Loss Of Keratocytes Research Articles

Long-term Outcomes of Penetrating Keratoplasty in Chronic and Delayed Mustard Gas Keratitis

To report the long-term outcomes of penetrating keratoplasty (PKP) in war victims with chronic and delayed mustard gas keratitis. This noncomparative interventional case series includes patients with advanced chronic or delayed mustard gas keratitis who had undergone PKP from 1989 to 2006. Best-corrected visual acuity (BCVA), graft clarity, episodes of graft rejection, duration of steroid use, and complications were evaluated. Histopathologic features of excised corneal buttons were also evaluated. Overall, 22 eyes of 19 patients underwent PKP. Mean age at the time of surgery was 41 +/- 4.6 years (range, 36-54 years), and mean follow-up duration was 40.9 +/- 48 months (range, 4-204 months). The graft remained clear in 17 (77.3%) eyes and failed in 5 (22.7%) eyes. Overall, 13 (59.1%) eyes experienced episodes of endothelial rejection, and 5 (22.7%) eyes had subepithelial immune rejection, 4 of which had simultaneous endothelial rejection. Fifteen (68.2%) eyes received topical steroids for >6 months. Fourteen (63.6%) eyes developed cataracts, leading to cataract extraction in 7 eyes. One eye developed steroid-induced glaucoma after multiple episodes of endothelial graft rejections. Mean preoperative BCVA was 1.92 +/- 0.63 logMAR, which improved to 1.04 +/- 0.65 logMAR (20/200) overall and 0.8 +/- 0.3 logMAR (20/120) in eyes with clear grafts (P < 0.001). Main histopathologic features of excised corneal buttons included corneal thinning and ulceration, loss of keratocytes, acute and chronic inflammation, stromal vascularization, and degenerative sequelae of long-standing inflammation. PKP in chronic or delayed-onset mustard gas keratitis should be considered as a high-risk graft; however, with appropriate management, graft clarity and visual outcomes may be favorable.

Prospective Evaluation of PermaVision Intracorneal Implants Using In Vivo Confocal Microscopy

To report effects of the PermaVision intracorneal lens at the cellular level using in vivo confocal microscopy. Four eyes implanted with intracorneal lenses beneath an IntraLase flap for correction of hyperopia were evaluated preoperatively and 1 to 6 months postoperatively. Intracorneal lenses were tolerated in three eyes with little or no haze observed clinically and good visual results. Minimal keratocyte activation was detected by confocal microscopy, and cell density was decreased posterior to the implants. Epithelial thinning was observed 1 month after implantation. Thickness stabilized by 6 months but remained thinner than baseline (33 +/- 2 microm vs 48 +/- 8 microm, P < .01). The fourth eye had a complicated course with early flap displacement followed by diffuse lamellar keratitis. Confocal microscopy revealed activated keratocytes throughout the anterior stroma. The implant was removed, and recovery was promising. Implantation of intracorneal lenses can induce side effects of epithelial thinning, keratocyte loss, and keratocyte activation.

Cellular effects of mitomycin-C on human corneas after photorefractive keratectomy

To investigate the effects of mitomycin-C (MMC) on epithelial and keratocyte cell kinetics after photorefractive keratectomy (PRK) using an in vitro human cornea model. Department of Academic Ophthalmology, Rayne Institute, St. Thomas' Hospital, London, United Kingdom. Twenty-four human eye-bank corneas were placed in a specially designed acrylic corneal holder and cultured using the air-interface organ culture technique for up to 4 weeks. The corneas were divided into 3 groups. Group 1 consisted of 8 human corneas that had -9.00 diopter (D) myopic PRK without MMC application. Group 2 consisted of 8 corneas that had -9.00 D PRK with MMC (0.2 microg/mL) application for 1 minute on the stromal surface after ablation. Group 3 consisted of 8 corneas that had -9.00 D PRK with 2-minute exposure to MMC (0.2 microg/mL). Temporal events in epithelial and keratocyte cell kinetics were evaluated using digital imaging, confocal microscopy, and light microscopy. Epithelial latency was significantly delayed with MMC application in Groups 2 and 3 (P<.001). Epithelial migration was delayed in Group 3 (2-minute exposure) compared to migration in Group 2 (P<.04), with a consequent delay in epithelial closure (P<.001). Group 3 corneas had poorly differentiated epithelium that was significantly thinner than in Groups 1 and 2 (P<.0001). A significant delay in keratocyte regeneration occurred after MMC application (P<.0005). At 4 weeks, the anterior stromal cell density was significantly lower in Group 3 than Group 2 (P<.001). There were no significant differences in the mid- and posterior stromal keratocyte density between the groups. Results suggest that epithelial healing after MMC is characterized by prolonged latency and decreased migration rate dependent on exposure time. Mitomycin C application did not result in increased loss of keratocytes, but it significantly delayed keratocyte repopulation in the anterior stroma. The use of MMC 0.2 microg/mL for 1 minute resulted in optimum modulation of healing characterized by reduced keratocyte activation with normal epithelial differentiation.

