醒脑静制剂对脑卒中模型大鼠的保护作用及机制研究
|
摘要
缺血性脑卒中是一种严重威胁人类健康和生命的疾病,尤其在老年人中多发。由于缺血性脑卒中具有高致残率和致死率,给个人、家庭及社会带来沉重负担。在既往报道中,绝大多数脑卒中患者即使脱离生命危险,也会伴有不可恢复的神经功能缺损,表现为口眼歪斜、肢体功能障碍、思维迟钝、记忆力减退、情绪不稳定、精神抑郁等。这些症状可能一直困扰患者,如不采用适当措施加以治疗和干预,可能会随着时间的推移而持续加重,给病人带来极大痛苦。
中药对于缺血性脑卒中的疗效已得到广泛认可,其作用靶点广泛,作用持久、温和,毒副作用小,在对症治疗的同时,也可对机体整体状态进行调节。我国医药资源丰富,经典名方、验方,经现代工艺制剂后,在确保临床疗效的同时,也扩大药物的应用范围,得到广泛认可。
醒脑静注射液由麝香、冰片、栀子、郁金等药物组成,能够清热泻火,凉血解毒,开窍醒脑。临床上用于常用于治疗神经系统损伤性疾病,如脑卒中、一氧化碳中毒、肝昏迷等急重症,尤其是对于急性缺血性脑病具有良好疗效,得到临床工作者的认可。然而,由于其剂型原因,醒脑静注射液不便于病人自行给药,使其不能长期应用于脑缺血恢复期的治疗,为其临床应用带来一定局限性。醒脑静口服制剂是注射液原方提取物,在确保疗效的基础上,可延长药物临床应用时间,且便于服用。经过前期筛选,目前制剂工艺已确定。并其对缺血性脑卒中模型大鼠具有一定作用保护作用,其对抗缺血性脑损伤的作用得到初步肯定。
本研究采用大脑中动脉血栓(MCAT)模型,通过比较两种醒脑静复方制剂(醒脑静注射液和醒脑静口服制剂)对局灶性脑缺血模型动物神经功能缺损评分和脑梗死体积的影响,明确口服制剂对大鼠缺血性脑损伤模型的保护作用。并采用永久性局灶性中动脉阻断脑缺血(pMCAO)大鼠模型,观察醒脑静口服制剂在脑缺血恢复早期(14 d)对缺血模型动物肢体运动、平衡能力相关指标,评价药物在缺血性脑损伤恢复早期的神经保护作用。另外,本研究中还采用转基因脑卒中动物模型——自发性脑卒中大鼠(SHRSP),评价醒脑静口服制剂对SHRSP血压、心率及一般体征的影响,并从对抗氧化性损伤的角度评价了醒脑静口服制剂对SHRSP的保护作用。
已有研究结果表明,醒脑静注射液对缺血性脑损伤具有明确保护作用,其作用机制与抗炎、抗氧化、改善能量代谢障碍等作用有关,但其对突触结构、功能的影响未见报道。本研究采用永久性局灶性中动脉阻断脑缺血(pMCAO)大鼠模型,从神经元病理改变、突触结构、功能角度,观察醒脑静注射液对脑缺血急性期神经元病理改变、突触结构改变、及突触功能相关蛋白表达的影响,探讨醒脑静注射液对局灶性脑缺血模型大鼠的保护作用机制。
本研究主要内容如下：
第一部分醒脑静口服制剂对局灶性脑缺血模型大鼠和自发性脑卒中大鼠的影响
第一节醒脑静口服制剂对局灶性脑缺血模型大鼠的保护作用
实验一两种醒脑静制剂对三氯化铁所致局灶性脑缺血模型大鼠脑损伤的保护作用
目的：观察两种醒脑静复方制剂对局灶性脑缺血模型大鼠的神经功能、脑梗死范围的影响,明确口服制剂在脑缺血急性期的脑保护作用。
方法：实验动物随机分为5组,即模型组、醒脑静注射液3、9mL/kg组(相当于临床人用量9倍、27倍)、醒脑静口服制剂40mg/kg、120mg/kg(每10 mL注射液生药量相当于口服制剂0.1289g,分别相当于临床人用量9倍、27倍)组,另设假手术组。造模前,醒脑静口服制剂组灌胃给药两天2 d,醒脑静注射液组腹腔注射给药,1次/d；第3 d给药1 h后,采用三氯化铁凝闭大脑中动脉,造成大鼠大脑中动脉血栓形成(MCAT)模型,造模6 h后给药1次。于造模后6h、24 h分别对大鼠进行神经功能缺损症状评分。并于造模24 h后,取脑,冠状切片,进行TTC染色,对白色梗死组织进行称重,以梗塞组织重量占总脑重量的百分比作为脑梗塞范围。
结果：实验结果显示,缺血后模型动物均表现出一定程度的神经功能缺损症状,表现为：身体蜷缩,身体平衡能力下降,向左侧旋转、倾倒,不能行走等。术后6 h,与模型组相比,醒脑静注射液9 mL/kg组神经功能缺损症状评分明显降低(P<0.05),醒脑静口服制剂也能够明显减轻缺血后神经功能损伤(P<0.05)；术后24h,与模型组相比,醒脑静3 mL/kg、9 mL/kg组神经功能缺损均明显减轻(P<0.05,P<0.01),醒脑静口服制剂120mg/kg组大鼠神经功能缺损症状明显减轻(P<0.05)。手术24 h后,缺血组动物脑组织可见不同程度的白色梗死灶。其中,模型组脑梗死组织可达14.9 g,经给药治疗,给药组脑梗死范围较模型组明显降低,醒脑静注射液3 mL/kg、9 mL/kg组均能够明显减小梗死范围(P<0.05,P<0.01)；醒脑静口服制剂40mg/kg、120mg/kg组能够显著减少梗死体积(P<0.01)。
结论：醒脑静口服制剂在缺血性脑损伤发生后24 h内能够改善缺血模型动物的神经功能缺损症状,减少缺血后脑组织梗死范围,且与醒脑静注射液组相比,效果无显著性差异。结果表明,醒脑静口服制剂对三氯化铁所致局灶性脑缺血模型大鼠具有保护作用。
实验二醒脑静口服制剂对永久性局灶性大脑中动脉阻断脑缺血(pMCAO)模型大鼠缺血损伤恢复早期神经功能的保护作用
目的：考察醒脑静口服制剂在脑缺血损伤恢复早期对pMCAO模型大鼠神经功能缺损的改善作用。
方法：采用线栓法制作pMCAO大鼠模型。术后3 d,对缺血动物进行神经功能缺损评分,根据评分结果将动物随机分为5组：模型组、醒脑静口服制剂12mg/kg组(相当于临床人用量的3.5倍)、醒脑静口服制剂25 mg/kg组(相当于临床人用量的7倍)、醒脑静口服制剂50 mg/kg (相当于临床人用量的14倍),以丁苯酞70 mg/kg组为阳性对照组,另设假手术组。给药组灌胃给服相应药物,假手术组与模型组灌胃给予等体积橄榄油。脑缺血后3 d至14 d灌胃给药,1次/d。分别于pMCAO后3d、6d、9d、12 d. 14 d,通过神经功能评分、横木行走实验、前肢触觉刺激实验、前肢抓握力量测试评价药物对缺血模型动物神经功能的保护作用。于14 d末次给药后,取动物脑组织,生化法测定脑组织中MDA含量及SOD活力。
结果：与假手术组相比,缺血模型大鼠表现出明显神经功能缺损症状。缺血后14 d,与模型组相比,醒脑静12 mg/kg,25 mg/kg和50 mg/kg组神经功能缺损症状明显改善,神经功能缺损症状评分降低(P<0.01),横木行走实验评分明显增加(P<0.05,P<0.01),撕除胶布潜伏期明显缩短(P<0.05,P<0.01),前肢抓握力量明显增加(P<0.01)。缺血14 d后,与假手术组相比,模型组大鼠脑组织中SOD活力明显下降,MDA含量明显增加,且与模型组相比,醒脑静12 mg/kg,25 mg/kg和50 mg/kg组脑组织MDA含量均明显降低(P<0.01)。
结论：醒脑静口服制剂在脑缺血恢复早期(14 d)对pMCAO模型大鼠具有神经保护作用,能够改善缺血动物神经功能缺损症状,减轻缺血后脑组织脂质过氧化损伤。
第二节醒脑静口服制剂对自发性脑卒中大鼠(SHRSP/Izm)的影响及抗氧化性损伤作用的研究
实验一醒脑静口服制剂对SHRSP机体状态的影响
目的：采用自发性脑卒中大鼠(SHRSP)模型,观察醒脑静口服制剂对于SHRSP血压、心率及一般体征的影响。
方法：SHRSP给予食盐负荷饮食(40 g/kg饮食),按体重随机分为4组：SHRSP组、维生素C组(ascorbic acid, AA)、醒脑静25 mg/kg组、醒脑静50 mg/kg组。AA组每日给予维生素C水溶液(1000 mg/d),醒脑静组灌胃给服相应剂量药物,SHRSP组灌胃等体积饮用水,1次/d,连续给药6周。每周监测大鼠体重变化情况,并于给药前(0周),给药第2、4、6周进行收缩压(SBP)和心率(HR)测定,及一般体征(包括进食量、饮水量、24小时尿量、24小时排便量)的监测。
结果：药前(第0周)各组动物体重、一般体征无差异。自给药第2周开始,给予食盐负荷后大鼠饮水量、排尿量、排便量明显增加,但与SHRSP组相比,AA组进食量明显减少(P<0.01),饮水量、排尿量、均显著降低(P<0.01),体重减轻(P<0.01)。给药第6周,各组进食量无差异,但AA组体重仍明显低于SHRSP组(P<0.01)。醒脑静组体重、进食量与SHRSP组相比均无显著性差异(P>0.05)。给药前,各组大鼠SBP无显著性差异；给予食盐负荷后,第2周,各组大鼠SBP均出现明显上升趋势；第4周,AA组、醒脑静高剂量组SBP较SHRSP组有下降趋势,但未表现出显著性差异；第6周此种趋势消失,除AA组SBP仍低于SHRSP组以外,其他各组SBP较模型组有所升高,但结果尚无统计学意义。给药第2周,各组HR有下降趋势,但从第4周至第
6周实验结束,HR呈现出上升趋势,且各组间无显著性差异。
结论：醒脑静口服制剂对SHRSP SBP及HR无显著影响,对动物体重增长、进食量、饮水量等一般体征无明显影响。
实验二醒脑静口服制剂对SHRSP氧化性损伤的影响
目的：观察醒脑静口服制剂对SHRSP氧化性损伤状态的影响,评价醒脑静口服制剂对SHRSP的保护作用。
方法：SHRSP给予盐负荷饮食(40 g/kg饮食),按体重随机分为4组：SHRSP组、维生素C组(ascorbic acid, AA)、醒脑静25 mg/kg组、醒脑静50 mg/kg组。AA组每日给予维生素c水溶液(1000 mg/d),醒脑静组灌胃给服相应剂量药物,SHRSP组灌胃给予等体积饮用水,1次/d,连续给药6周。大鼠于给药第2、4、6周采集血浆样本,生化法测定血中总体抗氧化状态(TAS, total antioxidant status);并于第6周给药结束后TBARS法测定脑组织、血浆中丙二醛(MDA)含量,ELISA法测定尿液8-羟基脱氧鸟苷(8-OHdG)含量,RT-PCR (?)去测定脑组织中SOD、TNF-αmRNA表达水平。
结果：给药第2周,与SHRSP组相比,AA组及醒脑静给药各组动物表现出自身抗氧化能力增加。醒脑静50 mg/kg组血浆TAS明显提高,抑制过氧化物能力增强(P<0.01)：第4周,AA组血浆TAS明显增加(P<0.05),醒脑静各剂量组TAS也较SHRSP组有所增加,但未表现出统计学差异；第6周,与SHRSP组相比,醒脑静50 mg/kg组血浆TAS均显著增加(P<0.01)。
给药第6周,与SHRSP组相比,AA组尿液中8-OHdG含量明显降低(P<0.05),其他给药组末见统计学差异；各组大鼠脑组织中MDA含量无显著性差异；SHRSP组血浆MDA含量较高,各给药组血浆MDA含量均有不同程度降低,其中醒脑静50 mg/kg组血浆MDA含量明显降低(P<0.05,P<0.01)。
与SHRSP组比较,给药第6周,给药组脑组织中SOD mRNA表达均增加,但至实验结束,尚未显示出统计学差异；醒脑静25 mg/kg组、50 mg/kg组TNF-a mRNA表达出现下降趋势,但尚无统计学意义。
结论：醒脑静口服制剂能够降低SHPSP体内脂质过氧化物含量,提高动物体内总体抗氧化能力,这种抗氧化能力的改善或许对SHPSP起保护作用。
第二部分醒脑静注射液的作用机制研究——醒脑静注射液对pMCAO模型大鼠突触可塑性的影响
实验一醒脑静注射液对pMCAO模型大鼠缺血神经元的保护作用
目的：观察醒脑静注射液对pMCAO模型大鼠在脑缺血急性期神经元的保护作用。
方法：采用线栓法建立pMCAO大鼠模型,缺血6 h后,模型动物随机分成5组：模型组、醒脑静1.2 mL/kg组(相当于临床人用量的3.5倍)、醒脑静2.5 mL/kg组(相当于临床人用量的7倍)、醒脑静5 mL/kg(当于临床人用量的14倍),以金纳多2.5 mL/kg组为阳性对照组,另设假手术组。给药组腹腔注射相应药物,假手术组与模型组腹腔注射等体积生理盐水。脑缺血后6 h至7 d给药,1次/d。采用苏木素-伊红染色(H&E staining)和尼氏染色(Nissl staining)(?)去观察醒脑静注射液对缺血后神经元病理改变的影响,采用透射电镜观察醒脑静注射液对脑缺血后神经元超微结构改变的影响。
结果：缺血2 d后,光镜下可见假手术组皮层神经元细胞形态圆整,排列整齐,神经元胞浆丰富,尼氏小体分布均匀；模型组脑组织皮层神经元大量变性坏死,核固缩深染,尼氏小体含量明显减少；与模型组相比,给药组皮层完整神经元数目增加,海马区神经元排列较为整齐,死亡神经元数目减少,神经元损伤程度明显减轻,尼氏小体脱失减轻。电镜下可见假手术组神经元细胞核形态规整,核膜光滑完整,核仁明显,染色质分布均匀,胞浆内细胞器丰富,线粒体成椭圆或圆形,脊清晰；模型组神经元核固缩,细胞周围呈现空泡状,细胞器少见；给药组细胞形态基本正常,核膜核仁清晰可见,且细胞器较为丰富。
缺血7 d后,模型组缺血周围区域仍可见大量神经元缺失,神经元变性死亡,尼氏小体脱失；与模型组相比,醒脑静注射液组神经元损失数目减少,结构完整、可辨认的神经元数量较多,尼氏体表达增加。电镜下可见缺血至7 d,模型组神经元固缩,核膜形态不规则,核内染色质浓缩、边集；醒脑静注射液组可见神经元形态基本正常,核膜光滑,核仁明显,且染色质均匀分布。
结论：醒脑静注射液在脑缺血急性期对神经元起保护作用,减轻因缺血造成的神经元死亡、尼氏小体脱失及神经元超微结构破坏。实验二醒脑静注射液对pMCAO模型大鼠缺血突触结构的影响
目的：观察醒脑静注射液对pMCAO模型大鼠在脑缺血急性期突触结构,及突触结构相关蛋白表达的影响。
方法：采用线栓法建立pMCAO大鼠模型,缺血6 h后,模型动物随机分成5组：模型组、醒脑静1.2 mL/kg组、醒脑静2.5 mL/kg组、醒脑静5 mL/kg,以金纳多2.5 mL/kg组为阳性对照组,另设假手术组。给药组腹腔注射给予相应药物,假手术组与模型组腹腔注射等体积生理盐水。脑缺血后6 h至7 d给药,1次/d。采用透射电镜观察pMCAO模型大鼠突触结构,免疫组织化学染色法观察缺血后突触结构相关蛋白的表达情况以及醒脑静注射液的干预作用。
结果：缺血2 d后,电镜下可见假手术组突触结构正常,突触前后膜结构清晰；模型组动物突触数量稀少,突触结构不清；给药组可见较多突触结构,形态丰富。缺血7 d后,模型组突触结构少见；给药组可见较为清晰突触结构,突触间隙可见,突触前膜靠近突触间隙一侧偶见突触小泡聚集。
与假手术组相比,缺血2 d后,模型组动物synapsin-Ⅰ、PSD-95在海马CA1区、CA3区、皮层均明显下降(P<0.01),α-synuclein在海马CA1区、CA3区、皮层表达均明显增加(P<0.01)。与模型组相比,synapsin-Ⅰ在醒脑静1.2 mL/kg组CA3区,2.5 mL/kg组CA3区和皮层,5 mL/kg组CA1区、CA3区、皮层表达明显升高(P<0.05,P<0.01);PSD-95在醒脑静1.2mL/kg组皮层,2.5 mL/kg组CA1区,5 mL/kg组CA1区和皮层表达明显升高(P<0.05,P<0.01);α-synuclein在醒脑静1.2 mL/kg组CA3区,2.5 mL/kg组皮层,醒脑静5 mL/kg组CA1区、CA3区和皮层表达均显著减少(P<0.05,P<0.01)。
缺血7d后,与假手术组相比,模型组synapsin-Ⅰ在海马CA1区、皮层表达仍显著降低(P<0.05,P<0.01), PSD-95在CA1区、CA3区和皮层表达仍显著降低(P<0.01),其中在CA1、CA3区表达略有上升,但与2d表达水平相比无显著性差异,α-synuclein在海马CA1区、CA3区和皮层表达仍显著增加(P<0.01)。与模型组相比,醒脑静1.2mL/kg、2.5 mL/kg、5 mL/kg组synapsin-Ⅰ在CA3、皮层表达仍有明显增加(P<0.05, P<0.01), PSD-95在醒脑静1.2 mL/kg组CA3、皮层表达有所上升(P<0.05,P<0.01), 2.5 mL/kg组CA1表达增加(P<0.05),5 mL/kg组CA1、CA3、皮层表达均有明显增加(P<0.05,P<0.01);α-synuclein在醒脑静1.2 mL/kg、2.5 mL/kg、5 mL/kg组CA1区、CA3区、皮层表达均有所下降(P<0.05,P<0.01)。
结论：脑缺血急性期,pMCAO模型大鼠突触结构破坏,与突触功能密切相关蛋白synapsin-Ⅰ、PSD-95、α-synuclein表达发生改变,表明突触结构、功能受到损伤。醒脑静注射液在缺血后2 d即能增加皮层、海马部位的PSD-95、synapsin-Ⅰ表达,并减少α-synuclein的积聚,这种作用持续到缺血后7 d仍然存在,表明醒脑静注射液在脑缺血急性期、亚急性期对神经元突触结构、功能具有保护作用。实验三醒脑静注射液对pMCAO大鼠突触功能相关蛋白表达的影响
目的：观察醒脑静注射液对pMCAO模型大鼠突触功能相关蛋白表达的影响。
方法：采用线栓法建立pMCAO大鼠模型,缺血6 h后,模型动物随机分成4组：模型组、醒脑静2.5 mL/kg组、醒脑静5 mL/kg,以金纳多2.5 mL/kg组为阳性对照组,另设假手术组。给药组腹腔注射给予相应药物,假手术组与模型组腹腔注射等体积生理盐水。脑缺血后6 h至7 d给药,1次/d。采用Western Blot (?)去观察缺血2 d、7 d后,pMCAO模型大鼠皮层、海马区域PSD-95、p-CaMKⅡ、NR2B蛋白的表达情况,以及醒脑静注射液对蛋白表达的影响。
结果：缺血后2 d,与假手术组相比,模型组PSD-95蛋白在皮层、海马表达均明显降低(P<0.01)；与模型组相比,给药组PSD-95蛋白明显增加(P<0.05,P<0.01)。缺血后7 d,与假手术相比,PSD-95蛋白的表达仍显著降低(P<0.05,P<0.01)；与模型组相比,醒脑静给药组皮层PSD-95蛋白的表达均有不同程度增加(P<0.05,P<0.01),金纳多组、醒脑静5.0 mL/kg组海马PSD-95蛋白表达也有增加,但未见统计学差异。
缺血后2 d,与假手术组相比,模型组p-CaMKⅡ蛋白在皮层、海马表达均明显降低(P<0.05)；与模型组相比,金纳多组p-CaMKⅡ蛋白表达明显增加(P<0.05)。缺血后7 d,模型组p-CaMKⅡ蛋白的表达与假手术组无显著性差异；与模型组相比,给药组p-CaMKⅡ蛋白的表达未见统计学差异。
缺血后2 d,与假手术组相比,模型组NR2B蛋白表达有所下降,其中在海马的表达显著降低(P<0.01)；与模型组相比,醒脑静给药组NR2B蛋白表达明显增加(P<0.05,P<0.01)。缺血后7 d,与假手术组相比,模型组NR2B蛋白在皮层、海马表达无显著性差异；但与模型组相比,醒脑静给药组表达水平仍有明显增加(P<0.05,P<0.01)。
结论：醒脑静注射液在缺血损伤急性期能够增加PSD-95、p-CaMKⅡ、NR2B蛋白的表达,对抗缺血引起的突触功能损伤。醒脑静注射液对突触的保护作用在脑缺血急性期(2d)作用较强,这或许是醒脑静注射液对抗急性缺血性脑损伤的机制之一。
Cerebral ischemic stroke is one of serious diseases that threaten human being, which with high incidence, especially in the senile. Due to its high morbidity and mortality, cerebral ischemic stroke imposes a heavy burden on individuals, families and society. In previous reports, the vast majority of patients with stroke suffer from unrecoverable neurological defects even when they are treated, such as limb dysfunction, mental retardation, memory loss, depression, and so on. These symptoms may have troubled the patients all the time. With no appropriate measures of treatment and intervention, they may continue and aggravate over time. Therefore, treatment in sub-acute phase is essential.
