Lisha Gan

Apoptosis in UV-exposed rabbit corneas.

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

Biosynthetic corneal implants for replacement of pathologic corneal tissue: performance in a controlled rabbit alkali burn model.

PURPOSE To evaluate the performance of structurally reinforced, stabilized recombinant human collagen-phosphorylcholine (RHCIII-MPC) hydrogels as corneal substitutes in a rabbit model of severe corneal damage. METHODS One eye each of 12 rabbits received a deep corneal alkali wound. Four corneas were implanted with RHCIII-MPC hydrogels. The other eight control corneas were implanted with either allografts or a simple cross-linked RHCIII hydrogel. In all cases, 6.25 mm diameter, 350 μm thick buttons were implanted by anterior lamellar keratoplasty to replace damaged corneal tissue. Implants were followed for nine months by clinical examination and in vivo confocal microscopy, after which implanted corneas were removed and processed for histopathological and ultrastructural examination. RESULTS Alkali exposure induced extensive central corneal scarring, ocular surface irregularity, and neovascularization in one case. All implants showed complete epithelial coverage by four weeks postoperative, but with accompanying suture-induced vascularization in 6 out of 12 cases. A stable, stratified epithelium with hemidesmosomal adhesion complexes regenerated over all implants, and subbasal nerve regeneration was observed in allograft and RHCIII-MPC implants. Initially acellular biosynthetic implants were populated with host-derived keratocytes as stromal haze subsided and stromal collagen was remodeled. Notably, RHCIII-MPC implants exhibited resistance to vascular ingrowth while supporting endogenous cell and nerve repopulation. CONCLUSIONS Biosynthetic implants based on RHC promoted cell and nerve repopulation in alkali burned rabbit eyes. In RHCIII-MPC implants, evidence of an enhanced resistance to neovascularization was additionally noted.

Leukocytes in the early events of corneal neovascularization.

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

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

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

Proliferating cell nuclear antigen : contradictory results regarding its presence in the lens

Abstract• Background: Cell division of the normal lens is believed to occur in the germinative zone of the lens epithelium only. The expression of proliferating cell nuclear antigen (PCNA), at least in its insoluble form, is restricted to cells shortly after and during DNA synthesis. • Method: To elucidate the location of the previously reported notable amounts of PCNA in lens epithelium, 6-μm sections of 10 New Zealand rabbit corneas were stained with specific antibodies. • Results: No PCNA-positive cells were detected in the lens epithelium, neither in the central zone nor in the germinative zone. • Conclusion: As the mitotic activity of lens epithelium is physiologically low (below 0.03% in adult rabbits), the likelihood of detecting mitotic figures in histologic sections is very limited. We hence conclude that PCNA may be expressed in the epithelia of the lens but is only occasionally visible in histological sections. Our most recent studies have confirmed this. The fixation process and the use of specific antibodies are decisive factors that may, when used correctly, allow specific localization PCNA that is involved in cell replication of lens epithelium. However, it has to be pointed out that the mitotic index of lens epithelium is age- and species-related, and hence so is the expression of PCNA, which makes comparison with other studies more difficult.