Sonic hedgehog信号通路在大鼠慢性高眼压视网膜神经节细胞损伤中的保护作用及机制研究
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摘要
第一部分慢性高眼压动物模型的建立和评估
目的：探讨建立稳定的大鼠慢性高眼压模型的方法。
方法：成年雄性SD大鼠(250g左右)60只,以右眼为高眼压眼,手术显微镜下分离上直肌两侧及鼻下方的3根巩膜上静脉,缝线结扎后缝合球结膜。左眼为对照眼,除不结扎静脉外,其它操作相同。分别于术后30min、1d、3d、5d、7d、10d、14d、28d、35d和42d用Tono-penXL笔式眼压计测量眼压并记录,测量时间为上午10点-11点。于术后2w和4w处死大鼠,处死前5天经上丘行DiI逆行标记RGCs,制作视网膜铺片,正置荧光显微镜观察并计数分析各组视网膜神经节细胞(retinal ganglion cells, RGCs)存活情况。结果：对照眼的平均眼压为13.4±0.27 mm,术后30min、1d和3d检测高眼压眼的眼压分别为21.4±0.25mmHg.25.5±0.27mmHg和23.4±0.19 mmHg,第5天达到最高值,为27.0±2.31 mmHg.随后眼压缓慢下降,第7d、10d、14d和28d分别测得眼压值为：26.4±2.01 mmHg.26.8±1.83 mmHg、25.6±0.21 mmHg和23.6±0.17 mmHg,上述各时间点测得的眼压数值与对照组相比均具有显著性差异(P<0.01)。术后第35d和42d时眼压降至正常范围,分别为14.7±0.31mmHg和15.9±0.26mmHg.视网膜铺片DiI逆行标记计数分析RGCs结果显示,对照眼RGCs的平均密度2201.45±124.67个／mm2,术后2周和4周,高眼压眼RGCs的密度显著减少,分别降到1974.30±108.81个／mm2和1732.96±89.14个/mm2,均明显少于对照眼(P<0.01)。
结论：结扎3条巩膜外静脉可以成功诱导SD大鼠慢性高眼压,并可使眼压稳定维持到4周,是目前慢性高眼压青光眼实验研究中较为便捷、实用和可靠的建立动物模型的手段。经大鼠上丘DiI逆行标记RGCs,结合形态学观察,可以有效示踪RGCs的形态和数量,是研究实验条件下RGCs数量变化的较为理想的方法。
第二部分大鼠慢性高眼压模型中视网膜Sonic Hedgehog(Shh)信号通路分子表达的研究
目的：研究Sonic Hedgehog及其下游信号通路分子Smoothened(Smo)、Patched(Ptc)和Glil在正常及慢性高眼压大鼠视网膜组织中的表达及变化。
方法：雄性SD大鼠60只,右眼为高眼压组,结扎3根巩膜上静脉；左眼为对照组,除不结扎静脉外,其它操作相同。建模成功的大鼠,分别于3d、1w、2w、4w各处死15只。其中3只用于视网膜Shh、受体Smo、Ptc以及核转录因子Glil的免疫荧光或免疫荧光双标染色,正置荧光显微镜观察呈现阳性荧光表达的细胞类型、数量及荧光强弱等；6只用于Western-blot方法定量检测视网膜组织中上述四种蛋白的表达情况。余下6只大鼠视网膜用于实时荧光定量逆转录聚合酶链反应(real-time fluorescent quantitative reversetranscription polymerase chain reaction, Real-time PCR),定量分析高眼压视网膜组织中Shh、Smo、Ptc和Glil的mRNA表达变化。Western-blot和Real-time PCR数据采用spss14.0软件包进行统计分析,进行正态性检验、方差齐性检验,描述性统计值以平均值±标准误(x±s)表示。两组之间配对资料的t检验,组间差异比较用One-way ANOVA,P＜0.05有统计学意义。
结果：免疫荧光双标显示：对照组大鼠视网膜Shh主要在RGCs表达,内丛状层也可见到弱阳性的表达；2w时,高眼压组在RGCs、内丛状层和外丛状层都可以检测到Shh表达,荧光强度明显增加。对照组Glil和Smo在RGCs的细胞质中有弱阳性表达,高眼压组Glil阳性荧光信号同时出现在RGCs细胞质和细胞核中。Real-time PCR结果显示1w、2w、4w高眼压组ShhmRNA分别是对照组的2.6±0.7、4.4±0.9和2.1±0.5倍,各时间点同对照组相比,差异有显著性(P<0.01), Smo和Gli1mRNA表达量于成模后1w开始升高,并于2w时到达高峰,随后又减少,与对照组相比,差异均有显著性(P<0.01)。Ptc的蛋白和mRNA表达量在高眼压组和对照组未见明显差异。Western-blot结果显示高眼压组在1w、2w和4w视网膜Shh蛋白分别是对照组的2.1±0.3、4.4±0.6和3.0±0.7倍(P<0.01),并于2w时达到高峰。视网膜Smo和Glil蛋白表达量在1w时开始增高,2w时到达高峰,随后下降。在1w、2w和4w时,Smo蛋白表达分别是对照组的3.2±0.4倍(P<0.05)、3.4±0.6倍(P<0.01)和1.4±0.2倍(P>0.05)；Glil蛋白表达量分别是对照组的1.6±0.1倍(P<0.05)、2.5±0.3倍(P<0.05)和2.0±0.5倍(P<0.05)。