Traditional Chinese medicines are effective for cerebral ischemia, which always act through various pathways. They are usually mild with lasting effect, with few side effects. They can treat both symptoms and causes of diseases, with regulation on generalized condition. In China, there are many famous classic formulas in ancient times, which can be prepared into Chinese patents by modern techniques. These patents are effective as ancient formulas in clinic, and can be applied for more symptoms because of their convenience for administration.
Xingnaojing Injection is commonly used in clinic, which is composed of she xiang (Moschus), bing pian (Borneolum Synthcticum), yu jin (Radix Curcumae Wenyujin), and zhi zi(Fructus Gradeniae). It is commonly applied to nervous system diseases, such as carbon monoxide poisoning, coma hepaticum, and so on. It is effective significantly for acute cerebral ischemia. However, injection is usually applied for acute symptoms. Because of its dosage form, it can not be administered out of hospitals. Development of peroral preparation of Xingnaojing injection will ensure its wide application. Peroral preparation of Xingnaojing is made from extract of identical Chinese medicinals of Xingnaojing injection. Based on previous study, effect of peroral preparation of Xingnaojing has been proved.
In this study, neuroprotective effect of Xingnaojing peroral preparation was evaluated in MCAT (Middle Cerebral Artery Thrombosis) model rats. We observed improvements in neuro-functions and the size of cerebral infarction to make sure both injection and peroral preparations have neuroprotective effect in acute phase of cerebral ischemia. In addition, we further explored action of peroral preparation in convalescence in pMCAO (permanent middle artery occlusion) model rats, and evaluated its effect as improvement on neurological defects and relief on oxidative stress. In addition, in this study, SHRSP was used to evaluate antioxidant action of Xingnaojing peroral preparation.
Studies have shown that the protection of Xingnaojing injection for ischemic injury is related to its anti-inflammatory, sedative, anti-oxidatant and other effects. However, whether its neuroprotective action is related to synaptic function has not been explored yet. We evaluated protective effect of Xingnaojing injection on neurons and synapse in acute and sub-acute phases in rat model of pMCAO. Post-ischemia synaptic structure and function was observed, and action of Xingnaojing Injection on synaptic structure and function was evaluated.
This study is composed of the two following parts:
1 Effect of Xingnaojing peroral preparation on rat model of focal cerebral ischemia and stroke-prone spontaneously hypertension rat
1.1 Neuroprotection of Xingnaojing peroral preparationin on rat model of focal cerebral ischemia
1.1.1 Protection from two preparations of on Xingnaojing on rat model of FeCl3-induced focal cerebral ischemia
Objective:it is to evaluate effects of preparations of Xingnaojing in acute phase of cerebral ischemia, and further demonstrate neuroprotection of Xingnaojing peroral preparation.
Methods:SD rats were randomly divided into model group, Xingnaojing injection 3 mL/kg group and 9 mL/kg group (equal to 9- and 27-time clinical dosage of adult),40 mg/kg group and 120 mg/kg group of peroral preparation, and the sham group. Rats were administered with peroral preparation two days earlier before the establishment of model, or intraperitoneally administered with Xingnaojing injection, once per day. One hour later administration on the third day, MCAT model in rats was established with FeCl3 Rats were administered with medicines 6 hours later after establishment of model. On 6 h and 24 h after establishment of model, scores on neurological defects were measured. And 24 hours after establishment of model, coronal sections of brain tissue was taken and stained with TTC. Weight of white infarction and whole brain tissues was measured, taking percentage weight of infarction to the total weight of brain as degree of cerebral infarct.
Results:after MCAT, rats got some neurological defects, such as curled body, poor balance, rotation and falling to the left side, inability to walk, and so on. Compared with model group, 9 mL/kg group of Xingnaojing Injection and 120 mg/kg group of Xingnaojing peroral preparation got lower score on neurological defects at 6 h post-MCAT (P<0.05); 24 h post-MCAT, Xingnaojing Injection 3 mL/kg and 9 mL/kg groups got significantly improved on neurological defects (P<0.05,P<0.01),120 mg/kg group of peroral preparation of Xingnaojing also got milder defects (P<0.01) than control group. In the sham group, there was no infarction on brain tissue, while white infracted area in the model group. In the medicated group, infarction size was more reduced than the model group. In the 3 mL/kg and 9 mL/kg groups of Xingnaojing Injection,120 mg/kg group of peroral preparation of Xingnaojing, infarction size was significantly smaller (P<0.05,P<0.01).
Conclusion:both Xingnaojing Injection and peroral preparation of Xingnaojing had significant neuroprotective effects within 24 hour after cerebral ischemia.
1.1.2 Protective effects of peroral preparation of Xingnaojing on neurological defects and oxidative damage in pMCAO model rats in early convalescence
Objective:it is to evaluate peroral preparation of Xingnaojing in pMCAO model rats in early convalescence
Method:pMCAO model was established with suture. Rats were randomly divided into model group, sham group, peroral preparation of Xingnaojing 12 mg/kg group,25 mg/kg group, and 50 mg/kg group, taking Butylphthalide 70 mg/kg group as positive control group. All animals were administered medicines 3-14 d post-ischemia, rats in the sham group were administered with same volume of oleum olivae. Neurological deficit scores, tactile stimulation experiments, beam walking test, strength test were operated to observe protective effects of peroral preparation of Xingnaojing on neural function in pMCAO model rats on day 3,6,9, 12, and 14; and measure SOD activity and MDA content in ischemic brain tissue.
Results:compared with the sham group, rats in the model group got obvious symptoms of neurological defects. After treatment, Xingnaojing 12 mg/kg group,25 mg/kg group, and 50 mg/kg group got significantly improvement on their neurological functions, manifesting improvement on scores of neurological defect (P<0.01), beam walking ability (P<0.05, P<0.01), and recovery of forelimbs strength (P<0.05,P<0.01), and enhancement of tactile sensitivity (P<0.01). Peroral preparation of Xingnaojing was able to significantly reduce the MDA content of ischemic brain tissue.
Conclusion:peroral preparation of Xingnaojing had protective action on pMCAO rat, by reducing oxidative damage.
1.2 Study on protection of Xingnaojing peroral preparation on SHRSP and related mechanism
1.2.1 Action of of Xingnaojing peroral preparation on general status in SHRSP
Objective:it is to explore impacts of peroral preparation of Xingnaojing on body weight, blood pressure, heart rate, and general condition in SHRSP.
Method:all SHRSP were given salt-loaded diet, and were randomly divided into SHRSP group, the AA group (vitamin C,1000 mg/d), Xingnaojing 25 mg/kg group and 50 mg/kg group. All rats were intragastrically administered with medicines for 6 weeks. Food intake, water intake, urine output, and defecation output within 24-hour were measured on the 2nd,4th and 6th weeks respectively.
Results:there were no significant differences on body weight and general condition between groups pre-medication (0 week). Since second week, given the salt-loaded food, water intake, urine output, the amount of defecation of SHRSP increased significantly. Compared with the SHRSP group, food intake, water intake, and urine output in AA group was significantly reduced (P<0.01), with great body weight loss (P<0.01). In the 6th week of medication, there were no significant differences on food intake between groups, but the body weight in the AA group was still significantly less than others'(P<0.01). There was no significant difference on body weight, food intake Xingnaojing-treated groups (P>0.05). Given salt-loaded food, SBP of rats significantly increased since 2nd week of treatment; in the 4th week, SBP in the AA group, and Xingnaojing 50 mg/kg group slightly reduced, but with no significant differences compared with SHRSP group. This tendency did not vanish unitl the 6th week, in which, SBP in AA group was lower than in the SHRSP group, while was slightly higher in the Xingnaoj ing-treated groups. However, there was no significant difference between groups. Since 2nd week of medication, HR decreased in every group slightly, while increased until 6th week, with no significant difference between groups.
Conclusion:peroral preparation of Xingnaojing had no significant impact on body weight, general condition, blood pressure, and heart rate.
2.2 Antioxidant action of Xingnaojing peroral preparation of in SHRSP
Objective:to investigate antioxidant action and related mechanism of Xingnaojing peroral preparation in SHRSP.
Method:all SHRSP given the high salt load diet were randomly divided into SHRSP group, the AA group (1000 mg/d), Xingnaojing 25 mg/kg, and 50 mg/kg group. Rats were administered with medicine for 6 weeks. Plasma TAS was measured respectively in 2nd,4th and 6th week, and MDA content in the brain tissue and plasma was measured on 6th week, and expression of SOD and TNF-αmRNA was measured by RT-PCR.
Results:in 2nd week, Xingnaojing peroral preparation was able to increase level of plasma
TAS. It had no significant action in 4th week. However, it was able to increase level of plasma TAS. It was able to reduce content of MDA in the plasma. There was only tendency of increased level of SOD and decreased TNF-αmRNA expression in medicated group, but no significant difference.
Conclusion:Xingnaojing peroral preparation of had protective action on SHRSP, which might relate to reduction on oxidative damage.
2 Study on protection of Xingnaojing injection on synapse and mechanism
2.1 Protection of Xingnaojing injection on neurons in pMCAO model rat Objective:to investigate neuronal protection of XNJ injection in pMCAO model rats in acute and sub-acute phase.
Methods:pMCAO model was established with suture. Rats were randomly divided into sham group, model group, XNJ Injection 1mL/kg,2.5 mL/kg, the 5 mL/kg groups, and taking Ginkgo injection 2.5 mL/kg group as control. Postoperative 6 h-7 d rats were administered respectively, once per day. H&E staining and Nissl staining was applied to observe morphology changes on neurons on 2 d and 7 d after ischemia. Microstructure of neurons was observed by transmission electron microscope.
Results:on 2nd d post-ischemia, in visual fields of optical microscope, cortical neurons in the sham group were round with complete form. It was rich in cytoplasm in neurons with plenty of Nissl bodies evenly distributed. In the model group, there were a great number of degenerated and necrotic neurons in the cortex, manifesting as shrunken and darkly stained nucleus, and great loss of Nissl bodies. In the treatment groups, there were more complete neurons in the cortex, with clearly arranged neurons in the hippocampus areas. There were less dead neurons, less damage to neurons, and less loss of Nissl bodies. In the field of transmission electron microscope, nuclear morphology in the sham group was clear with complete nuclear membrane, unabroken karyosome, and evenly distributed chromatin. There were plenty of organelles in the cytoplasm, of which itochondria was oval or round. However, in the model group, there were pycnotic neurons, around which, there were vacuolars. There was less organelle in the cytoplasm. In the treatment groups, morphology of neurons was normal with clear nuclear membrane and plenty of organelles.
Conclusion:in acute phase of cerebral ischemia, Xingnaojing Injection has protective effect on neurons by preventing neuronal death, loss of Nissl bodies, and ultrastructure damage of neurons induced by ischemia.
2.2 Effect of Xingnaojing injection on synaptic structure in pMCAO model rats
Objective:to investigate the effect of Xingnaojing injection on synaptic structure after cerebral ischemia at different time points.
Methods:pMCAO model was established by suture. Rats were randomly divided into model group, Xingnaojing Injection 1.2 mL/kg group,2.5 mL/kg group, and 5mL/kg group, taking Gingko injection 2.5mL/kg group as the control group, and the sham group. Rats were administered with medicines 6 h-7 d post-operation. On 2 d and 7 d after ischemia, synapse-related proteins, synapsin-Ⅰ, PSD-95, andα-synuclein were detected with immunohistochemical staining.
Results:compared with the sham group, on 2nd day post-ischemia, in the model group, expression of synapsin-Ⅰ, PSD-95 in hippocampal in CA1 and CA3 area, and cerebral cortex were significantly decreased (P<0.01);α-synuclein in hippocampal CA1 and CA3 area, and cortical expression were significantly increased (P<0.01). Compared with model group, expression of synapsin-Ⅰon CA3 area in Xingnaojing 1.2 mL/kg group, in CA3 and cortex of 2.5 mL/kg group, in CA1 area, CA3 area, and the cortex of 5 mL/kg group were significantly increased (P<0.05,P<0.01); PSD-95 expression was significantly increased in cortex and CA1 area in Xingnaojing 1.2 mL/kg group, CA1 area in 2.5 mL/kg group, and the cortex in 5 mL/kg group (P<0.05,P<0.01); expression of a-synuclein was decreased in the CA3 area in 1.2 mL/kg group, cortex in 2.5 mL/kg group, and CA1 and CA3 area in 5 mL/kg group (P<0.05.P<0.01).
Compared with the sham group, on 7th d post-ischemia, expression of synapsin-Ⅰin the model group in the CA1 area and cerebral cortex was still significantly lower (P<0.05, P<0.01), expression PSD-95 in CA1, CA3 areas and cortex remained significantly lower (P<0.01) than the sham group; expression ofα-synuclein in CA1 area, CA3 area, and cerebral cortex was still significantly increased (P<0.01). Compared with model group, expression of synapsin-Ⅰon CA3 area, and cerebral cortex in Xingnaojing 1.2 mL/kg,2.5 mL/kg,5 mL/kg groups were significantly increased (P<0.05,P<0.01); expression of PSD-95 on CA3 and cerebral cortex in Xingnaojing 1.2 mL/kg group was increased (P<0.05, P<0.01), in CA1 area in 2.5 mL/kg group was increased (P<0.05), in CA1, CA3 area, and cerebral cortex in 5 mL/kg group was increased significantly (P<0.05,P<0.01); expression ofα-synuclein in CA1, CA3, and cerebral cortex in Xingnaojing 1.2 mL/kg,2.5 mL/kg,5 mL/kg groups were decreased (P<0.05,P<0.01).
Conclusion:after pMCAO, expression of synaptic function related proteins, synapsin-Ⅰ, PSD-95,α-synuclein were significantly changed, indicating damage of synaptic function. On 2nd d after ischemia, Xingnaojing Injection was able to increase expression PSD-95 and synapsin-Ⅰ, and to reduce the accumulation ofα-synuclein. Part of this action lasted until 7th d post ischemia, indicating that Xingnaojing injection had a certain effect on neuronal synaptic function.
2.3 Effect of Xingnaojing injection on synaptic function in pMCAO model rats
Objective:it is to investigate the effect of Xingnaojing injection on synaptic structure after cerebral ischemia at different time points.
Methods:pMCAO model was established by suture. Rats were randomly divided into model group, Xingnaojing Injection 2.5 mL/kg group and 5mL/kg group, taking Gingko injection 2.5 mL/kg group as the control group, taking normal SD rats as the sham group. Rats were administered with medicines 6 h-7 d post-operation. At 2 d and 7 d after ischemia, expression PSD-95, p-CaMKⅡ, NR2B proteins were detected with western blotting.
Results:on 2nd d post-ischemia, in model group, PSD-95 protein expression in the cortex and hippocampus was significantly lower (P<0.01); compared with model group, in treatment group, protein expression of PSD-95 was significantly increased (P<0.05,P<0.01). On 7th day post-ischemia, compared with the sham group, protein expression of PSD-95 was still significantly lower (P<0.05,P<0.01); PSD-95 protein expression in cortex in treatment groups increased (P<0.05,P<0.01), PSD-95 protein expression also increased in Gingko group, Xingnaojing Injection 5 mL/kg group in hippocampal area, but there was no significant difference.
On 2nd d post ischemia, compared with the sham group, in model group, protein expression of p-CaMKII in the cortex and hippocampus areas significantly decreased (P<0.05); compared with model group, protein expression of p-CaMKⅡwas significantly increased in Gingko group (P<0.05). On 7th day after ischemia, in model group, there was no significant difference on protein expression p-CaMKII compared with the sham group. There was no significant difference on protein expression of p-CaMKII between the mdoel group and treatment groups.
At 2nd d post ischemia, in model group, protein expression of NR2B was significantly lower (P<0.01) than in the control group; compared with model group, NR2B protein expression in Xingnaojing treatment groups significantly increased (P<0.05,P<0.01). On 7th d post ischemia, there was no significant difference on NR2B protein expression between the sham group and model group. Compared with model group, the expression level of Xingnaojing treatment group has increased (P<0.05,P<0.01).
Conculsion:Xingnaojing injection was able to affect protein expression in both cortex and hippocampus areas in rat model of pMCAO in the acute and sub-acute phase. It was able to prevent protein loss related to ischemia-induced synaptic dysfunction; this might be one of the mechanisms for its neuroprotective effect.
