贝特舒滴眼液对兔急性高眼压模型视网膜神经节细胞的保护作用
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摘要
目的
应用贝特舒滴眼液对急性高眼压的兔眼进行治疗，通过视网膜神经细胞的凋亡检测和电镜观察超微结构的改变，来了解贝特舒滴眼液对青光眼视网膜神经节细胞的保护作用。
方法
大耳白兔12只，随机分成2组：给药组A和不给药组B。A组内再分2组，每只兔选一眼作高眼压灌注，为A_2组，对侧眼作为对照组A_1；同样，B组内也分2组，选一眼作急性高眼压模型组B_2，对侧眼作为对照组B_1。动物称重、麻醉后随机选一只眼，用连接生理盐水瓶及输液管的7号针头沿角膜缘作前房穿刺，升高输液瓶至距动物垂直距离为136cm处，此高度可形成100mmHg压力，维持60分钟。灌注前2小时，B组予贝特舒滴眼液点眼，灌注结束后按照说明予2滴/12小时，共3天；A组不予贝特舒治疗。3天后耳缘静脉注气法处死动物，摘取眼球，并去除角膜、虹膜、晶状体、玻璃体，把眼球壁固定、切片，各组分别进行视网膜神经细胞凋亡检测(用脱氧核糖核苷酸末端转移酶介导的缺口末端标记技术，即TUNEL技术)和电镜观察视网膜神经节细胞超微结构的变化，计数每个高倍镜视野中染色阳性的细胞数，经过统计学处理，比较各组的差异。
结 果
在A。组和B。组中，可以观察到染色阳性的细胞，细胞核呈棕
黄色，主要分布在视网膜的内核层和节细胞层人；与A。组没有找
到染色阳性细胞。每个高倍镜视野下，阳性细胞数经过统计学处
理，比较结果如下小。组与A。组比较，阳性细胞数减少O＝2．
666人咖＝2·57八P＜0．「L说明贝特舒滴眼液点眼有防止高
眼压导致视网膜神经节细胞凋亡发生的作用3B。与A；组比较，阳
性细胞数增多（t＝8．696，to。l5＝6．869，P＜0．001），说明贝特
舒滴眼液局部点眼不能完全阻止视网膜神经节细胞凋亡的发生；
A：与A；组比较，阳性细胞数增多（t＝10．826人。；。＝6．869，P＜
0．001入说明高眼压可以导致视网膜神经节细胞凋亡止；与B；组
比较，差异无显著性＊＝0，P一入说明贝特舒滴眼液无毒性作
用，不能导致视网膜神经节细胞凋亡；B。与B；组比较，阳性细胞
数增多（t＝8．696，to。；。＝6．869，P＜0．001），说明即使局部使
用贝特舒滴眼液，高眼压仍会导致视网膜神经节细胞凋亡的发生。
电镜观察的结果：A；人；组中视网膜神经节细胞核大，呈圆形或椭
圆形，有典型的双层核膜，胞质中粗面型内质网丰富，有颗粒状核
糖体附着，线粒体脊清晰k。组中线粒体高度肿胀，脊大部分消
失，空泡化，部分膜破裂，内腔出现细小致密物，粗面型内质网肿
胀，脱颗粒出。组少部分线粒体肿胀，部分脊肿胀变短，极少数有
空泡变，粗面型内质网末见明显扩张。
结 论
高眼压能导致视网膜神经节细胞发生凋亡，而贝特舒滴眼液
．t．
能部分地阻止其凋亡的发生，贝特舒滴眼液对视网膜神经细胞没
有毒性作用。
Objective
To treat rabbits eyes after acute ocular pressure with Betopic liquid, apoptosis in retinal ganglion cells being tested and cells'ultra-structure being observed, and understand the protection functions of Betopic eye liquid for glaucoma retinal ganglion cell.
Methods
12 Big ear rabbits were divided randomly into 2 groups: group A which were treated and group B which were not treated. In group A one eye of each rabbit was selected to be made a high ocular pressure model, it was group A2, the other one was control group A1. In group B, the acute ocular pressure group was B2, the other eye group was B1. Rabbit were weighed and anaesthetized and placed in a stereotaxic frame. The anterior chamber of one eye was cannulated with a injector needle through the limbus, which was connected a bottle of NS. To lift the bottle to 136 cm from the rabbit on a vertical distance which can cause a 100mmHg pressure, and maintain 60 mins. 2 hours before elevation of the ocular pressure in the anterior chamber , both eyes of rabbits in the group B were treated with 2 drops Betopic, 2 drops per 12 hours, lasting 3 days. Group A no treating. After 3 days, rabbits
were killed and apoptosis in retinal ganglion cells were tested ( with TUNEL technique) and cells'ultrastmcture were observed with electron microscope. To take count of the number of thapoptosis cells in every microscope view, and the data was treated with statistical calculation , then compare the difference in the four groups.
