摘要
芪丹中药提取物(QDPTP)是按照中医理论组方、经现代工艺加工而成的复方中药提取物。该组方中黄芪为君药,丹参、川芎和红花为臣药,银杏叶、三七和红景天为佐使药。已有研究表明QDPTP具有抗心肌缺血-再灌注损伤的作用。本课题分别采用大鼠局灶性和全脑缺血-再灌注模型,研究QDPTP的抗大鼠实验性脑缺血-再灌注损伤作用及机制,为此药的开发研究提供实验依据。
一、QDPTP抗脑缺血-再灌注损伤作用
1. QDPTP对局灶性脑缺血-再灌注大鼠神经功能的影响SD大鼠,给生理盐水(10ml/kg/d,7d,i.g.)、QDPTP低、中、高剂量(450、900、1800mg/kg/d,7d,i.g.),川芎嗪(33mg/kg/d,缺血前30min,i.v.)。阻塞右侧大脑中动脉造成局灶性缺血模型,2小时后再灌注,观察QDPTP对再灌注后神经功能的影响。模型组、QDPTP低、中、高剂量组和川芎嗪组的神经功能缺陷等级评分分别为3.7±1.2、1.4±0.5(vs模型组,P<0.01)、1.8±0.4(P<0.01)、1.8±0.5(P<0.01)和1.9±0.5(P<0.01)。此结果表明:QDPTP低、中、高剂量组均能显著改善缺血-再灌注引起的大鼠神经功能缺陷症状。
2. QDPTP对大鼠局灶性脑缺血-再灌注损伤脑梗死范围的影响在大鼠局灶性脑缺血-再灌注损伤模型,采用TTC染色,见模型组、QDPTP低、中、高剂量组和川芎嗪组的脑梗死范围分别为20.9±7.3(%)、6.6±4.4(%)、6.4±4.8(%)、4.4±2.6(%)、和6.6±4.3(%),所有用药组均明显缩小脑梗死范围(P<0.01,vs模型组)。
3. QDPTP对大鼠局灶性脑缺血-再灌注损伤脑含水量和脑指数的影响在大鼠局灶性脑缺血-再灌注模型,模型组动物脑组织含水量(81.8±0.8,%)较伪手术组明显增加(P<0.01);各给药组均明显降低局灶性缺血-再灌注引起的脑组织含水量升高(P<0.01或P<0.05),分别为:QDPTP低剂量(78.8±1.4,%)、中剂量(78.1±1.8,%)、高剂量(80.5±0.6,%)和川芎嗪(79.0±2.0,%)。与模型组比较,各给药组脑指数均表现出降低的趋势,但统计学意义不显著。
二、QDPTP抗脑缺血-再灌注损伤的机制研究
(一) QDPTP对大鼠全脑缺血-再灌注所致氧化损伤和炎症反应的影响
1.制备大鼠全脑缺血-再灌注损伤模型和实验分组采用Pulsinelli四血管闭塞的方法略加改良建立大鼠急性全脑缺血-再灌注损伤模型。SD大鼠,随机分为伪手术组、模型组(NS,10ml/kg/d,7d,i.g.)、QDPTP低、中剂量组(450、900mg/kg/d, 7d,i.g.),川芎嗪组(33mg/kg,i.v.,缺血前30min和再灌后各1次)。
2. QDPTP对全脑缺血-再灌注大鼠脑组织SOD活性和MDA含量的影响采用黄嘌呤氧化酶法测定SOD活力,采用硫代巴比妥显色法测定MDA含量,组织蛋白测定用考马斯亮蓝蛋白测定法。模型组中脂质过氧化产物MDA的含量(4.53±0.92nmol/mg prot)显著升高(P<0.01 vs伪手术组),过氧化物歧化酶SOD活性(79.4±7.3U/mg prot)显著降低(P<0.01 vs伪手术组);各给药组均能显著减少MDA含量(P<0.01 vs模型组),增加SOD活性(P<0.01 vs模型组),分别为:QDPTP低剂量(2.10±0.17,116.9±11.2)、中剂量(2.35±0.09,111.5±6.2)和川芎嗪(1.66±0.11,95.6±13.3)。
3. QDPTP对全脑缺血-再灌注大鼠脑组织及血清IL1-β含量的影响采用双抗体夹心酶联免疫吸附法测定。结果表明:模型组脑组织及血清IL1-β含量分别为1006.4±247.1 pg/ml和370.8±76.6 pg/ml,与之相比,QDPTP中剂量(550.7±99.8,268±64.6)组中脑组织及血清中IL1-β的含量显著降低(P<0.01),川芎嗪脑也明显减少组织IL1-β的含量(627.8±144.7,P<0.05)。(二) QDPTP对麻醉大鼠局部脑血流量和动脉血压的影响
采用激光多普勒血流仪测量大鼠软脑膜局部的血灌注量,其被视为局部的脑血流量(rCBF)。单次十二指肠给予生理盐水(10ml/kg)、QDPTP低、中、高剂量(450、900、1800mg/kg),尼莫地平(20mg/kg),复方丹参滴丸(60mg/kg),或静脉注射川芎嗪(33mg/kg),记录给药前后rCBF的变化。采用电生理记录仪监测给药前后外周动脉平均收缩压、平均舒张压、平均压的变化。结果表明:持续观察120min,溶剂对照组的脑血流量和血压各参数无明显变化。QDPTP低、中、高剂量、川芎嗪、尼莫地平和复方丹参滴丸均能不同程度增加大鼠局部脑血流(P<0.01或P<0.05),外周动脉血压出现不同程度的降低。
结论
1. QDPTP可减轻缺血-再灌注引起的神经功能损伤,减轻脑水肿,缩小脑梗死范围。
2. QDPTP抗脑缺血-再灌注损伤作用可能与抑制自由基损伤、减轻细胞因子介导的炎症反应、扩张脑血管增加脑血流等因素有关。
QDPTP is an compound extractive of several kinds of the traditional Chinese medicine including Astragalus mongholicus, salvia miltiorrhiza, szechwan lovage rhizome, Carthamus tinctorius and so forth. It has been proved that QDPTP protect heart damaged by ischemia / reperfusion (I/R). And the aims of this study were to confirm the protect effect of QDPTP in experimental cerebral ischemia / reperfusion model and further clarify the mechanisms of actions by using different experimental methods. The main results are as follows:
Part I QDPTP decreased cerebral injury induced by focal cerebral ischemia / reperfusion in rats
