缺血预处理对脑缺血再灌注损伤保护作用的实验研究

目的：通过观察Toll样受体4（Toll-like receptors,TLR4）和肿瘤坏死因子-α(TNF-α)的表达变化,探讨缺血预处理对大鼠再灌注损伤的保护作用并研究其意义。方法：将36只SPF级健康雄性SD大鼠随机分为缺血预处理组（CIP组n=12）、缺血再灌注组（I/R组n=12）和假手术组（Sham组n=12）;I/R组采用线栓法阻断大脑中动脉（MCAO）2h建立模型,CIP组先给予MCAO阻断10 min,再灌注72 h后给予与I/R组相同的处理,假手术组仅分离血管。均于术后1d取材检测脑梗死体积、用 Zea Longa评分标准进行神经功能评分以及通过荧光实时定量PCR（Real-time Quantitative polymerase chain reaction）法测定TLR4和TNF-α mRNA水平的表达。结果：缺血再灌注后1 d各组大鼠比较,CIP组大鼠脑梗死体积明显低于I/R组,差异有显著统计学意义（P＜0.05）,CIP组大鼠的神经功能缺损评分明显低于I/R组,二者比较有显著性差异（P<0.05）。TLR4、TNF-α mRNA在CIP组与I/R组的表达明显高于Sham组,差异有显著统计学意义（P＜0.05,P＜0.01）,而二者在CIP组的表达明显低于I/R组（P＜0.05）。结论：缺血预处理能改善由再灌注损伤引起的功能障碍,可能通过下调促炎因子的表达来减轻脑组织缺血再灌注损伤,从而减轻炎症反应。     

缺血后适应对大鼠缺血再灌注损伤时Bcl-2及GAP-43表达变化的影响

目的 观察缺血后适应对大鼠缺血再灌注损伤时Bcl-2和GAP-43表达变化的影响.方法 50只成年健康雄性SD大鼠随机分为缺血2h再灌注组（n=25）、缺血4.5 h后适应组（n=25）.线栓法制备大脑中动脉闭塞模型（MCAO）,Lumila Belayev12分、Zea Longa5分和悬吊实验术后24 h进行神经功能评分,TTC法染色后测量脑梗死体积,免疫组织化学方法和Western Blot检测Bcl-2及GAP-43蛋白.结果 与缺血2h再灌注组相比,缺血4.5 h后适应组神经功能评分和脑梗死体积差异没有统计学意义（P＞0.05）.缺血4.5 h后适应组Nogo-A表达降低,GAP-43表达增加,差异有统计学意义（P＜0.05）.结论 缺血后适应能够改善神经功能损伤,减小脑梗死体积,增加GAP-43的表示以及减少Nogo-A的表达,对缺血的脑组织起保护作用.     

干细胞因子联合抗白细胞介素17A抗体对小鼠脑缺血预后的影响

目的 探讨干细胞因子(stem cell factor,SCF)联合抗白细胞介素-17A抗体(anti-IL-17A)对小鼠脑缺血预后的影响.方法 通过线栓法对C57BL/6小鼠建立大脑中动脉栓塞(middle cerebral artery occlusion,MCAO)模型,60 min后取出线栓,恢复右侧大脑中动脉血流,建立脑缺血再灌注模型.小鼠脑缺血再灌注后分别给予腹腔注射生理盐水(normal saline,NS)、anti-IL-17A、SCF、anti-IL-17A+SCF.观察各组小鼠MCAO术后脑梗死体积、脑组织病理损伤和脑水肿程度、神经功能评分、神经细胞凋亡和炎症反应等变化.结果 与NS组相比,其余各治疗组脑梗死体积、脑组织病理损伤和脑水肿均显著减轻(P＜0.05),神经功能评分得到明显改善(P＜0.05),神经细胞凋亡减少(P＜0.05),且anti-IL-17A+ SCF组显著优于anti-IL-17A组和SCF组(P＜0.05).此外,anti-IL-17A还能明显减少脑组织IL-17A和IL-1 β的表达(P＜0.05).结论 SCF联合anti-IL-17A能更好地改善脑缺血损伤和促进神经功能恢复.     

