Nishida et al. 1995). In addition, TGFβ1 and 2 have beenshown to be present in human tear fluid (Gupta et al. 1996, Kokawa et al. 1996, Vesaluoma etal. 1997a). TGFβ1 and 2 are produced and TGFβ1 secreted by the human lacrimal gland,suggesting that the lacrimal gland may be one source of TGFβ in human tear fluid (Yoshino etal. 1996). TGF β 1 is involved in the regulation of keratocyte activation, myofibroblasttransformation, proliferation, chemotaxis, and wound healing after refractive surgery, and it isstrongly associated with excessive scarring (Jester et al. 1996, 1999, Andresen et al. 1997,Myers et al. 1997, Andresen and Ehlers 1998, MollerPedersen et al. 1998b, Jester et al. 2003).
Plateletderived growth factor (PDGF) is a cysteine knotcontaining dimer of 35 kDa composedof an A and B chain. It exists as isomers PDGFAA, PDGFAB, and PDGFBB (reviewed byJones and Kazlauskas 2001). PDGFBB is produced by corneal epithelial cells and is bound athigh levels in the epithelial basement membrane (reviewed by Wilson et al. 2001). PDGFreceptors are found in corneal fibroblasts (Li and Tseng 1996). PDGFBB has also been shown
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to have mitogenic, chemotactic, and migratory effects on corneal fibroblasts in vitro(Hoppenreijs et al. 1993, Andresen et al. 1997, Andresen and Ehlers 1998, Kamiyama et al.1998, Kim et al. 1999, Jester et al. 2002).
Tumor necrosis factorα (TNFα), on the other hand, is an inducer of apoptosis, which issupposed to play an important role in the first steps of corneal wound healing after PRK(Wilson et al. 1996a, 1996b, Helena et al. 1998). TGFβ1, TNFα, and PDGFBB are present inlow concentrations in human tear fluid, and PRK induces an increased release of TGFβ1, TNFα, and PDGFBB during the early days of wound healing (Gupta et al. 1996, Vesaluoma et al.1997a, 1997b, 1997c).
5. Corneal wound healing
Corneal wound healing is a complex physiologic sequence of events that contributes to reestablishment of normal function and clarity of the cornea. Interindividual variations in thewound healing process after all keratorefractive procedures are the major determinants ofsurgical outcome. The wound healing process plays a critical role in overcorrection,undercorrection, regression, and other complications as well as in corneal haze and refractiveinstability. Major challenges of corneal refractive surgery are to control the wound healingprocess more precisely and to promote more tissue regeneration than tissue fibrosis (Stramer etal. 2003, reviewed by Fini and Stramer 2005).
Interaction between the corneal epithelium and stroma is a wellknown phenomenon and acritical step in corneal wound healing. Removal of the epithelium induces disturbances in ATPcontent of anterior keratocytes (Herrman and Lebeau 1962), and the anterior stroma beneath theepithelial wound becomes acellular within the following 24 h (Nakayasu 1988, Campos et al.1994). Initially, the keratocyte loss was thought to be the result of mechanical trauma orosmotic changes (Dohlman et al. 1968). However, more recently, the loss of keratocytesfollowing epithelial removal has been shown to be mediated primarily by apoptosis (Wilson etal. 1996a, 1996b, Gao et al. 1997, Helena et al. 1998, Kim et al. 1999). Among others,interleukin – 1 (IL1 ) and TNF are released from the injured corneal epithelium and induceautocrine suicide in keratocytes by activating the Fas / Fasligand system (Wilson et al. 1996b,Mohan et al. 1997, reviewed by Wilson et al. 2001).
After stromal keratocyte loss due to apoptosis, stromal cells adjacent to the wound area becomeactivated and proliferate. The activated keratocytes migrate to the wound area (Hanna et al.1989, Del Pero et al. 1990, Zieske et al. 2001), and five days after wounding, these migrated
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keratocytes in the wound area undergo subsequent rounds of cell division and repopulate thewound area (Zieske et al. 2001).
Table 1. Characteristic differences in activated keratocytes.
FibroblastsMyofibroblasts /
Altered keratocytes
Presence in epithelial abrasions +
Presence in wounds penetrating Bowman’s layer + +
Association with wound fibrosis +
Biomechanical activity less active active
Expression of smooth muscle actin +
Expression of collagens, fibronectin, and other matrixproteins +
Expression of MMPs upregulated downregulated
The extent of the initial wound, especially whether or not the epithelial basement membrane isdisrupted, results in different phenotypes of activated keratocytes: fibroblasts or myofibroblasts/altered keratocytes (Table 1). Wounds that penetrate the basement membrane, such askeratectomy, lead to the presence of myofibroblasts in the wound area, while after pureepithelial abrasions with an intact basement membrane, myofibroblast are not present (MollerPedersen et al. 1998b, Zieske et al. 2001). Fibroblasts are less biomechanically active,synthesize matrix metalloproteinases (WestMays et al. 1997, BargagnaMohan et al. 1999),and are associated with corneal ulceration (Riley et al. 1995, Hargrave et al. 2002).Myofibroblasts, by contrast, are associated with wound fibrosis, synthesize collagen,fibronectin, and other matrix proteins (Ohji et al. 1993, Jester et al. 1996), and havedownregulated matrix metalloproteinase production (Girard et al. 1991, Fini et al. 1995, WestMays et al. 1999). Myofibroblasts express smooth muscle actin and have welldevelopedfocal adhesions, while fibroblasts do not express smooth muscle actin and have poor focaladhesions (Jester et al. 1995, 1996, Petridou et al. 2000). Myofibroblasts have strongbiomechanical involvement in matrix organization and wound contraction. Excess proliferationof stromal fibroblasts and myofibroblasts results in stromal hyperplasia and clinical haze,affecting optical transparency of the cornea (MollerPedersen et al. 1998a, 1998b). TGFseems to be the most essential cytokine in orchestrating the transformation process ofmyofibroblasts (Jester et al. 1997, 2003). However, in the cornea, the synergistic function of
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TGF and PDGF via integrin signaling pathways is needed for myofibroblast transformation(Jester et al. 2002).
The epithelial healing process requires epithelial cell proliferation, migration, anddifferentiation. The combination of cytokines and growth factors present in corneal cells andtear fluid regulates the early epithelial wound healing cascades. Paracrine cytokine trafficking inthe human cornea presents a unique stromal / epithelial interaction (Wilson et al. 1994a).
Cytokines responsible for epithelial proliferation are keratinocyte growth factor (KGF),hepatocyte growth factor (HGF), and epidermal growth factor (EGF) (Wilson et al. 1993,1994b, 1994c, Tervo et al. 1997). Epithelial proliferation is further stimulated by increasedlevels of PDGFBB (Li and Tseng 1997). Elevated production of KGF and HGF by keratocytesis present up to 7 days after corneal injury (Wilson et al. 1999a). In addition, increased reflextear fluid production results in higher availability of HGF (Tervo et al. 1997), PDGFBB(Vesaluoma et al. 1997c), and EGF (van Setten 1990). Enhanced expression of mRNA for EGFand HGF is present in the lacrimal gland after wounding, contributing to greater availability intear fluid (Wilson et al. 1999b). In addition, mRNAs for EGF and HGF receptors areconcurrently upregulated in the epithelium and keratocytes (Wilson et al. 1999a).
Migration of epithelial cells at the wound periphery is induced by HGF (Li and Tseng 1997)and EGF (Wilson et al. 1994c) on a provisional fibronectin matrix that accumulates around thewound (Maldonado and Furcht 1995). Fibronectin is not present in the normal corneal epithelialbasement membrane, but 8 h after wound healing it is detected in the epithelium (Fujikawa etal. 1984). Endogenously produced fibronectin by corneal epithelial cells promotes cell adhesion(Ohji et al. 1993). Elevated levels of fibronectin are also found in human tear fluid after PRK,promoting epithelial cell migration and attachment (Virtanen et al. 1995).
Once epithelial confluence is achieved, the epithelial cells are triggered to proliferate andsequentially differentiate to form a normal corneal stratified epithelium (Wilson et al. 1994c).Normal composition of the basement membrane is achieved by restoring collagens andlaminins, which occurs when the fibronectin matrix disappears (reviewed by Suzuki et al.2003).
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6. Antigenpresenting cells
In 1867, Engelman noted dendritic or polygonal cells in the corneal epithelium. One year later,a medical student Paul Langerhans observed dendritic cells in the skin epidermis. Similaritiesbetween corneal dendritic cells and Langerhans cells of the skin have since been proposed. Thecornea is an immune privileged tissue and has long been considered to lack antigenpresentingcells (APC) (Gillette et al. 1982). Today, APCs are known to play a critical role in cornealimmunology in both health and disease (Hamrah et al. 2002, Rosenberg et al. 2002, Hamrah etal. 2003a, 2003b, Zhivov et al. 2005, 2007, Mastropasqua et al. 2006).
Two distinct phenotypic populations of APCs in the normal cornea have been reported.Immature APCs, which do not express MHC class II antigen on their surface, tend to be locatedin the central corneal epithelium. Immature APCs have a large cell body with only a few shortprocesses, if any (Hamrah et al. 2003a). These APCs are able to capture the antigen, but areunable to present to lymphocytes. Mature APCs, by contrast, have a slender nucleated cell bodyfrom which mazes of long membrane processes extend that resemble dendrites of nerve cells.Mature APCs, expressing MHC class II antigen, are found in the peripheral corneal and limbalepithelium (Hamrah et al. 2002, 2003a, 2003b).
The evolution of corneal in vivo imaging techniques has provided new data regarding humancorneal APCs in living tissue. The density of APCs declines from the limbus to the center ofhealthy corneas (Hamrah et al. 2002, Zhivov et al. 2005, 2007, Mastropasqua et al. 2006). In thecorneal limbal epithelium, dendritic cells are found in virtually every healthy subject, whileonly 20–30% of healthy controls show APCs in the central cornea (Zhivov et al. 2005, 2007,Mastropasqua et al. 2006). More often APCs are present in the peripheral cornea, where theyshow signs of a mature phenotype with long slender dendritic processes, whereas immatureAPCs without dendrites typically predominate in the central cornea (Zhivov et al. 2005). Mostof the APCs are located at the level of subbasal cells or among the subbasal nerve plexus(Zhivov et al. 2005).
7. Excimer laser
The excimer laser used to sculpt the cornea has been the single most important advancement inthe field of refractive surgery. Excimer laser keratectomy involves remodeling the cornealstroma by tissue removal. The advantage of the excimer laser is its ability to remove tissue witha microscopic precision unattainable with other procedures.
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Excited dimers are molecules with bound upper states and weakly bound ground states. Thereaction of an excited rare gas atom with a halogen atom produces excited dimers that emitultraviolet radiation when decaying from the bound upper state to the rapidly dissociatingground state. Trokel et al. (1983) demonstrated that farUV laser emissions (between 150 and200 nm) can precisely remove corneal tissue without apparent thermal trauma to the adjacenttissue. According to several experimental studies, the 193 nm UV light from the argon fluoridelaser was established as the optimal wavelength with the least corneal transmission. The highenergy UV photons emitted by the argon fluoride laser caused less adjacent thermal trauma andcreated smoother ablation than longer wavelength lasers (Trokel et al. 1983).
8. Photorefractive keratectomy – PRK
Photorefractive keratectomy (PRK) is based on the use of excimer laser for accurate removaland resculpturing of corneal tissue in order to change the corneal curvature and refractive powerof the cornea. Before excimer laser photoablation, the epithelium must be removed eithermechanically with a blunt spatula or brush, or by excimer laser (Trokel et al. 1983, Seiler andWollensak 1991, Palllikaris et al. 1994, Gimbel et al. 1995).
Figure 3. Myopic PRK. After the epithelium is removed either mechanically or by excimer laser,Bowman’s layer, subbasal nerves, and the anterior stroma, including stromal nerves, are photoablatedby excimer laser. The depth of ablation depends on the magnitude of dioptric correction. Accordingly,stromal nerves are destroyed to a variable extent depending on ablation depth.