How has confocal microscopy helped us in refractive surgery?

To summarize the known uses of in-vivo confocal microscopy in refractive surgery, highlighting the current developments in the field. Examination of the cornea after laser in-situ keratomileusis demonstrated that the keratocyte density within the laser in-situ keratomileusis flap and anterior residual corneal bed continued to decline during the entire 3-year period of the study. The progressive loss of keratocytes in the flap and anterior portion of the residual corneal bed could have long-term implications in terms of corneal stability, refractive stability and cellular integrity after laser in-situ keratomileusis. Additional studies showed that the density of sub-basal nerves decreased by 90% 1 month after laser in-situ keratomileusis. At some point between 3 and 6 months after laser in-situ keratomileusis, the sub-basal nerves began to recover and by 2 years they had reached approximately 50% of their original preoperative density. Analysis of sub-basal nerve density after photorefractive keratectomy reported that the nerve density completely recovered to preoperative levels by 2 years. Other confocal microscopic studies demonstrated that the microscope can detect infectious organisms in vivo, without stains or dyes. The confocal microscope is a unique diagnostic instrument that can be used to evaluate corneal healing, long-term stability and to assess complications after refractive surgery. The ability of the device to view in-vivo cellular detail, microorganisms, inflammatory cells, epitheliod cells, fibrosis and measure the postoperative thickness of the residual corneal bed after laser in-situ keratomileusis, in a noninvasive manner, highlights the unique capabilities of this instrument.

Prevention of stromal cell loss after superfitial corneal injury in rabbits

For the study of the effects of trauma on the cornea, usually a rabbit eye is used as a model. The aim of this experimental study of corneal surface injury was to evaluate stromal cell loss after epithelial scraping and to indicate the possibilities of preventing it by applying contact lenses or topical heparin. The study was conducted on 21 Chinchilla gray rabbits, which were divided into five groups. The central portion measuring 6mm of corneal epithelium was removed from one eye of 18 experimental animals. Three rabbits in the fifth group, with intact corneas, served as controls. Postoperatively, the first experimental group (n=3) was treated for 24 hours with topically applied heparin. The second group (n=3) was treated for seven days with subconjunctivally applied heparin. In the third group, (n=6) soft contact lenses were placed for five days. The fourth group of (n=6), has not been treated after injury. During the first postoperative day, anterior stromal keratocytes were lost after epithelial removal, but the cell repopulation was completed within two weeks. Twenty four hours after deepithelization the corneas showed decreased density of keratocytes within the anterior stroma compared with the posterior storma. Topically applied heparin for 24 hours after deepithelization prevented keratocyte loss (p 0.05). The eyes treated with contact lenses had a significantly higher number of keratocytes than the control corneas and corneas treated with heparin. We were able to conclude that topical application of heparin during the first day and contact lens application during the first week, can minimize keratocyte loss and may thus be beneficial in the healing process.

