实验性脑出血急性期凝血酶与细胞骨架蛋白的关系及水蛭素的保护作用
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摘要
脑出血损伤机制是近年来研究的热点。实验表明，出血性脑损伤不仅由于血肿的占位效应及血肿对周围组织的直接破坏，继发性损伤也是出血性脑损伤的主要原因。研究表明，脑出血平均体积的血肿（人类54ml，相当于大鼠50ul血）在颅内容积缓冲的范围内，因而对颅内压或脑灌注压产生极小的影响，认为平均体积的血肿导致的脑水肿主要由于来自血块的生化物质而非容积效应。其中凝血酶在凝血级联反应中起关键作用，在脑出血后继发性损伤中的作用较为肯定，但其确切机制有待于进一步探讨。
细胞骨架是横越在细胞核和细胞质膜内侧面的一种纤维状蛋白基质，对细胞生命活动是必要的，如细胞形态的维持、运动性、胞内颗粒物质运输、有丝分裂、分泌加工过程等。细胞骨架由微管、微丝及中间纤维组成。微管的基本组成单位是微管蛋白，在体内，微管蛋白聚集成微管需要有MAP的参与。MAP促进微管蛋白聚集、抑制微管蛋白分解，是微管结构和功能的重要组成部分。其中MAP-2在脑内水平明显高于其它组织，MAP-2在神经元内的高水平表达，与树突及胞体的选择性结合及其对细胞骨架成分的调节作用均支持MAP-2作为神经元形态学调节因子的作用。因此，MAP-2结构损伤及功能障碍势必导致神经元形态及功能异常。
胶质纤维酸性蛋白（GFAP）是星形胶质细胞的特异蛋白和细胞骨架成分之一，是一种中间丝蛋白，是中间纤维的一种。损伤后星形胶质细胞被激活，反应性肥大、增生，GFAP表达增加。被激活的星形胶质细胞一方面具有保护作用，如可促进胶质瘢痕的
形成，防止再出血；在损伤区分泌多种神经营养因子，以促进损伤的修复。另一方面，过度的胶质化却可作为机械屏障妨碍髓鞘和轴索的再生，影响周围神经组织结构和功能的恢复；而且，肿胀的星形胶质细胞还可释放谷氨酸和一氧化氮，过高浓度的谷氨酸和一氧化氮具有神经毒性，是继发性脑损伤的重要原因之一；应激反应的星形胶质细胞还可以产生多种细胞因子和炎症介质，如白细胞介素-1β（IL-1β）和肿瘤坏死因子（TNF-α），引起血脑屏障开放，介导炎性反应，促进内皮细胞的坏死，故参与脑水肿的病理过程。可见，反应性星形胶质细胞增生在损伤后发挥着双刃剑的作用。
目的：本实验旨在从细胞骨架蛋白的角度进一步探讨脑出血后凝血酶在继发性脑损伤中的致病机制及其特异性抑制剂－水蛭素的保护作用。
方法：向体重为250~350g健康雄性大鼠尾壳核区（前囟前0.2mm，中线旁开4mm，垂直进针5.5mm）注入自体未抗凝动脉血50μl，制作大鼠实验性脑出血动物模型，将大鼠随机分为3组：1组：正常对照组；2组：单纯脑出血组，分别在术后6h、1d、2d、3d和7d处死动物，取脑；3组：水蛭素组（注血后5分钟向血肿局部注入10U水蛭素），分别在注药后3d和7d处死动物，取脑。上述每个时间点、每个观察指标（HE染色、脑组织含水量、免疫组化染色）各5只动物。HE染色定性观察脑出血后血肿周围组织的病理变化；脑组织含水量系定量测定脑出血后血肿周围组织水肿程度；MAP-2、GFAP免疫组化染色观察脑出血后的细胞骨架损伤。
结果：大体病理可见尾壳核区类圆形血肿，2~3d组可见血肿周围脑组织肿胀，侧脑室前角受压。光镜示从脑出血后6h开始血
肿周围组织水肿，神经细胞、胶质细胞肿胀，或细胞收缩、染色加深，细胞周围间隙变大，血管周围空隙亦变大。随脑出血后时间的延长，上述病理变化逐渐加重，3d组的组织水肿和细胞间隙空化最为明显，部分神经细胞坏死、消失。脑出血后脑组织含水量逐渐增加，从1d开始增加显著，与正常对照组相比P<0.05,3d达高峰，7d较3d组虽有所下降，但与正常对照组相比P仍然小于0.05。MAP-2、GFAP免疫组化染色结果表明：正常对照组MAP-2染色阳性细胞为神经元细胞，可见细胞轮廓清晰，突起纤细，突起较长且分枝数量多，MAP-2广泛分布于神经元突起部分，呈条索状；胞体内也有少量表达。脑出血后6h开始血肿周围MAP-2阳性细胞数目进行性减少，与正常对照组相比P<0.05，突起变形程度逐渐加重，且染色变淡，上述变化进行性加重，于3d达到最低，7d较3d组虽有所提高，但与正常对照组相比P仍然小于0.05。正常对照组GFAP阳性细胞数目少、染色浅，分布稀疏，胞体小，突起细短。脑出血后1d GFAP表达明显增强，与正常对照组相比P<0.05，出现肥大的星形胶质细胞，胞体变大，GFAP染色加深，突起增多、增粗且形状不规则，上述改变进行性加重，于7d达高峰。水蛭素组较相同时间点单纯脑出血组组织水肿和细胞坏死明显减轻；脑组织含水量降低（P<0.05）；MAP-2阳性细胞数量增多（P<0.05），变形程度减轻，染色加深；GFAP阳性细胞数目有所减少（P<0.05），细胞肥大、增生现象不明显。
结论：1. 本实验采用自体未抗凝动脉血注入法建立了可靠的实验性脑出血动物模型。2. 光镜示血肿周围的组织水肿和细胞间隙空化从脑出血后6h开始进行性加重，3d最为明显。脑出血后脑组织含水量逐渐增加，从1d开始增加显著，与正常对照组相比P<0.05，3d达高峰，7d较3d组虽有所下降，但与正常对照组相
比P仍然小于0.05。3. 脑出血后1周内，MAP-2的分解破坏或合成障碍标志着严重或致?
Intracerebral hemorrhage (ICH) is associated with high mortality and Morbidity. In addition to the mass effect and direct disruption, the secondary injury is also the main cause after ICH. An average-sized hematoma in humans (54ml) has little immediate effect on intracranial pressure or cerebral perfusion pressure because that volume is well with the volume-buffering capacity of the intracranial space. There is evidence that intracerebral blood causes brain injury through biochemical substances released from the hematoma that initiate formation of brain edema. Thrombin, released from the coagulation cascade after experimental ICH, leads to edema formation and disruption of blood-brain-barrier. The important role of thrombin after ICH is already affirmed, but its accurate mechanism is still unclear.
