大鼠高眼压视网膜缺血再灌注损伤实验研究及神经营养素受体p75的表达与意义
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摘要
目的：建立新型的大鼠高眼压视网膜缺血再灌注模型，通过光镜及电镜观察不同损伤的组织病理改变，复方丹参对视网膜缺血再灌注(RIR)的防护作用以及视网膜神经节细胞及内、外核层细胞凋亡的存在，并借助于免疫组织化学方法，初步研究p75在RIR损伤中的表达及意义。方法：SD大鼠74只，随机分为对照角膜损伤组，缺血组，缺血再灌注组，复方丹参防护组和生理盐水防护对照组。建立15kPa，90min高眼压视网膜缺血模型，再灌注1，4，12，24，72h后处死，普通光镜，透射电镜观察，免疫组织化学检测。结果：缺血再灌注组出现光镜和电镜下的病理改变，包括视网膜水肿，神经节细胞空泡变性，视网膜外节盘变形，内节线粒体轻度扩张，部分脱嵴，神经节细胞膜皱缩，染色质凝成块状，线粒体空泡化，外节盘溶解等。随着再灌注时间的延长，视网膜组织形态及超微结构损害加重。角膜损伤对照组无改变，复方丹参防护组损伤较生理盐水对照组明显减轻。缺血再灌注损伤后p75阳性表达，且随损伤的加重而增加。结论：高眼压视网膜缺血再灌注可造成视网膜结构严重损害，随再灌注时间延长而加重。复方丹参对缺血再灌注损伤有保护作用；在RIR模型中存在神经节细胞，内、外核层细胞凋亡；缺血再灌注损伤后视网膜内核层及神经节细胞出现p75阳性，表达随损伤的加重而增多，与视网膜细胞凋亡有关联。这些结果有助于视网膜缺血性损伤治疗方法的制定。
Objective : To establish a new animal model of ocular hypertension and observe the development process of retinal injury in ischemia - reperfusion state and investigate the protective effect of compound injection salvia in retinal ischemia reperfusion (RIR).To make sure apoptosis occur in retina and detect the expressin of the low-affinity neurotrophin receptor p75 in RIR .Method : 74 Sprague-Dawley rats were devided into five groups at random , containing cornia damaged group, ischemia group,ischemia-reperfusion group, salvia miltiorrhiza protect group and normal saline control group.60 of 60 rats subjected to 90 minutes of pressure-induced ischemia, then fixed for light and electron microscopy with reperfusion after 1,4,12 hours, and 1 or 3 days from the time ocular ischemia was relaxed. p75 was examined in the retina by immunohistochemical at light level, result : Neuroganglion fibrous layer showed vacuolation degeneration when ocular hypertension was maintained to 90 min .the ganglion cells of retina sho
wed that the karyltheca crimpled the karyotin agglomerated.the mitochondrion was vacuocated and the membranous disc of outer segment was damaged .The longer had ischemia-reperfusion continued, the more serious was the reperfusion injury .The histopathologic damage of retina in compound injection salvia group were not severe than that in the control group .The p75 was detected in the retinal ganglion cells , inner and out nuclear layer. During the time after the reperfusion started, especially after more than 12 hours, apoptotic signs became obvious and extended from the inner to the outer retina. These apoptotic findings could be seen with electron microscopy. By 3 days after the reperfusion, necrotic cells in the ganglion cell layer, and the inner and outer nuclear layer became more prominent than
apoptotic cells..Conclusion :RIR can make serious damage to retinal histopathology and ultrastructure. . mainline of compound injection salvia miltiorrhiza may be effected on protection from retinal damage induced by RIR. The low affinity neurotrophin receptor p75 seems to be involved in apoptosis after RIR . These results may provide a baseline for therapeutic strategy and the prognosis of ischemia-reperfusion injury in the retina.