小鼠脑缺血-再灌注损伤后泊洛沙姆188与银杏内酯B联合应用研究*

目的：探讨泊洛沙姆188与银杏内酯B联合应用对小鼠脑缺血-再灌注损伤的保护作用。方法：以线栓造成小鼠脑组织缺血状态，线栓移除前5min经尾静脉单次注射泊洛沙姆188（0.4g/kg），银杏内酯B（20mg/kg）或两药的混合溶液，24小时后评价小鼠神经症状并测定脑梗死面积和脑含水量。另一批小鼠在静脉注射给药后再每天腹腔注射上述等效剂量药物，24天后统计各组存活状况，绘制生存曲线；采用爬杆试验、悬挂试验测试小鼠肢体运动功能。结果：生理盐水组小鼠脑梗死面积为35.49%±8.31%，P188组、GB组以及合用组的脑梗死面积分别减少为27.76%±6.41%，25.51%±5.81%和21.16%±8.67%，P188与GB合用组比药物单用组的脑梗死面积更小，差异有统计学意义（P<0.05）。生理盐水组小鼠24天后的生存率为38.89%，P188组，GB组以及合用组小鼠的生存率分别为50%，53.57%和69.21%。P188与GB合用组与药物单用组相比，生存期明显延长，差异均有统计学意义（P<0.05）。P188组、GB组以及药物合用组T/turn分别为6.22秒,6.56秒和5.25秒，T/floor分别为15.50秒，14.88秒和13.25秒，P188和GB合用组与药物单用组相比，T/turn和T/floor均显著缩短，差异均有统计学意义（P<0.05）。与泊洛沙姆188和银杏内酯B单用相比，联合用药能进一步降低脑梗死面积，延长生存期，增强小鼠肢体的运动功能。结论：泊洛沙姆188与银杏内酯B合用能够增强对小鼠脑缺血-再灌注损伤的保护作用。     

重组人粒细胞集落刺激因子经鼻给药对脑梗死大鼠的脑保护作用

目的 探讨重组人粒细胞集落刺激因子(rhG-CSF)鼻腔给药对脑梗死大鼠的脑保护作用.方法 线栓法制备大鼠大脑中动脉阻塞模型,缺血2h再灌注,皮下及鼻腔给予 rhG-CSF(60 μg/kg).将动物分为正常组、假手术对照组、脑梗死组、脑梗死+皮下注射rhG-CSF组、脑梗死+经鼻生理盐水组、脑梗死+经鼻rhG-CSF组,每组6只.于术后3 d进行神经功能评分及FasL免疫荧光检测.结果 除正常组、假手术对照组外,其余各组动物均于术后出现不同程度的神经功能缺损,尤以脑梗死组、脑梗死+经鼻生理盐水组最为严重,神经功能评分最高[(10.20±1.85)分,(10.30±1.76)分],海马FasL阳性细胞数最多[(41.17±3.25)个,(41.00±2.76)个],差异无显著性( P ＞0.05),rhG-CSF皮下给药组大鼠神经功能评分降低[(5.67±1.32)分,P ＜0.01],海马FasL表达减少[(32.67±1.97)分,P ＜0.01],rhG-CSF经鼻给药组大鼠神经功能评分进一步降低[(4.00±0.93)分,P ＜0.05],海马FasL表达进一步减少[(19.50±1.05)个,P ＜0.01].结论 rhG-CSF经鼻给药能预防和修复脑梗死大鼠的神经功能缺损,通过抑制FasL表达发挥脑保护作用.     

重组人可溶性血栓调节蛋白对小鼠脑缺血/ 再灌注损伤的神经保护作用及机制研究

目的 研究重组人可溶性血栓调节蛋白（ART-123）对小鼠脑缺血/ 再灌注损伤的影响。方法 BALB/c 小鼠行4 h 大脑中动脉闭塞（MCAO）,再灌注时静脉注射Vehicle 或ART-123（1 或5 mg/kg）。再灌注24 h 后 评估小鼠脑梗死体积、运动协调、血浆高迁移率族蛋白（HMGB1）水平及出血量。结果 ART-123 可减少 MCAO 小鼠脑梗死体积,并呈剂量依赖性（F =4.843,P =0.038）。与Vehicle 组比较,ART-123（5 mg/kg）可 改善旋转试验中MCAO 小鼠运动协调（P =0.028）;降低血浆HMGB1 水平（P =0.000）。此外,Vehicle 组和 ART-123 组小鼠出血量差异无统计学意义（P >0.05）。结论 ART-123 可能通过抑制HMGB1 改善小鼠脑 缺血/ 再灌注损伤。     

电针预处理对脑缺血再灌注大鼠皮质神经元TRPC6表达的影响

目的观察电针预处理对脑缺血再灌注大鼠皮质神经元TRPC6表达的影响,探讨其对神经元可能的保护机制。方法将SD大鼠随机分为假手术组、模型组和电针预处理组,每组8只。采用大脑中动脉栓塞法制备脑缺血再灌注模型。电针预处理组在造模前取"百会"和"大椎"穴给予电针刺激。造模后24h进行神经功能评分并急性分离大鼠皮质神经元,通过蛋白免疫印迹(western blot)检测神经元TRPC6和Caspase-3蛋白表达,钙影像检测细胞内相对钙离子浓度。结果与假手术组比较,模型组大鼠的神经功能评分明显增高(P0.05),大脑皮质神经细胞TRPC6和Caspase-3蛋白表达明显增高(P0.05),神经元内钙离子浓度升高(P0.01);与模型组比较,电针预处理组能明显改善脑缺血再灌注后大鼠的神经功能症状(P0.05),抑制大脑皮质神经细胞TRPC6和Caspase-3蛋白表达(P0.05),并降低细胞内钙离子浓度(P0.01)。结论电针预处理对脑缺血再灌注大鼠脑损伤有保护作用,其机制可能与TRPC6下调,减少细胞钙离子内流从而抑制神经凋亡。     

