Corneal Epithelial Thickness Mapping in the Diagnosis of Ocular Surface Disorders Involving the Corneal Epithelium: A Comparative Study.

PURPOSE: The purpose of this study was to analyze the role of corneal epithelial thickness (ET) mapping provided by spectral domain optical coherence tomography in the diagnosis of ocular surface disorders (OSDs) involving the corneal epithelium. DESIGN: This was a retrospective comparative study. METHODS: Institutional settings are as follows. Study population includes 303 eyes with an OSD and 55 normal eyes (controls). Observation procedures include spectral domain optical coherence tomography with epithelial mapping in the central 6 mm. Main outcome measures include ET map classification (normal, doughnut, spoke-wheel, localized/diffuse, and thinning/thickening patterns) and ET data and statistics (minimum, maximum, and SD). A quantitative threshold was determined with receiver operating curves to distinguish pathological from normal corneas. Sensitivity and specificity of classification and quantitative data were calculated using all eyes to assess the ability to distinguish corneas with a given corneal disorder from other conditions. RESULTS: Classification of full agreement between 3 readers was obtained in 75.4% to 99.4% of cases. Main OSD features were keratoconus (135 eyes), doughnut pattern (sensitivity/specificity = 56/94%), and max-min ET ≥ 13 µm (84/43%); limbal deficiency (56 eyes), spoke-wheel pattern (66/98%), and max-min ET ≥ 14 µm (91/59%); epithelial basement membrane dystrophy (55 eyes), inferior thickening pattern (55/92%), and central ET > 56 µm (53/81%); dry eye (21 eyes), superior thinning pattern (67/88%), and minimal ET ≤ 44 µm (86/48%); pterygium (10 eyes), nasal thickening pattern (100/86%), and nasal ET > 56 µm (80/71%); and in situ carcinoma (11 eyes), max ET > 60 µm (91/60%), and ET SD >5 µm (100/58%). CONCLUSIONS: The epithelial map pattern recognition combined with quantitative analysis of ET is relevant for the diagnosis of OSDs and for distinguishing various OSDs from each other. Deep learning analysis of big data could lead to the fully automated diagnosis of these disorders.

Multimodal imaging of Hurler syndrome-related keratopathy treated with deep anterior lamellar keratoplasty.

BACKGROUND: Hurler syndrome-associated keratopathy is an exceedingly rare corneal disorder that requires corneal transplantation in advanced stages. Precise assessment of the corneal condition is necessary for deciding which type of keratoplasty (i.e., deep anterior lamellar or penetrating) should be proposed. We aimed to confront the results of multimodal imaging with those of histology in a case of Hurler syndrome-associated keratopathy. CASE PRESENTATION: A 16-year-old patient with Hurler's syndrome treated with hematopoietic stem cell transplantation was referred for decreased vision related to advanced keratopathy. The patient was treated with deep anterior lamellar keratoplasty (DALK) in both eyes with uncomplicated outcome. Visual acuity improved from 0.1 (20/200) preoperatively to 0.32 (20/63) and 0.63 (20/32) after transplantation. The corneal endothelial cell density was 2400 cells/mm2 in both eyes 3 years after transplantation. In vivo confocal microscopy (IVCM) and spectral domain optical coherence tomography (SD-OCT) were performed preoperatively. The corneal buttons retrieved during keratoplasty were processed for histology. In SD-OCT scans, corneal opacities appeared as diffuse stromal hyperreflectivity associated with increased corneal thickness. IVCM showed diffuse cytoplasmic granular hyperreflectivity and rounded/ellipsoid aspects of keratocytes, presence of small intracellular vacuoles, and hyperreflective epithelial intercellular spaces. Bowman's layer was thin and irregular. The corneal endothelium was poorly visualized but no endothelial damage was observed. Histology showed irregular orientation and organization of stromal lamellae, with the presence of macrophages whose cytoplasm appeared clear and granular. A perinuclear clear halo was visible within the epithelial basal cells. Bowman's layer featured breaks and irregularities. CONCLUSIONS: The observed corneal multimodal imaging features in mucopolysaccharidosis-related keratopathy were concordant with histology. Compared with standard histology, multimodal imaging allowed additional keratocyte features to be observed. It revealed both morphological and structural changes of all corneal layers but the endothelium. This information is essential for therapeutic management which should include DALK as the first-choice treatment in case of impaired visual acuity.