Objective : To establish a new animal model of ocular hypertension and observe the development process of retinal injury in ischemia - reperfusion state and investigate the protective effect of compound injection salvia in retinal ischemia reperfusion (RIR).To make sure apoptosis occur in retina and detect the expressin of the low-affinity neurotrophin receptor p75 in RIR .Method : 74 Sprague-Dawley rats were devided into five groups at random , containing cornia damaged group, ischemia group,ischemia-reperfusion group, salvia miltiorrhiza protect group and normal saline control group.60 of 60 rats subjected to 90 minutes of pressure-induced ischemia, then fixed for light and electron microscopy with reperfusion after 1,4,12 hours, and 1 or 3 days from the time ocular ischemia was relaxed. p75 was examined in the retina by immunohistochemical at light level, result : Neuroganglion fibrous layer showed vacuolation degeneration when ocular hypertension was maintained to 90 min .the ganglion cells of retina sho
wed that the karyltheca crimpled the karyotin agglomerated.the mitochondrion was vacuocated and the membranous disc of outer segment was damaged .The longer had ischemia-reperfusion continued, the more serious was the reperfusion injury .The histopathologic damage of retina in compound injection salvia group were not severe than that in the control group .The p75 was detected in the retinal ganglion cells , inner and out nuclear layer. During the time after the reperfusion started, especially after more than 12 hours, apoptotic signs became obvious and extended from the inner to the outer retina. These apoptotic findings could be seen with electron microscopy. By 3 days after the reperfusion, necrotic cells in the ganglion cell layer, and the inner and outer nuclear layer became more prominent than
apoptotic cells..Conclusion :RIR can make serious damage to retinal histopathology and ultrastructure. . mainline of compound injection salvia miltiorrhiza may be effected on protection from retinal damage induced by RIR. The low affinity neurotrophin receptor p75 seems to be involved in apoptosis after RIR . These results may provide a baseline for therapeutic strategy and the prognosis of ischemia-reperfusion injury in the retina.
引文
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4. Kijlstra A. The role of cytokines in ocular inflammation [J]. Br J Ophthalmol,1994 ; 78(3) : 885-7
5. Babu JS , Kanangat S, Rouse BT. T cell cytokine mRNA expression during he course of the immunopathologic ocular disease herpetic stromal keratitis [J]. J Immunol, 1995 ; 154(12) : 4822-9
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8. Bonomi L, Saettone MS,Bucci G. Methods to produce ocular hypertension in animals [J]. Ophthalmic Drug Deliv,1987 ; 11 (1) : 107-16
9. Kuang P G, Wu Wp, Liu Jx, et al. The effect of radix salviae miltiorrizae on substance P in cerebral ischemia: an animal experiment study[J] J Trad Chin Med,1991,11(2):123-7
10. Zhang X S, Kuang P G, Wu Wp, et al. The effect of radix salviae miltiorrhizae on peroxidation of low density lipoprotein due to copor dichloride [J] J Trad Chin Med. 1994,14(3): 195-201
11. Kuang P G, Xiang J. Effect of radix salviae miltiorrhizae on EAA and IAA during cerebral ischemia in gerbils: a microdialysis study [J] J Trad Chin Med.1994,14(1):195-50
12. Hu B, Yip HK, So KF. Localization of p75 neurotrophin receptor in the retina of the adult SD rat: an immunocytochemical study at light and electron microscopic levels.[J] Glia 1998 Oct;24(2): 187-97
13. Hu B, Yip HK, So KF. Expression of p75 neurotrophin receptor in the injured and regenerating rat retina. [J] Neuroreport 1999 Apr 26;10(6): 1293-7
14. Tomita H, Ishiguro S, Abe T, Tamal M. Increased expression of low-affinity NGF receptor in rat retinal Muller cells after ischemia and reperfusion. [J] Cell Struct Funct 1998 Aug;23(4):201-7