Clinical studies have shown relatively good safety, predictability, and refractive results in lowto moderate myopia (Seiler and Wollensak 1991, Gartry et al. 1992, McDonald et al. 1999).However, deeper ablation depths and higher corrections produce a greater incidence ofpostoperative haze and regression and less predictable refractive results (Seiler et al. 1992, Shahet al. 1998).
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8.1 Nerve regeneration after PRK
PRK photoablates Bowman’s layer, subbasal nerves, and anterior stromal nerves in the ablationzone, leaving sharply cut nerve trunks at the base and margin of the wound (Tervo et al. 1994,Trabucchi et al. 1994). The degree of stromal nerve destruction depends on the requiredablation depth. Considerable data exists on corneal nerve regeneration after PRK, includingboth experimental and human studies done by in vivo confocal microscopy (IVCM).
Experimental studies using histochemical methods have shown that nerve regeneration afterPRK is a biphasic process (Rozsa et al. 1983, Beuerman and Rozsa 1984, Tervo et al. 1994). Inthe first phase, the reinnervated subbasal plexus is formed by fine neurites that originate fromthe cut peripheral nerve plexus and extend centrally alongside the migrating epithelial cells(Rozsa et al. 1983, Beuerman and Rozsa 1984, Tervo et al. 1994). Neurofilamentimmunoreactivity can be observed as early as 24 h after the procedure (Trabucchi et al. 1994).The second phase of nerve regeneration is initiated by the degeneration of woundorientedneurites and the concomitant appearance of a second generation of stromal neurites thatultimately reestablish a new subbasal plexus (Rozsa et al. 1983, Beuerman and Rozsa 1984).The secondphase neurites originate from the transected stumps of stromal nerves at the woundbase and the wound margin (Rosza et al. 1983). The regenerating stromal nerves reach theepithelium and contribute to the formation of a new subbasal plexus 6 months after PRK (Tervoet al. 1994, Trabucchi et al. 1994). Trabucchi et al. (1994) observed a regenerated nerve plexusat 1–4 months after surgery that actually appeared thicker than normal. Ishikawa et al. (1994)noted a transient increase in intraepithelial nerves after PRK, the density returning to normallevels by about 7 months after surgery. Normal density of intraepithelial nerve endings wasreached 3 months after PRK (Tervo et al. 1994). However, the morphological alterations in theepithelial nerves persisted for as long as 12 months after the procedure, with stromal nervealterations also being present (Tervo et al. 1994).
In vivo confocal microscopy (IVCM) enables imaging of the living cornea, allowingobservation of the regeneration of subbasal nerves after PRK. The first regenerating subbasalnerves have been seen as early as one week after PRK (Linna and Tervo 1997). At one monthafter PRK, subbasal nerves were observed in only 1 of 18 eyes (Frueh et al. 1998). Partial or insome cases total recovery of subbasal nerves has been reported to occur at 812 months afterPRK (Corbett et al. 1996, Kauffman et al. 1996). However, even at 5 years after PRK, somecorneas do not achieve a normal pattern of subbasal nerve morphology, although the meansubbasal nerve density does not differ from that of normal controls (Moilanen et al. 2003). In arecent prospective longitudinal study, preoperative levels of nerve density were achieved 2years after myopic PRK, and the levels remained stable during the 5year followup (Erie et al.2005b).
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8.2 Corneal sensitivity after PRK
The early postoperative decline in corneal sensitivity is followed by a relatively fast recovery ofsensation in the following 3 months, when preoperative levels also in the central cornea areachieved (PerezSantonja et al. 1999, Matsui et al. 2001, Kumano et al. 2003, Lee et al. 2005).Interestingly, in some studies, no decrease in corneal sensitivity after PRK was observed duringthe followup (Kumano et al. 2003), while in another study a time period of up to 12 monthsafter PRK was needed to achieve normal sensitivity levels (Nejima et al. 2005). PRK patientsare characterized by higher tear fluid NGF levels 3 months postoperatively. Higher NGF levelscorrelated with faster recovery of corneal sensitivity (Lee et al. 2005). Loss of sensitivity afterPRK seems to be less severe than after LASIK, and the overall rate of recovery of sensitivity topreoperative levels is faster after PRK than LASIK (PerezSantonja et al. 1999, Matsui et al.2001, Kumano et al. 2003, Lee et al. 2005, Nejima et al. 2005).
9. Laser in situ keratomileusis LASIK
Ionnis Pallikaris was the first to describe laser in situ keratomileusis (LASIK) in 1990(Pallikaris et al. 1990). A hinged corneal flap, consisting of the epithelium, the subbasal nerveplexus, and the anterior stroma, is created with a microkeratome. After the flap is lifted, cornealsculpturing is performed on the exposed stromal bed, after which the corneal flap is replaced.The flap adheres spontaneously to the stromal bed, with no suturing required.
Figure 4. Myopic LASIK. A hinged flap, consisting of the epithelium, Bowman’s layer, subbasal nerves,and the anterior stroma, is created using a microkeratome. After the flap is lifted, the corneal stroma issculptured using excimer laser.
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LASIK is nowadays the most commonly performed procedure in refractive surgery and the firstchoice for the correction of refractive errors in the majority of patients (Duffey and Leaming2005). It is a relatively safe, predictable, and effective method for correcting low to moderatelyhigh (up to –15 D) myopia, offering many advantages over other existing procedures such asfast and painless recovery of vision, less regression, and less subepithelial haze (McDonald etal. 2001, Solomon et al. 2002, Sugar et al. 2002). However, patients subjected to LASIKsurgery often report dry eye symptoms postoperatively, and tear fluid abnormalities arefrequently described (Battat et al. 2001, Hovanesian et al. 2001, Toda et al. 2001, Albietz et al.2004a, De Paiva et al 2006, Shoja and Besharati 2007). These symptoms are the most commonadverse effects of LASIK, causing frustration for both patients and surgeons alike (Sugar et al.2002). LASIKassociated dry eye is believed to be attributable to the severing of the afferentcorneal nerves during the flap formation. Subbasal nerve bundles and superficial stromal nervebundles in the flap interface are cut by the microkeratome, with only nerves entering the flapthrough the hinge region being spared. Subsequent sculpturing of corneal stroma using excimerlaser severs stromal nerve fiber bundles (Linna et al. 1998, 2000, Lee et al. 2002, Brageeth andDua 2005, Erie et al. 2005b).
9.1 Nerve regeneration after LASIK
The regeneration of corneal nerves after LASIK has been investigated in experimental models(Latvala et al. 1996, Linna et al. 1998, Fukiage et al. 2007, reviewed by Tervo and Moilanen2003). Thin regenerating nerve fibers form connections with neighboring stromal nerve fibersand penetrate the most anterior acellular stromal layer to send subbasal nerve fibers to form thenerve terminals between epithelial cells. Regeneration of anterior stromal, subbasal, andepithelial nerve fibers occurs approximately 3 months after LASIK, while deep stromal nervesmay show abnormal morphology even 5 months after the procedure (Latvala et al. 1996, Linnaet al. 1998). Topically administered neurotrophic factor pituitary adenylate cyclaseactivatingpolypeptide (PACAP) has accelerated neural regeneration after LASIK in an experimentalmodel (Fukiage et al. 2007). Accordingly, topical neurotrophic factors PACAP and NGF seemto enhance the recovery of sensitivity after LASIK (Joo et al. 2004, Fukiage et al. 2007).However, lower tear fluid NGF levels after LASIK compared with PRK in human have beenaccompanied by slower recovery of sensation (Lee et al. 2005).
In vivo confocal microscopy studies have enabled imaging of the living human cornea in fourdimensions (x, y, z, and t [time]) and have produced abundant information on regeneration andmorphological changes in corneal nerves during the postLASIK healing process (Kauffmann etal. 1996, Slowik et al. 1996, Linna et al. 2000, Lee et al. 2002, PerezGomez and Efron 2003,Avunduk et al. 2004, Calvillo et al. 2004, Bragheeth and Dua 2005, Erie et al. 2005b).
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Degeneration of cut subbasal nerves manifests during approximately one week after surgery, atwhich time subbasal nerve density decreases by 90% (Linna et al. 2000, Lee et al. 2002,Calvillo 2004, Erie et al. 2005b). Thereafter, a slow regenerative process occurs andregenerating nerve fibers form connections with neighboring nerve fibers. Prospective studieshave shown that during the first year after LASIK, subbasal nerve fiber bundles graduallyregenerate, achieving numbers that nevertheless remain more than 50% lower than beforeLASIK (Lee et al. 2002). Total recovery of the subbasal plexus, especially its morphology,probably never occurs, and a significantly longer period of time than previously assumed seemsto be needed. Subbasal nerve density remained < 60% of preoperative levels at 3 years postLASIK (Calvillo et al. 2004), and nerve density near preoperative densities was not reacheduntil 5 years after LASIK (Erie et al. 2005b).
9.2 Corneal sensitivity after LASIK
An early loss of corneal sensitivity to coarse mechanical stimulation has been reported afterLASIK, followed by progressive recovery of sensitivity during the following postoperativemonths. Corneal sensitivity seems to be at its lowest 12 weeks after LASIK, and by 612months sensitivity has recovered to normal levels (Kim and Kim 1999, PerezSantonja et al.1999, Linna et al. 2000, Benitez del Castillo et al. 2001, Toda et al. 2001, Donnenfeld et al.2003, 2004, Michaeli et al. 2004, Bragheeth and Dua 2005, Lee et al. 2005). Recovery periodsof over 12 months have also been reported (Nejima et al. 2005). By contrast, the return of nearnormal sensitivity levels by 3 weeks postLASIK has also been described (Chuck et al. 2000).Studies comparing the recovery of corneal sensitivity after PRK and LASIK have utilized theCochetBonnet esthesiometer, which measures coarse mechanical sensation, but has certainlimitations in sensitivity and reproducibility (Murphy et al. 1998). In any case, after LASIK, theloss of sensitivity seems to be more intense and the time needed for recovery longer (PerezSantonja et al. 1999, Matsui et al. 2001, Kumano et al. 2003, Lee et al. 2005). Interestingly,lower tear fluid NGF levels in humans after LASIK, compared with PRK, have beenaccompanied by slower recovery of sensation. NGF is known to be a potent neurotrophic factor,and thus, tear fluid NGF is suggested to play a role in recovery of sensitivity as well as inregeneration of corneal nerves (Lee et al. 2005).
More recently, noncontact gas esthesiometers, which are more sensitive and reproducible thanmechanical esthesiometers, have been utilized in studies exploring the recovery of cornealsensitivity after LASIK (De Paiva and Pflugpfelder 2004, Gallar et al. 2004, Stapleton et al.2006). In contrast to studies utilizing mechanical esthesiometers, which report the greatestdecrease of sensitivity 12 weeks postLASIK (Linna et al. 2000, Donnenfeld et al. 2004), in theabove study, corneas were observed to be hypersensitive at 1 week postLASIK (Gallar et al.2004). This was followed by a significant decrease in sensitivity to mechanical stimuli during 3
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5 months after surgery. Corneal sensitivity was close to normal values by 2 years postLASIK(Gallar et al. 2004). Patients without dry eye at 140 months after LASIK presented withdecreased corneal sensitivity, while patients with LASIKassociated dry eye showed cornealhypersensitivity at 336 months (De Paiva and Pflugfelder 2004). Corneal hypersensitivityobserved in LASIKassociated dry eye patients was suggested to result from compromisedocular surface barrier function and hypersensitivity to air jet (De Paiva and Pflugfelder 2004).
Several factors influencing the severity of the postoperative decrease in corneal sensitivity andsubsequent recovery have been suggested, including ablation depth, hinge orientation (superioror nasal), hinge width, and flap thickness.
