低频超声辅助尿激酶溶栓治疗大鼠脑梗死的作用机制及对MMP-9和TIMP-1表达的影响
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摘要
研究背景：脑梗死是临床常见病多发病,具有高发生率、高致残率、高死亡率的特点,严重危害人类健康和增加社会及家庭的负担。研究脑梗死的发病机制、寻求有效的治疗方法,建立稳定的、可重复的、符合人类血栓栓塞性脑梗死的动物模型是十分必要的。常用的制作局灶性脑梗死模型的方法有线栓法、光化学诱导法等,这些模型能不同程度反映脑梗死的病理生理变化,但与人类卒中有很大不同。而血栓栓塞法制作的局灶性脑梗死模型更接近于人类缺血性卒中的病理过程。大脑中动脉是脑梗死的好发部位,其侧枝循环不丰富,因此普遍认为大脑中动脉栓塞模型是局灶性脑缺血的标准模型。
目的：建立符合人类血栓栓塞性脑梗死病理生理过程和适合用于溶栓研究的大鼠大脑中动脉闭塞动物模型。
方法：用健康成年Wistar雄性大鼠167只,体重280-320g。随机抽取21只作为假手术组,其余的应用自体血栓栓塞法制作大脑中动脉闭塞动物模型。模型制作：10%水合氯醛350mg／Kg腹腔麻醉,分离颈内动脉,颈外动脉,颈总动脉,然后从颈外动脉切口处逆行插入24号静脉留置针,假手术组注入生理盐水,其余组注入符合标准的血栓。大鼠苏醒后,应用NSS进行神经行为功能评分,大于7分为模型成功标准。将制模成功的大鼠随机分为梗死组、尿激酶组、超声+尿激酶组。尿激酶组在造模成功后3小时尾静脉注射尿激酶1.5万U,超声+尿激酶组在造模成功后3小时尾静脉注射尿激酶1万U,同时头颅部的相应部位给予1：5,800KHZ、1.2W／cm2的超声波照射30分钟。梗死组于尾静脉注射0.4ml生理盐水。24小时后每组分别进行取材,红四氮唑(TTC)染色,进行脑梗死体积测定。每组选1只治疗24小时后立即取脑,超微切片后,行透射电子显微镜检查。
结果：该研究共用Wistar大鼠167只,其中假手术组21只,用于造模146只,造模成功共63只,造模成功率约43.2%,随机分为梗死组、尿激酶组、超声+尿激酶组各21只。造模成功的大鼠表现为：左侧Horner征,角膜反射、惊恐反射、羽翼反射减弱或消失,右侧肢体瘫痪,爬行困难,不能直线爬行,原地绕圈,提起尾部时出现右上肢屈曲上抬,平衡障碍等,NSS评分>7分,认为造模成功。假手术组动物给药前后评分均为0；梗死组给药后24小时NSS评分为9.19±0.93,与给药前9.28±1.14比较,未见明显统计学差异(P＞0.05)。假手术组均未见梗死灶,梗死组均在左侧大脑半球可见不被染色的梗死灶,主要位于左侧大脑中动脉供血区,以皮质为主,基底节及海马也有受累,脑梗死体积为94.90±11.09 mm3。透射电镜显示：假手术组神经元无缺血表现,核大而圆,细胞器完整,梗死组可见基底节和皮层广泛神经元坏死表现,核膜溶解、消失,核碎裂；神经元正常结构严重破坏。
结论：通过颈外动脉逆行注入符合标准的自体血栓可成功建立大鼠大脑中动脉血栓栓塞性脑梗死动物模型。病理观察证实该模型符合人类脑梗死的基本特点,是适合用于溶栓研究的可靠的动物模型。
研究背景：脑梗死病理过程包括脑缺血的炎症反应,钙超载,自由基损害,兴奋性氨基酸毒性及缺血半暗带的神经细胞调亡。急性脑梗死时,脑组织缺血30-90分钟后,不可逆的脑损伤即开始出现,脑梗死最根本的治疗措施是使闭塞的脑血管早期再通,在缺血脑组织出现不可逆损害之前及时恢复供血,因此早期和超早期溶栓治疗可能是最有效,最有希望的治疗方法。目前尿激酶和r-tPA静脉溶栓是治疗超早期脑梗死的主要方法之一,由于溶栓时间窗短,较高的出血危险性,影响其在临床方面的广泛应用。超声溶栓是20世纪90年代发展起来的一项新技术,低频超声通过其机械作用、热学作用、空泡化作用,协助药物溶栓,已经成为近几年研究的热点之一。肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)主要是单核巨噬细胞产生,其它许多细胞,如神经系统的神经元、星形细胞和小胶质细胞受到刺激后也能产生TNF-α。TNF-α是一种炎性细胞因子,生物学活性广泛,在脑缺血时表达增加,增加血脑屏障通透性,促进内皮细胞黏附分子表达和多形核细胞、巨噬细胞、内皮细胞和神经胶质细胞释放炎症介质。核转录因子(nuclear factor k B, NF-k B)是Rel蛋白家族中的一组转录因子,是由多肽链p50和p65二个蛋白亚基组成同源或异源性二聚体。NF-κB作为一种多向性转录调节蛋白,能调节多种炎症因子的表达和细胞凋亡,在缺血中起了重要作用。
目的：通过观察应用低频超声辅助尿激酶静脉溶栓治疗大鼠脑梗死前后TNF-α,NF-κB的变化,探讨低频超声辅助尿激酶静脉溶栓治疗脑梗死过程中的作用机制。
方法：应用血栓栓塞法制备Wistar大鼠脑梗死模型,根据随机数字表随机平均分为3组(梗死组,尿激酶组,超声+尿激酶组),每组21只。大鼠苏醒后,采用NSS进行神经行为功能评分；尿激酶组在造模成功后3小时尾静脉注射尿激酶1.5万U,超声+尿激酶组在造模成功后3小时尾静脉注射尿激酶1万U,同时头颅部的相应部位给予1：5,800KHZ、1.2W／cm2的超声波照射30分钟。梗死组于尾静脉注射0.4ml生理盐水。24小时后每组分别进行灌注取材,采用TTC染色法测定脑梗死体积,采用免疫组化法监测TNF-α和NF-κβ的表达。
结果：每组大鼠均在治疗前和治疗24h后分别进行NSS评分。假手术组动物苏醒后活动正常,NSS评分均为0分；各组治疗前评分差异不明显(F=0.405,P＞0.05)；梗死组治疗前后NSS评分差异无统计学意义(t=0.288,P＞0.05)；尿激酶组治疗后24h评分明显低于溶栓前(t=7.840,P＜0.01)；超声+尿激酶组治疗后评分明显低于溶栓前(t=11.176,P＜0.01)。各组治疗后评分有显著差异(F=81.98,P＜0.01)；尿激酶组和超声+尿激酶组评分显著低于梗死组(q=12.387,q=17.633,P＜0.01),超声+尿激酶组低于尿激酶组(q=5.246,P＜0.05)；三组梗死灶体积差异显著(F=117.81,P＜0.01),梗死组的梗死灶体积为(94.90±11.09)mm3,主要位于左侧大脑中动脉供血区,以皮质为主,基底节及海马也有受累；尿激酶组梗死灶体积为(59.24±8.25)mm3,显著小于梗死组(q=15.87 P<0.01)；超声+尿激酶组梗死灶体积为(48.19±11.30)mm3,显著小于梗死组(q=20.76 P＜0.01)与尿激酶组(q=4.89,P＜0.05)。三组TNF-α表达差异显著(F=89.36,P＜0.01),尿激酶组和超声+尿激酶组TNF-α表达显著高于梗死组(q=18.001,14.004,P＜0.01),超声+尿激酶组低于尿激酶组(q=3.997,P＜0.05)；三组NF-κB表达差异显著(F=34.89,P＜0.01),尿激酶组和超声+尿激酶组NF-κB表达显著高于梗死组(q=11.619,q=7.654 P＜0.01),超声+尿激酶组低于尿激酶组(q=3.966,P＜0.05)。
