Rabbit corneal endothelial cell membrane potential

Membrane potential of rabbit corneal endothelial cells measured using microelectrodes was − 29.3 ± 0.8 mV, n = 45. (mean ± SEV1). Histological location of Lucifer Yellow dye iontophoresed out of the microelectrode confirmed that the microelectrode was located intracellularly, The Lucifer Yellow diffused five to six cell diameters away from the impaled cell indicating endothelial cell coupling. Depolarization by ouabain (10⁻⁴ M) and high extracellular potassium (potassium for sodium substitution) showed the cells to be responsive to changes in the bathing solution whilst impaled, that the cell membrane is more permeable to potassium than sodium and that membrane bound Na⁺-K⁺ -ATPase activity generates the transmembrane electrolyte gradients.     

Effect of indocyanine green intraocular stain on human and rabbit corneal endothelial structure and viability. An in vitro study

PURPOSE: To evaluate the direct effect of intraocular indocyanine green (ICG) on endothelial cell function, ultrastructure, and viability in human and rabbit corneas. SETTING: A laboratory evaluation study. METHODS: Paired human and rabbit corneas were mounted in an in vitro specular microscope for endothelial cell perfusion. One corneal endothelium was perfused with 25 mg ICG dissolved in 0.5 mL aqueous solvent in 4.5 mL balanced salt solution (BSS(R)) for 3 minutes followed by washout with a control solution. The percentage of ICG exposed to the corneal endothelium was 0.5%. The paired cornea was perfused with the same solution without ICG, followed by the washout. The corneas were fixed for scanning and transmission electron microscopy (TEM). In another group, the endothelial viability was determined using a live cell/dead cell assay. RESULTS: In rabbit corneas, the mean corneal swelling rate was 12.9 microm/h +/- 1.2 (SEM) in the ICG corneas and 2.8 +/- 1.9 microm/h in the controls. Scanning electron microscopy and TEM revealed a normal endothelial cell mosaic. The control electron micrographs were similar. In human corneas, the mean swelling rate was 19.1 +/- 2.8 microm/h in the ICG corneas and 19.2 +/- 2.6 microm/h in the controls. Scanning electron microscopy and TEM revealed intact junctions with slight cellular vacuolization, similar to that in the controls. In the live cell/dead cell subgroup, the mean damage was 17.3% +/- 1.7% in the ICG-exposed corneas and 22.0% +/- 8.9% in the controls. CONCLUSIONS: Three-minute exposure to ICG in BSS had no adverse effect on corneal endothelial function, ultrastructure, or viability in human and rabbit corneas. This study provides a safety profile for the corneal endothelium when ICG is used as an intraocular tissue stain in ophthalmic surgery.     

Cryopreservation of rabbit and cat corneas at -18 to -24 degrees C.

A simple method of corneal cryopreservation, in which corneas were frozen at -18 to -24 degrees C, was examined. Rabbit and cat corneas were placed successively in solutions of 50% fetal calf serum in McCarey-Kaufman medium with an increasing glycerol and glucose content. They were then frozen and stored in a -20 degrees C domestic freezer. Rabbit corneas stored in this way were examined in vitro by light and scanning electron microscopy, and both rabbit and cat corneas were also assessed after orthotopic allotransplantation into adult recipient animals. Functional corneal grafts were obtained with rabbit and cat tissue that had been cryopreserved for 3-4 weeks and 1 week, respectively. Endpoint analysis (by light and scanning electron microscopy) of grafts that had survived for 50 days indicated the presence of an intact corneal endothelial monolayer. The corneal endothelium slowly degenerated as the storage time was increased. Importantly, however, the endothelium appeared to withstand the freezing and thawing processes and we conclude that it may be possible to store corneas at temperatures above -196 degrees C, without the need for complex, low-temperature cryopreservation systems.     

碱烧伤角膜内皮细胞层的创伤修复—创伤闭合的动力学观察

在猫角膜建立碱烧伤的标准模型,观察了烧伤后内皮细胞层的创伤闭合过程。发现在创伤后第一周内皮层缺损达到闭合状态,4个月趋向稳定。结果提示猫角膜为研究角膜内皮细胞对碱烧伤的创伤反应和决断药物的疗效提供了一个理想的模型,并讨论了内皮细胞在碱烧伤修复过程中的重要作用。     

Gap junctional communication in the human corneal endothelium and epithelium

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.     