Deep ablations, thus greater corrections, result in a larger decrease in corneal sensitivity and alonger recovery (Kim and Kim 1999, Nassaralla et al. 2003, Bragheeth and Dua 2005, Shojaand Besharati 2007). Accordingly, ablation depth is a clear risk factor for developing dry eyeafter LASIK (De Paiva et al. 2006, Shoja and Besharati 2007).
Depending on the microkeratome used in LASIK, the hinge is positioned either superiorly ornasally. While long ciliary nerves run and penetrate the cornea at the 3 and 9 o’clock positions,it has been suggested that flaps with a superior hinge cause more severe nerve damage thanthose with a nasal hinge, which spares the medial nerve fibers. Seemingly in agreement withthis concept, eyes with a nasal hinge were found to have less dry eye symptoms (Donnenfeld etal. 2003) and better corneal sensitivity than eyes with a superior hinge during a 6monthpostoperative period (Donnenfeld et al. 2003, Vroman et al. 2005, Nassaralla et al. 2005).However, a prospective randomized clinical study found no difference in dry eye signs orsymptoms between patients treated with superiorly and those with nasally hinged flaps(Ghoreishi et al. 2005).
The narrow hinge of the flap resulted in a more pronounced decline in corneal sensitivity andmore severe dry eye than flaps with a broader hinge (Donnenfeld et al. 2004). The thickness ofthe flap has also been suggested to be an important factor in regaining corneal sensitivity; thinflaps with a nasally placed hinge were related to more rapid recovery (Nassaralla et al. 2005).
In conclusion, corneal mechanical sensitivity, measured with a CochetBonnet esthesiometer,decreases during the first postoperative weeks, and regeneration of nerves coincides with therecovery of sensitivity, with normal sensitivity levels typically being achieved 612 monthsafter LASIK. However, studies utilizing more sensitive noncontact gas esthesiometers suggestthat alterations in corneal sensitivity may persist for up to 24 months.
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10. Dry eye
A definition of dry eye was recently produced by the International Dry Eye WorkShop (DEWS2007): “Dry eye is a multifactorial disease of tears and ocular surface that results in symptomsof discomfort, visual disturbance, and tear film instability with potential damage to the ocularsurface. It is accompanied by increased osmolarity of the tear film and inflammation of theocular surface (Lemp et al. 2007).”
Large epidemiological studies have revealed the prevalence of dry eye at various ages rangefrom 5% to 34% (Smith et al. 2007). However, the definition of dry eye and the diagnostic testsand criteria varied markedly between these studies, and thus, caution is advised in making directcomparisons between the results.
Dry eye is classified into two etiopathogenic categories: 1) aqueous teardeficient dry eye and2) evaporative dry eye.
Aqueous teardeficient dry eye is further divided into SS dry eye and nonSS dry eye. NonSSdry eye includes 1) primary lacrimal gland deficiencies, e.g. agerelated dry eye; 2) secondarylacrimal gland deficiencies such as conditions with lacrimal gland infiltration, e.g. sarcoidosis,lymphoma, acquired immunodeficiency syndrome (AIDS), or graft versus host disease; 3)conditions associated with obstruction of lacrimal gland ducts, e.g. trachoma, cicatricalpemphigoid, erythema multiforme, and chemical and thermal burns; 4) conditions affectingsensory innervation, e.g. herpes simplex keratitis, herpes zoster ophthalmicus, penetratingkeratoplasty, PRK, LASIK, diabetes mellitus, and topical anesthetic abuse, as well assecretomotor innervation, e.g. damage to the VII cranial nerve and certain systemic medications(Lemp et al. 2007).
Evaporative dry eye may be intrinsic, with regulation of evaporative loss from the tear filmbeing directly affected by, for instance, meibomian lipid deficiency, poor lid congruity, wide lidaperture, and low blink rate. Extrinsic evaporative dry eye embraces those etiologies thatincrease evaporation by their pathological effects on the ocular surface, including vitamin Adeficiency, topical drug preservatives, contact lens wear, and ocular surface disease, e.g. allergy(Lemp et al. 2007).
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Figure 5. Lacrimal gland functional unit. Stimulation of the free nerve endings in the cornea generatesafferent nerve impulses that travel through the ophthalmic division of the trigeminal nerve to thesuperior salivary nucleus in the pons. The nerves synapse and the signal is integrated with cortical andother input in the pons. The efferent branch of the loop passes along the nervus intermedius to thepterygopalatine ganglion. Postganglionic fibers then terminate in the main and accessory (Wolfring andKrause) lacrimal glands. Increasing evidence suggests that nerve endings found around the Meibomianglands and conjunctival Goblet cells travel along the same route (Stern et al. 2004).
In the normal situation, the ocular surface, interconnecting nerves, and lacrimal glands form afunctional unit (Fig. 5) that controls the major components of the tear film and responds toenvironmental, endocrinological, and cortical influences. If any portion of this funtional unit iscompromised, lacrimal gland support to the ocular surface is impeded (Stern et al. 2004).
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11. Dry eye after refractive surgery
All keratorefractive surgical procedures, including PRK and LASIK, cause morphological andfunctional disturbances of corneal nerves. Dry eye is one of the most common complicationsafter refractive surgery (Hong and Kim 1997, Sugar et al. 2002). It is mainly thought to beattributable to the transection of afferent corneal nerves during the lamellar microkeratome cutin LASIK, and additional damage caused by excimer laser photoablation. The ocular surface,lacrimal glands, and interconnecting nerves form a functional unit (Stern et al. 2004), andimpairment of corneal nerves interrupts the cornea–trigeminal nerve–brain stem–facial nerve–lacrimal gland reflex arc, influencing both reflex and basal tear production.
The diagnostic criteria and treatment strategies for dry eye vary widely amongophthalmologists, and among cornea and dry eye specialists. Some clinicians consider clinicalsigns more essential than symptoms. Conversely, other clinicians value symptoms more highlyas early evidence of ocular disease. On the other hand, little correlation exists between thesymptoms and clinical test results in dry eye patients (reviewed by Pflugfelder et al. 2000 andBron 2001, Dogru et al. 2005). The clinical signs of LASIK dry eye include evaluation of tearfilm stability with application of fluorescein to the tear film to measure tear film breakup timeand positive vital staining of the ocular surface with lissamine green, fluorescein or rose bengal.Schirmer’s test with or without anesthesia is considered an important parameter at least for usein studies where statistical trends can be monitored. However, there is no consensus as to whichmethod is most useful or regarding diagnostic cutoffs (reviewed by Bron 2001). Symptoms canbe monitored using different validated questionnaires such as the ocular surface disease index(OSDI) questionnaire (Schiffman et al. 2000). Punctate epithelial keratopathy detected with rosebengal or fluorescein staining has been noted in 26% of eyes that have undergone LASIK(reviewed by Ang et al. 2001, Wilson 2001), but symptoms of ocular dryness and irritation werenoted in approximately half of the LASIK patients (Hovanesian et al. 2001).
In addition to decreased reflex and basal tear production, impairment of corneal nerves maylead to LASIKinduced neurotrophic epitheliopathy (LINE) (Wilson 2001, Wilson andAmbrosio 2001), where punctate epithelial microerosions are present on the corneal surface.However, LASIK patients who developed symptoms and signs of dry eye after the procedurehad no significant difference in tear production detected by Schirmer’s test with anesthesia frompatients with no symptoms or signs of dry eye at timepoints from 1 month to 6 months aftersurgery (Wilson 2001). Soreness of the eye to touch was observed at 6 months more often afterPRK than LASIK, affecting 26.8% and 6.7% of patients, respectively (Hovanesian et al. 2001).This was suggested to be related to symptoms of recurrent erosions.
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Other issues potentially contributing to dry eye symptoms after LASIK include differences incorneal curvature; while normal corneas show prolate profiles, i.e. steeper in the center, andhigh conventional myopic LASIK produces oblate corneas. These changes in corneal curvaturemay interfere with even tear distribution, but the mechanical rubbing due to the anatomicalchange may also be causative. Moreover, patients who seek refractive surgery are often contactlens intolerant and may have preclinical dry eye before surgery. In addition, mechanical traumacaused by the microkeratome suction ring to limbal Goblet cells has been suggested to play arole in LASIKassociated dry eye (Lenton and Albietz 1999). Diminished afferent input alsoresults in a decreased blinking rate, which appears to be involved in the pathogenesis ofLASIKassociated dry eye (Toda et al. 2001).
Risk factors for developing LASIKassociated dry eye are: female gender, dry eye beforesurgery (Albietz et al. 2004a), depth of photoablation, and preoperative refractive error(Nassaralla et al. 2003, Albietz et al. 2004a, De Paiva et al. 2006, Shoja and Besharati 2007).LASIK dry eye has been reported to develop more often in patients of Asian origin than inCaucasians (Albietz et al. 2005).
In addition to symptoms of dry eye, blurring of vision, and halos, dry eye has been associatedwith regression of refractive result in PRK (Corbett et al. 1996), hyperopic LASIK (Albietz etal. 2002), and myopic LASIK (Albietz et al. 2004b).
12. Sjögren’s syndrome
In 1928, Swedish ophthalmologist Henrik Sjögren (18991986) saw a patient complaining ofdry eyes, dryness of mouth, and pain in several joints. Henrik Sjögren was not the first to noticethe combination of xeroftalmia, xerostomia, and arthralgia. French ophthalmologist HenriGougerot published similar observations a few years earlier, in 1926. In 1933, Henrik Sjögrendefended his doctoral thesis “Zur Kentniss der Keratoconjunctivitis Sicca”, where he carefullydescribed 19 patients with a combination of dry eyes and dry mouth. The eponym “GougerotSjögren disease” appeared in the literature in the 1930s, but a decade later this was shortened toSjögren’s disease, mainly because of Sjögren’s ongoing interest in the syndrome.
Sjögren’s syndrome (SS) is a chronic, generalized autoimmune disease. Dry eye and dry mouthare its major clinical manifestations. In primary Sjögren’s syndrome (pSS), typical symptomsoccur in a pure form without an association with any other underlying autoimmune diseases.Secondary Sjögren’s syndrome (sSS) is associated with other autoimmune diseases such asrheumatoid arthritis or systemic lupus erythematosus (Fox et al. 2000, Fox and Stern 2002). Inaddition to sicca syndrome, patients frequently suffer from visceral manifestations, including
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autoimmune thyreoiditis, atrophic gastritis, renal tubular glomerular nephritis, autoimmunehepatitis, and interstitial cystitis.
12.1 Neurological manifestations in Sjögren’s syndrome
In 1933, Henrik Sjögren described a patient with trigeminal nerve involvement in his doctoralthesis. Since then, the trigeminal nerve has been identified to be the most commonly affectedcranial nerve in SS (Kaltreider and Talal 1969, reviewed by Kaplan et al. 1990). The overallprevalence of peripheral neuropathy, most commonly of the distal sensory symmetrical type,ranges from 10% to 30% in pSS; fortunately, these neuropathies are often subclinical(Gemignani et al. 1994, Olney 1998, Barendregt et al. 2001). In a Japanese study on pSSpatients, trigeminal neuropathy was observed in 50% (Tajima et al. 1997), while in a Finnishstudy only 4% of patients were reported to have trigeminal neuropathy (Hietaharju et al. 1990).Electrophysiological studies of the trigeminofacial and trigeminotrigeminal reflexes inpatients with trigeminal nerve involvement suggest lesions in the neurons of the Gasserianganglia rather than in the trigeminal axons (Valls–Sole et al. 1990). The pathophysiologicalmechanisms are unknown, but may involve vasculitis (Melgren et al. 1989) or lymphocyticinflammation of nerve cell ganglia (Griffin et al. 1990). Decreased corneal sensitivity in SSrelated dry eye has been observed with the aid of the CochetBonnet esthesiometer (Xu et al.1996).
