【作者】 张合亮；

【导师】 马廉亭； 龚杰；

【作者基本信息】 南方医科大学 ， 神经外科学， 2010， 博士

【摘要】 概述缺血性中风是以脑局部血液循环障碍、血流量减少为特征的一种病变,可造成不同程度的神经功能缺失。据统计,缺血性中风70%以上是由颅内或颅外大血管的急性阻塞引起。因此,尽快恢复缺血区的血流灌注是急性缺血性中风治疗的基本原则。溶栓治疗正是在此基础上建立起来的行之有效的方法,旨在通过闭塞动脉的再通来恢复缺血区的血流灌注,以拯救尚存活力的脑组织,改善病人预后。目前,以组织型纤溶酶原激活剂为基础的急性期溶栓治疗,是唯一通过美国食品及药物管理局认证的方法,已被越来越多的临床医生接受并应用于临床。然而,闭塞动脉溶栓再通后造成的缺血区过渡灌注,恶性脑水肿和症状性出血(再灌注损伤)却严重制约了该项措施的临床应用。特别是症状性出血,较高的发生率(6%)和严重的后果迫使医学工作者对这一方法重新审视。尽管试验结果再次证实了急性期溶栓治疗的疗效,但严格的治疗时间窗限制,却使临床上大多数病人不能从中受益,缺血再灌注损伤成为急性缺血性中风溶栓治疗应用的最大障碍。脑循环是包括颅内外血管在内的完整统一体,软脑膜动脉以上各级血管可产生整个脑循环系统50%以上的循环阻力,在血压出现波动时可以已通过动脉特有的肌源性反应调整循环阻力,维持循环稳定。血压依赖式舒缩反应是脑动脉自主调节的主要机制,是由管壁平滑肌细胞的舒缩活动完成的,血压上升平滑肌细胞收缩,血压下降平滑肌细胞舒张。急性脑缺血后,受累动脉肌源性反应受损,脑血管自主调节功能下降；而组织型纤溶酶原激活剂则可加重这种功能损伤。动脉收缩功能受损,动脉管腔不能相应收缩,使其丧失作为阻力血管对血流的限制作用,再灌注后将造成供血区的过渡灌注。动物实验证实,过渡灌注是梗塞动脉再通后的必发事件,与恶性脑水肿和继发出血密切相关,是缺血再灌注损伤发生的重要原因。控制性再灌注可在一定程度上补偿因缺血造成的动脉调节功能下降,限制缺血区的血流量,避免过渡灌注的发生,从而达到减轻或避免缺血再灌注损伤发生的目的。目前,控制性再灌注作为预防缺血再灌注损伤发生的有效措施已被广泛应用于许多组织、器官(如心肌、四肢、肺、肾和肠组织等)。然而,由于Willis动脉环的存在,局部脑血流的控制技术相对困难(特别是对于体型较小的动物),因此控制性再灌注在实验性脑缺血的治疗方面尚未见报道。临床和基础研究多采用降低系统血压的方法来减轻缺血后脑组织的过渡灌注,并且已在缺血再灌注损伤的预防上取得了一定的疗效。然而,系统血压的降低在缓解缺血区过渡灌注的同时,不可避免的会对其它脑区和其它组织器官造成影响,因此,更为理想的措施应该是局部血流限制技术。基于此,我们设计出了局部脑血流控制技术(部分再灌注技术),探讨部分再灌注对脑缺血再灌注损伤的保护性作用。本课题分二部分进行。第一部分研究了缺血和组织型纤溶酶原激活剂对脑动脉调节功能的影响,实验分二步进行：实验一对急性脑缺血后受累动脉自主调节功能的变化进行了研究；实验二研究了缺血联合组织型纤溶酶原激活剂对脑血管自主调节功能的影响。第二部分研究了血流限制技术联合组织型纤溶酶原激活剂对超时间窗大鼠血栓栓塞性脑梗死的治疗作用,实验分三步进行：实验一研究了部分再灌注对大鼠脑缺血后脑血流和继发出血的影响；实验二探讨了部分再灌注联合组织型纤溶酶原激活剂对局灶性脑缺血后脑血流和缺血再灌注损伤的影响；实验三研究了血流限制技术联合组织型纤溶酶原激活剂对超时间窗大鼠血栓栓塞性脑梗死的治疗作用。第一部分缺血和组织型纤溶酶原激活剂对脑动脉调节功能的影响实验一大鼠急性脑缺血后缺血区动脉调节功能的变化目的：研究急性脑缺血后缺血区动脉调节功能的变化,为部分再灌注技术的应用建立坚实的理论基础。方法：采用雄性Wistar大鼠制作局灶性脑缺血模型,2h后取受累侧大脑中动脉游离血管段,应用压力型小动脉测量仪测量压力和血管活性物质诱发的动脉直径变化,据此计算出膨胀性、肌源性张力及对5-羟色胺和乙酰胆碱的反应性,与对照组对比。结果：低压区(20mmHg、40mmHg和60mmHg时)缺血动脉肌源性张力增加(P<均0.01 vs.对照组),直径缩小(P<均0.05 vs.对照组)；高压区(100mmHg和120mmHg时),肌源性张力下降(P均<0.01 vs.对照组),动脉易膨胀(P均<0.01 vs.对照组),直径增大(P均<0.05 vs.对照组)；生理压力区(80mmHg),肌源性张力和动脉直径变化不明显(P均>0.05)。缺血后动脉对5羟色胺和乙酰胆碱反应性下降(5-HT:F=97.596, P=0.000; ACh:F=35.793, P=0.000)。结论：脑缺血可引起受累动自动调节功能下降,使其丧失作为阻力血管对血流的限制作用,再通后易出现下游微循环的过度灌注,易造成脑组织缺血再灌注损伤。实验二缺血联合组织型纤溶酶原激活剂对大鼠脑动脉调节功能的影响目的：研究缺血和组织型纤溶酶原激活剂对脑动脉调节功能的影响,探讨急性缺血性中风溶栓治疗后继发出血的机制,为部分再灌注联合组织型纤溶酶原激活剂治疗急性脑缺血建立坚实的理论基础。方法：动物随机分为假手术组(对照组,n=6)、组织型纤溶酶原激活剂灌注组(rtPA组,n=6)、缺血组(ISC组,n=8)和缺血并组织型纤溶酶原激活剂灌注组(rtPA-缺血组,n=8)。线栓法制作大鼠大脑中动脉梗塞模型,90min后游离术侧大脑中动脉,测量压力和血管活性物质诱发的动脉直径变化,据此计算出肌源性张力及对5-羟色胺和乙酰胆碱的反应性。结果：在压力由60mmHg升至120mmHg时,对照组肌源性反应强烈,肌源性张力明显增加(P=0.000)；而其它3组肌源性反应消失,肌源性张力下降(P均<0.05vs.其它3组)。对照组动脉直径最小(P均<0.05 vs.其它3组),rtPA-缺血组最大(P均<0.05 vs.其它3组)。与对照组相比,其它3组动脉对5羟色胺和乙酰胆碱的反应性下降(P均<0.05),rtPA-缺血组下降最明显(P<0.05 vs.其它3组)。结论：脑缺血可引起受累动脉自动调节功能下降,组织型纤溶酶原激活剂则可加重这种功能损伤。功能受损的动脉丧失其作为阻力血管对血流的调控作用,造成缺血区的过度灌注,血液通过断裂的微血管壁流出形成出血。第二部分血流限制技术联合组织型纤溶酶原激活剂对超时间窗大鼠血栓栓塞性脑梗死的治疗作用实验一部分再灌注对大鼠局灶性脑缺血后脑血流和继发出血的影响背景和目的：控制性再灌注作为治疗缺血再灌注损伤的措施,已被成功的应用于其它许多组织、器官。本文通过研究缺血后部分再灌注对脑血流和继发出血的影响,探讨控制性再灌注在脑缺血再灌注损伤中的治疗作用。方法：应用Wistar大鼠制作大脑中动脉急性缺血模型,3小时后随机分组,分别给予部分再灌注、完全再灌注和无灌注治疗。