The structure of the cytoplasm relies on the assembly and organization of a network of fibrous components, which includes microtubules, microfilaments, and intermediate filaments. The cytoskeleton is implicated in a wide variety of functions that are essential for cellular life including mitosis, maintenance of cell shape, motility, intracellular transport of organelles, and secretary processes. Microtubules have been isolated mainly from brain, where tubulin, a major microtubular component, is present in significantly larger amounts than in other tissues. Even though tubulin can self-assemble in vitro into microtubules under appropriate conditions, the presence of MAPs is required for in vivo assembly of tubulin. MAPs promote the assembly of tubulin and suppress its disassembly. MAP-2 is
present in larger amounts in brain than in other tissues. Microtubule-associated protein-2 is found mainly in dendrites and soma of neural cells in most regions of the central nervous system. The high levels of expression of these MAP-2 in neurons, their selective associations to either axonal or dendritic compartments, and their developmental regulation support a putative role of MAP-2 as mediators of neuronal morphogenesis.
Glial fibrillary acidic protein is a marker for astrocyte as well as one of the cytoskeleton components. There are densely distributed GFAP-positive cells around the blood clot, and the number and morphological features of GFAP-positive astrocytes changed as time went on after ICH. One hand, the activated astrocytes have protective functions, such as promoting the formation of glial scar to escape from reblooding; secreting various neural protective factors to accelerate the repairation of the injury. On the other hand, excessive glial reaction will affect the restoration of peripheral tissue structure and function because impair the regeneration of myelinated and axon; moreover, they will release glutamic acid and nitrous oxide which can destroy the nervous system; the glial reaction also produce inflammation medium, such as IL-1β and TNF-α resulting in the opening of the blood brain barrier, mediating inflammation reaction, accelerating the necrosis of endothelium to participate in the pathology of brain edema.
