【作者】 侯正玉；

【导师】 卞小芸； 宋秀君；

【作者基本信息】 河北医科大学 ， 眼科学， 2008， 硕士

【摘要】 目的:外伤性视神经病变(Traumatic Optic Neuropathy , TON)是指外力通过骨质或眼球的移动传递给视神经造成的间接损伤,是眼科常见致盲性眼病之一,这种损伤在闭合性脑部外伤中约占0. 5 %～5 %[1、2]。间接性外伤性视神经病变引起的视力损伤的程度各不相同,约50%的患者仅为“光感”或“无光感”视力。目前还没有确凿的治疗方案。近年来随着对细胞凋亡现象认识的深入及国内外大量视神经损伤与修复方面的研究,神经不可再生的观点越来越引起医学界质疑,神经再生问题的进一步研究有望引发医学研究领域的又一重大突破。国内外大量研究显示药物、手术、基因治疗等多种治疗方法均显示对视网膜神经节细胞(Retinal Ganglion Cells ,RGCs)的再生有一定促进作用。笔者通过大量查阅国内外权威文献,了解到视神经损伤后轴突的再生是一个多环节、多因素的过程,而α2-肾上腺素受体激动剂(α2-Selective Adrenergic Agonists ,α2-SAs)在动物实验中已被证实有确切疗效,但其具体作用机制仍不清楚,本课题通过制作大鼠视神经夹挫伤动物模型,应用α2-SAs(0.2%酒石酸溴莫尼定)进行干预,并通过免疫组织化学检测大鼠视网膜中bFGF、Bcl-2、Bax的表达,用透射电镜观察微观形态,以探讨该药在治疗视神经夹挫伤中的具体作用机制,为临床更好地治疗视神经损伤提供新的思路和策略,并增进神经再生问题方面的认识。方法1 90只雌性S-D大鼠随机分为正常组、未治疗组和治疗组,未治疗组和治疗组采用反向镊钳夹法制作大鼠视神经夹挫伤动物模型,其中治疗组大鼠在造模前及造模后给予0.2%酒石酸溴莫尼定滴眼液点眼,未治疗组及正常组不做任何处理。2分别于造模后l、3、5、7、14、21d通过氯胺酮肌肉注射麻醉未治疗组、治疗组大鼠及相应时间点正常组大鼠,摘出眼球固定于4%多聚甲醛溶液中,沿眼球赤道部剪开,弃去眼前节及眼内容物,取颞上象限距视神经2mm以内的视网膜组织切片并行HE染色,光镜下进行RGCs计数以观察RGCs的动态变化。3采用同样方法麻醉各组不同时间点用作免疫组织化学染色的大鼠,摘出眼球固定于4%多聚甲醛溶液中,取颞上象限距视神经2mm以内的视网膜组织行bFGF、Bcl-2、bax免疫组织化学染色,观察三种因子在各组大鼠视网膜中不同时间点的表达及差异。4分别于3、7、21d麻醉用于透射电镜观察的三组大鼠,迅速摘出眼球保存于2.5%戊二醛保存液中,取视神经周围部位视网膜组织送做透射电镜,观察三组大鼠视网膜不同时期的微观形态变化及差异。结果1光镜及透射电镜下观察到正常组大鼠视网膜各层细胞排列整齐致密,未治疗组视网膜各层细胞排列不规则,总厚度变薄,出现明显凋亡形态变化,治疗组形态变化较未治疗组有明显减轻。2 Bcl-2在正常组大鼠视网膜中的阳性反应主要出现于视神经节细胞层,未治疗组损伤后随损伤时间的延长,Bcl-2在视网膜中的表达逐渐增强,损伤后第7、14、21d时,呈强阳性反应;治疗组较未治疗组表达明显增加,有统计学差异(p<0.01)。3 Bax在正常组大鼠视网膜中有表达,阳性反应主要出现于神经节细胞层、内丛状层、内核层,未治疗组随视神经损伤时间的延长,Bax在3、5、7d未治疗组大鼠视网膜中的表达强度增强,且治疗组表达强度明显小于未治疗组,有统计学差异(p<0.05)。4 bFGF在正常组大鼠视网膜中主要分布于视神经节细胞层及内核层,未治疗组视神经节细胞层及内核层染色强度增加,外核层出现bFGF阳性细胞;治疗组较未治疗组阳性表达有明显增强,有统计学差异(p<0.05)。结论1 0.2%酒石酸溴莫尼定滴眼液明显减轻大鼠视神经夹挫伤后视网膜形态损伤变化,对大鼠视神经夹挫伤有较好的疗效。2 0.2%酒石酸溴莫尼定滴眼液点眼使视神经夹挫伤大鼠视网膜中bFGF表达明显增加;Bcl-2表达增加,Bax表达减少,Bcl-2/Bax比率明显增大,提示酒石酸溴莫尼定治疗大鼠视神经夹挫伤的作用机制可能与促进bFGF表达增加和抑制凋亡有关。更多还原