1小时后对各组脑血流、脑水肿和继发出血进行对比。结果：再通后,完全灌注组出现了早期的过渡灌注(127±16%),随之CBF快速下降,呈持续低灌注状态；部分再灌注组避免了早期的过渡灌注(85±17%)。再灌注1小时后,完全再灌注组半球血红素含量(689±196μg)和半球重量比(1.44±0.10)均高于对照组(323±17μg和1.20±0.08),差异具有统计学意义(P均<0.05)。与对照组相比,部分再灌注组并不加重继发出血(384±74μg)和脑水肿程度(1.30±0.12),差异不具统计学意义(P均>0.05)；而且,与对照组相比,明显减轻继发出血和脑水肿(P均<0.05)。结论：再灌注损伤是脑缺血发生后行闭塞动脉再通治疗的主要障碍,部分再灌注技术作为一种新颖的方法,可以有效阻止脑缺血后早期的过渡灌注,从而可以减轻继发出血和脑水肿的发生。实验二部分再灌注联合重组纤溶酶原激活剂对局灶性脑缺血后脑血流和缺血再灌注损伤的影响背景和目的：再灌注损伤是急性缺血性中风溶栓治疗的主要并发症,严重限制了溶栓治疗的临床应用。缺血后过渡灌注作为再灌注损伤的关键因素,在急性缺血性中风中经常发生,而且也是颈动脉狭窄病人行颈动脉内膜剥脱或血管内支架成形术术后常见并发症。有报道证实：在颈动脉内膜剥脱术中,施行“逐渐再血管化”技术,可以避免血流突然增加造成的过渡灌注,进而预防缺血再灌注损伤的发生。基于此种理论,我们设计应用部分再灌注技术,探讨部分再灌注技术联合重组纤溶酶原激活剂对大鼠大脑中动脉急性闭塞再通后脑血流、继发出血和脑水肿的影响。方法：制作缺血3小时的大鼠大脑中动脉急性梗死模型,采用单纯部分再灌注部分再灌注联合重组纤溶酶原激活剂、完全再灌注及完全再灌注联合重组纤溶酶原激活剂等方法治疗。并对各组脑血流,半球血红素含量和半球重量比进行比较。结果：部分再灌可以阻止缺血后早期的过渡灌注(部分再灌注各组vs.完全再灌注各组,P均<0.05)；减轻再通后脑水肿程度,与完全再灌注各组相比差异均具有统计学意义(P均<0.05)。与comp组相比,part组半球血红素含量明显下降(P=0.045)。而且,与完全再灌注联合重组纤溶酶原激活剂组相比,部分再灌注联合重组纤溶酶原激活剂组能显著改善后期低灌注状态(P=0.032)。结论：部分再灌注联合重组纤溶酶原激活剂可有效防止缺血3小时后再灌注相关的早期过渡灌注,缓解后期继发性低灌注,减轻继发出血和脑水肿程度。实验三血流限制技术联合重组纤溶酶原激活剂对超时间窗大鼠血栓栓塞性脑梗死的治疗作用目的：探讨血流限制技术联合重组纤溶酶原激活剂对超时间窗大鼠血栓栓塞性脑梗死的治疗作用方法：大鼠大脑中动脉血栓栓塞4小时后,分别应用血流限制技术联合重组纤溶酶原激活剂、重组纤溶酶原激活剂和生理盐水(对照组)治疗。治疗后6小时对神经功能缺损程度和脑梗死体积进行评估,对比。结果：丝线插入后即刻,par-rtPA组CBF较插入前明显改善(CBF t=-2.668, P=0.037).6小时后,血流限制技术联合重组纤溶酶原激活剂组脑梗死体积百分比显著低于单纯重组纤溶酶原激活剂组(5.4±2.6%vs.19.4±4.3％,P<0.05)和对照组(5.4±2.6%vs.13.3±3.1％,P<0.05).结论：血流限制技术联合重组纤溶酶原激活剂是一种治疗超时间窗血栓栓塞性脑梗死的有效方法。更多还原

【Abstract】 IntroductionIschemic stroke is a dynamic process triggered by the sudden loss of blood circulation to an area of the brain, resulting in a corresponding loss of neurologic function. It has been estimated that approximately 70% of ischemic strokes are caused by acute occlusion of a large intracranial or extracranial cerebral artery. Thus urgent reperfusion therapy, aimed at restoration or improvement of perfusion to the ischemic area, is a principle therapeutic target in the acute phase of ischemic stroke. Thrombolytic stroke therapy is based on the "recanalization hypothesis," i.e., that reopening of occluded vessels improves clinical outcome in acute ischemic stroke through regional reperfusion and salvage of threatened tissues. Indeed, intravenous administration of recombinant tissue plasminogen activator (rtPA) as a procedure for thrombolysis is currently the only FDA-approved medical therapy for treatment of patients with acute ischemic stroke. Though intravenous thrombolytic therapy for acute stroke is now generally accepted, it has also been challenged. Recanalization may exacerbate tissue injury by promoting reperfusion (hyperperfusion), excessive cerebral edema, and symptomatic intracerebral hemorrhage (SIH), producing a so-called "cerebral reperfusion injury". The SIH was seen in 6% of patients treated in the original National Institute of Neurological Disorders and Stroke trial that led to rtPA’s approval