不完全性脊髓缺血损伤动物模型的建立及价值

目的 探讨不完全性脊髓缺血损伤动物模型的建立方法,为不完全性脊髓缺血损伤机制研究提供理想的载体. 方法 24只新西兰大白兔按照随机数字表法分为对照组及3根组、4根组,每组8只.对照组用于排除麻醉和手术对运动诱发电位的影响;3根组、4根组分别结扎3根、4根腰动脉.各组麻醉后记录基线诱发电位,手术/结扎后30 min、2d、7d记录诱发电位;麻醉清醒后、手术/结扎后2d、7d进行运动功能评分;手术/结扎后7d后取缺血中心区标本进行HE染色,镜下观察. 结果 3根组动物结扎后30 min诱发电位波幅与对照组比较差异有统计学意义(P＜0.05),结扎后2d、7d与对照组比较差异无统计学意义(P＞0.05);4根组动物结扎后30 min、2d、7d3个时间点诱发电位波幅与对照组比较差异均有统计学意义(P＜0.05).3组动物手术/结扎后30min、2d、7d3个时间点的潜伏期与对照组比较差异均无统计学意义(P＞0.05).各组动物运动功能评分结果与诱发电位波幅变化一致. 结论 结扎3根腰动脉可以造成可逆性不完全性脊髓缺血损伤,结扎4根腰动脉可以造成不可逆性不完全性脊髓缺血损伤.     

兔子脊髓缺血动物模型建立的解剖学基础

目的了解左肾动脉与动脉分叉间腰动脉的自身解剖学特点及腰动脉与腹主动脉、脊椎的位置关系,为腰动脉阻断建立脊髓缺血动物模型提供解剖学基础。方法 10只新西兰大白兔红色乳胶灌注后显微镜下追踪解剖左肾动脉和动脉分叉间的5条腰动脉,测量其本干长度及左右分支到椎间孔的长度,同时观察腰动脉的变异情况,及其与腹主动脉、脊椎的位置关系。10只新西兰大白兔分为缺血组和对照组,对照组5只不结扎腰动脉,缺血组5只分别结扎左肾动脉与动脉分叉间所有腰动脉,12h后进行运动诱发电位评估,并取脊髓缺血中心区标本进行HE染色镜下观察。结果腰动脉主干长度(6.897±3.764)mm,直径(1.211±0.145)mm,左和右分支长度分别为(104.832±2.610)mm和(11.003±2.687)mm,下4腰动脉的直径为(1.461±0.333)mm,下4与其他腰动脉直径比较差异有统计学意义(P0.05);缺血组5条腰动脉结扎后,运动诱发电位波形完全消失,HE染色脊髓前角正常运动神经元基本消失,可见坏死和凋亡样改变;对照组假手术前后均可记录到稳定的诱发电位波形,HE切片可见正常脊髓组织结构完整。结论兔腰动脉呈"人字形"分支,变异情况少,手术结扎方便,适于动物模型的制作。     

线栓法制作大鼠局灶性脑缺血再灌注损伤模型的改进

目的 研究一种稳定、简便的大鼠局灶性脑缺血再灌注损伤模型制作方法。方法 采用健康SD雄性大鼠，线栓法制作局灶性脑缺血再灌注损伤模型．结扎颈总动脉（CCA）及颈外动脉（ECA），不结扎翼腭动脉，用5．0头皮针于CCA结扎的远端刺一小口插入栓塞线造成缺血，拔出线栓形成再灌注。通过神经功能缺损评分、红四氮唑（TTC）染色和病理学检查等方法评价该模型的可靠性。结果动物麻醉后一般只需15min左右即可完成手术，术后大鼠神经功能缺损明显，TTC染色示脑梗塞区苍白，病理学检查显示典型病理表现。结论该方法缺血效果明确可靠，术式简便，手术时间短，不需具备显微手术操作技巧，是一种理想的制作局灶性脑缺血再灌注损伤模型的方法。     