Development, structure, and bioengineering of the human corneal stroma: A review of collagen-based implants.

Bio-engineering technologies are currently used to produce biomimetic artificial corneas that should present structural, chemical, optical, and biomechanical properties close to the native tissue. These properties are mainly supported by the corneal stroma which accounts for 90% of corneal thickness and is mainly made of collagen type I. The stromal collagen fibrils are arranged in lamellae that have a plywood-like organization. The fibril diameter is between 25 and 35 nm and the interfibrillar space about 57 nm. The number of lamellae in the central stroma is estimated to be 300. In the anterior part, their size is 10-40 µm. They appear to be larger in the posterior part of the stroma with a size of 60-120 µm. Their thicknesses also vary from 0.2 to 2.5 µm. During development, the acellular corneal stroma, which features a complex pattern of organization, serves as a scaffold for mesenchymal cells that invade and further produce the cellular stroma. Several pathways including Bmp4, Wnt/ß-catenin, Notch, retinoic acid, and TGF-ß, in addition to EFTFs including the mastering gene Pax-6, are involved in corneal development. Besides, retinoic acid and TGF- ß seem to have a crucial role in the neural crest cell migration in the stroma. Several technologies can be used to produce artificial stroma. Taking advantage of the liquid-crystal properties of acid-soluble collagen, it is possible to produce transparent stroma-like matrices with native-like collagen I fibrils and plywood-like organization, where epithelial cells can adhere and proliferate. Other approaches include the use of recombinant collagen, cross-linkers, vitrification, plastically compressed collagen or magnetically aligned collagen, providing interesting optical and mechanical properties. These technologies can be classified according to collagen type and origin, presence of telopeptides and native-like fibrils, structure, and transparency. Collagen matrices feature transparency >80% for the appropriate 500-µm thickness. Non-collagenous matrices made of biopolymers including gelatin, silk, or fish scale have been developed which feature interesting properties but are less biomimetic. These bioengineered matrices still need to be colonized by stromal cells to fully reproduce the native stroma.

Long-Term Results of Cultured Limbal Stem Cell Versus Limbal Tissue Transplantation in Stage III Limbal Deficiency.

We aimed to evaluate efficiency and safety of transplantation of limbal stem cells (LSC) cultured on human amniotic membrane with no feeders and to compare cultured LSC with limbal tissue transplantation. Thirty eyes with stage III LSC deficiency were treated with autologous (autoLSC) or allogeneic (alloLSC) cultured LSC transplantation (prospective phase II clinical trial; average follow-up time, 72 months) or autologous (autoLT) or allogeneic (alloLT) limbal tissue transplantation (retrospective control group; average follow-up time, 132 months) between 1993 and 2014. The 5-year graft survival defined by absence of recurrence of the clinical signs of limbal deficiency was 71% for autoLSC, 0% for alloLSC, 75% for autoLT, and 33% for alloLT. Visual acuity improved by 9.2 lines for autoLSC and 3.3 lines for autoLT. It decreased by 0.7 lines for alloLSC and 1.9 lines for alloLT. Adverse events were recorded in 1/7 autoLSC, 7/7 alloLSC, 6/8 autoLT, and 8/8 alloLT patients. Corneal epithelial defect was the only adverse event recorded after autoLSC, whereas severe sight-threatening adverse events were recorded in the remaining three groups. Compared with failed grafts, successful grafts featured greater decrease in fluorescein staining, greater superficial vascularization-free corneal area, lower variability of the corneal epithelial thickness, and higher corneal epithelial basal cell density. Autologous cultured LSC transplantation was associated with high long-term survival and dramatic improvement in vision and was very safe. Autologous limbal tissue transplantation resulted in similar efficiency but was less safe. Cadaver allogeneic grafts resulted in low long-term success rate and high prevalence of serious adverse events. Stem Cells Translational Medicine 2019;8:1230&1241.