Corneal endothelial cytoskeletal changes in F-actin with aging, diabetes, and after cytochalasin exposure.

We investigated the changes in endothelial cytoskeletal F-actin that occur with aging, diabetes, and exposure to cytochalasin D. Rabbit corneas, human donor corneas (with or without polymegethism), and corneas of diabetic individuals were studied. Endothelial F-actin was stained using nitrobenzoxadiazole-phallacidin. Results of these experiments demonstrated that F-actin of the rabbit and human corneal endothelium was arranged in linear circumferential strands that formed a hexagonal array. After in vitro perfusion of cytochalasin D to the corneal endothelium, the F-actin became randomly distributed throughout the cytoplasm, the hexagonal shape of the endothelial cell was disrupted, and endothelial permeability to carboxyfluorescein increased. Changes in F-actin were also observed in the endothelium of the human corneas with polymegethism, and in donor tissue having had previous posterior chamber intraocular lens implantation. The corneas of diabetic individuals also showed marked irregular F-actin fibers crossing the endothelial cell cytoplasm. These abnormal patterns of F-actin may contribute in part to the polymegethism observed in the corneal endothelial cells and may be the result of constant stress in cell volume regulation, particularly in the corneas of diabetic individuals.     

Postnatal development of corneal endothelium

Comparison specular micrographs of infant and adult corneas from cats, cows, dogs, rabbits, and humans demonstrate that a large decrease in central endothelial cell density occurs during maturation of the cornea. Central endothelial cell counts of developing cat, dog, and rabbit corneas decrease rapidly during the first months of life. This rapid decline in endothelial cell density correlates with growth of the cornea to the adult size. Central endothelial cell counts of adult cat, cow, deer, dog, pig, rabbit, and human corneas are similar (2500 cells/mm2) despite a wide variation in corneal size. Comparison of observed endothelial cell counts with two hypothetical situations, one of unrestricted endothelial mitosis and the other of only endothelial hypertrophy, indicates that hypertrophy of individual cells is primarily responsible for achieving the adult cell density of 2500 cells/mm2 for these species. This observation is true for species that have a high adult endothelial mitotic capacity (rabbit) as well as those that do not (cat). The human cornea is a special case because the decline in central endothelial cell density indicates that a large apparent corneal endothelial cell loss (approximately 45%) occurs early in postnatal development.     

Corneal endothelial regeneration and tissue engineering

Human corneal endothelial cells (HCECs) have a limited proliferative capacity. Descemet stripping with automated endothelial keratoplasty (DSAEK) has become the preferred method for the treatment of corneal endothelial deficiency, but it requires a donor cornea. To overcome the shortage of donor corneas, transplantation of cultured HCEC sheets has been attempted in experimental studies. This review summarizes current knowledge about the mechanisms of corneal endothelial wound healing and about tissue engineering for the corneal endothelium. We also discuss recent work on tissue engineering for DSAEK grafts using cultured HCECs and HCEC precursor cell isolation method (the sphere-forming assay). DSAEK grafts (HCEC sheets) were constructed by seeding cultured HCECs on human amniotic membrane, thin human corneal stroma, and collagen sheets. The pump function of the HCEC sheets thus obtained was approximately 75%–95% of that for human donor corneas. HCEC sheets were transplanted onto rabbit corneas after DSAEK. While the untransplanted control group displayed severe stromal edema, the transplanted group had clear corneas throughout the observation period. The sphere-forming assay using donor human corneal endothelium or cultured HCECs can achieved mass production of human corneal endothelial precursors. These findings indicate that cultured HCECs transplanted after DSAEK can perform effective corneal dehydration in vivo and suggest the feasibility of employing the transplantation of cultured HCECs to treat endothelial dysfunction. Additionally, corneal endothelial precursors may be an effective strategy for corneal endothelial regeneration.     

