| Objective1. To explore the role of Na~+-K~+-ATPase activities and aquaporin-1 expression in edema developing by examining the alteration of Na~+-K~+-ATPase activities and aquaporin-1 expression in organ-cultured cornea.2. The animal model of rabbit bullous keratopathy (BK) was established by anterior chamber injection with 0.05% benzalkonium bromide. The Na~+-K~+-ATPase activities and the aquaporin-1 expression were detected to explore the role of Na~+-K~+-ATPase activities and aquaporin-1 expression in pathological mechanism of BK.3. To determine whether the activity of organ-cultured CEC post-operation could meet the demands of penetrating keratoplasty(PKP). PKP was performed for BK with the organ-cultured cornea, the transparent and thickness of cornea was observed.Methods1. The experiment was divided into 2 groups, 24 fresh rabbit corneas as the control group, while 24 corneas of organ culture preservation as the experimental group. The corneal thickness was measured with ultrasonic corneal pachymeter before enucleation and the corneal endothelial cell density was counted with non-contact specular microscope after enucleation in two groups. The experimental group corneas were preserved by organ culture for 4 weeks, the corneal thickness was measured with ultrasonic corneal pachymeter. Then every corneas were divided into half -chip, there are 48 half-chip total. It was divided into 4 groups, there are 12 half-chip in every groups. The corneal endothelial cell density of 12 half-chip were counted through Alizarin Red-Trypan blue staining; 12 half-clip corneas were fixed with 4% neutral formalin solution, HE staining was performed, the expression of AQP-1 in corneal stroma and corneal endothelial cell were detected through immunohistochemical staining; Na~+-K~+-ATPase activities in 12 half-clip corneas were examined with Na~+-K~+-ATPase kit; the expression of AQP-1 mRNA were detected through real-time fluorescent quantitation PCR. The corneal thickness and corneal endothelial cell density were compared before and after organ culture preservation in experiment groups. Na~+-K~+-ATPase activities and the expression of AQP-1 in immunohistochemical staining and real-time fluorescent quantitation PCR were compared with control group.2. The animal model of rabbit bullous keratopathy (BK) was established by anterior chamber injection with 0.05% benzalkonium bromide. The corneal thickness and corneal endothelial cell density were measured with ultrasonic corneal pachymeter and non-contact specular microscope separately before the animal models was established. The general situations of the eye were observed and the corneal thickness were measured with ultrasonic corneal pachymeter after the animal models was established. After a week, the corneas were removed after the experimental animals are put to death. The corneal endothelial cell density of 12 half-chip were counted through Alizarin Red-Trypan blue staining; 12 half-clip corneas were fixed with 4% neutral formalin solution , HE staining was performed, the expression of AQP-1 in corneal stroma and corneal endothelial cell were detected through immunohistochemical staining; Na~+-K~+-ATPase activities in 12 half-clip corneas were examined with Na~+-K~+-ATPase kit; the expression of AQP-1 mRNA were detected through real-time fluorescent quantitation PCR. The results were compared with control group.3. The animal model of rabbit BK was established by anterior chamber injection with 0.05% benzalkonium bromide. After a week, PKP was performed for BK with organ-cultured cornea preservation 4 weeks. The transparent of grafts was observed, the thickness of grafts were measured with ultrasonic corneal pachymeter. The grafts were removed after two weeks of PKP, the survival of grafts corneal endothelial cell were evaluated with Alizarin Red-Trypan blue staining.Results1. Cornea became thicker than ever evidently after organ culture storage in 4 weeks, the corneal thickness increased from 351.12±23.23μm up to 683.60±48.92μm; the CEC density decreased from 2742.61±124.02/mm2 to 2381.56±174.27 /mm2; Na~+-K~+-ATPase activities were 3.82±0.26U/mgprot in the fresh rabbit corneas and 3.57±0.26 U/mgprot in organ-cultured preservation corneas; HE staining showed that the corneal epithelium remained only 1~2 layers of cells, the regular columnar structure of the Basal cell disappeared, the edma was mild in corneal stroma, there are more wrinkles in Descemet's layer; The positive expression of AQP-1 immunohistochemical staining existed in the corneal endothelial cell and stroma, the positive staining lighted and the expression of AQP-1 decreased after organ-cultured preservation. Real-time quantitative PCR results showed that the expression of the AQP-1mRNA in corneal endothelial cell trended to reduce compared to before organ-cultured preservation.2. The BK cornea became opacity because of edema, and became thicker evidently. The corneal thickness reached to 696.17±43.54μm averagely in the seventh day; The CEC density decreased from 2876.00±164.65/mm2 to 646.58±126.72/mm2; Alizarin Red-Trypan blue staining showed that CEC flake off, lost their normal hexagonal appearance and become irregular, the cell membrane ruptured and dissolved; Na~+-K~+-ATPase activity decreased greatly to 1.03±0.28U/mgprot; HE staining showed that the endothelial cell layer was incomplete, CEC density decreased, part CEC loss, Bullous formed in subepithelial layer. The positive expression of AQP-1 immunohistochemistrical staining lighted in the corneal endothelial cell and stroma, there weren't positive expression in some area even. Real-time quantitative PCR results showed that the expression of the AQP-1mRNA in corneal endothelial cell trended to reduce greatly compared to the normal corneas.3. The grafts was mildly edema and basically transparent, the pupil and iris could be vaguely observed after 1 day of PKP; The Grafts were fully transparent, the pupil and iris could be clearly observed after 3 days of PKP; The Limbal and conjunctival congestion gradually reduce after 3 days, but mild congestive still was there. The corneal thickness became thin after 1 day and mostly restored to the thickness before preservation after 3 day; The grafts became slightly thinner than before preservation after 7 day and still thinner after 14 day. The graft CEC density after 14 days of PKP slightly decreased compared to after organ-cultured preservation, the irregular shape of CEC reduced also.Conclusions1.After organ culture storage, the cornea become thicker than ever, the CEC density decreases, Na~+-K~+-ATPase activity decreases slightly, the of AQP-1 expression reduces. the CEC density after storage can comply with the requirements of PKP; The corneal edema after organ culture storage is associated with the reduction of Na~+-K~+-ATPase activity and AQP-1 expression.2. The animal model of rabbit BK was established by anterior chamber injection with 0.05% benzalkonium bromide. The pathology of BK is associated with the reduction of Na~+-K~+-ATPase activity and AQP-1 expression.3. The rabbit corneal grafts are transparent after PKP with the organ-culture cornea. The organ-culture cornea can comply with the requirements of PKP for BK.
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