【作者】 崔红培；

【导师】 庄曾渊；

【作者基本信息】 中国中医科学院 ， 中西医结合临床， 2009， 博士

【摘要】 目的建立外伤性视神经病变(traumatic optic neuropathies,TON)大鼠模型,从视网膜神经节细胞(retinal ganglion cells,RGCs)存活状态、组织形态学、轴突修复影响因素三个方面观察血府逐瘀汤及其拆方对TON大鼠RGCs的影响,探讨其部分作用机制,为临床用药提供客观依据和指导。方法1.以钳夹伤大鼠视神经为TON动物模型,观察血府逐瘀汤及其拆方对大鼠RGCs存活状态的影响,计算存活率,筛选血府逐瘀汤最佳组方。2.采用组织病理学技术观察血府逐瘀汤最佳组方干预后3天、1周、2周、4周,钳夹伤大鼠视网膜厚度和视神经超微结构变化。3.采用免疫组织化学方法观察药物干预后3天、1周、2周、4周,生长相关蛋白GAP-43(growth associated protein 43,GAP-43)、Rho激酶(rho-associated coiled-coilforming protein kinase,ROCKⅡ)在钳夹伤大鼠视网膜、视神经组织中的表达情况。视网膜表达强度以平均光密度(average optical density,AOD)表示,视神经表达强度以积分光密度(integrated optical density,IOD)表示。结果1.血府逐瘀汤对TON大鼠RGCs存活率的影响各组RGCs平均存活率分别为:蒸馏水组62.84%±10.96%;灯盏细辛组75.76%±8.87%;血府逐瘀汤全方组89.18%±10.59%;血府逐瘀汤活血组69.68%±5.26%;血府逐瘀汤理气组66.95%±9.12%。血府逐瘀汤全方组RGCs存活率明显高于其它各组(P<0.01),血府逐瘀汤全方为最佳方药。2.血府逐瘀汤对TON大鼠视网膜视神经组织形态学的影响(1)视网膜厚度①3天视网膜内层(retinal length,RL)、神经纤维层(nerve fiber layer,NFL)、节细胞层(ganglion cell layer,GCL)、内丛状层(inner plexiform layer,IPL)和内核层(inner nuclearlayer,INL):各组均增加,组间同层比较没有显著性差异(P>0.05)。②1周RL和IPL:各组继续增加,组问同层比较没有显著性差异(P>0.05);NFL:各组轻度降低,组间同层比较没有显著性差异(P>0.05);GCL:治疗组和阳性对照组降低,模型组增加,治疗组明显低于模型组(P<0.05);INL:各组轻度增加,治疗组明显低于模型组(P<0.05)。③2周RL、IPL和INL:各组继续增加,组间同层比较没有显著性差异(P>0.05);NFL:各组均降低,组间没有显著性差异(P>0.05);GCL:各组均增加,阳性对照组低于模型组(P<0.05),治疗组和模型组没有显著性差异(P>0.05)。④4周RL、NFL和INL:各组明显降低,组间同层比较没有显著性差异(P>0.05);GCL:各组厚度降低,治疗组和阳性对照组均高于模型组(P<0.05);IPL:各组明显降低,治疗组高于模型组、阳性对照组(P<0.05)。(2)视神经超微结构①3天:模型组、治疗组均可见髓鞘板层分离、崩解,轴膜回缩,或外凸卷曲;轴突数目减少,部分轴突崩解呈空泡状,或退变形似蜂窝状;微丝微管数目明显减少,线粒体肿胀,嵴间隙增宽,嵴模糊不清,或完全崩解、消失。②1周:模型组、治疗组均可见髓鞘板层分离范围进一步扩大,结构疏松,部分向内、外突起,泡状解离,出现大量崩解产物;部分神经纤维塌陷形成“洋葱样”小体;微丝微管数目进一步减少,排列紊乱,线粒体肿胀,数目减少。③2周:模型组、治疗组上述改变继续加重,髓鞘呈薄膜样结构,泡状解离,巨噬细胞内见含有大量髓鞘碎片的吞噬泡;部分轴突空虚透明;微丝微管数目继续减少,线粒体溶解消失。胶质细胞增生明显,核质浓缩成块,胞质更为疏松,大部分线粒体溶解消失,存在大量吞噬了髓鞘碎屑的溶酶体。④4周:模型组髓鞘板层结构有所修复,比正常明显变薄,结构疏松;部分轴突恢复,少量变性消失;微管、微丝及线粒体数目有所增多,但较正常减少,线粒体结构模糊。胶质细胞形态不规则,胞核呈不规则形,染色质固缩成块,胞质内线粒体增多,可见大量胶质纤维。治疗组髓鞘板层结构明显修复,较模型组增厚、致密;大量轴突逐渐恢复,电子密度增高;微管、微丝数目明显较模型组增多,分布均匀,线粒体结构清晰。胶质细胞形态基本正常,胞核呈圆形、椭圆形,染色质分布较模型组均匀,胞质内见大量胶质纤维。3.血府逐瘀汤对RGCs修复环境的影响(1) GAP-43在钳夹伤大鼠视网膜、视神经中的表达①正常大鼠:视网膜:表达微弱,主要集中于GCL。视神经:表达微弱。②3天:视网膜:各组表达均增强,组间没有显著性差异(P>0.05)。视神经:各组表达均明显增强,组间没有显著性差异(P>0.05)。③1周:视网膜:各组表达持续增强,治疗组AOD值明显高于模型组、阳性对照组(P<0.05)。视神经:模型组减弱,治疗组和阳性对照组增强,二者没有显著性差异(P>0.05),均高于模型组(P<0.05)。④2周:视网膜:各组表达均减弱,治疗组AOD值低于阳性对照组(P<0.05),和模型组没有显著性差异(P>0.05)。视神经:模型组减弱,治疗组和阳性对照组增强,二者没有显著性差异(P>0.05),均高于模型组(P<0.05)。⑤4周:视网膜:各组表达均减弱,治疗组高于模型组(P<0.05),和阳性对照组没有显著性差异(P>0.05)。视神经:各组表达均减弱,组间比较没有显著性差异(P>0.05)。(2) ROCKⅡ在钳夹伤大鼠视网膜、视神经中的表达①正常大鼠:视网膜:表达微弱,主要集中在GCL和IPL。视神经:表达微弱。②3天:视网膜:模型组和阳性对照组明显增强,治疗组减弱,AOD值明显低于模型组(P<0.05)、阳性对照组(P<0.05)。视神经:各组表达明显增强,治疗组IOD值低于模型组(P<0.05),和阳性对照组没有显著性差异(P>0.05)。③1周:各组表达均减弱,组间比较没有显著性差异(P>0.05)。视神经:各组表达明显减弱,治疗组IOD值明显低于模型组(P<0.05)、阳性对照组(P<0.05)。④2周:视网膜:各组表达较7天时增强,治疗组最弱,三组没有显著性差异(P>0.05)。视神经:模型组、阳性对照组轻度减弱,治疗组明显减弱,IOD值明显低于模型组(P<0.05)、阳性对照组(P<0.05)。