中药对于缺血性脑卒中的疗效已得到广泛认可,其作用靶点广泛,作用持久、温和,毒副作用小,在对症治疗的同时,也可对机体整体状态进行调节。我国医药资源丰富,经典名方、验方,经现代工艺制剂后,在确保临床疗效的同时,也扩大药物的应用范围,得到广泛认可。
醒脑静注射液由麝香、冰片、栀子、郁金等药物组成,能够清热泻火,凉血解毒,开窍醒脑。临床上用于常用于治疗神经系统损伤性疾病,如脑卒中、一氧化碳中毒、肝昏迷等急重症,尤其是对于急性缺血性脑病具有良好疗效,得到临床工作者的认可。然而,由于其剂型原因,醒脑静注射液不便于病人自行给药,使其不能长期应用于脑缺血恢复期的治疗,为其临床应用带来一定局限性。醒脑静口服制剂是注射液原方提取物,在确保疗效的基础上,可延长药物临床应用时间,且便于服用。经过前期筛选,目前制剂工艺已确定。并其对缺血性脑卒中模型大鼠具有一定作用保护作用,其对抗缺血性脑损伤的作用得到初步肯定。
本研究采用大脑中动脉血栓(MCAT)模型,通过比较两种醒脑静复方制剂(醒脑静注射液和醒脑静口服制剂)对局灶性脑缺血模型动物神经功能缺损评分和脑梗死体积的影响,明确口服制剂对大鼠缺血性脑损伤模型的保护作用。并采用永久性局灶性中动脉阻断脑缺血(pMCAO)大鼠模型,观察醒脑静口服制剂在脑缺血恢复早期(14 d)对缺血模型动物肢体运动、平衡能力相关指标,评价药物在缺血性脑损伤恢复早期的神经保护作用。另外,本研究中还采用转基因脑卒中动物模型——自发性脑卒中大鼠(SHRSP),评价醒脑静口服制剂对SHRSP血压、心率及一般体征的影响,并从对抗氧化性损伤的角度评价了醒脑静口服制剂对SHRSP的保护作用。
已有研究结果表明,醒脑静注射液对缺血性脑损伤具有明确保护作用,其作用机制与抗炎、抗氧化、改善能量代谢障碍等作用有关,但其对突触结构、功能的影响未见报道。本研究采用永久性局灶性中动脉阻断脑缺血(pMCAO)大鼠模型,从神经元病理改变、突触结构、功能角度,观察醒脑静注射液对脑缺血急性期神经元病理改变、突触结构改变、及突触功能相关蛋白表达的影响,探讨醒脑静注射液对局灶性脑缺血模型大鼠的保护作用机制。
本研究主要内容如下：
第一部分醒脑静口服制剂对局灶性脑缺血模型大鼠和自发性脑卒中大鼠的影响
第一节醒脑静口服制剂对局灶性脑缺血模型大鼠的保护作用
实验一两种醒脑静制剂对三氯化铁所致局灶性脑缺血模型大鼠脑损伤的保护作用
目的：观察两种醒脑静复方制剂对局灶性脑缺血模型大鼠的神经功能、脑梗死范围的影响,明确口服制剂在脑缺血急性期的脑保护作用。
方法：实验动物随机分为5组,即模型组、醒脑静注射液3、9mL/kg组(相当于临床人用量9倍、27倍)、醒脑静口服制剂40mg/kg、120mg/kg(每10 mL注射液生药量相当于口服制剂0.1289g,分别相当于临床人用量9倍、27倍)组,另设假手术组。造模前,醒脑静口服制剂组灌胃给药两天2 d,醒脑静注射液组腹腔注射给药,1次/d；第3 d给药1 h后,采用三氯化铁凝闭大脑中动脉,造成大鼠大脑中动脉血栓形成(MCAT)模型,造模6 h后给药1次。于造模后6h、24 h分别对大鼠进行神经功能缺损症状评分。并于造模24 h后,取脑,冠状切片,进行TTC染色,对白色梗死组织进行称重,以梗塞组织重量占总脑重量的百分比作为脑梗塞范围。
结果：实验结果显示,缺血后模型动物均表现出一定程度的神经功能缺损症状,表现为：身体蜷缩,身体平衡能力下降,向左侧旋转、倾倒,不能行走等。术后6 h,与模型组相比,醒脑静注射液9 mL/kg组神经功能缺损症状评分明显降低(P<0.05),醒脑静口服制剂也能够明显减轻缺血后神经功能损伤(P<0.05)；术后24h,与模型组相比,醒脑静3 mL/kg、9 mL/kg组神经功能缺损均明显减轻(P<0.05,P<0.01),醒脑静口服制剂120mg/kg组大鼠神经功能缺损症状明显减轻(P<0.05)。手术24 h后,缺血组动物脑组织可见不同程度的白色梗死灶。其中,模型组脑梗死组织可达14.9 g,经给药治疗,给药组脑梗死范围较模型组明显降低,醒脑静注射液3 mL/kg、9 mL/kg组均能够明显减小梗死范围(P<0.05,P<0.01)；醒脑静口服制剂40mg/kg、120mg/kg组能够显著减少梗死体积(P<0.01)。
结论：醒脑静口服制剂在缺血性脑损伤发生后24 h内能够改善缺血模型动物的神经功能缺损症状,减少缺血后脑组织梗死范围,且与醒脑静注射液组相比,效果无显著性差异。结果表明,醒脑静口服制剂对三氯化铁所致局灶性脑缺血模型大鼠具有保护作用。
实验二醒脑静口服制剂对永久性局灶性大脑中动脉阻断脑缺血(pMCAO)模型大鼠缺血损伤恢复早期神经功能的保护作用
目的：考察醒脑静口服制剂在脑缺血损伤恢复早期对pMCAO模型大鼠神经功能缺损的改善作用。
方法：采用线栓法制作pMCAO大鼠模型。术后3 d,对缺血动物进行神经功能缺损评分,根据评分结果将动物随机分为5组：模型组、醒脑静口服制剂12mg/kg组(相当于临床人用量的3.5倍)、醒脑静口服制剂25 mg/kg组(相当于临床人用量的7倍)、醒脑静口服制剂50 mg/kg (相当于临床人用量的14倍),以丁苯酞70 mg/kg组为阳性对照组,另设假手术组。给药组灌胃给服相应药物,假手术组与模型组灌胃给予等体积橄榄油。脑缺血后3 d至14 d灌胃给药,1次/d。分别于pMCAO后3d、6d、9d、12 d. 14 d,通过神经功能评分、横木行走实验、前肢触觉刺激实验、前肢抓握力量测试评价药物对缺血模型动物神经功能的保护作用。于14 d末次给药后,取动物脑组织,生化法测定脑组织中MDA含量及SOD活力。
结果：与假手术组相比,缺血模型大鼠表现出明显神经功能缺损症状。缺血后14 d,与模型组相比,醒脑静12 mg/kg,25 mg/kg和50 mg/kg组神经功能缺损症状明显改善,神经功能缺损症状评分降低(P<0.01),横木行走实验评分明显增加(P<0.05,P<0.01),撕除胶布潜伏期明显缩短(P<0.05,P<0.01),前肢抓握力量明显增加(P<0.01)。缺血14 d后,与假手术组相比,模型组大鼠脑组织中SOD活力明显下降,MDA含量明显增加,且与模型组相比,醒脑静12 mg/kg,25 mg/kg和50 mg/kg组脑组织MDA含量均明显降低(P<0.01)。
结论：醒脑静口服制剂在脑缺血恢复早期(14 d)对pMCAO模型大鼠具有神经保护作用,能够改善缺血动物神经功能缺损症状,减轻缺血后脑组织脂质过氧化损伤。
第二节醒脑静口服制剂对自发性脑卒中大鼠(SHRSP/Izm)的影响及抗氧化性损伤作用的研究
实验一醒脑静口服制剂对SHRSP机体状态的影响
目的：采用自发性脑卒中大鼠(SHRSP)模型,观察醒脑静口服制剂对于SHRSP血压、心率及一般体征的影响。
方法：SHRSP给予食盐负荷饮食(40 g/kg饮食),按体重随机分为4组：SHRSP组、维生素C组(ascorbic acid, AA)、醒脑静25 mg/kg组、醒脑静50 mg/kg组。AA组每日给予维生素C水溶液(1000 mg/d),醒脑静组灌胃给服相应剂量药物,SHRSP组灌胃等体积饮用水,1次/d,连续给药6周。每周监测大鼠体重变化情况,并于给药前(0周),给药第2、4、6周进行收缩压(SBP)和心率(HR)测定,及一般体征(包括进食量、饮水量、24小时尿量、24小时排便量)的监测。
结果：药前(第0周)各组动物体重、一般体征无差异。自给药第2周开始,给予食盐负荷后大鼠饮水量、排尿量、排便量明显增加,但与SHRSP组相比,AA组进食量明显减少(P<0.01),饮水量、排尿量、均显著降低(P<0.01),体重减轻(P<0.01)。给药第6周,各组进食量无差异,但AA组体重仍明显低于SHRSP组(P<0.01)。醒脑静组体重、进食量与SHRSP组相比均无显著性差异(P>0.05)。给药前,各组大鼠SBP无显著性差异；给予食盐负荷后,第2周,各组大鼠SBP均出现明显上升趋势；第4周,AA组、醒脑静高剂量组SBP较SHRSP组有下降趋势,但未表现出显著性差异；第6周此种趋势消失,除AA组SBP仍低于SHRSP组以外,其他各组SBP较模型组有所升高,但结果尚无统计学意义。给药第2周,各组HR有下降趋势,但从第4周至第
6周实验结束,HR呈现出上升趋势,且各组间无显著性差异。
结论：醒脑静口服制剂对SHRSP SBP及HR无显著影响,对动物体重增长、进食量、饮水量等一般体征无明显影响。
实验二醒脑静口服制剂对SHRSP氧化性损伤的影响
目的：观察醒脑静口服制剂对SHRSP氧化性损伤状态的影响,评价醒脑静口服制剂对SHRSP的保护作用。
方法：SHRSP给予盐负荷饮食(40 g/kg饮食),按体重随机分为4组：SHRSP组、维生素C组(ascorbic acid, AA)、醒脑静25 mg/kg组、醒脑静50 mg/kg组。AA组每日给予维生素c水溶液(1000 mg/d),醒脑静组灌胃给服相应剂量药物,SHRSP组灌胃给予等体积饮用水,1次/d,连续给药6周。大鼠于给药第2、4、6周采集血浆样本,生化法测定血中总体抗氧化状态(TAS, total antioxidant status);并于第6周给药结束后TBARS法测定脑组织、血浆中丙二醛(MDA)含量,ELISA法测定尿液8-羟基脱氧鸟苷(8-OHdG)含量,RT-PCR (?)去测定脑组织中SOD、TNF-αmRNA表达水平。
结果：给药第2周,与SHRSP组相比,AA组及醒脑静给药各组动物表现出自身抗氧化能力增加。醒脑静50 mg/kg组血浆TAS明显提高,抑制过氧化物能力增强(P<0.01)：第4周,AA组血浆TAS明显增加(P<0.05),醒脑静各剂量组TAS也较SHRSP组有所增加,但未表现出统计学差异；第6周,与SHRSP组相比,醒脑静50 mg/kg组血浆TAS均显著增加(P<0.01)。
给药第6周,与SHRSP组相比,AA组尿液中8-OHdG含量明显降低(P<0.05),其他给药组末见统计学差异；各组大鼠脑组织中MDA含量无显著性差异；SHRSP组血浆MDA含量较高,各给药组血浆MDA含量均有不同程度降低,其中醒脑静50 mg/kg组血浆MDA含量明显降低(P<0.05,P<0.01)。
与SHRSP组比较,给药第6周,给药组脑组织中SOD mRNA表达均增加,但至实验结束,尚未显示出统计学差异；醒脑静25 mg/kg组、50 mg/kg组TNF-a mRNA表达出现下降趋势,但尚无统计学意义。
结论：醒脑静口服制剂能够降低SHPSP体内脂质过氧化物含量,提高动物体内总体抗氧化能力,这种抗氧化能力的改善或许对SHPSP起保护作用。
第二部分醒脑静注射液的作用机制研究——醒脑静注射液对pMCAO模型大鼠突触可塑性的影响
实验一醒脑静注射液对pMCAO模型大鼠缺血神经元的保护作用
目的：观察醒脑静注射液对pMCAO模型大鼠在脑缺血急性期神经元的保护作用。
方法：采用线栓法建立pMCAO大鼠模型,缺血6 h后,模型动物随机分成5组：模型组、醒脑静1.2 mL/kg组(相当于临床人用量的3.5倍)、醒脑静2.5 mL/kg组(相当于临床人用量的7倍)、醒脑静5 mL/kg(当于临床人用量的14倍),以金纳多2.5 mL/kg组为阳性对照组,另设假手术组。给药组腹腔注射相应药物,假手术组与模型组腹腔注射等体积生理盐水。脑缺血后6 h至7 d给药,1次/d。采用苏木素-伊红染色(H&E staining)和尼氏染色(Nissl staining)(?)去观察醒脑静注射液对缺血后神经元病理改变的影响,采用透射电镜观察醒脑静注射液对脑缺血后神经元超微结构改变的影响。
结果：缺血2 d后,光镜下可见假手术组皮层神经元细胞形态圆整,排列整齐,神经元胞浆丰富,尼氏小体分布均匀；模型组脑组织皮层神经元大量变性坏死,核固缩深染,尼氏小体含量明显减少；与模型组相比,给药组皮层完整神经元数目增加,海马区神经元排列较为整齐,死亡神经元数目减少,神经元损伤程度明显减轻,尼氏小体脱失减轻。电镜下可见假手术组神经元细胞核形态规整,核膜光滑完整,核仁明显,染色质分布均匀,胞浆内细胞器丰富,线粒体成椭圆或圆形,脊清晰；模型组神经元核固缩,细胞周围呈现空泡状,细胞器少见；给药组细胞形态基本正常,核膜核仁清晰可见,且细胞器较为丰富。
缺血7 d后,模型组缺血周围区域仍可见大量神经元缺失,神经元变性死亡,尼氏小体脱失；与模型组相比,醒脑静注射液组神经元损失数目减少,结构完整、可辨认的神经元数量较多,尼氏体表达增加。电镜下可见缺血至7 d,模型组神经元固缩,核膜形态不规则,核内染色质浓缩、边集；醒脑静注射液组可见神经元形态基本正常,核膜光滑,核仁明显,且染色质均匀分布。
结论：醒脑静注射液在脑缺血急性期对神经元起保护作用,减轻因缺血造成的神经元死亡、尼氏小体脱失及神经元超微结构破坏。实验二醒脑静注射液对pMCAO模型大鼠缺血突触结构的影响
目的：观察醒脑静注射液对pMCAO模型大鼠在脑缺血急性期突触结构,及突触结构相关蛋白表达的影响。
方法：采用线栓法建立pMCAO大鼠模型,缺血6 h后,模型动物随机分成5组：模型组、醒脑静1.2 mL/kg组、醒脑静2.5 mL/kg组、醒脑静5 mL/kg,以金纳多2.5 mL/kg组为阳性对照组,另设假手术组。给药组腹腔注射给予相应药物,假手术组与模型组腹腔注射等体积生理盐水。脑缺血后6 h至7 d给药,1次/d。采用透射电镜观察pMCAO模型大鼠突触结构,免疫组织化学染色法观察缺血后突触结构相关蛋白的表达情况以及醒脑静注射液的干预作用。
结果：缺血2 d后,电镜下可见假手术组突触结构正常,突触前后膜结构清晰；模型组动物突触数量稀少,突触结构不清；给药组可见较多突触结构,形态丰富。缺血7 d后,模型组突触结构少见；给药组可见较为清晰突触结构,突触间隙可见,突触前膜靠近突触间隙一侧偶见突触小泡聚集。
与假手术组相比,缺血2 d后,模型组动物synapsin-Ⅰ、PSD-95在海马CA1区、CA3区、皮层均明显下降(P<0.01),α-synuclein在海马CA1区、CA3区、皮层表达均明显增加(P<0.01)。与模型组相比,synapsin-Ⅰ在醒脑静1.2 mL/kg组CA3区,2.5 mL/kg组CA3区和皮层,5 mL/kg组CA1区、CA3区、皮层表达明显升高(P<0.05,P<0.01);PSD-95在醒脑静1.2mL/kg组皮层,2.5 mL/kg组CA1区,5 mL/kg组CA1区和皮层表达明显升高(P<0.05,P<0.01);α-synuclein在醒脑静1.2 mL/kg组CA3区,2.5 mL/kg组皮层,醒脑静5 mL/kg组CA1区、CA3区和皮层表达均显著减少(P<0.05,P<0.01)。
缺血7d后,与假手术组相比,模型组synapsin-Ⅰ在海马CA1区、皮层表达仍显著降低(P<0.05,P<0.01), PSD-95在CA1区、CA3区和皮层表达仍显著降低(P<0.01),其中在CA1、CA3区表达略有上升,但与2d表达水平相比无显著性差异,α-synuclein在海马CA1区、CA3区和皮层表达仍显著增加(P<0.01)。与模型组相比,醒脑静1.2mL/kg、2.5 mL/kg、5 mL/kg组synapsin-Ⅰ在CA3、皮层表达仍有明显增加(P<0.05, P<0.01), PSD-95在醒脑静1.2 mL/kg组CA3、皮层表达有所上升(P<0.05,P<0.01), 2.5 mL/kg组CA1表达增加(P<0.05),5 mL/kg组CA1、CA3、皮层表达均有明显增加(P<0.05,P<0.01);α-synuclein在醒脑静1.2 mL/kg、2.5 mL/kg、5 mL/kg组CA1区、CA3区、皮层表达均有所下降(P<0.05,P<0.01)。
结论：脑缺血急性期,pMCAO模型大鼠突触结构破坏,与突触功能密切相关蛋白synapsin-Ⅰ、PSD-95、α-synuclein表达发生改变,表明突触结构、功能受到损伤。醒脑静注射液在缺血后2 d即能增加皮层、海马部位的PSD-95、synapsin-Ⅰ表达,并减少α-synuclein的积聚,这种作用持续到缺血后7 d仍然存在,表明醒脑静注射液在脑缺血急性期、亚急性期对神经元突触结构、功能具有保护作用。实验三醒脑静注射液对pMCAO大鼠突触功能相关蛋白表达的影响
目的：观察醒脑静注射液对pMCAO模型大鼠突触功能相关蛋白表达的影响。
方法：采用线栓法建立pMCAO大鼠模型,缺血6 h后,模型动物随机分成4组：模型组、醒脑静2.5 mL/kg组、醒脑静5 mL/kg,以金纳多2.5 mL/kg组为阳性对照组,另设假手术组。给药组腹腔注射给予相应药物,假手术组与模型组腹腔注射等体积生理盐水。脑缺血后6 h至7 d给药,1次/d。采用Western Blot (?)去观察缺血2 d、7 d后,pMCAO模型大鼠皮层、海马区域PSD-95、p-CaMKⅡ、NR2B蛋白的表达情况,以及醒脑静注射液对蛋白表达的影响。
结果：缺血后2 d,与假手术组相比,模型组PSD-95蛋白在皮层、海马表达均明显降低(P<0.01)；与模型组相比,给药组PSD-95蛋白明显增加(P<0.05,P<0.01)。缺血后7 d,与假手术相比,PSD-95蛋白的表达仍显著降低(P<0.05,P<0.01)；与模型组相比,醒脑静给药组皮层PSD-95蛋白的表达均有不同程度增加(P<0.05,P<0.01),金纳多组、醒脑静5.0 mL/kg组海马PSD-95蛋白表达也有增加,但未见统计学差异。
缺血后2 d,与假手术组相比,模型组p-CaMKⅡ蛋白在皮层、海马表达均明显降低(P<0.05)；与模型组相比,金纳多组p-CaMKⅡ蛋白表达明显增加(P<0.05)。缺血后7 d,模型组p-CaMKⅡ蛋白的表达与假手术组无显著性差异；与模型组相比,给药组p-CaMKⅡ蛋白的表达未见统计学差异。
缺血后2 d,与假手术组相比,模型组NR2B蛋白表达有所下降,其中在海马的表达显著降低(P<0.01)；与模型组相比,醒脑静给药组NR2B蛋白表达明显增加(P<0.05,P<0.01)。缺血后7 d,与假手术组相比,模型组NR2B蛋白在皮层、海马表达无显著性差异；但与模型组相比,醒脑静给药组表达水平仍有明显增加(P<0.05,P<0.01)。
结论：醒脑静注射液在缺血损伤急性期能够增加PSD-95、p-CaMKⅡ、NR2B蛋白的表达,对抗缺血引起的突触功能损伤。醒脑静注射液对突触的保护作用在脑缺血急性期(2d)作用较强,这或许是醒脑静注射液对抗急性缺血性脑损伤的机制之一。
Cerebral ischemic stroke is one of serious diseases that threaten human being, which with high incidence, especially in the senile. Due to its high morbidity and mortality, cerebral ischemic stroke imposes a heavy burden on individuals, families and society. In previous reports, the vast majority of patients with stroke suffer from unrecoverable neurological defects even when they are treated, such as limb dysfunction, mental retardation, memory loss, depression, and so on. These symptoms may have troubled the patients all the time. With no appropriate measures of treatment and intervention, they may continue and aggravate over time. Therefore, treatment in sub-acute phase is essential.
Traditional Chinese medicines are effective for cerebral ischemia, which always act through various pathways. They are usually mild with lasting effect, with few side effects. They can treat both symptoms and causes of diseases, with regulation on generalized condition. In China, there are many famous classic formulas in ancient times, which can be prepared into Chinese patents by modern techniques. These patents are effective as ancient formulas in clinic, and can be applied for more symptoms because of their convenience for administration.
Xingnaojing Injection is commonly used in clinic, which is composed of she xiang (Moschus), bing pian (Borneolum Synthcticum), yu jin (Radix Curcumae Wenyujin), and zhi zi(Fructus Gradeniae). It is commonly applied to nervous system diseases, such as carbon monoxide poisoning, coma hepaticum, and so on. It is effective significantly for acute cerebral ischemia. However, injection is usually applied for acute symptoms. Because of its dosage form, it can not be administered out of hospitals. Development of peroral preparation of Xingnaojing injection will ensure its wide application. Peroral preparation of Xingnaojing is made from extract of identical Chinese medicinals of Xingnaojing injection. Based on previous study, effect of peroral preparation of Xingnaojing has been proved.
In this study, neuroprotective effect of Xingnaojing peroral preparation was evaluated in MCAT (Middle Cerebral Artery Thrombosis) model rats. We observed improvements in neuro-functions and the size of cerebral infarction to make sure both injection and peroral preparations have neuroprotective effect in acute phase of cerebral ischemia. In addition, we further explored action of peroral preparation in convalescence in pMCAO (permanent middle artery occlusion) model rats, and evaluated its effect as improvement on neurological defects and relief on oxidative stress. In addition, in this study, SHRSP was used to evaluate antioxidant action of Xingnaojing peroral preparation.
Studies have shown that the protection of Xingnaojing injection for ischemic injury is related to its anti-inflammatory, sedative, anti-oxidatant and other effects. However, whether its neuroprotective action is related to synaptic function has not been explored yet. We evaluated protective effect of Xingnaojing injection on neurons and synapse in acute and sub-acute phases in rat model of pMCAO. Post-ischemia synaptic structure and function was observed, and action of Xingnaojing Injection on synaptic structure and function was evaluated.
This study is composed of the two following parts:
1 Effect of Xingnaojing peroral preparation on rat model of focal cerebral ischemia and stroke-prone spontaneously hypertension rat
1.1 Neuroprotection of Xingnaojing peroral preparationin on rat model of focal cerebral ischemia
1.1.1 Protection from two preparations of on Xingnaojing on rat model of FeCl3-induced focal cerebral ischemia
Objective:it is to evaluate effects of preparations of Xingnaojing in acute phase of cerebral ischemia, and further demonstrate neuroprotection of Xingnaojing peroral preparation.
