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OBJECTIVE To investigate CYP4V2 gene variants and ocular clinical characteristics of Bietti corneoretinal crystalline dystrophy in China so as to provide more references for genotype and phenotype of BCD. METHODS Sixteen Chinese probands were recruited in Beijing Tongren Hospital in a retrospective study. All patients underwent CYP4V2 gene detection and ophthalmic clinical examinations. RESULTS CYP4V2 gene variants were detected in all patients. Eight variants were identified, and the most common one was c.802-8_810del17bpinsGC. Onset age of BCD was from 12 to 44 years, and the first symptoms mostly were decreased visual acuity or night blindness. Corneal crystalline depositions were observed in all patients and were found not only in epithelium and superficial stroma near the limbus but also in corneal endothelium. OCT showed atrophy of RPE in all patients, outer retinal tubulation in ten patients, macular edema in four patients, macular hole in three patients with one accompanied with retinal detachment, and choroidal neovascularization in one patient. CONCLUSION CYP4V2 gene variants were detected in all patients consistent with the genetic locus homogeneity of BCD, and c.802-8_810del17bpinsGC was the most common mutation. Corneal crystalline depositions were observed in all patients, which may be features of BCD and helpful for the diagnosis of BCD patients, especially those in the advanced stage without typical fundus crystalline depositions or without gene detection. However, considerable phenotypic variability was detected. Corneal crystalline deposits were observed not only in epithelium and superficial stroma but also in endothelium, which has not been reported before. This may provide further evidence for the variable phenotypic expression between affected individuals.

Purpose: To measure corneal endothelial cell (EC) quality and quantity following Descemet membrane (DM) stripping of human donor corneas and continued storage in organ culture medium containing dextran.Methods: DM stripping was performed in 30 organ cultured, corneoscleral discs. Corneas were divided into 3 groups of 10 corneas each. Baseline mean EC density (cells/mm2) was 2,372 (SD ± 259) in group 1, 2,540 (SD ± 266) in group 2, and 2,665 (SD ± 263) in group 3. Following subtotal DM stripping, culture was continued at 31°C for 24 hours (group 1), 72 hours (group 2), and 120 hours (group 3), respectively. EC density was measured before stripping and at the end of culture. At the end of culture, corneal EC morphology was graded using a scoring system and EC viability was measured by detection of adenosine triphosphate.Results: At the end of culture, mean EC density was 2,159 (SD ± 293) in group 1, 1,946 (SD ± 182) in group 2, and 2,047 (SD ± 225) in group 3. This constitutes an EC loss of 9,1% (SD...

Penetrating keratoplasty was the only therapeutic choice for the treatment of corneal endothelial decompensation until the introduction of evolutional endothelial keratoplasties, namely Descemet's stripping automated endothelial keratoplasty (DSAEK) and Descemet's membrane endothelial keratoplasty (DMEK). Although now in widespread use, DSAEK and DMEK still have associated problems, such as difficulty of the surgical technique, acute and chronic cell loss, and shortage of donor corneas. Therefore, regeneration of the corneal endothelium by tissue engineering techniques is being researched to overcome these problems. The concept of transplantation of cultured corneal endothelial cells (CECs) was proposed in the 1970s. However, cultivation of human CECs (HCECs) in sufficient quantity and with acceptable quality for clinical use has proven surprisingly difficult, and the development of methods for transplanting cultured HCECs has been necessary. Numerous research groups have developed culture protocols and techniques that are now bringing corneal endothelial regeneration closer to real-world therapy. For instance, we started a clinical trial in 2013 involving the injection of cultured HCECs into the anterior chamber of patients with corneal endothelial decompensation. This review outlines the rapid progression of this research field, including clinical trial results, and is also intended to identify topics that still require further research or discussion.

We aimed to evaluate the differences in the corneas of eyes with isolated iris coloboma.This study was designed as a prospective study. Among patients who applied to our hospital within the last 3-year period, those with isolated iris coloboma in one eye and a completely normal contralateral eye were included in the study. In addition to a complete ophthalmologic examination, all patients were scanned by Pentacam Scheimpflug camera in the dark, and a corneal endothelium cell count was taken using a Topcon Specular Microscope. Data entered using SPSS software were then evaluated by paired t-test; p0.05 value was considered statistically significant.Nine patients, seven men and two women, with a mean age of 35.77 ± 22.50 (range 8-60) years, were included in the study. In comparison to normal eyes, the colobomatous eyes were statistically found to have lower keratometry values in diopters (43.82 ± 2.17, 44.46 ± 1.86; p = 0.040), a greater negative asphericity (Q value) (-0.38 ± 0.07, -0.22 ± 0.20; p = 0.042), increased corneal thickness (569.78 ± 25.42, 537.33 ± 27.36; p0.001), decreased anterior chamber depth (2.50 ± 0.32, 2.81 ± 0.50; p = 0.005), a shallower angle (30.51 ± 10.68, 35.29 ± 9.99; p = 0.057), higher best-fit sphere (BFS) values of the anterior and posterior corneal surfaces (7.88 ± 0.39, 7.67 ± 0.37; p = 0.001; 6.40 ± 0.26, 6.23 ± 0.19; p = 0.006), larger pupil diameter (3.48 ± 0.49, 2.80 ± 0.34; p = 0.001), smaller horizontal corneal diameter (HCD) (11.06 ± 0.42; 11.26 ± 0.45; p = 0.009), and higher value of endothelial cell density (ECD) (3161 ± 431; 2923 ± 590; p = 0.031).We have determined that a number of factors such as keratometry values, particularly of the corneal endothelium, are different from those of normal eyes. These differences should be taken into account in the follow-up and treatment of eyes with iris coloboma.

To compare the outcomes of two nuclear fracture techniques used in phacoemulsification (stop-chop vs. retro-chop).A total of 131 eyes from 111 patients with severe cataracts and nucleosclerosis (of grades 4-5) were included. The patients were randomly assigned to undergo stop-chop or retro-chop nucleotomy during phacoemulsification. The Drysdale manipulator was used as the chopping instrument in both groups. The intraocular phacoemulsification parameters including phacoemulsification power and phacoemulsification time were compared across the two groups. The following parameters were compared postoperatively at 1, 2, 5, and 8 weeks using unpaired t test: corrected distance visual acuity (CDVA), corneal endothelial cell density (CD), central corneal thickness (CT).There were significant differences in the phacoemulsification parameters between the groups. Retro-chop group showed less phacoemulsification time (p0.05) and less phacoemulsification power (p0.05) than stop-chop group. There was also a significant difference in the rate of corneal endothelial cell loss between the two groups (p0.05). In contrast, there were no significant differences in the postoperative change of CT or CDVA between the groups.Retro-chop nucleotomy with a Drysdale nucleus manipulator is an effective and safe method. It reduces both the use of intraoperative ultrasound energy, and early postoperative corneal endothelial cell loss compared to other nucleotomy techniques.