Assessment of Stromal Keratocytes and Tear Film Inflammatory Mediators During Extended Wear of Contact Lenses

To monitor quantitative changes in stromal keratocyte density and the level of tear film inflammatory mediators following extended contact lens wear. Twenty-two subjects aged 32 +/- 11 years participated in this cross-sectional study. Eleven subjects had worn silicone hydrogel (Si-H) lenses on a 30-day continuous wear basis for 12 months. Eleven subjects had worn rigid gas permeable lenses on the same basis for 12 months. Eleven age-matched control subjects were also recruited. Ultrasound pachometry, confocal microscopy, and tear fluid sample collection were performed on all subjects. Tear samples were assayed for epidermal growth factor (EGF), hepatocyte growth factor (HGF) and interleukin (IL)-8. Corneal thickness was similar for all subject groups. Total keratocyte density was not different between the 3 groups; however, keratocyte density was lower for rigid lens wearers in the anterior to mid stroma and lower for Si-H lens wearers in the posterior stroma compared with control subjects. Rigid lens wearers exhibited an irregular keratocyte distribution across the corneal stroma. EGF concentration and rate of release was greater in the tears collected from the rigid lens wearers and Si-H lens wearers, and IL-8 concentration was higher in the samples collected from the rigid lens wearers compared with the samples collected from the control subjects. Mechanical stimulation of the corneal surface due to the physical presence of a contact lens and the consequent release of inflammatory mediators may account for a loss or redistribution of keratocytes.

In vitro human corneal model to investigate stromal epithelial interactions following refractive surgery

To develop an in vitro human corneal model to evaluate stromal epithelial interactions following corneal refractive surgical procedures. Department of Academic Ophthalmology, Rayne Institute, St. Thomas' Hospital, London, United Kingdom. Fifty-six human donor corneas procured from the eye bank were placed in a specially designed acrylic corneal holder and were cultured using the air-interface organ culture technique for up to 4 weeks. Corneal refractive surgical procedures such as a simple epithelial defect, 4 diopter (D) and 9 D photorefractive keratectomy (PRK), 4 D and 9 D laser-assisted subepithelial keratectomy (LASEK), and 9 D laser in situ keratomileusis (LASIK) were performed on the model. Temporal events in epithelial and keratocyte cell kinetics were evaluated using digital imaging, confocal microscopy, and light microscopy. Two-way analysis of variance and Student t tests were used to assess statistical significance. Epithelial healing following PRK was completed by 92 hours +/- 10 (SD) at a rate of 0.58 +/- 0.45 mm2/hour. In LASEK, the epithelial flap was replaced by regenerating peripheral epithelium that showed significant delay in epithelial closure (120 +/- 5 hours) with prolonged latency (24 +/- 4 hours, P<.0001) in comparison with PRK. The magnitude of keratocyte loss corresponded to ablation depth, and keratocyte regeneration was dependent on epithelial closure. In comparison, LASIK corneas showed a lesser percentage of keratocyte loss with poor recovery of keratocyte density in the stromal flap. Epithelial viability and keratocyte density were well preserved in the in vitro human model as observed in control corneas for up to 4 weeks. The temporal events in stromal epithelial interactions in the in vitro human model closely mimicked in vivo observations. The human model further avoided species-specific variations and provided a suitable test bed for evaluating newer algorithms and therapeutic regimens following refractive surgery.

Corneal Keratocyte Apoptosis Following Topical Intraoperative Mitomycin C in Rabbits

To determine the soaking duration and concentration effect of mitomycin C (MMC) on the corneal keratocyte following single intraoperative application. Two hundred ten pigmented rabbit eyes underwent mechanical epithelium debridement of the central 10-mm comea followed by soaking of six different solutions: MMC1A: 0.01% MMC x 1 minute; MMC1B: 0.01% MMC x 2 minutes; MMC2A: 0.02% MMC x 1 minute; MMC2B: 0.02% MMC x 2 minutes; BSS1: balanced salt solution (BSS) x 1 minute; and BSS2: BSS x 2 minutes. Thirty-five eyes were allocated into each group. Changes of the central comeal thickness, corneal clarity score, and keratocyte apoptosis were examined on days 0, 1, 2, 3, 5, 7, and 14. A significant increase of central corneal thickness and corneal clarity score was noted in all MMC treated groups. The central corneal thickness recovered to its baseline level whereas the corneal clarity score remained increased at 2 weeks. The changes were more significant in MMC2 than in MMC1 corneas. However, changes of central corneal thickness and corneal clarity score were more significant in MMC1B than in MMC1A, whereas no difference was noted between MMC2A and MMC2B. Significantly higher stromal keratocyte loss and keratocyte apoptosis, from superficial to deep corneal stroma, was noted in MMC1 and MMC2 groups up to 14 days postoperatively, in a soaking duration and dose-dependent manner. Single application of MMC on the corneal surface caused soaking concentration and duration dependent corneal edema and keratocyte apoptosis in this rabbit model.

