盐酸可乐宁对兔视网膜缺血损伤的保护作用的实验研究
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摘要
目的 缺血性视网膜损伤是眼科常见的一种临床表现，其病因繁多，如视网膜血管阻塞性疾病—视网膜静脉毛细血管供血不足，糖尿病视网膜病变，不规则动静脉短路，高血压视网膜病变，视网膜新生血管形成，视网膜脱离和玻璃体出血。此外，玻璃体切割及视网膜脱离手术等可以影响视网膜血流量，因此也可以引起视网膜缺血性损伤。视网膜组织是属于中枢神经系统的一部分，很多神经系统疾病都伴随有视网膜病变。近期，有大量研究指出谷氨酸(glutamate,Glu)等兴奋性神经递质有很强的兴奋性神经毒性。兴奋性氨基酸(excitatory amino acids,EAAs)——天门冬氨酸(aspartate,Asp)等在脑缺血过程中的释放，可能参与了低氧脑缺血损伤的机制，而α_2肾上腺素受体激动剂可以保护在缺血损伤中神经细胞的死亡。但其保护作用及机制还不是很清楚。
本文通过建立升高眼压致视网膜缺血的动物实验模型，采用兔视网膜缺血前腹腔内注射α_2肾上腺素受体激动剂——盐酸可乐宁0.5mg／kgwt，观察了用药组及未用药组的视网膜的结构、功能，及玻璃体的Glu浓度变化，并在缺血后1小时、第1天、第3天的变化并进行比较。从而探讨了盐酸可乐宁在视网膜缺血损伤的神经保护作用及机制。
方法 选取成年健康日本种大耳白家兔20只40只眼，将实验动物随机分为A、B两组，A组为未用药组，B为用药组即缺血前半小时腹腔内注射盐酸可乐宁0.5mg／kgwt。两组兔子均采取右眼为升高眼压视网膜缺血眼，左眼为前房穿刺但无灌注为对照眼，作为对照眼。缺血前、缺血后进行双眼视网膜电生理检查，缺血后第1天、第3天进行10×40倍光镜下视网膜组织病理学检查，并采用高压液相色谱仪进行玻璃体Glu浓度测定。视网膜电生理
硕士学位论文
检查采用缺血前、缺血后不同时间段的配对t检验，病理切片及玻璃体Glu
浓度的比较采用左右眼间及A、B两组间左右眼的不同的时间段进行组间比
较。
结果1.视网膜电生理检查，ERGb波波幅在A组右眼缺血后与缺血前相比
显著下降(P<0.05)，虽然在第1、3天略有恢复，但仍显著低于缺血前
(P<0.05)，而B组缺血后1小时ERGb波波幅略有下降，与左眼相比有显
著统计学差异(P<0.05)，在缺血后第1天，第3天逐渐恢复，且左右眼间
无统计学差异(P>0.05)，并且，A、B两组的右眼的ERGb波波幅在缺血
后任何时间相比都有显著的的统计学差异(P<0.05)。3OHZ闪烁光反应反
映的是视锥细胞的功能，即黄斑区的功能。因本实验视网膜缺血动物模型类
似于青光眼，故短暂视网膜缺血(45分钟)并未明显损害黄斑区的功能，
3OHz闪烁光反应波幅虽然在两组兔的右眼中在缺血后均略有下降，但与缺
血前相比，均无显著的统计学差异(P>0.05)。
2.视网膜病理切片检查，可见在缺血后第1天时，A组右眼视神经纤
维层厚度与左眼相比无统计学差异(P>0.05)，这可能与早期的水肿有关，
光镜检查可见神经纤维层有空泡和裂隙出现。而内颗粒层，内网状层厚度则
明显变薄(P<0.05)，神经节细胞数目减少(P<0.05)，神经节细胞及内颗
粒层细胞排列紊乱。至缺血后第3天，视网膜的组织病理学损伤更明显
(P<0.05)。而在B组右眼则可见视网膜病理切片各项观测指标在左右眼间无
显著差别(P>0.05)。此外，A、B两组的右眼的各项光镜下检查指标(除
缺血后1小时神经纤维层厚度)相比可见均有显著的统计学差异
(P<0 .05)。
3.玻璃体Glu浓度在缺血后第1天，缺血后第3天测定值在A、B组左
右眼中进行比较，可见在A组的右眼玻璃体Glu浓度与左眼相比显著升高
硕士学位论文
(P<0 .05)，且在缺血后第3天时升高更明显，这可能与视网膜神经细胞死
亡致Glu漏出到细胞外间隙所致。而在B组虽然在缺血后第3天玻璃体Glu
浓度略有升高，但与左眼相比并无显著差别(P>0 .05)，而A、B两组的右
眼玻璃体Gfu浓度相比在缺血后第1，3天时相比，均有显著的统计学差异
(P<0 .05)。
结论1.QZ肾上腺素受体激动剂—盐酸可乐宁可以在急性缺血损伤中保护
视网膜结构，表现为减轻视网膜内层的损伤—降低缺血所致的内颗粒层，
内丛状层、神经节纤维层厚度的变薄，减少神经节细胞的死亡。
2.QZ肾上腺素受体激动剂—盐酸可乐宁可以保护视网膜的功能，表
现为可保存ERGb波波幅。同时，升高眼压所致的视网膜缺血动物模型对
30Hz视网膜闪烁光反应的波幅无明显影响，即对黄斑区的功能无明显损
伤。
3.缺血所致的视网膜结构损伤与兴奋性神经递质Glu的释放有关，而
aZ肾上腺素受体激动剂—盐酸可乐宁可以通过降低Glu的释放，阻断这
种兴奋性毒性损伤。
Purpose Retinal ischemic insult is one of common ophthalmic clinic manifestations. There are many causes, such as obstructive retinopathies, venous capillary insufficiency, diabetic retinopathy; anomalous occular shunts,hypertensive retinopathy, retinal neovascularization, retinal detatchment and vitreous hemorrhage, and so on. In addition, vitrectomy and retinal detachment surgery, which can reduce retinal blood flow, can result in retinal ischemic insult. The retina is part of the central nervous system (CNS) and many neurological diseases accompany with retinal diseases. Recently, exprimental data indicate that excitatory amino acids (EAAs) -glutamate (Glu) can trigger excitotoxic insult. EAAs released during brain ischemia, which may be involved in hypoxic-ischemic brain injury. Moreover, some researchers also find that EAAs involved in the mechanism of retinal ischemia insult, and a 2-adrenoceptor agonists are neruropretective in ischemic insult. However, the neuroprotective effects and mechanism of a 2