To evaluate whether preoperative mannitolization can change ocular biometry and affect postoperative corneal endothelial cell density.Bilateral sequential cataract surgery was performed in 38 patients. Preoperative mannitolization was done in one eye of each subject. We checked the change in preoperative ocular biometry before and after intravenous mannitolization. We compared the postoperative corneal endothelial cell density between eyes with mannitolization and without mannitolization at postoperative week 1, 2, 5, and 8. We evaluated the relationship between change in ocular biometry and change in postoperative corneal endothelial cells in eyes that underwent preoperative mannitolization.After mannitolization, eyes exhibited decreased intraocular pressure, axial length (AL), and vitreous chamber depth (VCD) and increased anterior chamber depth (ACD) and lens position (LP) compared to before mannitolization (p0.05). Preoperative mannitolization has a tendency to decrease the intraoperative use of phaco energy in eyes with moderate nucleosclerosis. Eyes with preoperative mannitolization showed less loss of postoperative corneal endothelial cells than eyes without preoperative mannitolization (p0.05). The ACD, LP, and AL changes by mannitolization were all negatively correlated with corneal endothelial cell loss (p0.05).Preoperative mannitolization can decrease postoperative loss of corneal endothelial cells. The protective effect of preoperative mannitolization on the corneal endothelium may be due to the decreased need for phaco energy and changes in ocular biometry such as ACD, AL, and LP.

To evaluate whether the gentian violet staining of the anterior lens capsule during the cataract surgery is cytotoxic for the human lens epithelial cells, as an indirect indication of possible toxicity towards the corneal endothelium and the safety of gentian violet application.Two groups of anterior lens capsules obtained during the cataract surgery, gentian violet stained and non-stained, were incubated with fluorescent dye Fura-2. Their fluorescence, upon excitation at 360 and 380 nm, was imaged to monitor changes in free intracellular calcium concentration ([Ca(2+)]i) in response to pharmacological stimulation by acetylcholine. The [Ca(2+)]i homeostasis is the indicator of cellular function. The changes in [Ca(2+)]i were compared between the two groups.Epithelial cells responded to acetylcholine in both groups of capsules - gentian violet stained (n = 17) and non-stained ones (n = 33). No significant differences of the elicited responses were found in rise time (p = 0.89), decay time (p = 0.61) or amplitude of [Ca(2+)]i (p = 0.96 for 63× and p = 0.26 for 40× objectives) between the two groups of capsules (Student t test).The staining of the anterior lens capsule with gentian violet during phacoemulsification in concentration of 0.01%, does not have detectable cytotoxic effects, which would affect the [Ca(2+)]i homeostasis in lens epithelial cells. The data, if extrapolated to corneal endothelium, exposed to the same concentration, suggest that gentian violet in concentration of 0.01% is safe as an adjunct for capsule visualization in cataract surgery.

Purpose: To investigate temperature alterations in the aqueous humor and the corneal endothelium during phacoemulsification, and to evaluate the effect of ophthalmic viscosurgical devices in preventing a temperature rise at the corneal endothelium during phacoemulsification.Methods: Temperatures of the aqueous humor and corneal endothelium were measured using two temperature probes set in the anterior chamber during ultrasound oscillation in porcine eyes, with or without ophthalmic viscosurgical devices.Results: Without an ophthalmic viscosurgical device, temperatures in both the aqueous humor and the corneal endothelium rapidly rose during the ultrasound oscillation, while with an ophthalmic viscosurgical device, the temperature elevation of the corneal endothelium site was suppressed compared with the aqueous humor.Conclusion: Surgeons need to be especially aware of ophthalmic viscosurgical device retention during phacoemulsification, because it can protect the corneal endothelium from heat gene...

To compare the quality of corneal endothelium of precut Descemet-stripping automated endothelial keratoplasty (DSAEK) tissue when transported with and without the anterior lamellar corneal tissue (ALCT) when organ-culture corneal storage methods are used.After microkeratome-assisted excision of anterior corneal lamella, five pairs of corneas (10 eyes) were stored either with the ALCT on the stroma (five eyes) or with ALCT off the stroma (five eyes) in organ-culture medium. Three pairs (six matched corneas) were left in the transport medium for 24 h prior to the microkeratome cut. Two pairs (four matched corneas) were left in the transport medium for 48 h prior to the microkeratome cut. All cuts were performed using a 300 (four eyes) or 350 (six eyes) microns head. A vital dye assay (alizarin red S and trypan blue) was used to identify devitalized and necrotic endothelial cells.In all matched cases, there was no difference between the endothelial cell appearance with or without the anterior corneal lamella. In all cases, there was no evidence for trypan blue stained cells beyond that normally seen on acceptable transplantable corneas and there was no evidence of loss of cells or any lifting of Descemet's membrane.There is no difference between the quality of the donor endothelial cell appearance with ALCT-on or -off when the donor cornea is stored using the organ-culture system of corneal storage. Organ-culture storage system is a safe and effective system in regard to preparation and transport of donor lenticules for DSAEK.

To evaluate the activation of ERK, a MAP kinase, in the regulation of proliferation and migration of rabbit corneal endothelial cells during the healing process after scrape wound injury.Cultured rabbit corneal endothelial cells were used in this study. Immunocytochemistry and Western blot analysis were performed to demonstrate the phosphorylation of ERK after scrape injury. The injured cell sheets were treated with different concentrations of ERK inhibitor, PD98059. The wound closure at 24, 48, and 72 hr following injury was evaluated. In order to evaluate the role of proliferation during wound healing, immunocytochemistry with Ki67 and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay were performed. The role of cell migration in wound healing was evaluated by quantification with the time lapse microscopic video imaging system.Immunocytochemical and Western blot analysis revealed phosphorylation of ERK in the rabbit corneal endothelial cells after wounding. Inhibition of ERK phosphorylation by PD58059 resulted in a dose-dependent delay of wound closure. The inhibition of wound healing became significant from 48 to 72 hr in cells treated with 10 and 50 micro M of PD58059 (p0.05). ERK inhibition suppressed cell proliferation as indicated by Ki67 immunostaining and MTS assay. The time lapse microscopic video imaging analysis indicated delayed cell migration caused by PD98059.ERK activation regulates wound repair of rabbit corneal endothelial cells. Both cell proliferation and migration are involved in this process.