Incidence of Intraocular Pressure Elevation and Glaucoma after Lamellar versus Full-Thickness Penetrating Keratoplasty.

PURPOSE: To analyze the cumulated incidence of glaucoma after penetrating keratoplasty (PK), anterior lamellar keratoplasty (ALK), and endothelial keratoplasty (EK). DESIGN: Cohort study. Data were recorded prospectively and analyzed retrospectively. PARTICIPANTS: A total of 1657 consecutive eyes of 1657 patients undergoing corneal transplantation between 1992 and 2013. METHODS: Penetrating keratoplasty (date range, 1992-2013), ALK (date range, 2002-2013), and Descemet's stripping automated EK (date range, 2006-2013). MAIN OUTCOME MEASURES: Postoperative intraocular pressure (IOP), glaucoma treatments, and glaucoma-related loss of vision (loss of central visual function resulting in absence of light perception or light perception limited to the temporal visual field). Cox proportional hazard regression model was used to analyze risk factors for glaucoma after keratoplasty. RESULTS: The 10-year cumulated incidence of elevated IOP and elevated IOP requiring treatment was 46.5% and 38.7%, respectively. In multivariate analysis, 4 variables were significantly associated with a higher incidence of elevated IOP requiring treatment after keratoplasty: preoperative glaucoma or IOP >20 mmHg (adjusted hazard ratio [HR], 1.56; P < 0.001), penetrating keratoplasty (PK) (adjusted HR, 1.12 vs. ALK and 1.10 vs. EK; P < 0.001), postoperative lens status (adjusted HR vs. phakic eyes: 1.15 for posterior chamber intraocular lens, 1.43 for anterior chamber intraocular lens [IOL], 2.83 for aphakic eyes; P < 0.001), and IOL exchange or removal during surgery (adjusted HR, 1.48; P < 0.001). Recipient age, preoperative diagnosis, filtering surgery before keratoplasty, vitrectomy associated with keratoplasty, and filtering surgery associated with keratoplasty were significantly associated with a higher incidence of elevated IOP requiring treatment after keratoplasty in univariate analysis but not in multivariate analysis. The 10-year probability of loss of vision related to glaucoma was 1.0% after EK, 2.1% after ALK, and 3.6% after PK (P = 0.036). CONCLUSIONS: The incidence of elevation of IOP after keratoplasty and development of glaucoma are significantly decreased with ALK and EK compared with PK. We believe this is due to diminished surgery-induced damage to the anterior chamber angle and trabecular meshwork, and reduced postoperative use of steroids.

Femtosecond-Assisted Arcuate Keratotomy for the Correction of Postkeratoplasty Astigmatism: Vector Analysis and Accuracy of Laser Incisions.

PURPOSE: To assess the clinical and refractive outcomes of femtosecond-assisted arcuate keratotomy in postkeratoplasty patients, and the accuracy of the incisions, using optical coherence tomography. METHODS: This is a retrospective study of patients with high postkeratoplasty astigmatism. Patients with a minimum of 4 diopters (D) of postkeratoplasty regular astigmatism were included. The main outcome measures were corrected distance visual acuity, keratometry, corneal topography, and the depth of corneal incisions. Arcuate keratotomy procedures were performed using the IntraLase Femtosecond laser. The depth of keratotomies was set to 75% of the thinnest pachymetry. RESULTS: Twenty eyes of 20 patients were recruited in this study. The mean age at surgery was 51 years, and the mean follow-up period was 17 ± 7.9 months. The corrected distance visual acuity improved significantly from 20/60 preoperatively to 20/41 after surgery (P = 0.004). The mean preoperative and postoperative spherical equivalents were -4.34 ± 2.91 D and -4.44 ± 3.64 D, respectively (P = 0.49). The mean keratometric cylinder decreased from 9.45 ± 2.97 D (range, 4.2-15.2 D) to 4.64 ± 2.79 D (range, 1.4-11.8 D) (P < 0.001). There was no statistical difference between the mean surgical-induced astigmatism and the mean target-induced astigmatism (P = 0.313). The mean difference between the scheduled and actual incision depth was 10.5 ± 22.2 µm (P = 0.057). No complications occurred during the procedures. CONCLUSIONS: Femtosecond-assisted keratotomy seems to be a safe and efficient technique for the reduction of large amounts of corneal astigmatism. Although overcorrection and undercorrection may occur, the visual outcome is satisfactory. Optical coherence tomography analysis reports a good predictability of the depth of incisions.