结论：尿激酶在脑梗死超早期有肯定的溶栓作用。低频超声具有显著的协助尿激酶溶栓作用,可以减少尿激酶用量,达到与大量尿激酶溶栓的同等效果。TNF-α与NF-κB共同在脑梗死后缺血损伤中起着重要作用。
研究背景：基质金属蛋白酶(matrix metalloproteinases, MMPs), MMPs是一类Zn2+离子依赖的蛋白水解酶,参与细胞外基质的代谢。MMP-9又称明胶酶-B,是基质金属蛋白酶的一种,主要由中性粒细胞、单核细胞、血管内皮细胞合成与分泌。生理条件下MMP-9的活性受到严格的调控,当其以酶原的形式从胞内分泌到胞外后,经酶解过程而激活。MMP-9可能降解血管外基质(ECM)的关键成分如Ⅳ胶原,破坏血管,导致渗漏和破裂。在脑缺血及溶栓治疗过程中,MMP-9可能通过降解ECM,改变血管通透性,损伤血脑屏障,参与血管源性脑水肿、参与了溶栓引起的脑缺血／再灌注损伤、出血过程。
目的：通过研究大鼠局灶性脑缺血应用溶栓前后MMP-9及其抑制剂体TIMP-1表达的变化情况,探讨MMP-9在脑梗死缺血损伤和溶栓后出血性转化中的作用。
方法：应用血栓栓塞法制备Wistar大鼠脑梗死模型,分别给予尿激酶、尿激酶+低频超声治疗,采用NSS评价治疗前后神经功能,采用TTC测定脑梗死体积,采用免疫组化法监测MMP-9和TIMP-1蛋白的表达,应用原位杂交法测定MMP-9和TIMP-1 mRNA的表达。
结果：尿激酶组和超声+尿激酶组MMP-9蛋白表达均显著高于梗死组(F=54.46,q=14.443,q=9.883,P<0.01),超声+尿激酶组低于尿激酶组(q=4.560,P<0.05)。尿激酶组和超声+尿激酶组MMP-9 mRNA表达均显著高于梗死组(F=98.03,q=18.612,q=15.199,P<0.01),超声+尿激酶组低于尿激酶组(q=3.412,P＜0.05)。尿激酶组和超声+尿激酶组TIMP-1蛋白表达均显著高于梗死组(F=47.15,q=13.091,q=10.13,P<0.01)。超声+尿激酶组高于尿激酶组(q=2.953,P＜0.05)。尿激酶组和超声+尿激酶组TIMP-1 mRNA表达均显著高于梗死组(F=71.77,q=10.029,q=16.843,P<0.01)。超声+尿激酶组高于尿激酶组(q=6.814,P＜0.05)。
结论：MMP-9和TIMP-1的平衡紊乱可能在脑梗死后溶栓后脑缺血损伤和出血转化中起着重要作用。通过抑制MMP-9活性或应用其抑制剂是减轻脑缺血损伤,减少脑梗死溶栓后的出血并发症的新途径。
Background:Cerebral infarction is a clinical common and frequently-occurring disease with high incidence, high morbidity and high mortality, characterized by a serious threat to human health and increase the burden on society and the family. To establish a stable, repeatable animal model consistent with human thromboembolic cerebral infarction is very necessary for studing the pathogenesis of cerebral infarction and seeking effective treatment methods. Commonly used method of producing focal cerebral infarction model are cable bolt method and photochemical induction method. These models can reflect varying degrees of changes in the pathophysiology of cerebral infarction, but very different human stroke. The thrombus embolization produced by focal cerebral infarction model is more close to the human pathological process of ischemic stroke. The middle cerebral artery is the most frequent sites of cerebral infarction and its collateral circulation is not rich。it was generally accepted that the middle cerebral artery occlusion model is the standard model of focal cerebral ischemia.
Objective:To establish a middle cerebral artery occlusion model consistent with the human cerebral thromboembolic pathophysiological processes and suitable for thrombolysis study.
