缺血再灌注大鼠脑内代谢型谷氨酸受体1和代谢型谷氨酸受体5的mRNA水平变化

目的:利用缺血再灌注大鼠模型研究代谢型谷氨酸受体1(mGluRl)和代谢型谷氨酸受体5(mGluR5)的mRNA表达水平在缺血再灌注后的变化,探讨其在缺血后脑损伤中的作用。方法:采用线栓法制备缺血再灌注大鼠模型,运用RT-PCR技术半定量分析脑组织内mGluRl和mGluR5的mRNA表达水平。结果:以β-Actin为参照,实验组缺血2h再灌注24h后缺血侧mGluRl和mGluR5的mRNA相对值较假手术对照组均显著上升(P<0.05);非缺血侧两者的mRNA相对值与假手术对照组相比,无显著差异(P/0.05)。结论:代谢型谷氨酸受体l和代谢型谷氨酸受体都参与了缺血再灌注后的病理过程。     

代谢型谷氨酸受体在脑缺血耐受中的研究进展

代谢型谷氨酸受体（metabotropicglutamatereceptors。mGluRs）是脑内广泛存在的受体。通过与G蛋白偶联调节膜上离子通道、第二信使生成、参与诸多的生理及病理过程。目前为止已克隆出8种mGhRs，分别为mGluRl，mGluR2。mGluR3，mGluR4，mGluR5，mGluR6。mGluR7。mGluR8。其中．多种mGluRs又有剪接变异。根据氨基酸的序列相似性，信号转导机制及激动剂的选择性不同。这些mGluRs又分为G-Ⅰ，GⅡ。GⅢ三组：G-Ⅰ组（mGluRl，mGluR5）主要通过活化磷脂酶C（PLC），将膜内的磷酸肌醇（P1）水解；G-Ⅱ组（mGhR2。mGluR3）和GIⅡ组（mGluR4,mGluR6,mGluR7,mGluR8）受体激活时，抑制由毛候萜（forskolin）诱发的胞内cAMP累积。mGluRs可作为突触后成分或者突触前自身受体而调节中枢神经系统的突触传递。不同的mGluRs的转导机制不同。因此mGluRs对谷氨酸介导的突触传递可以是增强作用。可以表现为抑制效应，并参与调节膜上酶及离子受体的功能及参与海马突触可塑性，影响海马突触传递效能，影响神经系统的发育、突触形成。现将这些受体在脑缺血缺氧及脑耐受形成中的作用做一综述。     

N—甲基—D—天门冬氨酸受体拮抗剂对猫创伤性脑水肿的影响

本实验观察了竞争性Ｎ－甲基－Ｄ－门冬氨酸（ＮＭＤＡ）受体拮抗剂Ｄ－（－）－２－氨基－７－磷庚酸酯（Ｄ－ＡＰ７）对猫创伤性脑水肿的影响。结果发现Ｄ－ＡＰ７可明显减轻脑含水量，改善脑Ｎａ＾＋，Ｋ＾＋，Ｃａ＾＋＾＋，Ｍｇ＾＋＾＋含量，提示兴奋性氨基酸与创伤性脑水肿有关，其中ＮＭＤＡ受体可能起主要作用，本文探讨了ＮＭＤＡ受体损拮抗剂对创伤性脑水肿的保护机理。     

N－甲基－D－天门冬氨酸受体拮抗剂对猫创伤性脑水肿的影响

本实验观察了竞争性Ｎ－甲基－Ｄ－天门冬氨酸（ＮＭＤＡ）受体拮抗剂Ｄ－（－）－２－氨基－７－磷庚酸酯（Ｄ－ＡＰ７）对猫创伤性脑水肿的影响。结果发现Ｄ－ＡＰ７可明显减轻脑含水量，改善脑Ｎａ＋、Ｋ＋、ｃａ＋＋、Ｍｇ＋＋含量，提示兴奋性氨基酸与创伤性脑水肿有关，其中ＮＭＤＡ受体可能起主要作用，本文探讨了ＮＭＤＡ受体拮抗剂对创伤性脑水肿的保护机理。     

促代谢型谷氨酸受体1亚型信号转导介导长时程抑制

人的经历，如所见、所闻、所为，都能引起大脑神经元之间连接强度的持久改变，并且这些改变是人类大脑编译信息的方式。最新研究发现一种可能与成瘾行为有关的记忆存储的生物化学机制。[第一段]     