Purpose: Gene delivery at high efficiency is crucial for cornea endothelial cell gene therapy. This study investigated the efficiency of gene transfer by recombinant adeno-associated virus (rAAV) in an organ culture system.Methods: Human cornea tissue was exposed to rAAV delivering green fluorescent protein (ss-rAAV2-CMV-GFP) for one hour and then cultured at 31ˆC for 2 weeks in a medium supplemented with growth factors. Endothelial cells expressing GFP gene were then identified.Results: High-efficiency gene transfer was found in over 90% of endothelial cells. Gene expression could be detected within 24 hours and remained stable up to 2 weeks in the organ culture system.Conclusions: The high-delivery efficiency and rapid induction of gene expression indicate that rAAV is a promising vector for cornea endothelial cell gene therapy for ocular diseases. Organ culture at 31ˆC using culture medium supplemented with growth factors significantly facilitates gene transfer into human corneal endothelium.

Gap junctional communication in the epithelium and endothelium of human corneas was studied. The influence of corneal storage on endothelial gap junctions was also examined.Donor human corneal cells were injected with carboxyfluorescein while surrounding cells were monitored for traces of fluorescence. Dye-spread coefficients were measured in corneal endothelial cells. Western blot and immunohistochemical analysis of the endothelium and epithelium was employed to determine if connexin 43 was present.Dye coupling occurs in both the epithelium and endothelium of the human cornea. Epithelial dye coupling was extensive in the basal layers but less apparent in the superficial layers. Endothelial dye coupling was similar to that reported for rabbit corneas. Western blot and immunohistochemical analyses demonstrated the presence of connexin 43 in both cell types.Gap junctional communication occurs in the endothelium and epithelium of human corneas, and both cell types express connexin 43. These results are similar to previous rabbit studies, thereby strengthening the use of the rabbit cornea as a gap junction model.

Topical or intracameral administration of H-7 doubles outflow facility and reduces intraocular pressure in cynomolgus monkeys, by relaxing and expanding the trabecular meshwork (TM) and Schlemm's canal (SC). Since H-7 may have anti-glaucoma potential, we determined its effects on the corneal endothelium and ciliary epithelium for safety considerations.Following topical H-7, aqueous humor flow (AHF), corneal endothelial transfer coefficient (k(a)) and anterior chamber (AC) entry of i.v. fluorescein were measured by fluorophotometry; AC aqueous protein concentration ([Protein](AC)) was determined by Lowry assay; and corneal thickness and endothelial cell density and morphology were measured by ultrasonic pachymetry and specular microscopy respectively. Following intracameral H-7, specular and/or light and electron microscopy of the corneal endothelium or ciliary epithelium were performed.Following unilateral topical H-7: (1) AHF and k(a) were essentially unchanged at 0.5--3.0, 3.5--6.0, and 0.5--6.0 hr, with an insignificant increase from 0.5--1.5 hr; (2) [Protein]( AC) was insignificantly increased at 1-1.5 hr but had returned to baseline by 2.5 hr; (3) entry of i.v. fluorescein into aqueous or cornea was modestly and transiently increased; (4) the central cornea thickened significantly at 1--2.5 hr, gradually returning to baseline 2.5 hr after H-7, while peripheral corneal thickness was less affected; (5) corneal endothelial cell borders became indistinct by 1 hr, but cell morphology was recovering by 3--5 hr and had completely returned to normal by 24 hr; (6) corneal endothelial cell density was unchanged at 5--24 hr. Following intracameral H-7, no significant changes were observed in corneal endothelial cell density or morphology by specular microscopy, nor in corneal endothelial or ciliary epithelial morphology by light and electron microscopy.A facility-effective intracameral dose of H-7 had no discernible structural effect on the corneal endothelium or ciliary epithelium. It is not yet clear whether carefully chosen topical doses of H-7 or analogues can enhance outflow facility without meaningfully affecting the cornea and ciliary processes.

Chloride and bicarbonate are necessary for maintenance of fluid transport by the corneal endothelium, however there is little information on the identity of anion transport proteins that could serve as anion efflux mechanisms in endothelial cells. Therefore, we ask whether mRNA for the anion transport related proteins, CFTR, CLC-2, ClC-3, ClC-5 and AE2, are expressed in human, bovine or rabbit corneal endothelium.RT-PCR was performed for CFTR, CLC-2, ClC-3, ClC-5 and AE2 using total RNA from fresh human, bovine and rabbit corneal endothelium as well as cultured bovine corneal endothelial cells (CBCEC). Specificity of PCR products was confirmed by sequencing.RT-PCR analysis gave positive bands at the predicted size for CLC-3 and CLC-5 from fresh human, rabbit and bovine as well as CBCEC. However, for CLC-2, no band was apparent around the predicted size from fresh and cultured corneal endothelium. A band at the predicted size was obtained for CFTR from fresh human, rabbit and bovine endothelium, as well as from CBCEC. RT-PCR analysis for AE2 produced specific bands from fresh human, rabbit and bovine corneal endothelium, but no positive band was obtained from CBCEC. Sequencing analysis further confirmed the identities of CLC-3, CLC-5, CFTR and AE2 in corneal endothelium.CFTR, CLC-3 and ClC-5 are expressed in fresh and cultured corneal endothelial cells. However, consistent with previous immunoblots studies, AE2 is only expressed in fresh corneal endothelium. These results have implications for modeling possible apical anion efflux mechanisms in corneal endothelium.

Corneal endothelium plays an important role in the maintenance of corneal transparency and stability. The purpose of this study was to evaluate the performance of the TOPCON SP-2000P and IMAGEnet system in terms of (a) the difference in results between automated endothelial cell analysis and retraced cell analysis, (b) the differences in the endothelial cell analysis when using the first image and the clearest of three images taken with the SP-2000P, (c) the repeatability and reproducibility of the IMAGEnet system in the determination of corneal endothelial cell variables, and (d) the repeatability and reproducibility of the SP-2000P in capturing endothelial cell images for endothelial cell analysis.Two experiments were performed. Twenty male subjects participated in the first experiment in which endothelial images were captured by examiner SWC and endothelial cell analysis was performed by two examiners, SWC (twice on two different days) and PC (once only). Nineteen male subjects participated in the second experiment in which endothelial cell images were captured by examiners SWC and PC on Visit 1 and by examiner SWC on Visit 2. Endothelial cell analysis was performed by examiner SWC only. The 95% limits of agreements (95%LA) and the intraclass correlation coefficient (ICC) between tested parameters were determined.Automated cell analysis significantly underestimated average cell size (ACS) and hexagonality, and overestimated endothelial cell density (ECD) and coefficient of variance of cell size (CV) (p0.05). No statistically significant differences were found in the endothelial cell variables between the first and the clearest images (p0.05). There were no statistically significant intra- or inter-examiner differences in any of the endothelial variables determined (p0.05). For a set of endothelial cell images, the reproducibility and repeatability of the endothelial cell variables determined with the IMAGEnet system were good, with ICC0.9. No significant inter-visit or inter-examiner differences were found (p0.05). Reliability was good for the determination of ECD and ACS (ICC0.9) but poor or average for the determination of CV and hexagonality (ICC: 0.4-0.8).We suggest the use of the clearest image with the retraced method for endothelial cell analysis with the IMAGEnet system, as the variability in hexagonality results between the first and the clearest images can be clinically significant. For the same set of images captured by one examiner, the TOPCON IMAGEnet system was reliable in determining ECD and ACS, and was fairly reliable in determining hexagonality and CV. When images were captured on different days or by different examiners, the TOPCON SP-2000P with the IMAGEnet system gives repeatable and reproducible values for ECD and ACS only. Caution should therefore be exercised when using CV and hexagonality to monitor endothelial changes in comparative studies.