栓线法大鼠局灶性脑缺血模型的研究

采用颈部旁侧手术入路，结扎右侧颈总动脉、颈外动脉及其分支后颈内动脉栓线法制作大鼠大脑中动脉闭塞再灌流损伤模型。闭塞成功率９４．１％，动物偏瘫症状评分在缺血２ｈ再灌流３ｄ后趋于稳定，病理改变以尾壳核损害最重。再灌流７ｄ脑梗塞体积为９７．８±９．４ｍｍ３，动物死亡率５９．４％。缺血２ｈ后再灌流先出现过渡灌注，而后呈持续性低灌注。本文模型勿需开颅，缺血效果可靠，对局灶性脑缺血的研究具有实用价值。     

实验性局灶性脑缺血不同脑区VEGF、VEGFR-1、2表达及意义

目的 通过探索血管内皮生长因子及其受体在局灶性脑缺血中的表达，进而探求血管内皮生长因子在局灶性脑缺血中的作用。方法 应用左侧颈总动脉结扎加缺氧诱导的方法，建立SD大鼠永久大脑中动脉闭塞模型，应用免疫组化方法检测血管内皮生长因子及其受体(VEGF、VEGFR-1、2)的表达，同时观察局灶性脑缺血后血管型成情况。结果 VEGF及VEGFR-1、2在局灶性脑缺血后6h表达增强，24h达高峰，在1周、2周恢复到对照水平。VEGF主要在缺血半影区(IP)神经元、胶质细胞及血管内皮细胞表达；VEGFR-1、2主要在缺血半影区血管内皮细胞表达。在缺血后48h半影区周边出现血管增生，1周后达高峰。结论 在局灶性脑缺血早期VEGF及VEGFR-1、2在神经细胞、胶质细胞、血管内皮细胞等均有表达，可促进缺血半影区的血管增生，对改善缺血半影区血供有重要作用。     

椎基底动脉系缺血时植物神经作用的实验研究

目的 通过不同方法建立椎基镀动脉系缺血动物模型，对缺血病灶区超微结构进行研究，比较与血管伴行的交感神经损伤与否、病灶内的病理形成的差异，探讨交感神经在缺血时的作用机理。方法 通过电凝和直接栓塞兔小脑后下动脉，建立不同的椎基底动脉系缺血的模型，对模型进行动态的脑干诱发电位监测，并以光镜、透射电镜检查病灶内的病理变化，结果 病理检查显示电凝损害了闭塞部位的植物神经后，沿血管走行的对血管起支配作用的植物神经部分发生溃变，梗塞灶内充血性、缺血性梗塞同时存在，而栓塞组梗塞灶内毛细血管旁的交感神经纤维形态完整，梗塞均表现为缺血性，动态的脑干听觉诱发电位检查发现，由于支配血管的交感神经损害，发生了脑干的继发性损害，缺血性损害范围增大，预后恶化，结论 交感神经对脑血管的支配作用在缺血性中风的病理过程中起着重要的作用。它维持了正常情况下脑血管的张力，在 缺血、缺氧时能有效地增加脑血流，限制局灶性梗塞范围的扩大，对邻近的脑组织起着保护性作用。     