1. The effects of QDPTP on the neurological deficits scores after I/R in rats
Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) for 2 hours and followed reperfusion for 4 hours. After that, we estimated deficit scores grading on a scale of 0-5. QDPTP (450, 900, 1800mg/kg) and Lig (33mg/kg) were found significantly decreasing neurological deficit scores after I/R (P<0.01).
2. The effects of QDPTP on cerebral infarction size after I/R in rats
The cerebral infarction size was measured by TTC staining technique after I/R. QDPTP (450, 900, 1800mg/kg) and Lig (33mg/kg) evidently decreased the cerebral infarction size after I/R (P<0.01).
3. The effects of QDPTP on cerebral water contents and cerebral index after I/R in rats
We measured the cerebral water contents with wet/dry weighing method and surveryed cerebral index after I/R in rats. The results showed that QDPTP (450, 900, 1800mg/kg) groups and Lig (33mg/kg) cut down both cerebral water contents (P<0.01 or P<0.05) and cerebral index.
Part II Mechanism of protection of QDPTP from cerebral ischemia/ reperfusion in rats
1. The effects of QDPTP on oxidative damage and inflammatory reaction after global cerebral I/R in rats
A. The effects of QDPTP on cerebral SOD activities and MDA contents after global cerebral I/R in rats
The model of global ischemia / reperfusion was established by improved Pulsinelli’s method for 20 minutes’occlusion and 24 hours’reperfusion. The activities of SOD and the contents of MDA in cerebral tissue after I/R were tested respectively. Comparing with NS in model group, QDPTP (450, 900mg/kg) lowered the contents of MDA (P<0.01) and reserved the activities of SOD(P<0.01). These effects of QDPTP (450, 900mg/kg) were comparable with Lig (33mg/kg).
②The effects of QDPTP on IL1-βcontents of cerebral tissue and blood serum after global cerebral I/R in rats
In the model group the IL1-βcontents of cerebral tissue and blood serum messured by ELISA were markedly increased after global cerebral I/R in rats. Comparing with the model group, the IL1-βcontents of cerebral tissue and blood serum decreased significantly in QDPTP (900mg/kg) group (P<0.01) and Lig (33mg/kg) (P<0.05) .
2. The effects of QDPTP on cerebral blood flow and artery blood pressure in anesthetized rats
The regional cerebral blood flow (rCBF) in cerebral pia mater was examined by Laser Doppler Flowmeter and the artery blood pressure (including mean systolic pressure, mean diastolic pressure and mean blood pressure) were monitored by the electrophysiological polygraph before and after drug administration. Comparing with the solvent, QDPTP (450, 900, 1800mg/kg), Lig (33mg/kg), Nim (20mg/kg) and DSDW (60mg/kg) expanded rCBF (P<0.01 or P<0.05) and simultaneously decreased the artery blood pressure (P<0.01 or P<0.05).