氯胺酮咪唑安定麻醉对大鼠局灶性脑缺血损伤的影响

目的比较氯胺酮联合咪唑安定麻醉与单纯氯胺酮麻醉对局灶性脑缺血损伤的影响。方法30只SD大鼠在氯胺酮或氯胺酮-咪唑安定麻醉下制作永久性大脑中动脉阻塞模型。术后4h行神经病学评分、24h测量梗塞体积、72h进行TUNEL染色检测半暗带内的细胞凋亡。结果术后4h神经病学评分在氯胺酮-咪唑安定组和氯胺酮组间无显著性差异(1.74±0.52vs1.57±0.65,P>0.05),但氯胺酮-咪唑安定组大鼠的脑梗塞体积和半暗带内凋亡细胞密度明显低于氯胺酮组大鼠的相应参数(梗塞体积:24.1%±4.63%vs38.48%±4.18%;凋亡细胞密度:178±23个/mm2vs258±15个/mm2,P<0.05)。结论氯胺酮麻醉中联合应用咪唑安定对大鼠局灶性脑缺血损伤具有保护作用。     

远隔缺血预适应对大鼠局灶性脑缺血后诱导型一氧化氮合酶的影响

目的：研究远隔缺血预适应（ remote ischemic preconditioning,RIPC）对大鼠脑缺血再灌注后诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS）表达的影响,以探讨 RIPC 保护脑缺血损伤的相关机制。方法应用线栓法制作大脑中动脉栓塞（middle cerebral artery occlusion,MCAO）模型,将21只健康雄性 SD 大鼠采用数字表法随机分为3组：假手术组（sham,n=7）,MCAO 组（n=7）,RIPC＋MCAO 组（n =7）。在 MCAO 手术前连续3 d,进行远隔肢体缺血预适应处理（双侧股动脉夹闭10 min/放开10 min,每天3次）。在缺血2 h-再灌注24 h 后进行神经功能评分,TTC 染色检测脑梗死体积,采用 Western blotting 方法检测梗死侧脑组织的 iNOS 蛋白表达水平。结果1）在 MCAO 手术过程中,3组实验动物的生理指标均在正常范围内,且组间差异无统计学意义。与 sham 组相比,MCAO 组大鼠再灌注24 h 时神经功能缺损评分显著升高、脑梗死体积明显增大（P〈0.05）。而 RIPC＋MCAO 组大鼠神经功能较 MCAO 组大鼠得到明显改善、脑梗死体积显著减少（P〈0.05）。2）再灌注24 h 时,与 sham 组相比,MCAO 组大鼠脑梗死侧 iNOS 蛋白表达显著增加（P〈0.05）。与 MCAO 组相比,RIPC＋MCAO 组大鼠脑梗死侧 iNOS 蛋白表达显著降低（P〈0.05）。结论 RIPC 处理对大鼠脑缺血损伤具有保护作用,其机制与降低脑缺血大鼠脑内 iNOS 蛋白表达有关。     

Experimental Study on the Protection of Agrimony Extracts from Different Extracting Methods against Cerebral Ischemia-Reperfusion Injury