Previous studies suggest that proteins associated with the apical junctional complex (AJC) play essential roles in the development, maintenance and regulation of barrier function in transport epithelium and vascular endothelium. The goal of this study is to identify and determine the spatial organization of several major AJC-associated proteins in normal human and feline corneal endothelium.Fresh corneal tissue was obtained from 4 recipient buttons removed during penetrating keratoplasty (two from keratoconus patients, and two from patients with post-traumatic stromal scarring) as well as from 16 cat eyes. En bloc double- and triple-labeling of corneas was performed using phalloidin, and mouse, rat or rabbit antibodies to ZO-1, occludin, pan-cadherin, alpha-catenin, beta-catenin and plakoglobin (gamma-catenin). The 3-D localization of the proteins was then determined in situ using laser confocal microscopy.Similar staining patterns were obtained for the corneal endothelium of normal cat corneas and fresh human buttons. Apically, f-actin was arranged into dense peripheral bands (DPB) in individual cells that were separated from those in adjacent cells. Diffuse phalloidin staining also extended from the DPB into the cytoplasm apically. Although weaker, phalloidin staining also appeared to be associated with the basolateral cell borders. The adherens junction protein, cadherin, formed a thin pericellular band at the apical cell junctions between the DPB. In addition, cadherin staining also appeared to extend along the basolateral cell borders in a convoluted pattern. Staining for alpha-catenin, beta-catenin and plakoglobin each showed a nearly identical organization as cadherin. ZO-1 formed a single apical band that was localized between the DPB; however, no basolateral ZO-1 staining was detected. Interestingly, the distribution of ZO-1 was discontinuous around the cell, with the largest gaps occurring at the Y-junctions between adjacent endothelial cells. Positive staining for occludin was not detected in either human or feline corneal endothelium.The composition and organization of the AJC of corneal endothelium appears to be different from that of classical transport epithelia; these findings may be related to functional differences between these two cell types.

Insulin-like growth factor binding proteins (IGFBPs) may modulate insulin-like growth factor-I (IGF-I) action and are important regulating factors in the IGF system. Our aim was to determine the presence of IGFBP-2 and -5 in the anterior compartment of the eye and to compare the histological sites of these IGFBP proteins with the respective IGFBP mRNAs.To investigate this, immunohistochemistry was used to detect the presence of IGFBP-2 and -5 proteins, and in situ hybridization was used to determine the presence of IGFBP-2 and -5 mRNAs. The studies were performed in normal adult male Sprague-Dawley rats. Immunohistochemistry was performed by the immunoperoxidase method with polyclonal antibovine-IGFBP-2 and antihuman-IGFBP-5 antibodies. In situ hybridization was performed using 35S-radiolabelled riboprobes.IGFBP-2 mRNA and protein were demonstrated in the outer non-pigmented ciliary epithelium, the corneal germinal epithelium and the corneal endothelium. IGFBP-2 mRNA was detected in these same histological layers of the ciliary processes and the cornea. IGFBP-5 mRNA localized to the stroma and also to the inner pigmented ciliary epithelium and IGFBP-5 protein was demonstrated in the adjacent outer nonpigmented ciliary epithelium. IGFBP-5 mRNA and protein were not demonstrated in the cornea. IGFBPs-2 and -5 were not demonstrated by immunohistochemistry in other structures of the eye.We have shown co-localization of IGFBP-2 mRNA and protein and adjacent cellular localization of IGFBP-5 mRNA and proteins in the anterior compartment of the eye. The presence of IGFBP-2 and -5 in the outer ciliary epithelium suggests secretion into the aqueous humour, where they may enhance trapping of IGF-1 which may be important for lens and corneal cell survival. Our studies outlining the site of locally synthesized IGFBPs suggests specific roles in regulation of IGFs and highlights the potential importance of the IGF system in the eye.

In vitro expansion of human corneal endothelial precursor (HCEP) cells has been reported via production of cell aggregated spheres. However, to translate this procedure in human patients warrants maintaining the position of the eyeballs facing down for 36 h, which is not feasible. In this study, we report a method using a nanocomposite (NC) gel sheet to accomplish the integration of HCEP cells to the endothelium of cadaver bovine's eyes.HCEP cells were isolated from the corneal endothelium of a cadaver human eye and then expanded using a thermoreversible gelation polymer (TGP) as reported earlier. For the study, three cadaver bovine eyes were used. The NC gel sheets were inserted into the bovine eyes', aligned and suture-fixed in position under the host endothelium. HCEP cells previously expanded in the TGP were harvested and injected using a 26-gauge syringe between the endothelium and the NC gel sheet. The eyes were left undisturbed for three hours following which the NC gel sheets were gently removed. The corneas were harvested and subjected to histopathological studies.Histopathological studies showed that all the three corneas used for NC gel sheet implantation showed the presence of engrafted HCEP cells, seen as multi-layered cells over the native endothelium of the bovine cornea. Examination of the NC gel sheets used for implantation showed that only very few corneal endothelial cells remained on the sheets amounting to what could be considered negligible.The use of the NC gel sheet makes HCEP cell transplantation feasible for human patients. Further in vitro basic studies followed by translational studies are necessary to bring this method for clinical application in appropriate indications.