Keratocyte loss in corneal infection through apoptosis: a histologic study of 59 cases

BackgroundKeratocyte loss by apoptosis following epithelial debridement is a well-recognized entity. In a study of corneal buttons obtained from patients of corneal ulcer undergoing therapeutic keratoplasty, we observed loss of keratocytes in the normal appearing corneal stroma, surrounding the zone of inflammation. Based on these observations, we hypothesized that the cell loss in the inflammatory free zone of corneal stroma is by apoptosis that could possibly be a non-specific host response, independent of the nature of infectious agent.MethodsTo test our hypothesis, in this study, we performed Terminal deoxyribonucleotidyl transferase-mediated d-Uridine 5" triphosphate Nick End Labelling (TUNEL) staining on 59 corneal buttons from patients diagnosed as bacterial, fungal, viral and Acanthamoeba keratitis. The corneal sections were reviewed for morphologic changes in the epithelium, stroma, type, degree and depth of inflammation, loss of keratocytes in the surrounding stroma (posterior or peripheral). TUNEL positivity was evaluated in the corneal sections, both in the zone of inflammation as well as the surrounding stroma. A correlation was attempted between the keratocyte loss, histologic, microbiologic and clinical features.ResultsThe corneal tissues were from 59 patients aged between 16 years and 85 years (mean 46 years) and included fungal (22), viral (15), bacterial (14) and Acanthamoeba (8) keratitis. The morphological changes in corneal tissues noted were: epithelial ulceration (52, 88.1%), destruction of Bowman's layer (58, 99%), mild to moderate (28; 47.5%) to severe inflammation (31; 52.5%). Morphologic evidence of disappearance or reduced number of keratocytic nuclei in the corneal stroma was noted in 49 (83%) cases; while the TUNEL positive brown cells were identified in all cases 53/54 (98%), including cases of fungal (19), bacterial (14), viral (13), and Acanthamoeba keratitis. TUNEL staining was located mostly in the deeper stroma and in few cases the peripheral stroma. TUNEL positivity was also noted with the polymorphonuclear infiltrates and in few epithelial cells (10 of 59, 17%) cases, more with viral infections (6/10; 60%).ConclusionsWe report apoptotic cell death of keratocytes in the corneal stroma in infectious keratitis, a phenomenon independent of type of infectious agent. The inflammatory cells in the zone of inflammation also show evidence of apoptotic cell death. It could be speculated that the infective process possibly triggers keratocyte loss of the surrounding stroma by apoptosis, which could possibly be a protective phenomenon. It also suggests that necrotic cell death and apoptotic cell deaths could occur simultaneously in infective conditions of the cornea.

Protective role of corneal epithelium against ultraviolet radiation damage

It is known that the corneal epithelium strongly absorbs ultraviolet radiation (UVR). The aim of the present study was to examine the protective role of corneal epithelium against UVR damage by comparing the biological effect of UVR exposure on whole corneas with that on de-epithelialized corneas. Six New Zealand albino rabbit corneas were exposed to UVR centred around 280 nm at a dose that causes biomicroscopically significant keratitis (012 J/cm(2)). Three corneas underwent manual de-epithelialization prior to UVR exposure. A control group of three rabbits underwent only manual de-epithelialization. The animals were killed 76 hours after treatment. The corneas were stained with haematoxylin and evaluated by light microscopy. Corneas that underwent only the exposure to UVR showed a loss of epithelial cells in the treated area. No damage to keratocytes or the stroma was detected. Corneas that underwent manual de-epithelialization showed a loss of epithelial cells, and also keratocytes in the anterior quarter of the corneal stroma. However, corneas that were exposed to UVR after manual de-epithelialization showed very deep stromal damage. The keratocytes disappeared through the entire thickness of the stroma in the UVR-exposed area. Exposure to UVR at 280 nm alone does not result in any deep damage to the corneal stroma and keratocytes. Manual de-epithelialization causes the disappearance of anterior keratocytes. However, the stromal damage caused by UVR in the de-epithelialized corneas was very deep. The corneal epithelium serves to protect the deeper corneal structures against UVR damage, probably by absorbing a substantial amount of the UVR energy applied to the eye.