Objective To study the protective effect of agrimony extracts from different extracting methods on cerebral ischemia-reperfusion injury in rats, in order to optimize the extraction scheme of agrimony. Methods Male rats were randomly assigned into seven groups: 1. Sham-operated group, 2. Untreated MCAO group (MCAO), 3. Petroleum ether extract of Agrimonia pilosa treated MCAO group (PEA), 4. Ethyl acetate extract of Agrimonia pilosa treated MCAO group (EAEA), 5. Ethanol extract of Agrimonia pilosatreated MCAO group (EEA), 6. Water extract of Agrimonia pilosatreated MCAO group (WEA), 7. Nimodipine treated MCAO group (NP). Intragastrical drug administration (i.g) was performed at 0 and 6 hours after MCAO. Neurological function tests were performed after reperfusion for 24 hours, then the brain was removed for the evaluations of the cerebral infarction volume (percentage of total brain volume) by immunohistochemistry, histological changes (hematoxylin-eosin staining), Na+/K+-ATPase, Ca2+-ATPase (modified method of Svoboda and Mosinger), mRNA expression of Tumor suppressor gene (P53) and hot shock protein (HSP70) (quantitative real-time PCR). Results The neurological function of MCAO group had significantly higher scores than the sham group (P<0.01). The WEA group showed a significantly lower neurological score than the MCAO group (P<0.05), indicating the protective effect of WEA on neurological deficits. The mean infarction volumes of WEA (13.5±6.6%,F=4.75,P<0.01), EEA (19.90±6.90%,F=5.23,P<0.01), PEA (20.40±5.30%, F=4.68, P<0.01) and EAEA (22.50±10.50%,F=6.25,P<0.05) group were all significantly smaller than that of MCAO group (29.40±6.50%). HE staining demonstrated that, compared to the treated groups, the infarcted cerebral tissue of MCAO group had more swelling neural cells, lighter stained nucleus, fewer and irregularly distributed neurons. The activity of Na+/K+-ATPase and Ca2+-ATPase reduced in the MCAO group (3.67±0.48 U/mg, 1.28±0.26 U/mg, respectively), and were significantly higher in WEA group (7.56±0.85 U/mg,F=12.65, P=0.010; 3.59±0.22 U/mg,F=8.32,P=0.041, respectively). The MCAO group showed significantly elevated P53 and HSP70 mRNA expressions compared to the sham group (P<0.01,P<0.05). P53 mRNA expressions in Agrimony extracts treated groups were significantly lower than that of the MCAO group (allP<0.01), with the WEA group showing the greatest difference from MCAO group. The HSP70 mRNA level of the treated groups were not significantly different from that of the MCAO group. Conclusions Treatmentusingwater extracts of agrimony can promote the best functional and metabolic recovery for rat model of cerebral ischemia-reperfusion injury, which maybe relate with the upregulation of energy metabolism in nerve cells after MCAO.     

活血化瘀系列方对脑缺血再灌注损伤的保护作用及机制研究

目的 :探讨活血化瘀系列方 (活血汤、活血益气汤、活血养阴汤 )对大鼠脑缺血再灌注损伤的保护作用及其机制。方法 :采用大脑中动脉阻断法制备大鼠脑缺血再灌注模型 ,缺血 6 0 min,再灌注 3d。观察药物对脑缺血再灌注引起的神经功能缺损症状及病理损伤的影响 ,检测脑组织一氧化氮合酶 (NOS)、超氧化物歧化酶 (SOD)活性的变化和丙二醛 (MDA)含量的变化 ,免疫印迹分析 NMDA受体亚单位蛋白 NR1和内皮型 NOS(e NOS)的表达。结果 :活血化瘀系列方及尼莫地平能显著改善神经功能缺失症状 ,减小梗死体积和脑水肿面积 ,减少神经元损伤 (P<0 .0 5 ) ,组间没有显著性差异 ;活血化瘀系列方和尼莫地平不同程度降低脑组织内 NOS活性和 MDA含量 ,抑制NR1表达 ,增加 SOD的活性 ,但对 e NOS的表达均无明显影响。结论 :活血化瘀系列方对大鼠脑缺血再灌注损伤有保护作用 ,其机制可能与降低脑组织内 NOS活性 ,降低 NR1的表达 ,减少 MDA含量和增加 SOD活性有关。     

T细胞死亡相关基因8对大鼠局灶性脑缺血再灌注损伤的影响

目的: 探讨活化T细胞死亡相关基因8（T cell death associated gene 8,TDAG8）对大鼠局灶性脑缺血再灌注损伤的影响。方法: 将健康成年雄性SD大鼠132只采用随机数字表法分为6组：对照组（n=18）,缺血再灌注组（I/R组,n=42）,TDAG8激动剂BTB09089组（BTB组,n=18）,空白载体组（空载体组,n=18）,TDAG8-siRNA阴性对照组（siRNA阴性组,n=18）和TDAG8-siRNA组（siRNA组,n=18）。采用线栓法短暂性阻塞大鼠大脑中动脉（middle cerebral artery occlusion,MCAO）制备大鼠局灶性脑I/R模型。BTB组、空载体组、siRNA阴性组和siRNA组于缺血前72 h、48 h 和24 h分别经侧脑室注射20 μmol/L的BTB09089、空白转染试剂jetPEI、4 μg siRNA阴性对照和4 μg TDAG8-siRNA各8 μL,其中空载体组、siRNA阴性组和siRNA组同时均注入等量转染试剂jetPEI。于再灌注6 h时每组取6只大鼠断头取脑,蛋白质印迹法检测缺血半暗带TDAG8、cleaved caspase-3、Bcl-2和TDAG8下游相关蛋白p-Akt、p-CREB的表达水平;于再灌注24 h和72 h时每组分别取6只大鼠评价神经功能并测定脑梗死体积百分比。结果： 与对照组比较,其他5组再灌注6 h时缺血半暗带TDAG8、cleaved caspase-3、p-Akt和p-CREB表达均上调（均P<0.05）,Bcl-2表达下调（P<0.05）,再灌注24 h和72 h时脑梗死体积百分比明显升高、神经功能缺陷评分显著降低（均P<0.05）;与I/R组比较,BTB组再灌注6 h时 TDAG8、Bcl-2、p-Akt和p-CREB水平上调（均P<0.05）,cleaved caspase-3表达下调（P<0.05）,再灌注24 h和72 h时脑梗死体积百分比显著降低,神经功能缺陷评分明显升高（均P<0.05）;与siRNA阴性组比较,siRNA组再灌注6 h时TDAG8、Bcl-2、p-Akt和p-CREB水平下调（P<0.05）,cleaved caspase-3表达上调（P<0.05）,再灌注后24 h和72 h脑梗死体积百分比显著升高,而神经功能缺陷评分明显降低（P<0.05）。结论： 活化TDAG8可能通过Akt信号通路调控短暂性大鼠MCAO诱导的脑缺血再灌注损伤。     