⑤4周:视网膜:各组表达继续增强,治疗组最弱,AOD值明显低于模型组(P<0.05),和阳性对照组没有显著性差异(P>0.05)。视神经:各组表达均增强,治疗组IOD值明显低于模型组(P<0.05)、阳性对照组(P<0.05)。结论1.血府逐瘀汤全方能够明显提高TON大鼠RGCs存活率,优于单纯的活血药、理气药。2.血府逐瘀汤能够在一定时间一定程度上缓解TON大鼠视网膜组织水肿,延缓视网膜萎缩变薄,保护视神经超微结构,为轴浆运输功能的恢复提供物质基础和载体。3.血府逐瘀汤能在一定程度上增强组织中GAP-43的表达,同时,强烈抑制ROCKⅡ的表达,阻断Rho/ROCK信号转导通路,促进轴突修复。4.血府逐瘀汤对RGCs的保护体现在多个环节:提高RGCs存活率;维护、构建正常的组织结构;改善微环境,促进轴突修复。5.用玄府理论解释外伤性视神经病变的病因病机,据此建立活血开玄法和理气开玄法并用的治则,得到了良好印证,为该病的临床治疗提供了一个新的思路。更多还原

【Abstract】 ObjectiveTo evaluate the effects of Xuefu Zhuyu Decoction on traumatic optic neuropathies（TON） after optic nerve injury in rats through the survival ratio of RGCs,the tectology of the retina and optic nerve,the microenvironments of optic nerve restoration,and to investigate the possible mechanisms,which may be valuable as therapeutic agents for TON,and also to provide the new thoughts and methods for the study of Traditional Chinese Medicines’s effect on TON.Methods1.To observe the impacts of distilled water,Erigeron,Xuefu Zhuyu Decoction on RGCs after optic nerve injury in rats and selecte the best prescription.2.To observe the retinal thickness and the optic nerve ultrastructure at different times.3.To evaluate the effects of Xuefu Zhuyu Decoction on the microenvironments of the injured optic nerve restoration.The expressions of growth associated protein 43（GAP-43） and rho-associated coiled-coil forming protein kinase（ROCKⅡ） were detected on the 3^rd day and the 1^st,the 2^nd,the 4^th week.Results1.The effect of Xuefu Zhuyu Decoction on RGCsThe average survival ratio of RGCs:The RGCs’ survival ratios of Group K（Distilled Water）,Group D（Erigeron）,Group Z（Xuefu Zhuyu Decoction）,Group H（Huoxue Decoction） and Group L（Liqi Decoction） were respectively 62.84%±10.96%,75.76%±8.87%,89.18%±10.59%,69.68%±5.26%and 66.95%±9.12%.The RGCs’ survival ratio of Group Z was significantly higher than other groups（P<0.01）.2.The effect of Xuefu Zhuyu Decoction on the tectology of the retina and optic nerve（1）Retinal thickness①The 3^rd day Inner retina（retinal length,RL）,nerve fiber layer（nerve fiber layer,NFL）,ganglion cell layer（ganglion cell layer,GCL）,inner plexiform layer（inner plexiform layer,IPL） and INL （inner nuclear layer,INL）:Each group increased,and there was no significant difference in the same layers（P>0.05）.②The 1^st weekRL and the IPL:All groups continued to increase,and there was no significant difference in the same layers（P>0.05）.NFL:Slightly lower in each group,there was no significant difference in the same layers（P>0.05）.GCL:Treatment group and positive control group reduced,and treatment group was significantly lower than model group（P<0.05）.INL:Slight increase in each group,treatment group was significantly lower than model group（P<0.05）.