Long-term outcome of iron-endocytosing cultured corneal endothelial cell transplantation with magnetic attraction

The long-term efficacy and safety of transplanting iron-endocytosing cultured corneal endothelial cells (CECs) with magnetic attraction were evaluated. Rabbit corneas were subjected to cryo-injury to detach CECs. Cultured rabbit CECs (RCEC) were exposed to spherical iron powder and then injected into the anterior chamber, after which a neogium magnet was fixed on the eyelid for 24 hr to attract the cells to Descemet's membrane (RCEC-iron group, n=4). An RCEC group (cryo-injury and injection of normal cultured RCEC, n=4) and a Cryo group (cryo-injury without injection of RCEC, n=4) served as controls. Intraocular pressure was measured for 12 months after surgery. Corneal findings on slit lamp biomicroscopy, RCEC density, the electro-retinogram (ERG), and residual iron in the ocular tissues were evaluated at final assessment. Intraocular pressure did not increase in any group throughout 12 months of observation. At the final assessment, the average corneal edema score of the RCEC-iron group was significantly lower than that of the RCEC or Cryo groups (p=0.021). The average CEC density of the RCEC-iron group was 2581+/-230 cells mm(-2) (mean+/-SD), whereas no CECs were observed on the inner surface of the central cornea in the RCEC and Cryo groups. No significant differences of the ERG (a- and b-wave amplitudes, and b-wave/a-wave ratio) were detected among the groups. Iron powder was not detected by Berlin blue staining in the ocular tissues of the RCEC-iron group. Apoptotic cells were not observed in the endothelium by terminal transferase-mediated nick-end labeling. Transplanted iron-endocytosing RCEC remained viable for 12 months after surgery. There were no detectable ocular complications after the transplantation of iron-endocytosing cultured RCEC. Magnetic attachment of iron-endocytosing CECs can be an effective and safe method for corneal endothelial repair.     

Corneal endothelial damage in congenital and juvenile glaucoma

Corneal changes are typical for infantile glaucoma. In newborns, Descemet's membrane and the stroma are still soft, and distend when intraocular pressure rises. Cell counts were performed under the reflecting microscope to determine whether the endothelium is affected when corneal diameter increases and changes occur in Descemet's membrane. The mean endothelial cell density of 20 patients with congenital or secondary juvenile glaucoma, aged between 4 and 29 years, was found to be 2780/mm2. Even though increased corneal diameter was the most common primary symptom of buphthalmos, there were no relationships between corneal diameter on the one hand and postoperative endothelial cell density, visual outcome, or postoperative pressure on the other. In children who had had corneal edema followed by ruptures of Descemet's membrane the number of endothelial cells was significantly reduced, visual outcome was worse, and there were more problems with regulation of intraocular pressure. Lower-than-average endothelial cell densities were also found in some patients with secondary glaucoma.     

Advanced glycation end products in Descemet's membrane and their effect on corneal endothelial cell

PUPPOSE: The purpose of this study was to evaluate the effect of advanced glycation end products (AGEs) in Descemet's membrane on the attachment and spreading of the corneal endothelial cells. METHODS: An anti-AGEs monoclonal antibody (6D12), which recognizes a N(epsilon)-carboxymethyl lysine (CML)-protein adduct as an epitope, was used for immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). Fresh bovine Descemet's membrane was incubated for 4 weeks in the buffered solution with 500 mM of glucose-6-phosphate (G-6-P). In the incubated Descemet's membrane, the immunohistochemical localization of CML was examined. Type I collagen-, type IV collagen-, fibronectin-, or laminin-coated 96-well plates were glycated by G-6-P. The amount of CML was determined by ELISA using 6D12. Cultured bovine corneal endothelial cells were seeded onto glycated or non-glycated extracellular matrix (ECM) in 96-well plates and allowed to attach for 3 hours. The number and the surface area of the attached cells were examined. RESULTS: Immunoreactivity to CML was detected in Descemet's membrane incubated in the buffered solution containing G-6-P. Glycation of fibronectin and laminin decreased the number and the surface area of the attached corneal endothelial cells. Aminoguanidine in the incubation mixture inhibited CML formation of ECM components and increased the number and the surface area of the attached corneal endothelial cells in a dose-dependent manner. CONCLUSIONS: AGE formation on fibronectin and laminin attenuated the attachment and spreading of the corneal endothelial cells. AGEs' formation in Descemet's membrane may be responsible for the corneal endothelial cell loss with aging and corneal endothelial abnormalities in diabetic patients     