and was subsequently confirmed in multiple postmarketing studies. Ischemic/reperfusion injury is the major obstacle toward the goal of extending this treatment modality to a higher proportion of patients with acute ischemic stroke.The cerebral circulation is a unique vascular bed in that the extracranial and large intracranial pial vessels contribute significantly to cerebrovascular resistance and respond myogenically to changes in perfusion pressure. The contractile response of the cerebral arteries to pressure contributes to autoregulation of cerebral blood flow (CBF) and is facilitated by vascular smooth muscle that contracts to increased pressure and relaxes in response to decreased pressure. It has been demonstrated that reperfusion following transient cerebral ischemia causes autoregulatory loss and hyperperfusion due to a diminished CVR that exposes the microcirculation to excessive perfusion pressure. In addition, treatment with tissue plasminogen activator significantly diminished myogenic reactivity in isolated cerebral arteries, a result that was additive if arteries were exposed to ischemia. Several animal studies suggest that the restoration of cerebral circulation consistently results in a hyperperfusion phase. Hyperperfusion may contribute to the development of reperfusion injury by causing brain edema or hemorrhage.Controlled reperfusion as an efficiency strategy has been used successfully in many organs and tissues for preventing reperfusion jury, such as pulmonary injury, myocardium, limb and kidney. However, since it is difficult to control CBF of a certain region without affecting other areas in cerebral circulation than in others, due to the Willis Cycle, this method was not studied in treatment of experimental cerebral ischemia. Based on this, we developed a partial reperfusion technique which can limit the focal cerebral blood flow to test whether controlled reperfusion reduces the cerebral ischemic/reperfusion injury and improves neurological deficit, and the potential mechanisms involved will also be discussed.This paper includes two parts. Effects of ischemia and rtPA on cerebrovascular autoregulation were studied in part one, which including 2 experients:In first experiment, changes of affected artery autoregulation after focal cerebral ischemia were studied. In second experiment, combined effects of cerebral ischemia and tissue plaminogen activator on cerebrovascular autoregulation were investigated. Therapeutic effects of partial reperfusion technique combined with rtPA on experimental cerebral ischemia were studied in part two, which including 3 experiments:In first experiment, we investigated wherther partial reperfusion is effective on preventing reperfusion injury following experimental cerebral ischemia. In second experiment, combined effects of partial reperfusion and tissue