镁剂对大鼠蛛网膜下腔出血后血管内皮生长因子表达的影响
摘要
前言
蛛网膜下腔出血(subarachnoid hemorrhage，SAH)是指脑底部或脑表面的血管破裂，血液直接流入蛛网膜下腔的一种常见的非外伤性出血的急性脑血管疾病。本病死亡率与致残率均较高，其预后影响因素主要为脑积水的出现、再出血、血管痉挛CVS和迟发性缺血DCI的发生等，以上称为继发性脑损害。因此，有效地防治继发性脑损害是改善SAH总体预后的关键性环节。近年来的研究表明，VEGF参与了该病继发性病理损伤过程，在继发性脑损伤中起重要的保护性作用，而在蛛网膜下腔出血后硫酸镁对VEGF的表达及作用尚无人研究。研究硫酸镁对VEGF在蛛网膜下腔出血病程中的表达及作用的规律，对于进一步加深对蛛网膜下腔出血后病理损伤的认识，探讨有效的治疗途径，促进神经功能恢复，有效地防治继发性脑损害有重要临床意义。本研究旨在应用蛛网膜下腔出血大鼠模型，观察蛛网膜下腔出血后大脑不同时间点的VEGF表达变化情况，以探讨蛛网膜下腔出血后硫酸镁对VEGF的时程变化规律及作用，为临床应用硫酸镁治疗蛛网膜下腔出血提供理论上的依据。
材料和方法
一、实验动物
雄性SD大鼠体重280-320克。共96只
二、实验试剂
10％水合氯醛肝素0.9％氯化钠APES多聚赖氨酸乙醇丙酮苏木H_2O_2 SABC免疫组化试剂盒
三、实验器材
天平、手术刀、剪、1ml注射器、500ml容器+输液管、托盘、10％水合氯醛、酒精灯、试管、4号针头、超净工作台烘箱、恒温培养箱、4℃冰箱、压力锅、载玻片、盖玻片、封片胶、切片架、染色缸、湿盒、微量移液器、加样头
TS-12F生物组织自动脱水机SAKURA Tissue-Tek TEC包埋机OLYMPUSB201显微镜成像系统
实验方法
一、将96只雄性SD大鼠随机分为SAH组、假手术组(PO)、SAH加镁组、假手术加镁组
二、枕大池注射自体血法制备蛛网膜下腔出血大鼠模型
三、免疫组化
取材、固定、组织块的脱水、透明、石蜡包埋切片染色封片
用免疫组化技术对大脑皮质，海马，血管进行VEGF标记。
四、分析统计
观察脑损伤后不同时段处死标本中VEGF的表达，用OLYMPUS／BX51／Evolution MP5.0摄像系统采集图像，MetaMorph显微图像系统分析，测量结果以阳性细胞的积分光密度(intergrated OD)表示
实验结果
假手术组和假手术加Mg组均可见到少量VEGF的表达，两者差异无显著性。在蛛网膜下腔出血组24小时明显表达，3天表达强度进一步增高，7天达高峰，14天下降至正常。其阳性细胞主要分布在大脑皮层，基底节区、海马等部位也有较多。其细胞类型主要为神经细胞、胶质细胞、血管内皮细胞等。在SAH后的不同时间点免疫组织化学结果为：SAH加Mg组大鼠VEGF的表达明显高于SAH组。SAH加Mg组与SAH组VEGF表达差别有显著的统计学意义。
讨论
Senger等在1983年发现了血管通透因子并且克隆出了相关基因，然Ferrara等在1989年将其改名为血管内皮生长因子。具有促进血管新生的作用正常情况下，血管内皮生长因子在个体胚胎发育过程中有广泛的表达，如胚胎和出生后脑室的神经外胚层、脑垂体、肾脏、肾上腺、卵巢、子宫内膜和胎盘等正常组织。而在正常成人脑组织或脑血管的生长处于静止状态，一般无VEGF的表达。1992年Sweiki等发现缺氧可诱导血管内皮生长因子的表达。随后一些学者的实验指出血管内皮生长因子的表达是脑损伤后机体一种内源性防御机制，即内源性血管内皮生长因子对损伤脑组织可能具有保护作用，近年来发现它还具有神经再生和直接神经保护等有益作用。
在对SAH引起的继发脑损害的研究中发现，VEGF参与了该病继发性病理损伤过程，在继发性脑损伤中起重要的保护性作用。
