实验性大鼠颅脑损伤后GFAP、SOD和NO的变化研究
摘要
本实验采用免疫组织化学技术并结合一般病理形态学观察及生物化学方法，研究大鼠实验性颅脑损伤后脑组织胶质纤维酸性蛋白(glial fibriliary acidic protein，GFAP)与血清中超氧化物歧化酶(superoxide dismutase，SOD)、一氧化氮(nitric oxide，NO)的表达变化与损伤时间的关系。实验组用针刺法造成大鼠右顶叶脑挫伤后，按30min，1h，3h，6h，12h，1d，3d，7d，14d，21d不同时间分批处死动物并立即取出全脑，同时颈静脉取血2ml，正常对照组不造成颅脑损伤，直接处死动物后立即取材。将脑组织以挫伤灶为中心，旁开1mm行大脑全冠状切面取材，常规石蜡包埋，4μm切片，作免疫组化GFAP染色观察，同时行HE染色对照，采用计算机彩色图像分析技术对染色结果作定量测定；将所取静脉血离心，测定血清中SOD和NO的变化情况。
结果表明：
1．伤后30min即可见GFAP阳性细胞散在于损伤区周围，但突起较短，染色较浅，伤后3d组在损伤区周围GFAP阳性细胞增多，胞体增大，染色加深，伤后7d组阳性细胞数量减少，着色变浅，突起变短，21d时受伤组与正常对照组基本看不出明显差别。
2．分析受伤侧与非受伤侧各时间段之间GFAP阳性细胞平均光密度和阳性率的差别，各时间段与3d组均有明显的差别，而双侧大脑各时间段除3d组外均与正常对照组无显著性差别。
3．所有观察对象双侧大脑均可见GFAP阳性表达，且各时间段受伤侧与非受伤侧之间无显著性差别。
4．SOD活力在伤后30min即开始下降，至7d时降至最低，然后逐渐上升，至21d时基本恢复正常。
5．NO浓度在伤后3h降至最低，然后上升，至7d时升至最高，然后下降，21d时基本恢复正常。
从以上结果分析表明，脑挫伤后GFAP及血清中SOD活力和NO浓度的变化有一定的时间规律性，可用于颅脑损伤的时间推断，且三者存在一定的相关性，联合应用更能提高推测的准确性。
In this study, immunohistochemical method was performed to observe the change of glial fibriliary acidic protein(GFAP) staining in the brain of rat after experimental brain injury, at the same time, we also observed the transformation of blood SOD and NO levels of the experimental models. The right parietal cortex contusion was made with stab wound and the rat was killed at various survival time( 30min, Ih, 3h, 6h, 12h,ld, 3d, 7d, 14d, 21d). Paraffin sections(4 μ m) were cut coronally of the whole brain and stained with antibody of GFAP, the hemotoxylin eosin(HE) stain was done in the mean time. The staining results was measured quantitatively with computer imaging analysis system. The SOD and NO of blood from jugular vein after centrifuging were also measured.
The results demonstrated that:
1. At 30min after injury, GFAP positive astrocytes around the wound were already deteched, but tubers were short and staining was light; at 3d after injury, the positive astrocytes were increased, the bodies were bigger and the staining was heavier than before; at 7d after injury, the amount, the tuber, the staining were decreased, no obvious difference was deteched between injuried and normal rats till 21d after injury.
2. The difference of every time group of GFAP positive cells in both sides of brain was analysed, at 3d after injury, the difference was distinct, at the other survival time after injury, there was no obvious difference with the normal control group.
3. The GFAP positive cells were observed in the two sides of the brain and no obvious difference between injuried and non-injuried sides was deteched.
4. At 30min after injury, the activity of SOD decreased, came to the lowest point at the time of 7d, then increased, at 21 days after injury, there was no obvious difference with the normal control.
5. The density of NO came to lowest at the time of 3h, then increased, came to peak at 7 days; at 21 days after injury, there was no obvious difference with the normal control.