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AIMS OF THE STUDY
The role of corneal innervation in dry eye following corneal refractive surgery and in Sjögren’ssyndromerelated dry eye was investigated. The following goals were set:
1. To examine the role of tear fluid cytokines, regeneration of subbasal nerves, and subepithelial haze after PRK using IVCM (I).
2. To examine the recovery of corneal sensitivity using a novel noncontactesthesiometer in patients who had undergone correction of high myopia by LASIKand to assess the relationship between dry eye symptoms and sensory recovery (II).
3. To examine the alterations in corneal nerve morphology in primary Sjögren’ssyndrome (III).
4. To examine the relationship between corneal nerve morphology and cornealsensitivity in primary Sjögren’s syndrome patients (IV).
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SUBJECTS AND METHODS
1. Subjects
These studies were carried out according to the tenets of the Declaration of Helsinki at HelsinkiUniversity Eye Hospital. Research protocols were approved by the Ethics Review Committee ofHelsinki University Eye Hospital. All patients and controls were informed both orally and witha written brochure about the studies. Informed consent was obtained from all participants.
Table 2. Demographic characteristics of subjects enrolled in studies IIV.Patients
nFemale
%Patients
ageControls
nFemale
%Controls
age
I PRK 20 80 30.7 ± 5.9
II LASIK 20 70 34.0 ± 7.4 10 60 39.8 ± 10.4
III pSS 10 90 50.1 ± 13.5 10 90 48.3 ± 14.5
IV pSS 20 95 54.5 ± 7.0 10 90 50.2 ± 4.6
Data presented as mean ± SD.
1.1 PRK patients (I)
The study evaluated 20 eyes of 20 patients (16 females and 4 males, mean age 30.7 ± 5.9 years)scheduled for myopic PRK. The spherical equivalent (SE) of the intended correction was 4.7 ±1.5 D (range 2.75 D to 9.00 D). Astigmatic correction was performed on 12 patients. Theintended cylinder correction was 0.73 ± 0.27 D (range 0.50 to 1.50 D).
1.2 LASIK patients and controls (II)
The study evaluated 30 eyes of 30 subjects. Twenty eyes of 20 patients (14 females and 6males, mean age 34.0 ± 7.4 years) who had undergone > 10 D myopic LASIK 25 years earlierwere included in the study, and 10 eyes of 10 healthy volunteers (6 females and 4 males, meanage 39.8 ± 10.4 years) served as a control group. The mean followup time was 44.2 ± 11.3months (range 2358 months). A cohort of patients who met the inclusion criteria was selectedfrom the hospital database. These patients were sent an invitation to an additional followupexamination. The inclusion criteria were 1) high myopic correction (SE > 10D) with or without
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astigmatic correction and 2) time interval of 2 years or more after the last LASIK surgery.Exclusion criteria were 1) retreatments, and 2) patients with a history of any other ophthalmicsurgical operations. The first 20 consecutive volunteers were included in the study.
1.3 Primary Sjögren’s syndrome patients and controls (III and IV)
Study III evaluated 20 eyes of 20 subjects. Ten patients with pSS (9 females and 1 male, meanage 50.1 ± 13.5 years) were recruited from the Department of Rheumatology, HelsinkiUniversity Central Hospital, where they had been thoroughly examined by a rheumatologist.Ten age and sexmatched healthy controls (9 females and 1 male, mean age 48.3 ± 14.5 years)served as a control group. The mean duration of dry eye symptoms was 16.3 ± 8.2 years, but theaverage time elapsed from diagnosis was only 8.0 ± 4.6 years.
Study IV evaluated 30 eyes of 30 subjects. Twenty patients with pSS (19 females and 1 male,mean age 54.5 ± 7.0 years) and ten age and sexmatched healthy controls (9 females and 1male, mean age 50.2 ± 4.6 years) participated. The mean duration of dry eye symptoms was18.3 ± 10.0 years, and the average time interval between first symptoms and diagnosis was 10.3± 10.3 years.
SS diagnosis in both studies (III and IV) was made according to American–European consensuscriteria (Vitali et al. 2002). The diagnosis was set when at least 4 of the 6 following criteriawere met, with either item 4 (histopathology) or item 6 (serology) being present. Diagnosis wasalso set if any 3 of the 4 objective criteria were present (i.e. items 3, 4, 5, and 6).
1. Ocular symptoms (daily, persistent, troublesome dry eyes for more than 3 months and/orrecurrent sensation of sand or gravel in the eyes and/or use of tear substitutes more than 3 timesa day).
2. Oral symptoms (daily feeling of dry mouth for more than 3 months and/or recurrently orpersistently swollen salivary glands or the need to frequently drink liquids to aid swallowing ofdry food).
3. Ocular signs (Schirmer’s I test, performed without anesthesia [< 5 mm in 5 min] and/or Rosebengal score or other ocular dye score [> 4 according to van Bijsterveld’s scoring system]).
4. Histopathology (focal lymphocytic sialadenitis in minor salivary glands).
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5. Salivary gland involvement (unstimulated whole salivary flow [< 1.5 ml in 15 min] and/orparotid sialography showing the presence of diffuse sialectasias and/or salivary scintigraphyshowing delayed uptake, reduced concentration, and/or delayed excretion of tracer).
6. Autoantibodies (antibodies to Ro [SSA] and/or La [SSB]).
Patient were excluded if they had past head and neck radiation treatment, hepatitis C infection,acquired immunodeficiency syndrome (AIDS), preexisting lymphoma, sarcoidosis, graft versushost disease, or if they used anticholinergic drugs.
2. Methods
2.1 Clinical examination
Ophthalmic examinationAll patients were evaluated for uncorrected visual acuity (UCVA) and best spectaclecorrectedvisual acuity (BSCVA) and manifest refractions and underwent tonometry, slitlampexamination, and dilated funduscopic examination.
Schirmer’s testSchirmer’s test was performed using a Schirmer test strip (Clement Clarke International Ltd.,Harlow, United Kingdom). Two drops of oxybuprocaine hydrochloride 4 mg/ml (Oftan®
Obucain, Santen Oy, Tampere, Finland) were then administered to prevent reflex tearing. Thestrip was positioned at the intersection of the temporal third with the nasal twothirds of thelower eyelid, and the patient waited for 5 min with eyes closed until the strips were removed,and the length of the moistened area was measured.
Tear breakup time (BUT)To measure BUT, 1 drop of oxybuprocaine hydrochloride 3 mg/ml and sodium fluorescein dye1.25 mg/ml (Oftan® Flurecain, Santen Oy, Tampere, Finland) was instilled in the lowerconjunctival sac with a micropipette. The tear film was observed under cobaltbluefilteredlight. The interval between the last complete blink and the first appearance of randomlydistributed dry spots was measured. The average of three measurements was calculated.
Grading of corneal fluorescein stainingGrading of corneal fluorescein staining was performed as recommended by Lemp (1995). Thecornea was divided into five areas: central, upper, temporal, inferior, and nasal. Punctate
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corneal staining was recorded using a standardized grading system of 03 for each of the fiveareas, resulting in a scale of 015.
Clinical haze gradingClinical corneal haze was estimated with a slit lamp according to the Fantes’ scale (Fantes et al.1990). Criteria for corneal opacity grading were as follows: grade 0, totally clear; grade 0.5, atrace or a faint corneal haze; grade 1, haze of minimal density seen with difficulty with directand diffuse illumination; grade 2, mild haze easily visible with direct focal slit illumination;grade 3, moderately dense opacity partially obscuring the iris details; and grade 4, severelydense opacity completely obscuring details of intraocular structures.
CMTF haze estimateConfocal microscopy through focusing (CMTF) scans were obtained as previously described (Liet al. 1997, MollerPedersen et al. 1997). Using the custom software, the CMTF data weredigitized onto the PC, intensity profile curves were calculated, and a quantitative estimate of theincreased backscattering from the subepithelial haze (CMTFhaze estimate) was achieved. Thethickness of the haze area was also calculated. One to four acceptable CMTF scans wereproduced for each eye. Mean values of the measurements were used for all statistical calculations.
Subjective dry eye symptomsSubjective symptoms were evaluated using ocular surface disease index (OSDI), which is areliable and validated 12item questionnaire for assessing subjective symptoms in ocular surfacediseases and the impact on visual functioning (Schiffmann et al. 2000). In addition, visualanalog scale (VAS) was used to evaluate global severity of ocular discomfort. The subjectswere asked to evaluate severity of ocular symptoms during the past month and to place a markwith a pencil on a 100mm scale, with the left end representing minimal symptoms (0 mm) andright end maximal symptoms (100 mm).
2.2 Tear fluid analysis (I)
Tear fluid collectionAll tear fluid samples were collected with a scaled 5 or 25µl firepolished microcapillary tube asdescribed previously (van Setten et al. 1989). Tears were collected preoperatively (day 0), on thesecond postoperative day (day 2) and 3 months postoperatively. The samples were immediatelytransferred to Eppendorf tubes and stored at 70oC until assessed. Tear fluid flow in the collectioncapillary (µl/min) was calculated by dividing the volume of the tear fluid sample by the tear fluidcollection time. The tear flowcorrected concentration, i.e. rate of release, was calculated bymultiplying the concentration in the sample (ng/l) by the tear fluid flow in the collection capillary(µl/min) (Vesaluoma et al. 1997a). As the interindividual variations in the tear fluid cytokine
37
concentrations were high, changes (%) in the cytokine concentrations and rates of release inpreoperative vs. postoperative samples were also calculated.
Cytokine immunoassaysThe concentrations of TGF 1 in tear fluid were measured by an enzyme immunoassay(Vesaluoma et al. 1997a). Microtiter plates (Maxisorp, Nunc Intermed, Denmark) were coated withmonoclonal mouse antiTGFβ1, β2, and β3 (Genzyme Diagnostics, Cambridge, MA, USA) 0.1µg/well in 0.05 M Na2CO3 buffer, pH 9.2, overnight at 4°C. After washing the wells with 0.05 Mphosphatebuffered saline, pH 7.3, containing 0.05% Tween 20, 100 µl of acidactivated andneutralized (0.1 M HCL, 4°C, 1 h) standard dilutions (natural human TGFβ1, Code BDP 1, R &D Systems, London, UK) and samples (final dilutions 18fold) were added to the wells andincubated overnight at 4°C. The unbound material was removed with the above washing buffer,and 100 µl of 1000fold diluted antibodies to human TGFβ1 (Code 27.283.29, Jansen Biochimica,Beerse, Belgium) conjugated with alkaline phosphatase was added and incubated at 37°C for 2 h.After washing, the amount of alkaline phosphatase fixed to the tubes was determined indiethanolamine (1.0 mol/l)magnesium chloride (0.5 mol/l) buffer, pH 10.0, using pnitrophenylphosphate as a substrate for 1 h at room temperature. The absorbance of the pnitrophenolate liberated was measured at 405 nm with a 340 ATC microtitration plate reader (SLT,Labinstruments, Vienna, Austria). The detection limit for the assay was 5 ng/l.