硫酸镁中的Mg~(2+)为天然的Ca~(2+)拮抗剂，是目前较为确定的内源性保护因子，可通过非竞争性阻断NMDA受体，抑制兴奋性氨基酸释放，阻断电压门控钙通道，减少自由基的生成，松弛血管平滑肌，拮抗内皮素21等，因而有可能起到防治CVS、DCI和再出血的作用。
硫酸镁对作为神经保护剂对SAH的治疗作用在动物实验中已得到证实，而在蛛网膜下腔出血后硫酸镁对VEGF的表达及作用尚无人研究。本实验组用免疫组化及VEGF免疫反应阳性神经元的统计分析发现SAH后VEGF表达增加，而加用硫酸镁SAH组较SAH组更高，表明硫酸镁能促进SAH后脑内VEGF的表达，改善SAH总体预后。但其具体机制仍需进一步研究。
本实验应用硫酸镁剂量是通过文献及预实验摸索得来的。
结论
硫酸镁促进了VEGF的表达，其通过促进VEGF的表达参与了继发性脑损伤的保护机制
Objective: To explore effects of MgSO_4 on expression of VEGF after subarachnoidhemorrhage of SD rats
Motheds: Ninety-six SD rats were divided randomly into four groups
Including: presduo-operated group; SAH model group; presduo-operatedgroups dealedwith MgSO_4, SAH model group dealed with MgSO_4.. VEGF were measured by radioimmunoassay respectively at different time.
Results: To compared with that in SAH model group,.The levels of expression ofVEGF were increased in SAH model groups dealed with MgSO_4.
Conclusions: Magnesium can increase the levels of expression of VEGF
蛛网膜下腔出血(subarachnoid hemorrhage，SAH)是指脑底部或脑表面的血管破裂，血液直接流入蛛网膜下腔的一种常见的非外伤性出血的急性脑血管疾病。本病死亡率与致残率均较高，其预后影响因素主要为脑积水的出现、再出血、血管痉挛CVS和迟发性缺血DCI的发生等，以上称为继发性脑损害。因此，有效地防治继发性脑损害是改善SAH总体预后的关键性环节。近年来的研究表明，VEGF参与了该病继发性病理损伤过程，在继发性脑损伤中起重要的保护性作用，而在蛛网膜下腔出血后硫酸镁对VEGF的表达及作用尚无人研究。研究硫酸镁对VEGF在蛛网膜下腔出血病程中的表达及作用的规律，对于进一步加深对蛛网膜下腔出血后病理损伤的认识，探讨有效的治疗途径，促进神经功能恢复，有效地防治继发性脑损害有重要临床意义。本研究旨在应用蛛网膜下腔出血大鼠模型，观察蛛网膜下腔出血后大脑不同时间点的VEGF表达变化情况，以探讨蛛网膜下腔出血后硫酸镁对VEGF的时程变化规律及作用，为临床应用硫酸镁治疗蛛网膜下腔出血提供理论上的依据。
材料和方法
一、实验动物
雄性SD大鼠体重280-320克。共96只
二、实验试剂
10％水合氯醛肝素0.9％氯化钠APES多聚赖氨酸乙醇丙酮苏木H_2O_2 SABC免疫组化试剂盒
三、实验器材
天平、手术刀、剪、1ml注射器、500ml容器+输液管、托盘、10％水合氯醛、酒精灯、试管、4号针头、超净工作台烘箱、恒温培养箱、4℃冰箱、压力锅、载玻片、盖玻片、封片胶、切片架、染色缸、湿盒、微量移液器、加样头