The results of this study indicate there is a rule that the expression of GFAP positive
staining changes and the transformation of SOD and NO of serum with prolonging survival time after brain injury, which will be of great value in estimation of brain trauma time. Especially, the three is relative to each other, which may will promote the accuracy of estimation if we apply the three in the same time.
结果表明：
1．伤后30min即可见GFAP阳性细胞散在于损伤区周围，但突起较短，染色较浅，伤后3d组在损伤区周围GFAP阳性细胞增多，胞体增大，染色加深，伤后7d组阳性细胞数量减少，着色变浅，突起变短，21d时受伤组与正常对照组基本看不出明显差别。
2．分析受伤侧与非受伤侧各时间段之间GFAP阳性细胞平均光密度和阳性率的差别，各时间段与3d组均有明显的差别，而双侧大脑各时间段除3d组外均与正常对照组无显著性差别。
3．所有观察对象双侧大脑均可见GFAP阳性表达，且各时间段受伤侧与非受伤侧之间无显著性差别。
4．SOD活力在伤后30min即开始下降，至7d时降至最低，然后逐渐上升，至21d时基本恢复正常。
5．NO浓度在伤后3h降至最低，然后上升，至7d时升至最高，然后下降，21d时基本恢复正常。
从以上结果分析表明，脑挫伤后GFAP及血清中SOD活力和NO浓度的变化有一定的时间规律性，可用于颅脑损伤的时间推断，且三者存在一定的相关性，联合应用更能提高推测的准确性。
In this study, immunohistochemical method was performed to observe the change of glial fibriliary acidic protein(GFAP) staining in the brain of rat after experimental brain injury, at the same time, we also observed the transformation of blood SOD and NO levels of the experimental models. The right parietal cortex contusion was made with stab wound and the rat was killed at various survival time( 30min, Ih, 3h, 6h, 12h,ld, 3d, 7d, 14d, 21d). Paraffin sections(4 μ m) were cut coronally of the whole brain and stained with antibody of GFAP, the hemotoxylin eosin(HE) stain was done in the mean time. The staining results was measured quantitatively with computer imaging analysis system. The SOD and NO of blood from jugular vein after centrifuging were also measured.
The results demonstrated that:
1. At 30min after injury, GFAP positive astrocytes around the wound were already deteched, but tubers were short and staining was light; at 3d after injury, the positive astrocytes were increased, the bodies were bigger and the staining was heavier than before; at 7d after injury, the amount, the tuber, the staining were decreased, no obvious difference was deteched between injuried and normal rats till 21d after injury.
2. The difference of every time group of GFAP positive cells in both sides of brain was analysed, at 3d after injury, the difference was distinct, at the other survival time after injury, there was no obvious difference with the normal control group.
3. The GFAP positive cells were observed in the two sides of the brain and no obvious difference between injuried and non-injuried sides was deteched.
4. At 30min after injury, the activity of SOD decreased, came to the lowest point at the time of 7d, then increased, at 21 days after injury, there was no obvious difference with the normal control.
5. The density of NO came to lowest at the time of 3h, then increased, came to peak at 7 days; at 21 days after injury, there was no obvious difference with the normal control.
The results of this study indicate there is a rule that the expression of GFAP positive
staining changes and the transformation of SOD and NO of serum with prolonging survival time after brain injury, which will be of great value in estimation of brain trauma time. Especially, the three is relative to each other, which may will promote the accuracy of estimation if we apply the three in the same time.