Objective: To explore the role of thrombin in the pathological mechanism of ICH by observing the expression of MAP-2 and GFAP in the perihematomal brain regions, and the protective mechanism of
hirudin.
Methods: The rat was positioned in a stereotactic frame, rat models of ICH were made by infusion of autologous blood into the right neucleus caudatus (coordinates: 0.2mm anterior, 5.5mm ventral, and 4mm lateral to the bregma). Large Wistar rats were divided randomly into three groups. Group 1: normal control group. Group 2 : pure intracerebral hemorrhage group, the animals were sacrificed respectively on the 6h , 1d, 2d, 3d, and 7d after blood infusion. Group 3: hirudin group (the administration of hirudin after blood infusion), the animals were sacrificed respectively on the 3d, and 7d after the administration of hirudin. Each group and each time phase included 5 rats respectively. Brains were examined by water content and conventional histologic and immunohistologic
细胞骨架是横越在细胞核和细胞质膜内侧面的一种纤维状蛋白基质，对细胞生命活动是必要的，如细胞形态的维持、运动性、胞内颗粒物质运输、有丝分裂、分泌加工过程等。细胞骨架由微管、微丝及中间纤维组成。微管的基本组成单位是微管蛋白，在体内，微管蛋白聚集成微管需要有MAP的参与。MAP促进微管蛋白聚集、抑制微管蛋白分解，是微管结构和功能的重要组成部分。其中MAP-2在脑内水平明显高于其它组织，MAP-2在神经元内的高水平表达，与树突及胞体的选择性结合及其对细胞骨架成分的调节作用均支持MAP-2作为神经元形态学调节因子的作用。因此，MAP-2结构损伤及功能障碍势必导致神经元形态及功能异常。
胶质纤维酸性蛋白（GFAP）是星形胶质细胞的特异蛋白和细胞骨架成分之一，是一种中间丝蛋白，是中间纤维的一种。损伤后星形胶质细胞被激活，反应性肥大、增生，GFAP表达增加。被激活的星形胶质细胞一方面具有保护作用，如可促进胶质瘢痕的
形成，防止再出血；在损伤区分泌多种神经营养因子，以促进损伤的修复。另一方面，过度的胶质化却可作为机械屏障妨碍髓鞘和轴索的再生，影响周围神经组织结构和功能的恢复；而且，肿胀的星形胶质细胞还可释放谷氨酸和一氧化氮，过高浓度的谷氨酸和一氧化氮具有神经毒性，是继发性脑损伤的重要原因之一；应激反应的星形胶质细胞还可以产生多种细胞因子和炎症介质，如白细胞介素-1β（IL-1β）和肿瘤坏死因子（TNF-α），引起血脑屏障开放，介导炎性反应，促进内皮细胞的坏死，故参与脑水肿的病理过程。可见，反应性星形胶质细胞增生在损伤后发挥着双刃剑的作用。
目的：本实验旨在从细胞骨架蛋白的角度进一步探讨脑出血后凝血酶在继发性脑损伤中的致病机制及其特异性抑制剂－水蛭素的保护作用。
方法：向体重为250~350g健康雄性大鼠尾壳核区（前囟前0.2mm，中线旁开4mm，垂直进针5.5mm）注入自体未抗凝动脉血50μl，制作大鼠实验性脑出血动物模型，将大鼠随机分为3组：1组：正常对照组；2组：单纯脑出血组，分别在术后6h、1d、2d、3d和7d处死动物，取脑；3组：水蛭素组（注血后5分钟向血肿局部注入10U水蛭素），分别在注药后3d和7d处死动物，取脑。上述每个时间点、每个观察指标（HE染色、脑组织含水量、免疫组化染色）各5只动物。HE染色定性观察脑出血后血肿周围组织的病理变化；脑组织含水量系定量测定脑出血后血肿周围组织水肿程度；MAP-2、GFAP免疫组化染色观察脑出血后的细胞骨架损伤。
结果：大体病理可见尾壳核区类圆形血肿，2~3d组可见血肿周围脑组织肿胀，侧脑室前角受压。光镜示从脑出血后6h开始血
肿周围组织水肿，神经细胞、胶质细胞肿胀，或细胞收缩、染色加深，细胞周围间隙变大，血管周围空隙亦变大。随脑出血后时间的延长，上述病理变化逐渐加重，3d组的组织水肿和细胞间隙空化最为明显，部分神经细胞坏死、消失。脑出血后脑组织含水量逐渐增加，从1d开始增加显著，与正常对照组相比P<0.05,3d达高峰，7d较3d组虽有所下降，但与正常对照组相比P仍然小于0.05。MAP-2、GFAP免疫组化染色结果表明：正常对照组MAP-2染色阳性细胞为神经元细胞，可见细胞轮廓清晰，突起纤细，突起较长且分枝数量多，MAP-2广泛分布于神经元突起部分，呈条索状；胞体内也有少量表达。脑出血后6h开始血肿周围MAP-2阳性细胞数目进行性减少，与正常对照组相比P<0.05，突起变形程度逐渐加重，且染色变淡，上述变化进行性加重，于3d达到最低，7d较3d组虽有所提高，但与正常对照组相比P仍然小于0.05。正常对照组GFAP阳性细胞数目少、染色浅，分布稀疏，胞体小，突起细短。脑出血后1d GFAP表达明显增强，与正常对照组相比P<0.05，出现肥大的星形胶质细胞，胞体变大，GFAP染色加深，突起增多、增粗且形状不规则，上述改变进行性加重，于7d达高峰。水蛭素组较相同时间点单纯脑出血组组织水肿和细胞坏死明显减轻；脑组织含水量降低（P<0.05）；MAP-2阳性细胞数量增多（P<0.05），变形程度减轻，染色加深；GFAP阳性细胞数目有所减少（P<0.05），细胞肥大、增生现象不明显。