Aging changes of mouse corneal endothelium and Descemet's membrane

The purpose of this study was to characterize age-associated changes in the corneal endothelium and Descemet's membrane (DM) in C57BL/6 mice, an inbred strain commonly used as a genetic disease model. Corneas from mice aged 2 weeks to 24 months were studied. Light microscopy was used to assess central endothelial cell density, area, and morphology. Transmission electron microscopy was used to assess thickness and ultrastructural features of DM. Central corneal endothelial cell density showed a rapid decline from 5,232+/-892 cells/mm(2) (mean+/-S.D.) at 2 weeks to 2,532+/-112 cells/mm(2) at 16 weeks of age. Thereafter, cell density declined more slowly, reaching 2,004+/-134 cells/mm(2) at 24 months of age. DM thickness showed an approximately linear increase from 1.12+/-0.22 microm (mean+/-S.D.) at 2 weeks to 4.19+/-1.17 microm at 24 months of age. DM in 2 and 6 week age groups was composed entirely of material with an electron dense, periodic banding pattern. Sixteen week, 12 month, and 24 month age groups exhibited an additional, progressively thicker, homogeneous layer lacking periodic banding. The observed age-dependent thickening of DM was predominantly due to accumulation of the posterior, non-banded layer. In C57BL/6 mice, central endothelial cell density declines with age and DM progressively thickens due to accumulation of a posterior, non-banded portion. These age-associated changes are strikingly similar to observations in humans and thus further support the potential usefulness of the mouse model for studying disorders of the corneal endothelium and Descemet's membrane.     

Transplantation of corneas reconstructed with cultured adult human corneal endothelial cells in nude rats

PURPOSE: The feasibility of corneal reconstruction with cultured adult human corneal endothelial cells (HCEC) was examined in a nude rat model. METHODS: Endothelial cells were removed from the corneas of Lewis rats using a sterile cotton swab. Cultured adult HCEC labelled with a fluorescent marker chloromethyl-benzamidodialkylcarbocyanine (CM-Dil) were seeded onto the denuded Descemet's membrane. Then the corneas were centrifuged, incubated for 2 days, and transplanted into the eyes of nude rats using the penetrating keratoplasty technique (HCEC group). Control nude received corneas denuded of endothelium and without HCEC. The operated eyes were observed for 28 days after transplantation, and then were subjected to histological and fluorescein microscopic examination. RESULTS: The mean corneal thickness was significantly smaller in the HCEC group than in the control group throughout the observation period. The corneal endothelial cell density of the grafts at 28 days postoperatively ranged from 2425 to 3250 cells mm(-2) (mean+/-sd, 2744+/-337 cells mm(-2)). Fluorescein microscopy at 28 days after surgery showed numerous DiI-labelled cells on the posterior corneal surface in the HCEC group. Frozen sections showed a monolayer of DiI-labelled cells on Descemet's membrane. CONCLUSIONS: Cultured adult HCEC function well and maintain corneal transparency for 1 month after transplantation in nude rats.     

Surgical detachment of Descemet's membrane and endothelium imaged over time by in vivo confocal microscopy

A 90-year-old woman developed a large circular capsulorhexis-like defect in Descemet's membrane as a complication of small incision cataract surgery. Nine months post-surgery, in vivo confocal microscopic examination of the temporal mid-peripheral cornea revealed an endothelial cell density of 934 +/- 69 cells/mm2 (normal range 1566-3088 cells/mm2). Endothelial pigmented deposits were visible as scattered hyper-reflective areas on the posterior endothelial surface. Descemet's folds were also noted. In vivo confocal microscopy performed 3 years later showed the temporal mid-peripheral corneal endothelial density (in the region of the break) was 948 +/- 66 cells/mm2. A reduction of endothelial polymegathism and pleomorphism was observed. Imaging in the region of the temporal portion of the original Descemet's defect showed well-defined linear structures with hyper-reflective edges. Compared to 3 years previously, the cornea at the level of Descemet's membrane appeared to have greater reflectivity. This case demonstrates how microstructural changes in the cornea can be described and analysed over time with the assistance of in vivo confocal microscopy.     