TS-12F生物组织自动脱水机SAKURA Tissue-Tek TEC包埋机OLYMPUSB201显微镜成像系统
实验方法
一、将96只雄性SD大鼠随机分为SAH组、假手术组(PO)、SAH加镁组、假手术加镁组
二、枕大池注射自体血法制备蛛网膜下腔出血大鼠模型
三、免疫组化
取材、固定、组织块的脱水、透明、石蜡包埋切片染色封片
用免疫组化技术对大脑皮质，海马，血管进行VEGF标记。
四、分析统计
观察脑损伤后不同时段处死标本中VEGF的表达，用OLYMPUS／BX51／Evolution MP5.0摄像系统采集图像，MetaMorph显微图像系统分析，测量结果以阳性细胞的积分光密度(intergrated OD)表示
实验结果
假手术组和假手术加Mg组均可见到少量VEGF的表达，两者差异无显著性。在蛛网膜下腔出血组24小时明显表达，3天表达强度进一步增高，7天达高峰，14天下降至正常。其阳性细胞主要分布在大脑皮层，基底节区、海马等部位也有较多。其细胞类型主要为神经细胞、胶质细胞、血管内皮细胞等。在SAH后的不同时间点免疫组织化学结果为：SAH加Mg组大鼠VEGF的表达明显高于SAH组。SAH加Mg组与SAH组VEGF表达差别有显著的统计学意义。
讨论
Senger等在1983年发现了血管通透因子并且克隆出了相关基因，然Ferrara等在1989年将其改名为血管内皮生长因子。具有促进血管新生的作用正常情况下，血管内皮生长因子在个体胚胎发育过程中有广泛的表达，如胚胎和出生后脑室的神经外胚层、脑垂体、肾脏、肾上腺、卵巢、子宫内膜和胎盘等正常组织。而在正常成人脑组织或脑血管的生长处于静止状态，一般无VEGF的表达。1992年Sweiki等发现缺氧可诱导血管内皮生长因子的表达。随后一些学者的实验指出血管内皮生长因子的表达是脑损伤后机体一种内源性防御机制，即内源性血管内皮生长因子对损伤脑组织可能具有保护作用，近年来发现它还具有神经再生和直接神经保护等有益作用。
在对SAH引起的继发脑损害的研究中发现，VEGF参与了该病继发性病理损伤过程，在继发性脑损伤中起重要的保护性作用。
硫酸镁中的Mg~(2+)为天然的Ca~(2+)拮抗剂，是目前较为确定的内源性保护因子，可通过非竞争性阻断NMDA受体，抑制兴奋性氨基酸释放，阻断电压门控钙通道，减少自由基的生成，松弛血管平滑肌，拮抗内皮素21等，因而有可能起到防治CVS、DCI和再出血的作用。
硫酸镁对作为神经保护剂对SAH的治疗作用在动物实验中已得到证实，而在蛛网膜下腔出血后硫酸镁对VEGF的表达及作用尚无人研究。本实验组用免疫组化及VEGF免疫反应阳性神经元的统计分析发现SAH后VEGF表达增加，而加用硫酸镁SAH组较SAH组更高，表明硫酸镁能促进SAH后脑内VEGF的表达，改善SAH总体预后。但其具体机制仍需进一步研究。
本实验应用硫酸镁剂量是通过文献及预实验摸索得来的。
结论
硫酸镁促进了VEGF的表达，其通过促进VEGF的表达参与了继发性脑损伤的保护机制
Objective: To explore effects of MgSO_4 on expression of VEGF after subarachnoidhemorrhage of SD rats
Motheds: Ninety-six SD rats were divided randomly into four groups
Including: presduo-operated group; SAH model group; presduo-operatedgroups dealedwith MgSO_4, SAH model group dealed with MgSO_4.. VEGF were measured by radioimmunoassay respectively at different time.