结论：大鼠慢性高眼压动物模型视网膜组织中Shh及Smo、Gli1表达水平升高,Glil在RGC中呈现核转位表达。提示慢性高眼压时视网膜Shh信号转导通路激活,可能参与慢性高眼压视网膜视神经损伤修复的生理病理过程。
第三部分：Shh信号转导通路对大鼠慢性高眼压RGCs损伤的作用及机制研究
目的：研究Shh信号转导通路对大鼠慢性高眼压RGCs损伤的作用,并探讨其可能分子机制。
方法：成年雄性SD大鼠以右眼结扎3条巩膜上静脉诱导高眼压形成。按照玻璃体腔内注射药物或浓度的不同将大鼠随机分为9组：不注射组、10μg/ml Shh-N组、50μg/ml Shh-N组、100μg/ml Shh-N组、1.0μg/ml cyclopamine (Shh阻滞剂)组、5.0μg/ml cyclopamine组、5.0μg/ml tomatidine组(与Cyclopamine有相似的化学结构,但不能抑制Shh信号转导通路,)、0.1M磷酸盐缓冲液(phosphate-buffered saline, PBS)组、和45%(W/V)2-羟丙基-环糊精(2-hydroxypropyl-cyclodextrin, HBC)组,注射体积均为2μl。每周重复注射一次。检测时间点为玻璃体腔注射后1w、2w和4w。在动物处死前5d,行DiI逆行标记RGCs。制作全视网膜铺片正置荧光显微镜观察RGCs的存活及数量,并提取视网膜蛋白和总RNA行Western-blot和Real-time PCR检测信号通路中各成分含量的变化。
结果：眼压升高后2w和4w时,玻璃体腔内注射100μg/ml Shh-N组RGCs丧失率为分别为4.54±0.36%和9.67±0.31%,与PBS组(15.26±1.57%,22.58±1.97%)相比差异有显著性(P<0.01)；注射Shh-N50μg/ml RGCs丧失率分别为7.31±0.39%和12.67±0.29%,与PBS组(15.26±1.57%,22.58±1.97%)相比差异也有显著性(P<0.05);注射10μg/ml Shh-N组RGCs丧失率与PBS组相比无显著性差异。玻璃体腔注射1.0μg/ml Cyclopamine组RGCs丧失率分别为19.29±0.43%(P<0.05)和26.4±2.04%(P<0.05),5.0μg/ml Cyclopamine组RGC丧失率分别为25.2±0.29%和30.7±0.31%,与HBC组相比差异均有显著性(P<0.01)。注射tomatidine组与注射PBS组相比,RGCs的丧失率无统计学差异。HBC组与未注射组RGCs数量无统计学差异。Shh对损伤RGC的保护作用及Cyclopamine的阻断效应均呈剂量相关性,高浓度组效果更加显著。玻璃体腔注射Shh-N 2w后,Western Blot结果显示高眼压眼视网膜Smo和Glil蛋白表达量增加,Real-timePCR结果：Smo mRNA和Gli1 mRNA分别为对照组的1.52±0.16%倍和1.96±0.31%倍。注射Cyclopamine后,高眼压眼Smo和Glil蛋白和mRNA表达量分别降低。
结论外源性和内源性Shh都可以在高眼压引起的RGCs损伤中发挥保护作用,并呈剂量相关性,以高浓度为显著。Shh可能通过释放Smo,激活转录因子Glil发挥神经保护作用。
Part1 Establishment of chronic ocular hypertension model in rats
Purpose: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 rats
Purpose: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 hypertension
Purpose:To study the function of Sonic hedgehog signaing in retinal ganglion cells following chronic ocular hypertension
Methods: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.