Conclusion
1. QDPTP had protective effects on the brain damaged by acute local and global cerebral ischemia/reperfusion in rats.
2. QDPTP improved neurological function and decreased the infarction size and cerebral edema induced by acute local cerebral ischemia /reperfusion in rats.
3. The mechanism of anti-cerebral injury of QDPTP in cerebral ischemia / reperfusion model may be related to the inhibition of the damages caused by oxidative free radicals, the extenuation of inflammatory reaction mediated by cytokines, the extension of cerebral vessels and the increase of cerebral blood flow.
引文
1. Nagy K, Domoki F, Bari F. Ischemic preconditioning in the brain. Ideggyogy Sz. 2005,58(9-10):305-13.
2. Graf R. Letter of apology. Cerebral ischemia and reperfusion: the pathophysiologic concept as a basis for clinical therapy. J Cereb Blood Flow Metab. 2004 ,24(4):351-71.
3. Philip R. Weinstein. MD, et al. Molecular Identification of the Ischemic Penumbra. Stroke. 2004(35):2666-2673.
4. 蒋晓江 , 李书林 . 缺血缺氧性脑病的神经保护 , 中国临床康复 , 2004, 8(28):6163-6165.
5. Janardhan V, Qureshi AI. Mechanisms of ischemic brain injury. Curr Cardiol Rep. 2004, Mar6(2):117-23.
6. 戴嫣. 中医药对缺血性中风的治疗及研究概况. 现代中西医结合杂志. 2004,13 (14):1945-1947.
7. Warner DS. Pharmacologic protection from ischemic neuronal injury. J Neurosurg Anesthesiol. 2004, 16(1):95-7.
8. 吴正国, 中药对缺血性脑损伤保护作用的研究进展, 国外医学中医中药分册2004, 26(2):70-73.
9. 储国祥, 陈修. 脑缺血和脑血流实验方法[A]. 见徐叔云, 卞如濂, 陈修主编, 药理实验方法学[M]. 第 3 版. 北京: 人民卫生出版社, 2002 : 1060-1072.
1. Longa EZ, Weinstein PR, Carlson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats [J ]. Stroke , 1989 ,20 (1) :842911.
2. 徐叔云, 卞如濂等. 药理实验方法学. 第三版. 北京:人民卫生出版社. 2002;1066~7.
3. 刘敬霞, 李建生. 线栓法制备大鼠局灶性脑缺血模型的进展及评价. 中国比较医学杂志, 2004, 14 (6).
4. Markgraf CG, Kraydieh S, et al. Comparative histopathologic consequence of photothrombotic occlusion of the distal middle cerebral artery in Sprague2Dawley and Wistar rats[J ] . Stroke ,1993 ,24(2) :286 - 293.
5. 蒋杞英, 胡艳秋. 雌激素对局灶性脑缺血大鼠脑梗死体积与 Bd-2 蛋白表达的影响. 中国临床康复. 2005, 9 (41): 68-9.
6. Gibson CL, Gray LJ, et al. Estrogens and experimental ischemic stroke: a systematic review. J Cereb Blood Flow Metab. 2006 Jan 25.
7. 曹霞, 曹秉振等. 线栓法复制局灶性脑缺血模型影响因素探讨[J ]. 中国病理生理学杂志, 2000 ,16(1) :107 - 109.
8. Philip R. Weinstein. MD, et al. Molecular Identification of the Ischemic Penumbra. Stroke. 2004(35):2666-2673.
9. 杨允东, 关宁等, 大鼠脑缺血模型血脑屏障结构和功能状态的研究. 锦州医学院学报, 2005, 26 (5) : 23-26.
10. 王耀明, 莫雪安. 脑缺血再灌注后大鼠血脑屏障通透性的免疫组织化学研究. 中国病理生理杂志. 2000 ,16(8) : 755-756.
11. Faubel S, Edelstein CL. Caspases as drug targets in ischemic organ injury. Curr Drug Targets Immune Endocr Metabol Disord. 2005 5(3):269-87.
12. 杨牧祥, 于文涛等. 中风康对局灶性脑缺血大鼠脑水肿的影响, 中国全科医学, 2005, 8 (18) : 1500-1.
1.Pulsinelli WA, Brierley JB. A new model of bilateral hemispheric ischemic in unanesthetized rat [J]. Stroke , 1979 , 10 :267-272.
2.贾栋, 高国栋. 大鼠全脑缺血/再灌注模型之比较研究. 神经病学与精神病学:卒中与神经疾病. 1999,(3).
3.Traystman RJ ,Kirsch JR ,Koehler RC. Oxygen radical mechanisms of brain injury following ischemia and reperfusion. J A ppl Physicol ,1991 ;71 (4) :1185～95.