Results: To compared with that in SAH model group,.The levels of expression ofVEGF were increased in SAH model groups dealed with MgSO_4.
Conclusions: Magnesium can increase the levels of expression of VEGF
引文
1 Van den Bergh WM, Albrecht KW, Berkelbach vander Sp renkel JW, et aLMagnesium therapy after neurysmal subarachnoid hemorrhage a dose finding study for long term treatment. Acta Neurochir (Wien), 2003,145: 195 -199.
2 Senger DR ,Galli SJ, Dvorak AM, et al.Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid.Science 1983;219(4587):983-5
3 Ferrara N, WJ, Pituitary follicular cells secrete a novel heparin-binding growh factorspecific for vascular endothelial cells.Biochem Biophys Res Commun 989;161(2):851-8
4 Marti HH,Risauw Systemic hypoxia changes the organ-specific distribution of vascular endothelial growh factor and its receptors.Proc Natl Acad Sci USA1998;95(26): 15809-14
5 Pozzati E, Giangaspero F, marliani F, et al. Occult cerebrovascular malformations after irradiation[J]. Neurosurgery,1996,39(4):677-684
6 Shweiki D,Itlin A,Soffer D,et al. vascular endothelial growh factor induced by hypoxia may mediate hypoxia-initiated angigenesis.Nature 1992;359(6398):843-5
7 Hayashi T, Abe K, ItoyamaY.Reduction of ischemic damage by application of vascular endothelial growh factor un rat brain after transeni ischemia. J Cereb Blood Flow Metab 1998; 18(8)887-95
8 Marti HJ, Bernaudin M , Bellail A,et al. hypoxia- induced vascular endothelial growh factor expression after cerebral isghemia.Am J Pathol 2000;156(3):965-76
9 Neufeld G,Cohen T,Gengrinovitch S,et al.Vascular endothelial growth factor (VEGF) and its receptors.FASEB J 1999;13(1):9-22
10 Jin K, Mao XO , Batteur SP,et al .Caspase-3 and the regulation of hypoxic neuronal death by vascular endothelial growth factor.Neuroscience 2001;108 (2):351-8
11 YangZJ,BaoWL Qiu MH ,et al.Role of vascular endothelial growth factor in neurnal CNA damage and repair in rat brain following a trannsient cerebral ischemia . J Neurosci Res 2002;70(2:140-9)
12 Carmeliet P , Storkebaum E .Vascular and neuronal effects of VEGF in the nervous sysstenrimplications for neurological disorders. Semin Cell Dev Biol 2002; 13(1):39-53
13 Pekala P, marlow M, Heuvelman D,et al.Regulation of hexose transport in aoric endothelial cells by insulin.J Biol Chem 1999;265(30): 18051-4
14 Jin KL, MaoXO, GreenbergDA. Vascular endothelial growth factor. Direct neuro protective effect in in vitro ischemia.Proc Natl Acad Sci USA2000;97(18): 10242-7
15 Jin KL, Mao XO, Gteenberg DA.Vascular endothelial growth factor rescues HN33 neural cells from death induced by serum withdrawal JMol Neurosci 2000; 14(3) 197-203
16 Rosenstein JM,Mani N,Khaibullina A,et al.Neurotrophic effects of vascular endothelial growth factor on organotypic cortical explants and primary cortical neurons. JNeurosci 2003;23(35): 11036-44
17 khaibullina AA,Rosenstein JM,Krum JM.Vascular endothelial growth factor promotes neurite maturation in primary CNS neuronal cultures. Brain Res Dev Brain Res 2004;148(1):59-68
18 Manoonkitiwongsa PS,Schultz RL, McCreery DB, et al.Neuroprotection of ischemic brain by vascular endothelial growth factor is critically dependent on proper dosage and may be compromised by angiogenesis. J Cereb Blood Flow Metab 2004; 24(6); 693-702
19 Wang Y,Kilic E, Kilic U,et al .VEGF overexpression induces post-ischaemic neuro protection, but facilitates haemodynamic steal phenomena.Brain 2005; 128(Pt 1): 52-63
20 Jarus2Dziedzic K, Bogucki J, Zub W. The influence of rup tured cerebral aneurysm localization on the blood flow velocity evaluated by transcranial Doppler ultrasonography. Neurol Res, 2001, 23: 23-28.