The corneal stroma during contact lens wear

Recent technological advances have lead to novel descriptions of the microanatomy of the corneal stroma. In the first section of this review, these findings and the role they play in the maintenance of vital properties such as corneal transparency, mechanical strength, homeostasis, wound-healing response and metabolism are described. In the second part, contact lens induced stromal alterations such as acidosis, oedema, striae, thinning and opacities are reviewed as well as the more recently described phenomenon of microdot deposits and keratocyte loss with an emphasis on how lens wearing stromal effects can be minimised.

Uncoupling Keratocyte Loss of Corneal Crystallin from Markers of Fibrotic Repair

Corneal crystallins are lost from resident cells of the corneal stroma during wound repair, and this is associated with a loss of cell transparency. The goal of this study was to identify factors inducing loss of the corneal crystallin transketolase (TKT). A cell culture model of freshly isolated rabbit corneal keratocytes was used. Fibrotic markers included cell proliferation, adoption of a "fibroblastic" spindle-shaped morphology associated with cytoskeletal rearrangement, loss of TKT, and expression of alpha-smooth muscle actin (alpha-sm actin), a marker for the myofibroblast. When freshly isolated keratocytes were cultured in the continuous presence of 10% calf serum, the high level of intracellular TKT protein was reduced dramatically within 24 to 48 hours. In contrast, TKT protein was retained in cells maintained in the absence of serum. When cells were prevented from proliferating by exposure to serum for <24 hours or by continuously exposing to serum at a contact-inhibiting plating density, TKT loss was inhibited. TKT loss was induced by treatment of serum-free cultures with the serum cytokines platelet-derived growth factor or basic fibroblast growth factor, both of which also stimulated keratocyte proliferation, although not other changes associated with fibrosis. However, TKT loss was not induced by treatment of serum-free cultures with a third serum cytokine, transforming growth factor- (TGF)-beta, even though TGF-beta stimulated cell proliferation at low doses and induced the fibroblastic spindle-shape and express alpha-sm actin at high doses. TKT loss in corneal keratocytes can be induced by PDGF or bFGF and this loss can be uncoupled from other fibrotic markers. Targeting these cytokines or the signaling pathways that they activate could enable retention of corneal crystallin in stromal cells during repair, a more regenerative outcome. The result would be enhanced clarity of the cornea.

Role of keratocyte loss on corneal wound repair after LASIK.

To investigate whether an initial keratocyte loss intensifies central corneal wound repair after LASIK in rabbits. New Zealand White rabbits received either conventional LASIK (-8 D, 6-mm diameter) or LASIK combined with a 7-mm diameter, epithelial denudation (LASIK-scrape). Animals were examined during 4 months by slit lamp and in vivo confocal microscopy to monitor changes in central corneal morphology, light backscattering (haze), and sublayer thickness. At various time points, corneas were processed for histology and stained for nuclei; F-actin; ED-A fibronectin; alpha-smooth muscle actin; TGF-beta1, -beta2, and -beta receptor II; and connective tissue growth factor (CTGF). In vivo confocal microscopy identified no major acellular zones or changes in cell morphology or reflectivity after conventional LASIK. By contrast, a complete loss of keratocytes was observed in the anterior 77 +/- 25 microm stroma 1 week after LASIK-scrape. Highly reflective, migratory fibroblasts gradually repopulated the acellular zone, and by week 8, quiescent-appearing keratocytes were observed throughout the stroma. Correspondingly, stromal light backscattering peaked at 2 weeks after LASIK-scrape (2200 +/- 620 U) followed by a decline to approximately 60 U from week 8; comparable to the slightly increased reflectivity (approximately 50 U) observed after conventional LASIK (ns). Stromal thickness appeared stable 8 weeks after both LASIK and LASIK-scrape, after a regrowth of 13 +/- 3 and 20 +/- 11 microm, respectively (ns). In addition, both procedures induced a minor and comparable epithelial hyperplasia of 4 +/- 2 and 7 +/- 5 microm, respectively (ns). No myofibroblast transformation or TGF-beta growth factor expression was observed below the flap after either treatment. LASIK-scrape induces an anterior keratocyte loss, leading to development of temporary haze during cell repopulation. However, 8 weeks after both LASIK and LASIK-scrape, only a slightly increased reflectivity is noted at the interface. Corneal thickness is stable by week 8, and stromal regrowth and epithelial hyperplasia are comparable after both treatments. Thus, an initial loss of stromal keratocytes does not appear to intensify corneal wound repair after LASIK.