To evaluate the protective effect of Biolon (sodium-hyaluronate 1%) use on corneal endothelium, during donor's corneal harvesting.A prospective, randomized, double blind, comparative study was performed on 120 corneas donated from 60 donors. One cornea from each donor (n = 60) was harvested using intracameral injection of ophthalmic viscosurgical device (OVD), and the fellow cornea with no use of OVD (n = 60, control group). Endothelial cell density (ECD) values were obtained at the center of each corneal graft with a specular microscope one day after harvesting.ECD in the OVD group were 2839 ± 412.5 cells/mm(2) and in the control group 2748 ± 429.7 cells/mm(2). (p = 0.03). Distribution curve of the difference in ECD between the OVD and no OVD eyes showed that 47% of the donors had 100 cells/mm2 difference, and 32% of donors had200 cells/mm(2) difference in favor of the OVD group.Intracameral OVD injection during corneal graft harvesting may protect the endothelium from the mechanical damage induced during the procedure. Further investigation is needed before routine the use of intracameral OVD should be considered in this setting.

Descemet's membrane, the specialised basement membrane of the corneal endothelium, contains a form of extracellular matrix described as wide-spaced collagen. In healthy human Descemet's membrane, wide-spaced collagen forms a highly ordered array in a region called the anterior banded zone. However, in corneal endotheliopathies such as Fuchs' endothelial dystrophy and the iridocorneal-endothelial syndrome large amounts of wide-spaced collagen are deposited posterior to Descemet's membrane in a grotesque parody of the anterior banded zone termed a posterior collagenous layer. The purpose of this study was to identify the composition of the wide-spaced collagen found in the Descemet's membrane of normal and diseased human corneas. Tissue from three normal human corneas, three from patients with Fuchs' endothelial dystrophy and five from patients with the iridocorneal-endothelial syndrome was prepared for immuno-electron microscopy by freezing or embedding in Lowicryl K4M resin. Immunocytochemistry on ultrathin sections was performed with antibodies to collagen Types I, III, V, VI and VIII, fibronectin, laminin, P component and tenascin. Ultrastructural labelling of the wide-spaced collagen in the anterior banded zone of normal and diseased corneas and also of the wide-spaced collagen in the posterior collagenous layer of all the diseased corneas was demonstrated with antibody to collagen Type VIII. Wide-spaced collagen was not labelled by any of the other antibodies used. Large amounts of Type VIII collagen are present in discrete regions of healthy and diseased Descemet's membrane. The deposition of Type VIII collagen may significantly influence the pathobiology of the corneal endotheliopathies.

Contact lens wear causes significant epithelial and stromal acidosis. In this study, we tested whether lens wear can cause endothelial acidosis as well. Rabbit corneas were isolated and perfused in vitro. The endothelial intracellular pH (pHi) was measured with a pH sensitive fluorescent probe (BCECF). Three conditions were examined: 1) Polymethylmethacrylate (PMMA) and rigid gas-permeable (RGP) contact lens wear using a range of oxygen transmissibility (Dk/L) from 0 to 121, 2) epithelial hypoxia produced by exposure to oligomycin/sodium azide solution or epithelial perfusion with 100% N2 equilibrated Ringer's solution, and 3) epithelial exposure to Ringer's equilibrated with 5% CO2, balance air. PMMA and RGP contact lens wear acidified endothelial cells by 0.23 +/- 0.01 (n = 23) and 0.11 +/- 0.01 pH units (n = 23), respectively, within twenty min of lens insertion. Epithelial hypoxia, induced by sodium azide and oligomycin, reversibly acidified the endothelium by 0.04 +/- 0.01 pH units (n = 4). However, epithelial hypoxia induced by perfusion with 100% N2 equilibrated Ringer's did not have a significant effect on endothelial pHi. Introduction of 5% CO2 to the epithelium, acidified the endothelium by 0.15 +/- 0.02 pH units (n = 7) within 10 min. We conclude that contact lens wear can significantly acidify corneal endothelial cells. The endothelial pHi change is caused almost exclusively by a build up of CO2 behind the lens; hypoxia having very little contribution. As expected, RGP contact lenses induced less endothelial acidosis than PMMA controls.

Descemet's membrane consists of two zones, the 'anterior banded zone' which contains wide-spaced collagen and the amorphous 'posterior non-banded zone'. It is attached anteriorly to the corneal stroma by a narrow transitional zone termed the 'interfacial matrix'. The distribution of fibronectin and P component within the different layers of Descemet's membrane was investigated using an ultrastructural immunogold technique. Seven normal human corneas from an eye bank and one specimen from an orbital exenteration were examined. Fibronectin was predominantly present in the posterior part of the posterior non-banded zone and in the anterior banded zone. The anterior part of the posterior non-banded zone contained less fibronectin. P component was present throughout the anterior banded and posterior non-banded zones. There was a sharp demarcation at the interfacial matrix since neither substance was observed in the corneal stroma. The differences shown in the distribution of fibronectin and P component within Descemet's membrane may have resulted from their binding to other substances or alternatively from differences in the quantities laid down during the evolution of this basement membrane.

Specular microscopic studies indicate that the size (polymegathism) and shape (pleomorphism) of the hexagonal corneal endothelial cells change in diabetics. Similar morphometric changes of the corneal endothelium have also been experimentally observed in diabetic rats as well as in diabetic and galactose-fed dogs and concomitant administration of aldose reductase inhibitors reduced these morphological changes. The purpose of this study was to examine whether corneal endothelial changes in galactose-fed dogs are reversible by the marked reduction of galactitol production after stopping prolonged galactose feeding. Ten control dogs were fed a normal diet, while 48 dogs were fed a diet containing 30% galactose. The galactose diet was removed from 15 dogs after 24 months at which time pericyte ghosts in the retina had developed and another 15 dogs were removed from the galactose diet after 31 months when retinal microaneurysms had developed. Eighteen dogs remained on galactose diet throughout the study (38 months). Specular microscopy was conducted on members of all groups after 38 months of study and the photographs were analyzed in masked fashion on the Bambi image analysis system. The evaluation of the corneal endothelial cells revealed significant differences in the cell size and density between galactose-fed dogs in the three groups and normal, age-matched control dogs. Corneal endothelial changes were not significantly reduced in dogs fed galactose for either 24 months or 31 months and then fed a normal diet for 14 and 7 months, respectively, indicating that amelioration of endothelial cell changes requires therapy prior to the advent of endothelial morphologic changes.

The proteinase inhibitor alpha 1-antichymotrypsin is present in the epithelial, stromal and endothelial layers of the human cornea. This was determined by immunolocalization in corneal sections and by Western blot analysis of extracts from the three separated layers. The inhibitor was quantified in the extracts by immunodot blot analysis. The levels observed were 1.3 +/- 0.3 microgram/cornea for the epithelial layer, 22.8 +/- 3.8 micrograms/cornea for the stromal layer and an average of 0.17 micrograms/cornea for the endothelial layer. alpha 1-Antichymotrypsin is being synthesized by the cornea. Metabolically labeled inhibitor was immunoprecipitated from the three layers following organ culture of the intact cornea. Two major forms were detected. These were the native, mature 64 kDa form and a 50 kDa form which is either a degradation product or an incompletely glycosylated form. These results indicate that the cornea has the ability to locally control degradation through synthesis of this inhibitor. Local synthesis of this inhibitor releases the cornea from total dependance upon the vascular system for its supply of alpha 1-antichymotrypsin.