Methods:167 healthy adult male Wistar rats weighing 280-320g were choosed. 21 rats selected as the sham operation group randomly. The rest were used to make animal models of middle cerebral artery occlusion by Applicatiing autologous blood clot embolization. Model Making:First anesthesiaing rats by intraperitoneal injecting 10% chloral hydrate of animals, separating the internal carotid artery, external carotid artery, carotid artery, and then inserted retrograde venous catheter from the external carotid artery, sham-operated group injected with normal saline.The remaining group of injected standard thrombosis. After the rats regained consciousness, to applicating NSS for neurobehavioral function score, criteria of success model is more than 7. The success of the system model rats were randomly divided into infarction, urokinase group, ultrasound+UK group. After the success in modeling 3 hours, urokinase group was injected urokinase-15000 U through the tail vein, ultrasonic+urokinase group was injected with urokinase 10000 U, while giving the Department of ultrasonic irradiation in the head for 30 minutes. Infarction group was injected 0.4ml saline through the tail vein. Infarct volume in each group separately was determined by TTC staining after 24 hours. One rat in each group was selected for the AMD chips, line transmission electron microscope after 24 hours immediately.
Results:The study shared 167 Wistar rats, in which sham operation group was 21,146 rats for making model,63 successful model were produced, the success rate was about 43.2%.These successful models were randomly divided into infarction, urokinase group, Ultrasound+UK group. The successful modeling of rats were as follows:the left Horner syndrome, corneal reflex, panic reflex, reflex wings weakening or disappearance of the right limb paralysis, crawling difficulties and can not crawl a straight line, in-situ circling, speaking of the right upper limb flexion occurs when the tail swarmed, balance disorders. NSS score of sham-operated group were 0 before and after administration; NSS score of infarction group before and 24 hours after administration were9.19±0.93 and 9.28±1.14, there was no significant statistical difference (P> 0.05). Sham-operated group showed no infarction, infarction were visible in the left cerebral hemisphere of infarction groups. The main cortex, basal ganglia and hippocampus weree mainly involved.infarct size was 94.90±11.09 mm3. Transmission electron microscopy revealed:neurons in sham-operated group was normal with the performance of the large round neuons. Basal ganglia and cortex infarction was seen in infarction group with extensive neuronal necrosis performance.
Conclusion:Pathological observation confirmed that the middle cerebral artery thromboembolism model in rat according with the basic characteristics of the human cerebral infarction, is a reliable animal model suitable for thrombolysis research.