结论：1. 本实验采用自体未抗凝动脉血注入法建立了可靠的实验性脑出血动物模型。2. 光镜示血肿周围的组织水肿和细胞间隙空化从脑出血后6h开始进行性加重，3d最为明显。脑出血后脑组织含水量逐渐增加，从1d开始增加显著，与正常对照组相比P<0.05，3d达高峰，7d较3d组虽有所下降，但与正常对照组相
比P仍然小于0.05。3. 脑出血后1周内，MAP-2的分解破坏或合成障碍标志着严重或致?
Intracerebral hemorrhage (ICH) is associated with high mortality and Morbidity. In addition to the mass effect and direct disruption, the secondary injury is also the main cause after ICH. An average-sized hematoma in humans (54ml) has little immediate effect on intracranial pressure or cerebral perfusion pressure because that volume is well with the volume-buffering capacity of the intracranial space. There is evidence that intracerebral blood causes brain injury through biochemical substances released from the hematoma that initiate formation of brain edema. Thrombin, released from the coagulation cascade after experimental ICH, leads to edema formation and disruption of blood-brain-barrier. The important role of thrombin after ICH is already affirmed, but its accurate mechanism is still unclear.
The structure of the cytoplasm relies on the assembly and organization of a network of fibrous components, which includes microtubules, microfilaments, and intermediate filaments. The cytoskeleton is implicated in a wide variety of functions that are essential for cellular life including mitosis, maintenance of cell shape, motility, intracellular transport of organelles, and secretary processes. Microtubules have been isolated mainly from brain, where tubulin, a major microtubular component, is present in significantly larger amounts than in other tissues. Even though tubulin can self-assemble in vitro into microtubules under appropriate conditions, the presence of MAPs is required for in vivo assembly of tubulin. MAPs promote the assembly of tubulin and suppress its disassembly. MAP-2 is
present in larger amounts in brain than in other tissues. Microtubule-associated protein-2 is found mainly in dendrites and soma of neural cells in most regions of the central nervous system. The high levels of expression of these MAP-2 in neurons, their selective associations to either axonal or dendritic compartments, and their developmental regulation support a putative role of MAP-2 as mediators of neuronal morphogenesis.