七氟醚预处理对局灶性脑缺血再灌注大鼠T细胞死亡耦联基因8表达的影响

目的：观察七氟醚预处理对局灶性脑缺血再灌注大鼠缺血半暗带内T细胞死亡耦联基因8（TDAG8）表达的影响。方法：选取54只成年雄性SD大鼠,随机分为对照组、再灌注组和七氟醚组,每组18只。采用大脑中动脉内线栓阻断法（middle cerebral artery occlusion,MCAO）制造大鼠局灶性脑缺血再灌注模型。缺血2h后再灌注48h。七氟醚组在造模前吸入最低肺泡有效浓度（MAC）为1的七氟醚30min,对照组和再灌注组则吸入O2。大鼠行神经行为学评分后处死。取大脑行2,3,5-氯化三苯基四氮唑（TFC）染色,计算脑梗死容积百分比,采用蛋白印迹法和RT—PCR法检测脑缺血半暗带内的TDAG8蛋白和mRNA的表达变化。结果：①与对照组相比,再灌注组和七氟醚组脑梗死容积百分比均显著升高（P均〈0．05）;七氟醚组脑梗死容积百分比较再灌注组显著降低（P〈0．05）。②与对照组相比,再灌注组和七氟醚组TDAG8蛋白和mRNA表达均显著升高（P均〈0．05）;七氟醚组较TDAG8的表达再灌注组明显降低（P〈0．05）。结论：七氟醚预处理可降低TDAG8的表达,对脑缺血再灌注损伤具有一定保护作用。     

Neuroprotective Effects of Grape Seed Procyanidin Extract on Ischemia-Reperfusion Brain Injury

Objective Oxidative stress (OS) plays a crucial role in ischemic stroke. Grape seed procyanidin extract (GSPE) was reported to be a critical regulator of OS. We hypothesized that GSPE might also be protective in ischemia-reperfusion brain injury. This study aimed to explore whether GSPE administration can protect mice from ischemia-reperfusion brain injury. Methods Transient middle cerebral artery occlusion (MCAO) was conducted followed by reperfusion for 24 hours to make ischemia-reperfusion brain injury in mice that received GSPE (MCAOG, n=60) or normal saline (MCAONS, n=60). Sham-operated mice (GSPE group and normal saline group) were set as controls. The neurological severity score (NSS) was used to evaluate neural function impairment 1 hour, 24 hour, 3 days and 7 days after MCAO. Mice underwent brain T2WI imaging with a 3T animal MRI scanner 24 hours after reperfusion, and the stroke volume of brains were calculated according to abnormal signal intensity. Immunohistopathological analysis of brain tissues at 24 h after reperfusion was performed for neuronal nuclear antigen (NeuN), CD34, Bcl-2, and Bax. Glutathione peroxidation (GSH-Px) activity and the level of malonaldehyde (MDA) of brain tissue were also examined. The above indexes were compared among the groups statistically. Results Significant functional improvement was observed 24 hours after MCAO in MCAOG group compared to MCAONS group (P<0.05). MCAOG group had smaller cerebral stroke volume (22.46 ± 11.45 mm3vs. 47.84±9.06 mm3,P<0.05) than MCAONS group 24 hours after MCAO. More mature NeuN-immunoreactive neurons and more CD34-positive cells in peri-infarct zones were observed in brain tissue of MCAOG mice 24 h after MCAO than that of MCAONS mice (bothP<0.05). MCAONS mice had significantly higher number of Bax-positive cells in brain tissue than MCAOG (P<0.05). The mean MDA level was significantly lower (P<0.05) and the GSH-Px activity was significantly higher (P<0.05) in brains of MCAOG mice compared to those of MCAONS mice. Conclusion GSPE administration protects mice from ischemia-reperfusion brain injury through attenuating oxidative stress and apoptosis, promoting angiogenesis, and activating antioxidant enzyme GSH-Px. GSPE may represent a new therapeutical direction for the treatment of ischemia-reperfusion brain injury.     