Decreased keratocyte death after laser-assisted subepithelial keratectomy and photorefractive keratectomy in rabbits

To compare keratocyte loss in the corneal stroma after laser-assisted subepithelial keratectomy (LASEK) and photorefractive keratectomy (PRK) in rabbits. Department of Ophthalmology, University of Essen, Essen, and the Institute of Anatomy, University of Bochum, Bochum, Germany. Laser-assisted subepithelial keratectomy and PRK were performed in rabbits and studied 1, 3, 10, and 20 days after surgery. Excimer photoablation was done unilaterally with a 6.0 mm ablation zone and an 80 microm depth, equivalent to -6.0 diopters. Keratocyte death was analyzed using DNA fragmentation-detecting terminal deoxynucleotidyl transferase deoxy-UTR-nick end labeling (TUNEL) assay and transmission electron microscopy. Numerous TUNEL-positive keratocytes occurred 1 day after PRK; the number decreased significantly after 3 days. After LASEK, significantly fewer TUNEL-positive keratocytes were noted at the early time points (P<.001 at 1 day; P< or =.05 at 3 days). At 10 days, the number of TUNEL-positive keratocytes decreased in both groups but remained significantly higher after PRK than after LASEK (P<.001). Twenty days after both procedures, no significant signs of keratocyte death were found in the corneal stroma. Transmission electron microscopy revealed few apoptotic keratocytes after LASEK. After PRK, apoptotic keratocytes, characterized by chromatin condensation, apoptotic bodies, and cell shrinkage, were scattered in the stroma. The ultrastructural findings confirmed the results obtained with the TUNEL assay. Laser-assisted subepithelial keratectomy induced significantly less apoptotic keratocyte death than PRK and promoted wound healing in the acute phase after photoablation. This procedure may offer the possibility of treating higher myopia with a decreased risk for developing wound healing-related complications known to occur after PRK.

Prevention of experimental diffuse lamellar keratitis using a novel platelet-activating factor receptor antagonist

Purpose: To determine whether a novel platelet-activating factor (PAF) antagonist prevents experimentally induced diffuse lamellar keratitis (DLK) after laser in situ keratomileusis (LASIK). Settings : Department of Ophthalmology and Neuroscience Center of Excellence, Louisiana State University Health Science Center, New Orleans, Louisiana, USA. Methods: Twenty eyes of 10 New Zealand albino rabbits were used. The left eyes were treated with a peribulbar injection of 0.5 mL of PAF receptor antagonist LAU 0901 (2,4,6-trimethyl-1,4-dihydropyridine-3,5-dicarboxylic acid ester) dissolved in 20 hydroxypropyl B cyclodextrin (30 μg /mL). Two rabbits were treated with a peribulbar injection of 0.5 mL of vehicle (cyclodextrin) alone and served as controls. A corneal flap was cut in all eyes, and the interface was exposed to Pseudomonas aeruginosa endotoxin. The left eyes were additionally treated with 1 drop of LAU 0901 4 times a day. Rabbits were killed on postoperative days 1, 2, 3, 5, and 8. The eyes were enucleated and processed for histopathology and immunohistochemical examination. Results: Corneas not treated with LAU 0901 and controls showed a severe inflammatory response in the flap margin and stromal interface, characterized by loss of keratocytes, activation of adjacent keratocytes and transformation to myofibroblasts, infiltration of polymorphonuclear leukocytes and monocytes, and presence of epithelial cells with necrosis and melting of adjacent stroma. Corneas of rabbits treated with LAU 0901 showed minimal loss of keratocytes and myofibroblast transformation, minimal inflammatory cell infiltration, and minimal presence of epithelial cells in the interface. Conclusion: Induction of DLK was blocked by a PAF receptor antagonist in rabbit eyes. The histopathological evaluation and immunohistochemical studies showed that treatment with LAU 0901 blocked keratocyte apoptosis, transformation of fibroblasts to myofibroblasts and migration to the wound site, and chemotaxis of inflammatory cells, inhibiting the inflammatory response and promoting adequate healing of the flap interface and adjacent stroma.