PDGFBB concentrations were measured by a sandwich enzyme immunoassay (Vesaluoma et al.1997b). Microtiter plates (MaxisorpTM, Nunc Instrumed, Denmark) were coated with goat IgGtype antibody to highly purified Escherichia coliderived recombinant human PDGFBB (CodeAB220Na, R & D Systems Europe Ltd., Abingdon, UK) (0.25 µg/well) in 0.05 M Na2CO3
buffer, pH 9.2, overnight at 4°C. After washing the wells with 350 µl of water, 100 µl ofrecombinant human PDGFBB homodimer (Code 203801, Genzyme Diagnostics, Cambridge,MA, USA) as a standard (serial dilutions from 1000 to 5 ng/l) or 5 to 20fold diluted tears wereadded to the wells and incubated at 24°C for 60 min on a horizontal rotating table (80 rpm). Theunbound material was removed and the wells were washed twice with saline containing 0.05%Tween 20. One hundred microliters of 100fold diluted rabbit antibody to human PDGFBB (CodeZP215, Genzyme Diagnostics, Cambridge, MA, USA) was added and incubated for 1 h at roomtemperature as above. After washing, 100 µl of 500fold diluted alkaline phosphatase conjugatedswine antibody to rabbit IgG (Code 67850, Orion Diagnostica, Espoo, Finland) was added andincubated as above. The plates were then washed three times with 350 µl of washing solution. Theamount of alkaline phosphatase fixed to the tubes was determined in 1.0 M diethanolamine0.5 MMgCl2 buffer, pH 10.0 (Code 170057, Reagena Ltd., Kuopio, Finland) at room temperature for 3 hin the dark, using pnitrophenolphosphate (Code 104105, Sigma Chemical Co., St. Louis, MO,USA) as a substrate. The absorbance of pnitrophenolate liberated was measured at 405 nm with a
38
340 ATC microtitration plate reader (SLTLab Instruments, Vienna, Austria). The absorbance ofthe 0 ng/l standard was subtracted from all the other absorbances, and the delta absorbances wereused for calculations. The detection limit of the assay was 20 ng/l.
TNFα concentrations were determined by a double antibody radioimmunoassay developed formeasuring of serum TNF as described by Vesaluoma et al. (1997b). A volume of 10 µl of tear fluidsamples was first diluted by adding assay buffer to reach a final volume of 100 µl. TNFα fromtears competed with a fixed amount of 125 Ilabeled TNFα (10 000 counts/min for 50 µl) for thebinding sites of 30 000fold diluted specific rabbit antibodies. The bound TNFα was precipitatedwith Sepharosebound antirabbit IgG and centrifuged, and the radioactivity of the pellets was thencounted. E. coliderived recombinant human TNF α (Code TNFH, Genzyme Diagnostics,Cambridge, MA, USA) was used as a standard. This had a molecular weight of 36 kDa, and aspecific activity > 1 x 107 U/mg of protein, as measured by bioassay with mouse L 929 cells.Rabbit antiserum to human TNFα (Code P300A, Endogen, MA, USA) showed < 1% crossreaction with lymphotoxins. The detection limit of the assay was 10 ng/l.
2.3 In vivo confocal microscopy – IVCM (I, III, and IV)
Tandem scanning in vivo confocal microscopy (I and III)
A tandem scanning confocal microscope (TSCM, Model 165A, Tandem Scanning Corp., Reston,VA, USA) was used to examine the central cornea. A 24X, 0.6 NA variable working distanceobjective lens was used. The fieldofview with this lens is 450 x 360 µm, and the zaxisresolution 9 µm. Images were detected by using a lowlightlevel camera (model VE1000; DageMTI, Michigan, IN, USA) and recorded on SVHS tape. Video images of interest were digitizedusing a PCbased imaging system with custom software (University of Texas, SouthwesternMedical Center at Dallas, Dallas, TX, USA).In addition, confocal microscopy through focusing (CMTF) scans were obtained. Intensity profilecurves for digitized CMTF data were calculated. From each CMTF scan, epithelial, Bowman’slayer, and total corneal thicknesses were measured. The mean values of each individual’smeasurements were used for the statistical analysis.
After the layers of the central cornea had been scanned with the confocal microscope, specialattention was paid to the morphology of the surface and basal epithelium. The microscope wasthen focused beneath the basal epithelium to evaluate the subbasal nerve plexus in detail.Afterwards, images were digitized from the SVHS video tape, and the number of nerve fibers inthe image was determined using pointcounting principles. The number of nerve fibers in the
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image with the most subbasal nerve fibers was used as a measure of nerve density. Nervemorphology was assessed from the SVHS video tape and digitized images. The morphology ofthe nerve fibers was evaluated, with attention being paid to the following aspects: thickness(fine and faint, normal, thick), beading, sidebranching, and sprouting. It must, however, benoted that resolution of confocal microscopy enables counting of nerve fiber bundles, but not ofsingle fibers or terminals.
Scanning slit in vivo confocal microscopy (ConfoScan 3) (IV)Corneal in vivo morphology was evaluated using an in vivo scanning slit confocal microscope(ConfoScan 3, software version 3.4, Nidek Technologies Slr, Vigonza, Italy) equipped with aAchroplan 40 x objective (Carl Zeiss Meditec AG, Jena, Germany). A topical anesthetic wasinstilled in the lower conjunctival fornix of both eyes before examination (oxybuprocainehydrochloride 4 mg/ml, Oftan® Obucain, Santen Oy, Tampere, Finland). The subject wascomfortably seated in front of the microscope with the aid of chin and forehead rests and askedto look straight ahead, while a fixation target was not available. A drop of ophthalmic lubricantgel (2 mg/g carbomere, Viscotears®, CIBA Vision Europe Ltd., Southhampton, UK) wasapplied on the objective tip to serve as a coupling media. The microscope is supplied withautomatic alignment software that was used to center the objective. Automated mode was usedto obtain fullthickness scans from the central cornea. During a scan, the instrument recordedimages at 25 frames/s as the focal plane advanced anteriorly 4.08.0 µm between frames; scanswere repeated until 350 frames were recorded. The best focused confocal microscopic imagesfrom the central cornea were used for nerve density calculations with software supplied by themanufacturer. The field size of the microscope was 422 µm × 322 µm (0.136 mm2) based oncalculations from a calibration slide.
2.4 Noncontact esthesiometry (II)
Central corneal mechanical sensitivity was assessed with a modified Belmonte Noncontactesthesiometer developed by the Cooperative Research Center for Eye Research andTechnology, Sydney, Australia, and based on an instrument previously designed by Dr. CarlosBelmonte, which has been used extensively to assess corneal sensation in animals and humans(Belmonte et al. 1999, Acosta et al. 2001). The instrument is mounted on the frame of an airtonometer and has a box controller and two gas tanks, containing 100% air and 100% CO2,respectively. The tip of the esthesiometer is adjusted to a distance of 4 mm in front of thecornea, using a focusing mechanism.
Subjects were seated in front of the gas esthesiometer, with their chin placed in a cup and theirforehead against a band. An audible click produced by the opening of the gas valve identified
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the onset of the stimulus. After each pulse, the subject was asked to report whether the stimuluswas felt independently of the sensation evoked. Sequential stimulus was only performed afterthe patient reported no sensation from the previous stimulation. Subjects were told to blinkfreely between stimuli.
Mechanical stimulation consisted of a series of pulses of warmed air (constant temperature of42°C at the tip of the probe), with flow varying from 0 to 160 ml/min, applied to the centralcorneal surface. Sensation caused by the mechanical stimulation was determined using thevisual analog scale. Briefly, a series of 2s pulses were applied randomly in 20 ml/min steps,from 160 ml/min to 20 ml/min. After a negative answer, the next stimulus was 10 ml/minhigher until the lowest positive value was achieved. The lowest airflow that elicited a response,even weakly, was recorded as the mechanical threshold.
2.5 PRK (I)
PRK was performed after surgical abrasion of the epithelium (diameter 6.5 mm) using a BeaverEye Blade (Becton Dickinson, Franklin Lakes, NJ, USA). Sixmillimeterwide PRKs of varyingablation depths were performed using a VisX 20/20 excimer laser (VisX Co., Sunnyvale, CA,USA) or a NIDEK EC 5000 excimer laser (Nidek, Gamagoni, Aichi, Japan). The mean ablationdepth was 60.7 ± 14.1 µm (range 3693 µm).
Eyepatching and postoperative medicationEach eye was pressurepatched for 3 days following PRK. In the morning of the first and/or secondpostoperative day, the patch was removed and the lids were gently cleaned with a paper wipe.After waiting for about 30 s, the tear fluid sample was collected, after which chloramphenicolointment (Oftan Chlora; Santen, Tampere, Finland) was applied and the eye repatched. In additionto the ointment twice a day for 4 days, the postoperative medication included fluorometholonedrops (LiquifilmFML; Allergan, Irvine, CA, USA) starting on the fourth postoperative day threetimes a day for 13 months, oral diclofenac sodium 25 mg (Voltaren; CibaGeigy, Basel,Switzerland) 30 min before the operation and 23 times a day for the first days after PRK, and oraldiazepam 510 mg (Diapam; Orion, Helsinki, Finland) for the first two postoperative nights.
2.6 LASIK (II)
The corneal flap was created with an automated Hansatome microkeratome (Hansatome, modelHT 230, Chiron Vision, Hansa Research & Developement, Inc., Miami, FL, USA). Theintended thickness of the flap was either 160 or 180 µm, with a superiorly located hinge.Subsequent laser ablation was performed using a VisX Star S2 (VisX Co., Sunnyvale, CA,USA) excimer laser. The patients were prescribed topical ofloxacin 3 mg/ml (Exocin; Allergan
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Pharmaceuticals Ltd., Westport, Ireland) three times a day for 7 days and fluorometholone 1mg/ml, (LiquifilmFML, Allergan Pharmaceuticals Ltd., Westport, Ireland) two times a dayfor 7 days starting on day 2. Nonpreserved artificial tears were to be used four times a day forat least 6 months after the operation.
2.7 Statistical analyses
Statistical comparisons of the mean between groups were performed either with the ttest orwith the Mann–Whitney Utest for normally distributed or skewed data, respectively, usingSPSS for Windows (version 8.0 11.0, SPSS Inc., Chicago, IL, USA). Normality of the data wastested using the ShaphiroWilk test or the KolmogorovSmirnov test as appropriate. In addition,histograms were used to help in testing normality of samples. Bonferroni corrections wereapplied in multivariate analysis. Bivariate correlations were examined using Pearson’s (r) orSpearman’s (rho) correlation test. All values are given as mean + standard deviation (SD) ormedian and interquartile range (IQR) for normally distributed or skewed data, respectively. Pvalues of less than 0.05 were considered statistically significant.
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RESULTS
1. Tear fluid cytokines, haze, and nerve regeneration after PRK (I)
1.1 Tear fluid cytokines after PRK
The tear fluid flow in the collection capillary was (mean ± SD) 9.6 ± 11.1 µl/min (0.742.9µl/min) preoperatively, 43.0 ± 30.7 µl/min (7.1125.0 µl/min; P < 0.001) on day 2, and 8.8 ±8.2 µl/min (0.733.3 µl/min; P = 0.003) at 3 months postoperatively. A weak positivecorrelation (r = 0.498, P = 0.03) was observed between ablation depth and tear fluid flow onday 2. However, at 3 months postoperatively, no correlation existed between these parameters(rho = 0.285, P = 0.237). Cytokine concentrations and rates of release are given in Table 3.
1. 2 IVCM three months after PRK
Confocal images can be found in the original articles (I, III, and IV) at the end of this thesis.
Surface epithelial cells, posterior stromal keratocytes, and endothelial cells of all corneaspresented with a normal shape and reflectivity (images not shown). The basal epithelial cell areaof two corneas showed pathological findings. The basal epithelial cells of a cornea with ahistory of recurrent erosions presented with presumably intracellular deposits (Rosenberg et al.2000). Similar deposits could be discerned among subepithelial haze resulting from highlyreflective keratocyte nuclei and visible keratocyte processes (I / Fig. 1A). Another corneashowed highly reflective particles, round to oval in shape, in the subepithelial area (I / Fig. 1B).The basal epithelial cells of all other patients appeared normal, and the Bowman’s layer wasabsent in all corneas. The epithelial thickness varied from 25 to 49 µm (39 ± 7 µm), thus, theepithelium was thinner than in normal corneas (Li et al. 1997).