-adrenoceptor agonists
remain unclear.
In this experiment, transient retinal ischemia was induced in rabbits by raising intraocular pressure. Before ischemia rabbits were injected intraperitoneally chlonidine hydrochloride ( 0.5mg/kg wt) , we compared the control and treatment groups by studying the retinal elctrophysiological, histological, and vitreal glutamate changes 1 hour, Iday, and 3 day after retinal ischemia. So we will further confirm clonidine's neuroprotective effects and mechanism.
Method Twenty healthy adult rabbits (forty eyes) were randomly divided into two groups, control and treatment groups, and the treatment group were injected intraperitoneally clonidine hydrochloride ( 0.5mg/kg wt ) before ischemia. Elevating the intraocular pressure in all right eyes produced the retinal ischemia, and the left eyes were sham-operated. We examed the retinal electrophysiology of both eyes before and after ischemia, histology of rabbit retina 1 day and 3 day after ischemia, analyzed vitreous humor glutamate levels between control and clonidine-treated eyes, and compared results in different time. Result 1. Electrophysiological examination show significant reduction of ERG b-wave amplitude in right eyes of control group (P<0.05) , although there are gradual recovery 1 day and 3 day after ischemia, the amplitude is still significantly lower than that before ischemia. There is no significant reduction of b-wave amplitude in right eyes except 1 hour after ischemia compared with left eyes in treatment groups. In addition, the amplitude of 30 Hz flicker ERGs didn't significantly reduced in both groups (P>0.05) , although it has a mild reduction after ischemia.
2. Retinal histological section examination show inner nuclear layer, inner plexiform layer and ganglion fiber layer became significantly thinner 1 day and 3 day after ischemia than those before ischemia (P<0.05) , but the ganglion fiber layer's thickness didn't significantly reduced 1 hour after ischemia because mild edema (P>0.05) . Meanwhile the ganglion cell count became decreased after ischemia (P<0.05) . The inner nuclear layer cells and ganglion cells became disordered. On the contrary, there is significant difference between right and left eyes in treatment group, also between right eyes of control and treatment
groups except the ganglion fiber layer thickness after 1 hour after ischemia (P<0.05) .
3. Vitreous humor glutamate analysis show the right eye vitreous glutamate concentration significantly increased compared with left eyes in control group 1 day and 3 day after ischemia (P<0.05) , and much higher hi 3 day after ischemia which may be caused by the death of retinal neural cells hi which the glutamate leaked into extracellular space. The vitreal glutamate didn't significantly increase in treatment group, though it mildly increased in 3 day after ischemia.