引文
[1]．冯家笙、徐慧君、武义鸣等．中枢神经系统损伤后不同时期星形胶质细胞的变化．解剖学报[J]，1996，27(1)
[2]．朱长庚．星形胶质细胞．解剖学报[J]，1990，21(4)
[3]. Okimura Y, Tanno H, Fududa K, et al. Reactive astrocytes in acute stage after experimental brain injury. :relationship to extravasated plasma protein and expression of heat shock protein. J Neurotrauma[J], 1996,13(7): 385
[4]. Hozumi I, Chiu FC, Norton WT. Biochemical and immunocytochemical changes in glial fibriliary acidic protein after stab wounds. Brain Res[J], 1990,524:64
[5]. Carsten Culmsee, Ralf K Stumm, Martin K-H Schafer, et al. Clenbuterol induces growth factor mRNA, activates astrocytes, and protects rat brain tissue against ischemic damage. European Journal of Pharmacology[J], 1999,379: 33
[6]．邓平、徐小虎、祝家镇．大白鼠脑干损伤后脑组织中GFAP免疫组化改变的初步研究．法医学杂志[J]，1996，12(3)
[7]. Pasquale A. Cancilla,M.D., James Bready, B.A., Judith Berliner, Ph.D., et al. Expression of mRNA for glial fibriliary acidic protein after experimental cerebral injury. Journal of Neuropathology and Experimental Neurology[J], 1992,51,3: 560
[8]．邓平、徐小虎、祝家镇．从胶质原纤维酸性蛋白阳性细胞数推断脑干损伤时间．中山医科大学学报[J]，1997，18(4)
[9]. Holmin S, Schalling M, Hojeberg B, et al. Delayed cytokine expression in rat brain following experimental contusion. J Neurosurg[J], 1997,86(3):493
[10]．王克万、杨志焕、王正国等．大鼠颅脑损伤后星形细胞早期反应特点。创伤外科杂志[J]，1999，1(3)
[11]．李学君，孙凤艳．羟自由基和脑缺血[J]．中国药理学通报，1998，14(10)
[12] Tkeda. Oxygen-free radicals in fraumatic brain oedema, Neuro-Res, 1989,11:213
[13]. Jonathan W, Graham M. Management of severe head injury. Contemp Neurosurg,
1996,18(3):1
[14]. Dawson DA. Inhibition of nitric oxide sythesis does not reduce infract volume in rat model of focal cerebral ischacmia. [J]. Neuro Sci lett, 1992,142(2) 151-154
[15]. Lipton SA, Choi YB, Pan. ZH, et al. A redox-based mechanism for the neuroprotective and neuroplest-ructive effects of nitric and related nitrosocompound [J] Nature, 1993, 364: 626-632
[16]．段亚清、王晓峰、李占国等．大鼠脑震荡海马区SOD、NO含量的变化．白求恩医科大学学报，1995，21(3)
[17]．剡海宇、徐苏、张能斌等．颅脑损伤患者早期血浆心钠素精氨酸加压素超氧化物歧化酶和内皮素含量变化及其临床意义．中国危重病急救医学，1998，10(4)
[18] 胡侦明，劳汉昌，张宝华等．脊髓损伤早期三七总皂甙抗氧自由基作用的实验研究．中国脊柱脊髓杂志，1996，6(4)：164
[19] 孙树清，吴中学，张友平等．脑损伤后脑组织自由基变化的实验研究．首都医科大学学报，2002，23(3)
[20] Niwa K, Lindawer V, Villringer A. Blockde of nitric oxide synthesis in rats strongly attenuates the CBF response to exreacellular acidosis. J Cereb Blood Flow Metab,1993,13: 535-539
[21] 刘杰波．炎症因子诱导星形胶质细胞凋亡及机制探讨．中国当代儿科杂志，2003，5(4)
[22] 李红丽，蔡文琴，邓晓林等．大鼠不同类型脑损伤后NO表达变化的比较．第三军医大学学报，2000，22(7)
[23] 范有明，高钰琦，张国兵等．缺氧对大鼠大脑皮层星形胶质细胞iNOSmRNA表达的影响．中国应用生理学杂志，2003，19(1)
[24] 吕晓红，王岩，李效宁等．急性脑梗塞病人血浆中一氧化氮与相关因素关系的研究．白求恩医科大学学报，2000，26(1)
[25] 赵宝路、陈惟昌．生物化学与生物物理学进展．1993，20：409-412
[26] 于如同，官鹏，高立达等．氧化应激对体外神经元一氧化氮释放量的影响．中国病理生理杂志，2001，17(5)：469-470