The subbasal nerve plexus was absent in two corneas, and 18 corneas showed 15 (2.1 ± 1.4)regenerating nerve fiber bundles (I / Fig. 1C). Subepithelial haze was observed in all corneas,and in two cases it was very highly reflecting and appeared vacuolized (I / Fig. 1D). The firstanterior keratocytes exhibited brightly reflecting nuclei and thickened keratocyte processes assigns of ongoing keratocyte activation (I / Fig. 1D). In one of these two corneas, the subbasalnerve plexus was absent (I / Fig. 1D). In the other 18 corneas, haze was more subtle, andsubbasal nerve fiber bundles were visible (I / Fig. 1C), except in one case. The mean CMTFhaze estimate was 506 ± 402 U, and the thickness of the haze area was 40.9 ± 9.2 µm. With theslitlamp, haze was mild (0.38 ± 0.39; range 0 – 1, (scale +0 to +4)) in all eyes. In general, theCMTF haze estimates were relatively low, in accordance with the clinical scoring.
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Table 3. Concentrations and releases of PDGFBB, TGF 1, and TNF after PRK (I).
Release Day 0
Concentration Day 0
ConcentrationDay 2
ConcentrationMonth 3
ReleaseDay 2
ReleaseMonth 3
(ng /l) (ng/l) (ng/l) (pg/min) (pg/min) (pg/min)PDGFBB
Below detectionlimit (n)
(13/19) (7/19) (10/20) (13/19) (7/19) (10/19)
Median 20 1230 660 0.02 51 2Mean 1700 7280 2920 13 301 7SD 4330 13500 5900 41 553 14Min 20 20 20 0.02 0.02 0.02Max 17780 56950 21200 180 2373 59Pvalue 0.075 0.248 0.008* 0.721
TGF 1Below detectionlimit (n)
(12/19) (13/19) (7/20) (12/19) (13/19) (7/19)
Median 5 5 255 0.005 0.005 1Mean 820 210 840 1 5 3SD 2850 440 1500 2 10 4Min 5 5 5 0.005 0.005 0.005Max 12560 1720 6730 10 36 17Pvalue 0.445 0.198 0.203 0.116
TNFBelow detectionlimit (n)
(0/19) (0/19) (0/20) (0/19) (0/19) (0/19)
Median 1760 920 770 7 27 4Mean 1920 1020 990 10 37 5SD 1560 480 970 8 27 5Min 120 240 90 2 11 0,8Max 6910 1930 4150 30 120 18Pvalue 0.039* 0.002* 0.000** 0.085
* = P < 0.05, ** = P < 0.01
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1.3 Correlations between IVCM data and cytokines (I)
The number of subbasal nerve fiber bundles was positively correlated with epithelial thickness(r = 0.58, P = 0.007). However, the number of subbasal nerve fiber bundles was not correlatedwith the CMTF haze estimate (rho = 0.281, P = 0.230) or the thickness of the haze area (r = 0.398, P = 0.082). Ablation depth showed no correlation with nerve count (r = 0.240, P =0.308), CMTF haze estimate (rho = 0.041, P = 0.865), or thickness of the haze area (r = 0.006,P = 0.979). The CMTF haze estimate, as expected, showed a strong correlation with thethickness of the haze area (rho = 0.783, P = 0.000).
No correlations were found between cytokine concentrations or rates of release at any timepoints and the CMTF haze estimate at 3 months. The magnitude of the changes from day 0 today 2 in the concentrations or rates of release of the three cytokines was not correlated with theCMTF haze estimate at 3 months (data not shown).
2. Dry eye and corneal sensitivity after high myopic LASIK (II)
2.1 Subjective symptoms
The majority of patients (55%) reported dry eye symptoms when asked a simple question: Doyou have dry eyes? Ocular surface disease index (OSDI) score, indicating degree of dry eyesymptoms, was significantly higher in LASIK patients (18.6 ± 6.4%) than in normal controls(7.5 ± 5.7%; P = 0.022). However, almost all LASIK patients (95%) were satisfied with theoverall outcome and would have chosen the operation again. The only patient who was notentirely satisfied had undergone epithelial erosion during the surgery and subsequent diffuselamellar keratitis (DLK) in both eyes. A predisposing factor seemed to be a loose epitheliumdue to basement membrane dystrophy, although the patient had a negative history of erosionsand the preoperative examination revealed no abnormalities.
2.2 Objective dry eye tests
We found no difference in objective dry eye tests. Schirmer’s test values did not differsignificantly between patients and controls (14.4 ± 8.9 mm and 9.0 ± 4.2 mm, respectively, P =0.066), although LASIK patients showed slightly higher scores contrary to expectations. TearBUTs were similar in patients and controls (15.9 ± 11.2 s and 14.0 ± 10.0 s, respectively, P =0.505). None of the patients or controls showed corneal fluorescein staining.
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2.3 Corneal sensitivity
The mean corneal sensitivity thresholds did not differ between the groups, being 73.5 ± 29.6ml/min in LASIK patients and 78.0 ± 18.7 ml/min in controls (P = 0.666). No correlations werepresent between corneal sensitivity threshold values and OSDI scores. Even when patients weredivided into two groups: 1) patients claiming to have dry eyes and 2) patients claiming not tohave dry eyes, we found no significant difference between sensitivity threshold values. Nosignificant correlation existed between corneal sensitivity thresholds and objective dry eye signs(Schirmer’s test or BUT).
3. Corneal morphology and sensitivity in primary Sjögren’s syndrome (III and IV)
3.1 Subjective symptoms (IV)
OSDI and VAS scores were significantly higher in pSS patients than in controls (Table 5), witha strong positive correlation between these two measures of ocular discomfort (rho 0.92, P <0.01), suggesting that VAS can be used to assess severity of dry eye symptoms in a relativelytimeeffective manner.
3.2 Objective dry eye tests (III and IV)
Objective dry eye test values (Schirmer’s test, tBUT) were lower in pSS patients than incontrols (Tables 4 and 5). Corneal fluorescein was observed in the majority of pSS patients,while none of the controls showed corneal fluorescein staining (Tables 4 and 5).
Table 4. Characteristics of pSS patients and controls (III).
pSS Controls Pvalue
Number of patients 10 10
Nerve count (nerves/frame) 5.4 ± 1.8 5.0 ± 1.4 0.584†
Schirmer’s test (mm) 3.0 ± 2.8 7.8 ± 4.8 0.015†
Corneal fluorescein staining % 60 0
Data presented as mean ± SD, † = ttest, Corneal fluorescein staining % = percentage of patientsshowing corneal fluorescein staining.
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Table 5. Characteristics of pSS patients and controls (IV).
pSS Controls Pvalue
Number of patients 20 10
Corneal fluorescein staining 1.5 (0.04.0) 0.002‡
Schirmer’s test (mm) 2.5 (1.04.8) 9.0 (3.010.0) 0.012‡
Tear breakup time (s) 5.0 (3.06.0) 7.0 (5.812.0) 0.006‡
Mechanical threshold (ml/min) 54.5 + 40.1 85.0 + 24.6 0.036†
Chemical threshold (% CO2) 29.2 ± 24.0 28.8 ± 14.6 0.960†
Nerve count (nerves/frame) 5.9 ± 2.2 6.1 ± 2.5 0.782†
Stromal nerve thickness (µm) 7.9 (6.413.3) 5.7 (3.77.3) 0.018‡
VAS (0100 mm) 61.0 (23.080.5) 2.5 (0.08.8) 0.000‡
OSDI (0100 %) 37.5 (20.860.8) 5.3 (0.012.5) 0.000‡
Data presented as mean ± SD or median and (interquartile range), Mechanical threshold = Cornealmechanical sensitivity detection threshold, Chemical threshold = Corneal chemical sensitivity detectionthreshold, OSDI = Ocular Surface Disease Index, VAS = Visual Analog Scale, † = ttest, ‡ = MannWhitney U test.
3.3 Corneal sensitivity (IV)
The mean mechanical sensitivity detection threshold was lower in pSS patients, implicatingcorneal mechanical hypersensitivity in pSS dry eye. However, no difference was found inchemical sensitivity detection thresholds (Table 5).
3.4 IVCM in primary Sjögren’s syndrome (III and IV)
Confocal images can be found in the original articles (III and IV) at the end of this thesis.
The surface epithelium was irregular or patchy in the majority of SS corneas (III / Fig. 1A),whereas control corneas showed normal morphology (III / Fig. 1B). Basal epithelial cellsappeared normal in all subjects.
The subbasal nerve density did not differ between SS and control groups (Tables 4 and 5), asreflected in long nerve fiber bundles per microscopic field. The normal subbasal nerve plexus of
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a healthy control subject is shown in IV / Figure 1. Nerve sprouting or a nerve growth conelikepattern (III / Figs 2A and 2B; IV / Fig. 2), presumably indicating neural regeneration, was foundat the level of the subbasal nerve plexus in 2040% of patients. One SS patient with severeperipheral neuropathy showed very thin subbasal nerve fiber bundles without beading (III / Fig.2C). Control subjects, by contrast, had relatively thick subbasal nerve fibers. Only a singlehighly reflective nerve fiber bundle was observed in one of the control subjects. In addition,subbasal nerve fiber bundles showed abnormal tortuosity in 2/10 eyes of the SS group (III / Fig.3B); no such abnormality was seen in the control group (III / Fig. 3D). Interindividual variationin the density of the subbasal nerve plexus was wide, ranging between 4 and 9 long nerve fiberbundles per microscopic field. Round particles, measuring approximately 1112 µm in diameter,possibly cells of inflammatory origin, were found at the level of the subbasal nerve plexus ofone SS cornea (III / Fig. 3A).
Anterior keratocytes revealed signs of activation, hyperreflectivity, or visible processes in 5/10SS corneas (III / Fig. 1C). Interestingly, three of these corneas also showed nerve sprouting (III/ Figs. 1A and 1B).
The stromal nerves appeared significantly thicker in pSS (IV / Figs 4 and 5) than in controls (IV/ Fig. 3; Table 5), possibly indicating neural regeneration. In one patient, an extremely thickstromal nerve measuring 43 µm in diameter was observed (IV / Fig. 5); this patient alsopresented with APCs among subbasal nerves and a very low mechanical sensory threshold (10ml/min), implicating severe corneal hypersensitivity.
Mature APCs with typical branching and long dendritic extensions were observed among thesubbasal plexus of the central cornea in 35% (7/20) of patients (IV / Fig. 6.) but in only 10%(1/10) of controls. In patients in whom APCs were observed (n = 7), their average density in thecentral cornea was 174 ± 26 cells/mm2. The number of mature APCs in the central cornea ofthese patients displayed a strong positive correlation with the subjective symptom score (r =0.847, P = 0.016). Patients with APCs and/or nerve growth cones otherwise did not differ fromother patients with respect to subjective symptoms, objective signs, or other features of nervemorphology.
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DISCUSSION
1. Tear fluid cytokines and haze after PRK (I)
The cytokines TGF 1, TNF , and PDGFBB were chosen to be analyzed in tear fluid for theirpotential influence on corneal keratocytes and stromal wound healing, including keratocyterepopulation of the ablated area after initial keratocyte apoptosis, keratocyte activation, andsubsequent production of the ECM (Ohji et al. 1993, Jester et al. 1996, 2002, Wilson et al.1996a, Andresen et al. 1997, Myers et al. 1997, Andresen and Ehlers 1998, reviewed by Jesteret al. 1999). However, of the measured cytokines, only TNF was readily measurable in allsamples at all timepoints. TGF 1 and PDGFBB were detected in 8/20 and 7/20 preoperativesamples, in 6/19 and 13/19 samples on day 2, and in 7/20 and 10/20 samples at 3 months,respectively.
Several potential sources exist for tear fluid cytokines, including the main and accessorylacrimal glands, corneal epithelial and stromal cells, conjunctival or inflammatory cells, theECM, and leakage from conjunctival vessels. Corneal wounding seems to result in increasedlacrimal gland mRNA levels for TNF , HGF, KGF, and EGF (Thompson et al. 1994, Wilsonet al. 1999b). Whether the PRK wound induces cytokine production in the human lacrimalgland is unknown.