Methods:SD rats were randomly divided into model group, Xingnaojing injection 3 mL/kg group and 9 mL/kg group (equal to 9- and 27-time clinical dosage of adult),40 mg/kg group and 120 mg/kg group of peroral preparation, and the sham group. Rats were administered with peroral preparation two days earlier before the establishment of model, or intraperitoneally administered with Xingnaojing injection, once per day. One hour later administration on the third day, MCAT model in rats was established with FeCl3 Rats were administered with medicines 6 hours later after establishment of model. On 6 h and 24 h after establishment of model, scores on neurological defects were measured. And 24 hours after establishment of model, coronal sections of brain tissue was taken and stained with TTC. Weight of white infarction and whole brain tissues was measured, taking percentage weight of infarction to the total weight of brain as degree of cerebral infarct.
Results:after MCAT, rats got some neurological defects, such as curled body, poor balance, rotation and falling to the left side, inability to walk, and so on. Compared with model group, 9 mL/kg group of Xingnaojing Injection and 120 mg/kg group of Xingnaojing peroral preparation got lower score on neurological defects at 6 h post-MCAT (P<0.05); 24 h post-MCAT, Xingnaojing Injection 3 mL/kg and 9 mL/kg groups got significantly improved on neurological defects (P<0.05,P<0.01),120 mg/kg group of peroral preparation of Xingnaojing also got milder defects (P<0.01) than control group. In the sham group, there was no infarction on brain tissue, while white infracted area in the model group. In the medicated group, infarction size was more reduced than the model group. In the 3 mL/kg and 9 mL/kg groups of Xingnaojing Injection,120 mg/kg group of peroral preparation of Xingnaojing, infarction size was significantly smaller (P<0.05,P<0.01).
Conclusion:both Xingnaojing Injection and peroral preparation of Xingnaojing had significant neuroprotective effects within 24 hour after cerebral ischemia.
1.1.2 Protective effects of peroral preparation of Xingnaojing on neurological defects and oxidative damage in pMCAO model rats in early convalescence
Objective:it is to evaluate peroral preparation of Xingnaojing in pMCAO model rats in early convalescence
Method:pMCAO model was established with suture. Rats were randomly divided into model group, sham group, peroral preparation of Xingnaojing 12 mg/kg group,25 mg/kg group, and 50 mg/kg group, taking Butylphthalide 70 mg/kg group as positive control group. All animals were administered medicines 3-14 d post-ischemia, rats in the sham group were administered with same volume of oleum olivae. Neurological deficit scores, tactile stimulation experiments, beam walking test, strength test were operated to observe protective effects of peroral preparation of Xingnaojing on neural function in pMCAO model rats on day 3,6,9, 12, and 14; and measure SOD activity and MDA content in ischemic brain tissue.
Results:compared with the sham group, rats in the model group got obvious symptoms of neurological defects. After treatment, Xingnaojing 12 mg/kg group,25 mg/kg group, and 50 mg/kg group got significantly improvement on their neurological functions, manifesting improvement on scores of neurological defect (P<0.01), beam walking ability (P<0.05, P<0.01), and recovery of forelimbs strength (P<0.05,P<0.01), and enhancement of tactile sensitivity (P<0.01). Peroral preparation of Xingnaojing was able to significantly reduce the MDA content of ischemic brain tissue.
Conclusion:peroral preparation of Xingnaojing had protective action on pMCAO rat, by reducing oxidative damage.
1.2 Study on protection of Xingnaojing peroral preparation on SHRSP and related mechanism
1.2.1 Action of of Xingnaojing peroral preparation on general status in SHRSP
Objective:it is to explore impacts of peroral preparation of Xingnaojing on body weight, blood pressure, heart rate, and general condition in SHRSP.
Method:all SHRSP were given salt-loaded diet, and were randomly divided into SHRSP group, the AA group (vitamin C,1000 mg/d), Xingnaojing 25 mg/kg group and 50 mg/kg group. All rats were intragastrically administered with medicines for 6 weeks. Food intake, water intake, urine output, and defecation output within 24-hour were measured on the 2nd,4th and 6th weeks respectively.
Results:there were no significant differences on body weight and general condition between groups pre-medication (0 week). Since second week, given the salt-loaded food, water intake, urine output, the amount of defecation of SHRSP increased significantly. Compared with the SHRSP group, food intake, water intake, and urine output in AA group was significantly reduced (P<0.01), with great body weight loss (P<0.01). In the 6th week of medication, there were no significant differences on food intake between groups, but the body weight in the AA group was still significantly less than others'(P<0.01). There was no significant difference on body weight, food intake Xingnaojing-treated groups (P>0.05). Given salt-loaded food, SBP of rats significantly increased since 2nd week of treatment; in the 4th week, SBP in the AA group, and Xingnaojing 50 mg/kg group slightly reduced, but with no significant differences compared with SHRSP group. This tendency did not vanish unitl the 6th week, in which, SBP in AA group was lower than in the SHRSP group, while was slightly higher in the Xingnaoj ing-treated groups. However, there was no significant difference between groups. Since 2nd week of medication, HR decreased in every group slightly, while increased until 6th week, with no significant difference between groups.
Conclusion:peroral preparation of Xingnaojing had no significant impact on body weight, general condition, blood pressure, and heart rate.
2.2 Antioxidant action of Xingnaojing peroral preparation of in SHRSP
Objective:to investigate antioxidant action and related mechanism of Xingnaojing peroral preparation in SHRSP.
Method:all SHRSP given the high salt load diet were randomly divided into SHRSP group, the AA group (1000 mg/d), Xingnaojing 25 mg/kg, and 50 mg/kg group. Rats were administered with medicine for 6 weeks. Plasma TAS was measured respectively in 2nd,4th and 6th week, and MDA content in the brain tissue and plasma was measured on 6th week, and expression of SOD and TNF-αmRNA was measured by RT-PCR.
Results:in 2nd week, Xingnaojing peroral preparation was able to increase level of plasma
TAS. It had no significant action in 4th week. However, it was able to increase level of plasma TAS. It was able to reduce content of MDA in the plasma. There was only tendency of increased level of SOD and decreased TNF-αmRNA expression in medicated group, but no significant difference.
Conclusion:Xingnaojing peroral preparation of had protective action on SHRSP, which might relate to reduction on oxidative damage.
2 Study on protection of Xingnaojing injection on synapse and mechanism
2.1 Protection of Xingnaojing injection on neurons in pMCAO model rat Objective:to investigate neuronal protection of XNJ injection in pMCAO model rats in acute and sub-acute phase.
Methods:pMCAO model was established with suture. Rats were randomly divided into sham group, model group, XNJ Injection 1mL/kg,2.5 mL/kg, the 5 mL/kg groups, and taking Ginkgo injection 2.5 mL/kg group as control. Postoperative 6 h-7 d rats were administered respectively, once per day. H&E staining and Nissl staining was applied to observe morphology changes on neurons on 2 d and 7 d after ischemia. Microstructure of neurons was observed by transmission electron microscope.
Results:on 2nd d post-ischemia, in visual fields of optical microscope, cortical neurons in the sham group were round with complete form. It was rich in cytoplasm in neurons with plenty of Nissl bodies evenly distributed. In the model group, there were a great number of degenerated and necrotic neurons in the cortex, manifesting as shrunken and darkly stained nucleus, and great loss of Nissl bodies. In the treatment groups, there were more complete neurons in the cortex, with clearly arranged neurons in the hippocampus areas. There were less dead neurons, less damage to neurons, and less loss of Nissl bodies. In the field of transmission electron microscope, nuclear morphology in the sham group was clear with complete nuclear membrane, unabroken karyosome, and evenly distributed chromatin. There were plenty of organelles in the cytoplasm, of which itochondria was oval or round. However, in the model group, there were pycnotic neurons, around which, there were vacuolars. There was less organelle in the cytoplasm. In the treatment groups, morphology of neurons was normal with clear nuclear membrane and plenty of organelles.
Conclusion:in acute phase of cerebral ischemia, Xingnaojing Injection has protective effect on neurons by preventing neuronal death, loss of Nissl bodies, and ultrastructure damage of neurons induced by ischemia.
2.2 Effect of Xingnaojing injection on synaptic structure in pMCAO model rats
Objective:to investigate the effect of Xingnaojing injection on synaptic structure after cerebral ischemia at different time points.
Methods:pMCAO model was established by suture. Rats were randomly divided into model group, Xingnaojing Injection 1.2 mL/kg group,2.5 mL/kg group, and 5mL/kg group, taking Gingko injection 2.5mL/kg group as the control group, and the sham group. Rats were administered with medicines 6 h-7 d post-operation. On 2 d and 7 d after ischemia, synapse-related proteins, synapsin-Ⅰ, PSD-95, andα-synuclein were detected with immunohistochemical staining.
Results:compared with the sham group, on 2nd day post-ischemia, in the model group, expression of synapsin-Ⅰ, PSD-95 in hippocampal in CA1 and CA3 area, and cerebral cortex were significantly decreased (P<0.01);α-synuclein in hippocampal CA1 and CA3 area, and cortical expression were significantly increased (P<0.01). Compared with model group, expression of synapsin-Ⅰon CA3 area in Xingnaojing 1.2 mL/kg group, in CA3 and cortex of 2.5 mL/kg group, in CA1 area, CA3 area, and the cortex of 5 mL/kg group were significantly increased (P<0.05,P<0.01); PSD-95 expression was significantly increased in cortex and CA1 area in Xingnaojing 1.2 mL/kg group, CA1 area in 2.5 mL/kg group, and the cortex in 5 mL/kg group (P<0.05,P<0.01); expression of a-synuclein was decreased in the CA3 area in 1.2 mL/kg group, cortex in 2.5 mL/kg group, and CA1 and CA3 area in 5 mL/kg group (P<0.05.P<0.01).
Compared with the sham group, on 7th d post-ischemia, expression of synapsin-Ⅰin the model group in the CA1 area and cerebral cortex was still significantly lower (P<0.05, P<0.01), expression PSD-95 in CA1, CA3 areas and cortex remained significantly lower (P<0.01) than the sham group; expression ofα-synuclein in CA1 area, CA3 area, and cerebral cortex was still significantly increased (P<0.01). Compared with model group, expression of synapsin-Ⅰon CA3 area, and cerebral cortex in Xingnaojing 1.2 mL/kg,2.5 mL/kg,5 mL/kg groups were significantly increased (P<0.05,P<0.01); expression of PSD-95 on CA3 and cerebral cortex in Xingnaojing 1.2 mL/kg group was increased (P<0.05, P<0.01), in CA1 area in 2.5 mL/kg group was increased (P<0.05), in CA1, CA3 area, and cerebral cortex in 5 mL/kg group was increased significantly (P<0.05,P<0.01); expression ofα-synuclein in CA1, CA3, and cerebral cortex in Xingnaojing 1.2 mL/kg,2.5 mL/kg,5 mL/kg groups were decreased (P<0.05,P<0.01).
Conclusion:after pMCAO, expression of synaptic function related proteins, synapsin-Ⅰ, PSD-95,α-synuclein were significantly changed, indicating damage of synaptic function. On 2nd d after ischemia, Xingnaojing Injection was able to increase expression PSD-95 and synapsin-Ⅰ, and to reduce the accumulation ofα-synuclein. Part of this action lasted until 7th d post ischemia, indicating that Xingnaojing injection had a certain effect on neuronal synaptic function.
2.3 Effect of Xingnaojing injection on synaptic function in pMCAO model rats
Objective:it is to investigate the effect of Xingnaojing injection on synaptic structure after cerebral ischemia at different time points.
Methods:pMCAO model was established by suture. Rats were randomly divided into model group, Xingnaojing Injection 2.5 mL/kg group and 5mL/kg group, taking Gingko injection 2.5 mL/kg group as the control group, taking normal SD rats as the sham group. Rats were administered with medicines 6 h-7 d post-operation. At 2 d and 7 d after ischemia, expression PSD-95, p-CaMKⅡ, NR2B proteins were detected with western blotting.
Results:on 2nd d post-ischemia, in model group, PSD-95 protein expression in the cortex and hippocampus was significantly lower (P<0.01); compared with model group, in treatment group, protein expression of PSD-95 was significantly increased (P<0.05,P<0.01). On 7th day post-ischemia, compared with the sham group, protein expression of PSD-95 was still significantly lower (P<0.05,P<0.01); PSD-95 protein expression in cortex in treatment groups increased (P<0.05,P<0.01), PSD-95 protein expression also increased in Gingko group, Xingnaojing Injection 5 mL/kg group in hippocampal area, but there was no significant difference.
On 2nd d post ischemia, compared with the sham group, in model group, protein expression of p-CaMKII in the cortex and hippocampus areas significantly decreased (P<0.05); compared with model group, protein expression of p-CaMKⅡwas significantly increased in Gingko group (P<0.05). On 7th day after ischemia, in model group, there was no significant difference on protein expression p-CaMKII compared with the sham group. There was no significant difference on protein expression of p-CaMKII between the mdoel group and treatment groups.
At 2nd d post ischemia, in model group, protein expression of NR2B was significantly lower (P<0.01) than in the control group; compared with model group, NR2B protein expression in Xingnaojing treatment groups significantly increased (P<0.05,P<0.01). On 7th d post ischemia, there was no significant difference on NR2B protein expression between the sham group and model group. Compared with model group, the expression level of Xingnaojing treatment group has increased (P<0.05,P<0.01).
Conculsion:Xingnaojing injection was able to affect protein expression in both cortex and hippocampus areas in rat model of pMCAO in the acute and sub-acute phase. It was able to prevent protein loss related to ischemia-induced synaptic dysfunction; this might be one of the mechanisms for its neuroprotective effect.
引文
[1]张路晗,向金莲,程睿,等.醒脑静注射液的药效学研究[J].华西药学杂志,2001,16(6)：429-431.
[2]许风雷,高丽霞,吴泰相,等.醒脑静注射液治疗脑梗塞临床疗效及安全性随机对照试验的系统评价[J].中国循证医学杂志,2005,5(7)：549-554.
[3]林森,吴波,刘呜.醒脑静注射液治疗脑出血的系统评价[J].中国循证医学杂志,2008,8(2)：93-96.
[4]庄伟端,欧利民,郑俊忠.等.醒脑静注射液对急性脑梗死意识障碍患者的影响[J].中国中西医结合急救杂志.2001,8(6)：381.
[5]杜守颖,陆洋,姚宗玲,等.醒脑静中各成分对栀子提取物中栀子苷大鼠鼻腔吸收的影响[J].中国实验方剂学杂志,2012,18(4)：146-150.
[6]董六一,魏倩.醒脑静乳剂对家兔失血性休克及感染性休克的保护作用fJl.中国实验方剂学杂志,2010,16(1)：98-101.
[7]唐兴荣,张水冰,余尚贞.脑立苏冲剂对脑缺血模型大鼠的影响[J].广州中医药大学学报,2005,22(5)：383.
[8]蒋振亚,李常度,周东,等.麝香对大鼠实验性脑缺血神经元的损伤的保护作用[J].中国医科大学学报,2007,38(5)：456.
[9]方永奇,李翎.醒脑开窍中药治疗脑病的共性作用概况[J].广州中医药大学学报, 2008,25(5)：470-473.
[10]中国医学科学院药物研究所编.中草药现代研究[M].北京：北京医科大学中国协和医科大学联合出版社,1995：206.
[11]朱长庚.神经解剖学[M].北京：人民卫生出版社,2002：1033.
[12]Mullen KD, Szauter KM, Mc Cullough AJ. Blood-Brain-Barrier (BBB) permeability [J]. Hepatology,1998,8 (2):440.
[13]刘金江,陈华德.血脑屏障与中医药研究[J].浙江中医学院学报,1999,4(23)：2-5.
[14]陈文垲,黄玉芳,王海东.麝香“归经入脑”的实验研究[J].中西医结合学报,2007,(4)：288-291.
[15]莫启忠,宫斌,茅惠明,等.H-麝香酮在体内的吸收分布和排泄：中药麝香芳香开窍机制的初步探讨[J].中成药研究,1984,(7)：1.
[16]曹婉如,林素青,赵蔚君,等.麝香和麝香酮缩短大鼠戊巴比妥钠睡眠时间的探讨[J].中草药,1980,11(7)：309-312.
[17]蒋振亚,李常度.麝香对大鼠实验性脑缺血神经元损伤的保护作用[J].中国中医药科技,2001,8(2)：97-98.
[18]魏楚蓉,伍赶球.冰片的药理作用及其机制研究进展[J].国际病理科学与临床杂志,2010,30(4)：447-451.
[19]梁美蓉,刘启德,黄天来,等.冰片在大鼠血清和脑组织中的药代动力学特征[J].中药新药与临床药理,1993,4(4)：38-41.
[20]陈艳明,王宁生.冰片对血脑屏障体外模型细胞间紧密连接和细胞吞饮囊泡的影响[J].中国中西医结合杂志,2004,24(7)：632-634.
[21]赵保胜,宓穗卿,张银卿.冰片对大鼠脑微血管内皮细胞ICAM-1表达量的影响[J].中药新药与临床药理,2001,12(2)：88-92.
[22]方永奇,邹衍衍,李羚,等.芳香开窍药和祛痰药对中枢神经兴奋性的影响[J].中医药研究,2002,18(3)：40.
[23]薛丽,谌小维,樊宏孝,等.冰片对长时连续作业大鼠前额叶皮层单胺类递质水平的影响[J].第三军医大学学报,2006,28(18)：1867-1869.
[24]胡利民,张艳军,王威,等.冰片与丹酚酸B和三七总皂苷配伍对局灶性脑缺血再灌注大鼠脑VEGF mRNA表达的影响[J].中国中西医结合急救杂志,2005,12(5)：263-266.
[25]何晓静,菅凌燕,刘玉兰.冰片注射液对小鼠实验性脑缺血的保护作用[J].新乡医学院学报,2006,23(1)：23-25.
[26]何晓静,吕庆杰,刘玉兰.冰片注射液对缺血再灌注大鼠脑内炎症反应的影响[J].华西药学杂志,2006,21(6)：523-526.
[27]杨蕾,修春,王宁生.冰片对大鼠血小板胞浆内游离钙离子浓度的影响[J].时珍国 医国药,2010,21(1)：1-3.
[28]杨蕾,李伟荣,宓穗卿,等.冰片对三氯化铁诱导的大鼠动脉血栓形成的抑制作用及机制[J].中国实验方剂学杂志,2010,16(6)：164-166.
[29]刘养凤,张军平,张伯礼,等.冰片对大鼠下丘脑单胺类神经递质的影响[J].中国中医药信息杂志,2004,11(2)：122-124.
[30]上海中医学院基础部同位素室.3H-冰片在机体内的吸收、分布和排泄[J].中成药研究,1981,3(5)：81.
[31]陈艳明,王宁生.冰片对P-糖蛋白的影响[J].中药新药与临床药理,2003,14(2)：96-99.
[32]刘养凤,张军平,张伯礼.冰片对大鼠下丘脑单胺类神经递质的影响[J].中国中医药信息杂志,2004,11(2)：122-124.
[33]李伟荣,姚丽梅,宓穗卿,等.冰片对大鼠下丘脑组胺、5-羟色胺含量的影响[J].中药材,2004,27(12)：937-939.
[34]贾钰铭,丁正,曾令源,等.冰片促进顺铂透过血脑屏障的实验研究[J].四川医学,2004,25(2)：156-157.
[35]刘煜德,孙丹邈,黄小洵,等.冰片对BMSCs透过血脑屏障的影响[J].亚太传统医药,2007,3(7)：46-48.
[36]陈瑞玲,赵志刚,张星虎,等.冰片对丙戊酸钠透过血脑屏障的影响[J].中国康复理论与实践,2007,14(2)：151-153.
[37]肖玉强,张良玉,唐海涛,等.冰片促进砷剂透过血脑屏障实验研究[J].中华神经外科疾病研究杂志,2007,6(3)：244-246.
[38]王勇,张忠义,徐峰,等.冰片对川芎嗪血药浓度和在脑中分布的影响[J].中国药业,2006,15(1)：30-31.
[39]赵保胜,刘启德.冰片促血脑屏障开放与病理性开放的比较[J].中药新药与临床药理,2002,13(5)：287-288.
[40]牟家琬,杨胜华,孙玉梅,等.龙脑与异龙脑的体外抗菌作用的研究[J].华西药学杂志,1989,4(1)：20-22.
[41]江光池,等.龙脑和异龙脑的抗炎作用[J].华西药学杂志,1990,5(3)：190.
[42]江光池,等.龙脑和异龙脑对小鼠和家兔的药理作用[J].华西药学杂志,1989,4(1)：23.
[43]彭婕,钱之玉,刘同征,等.栀子提取物京尼平苷和西红花苷利胆作用的研究[J].中国中药杂志,2003,2(11)：105-108.
[44]王钢力,陈德昌,赵淑杰.栀子属植物化学成分研究进展.中国中药杂志,1996,21(2)：67-69.