Glial fibrillary acidic protein is a marker for astrocyte as well as one of the cytoskeleton components. There are densely distributed GFAP-positive cells around the blood clot, and the number and morphological features of GFAP-positive astrocytes changed as time went on after ICH. One hand, the activated astrocytes have protective functions, such as promoting the formation of glial scar to escape from reblooding; secreting various neural protective factors to accelerate the repairation of the injury. On the other hand, excessive glial reaction will affect the restoration of peripheral tissue structure and function because impair the regeneration of myelinated and axon; moreover, they will release glutamic acid and nitrous oxide which can destroy the nervous system; the glial reaction also produce inflammation medium, such as IL-1β and TNF-α resulting in the opening of the blood brain barrier, mediating inflammation reaction, accelerating the necrosis of endothelium to participate in the pathology of brain edema.
Objective: To explore the role of thrombin in the pathological mechanism of ICH by observing the expression of MAP-2 and GFAP in the perihematomal brain regions, and the protective mechanism of
hirudin.
Methods: The rat was positioned in a stereotactic frame, rat models of ICH were made by infusion of autologous blood into the right neucleus caudatus (coordinates: 0.2mm anterior, 5.5mm ventral, and 4mm lateral to the bregma). Large Wistar rats were divided randomly into three groups. Group 1: normal control group. Group 2 : pure intracerebral hemorrhage group, the animals were sacrificed respectively on the 6h , 1d, 2d, 3d, and 7d after blood infusion. Group 3: hirudin group (the administration of hirudin after blood infusion), the animals were sacrificed respectively on the 3d, and 7d after the administration of hirudin. Each group and each time phase included 5 rats respectively. Brains were examined by water content and conventional histologic and immunohistologic
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Folkerts MM, Berman RF, Muizelaar JP, et al. Disruption of MAP-2 immunostaining in rat hippocampus after traumatic brain injury[J]. J Neurotrauma, 1998, 15:349-363.
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Markwardt F. The development of hirudin as an antithrombotic drug[J]. Thromb Res, 1994, 74(1): 1-8.
方琪, 包仕尧, 许峥, 等. 凝血酶与脑水肿的实验研究[J]. 江苏医药杂志, 2002, 28(11):817-819.
Fenton JW, Villanueva GB, Ofosu FA, et al. Thrombin inhibition by hirudin: how hirudin inhibits thrombin[J]. Haemost. 1991, 21 (1):27-35.
Moller T, Hanisch UK, Ransom BR. Thrombin-induced activation of cultured rodent microglia[J]. J Neurochem, 2000, 75(4): 1539-1547.
莫永炎, 姜勇, 陈瑗. 脑星形胶质细胞生物学功能研究进展[J]. 生理科学进展, 2002, 33(1):17-19.
Holmin S, Scballing M, Hojeberg B, et al. Delayed cytokine expression in rat brain following experimental conlusion[J]. J Neurosurg, 1997, 86(3):493-497.
Kubo Y, Suzuki M, Kudo A, et al. Thrombin inhibitor ameliorates secondary damage in rat brain injury: suppression of inflammatory cells and vimentin-positive astrocytes[J]. J Neurotrauma, 2000, 17(2): 163-72.
Pekny M, Johansson CB, Eliasson C, et al. Abnormal reaction to
central nervous system injury in mice lacking glial fibrillary acidic protein and vimentin[J]. J Cell Biol, 1999, 145(3): 503-514.
Condorell DF. GFAP beta mRNA expression in the normal rat brain and neuronal injury[J]. Neurochem Res, 1999, 24(5): 709-714.
Fields RD, Stevens-Graham B. New insights into neuron-glial communication[J]. Science, 2002, 298(5593): 556-562.
Huang W, Hui YN, LU R. Immunohistochemical study of the astrocytes and axons responses to crush injury in optic nerve[J]. J Fourth Ml Med Univ, 2002, 22(6): 481-484
Figueroa BE, Keep RF. Plasminogen activators potentiate thrombin-induced brain injury[J]. Stroke, 1998, 29(6): 1202-1213.