1 GulesI, SatohM, Clower BR,etal Comparison of three rat models of cerebral vasospasm [J]. Am J Physiol Heart CircPhysiol, 2002; 283(6): 2551-2559.
2 Meguro T, Clower BR, Carpenter R, et al. Improved ratmodel for cerebral vasospasm studies [J]. Neurol Res, 2001;23(7): 761-766.
3 Suzuki H, Kanamaru K, Tsunoda H, et al. Heme oxygenase-1 gene induction as an intrinsic regulation against delayed cerebral vasospasm in rats [J]. J Clin Invest, 1999; 104(1): 59-66.
4 Harada S, Kamiya K, Masago A, et al. Subarachnoid hemorrhageinduces c-fos, c-jun and hsp70 mRNA expression inrat brain [J]. Neuroreport, 1997; 8(15): 3399-3404.
5 Megyesi JF, Votlrath B, Cook DA, et al. In vivo animalmodels of cerebral vasospasm: a review [J]. Neurosurgery,2000; 46(2): 448-461.
6 Bederson JB, Germano IM, Guarino L. Cortical blood flowand cerebral perfusion pressure in a new noncraniotomymodel of subarachnoid hemorrhage in the rat [J]. Stroke,1995; 26(6): 1086-1092.
7 Veelken JA, Laing RJ, Jakubowski J. The Sheffield modelof subarachnoid hemorrhage in rats [J]. Stroke, 1995; 26(7): 1279-1284.
8 Carpenter RC, Miao L, Miyagi Y, et al. Altered expressionof P2 receptor mRNAs in the basilar artery in a rat double hemorrhage model [J]. Stroke, 2001; 32(2): 516-522.
9 Ono S, DateI,OnodaK,et al. Time course of the diameterof the major cerebral arteries after subarachnoid hemorrhageusing corrosion cast technique [J]. Neurol Res, 2003; 25(4):383-389.
10 Ram Z, Sahar A, Hadani M. Vasospasm due to massive subarachnoid Haemorrhage a rat model [J]. Acta Neurochir (Wien), 1991; 110(3-4): 181-184.
11 Piepgras A, Thome C, Schmiedek P. Characterization of ananterior circulation rat subarachnoid hemorrhage model [J] . Stroke, 1995; 26(12): 2347-2352.