Development of human corneal epithelium on organized fibrillated transparent collagen matrices synthesized at high concentration.

Several diseases can lead to opacification of cornea requiring transplantation of donor tissue to restore vision. In this context, transparent collagen I fibrillated matrices have been synthesized at 15, 30, 60 and 90 mg/mL. The matrices were evaluated for fibril organizations, transparency, mechanical properties and ability to support corneal epithelial cell culture. The best results were obtained with 90 mg/mL scaffolds. At this concentration, the fibril organization presented some similarities to that found in corneal stroma. Matrices had a mean Young's modulus of 570 kPa and acellular scaffolds had a transparency of 87% in the 380-780 nm wavelength range. Human corneal epithelial cells successfully colonized the surface of the scaffolds and generated an epithelium with characteristics of corneal epithelial cells (i.e. expression of cytokeratin 3 and presence of desmosomes) and maintenance of stemness during culture (i.e. expression of ΔNp63α and formation of holoclones in colony formation assay). Presence of cultured epithelium on the matrices was associated with increased transparency (89%).

Deep sclerectomy versus trabeculectomy in eyes with penetrating keratoplasty.

PURPOSE: The aim of this study was to compare deep sclerectomy to trabeculectomy in eyes with penetrating keratoplasty (PK). METHODS: In a retrospective comparative case series, 32 consecutive deep sclerectomies (deep sclerectomy group) and 32 matched trabeculectomies (trabeculectomy group) were performed in eyes with PK. Control cases were matched for the timing of glaucoma surgery, number of previous glaucoma surgical procedures, corneal disease, and lens status. The main outcome measures were the success rate of glaucoma surgery and graft survival. Intraocular pressure, graft transparency, and postoperative complications were recorded. The criteria for glaucoma surgery failure were a postoperative intraocular pressure higher than 21 mm Hg or a decrease lower than 30%. RESULTS: The average follow-up time of glaucoma surgery was 29 ± 30 months. No significant differences were observed between both groups for all baseline variables and postoperative follow-up time. The success rate of glaucoma surgery was, respectively, 76% and 44% at 1 and 5 years in the deep sclerectomy group and 69% and 49% in the trabeculectomy group (P = 0.69). The graft survival estimates were, respectively, 100% and 73% at 1 and 5 years in the deep sclerectomy group and 87% and 40% in the trabeculectomy group (P = 0.02). Nonimmune postoperative events and nonimmune graft failures were significantly more frequent in the trabeculectomy group compared with the deep sclerectomy group (P = 0.04). CONCLUSIONS: Graft survival was higher in eyes with deep sclerectomy compared with trabeculectomy. Deep sclerectomy seems as efficient as, but safer than, trabeculectomy and could be performed as a first-choice treatment in the absence of major peripheral anterior synechiae.

Effects of isoproterenol and cholera toxin on human limbal epithelial cell cultures.