Conclusion 1. a 2-adrenoceptor agonist-clonidine hydrochloride can protect retinal structure in acute ischemia insult, which can attenuate inner layer insult by preventing
本文通过建立升高眼压致视网膜缺血的动物实验模型，采用兔视网膜缺血前腹腔内注射α_2肾上腺素受体激动剂——盐酸可乐宁0.5mg／kgwt，观察了用药组及未用药组的视网膜的结构、功能，及玻璃体的Glu浓度变化，并在缺血后1小时、第1天、第3天的变化并进行比较。从而探讨了盐酸可乐宁在视网膜缺血损伤的神经保护作用及机制。
方法 选取成年健康日本种大耳白家兔20只40只眼，将实验动物随机分为A、B两组，A组为未用药组，B为用药组即缺血前半小时腹腔内注射盐酸可乐宁0.5mg／kgwt。两组兔子均采取右眼为升高眼压视网膜缺血眼，左眼为前房穿刺但无灌注为对照眼，作为对照眼。缺血前、缺血后进行双眼视网膜电生理检查，缺血后第1天、第3天进行10×40倍光镜下视网膜组织病理学检查，并采用高压液相色谱仪进行玻璃体Glu浓度测定。视网膜电生理
硕士学位论文
检查采用缺血前、缺血后不同时间段的配对t检验，病理切片及玻璃体Glu
浓度的比较采用左右眼间及A、B两组间左右眼的不同的时间段进行组间比
较。
结果1.视网膜电生理检查，ERGb波波幅在A组右眼缺血后与缺血前相比
显著下降(P<0.05)，虽然在第1、3天略有恢复，但仍显著低于缺血前
(P<0.05)，而B组缺血后1小时ERGb波波幅略有下降，与左眼相比有显
著统计学差异(P<0.05)，在缺血后第1天，第3天逐渐恢复，且左右眼间
无统计学差异(P>0.05)，并且，A、B两组的右眼的ERGb波波幅在缺血
后任何时间相比都有显著的的统计学差异(P<0.05)。3OHZ闪烁光反应反
映的是视锥细胞的功能，即黄斑区的功能。因本实验视网膜缺血动物模型类
似于青光眼，故短暂视网膜缺血(45分钟)并未明显损害黄斑区的功能，
3OHz闪烁光反应波幅虽然在两组兔的右眼中在缺血后均略有下降，但与缺
血前相比，均无显著的统计学差异(P>0.05)。
2.视网膜病理切片检查，可见在缺血后第1天时，A组右眼视神经纤
维层厚度与左眼相比无统计学差异(P>0.05)，这可能与早期的水肿有关，
光镜检查可见神经纤维层有空泡和裂隙出现。而内颗粒层，内网状层厚度则
明显变薄(P<0.05)，神经节细胞数目减少(P<0.05)，神经节细胞及内颗
粒层细胞排列紊乱。至缺血后第3天，视网膜的组织病理学损伤更明显
(P<0.05)。而在B组右眼则可见视网膜病理切片各项观测指标在左右眼间无
显著差别(P>0.05)。此外，A、B两组的右眼的各项光镜下检查指标(除
缺血后1小时神经纤维层厚度)相比可见均有显著的统计学差异
(P<0 .05)。
3.玻璃体Glu浓度在缺血后第1天，缺血后第3天测定值在A、B组左
右眼中进行比较，可见在A组的右眼玻璃体Glu浓度与左眼相比显著升高
硕士学位论文
(P<0 .05)，且在缺血后第3天时升高更明显，这可能与视网膜神经细胞死
亡致Glu漏出到细胞外间隙所致。而在B组虽然在缺血后第3天玻璃体Glu
浓度略有升高，但与左眼相比并无显著差别(P>0 .05)，而A、B两组的右
眼玻璃体Gfu浓度相比在缺血后第1，3天时相比，均有显著的统计学差异
(P<0 .05)。
结论1.QZ肾上腺素受体激动剂—盐酸可乐宁可以在急性缺血损伤中保护
视网膜结构，表现为减轻视网膜内层的损伤—降低缺血所致的内颗粒层，
内丛状层、神经节纤维层厚度的变薄，减少神经节细胞的死亡。
2.QZ肾上腺素受体激动剂—盐酸可乐宁可以保护视网膜的功能，表
现为可保存ERGb波波幅。同时，升高眼压所致的视网膜缺血动物模型对
30Hz视网膜闪烁光反应的波幅无明显影响，即对黄斑区的功能无明显损
伤。
3.缺血所致的视网膜结构损伤与兴奋性神经递质Glu的释放有关，而
aZ肾上腺素受体激动剂—盐酸可乐宁可以通过降低Glu的释放，阻断这
种兴奋性毒性损伤。
Purpose Retinal ischemic insult is one of common ophthalmic clinic manifestations. There are many causes, such as obstructive retinopathies, venous capillary insufficiency, diabetic retinopathy; anomalous occular shunts,hypertensive retinopathy, retinal neovascularization, retinal detatchment and vitreous hemorrhage, and so on. In addition, vitrectomy and retinal detachment surgery, which can reduce retinal blood flow, can result in retinal ischemic insult. The retina is part of the central nervous system (CNS) and many neurological diseases accompany with retinal diseases. Recently, exprimental data indicate that excitatory amino acids (EAAs) -glutamate (Glu) can trigger excitotoxic insult. EAAs released during brain ischemia, which may be involved in hypoxic-ischemic brain injury. Moreover, some researchers also find that EAAs involved in the mechanism of retinal ischemia insult, and a 2-adrenoceptor agonists are neruropretective in ischemic insult. However, the neuroprotective effects and mechanism of a 2
-adrenoceptor agonists
remain unclear.