No correlations were present between pre or postoperative tear fluid levels of TGF 1, TNF ,or PDGFBB and subepithelial postPRK haze estimated at 3 months by IVCM. Nor did eyeswith elevated in cytokine concentrations present with higher CMTF haze estimates. The clinicalhealing of all eyes proceeded without problems, and the maximal clinical haze observed at 3months was scored 1 on a scale from +0 to +4. One of the limitations of the study was the smallsize of the patient group, with no severe complications, such as dense haze (score +3 or +4)occurring in this group. The CMTF haze estimates were also low, which might have contributedto the result. Nevertheless, the observed high cytokine concentrations, e.g. of TGF 1, inindividual eyes without haze formation may suggest that tear fluid analysis is not optimal forscreening potential candidates for haze formation even had the patient cohort been morerepresentative in terms of different degrees of subepithelial haze.
2. Nerve regeneration after PRK (I)
During PRK the anterior stromal nerve trunks and subbasal nerve plexus are ablated. Properinnervation has long been known to be necessary for normal corneal epithelial regeneration(Beuerman and Schimmelpfennig 1980). In addition, individual epithelial cells and possibly
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also keratocytes are innervated (Muller et al. 1996). It is, therefore, to be expected that properinnervation is important for the physiological status of epithelial cells and quiescent keratocytes.Intriguingly, innervation might have a direct effect on corneal wound healing. To myknowledge, I am the first to demonstrate that the degree of subbasal nerve plexus regenerationafter PRK in human in vivo cornea positively correlates with restoration of the thickness ofcorneal epithelium. This is an additional piece of indirect evidence for the trophic andregulatory functions of corneal innervation. My observation is in line with earlier in vitro orexperimental findings showing that innervation has an influence on epithelial proliferation(Araki et al. 1994, Nakamura et al. 1997). Whether nerves also exert an effect on the healing ofstromal tissue remains obscure. No significant correlation was observed between the number ofsubbasal nerve fiber bundles and the CMTF haze estimate or the thickness of the haze area.
After the initial anterior keratocyte loss after PRK, the area is repopulated with new migratorykeratocytes (reviewed by Fini and Stramer 2005), which will rapidly transform to alteredkeratocytes that produce ECM (MollerPedersen et al. 1997). The return of keratocytes toquiescence takes months (Linna and Tervo 1997, Bohnke et al. 1998, Frueh et al. 1998), and thetiming coincidences with the gradual disappearance of the subepithelial haze (as observed byslit lamp) and regeneration of innervation. MollerPedersen et al. (1997) showed delicatesubbasal nerve fibers in 9/17 patients at 1 month after PRK, whereas according to Frueh et al.(1998) only 1/18 corneas presented with subbasal nerves at that time. In their study, 7/18corneas showed nerve regeneration by 4 months and 13/18 corneas by 12 months. Bohnke et al.(1998) examined 15 eyes at 843 months postoperatively and found regenerated nerve fibers inall eyes. In my study, only two corneas showed a total absence of a subbasal nerve plexus at 3months, whereas in the other 18 corneas at least single nerve fiber bundles were observed in thecentral area, although the branching pattern was not normal by 3 months. However, recovery ofcorneal nerve density to preoperative levels took 2 years after PRK (Erie et al. 2005b). Thereare certain limitations in grading of neural regeneration, e.g. dense haze impedes theobservation of thin nerve fiber bundles. The resolution of confocal microscopy equipment alsohas a limited threshold.
Regrowth of nerve fibers into the ablated area is also essential for restoration of cornealsensitivity after refractive surgery. After PRK, the central sensitivity, measured using a CochetBonnet esthesiometer, returns to the preoperative level by 3 months (PerezSantonja et al. 1999,Matsui et al. 2001, Lee et al. 2005). Until then, the cornealacrimal gland reflex arc, whichregulates tear fluid release from the lacrimal glands, is supposed to be damaged, and severalpatients complain about dryness of the ocular surface for the first few months after PRK.
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3. Corneal sensitivity after high myopic LASIK (II)
Corneal mechanical sensitivity detection thresholds over the long term after high myopicLASIK did not differ from those of control subjects. Accordingly, the results of objective dryeye tests were similar to those of controls. However, the patients reported ongoing oculardiscomfort and symptoms resembling dry eye (photophobia, grittiness, foreign body sensation,and ocular irritation) significantly more than age and sexmatched control subjects. Loss ofsensitivity and recovery of sensitivity after LASIK have been shown to correlate with highcorrections (Kim and Kim 1999, Nassaralla et al. 2003, Bragheeth and Dua 2005, Shoja andBesharati 2007). It is therefore logical to presume that corneal nerves are implicated in thisprocess in some way. On the other hand, high correction seems to be one of the risk factors forpatients developing socalled chronic dry eye syndrome after LASIK (reviewed by Albietz andLenton. 2004, De Paiva et al. 2006, Shoja and Besharati 2007).
De Paiva and Pflugfelder (2004) found decreased corneal sensitivity after LASIK in patientswithout dry eye. They also observed corneal hypersensitivity in patients with postLASIK dryeye compared with normal controls. The LASIK group consisted of 20 patients 140 monthsafter myopic or hyperopic correction; data for refractive corrections were not provided. ThepostLASIK dry eye group included six patients who reported dry eye symptoms and had ocularsigns compatible with dry eye after a mean interval of 12 months (range 3–36 months)following myopic LASIK. The authors suggested that corneal hypersensitivity observed in dryeye patients is due to compromised ocular surface barrier function and hypersensitivity to airjet. Some of the differences between their study and my own may be explained by 1) I includedonly patients with high myopic corrections, and 2) the mean interval after surgery in my studywas longer (44.2 ± 11.3 months, range 2358 months).
In addition to decreased reflex and basal tear production, impairment of corneal nerves maylead to LASIKinduced neurotrophic epitheliopathy (LINE) (Wilson 2001, Wilson andAmbrosio 2001, reviewed by Ambrosio et al. 2007), where punctate epithelial microerosionscan be detected on the corneal surface. However, patients who developed symptoms and signsof dry eye after LASIK had no significant difference in tear production detected by Schirmer’stest with anesthesia from patients who had no symptoms or signs of dry eye at timepoints from1 month to 6 months after surgery (Wilson 2001). LASIKassociated dry eye has beensuggested to represent a neurotrophic epitheliopathy rather than an actual dry eye syndrome(Wilson 2001). This is supported by the data presented in my study of high myopic LASIKpatients. The majority of patients reported ocular discomfort resembling dry eye symptoms,although clinical dry eye tests were normal and corneal sensitivity levels were within normallimits. These symptoms of ocular discomfort could be in part be derived from aberrantlyregenerated corneal nerves.
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LASIK DRY EYE HYPOTHESISLASIK patients complain
about ongoing dry eye symptoms
ALTHOUGH...
Some of the symptoms may becaused by aberrantly
regenerated nerve fibers
?Objective clinicaldry eye tests are normal
Mechanical sensitivityis normal
Figure 6. LASIK dry eye hypothesis: in the long term (25 years) after high myopic LASIK, the majorityof patients complain about ongoing dry eye –like symptoms. However, clinical dry eye tests are normal,and corneal mechanical sensitivity is comparable with that of normal controls. Some of these symptomsmay thus be derived from aberrantly regenerated nerve fibers, representing a form of cornealneuropathy rather than conventional dry eye disease.
Other issues possibly contributing to dry eye symptoms after LASIK include differences incorneal curvature; while normal corneas show prolate profiles, i.e. are steeper in the center, highconventional myopic LASIK produces oblate corneas. These changes in corneal curvature mayinterfere with even tear distribution, but mechanical rubbing due to the actual anatomicalchange may also be causative. Mechanical trauma caused by the microkeratome suction ring to
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limbal Goblet cells has been suggested to play a role in LASIK dry eye (Shin and Lee 2006).Diminished afferent input from corneal nerves also results in a decreased blinking rate, whichmay be involved in the pathogenesis of LASIK dry eye (Toda et al. 2001). Moreover, patientswho seek refractive surgery are often contact lensintolerant and may have preclinical dry eyebefore surgery. Unfortunately, my study was crosssectional and I was not able to conductpresurgical examinations.
Lay patients are unable to differentiate between symptoms of ocular dryness and "sore eyes"due to other basic conditions. In the end, however, the most important aspect is the subjectivesymptoms experienced by patients. Interestingly, patients report relief in symptoms when usingartificial tears, which may be explained by lowering the frictional forces between lids and theocular surface, and thus, the corneal afferent nerves being less activated.
Some of the symptoms experienced may be derived from aberrantly regenerated corneal nervesafter LASIK surgery. LASIK dry eye could in fact be a sort of “phantom pain”, representing aform of corneal neuropathy rather than conventional dry eye syndrome. This new insight intothe pathogenesis of LASIK dry eye syndrome as a form of corneal neuropathy has clinicalimplications and also offers novel therapeutic possibilities. Patients should be informed of thepossibility of developing chronic dry eye symptoms, especially after deep ablations. In severecases of disabling chronic pain after LASIK, when conventional therapeutic possibilities fail tooffer relief, consultation of a physician specialized in pain treatment is recommended.
4. Corneal sensitivity in primary Sjögren’s syndrome (IV)
Corneal sensitivity detection thresholds to mechanical air jet stimuli were significantly decreasedin patients with pSS dry eye, implicating corneal hypersensitivity. My finding is in line withearlier observations by De Paiva and Pflugfelder (2004), who also found decreased mechanicalsensitivity detection thresholds in dry eye measured using a similar modified Belmontenoncontact gas esthesiometer. By contrast, Bourcier et al. (2005) and Benitez Del Castillo et al.(2007) reported increased corneal sensitivity detection thresholds in dry eye patients, suggestingcorneal hypoesthesia.
Many variables, such as selection of patients and controls and disease severity, can influence theresults. For example, in the Bourcier study, only onethird of patients had a diagnosis of pSS orsSS. I included only patients with a diagnosis of pSS and age and sexmatched controls. Inaddition, variability among noncontact gas esthesiometers is inevitable and may explain part ofthe discrepancy. We and De Paiva and Pflugfelder (2004) used a modified Belmonteesthesiometer, while Bourcier et al. and Benitez Del Castillo et al. utilized the original Belmonteesthesiometer. Differences in the size of the tip, the tip distance to the cornea, and the size of the
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corneal area stimulated by the air jet are important factors. Moreover, therapeutic approaches intreating dry eye obviously differ. For instance, different use of topical and/or systemic antiinflammatory pharmaceuticals during the course of the disease may affect the results.
Decreased corneal sensitivity in dry eye utilizing the CochetBonnet esthesiometer was observedby Xu et al. (1996) and Villani et al. (2007). Different results in these cases might be explainedby different research methodologies, as the CochetBonnet esthesiometer uses mechanicalcontact probe stimulation with some limitations in sensitivity and reproducibility. In addition, amechanical contact probe and a noncontact air jet stimulate corneal afferent sensory nerveendings differently.
De Paiva and Pflugfelder (2004) suggested that the corneal hypersensitivity observed in dry eyeis due to compromised ocular surface barrier function. In my study, I found a positive correlationbetween corneal mechanical sensitivity and increased corneal fluorescein staining, supportingthis part of their hypothesis. In addition, I observed a positive correlation between cornealmorphology, corneal mechanical (hyper)sensitivity, and subjective symptoms, indicating that inpatients with the most severe ocular symptoms weak stimuli are recognized and may even elicitpainful sensations. The findings of altered corneal nerve morphology may offer anotherexplanation for the observed corneal mechanical hypersensitivity.