目的：探讨建立稳定的大鼠慢性高眼压模型的方法。
方法：成年雄性SD大鼠(250g左右)60只,以右眼为高眼压眼,手术显微镜下分离上直肌两侧及鼻下方的3根巩膜上静脉,缝线结扎后缝合球结膜。左眼为对照眼,除不结扎静脉外,其它操作相同。分别于术后30min、1d、3d、5d、7d、10d、14d、28d、35d和42d用Tono-penXL笔式眼压计测量眼压并记录,测量时间为上午10点-11点。于术后2w和4w处死大鼠,处死前5天经上丘行DiI逆行标记RGCs,制作视网膜铺片,正置荧光显微镜观察并计数分析各组视网膜神经节细胞(retinal ganglion cells, RGCs)存活情况。结果：对照眼的平均眼压为13.4±0.27 mm,术后30min、1d和3d检测高眼压眼的眼压分别为21.4±0.25mmHg.25.5±0.27mmHg和23.4±0.19 mmHg,第5天达到最高值,为27.0±2.31 mmHg.随后眼压缓慢下降,第7d、10d、14d和28d分别测得眼压值为：26.4±2.01 mmHg.26.8±1.83 mmHg、25.6±0.21 mmHg和23.6±0.17 mmHg,上述各时间点测得的眼压数值与对照组相比均具有显著性差异(P<0.01)。术后第35d和42d时眼压降至正常范围,分别为14.7±0.31mmHg和15.9±0.26mmHg.视网膜铺片DiI逆行标记计数分析RGCs结果显示,对照眼RGCs的平均密度2201.45±124.67个／mm2,术后2周和4周,高眼压眼RGCs的密度显著减少,分别降到1974.30±108.81个／mm2和1732.96±89.14个/mm2,均明显少于对照眼(P<0.01)。
结论：结扎3条巩膜外静脉可以成功诱导SD大鼠慢性高眼压,并可使眼压稳定维持到4周,是目前慢性高眼压青光眼实验研究中较为便捷、实用和可靠的建立动物模型的手段。经大鼠上丘DiI逆行标记RGCs,结合形态学观察,可以有效示踪RGCs的形态和数量,是研究实验条件下RGCs数量变化的较为理想的方法。
第二部分大鼠慢性高眼压模型中视网膜Sonic Hedgehog(Shh)信号通路分子表达的研究
目的：研究Sonic Hedgehog及其下游信号通路分子Smoothened(Smo)、Patched(Ptc)和Glil在正常及慢性高眼压大鼠视网膜组织中的表达及变化。
方法：雄性SD大鼠60只,右眼为高眼压组,结扎3根巩膜上静脉；左眼为对照组,除不结扎静脉外,其它操作相同。建模成功的大鼠,分别于3d、1w、2w、4w各处死15只。其中3只用于视网膜Shh、受体Smo、Ptc以及核转录因子Glil的免疫荧光或免疫荧光双标染色,正置荧光显微镜观察呈现阳性荧光表达的细胞类型、数量及荧光强弱等；6只用于Western-blot方法定量检测视网膜组织中上述四种蛋白的表达情况。余下6只大鼠视网膜用于实时荧光定量逆转录聚合酶链反应(real-time fluorescent quantitative reversetranscription polymerase chain reaction, Real-time PCR),定量分析高眼压视网膜组织中Shh、Smo、Ptc和Glil的mRNA表达变化。Western-blot和Real-time PCR数据采用spss14.0软件包进行统计分析,进行正态性检验、方差齐性检验,描述性统计值以平均值±标准误(x±s)表示。两组之间配对资料的t检验,组间差异比较用One-way ANOVA,P＜0.05有统计学意义。
结果：免疫荧光双标显示：对照组大鼠视网膜Shh主要在RGCs表达,内丛状层也可见到弱阳性的表达；2w时,高眼压组在RGCs、内丛状层和外丛状层都可以检测到Shh表达,荧光强度明显增加。对照组Glil和Smo在RGCs的细胞质中有弱阳性表达,高眼压组Glil阳性荧光信号同时出现在RGCs细胞质和细胞核中。Real-time PCR结果显示1w、2w、4w高眼压组ShhmRNA分别是对照组的2.6±0.7、4.4±0.9和2.1±0.5倍,各时间点同对照组相比,差异有显著性(P<0.01), Smo和Gli1mRNA表达量于成模后1w开始升高,并于2w时到达高峰,随后又减少,与对照组相比,差异均有显著性(P<0.01)。Ptc的蛋白和mRNA表达量在高眼压组和对照组未见明显差异。Western-blot结果显示高眼压组在1w、2w和4w视网膜Shh蛋白分别是对照组的2.1±0.3、4.4±0.6和3.0±0.7倍(P<0.01),并于2w时达到高峰。视网膜Smo和Glil蛋白表达量在1w时开始增高,2w时到达高峰,随后下降。在1w、2w和4w时,Smo蛋白表达分别是对照组的3.2±0.4倍(P<0.05)、3.4±0.6倍(P<0.01)和1.4±0.2倍(P>0.05)；Glil蛋白表达量分别是对照组的1.6±0.1倍(P<0.05)、2.5±0.3倍(P<0.05)和2.0±0.5倍(P<0.05)。