5-Fluorouracil (5-FU) and Mitomycin C (MMC) are used as adjunct chemotherapy during glaucoma filtering surgery to suppress conjunctival fibroblast proliferation. Since part of these agents may gain access to the anterior chamber and cause cytotoxicity to the corneal endothelium we set up an in vitro system to establish a dose-response effect. Cytotoxicity of MMC and 5-FU was quantified using Mosmann's colorimetric assay in a bovine endothelial cell culture system. In this assay the respiratory activity of the cells is used as a marker for cell viability. After incubation for 5 minutes the 3.0 mg/ml concentration of MMC showed endothelial cytotoxicity, whereas no endothelial toxicity of 5-FU was noted in concentrations up to 50 mg/ml. After incubation for 30 minutes endothelial cytotoxicity was demonstrated for 50 mg/ml of 5-FU and 1 mg/ml of MMC. After an exposure-time of 60 minutes the toxicity level remained 50 mg/ml for 5-FU but decreased to 0.5 mg/ml for MMC. We conclude that with respect to the clinically used concentrations and methods of application of 5-FU and MMC in vivo endothelial toxicity is not to be expected. However, in cases of accidental access of MMC to the anterior eye chamber and following a reduction of aqueous turnover rate the safety of MMC is unwarranted.

The Na(+)- and K(+)-dependent ATPase (Na,K-ATPase) expressed in the basolateral membrane of corneal endothelial cells plays an important role in the pump function of the corneal endothelium. We investigated the possible role of dexamethasone in the regulation of Na,K-ATPase activity and pump function in corneal endothelial cells.Confluent monolayers of mouse corneal endothelial cells were exposed to dexamethasone. ATPase activity of the cells was evaluated by spectrophotometric measurement of phosphate released from ATP with the use of ammonium molybdate, with Na,K-ATPase activity being defined as the portion of total ATPase activity sensitive to ouabain. Pump function of the cells was measured with the use of an Ussing chamber, with the pump function attributable to Na,K-ATPase activity being defined as the portion of the total short-circuit current sensitive to ouabain. Western blot analysis was examined to measure the expression of the Na,K-ATPase alpha(1)-subunit.Dexamethasone (1 or 10 microM) increased the Na,K-ATPase activity and pump function of the cultured cells. These effects of dexamethasone were blocked by cycloheximide, a protein synthesis inhibitor. Western blot analysis also indicated that dexamethasone increased the expression of the Na,K-ATPase alpha(1)-subunit, whereas it decreased the expression of the phospho-Na,K-ATPase alpha(1)-subunit.Our results suggest that dexamethasone stimulates Na,K-ATPase activity in mouse corneal endothelial cells. The effect of dexamethasone activation in these cells is mediated by Na,K-ATPase synthesis and increase in an enzymatic activity by dephosphorylation of Na,K-ATPase alpha(1)-subunits.

Examine the mechanism of bicarbonate maintenance of cell-to-cell coupling in rabbit corneal epithelium and endothelium.Carboxyfluorescein was microinjected into rabbit corneal epithelial and endothelial cells. Adjacent cells were observed for fluorescence. Bathing solutions were buffered with bicarbonate, HEPES, phosphate, or acetate-citrate. The influence of intracellular pH and transmembrane voltage (V m ) were examined.Bicarbonate was the only buffer to increase dye coupling. Substitution of bicarbonate structural analogs bisulfite and carbamate in a HEPES-buffered solution increased dye coupling in both cell types. Intracellular pH and V m alterations in corneal epithelial cells bathed in HEPES vs. bicarbonate buffered media had no significant effects on dye coupling.Bicarbonate increases intercellular communication in the corneal epithelium and endothelium. This effect appears to result from an interaction of the bicarbonate molecule (or one of its structural analogs) with either gap junction proteins or an intermediary. We also demonstrate the presence of Cx43 in the rabbit corneal endothelium.

To determine the influence of Prostaglandin (PG) E2 on transforming growth factor (TGF)-beta2-mediated inhibitory effects on the proliferation of corneal endothelial cells (CE).The PGE2 and cell proliferation assays were performed using cultured rabbit corneal endothelium. A PGE2-specific enzyme immunoassay was used to check PGE2 synthesis in supernatants of cells cultured with and without added TGF-beta2 and/or indomethacin. To evaluate the inhibitory effects of PGE2 and TGF-beta2 on CE proliferation, the number of cells grown with exogenous PGE2, or TGF-beta2 with or without indomethacin pretreatment was determined.TGF-beta2, 0.5 to 50 ng/ml, increased the PGE2 secretion of CE dose-dependently in a time-dependent manner. Indomethacin (or =0.1 microg/ml) inhibited this PGE2 secretion to a low level (around 5-10 ng/ml) in the presence or absence of exogenous TGF-beta2. Both exogenous TGF-beta2 and PGE2 inhibited CE proliferation dose-dependently over a wide range of concentrations. Indomethacin reversed the inhibitory effects of TGF-beta2 but not those of exogenous PGE2. In the medium supplemented with indomethacin, even in the presence of 50 ng/ml of TGF-beta2, CE growth did not differ from control cultures.TGF-beta2 stimulates PGE2 synthesis in CE and inhibits CE proliferation in a dose-dependent manner. Indomethacin extinguishes the inhibitory effects of TGF-beta2 on CE proliferation but not the effect of exogenous PGE2. These data suggest that the antiproliferative effects of TGF-beta2 on CE may be possibly due to TGF-beta2-induced synthesis of PG, most likely PGE2.Inhibition of endogenous prostaglandins synthesis by indomethacin extinguished the inhibitory effects of Transforming Growth Factor-beta2 on corneal endothelium proliferation but not exogenous prostaglandin E2. It suggesting that TGF-beta2-induced autocrine synthesis of PGs, most likely PGE2, may be responsible for the anti-proliferative effects of TGF-beta2 on corneal endothelium.