Keratocyte apoptosis after corneal collagen cross-linking using riboflavin/UVA treatment.

Combined riboflavin/UVA treatment inducing collagen cross-links in the cornea has been shown to increase the biomechanical rigidity of the cornea and has been used successfully in the treatment of progressive keratoconus. The current study was undertaken to investigate the possible cytotoxic effect of combined riboflavin/UVA treatment on corneal keratocytes in vivo. Thirty-four New Zealand white rabbits were treated with 0.1% riboflavin solution and surface UVA irradiances ranging from 0.75 to 4 mW/cm2 (1.35- 7.2 J/cm2) for 30 minutes. The animals were euthanized either 4 (n = 6) or 24 (n = 28) hours postoperatively. Four additional control eyes underwent epithelial debridement alone. The corneas of the enucleated eyes were evaluated in routine histologic sections. In addition, the TUNEL technique and transmission electron microscopy were used for the detection of keratocyte apoptosis. In the control eyes with corneal epithelial debridement only, apoptotic keratocytes were found in the anterior 50 microm of the corneal stroma 4 hours postoperatively. However, riboflavin/UVA-induced apoptosis was only visible in the rabbit eyes enucleated 24 hours postoperatively. In these eyes, we found apoptosis of keratocytes down to a variable stromal depth depending on the applied UVA irradiance. A cytotoxic UVA irradiance for keratocytes in the range of 0.5-0.7 mW/cm2 could be deduced. Riboflavin/UVA treatment leads to a dose-dependent keratocyte damage that can be expected in human corneas down to a depth of 300 microm using a surface UVA dose of 5.4 J/cm2. Future studies should be done to examine the keratocyte repopulation and exclude possible adverse sequelae of keratocyte loss like stromal scarring or thinning.

Improved Preparation and Preservation of Human Keratoplasty Lenticules

Purpose: To improve the preparation of lenticules from human cornea and to obtain their preservation without loss of viable keratocytes. Methods: The epithelium was manually removed after bathing the surface of the cornea with a solution of trypsin and EDTA. Lenticules were prepared by microkeratome resection and viable keratocytes were visualized by staining with thiazolyl blue (MTT). Results: The pretreatment with trypsin-EDTA allowed the removal of the epithelium without damage to the keratocytes and the stroma. When these lenticules were incubated in Optisol-GS for 7 days at 4°C, they showed a limited thickness increase and a preservation of keratocyte viability. Conclusion: This procedure allows the preparation of lenticules with viable keratocytes that can be preserved in the cold for at least 1 week.

Confocal microscopic observations of stromal keratocytes during extended contact lens wear

Background: The aim of this study was to monitor changes in keratocyte density during extended contact lens wear and to explore the possible role of hypoxia and oedema in any changes observed. Methods: Twenty‐three neophyte myopic subjects wore a high Dk/t lens (Pure Vision) in one eye and a low Dk/t lens (Acuvue 2) in the other eye on an extended wear basis for six months. Slit‐scanning confocal microscopy and ultrasonic pachometry were performed on both eyes at baseline (before lens wear), after three and six months of lens wear, and one week after cessation of lens wear (the ‘post‐cessation’ visit). Results: No differences were established between die two lenses or between the three study visits for anterior stromal keratocyte density (KD). Posterior stromal KD was similar for the two lenses throughout the study. However, there was an overall drop in posterior KD of 14 per cent in bodl eyes at the six‐month visit, compared to die initial visit. Posterior KD at die six‐mondi visit was no different from diat at die post‐cessation visit. Corneal thickness was similar for die two lenses at die initial and post‐cessation visits, but was diree per cent greater for the eye wearing die Acuvue 2 lens at die six‐month visit. Conclusion: Extended contact lens wear causes a loss of keratocytes; diis loss cannot be attributed to contact lens‐induced hypoxia and/or oedema, or to an artifact of confocal microscopy relating to presence of residual oedema. It is postulated diat lens‐induced keratocyte loss may be related to die physical presence of contact lenses creating some mechanical effect.