[45]张小燕,张占军,王忠,等.栀子苷对局灶性脑缺血大鼠脑组织基因表达谱的影响.中国中西医结合杂志,2005,25(1)：42-44.
[46]朱晓磊,张娜,李澎涛,等.栀子苷阻抑脑缺血损伤级联反应的作用环节探讨.中国中药杂志,2004,29(11)：1065-1068.
[47]Yamazaki M, Chiba K, Yoshikawa C. Genipin suppresses A23187-induced cytotoxicity in neuro2a cells [J]. BIOL PHARMBULL,2009,32 (6):1043-1046.
[48]朱晓磊,张娜,李澎涛,等.胆酸、桅子昔配伍对脑缺血时脑组织TNF-α, IL-1β和ICAM-1含量的影响[J].北京中医药大学学报,2004,27(6)：26-29.
[49]李伟华,朱陵群,王硕仁,等.黄芩苷、栀子苷对神经细胞缺氧缺塘／再灌注损伤的保护作用[J].中国药学杂志,2004,39(5)：344-346.
[50]李传云,李澎涛,潘彦舒,等.牛黄、栀子配伍对大鼠局灶性脑缺血再灌注不同时段脂质过氧化损伤的影响[J].中国医药学报,2004,19(9)：528-530.
[51]李宝莉,陈雅慧,杨暄,等.栀子油的提取和对中枢神经系统的作用[J].第四军医大学学报,2008,29(23)：2152-2155.
[52]郝洪谦,孙兵,郑开俊,等.郁金二酮对家猫睡眠节律电活动的调制作用[J].中草药,1994,25(8)：423-424.
[53]饶旺福,饶淑珍.中医治疗中风病进展[J].江西中医药,1999,30(3)：53-56.
[54]刘岩,王建,姚洪武,等.开窍药对缺血缺氧损伤PC12细胞的影响及其作用机制[J].中药药理与临床,2010,26(4)：35-38.
[55]夏鑫华,刘亚敏,赵光峰.麝香配伍冰片对大鼠局灶性脑缺血再灌注损伤的脑保护研究[J].中国实验方剂学杂志,2009,15(2)：42-45.
[56]倪彩霞,曾南,许福会,等.芳香开窍药对脑缺血再灌注损伤大鼠血脑屏障影响的实验研究[J].中国中药杂志,2011,36(18)：2562-2566.
[57]苗明三.醒脑静注射液对中枢神经系统的影响[J].中药药理与临床,2004,20(3)：42.
[58]Khan M, Elango C, Ansari MA, et al. Caffeic acid phenethyl ester reduces neurovascular inflammation and protects rat brain following transient focal cerebral ischemia [J]. Neurvchem,2007,102 (2):365-377.
[59]Wang Q, Tang XN, Midori A. The inflammatory response in stroke [J]. Neuroimmunol, 2007,184 (1-2):53-68.
[60]Williams AJ, Dave JR, Tortella FC. Neuroprotection with the proteasome inhibitor MLN519 in focal ischemic brain injury:relation to nuclear factor κB (NF-κB), inflammatory gene expression, and leukocyte infiltration [J]. Neurochem Int,2006,49 (2): 106-112.
[61]王万铁,陈寿权,等.醒脑静注射液对脑缺血-再灌注损伤家兔血清白介素-8的影响[J].温州医学院学报,2003,33(1)：1-3.
[62]陈寿权,王万铁,王明山,等.醒脑静对家兔脑缺血再灌流时TNF、IL-1、IL-6水平及脑超微结构影响的实验研究[J].中国急救医学,2000,20(11)：637-639.
[63]沈思钰,傅晓东,陈伟华,等.醒脑静对脑缺血大鼠神经保护作用与氨基酸受体表达的关系[J].中国临床康复,2004,8(4)：686-687.
[64]万文成,李杰芬,罗海燕,等.醒脑静对大鼠皮层神经细胞的保护作用[J].广州中医药大学学报,2002,19(2)：125-126.
[65]张红波,代建峰,张宾辉.醒脑静抗鼠脑缺血-再灌注损伤的实验研究.浙江中西医结合杂志,2006,16(6)：351-353.
[66]周红宇,胡国新,陈言,等.醒脑静注射液对脑缺血再灌注小鼠记忆功能的保护作用.温州医学院学报,2002,32(3)：140-142.
[67]傅强,崔华雷,孙中吉,等.醒脑静注射液对脑缺血再灌注诱导的脑神经细胞凋亡防治作用的实验研究.中国中西医结合急救杂志,2000,7(3)：144-146.
[68]戴永建,戢翰升.醒脑静注射液对大鼠脑损伤后细胞凋亡及相关蛋白表达的影响.中国临床神经外科杂志,2006,(9)：551-553
[1]Renato M.E. Sabbatini. Neurons and Synapses:The History of Its Discovery [J]. Brain & Mind Magazine,2003,17, April-July
[2]Janz R, Sudhof TC, Hammer RE, et al. Essential roles in synaptic plasticity for synaptogyrin I and synaptophysin [J]. Neuron,1999,24 (3):687-671.
[3]Bolay H, Gursoy Ozdemir Y, Sara Y. Persistent defect in transmitter release and synapsin phosphorylation in cerebral cortex after transient moderate ischemic injury [J]. Stroke, 2002,33(5):1369-1375.
[4]张艳玲,李露斯,肖桃元,等.血管性痴呆大鼠的突触超微结构改变及结构参数分析[J].第三军医大学学报,2002,24(3)：264-266.
[5]Coleman PD, Yao PJ. Synaptic slaughter in Alzheimer's disease [J]. Neurobiol Aging, 2003,24(8):1023-1027.
[6]Landis DM, Hall AK, Weinstein LA, et al. The organization of cytoplasm at presynaptic active zone of a central nervous system synapse [J]. Neuron,1988,1 (3):201-209.
[7]Zhen M, Jin Y. Presynaptic terminal differentiation:transport and assembly [J]. CURR OPINNEUROBIOL,2004,14 (3):280.
[8]胡人义.突触的超微结构.复旦神经生物学讲座,1991,7：39.
[9]Colonnier M. Synaptic patterns on different cell types in the different laminae of the cat visual cortex. An electron microscope study [J]. BRAIN RES,1968,9 (2):268-287.
[10]Ziff EB. Enlightening the postsynaptic density [J]. Neuron,1997,19 (6):1163-1173.
[11]Papadia, S., Hardingham, G.E, et al. The dichotomy of NMDA receptor signaling [J]. NEUROSCIENTIST,2007,13 (6):572-579.
[12]Paoletti, P., Neyton, J. NMDA receptor subunits:function and pharmacology [J]. CURR OPIN PHARMACOL,2007,7:39-47.
[13]Dingledine R, Borges K, Bowie D, et al. The glutamate receptor ion channels [J]. PHARMACOL REV,1999,51 (1):7-61
[14]Cousins SL, Kenny AV, Stephenson FA. Delineation of additional PSD-95 binding domains with in NMDA receptor NR2 subunits reveals differences between NR2A /PSD-95 and NR2B/PSD-95 association [J]. NEUROSCIENCE, 2009,158:89-95.
[15]Kennedy, M.B. Signal-processing machines at the postsynaptic density [J]. SCIENCE, 2000,290,750-754.
[16]Xu, W.F. PSD-95-like membrane associated guanylate kinases (PSD-MAGUKs) and synaptic plasticity [J]. Curr Opin Neurobiol.2011,21:306-312.
[17]Carter A G, Regehr W G. Prolonged synaptic currents and glutamate spillover at the parallel fiber to stellate cell synapse [J]. NEUROSCIENCE,2000,20 (12):4423-4434.
[18]Chen S, Diamond J S. Synaptically released glutamate activates extrasynaptic NMDA receptors on cells in the ganglion cell layer of rat retina [J]. NEUROSCIENCE,2002,22 (6):2165-2173.
[19]Lalo U, Pankratov Y, Kirchhoff F, et al. NMDA receptors mediate neuron-to-glia signaling in mouse cortical astrocytes [J]. NEUROSCIENCE,2006,26 (10):2673-2683.
[20]Salter M G, Fern R. NMDA receptors are expressed in developing oligodendrocyte processes and mediate injury [J]. Nature,2005,438 (7071):1167-1171.
[21]Essin K, NistriA, Magazanik L. Evaluation of GluR2 subunit involvementin AMPA receptor function neonatal rat hypoglossal motoneurons [J]. EurJNeurosci,2002,15 (2): 1899-1906.
[22]Koike K, TsukadaS, Tsuzuki K, et al. Regulation of kinetic properties ofGluR2 AMPA receptor channels by alternative splicing [J]. JNEUROSCI 2000,20 (6):2166-2174.
[23]Partin KM. Domain interactions regulating AMPA receptor desensitization [J]. J NEUROSCI,2001,21 (6):1939-1948.
[24]Steiner P, Sarria J C, Glauser L, et al. Modulation of receptor cycling by neuron enriched endosomal protein of 21 kD [J]. J CELL BIOL,2002,157 (7):1197-1209.
[25]Ozawa S, Kamiya H, Tsuzuki K. Glutamate receptors in the mammalian central nervous system [J]. ProgNeurobiol,1998,54 (5):581-618.
[26]Scannevin RH, Huganir RL. Postsynaptic organization and regulation of excitatory synapse [J]. Nat Rev Neurosci,2000,1 (2):133-141.
[27]Peters A, Palay SL, Webster Hd. The fine structure of the nervous system:neurons and their supporting cells.3rded [M]. New York:Oxford University Press,1991:169.
[28]Yang H W. Appenteng K, Batten T F. Ultrastructural subtypesof glutamate immuno reactive terminals on rat trigeminal motoneurones and their relationships with GABA immunoreactive terminals [J]. Exp Brain Res,1997,114 (1):99-1161.
[29]Siekevitz P. The postsynaptic density:A possible role in long lasting effects in the central nervous system [M]. ProcNatlAcademic science,1985,82:3494-3498.
[30]何祥,冯龚.电刺激对脑梗死大鼠突触界面结构的影响[J].中国临床康复,2004,8(31)：6900-6901.
[31]Rao A, Kim E, Sheng M, Craig AM. Heterogeneity in the molecular composition of excitatory postsynaptic sites during development of hippocampal neurons in culture [J]. Neuro-science,1998,18 (4):1217-1229.
[32]Morris R. Developments of a water-maze procedure for studying spatial learning in the rat [J]. JNeurosci Methods,1984,11 (1):47-60.
[33]Haraguchi K, Satoh K, Yanai H, et al. The hDL G-associated protein DAP interacts with dynein light chain and neuronal nitric oxide synthase [J]. Genes Cells,2000,5:905-911.
[34]韩太真,吴馥梅.学习与记忆的神经生物学[M].第1版.北京：北京医科大学中国协和医科大学联合出版社,1998：2201.
[35]JanzR, Sudhof TC, Hammer RE, et al. Essential roles in synaptic plasticity for synaptogyrin I and synaptophysin [J]. Neuron,1999,24 (3):687-691.
[36]Bolay H, Gursoy-Ozdemir Y, Sara Y. Persistent defect intransmitter release and synapsin phosphorylation in cerebralcortex after transient moderate ischemic injury. Stroke,2002, 33 (5):1369-1375.
[37]王慧娟,李陈莉,赵秀丽,等.大鼠脑缺血后突触超微结构的变化[J].解剖学杂志,2003,12(2)：587-590.
[38]赵昱,马洪骏,李陈莉,等.大鼠脑缺血/再灌注后脑组织硝基络氨酸的表达及超微结构的改变[J].苏州大学学报(医学版),2003,23：154-158.
[39]Vanlookeren Campagne M, Destreicher AB. Ultrastructuraldoublecocalization of B250/GAP43 and synaptophysin (P38) in the neonatal and adult rat hippocampus [J]. J Neurocytol,1990,19 (6):948-952.
[40]王慧娟,苏丽英,李陈莉,等.大鼠脑缺血后突触体素与突触密度的关系[J].解剖学杂志,2004,27(6)：642-645.
[41]Kojiro K, Satoshi G, Shinji N, et l. Changes of immunore-activity for synaptophysin following A transient cerebral ischemia in Rhe Rat Striatum [J]. Brain Research,1993, 616:320.
[42]Stroemer RP, Kent TA, Hulsebosch CE. Enhanced neuocortical neural sprouting, synaptogenesis, and behavioral recovery with D2 amphetamine therapy after neocortical infarction in rats [J]. Stroke,1998,29:2381-2395.
[43]邹凡,杨万同,田峻,等.当归注射液对全脑缺血后神经再塑影响的实验研究[J].武汉大学学报(医学版),2004,25(1)：38-41.
[44]Luo Y, Cao G, Pei W, et al. Induct ion of caspase-activated deoxyribonuclease activity after focal cerebral ischemia and reperfusion [J]. J Cereb Blood Flow Metab,2002,22 (1): 15-20.
[45]Swartz KJ. Modulat ion of Ca2+ channels by kinase C in rat central and peripheral neurons:distribut ion of G protein- mediated inhibit ion [J]. Neuron,1993,11 (2): 305-320.
[46]TominagaT, Kure S, Narisawa K, et al. Endonuclease activation following focal ischemic injury in the rat brain [J]. Brain Res,1993,608 (1):21-26.
[47]Schmidt Kastner R, Bedard A, Hakin A. Transient expression of GAP43 within the hippocampus after global brain ischemia in rats [J]. Cell Tissue Res,1997,288 (2): 225-238.
[48]Soderling TR. Structure and regulation of calcium/ calmodulin- dependent protein kinase Ⅱ and Ⅳ[J]. Biochem Biophys Acta,1996,1297:131.
[49]李燕华,李吕力,王铁建,等.大鼠脑缺血后P- CaMK Ⅱ的表达与Ca2+浓度的关系.中国急救医学,2006,26(6)：436-438.
[50]Xia Z, Storm DR. The role of calmodulin as a signal integrator for synaptic plasticity [J]. Nat Rev Neurosc,2005; 6 (4):267-276.
[51]Ikeda J, Terakawa S, Muyota S, et al. Nuclear disintegration as a leading step of glutamate excitotoicity in brain neurons [J]. J Neurosci Res,1996,43 (5):2613-2622.
[52]Belayev L, Busto R, Zhao W, et al. HU-211, a novel noncompetitive N-methyl D-aspartate antagonist, improves neurological deficit and reduces infarct volume after reversible focal cerebral ischemia in the rat [J]. Stroke,1995,26:2313-2319.
[53]Regan R F, Panter S S. Hemoglobin potentiates excito toxic injury in cortical cell culture [J]. Neuro trauma,1996,13 (3):223-231.
[54]Olney J W, Ho O L, Rhee V, et al. Neurotoxic effects of glutamate [J]. New Engl J Med, 1973,289:1374-1375.
[55]Barth A, Bath L, Newell DW. Combination therapy of MK-801 and aphenyl-terbuthyl-nitroneenchances protections against ischemic neuronal damage in organotypichippocampals slice cultures [J]. Expneeurol,1996,141 (2):330.
[56]徐科.神经生物学纲要[M].北京：科学技术出版社,2001,199-202.
[57]Gurd JW, Bissoon N, Beesley PW, et al. Differential effects of hypoxia-ischemia on subunit expression and tyrosine phosphorylation of the NMDA receptor in 7- and 21-day-old rats [J]. JNeurochemi,82 (4):848-856.
[58]Cai Z, Rhodes PG. Intrauterine hypoxia-ischemia alters expression of NMDA receptor in the young rat brain [J]. Neurochem Res,2001,26 (5):487-495.
[59]徐铁军,樊红彬,张凤真,等.前脑缺血再灌流后大鼠海马NMDA受体亚单位NR2A和NR2B蛋白质表达的变化[J].神经解剖学杂志,2002,18(2)：110-116.
[60]Small DL, Poulter MO, Buchan AM, et al. Alternation in NMDAR subunit RNA expression in vulnerable and resistent regions of in vitro ischemic rat hippocampal slice [J]. NeurosciLett,1997,232 (2):87-90.
[61]Martone ME, Jones YZ, Young SJ, et al. Modification of postsynaptic densities after transient ischemia:aquantitative and thress-dimensional ultra-structural study [J].J Neurosci,1999,19:1988-1997.
[62]Takagi N, Cheung HH, Bissoon N, et al. The effect of transient global ischemia on the interaction of Src and Fyn with NMDA receptor and postsynaptic densities:possible involvement of SH2 domains [J]. J CerebBloodFlowMetab,1999,19:880-888.
[63]刘宝松,廖维宏,陈恒胜,等.缺血损伤后突触后膜GluR2含量变化及机制[J].第三军医大学学报,2004,26(22)：2051-2053.
[64]Kemp J A, McKernan R M. NMDA receptor pathways as drug targets [J]. Nat Neurosci, 2002,5 Suppl 1039-1042.
[65]Mott D D, Doherty J J, Zhang S, et al. Phenylethanolamines inhibit NMDA receptors by enhancing proton inhibition [J]. Nat Neurosci,1998,1 (8):659-667.
[66]朱兆洪,丁柱,贺顺龙,等.黄芪益母草注射液对脑缺血大鼠神经元突触结构的保护作用[J].中国中西医结合急救杂志,2003,10(2)：102-104.
[67]刘晓梅,位学海,刘月霞,等.银杏叶提取物对局灶性脑缺血-再灌注大鼠突触素表达的影响[J].黑龙江医药科学,2007,30(2)：4-6.
[68]邓学军,孙圣刚,梅元武.白藜芦醇苷保护脑海马区缺血神经元的机制探讨[J].现代康复,2001,5(7)：52.
[69]景玉宏,冯慎远,汤晓琴.石菖蒲对学习记忆的影响及突触机制[J].中国中医基础医学杂志,2002,8(6)：38-40.
[70]刘晓梅,田国忠.红景天苷对局灶性脑缺血再灌注大鼠突触超微结构的影响[J].解剖学研究,2009,31(5)：353-355.
[71]朱晓磊,张娜,李澎涛,等.胆酸、栀子苷配伍对脑缺血时脑组织TNF-α、IL-1β和ICAM-1含量的影响[J].北京中医药大学学报,2004,27(6)：26-29
[1]许风雷,高丽霞,吴泰相,等.醒脑静注射液治疗脑梗塞临床疗效及安全性随机对照试验的系统评价[J].中国循证医学杂志,2005,5(7)：549-554.
[2]林森,吴波,刘呜.醒脑静注射液治疗脑出血的系统评价[J].中国循证医学杂志,2008,8(2)：93-96.
[3]庄伟端,欧利民,郑俊忠.等.醒脑静注射液对急性脑梗死意识障碍患者的影响[J].中国中西医结合急救杂志.2001,8(6)：381.
[4]魏江磊,邵念方.醒脑静注射液治疗缺血性卒中临床疗效的研究[J].中西医结合实用临床急救,1999,6(5)：197-200.
[5]张万增,郁亚琴.醒脑静注射液在急性脑梗死治疗中的作用[J].中西医结合实用临床急救,1998,5(9)：394-396.
[6]吴玉生,张文高,郑国玲,等.醒脑静注射液对急性脑缺血大鼠炎症反应及病理的影响[J].山东大学学报(医学版),2007,45(7)：746-748.
[7]王万铁,陈寿权,王卫,等.醒脑静注射液对实验性脑缺血及再灌注损伤中氧自由基水平的影响[J].中医药学刊,2004,22(3)：434-435.
[8]刘洪章,马志伟,刘毅,等.醒脑静注射液对急性脑损伤患者MDA, SOD, TNF-α, IL-8的影响[J].中国实验方剂学杂志,2011,17(13)：225-226.
[9]马勇,杜守颖,黎迎,等.醒脑静固体制剂中挥发性成分环糊精包合物制备工艺研究[J].实验方剂学,2011,17(14)：7-11.
[10]高琦瑛,陈岫.银杏叶提取物对局灶性脑缺血模型大鼠的保护作用.中国药物应用与监测,2005,(3)：12-15.
[11]Tamura A, Graham DI, McCulloch J, et al. Focal cerebral ischemia in the rats:1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion [J]. J Cereb Blood Flow Metab,1981,1 (1):53-60.
[12]曹霞,曹秉振,郭述苏.大鼠大脑中动脉局灶性脑缺血模型的时间梗死体积相关研究[J].解放军医学杂志,2000,25(2)：123-124.
[13]Siesjo BK, Sharpe LG. Feigin R. Glutamate-induced brain damage in infant primates [J]. JNeurpathol Exp Neurol,1992,77:169-184.
[14]Linnik MD, Zobrist RH, Hatfield MD. Evidence supporting a role of programmed cell death in focal cerebral ischemia in rats [J]. Stroke,1993,24:2002-2009.
[15]Hara H, Sukamoto T, Kogure K. Mechanism and pathogensis of ischemia-induced neuronal damage [J]. PROG NEUROBIOL,1993,40:645-670.