[27] Dewitt DS, Smith TG, Deyo DJ, et al. Posttraumatic hypofusion in rats is prevented or reversed by treatment with L-arginine or superoxide dismutase. J Cereb Blood Flow Metab, 1995,15:s715-s720
[28] Bolanos JP, Penchem S, Heales SJR, et al. NO mediated inhibition of the mitochondrial respiratory chain in cultured astrocytes. J Neurochemistry, 1994,63:910-916
[29] Rosengren L, Wikkelso C, Hagberg L. Asensitive ELISA for glial acidic protein: application in CSF of adults.[J] J Neurosci Methods. 1994,51: 197-204
[30] Wieneke J.A, Herman P.M, Henk Nijzing. Measurement of glial fibrillary acidic protein in blood: an analytical method. [J] Clinica Chimica Acta. 2002,326: 151-154
[31] 方燕南、黄如训．星形细胞及胶质纤维酸性蛋白在脑梗死中的作用．国外医学脑血管疾病分册[J]，2000，(6)，12
[32] Petito CK, Chung MC, Verkhovsky LM, et al. Brain glutamine synthetase increase following cerebral ischemia in the rat. Brain Res[J], 1992,569: 275-280
[33] 杨忠、李红丽、蔡文琴等．两种不同脑损伤后星形胶质细胞反应性变化的比较研究．第三军医大学学报[J]，2001，23，9
[34] Hill SJ, Barbarese E, McIntosh TK. Regional heterogeneity in the response of astrocytes following traumatic brain injury in the adult rat.[J]. J Neuropathol Exp Neurol [J],1996, 55(12): 1221-1229
[35] Pulsinelli WA, Buchan AM. The four-vessel occlusion rat model: method for complete occlusion of vertebral arteries and control of collateral circulation[J]. Stroke[J], 1988(19): 913-914
[2]．朱长庚．星形胶质细胞．解剖学报[J]，1990，21(4)
[3]. Okimura Y, Tanno H, Fududa K, et al. Reactive astrocytes in acute stage after experimental brain injury. :relationship to extravasated plasma protein and expression of heat shock protein. J Neurotrauma[J], 1996,13(7): 385
[4]. Hozumi I, Chiu FC, Norton WT. Biochemical and immunocytochemical changes in glial fibriliary acidic protein after stab wounds. Brain Res[J], 1990,524:64
[5]. Carsten Culmsee, Ralf K Stumm, Martin K-H Schafer, et al. Clenbuterol induces growth factor mRNA, activates astrocytes, and protects rat brain tissue against ischemic damage. European Journal of Pharmacology[J], 1999,379: 33
[6]．邓平、徐小虎、祝家镇．大白鼠脑干损伤后脑组织中GFAP免疫组化改变的初步研究．法医学杂志[J]，1996，12(3)
[7]. Pasquale A. Cancilla,M.D., James Bready, B.A., Judith Berliner, Ph.D., et al. Expression of mRNA for glial fibriliary acidic protein after experimental cerebral injury. Journal of Neuropathology and Experimental Neurology[J], 1992,51,3: 560
[8]．邓平、徐小虎、祝家镇．从胶质原纤维酸性蛋白阳性细胞数推断脑干损伤时间．中山医科大学学报[J]，1997，18(4)
[9]. Holmin S, Schalling M, Hojeberg B, et al. Delayed cytokine expression in rat brain following experimental contusion. J Neurosurg[J], 1997,86(3):493
[10]．王克万、杨志焕、王正国等．大鼠颅脑损伤后星形细胞早期反应特点。创伤外科杂志[J]，1999，1(3)
[11]．李学君，孙凤艳．羟自由基和脑缺血[J]．中国药理学通报，1998，14(10)