建立在体角膜内皮细胞损伤后再生动物模型的实验研究

目的探讨建立在体角膜内皮损伤动物模型的方法及其损伤后再生的潜能。 方法选取2周龄的正常六角龙鱼30只。采用数字表法随机分为正常观察组、NaOH损伤组及机械损伤组,各10只。采用活体共聚焦显微镜检查、组织病理学检查、茜素红-曲利苯蓝内皮染色和氯化金角膜神经染色等方法观察。对正常观察组六角龙鱼,观察其眼球和角膜正常结构;通过NaoH溶液和器械两种方法损伤角膜内皮层,建立角膜内皮细胞损伤模型,观察其角膜内皮细胞再生的情况。对4个时间点角膜内皮细胞的密度进行正态性检验和方差齐性检验,使用重复测量方差分析对不同组别、不同时间点角膜内皮细胞密度的均值进行比较,事前进行Mauchly球形检验,如果检验结果显著,采用多元方差分析,否则选用校正自由的F检验。 结果六角龙鱼角膜厚度约为(75.75±7.51)μm,角膜上皮细胞层较厚,约占角膜厚度的一半。经NaoH溶液和器械两种方法损伤其角膜内皮细胞后,六角龙鱼角膜内皮细胞密度明显降低。NaoH损伤组和机械损伤组在不同时间点,六角龙鱼角膜内皮细胞密度的比较,具有统计学意义(F＝31.38,51.77;P<0.05)。而各个时间点间的差异,两组均无统计学意义(F＝1.37,2.67,0.70,4.14;P>0.05)。在损伤后3 d时,NaoH损伤组六角龙鱼角膜内皮细胞的密度为(128±14)个/mm²,机械损伤组为(113±11)个/mm²。与损伤前比较,其角膜内皮细胞密度的差异均有统计学意义(t＝19.39,8.78;P<0.05);在损伤后7 d时,NaoH损伤组六角龙鱼角膜内皮细胞的密度为(157±20)个/mm²,机械损伤组为(169±19)个/mm²。与损伤后3 d时比较,其角膜内皮细胞密度均有所恢复,差异均有统计学意义(t＝3.75,8.07;P<0.05);在损伤后14 d时,可见NaoH损伤组六角龙鱼角膜内皮细胞的密度为(198±17)个/mm²,机械损伤组为(223±17)个/mm²。与损伤后3 d时和7 d时比较,均有明显恢复,差异均有统计学意义(t＝10.05,8.07;P<0.05)和(t＝4.94,6.70;P<0.05)。随着时间延长,两组六角龙鱼角膜内皮细胞的密度均逐渐恢复。在伤后14 d时,其角膜内皮细胞形态、大小和密度均基本恢复正常状态。 结论使用NaoH溶液和器械两种方法均可成功建立六角龙鱼角膜内皮细胞损伤的动物模型。初步观察结果表明,其角膜内皮细胞具有一定的再生潜能。此方法可为角膜内皮细胞损伤后再生研究提供一种新的动物模型。     

人表皮生长因子对体外培养兔角膜内皮细胞影响的实验研究

目的：明确人表皮生长因子（ｈｕｍａｎ Ｅｐｉｄｅｒｍａｌ Ｇｒｏｗｔｈ Ｆａｃｔｏｒ,ｈＥＧＦ）对角膜内皮细胞的影响。方法：采用体外培养的兔角膜内皮细胞,观察接种后不同时点ｈＥＧＦ对其生长状态的影响;兔角膜片内皮损伤模型,离体培养后行Ｈ－Ｅ染色和Ｈ＾３－ＴｄＲ掺入放射自显影,观察ｈＥＧＦ对其修复的影响。结果：兔角膜内皮细胞体外培养ｈＥＧＦ组细胞数高于对照组,并使细胞形态产生纺锤形改变;角膜内皮细胞     

Changes in corneal endothelial apical junctional protein organization after corneal cold storage