[1]Koizumi J, Yoshida Y, Nakazawa T, et al. Experimental studies of ischemic brain edema: A new experimental model of cerebral embolism in rats in which recirculation can be introduced [J]. Stroke,1986,8:1-8.
[2]Nagasawa H, Kogure K. Correlation between cerebral blood flow and histologic changes in a new rat model of middle cerebral artery occlusion [J]. Stroke,1989,20 (8): 1037-1043.
[3]王旋,顾振纶,秦振红,等.银杏内酯A和B混合物对大鼠永久性局灶性脑缺血的保护作用[J].中草药,2007,38(2)：241-244.
[4]陈红,马雷,李丽.大鼠MCAO模型线拴插入深度对脑梗死体积的影响[J].中国比较医学杂志,2006(9)：566-566.
[5]董六一,魏倩.醒脑静乳剂对家兔失血性休克及感染性休克的保护作用[J].中国实验方剂学杂志,2010,16(1)：98-101.
[6]Robin L. Roof, Gerald P. Schielke, XiaodanRen, et al. A Comparison of Long-Term Functional Outcome After 2 Middle Cerebral Artery Occlusion Models in Rats [J]. Stroke, 2001,32:2648-2657.
[7]李翎,方永奇,邹衍衍,等.神昌注射液对脑栓塞大鼠血小板活化和血栓形成的影响[J].中国实验方剂学杂志,2010,16(5)：175-177.
[8]刘小光,冯亦璞.丁基苯酞对局部脑缺血大鼠行为和病理改变的保护作用[J].药学学报,1995,30(12)：896-903.
[9]R. L. Macdonald and M. Stoodley. Pathophysiology of cerebral ischemia [J]. Neurol Med Chir,1998,38:1-11.
[10]Halliwell, B. Oxidative stress, nutrition and health. Experimental strategies for optimization of nutritional antioxidant intake in humans [J]. Free Rad. Res.1996,25: 57-74.
[11]Ikonomidou C, KaindlAM.Neuronal death and oxidative stress in the developing brain [J]. ARS,2011,14(8):1535-1550.
[12]J.W. Schmidley. Free radicals in central nervous system ischemia [J]. Stroke,1990,21: 1086-1090.
[13]Ikeda Y, Long DM. The molecular basis of brain injury and brain edema:the role of oxygen free radicals [J]. Neurosurgery,1990,27 (1):1-11.
[14]McCord JM. Oxygen-derived free radicals in post-ischemic tissue injury [J]. N Engl J Med,1985,312:159-163.
[1]Katsumi Ikeda, Hiroko Negishi, Yukio Yamori. Antioxidant nutrients and hypoxia/ischemia brain injury in rodents. Toxicology,2003,189:55-61.
[2]Hayashi K., Koyama M., Kido H., et al. Preventative and therapeutic effects of AE0047 on renal injury in stroke-prone spontaneously hypertensive rats. Clin. Exp. Pharmacol. Physiol,1997,24,831-840.
[3]Ogata J., Fujishima M., Tamaki K., et al. Stroke-prone spontaneously hypertensive rats as an experimental model of malignant hypertension. A pathological study. Virchows Arch A Pathol Anat Histol.1982:394 (3):185-94.
[4]吴汉宁,孟秋云,徐林,等.高血压病患者血压各参数与其发生冠心病脑卒中的相关性研究.中国现代医药杂志,2004,6(6)：33-35.
[5]Higashino H, Suzuki A. Upper shift of the thermocenter causes hyperthermia in SHRSP. Acta Med Kinki Univ,1995,20:279-289.
[6]王文,刘力生,张秀娥,等.尼莫地平对盐负荷SHRsp脑卒中的预防作用,中国循环杂志,1989,4：146-148.
[1]Paniker NV, Srivas tava SK, Beu tler E. Glutathuone metabolism of the cells:Effect of glutathione reductase deficiency on the stimulation of hexose monophosphate shunt under oxidative stress [J]. Biochim Biophys Acta,1970,215 (3):456-460.
[2]ChadeAR, Lerman A, Lerman LO. Kidney in early atherosc lerosis [J]. Hypertension, 2005,45:1042-1049.
[3]Babior BM, Takeuchic C, Rued IJ, et al. Investigating antibody-catalyzed ozone generation by human neutrophils [J]. Proc Natl Acad Sci U.S.A.,2003,100 (6): 3031-3034.
[4]Chang, C.Y., Lai, Y.C., Cheng, T.J., et al. Plasma levels of antioxidant vitamins, selenium, total sulfhydryl groups and oxidative products in ischemic-stroke patients as compared to matched controls in Taiwan. Free Rad Res.1998,28,15-24.
[5]Dringen R. Metabolism and functions of glutathione in brain [J]. Prog Neurobiol,2000, 62:649-671.
[6]Winkler SR, Manoz- Ruizl. Mechanism of action of mannitol [J]. Surg Neurol,1995,43: 59.
[7]McIntyre M., Hamilton C. A., Rees D. D., et al. Sex differences in the abundance of endothelial nitric oxide in a model of genetic hypertension [J]. Hypertension,1997,30 (6): 1517-1524.
[8]Higashino Hideaki, Maeda Kana, Kawamoto Masazumi, et al. Beneficial effects of long-term administration of ONO-3144, a free radical scavenger, on stroke-prone SHR [J]. Acta Pharmacol Sin,2002,23 (5):393-398.
[9]Gemba, T., Matsunaga, K., Ueda, M., et al. Changes inextracellular concentration of amino acids in the hippocampus during cerebral ischemia in stroke-prone SHR, stroke-resistant SHR and normotensive rats [J]. Neurosci. Lett.,1992,135:184-188.
[10]Tagami, M., Yamagata, K., Ikeda, K., et al. Genetic vulnerability of cortical neurons isolated from stroke-prone spontaneously hypertensive rats in hypoxia and oxygen reperfusion [J]. Hypertens. Res.,1999a,22:23-29.
[11]Sung Gu Han, Yangho Kim, Michael L. Kashon, et al. Correlates of Oxidative Stress and Free-Radical Activity in Serum from Asymptomatic Shipyard Welders [J]. Am JRespir Crit Care Med,2005,172(12):1541-1548.
[12]王显苏,朱虹.酶制剂在葡萄酒酿造中的应用[J].酿酒科技,2009,(7)：52-55.
[13]Loft S., Vistisen K., Sagara M., et al.Oxidative DNA damage estimated by 8-hydroxydeoxyguanosine excretion in humans:influence of smoking, gender and body mass index [J]. Carsinogenesis,1992,13:2241-2247.
[14]Han, D., Loukianoff, S., McLaughlin, L. et al. Handbook of Oxidants and Antioxidants in Exercise [J]. Elsevier, Amsterdam,2000, pp.433-484.
[15]梅素容,吴达,高晓丹,等.尿中8-羟基脱氧鸟苷作为肿瘤疗效评价生物标记物的研究[J].华中科技大学学报(医学版),2007,36(1)：19-22.
[16]鲁燕,陆轶群,成兴波.2型糖尿病患者血清8-OHdG与胰岛素抵抗和颈动脉内膜中层 厚度的相关性[J].江苏医药,2011,37(10)：1175-1177.
[17]Halliwell, B., Chirico, S.,1993. Lipid peroxidation:its mechanism, measurement, and significance [J]. Am. J. Clin. Nutr,57 (5),715S-724.
[18]Ikonomidou C, KaindlAM.Neuronal death and oxidative stress in the developing brain [J]. ARS,2011,14(8):1535-1550.
[19]J.W. Schmidley. Free radicals in central nervous system ischemia [J]. Stroke,1990,21: 1086-1090.
[20]Ikeda Y, Long DM. The molecular basis of brain injury and brain edema:the role of oxygen free radicals [J]. Neurosurgery,1990,27 (1):1-11.
[21]Powers, S.K., Lennon, S.L.. Analysis of cellular response to free radicals:focus on exercise and skeletal muscle [J]. Proc. Nutr. Soc.,1999,58:1025-1033.
[22]Uno H, Matsuyama T, Akita H, et al. Induction of tumor necrosis factor- alpha in the mouse hippocampus following transient forebrain ischemia [J]. JCereb Blood Flow Metab,1997,17 (5):491-499.
[23]Nawashiro H, Martin D, Hallenbeck JM, et al. Neuroprotective effects of TNF binding protein in focal cerebral ischemia [J]. Brain Res,1997,778 (2):265-271.
[24]Barone FC, Arvin B, White RF, et al. Tumor necrosis factor alpha:A mediator of focal ischemic brain injury [J]. Stroke,1997,28 (6):1233-1244.第一部分研究小结1.MCAT模型大鼠在脑缺血损伤后表现出神经功能缺损症状,如身体向健侧倾斜、
旋转,瘫痪,不能行走,同时,动物精神状态低迷,进食减少,部分动物死亡。说明
MCAT模型建立成功。2.以神经功能缺损症状评分和脑梗死体积作为指标,评价两种醒脑静制剂对MCAT
大鼠的脑保护作用。结果表明,醒脑静注射液、口服制剂在缺血后6h、24 h均能明显
改善模型动物的神经功能缺损症状,并能够明显缩小脑梗死范围。3.pMCAO模型是常用的脑缺血动物模型,操作简便、可复制性强。手术过程中不
需要开颅,避免开颅对动物颅内环境的影响,常用于评价缺血性脑损伤的病理生理过程
以及药物对这些过程的干预作用。4.醒脑静口服制剂在脑缺血恢复早期能够改善pMCAO模型大鼠的神经功能缺损
症状,降低神经功能缺损评分,增加缺血动物前肢触觉敏感度,增加前肢拉力,促进动
物平衡能力的恢复,减轻脂质过氧化物对脑组织的损伤。
[1]辛世萌,刘远洪,聂志余,等.栓线长度、直径、及大鼠体重与栓线法大鼠局灶性脑缺血模型关系的研究[J].大连医科大学学报,2000,22(2)：105-107.
[2]Robin L. Roof, Gerald P. Schielke, XiaodanRen, et al. A Comparison of Long-Term Functional Outcome After 2 Middle Cerebral Artery Occlusion Models in Rats [J]. Stroke, 2001,32:2648-2657.
[3]刘明,孙建宁,董世芬,等.梓醇对永久性脑缺血损伤大鼠恢复早期运动感觉功能和能量代谢的影响[J].北京中医药大学学报,2011,34(4)：245-249.
[4]蒋文华.神经解剖学[M].上海：复旦大学出版社,2004：54-55.
[1]张翠玲,刘立,任宇虹,等.脑缺血性损伤后的皮层突触再生与运动功能恢复[J].第四军医大学学报,2001,22(12)：1122-1147.
[2]Craven SE, Bredt DS. PDZ proteins organize synaptic signaling pathways [J]. Cell,1998, 93:495-498.
[3]Gardoni F, Schrama LH, Kamal A, et al. Hippocampal synaptic plasticity involves competition between Ca2+/calmodulin-dependent protein kinase Ⅱ and postsynaptic density 95 for binding to the NR2A subunit of the NMD A receptor [J]. J Neuro sci,2001, 21:1501-1509.
[4]Martone M E, Jones Y Z, Young S J, et al. Modification of postsynaptic densities after transient cerebral ischemia:a quantitative and three dimensional ultra-structural study [J]. J Neuro sci,1999,19 (6):1988-1997.
[5]Hu BR, Park M, Martone M E, et al. Assembly of proteins to postsynaptic densities after transient cerebral ischemia [J]. J Neuro sci,1998,18(2):625-633.
[6]Takagi N, Logan R, Teves L, et al. Altered interaction between PSD 95 and the NMDA receptor following transient global ischemia [J]. J Neuro Chem.,2000,74 (1):169-178.
[7]Stroemer RP, Kent TA, Hulsebosch CE. Enhanced neocortical neural sprouting, synaptogenesis, and behavioral recovery with D2 abnphetanube therapy after necortical infarction in rats [J]. Stroke,1998,29:2391-2395.
[8]梁燕玲,张苏明,许康.短暂脑缺血再灌注后突触蛋白Ⅰ表达与细胞凋亡[J].中国临床康复,2005,9(37)：54-56.
[9]Al-Wandi A, Ninkina N, Millership S, et al. Absence of a-synuclein affects dopamine metabolism and synaptic markers in the striatum of aging mice [J]. Neurobiol Aging, 2010,31:796-804.
[10]Baba M, Nakajo S, Tu PH, et al. Aggregation of alpha-synuclein in Lewy bodies of sporadic Parkinson's disease and dementia with Lewy bodies [J]. Am J Pathol,1998,152: 879-884
[11]Hashimoto M, Rockenstein E, Crews L, et al. Role of protein aggregation in mitocjondrial dysfunction and neurodegenetation in Alzheimer's and Parkinson's disease [J]. Neuro Molecular Med,2003,4:21-35.
[12]Forloni G, Bertani I, Calella AM, et al. Alpha-syn and Parkinson's disease:selective neurodegenerative effect of alpha-syn fragment on dopaminergic neurons in vitro and in vivo [J]. Ann Neurol,2000,47 (5):632-640.
[13]Isin Unal-Cevik, Yasemin Gursoy-Ozdemir, Muge Yemisci, et al. Alpha-synuclein aggregation induced by brief ischemia negatively impacts neuronal survival in vivo:a study in [A30P] alpha-synuclein transgenic mouse [J]. JCEREBR BLOOD FMET,2011, 31:913-923.
[14]刘罡,吴毅,胡永善,等.跑台训练对脑缺血大鼠脑组织超微结构及突触素表达的影 响[J].中国康复医学杂志,2008,23(10)：872-874.
[15]叶灵静,徐晓虹,王亚民,等.丰富环境对脑缺血大鼠突触界面结构修饰和PSD-95基因表达的影响[J].心理学报,2008,40(6)：709-716.
[16]庄伟端,欧利民,郑俊忠.等.醒脑静注射液对急性脑梗死意识障碍患者的影响[J].中国中西医结合急救杂志.2001,8(6)：381.
[17]夏鑫华,刘亚敏,赵光峰,等.麝香配伍冰片对局灶性脑缺血再灌注大鼠神经元突触超微结构的影响[J].江西中医学院学报,2008,20(1)：56-58.
[18]王珏,程安龙,吕善庆,等.醒脑静治疗急性脑血管病的临床观察[J].脑与神经疾病杂志,2000,8(2)：115.
[1]张翠玲,刘立,任宇虹,等.脑缺血性损伤后的皮层突触再生与运动功能恢复[J].第四军医大学学报,2001,22(12)：1122-1147.
[2]Gardoni F, Schrama LH, Kamal A, et al. Hippocampal synaptic plasticity involves competition between Ca2+/calmodulin-dependent protein kinase Ⅱ and postsynaptic density 95 for binding to the NR2A subunit of the NMDA receptor [J]. J Neuro sci,2001, 21:1501-1509.
[3]Martone M E, Jones Y Z, Young S J, et al. Modification of postsynaptic densities after transient cerebral ischemia:a quantitative and three dimensional ultra-structural study [J]. J Neuro sci,1999,19(6):1988-1997.
[4]Hu BR, Park M, Martone M E, et al. Assembly of proteins to postsynaptic densities after transient cerebral ischemia [J]. J Neuro sci,1998,18 (2):625-633.
[5]Takagi N, Logan R, Teves L, et al. Altered interaction between PSD 95 and the NMDA receptor following transient global ischemia [J]. J Neuro chem,2000,74 (1):169-178.
[6]Luo Y, Cao G, Pei W, et al. Induct ion of caspase- activated deoxyribonuclease activity after focal cerebral ischemia and reperfusion [J]. J Cereb Blood Flow Metab,2002,22(1): 15-20.
[7]Schmidt Kastner R, Bedard A, Hakin A. Transient expression of GAP43 within the hippocampus after global brain ischemia in rats. Cell Tissue Res,1997,288 (2):225-238.
[8]Soderling TR. Structure and regulation of calcium/calmodulin- dependentprotein kinase Ⅱ and Ⅳ[J].BiochemBiophys Acta,1996,1297:131-138.
[9]李燕华,李吕力,王铁建,等.大鼠脑缺血后P- CaMKⅡ的表达与Ca2+浓度的关系[J].中国急救医学,2006,26(6)：436-438.
[10]Xia Z, Storm DR. The role of calmodulin as a signal integrator for synaptic plasticity [J]. Nat Rev Neurosc,2005,6 (4):267-276.
[11]孙臣友,方周溪,戚双双,等.莪素油对慢性低氧大鼠学习与记忆和海马p-CaMKⅡ表达的影响[J].中国病理生理杂志,2008,24(11)：2156-2161.
[12]Hattori H, Wasterlain CG. Excitatory amino acids in the developing brain:ontogeny, plasticity, and excitotoxicity [J]. Pediatr Neurol,1990,6 (4):219-228.
[13]Hollnann M, Heinemann S. Cloned glutamate receptor [J]. Ann Rev Neurosci,1994,17 (3):31-108.
[14]刘志安,徐铁军,张凤,等.NMDA受体亚单元NR2A和NR2B mRNA在缺血大鼠海马的表达及其与细胞凋亡的关系[J].中国神经科学杂志,2004,20(2)：129-134.
[15]陈泽斌,段妍君,袁芳.急性脑缺血再灌注大鼠顶皮质NMDA受体亚单位NR2A/B蛋白的表达变化[J].湖北中医学院学报,2005,7(3)：3-5.
[2]许风雷,高丽霞,吴泰相,等.醒脑静注射液治疗脑梗塞临床疗效及安全性随机对照试验的系统评价[J].中国循证医学杂志,2005,5(7)：549-554.
[3]林森,吴波,刘呜.醒脑静注射液治疗脑出血的系统评价[J].中国循证医学杂志,2008,8(2)：93-96.
[4]庄伟端,欧利民,郑俊忠.等.醒脑静注射液对急性脑梗死意识障碍患者的影响[J].中国中西医结合急救杂志.2001,8(6)：381.
[5]杜守颖,陆洋,姚宗玲,等.醒脑静中各成分对栀子提取物中栀子苷大鼠鼻腔吸收的影响[J].中国实验方剂学杂志,2012,18(4)：146-150.
[6]董六一,魏倩.醒脑静乳剂对家兔失血性休克及感染性休克的保护作用fJl.中国实验方剂学杂志,2010,16(1)：98-101.
[7]唐兴荣,张水冰,余尚贞.脑立苏冲剂对脑缺血模型大鼠的影响[J].广州中医药大学学报,2005,22(5)：383.
[8]蒋振亚,李常度,周东,等.麝香对大鼠实验性脑缺血神经元的损伤的保护作用[J].中国医科大学学报,2007,38(5)：456.
[9]方永奇,李翎.醒脑开窍中药治疗脑病的共性作用概况[J].广州中医药大学学报, 2008,25(5)：470-473.
[10]中国医学科学院药物研究所编.中草药现代研究[M].北京：北京医科大学中国协和医科大学联合出版社,1995：206.
[11]朱长庚.神经解剖学[M].北京：人民卫生出版社,2002：1033.
[12]Mullen KD, Szauter KM, Mc Cullough AJ. Blood-Brain-Barrier (BBB) permeability [J]. Hepatology,1998,8 (2):440.
[13]刘金江,陈华德.血脑屏障与中医药研究[J].浙江中医学院学报,1999,4(23)：2-5.
[14]陈文垲,黄玉芳,王海东.麝香“归经入脑”的实验研究[J].中西医结合学报,2007,(4)：288-291.
[15]莫启忠,宫斌,茅惠明,等.H-麝香酮在体内的吸收分布和排泄：中药麝香芳香开窍机制的初步探讨[J].中成药研究,1984,(7)：1.
[16]曹婉如,林素青,赵蔚君,等.麝香和麝香酮缩短大鼠戊巴比妥钠睡眠时间的探讨[J].中草药,1980,11(7)：309-312.
[17]蒋振亚,李常度.麝香对大鼠实验性脑缺血神经元损伤的保护作用[J].中国中医药科技,2001,8(2)：97-98.
[18]魏楚蓉,伍赶球.冰片的药理作用及其机制研究进展[J].国际病理科学与临床杂志,2010,30(4)：447-451.
[19]梁美蓉,刘启德,黄天来,等.冰片在大鼠血清和脑组织中的药代动力学特征[J].中药新药与临床药理,1993,4(4)：38-41.
[20]陈艳明,王宁生.冰片对血脑屏障体外模型细胞间紧密连接和细胞吞饮囊泡的影响[J].中国中西医结合杂志,2004,24(7)：632-634.
[21]赵保胜,宓穗卿,张银卿.冰片对大鼠脑微血管内皮细胞ICAM-1表达量的影响[J].中药新药与临床药理,2001,12(2)：88-92.
[22]方永奇,邹衍衍,李羚,等.芳香开窍药和祛痰药对中枢神经兴奋性的影响[J].中医药研究,2002,18(3)：40.
[23]薛丽,谌小维,樊宏孝,等.冰片对长时连续作业大鼠前额叶皮层单胺类递质水平的影响[J].第三军医大学学报,2006,28(18)：1867-1869.