代谢型谷氨酸受体1阻滞剂LY367385对NMDA所致神经元损伤的保护作用

目的探讨代谢型谷氨酸受体1在神经系统兴奋性损伤中的作用。方法用代谢型谷氨酸受体1特异性阻滞剂LY367385预处理神经元细胞1 h,加入N-甲基-D-门冬氨酸(NMDA)造成兴奋性损伤;采用甲基噻唑基四唑(MTT)比色实验检测细胞活性; LDH试剂盒检测培养基中乳酸脱氢酶(LDH)活性;转移酶介导的三磷酸脱氧鸟苷-生物素刻痕末端标记(TUNEL)法检测细胞凋亡指数;免疫共沉淀和免疫印迹实验检测NMDA受体2B亚基(NR2B)和突触后致密物质95(PSD95)相互作用。结果 MTT、LDH和TUNEL实验显示,LY367385能减轻NMDA造成的神经元损伤(P 0. 05);免疫共沉淀和免疫印迹实验显示,NMDA受体与PSD95存在相互关系,LY367385可以减轻兴奋性损伤造成的NMDAR-PSD95表达增高(P 0. 05)。结论 LY367385能够明显减轻NMDA造成的神经元损伤,其机制可能与阻断代谢型谷氨酸受体1可以减少NMDA受体与PSD95连接相关。     

大鼠代谢型谷氨酸受体1亚型基因片段的克隆及其在小脑的表达

目的：克隆大鼠代谢型谷氨酸受体1亚型（mGluR1)基因特异片段，制备cDNA探针。方法：从Wistar大鼠小脑中提取总RNA，以RT－PCR方法得到预期的599bp条带，将这一片段克隆到pGEM-T easy载体上，经酶切鉴定正确后送测序。将重组质粒经限制性内切酶酶切制备成线性模板，通过体外转录的方法 合成地高辛标记的mGluR1cRNA正义及反义探针。取成年Wistar大鼠小脑组织进行原位杂交实验，以检测探针的可靠性。结果：测序证实用RT－PCR的方法获得了mGluR1基因特异片段，成功地构建了pGEM-TmGluR1重组质粒。根据斑点杂交实验结果计算出正义、反义探针浓度分别为10ng/μl及30ng/μl。原位杂交实验的结果显示，用mGluR1反义探针进行杂交的阳性信号主要分布在大鼠小脑蒲肯野氏细胞胞浆，用正义探针杂交无阳性信号。结论：本实验克隆了mGluR1基因特异片段，并制备了cRNA探针，并用大鼠小脑进行的原位杂交实验显示，此探针灵敏度高，特异性好。     

局灶脑缺血大鼠代谢型谷氨酸受体5mRNA表达及意义

目的观察局灶脑缺血大鼠脑组织中代谢型谷氨酸受体5mRNA(metabotropic glutamate receptor 5,mGluR5 mRNA)在脑缺血不同时段的表达及其动态变化的规律,探讨其在急性脑缺血中变化的意义。方法55只雄性Wistar大鼠随机分为正常对照组、脑缺血1、3、6、12及24h组(手术组)及其相对应的假手术对照组,采用线栓法制备大鼠MCAO模型,至上述规定时间点后取大脑组织,利用地高辛标记寡聚核苷酸探针原位杂交技术判断神经元阳性表达的强弱,用计算机图像分析系统检测各组mGluR5 mRNA表达的灰度值及阳性细胞数。结果各手术组mGluR5 mRNA表达均高于假手术对照组及正常对照组,与正常对照组相比,缺血1h其表达即有升高,至6h达高峰。而假手术对照组与正常对照组相比无统计学意义。结论急性脑缺血可引起mGluR5 mRNA表达上调,提示mGluR5参与了局灶性脑缺血损伤,可能促进了缺血性脑血管病的发展。     

代谢型谷氨酸受体及其拮抗剂MCPG对弥漫性脑损伤大鼠的影响

目的:观察代谢型谷氨酸受体拮抗剂MCPG对弥漫性脑损伤大鼠的影响。方法:使用Marmarou的动物模型,55只动物分为对照、损伤和MCPG组。给予MCPG组立体定向脑室注射MCPG lumol/5ul。分别在伤后1、4、8、12、24小时测定脑组织含水量、递质性氨基酸(Glu、GABA)及离子Ca~(2+)、Mg~(2+)含量。结果:伤后脑组织含水量、GABA、Ca~(2+)升高,Glu、Asp及Mg~(2+)含量下降。MCPG组较损伤组各项指标均有明显的好转。结论:MCPG能有效的抑制代谢型谷氨酸受体激活导致的继发性损害。     