Background:pathological processes of cerebral infarction includes inflammation, calcium overload, free radical damage, excitatory amino acid toxicity and neural cell apoptosis of ischemic penumbra. Irreversible brain damage that is beginning to emerge after cerebral ischemia 30-90 minutes. the most fundamental measure of cerebral infarction treatment is to make an early cerebral artery recanalization in ischemic brain tissue before irreversible damage. So early and ultra-early thrombolytic therapy may be the most effective and most promising treatment of cerebral infarction. Currently urokinase and r-tPA intravenous thrombolysis is considered as one of the principal treatment of acute cerebral infarction, but the shorter thrombolysis time window and the higher risk of bleeding affect the clinical application. Ultrasound thrombolysis is a new technology. low-frequency ultrasound helping drug thrombolysis through its mechanical effects, thermal effects, vacuolization role has become one of the hot research in recent years. Tumor necrosis factor-α(TNF-α) are produced mainly by monocyte-macrophage cells, many other cells, such as the nervous system neurons, astrocytes and microglia cells,can produce TNF-αafter being stimulated. TNF-αis an inflammatory cytokine with multiple biological activity,increased expression in the brain ischemia, increasing blood-brain barrier permeability, promoting endothelial cell adhesion molecule expression, the release of inflammatory mediators.Nuclear factor kB (NF-kB) is a group of transcription factors in Rel protein family, which is composed of p50 and p65 polypeptide chains. NF-kB, as a pleiotropic transcriptional regulatory protein, can regulate the expression of multiple inflammatory factors and apoptosis in ischemia.
Objective:To investigate the mechanism of the low-frequency ultrasound-enhanced intravenous thrombolysis with urokinase in cerebral infarction rats by observing the changes of TNF-αand NF-kB.
Methods:Wistar rats were prepared for thromboembolic stroke model. These successful models were randomly divided into 3 groups:infarction group, urokinase(UK) group, Ultrasound+UK group. After the rats regained consciousness, to applicating NSS for neurobehavioral function score, more than 7 is criteria of success model. After the success in modeling 3 hours, urokinase group was injected urokinase15000 U through the tail vein, ultrasonic+urokinase group was injected with urokinase 10000 U, while giving the Department of ultrasonic irradiation in the head for 30 minutes. Infarction group was injected 0.4ml saline through the tail vein. Infarct volume in each group separately was determined by TTC staining after 24 hours. Expressions of TNF-αand NF-kB were detected by immunohistochemistry.
Results:The rats were checked nerve functions through the NSS score before and post-treatment 24h. Sham-operated group NSS score was 0 points; pre-treatment score in each group was not significantly different (F= 0.405, P> 0.05); there were no significant difference in NSS score between before and after treatment in infarction group(t= 0.288, P> 0.05); NSS scores of urokinase group and ultrasound+UK group 24h after treatment were significantly lower than those of before (t= 7.84, t= 11.176,P<0.01). There were significantly different in the infarct volume of the three groups (F= 117.81, P<0.01), infarct volume of infarction group is (94.90±11.09) mm3, mainly located in the left middle cerebral artery area;infarct volume of UK group (59.24±8.25) mm3, significantly smaller than the infarction group (P<0.01); infarct volume of ultrasound+UK group (48.19±11.30) mm3, significantly smaller than those of infarction group and UK group (P<0.01). TNF-αexpression in both ultrasound+UK group and UK group were significantly higher than that in infarction group (P<0.01), NF-αexpression in ultrasound+UK group was lower than that in UK group (P<0.05). NF-kB expression in both ultrasound+UK group and UK group were significantly higher than that in infarction group (P<0.01), NF-kB expression in ultrasound+UK group was lower than that in UK group (P<0.05).
Conclusion:This study confirmed the urokinase thrombolysis effect in cerebral infarction. Low-frequency ultrasound has a significant role in assisting urokinase thrombolysis,can reduce the dosage of urokinase, and reduce the risk of bleeding complications. TNF-αand NF-kB play an important role on ischemic damage after acute cerebral infarction
Background:Matrix metalloproteases (MMPs) are a family of zinc-binding proteolytic enzymes involved in remodeling of the extracellular matrix The extracellular matrix molecules that contain collagen-Ⅳ, laminin, fibronectin and other glycoproteins constitute the basement membrane underlying the vasculature and play a critical role in maintaining the integrity of the blood-brain barrier (BBB).MMPs (MMP-9 in particular) have been previously implicated in the pathogenesis of stroke. Activation of MMP-9 has been shown to increase inflammatory infiltration, BBB permeability,brain edema, infarction volume via the degradation of collagen and laminin.
Objective:To study MMP-9 and its inhibitor TIMP-1 expression changes in focal cerebral infarction after application of thrombolytic therapy and to explore the role of MMP-9 on ischemia/reperfusion injury and hemorrhagic transformation.
Methods:Wistar rats were prepared for thromboembolic stroke model. These successful models were randomly divided into infarction, urokinase(UK) group, Ultrasound+UK group. After the success in modeling 3 hours, each group were given urokinase, urokinase+low-frequency ultrasound therapy seperatly. Nerve function before and after treatment were evaluated by using NSS, Infarct volume was measured using TTC staining. MMP-9 and TIMP-1 protein expressions were dectected by using immunohistochemistry, and MMP-9 and TIMP-1 mRNA expression were dectected by using in situ hybridization.
Results:MMP-9 protein and mRNA expression in urokinase group and ultrasound+ UK group, were significantly higher than that of infarction group (F=54.46, F=98.03,, P<0.01).MMP-9 protein and mRNA expression in ultrasound+urokinase group was lower than that of urokinase group (P<0.05). TIMP-1 protein and TIMP-1 mRNA expression in UK group and ultrasound+UK group were significantly higher than that of infarction group (F=47.15, F=71.77,P<0.01).TIMP-1 protein and TIMP-1 mRNA expression in ultrasound+UK group was higher than that of the UK group (P <0.05).