Results
The apoptosis cells can be observed in group A2 and group B2,
not in group A1 and B1. The apoptosis cells are colorated brown, mainly in inner granule layer and ganglion cells layer. The data was calculated in statistical statistics, the result is; The number of apoptosis in retinal ganglion cells decreases in this sequence: A2, B2, A1 and B1. (P<0. 05). As Electron microscope display, the ultrastruc-ture in group A1 and B1 is normal; A large, dual membrane -bound, usually spherical nucleus is in retinal ganglion cell, plenty of endo-plasmic reticulum ( ER) in protoplast, and mitochondrial crista is clear. In group B2, partial mitochondrial crista swell, vacuolar degeneration and endoplasmic reticulum(ER) dilate partially. In group A2 most mitochondrion are vacuolar degeneration, membranes break, ER dilate.
Conclusion
High ocular pressure can cause retinal ganglion cells apoptosis, while Betopic liquid can partially prevent apoptosis, and Betopic liquid has no toxicity to the retinal nerve cells.
应用贝特舒滴眼液对急性高眼压的兔眼进行治疗，通过视网膜神经细胞的凋亡检测和电镜观察超微结构的改变，来了解贝特舒滴眼液对青光眼视网膜神经节细胞的保护作用。
方法
大耳白兔12只，随机分成2组：给药组A和不给药组B。A组内再分2组，每只兔选一眼作高眼压灌注，为A_2组，对侧眼作为对照组A_1；同样，B组内也分2组，选一眼作急性高眼压模型组B_2，对侧眼作为对照组B_1。动物称重、麻醉后随机选一只眼，用连接生理盐水瓶及输液管的7号针头沿角膜缘作前房穿刺，升高输液瓶至距动物垂直距离为136cm处，此高度可形成100mmHg压力，维持60分钟。灌注前2小时，B组予贝特舒滴眼液点眼，灌注结束后按照说明予2滴/12小时，共3天；A组不予贝特舒治疗。3天后耳缘静脉注气法处死动物，摘取眼球，并去除角膜、虹膜、晶状体、玻璃体，把眼球壁固定、切片，各组分别进行视网膜神经细胞凋亡检测(用脱氧核糖核苷酸末端转移酶介导的缺口末端标记技术，即TUNEL技术)和电镜观察视网膜神经节细胞超微结构的变化，计数每个高倍镜视野中染色阳性的细胞数，经过统计学处理，比较各组的差异。
结 果
在A。组和B。组中，可以观察到染色阳性的细胞，细胞核呈棕
黄色，主要分布在视网膜的内核层和节细胞层人；与A。组没有找
到染色阳性细胞。每个高倍镜视野下，阳性细胞数经过统计学处
理，比较结果如下小。组与A。组比较，阳性细胞数减少O＝2．
666人咖＝2·57八P＜0．「L说明贝特舒滴眼液点眼有防止高
眼压导致视网膜神经节细胞凋亡发生的作用3B。与A；组比较，阳
性细胞数增多（t＝8．696，to。l5＝6．869，P＜0．001），说明贝特
舒滴眼液局部点眼不能完全阻止视网膜神经节细胞凋亡的发生；
A：与A；组比较，阳性细胞数增多（t＝10．826人。；。＝6．869，P＜
0．001入说明高眼压可以导致视网膜神经节细胞凋亡止；与B；组
比较，差异无显著性＊＝0，P一入说明贝特舒滴眼液无毒性作
用，不能导致视网膜神经节细胞凋亡；B。与B；组比较，阳性细胞
数增多（t＝8．696，to。；。＝6．869，P＜0．001），说明即使局部使
用贝特舒滴眼液，高眼压仍会导致视网膜神经节细胞凋亡的发生。
电镜观察的结果：A；人；组中视网膜神经节细胞核大，呈圆形或椭
圆形，有典型的双层核膜，胞质中粗面型内质网丰富，有颗粒状核
糖体附着，线粒体脊清晰k。组中线粒体高度肿胀，脊大部分消
失，空泡化，部分膜破裂，内腔出现细小致密物，粗面型内质网肿
胀，脱颗粒出。组少部分线粒体肿胀，部分脊肿胀变短，极少数有
空泡变，粗面型内质网末见明显扩张。
结 论
高眼压能导致视网膜神经节细胞发生凋亡，而贝特舒滴眼液
．t．
能部分地阻止其凋亡的发生，贝特舒滴眼液对视网膜神经细胞没
有毒性作用。
Objective
To treat rabbits eyes after acute ocular pressure with Betopic liquid, apoptosis in retinal ganglion cells being tested and cells'ultra-structure being observed, and understand the protection functions of Betopic eye liquid for glaucoma retinal ganglion cell.
Methods
12 Big ear rabbits were divided randomly into 2 groups: group A which were treated and group B which were not treated. In group A one eye of each rabbit was selected to be made a high ocular pressure model, it was group A2, the other one was control group A1. In group B, the acute ocular pressure group was B2, the other eye group was B1. Rabbit were weighed and anaesthetized and placed in a stereotaxic frame. The anterior chamber of one eye was cannulated with a injector needle through the limbus, which was connected a bottle of NS. To lift the bottle to 136 cm from the rabbit on a vertical distance which can cause a 100mmHg pressure, and maintain 60 mins. 2 hours before elevation of the ocular pressure in the anterior chamber , both eyes of rabbits in the group B were treated with 2 drops Betopic, 2 drops per 12 hours, lasting 3 days. Group A no treating. After 3 days, rabbits
were killed and apoptosis in retinal ganglion cells were tested ( with TUNEL technique) and cells'ultrastmcture were observed with electron microscope. To take count of the number of thapoptosis cells in every microscope view, and the data was treated with statistical calculation , then compare the difference in the four groups.