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17. Rothman SM , Fulling KH, Nelson JS. Sickle cell anemia and central nervous system infarction: a neuropathological study. [J]Ann Neurol, 1986 Dec;20(6):684-90
18. Choi DW, Rothman SM. The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death. [J]Annu Rev Neurosci 1990; 13:171-82
19. Olney JW. Excitotoxic amino acids and neuropsychiatric disorders. [J]Annu Rev Pharmacol Toxicol 1990;30:47-71
20. Andine P, Jacobson I, Hagberg H. Calcium uptake evoked by electrical stimulation is enhanced postischemically and precedes delayed neuronal death in CA1 of rat
hippocampus: involvement of N-methyl-D-aspartate receptors.[J] J Cereb Blood Flow Metab 1988 Dec;8(6):799-807
21. Lodge D. The Pharmacology of Excitatory Amino Acids. Cambridge : Esevier,1991:68-74
22. Olney JW. Glutamate-induced neuronal necrosis in the infant mouse hypothalamus.An electron microscopic study. [J] J Neuropathol Exp Neurol 1971 Jan;30(1):75-90
23. Olney JW, Ho OL, Rhee V. Cytotoxic effects of acidic and sulphur containing amino acids on the infant mouse central nervous system.[J] Exp Brain Res 1971;14(1):61-76
24. Weiler R. Neurobiology of the Inner Retina. Berlin : Springer, 1989:1-4
25. Osborne NN. Progress in Retinal Research. Oxford : Pergamon Press,1990:9:399
26. Zeevalk GD, Nicklas WJ. Chemically induced hypoglycemia and anoxia:relationship to glutamate receptor-mediated toxicity in retina. [J] J Pharmacol Exp Ther 1990 Jun;253(3): 1285-92
27. Romano C, Price M, Bai HY, Olney JW. Neuroprotectants in Honghua: glucose attenuates retinal ischemic damage. [J]Invest Ophthalmol Vis Sci, 1993 Jan;34(1):72-80
28. Cao W, Zaharia M, Drumheller A, Casanova C, Lafond G, Brunette JR, Jolicoeur F.Effects of dextromethorphan on ischemia induced electroretinogram changes in rabbit. [J]Curr Eye Res 1994 Feb; 13(2):97-102
29. 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-4-yl)propionate and kalnate in cultured cerebral cortical neurons. [J]Proc Natl Acad Sci USA 1992 Apr 1 ;89(7):2590-4
30. Rothman SM. The neurotoxicity of excitatory amino acids is produced by passive chloride influx.[J] J Neurosci 1985 Jun;5(6): 1483-9
31. Olney JW, Price MT, Samson L, Labruyere. The role of specific ions in glutamate neurotoxicity.[J] Neurosci Lett 1986 Mar 28;65(1):65-71
32. Szabo ME, Droy-Lefaix MT, Doly M, Braquet.Ischaemia-and reperfiusion-induced Na+, K+, Ca2+and Mg2+shifts in rat retina: effects of two free radical scavengers,SOD and EGB 761. [J] Exp Eye Res 1992 Jul;55(1):39-45
33. Zhang H,Komano H, Fuller RS, Gandy SE, Frail DE. Proteolytic processing and secretion of human beta-amyloid precursor protein in yeast. Evidence for a yeast secretase activity.[J] J Biol Chem 1994 Nov 11 ;269(45):27799-802
34. Downey JM. Free radicals and their involvement during long-term myocardial ischemia and reperfusion. [J] Annu Rev Physiol 1990;52:487-504
35. Lucchesi BR, Mullane KM. Leukocytes and ischemia-induced myocardial injury. [J] Annu Rev Pharmacol Toxicol 1986;26:201-24
36. Halliwell B. Drug antioxidant effects. A basis for drug selection? [J]Drugs 1991 Oct;42(4):569-605
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38. Alberts B et al. Molecular Biology of the Cell, 2nd ed , New York and London:Garland Publishing Inc, 1989:61
39. Warner HR et al. Mordem Biological Theories of Aging. New York: Raven Press,1987:113
40. Emerit I et al. Antioxidants in Therapy and Preventative Medicine. New York: Plenum Press, 1990:235
41. Zhao BL, Jiang W, Hou JW, et al.Scavenging effects of salvia miltiorrhiza on free radicals and its protection for myocardial mltotchondrial membranes from ischemiareperfusion injury.[J] Biochem Mol Biol Int, 1996,38(6) : 1171