Conclusion:Balance between MMP-9 and TIMP-1 may be plays an important role on acute cerebral infarction following thrombolytic therapy. Cerebral ischemia/ reperfusion injury and hemorrhagic transformation maybe be reduced by inhibiting MMP-9 activity or application of their inhibitors.It is expected to become a new treatment for cerebral infarction.
目的：建立符合人类血栓栓塞性脑梗死病理生理过程和适合用于溶栓研究的大鼠大脑中动脉闭塞动物模型。
方法：用健康成年Wistar雄性大鼠167只,体重280-320g。随机抽取21只作为假手术组,其余的应用自体血栓栓塞法制作大脑中动脉闭塞动物模型。模型制作：10%水合氯醛350mg／Kg腹腔麻醉,分离颈内动脉,颈外动脉,颈总动脉,然后从颈外动脉切口处逆行插入24号静脉留置针,假手术组注入生理盐水,其余组注入符合标准的血栓。大鼠苏醒后,应用NSS进行神经行为功能评分,大于7分为模型成功标准。将制模成功的大鼠随机分为梗死组、尿激酶组、超声+尿激酶组。尿激酶组在造模成功后3小时尾静脉注射尿激酶1.5万U,超声+尿激酶组在造模成功后3小时尾静脉注射尿激酶1万U,同时头颅部的相应部位给予1：5,800KHZ、1.2W／cm2的超声波照射30分钟。梗死组于尾静脉注射0.4ml生理盐水。24小时后每组分别进行取材,红四氮唑(TTC)染色,进行脑梗死体积测定。每组选1只治疗24小时后立即取脑,超微切片后,行透射电子显微镜检查。
结果：该研究共用Wistar大鼠167只,其中假手术组21只,用于造模146只,造模成功共63只,造模成功率约43.2%,随机分为梗死组、尿激酶组、超声+尿激酶组各21只。造模成功的大鼠表现为：左侧Horner征,角膜反射、惊恐反射、羽翼反射减弱或消失,右侧肢体瘫痪,爬行困难,不能直线爬行,原地绕圈,提起尾部时出现右上肢屈曲上抬,平衡障碍等,NSS评分>7分,认为造模成功。假手术组动物给药前后评分均为0；梗死组给药后24小时NSS评分为9.19±0.93,与给药前9.28±1.14比较,未见明显统计学差异(P＞0.05)。假手术组均未见梗死灶,梗死组均在左侧大脑半球可见不被染色的梗死灶,主要位于左侧大脑中动脉供血区,以皮质为主,基底节及海马也有受累,脑梗死体积为94.90±11.09 mm3。透射电镜显示：假手术组神经元无缺血表现,核大而圆,细胞器完整,梗死组可见基底节和皮层广泛神经元坏死表现,核膜溶解、消失,核碎裂；神经元正常结构严重破坏。
结论：通过颈外动脉逆行注入符合标准的自体血栓可成功建立大鼠大脑中动脉血栓栓塞性脑梗死动物模型。病理观察证实该模型符合人类脑梗死的基本特点,是适合用于溶栓研究的可靠的动物模型。
研究背景：脑梗死病理过程包括脑缺血的炎症反应,钙超载,自由基损害,兴奋性氨基酸毒性及缺血半暗带的神经细胞调亡。急性脑梗死时,脑组织缺血30-90分钟后,不可逆的脑损伤即开始出现,脑梗死最根本的治疗措施是使闭塞的脑血管早期再通,在缺血脑组织出现不可逆损害之前及时恢复供血,因此早期和超早期溶栓治疗可能是最有效,最有希望的治疗方法。目前尿激酶和r-tPA静脉溶栓是治疗超早期脑梗死的主要方法之一,由于溶栓时间窗短,较高的出血危险性,影响其在临床方面的广泛应用。超声溶栓是20世纪90年代发展起来的一项新技术,低频超声通过其机械作用、热学作用、空泡化作用,协助药物溶栓,已经成为近几年研究的热点之一。肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)主要是单核巨噬细胞产生,其它许多细胞,如神经系统的神经元、星形细胞和小胶质细胞受到刺激后也能产生TNF-α。TNF-α是一种炎性细胞因子,生物学活性广泛,在脑缺血时表达增加,增加血脑屏障通透性,促进内皮细胞黏附分子表达和多形核细胞、巨噬细胞、内皮细胞和神经胶质细胞释放炎症介质。核转录因子(nuclear factor k B, NF-k B)是Rel蛋白家族中的一组转录因子,是由多肽链p50和p65二个蛋白亚基组成同源或异源性二聚体。NF-κB作为一种多向性转录调节蛋白,能调节多种炎症因子的表达和细胞凋亡,在缺血中起了重要作用。
目的：通过观察应用低频超声辅助尿激酶静脉溶栓治疗大鼠脑梗死前后TNF-α,NF-κB的变化,探讨低频超声辅助尿激酶静脉溶栓治疗脑梗死过程中的作用机制。
方法：应用血栓栓塞法制备Wistar大鼠脑梗死模型,根据随机数字表随机平均分为3组(梗死组,尿激酶组,超声+尿激酶组),每组21只。大鼠苏醒后,采用NSS进行神经行为功能评分；尿激酶组在造模成功后3小时尾静脉注射尿激酶1.5万U,超声+尿激酶组在造模成功后3小时尾静脉注射尿激酶1万U,同时头颅部的相应部位给予1：5,800KHZ、1.2W／cm2的超声波照射30分钟。梗死组于尾静脉注射0.4ml生理盐水。24小时后每组分别进行灌注取材,采用TTC染色法测定脑梗死体积,采用免疫组化法监测TNF-α和NF-κβ的表达。
