| Objectives1. To detect the day-night rhythm of rats' normal intraocular pressure(IOP) , and to establish stable chronic ocular hypertension models of rats.2. To establish Cop-1-immunized models of rats with chronic ocular hypertension, and to investigate the morphologic changes of all layers in glaucomatous rat's retina ,and to assess the role of autoimmunization induced by Cop-1 in protecting glaucomatous rat's retina against ocular hypertension.3. To investigate how Cop-1 -induced-autoimmunization contributes to the phosphorylation of extracellular signal-regulated protein kinase(ERK) in glaucomatous rat's retinal Muller cells and the involvement of ERK pathway in protection of retinal ganglion cells against death from glaucoma4. To characterize the expression of interleukin-6 (IL-6) in Cop-1-immunized glaucomatous rat's retina, and to evaluate the neuroprotection of IL-6 for retinal ganglion cells.Methods1. Intraocular pressure(IOP) was measured twice a day by using a handheld tonometer(Tonopen-XL), respectively at 7 a.m an 10 p.m. Datum were compared using paired t-test.2. Animals were deeply anesthetized by intraperitoneal injection of 1 % amobarbital sodium, and an increase in IOP was achieved by cauterizing 2 episcleral veins. After treatment, IOP was measured by using a handheld tonometer(Tonopen-XL), respectively at 0 min, 30 min, 1h, 12h, 1d, 1wk, 1 month and 2 months. Then the mean IOP at different time was described in a line graph.3. After 2 episcleral veins were cauterized, animals were immunized with 200ug of Cop-1 emulsified with an equal volume of complete Freund's adjuvant(CFA) containing 5mg/ml Mycobacterium tuberculosis. The emulsion was injected into the hind legs of rats. Animals were immunized again with the above antigen emulsified with an equal volume of incomplete Freund's adjuvant as a booster, respectively 2 wk and 4wk after the first immunization. 8wk after the first treatment, the specific antibodies of Cop-1 were detected by Enzyme-Linked Immunosorbnent Assay (ELISA).4. Female Sprague Dawley rats were divided into 3 groups: normal group, glaucomatous group , Cop-1-immunized group.8wk after treatment, animals were killed and their eyes were removed and fixed in 4% paraformaldehyde at 4 C. Eye sections were cut to a thickness of 18um, then mounted onto gelatin-coatedslides and stored at -20C until needed. Frozen sections were thawed for 2h at roomtemperature before they were stained with hematoxylin and eosin. TUNEL staining was carried out to detect apoptotic cells in rats' retinas.5. Sections were immunohistochemically stained for phosphorylated ERK, glial fibrillary acidic protein(GFAP), Vimentin and IL-6 in rats' retinas of different groups. Immunofluorescent double-labeling was performed to identify in which cells in the retina ERK pathway was activated.Results1. Normal IOP of rats at daytime ranged from 1.06 kPa to 1.99 kPa while that at night varied from 1.73 kPa to 2.66 kPa. The mean IOP at daytime and night was (1.46+0.25)kPa and (1.86 + 0.23)kPa, respectively. The former was higher than the latter (P<0.01).2. 8wk after 2 episcleral veins were cauterized, the mean IOP of glaucomatous eyes was (4.04 + 0.2 l)Kpa and higher than that of control eyes. 8wk after autoimmunization induced by Cop-l,the specific antibodies of Cop-1 were identified in blood serums of Cop-1-immunized glaucomatous rats.3. Staining with hematoxylin and eosin demonstrated that the thickness of glaucomatous ratina was significantly thinner than that of normal retina. TUNEL-positive cells in glaucomatous retina was much more than that in normal retina. The number of TUNEL-positive cells in Cop-1-immunized glaucomatous retina was reducing.4. There was an increasing immumoreactivity of ERK, GFAP Vimentin. IL-6 in the retina of glaucomatous rats andCop-1-immunized glaucomatous rats. The results of immunofluorescent double-labeling showed tha... |
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