建立椎管内静脉高压动物模型的手术方法及稳定性评价*

背景：构建稳定的脊髓静脉高压动物模型可为临床研究脊髓血管病变提供可靠的平台,课题组前期已成功构建了兔脊髓静脉高压模型。 目的：在前期研究基础上改进手术方法和围手术期处理,建立长期稳定的兔脊髓静脉高压模型。 方法：48只新西兰大白兔随机分为急性期组、短期组、中期组、长期组,每组12只兔又分为模型兔8只和假手术兔4只。模型兔通过开腹侧侧吻合兔左肾动静脉,形成动静脉瘘,结扎后腔静脉远端和近端导致动脉血异常引流至腰静脉、椎管内静脉丛形成脊髓淤血和脊髓静脉高压。采用Jacobs法对后肢功能评级,Reuter法评估脊髓感觉运动反射功能,每组动物到期后对模型兔行MRI扫描、经股动脉DSA检查动静脉瘘口的通畅,并灌注解剖取脊髓行病理学检查以判定模型符合要求。 结果与结论：32只模型兔存活29只,生存率91%,动静脉瘘口通畅率79%,但长期组瘘口通畅率低,仅29%。术后模型后肢运动、感觉功能均有所减退。模型兔脊髓MRI表现为脊髓相应阶段的水肿;病理检查在光镜下见脊髓实质内淋巴细胞的浸润、胶质细胞的增生、髓内血管的玻璃样变性和神经元细胞变性,符合脊髓静脉高压的脊髓病理变化;透射电镜观察可见髓鞘板层的松散、薄髓纤维内线粒体数目增加和神经元的固缩。提示成功建立模拟人类脊髓血管畸形的脊髓静脉高压机制导致脊髓损伤的动物模型,该模型的可靠性和稳定性较高。     

兔大脑中动脉梗死模型的建立

目的：建立一种简单、实用的兔大脑中动脉阻塞（MCAO）模型。方法：20只新西兰大白兔，随机分为A、B两组，A组为电凝阻断右侧大脑中动脉形成局灶性脑梗死模型，B组为结扎右侧大脑中动脉形成局灶性脑梗死模型。利用MRI、TTC染色和光镜评价MCA闭塞效果。A、B两组动物模型制成后10小时进行SE序列冠状位检查：T1WI和T2WI。结果：A、B两组动物MCAO后10小时MRI、TTC染色和光镜检查均证实脑梗死存在。MCAO后10小时脑MRI均显著MCA供血区脑组织呈长T2信号。A组方法制备兔MCAO操作简单易学，手术时间短，1h；B组方法操作复杂，手术时间长，2h。结论：用电凝阻断法制备兔大脑中动脉阻塞模型是一种较理想的方法。     

局灶性脑缺血耐受和星形胶质细胞反应

目的　研究短暂性局灶性脑缺血预处理对永久性局灶性脑缺血的保护作用 ,及最佳预处理时间剂量 ,并探讨星形胶质细胞在脑缺血耐受中的反应。方法　采用开颅方法阻断大鼠大脑中动脉 ,通过观察大鼠脑梗死后神经功能损伤状况、脑梗死体积分析及病理形态学变化 ,评价不同的缺血预处理时间剂量 (10分钟、2 0分钟、30分钟 )对永久性局灶性脑缺血的保护作用。采用胶质纤维酸性蛋白 (GFAP)免疫组化法观察星形胶质细胞在脑缺血耐受中的反应。结果　与对照组相比 ,缺血预处理 2 0分钟未引起明显的神经元损伤 ,但使永久性局灶性脑缺血后神经功能损伤减轻 ,梗死体积明显减小 (P <0 .0 1)。免疫组化显示 ,2 0分钟缺血预处理组及重复缺血组星形胶质细胞在损伤预处理侧广泛激活。结论　 2 0分钟局灶性脑缺血预处理能够有效诱导脑缺血耐受。星形胶质细胞的激活可能与脑缺血耐受中神经元的存活相关。     

兔大脑中动脉梗死模型的建立

目的 :建立一种简单、实用的兔大脑中动脉阻塞 (MCAO)模型。方法 :2 0只新西兰大白兔 ,随机分为A、B两组 ,A组为电凝阻断右侧大脑中动脉形成局灶性脑梗死模型 ,B组为结扎右侧大脑中动脉形成局灶性脑梗死模型。利用MRI、TTC染色和光镜评价MCA闭塞效果。A、B两组动物模型制成后 10小时进行SE序列冠状位检查 :T1WI和T2 WI。结果 :A、B两组动物MCAO后 10小时MRI、TTC染色和光镜检查均证实脑梗死存在。MCAO后 10小时脑MRI均显示MCA供血区脑组织呈长T2 信号。A组方法制备兔MCAO操作简单易学 ,手术时间短 ,1h ;B组方法操作复杂 ,手术时间长 ,2h。结论 :用电凝阻断法制备兔大脑中动脉阻塞模型是一种较理想的方法     