42．周武雄，钟慈声，袁伟，等。血管吻合术后过氧化氢的形成及丹参素对超氧自由基的作用[J]上海医科大学学报，1998，25(2)：115
43．杨卫东，朱鸿良，赵宝路，等。丹参的氧自由基清除作用。[J]中国药理学报，1990，6(2)：118
44．幸志强，曾旭初，易春涛，等。丹参对冠心病患者血清脂质过氧化物及超氧化物岐化酶的影响。[J]中国中西医结合杂志，1996，16(15)287
45．黄忠耀，廖崇先，陈道中。丹参对体外循环中肺内氧自由基产生的影响。[J]中国中西医结合杂志，1996，16(8)：451
46．徐罗铃，吴绮，王伯瑶，等。丹参酮对人中性粒细胞氧自由基释放的抑制效应。[J]中国病理生理杂志，1994，10(3)：635
47．张力，王孝铭，朱世军，等。外源O_2对心肌线粒体跨膜电位和质子转位的作用。[J]中国病理生理杂志，1996，12(2)：181
48．苏晓华，王孝铭，焦选茂，等。大鼠心肌缺血再灌注线粒体内膜体电子质子偶联和氧化磷酸化变化及丹参素的作用。[J]中国病理生理杂志，1996，12(2)：186
49．卢漪萍，史陈让，吴在德，等。丹参防治肝缺血再灌注期肝细胞内钙超载的实验与临床研究。[J]中华外科杂志，1996，34(2)：98
50. Smith WL, Marnett LT, Dewit DL. Prostaglandin and thromboxane biosynthesis [J] Pharmacol Ther, 1991,49(3): 153
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2. Vorwer CK, Hyman BT, Miler JW, Husain D, et al. The role of neuronal and endothelial nitric oxide sythase in retinal excitotoxiciy [J]. Invest Ophthalmol Vis Sci, 1997;389(9):2038-44
3. Hattori Y, Oka M, Kasai K. Lipopolysaccharide treament in vivo induces tissue expression of GTP cyclohydrolase I mRNA. [J] FEBSLett, 1995;368(2) : 336-8
4. Kijlstra A. The role of cytokines in ocular inflammation [J]. Br J Ophthalmol,1994 ; 78(3) : 885-7
5. Babu JS , Kanangat S, Rouse BT. T cell cytokine mRNA expression during he course of the immunopathologic ocular disease herpetic stromal keratitis [J]. J Immunol, 1995 ; 154(12) : 4822-9
6. Masanori H, Kazuaki M, Kano H et al. Roles of constitutive nitric oxide synthase in postisehemic rat retina [J]. Invest Ophthalmol Vis Sci, 1999 ; 40(2) : 450-8
7. Zhu MD, Cai FY.The effect of Salviae Miltiorrhizae Co. on the retrograde axoplasmic trnasrt in the optic nerve of rabbits with chronec IOP elevation [J].Chin J Ophthalmol,1991; 27(3) :174-8
8. Bonomi L, Saettone MS,Bucci G. Methods to produce ocular hypertension in animals [J]. Ophthalmic Drug Deliv,1987 ; 11 (1) : 107-16
9. Kuang P G, Wu Wp, Liu Jx, et al. The effect of radix salviae miltiorrizae on substance P in cerebral ischemia: an animal experiment study[J] J Trad Chin Med,1991,11(2):123-7
10. Zhang X S, Kuang P G, Wu Wp, et al. The effect of radix salviae miltiorrhizae on peroxidation of low density lipoprotein due to copor dichloride [J] J Trad Chin Med. 1994,14(3): 195-201
11. Kuang P G, Xiang J. Effect of radix salviae miltiorrhizae on EAA and IAA during cerebral ischemia in gerbils: a microdialysis study [J] J Trad Chin Med.1994,14(1):195-50
12. Hu B, Yip HK, So KF. Localization of p75 neurotrophin receptor in the retina of the adult SD rat: an immunocytochemical study at light and electron microscopic levels.[J] Glia 1998 Oct;24(2): 187-97
13. Hu B, Yip HK, So KF. Expression of p75 neurotrophin receptor in the injured and regenerating rat retina. [J] Neuroreport 1999 Apr 26;10(6): 1293-7
14. Tomita H, Ishiguro S, Abe T, Tamal M. Increased expression of low-affinity NGF receptor in rat retinal Muller cells after ischemia and reperfusion. [J] Cell Struct Funct 1998 Aug;23(4):201-7
15. Harada T, Harada C, Nakayama N, Okuyama S, Yoshida K, Kohsaka S, Matsuda H,Wada K. Modification of glial-neuronal cell interactions prevents photoreceptor apoptosis during light-induced retinal degeneration. [J] Neuron. 2000: May ; 26(2):285-6
16. Ding J, Hu B, Tang LS, Yip HK. Study of the role of the low-affinity neurotrophin receptor p75 in naturally occurring cell death during development of the rat retina.[J] Dev Neurosci 2001;23(6):390-8
17. Rothman SM , Fulling KH, Nelson JS. Sickle cell anemia and central nervous system infarction: a neuropathological study. [J]Ann Neurol, 1986 Dec;20(6):684-90
18. Choi DW, Rothman SM. The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death. [J]Annu Rev Neurosci 1990; 13:171-82
19. Olney JW. Excitotoxic amino acids and neuropsychiatric disorders. [J]Annu Rev Pharmacol Toxicol 1990;30:47-71