吡格列酮对家兔局灶性脑缺血再灌注损伤细胞凋亡的影响

目的探讨吡格列酮对脑缺血再灌注损伤家兔细胞凋亡的影响及其机制。方法40只雄性家兔随机分为5组：①假手术组（S组,n=8）;②模型组（M组,n=8）;③吡格列酮组（P组,n=8）;（4）GW9662＋吡格列酮组（GP组,n=8）;（5）DMSO组（D组,n=8）。除S组外,其余各组均通过线栓法建立家兔大脑中动脉阻塞（MCAO）再灌注损伤模型。于术前30min分别给予相应处理。缺血120min再灌注24h后,对家兔进行神经功能评分;HE染色观察病理形态学。TUNEL检测神经细胞凋亡。结果①与S组比较,M组神经功能评分明显增加（P〈0．05）;与M组比较,P组神经功能评分明显降低（P〈0．05）。②与S组比较,M组凋亡细胞明显增加（P〈0．05）;与M组比较,P组凋亡细胞明显降低（P〈0．05）。结论吡格列酮可通过减轻形态学改变、抗神经细胞凋亡,对神经细胞发挥保护作用。     

联合应用缺血后处理、远隔缺血后处理和纳洛酮后处理对大鼠脑缺血-再灌注损伤的影响

目的 评价联合应用缺血后处理、远隔缺血后处理和纳洛酮后处理对大鼠局灶性脑缺血-再灌注损伤的影响.方法 将110只大鼠随机分为5组(n=22),通过阻塞右侧大脑中动脉90 min和再灌注24 h实施局灶性脑缺血.再灌注.Ⅰ组为对照组;Ⅱ组为缺血后处理组,再灌注开始时实施3次30 s的缺血和再灌注;Ⅲ组为远隔缺血后处理组,再灌注开始前实施5 min的右侧股动脉缺血;Ⅳ组为纳洛酮后处理组,再灌注开始时腹腔注射纳洛酮10 mg/kg;Ⅴ组为联合应用组.再灌注2 h和24 h时测定大鼠的神经功能障碍评分(NDS);再灌注24 h时,测定脑梗死区面积(n=10)、脑组织微管相关蛋白2(MAP2)表达(n=6)和脑组织血浆容量、血管直径和节段长度(n=6).结果 观察期所有时间点的心率和平均动脉压(MAP)组间比较差异均无统计学意义(均P＞0.05).再灌注24 h后,Ⅰ～Ⅴ组的缺血侧脑梗死面积与同侧大脑半球面积的比值(即脑梗死严重程度)分别是43%±6%、31%±4%、32%±5%、28%±6%和21%±7%.与Ⅰ组比较,Ⅱ～Ⅴ组的NDS和脑梗死严重程度均低(均P＜0.05),MAP2表达、血浆容量、血管直径和节段长度均高,但上述指标在Ⅱ组、Ⅲ组和Ⅵ组之间比较差异均无统计学意义(均P＞0.05).与Ⅰ组、Ⅱ组、Ⅲ组和Ⅳ组比较,Ⅴ组的NDS评分和脑梗死程度均低(均P＜0.05),MAP2表达和血浆容量显著高(均P＜0.05),但是缺血侧脑组织的血管直径和节段长度在Ⅱ组、Ⅲ组Ⅵ组和Ⅴ组之问差异均无统计学意义(均P＞0.05).结论 在局灶性脑缺血-再灌注损伤大鼠,缺血后处理、远隔缺血后处理和纳洛酮后处理均具有明显的神经保护作用,表现为脑梗死严重程度降低和神经功能障碍改善.联合应用3种后处理措施可获得增强的神经保护效应.

