Artificial corneas: a regenerative medicine approach.

Corneal substitutes are being developed to address the shortage of human donor tissues as well as the current disadvantages in some clinical indications, which include immune rejection. In the past few years, there have been significant developments in bioengineered corneas that are designed to replace part or the full thickness of damaged or diseased corneas that range from keratoprostheses that solely address the replacement of the cornea's function, through tissue-engineered hydrogels that permit regeneration of host tissues. We describe examples of corneal substitutes that encourage regeneration of the host tissue. We also contend that it is unlikely that there will be a single "one-size-fits-all" corneal substitute for all indications. Instead, there will most likely be a small range of corneal substitutes ranging from prostheses to tissue-engineered matrix substitutes that are tailored to different clusters of clinical indications. The tissue-engineered matrices can either be produced as sterile acellular matrices, or complete with functional cells, ready for implantation.

Visual outcome in corneal grafts: a preliminary analysis of the Swedish Corneal Transplant Register.

AIM: To assess visual outcome and the incidence of complications at 2 years postoperatively in corneal grafts reported to the Swedish Corneal Transplant Register. METHODS: Preoperative and 2 year follow up data were submitted to the Swedish Corneal Transplant Register by surgeons in eight corneal transplant clinics in Sweden. Preoperative data on 1957 grafts and 520 grafts with 2 year follow up were included in the analysis. Data were analysed by multiple linear and logistic regression methods, as appropriate. RESULTS: The major diagnostic categories were keratoconus (29%), bullous keratopathy (21%), and "other diagnosis" (32%). Fuchs' endothelial dystrophy and stromal dystrophies accounted for 15% and 3% of grafts, respectively. At 2 years the overall incidence of complications, other than rejection and regrafting, was 26%, with an increasing frequency from keratoconus < Fuchs' dystrophy < bullous keratopathy < "other diagnosis." Rejection was observed in 15% of grafts and was more likely in the bullous keratopathy (OR 3.1, 95% CI 1.1 to 9.0, p=0.04) and "other diagnosis" (OR 2.6, 95% CI 1.1 to 5.9, p=0.03) groups. Regrafting, which occurred in 10% of cases, was not influenced by diagnosis, but it was related to the incidence of rejection (OR 14.8, 95% CI 6.1 to 35.9, p<0.001) and other complications (OR 4.4, 95% CI 1.9 to 10.4, p=0.001), and to the presence of other sight threatening pathology in the eye (OR 3.6, 95% CI 1.3 to 9.9, p=0.01). Visual acuity was improved in a high proportion of the patients, especially those with keratoconus and Fuchs' dystrophy where, respectively, 86% and 54% of grafts achieved a visual acuity of > or =0.5 at 2 years, compared with only 31% with bullous keratopathy and 35% in the "other diagnosis" group. 60% of grafts for keratoconus and Fuchs' dystrophy achieved a visual acuity equal to or better than the other eye. Postoperative astigmatism was higher in the bullous keratopathy (p=0.01) group. Patients with high astigmatism benefited from refractive surgery, showing a reduction from 7.9 (95%CI 6.9, 8.7) to 3.2 (95% CI 2.6, 3.9) dioptres (p<0.001). A centre effect was evident in visual outcome. CONCLUSION: The overall incidence of complications was related to diagnosis. Complications other than rejection and regrafting were most likely in the "other diagnosis" group, and further analysis of this group is therefore planned. The best improvement in visual acuity and the lowest astigmatism were achieved in the keratoconus and Fuchs' dystrophy groups; but the influence of diagnosis on astigmatism was small and, overall, the statistical model accounted for only 8% of the variability in astigmatism. Refractive surgery was, however, effective in reducing astigmatism. It is hoped that a better understanding of the factors that determine the visual outcome of grafts will emerge from future analyses of the Swedish Corneal Transplant Register, helping to refine the criteria for patient selection and to guide clinical practice.

Cellular proliferation and leukocyte infiltration in the rabbit cornea after photorefractive keratectomy.