20. Andine P, Jacobson I, Hagberg H. Calcium uptake evoked by electrical stimulation is enhanced postischemically and precedes delayed neuronal death in CA1 of rat
hippocampus: involvement of N-methyl-D-aspartate receptors.[J] J Cereb Blood Flow Metab 1988 Dec;8(6):799-807
21. Lodge D. The Pharmacology of Excitatory Amino Acids. Cambridge : Esevier,1991:68-74
22. Olney JW. Glutamate-induced neuronal necrosis in the infant mouse hypothalamus.An electron microscopic study. [J] J Neuropathol Exp Neurol 1971 Jan;30(1):75-90
23. Olney JW, Ho OL, Rhee V. Cytotoxic effects of acidic and sulphur containing amino acids on the infant mouse central nervous system.[J] Exp Brain Res 1971;14(1):61-76
24. Weiler R. Neurobiology of the Inner Retina. Berlin : Springer, 1989:1-4
25. Osborne NN. Progress in Retinal Research. Oxford : Pergamon Press,1990:9:399
26. Zeevalk GD, Nicklas WJ. Chemically induced hypoglycemia and anoxia:relationship to glutamate receptor-mediated toxicity in retina. [J] J Pharmacol Exp Ther 1990 Jun;253(3): 1285-92
27. Romano C, Price M, Bai HY, Olney JW. Neuroprotectants in Honghua: glucose attenuates retinal ischemic damage. [J]Invest Ophthalmol Vis Sci, 1993 Jan;34(1):72-80
28. Cao W, Zaharia M, Drumheller A, Casanova C, Lafond G, Brunette JR, Jolicoeur F.Effects of dextromethorphan on ischemia induced electroretinogram changes in rabbit. [J]Curr Eye Res 1994 Feb; 13(2):97-102
29. 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-4-yl)propionate and kalnate in cultured cerebral cortical neurons. [J]Proc Natl Acad Sci USA 1992 Apr 1 ;89(7):2590-4
30. Rothman SM. The neurotoxicity of excitatory amino acids is produced by passive chloride influx.[J] J Neurosci 1985 Jun;5(6): 1483-9
31. Olney JW, Price MT, Samson L, Labruyere. The role of specific ions in glutamate neurotoxicity.[J] Neurosci Lett 1986 Mar 28;65(1):65-71
32. Szabo ME, Droy-Lefaix MT, Doly M, Braquet.Ischaemia-and reperfiusion-induced Na+, K+, Ca2+and Mg2+shifts in rat retina: effects of two free radical scavengers,SOD and EGB 761. [J] Exp Eye Res 1992 Jul;55(1):39-45
33. Zhang H,Komano H, Fuller RS, Gandy SE, Frail DE. Proteolytic processing and secretion of human beta-amyloid precursor protein in yeast. Evidence for a yeast secretase activity.[J] J Biol Chem 1994 Nov 11 ;269(45):27799-802
34. Downey JM. Free radicals and their involvement during long-term myocardial ischemia and reperfusion. [J] Annu Rev Physiol 1990;52:487-504
35. Lucchesi BR, Mullane KM. Leukocytes and ischemia-induced myocardial injury. [J] Annu Rev Pharmacol Toxicol 1986;26:201-24
36. Halliwell B. Drug antioxidant effects. A basis for drug selection? [J]Drugs 1991 Oct;42(4):569-605
37. Rice-Evans CA et al. Free Radical Damage and Its Control. Amsterdam: Elseveer,1994
38. Alberts B et al. Molecular Biology of the Cell, 2nd ed , New York and London:Garland Publishing Inc, 1989:61
39. Warner HR et al. Mordem Biological Theories of Aging. New York: Raven Press,1987:113
40. Emerit I et al. Antioxidants in Therapy and Preventative Medicine. New York: Plenum Press, 1990:235
41. Zhao BL, Jiang W, Hou JW, et al.Scavenging effects of salvia miltiorrhiza on free radicals and its protection for myocardial mltotchondrial membranes from ischemiareperfusion injury.[J] Biochem Mol Biol Int, 1996,38(6) : 1171
42．周武雄，钟慈声，袁伟，等。血管吻合术后过氧化氢的形成及丹参素对超氧自由基的作用[J]上海医科大学学报，1998，25(2)：115
43．杨卫东，朱鸿良，赵宝路，等。丹参的氧自由基清除作用。[J]中国药理学报，1990，6(2)：118
44．幸志强，曾旭初，易春涛，等。丹参对冠心病患者血清脂质过氧化物及超氧化物岐化酶的影响。[J]中国中西医结合杂志，1996，16(15)287
45．黄忠耀，廖崇先，陈道中。丹参对体外循环中肺内氧自由基产生的影响。[J]中国中西医结合杂志，1996，16(8)：451
46．徐罗铃，吴绮，王伯瑶，等。丹参酮对人中性粒细胞氧自由基释放的抑制效应。[J]中国病理生理杂志，1994，10(3)：635
47．张力，王孝铭，朱世军，等。外源O_2对心肌线粒体跨膜电位和质子转位的作用。[J]中国病理生理杂志，1996，12(2)：181
48．苏晓华，王孝铭，焦选茂，等。大鼠心肌缺血再灌注线粒体内膜体电子质子偶联和氧化磷酸化变化及丹参素的作用。[J]中国病理生理杂志，1996，12(2)：186
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