In this experiment, transient retinal ischemia was induced in rabbits by raising intraocular pressure. Before ischemia rabbits were injected intraperitoneally chlonidine hydrochloride ( 0.5mg/kg wt) , we compared the control and treatment groups by studying the retinal elctrophysiological, histological, and vitreal glutamate changes 1 hour, Iday, and 3 day after retinal ischemia. So we will further confirm clonidine's neuroprotective effects and mechanism.
Method Twenty healthy adult rabbits (forty eyes) were randomly divided into two groups, control and treatment groups, and the treatment group were injected intraperitoneally clonidine hydrochloride ( 0.5mg/kg wt ) before ischemia. Elevating the intraocular pressure in all right eyes produced the retinal ischemia, and the left eyes were sham-operated. We examed the retinal electrophysiology of both eyes before and after ischemia, histology of rabbit retina 1 day and 3 day after ischemia, analyzed vitreous humor glutamate levels between control and clonidine-treated eyes, and compared results in different time. Result 1. Electrophysiological examination show significant reduction of ERG b-wave amplitude in right eyes of control group (P<0.05) , although there are gradual recovery 1 day and 3 day after ischemia, the amplitude is still significantly lower than that before ischemia. There is no significant reduction of b-wave amplitude in right eyes except 1 hour after ischemia compared with left eyes in treatment groups. In addition, the amplitude of 30 Hz flicker ERGs didn't significantly reduced in both groups (P>0.05) , although it has a mild reduction after ischemia.
2. Retinal histological section examination show inner nuclear layer, inner plexiform layer and ganglion fiber layer became significantly thinner 1 day and 3 day after ischemia than those before ischemia (P<0.05) , but the ganglion fiber layer's thickness didn't significantly reduced 1 hour after ischemia because mild edema (P>0.05) . Meanwhile the ganglion cell count became decreased after ischemia (P<0.05) . The inner nuclear layer cells and ganglion cells became disordered. On the contrary, there is significant difference between right and left eyes in treatment group, also between right eyes of control and treatment
groups except the ganglion fiber layer thickness after 1 hour after ischemia (P<0.05) .
3. Vitreous humor glutamate analysis show the right eye vitreous glutamate concentration significantly increased compared with left eyes in control group 1 day and 3 day after ischemia (P<0.05) , and much higher hi 3 day after ischemia which may be caused by the death of retinal neural cells hi which the glutamate leaked into extracellular space. The vitreal glutamate didn't significantly increase in treatment group, though it mildly increased in 3 day after ischemia.