5. Corneal nerves in primary Sjögren’s syndrome (II and IV)
Corneal subbasal nerve density was similar between pSS patients and controls (III and IV),suggesting that this may not per se explain the difference seen in corneal sensation. Previouslypublished data concerning corneal nerve density in dry eye have been conflicting. Increasedsubbasal nerve counts in aqueous teardeficient patients were observed by Zhang et al. (2005),whereas decreased nerve counts were reported by Benitez Del Castillo et al. (2004), and (2007)and Villani et al. (2007). However, in the Benitez Del Castillo studies, a statistically significantdifference was found between the SS group and the control group composed of youngersubjects, but not between the SS group and the control group of older subjects. Althoughcorneal nerve density is easily calculated and quantified, other properties of the corneal nerves,such as morphological alterations and inflammatory findings, seem to play a more importantrole than nerve density, which may be increased, decreased, or similar compared with controls.
Although corneal subbasal nerve density showed no difference, several morphologicalalterations were present in corneal nerves. Nerve growth conelike structures in subbasal nerveswere found in 2040% of our patients (III / Figs. 2A and 2B; IV / Fig. 2). These alterations mayimplicate ongoing nerve sprouting, and may result from attempts of the inflammationinjured
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nerve fibers to regenerate. Inflammation is recognized as a critical factor in the pathogenesis ofdry eye, and the upregulation of neurotrophins, e.g. nerve growth factor (NGF), duringinflammation has been shown in many studies. NGF is known to induce axonal regeneration,nerve sprouting, and when overexpressed even hypertrophy of the peripheral nervous system(Albers et al. 1994). On the other hand, focally applied neutralizing antibodies to neurotrophicfactors reduce collateral axonal branching after peripheral nerve lesion (Streppel et al. 2002).NGF is expressed in alphasmooth muscle actinpositive dermal myofibroblasts (Hasan et al.2000), which are comparable with corneal altered keratocytes. Altered keratocytes developduring the corneal wound healing process (reviewed by Jester et al. 1999), and in variousinflammatory conditions (reviewed by Petrol et al. 1998, Rosenberg et al. 2002). I observedaltered anterior keratocytes with signs of activation: hyperreflectivity or visible processes, in5/10 SS corneas (III / Fig. 1C). Interestingly, the majority of these corneas also showed nervegrowth conelike structures. Presumably, these altered keratocytes are the source of NGF in SSdry eye. Recently, it has been shown that patients with dry eye present with elevated tear fluidlevels of NGF (Lee et al. 2006), and topical antiinflammatory treatment with prednisolonedecreases tear fluid NGF levels and markedly alleviates dry eye symptoms (Lee et al. 2006).
Stromal nerves appeared significantly thicker in SS patients than in controls. A similar findingwas reported by Benitez Del Castillo et al. 2004. The implication of the thickening of stromalnerves remains unclear. However, it has earlier been shown using immunoelectron microscopyand morphometry that nerve fibers regenerating after an inflammatory insult are significantlythicker than normal healthy nerve fibers (Imai et al. 1997, Niissalo et al. 2002). Possibly,stromal nerves are thickened secondary to chronic ocular surface inflammation or this findingmay represent a form of neural regeneration.
Antigenpresenting cells (APCs) play a critical role in corneal immunology in health anddisease (Hamrah et al. 2002, 2003a, 2003b). APCs have been observed in living corneas bymodern IVCM (Rosenberg et al. 2000a, 2002, Zhivov et al. 2005, 2007, Mastropasqua et al.2006). The density of APCs declines from the limbus to the center in healthy corneas (Hamrahet al. 2002, 2003b, Zhivov et al. 2005, 2007, Mastropasqua et al. 2006). In the corneal limbalepithelium, dendritic cells are present in virtually all healthy subjects (Mastropasqua et al.2006), while in the central cornea only some 2030% of healthy controls show APCs (Zhivov etal. 2005, 2007, Mastropasqua et al. 2006). APCs are more often found in the peripheral cornea,where they show signs of having a mature phenotype with long slender dendritic processes,while immature APCs without dendrites typically predominate in the central cornea (Zhivov etal. 2005). We focused on the central cornea and observed APCs in 35% of patients and in 10%of controls. The density of the APCs in the central cornea in pSS patients was significantlyhigher than that reported in healthy subjects (Zhivov et al. 2005, 2007, Mastropasqua et al.2006). The high APC density together with the mature phenotype of these cells in the central
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cornea (IV / Fig. 6) suggest that these cells are actively involved in the local pathomechanism asantigen processing and presenting cells. This may contribute to the perpetuation of dry eye andeven lacrimal gland adenitis, as it has recently been shown that desiccating stress exposesantigenic epitopes shared by the ocular surface and lacrimal glands (Niederkorn et al. 2006).
Primary Sjögren’s syndrome patients presented with corneal mechanical hypersensitivity,although their corneal nerve density did not differ from that of controls. Corneal sensitivityappeared to correlate with subjective symptoms and objective ocular signs, but not with cornealnerve density. However, alterations in corneal nerve morphology (nerve sprouting andthickened stromal nerves) and an increased number of APCs, implicating the role ofinflammation, were observed. I hypothesize that chronic inflammation and diminished volumeof tear fluid, enriched in proinflammatory cytokines such as IL1 and IL6 (Tishler et al. 1998,Solomon et al. 2001), lead to keratocyte activation, which is followed by synthesis of NGF orother neuronal growth factors. NGF and its highaffinity receptor TrkA are expressed in thehuman corneal stroma and epithelium (Lambiase et al. 2000, You et al. 2000). In addition tohypertrophy of neural tissue (Albers et al. 1994), NGF facilitates transmission of pain(hyperalgesia), possibly due to its effect on ion channels (Lewin et al. 1993, reviewed by Apfel2000, Gould et al. 2000). Overexpression of neuronal growth factors could explain not only theobserved changes in corneal nerves and corneal hypersensitivity but also the tenderness – oreven chronic pain and ocular hyperalgesia often observed in these patients.
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SJÖGREN’S SYNDROMEHYPOTHESIS
Hyperalgesia
Nerve sproutingDegeneration ofepithelium and nervesKeratocyte activation
Neurotrophins
Chronic ocularsurface inflammation Langerhan’s cells IL1, IL6, etc
Figure 7. Sjögren’s syndrome hypothesis: ongoing ocular surface inflammation along with diminishedtear fluid volume, enriched with proinflammatory cytokines, leads to degeneration of the surfaceepithelium and subbasal nerves. This results in activation of altered keratocytes, followed by synthesis ofNGF or other neuronal growth factors. Overexpression of neuronal growth factors could explain notonly the observed changes in corneal nerves and corneal hypersensitivity but also the tenderness – oreven the chronic pain and ocular hyperalgesia often observed in these patients.
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SUMMARY AND CONCLUSIONS
Pre or postoperative levels of the tear fluid cytokines TGF 1, TNF and PDGFBB did notcorrelate with the development of corneal haze, as estimated by IVCM at 3 months after PRK.However, it is noteworthy that none of our patients showed severe grade 3 or 4 haze. Subbasalnerve fibers were observed in the central cornea in 18/20 eyes. A positive correlation wasobserved between the regeneration of subbasal innervation (based on the IVCM nerve count at3 months after PRK) and the thickness of regenerated epithelium, possibly reflecting the trophiceffect of corneal nerves on epithelium. However, the CMTF haze estimate did not correlate withregeneration of the subbasal nerve plexus.
The majority of patients reported ongoing dry eye symptoms long after high myopic LASIK,although objective clinical signs were not demonstrable. No difference in corneal sensitivitywas observed between patients and controls. I assume that at least some of the symptoms maybe derived from aberrantly regenerated corneal nerves after LASIK surgery, thus representing aform of corneal neuropathy rather than conventional dry eye.
Primary Sjögren’s syndrome patients presented with corneal mechanical hypersensitivity,although their corneal subbasal nerve density did not differ from that of controls. Cornealmechanical sensitivity appeared to correlate with subjective symptoms and objective ocularsigns, but not with corneal nerve density. However, alterations in corneal nerve morphology(nerve sprouting and thickened stromal nerves) and an increased number of APCs amongsubbasal nerves, implicating the role of inflammation, were observed. We suggest that thesefindings offer an explanation for the corneal hypersensitivity or even ocular hyperalgesia oftenobserved in these patients.
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ACKNOWLEDGMENTS
This study was carried at the Department of Ophthalmology, Helsinki University CentralHospital, from 2000 to 2007. I am sincerely grateful to Professor Leila Laatikainen and hersuccessor, Professor Tero Kivelä, the current head of the Department of Ophthalmology, forproviding excellent working facilities.
My deepest gratitude goes to my supervisor, Professor Timo Tervo, with whom I have hadnumerous inspiring conversations on scientific issues as well as on other common subjects ofinterest. Without his innovative ideas, my thesis would not be the same.
I am sincerely grateful to Professor Yrjö T. Konttinen. I have been privileged to be able tocollaborate with him and to share thoughts and ideas 24 hours a day, 365 days a year, literally.His consistent enthusiasm and positive thinking were instrumental to the completion of thisthesis.
I am deeply grateful to Docent Minna Vesaluoma. Without her, I would not have been able tostart my scientific career in 1998.
My thanks also belong to my collaborators and colleagues Carola GrönhagenRiska, MaaretHelintö, Juha Holopainen, Eija Kaila, Sissi Katz, Liisa Konttinen, Maria Lamberg, NinaLindbohm, Jukka Moilanen, Waldir Neira, AnnaMaija Teppo, Tuuli Valle, and Steven Wilson.
Professors Hannu Uusitalo and MarjaLiisa Vuori, the official reviewers of the thesis, arethanked for constructive criticism that vastly improved the manuscript.
I am deeply grateful to my authoreditor Carol Ann Pelli for editing the language of thismanuscript, and also to architect Anna Pakkala for drawing the pictures of my thesis.
Special thanks go to my teacher of ophthalmology in medical school in 1997, Docent PaulaSummanen. She inspired my interest in ophthalmology at the beginning of my career. I alsoowe special thanks to Jouko Larinkari, MD and Tapio Stenborg, MD, for their encouragementand for teaching me the basics of ocular surgery and ophthalmology.
I am very grateful to Richard C. Troutman, MD, and the International Society of RefractiveSurgery of the American Academy of Ophthalmology for the prestigious Troutman Award in2007. I was privileged to meet Dr. Troutman, a pioneer in microsurgery of the eye, and reallyappreciate his encouragement.
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Scientific work has brought an enormous amount of joy and contentment into my life. I cannotimagine my years as an ophthalmic resident without being involved in academic research.Several interesting congresses around the world and many new friends are but a few examples.Research has opened my eyes in so many ways and given me courage to make my owndecisions, not always blindly accepting information written by others.
I am deeply grateful to have wonderful parentsinlaw, Arja and Tapani Tuisku, who havealways been there for me and lent a helping hand.
I have been blessed with loving parents, my mother MarjaLeena and my late father SeppoTuominen. They have always been supportive and understanding and have encouraged me tofollow in their footsteps as an MD. I remember discussions between my father and myself as alittle boy about the definition of an academic dissertation. It was not easy to understand whypeople have to argue about things – why can’t they just agree? It took some thirty years for meto discover the answer. I also wish to thank my sister Anna and my brothers Jouni and Joonasfor their interest in my project.
This work is dedicated to my family, to whom I owe my warmest thanks, especially to mywonderful wife Johanna, who has encouraged and sometimes even pressured me to finish thisthesis. She has always taken such good care of us boys (see below), although it has sometimesrequired exceptionally long nerve fibers. My two lively sons, Tom (4 years) and Matias (3years), have brought an enormous amount of energy and joy into my life. Unfortunately, I havenot been able to do things with them as much as I would have liked. Thankfully, though, theyunderstand that we are not always able to do our men’s jobs, such as fixing things with ourdifferent tools, because daddy must sometimes leave to fix eyes...
This work was supported by the Mary and Georg C. Ehrnrooths Foundation, the Finnish EyeFoundation, the Finnish Medical Society Duodecim, the Finnish Eye and Tissue BankFoundation, the Friends of the Blind Foundation, the Paulo Foundation, and State EVO grants.
Helsinki, January 2008
Ilpo S. Tuisku
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