PURPOSE: To map the proliferative activity of corneal cells during wound healing following photorefractive keratectomy (PRK) and to compare two markers for proliferation. METHODS: PRK, 5- mm in diameter with a -6 D setting, was performed in one eye of 28 New Zealand White Rabbits. The rabbits were sacrificed at time points between 12 hours and three months after surgery. The treated and fellow corneas were fixed in 10% formaldehyde, paraffin embedded, and immunohistochemically stained for proliferate cell nuclear antigen (PCNA) and at one time point, 1 week, also for Ki-67. RESULTS: Following initial sliding of the epithelial cells, the proliferative activity in the wound area starts in the leading edge (24 hours) and is spread towards the periphery. The proliferative activity peaks after one week and subsides during the following two weeks. Early (24 hours) proliferative activity is also seen in the limbal epithelium which peaks after three days. The keratocytes express PCNA in the peripheral stroma 48 hours after injury. They then also migrate to repopulate the stroma under the wound area. The expression period lasts 1 week and subsides the following week. Leukocytes are found in the wound as early as 12 hours after injury. The cells disappear around the time of epithelial wound closure, i.e. after 3 days. The two proliferative markers PCNA and KI 67 show a similar distribution after surgery. CONCLUSION: Epithelial proliferative activity starts earlier after injury, and is preceded by leukocyte presence in the wound. The PCNA expression starts later in the keratocytes but lasts somewhat longer (3 weeks). PCNA expression appears more efficient than Ki-67 to show proliferative activity of slow cycling cells in the cornea

Wound healing response in the presence of stromal irregularities after excimer laser treatment.

PURPOSE: To trace the fate of stromal irregularities after excimer laser treatment and to increase our knowledge of the reasons why surface irregularities in the ablation bed cause inferior postoperative results. METHODS: Twelve New Zealand White rabbits received a transepithelial photoablation to a preset depth of 60 microm. An electron microscopy specimen grid was then placed on the denuded stroma and another 20 microm ablation was applied in order to produce surface irregularities. Another six rabbits received a plano transepithelial photoablation to a preset depth of 80 microm. The treated corneas were harvested at various timepoints and differentially further processed for microradiography, hematoxylin-eosin -, hyaluronan (HA)- and leukocyte protein L1 staining. RESULTS: In the grid treated corneas the subepithelial mesh pattern is clearly discernible after 1 week, and after 4 weeks it is replaced by a subepithelial layer containing HA and water. The thinning of this layer between 1 and 12 weeks is statistically significant (p<0.05). After 4 and 8 week the plano treated corneas only exhibit some subepithelial HA- and water accumulation. After 1 day the grid treated corneas show an extensive stromal infiltration of leukocytes. In the plano treated corneas the leukocytes mainly remain on the surface. CONCLUSIONS: During the healing process stromal irregularities are flattened, leaving a homogeneous zone with increased water content. This subepithelial layer is rarefying as new subepithelial tissue is forming. Postablational irregularities induce a more pronounced healing reaction when compared to a smooth ablation surface. Leukocyte infiltration seems to play a role in this process.

Short-term impact of corticosteroids on hyaluronan and epithelial hyperplasia in the rabbit cornea after photorefractive keratectomy.

PURPOSE: To investigate the impact of corticosteroids on subepithelial hyaluronan deposition and corneal epithelium thickness in the first 10 days after photorefractive keratectomy (PRK) and to analyze a possible contralateral effect of corticosteroids. METHODS: Thirty-two New Zealand white rabbits were assigned into two groups and had a transepithelial 5.0-mm diameter, 8.00-diopter myopic PRK performed on one eye. The corticosteroid treatment group (16 animals) received 0.1 mL of methylprednisolone 4% subconjunctivally on the operation table, followed by 0.1% dexamethasone eye drops six times a day during the postoperative period. The sodium chloride (NaCl) treatment group received topical isotonic NaCl eye drops six times a day. In each treatment group, eight animals were killed after 3 and 9 days, respectively. The harvested specimens were stained for hyaluronan and the epithelial thickness was measured. RESULTS: In contrast to the epithelial thickness, the subepithelial hyaluronan did not show a significant increase during the observation period. The corticosteroid treated group showed at both time-points significantly less subepithelial hyaluronan formation as well as a significantly thinner epithelium, when compared with the NaCl-treated group. At 9 days, the corticosteroid-treated group showed a mild epithelial hyperplasia in only one of eight eyes, whereas this was a common finding in the NaCl-treated group. We detected no hyaluronan deposits in any contralateral-untreated eye, and the epithelial thickness did not differ significantly between any of the four contralateral-untreated eye groups. CONCLUSIONS: Corticosteroid medication during the first 10 days after operation reduces the amount of subepithelial hyaluronan production and inhibits the epithelial proliferation, and epithelial hyperplasia is prevented. Neither a contralateral hyaluronan deposition nor a contralateral corticosteroid effect could be detected.