4.贾玉洁, 闵连秋等. 原花青素对大鼠局灶性脑缺血的保护作用. 中国脑血管病杂志. 2005, 2 (12) : 558~560.
5.Beridze MZ, Urushadze IT, Shakarishvili RR. Mechanisms of delayed neuronal death in experimental acute cerebral ischemia. [J] Zh Nevrol Psikhiatr Im S S Korsakova. 2001;Suppl3:35-40.
6.罗正曜, 刘双等. 自由基与疾病[A]. 见方允中, 郑荣梁主编, 自由基生物学的理论与应用[M]. 北京: 科学出版社, 2002 : 592-620.
7.Sun-Ok Kim, In Sun Cho, Hee Kyoung Gu, et al. KR-31378 protects neurons from ischemia–reperfusion brain injury by attenuating lipid peroxidation and glutathione loss.[J] European Journal of Pharmacology, 2004 (487): 81– 91
8.Ron N.Apte, Elena Voronov. Interleukin-1αmajor pleiotropic cytokine in tumor-host interactions.[J] Cancer Biology, Vol.12,2002: 277-290.
9.Basu A, Lazovic J, Krady JK, et al. Interleukin-1 and the interleukin-1 type 1 receptor are essential for the progressive neurodegeneration that ensues subsequent to a mildhypoxic/ischemic injury. [J] J Cereb Blood Flow Metab. 2005 Jan; 25(1):17-29.
10.Dirnag IU, Ladecola C, Moskowitz MA. Pathobiology of ischaemic stroke: an integrated view [J]. Trends Neurosci. 1999, 22(9): 391-397.
11.Oprica M, Eriksson C, Schultzberg M. Inflammatory mechanisms associated with brain damage induced by kainic acid with special reference to the interleukin-1 system[J]. J Cell Mol Med. 2003 Apr-Jun; 7(2):127-40.
12.肖伟忠, 孙圣刚等. 环孢素A对大鼠脑缺血-再灌注后 IL-1β表达的影响.卒中与神经疾病. 2004, 11 (5) : 289.
13.屠永华, 李岚. 炎性细胞因子与脑缺血. 中国临床康复. 2003, 7 (1) : 49-50.
1.王斌, 张爱霞等. TMB-8 抑制 5-HT 和 KCl 引起大鼠脑血流量减少. 药学学报. 2003, 38 (5): 342-345.
2.王文远, 赵建明等. 平衡针法对局灶性脑缺血大鼠血清 TNF-、sICAM-1 的响及其相关性的分析. 针刺研究. 2004, 29 (4): 170-173.
3.Sharbrough FW, Messick JM Jr, Sundt TM J r. Correlation of continuous electroencephalograms with cerebral blood flow measurements buring carotid endarterectomy. S troke, 1973, 4(4): 674- 683.
4.Branston NM , Strong AJ, Symon L. Extracellular potassium activity, evoked potential and tissue blood flow. Relationships during progressive ischem ia inbaboon cerebral cortex. J N eu rol S ci, 1977, 32(2): 305-321.
5.Mohamed AA, Gotoh O, Graham D I, Osborne KA ,McCulloch J , Mendelow AD. Effect of pretreatment with the calcium antagonist nimodipine on local cerebral blood flow and histopathology after middle cerebral artery occlusion. A nn N eu rol, 1985, 18(6): 705-711.
6.阎超华, 张均田等. 丁基苯酞对正常及局部脑缺血大鼠纹状体脑血流影响. 中国药理毒理学杂志, 1998, 12 (1) : 36-39.
7.常助飞, 王忠诚等. 激光多普勒血流测量及其在神经外科的应用[J]. 国外医学脑血管疾病分册, 1996, 4 (2) : 95-97. Chang PF, Wang ZC, Luan GM. Laser Doppler flowmetry and its using in neurosurgery [J]. cerebrovascular diseases foreign medical sciences, 1996; 4(2).95-97(Chinese).
8.Yumin Luoa, Zhen Qin, Astragaloside IV protects against ischemic brain injury in a murine model of transient focal ischemia[J]. NeuroscienceLetters,363 (2004):218–223.
9.夏玉叶, 闵旸等. 羟基红花黄色素 A 对大鼠脑缺血损伤的神经保护作用. 中国医药工业杂志, 2005, 36 (12) : 760-762.
10.安祯祥. 治疗缺血性中风病的中药药理研究现状及前景展望, 贵阳中医学院学报, 2002, 24 (1) : 60-62.
1. Cipolla MJ, McCall AL, Lessov N, Porter JM, Kontos H. Reperfusion decreases myogenic reactivity and alters middle cerebral artery function after focal cerebral ischemia in rats. Stroke. 1997;28:176–180.