plasminogen activator on cerebral blood flow and reperfusion injury after transient cerebral ischemia were studied. In third experiment, we confirmed that partial reperfusion reduces ischemia/reperfusion injury following delayed thrombolysis in rat embolic stroke model.Part-ⅠEffects of ischemia and rtPA on cerebrovascular autoregulationExperiment-1 Changes of affected artery autoregulation after focal cerebral ischemiaObjective:To investigate the effect of cerebral ischemia on affected artery autoregulation, and evaluate the probably mechanisms of ischaemia-reperfusion injury.Methods:Intraluminal suture ischemic model was used by occlusion of left middle cerebral artery in rats. Two hours later, the middle cerebral artery segments were isolated from both ischemia and control groups for measurement of changes in vessel diameter induced by increasing pressure and vasoactive compounds. And then, distensibility, myogenic tone, reactivity to 5-HT and ACh were calculated and compared between groups.Results:In lower pressure range (20mmgHg,40mmHg and 60mmHg) ischemic vessels showed an increased myogenic tone (P<0.01 vs control group, for all) and decreased diameter (P<0.05 vs control group, for all); In higher pressure range (100mmgHg and 120mmHg), ischemic vessels showed an increased diameter(P<0.05 vs control group, for both), distensibility (P<0.01 vs control group, for both) and decreased myogenic tone(P<0.01 vs control group, for both); In normal pressure range (80mmHg), myogenic tone and diameter were not altered after ischemia (P>0.05 vs control group, for both). Both groups constricted to 5-HT and dilated to ACh, however, the response was significantly diminished after ischemia(5-HT: F=97.596, P=0.000; ACh:F=35.793, P=0.000).Conclusion:These findings demonstrate that contractile and diastolic function of affected artery was impaired after ischemia, a result that may contribute to ischemia-reperfusion injury by losing upstream cerebrovascular resistance and increasing perfusion on the microcirculation.Experimental-2 Combined effects of ischemia with tissue plaminogen activator on cerebrovascular autoregulationObjective:To investigate the combined effects of ischemia and tissue plasminogen activator on vasoactive function of affected arteries and evaluate the probably mechanisms of the secondary hemorrhage after thrombolysis.Methods:Intraluminal suture ischemic model was used by occlusion of right middle cerebral artery in rats. Ninety minutes later, the middle cerebral artery segments were isolated for measurement of changes in vessel diameter induced by increasing pressure and vasoactive compounds. Arteries were either nonischemic (control; n=6), nonischemic and perfused with rtPA for 50-60mins (rtPA; n=6), ischemic (ISC; n=8), or ischemic and perfused with rtPA for 50-60mins (rtPA-ISC; n=8). And then, myogenic