角膜内皮细胞移植

角膜内皮细胞在体外培养的条件下可以在同种异体或异种角膜后弹力层上成功生长,培养的上皮细胞的生长情况及特性与供体年龄、内皮细胞来源部位、载体材料、培养条件、以及是否应用生长因子有密切关系。人角膜内皮细胞培养的成功,以及在非人类的灵长目动物眼上的成功移植,为内皮移植技术的治疗作用提供了实验基础。培养传代的角膜内皮细胞移植到活体上后具有正常内皮细胞的功能,可以作为穿透性角膜移植的替代方法之一加以选择。     

Cultivated corneal endothelial cell sheet transplantation in a primate model

PURPOSE: To examine the feasibility of cultivated corneal endothelial cell transplantation in a primate model. METHODS: Monkey corneal endothelial cells (MCECs) obtained from three cynomolgus monkeys were cultivated, with subcultures grown on collagen type I carriers for 4 weeks. The corneal endothelium of the right eye of six monkeys was mechanically scraped, after which a cultivated MCEC sheet was brought into the anterior chamber of four eyes and fixed to Descemet's membrane by air. As the control, a collagen sheet without MCECs was transplanted into one eye of one monkey, and a suspension of cultivated MCECs was injected into the anterior chamber in one eye. RESULTS: Cultivated MCECs produced a confluent monolayer of closely attached hexagonal cells that showed both ZO-1 and Na(+)-K(+) ATPase expression. In the early postoperative period MCEC sheets were attached to Descemet's membrane and corneal clarity was recovered. The recovered clarity was accompanied by a decrease in corneal thickness. Fluorescein DiI labeled donor corneal endothelial cells were detected on the host cornea on postoperative day 7. Six months after transplantation MCEC-transplanted corneas remained clear, and hexagonal cells were observed by in vivo specular microscopy with a density of 1992 to 2475 cells/mm(2). Control eyes showed irreversible bullous keratopathy that precluded pachymetry and specular microscopy. CONCLUSIONS: A model of cultivated corneal endothelial transplantation for corneal endothelial dysfunction was established in primates whose corneal endothelial cells have less proliferative capacity in vivo. Our results suggest that this is a useful model for long-term observation in advance of the future clinical application of cultivated corneal endothelial transplantation.     

Pseudokeratoconus in trisomy 21 and posterior polymorphous corneal dystrophy

The authors examined three members of a family with an autosomal dominant trait of posterior polymorphous corneal dystrophy of varying expressivity. The 67-year-old white mother had a visual acuity of 20/30, with only discrete irregularities at the level of Descemet's membrane. The daughter developed bullous keratopathy with polymorphous ring-shaped opacities in the central area of Descemet's membrane early, in her 34th year. The 25-year-old son, who also had Down's syndrome, presented with the clinical symptoms of acute keratoconus. Light microscopy revealed a thickened, multilaminated Descemet's membrane with vesicles, breaks, and dislocation of endothelial cells into the deep stroma. Transmission electron microscopy showed a normal anterior, ribbonlike portion of Descemet's membrane and a fibroblastic differentiation of the corneal endothelial cells.     

Culture of human corneal endothelial cells isolated from corneas with Fuchs endothelial corneal dystrophy

The purpose of this study was to assess the feasibility of initiating primary cultures of corneal endothelial cells from patients suffering from Fuchs endothelial corneal dystrophy (FECD; MIM# 1036800). We also evaluated which conditions yielded the best results for culture. Twenty-nine patients undergoing Descemet stripping automated endothelial keratoplasty consented to the use of their excised Descemet's membrane for this study. Out of the 29 specimens, 18 successfully initiated a culture. Cell morphology varied between endothelial (rounded, slightly elongated cells, n?=?12) and fibroblastic-like (thin and very elongated cells, n?=?6). These differences in cell morphology were also observed with the normal human corneal endothelial cell cultures. The cultures that initially presented an endothelial morphology maintained their shape in subcultures. Clusterin expression was similar in FECD and normal endothelial cells. Transmission electron microscopy of FECD Descemet's membranes showed a high degree of various abnormalities generally found in this disease, such as a thickened Descemet's membrane, presence of a posterior banded layer, presence of a fibrillar layer and striated bodies of various sizes and periodicities. Patient's age was predictive of culture success, all younger FECD donors generating cultures of endothelial morphology. The absence of a fibrillar layer was also a factor associated with greater success. Culture success was not dependent on specimen size, specimen pigmentation, or patient's preoperative central corneal thickness. In conclusion, this paper shows for the first time that central Descemet's membranes of patients suffering from FECD possess proliferative endothelial cells that can be isolated and cultured without viral transduction, opening the way for new in?vitro studies of this disease.     