2. Faraci FM. Vascular protection. Stroke. 2003;34:327–329.
3. Kidwell CS, Liebeskind DS, Starkman S, Saver JL. Trends in acute ischemic stroke trials through the 20th century. Stroke. 2001;32: 1349–1359.
4. Zachary I, Mathur A, Yla-Herttuala S, Martin J. Vascular protection: a novel nonangiogenic cardiovascular role for vascular endothelial growth factor. Arterioscler Thromb Vasc Biol. 2000;20:1512–1520.
5. Faggiotto A, Paoletti R. State-of-the-art lecture. Statins and blockers of the renin-angiotensin system: vascular protection beyond their primary mode of action. Hypertension. 1999;34:987–996.
6. Shah PK, Kaul S, Nilsson J, Cercek B. Exploiting the vascular protective effects of high-density lipoprotein and its apolipoproteins: an idea whose time for testing is coming, part II. Circulation. 2001;104:2498–2502.
7. Schiffrin EL. Effects of antihypertensive drugs on vascular remodeling: do they predict outcome in response to antihypertensive therapy? Curr Opin Nephrol Hypertens. 2001;10:617–624.
8. Tzemos N, Lim PO, MacDonald TM. Nebivolol reverses endothelial dysfunction in essential hypertension: a randomized, double-blind, crossover study. Circulation. 2001;104:511–514.
9. Ferro A. Statins and vascular protection: a “radical” view. J Hypertens. 2002;20:359–361.
10. Vaughan DE. AT(1) receptor blockade and atherosclerosis: hopeful insights intovascular protection. Circulation. 2000;101:1496–1497.
11. Lammert E, Cleaver O, Melton D. Induction of pancreatic differentiation by signals from blood vessels. Science. 2001;294:564–567.
12. Matsumoto K, Yoshitomi H, Rossant J, Zaret KS. Liver organogenesis promoted by endothelial cells prior to vascular function. Science. 2001; 294:559–563.
13. Dirnagl U, Iadecola C, Moskowitz MA. Pathobiology of ischemic stroke: an integrated view. Trends Neurosci. 1999;22:391–397.
14. Paulson OB, Strandgaard S, Edvinsson L. Cerebral autoregulation. Cerebrovasc Brain Metab Rev. 1990;2:161–192.
15. Strandgaard S, Paulson OB. Pathophysiology of stroke. J Cardiovasc Pharmacol. 1990;15(suppl 1):S38–S42.
16. Cipolla MJ, Curry AB. Middle cerebral artery function after stroke: the threshold duration of reperfusion for myogenic activity. Stroke. 2002;33: 2094–2099.
17. Cipolla MJ, Osol G, Kontos HA. Vascular smooth muscle actin cytoskeleton in cerebral artery forced dilatation. Stroke. 1998;29: 1223–1228.
18. Cipolla MJ, Lessov N, Hammer ES, Curry AB. Threshold duration of ischemia for myogenic tone in middle cerebral arteries: effect on vascular smooth muscle actin. Stroke. 2001;32:1658–1664.
19. Cipolla MJ, Lessov N, Clark WM, Haley EC Jr. Postischemic attenuation of cerebral artery reactivity is increased in the presence of tissue plasminogen activator. Stroke. 2000;31:940–945.
20. Lipton P. Ischemic cell death in brain neurons. Physiol Rev. 1999;79: 1431–1568.
21. Kontos CD, Wei EP, Williams JI, Kontos HA, Povlishock JT. Cytochemical detection of superoxide in cerebral inflammation and ischemia in vivo. Am J Physiol. 1992;263:H1234–H1242.
22. Kontos HA. Oxygen radicals in cerebral ischemia: the 2001 Willis Lecture. Stroke. 2001;32:2712–2716.
23. Nelson CW, Wei EP, Povlishock JT, Kontos HA, Moskowitz MA. Oxygen radicalsin cerebral ischemia. Am J Physiol. 1992;263: H1356–H1362.
24. Weisbrot-Lefkowitz M, Reuhl K, Perry B, Chan PH, Inouye M, Mirochnitchenko O. Overexpression of human glutathione peroxidase protects transgenic mice against focal cerebral ischemia/reperfusion damage. Brain Res Mol Brain Res. 1998;53:333–338.
25. Zhao Q, Pahlmark K, Smith ML, Siesjo BK. Delayed treatment with the spin trap alpha-phenyl-N-tert-butyl nitrone (PBN) reduces infarct size following transient middle cerebral artery occlusion in rats. Acta Physiol Scand. 1994;152:349–350.