Abstract：

Objective To assess the effects of ischemic postconditioning, remote ischemic postconditioning and naloxone postconditioning on focal cerebral ischemia-reperfusion injury in rats.Methods A total of 110 adult SD rats were randomly divided into 5 groups (n =22 each). The focal cerebral ischemia-reperfusion injury was induced by a 90-minute occlusion of right middle cerebral artery (MCA) and a 24-hour reperfusion sequentially. Group 1 was of ischemia-reperfusion control; Group 2 ischemic postconditioning induced by three 30-second cycles of MCA occlusion followed by a 30-second reperfusion; Group 3 remote ischemic postconditioning performed via a transient occlusion of right femoralartery at 5 min before the initiatlon of reperfusion:Group 4 naloxone posteonditioning with naloxone 10 mg/kg intraperitoneaUy injected at the initiation of reperfusion;Group 5 combined ischemic,remote ischernic & naloxone postconditioning performed simultaneously in accordance with the methods used in Groups 2,3 & 4.The neumlogie deftcit scores(NDS)were obtained at 2 h & 24 h post-reperfusion.At 24 h post-reperfusion.the anesthetized rat was sacrificed by decapitation and the brain rapidly extracted to asseSS the size ofcerebral infaret(n=10),detect the cerebral expression of microtubule-associated protein2(MAP2)(n=6),measure the plasma volume of cerebral tissues and quantify the diameter and segment artery at 5 min before the initiation of reperfusion; Group 4 naloxone postconditioning with naloxone 10 mg/kg intraperitoneally injected at the initiation of reperfusion; Group 5 combined ischemic, remote ischemic & naloxone postconditioning performed simultaneously in accordance with the methods used in Groups 2, 3 & 4. The neurologic deficit scores ( NDS) were obtained at 2 h & 24 h post-reperfusion. At 24 h post-reperfusion, the anesthetized rat was sacrificed by decapitation and the brain rapidly extracted to assess the size of cerebral infarct (n = 10), detect the cerebral expression of microtubule-associated protein2 ( MAP2) (n =6) , measure the plasma volume of cerebral tissues and quantify the diameter and segment length of cerebral microvessel (n = 6 ). Results There were no significant differences in the heart rate (HR) and mean arterial pressure (MAP) among the above five groups at all observed time points (P ＞ 0. 05). At 24 h post-reperfusion, the percentage of ischemic cerebral infarct size was 43% ±6% , 31% ±4% , 32% ±5% , 28% ±6% & 21% ±7% in ipsilateral hemisphere area (i. e. , cerebral infarct severity)in Groups 1-5 respectively. Compared with Group 1, the levels of NDS and cerebral infarct severity significantly decreased at ischemic side in Groups 2-5 ( P ＜ 0. 05 ). And the cerebral expression of MAP2,plasma volume of cerebral tissues, diameter and segment length of cerebral microvessel significantly increased at the ischemic side (all P＜0. 05). However, there were no significant differences in the abovementioned parameters at ischemic side among Groups 2, 3 and 4 (all P ＞0. 05). The parameters of NDS,cerebral infarct severity, cerebral expression of MAP2 and plasma volume of cerebral tissues in the ischemic side significantly increased in Group 5 compared with Groups 1,2,3 and 4 (all P ＜ 0. 05). The diameter and segment length of cerebral microvessel at ischemic side were not different among Groups 2,3,4 and 5 (all P＞0. 05). Conclusion In focal cerebral ischemia-reperfusion rats, ischemic, remote ischemic and naloxone postconditioning may produce significant neuroprotective effects of reduced cerebral infarct severity and improved neurologic dysfunctions. A combination of three postconditioning approaches enhances the above neuroprotective effects.     

CIB1沉默表达对大鼠脑缺血-再灌注损伤后Caspase-3 mRNA表达影响

目的探讨钙离子结合蛋白（calcium and integrin-binding protein,CIB1）双链RNA（CIB1.siRNA）对大鼠脑缺血-再灌注损伤的作用.方法 SD大鼠120只,随机分成三组（每组40只）：假手术组（S组）、局灶性脑缺血-再灌注组（A组）、局灶性脑缺血-再灌注＋CIB1-siRNA组（B组）.A组、B组行大脑中动脉阻断,阻断1 h再开放,制备大鼠局灶性脑缺血-再灌注模型,B组在缺血前即刻、16、24 h水压法尾静脉注射CIB1-siRNA 2.5 mg/kg（用PBS稀释至10 mL）,S组给予等量PBS,S组只作切口,不作缺血再灌注.S组、B组、A组分别于再灌注1、5、11、23、47 h各处死8只大鼠,断头取脑,计算脑梗塞体积比,用RT-PCR法测定脑缺血半影区Caspase-3 mRNA表达水平.结果 B组、A组再灌注23 h时脑梗塞体积比达到高峰,再灌注47 h脑梗塞体积比均高于S组（P〈0.01）;与A组比较,B组脑梗塞体积比增加（P〈0.01）.A组和B组再灌注5 h时Caspase-3 mRNA表达高峰,B组各时间段Caspase-3 mRNA表达均强于A组.结论 CIB1-siRNA预先给药可加重大鼠脑缺血再灌注损伤.     