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.     

Contact lens intolerance induced by DM rupture and secondary endothelial degeneration. A specular microscopic study

Breaks or tears in the endothelium and Descemet's membrane (DM) are easily noticed in exact biomicroscopical ophthalmological examination. We present 2 patients with a large unilateral horizontal rupture in Descemet's membrane and abnormal corneal endothelia. In the area of the rupture the endothelial mosaic was highly abnormal, and there was no cell reflex. At the site of the rupture and at the periphery of the cornea of the affected eye the endothelial cell density was distinctly lowered, with pleomorphism and polymegathism. In an eye with a DM rupture and degenerated endothelium, difficulties in contact lens wear seem to be due to the hypoxia caused by soft contact lenses. For such patients, only gas-permeable hard lenses with short wearing times should be recommended. Check-ups should be frequent and meticulous. To ensure that endothelial degeneration is noticed well before corneal damage is clinically evident, the endothelial cells must be counted and changes sought for by specular microscopy.     

Endothelial cell repopulation after intracameral acetic acid irrigation

A significant, but transient corneal edema followed inadvertent intracameral irrigation of acetic acid in two patients. Since postsurgical corneal edema generally indicates corneal endothelial dysfunction, the effects of acetic acid on corneal endothelium were studied in the cat where the endothelial regenerative capacity is thought to be comparable to that of humans. The anterior chambers of feline eyes were irrigated with 0.25% acetic acid or balanced salt solution in a double-masked manner. Marked corneal opacification and thickening were evident following exposure to the acetic acid but did not occur following irrigation with balanced salt solution alone. Following intracameral acetic acid irrigation, a few endothelial cells were found in the anterior chamber angle but could not be found on Descemet's membrane. Over an 8-week period gradual repopulation of Descemet's membrane by endothelial cells occurred, first in the periphery and later in the central cornea, coincident with resolution of corneal edema.     

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

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

Herpes simplex stromal and endothelial keratitis. Granulomatous cell reactions at the level of Descemet's membrane, the stroma, and Bowman's layer

Fifty-three (25%) of 215 keratectomy specimens of patients with herpes simplex stromal keratitis displayed granulomatous reactions at the level of Descemet's membrane (50/53), midstroma (13/53), and Bowman's layer (5/53). Using an immunoperoxidase technique, herpes simplex virus (HSV) antigens were detected in keratocytes, endothelial cells, and foci of epithelioid histiocytes and multinucleated giant cells around Descemet's membrane. Both granulomatous reactions and HSV antigens were identified significantly more often in specimens with ulcerative necrotizing stromal keratitis than in those from patients with stromal scarring or nonulcerative nonnecrotizing keratitis (P less than 0.00001 and P less than 0.005, respectively). Herpes simplex virus antigens also were present in endothelial cells adjacent to foci of granulomatous reactions around Descemet's membrane in association with disciform stromal scarring. To our knowledge, this is the first demonstration of HSV antigens in human corneal endothelial cells and in the granulomatous reactions at the level of Descemet's membrane.     