26. Murakami K, Kondo T, Kawase M, Li Y, Sato S, Chen SF, Chan PH. Mitochondrial susceptibility to oxidative stress exacerbates cerebral infarction that follows permanent focal cerebral ischemia in mutant mice with manganese superoxide dismutase deficiency. J Neurosci. 1998;18: 205–213.
27. Clark WM, Rinker LG, Lessov NS, Lowery SL, Cipolla MJ. Efficacy of antioxidant therapies in transient focal ischemia in mice. Stroke. 2001; 32:1000–1004.
28. De La Cruz JP, Guerrero A, Gonzalez-Correa JA, Arrebola MM, de la Cuesta SF. Antioxidant effect of acetylsalicylic and salicylic acid in rat brain slices subjected to hypoxia. J Neurosci Res. 2004;75:280–290.
29. Matsuo Y, Mihara S, Ninomiya M, Fujimoto M. Protective effect of endothelin type A receptor antagonist on brain edema and injury after transient middle cerebral artery occlusion in rats. Stroke. 2001;32: 2143–2148.
30. Macrae IM, Robinson MJ, Graham DI, Reid JL, McCulloch J. Endothelin-1-induced reductions in cerebral blood flow: dose dependency, time course, and neuropathological consequences. J Cereb Blood Flow Metab. 1993;13:276–284.
31. Stanimirovic DB, Bertrand N, McCarron R, Uematsu S, Spatz M. Arachidonic acid release and permeability changes induced by endothelins in human cerebromicrovascular endothelium. Acta Neurochir Suppl (Wien). 1994;60:71–75.
32. Zhang Z, Chopp M. Vascular endothelial growth factor and angiopoietins in focal cerebral ischemia. Trends Cardiovasc Med. 2002;12:62–66.
33. Yancopoulos GD, Davis S, Gale NW, Rudge JS, Wiegand SJ, Holash J.Vascular-specific growth factors and blood vessel formation. Nature. 2000;407:242–248.
34. Gursoy-Ozdemir Y, Bolay H, Saribas O, Dalkara T. Role of endothelial nitric oxide generation and peroxynitrite formation in reperfusion injury after focal cerebral ischemia. Stroke. 2000;31:1974–1980.
35. Zhang ZG, Zhang L, Croll SD, Chopp M. Angiopoietin-1 reduces cerebral blood vessel leakage and ischemic lesion volume after focal cerebral embolic ischemia in mice. Neuroscience. 2002;113:683–687.
36. Thurston G, Rudge JS, Ioffe E, Zhou H, Ross L, Croll SD, Glazer N, Holash J, McDonald DM, Yancopoulos GD. Angiopoietin-1 protects the adult vasculature against plasma leakage. Nat Med. 2000;6:460–463.
37. Hayashi T, Noshita N, Sugawara T, Chan PH. Temporal profile of angiogenesis and expression of related genes in the brain after ischemia. J Cereb Blood Flow Metab. 2003;23:166–180.
38. Rothwell NJ, Hopkins SJ. Cytokines and the nervous system II: actions and mechanisms of action. Trends Neurosci. 1995;18:130–136.
39. Nawashiro H, Tasaki K, Ruetzler CA, Hallenbeck JM. TNF-alpha pretreatment induces protective effects against focal cerebral ischemia in mice. J Cereb Blood Flow Metab. 1997;17:483–490.
40. Sairanen T, Carpen O, Karjalainen-Lindsberg ML, Paetau A, Turpeinen U, Kaste M, Lindsberg PJ. Evolution of cerebral tumor necrosis factor-alpha production during human ischemic stroke. Stroke. 2001;32: 1750–1758.
41. Vila N, Castillo J, Davalos A, Chamorro A. Proinflammatory cytokines and early neurological worsening in ischemic stroke. Stroke. 2000;31: 2325–2329.
42. Lindsberg PJ, Hallenbeck JM, Feuerstein G. Platelet-activating factor in stroke and brain injury. Ann Neurol. 1991;30:117–129.
43. Gong C, Qin Z, Betz AL, Liu XH, Yang GY. Cellular localization of tumor necrosis factor alpha following focal cerebral ischemia in mice. Brain Res. 1998;801:1–8.
44. Tilton RG, Berens KL. Functional role for selectins in the pathogenesis of cerebral ischemia. Drug News Perspect. 2002;15:351–357.
45. Hess DC, Howard E, Cheng C, Carroll J, Hill WD, Hsu CY. Hypertonic mannitol loading of NF-_B transcription factor decoys in human brain microvascular endothelial cells blocks upregulation of ICAM-1 editorial comment. Stroke. 2000;31:1179–1186.