Leukocytes in the early events of corneal neovascularization.

PURPOSE: To study the early events in corneal neovascularization after alkali injury and their relationship to the presence and absence of leukocytes. METHODS: A standardized 5.5-mm diameter penetrating central corneal alkali wound was induced in one eye in each of ten New Zealand white rabbits (2.5 kg). In five of the ten rabbits, 1.5 mL 5% fucoidin was given intravenously every 2 hours to prevent leukocytes from leaving the blood stream. Presence of hyaluronan (HA) and proliferating cell nuclear antigen (PCNA) in the corneas were analyzed using immunohistochemical staining 36 hours after injury. RESULTS: In the alkali wounded corneas, HA was expressed intensively in the limbal area where a massive infiltration of leukocytes was seen. PCNA was expressed in the vascular endothelium as well as in the corneal cells. In the leukocyte-free corneas, HA staining intensity and distribution were the same as in uninjured corneas. No positive PCNA staining was seen in the vascular endothelial cells in these corneas. CONCLUSIONS: Extravasated leukocytes in the alkali-burned corneas caused enhanced production of HA and proliferation of vascular endothelial cells.

Repeated UVR exposures cause keratocyte resistance to apoptosis and hyaluronan accumulation in the rabbit cornea.

PURPOSE: To evaluate hyaluronan (HA) production and level of apoptosis of corneal cells after repeated UVR exposures. METHODS: Fifteen albino rabbit corneas were exposed to 310 nm UVR at a dose that causes biomicroscopically significant keratitis (0.47 J/cm2). Nine rabbits received a single dose of UVR. Six rabbits were irradiated 3 times at 7-day intervals. Rabbits exposed to the single dose of UVR, were sacrificed 24 hours, 7 and 14 days after irradiation. Rabbits exposed to the repeated doses of UVR, were sacrificed 24 hours and 14 days after the last irradiation. The corneal tissue specimens were processed for histological analysis using specific staining for HA, and the TdT-dUTP terminal nick-end labeling (TUNEL) assay. RESULTS: Corneas exposed to a single UVR dose showed extensive positive TUNEL staining 24 hours after exposure. Almost all basal epithelial cells, keratocytes throughout the entire thickness of the stroma, and endothelial cells were TUNEL-positive. No HA was found 24 hours after exposure. Extracellular HA staining of high intensity was found at day 7 throughout the entire central stroma, except the anterior one-fourth. At day 14 only a faint HA staining was detected in the posterior stroma, close to Descemet's membrane. Corneas exposed to repeated UVR doses showed at 24 hours positive TUNEL staining only in epithelial cells and in very few stromal cells. The majority of stromal cells and endothelial cells were unaffected. At the same time HA staining of very high intensity was found both at 24 hours and day 14, and it was evenly distributed throughout the entire thickness of the stroma. CONCLUSION: Repeated UVR exposures lead to increased production and accumulation of HA in the corneal stroma. The repopulated keratocytes are much more resistant to apoptosis than the native ones. HA accumulation may be a sign of long-term changes in the cornea.

Expression of matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) in inflammation-associated corneal neovascularization.

Matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) are all implicated in the development of neovascularization. To investigate the possible role of these factors in corneal neovascularization we have analysed the expression of MMP-2, MMP-9 and VEGF in a rat model of inflammation-associated corneal neovascularization. In this model, corneal neovascularization was induced in Long-Evans rats by krypton laser photocoagulation whereafter eyes were enucleated at 1, 4, 7, 10 and 20 days. Slit-lamp biomicroscopy and histologic analysis revealed a gradual development of corneal neovascularization that peaked 7-10 days after treatment when newly formed vessels could be seen throughout the corneal surface reaching deep into the stroma. Antisense and sense riboprobes were generated using DNA complementary to MMP-2, MMP-9 and VEGF, and mRNA expression was analysed using in situ hybridization. The expression of MMP-2 and MMP-9 in untreated corneas was low or absent whereas VEGF was weakly expressed in the corneal epithelium. MMP-2 expression was increased during corneal neovascularization and was mainly localized to the cells infiltrating areas of new vessel formation. Many of these cells appeared to be inflammatory cells. VEGF expression had a similar overall distribution to MMP-2 during neovascularization with the exception that its expression in the corneal epithelium remained and even increased slightly. MMP-9 was prominently expressed at the border of regenerating corneal epithelium in areas with epithelial wounding but was not detected in the vascularized stroma. Together, the results of the present study support a role for MMP-2 and VEGF in inflammation-associated corneal neovascularization whereas MMP-9 instead appears to be involved in corneal epithelial wound-healing.

Wound healing after photorefractive keratectomy.

For more than 15 years, the excimer laser has been used as a surgical instrument on the cornea. Photorefractive keratectomy (PRK) followed radial keratotomy as researchers sought a more precise technique. In PRK, precision turned out to depend on surgical technique as well as the wound-healing process, with the 2 factors interdependent. The PRK technique has evolved toward a large diameter, flat ablation curvatures, and an even surface. The role of such factors as cytokines and interleukins has become more clear in the past 10 years. However, understanding the wound-healing process becomes more complicated with increasing know edge. Learning the contributing factors and performing trials with new drugs and antibodies to modulate wound healing have shown positive results on the experimental level. Patient selection based on the concentration of epidermal growth factor in tears may be another way to increase PRK s precision. The PRK technique has taught much about wound healing. For the technique to be competitive, increased precision, particularly in eyes with high myopia, is needed. Two other factors are imperative: controlling postoperative pain and decreasing visual rehabilitation time.

Apoptosis in UV-exposed rabbit corneas.

PURPOSE: Apoptosis was studied in rabbit corneas as a possible mechanism of cell death after photokeratitis induced by different UV wavelengths. METHOD: Fourteen albino rabbit corneas were exposed to 280- and 310-nm UV radiation (UVR) in 10-nm full wavebands at doses that cause biomicroscopically significant keratitis (0.12 J/cm2 for 280 nm and 0.47 J/cm2 for 310 nm). Animals were killed 24 and 76 h after exposure. Corneas were processed for light and transmission electron microscopy and in situ end labeling of fragmented DNA by using a modification of the TUNEL technique. RESULTS: Corneas exposed to 280-nm UVR showed TUNEL-positive staining only in epithelial cells and superficial keratocytes at 24 and 76 h after irradiation. Twenty-four hours after 310-nm UVR exposure, TUNEL-positive staining was present in the epithelial cells, keratocytes throughout the entire thickness of the central stroma, and in endothelial cells. Seventy-six hours after exposure to 310-nm UVR, keratocytes disappeared throughout the whole thickness of the damaged stroma. Only a few epithelial cells were TUNEL positive at that time. Transmission electron microscopy (TEM) verified the occurrence of apoptotic nuclei and cells. CONCLUSION: Apoptosis appears to be a mechanism of corneal cell death after UVR. The 310-nm UVR caused more extensive damage to the corneal stroma and endothelium than did the 280-nm UVR.

One spatial filter limits speed of detecting low and middle frequency gratings.

Reaction times for detecting sinusoidal gratings depend jointly on grating contrast and spatial frequency. We examine whether the effect of spatial frequency results from low-pass filtering in a single channel or reflects processing of different frequencies by two or more different processing streams. Observers performed a speeded two-alternative spatial forced-choice detection. Errors and reaction times were measured. Contrasts varied from 0.05 to 0.67, and spatial frequencies from 0.72 to 6.51 cpd. No effect of uncertainty about spatial frequency was found, arguing against multiple channels. The data are well fit by a single channel model driven by a low pass filter.

Influence of topical treatment on epithelial wound healing and pain in the early postoperative period following photorefractive keratectomy.