Morphogenesis of rabbit corneal endothelium

We studied ultrastructurally the development of rabbit corneal endothelium from the 13th day of gestation to 3 days after birth. Precursor corneal endothelial cells, stromal cells, and a vascular network migrate in close association with each other between the developing corneal and lens epithelia. During development, newly deposited extracellular fibrous matrices separate the prospective endothelium from the capillaries and corneal stroma. The extracellular matrix between the apical endothelial surface and the vascular network loses its fibrous appearance early in development. Simultaneously, randomly organized fibrils are deposited on the basal endothelial surface facing the stroma. These fibrils, gradually obscured by the deposition of a nonfibrous component, eventually become part of Descemet's membrane. Early in development, prospective endothelial cells cannot be distinguished morphologically from the overlying corneal stromal cells. Morphologic differentiation of the endothelial cell is characterized by the formation of sinuous lateral borders that interdigitate with those of adjacent cells to form a continuous single-cell layer of tissue. The basal endothelial membrane forms a pitted surface, distinguishing it from the apical cell membrane. Intercellular junctions between lateral membranes, a cilium projecting into the anterior chamber, and deposition of Descemet's membrane on the basal endothelial surface contribute to the polarization of the endothelium. Throughout most of corneal development the vascular pupillary membrane maintains a close association with the apical surface of the differentiating endothelium. We conclude that fetal corneal endothelium develops within a complex extracellular matrix environment and in proximity to the underlying vascular network. These structures play an important role in the morphogenesis of corneal endothelium.     

Morphological study of corneal edema after anterior segment ischemia

This experiment was performed to investigate the correlation between morphological changes of the corneal endothelium and clinical corneal edematous opacities after a procedure causing obstruction of bilateral long posterior ciliary arteries of the albino-rabbits. Shortly after the procedure, corneal endothelium became irregular on the anterior-chamber side and there were numerous lysosomal granules in the cytoplasm of corneal endothelial cells. On the third day, endothelial cells became necrotic and collapsed and a part of the cells peeled into the anterior chamber. Fibroblast-like cells and inflammatory cells were piled up in severely damaged areas of the endothelium. On the 11th day, the corneal endothelium developed again as a single layered structure on Descemet's membrane. It's seemed that corneal edema was induced as a result of severe damage corneal endothelium exposed to biochemical mediators such as prostaglandins released into the aqueous humor because the severity of corneal edema indicated some relation to changes in the concentration of prostaglandins in aqueous humor after anterior segmental ischemia.     

Spontaneous resolution of corneal oedema following Descemet's detachment

We report a case of spontaneous resolution of corneal oedema in the presence of a large persistent Descemet's detachment in a 71-year-old woman. Detachment of Descemet's membrane occurred during phacoemulsification surgery and produced significant corneal oedema. The patient declined surgical repair. Six months later the corneal oedema resolved and in vivo confocal microscopy showed endothelial cells on the posterior stroma in the region of the detachment. Endothelial cell hypertrophy, migration and redistribution were thought to be responsible for reversal of the corneal oedema. Experimental evidence and clinical studies raise the possibility that limited endothelial cell proliferation may occur.     

Corneal endothelial safety of intracameral preservative-free 1% xylocaine

PURPOSE: To evaluate the effect of intracameral preservative-free 1% xylocaine on the corneal endothelium as an adjuvant to topical anaesthesia during phacoemulsification and Acrysof foldable IOL implantation. MATERIAL & METHODS: This is a prospective, controlled, randomised, double-masked study. 106 patients with soft to moderately dense (Grade 1-3) senile cataract and corneal endothelial cell density of >1500/mm2 were randomised to the xylocaine group (n=53) and control group(n=53). Central endothelial specular microscopy and ultrasound corneal pachymetry were performed preoperatively. On the first postoperative day the eyes were evaluated for corneal oedema and Descemet's folds. Ultrasound corneal pachymetry was performed at 1, 3 and 12 months. Specular microscopy was performed at 3 and 12 months. Cell loss was expressed as a percentage of preoperative cell density. Six patients could not complete one year follow-up. Chi-square and paired t test (2 tail) statistical tests were applied for analysis. RESULTS: Four (7.54%) patients in the xylocaine group and 5 (9.43%) in the control group had a few Descemet's folds associated with mild central stromal oedema. Corneal thickness increased from 549.3micro +/- 37.2micro to 555.5micro +/- 36.5micro in the xylocaine group and from 553.1micro +/- 36.2micro to 559.3micro +/- 40.5micro in the control group at the one-month postoperative visit. Thickness returned to the preoperative level in xylocaine group 549.6micro +/- 34.5micro and control group 554.7micro +/- 41.1micro at three months. (P=0.484) The percentage of cell loss was 4.47 +/- 2.53% in the xylocaine group and 4.49 +/- 3.09% in the control group at one year. (P=0.97) CONCLUSION: Intracameral preservative-free 1% xylocaine does not appear to affect corneal endothelium adversely during phacoemulsification.