结论：大鼠慢性高眼压动物模型视网膜组织中Shh及Smo、Gli1表达水平升高,Glil在RGC中呈现核转位表达。提示慢性高眼压时视网膜Shh信号转导通路激活,可能参与慢性高眼压视网膜视神经损伤修复的生理病理过程。
第三部分：Shh信号转导通路对大鼠慢性高眼压RGCs损伤的作用及机制研究
目的：研究Shh信号转导通路对大鼠慢性高眼压RGCs损伤的作用,并探讨其可能分子机制。
方法：成年雄性SD大鼠以右眼结扎3条巩膜上静脉诱导高眼压形成。按照玻璃体腔内注射药物或浓度的不同将大鼠随机分为9组：不注射组、10μg/ml Shh-N组、50μg/ml Shh-N组、100μg/ml Shh-N组、1.0μg/ml cyclopamine (Shh阻滞剂)组、5.0μg/ml cyclopamine组、5.0μg/ml tomatidine组(与Cyclopamine有相似的化学结构,但不能抑制Shh信号转导通路,)、0.1M磷酸盐缓冲液(phosphate-buffered saline, PBS)组、和45%(W/V)2-羟丙基-环糊精(2-hydroxypropyl-cyclodextrin, HBC)组,注射体积均为2μl。每周重复注射一次。检测时间点为玻璃体腔注射后1w、2w和4w。在动物处死前5d,行DiI逆行标记RGCs。制作全视网膜铺片正置荧光显微镜观察RGCs的存活及数量,并提取视网膜蛋白和总RNA行Western-blot和Real-time PCR检测信号通路中各成分含量的变化。
结果：眼压升高后2w和4w时,玻璃体腔内注射100μg/ml Shh-N组RGCs丧失率为分别为4.54±0.36%和9.67±0.31%,与PBS组(15.26±1.57%,22.58±1.97%)相比差异有显著性(P<0.01)；注射Shh-N50μg/ml RGCs丧失率分别为7.31±0.39%和12.67±0.29%,与PBS组(15.26±1.57%,22.58±1.97%)相比差异也有显著性(P<0.05);注射10μg/ml Shh-N组RGCs丧失率与PBS组相比无显著性差异。玻璃体腔注射1.0μg/ml Cyclopamine组RGCs丧失率分别为19.29±0.43%(P<0.05)和26.4±2.04%(P<0.05),5.0μg/ml Cyclopamine组RGC丧失率分别为25.2±0.29%和30.7±0.31%,与HBC组相比差异均有显著性(P<0.01)。注射tomatidine组与注射PBS组相比,RGCs的丧失率无统计学差异。HBC组与未注射组RGCs数量无统计学差异。Shh对损伤RGC的保护作用及Cyclopamine的阻断效应均呈剂量相关性,高浓度组效果更加显著。玻璃体腔注射Shh-N 2w后,Western Blot结果显示高眼压眼视网膜Smo和Glil蛋白表达量增加,Real-timePCR结果：Smo mRNA和Gli1 mRNA分别为对照组的1.52±0.16%倍和1.96±0.31%倍。注射Cyclopamine后,高眼压眼Smo和Glil蛋白和mRNA表达量分别降低。
结论外源性和内源性Shh都可以在高眼压引起的RGCs损伤中发挥保护作用,并呈剂量相关性,以高浓度为显著。Shh可能通过释放Smo,激活转录因子Glil发挥神经保护作用。
Part1 Establishment of chronic ocular hypertension model in rats
Purpose: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 rats
Purpose: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 hypertension
Purpose:To study the function of Sonic hedgehog signaing in retinal ganglion cells following chronic ocular hypertension
Methods: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.
引文
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