[24]胡利民,张艳军,王威,等.冰片与丹酚酸B和三七总皂苷配伍对局灶性脑缺血再灌注大鼠脑VEGF mRNA表达的影响[J].中国中西医结合急救杂志,2005,12(5)：263-266.
[25]何晓静,菅凌燕,刘玉兰.冰片注射液对小鼠实验性脑缺血的保护作用[J].新乡医学院学报,2006,23(1)：23-25.
[26]何晓静,吕庆杰,刘玉兰.冰片注射液对缺血再灌注大鼠脑内炎症反应的影响[J].华西药学杂志,2006,21(6)：523-526.
[27]杨蕾,修春,王宁生.冰片对大鼠血小板胞浆内游离钙离子浓度的影响[J].时珍国 医国药,2010,21(1)：1-3.
[28]杨蕾,李伟荣,宓穗卿,等.冰片对三氯化铁诱导的大鼠动脉血栓形成的抑制作用及机制[J].中国实验方剂学杂志,2010,16(6)：164-166.
[29]刘养凤,张军平,张伯礼,等.冰片对大鼠下丘脑单胺类神经递质的影响[J].中国中医药信息杂志,2004,11(2)：122-124.
[30]上海中医学院基础部同位素室.3H-冰片在机体内的吸收、分布和排泄[J].中成药研究,1981,3(5)：81.
[31]陈艳明,王宁生.冰片对P-糖蛋白的影响[J].中药新药与临床药理,2003,14(2)：96-99.
[32]刘养凤,张军平,张伯礼.冰片对大鼠下丘脑单胺类神经递质的影响[J].中国中医药信息杂志,2004,11(2)：122-124.
[33]李伟荣,姚丽梅,宓穗卿,等.冰片对大鼠下丘脑组胺、5-羟色胺含量的影响[J].中药材,2004,27(12)：937-939.
[34]贾钰铭,丁正,曾令源,等.冰片促进顺铂透过血脑屏障的实验研究[J].四川医学,2004,25(2)：156-157.
[35]刘煜德,孙丹邈,黄小洵,等.冰片对BMSCs透过血脑屏障的影响[J].亚太传统医药,2007,3(7)：46-48.
[36]陈瑞玲,赵志刚,张星虎,等.冰片对丙戊酸钠透过血脑屏障的影响[J].中国康复理论与实践,2007,14(2)：151-153.
[37]肖玉强,张良玉,唐海涛,等.冰片促进砷剂透过血脑屏障实验研究[J].中华神经外科疾病研究杂志,2007,6(3)：244-246.
[38]王勇,张忠义,徐峰,等.冰片对川芎嗪血药浓度和在脑中分布的影响[J].中国药业,2006,15(1)：30-31.
[39]赵保胜,刘启德.冰片促血脑屏障开放与病理性开放的比较[J].中药新药与临床药理,2002,13(5)：287-288.
[40]牟家琬,杨胜华,孙玉梅,等.龙脑与异龙脑的体外抗菌作用的研究[J].华西药学杂志,1989,4(1)：20-22.
[41]江光池,等.龙脑和异龙脑的抗炎作用[J].华西药学杂志,1990,5(3)：190.
[42]江光池,等.龙脑和异龙脑对小鼠和家兔的药理作用[J].华西药学杂志,1989,4(1)：23.
[43]彭婕,钱之玉,刘同征,等.栀子提取物京尼平苷和西红花苷利胆作用的研究[J].中国中药杂志,2003,2(11)：105-108.
[44]王钢力,陈德昌,赵淑杰.栀子属植物化学成分研究进展.中国中药杂志,1996,21(2)：67-69.
[45]张小燕,张占军,王忠,等.栀子苷对局灶性脑缺血大鼠脑组织基因表达谱的影响.中国中西医结合杂志,2005,25(1)：42-44.
[46]朱晓磊,张娜,李澎涛,等.栀子苷阻抑脑缺血损伤级联反应的作用环节探讨.中国中药杂志,2004,29(11)：1065-1068.
[47]Yamazaki M, Chiba K, Yoshikawa C. Genipin suppresses A23187-induced cytotoxicity in neuro2a cells [J]. BIOL PHARMBULL,2009,32 (6):1043-1046.
[48]朱晓磊,张娜,李澎涛,等.胆酸、桅子昔配伍对脑缺血时脑组织TNF-α, IL-1β和ICAM-1含量的影响[J].北京中医药大学学报,2004,27(6)：26-29.
[49]李伟华,朱陵群,王硕仁,等.黄芩苷、栀子苷对神经细胞缺氧缺塘／再灌注损伤的保护作用[J].中国药学杂志,2004,39(5)：344-346.
[50]李传云,李澎涛,潘彦舒,等.牛黄、栀子配伍对大鼠局灶性脑缺血再灌注不同时段脂质过氧化损伤的影响[J].中国医药学报,2004,19(9)：528-530.
[51]李宝莉,陈雅慧,杨暄,等.栀子油的提取和对中枢神经系统的作用[J].第四军医大学学报,2008,29(23)：2152-2155.
[52]郝洪谦,孙兵,郑开俊,等.郁金二酮对家猫睡眠节律电活动的调制作用[J].中草药,1994,25(8)：423-424.
[53]饶旺福,饶淑珍.中医治疗中风病进展[J].江西中医药,1999,30(3)：53-56.
[54]刘岩,王建,姚洪武,等.开窍药对缺血缺氧损伤PC12细胞的影响及其作用机制[J].中药药理与临床,2010,26(4)：35-38.
[55]夏鑫华,刘亚敏,赵光峰.麝香配伍冰片对大鼠局灶性脑缺血再灌注损伤的脑保护研究[J].中国实验方剂学杂志,2009,15(2)：42-45.
[56]倪彩霞,曾南,许福会,等.芳香开窍药对脑缺血再灌注损伤大鼠血脑屏障影响的实验研究[J].中国中药杂志,2011,36(18)：2562-2566.
[57]苗明三.醒脑静注射液对中枢神经系统的影响[J].中药药理与临床,2004,20(3)：42.
[58]Khan M, Elango C, Ansari MA, et al. Caffeic acid phenethyl ester reduces neurovascular inflammation and protects rat brain following transient focal cerebral ischemia [J]. Neurvchem,2007,102 (2):365-377.
[59]Wang Q, Tang XN, Midori A. The inflammatory response in stroke [J]. Neuroimmunol, 2007,184 (1-2):53-68.
[60]Williams AJ, Dave JR, Tortella FC. Neuroprotection with the proteasome inhibitor MLN519 in focal ischemic brain injury:relation to nuclear factor κB (NF-κB), inflammatory gene expression, and leukocyte infiltration [J]. Neurochem Int,2006,49 (2): 106-112.
[61]王万铁,陈寿权,等.醒脑静注射液对脑缺血-再灌注损伤家兔血清白介素-8的影响[J].温州医学院学报,2003,33(1)：1-3.
[62]陈寿权,王万铁,王明山,等.醒脑静对家兔脑缺血再灌流时TNF、IL-1、IL-6水平及脑超微结构影响的实验研究[J].中国急救医学,2000,20(11)：637-639.
[63]沈思钰,傅晓东,陈伟华,等.醒脑静对脑缺血大鼠神经保护作用与氨基酸受体表达的关系[J].中国临床康复,2004,8(4)：686-687.
[64]万文成,李杰芬,罗海燕,等.醒脑静对大鼠皮层神经细胞的保护作用[J].广州中医药大学学报,2002,19(2)：125-126.
[65]张红波,代建峰,张宾辉.醒脑静抗鼠脑缺血-再灌注损伤的实验研究.浙江中西医结合杂志,2006,16(6)：351-353.
[66]周红宇,胡国新,陈言,等.醒脑静注射液对脑缺血再灌注小鼠记忆功能的保护作用.温州医学院学报,2002,32(3)：140-142.
[67]傅强,崔华雷,孙中吉,等.醒脑静注射液对脑缺血再灌注诱导的脑神经细胞凋亡防治作用的实验研究.中国中西医结合急救杂志,2000,7(3)：144-146.
[68]戴永建,戢翰升.醒脑静注射液对大鼠脑损伤后细胞凋亡及相关蛋白表达的影响.中国临床神经外科杂志,2006,(9)：551-553
[1]Renato M.E. Sabbatini. Neurons and Synapses:The History of Its Discovery [J]. Brain & Mind Magazine,2003,17, April-July
[2]Janz R, Sudhof TC, Hammer RE, et al. Essential roles in synaptic plasticity for synaptogyrin I and synaptophysin [J]. Neuron,1999,24 (3):687-671.
[3]Bolay H, Gursoy Ozdemir Y, Sara Y. Persistent defect in transmitter release and synapsin phosphorylation in cerebral cortex after transient moderate ischemic injury [J]. Stroke, 2002,33(5):1369-1375.
[4]张艳玲,李露斯,肖桃元,等.血管性痴呆大鼠的突触超微结构改变及结构参数分析[J].第三军医大学学报,2002,24(3)：264-266.
[5]Coleman PD, Yao PJ. Synaptic slaughter in Alzheimer's disease [J]. Neurobiol Aging, 2003,24(8):1023-1027.
[6]Landis DM, Hall AK, Weinstein LA, et al. The organization of cytoplasm at presynaptic active zone of a central nervous system synapse [J]. Neuron,1988,1 (3):201-209.
[7]Zhen M, Jin Y. Presynaptic terminal differentiation:transport and assembly [J]. CURR OPINNEUROBIOL,2004,14 (3):280.
[8]胡人义.突触的超微结构.复旦神经生物学讲座,1991,7：39.
[9]Colonnier M. Synaptic patterns on different cell types in the different laminae of the cat visual cortex. An electron microscope study [J]. BRAIN RES,1968,9 (2):268-287.
[10]Ziff EB. Enlightening the postsynaptic density [J]. Neuron,1997,19 (6):1163-1173.
[11]Papadia, S., Hardingham, G.E, et al. The dichotomy of NMDA receptor signaling [J]. NEUROSCIENTIST,2007,13 (6):572-579.
[12]Paoletti, P., Neyton, J. NMDA receptor subunits:function and pharmacology [J]. CURR OPIN PHARMACOL,2007,7:39-47.
[13]Dingledine R, Borges K, Bowie D, et al. The glutamate receptor ion channels [J]. PHARMACOL REV,1999,51 (1):7-61
[14]Cousins SL, Kenny AV, Stephenson FA. Delineation of additional PSD-95 binding domains with in NMDA receptor NR2 subunits reveals differences between NR2A /PSD-95 and NR2B/PSD-95 association [J]. NEUROSCIENCE, 2009,158:89-95.
[15]Kennedy, M.B. Signal-processing machines at the postsynaptic density [J]. SCIENCE, 2000,290,750-754.
[16]Xu, W.F. PSD-95-like membrane associated guanylate kinases (PSD-MAGUKs) and synaptic plasticity [J]. Curr Opin Neurobiol.2011,21:306-312.
[17]Carter A G, Regehr W G. Prolonged synaptic currents and glutamate spillover at the parallel fiber to stellate cell synapse [J]. NEUROSCIENCE,2000,20 (12):4423-4434.
[18]Chen S, Diamond J S. Synaptically released glutamate activates extrasynaptic NMDA receptors on cells in the ganglion cell layer of rat retina [J]. NEUROSCIENCE,2002,22 (6):2165-2173.
[19]Lalo U, Pankratov Y, Kirchhoff F, et al. NMDA receptors mediate neuron-to-glia signaling in mouse cortical astrocytes [J]. NEUROSCIENCE,2006,26 (10):2673-2683.
[20]Salter M G, Fern R. NMDA receptors are expressed in developing oligodendrocyte processes and mediate injury [J]. Nature,2005,438 (7071):1167-1171.
[21]Essin K, NistriA, Magazanik L. Evaluation of GluR2 subunit involvementin AMPA receptor function neonatal rat hypoglossal motoneurons [J]. EurJNeurosci,2002,15 (2): 1899-1906.
[22]Koike K, TsukadaS, Tsuzuki K, et al. Regulation of kinetic properties ofGluR2 AMPA receptor channels by alternative splicing [J]. JNEUROSCI 2000,20 (6):2166-2174.
[23]Partin KM. Domain interactions regulating AMPA receptor desensitization [J]. J NEUROSCI,2001,21 (6):1939-1948.
[24]Steiner P, Sarria J C, Glauser L, et al. Modulation of receptor cycling by neuron enriched endosomal protein of 21 kD [J]. J CELL BIOL,2002,157 (7):1197-1209.
[25]Ozawa S, Kamiya H, Tsuzuki K. Glutamate receptors in the mammalian central nervous system [J]. ProgNeurobiol,1998,54 (5):581-618.
[26]Scannevin RH, Huganir RL. Postsynaptic organization and regulation of excitatory synapse [J]. Nat Rev Neurosci,2000,1 (2):133-141.
[27]Peters A, Palay SL, Webster Hd. The fine structure of the nervous system:neurons and their supporting cells.3rded [M]. New York:Oxford University Press,1991:169.
[28]Yang H W. Appenteng K, Batten T F. Ultrastructural subtypesof glutamate immuno reactive terminals on rat trigeminal motoneurones and their relationships with GABA immunoreactive terminals [J]. Exp Brain Res,1997,114 (1):99-1161.
[29]Siekevitz P. The postsynaptic density:A possible role in long lasting effects in the central nervous system [M]. ProcNatlAcademic science,1985,82:3494-3498.
[30]何祥,冯龚.电刺激对脑梗死大鼠突触界面结构的影响[J].中国临床康复,2004,8(31)：6900-6901.
[31]Rao A, Kim E, Sheng M, Craig AM. Heterogeneity in the molecular composition of excitatory postsynaptic sites during development of hippocampal neurons in culture [J]. Neuro-science,1998,18 (4):1217-1229.
[32]Morris R. Developments of a water-maze procedure for studying spatial learning in the rat [J]. JNeurosci Methods,1984,11 (1):47-60.
[33]Haraguchi K, Satoh K, Yanai H, et al. The hDL G-associated protein DAP interacts with dynein light chain and neuronal nitric oxide synthase [J]. Genes Cells,2000,5:905-911.
[34]韩太真,吴馥梅.学习与记忆的神经生物学[M].第1版.北京：北京医科大学中国协和医科大学联合出版社,1998：2201.
[35]JanzR, Sudhof TC, Hammer RE, et al. Essential roles in synaptic plasticity for synaptogyrin I and synaptophysin [J]. Neuron,1999,24 (3):687-691.
[36]Bolay H, Gursoy-Ozdemir Y, Sara Y. Persistent defect intransmitter release and synapsin phosphorylation in cerebralcortex after transient moderate ischemic injury. Stroke,2002, 33 (5):1369-1375.
[37]王慧娟,李陈莉,赵秀丽,等.大鼠脑缺血后突触超微结构的变化[J].解剖学杂志,2003,12(2)：587-590.
[38]赵昱,马洪骏,李陈莉,等.大鼠脑缺血/再灌注后脑组织硝基络氨酸的表达及超微结构的改变[J].苏州大学学报(医学版),2003,23：154-158.
[39]Vanlookeren Campagne M, Destreicher AB. Ultrastructuraldoublecocalization of B250/GAP43 and synaptophysin (P38) in the neonatal and adult rat hippocampus [J]. J Neurocytol,1990,19 (6):948-952.
[40]王慧娟,苏丽英,李陈莉,等.大鼠脑缺血后突触体素与突触密度的关系[J].解剖学杂志,2004,27(6)：642-645.
[41]Kojiro K, Satoshi G, Shinji N, et l. Changes of immunore-activity for synaptophysin following A transient cerebral ischemia in Rhe Rat Striatum [J]. Brain Research,1993, 616:320.
[42]Stroemer RP, Kent TA, Hulsebosch CE. Enhanced neuocortical neural sprouting, synaptogenesis, and behavioral recovery with D2 amphetamine therapy after neocortical infarction in rats [J]. Stroke,1998,29:2381-2395.
[43]邹凡,杨万同,田峻,等.当归注射液对全脑缺血后神经再塑影响的实验研究[J].武汉大学学报(医学版),2004,25(1)：38-41.
[44]Luo Y, Cao G, Pei W, et al. Induct ion of caspase-activated deoxyribonuclease activity after focal cerebral ischemia and reperfusion [J]. J Cereb Blood Flow Metab,2002,22 (1): 15-20.
[45]Swartz KJ. Modulat ion of Ca2+ channels by kinase C in rat central and peripheral neurons:distribut ion of G protein- mediated inhibit ion [J]. Neuron,1993,11 (2): 305-320.
[46]TominagaT, Kure S, Narisawa K, et al. Endonuclease activation following focal ischemic injury in the rat brain [J]. Brain Res,1993,608 (1):21-26.
[47]Schmidt Kastner R, Bedard A, Hakin A. Transient expression of GAP43 within the hippocampus after global brain ischemia in rats [J]. Cell Tissue Res,1997,288 (2): 225-238.
[48]Soderling TR. Structure and regulation of calcium/ calmodulin- dependent protein kinase Ⅱ and Ⅳ[J]. Biochem Biophys Acta,1996,1297:131.
[49]李燕华,李吕力,王铁建,等.大鼠脑缺血后P- CaMK Ⅱ的表达与Ca2+浓度的关系.中国急救医学,2006,26(6)：436-438.
[50]Xia Z, Storm DR. The role of calmodulin as a signal integrator for synaptic plasticity [J]. Nat Rev Neurosc,2005; 6 (4):267-276.
[51]Ikeda J, Terakawa S, Muyota S, et al. Nuclear disintegration as a leading step of glutamate excitotoicity in brain neurons [J]. J Neurosci Res,1996,43 (5):2613-2622.
[52]Belayev L, Busto R, Zhao W, et al. HU-211, a novel noncompetitive N-methyl D-aspartate antagonist, improves neurological deficit and reduces infarct volume after reversible focal cerebral ischemia in the rat [J]. Stroke,1995,26:2313-2319.
[53]Regan R F, Panter S S. Hemoglobin potentiates excito toxic injury in cortical cell culture [J]. Neuro trauma,1996,13 (3):223-231.
[54]Olney J W, Ho O L, Rhee V, et al. Neurotoxic effects of glutamate [J]. New Engl J Med, 1973,289:1374-1375.
[55]Barth A, Bath L, Newell DW. Combination therapy of MK-801 and aphenyl-terbuthyl-nitroneenchances protections against ischemic neuronal damage in organotypichippocampals slice cultures [J]. Expneeurol,1996,141 (2):330.
[56]徐科.神经生物学纲要[M].北京：科学技术出版社,2001,199-202.
[57]Gurd JW, Bissoon N, Beesley PW, et al. Differential effects of hypoxia-ischemia on subunit expression and tyrosine phosphorylation of the NMDA receptor in 7- and 21-day-old rats [J]. JNeurochemi,82 (4):848-856.
[58]Cai Z, Rhodes PG. Intrauterine hypoxia-ischemia alters expression of NMDA receptor in the young rat brain [J]. Neurochem Res,2001,26 (5):487-495.
[59]徐铁军,樊红彬,张凤真,等.前脑缺血再灌流后大鼠海马NMDA受体亚单位NR2A和NR2B蛋白质表达的变化[J].神经解剖学杂志,2002,18(2)：110-116.
[60]Small DL, Poulter MO, Buchan AM, et al. Alternation in NMDAR subunit RNA expression in vulnerable and resistent regions of in vitro ischemic rat hippocampal slice [J]. NeurosciLett,1997,232 (2):87-90.
[61]Martone ME, Jones YZ, Young SJ, et al. Modification of postsynaptic densities after transient ischemia:aquantitative and thress-dimensional ultra-structural study [J].J Neurosci,1999,19:1988-1997.
[62]Takagi N, Cheung HH, Bissoon N, et al. The effect of transient global ischemia on the interaction of Src and Fyn with NMDA receptor and postsynaptic densities:possible involvement of SH2 domains [J]. J CerebBloodFlowMetab,1999,19:880-888.
[63]刘宝松,廖维宏,陈恒胜,等.缺血损伤后突触后膜GluR2含量变化及机制[J].第三军医大学学报,2004,26(22)：2051-2053.
[64]Kemp J A, McKernan R M. NMDA receptor pathways as drug targets [J]. Nat Neurosci, 2002,5 Suppl 1039-1042.
[65]Mott D D, Doherty J J, Zhang S, et al. Phenylethanolamines inhibit NMDA receptors by enhancing proton inhibition [J]. Nat Neurosci,1998,1 (8):659-667.
[66]朱兆洪,丁柱,贺顺龙,等.黄芪益母草注射液对脑缺血大鼠神经元突触结构的保护作用[J].中国中西医结合急救杂志,2003,10(2)：102-104.
[67]刘晓梅,位学海,刘月霞,等.银杏叶提取物对局灶性脑缺血-再灌注大鼠突触素表达的影响[J].黑龙江医药科学,2007,30(2)：4-6.