To determine if factors present in the aqueous humor (AH) protect the corneal endothelium from apoptosis.Mouse and human corneal endothelial cells were cultured in vitro, and apoptosis was induced by nutrient deprivation. AH and supernatant from iris/ciliary body (I/CB) cell cultures were tested for their effect on corneal endothelial cell apoptosis. The effect of I/CB supernatant on Fas, Bax, and Bcl-2 gene transcription was evaluated by Northern blotting. I/CB supernatant was subjected to proteinase analysis to identify the apoptosis inhibitory factor(s).Rabbit AH and supernatant from mouse I/CB cell cultures inhibited the apoptosis of mouse and human immortalized corneal endothelial cell lines. The inhibition of apoptosis was associated with an upregulation of Bcl-2 gene transcription and Bcl-2 protein expression. Bax gene expression was not significantly affected by I/CB cell supernatant. Induction of apoptosis by stimulation of the Fas receptor was unaffected by I/CB cell supernatant. Protease analyses indicated that the apoptosis inhibitory factor was a protein.The inability of corneal endothelial cells to undergo mitosis renders the corneal endothelium vulnerable to loss of physiological function through cellular attrition. However, a protein(s) produced by I/CB cells and present in the AH, upregulates Bcl-2 gene transcription and protects the corneal endothelial cells from apoptosis.

To determine the minimum number of cells required to establish a confluent monolayer of retinal pigment epithelium (RPE) with an epitheloid morphology in vitro.Primary or passaged human RPE were harvested by trypsinization from 6 donors and plated onto bovine corneal endothelium extracellular matrix-coated tissue culture plastic in 96-well plates. Plating densities ranged from 1 to 66,000 viable cells/well (0.03-2062 viable cells/mm2) for primary cells or 1 to 100,000 viable cells/well (0.03-3112 viable cells/mm2) for passaged cells. The time required to reach confluence was determined by monitoring the cultures daily until they reached confluence. Mean cell area and circularity index at confluence was calculated to determine the effect of different plating densities on final RPE morphology.Primary RPE plated at densities above 10 viable cells/mm2 (320 cells/well) and passaged RPE plated above 2 viable cells/mm2 (64 cells/well) reached confluence on every occasion. There was a negative correlation between the plating density and time required to reach confluence. Plating densities above 3 viable cells/mm2 (96 cells/well) and 50 viable cells/mm2 (1600 cells/well) yielded smaller, rounder cells at confluence for primary and passaged RPE, respectively.As few as 96 primary RPE cells and 1600 passaged RPE are required to obtain a confluent, 6mm (4-disc diameter) patch of RPE in vitro. This suggests that autologous RPE grafts can be prepared with high efficiency for subsequent transplantation into the subretinal space in vivo.

Purpose. A study was made of the distribution of keratan sulphate in the human anterior chamber. Methods. The monoclonal antibody, 5-D-4, was used in immuno-electron microscopy to visualise keratan sulphate distribution in the anterior chamber of 16 normal eyes, 7 Fuchs' dystrophy corneas, and a macular dystrophy cornea. Results. Keratan sulphate was detected in normal human aqueous humour and also on the surface of trabecular cells in the uveal meshwork. Normal corneal stroma showed an increase in keratan sulphate labelling from anterior to posterior, with marked labelling in the posterior region of Descemet's membrane. The apical surface of the corneal endothelium labelled positively, but showed considerable variation in the level of labelling from cell to cell. The macular dystrophy cornea had the classic histopathological features of a type I case, including a highly abnormal Descemet's membrane. No keratan sulphate was detected in the macular dystrophy patient's corneal stroma or serum. The Fuchs' endothelial dystrophy corneas showed a normal distribution of keratan sulphate labelling in the stroma. The Fuchs' endothelial cells labelled for keratan sulphate but were highly abnormal in appearance, often exhibiting long filopodia and appearing to be actively migrating. Conclusions. This work has shown that keratan sulphate has a much wider distribution than was previously believed. The detection of keratan sulphate on the trabecular and endothelial cell surfaces also suggests a possible role for this molecule in cell adhesion and/or migration.

In vivo repair of the adult human corneal endothelium occurs mainly by movement of cells into the wound defect rather than by cell division. Two forms of cell movement contribute to endothelial wound repair: migration of individual cells into the defect and spreading of the confluent monolayer into the wound area. This laboratory has developed a tissue culture model using rabbit corneal endothelial cells pretreated with the mitotic inhibitor 5-fluorouracil to mimic the relatively amitotic state of human corneal endothelium in vivo. This model permits study of the effects of growth factors and other agents on individual cell migration and monolayer spreading in response to wounding. mRNA for epidermal growth factor (EGF) and its receptor has been detected in cultured corneal endothelial cells and EGF receptors have been detected on human corneal endothelial cells in situ, suggesting that this growth factor may act in an autocrine manner. Prostaglandin E2 (PGE2) is synthesized by cultured corneal endothelial cells and is present in relatively high quantity in aqueous humor in response to corneal wounding and to inflammation in the anterior chamber. Although corneal endothelial cells may be exposed to both EGF and PGE2, little is known about their effects on monolayer repair. The current study compared the effects of PGE2 alone, EGF alone, and both agents in combination on individual cell migration and monolayer spreading using the wound model system and also determined the effect of EGF on PGE2 secretion using a commercial immunoassay. A 15 min exposure of wounded cultures to exogenous PGE2 stimulated individual cell migration and suppressed monolayer spreading.(ABSTRACT TRUNCATED AT 250 WORDS)

During phacoemulsification cavitation bubbles are formed. These bubbles are believed to be one source of damage to corneal endothelium seen after phacoemulsification. Free radicals are induced whenever cavitation bubbles implode. The aim of this study was to confirm the initiation of free radicals by phacoemulsification and to correlate the power of ultrasound in the phacoemulsification process to the amount of free radicals formed, using both in vitro and in vivo techniques. The formation of free radicals was determined by adding luminol to a buffer and measuring the chemoluminescence in vitro and in rabbit eyes (Lumacounter 2080 or a single-photon-counting apparatus) during phacoemulsification. The data obtained show that free radicals are formed during phacoemulsification and that the amount of free radicals correlates with the power of ultrasound. Furthermore, the radical formation could be inhibited by the radical scavengers SOD, Healon and Healon GV. These results were achieved both in vitro in the test tube and in vivo in rabbit eyes. By showing that the addition of SOD to the irrigation buffer during phacoemulsification decreases the corneal endothelial cell damage, we show that free radicals could have a role in postoperative complications seen clinically.