Curcumin protects against ischemic spinal cord injury The pathway effect

Inducible nitric oxide synthase and N-methyI-D-aspartate receptors have been shown to participate in nerve cell injury during spinal cord ischemia. This study observed a protective effect of curcumin on ischemic spinal cord injury. Models of spinal cord ischemia were established by ligating the lumbar artery from the left renal artery to the bifurcation of the abdominal aorta. At 24 hours after model establishment, the rats were intraperitoneally injected with curcumin, Reverse transcrip- tion-polymerase chain reaction and immunohistochemical results demonstrated that after spinal cord ischemia, inducible nitric oxide synthase and N-methyI-D-aspartate receptor mRNA and protein expression significantly increased. However, curcumin significantly decreased inducible nitric oxide synthase and N-methyI-D-aspartate receptor mRNA and protein expression in the ischemic spinal cord. Tadov scale results showed that curcumin significantly improved motor function of the rat hind limb after spinal cord ischemia. The results demonstrate that curcumin exerts a neuroprotective ef- fect against ischemic spinal cord injury by decreasing inducible nitric oxide synthase and N-methyI-D-aspartate receptor expression.     

Spinal cord ischemia. Development of a model in the mouse

BACKGROUND AND PURPOSE: Spinal cord ischemia with resulting paraplegia is a devastating complication of thoracoabdominal aortic surgery. Experimental models of spinal cord ischemia have been developed in primate, dog, pig, rabbit, and rat with variable reproducibility, but none has been developed in mouse. Because genetically engineered mice have become important to examine the impact of specific genes in ischemic pathophysiology, we sought to develop a reproducible mouse model of spinal cord ischemia. METHODS: C57BL/6NCrlBR mice were subjected to cross-clamping of the aortic arch, left subclavian artery, and internal mammary artery for 9 minutes (group A; n=8) or 11 minutes (group B; n=29) followed by reperfusion for 24 or 48 hours. Mean distal arterial blood pressure (left femoral artery) and lumbar (L1) spinal cord blood flow (laser-Doppler flowmetry) were measured for the duration of the procedure. The arterial blood supply of the spinal cord was visualized by intravascular perfusion of carbon black ink. We evaluated motor function in the hind limbs at 0, 1, 3, 6, and 24 hours after reperfusion using a rating scale of 0 (normal function) to 6 (total absence of movement). Spinal cord histopathology was evaluated after 24 and 48 hours of reperfusion by Luxol fast blue-hematoxylin and eosin. RESULTS: The vascular anatomy of the mouse and human spinal cord appeared similar in that blood was supplied by 1 anterior and 2 posterior spinal arteries and heterosegmental radicular arteries. During combined occlusion of aortic arch and left subclavian artery, mean distal arterial blood pressure dropped to 10+/-5 mm Hg, and spinal cord blood flow at the L1 level decreased to 27+/-7% of baseline. All animals recovered from anesthesia with acute paraplegia. Animals in the 9-minute group (group A) showed steady recovery of hind limb function over the ensuing 24 hours, whereas the majority (80%) in the 11-minute group (group B) remained paralyzed with maximum deficit throughout the postoperative period. Mortality was 0% and 21% in groups A and B, respectively. Maximal ischemic damage was observed at the lower thoracic and higher lumbar spinal levels in both groups. In group A (9 minutes), tissue damage was mild, affecting predominantly dorsal horns and intermediate gray matter, whereas ventral horns were minimally involved. All mice in group B (11 minutes) showed extensive gray matter lesions particularly involving dorsal horns and intermediate areas; in ventral horns, >50% of motor neurons died. White matter lesions were present in the most severely damaged cords only. CONCLUSIONS: Spinal cord ischemia caused by aortic arch plus left subclavian artery cross-clamping provides a mouse model useful for the study of spinal cord injury and of potential relevance to the complications following thoracoabdominal aortic surgery in humans.     