Results
The apoptosis cells can be observed in group A2 and group B2,
not in group A1 and B1. The apoptosis cells are colorated brown, mainly in inner granule layer and ganglion cells layer. The data was calculated in statistical statistics, the result is; The number of apoptosis in retinal ganglion cells decreases in this sequence: A2, B2, A1 and B1. (P<0. 05). As Electron microscope display, the ultrastruc-ture in group A1 and B1 is normal; A large, dual membrane -bound, usually spherical nucleus is in retinal ganglion cell, plenty of endo-plasmic reticulum ( ER) in protoplast, and mitochondrial crista is clear. In group B2, partial mitochondrial crista swell, vacuolar degeneration and endoplasmic reticulum(ER) dilate partially. In group A2 most mitochondrion are vacuolar degeneration, membranes break, ER dilate.
Conclusion
High ocular pressure can cause retinal ganglion cells apoptosis, while Betopic liquid can partially prevent apoptosis, and Betopic liquid has no toxicity to the retinal nerve cells.
引文
1 王宝平，蒋幼芹，黄佩刚，等．兔实验性急性高眼压模型视网膜谷氨酸的变化．中华眼科杂志，2000，36：378～380．
2 Dreyer EB. AT proposed role for excitotoxicity in glaucoma. J Glaucoma, 1998; 7: 62-67.
3 归东梅，高殿文，徐洪斌，等．急性高眼压状态大鼠视网膜一氧化氮及其合酶变化的研究。中华眼底病杂志，2001，17：230～233．
4 Dawson TM, Dawson VL. Nitric oxide synthase: role as a transmitter/mediator in the brain and endocrine system. Annu Rev Med, 1996; 47: 219～227.
5 Choi DW, Koh JY, Peters S et al. Pharmacology of glutamate neurotoxicity in cortical cell culture: attenuation by NMDA antagonists. J Neurosci, 1988; 81: 185～196.
6 Siesjo BK. Pathophysiology and treatment of focal cerebral ischemia. Part Ⅱ: Mechanisms of damage and treatment. J Neurosurg, 1992; 77: 337～354.
7 Osborne NN, Chidlow G, Nash MS. et al. The potential of neuroprotection in glaucoma treatment. Curr Opin Ophthalmol, 1999; 10: 82～92.
8 Osborne NN, Cazevieille C, Carvalho AL et al. In vivo and in vitro experiments show that betaxolol is a retinal neuroprotective agent. Brain Res, 1997; 751: 113～123.
9 Camilleri - Broet S, Vanderwerff H, Caldwell E et al. Distinct alterations in mitochondrial mass and function characterize different models of apoptosis. Exp Cell Res, 1998; 239: 277～292.
10 Quigley HA, Nickells RW, Kerrigan LA et al. Retinal ganglion cell death in experimental glaucoma and after axotomy occurs by apoptosis. Invest Ophthalmol Vis Sci, 1995; 36: 774～786.
2 Dreyer EB. AT proposed role for excitotoxicity in glaucoma. J Glaucoma, 1998; 7: 62-67.
3 归东梅，高殿文，徐洪斌，等．急性高眼压状态大鼠视网膜一氧化氮及其合酶变化的研究。中华眼底病杂志，2001，17：230～233．
4 Dawson TM, Dawson VL. Nitric oxide synthase: role as a transmitter/mediator in the brain and endocrine system. Annu Rev Med, 1996; 47: 219～227.
5 Choi DW, Koh JY, Peters S et al. Pharmacology of glutamate neurotoxicity in cortical cell culture: attenuation by NMDA antagonists. J Neurosci, 1988; 81: 185～196.
6 Siesjo BK. Pathophysiology and treatment of focal cerebral ischemia. Part Ⅱ: Mechanisms of damage and treatment. J Neurosurg, 1992; 77: 337～354.
7 Osborne NN, Chidlow G, Nash MS. et al. The potential of neuroprotection in glaucoma treatment. Curr Opin Ophthalmol, 1999; 10: 82～92.
8 Osborne NN, Cazevieille C, Carvalho AL et al. In vivo and in vitro experiments show that betaxolol is a retinal neuroprotective agent. Brain Res, 1997; 751: 113～123.
9 Camilleri - Broet S, Vanderwerff H, Caldwell E et al. Distinct alterations in mitochondrial mass and function characterize different models of apoptosis. Exp Cell Res, 1998; 239: 277～292.
10 Quigley HA, Nickells RW, Kerrigan LA et al. Retinal ganglion cell death in experimental glaucoma and after axotomy occurs by apoptosis. Invest Ophthalmol Vis Sci, 1995; 36: 774～786.