Specific and high affinity binding of the potent muscarinic cholinergic antagonist, [3H]quinuclidinylbenzilate ([3H]QNB) was observed using intact native and cultured adult human corneal endothelium (HCE). Specific binding was proportional to radioligand concentration between 0.03 and 5 nM, indicating a maximal binding capacity (Bmax) of 130 fmol of [3H]QNB/mg protein and a dissociation constant (Kd) of 0.3 nM. Atropine competed effectively with [3H]QNB for binding sites; requiring 3 nM to inhibit 50% of the binding of 1 nM [3H]QNB. Carbachol also competed with [3H]QNB at higher concentrations, but nicotine did not affect [3H]QNB binding at levels up to 1 nM. [3H]QNB binding was also observed in cultured cells of adult human, rabbit, and bovine corneal endothelium. Native and cultured HCE were affinity labelled using tritium-labelled propylbenzilylcholine mustard (PBCM). Separation of the proteins in affinity labelled native and cultured tissue by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that only one protein in each preparation, of 60 and 55 kilodaltons (kDa), respectively, was specifically radiolabelled. These data indicate that the corneal endothelium of human and several animal species exhibit muscarinic cholinoceptors.

In these experiments we characterize rabbit and bovine corneal endothelia cell cultured on microporous membrane filters (0.6cm2). Cell cultured bovine or rabbit corneal endothelial cells (subcultures 1-3) were seeded onto Millicell-HA filter inserts. Electrical resistance measured across the cultured monolayers increased steadily through 14 days of culture, reaching 34.2 +/- 0.8 ohm-cm2 (mean +/- SE) for rabbit cells and 33.1 +/- 1.1 ohm-cm2 for bovine cells. Alizarin red staining of the monolayers showed a polygonal morphology comparable to that observed in situ. Transmission electron microscopy showed well developed apical junctional complexes and flaps. Exposure of the monolayers to calcium-free medium resulted in the disruption of intercellular junctions, rounding-up of the cells and a decrease in electrical resistance (to near 0). Transmonolayer fluxes of inulin and dextran correlated well to the resistance measurements. Results of this study demonstrate that corneal endothelium, both bovine and rabbit, grown on filter inserts is comparable in morphology and ultrastructure to corneal endothelium in situ. The cells cultured in this system form functional apical junctional complexes that effect a barrier function comparable to that of the endothelium in situ.

Trabecular meshwork cells from an inbred strain of mice (A/J) were established in tissue culture. Within 1 hour of enucleation, tissue containing the cornea and the chamber angle was excised and placed in tissue culture. Two to five days later, three cell types grew from the explants. Two of these cell types, corneal endothelium and fibroblasts, grew together, with the fibroblasts preferentially spreading on top of the endothelial cells. The trabecular meshwork cells extended from the explant as a distinct morphological type. The corneal endothelium and its associated fibroblasts were then removed from the culture flask with a sterile cotton swab, leaving a monolayer of pure trabecular meshwork cells. These cells required 3-4 weeks to reach confluency and could be passaged five times. They were actively phagocytic in culture and exhibited immunoreactivity to antibodies against two extracellular matrix components, laminin and collagen type IV. Mouse trabecular meshwork cells also expressed receptors for acetylated low-density lipoprotein, a property shared by trabecular meshwork cells derived from other species. The availability of trabecular meshwork cells from an inbred strain of mice will facilitate future in vivo functional studies of these cells in a syngeneic system, as well as investigations of potential immunoregulatory properties of the trabecular meshwork.

Previous studies have suggested that the plasminogen activator (PA)/plasmin system has important roles in the pathogenesis of epithelial defects and stromal ulceration. The current studies were performed to localize PA species and identify them as tissue-type PA (tPA) or urokinase-like PA (uPA) as the two have distinct regulatory properties potentially related to the mechanisms of defect formation and ulceration. To determine the locations and types of PA species, antibodies to tPA or to uPA or the drug amiloride (a drug that inhibits uPA but not tPA) were incorporated into fibrin/fibronectin (Fn) clots overlying frozen sections to block regional fibrinolysis. Normal rabbit eyes showed tPA activity in association with corneal epithelium, corneal endothelium, and ciliary body/iris. After epithelial scrape or alkali burn, corneal tPA activity was detected initially in the defect zone colinear with fibrin/Fn and was symmetrical to resurfacing epithelium. The observation that initial fibrinolysis occurs in the defect zone, known to contain fibrin/Fn, suggests that tPA from blood (limbal vascular endothelium) and/or from corneal epithelium has become bound to (and activated on) the fibrin/Fn. PA activity was also associated with the leading edges of migrating epithelium post-scrape and post-burn and was not inhibited by antibodies to either tPA or uPA but was inhibited by amiloride. After complete closure of the primary defect post-scrape, only tPA appeared to be associated with the epithelium in that all PA activity was inhibited by antibodies to tPA. The observation that leading edge activity post-burn, in correlation with the formation of secondary defects, continues to be inhibitable by amiloride but not by antibodies to tPA suggests that uPA remains abnormally on the leading edge, and that sustained uPA activity in that location results in inappropriate degradation of subepithelial fibrin/Fn to result in a defect. Successful regulation of uPA activity at the leading edge of corneal epithelium post-burn would be expected to be useful therapeutically in the healing of epithelial defects and the prevention of stromal ulceration.

The physiology and anatomy of the cornea of the New Zealand white rabbit were studied from birth to young adulthood (3 months). The main objective of the study was to follow the ontogeny of the corneal endothelium and correlate its maturation with the establishment of stromal transparency. With maturity, central corneal thickness increases as do corneal diameter and surface area. Endothelial morphology undergoes marked changes including an increase in cell hexagonality and cell surface area, along with a decrease in cell density and coefficient of variation of cell area. Corneal hydration decreases from a high value at birth to the adult level by 20 days after birth, the time of the onset of stromal transparency. By transmission electron microscopy, corneas of newborn rabbits exhibit an endothelium of irregular cell height with some overlap at the bases of adjacent cells. Apical junctions are incomplete in the neonates. With time the endothelium thins and cells becomes more regular in height, overlap of adjacent cells diminishes, and apical junctions develop. Descemet's membrane is thin in newborns and thickens and becomes more homogenous in appearance with maturation. The abundance of Na/K ATPase pump sites per endothelial cell, as determined by 3H-ouabain binding, increases progressively with age even after the establishment of corneal transparency at 20 days. Scatchard and LIGAND analyses of 3H-ouabain binding data indicate that there is a progressive increase in Bmax with no change in the KD from 7 days to 3 months.

A mounting block for in vitro perfusion of the cat cornea is described. Using this apparatus and the techniques of Araie, the permeability (Pac) of the normal cat corneal endothelium to carboxyfluorscein was determined to be 2.5 plusmn; 0.2 × 10-4 cm/min. To assess the sensitivity of this technique in determining changes in Pac associated with alterations in endothelial, morphology, three cats underwent 2 successive unilateral, central, 10 mm diameter circular areas of endothelial debridement 6 weeks apart. Six weeks following the second wounding all 3 animals underwent morphometric analysis and Pac determination. A trend toward an elevation in Pac with extreme reductions in cell density was observed.