基于扩散张量成像初探苍艾挥发油对大鼠脑缺血再灌注损伤的神经保护作用

  目的  利用MRI扩散张量成像技术研究经鼻吸入苍艾挥发油对大鼠脑缺血再灌注损伤模型的神经保护作用。  方法  24只健康成年雄性 SD 大鼠,按照随机分配原则,分为假手术组、中动脉阻闭再灌注（middle cerebral artery occlusion, MCAO）组和苍艾挥发油（volatile oil of Cang Ai, VOCA）组。采用Zea Longa评分于造模后即刻和造模后第7天对各组大鼠神经功能损伤程度进行评估。用 7.0T磁共振对3组大鼠在造模成功后3 h、3 d、7 d行冠状位扩散张量成像（diffusion tensor imaging, DTI）扫描,对最大梗死层面的纹状体区和运动平层区DTI的表观扩散系数（apparent diffusion coefficient, ADC）和各向异性分数（fractional anisotropy, FA）进行测量,然后计算相对表观扩散系数(rADC)和相对各向异性分数（rFA）。造模7 d后采用 TTC 染色法对各组大鼠脑梗死体积进行评估,并对rFA、rADC与神经功能缺损评分进行相关性分析。  结果  假手术组苏醒后无神经功能缺损,MCAO组与VOCA组均有不同程度神经功能缺损,造模后7 d时VOCA组神经功能缺损评分小于MCAO组（P<0.05）。DTI扫描结果显示,造模后3 h以及3 d,VOCA组大鼠纹状体rADC值均高于MCAO组（P<0.05）,7 d时3组无明显差异（P>0.05）。VOCA组运动皮层区rADC值在造模后3 h高于MCAO组（P<0.01）,3 d、7 d时差异无统计学意义。VOCA组纹状体rFA值在造模后3 d和7 d均高于MCAO组（P<0.05）。MCAO组与VOCA组大鼠运动皮层区rFA值在3个时间点的组间差异均无统计学意义。TTC染色结果显示假手术组无梗死区,VOCA组脑梗死体积小于MCAO组（P<0.05）。相关性分析显示MCAO组和VOCA组大鼠纹状体rFA值（r=?0.847）、运动皮层rADC值（r=?0.647）、运动皮层rFA值（r=?0.660）与神经功能缺损评分均呈明显相关性（P<0.05）。  结论  VOCA在脑缺血再灌注后能有效通过降低缺血区细胞的毒性水肿及加快神经纤维束恢复来保护MCAO大鼠的神经功能。rFA、rADC值可作为评价脑缺血再灌注后神经功能恢复的有效指标。     

CIB1-siRNA对大鼠脑缺血-再灌注损伤后凋亡相关蛋白Caspase-3表达影响

目的探讨钙离子结合蛋白（calcium and integrin-binding protein,CIB1）双链RNA（CIB1.siRNA）对大鼠脑缺血-再灌注损伤的作用.方法 SD大鼠120只,体重290～310 g,随机分成三组：假手术组（S组,n=40）、局灶性脑缺血-再灌注组（A组,n=40）、局灶性脑缺血-再灌注＋CIB1-siRNA组（B组,n=40）.A组、B组行大脑中动脉阻断,阻断1 h再开放,制备大鼠局灶性脑缺血-再灌注模型,B组在缺血前即刻、16、24 h水压法尾静脉注射CIB1-siRNA 2.5 mg/kg（用PBS稀释至10 mL）,S组给予等量PBS,S组只作切口,不作缺血再灌注.S组、B组、A组分别于再灌注1、5、11、23、47 h各处死8只大鼠,断头取脑,计算脑梗塞体积比,用RT-PCR法测定脑缺血半影区Caspase-3 mRNA表达水平.结果 B组、A组再灌注23 h时脑梗塞体积比达到高峰,再灌注47 h时B组、A组脑梗塞体积比均高于S组（P〈0.01）;与A组比较,B组脑梗塞体积比增加（P〈0.01）.A组和B组再灌注23 h时Caspase-3表达高峰,B组各时间段Caspase-3表达强于A组.结论 CIB1-siRNA预先给药可加重大鼠脑缺血-再灌注损伤.     

参附注射液对脑缺血再灌注大鼠MDA、SOD、TXB2及6-keto-PGF1a的影响及意义?

  目的观察参附注射液对局灶性脑缺血-再灌注大鼠脑组织中超氧化物歧化酶（SOD）活性及丙二醛（MDA）、血栓素B2(TXB2)、6-酮-前列腺素F1a（6-keto-PGF1a）含量的影响,探讨参附注射液对脑的保护机制。方法清洁级雄性SD大鼠60只,随机分为3组假手术组（Sham组,n=20）、脑缺血再灌注组（IR组,n=20）、参附注射液预处理组（SFI组,n=20）。采用线栓法制备大鼠大脑中动脉闭塞后再灌注(MCAOR) 模型, 观察大鼠MCAOR 时神经功能状态,TTC 染色测脑梗死面积, 同时测定血浆中MDA、SOD、TXB2、6-keto-PGF1a的变化。结果参附注射液能有效改善MCAOR大鼠神经功能缺失症状,明显减小梗死灶。IR组与假手术组比较,血清中SOD活性降低,MDA、TXB2含量增加,6-keto-PGF1a含量降低（P<0.05）;参附治疗组与IR组比较,SOD活性增高,MDA、TXB2含量下降,6-keto-PGF1a含量增高（P<0.05）。结论参附注射液对脑缺血再灌注损伤引发的自由基损伤有保护作用,并且能够纠正TXB2/6-keto-PGF1a平衡,达到对脑缺血再灌注大鼠的脑保护作用。