PURPOSE: To evaluate the effect of topical treatment given immediately after photorefractive keratectomy. METHODS: A prospective, randomized, double-blind clinical trial was performed on 60 eyes from 60 patients to evaluate the effects of topical diclofenac, dexamethasone and placebo (BSS) on epithelial wound healing, inflammation and duration of pain in the 3 days following surgery. RESULTS: Topical diclofenac and dexamethasone as one group significantly retarded epithelial healing and presented a higher number of central islands than the placebo, 3 days after surgery. Topical diclofenac retarded epithelial healing significantly more than dexamethasone. Anterior chamber flare was elevated after surgery without significant differences between treatment groups. Diclofenac showed a tendency to reduce the duration of pain following the procedure, although not significantly. CONCLUSIONS: Postoperative topical diclofenac and dexamethasone used during the first three days following photorefractive keratectomy may affect the epithelial healing and early visual rehabilitation.

Corneal stem cell grafting after chemical injury.

PURPOSE: To evaluate the clinical and histological outcome after corneal stem cell grafting in unilateral chemical burns in eight consecutive patients. METHODS: The visual performance and degree of irritation were evaluated following autologous corneal stem cell grafting. Scar tissue overlying the injured corneas as well as two corneal buttons obtained at penetrating graft performed a year or more after the stem cell graft were evaluated histologically. RESULTS: Seven of the 8 grafted eyes obtained useful vision postoperatively. Two of these eyes had undergone a penetrating graft following initial surgery. The chronic irritation before surgery was significantly reduced. In one eye a penetrating graft was opacified due to a late developing scar entropion. CONCLUSIONS: Autologous corneal stem cell grafting proved successful in restoring vision and reduce irritation in unilateral chemical burns. Histological examination indicates that the conjunctival overgrowth is replaced by regular corneal epithelium.

Effect of leukocytes on corneal cellular proliferation and wound healing.

PURPOSE: To establish whether fucoidin, by blocking the adhesion of leukocytes on the limbal vascular endothelium, prevents extravasation of the cells from the blood stream into the limbal stroma and the wounded area after corneal injury. Successful leukocyte blocking enabled investigation of the influence of leukocytes on corneal cellular proliferation after corneal wounding. METHODS: Thirty-two New Zealand White rabbits were used. Photorefractive keratectomy (PRK) and a standardized alkali corneal wound were used as models in two sets of experiments. In half of the injured rabbits fucoidin was used to prevent leukocytes from leaving the local vessels. The efficiency of the blocking technique was evaluated by counting the number of leukocytes in the limbal and wounded corneal areas. Proliferating cell nuclear antigen (PCNA) was used as a marker for proliferative activity. RESULTS: The infiltration of leukocytes into the limbus and the cornea after PRK and alkali injuries can be blocked by fucoidin. The healing rate of corneal epithelium after alkali burn was retarded in the absence of leukocytes. PCNA expression was enhanced in the presence of leukocytes. Fucoidin per se had no influence on corneal cell proliferation and wound healing. CONCLUSIONS: Polymorphonuclear leukocytes (PMNs) can be prevented from entering the cornea in vivo by fucoidin after PRK and after alkali burn. The corneal epithelial healing rate is delayed in the absence of PMNs in vivo, and PCNA expression increases in the presence of leukocytes.

Plano and refractive keratectomy. Comparison of the wound healing response.

PURPOSE: To compare the extension of the subepithelial low dry mass layer after plano and refractive keratectomy, in order to know more about the factors that influence the corneal wound healing response. METHODS: Thirty rabbits assigned into two groups were treated on one eye each with plano and refractive keratectomy, respectively. In each group five rabbits were sacrificed after 10 days, 4 weeks and 21 weeks, respectively. The corneal buttons were excised, freeze-sectioned and freeze-dried. On the freeze-dried sections microradiography was performed. In the treated eyes the superficial stroma showed qualitatively underneath the epithelium areas with low dry mass, whose extension was measured by a modified planimetry. RESULTS: The difference between the extension of subepithelial low dry mass areas in the plano and the refractive keratectomy group was small but statistically significant at 4 weeks. Within the refractive group the extension of low dry mass was significantly higher at 4 weeks compared to 21 weeks. CONCLUSION: A flat surface ablation may cause a less pronounced healing response when compared to a steep curve refractive treatment.