tone, reactivity to 5-HT and ACh were calculated.Results:Control arteries increased myogenic tone from 60mmHg to 120mmHg (P=0.000) and responded myogenic reactivity, all other groups decreased tone at 120mmHg (P<0.05 vs.control for all) and lost myogenic reactivity; Diameters of control arteries showed the least (P<0.05 vs.all three others), rtPA-ISC group showed the largest (P<0.05vs.all three others); All arteries constricted to 5-HT and dilated to ACh, however, the response was diminished in all groups compared with control (P<0.05 for all other groups); rtPA-ISC responded the least (P<0.05 vs. all other groups).Conclusion:These findings demonstrate that vasoactive function of affected artery was impaired after ischemia, and the impaired function was increased in the presence of tissue plasminogen activator. A result that may contribute to intracerebral heamorrhage by losing upstream cerebrovascular resistance and increasing perfusion on the microcirculation after thrombolysis.Part-ⅡTherapeutic effects of partial reperfusion technique combined with rtPA on experimental cerebral ischemiaExperiment-1 Effects of partial reperfusion on cerebral blood flow and the secondary heamorrhage after focal cerebral ischemia in rats Background and objective:Controlled reperfusion as an efficiency strategy has been used successfully in other organs and tissues for preventing reperfusion jury. In the present study, we investigated whether partial reperfusion is effective on preventing reperfusion injury following experimental cerebral ischemia.Methods:A total of 19 Male Wistar rats who suffered 3 hours cerebral ischemia were treated with partial reperfusion, complete reperfusion or no reperfusion (control).Results:A transient postischemic hyperperfusion (127±16%) followed by sustained reduction in CBF was recorded by laser Doppler flowmetry in complete reperfusion group, and partial reperfusion inhibited this postischemic hyperperfusion (85±17%). After 1 h of reperfusion, complete reperfusion produced a significant intracerebral heamorrhage (689±196μg vs.323±17μg; P<0.05) and brain edema (1.44±0.10 vs. 1.20±0.08; P<0.05) compared with control, and partial reperfusion didn’t exacerbate intracerebral haemorrhage (384±74μg) and brain edema (1.30±0.12) compared with control group (P>0.05, for both), it furthermore reduced the complete reperfusion related haemorrhage and brain edema (P<0.05 vs. complete reperfusion, for both).Conclusion:Reperfusion injury is the major obstacle of recanalization therapy for patients with acute ischemic stroke. This study demonstrats that partial reperfusion is an effective strategy for prevention of postischemic hyperperfusion and the secondary hemorrhage and brain edema.Experiment-2 Combined effects of partial reperfusion and tissue plasminogen activator on cerebral blood flow and reperfusion injury