③The 2^nd weekRL,IPL and INL:All groups continued to increase with no significant difference in the same layers（P>0.05）.NFL:All groups decreased with no significant difference whin all groups（P>0.05）.GCL:All groups decreased with no difference between treatment group and model group（P>0.05）,and positive control group was lower than model group（P<0.05）.④The 4^th weekRL,NFL and INL:All groups reduced significantly with no statistic difference whin all groups（P>0.05）.GCL:All groups reduced,and treatment group and positive control group were higher than model group（P<0.05）.IPL:All groups reduced significantly,and treatment group was higher than model group,positive control group（P<0.05）.（2）Ultrastructure of optic nerve①The 3^rddayModel group and Treatment group:Lamellar separation of myelin can be seen.Membrane collapsed,axis retracted,or capreomycin convexed.The number of axons reduced,and parts of them collapse.The numbers of microfilaments and microtubules were both reduced significantly.The mitochondrions were swelled,crest gap widened,or totally collapsed and disappeared.②The 1^st weekModel group and Treatment group:Lamellar separation of myelin can be seen further expansion than before.Parts of nerve fibers collapsed and formed "onion-like body".The numbers of microtubules and microfilaments further reduced,with disorder,mitochondrial swelling,a decrease in the number.③The 2^nd weekModel group and Treatment group:All continued to add to the above changes.The myelins bacame thiner and dissociated bubbly.There were many macrophages containing myelin debris.Some axons were transparent.The numbers of microtubules and microfilament continued to decline and the mitochondria dissolved and disapped.Glial cell proliferated,and the nuclear concentrated into a block.The majority of Mitochondrias disappeared and the lysosomes swallowed up a large number of myelin debris can be seen.④The 4^th weekModel group:The lamellar structure of myelin repaird and was thiner than normal.Parts of the axon restorated and small of them disappeared.The microtubules,microfilaments and mitochondrias increased.The modality of glial cells was irregular and a large number of glial fibrillary can be seen.Treatment group:The lamellar structure of myelin repaired obviously than model group.A large number of axons gradually restored,the electron density increased.Treat group was significantly more than treat group in the numbers of microtubule, microfilament.The mitochondria structure were clear.The glial cells were almost normal and a large number of glial fibrillary in the cytoplasm can be seen.3.The effects of Xuefu Zhuyu Decoction on the microenvironment of optic nerve restoration（1） The expression of GAP-43 in the retina and optic nerve①Normal retina:The expression was weak,and mainly concentrated in the GCL. Normal optic nerve:the expression is weak.