[68]邓学军,孙圣刚,梅元武.白藜芦醇苷保护脑海马区缺血神经元的机制探讨[J].现代康复,2001,5(7)：52.
[69]景玉宏,冯慎远,汤晓琴.石菖蒲对学习记忆的影响及突触机制[J].中国中医基础医学杂志,2002,8(6)：38-40.
[70]刘晓梅,田国忠.红景天苷对局灶性脑缺血再灌注大鼠突触超微结构的影响[J].解剖学研究,2009,31(5)：353-355.
[71]朱晓磊,张娜,李澎涛,等.胆酸、栀子苷配伍对脑缺血时脑组织TNF-α、IL-1β和ICAM-1含量的影响[J].北京中医药大学学报,2004,27(6)：26-29
[1]许风雷,高丽霞,吴泰相,等.醒脑静注射液治疗脑梗塞临床疗效及安全性随机对照试验的系统评价[J].中国循证医学杂志,2005,5(7)：549-554.
[2]林森,吴波,刘呜.醒脑静注射液治疗脑出血的系统评价[J].中国循证医学杂志,2008,8(2)：93-96.
[3]庄伟端,欧利民,郑俊忠.等.醒脑静注射液对急性脑梗死意识障碍患者的影响[J].中国中西医结合急救杂志.2001,8(6)：381.
[4]魏江磊,邵念方.醒脑静注射液治疗缺血性卒中临床疗效的研究[J].中西医结合实用临床急救,1999,6(5)：197-200.
[5]张万增,郁亚琴.醒脑静注射液在急性脑梗死治疗中的作用[J].中西医结合实用临床急救,1998,5(9)：394-396.
[6]吴玉生,张文高,郑国玲,等.醒脑静注射液对急性脑缺血大鼠炎症反应及病理的影响[J].山东大学学报(医学版),2007,45(7)：746-748.
[7]王万铁,陈寿权,王卫,等.醒脑静注射液对实验性脑缺血及再灌注损伤中氧自由基水平的影响[J].中医药学刊,2004,22(3)：434-435.
[8]刘洪章,马志伟,刘毅,等.醒脑静注射液对急性脑损伤患者MDA, SOD, TNF-α, IL-8的影响[J].中国实验方剂学杂志,2011,17(13)：225-226.
[9]马勇,杜守颖,黎迎,等.醒脑静固体制剂中挥发性成分环糊精包合物制备工艺研究[J].实验方剂学,2011,17(14)：7-11.
[10]高琦瑛,陈岫.银杏叶提取物对局灶性脑缺血模型大鼠的保护作用.中国药物应用与监测,2005,(3)：12-15.
[11]Tamura A, Graham DI, McCulloch J, et al. Focal cerebral ischemia in the rats:1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion [J]. J Cereb Blood Flow Metab,1981,1 (1):53-60.
[12]曹霞,曹秉振,郭述苏.大鼠大脑中动脉局灶性脑缺血模型的时间梗死体积相关研究[J].解放军医学杂志,2000,25(2)：123-124.
[13]Siesjo BK, Sharpe LG. Feigin R. Glutamate-induced brain damage in infant primates [J]. JNeurpathol Exp Neurol,1992,77:169-184.
[14]Linnik MD, Zobrist RH, Hatfield MD. Evidence supporting a role of programmed cell death in focal cerebral ischemia in rats [J]. Stroke,1993,24:2002-2009.
[15]Hara H, Sukamoto T, Kogure K. Mechanism and pathogensis of ischemia-induced neuronal damage [J]. PROG NEUROBIOL,1993,40:645-670.
[1]Koizumi J, Yoshida Y, Nakazawa T, et al. Experimental studies of ischemic brain edema: A new experimental model of cerebral embolism in rats in which recirculation can be introduced [J]. Stroke,1986,8:1-8.
[2]Nagasawa H, Kogure K. Correlation between cerebral blood flow and histologic changes in a new rat model of middle cerebral artery occlusion [J]. Stroke,1989,20 (8): 1037-1043.
[3]王旋,顾振纶,秦振红,等.银杏内酯A和B混合物对大鼠永久性局灶性脑缺血的保护作用[J].中草药,2007,38(2)：241-244.
[4]陈红,马雷,李丽.大鼠MCAO模型线拴插入深度对脑梗死体积的影响[J].中国比较医学杂志,2006(9)：566-566.
[5]董六一,魏倩.醒脑静乳剂对家兔失血性休克及感染性休克的保护作用[J].中国实验方剂学杂志,2010,16(1)：98-101.
[6]Robin L. Roof, Gerald P. Schielke, XiaodanRen, et al. A Comparison of Long-Term Functional Outcome After 2 Middle Cerebral Artery Occlusion Models in Rats [J]. Stroke, 2001,32:2648-2657.
[7]李翎,方永奇,邹衍衍,等.神昌注射液对脑栓塞大鼠血小板活化和血栓形成的影响[J].中国实验方剂学杂志,2010,16(5)：175-177.
[8]刘小光,冯亦璞.丁基苯酞对局部脑缺血大鼠行为和病理改变的保护作用[J].药学学报,1995,30(12)：896-903.
[9]R. L. Macdonald and M. Stoodley. Pathophysiology of cerebral ischemia [J]. Neurol Med Chir,1998,38:1-11.
[10]Halliwell, B. Oxidative stress, nutrition and health. Experimental strategies for optimization of nutritional antioxidant intake in humans [J]. Free Rad. Res.1996,25: 57-74.
[11]Ikonomidou C, KaindlAM.Neuronal death and oxidative stress in the developing brain [J]. ARS,2011,14(8):1535-1550.
[12]J.W. Schmidley. Free radicals in central nervous system ischemia [J]. Stroke,1990,21: 1086-1090.
[13]Ikeda Y, Long DM. The molecular basis of brain injury and brain edema:the role of oxygen free radicals [J]. Neurosurgery,1990,27 (1):1-11.
[14]McCord JM. Oxygen-derived free radicals in post-ischemic tissue injury [J]. N Engl J Med,1985,312:159-163.
[1]Katsumi Ikeda, Hiroko Negishi, Yukio Yamori. Antioxidant nutrients and hypoxia/ischemia brain injury in rodents. Toxicology,2003,189:55-61.
[2]Hayashi K., Koyama M., Kido H., et al. Preventative and therapeutic effects of AE0047 on renal injury in stroke-prone spontaneously hypertensive rats. Clin. Exp. Pharmacol. Physiol,1997,24,831-840.
[3]Ogata J., Fujishima M., Tamaki K., et al. Stroke-prone spontaneously hypertensive rats as an experimental model of malignant hypertension. A pathological study. Virchows Arch A Pathol Anat Histol.1982:394 (3):185-94.
[4]吴汉宁,孟秋云,徐林,等.高血压病患者血压各参数与其发生冠心病脑卒中的相关性研究.中国现代医药杂志,2004,6(6)：33-35.
[5]Higashino H, Suzuki A. Upper shift of the thermocenter causes hyperthermia in SHRSP. Acta Med Kinki Univ,1995,20:279-289.
[6]王文,刘力生,张秀娥,等.尼莫地平对盐负荷SHRsp脑卒中的预防作用,中国循环杂志,1989,4：146-148.
[1]Paniker NV, Srivas tava SK, Beu tler E. Glutathuone metabolism of the cells:Effect of glutathione reductase deficiency on the stimulation of hexose monophosphate shunt under oxidative stress [J]. Biochim Biophys Acta,1970,215 (3):456-460.
[2]ChadeAR, Lerman A, Lerman LO. Kidney in early atherosc lerosis [J]. Hypertension, 2005,45:1042-1049.
[3]Babior BM, Takeuchic C, Rued IJ, et al. Investigating antibody-catalyzed ozone generation by human neutrophils [J]. Proc Natl Acad Sci U.S.A.,2003,100 (6): 3031-3034.
[4]Chang, C.Y., Lai, Y.C., Cheng, T.J., et al. Plasma levels of antioxidant vitamins, selenium, total sulfhydryl groups and oxidative products in ischemic-stroke patients as compared to matched controls in Taiwan. Free Rad Res.1998,28,15-24.
[5]Dringen R. Metabolism and functions of glutathione in brain [J]. Prog Neurobiol,2000, 62:649-671.
[6]Winkler SR, Manoz- Ruizl. Mechanism of action of mannitol [J]. Surg Neurol,1995,43: 59.
[7]McIntyre M., Hamilton C. A., Rees D. D., et al. Sex differences in the abundance of endothelial nitric oxide in a model of genetic hypertension [J]. Hypertension,1997,30 (6): 1517-1524.
[8]Higashino Hideaki, Maeda Kana, Kawamoto Masazumi, et al. Beneficial effects of long-term administration of ONO-3144, a free radical scavenger, on stroke-prone SHR [J]. Acta Pharmacol Sin,2002,23 (5):393-398.
[9]Gemba, T., Matsunaga, K., Ueda, M., et al. Changes inextracellular concentration of amino acids in the hippocampus during cerebral ischemia in stroke-prone SHR, stroke-resistant SHR and normotensive rats [J]. Neurosci. Lett.,1992,135:184-188.
[10]Tagami, M., Yamagata, K., Ikeda, K., et al. Genetic vulnerability of cortical neurons isolated from stroke-prone spontaneously hypertensive rats in hypoxia and oxygen reperfusion [J]. Hypertens. Res.,1999a,22:23-29.
[11]Sung Gu Han, Yangho Kim, Michael L. Kashon, et al. Correlates of Oxidative Stress and Free-Radical Activity in Serum from Asymptomatic Shipyard Welders [J]. Am JRespir Crit Care Med,2005,172(12):1541-1548.
[12]王显苏,朱虹.酶制剂在葡萄酒酿造中的应用[J].酿酒科技,2009,(7)：52-55.
[13]Loft S., Vistisen K., Sagara M., et al.Oxidative DNA damage estimated by 8-hydroxydeoxyguanosine excretion in humans:influence of smoking, gender and body mass index [J]. Carsinogenesis,1992,13:2241-2247.
[14]Han, D., Loukianoff, S., McLaughlin, L. et al. Handbook of Oxidants and Antioxidants in Exercise [J]. Elsevier, Amsterdam,2000, pp.433-484.
[15]梅素容,吴达,高晓丹,等.尿中8-羟基脱氧鸟苷作为肿瘤疗效评价生物标记物的研究[J].华中科技大学学报(医学版),2007,36(1)：19-22.
[16]鲁燕,陆轶群,成兴波.2型糖尿病患者血清8-OHdG与胰岛素抵抗和颈动脉内膜中层 厚度的相关性[J].江苏医药,2011,37(10)：1175-1177.
[17]Halliwell, B., Chirico, S.,1993. Lipid peroxidation:its mechanism, measurement, and significance [J]. Am. J. Clin. Nutr,57 (5),715S-724.
[18]Ikonomidou C, KaindlAM.Neuronal death and oxidative stress in the developing brain [J]. ARS,2011,14(8):1535-1550.
[19]J.W. Schmidley. Free radicals in central nervous system ischemia [J]. Stroke,1990,21: 1086-1090.
[20]Ikeda Y, Long DM. The molecular basis of brain injury and brain edema:the role of oxygen free radicals [J]. Neurosurgery,1990,27 (1):1-11.
[21]Powers, S.K., Lennon, S.L.. Analysis of cellular response to free radicals:focus on exercise and skeletal muscle [J]. Proc. Nutr. Soc.,1999,58:1025-1033.
[22]Uno H, Matsuyama T, Akita H, et al. Induction of tumor necrosis factor- alpha in the mouse hippocampus following transient forebrain ischemia [J]. JCereb Blood Flow Metab,1997,17 (5):491-499.
[23]Nawashiro H, Martin D, Hallenbeck JM, et al. Neuroprotective effects of TNF binding protein in focal cerebral ischemia [J]. Brain Res,1997,778 (2):265-271.
[24]Barone FC, Arvin B, White RF, et al. Tumor necrosis factor alpha:A mediator of focal ischemic brain injury [J]. Stroke,1997,28 (6):1233-1244.第一部分研究小结1.MCAT模型大鼠在脑缺血损伤后表现出神经功能缺损症状,如身体向健侧倾斜、
旋转,瘫痪,不能行走,同时,动物精神状态低迷,进食减少,部分动物死亡。说明
MCAT模型建立成功。2.以神经功能缺损症状评分和脑梗死体积作为指标,评价两种醒脑静制剂对MCAT
大鼠的脑保护作用。结果表明,醒脑静注射液、口服制剂在缺血后6h、24 h均能明显
改善模型动物的神经功能缺损症状,并能够明显缩小脑梗死范围。3.pMCAO模型是常用的脑缺血动物模型,操作简便、可复制性强。手术过程中不
需要开颅,避免开颅对动物颅内环境的影响,常用于评价缺血性脑损伤的病理生理过程
以及药物对这些过程的干预作用。4.醒脑静口服制剂在脑缺血恢复早期能够改善pMCAO模型大鼠的神经功能缺损
症状,降低神经功能缺损评分,增加缺血动物前肢触觉敏感度,增加前肢拉力,促进动
物平衡能力的恢复,减轻脂质过氧化物对脑组织的损伤。
[1]辛世萌,刘远洪,聂志余,等.栓线长度、直径、及大鼠体重与栓线法大鼠局灶性脑缺血模型关系的研究[J].大连医科大学学报,2000,22(2)：105-107.
[2]Robin L. Roof, Gerald P. Schielke, XiaodanRen, et al. A Comparison of Long-Term Functional Outcome After 2 Middle Cerebral Artery Occlusion Models in Rats [J]. Stroke, 2001,32:2648-2657.
[3]刘明,孙建宁,董世芬,等.梓醇对永久性脑缺血损伤大鼠恢复早期运动感觉功能和能量代谢的影响[J].北京中医药大学学报,2011,34(4)：245-249.
[4]蒋文华.神经解剖学[M].上海：复旦大学出版社,2004：54-55.
[1]张翠玲,刘立,任宇虹,等.脑缺血性损伤后的皮层突触再生与运动功能恢复[J].第四军医大学学报,2001,22(12)：1122-1147.
[2]Craven SE, Bredt DS. PDZ proteins organize synaptic signaling pathways [J]. Cell,1998, 93:495-498.
[3]Gardoni F, Schrama LH, Kamal A, et al. Hippocampal synaptic plasticity involves competition between Ca2+/calmodulin-dependent protein kinase Ⅱ and postsynaptic density 95 for binding to the NR2A subunit of the NMD A receptor [J]. J Neuro sci,2001, 21:1501-1509.
[4]Martone M E, Jones Y Z, Young S J, et al. Modification of postsynaptic densities after transient cerebral ischemia:a quantitative and three dimensional ultra-structural study [J]. J Neuro sci,1999,19 (6):1988-1997.
[5]Hu BR, Park M, Martone M E, et al. Assembly of proteins to postsynaptic densities after transient cerebral ischemia [J]. J Neuro sci,1998,18(2):625-633.
[6]Takagi N, Logan R, Teves L, et al. Altered interaction between PSD 95 and the NMDA receptor following transient global ischemia [J]. J Neuro Chem.,2000,74 (1):169-178.
[7]Stroemer RP, Kent TA, Hulsebosch CE. Enhanced neocortical neural sprouting, synaptogenesis, and behavioral recovery with D2 abnphetanube therapy after necortical infarction in rats [J]. Stroke,1998,29:2391-2395.
[8]梁燕玲,张苏明,许康.短暂脑缺血再灌注后突触蛋白Ⅰ表达与细胞凋亡[J].中国临床康复,2005,9(37)：54-56.
[9]Al-Wandi A, Ninkina N, Millership S, et al. Absence of a-synuclein affects dopamine metabolism and synaptic markers in the striatum of aging mice [J]. Neurobiol Aging, 2010,31:796-804.
[10]Baba M, Nakajo S, Tu PH, et al. Aggregation of alpha-synuclein in Lewy bodies of sporadic Parkinson's disease and dementia with Lewy bodies [J]. Am J Pathol,1998,152: 879-884
[11]Hashimoto M, Rockenstein E, Crews L, et al. Role of protein aggregation in mitocjondrial dysfunction and neurodegenetation in Alzheimer's and Parkinson's disease [J]. Neuro Molecular Med,2003,4:21-35.
[12]Forloni G, Bertani I, Calella AM, et al. Alpha-syn and Parkinson's disease:selective neurodegenerative effect of alpha-syn fragment on dopaminergic neurons in vitro and in vivo [J]. Ann Neurol,2000,47 (5):632-640.
[13]Isin Unal-Cevik, Yasemin Gursoy-Ozdemir, Muge Yemisci, et al. Alpha-synuclein aggregation induced by brief ischemia negatively impacts neuronal survival in vivo:a study in [A30P] alpha-synuclein transgenic mouse [J]. JCEREBR BLOOD FMET,2011, 31:913-923.
[14]刘罡,吴毅,胡永善,等.跑台训练对脑缺血大鼠脑组织超微结构及突触素表达的影 响[J].中国康复医学杂志,2008,23(10)：872-874.
[15]叶灵静,徐晓虹,王亚民,等.丰富环境对脑缺血大鼠突触界面结构修饰和PSD-95基因表达的影响[J].心理学报,2008,40(6)：709-716.
[16]庄伟端,欧利民,郑俊忠.等.醒脑静注射液对急性脑梗死意识障碍患者的影响[J].中国中西医结合急救杂志.2001,8(6)：381.
[17]夏鑫华,刘亚敏,赵光峰,等.麝香配伍冰片对局灶性脑缺血再灌注大鼠神经元突触超微结构的影响[J].江西中医学院学报,2008,20(1)：56-58.
[18]王珏,程安龙,吕善庆,等.醒脑静治疗急性脑血管病的临床观察[J].脑与神经疾病杂志,2000,8(2)：115.
[1]张翠玲,刘立,任宇虹,等.脑缺血性损伤后的皮层突触再生与运动功能恢复[J].第四军医大学学报,2001,22(12)：1122-1147.
[2]Gardoni F, Schrama LH, Kamal A, et al. Hippocampal synaptic plasticity involves competition between Ca2+/calmodulin-dependent protein kinase Ⅱ and postsynaptic density 95 for binding to the NR2A subunit of the NMDA receptor [J]. J Neuro sci,2001, 21:1501-1509.
[3]Martone M E, Jones Y Z, Young S J, et al. Modification of postsynaptic densities after transient cerebral ischemia:a quantitative and three dimensional ultra-structural study [J]. J Neuro sci,1999,19(6):1988-1997.
[4]Hu BR, Park M, Martone M E, et al. Assembly of proteins to postsynaptic densities after transient cerebral ischemia [J]. J Neuro sci,1998,18 (2):625-633.
[5]Takagi N, Logan R, Teves L, et al. Altered interaction between PSD 95 and the NMDA receptor following transient global ischemia [J]. J Neuro chem,2000,74 (1):169-178.
[6]Luo Y, Cao G, Pei W, et al. Induct ion of caspase- activated deoxyribonuclease activity after focal cerebral ischemia and reperfusion [J]. J Cereb Blood Flow Metab,2002,22(1): 15-20.
[7]Schmidt Kastner R, Bedard A, Hakin A. Transient expression of GAP43 within the hippocampus after global brain ischemia in rats. Cell Tissue Res,1997,288 (2):225-238.
[8]Soderling TR. Structure and regulation of calcium/calmodulin- dependentprotein kinase Ⅱ and Ⅳ[J].BiochemBiophys Acta,1996,1297:131-138.
[9]李燕华,李吕力,王铁建,等.大鼠脑缺血后P- CaMKⅡ的表达与Ca2+浓度的关系[J].中国急救医学,2006,26(6)：436-438.
[10]Xia Z, Storm DR. The role of calmodulin as a signal integrator for synaptic plasticity [J]. Nat Rev Neurosc,2005,6 (4):267-276.
[11]孙臣友,方周溪,戚双双,等.莪素油对慢性低氧大鼠学习与记忆和海马p-CaMKⅡ表达的影响[J].中国病理生理杂志,2008,24(11)：2156-2161.
[12]Hattori H, Wasterlain CG. Excitatory amino acids in the developing brain:ontogeny, plasticity, and excitotoxicity [J]. Pediatr Neurol,1990,6 (4):219-228.
[13]Hollnann M, Heinemann S. Cloned glutamate receptor [J]. Ann Rev Neurosci,1994,17 (3):31-108.
[14]刘志安,徐铁军,张凤,等.NMDA受体亚单元NR2A和NR2B mRNA在缺血大鼠海马的表达及其与细胞凋亡的关系[J].中国神经科学杂志,2004,20(2)：129-134.
[15]陈泽斌,段妍君,袁芳.急性脑缺血再灌注大鼠顶皮质NMDA受体亚单位NR2A/B蛋白的表达变化[J].湖北中医学院学报,2005,7(3)：3-5.