| Part1 Establishment of chronic ocular hypertension model in ratsPurpose:To establishment consistent chronic ocular hypertension glaucoma model in rats.Methods:Sixty adult male Sprague Dawley (SD) rats weighting approximately 250g. Unilateral intraocular pressure (IOP) elevation was induced in the right eye by ligating 3 episcleral veins. The contralateral eyes, which served as controls, were sham-operated by isolating the veins in a similar manner without any ligation. IOPs were measured in both eyes under general anesthesia using Tono-pen XL tonometer at the following times after surgey:30 minutes, 1day,3 days,5days,7days, 10day, 14days,21days,28days,35days and 42days. Animals with IOPs returned to normal were excluder from the study. Retinal ganglion cells(RGCs)were retrogradely labled with a DiI fluorescent tracer at 5 days before sacrifice. At 2w and 4w after operation, the eyes were then enucleated, and the retinas were prepared as flatmounts. DiI-labeled RGCs were visualized with a fluorescence microscope.Result:The IOP in control eyes was 13.4±0.27 mm Hg and remained constant throughout the experiment. At 30 minutes,1day and 3 days after operation, the IOP of high intraocular pressure group eyes was 21.4±0.25mmHg,25.5±0.27mmHg and 23.4±0.19 mmHg respectively.The highest IOP reached 27±2.31 mm Hg at 5days and was significantly greater than the IOP of control eyes. At the following days of 7days,10days,14days and 28days, the IOP was 26.4±2.01 mmHg,26.8±1.83 mmHg,25.6±0.21 mmHg and 23.6±0.17 mmHg, respectively, and reached to normal at 35days(14.7±0.31mmHg) and 42days(15.9±0.26mmHg)。Retinal flat mount revealed that the density of the RGCs was decreased to 1974.30±108.81 clls/mm2 and 1732.96±89.14 cells/mm2 at 2 and 4 weeks after IOP elevation, and was significantly decreased than the control eyes (p< 0.01).Concousion:A consistent chronic ocular hypertension glaucoma model can be established by ligating 3 episcleral veins. DiI retrograde labeling could be an ideal measure in the research of RGCs.Part 2 Experssion of Sonic Hedgehog signaing in chronic ocular hypertension in ratsPurpose:To study changes in the expression of Sonic Hedgehog (Shh) and related molecules Smoothened(Smo),Patched(Ptc) and Gli1 in the rats' retina of chronic ocular hypertension.Methods:Sixty adult male SD rats weighting approximately 250g. Unilateral intraocular pressure (IOP) elevation was induced in the right eye by ligating 3 episcleral veins. The contralateral eyes which served as controls, were sham-operated. Retinal expression of Shh, its receptor Ptc,Smo and transcription factor Gli1 protein and mRNA was determined by immunohistochemistry, western blotting, and real-time fluorescent quantitative reversetranscription polymerase chain reaction(Real-time PCR). Statistical analysis was performed using SPSS software Ver.14.0 for Windows.Two groups were compared using paired t-tests. Multiple groups were compared using a one-way ANOVA with Duncan's multiple pairwise comparison tests. Data were shown as mean±SD. Values of p less than 0.05 were considered significant.Result:Shh protein expression in both normal and hypertensive rat eyes using immunohistochemistry. In the control eyes, Shh was mainly detected in the RGCs. Weak Shh staining was also present in the inner plexiform layers (IPL). Smo and Gli1 were immunohistochemically detected in both normal and hypertensive rat eyes. Gli1 protein is observed in the cell cytoplasm of RGCs in the control eyes, whereas nuclear translocation of Gli1 was detected in RGCs of chronic ocular hypertension eyes. Real-time PCR analysis shows that retinas from the elevated IOP group had 2.1-to 4.4-fold greater Shh expression than control retinas (p< 0.05), with Shh expression reaching a peak at 2 weeks after IOP elevation. So did Gli1 and Smo mRNA expression. However, Ptc mRNA up-regulation was not detected. Western blot analysis shows that at 1w,2w and 4w after operation, retinas from the elevated IOP group had 2.1±0.3,4.4±0.6 and 3.0±0.7 fold greater Shh protein expression than control retinas,3.2±0.4 (P<0.05),3.4±0.6 (P＜0.01) and 1.4±0.2(P>0.05) fold greater Smo protein expression than control retinas, and 1.6±0.1 (P<0.05) 2.5±0.3(P<0.05),2.0±0.5(P<0.05)fold greater Gli1 protein expression than control retinas. In contrast, no differences were detected in Ptc expression between hypertensive and control retinas.Conclusion:Shh,Smo and Glil are up-regulated in a time-dependent manner in retinas exposed to ocular hypertension. Changes in the expression of Shh and its downstream components, as well as the nuclear translocation of Glil in hypertensive retina, suggest an involvement of Shh pathway in the chronic intraocular hypertension.Part3 The effect of Sonic hedgehog in retinal ganglion cells following chronic ocular hypertensionPurpose:To study the function of Sonic hedgehog signaing in retinal ganglion cells following chronic ocular hypertensionMethods:Intraocular pressure elevation in adult rat was induced by ligating 3 episcleral veins. Exogenous Shh and its inhibitor cyclopamine and tomatidine were intravitreally injected to examine their effects on RGC survival after ocular hypertension.The rats were randomly divided into 9 groups:untreated group,10μg/ml Shh-N group,50μg/ml Shh-N group,100μg/ml Shh-N group,1.0μg/ml cyclopamine group,5.0μg/ml cyclopamine group,5.0μg/ml tomatidine group,phosphate-buffered saline (PBS) group and 2-hydroxypropyl-cyclodextrin (HBC) group. Each group received intravitreal application of 2μl. At 1w,2w and 4w after the operation, the animals were killed, before sacrifice, RGCs were retrogradely labeled by injecting DiI into the superior colliculi of the brain. Retinal flat mount photographs were assessed for the number of survival RGCs. Shh pathway components mediating neuroprotective effects were characterized using western blotting and Real-time PCR.Results:RGCs loss at 2 and 4 weeks after IOP elevation, was significantly reduced by intravitreally injected 100μg/ml Shh-N (4.54±0.36%and 9.67±0.31%) and 50μg/ml Shh-N (7.31±0.39%and 12.67±0.29%) (p< 0.01, versus PBS-treated groups). In contrast, cyclopamine increased RGC loss. (25.2±0.29%at 2 weeks and 30.7±0.31%at 4 weeks) (p< 0.05, versus HBC-treated groups). No differences were detected in RGCs loss between tomatidine-treated groups and PBS group. At 2 weeks after intravitreally injected of Shh-N, Western blot analysis shows that Smo and Gli1 protein expression increased. Real-time PCR analysis shows that the mRNA levels of Smo and Gli1 were significantly up-regulated (1.52±0.16%and 1.96±0.31% fold greater than HBC group). In contrast, intravitreal cyclopamine administration decreased retinal expression of both Smo and Gli1 protein and mRNA.Conclusions:Shh has neuroprotective effects on damaged RGCs in a rat chronic hypertension model. Shh may exert neuroprotective effects by relieving the inhibition of Smo and subsequently activating Gli1.
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