PURPOSE: Understanding the mechanisms regulating corneal endothelial permeability during storage and recovery is of critical importance both to improving Eye Banking practices and preventing corneal transplant failure. The goal of this study was to determine the effects of cold storage on the organization of apical junctional complex (AJC) proteins and their relationship to F-actin in corneal endothelium. METHODS: Immunostaining using antibodies to the AJC proteins, ZO-1, cadherin, and alpha- and beta-catenin was performed on 16 eye bank corneas and four cat corneas after 2-8 days of storage at 4 degrees C in Optisol-GS, and compared with fresh corneas. The 3-D in situ localization of the AJC proteins was then determined by using laser confocal microscopy. AJC organization also was assessed after stored human corneas were further incubated at 37 degrees C in Optisol-GS or in serum-free media. RESULTS: In normal human and cat corneas, F-actin was organized into dense peripheral bands (DPBs) along the apical cell border. The tight-junction protein, ZO-1, and the adherens junction proteins, cadherin and alpha- and beta-catenin, each formed a uniquely discontinuous hexagonal apical band with the largest gaps occurring at the Y-junctions between adjacent endothelial cells. In stored eye bank and cat corneas, cells lost their normal hexagonal F-actin staining pattern and appeared rounded and distorted, with increased cytoplasmic staining and incomplete and condensed DPBs. Similar distortions were observed in the apical bands of cadherin, catenin, and ZO-1 staining between endothelial cells. Gaps in staining at the endothelial Y-junctions were significantly enlarged; corresponding gaps also were observed with phalloidin staining. These changes were reversed after overnight incubation at 37 degrees C in either serum-free media or Optisol-GS. Quantitative analysis demonstrated a significant increase in the size of the Y-junctional gaps (p < 0.0001) after cold storage of cat corneas as compared with fresh corneas. CONCLUSION: These results suggest that disruption of the F-actin cytoskeleton and AJC may explain, in part, the loss of function (corneal swelling) after prolonged cold storage.     

Expression of adhesion molecule CD44 on human corneas

AIMS—This study was undertaken to confirm the distribution and expression of the molecule CD44 on human corneas under normal and pathological conditions.
METHODS—Fifty eight corneal buttons from adult patients suffering from various corneal diseases and four normal corneas were included in this study. Frozen sections were stained immunohistochemically with monoclonal antibodies against human CD44 using an APAAP method and observed under a light microscope.
RESULTS—In normal corneas CD44 was predominantly expressed on the membranes of basal epithelial cells and on the keratocytes, as well as on the vascular endothelial cells of the corneal limbi, but was not expressed on corneal endothelial cells. Enhanced expression of CD44 was observed on the epithelium of corneas with inflammation and allograft rejection. In a number of abnormal conditions including allograft rejection, corneal trauma, primary and secondary corneal endothelial decompensation the remaining endothelial cells stained positively for CD44. However, in some corneas of keratitis, keratoconus, and dystrophy the endothelium which appeared relatively integral in morphology and amount remained CD44 negative.
CONCLUSIONS—These results suggest that CD44, the hyaluronate receptor, may play an important role in corneal cell-cell and cell-matrix interactions. Its regulation is closely related to corneal inflammatory reactions. The induction of CD44 on corneal endothelium might play a potential role in compensatory processes when corneal endothelial cells are injured.

     

The coating of bovine and rabbit corneas denuded of their endothelium with bovine corneal endothelial cells.