【Abstract】 Objective: Traumatic optic neuropathy (TON) is an common ophthalmopathy that can leading to blind which can be indirectly caused by the extra-power passing from bone or eye ball. This kind of injury occupies 0.5%～5% in the closed brain trauma[1、2]. Degrees of the visual acuity damage indirectly caused by the TON differ from each other, however, about 50% of them are“light perception”or“non-light perception”. There is still no surely useful treatment plan for treating this kind of disease at present. With the development of the research on cell apoptosis and the optic nerve trauma and renovation, the view that nerve can not regeneration evoks more and more doubts in medical field. The continuing of research on the nerve regenation may initiates another breakthrough in medical domain.Many researches in our nation and overseas indicate that lots of new treatment methods show invariably effection in promoting the regeneration of the RGCs, such as medicine therapy, operation therapy and gene therapy.Author refers to lots of authority documents in domestic or overseas, and understands that the regeneration of the axonal after the optic nerve trauma is an complicated process including multiple factors and elements, andα2-selective adrenergic agonists (α2-SAs) has been proved to have evident effection in animal experiment, but its concrete mechanism of action is still unknown. This topic aims to research the concrete mechanism of action of the 0.2%brimonidine tartrate in therapying the optic nerve crush injury rat from immunohistochemistry of the Bcl-2, Bax, bFGF and the transmission electron microscope (TEM). We expect that this research may provide us some new strategies for treating optic nerve trauma and expand our recognition on nerve regeneration.Methods1 90 female adult Sprague-Dawley rats were randomly divided into normal group, untreated group and treated group. The untreated and treated groups received an optic nerve crush injury by reverse pliers; Then the treated group was gieven drops of 0.2%brimonidine tartrate topically before and after the crush injury;The untreated and normal groups received none intervention.2 All three groups were anesthetized with ketamine by 1, 3, 5, 7, 14, 21d after the optic nerve crush injury,and all the eye balls were enucleate and fixed into 4% paraformaldehyde for 72 hours,then the eye balls were sheared along the ambitus ,eye protomerite and eye content were removed.The part of temple upper quadrant rat retina which leave optic papillary within 2mm was selected to cutting sheet and HE dyeing,then RGCs number were counted out under the light microscope.3 Rats for immunohistochemistry were anesthetized with the same method , and all the eye balls were enucleate and fixed into 4% paraformaldehyde ,then the part of temple upper quadrant rat retina that leave the optic papillary within 2mm was selected for Bcl-2,Bax,bFGF immunohistochemistry staining.4 Rats from three groups for TEM were anesthetized by 3,7,21d,and eye balls were enucleate quickly and fixed into 2.5% glutaraldehyde. Retina tissue around the optic papillary was selected for TEM in order to observe the microcosmic morphologic change among three groups.Results1 Bax expression was observed in normal group rats retina and its potitive staining mainly located at ganglion cell layer, inner plexiform layer,inner nuclear layer.The expression of Bax was gradually strengthened by 3, 5, 7d in untreated group.The expression of Bax in untreated group is higher than that in treated group,and the statistics difference is significant (p<0.05) .2 The positive expression of Bcl-2 in normal group rat retina was mainly located at ganglion cell layer, The expression of Bcl-2 was gradually strengthened in untreated group and showed strong positive action by 7, 14, 21d. The expression of Bcl-2 in treated group is higher than that in untreated group,and the statistics difference is significant (p<0.01) . 3 The positive expression of bFGF in normal group rat retina was mainly located at ganglion cell layer and inner nuclear layer.The staining in ganglion cell layer and inner nuclear layer were increased in untreated group ,and positive cells were observed in outer nuclear layer. The expression of bFGF in treated group is higher than that in untreated group,and the statistics difference is significant (p<0.01) .4 Every sheet of cells in normal group rats retina lined up in order under the light microscope and TEM;Cells in untreated group lined up irregularly,the whole thickness thinningzed,and manifest apoptosis morphologic change was observed.Mohphologic change in treated group was lessened obviously.Conclusions1 0.2%brimonidine tartrate relieves the apoptosis morphologic change and elevate survival rate of RGCs, showing obvious effect in threapying rat optic nerve crush injury.2 0.2% brimonidine tartrate elevates the expression of bFGF、Bcl-2 ,cuts down the expression of the Bax in optic nerve crush injury rat retina,this hints that the therapeutic mechanism of 0.2% brimonidine tartrate may relevants with its effects of promoting the expression of bFGF and inhibiting apoptosis.更多还原