结果：每组大鼠均在治疗前和治疗24h后分别进行NSS评分。假手术组动物苏醒后活动正常,NSS评分均为0分；各组治疗前评分差异不明显(F=0.405,P＞0.05)；梗死组治疗前后NSS评分差异无统计学意义(t=0.288,P＞0.05)；尿激酶组治疗后24h评分明显低于溶栓前(t=7.840,P＜0.01)；超声+尿激酶组治疗后评分明显低于溶栓前(t=11.176,P＜0.01)。各组治疗后评分有显著差异(F=81.98,P＜0.01)；尿激酶组和超声+尿激酶组评分显著低于梗死组(q=12.387,q=17.633,P＜0.01),超声+尿激酶组低于尿激酶组(q=5.246,P＜0.05)；三组梗死灶体积差异显著(F=117.81,P＜0.01),梗死组的梗死灶体积为(94.90±11.09)mm3,主要位于左侧大脑中动脉供血区,以皮质为主,基底节及海马也有受累；尿激酶组梗死灶体积为(59.24±8.25)mm3,显著小于梗死组(q=15.87 P<0.01)；超声+尿激酶组梗死灶体积为(48.19±11.30)mm3,显著小于梗死组(q=20.76 P＜0.01)与尿激酶组(q=4.89,P＜0.05)。三组TNF-α表达差异显著(F=89.36,P＜0.01),尿激酶组和超声+尿激酶组TNF-α表达显著高于梗死组(q=18.001,14.004,P＜0.01),超声+尿激酶组低于尿激酶组(q=3.997,P＜0.05)；三组NF-κB表达差异显著(F=34.89,P＜0.01),尿激酶组和超声+尿激酶组NF-κB表达显著高于梗死组(q=11.619,q=7.654 P＜0.01),超声+尿激酶组低于尿激酶组(q=3.966,P＜0.05)。
结论：尿激酶在脑梗死超早期有肯定的溶栓作用。低频超声具有显著的协助尿激酶溶栓作用,可以减少尿激酶用量,达到与大量尿激酶溶栓的同等效果。TNF-α与NF-κB共同在脑梗死后缺血损伤中起着重要作用。
研究背景：基质金属蛋白酶(matrix metalloproteinases, MMPs), MMPs是一类Zn2+离子依赖的蛋白水解酶,参与细胞外基质的代谢。MMP-9又称明胶酶-B,是基质金属蛋白酶的一种,主要由中性粒细胞、单核细胞、血管内皮细胞合成与分泌。生理条件下MMP-9的活性受到严格的调控,当其以酶原的形式从胞内分泌到胞外后,经酶解过程而激活。MMP-9可能降解血管外基质(ECM)的关键成分如Ⅳ胶原,破坏血管,导致渗漏和破裂。在脑缺血及溶栓治疗过程中,MMP-9可能通过降解ECM,改变血管通透性,损伤血脑屏障,参与血管源性脑水肿、参与了溶栓引起的脑缺血／再灌注损伤、出血过程。
目的：通过研究大鼠局灶性脑缺血应用溶栓前后MMP-9及其抑制剂体TIMP-1表达的变化情况,探讨MMP-9在脑梗死缺血损伤和溶栓后出血性转化中的作用。
方法：应用血栓栓塞法制备Wistar大鼠脑梗死模型,分别给予尿激酶、尿激酶+低频超声治疗,采用NSS评价治疗前后神经功能,采用TTC测定脑梗死体积,采用免疫组化法监测MMP-9和TIMP-1蛋白的表达,应用原位杂交法测定MMP-9和TIMP-1 mRNA的表达。
结果：尿激酶组和超声+尿激酶组MMP-9蛋白表达均显著高于梗死组(F=54.46,q=14.443,q=9.883,P<0.01),超声+尿激酶组低于尿激酶组(q=4.560,P<0.05)。尿激酶组和超声+尿激酶组MMP-9 mRNA表达均显著高于梗死组(F=98.03,q=18.612,q=15.199,P<0.01),超声+尿激酶组低于尿激酶组(q=3.412,P＜0.05)。尿激酶组和超声+尿激酶组TIMP-1蛋白表达均显著高于梗死组(F=47.15,q=13.091,q=10.13,P<0.01)。超声+尿激酶组高于尿激酶组(q=2.953,P＜0.05)。尿激酶组和超声+尿激酶组TIMP-1 mRNA表达均显著高于梗死组(F=71.77,q=10.029,q=16.843,P<0.01)。超声+尿激酶组高于尿激酶组(q=6.814,P＜0.05)。
结论：MMP-9和TIMP-1的平衡紊乱可能在脑梗死后溶栓后脑缺血损伤和出血转化中起着重要作用。通过抑制MMP-9活性或应用其抑制剂是减轻脑缺血损伤,减少脑梗死溶栓后的出血并发症的新途径。
Background:Cerebral infarction is a clinical common and frequently-occurring disease with high incidence, high morbidity and high mortality, characterized by a serious threat to human health and increase the burden on society and the family. To establish a stable, repeatable animal model consistent with human thromboembolic cerebral infarction is very necessary for studing the pathogenesis of cerebral infarction and seeking effective treatment methods. Commonly used method of producing focal cerebral infarction model are cable bolt method and photochemical induction method. These models can reflect varying degrees of changes in the pathophysiology of cerebral infarction, but very different human stroke. The thrombus embolization produced by focal cerebral infarction model is more close to the human pathological process of ischemic stroke. The middle cerebral artery is the most frequent sites of cerebral infarction and its collateral circulation is not rich。it was generally accepted that the middle cerebral artery occlusion model is the standard model of focal cerebral ischemia.