[12] Tkeda. Oxygen-free radicals in fraumatic brain oedema, Neuro-Res, 1989,11:213
[13]. Jonathan W, Graham M. Management of severe head injury. Contemp Neurosurg,
1996,18(3):1
[14]. Dawson DA. Inhibition of nitric oxide sythesis does not reduce infract volume in rat model of focal cerebral ischacmia. [J]. Neuro Sci lett, 1992,142(2) 151-154
[15]. Lipton SA, Choi YB, Pan. ZH, et al. A redox-based mechanism for the neuroprotective and neuroplest-ructive effects of nitric and related nitrosocompound [J] Nature, 1993, 364: 626-632
[16]．段亚清、王晓峰、李占国等．大鼠脑震荡海马区SOD、NO含量的变化．白求恩医科大学学报，1995，21(3)
[17]．剡海宇、徐苏、张能斌等．颅脑损伤患者早期血浆心钠素精氨酸加压素超氧化物歧化酶和内皮素含量变化及其临床意义．中国危重病急救医学，1998，10(4)
[18] 胡侦明，劳汉昌，张宝华等．脊髓损伤早期三七总皂甙抗氧自由基作用的实验研究．中国脊柱脊髓杂志，1996，6(4)：164
[19] 孙树清，吴中学，张友平等．脑损伤后脑组织自由基变化的实验研究．首都医科大学学报，2002，23(3)
[20] Niwa K, Lindawer V, Villringer A. Blockde of nitric oxide synthesis in rats strongly attenuates the CBF response to exreacellular acidosis. J Cereb Blood Flow Metab,1993,13: 535-539
[21] 刘杰波．炎症因子诱导星形胶质细胞凋亡及机制探讨．中国当代儿科杂志，2003，5(4)
[22] 李红丽，蔡文琴，邓晓林等．大鼠不同类型脑损伤后NO表达变化的比较．第三军医大学学报，2000，22(7)
[23] 范有明，高钰琦，张国兵等．缺氧对大鼠大脑皮层星形胶质细胞iNOSmRNA表达的影响．中国应用生理学杂志，2003，19(1)
[24] 吕晓红，王岩，李效宁等．急性脑梗塞病人血浆中一氧化氮与相关因素关系的研究．白求恩医科大学学报，2000，26(1)
[25] 赵宝路、陈惟昌．生物化学与生物物理学进展．1993，20：409-412
[26] 于如同，官鹏，高立达等．氧化应激对体外神经元一氧化氮释放量的影响．中国病理生理杂志，2001，17(5)：469-470
[27] Dewitt DS, Smith TG, Deyo DJ, et al. Posttraumatic hypofusion in rats is prevented or reversed by treatment with L-arginine or superoxide dismutase. J Cereb Blood Flow Metab, 1995,15:s715-s720
[28] Bolanos JP, Penchem S, Heales SJR, et al. NO mediated inhibition of the mitochondrial respiratory chain in cultured astrocytes. J Neurochemistry, 1994,63:910-916
[29] Rosengren L, Wikkelso C, Hagberg L. Asensitive ELISA for glial acidic protein: application in CSF of adults.[J] J Neurosci Methods. 1994,51: 197-204
[30] Wieneke J.A, Herman P.M, Henk Nijzing. Measurement of glial fibrillary acidic protein in blood: an analytical method. [J] Clinica Chimica Acta. 2002,326: 151-154
[31] 方燕南、黄如训．星形细胞及胶质纤维酸性蛋白在脑梗死中的作用．国外医学脑血管疾病分册[J]，2000，(6)，12
[32] Petito CK, Chung MC, Verkhovsky LM, et al. Brain glutamine synthetase increase following cerebral ischemia in the rat. Brain Res[J], 1992,569: 275-280
[33] 杨忠、李红丽、蔡文琴等．两种不同脑损伤后星形胶质细胞反应性变化的比较研究．第三军医大学学报[J]，2001，23，9
[34] Hill SJ, Barbarese E, McIntosh TK. Regional heterogeneity in the response of astrocytes following traumatic brain injury in the adult rat.[J]. J Neuropathol Exp Neurol [J],1996, 55(12): 1221-1229
[35] Pulsinelli WA, Buchan AM. The four-vessel occlusion rat model: method for complete occlusion of vertebral arteries and control of collateral circulation[J]. Stroke[J], 1988(19): 913-914