Conclusion 1. a 2-adrenoceptor agonist-clonidine hydrochloride can protect retinal structure in acute ischemia insult, which can attenuate inner layer insult by preventing
引文
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26． Quigley HA, Green WR. The histology of human glaucoma cupping and optic nerve damage. Clinic ophthalmologic correlation in 21 eyes. Ophthalmology, 1979, 86: 1803-1827
27． Louzada-Junior P, Dias JJ, Santos WF, et al. Glutamate release in experimental ischemia of the retina: an approach using microdialysis. J Neurochem, 1992, 59(1) : 358-363
28． Johnson NF, Foulds WS. The effects of total acute ischemia on the structure of the rabbit retina. Exp Eye Res, 1978, 27(1) : 45-59
29． Szabo ME, Droy-Lefaix MT, Doly M, et al. Ischemia and reperfusion-induced histologic changes in the rat retina. IOVS, 1991,32: 1471-1478
30． Takahashi K, Lam TT, Edward DP, et al. Protective effects of flunarizine on ischemic injury in the rat retina. Arch Ophthalmol, 1992, 110: 862-870
31． Lafuente MP, Villegas-Perez, Sobrado-Calvo P, et al. Neuoprotective effects of a 2-selective adrenergic agonists against ischemia-induced retinal ganglio-n cell death. IOVS, 2001, 42(9) : 2075-2084
32． Holopigian K. Evidence for photoreceptor changes in patients with DR.IOVS, 1997, 38(11) : 2355-65
33． Nayak.MS and Marmor MF. Post-ischemia ERG recovery is influenced by temperature. Curr Eye Res, 1995, 14: 81-85
34． Miyazaki M, Nazarali AJ, Boisvet DP, et al. Inhibition of ischemia-induced brain catecholamine attentions by clonidine. Brain Research, 1989, 22: 207-211
35． Zhang X, Kindel GH, Wulfert E, et al. Effects of immobilization stress on hi-ppocampal monoamine release: Modification by mivazerol,a new a 2-adreno ceptor agonist. Neuropharmacology, 1995, 34(12) : 1667-1672
36． Hoffman WE, Kochs E, Werner C, et al. Dexmedetomidine improve neurologic outcome from incomplete ischemia in the rat. Anesthesiology, 1991, 75: 328-332
37． Maier C, Steinberg GK, Sun GH, et al. Neuroprotection by the a 2-adrenoce ptor agonist dexmedetomidine. Anesthesiology, 1993,79: 306-312
38． Halonen T, Kotti T, Tuunanen J, et al. a 2-adrenoceptor agonist, dexmedetomidine, protects against kainic acid-induced convulsions and neuronal damage. Brain Res, 1995, 693: 217-224
39． Kuhmonen J, Pokorny J, Miettinen R, et al. Neuroprotective effects of dexm-ed etomidine in the gerbil hippocampus after transient global ischemia. Anesthesiology, 1997, 87: 371-377
40． Engelhard K, Werner C, Kaspar O, et al. Effects of the alpha2-adrenoceptor agonist dexmedetomidine on cerebral neurotransimitter concentations during cerebral ischemia in rats. Anesthesiology, 2002,96(2) : 450-457
41． 李凤鸣《眼科全书》，人民卫生出版社，1996:1841-1844
42． Miller RF, Slaughter MM. Excitatory amino-acid receptors in the vertebrate retina. In retinal transmitters and modulators: models of the brain. CRC Press: Boca Raton, 1985,2: 123
43． Bloomfield SA, Dowlling JE. Roles of aspartate and glutamate in synaptic transmission in rabbit retina: I .Outer plexiform layer. J Neurophysiol, 1995, 53: 699-713．
44． Olney JW. The toxic effects of glutamate and related compounds in the retina and brain. Retina, 1982, 2: 341-359
45． Ulshafer RJ, Sherry DM, Dawson R Jr, et al. Excitatory amino acid involvement in retinal degeneration. Brain Res, 1990, 531: 350-354
46． Vorwerk CK, Lipton SA, Zurakowski D, et al. Chronic low-dose glutamate is toxic to retinal ganglion cells,Toxcity blocked by memantine. IOVS, 1996, 37: 1618-1624
47． Frandsen A, Schousboe A. Mobilization of dantrolene-sensitive intracellular calcium pools is involved in the cytotoxicity induced by quisqualate and N-methyl-D-aspartate but not by 2-amino-3-(3-hydroxy-5-methylisoxazol-yl) protionate and kainate in cultured cerebral cortical neurons. Proc Natl Acad Sci, 1992, 89(7) : 2590-2594