经皮局部低温治疗兔脊髓损伤后病理与运动诱发电位的研究

目的 :探讨经皮局部低温对兔脊髓损伤的治疗作用。方法 :18只健康家兔随机均分为常温未伤组 ,常温致伤组 ,低温致伤组。保留椎板显露脊髓 ,以Allen法制作脊髓损伤模型。术毕 ,低温致伤组立即予经皮局部低温治疗 ,其他组不予治疗。各组动物分别于术前 ,术后 6、2 4h ,3d ,1、2、4周 7个时间点行Tarlov评分和MEP检查 ,并取材行病理分级。结果 :低温致伤组动物在各个时间点较常温致伤组Tarlov评分分值提高 ,MEP的N1波潜伏期缩短 ,病理损害轻。结论 :经皮局部低温对兔脊髓损伤有治疗作用     

细胞外三磷酸腺苷对大鼠脊髓损伤后胶质纤维酸性蛋白表达的影响

目的 研究细胞外三磷酸腺苷(ATP)对大鼠脊髓损伤后胶质纤维酸性蛋白(GFAF)表达和运动功能恢复的影响.方法 健康成年Wistar大鼠66只按照随机数字表法取6只作为正常对照组,余60只制作成脊髓打击伤动物模型,并再按照随机数字表法分为两组:ATP组(A组,给予ATP注射)和对照组(B组,给予等量生理盐水注射),每组30只大鼠.伤后1、3、7、14和28 d取材,应用免疫组织化学方法观察GFAP的表达,采用计算机图像分析系统进行半定量分析;并用改良的Tarlov评分观察大鼠脊髓损伤后运动功能的恢复情况.结果 大鼠脊髓损伤后GFAP的表达呈进行性升高,损伤后14 d达高峰;在损伤后7、14和28 d,A组大鼠GFAP的表达明显强于B组;脊髓损伤后14 d和28 d,A组大鼠改良的Tarlov评分明显大于B组;以上差异均有统计学意义(P<0.05).结论 细胞外ATP能促进大鼠损伤脊髓表达GFAP,并有助于大鼠脊髓损伤后运动功能的恢复.     

皮层体感诱发电位在不同水平脊髓缺血中的变化

目的 为术中应用皮层体感诱发电位(CSEP)监测脊髓功能,预防术后运动功能障碍提供理论依据. 方法 33只新西兰大白兔采用随机数字表法分为6组,对照组8只用于排除麻醉和手术对诱发电位的影响,余25只根据结扎左肾动脉和动脉分叉间节段性脊髓供血动脉的条数分为5组(n=5),分别为1～5根组.记录各组动物麻醉后基线诱发电位,血管结扎后急性期诱发电位,结扎后30 min、2 d后诱发电位.动物麻醉清醒后、血管结扎后2 d时进行运动功能评分,并取缺血中心区标本行HE染色. 结果 潜伏期对缺血性损伤不敏感,实验组与对照组差异无统计学意义(P>O.05);波幅变化复杂,对运动功能特异性差,2、3、4根组均观察到急性期波幅先降低又逐渐恢复趋势,波幅的变化可以反映脊髓的病理损害程度和运动功能. 结论 缺血急性期CSEP波幅变化复杂,对运动功能特异性差,波形记录的信号需要平均化过程,造成结果 解释的延迟,术中应联合运动诱发电位对脊髓功能进行监测.     