Confocal microscopic observations of the human cornea following overnight contact lens wear

Background: Striae and folds are observed with a slidamp biomicroscope in the cornea following overnight contact lens wear. These phenomena are poorly understood. The aim of this study is to employ confocal microscopy to observe and document these and other morphological changes in die human cornea following overnight contact lens wear. Methods: Slitlamp biomicroscopy, slit‐scanning confocal microscopy and ultrasonic pachometry were performed on both eyes of 13 subjects (3M, 10F, age 24 ± 3 years) before and after eight hours overnight wear of a ‐3.00 D Bausch & Lomb one day disposable soft contact lens (Dk/t = 15.1 times 10‐9 (cm/sec) x {ml O2/ml x mmHg)) in one eye; the other non‐lens‐wearing eye acted as a control. Results: Following sleep, both corneas were swollen (lens‐wearing eye 11.8 ± 3.8 per cent; control eye 2.1 ± 1.9 per cent) and the stroma of both corneas displayed an apparent reduction in keratocyte density (lens‐wearing eye 21 per cent; control eye 10 per cent). Folds were observed with the slidamp biomicroscope and long, straight, dark, orthogonal lines were observed widi die confocal microscope, in the posterior stroma of the oedematous lens‐wearing eyes. Such features were not observed in die control eyes. The keratocytes appeared less distinct with greater levels of corneal oedema. Conclusion: The apparent loss of keratocytes following overnight lens wear is an optical artefact that can be explained in terms of corneal oedema causing volumetric tissue expansion and a loss of optical clarity, which hampers keratocyte detection. These findings place the onus on researchers postulating a loss of stromal keratocytes following clinical interventions, such as contact lens wear, to account for the effects of oedema.

Pathology of ocular irritation with bleaching agents in the rabbit low-volume eye test.

Despite differences in the processes leading to tissue damage, the ocular irritation response to various surfactants, two concentrations of an acid and an alkali, and an acetone, alcohol, aromatic amine, and aldehyde has been shown to depend on the extent of initial injury. The purpose of this study was to assess the extent to which this fundamental relationship exists for bleaching agents in the rabbit low-volume eye test. Ten microl of sodium perborate monohydrate (NaBO3), sodium hypochlorite (NaOCl), 10% hydrogen peroxide (H2O2), and 15% H2O2 was applied directly to the cornea of the right eye of each rabbit. Macroscopic assessments for irritation were made 3 hours after dosing and periodically until 35 days. Light microscopic examinations were conducted on tissues obtained at 3 hr and on 1, 3, and 35 days. In vivo confocal microscopy (CM) and measurements of dead corneal epithelial cells and keratocytes at 3 hours and 1 day were used to characterize quantitatively initial corneal injury, while in vivo CM performed at 3 hours and 1, 3, 7, 14, and 35 days was used to characterize quantitatively the corneal changes over time. The changes with NaBO3 and NaOCl were consistent with mild irritancy. For both, corneal injury was limited to the epithelium and superficial stroma. The changes with 10% H202 and 15% H2O2 were consistent with severe irritation. Both concentrations affected the epithelium and deep stroma, with 15% H2O2 also at times affecting the endothelium. However, unlike other irritants previously studied, with 10% H2O2 and 15% H2O2 there was an incongruity between the extent of epithelial and stromal injury, with stromal injury being more extensive than epithelial injury. A similar, although less dramatic, effect was observed with NaBO3. Additionally, there was still significant keratocyte loss at 35 days with 10% H2O2 and 15% H2O2 even though the eyes at times were considered to be macroscopically normal. These observations highlight the need to include both epithelial and stromal components in an ex vivo or in vitro alternative assay. In conclusion, these results continue to support and extend our hypothesis that ocular irritation is principally defined by the extent of initial injury despite clear differences in the means by which irritants cause tissue damage. Importantly, we have identified unique differences in the ocular injury and responses occurring with bleaching agents that are important to consider in the development and validation of alternative ocular irritation tests to characterize a broad range of materials differing in type and irritancy.