PURPOSE: Cholera toxin and isoproterenol (ß-adrenergic receptor agonist) are largely used to enhance cell proliferation. The aim of the study was to assess the effects of cholera toxin and isoproterenol on growth and differentiation of cells cultured from human superficial limbal explants. METHODS: Limbal epithelial cells were cultured from superficial limbal explantsin basal medium either supplemented with cholera toxin or isoproterenol for 3 weeks. Growth kinetics and morphometry were studied by light and confocal microscopy. Progenitor and differentiated epithelial cell markers were studied by immunocytochemistry, flow cytometry, Colony Formation Assay, and reverse transcription and polymerase chain reaction. RESULTS: Cell proliferation was significantly higher with 0.5 µg/ml (p = 0.049), 1 µg/ml (p = 0.005), and 2 µg/ml (p = 0.008) isoproterenol whereas, cholera toxin and 4 µg/ml isoproterenol did not significantly increase cell proliferation. Multilayered epithelial cell sheets were obtained in all culture conditions. Addition of isoproterenol resulted in smaller cell size (p < 0.05) 14 days after cells were cultured, whereas cholera toxin had no effects. Strong expression of cytokeratins 3 and 4/5/6/8/10/13/18 and lower expression of cytokeratin 19, vimentin, and Delta N p63α were observed after 3 weeks of culture with no significant differences in the percentage of positive cells according to culture medium. Colony-forming efficiencies were observed after 2 weeks in all culture condition but not after 3 weeks. CONCLUSION: Isoproterenol was more efficient than cholera toxin for enhancing cell proliferation and resulted in smaller cell size. It appears to be useful and safe for growing human limbal epithelial progenitors from limbal explants with no feeders before transplantation to patients with limbal deficiency.

Infectious keratitis in severe limbal stem cell deficiency: characteristics and risk factors.

PURPOSE: To evaluate the incidence, clinical and microbiological characteristics and risk factors of infectious keratitis in patients with limbal stem cell deficiency (LSCD). METHODS: Retrospective comparative case series of 35 patients with severe LSCD. RESULTS: The mean follow-up time was 46 months. Infectious keratitis were mainly caused by Gram positive bacteria (94%). Only 7 infections (37%) healed under fortified adapted antibiotics. In 8 cases (42%), amniotic membrane transplantation was required and in 4 cases (21%) «à chaud¼ keratoplasty was performed. Significant risk factors associated with infectious keratitis were: soft contact lens extended wear, history of persistent epithelial defects, number of quadrants of corneal vascularization, re-epithelialization time after amniotic membrane or corneal transplantation, and use of corticosteroid or cyclosporin eye drops. CONCLUSION: Infectious keratitis in LSCD is frequent and severe. The restoration of the epithelial barrier integrity and a careful use of therapeutic contact lenses may help to prevent infection.

Morphometry of corneal epithelial cells on normal eyes and after anterior lamellar keratoplasty.

PURPOSE: To analyze morphometry of corneal epithelial cells in normal eyes and after anterior lamellar keratoplasty (ALK) using confocal microscopy. METHODS: This retrospective comparative case series included 29 eyes of patients who had undergone ALK (ALK group) and 29 subjects before refractive surgery with normal corneas (control group). The Heidelberg Retina Tomograph II with the Cornea Module was used. Images were recorded and analyzed with Image J software. Morphometric parameters used to analyze epithelial cells were cell area, coefficient of variation of cell area, perimeter, circularity, and Feret diameter. RESULTS: Confocal microscopy was performed between 3 and 70 months after surgery. For basal epithelial cells, no significant differences between right eyes and left eyes were found in normal eyes for all morphometric parameters (P < 0.05 and rs > 0.40). The mean epithelial cell area increased during central and vertical migration (migration of epithelial cells from the basal stratum to the outermost layer) (P < 0.001). The mean basal cell area in the central zone was significantly higher (P < 0.001) after ALK in the ALK group than that in the control group. After ALK, the mean basal cell area decreased with postoperative time (rS = -0.78; P < 0.001) and returned to normal 24 months after transplantation (P = 0.27). Comparison of both groups showed significant differences in cell perimeter, circularity, and Feret diameter of basal cells and intermediate cells (P ≤ 0.001). CONCLUSION: Morphometric analysis of the corneal basal epithelium seems to be a relevant tool to assess the epithelial condition.

Predicted long-term outcome of corneal transplantation.