Therapeutic efficacy of SJA6017, a calpain inhibitor, in rat spinal cord injury

Apoptosis is an important element of the secondary processes that occur after spinal cord injury. Calpain and caspases are key proteases in apoptotic cell death. We evaluated the neuroprotective effects of SJA6017 (a calpain inhibitor) and measured functional recovery in a rat spinal cord injury model. Thirty Wistar albino rats were divided into three groups of 10 animals each: sham-operated (group 1), trauma control (group 2) and trauma-plus-SJA6017 treatment (group 3). Spinal cord trauma was produced in the thoracic region of the animals. Rats in group 3 received SJA6017 1 min after trauma. Treatment efficacy was evaluated after injury using light microscopy and TUNEL staining. Neurological performance was assessed using an inclined plane and a modified version of the Tarlov's grading scale. Group 2 rats showed moderate trauma with widespread edema, hemorrhage, vascular thrombi and necrosis 24 h after injury. Group 3 rats had significantly reduced tissue injury and apoptosis. Tarlov scores revealed that group 3 rats also had ameliorated recovery of limb function. Our results demonstrate that treatment with SJA6017 reduces apoptotic cell death, preserves spinal cord tissue and improves functional outcome. Treating calpain-induced apoptosis with this agent may be a feasible therapeutic strategy for patients with spinal cord injury.     

Epidural perfusion cooling protects against spinal cord ischemia in rabbits

The protective effect of regional epidural spinal cord cooling was evaluated in a rabbit spinal cord ischemia model. Hypothermia was performed by the continual perfusion of 2–4°C cold saline in the epidural space around the ischemic lumbar segments, 4 min before and during ischemia. The spinal cord was deeply hypothermic (21°C) throughout the whole ischemie period. Ischemia was induced by the occlusion of the abdominal aorta for 40 min under normothermic or hypothermie conditions. Recovery of motor and sensory functions, spinal cord-evoked potentials, and motor-evoked potentials were then evaluated up to 24 h postischemia. After this period, choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities were measured, in particular, zones of the lumbar spinal cord. AChE was also investigated histochemically. Animals in the normothermic group displayed fully developed spastic paraplegia with near complete loss of spinal somatosensory and motor-evoked potentials. AChE histochemistry showed extensive necrotic changes affecting lumbosacral gray matter. These changes corresponding with the pronounced losses of ChAT and AChE activities indicated irreversible injury of the spinal cord. In contrast, after hypothermic ischemia, animals survived without any sign of neurological impairment with almost full recovery of the spinal cord-evoked potentials. ChAT and AChE activities in the gray matter showed near control values corresponding with histochemical analysis of fully preserved gray matter. Hypothermia under the present experimental conditions efficiently protected the spinal cord against ischemic injury.     

NMDAR1、Caspase-3在脊髓缺血性损伤的表达特点及相关性研究

目的探讨脊髓缺血后N-甲基-D-天门冬氨酸受体1（N-methyl-D-aspartatereceptor,NMDARl）、Caspase-3的表达变化规律及意义。方法40只新西兰大白兔采用结扎腰动脉建立脊髓缺血模型,于缺血后6h、12h、24h、48h、72h取缺血脊髓标本,运用反转录聚合酶链反应（RT—PCR）及蛋白印记技术在基因和蛋白水平检测NMDAR1、Caspase-3在不同缺血时间的表达变化规律,同时运用免疫组织化学技术观察NMDAR1、Caspase-3在细胞中的表达定位情况;用SPSS统计分析。结果RT-PCR、蛋白印记显示缺血组与对照组比较NMDAR1、Caspase-3表达增加,并且有随缺血时间的延长表达逐渐增高的趋势,相关性分析显示NMDAR1、caspase-3在mRNA（r=0．947,P〈0．005）、蛋白（r=0．984,P〈0．005）水平呈正相关;免疫组化结果发现,NMDAR1、Caspase-3主要在细胞浆中表达。结论脊髓缺血时NMDAR1、caspase-3随缺血时间延长表达增加,NMDAR1、Caspase-3共同参与脊髓缺血性损伤过程,其表达与缺血时间有关。