46. Mocco J, Choudhri T, Huang J, Harfeldt E, Efros L, Klingbeil C, Vexler V, Hall W, Zhang Y, Mack W, Popilskis S, Pinsky DJ, Connolly ES Jr. HuEP5C7 as a humanized monoclonal anti-E/P-selectin neurovascular protective strategy in a blinded placebo-controlled trial of nonhuman primate stroke. Circ Res. 2002;91:907–914.
47. D’Ambrosio AL, Pinsky DJ, Connolly ES. The role of the complement cascade in ischemia/reperfusion injury: implications for neuroprotection. Mol Med. 2001;7:367–382.
48. Di Napoli M. Systemic complement activation in ischemic stroke. Stroke. 2001;32:1443–1448.
49. Huang J, Kim LJ, Mealey R, Marsh HC Jr, Zhang Y, Tenner AJ, Connolly ES Jr, Pinsky DJ. Neuronal protection in stroke by an sLexglycosylated complement inhibitory protein. Science. 1999;285:595–599.
50. del Zoppo GJ. In stroke, complement will get you nowhere. Nat Med. 1999;5:995–996.
51. Rosenberg GA, Kornfeld M, Estrada E, Kelley RO, Liotta LA, Stetler-Stevenson WG. TIMP-2 reduces proteolytic opening of blood-brain barrier by type IV collagenase. Brain Res. 1992;576:203–207.
52. Rosenberg GA, Estrada EY, Dencoff JE. Matrix metalloproteinases and TIMPs are associated with blood-brain barrier opening after reperfusion in rat brain. Stroke. 1998;29:2189–2195.
53. Rosenberg GA, Cunningham LA, Wallace J, Alexander S, Estrada EY, Grossetete M, Razhagi A, Miller K, Gearing A. Immunohistochemistry of matrix metalloproteinases in reperfusion injury to rat brain: activation of MMP-9 linked tostromelysin-1 and microglia in cell cultures. Brain Res. 2001;893:104–112.
54. Heo JH, Lucero J, Abumiya T, Koziol JA, Copeland BR, del Zoppo GJ. Matrix metalloproteinases increase very early during experimental focal cerebral ischemia. J Cereb Blood Flow Metab. 1999;19:624–633.
55. Asahi M, Asahi K, Jung JC, del Zoppo GJ, Fini ME, Lo EH. Role for matrix metalloproteinase 9 after focal cerebral ischemia: effects of gene knockout and enzyme inhibition with BB-94. J Cereb Blood Flow Metab. 2000;20:1681–1689.
56. Chang DI, Hosomi N, Lucero J, Heo JH, Abumiya T, Mazar AP, del Zoppo GJ. Activation systems for latent matrix metalloproteinase-2 are upregulated immediately after focal cerebral ischemia. J Cereb Blood Flow Metab. 2003;23:1408–1419.
57. del Zoppo GJ, Schmid-Schonbein GW, Mori E, Copeland BR, Chang CM. Polymorphonuclear leukocytes occlude capillaries following middle cerebral artery occlusion and reperfusion in baboons. Stroke. 1991;22: 1276–1283.
58. Iadecola C. Bright and dark sides of nitric oxide in ischemic brain injury. Trends Neurosci. 1997;20:132–139.
59. Lerouet D, Beray-Berthat V, Palmier B, Plotkine M, Margaill I. Changes in oxidative stress, iNOS activity and neutrophil infiltration in severe transient focal cerebral ischemia in rats. Brain Res. 2002;958:166–175.
60. Zhu DY, Deng Q, Yao HH, Wang DC, Deng Y, Liu GQ. Inducible nitric oxide synthase expression in the ischemic core and penumbra after transient focal cerebral ischemia in mice. Life Sci. 2002;71:1985–1996.
61. Croll SD, Wiegand SJ. Vascular growth factors in cerebral ischemia. Mol Neurobiol. 2001;23:121–135.
62. Plate KH, Beck H, Danner S, Allegrini PR, Wiessner C. Cell type specific upregulation of vascular endothelial growth factor in an MCA-occlusion model of cerebral infarct. J Neuropathol Exp Neurol. 1999;58:654–666.
63. Huang Z, Chen K, Huang PL, Finklestein SP, Moskowitz MA. bFGF ameliorates focal ischemic injury by blood flow-independent mechanisms in eNOS mutant mice.Am J Physiol. 1997;272:H1401–H1405.
64. Ay H, Ay I, Koroshetz WJ, Finklestein SP. Potential usefulness of basic fibroblast growth factor as a treatment for stroke. Cerebrovasc Dis. 1999;9:131–135.
65. Beck H, Acker T, Wiessner C, Allegrini PR, Plate KH. Expression of angiopoietin-1, angiopoietin-2, and tie receptors after middle cerebral artery occlusion in the rat. Am J Pathol. 2000;157:1473–1483.