A method for coating corneas denuded of their endothelium has been developed. The attachment and spreading of cultured bovine corneal endothelial cells seeded upon the Descemet's membrane of corneal buttons previously denuded of their endothelium by delicately sweeping the endothelial side with a cotton swab have been analyzed. Confluent cultures of bovine corneal endothelial cells were exposed to trypsin to disrupt the cell monolayer into single cells. Increasing concentrations of endothelial cells ranging from 2·5 × 10⁴ to 3 × 10⁵ cells were then seeded on the denuded Descemet's membrane of 11 mm bovine corneal buttons. When the corneal buttons were stained with alizarin red after an incubation period of 8 hr at 37°C, the best coating was observed with 10⁵ seeded cells. In this case, no areas of denudation could be seen and the cells were clearly apposed to one another, thereby reconstituting an endothelial cell monolayer. The cultured bovine corneal endothelial cells seeded on denuded Descemet's membranes plated extremely rapidly. By 15 min, 80% of Descemet's membrane was covered with a monolayer of endothelial cells and by 30 min all of Descemet's membrane was covered.The plating of bovine corneal endothelial cells on denuded Descemet's membrane was a direct function of the trypsin concentration to which they were first exposed. Cells first treated with 0·05% trypsin plated poorly 1 hr after being seeded on a denuded Descemet's membrane. Better plating efficiency was achieved with cells first exposed respectively to 0·025% and 0·01% trypsin. The best results were consistently obtained with cells first dissociated with 0·005% trypsin.Although serum is required in vitro for the attachment of normal cells to tissue culture dishes, it was not required for the attachment of corneal endothelial cells to the denuded Descemet's membrane. Cultured corneal endothelial cells plated equally well in the presence or absence of serum. Similar results were obtained when the cells were suspended in aqueous humor instead of in tissue culture medium.When denuded rabbit corneas were used as a substrate instead of bovine corneas, all the parameters studied for the attachment and spreading of bovine corneal endothelial cells seeded on bovine corneas (cell density, time, and medium) lead to similar conclusions with respect to the interactions between corneal endothelial cells and rabbit Descemet's membrane.     

Changes in nuclear DNA content and cell size of injured human corneal endothelium

To understand how human corneal endothelium compensates for cell loss, nuclear DNA-cytofluorometry and cell morphometry were carried out on injured corneal endothelium. The examined corneas included two cases of keratoconus complicated with acute hydrops and one without acute hydrops, two cases of herpetic keratitis, one case of post-intracapsular cataract extraction (post-ICCE) and one case of luetic keratitis. The endothelial cell layer was separated from Descemet's membrane and double-stained with Rhodamine-labeled wheat germ agglutinin-lectin (WGA) and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI). The area of each cell was measured with a color image analyser and compared with its cytofluorometric nuclear DNA content. The endothelium in apparently intact regions of the diseased corneas showed the same DNA-ploidy pattern and cell area as the physiological corneas. However, endothelial cells in injured regions had greater area, even in diploidy, than in presumably normal ones and showed a larger number of hyperploid cells ranging from 4C to 36C. Hyperploid cells consisted of many multinucleates and few polyploidies and had extremely large and bizarre cytoplasm. All injured corneas were accompanied by cells with numerous micronuclei. A few asymmetrical 4C-binucleates (with DNA values such as 1.3 plus 2.6C) appeared in the case of the post-ICCE. It is concluded that damage to human corneal endothelial cells in vivo results in cell enlargement with or without DNA synthesis. Those changes appear more severe in diseased corneas than in the situation of physiological aging which we have reported previously. In severe cases, micronuclei, polyploid cells and multinucleated giant cells are frequent, thereby suggesting a possible long-persistent metabolic impairment of the endothelium after severe damage to the cornea.     

Effects of posterior chamber lens implantation on the endothelium of transplanted corneas

AIMS—The morphological changes of the corneal endothelium after posterior chamber lens implantation in the transplanted corneas were investigated.
METHODS—36 patients underwent extracapsular cataract extraction with posterior chamber lens implantation. Among these, penetrating keratoplasty had been performed in 18 patients before cataract surgery. The indications for penetrating keratoplasty in these cases included keratoconus, herpetic keratitis, and macula cornea. 18 cataract patients with normal corneas were also studied as controls. The central corneal endothelium in each subject was examined with a wide field specular microscope at a few days before and 3 months after cataract surgery.
RESULTS—Although the transplanted corneas showed lower endothelial cell densities, marked polymegethism, and pleomorphism in the baseline variables, the endothelial morphological changes in the transplanted corneas after posterior chamber lens implantation were comparable with those in the normal corneas. Also, there was no clinical evidence, especially, of corneal epithelial and/or endothelial rejections and corneal decompensation in all corneas.
CONCLUSION—Even though the transplanted corneas have a lower endothelial cell density and marked polymegethism, it is believed that cataract surgery does not induce corneal decompensation in cases where the peripheral recipient endothelium can be considered to have normal morphology.