OBJECTIVE: To analyze graft survival and the outcome of the corneal endothelium after corneal transplantation in a single model to predict the long-term prognosis of these grafts. DESIGN: Cohort study. Data were recorded prospectively and then analyzed retrospectively. PARTICIPANTS: One thousand one hundred forty-four consecutive eyes of 1144 patients who underwent corneal transplantation between 1992 and 2006. INTERVENTIONS: Penetrating keratoplasty and deep anterior lamellar keratoplasty. MAIN OUTCOME MEASURES: Slit-lamp examination and wide-field specular microscopy results. A joint analysis of endothelial cell loss and time to graft failure was undertaken. From midterm simultaneous analysis of graft survival and endothelial cell loss, long-term graft survival was predicted. RESULTS: The observed 5- and 10-year graft survival estimates were, respectively, 74% and 64%. The average endothelial cell density (cell loss) was 2270 cells/mm(2) before surgery, 1058 cells/mm(2) (-53%) during the sixth postoperative year, and 865 cells/mm(2) (-61%) during the 10th postoperative year. Overall, the predicted graft survival estimate was 27% at 20 years and 2% at 30 years. Both observed and predicted graft survival were higher in patients who had undergone lamellar keratoplasty than in patients who had undergone penetrating keratoplasty and had normal recipient endothelium and higher in patients who had undergone penetrating keratoplasty and had normal recipient endothelium than in patients who had undergone penetrating keratoplasty and had impaired recipient endothelium. CONCLUSIONS: For corneal diseases involving the endothelium, penetrating keratoplasty seems to be a good therapeutic approach in elderly patients because the graft life-span may be similar to the patient life expectancy. Conversely, for younger patients, penetrating keratoplasty is only a midterm therapeutic approach. For corneal diseases not involving the endothelium, deep anterior lamellar keratoplasty seems to be a promising therapeutic approach with higher long-term expected survival.

Donor selection, retrieval and preparation of donor tissue. Donor selection.

Corneal transplantation safety is widely dependent on clinical donor selection. Donor-to-host transmission of rabies and Creutzfeldt-Jakob disease is well established, and it is lethal for the recipient. Taking into consideration this latter figure, contraindications to ocular tissue transplantation include not only rabies, contact with rabies virus, spongiform encephalitis, family history of spongiform encephalitis, recipients of human pituitary-derived hormones before 1987, surgery using dura mater and brain/spinal surgery before 1992, but also CNS diseases of unknown etiology or those with unknown risk of transmission. It has been established that hepatitis B virus and herpes simplex virus can be transmitted by corneal transplantation, and both diseases are contraindications to transplantation. HIV infection, syphilis, hepatitis C, hepatitis A, tuberculosis, HTLV-1 and -2 infection, active leprosy, active typhoid, smallpox and active malaria are also contraindications to ocular tissue transplantation even if no evidence of donor-to-recipient transmission has been demonstrated. A history of corneal refractive surgery in the donor eye, ocular inflammation, retinoblastoma, and malignant tumors of the anterior segment are contraindications to keratoplasty.

Graft reepithelialization after penetrating keratoplasty using organ-cultured donor tissue.

OBJECTIVE: To analyze graft reepithelialization after penetrating keratoplasty using organ-cultured donor tissue. DESIGN: Retrospective observational cohort study. PARTICIPANTS: One thousand one hundred thirty-eight consecutive penetrating keratoplasties (1003 patients) performed between 1992 and 2004 were analyzed, and 1003 were included (1 graft per patient was included). METHODS: Slit-lamp examination after fluorescein staining. MAIN OUTCOME MEASURE: Graft reepithelialization time was recorded. RESULTS: The average graft reepithelialization time was 4.6+/-13.2 days (range, 1-210 days; median, 2.5 days). Complete corneal epithelial healing was obtained in 1 day in 28.5% of patients, in 3 days in 65.8%, in 7 days in 93.6%, and in 14 days in 97.0%. Postoperative chronic epithelial defects occurred in 3.0% of patients. In univariate analysis, death-to-storage time, storage time, deswelling time, rejection risk, trephination size, suture method, use of dexamethasone ointment during surgery, use of sodium hyaluronate during surgery, and use of high postoperative steroid regimen significantly influenced the graft reepithelialization time (P<0.05). In multiple regression, only the rejection risk (beta = 0.07; P = 0.045), use of sodium hyaluronate at the end of surgery (beta = -0.17; P = 0.001), and steroid regimen (beta = 0.09; P = 0.022) significantly influenced the graft reepithelialization time. CONCLUSIONS: The use of sodium hyaluronate at the end of surgery to coat the ocular surface shortened graft reepithelialization time after penetrating keratoplasty using organ-cultured donor tissue. Graft reepithelialization time was longer in high-risk recipients, and it increased with high dexamethasone eyedrops regimen.