12 Kassell NF, Torner JC, Haley EC Jr, et al. The internationalcooperative study on the timing of aneurysm surgery. Part 1 :overall management results [J]. J Neurosurg, 1990; 73(1)：18-36．
2 Senger DR ,Galli SJ, Dvorak AM, et al.Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid.Science 1983;219(4587):983-5
3 Ferrara N, WJ, Pituitary follicular cells secrete a novel heparin-binding growh factorspecific for vascular endothelial cells.Biochem Biophys Res Commun 989;161(2):851-8
4 Marti HH,Risauw Systemic hypoxia changes the organ-specific distribution of vascular endothelial growh factor and its receptors.Proc Natl Acad Sci USA1998;95(26): 15809-14
5 Pozzati E, Giangaspero F, marliani F, et al. Occult cerebrovascular malformations after irradiation[J]. Neurosurgery,1996,39(4):677-684
6 Shweiki D,Itlin A,Soffer D,et al. vascular endothelial growh factor induced by hypoxia may mediate hypoxia-initiated angigenesis.Nature 1992;359(6398):843-5
7 Hayashi T, Abe K, ItoyamaY.Reduction of ischemic damage by application of vascular endothelial growh factor un rat brain after transeni ischemia. J Cereb Blood Flow Metab 1998; 18(8)887-95
8 Marti HJ, Bernaudin M , Bellail A,et al. hypoxia- induced vascular endothelial growh factor expression after cerebral isghemia.Am J Pathol 2000;156(3):965-76
9 Neufeld G,Cohen T,Gengrinovitch S,et al.Vascular endothelial growth factor (VEGF) and its receptors.FASEB J 1999;13(1):9-22
10 Jin K, Mao XO , Batteur SP,et al .Caspase-3 and the regulation of hypoxic neuronal death by vascular endothelial growth factor.Neuroscience 2001;108 (2):351-8
11 YangZJ,BaoWL Qiu MH ,et al.Role of vascular endothelial growth factor in neurnal CNA damage and repair in rat brain following a trannsient cerebral ischemia . J Neurosci Res 2002;70(2:140-9)
12 Carmeliet P , Storkebaum E .Vascular and neuronal effects of VEGF in the nervous sysstenrimplications for neurological disorders. Semin Cell Dev Biol 2002; 13(1):39-53
13 Pekala P, marlow M, Heuvelman D,et al.Regulation of hexose transport in aoric endothelial cells by insulin.J Biol Chem 1999;265(30): 18051-4
14 Jin KL, MaoXO, GreenbergDA. Vascular endothelial growth factor. Direct neuro protective effect in in vitro ischemia.Proc Natl Acad Sci USA2000;97(18): 10242-7
15 Jin KL, Mao XO, Gteenberg DA.Vascular endothelial growth factor rescues HN33 neural cells from death induced by serum withdrawal JMol Neurosci 2000; 14(3) 197-203
16 Rosenstein JM,Mani N,Khaibullina A,et al.Neurotrophic effects of vascular endothelial growth factor on organotypic cortical explants and primary cortical neurons. JNeurosci 2003;23(35): 11036-44
17 khaibullina AA,Rosenstein JM,Krum JM.Vascular endothelial growth factor promotes neurite maturation in primary CNS neuronal cultures. Brain Res Dev Brain Res 2004;148(1):59-68
18 Manoonkitiwongsa PS,Schultz RL, McCreery DB, et al.Neuroprotection of ischemic brain by vascular endothelial growth factor is critically dependent on proper dosage and may be compromised by angiogenesis. J Cereb Blood Flow Metab 2004; 24(6); 693-702
19 Wang Y,Kilic E, Kilic U,et al .VEGF overexpression induces post-ischaemic neuro protection, but facilitates haemodynamic steal phenomena.Brain 2005; 128(Pt 1): 52-63
20 Jarus2Dziedzic K, Bogucki J, Zub W. The influence of rup tured cerebral aneurysm localization on the blood flow velocity evaluated by transcranial Doppler ultrasonography. Neurol Res, 2001, 23: 23-28.
1 GulesI, SatohM, Clower BR,etal Comparison of three rat models of cerebral vasospasm [J]. Am J Physiol Heart CircPhysiol, 2002; 283(6): 2551-2559.
2 Meguro T, Clower BR, Carpenter R, et al. Improved ratmodel for cerebral vasospasm studies [J]. Neurol Res, 2001;23(7): 761-766.
3 Suzuki H, Kanamaru K, Tsunoda H, et al. Heme oxygenase-1 gene induction as an intrinsic regulation against delayed cerebral vasospasm in rats [J]. J Clin Invest, 1999; 104(1): 59-66.
4 Harada S, Kamiya K, Masago A, et al. Subarachnoid hemorrhageinduces c-fos, c-jun and hsp70 mRNA expression inrat brain [J]. Neuroreport, 1997; 8(15): 3399-3404.
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