Messenger RNA levels for genes involved in extracellular matrix from human corneal scrapings before and after photorefractive keratectomy.

PURPOSE: To measure the levels of mRNA for genes important in cellular and extracellular matrix regulation in human corneal epithelium before and after photorefractive keratectomy (PRK) in order to explain myopic regression following surgery. METHODS: Scrapings from 26 normal corneas before a first photorefractive keratectomy were randomly pooled in two samples of 16 and 10 scraping, respectively, and compared to another 23 scrapings from corneas with myopic regression after a previous photorefractive keratectomy, also randomly pooled in another 2 samples of 16 and 7 scrapings each. The scrapings were analysed for seven different messenger RNAs involved in extracellular matrix using competition-based quantitative reverse-transcription polymerase chain reaction. RESULTS: Messenger RNAs for TGFalpha (Transforming growth factor-alpha), TGFbeta1 (Transforming growth factor-beta1), EGF-R (Epidermal growth factor-receptor) and TIMP1 (Tissue inhibitor metalloproteinase-1) were present in all samples. No mRNA for MMP9 (Metalloproteinase 9) or MMP2 (Metalloproteinase 2) were detected in any sample. Messenger RNA for collagen (alpha1) III was present in one sample following photorefractive keratectomy. CONCLUSIONS: The detection and measurement of levels of messenger RNA for selected growth factors, receptors, metalloproteinases and extracellular matrix proteins in ex vivo samples of human corneal epithelium is important and possible with a modified polymerase chain reaction technique. Messenger RNAs for Collagen III and for TGF-beta1 were elevated in one sample after photorefractive keratectomy.

Phototherapeutic keratectomy for map-dot-fingerprint corneal dystrophy.

PURPOSE: To evaluate the excimer laser as a surgical instrument to treat map-dot-fingerprint (MDF) dystrophies. METHODS: Thirty eyes (24 patients) with MDF dystrophy were treated with phototherapeutic keratectomy (PTK). The treatment goal was either to improve vision (24 eyes) or to heal recurrent corneal erosions or both (10 eyes). Besides long-standing reduction in visual acuity (17 eyes), associated symptoms were fluctuating visual acuity and clinical refraction (12 eyes) and monocular diplopia (eight eyes). In 14 eyes, two or three symptoms were present, whereas 16 eyes only had one symptom. The mean age was 54 years (range, 36-79 years), and there were 14 male and 10 female subjects. Mean follow-up was 30 months (range, 12-70 months). RESULTS: In 14 of 17 eyes with long-standing reduction in visual acuity, best spectacle-corrected Snellen visual acuity (BSCVA) improved by two lines or more. All eyes with fluctuating visual acuity/clinical refraction stabilized 1-3 months after PTK. Monocular diplopia or "ghost images" disappeared in all eyes after treatment. In one of 10 eyes with recurrent corneal erosions, there was one recurrence during the follow-up period. All eyes healed shortly after treatment. No recurrence of corneal dystrophic changes was seen in the ablation zone at the final follow-up (mean, 30 months). Dystrophic changes could, however, still be seen outside the treatment zone in 50% of the eyes, but were asymptomatic in all eyes. The mean refractive change was 0.34 +/- 1.05 (mean +/- SD) diopters. CONCLUSION: In this study, excimer laser photoablation was shown to be an effective, safe, and stable choice of treatment for map-dot-fingerprint dystrophy. A refractive change, as hyperopic shift, can be an adverse side effect in some individual cases.

Cellular response and reactive hyaluronan production in UV-exposed rabbit corneas.