Objective:To establish a middle cerebral artery occlusion model consistent with the human cerebral thromboembolic pathophysiological processes and suitable for thrombolysis study.
Methods:167 healthy adult male Wistar rats weighing 280-320g were choosed. 21 rats selected as the sham operation group randomly. The rest were used to make animal models of middle cerebral artery occlusion by Applicatiing autologous blood clot embolization. Model Making:First anesthesiaing rats by intraperitoneal injecting 10% chloral hydrate of animals, separating the internal carotid artery, external carotid artery, carotid artery, and then inserted retrograde venous catheter from the external carotid artery, sham-operated group injected with normal saline.The remaining group of injected standard thrombosis. After the rats regained consciousness, to applicating NSS for neurobehavioral function score, criteria of success model is more than 7. The success of the system model rats were randomly divided into infarction, urokinase group, ultrasound+UK group. After the success in modeling 3 hours, urokinase group was injected urokinase-15000 U through the tail vein, ultrasonic+urokinase group was injected with urokinase 10000 U, while giving the Department of ultrasonic irradiation in the head for 30 minutes. Infarction group was injected 0.4ml saline through the tail vein. Infarct volume in each group separately was determined by TTC staining after 24 hours. One rat in each group was selected for the AMD chips, line transmission electron microscope after 24 hours immediately.
Results:The study shared 167 Wistar rats, in which sham operation group was 21,146 rats for making model,63 successful model were produced, the success rate was about 43.2%.These successful models were randomly divided into infarction, urokinase group, Ultrasound+UK group. The successful modeling of rats were as follows:the left Horner syndrome, corneal reflex, panic reflex, reflex wings weakening or disappearance of the right limb paralysis, crawling difficulties and can not crawl a straight line, in-situ circling, speaking of the right upper limb flexion occurs when the tail swarmed, balance disorders. NSS score of sham-operated group were 0 before and after administration; NSS score of infarction group before and 24 hours after administration were9.19±0.93 and 9.28±1.14, there was no significant statistical difference (P> 0.05). Sham-operated group showed no infarction, infarction were visible in the left cerebral hemisphere of infarction groups. The main cortex, basal ganglia and hippocampus weree mainly involved.infarct size was 94.90±11.09 mm3. Transmission electron microscopy revealed:neurons in sham-operated group was normal with the performance of the large round neuons. Basal ganglia and cortex infarction was seen in infarction group with extensive neuronal necrosis performance.
Conclusion:Pathological observation confirmed that the middle cerebral artery thromboembolism model in rat according with the basic characteristics of the human cerebral infarction, is a reliable animal model suitable for thrombolysis research.
Background:pathological processes of cerebral infarction includes inflammation, calcium overload, free radical damage, excitatory amino acid toxicity and neural cell apoptosis of ischemic penumbra. Irreversible brain damage that is beginning to emerge after cerebral ischemia 30-90 minutes. the most fundamental measure of cerebral infarction treatment is to make an early cerebral artery recanalization in ischemic brain tissue before irreversible damage. So early and ultra-early thrombolytic therapy may be the most effective and most promising treatment of cerebral infarction. Currently urokinase and r-tPA intravenous thrombolysis is considered as one of the principal treatment of acute cerebral infarction, but the shorter thrombolysis time window and the higher risk of bleeding affect the clinical application. Ultrasound thrombolysis is a new technology. low-frequency ultrasound helping drug thrombolysis through its mechanical effects, thermal effects, vacuolization role has become one of the hot research in recent years. Tumor necrosis factor-α(TNF-α) are produced mainly by monocyte-macrophage cells, many other cells, such as the nervous system neurons, astrocytes and microglia cells,can produce TNF-αafter being stimulated. TNF-αis an inflammatory cytokine with multiple biological activity,increased expression in the brain ischemia, increasing blood-brain barrier permeability, promoting endothelial cell adhesion molecule expression, the release of inflammatory mediators.Nuclear factor kB (NF-kB) is a group of transcription factors in Rel protein family, which is composed of p50 and p65 polypeptide chains. NF-kB, as a pleiotropic transcriptional regulatory protein, can regulate the expression of multiple inflammatory factors and apoptosis in ischemia.