②The 3^rd day:Retina and Optic Nerve:The expression of all groups increased obviously and there was no significant difference whin groups（P>0.05）.③The 1^st week:Retina:All groups increased obviously.The AOD of treat group was higher than model group,positive control group obviously（P<0.05）.Optic Nerve:Model group decreased,treatment group and positive control group increased.Their IOD were obviously higher than model group（P<0.05）,and there was no significant difference between them（P>0.05）. ④The 2^nd week:Retina:The expression of every group decreased.The AOD of treatment group was lower than positive contol group（P<0.05）,and there was no significant difference between treat group and model group（P>0.05）.Optic nerve:Model group decreased, treatment group and positive contol group increased with no significant difference between them（P>0.05）.But their were both higher than model group（P<0.05）.⑤The 4^th week:Retina:The expression in all groups decreased.Treatment group was higher than model group（P<0.05）.Optic nerve:The expression in all groups decreased,and there was no statistic difference whin all groups（P>0.05）.（2）The expression of ROCKⅡin the retina and optic nerve①Normal retina:The expression was weak,and mainly concentrated in the GCLand ipl. Normal optic nerve:The expression was weak.②The 3^rd day:Retina:The expression of model group and positive control group increased.Treat group decreased.Optic Nerve:All groups increased obviously,The IOD of treat group was lower than model group（P0.05）.③The 1^st,2^nd week:The expression of all groups decreased and there was no diference whin groups（P>0.05）.Optic nerve:Treat group was obviously lower than model and positive control group（P<0.05）.④The 4^th week:Retina:All groups increased.Treat group was lower than model group.Optic nerve:All groups increased.Treat group was lower than model group and positive group（P<0.05）.Conclusion1.Xuefuzhuyu Decoction can increase the survival ratio of RGCs significantly.It can be used to treat traumatic optic neuropathies.2.Xuefuzhuyu Decoction can prevent retinal tissue edema and atrophy,and protect the microstructures of optic nerve,which is valuable for restorating the transportation of axonal plasm.3.The retina and optic nerve can self-repair by regulating the expression level of GAP-43 and ROCKⅡ.Xuefuzhuyu Decoction can increase the expression of GAP-43 and decrease the expression of ROCKⅡ.4.Xuefuzhuyu Decoction can treat TON efficiently by improving the survival ratio of RGCs,maintaining or building the structures,accelerating transportation of axonal plasm,and improving the microenvironment for optic nerve fiber to survive or regenerate.更多还原