48． Schwartz M, Belkin M, Yoles E, et al. Potential treatment modalities for glau-comatous neuropathy:neuroprotection and neuroregeneration. J Glaucoma, 1996, 5: 427-432
49． Marc RE, Liu W-L S, Kalloniatis M, et al. Patterns of glutamate immunoreac-tivity in the goldfish retina. J Neurosci, 1990,10: 4006-4034
50． Erecinsk M, Silver LA. Metabolism and role of glutamate in mammalian brain. Prog Neurobiol, 1990,35: 245-96
51． Hansen AJ and Nedergaard M. Brain ion hoeostasis in cerebral ischemia. Neurochem Pathol, 1988,9: 195-209
52． Billups B, Attwell D. Modulation of non-vesicular glutamate release by pH. Nature, 1996,379: cells isolated from the salamander(Amblystoma)retina. J Physiol 1991;436: 169-193
53． Barbour B, Brew H and Attwell D. Electrogenic uptake of glutamate and aspartate into glial cells isolated from the salamander(Amblystoma)retina. J Physiol 1991;436:169-193
54． Von Blankenfeld G and Kettenmann H. Glutamate and GABA receptors in vertebrate glial cells. Molecular Neurobiology, 1991, 5: 31-43
55． Talor R. Quick on the uptake: Brain glutamate transporters cloned. J NIH Res, 1993, 5: 55-60
56． Guastella J, Nelson N, Nelson H, et al. Cloning and expression of a rat brain GAB A transporter. Science, 1990, 249: 1303-1306
57． Rothsein JD, Martin LJ and Kuncl RW. Decreased glutamate transport by the brain and spinal cord in amyotrophic lateral sclerosis. N Engl J Med, 1992, 326: 1464-1468
58． Berkman MZ, Zirh TA, Berkman K., et al. Tizanidine is an effective agent in the prevention of focal cerebral ischemia in rats: An experimental study. Surg Neural, 1998, 50: 264-270
59． Talke P and Bidder PE. Effects of dexmedetomidine on hypoxia-evoked glutamate release and glutamate receptor activity in hippocampal slices. Anesthesiology, 1996, 85: 551-557
60． Wen R, Cheng T, Li Y, et al. Alpha2-adrenergic agonists induce basic fibrob-last growth factor expression in photoreceptors in vivo and ameliorate light damage. J Neurosci, 1996, 16: 5986-5992
61． Peng M, Li Y, Luo Z, et al. Alpha2-adrenergic agonists selectively activate extracellular signal-regulated kinases in Muller cells in vivo. IOVS, 1998, 39: 1721-1726
2． Osborne NN and Larsen AK. Antigens associated with specific retinal cells are affected by ischemia and caused by raised intraocular pressure: effect of glutamate antagonists. Neurochem Int,1996,29(3) : 263-270
3． Chao H-M, Osborne NN. Topically applied clonidine protects the rat retina from ischemia/reperfusion by stimulating a 2-adrenoceptors and not by an action on imidazoline receptors and not by an action on imidazoline receptors. Brain Research, 2001,904: 126-136
4． Cao W, Drumheller A, Zaharia M, et al. Effects of experimentally induced ischemia on dopamine metabolism in rabbit retina. IOVS,1993, 34(11) : 3140-3146
5． 李凤鸣《眼科全书》，人民卫生出版社，1996:334-387
6． Watkins JC. Excitatory amino acids in kainic acid as a tool in neurobiology (E.G.McGeer, J.W.Ohiey, and P.L.McGeer.Eds), pp. 37-69． Raven press, New York.
7． Watkins JC, and Evans RH. Excitatory amino acid transmitters. Annu Rev Phannacol Toxico u mol/L, 1981,21: 165-204
8． Olney JW.O.L.HO and Khee V. Cytotoxic effects of acidic and sulphur-con taining amino acids on the infant mouse central nervous system. Exp Brain Res, 1971,14: 61-76．
9． Monaghan DT, Bridges RJ, Cotman CW. The excitation amino acid receptors: their classes, pharmacology, and distinct properties in the function of the central nervous system. Ann Rev Pharmacol Toxicol,1989,29: 365-402
10 Farooqui AA, Horrocks LA. Excitatory amino acid receptors, neural membrane phospholipid metabolism and neurological disorders. Brain Res Brain Res Rev. 1991,16:171-191
11． Bamstable CT. Glutamate and GABA in the retinal circuity. Curr Opin Neurobiol, 1993,13: 520-525
12． Perlman JI, McCole SM, Pulluru P, et al. Disturbances in the distribution of neurotransmitters in the rat retina after ischemia. Curr Eye Res, 1996, 15(6) : 589-596．