after transient cerebral ischemia in rats Background and objective:Reperfusion injury is the major obstacle of thrombolysis therapy for patients with acute ischemic stroke. Postischemic hyperperfusion as a risk factor of reperfusion injury is recognized as a frequent occurrence in stroke and a severe complication of endarterectomy. Stepwise revascularization which prevents abrupt improvement in reperfusion is an efficiency strategy for preventing hyperperfusion and the secondary reperfusion injury in endarterectomy procedures. Based on the same principle, blood flow limitation technique was introduced and used to determine the combined effects of partial reperfusion and tissue plasminogen activator on cerebral blood flow and the development of the secondary haemorrhage and brain edema after transient cerebral ischemia.Methods:Rats who suffered 3 h middle cerebral artery occlusion were treated with partial reperfusion (part), partial reperfusion with tissue plasminogen activator (part-rtPA), complete reperfusion (comp) and complete reperfusion with tissue plasminogen activator (comp-rtPA). Cerebral blood flow, secondary haemorrhage and brain edema were compared among the groups.Results:Partial reperfusion significantly inhibited the early postischemic hyperperfusin (partial groups vs. complete groups, P<0.05 for all) and reduced brain edema (partial groups vs. complete groups, P<0.05 for all). Compared with comp group, part group reduced the secondary hemorrhage (P=0.045). Furthermore, part-rtPA also improved the subsequent hypoperfusion compared with comp-rtPA group (P=0.032).Conclusion:Partial reperfusion combined with tissue plasminogen administration is an efficiency therapeutic strategy for 3 hour’s cerebral ischemia in rats. Experiment-3 Therapeutic effect of controlled reperfusion combined with tissue plasminogen activator on delayed embolic stroke in ratsObjective:To study the effects of controlled reperfusion combined with tissue plasminogen activator on delayed embolic stroke in rats.Methods:Controlledl reperfusion technique was used combined with tissue plasminogen activator to treat rats who suffered 4 h embolic stroke. Neurological function and infarct volume were evaluated and compared with rtPA (just using tissue plasminogen activator) and control group (saline alone) at 6 h after treatment.Results:Partial reperfusion combined tissue plasminogen activator group showed a improved (CBF t=-2.668, P=0.037) than before suture insertion, showed a better neurological function (9±5 vs.14±4, P<0.05) and a lower infarct volume% (5.4±2.6% vs.19.4±4.3%, P<0.05) than rtPA group, and also showed a lower infarct volume%(5.4±2.6% vs.13.3±3.1%, P<0.05) compared control group.Conclusion:Partial reperfusion combined with rtPA administration is an efficiency therapeutic strategy for delayed embolic stroke in rats.更多还原