     

Human and rabbit corneal endothelial permeability after different chemical forms of glutathione

This study aimed to compare the effects of reduced glutathione (GSH) with those of S-(1,2-dicarboxyethyl)glutathione (DCE-GS) (in rabbits and humans), and different concentrations of the latter (in humans), on corneal endothelial permeability when added to solutions bathing the isolated cornea. Inulin/dextran permeability was determined from stromal- to endothelial-facing surfaces of de-epithelialized corneas. The bathing solution was modified Opeguard(R)-MA (MOMA), an ocular irrigating solution, to which either GSH or another intrinsic tripeptide, DCE-GS, was added. Paired corneas were used to compare either different combinations of GSH with DCE-GS (rabbit or human) or various concentrations of DCE-GS from 0.25 to 2.0 mM (human). Endothelial cyclic AMP levels were determined in cultured rabbit cells. MOMA alone resulted in approximately the same permeability as MOMA + 0.3 mM GSH while the use of 2 mM DCE-GS significantly reduced rabbit (40% maximum, p < 0.00001) and human (30% maximum, p < 0.01) corneal permeability. Human corneal endothelial permeability remained reduced through a range of concentrations of DCE-GS from 0.25 to 2.0 mM DCE-GS. Tissue-cultured rabbit corneal endothelium showed an increase in cyclic AMP after DCE-GS or GSH. DCE-GS potentially offers a viable alternative to GSH for inclusion in ocular irrigating or corneal preservative solutions since it maintains human corneal endothelial permeability at a lower, stable value relative to non-DCE-GS-containing solutions.     

Penetrating keratoplasty in the cat. A clinically applicable model

A series of 28 consecutive penetrating keratoplasties were performed on adult cats. Donor corneas (n = 14) were maintained in culture medium for 14--24 hours prior to transplantation. Rotational autografts (n = 7) were used to control for cell loss caused by culture maintenance as well as for the effects of surgery. Additional homografts (n = 7) were transplanted following removal of the corneal endothelium to study the extent of host corneal endothelial cell regeneration. Pre- and post-operative endothelial cell counts of the homografts made from specular micrographs demonstrated an average cell loss of 30% one month following surgery. A similar 30% average cell loss was present in the rotational autografts. Clinically, both homografts and autografts remained clear and were near normal in thickness. Homografts lacking endothelium exhibited persistent, severe edema that correlated with the inability of the host corneal endothelium to resurface the graft. Clinical and morphologic evidence of mild homograft rejection as observed in 15% of the animals that received normal homografts. Corneal endothelial cell loss following penetrating keratoplasty in the cat approximates that observed following the same procedure in the human. Additionally, regenerative capacity of the corneal endothelium in the cat, like that of the human, is limited. These features suggest that this cooperative, hardy animal is an excellent model in which to study many aspects of corneal transplantation that have direct application to the treatment of human corneal disease.     

The spatial organization of apical junctional complex-associated proteins in feline and human corneal endothelium

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.     

Heterologous transplantation versus enhancement of human corneal endothelium

The ability to successfully transplant human corneal endothelium would offer a significant advance in the treatment of many corneal diseases. To investigate the feasibility of this, we established cultures of endothelial cells derived from neonatal human corneas. Eye bank donor corneas were either enhanced with a suspension of cultured endothelial cells or underwent endothelial cell removal and subsequent replacement with cultured endothelium. Following a 48-h incubation, the corneas were transplanted into the eyes of nonhuman primates. Over a 12-month period, 67% of the corneas with complete endothelial cell replacement thinned and remained clear, with a mean corneal thickness of 0.57 mm. Enhanced corneal buttons demonstrated a significantly lower success rate (35%), with opacified and thickened corneas. Control eyes in which the native endothelium was removed demonstrated advanced corneal edema and vascularization, with a mean corneal thickness in excess of 1 mm. By utilizing established tissue-culture techniques, we have demonstrated that human corneal endothelium, when cultured and subsequently transplanted, retains its in vivo pump function. Although further studies are warranted, these results indicate that transplanted human corneal endothelial cells can function normally and suggest the possibility of endothelial cell replacement for therapeutic purposes.