13． Lam TT, Siew E, Chu R, Tso MOM. Ameliorative effect of MK-801 on retinal ischemia. J Ocul Pharmacol Ther, 1977, 13: 129-137
14． Yoon YH, Marmor MF. Dextromethorphan protects retina against ischemic injury in vivo. Arch Ophthalmol, 1989, 107: 409-411
15． Osborne NN, Schwartz M, Pergande G. Protection of rabbit retina against ischemia injury by flupirtine. IOVS, 1996, 37: 274-280
16． Osbome NN. Memantine reduces alterations to the mammalian retina in situ ,induced by ischemia. Vis Neurosci, 1999, 16: 45-52
17． Lagreze WA, KnOrle R, Bach M, et al. Memantine is neuroprotective in a rat model of pressure-induced retinal ischemia. IOVS, 1998,39(16) : 1063-1067
18． Laudenbach V, Mantz J, Lagercrantz H, et al.Effects of a 2-adrenoceptor agonists on perinatal excitotoxic brain injury. Anesthesiology, 2002, 96: 134-141
19． Donello JE, Padillo EV, Webster ML, et al. a 2-adrenoceptor agonists inhibit vitreal glutamate and aspartate accumulation and presence retinal function after transient ischemia. J Pharmacol Exp Ther, 2001,296(1) : 216-223
20． 周跃华 缺血性视网膜损伤机制的研究《国外医学 眼科学分册》，1999, 23 (2) : 105-109
21． Roth S, Park SS, Sikorski CW, et al. Concentrations of adenosine and its metabolites in the rat retina/choroid during reperfusion after ischemia. Curr Eye Res, 1997, 16: 875-885
22． Osbome NN, Larsen A, and Barnett NL. Influence of excitatory amino acids and ischemia on rat retinal choline acetyltransferase containing cells. IOVS, 1995,36(8) : 1692-1700
23． Mosinger JL, Price MT, Bai HY, et al. Blockade of both NMD A and non-NMDA receptors is required for optimal protection against ischemic neuron-al degeneration in the in vivo adult mammalian retina. Exp Neuro, 1991,113: 10-17
24． Shay J, Ames A. Retina subjected to components of ischemia in vitro, selective vulnerability and minimum lethal exposure of neurons and glia to oxygen and/or glucose deprivation and to loss of exchange with incubating medium. Arch Neurol, 1976,13: 715-721
25． Hoskins HD, Kass MA. Becher-Shaffer's diagnosis and therapy of the glaucomas, 6th edn. Mosby, st.Louis, pp 178-186
26． Quigley HA, Green WR. The histology of human glaucoma cupping and optic nerve damage. Clinic ophthalmologic correlation in 21 eyes. Ophthalmology, 1979, 86: 1803-1827
27． Louzada-Junior P, Dias JJ, Santos WF, et al. Glutamate release in experimental ischemia of the retina: an approach using microdialysis. J Neurochem, 1992, 59(1) : 358-363
28． Johnson NF, Foulds WS. The effects of total acute ischemia on the structure of the rabbit retina. Exp Eye Res, 1978, 27(1) : 45-59
29． Szabo ME, Droy-Lefaix MT, Doly M, et al. Ischemia and reperfusion-induced histologic changes in the rat retina. IOVS, 1991,32: 1471-1478
30． Takahashi K, Lam TT, Edward DP, et al. Protective effects of flunarizine on ischemic injury in the rat retina. Arch Ophthalmol, 1992, 110: 862-870
31． Lafuente MP, Villegas-Perez, Sobrado-Calvo P, et al. Neuoprotective effects of a 2-selective adrenergic agonists against ischemia-induced retinal ganglio-n cell death. IOVS, 2001, 42(9) : 2075-2084
32． Holopigian K. Evidence for photoreceptor changes in patients with DR.IOVS, 1997, 38(11) : 2355-65
33． Nayak.MS and Marmor MF. Post-ischemia ERG recovery is influenced by temperature. Curr Eye Res, 1995, 14: 81-85
34． Miyazaki M, Nazarali AJ, Boisvet DP, et al. Inhibition of ischemia-induced brain catecholamine attentions by clonidine. Brain Research, 1989, 22: 207-211
35． Zhang X, Kindel GH, Wulfert E, et al. Effects of immobilization stress on hi-ppocampal monoamine release: Modification by mivazerol,a new a 2-adreno ceptor agonist. Neuropharmacology, 1995, 34(12) : 1667-1672
36． Hoffman WE, Kochs E, Werner C, et al. Dexmedetomidine improve neurologic outcome from incomplete ischemia in the rat. Anesthesiology, 1991, 75: 328-332
37． Maier C, Steinberg GK, Sun GH, et al. Neuroprotection by the a 2-adrenoce ptor agonist dexmedetomidine. Anesthesiology, 1993,79: 306-312