66. Lin TN, Wang CK, Cheung WM, Hsu CY. Induction of angiopoietin and tie receptor mRNA expression after cerebral ischemia-reperfusion. J Cereb Blood Flow Metab. 2000;20:387–395.
67. Laitinen M, Zachary I, Breier G, Pakkanen T, Hakkinen T, Luoma J, Abedi H, Risau W, Soma M, Laakso M, Martin JF, Yla-Herttuala S. VEGF gene transfer reduces intimal thickening via increased production of nitric oxide in carotid arteries. Hum Gene Ther. 1997;8:1737–1744.
68. Servos S, Zachary I, Martin JF. VEGF modulates NO production: the basis of a cytoprotective effect? Cardiovasc Res. 1999;41:509–510.
69. Tsurumi Y, Murohara T, Krasinski K, Chen D, Witzenbichler B, Kearney M, Couffinhal T, Isner JM. Reciprocal relation between VEGF and NO in the regulation of endothelial integrity. Nat Med. 1997;3:879–886.
70. Wheeler-Jones C, Abu-Ghazaleh R, Cospedal R, Houliston RA, Martin J, Zachary I. Vascular endothelial growth factor stimulates prostacyclin production and activation of cytosolic phospholipase A2 in endothelial cells via p42/p44 mitogen-activated protein kinase. FEBS Lett. 1997;420: 28–32.
71. Chen J, Zhu RL, Nakayama M, Kawaguchi K, Jin K, Stetler RA, Simon RP, Graham SH. Expression of the apoptosis-effector gene, Bax, is upregulated in vulnerable hippocampal CA1 neurons following global ischemia. J Neurochem. 1996;67:64–71.
72. Hara A, Iwai T, Niwa M, Uematsu T, Yoshimi N, Tanaka T, Mori H. Immunohistochemical detection of Bax and Bcl-2 proteins in gerbil hippocampusfollowing transient forebrain ischemia. Brain Res. 1996; 711:249–253.
73. Sairanen TR, Lindsberg PJ, Brenner M, Siren AL. Global forebrain ischemia results in differential cellular expression of interleukin-1beta (IL-1beta) and its receptor at mRNA and protein level. J Cereb Blood Flow Metab. 1997;17:1107–1120.
74. Saito T, Nagao T, Okabe M, Saito K. Neurochemical and histochemical evidence for an abnormal catecholamine metabolism in the cerebral cortex of the Long-Evans cinnamon rat before excessive copper accumulation in the brain. Neurosci Lett. 1996;216:195–198.
75. Abid MR, Guo S, Minami T, Spokes KC, Ueki K, Skurk C, Walsh K, Aird WC. Vascular endothelial growth factor activates PI3K/Akt/Forkhead signaling in endothelial cells. Arterioscler Thromb Vasc Biol. 2004;24:294–300.
76. Zachary I. VEGF signaling: integration and multi-tasking in endothelial cell biology. Biochem Soc Trans. 2003;31:1171–1177.
77. Cai J, Ahmad S, Jiang WG, Huang J, Kontos CD, Boulton M, Ahmed A. Activation of vascular endothelial growth factor receptor-1 sustains angiogenesis and Bcl-2 expression via the phosphatidylinositol 3-kinase pathway in endothelial cells. Diabetes. 2003;52:2959–2968.
78. The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group. Tissue plasminogen activator for acute ischemic stroke. N Engl J Med. 1995;333:1581–1587.
79. del Zoppo GJ, Higashida RT, Furlan AJ, Pessin MS, Rowley HA, Gent M. PROACT: a phase II randomized trial of recombinant pro-urokinase by direct arterial delivery in acute middle cerebral artery stroke. Stroke. 1998;29:4–11.
80. Sherman DG, Atkinson RP, Chippendale T, Levin KA, Ng K, Futrell N, Hsu CY, Levy DE. Intravenous ancrod for treatment of acute ischemic stroke: the STAT Study: a randomized controlled trial. Stroke Treatmentwith Ancrod Trial. JAMA. 2000;283:2395–2403.
81. Gladstone DJ, Black SE, Hakim AM. Toward wisdom from failure: lessons from neuroprotective stroke trials and new therapeutic directions. Stroke. 2002;33:2123–2136.
82. Jonas S, Aiyagari V, Vieira D, Figueroa M. The failure of neuronal protective agents versus the success of thrombolysis in the treatment of ischemic stroke. The predictive value of animal models. Ann N Y Acad Sci. 2001;939:257–267.
83. George SJ. Therapeutic potential of matrix metalloproteinase inhibitors in atherosclerosis. Expert Opin Investig Drugs. 2000;9:993–1007.