Corneal epithelial cultures generated from organ-cultured limbal tissue: factors influencing epithelial cell growth.

PURPOSE: To explore the in vitro proliferative potential of human limbal epithelial cells after 31 degrees C organ-culture storage and to investigate putative factors influencing it. METHODS: 185 cultures of limbal explants were carried-out either from full-thickness explants (n = 102) or from enzymatically dissociated cells (n = 83) seeded on a feeder layer of human keratocytes. Epithelial outgrowth was assessed by phase contrast microscopy using a computerized image analysis software. Cell phenotype was evaluated by transmission electron microscopy and immunocytology. Univariate and multivariate analysis were performed to determine factors influencing epithelial growth in culture. RESULTS: An epithelial outgrowth of 100 square mm or more was observed in 52% of cultures, (average growth area: 440 +/- 256 mm at three weeks). Corneal epithelial phenotype was confirmed by transmission electron microscopy, and cytokeratin pattern. Cytokeratine 19, deltaNp63, nestin and vimentin positive staining revealed undifferentiated epithelial cells in both explant and cell suspension cultures at three weeks. Short death to cornea retrieval time (p < 0.03) and female donors (p < 0.01) were associated with higher cell growth. Enzymatic treatment of explants by trypsin, but not dispase, decreased cell proliferation at two (p < 0.03) and three weeks (p < 0.04). Donor age, duration of corneal storage, and source of the explant did not influence the cell growth. CONCLUSION: Organ-culture conditions can preserve limbal cell mitotic potential if limbal tissue is excised early after circulatory arrest. Human keratocytes can be used as a feeder layer allowing epithelial cells to maintain poorly differentiated phenotype in culture. Further investigations are needed to explain the influence of the donor sex on epithelial cell growth in culture.

Culture-proven herpetic keratitis after penetrating keratoplasty in patients with no previous history of herpes disease.

OBJECTIVE: To report three cases of herpetic infection in recipients of organ-cultured donor corneas among 586 consecutive corneal transplantation procedures. METHODS: Three patients with no history of symptomatic herpes infection underwent corneal transplantation for keratoconus (2 patients) and Fuchs dystrophy (1 patient). Two patients developed keratouveitis and primary graft failure. The third patient developed dendritic keratitis in the graft. Culture of corneal scrapings and the patient's bandage contact lens were positive for herpes simplex virus type 1 (HSV-1). Donor and recipient sera were tested for HSV serology by EIA. Recipient corneal buttons were studied by means of transmission electron microscopy and immunohistochemistry. The three HSV-1 strains were genotyped by sequencing part of a variable antigenic domain of glycoprotein B (gB). RESULTS: None of the donor corneas showed endothelial cell necrosis after organ culture. All keratoplasties performed with the three mate donor corneas had an uncomplicated course. All three donor sera were positive for HSV. Preoperative recipient sera were positive for HSV. Analysis of the recipient corneal buttons showed no evidence of herpetic infection. Sequence analysis revealed three different gB genotypes. CONCLUSION: Ascertaining that a postoperative herpetic infection in a corneal transplant originates from the donor tissue is still difficult. Although some features of the reported cases suggest donor-to-host transmission of herpes simplex virus, the recipients could have been the source of the virus.