38． Halonen T, Kotti T, Tuunanen J, et al. a 2-adrenoceptor agonist, dexmedetomidine, protects against kainic acid-induced convulsions and neuronal damage. Brain Res, 1995, 693: 217-224
39． Kuhmonen J, Pokorny J, Miettinen R, et al. Neuroprotective effects of dexm-ed etomidine in the gerbil hippocampus after transient global ischemia. Anesthesiology, 1997, 87: 371-377
40． Engelhard K, Werner C, Kaspar O, et al. Effects of the alpha2-adrenoceptor agonist dexmedetomidine on cerebral neurotransimitter concentations during cerebral ischemia in rats. Anesthesiology, 2002,96(2) : 450-457
41． 李凤鸣《眼科全书》，人民卫生出版社，1996:1841-1844
42． Miller RF, Slaughter MM. Excitatory amino-acid receptors in the vertebrate retina. In retinal transmitters and modulators: models of the brain. CRC Press: Boca Raton, 1985,2: 123
43． Bloomfield SA, Dowlling JE. Roles of aspartate and glutamate in synaptic transmission in rabbit retina: I .Outer plexiform layer. J Neurophysiol, 1995, 53: 699-713．
44． Olney JW. The toxic effects of glutamate and related compounds in the retina and brain. Retina, 1982, 2: 341-359
45． Ulshafer RJ, Sherry DM, Dawson R Jr, et al. Excitatory amino acid involvement in retinal degeneration. Brain Res, 1990, 531: 350-354
46． Vorwerk CK, Lipton SA, Zurakowski D, et al. Chronic low-dose glutamate is toxic to retinal ganglion cells,Toxcity blocked by memantine. IOVS, 1996, 37: 1618-1624
47． Frandsen A, Schousboe A. Mobilization of dantrolene-sensitive intracellular calcium pools is involved in the cytotoxicity induced by quisqualate and N-methyl-D-aspartate but not by 2-amino-3-(3-hydroxy-5-methylisoxazol-yl) protionate and kainate in cultured cerebral cortical neurons. Proc Natl Acad Sci, 1992, 89(7) : 2590-2594
48． Schwartz M, Belkin M, Yoles E, et al. Potential treatment modalities for glau-comatous neuropathy:neuroprotection and neuroregeneration. J Glaucoma, 1996, 5: 427-432
49． Marc RE, Liu W-L S, Kalloniatis M, et al. Patterns of glutamate immunoreac-tivity in the goldfish retina. J Neurosci, 1990,10: 4006-4034
50． Erecinsk M, Silver LA. Metabolism and role of glutamate in mammalian brain. Prog Neurobiol, 1990,35: 245-96
51． Hansen AJ and Nedergaard M. Brain ion hoeostasis in cerebral ischemia. Neurochem Pathol, 1988,9: 195-209
52． Billups B, Attwell D. Modulation of non-vesicular glutamate release by pH. Nature, 1996,379: cells isolated from the salamander(Amblystoma)retina. J Physiol 1991;436: 169-193
53． Barbour B, Brew H and Attwell D. Electrogenic uptake of glutamate and aspartate into glial cells isolated from the salamander(Amblystoma)retina. J Physiol 1991;436:169-193
54． Von Blankenfeld G and Kettenmann H. Glutamate and GABA receptors in vertebrate glial cells. Molecular Neurobiology, 1991, 5: 31-43
55． Talor R. Quick on the uptake: Brain glutamate transporters cloned. J NIH Res, 1993, 5: 55-60
56． Guastella J, Nelson N, Nelson H, et al. Cloning and expression of a rat brain GAB A transporter. Science, 1990, 249: 1303-1306
57． Rothsein JD, Martin LJ and Kuncl RW. Decreased glutamate transport by the brain and spinal cord in amyotrophic lateral sclerosis. N Engl J Med, 1992, 326: 1464-1468
58． Berkman MZ, Zirh TA, Berkman K., et al. Tizanidine is an effective agent in the prevention of focal cerebral ischemia in rats: An experimental study. Surg Neural, 1998, 50: 264-270
59． Talke P and Bidder PE. Effects of dexmedetomidine on hypoxia-evoked glutamate release and glutamate receptor activity in hippocampal slices. Anesthesiology, 1996, 85: 551-557
60． Wen R, Cheng T, Li Y, et al. Alpha2-adrenergic agonists induce basic fibrob-last growth factor expression in photoreceptors in vivo and ameliorate light damage. J Neurosci, 1996, 16: 5986-5992
61． Peng M, Li Y, Luo Z, et al. Alpha2-adrenergic agonists selectively activate extracellular signal-regulated kinases in Muller cells in vivo. IOVS, 1998, 39: 1721-1726