PURPOSE: The distribution of hyaluronan (HA) and the cellular response after photokeratitis induced by different ultraviolet (UV) wavelengths in the rabbit cornea was examined to help understand the mechanism of corneal injury and repair after UV damage. HA is a high molecular weight disaccharide polymer capable of binding considerable amounts of water. It is not normally found in the rabbit corneal stroma. The production of HA represents a generalized corneal response to injury. METHODS: Twenty-four albino rabbit corneas were exposed to 270, 290, and 310 nm of UV radiant energy in 8-nm full wavebands in doses producing biomicroscopically significant keratitis (three corneal thresholds for keratitis (Hc): 0.016 J/cm2 for 270 nm, 0.04 J/cm2 for 290 nm, and 0.14 J/cm2 for 310 nm) and in subkeratitis doses (0.7 Hc: 0.004 J/cm2 for 270 nm, 0.008 J/cm2 for 290 nm, and 0.03 J/cm2 for 310 nm). The rabbits exposed to 270 and 290 nm of UV radiation were sacrificed 3 days after exposure. The rabbits exposed to 310 nm of UV radiation were sacrificed 3, 7, and 14 days after exposure, respectively. The corneal tissue specimens were double stained and examined morphologically and histochemically for HA by light microscopy. RESULTS: Evaluation of corneas exposed to 270 and 290 nm of UV radiant energy in both subkeratitis and keratitis doses and those corneas exposed to 310 nm of radiant energy in subkeratitis dose showed neither stromal changes nor production of HA by corneal cells. Corneas exposed to 310 nm of UV radiant energy in keratitis dose at 3 days after exposure showed disappearance of keratocytes in entire thickness of central cornea. Cells bordering this damaged area were staining for HA. By 7 days after exposure almost the whole damaged area, except one fourth of anterior stroma, was repopulated by new keratocytes staining positive for HA. The corneal structures became normal and HA almost completely disappeared 14 days after exposure. CONCLUSIONS: A keratitis dose of 310 nm of UV light irradiation is needed to cause keratocyte damage. A keratitis dose of the shorter wavelengths does not cause keratocyte cell damage at the light microscopic level. The keratocyte production of HA appears to be a sign of cell readiness to repopulate the damaged stroma devoid of keratocytes.

Assessment of high and low contrast visual acuity after photorefractive keratectomy for myopia.

BACKGROUND: Our aim was to assess visual acuity using standardized charts and illumination conditions after photorefractive keratectomy. METHODS: High and low contrast visual acuity were measured on Bailey-Lovie logarithm of the minimum angle of resolution (LogMAR) charts under high and low illumination conditions on 105 photorefractive keratectomy patients who had been treated with the Summit (N = 60) or the VISX (N = 45) excimer laser. RESULTS: Best corrected visual acuity was reduced in the treated eye compared with the untreated control eye under all test conditions, with the greatest differences under conditions of low contrast and low illumination. Reduction of acuity under low contrast and low illumination was related to small optic zone sizes and steep ablation edge profiles found in Summit-treated eyes. In the VISX-treated eyes, high contrast acuity was reduced in the presence of central topographical irregularities, subepithelial haze, and higher myopic corrections. CONCLUSIONS: Testing conditions such as those described here may be useful in quantifying vision degradation in suboptimal viewing conditions and among patients with vague complaints.

Expression of proliferating cell nuclear antigen in corneas kept in long term culture.

PURPOSE: To investigate the proliferative activity of the donor corneal cells and to examine how this property changed during long term culture. METHOD: Fourteen human corneas from donors (ages from 50-91) were cultured in the medium (MEM+8% FBS with or without dextran). The proliferating status of corneal cells was evaluated by immunohistochemical staining of proliferating cell nuclear antigen (PCNA) in the cells. Three corneas at each time point were fixed in paraformalin at day 0, day 3 and after 3 weeks cultured in medium as well as 3 weeks plus 2 or 5 days in fresh medium with 8% dextran. Paraffin-embedded corneas were sectioned to 4 microm and stained with antibody PC 10 against PCNA. The number of PCNA positive cells was identified under light microscope. RESULT: Prior to organ culture only basal limbal epithelial cells stained positive for PCNA. After 3 days in culture 50 percent of the epithelial cells were positive as were several keratocytes and some endothelial cells in the peripheral corneas. After 21 days no cells showed proliferative activity. After 21 days in culture and 5 days in fresh deswelling medium the essentially monolayered epithelium stained positively in the limbal area. The proliferative activity of the keratocytes in the anterior stroma was extensive. Endothelial cells stained positive in the peripheral cornea. CONCLUSION: Limbal epithelial cells appear to survive in the organ culture. The corneas may be worth evaluating as sources of stem cells for grafting. Likewise, the keratocytes survive organ culture and can be induced to proliferate after a change to fresh medium. The endothelium is stimulated to proliferate in organ culture and in fresh medium after long term storage.