Objective:To investigate the mechanism of the low-frequency ultrasound-enhanced intravenous thrombolysis with urokinase in cerebral infarction rats by observing the changes of TNF-αand NF-kB.
Methods:Wistar rats were prepared for thromboembolic stroke model. These successful models were randomly divided into 3 groups:infarction group, urokinase(UK) group, Ultrasound+UK group. After the rats regained consciousness, to applicating NSS for neurobehavioral function score, more than 7 is criteria of success model. After the success in modeling 3 hours, urokinase group was injected urokinase15000 U through the tail vein, ultrasonic+urokinase group was injected with urokinase 10000 U, while giving the Department of ultrasonic irradiation in the head for 30 minutes. Infarction group was injected 0.4ml saline through the tail vein. Infarct volume in each group separately was determined by TTC staining after 24 hours. Expressions of TNF-αand NF-kB were detected by immunohistochemistry.
Results:The rats were checked nerve functions through the NSS score before and post-treatment 24h. Sham-operated group NSS score was 0 points; pre-treatment score in each group was not significantly different (F= 0.405, P> 0.05); there were no significant difference in NSS score between before and after treatment in infarction group(t= 0.288, P> 0.05); NSS scores of urokinase group and ultrasound+UK group 24h after treatment were significantly lower than those of before (t= 7.84, t= 11.176,P<0.01). There were significantly different in the infarct volume of the three groups (F= 117.81, P<0.01), infarct volume of infarction group is (94.90±11.09) mm3, mainly located in the left middle cerebral artery area;infarct volume of UK group (59.24±8.25) mm3, significantly smaller than the infarction group (P<0.01); infarct volume of ultrasound+UK group (48.19±11.30) mm3, significantly smaller than those of infarction group and UK group (P<0.01). TNF-αexpression in both ultrasound+UK group and UK group were significantly higher than that in infarction group (P<0.01), NF-αexpression in ultrasound+UK group was lower than that in UK group (P<0.05). NF-kB expression in both ultrasound+UK group and UK group were significantly higher than that in infarction group (P<0.01), NF-kB expression in ultrasound+UK group was lower than that in UK group (P<0.05).
Conclusion:This study confirmed the urokinase thrombolysis effect in cerebral infarction. Low-frequency ultrasound has a significant role in assisting urokinase thrombolysis,can reduce the dosage of urokinase, and reduce the risk of bleeding complications. TNF-αand NF-kB play an important role on ischemic damage after acute cerebral infarction
Background:Matrix metalloproteases (MMPs) are a family of zinc-binding proteolytic enzymes involved in remodeling of the extracellular matrix The extracellular matrix molecules that contain collagen-Ⅳ, laminin, fibronectin and other glycoproteins constitute the basement membrane underlying the vasculature and play a critical role in maintaining the integrity of the blood-brain barrier (BBB).MMPs (MMP-9 in particular) have been previously implicated in the pathogenesis of stroke. Activation of MMP-9 has been shown to increase inflammatory infiltration, BBB permeability,brain edema, infarction volume via the degradation of collagen and laminin.
Objective:To study MMP-9 and its inhibitor TIMP-1 expression changes in focal cerebral infarction after application of thrombolytic therapy and to explore the role of MMP-9 on ischemia/reperfusion injury and hemorrhagic transformation.
Methods:Wistar rats were prepared for thromboembolic stroke model. These successful models were randomly divided into infarction, urokinase(UK) group, Ultrasound+UK group. After the success in modeling 3 hours, each group were given urokinase, urokinase+low-frequency ultrasound therapy seperatly. Nerve function before and after treatment were evaluated by using NSS, Infarct volume was measured using TTC staining. MMP-9 and TIMP-1 protein expressions were dectected by using immunohistochemistry, and MMP-9 and TIMP-1 mRNA expression were dectected by using in situ hybridization.
Results:MMP-9 protein and mRNA expression in urokinase group and ultrasound+ UK group, were significantly higher than that of infarction group (F=54.46, F=98.03,, P<0.01).MMP-9 protein and mRNA expression in ultrasound+urokinase group was lower than that of urokinase group (P<0.05). TIMP-1 protein and TIMP-1 mRNA expression in UK group and ultrasound+UK group were significantly higher than that of infarction group (F=47.15, F=71.77,P<0.01).TIMP-1 protein and TIMP-1 mRNA expression in ultrasound+UK group was higher than that of the UK group (P <0.05).
Conclusion:Balance between MMP-9 and TIMP-1 may be plays an important role on acute cerebral infarction following thrombolytic therapy. Cerebral ischemia/ reperfusion injury and hemorrhagic transformation maybe be reduced by inhibiting MMP-9 activity or application of their inhibitors.It is expected to become a new treatment for cerebral infarction.
引文
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