N-Methyl-D-Aspartate受体拮抗剂对脑缺血后成年大鼠神经干细胞内源性激活的实验研究

目的　研究N - Methyl- D- Aspartate(NMDA)受体拮抗剂MK- 80 1对脑缺血后神经干细胞(NSC)激活的作用。方法　将4 0只SD大鼠分成对照组和实验组,两组大鼠均采用传统线栓法作成大脑中动脉缺血再灌注模型,实验组大鼠腹腔注射MK- 80 1,对照组腹腔注射生理盐水,通过免疫组织化学技术标记鼠脑海马齿状回颗粒细胞层(SGZ)、室管膜下层(SVZ)及梗死皮质周边区注射后第3、7、11、18天的Brdu、Nestin阳性细胞数。结果　对照组大鼠Brdu、Nestin阳性细胞7d在SGZ出现一小高峰,然后迅速下降,11d阳性细胞甚少,梗死皮质周边区更少;而实验组Brdu、Nestin阳性细胞3d在SVZ明显表达,7～11d在SGZ区达高峰,并可持续至18d,同样梗死皮质区Brdu、Nestin阳性细胞7～18d表达明显,两组比较,有统计学意义(P<0 .0 1)。结论　NMDA受体拮抗剂MK- 80 1在脑缺血后,能促进NSC的增殖、分化。     

神经生长因子对急性缺血性脑水肿和内皮素变化的实验研究

本实验应用放射免疫测定法和密度梯度法观察了神经生长因子对Ｗｉｓｔａｒ大鼠缺血皮层和血浆内皮素（ＥＴ－１）和脑皮层比重的影响，结果表明，脑缺血前后１０ｄ分别每天给予不同剂量的神经生长因子（２ｍｇ／ｋｇ，１ｍｇ／ｋｇ，２ｍｇ／ｋｇ，３ｍｇ／ｋｇ）能不同程度地影响脑缺血皮层中内皮素的变化，缺血性脑水肿也相应地得到改善，且呈量效依赖关系，为该药治疗脑血管病提供其理论依据。     

白细胞介素1受体拮抗剂对大鼠外伤性脑水肿的治疗作用

目的 观察白细胞介素1受体拮抗剂(IL—1ra)对于外伤性脑水肿的影响。方法 液压伤致大鼠外伤性脑水肿。伤后脑室注射IL-1ra。伤后24h磁共振，脑组织干湿重，HE病理图像分析及电镜等方法观察鼠脑水肿情况。结果 经IL-1ra治疗，与创伤组或治疗对照组相比在磁共振T2加权像上可以水肿减轻；创伤部位脑含水量减低；病理切片可见相同的变化；超微病理发现IL—1ra具有保护作用。结论 IL-1ra对于外伤性脑水肿有治疗作用，内源性IL-1参与了外伤性脑水肿的发生。     

Protective effect of LY379268, a selective group II metabotropic glutamate receptor agonist, on dizocilpine-induced neuropathological changes in rat retrosplenial cortex

In the present study, we examined the effects of LY379268, the group II metabotropic glutamate receptor (mGluR) agonist, on the neuropathological changes in the rat retrosplenial cortex induced by noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine ((+)-MK-801). Administration of LY379268 (1, 3, 10 mg/kg, i.p.) reduced dizocilpine (0.5 mg/kg, i.p.)-induced neuropathological changes in the retrosplenial cortex, in a dose-dependent manner. Co-administration of LY379268 (10 mg/kg, i.p.) with group II mGluR antagonist LY341495 (5 mg/kg, i.p.) blocked the effects of LY379268. Furthermore, LY379268 (10 mg/kg, i.p.) significantly reduced the expression of heat shock protein HSP-70, a marker of reversible neuronal injury, in the rat retrosplenial cortex after administration of dizocilpine (0.5 mg/kg, i.p.). Moreover, pretreatment with LY379268 (10 mg/kg, i.p.) significantly suppressed the increase in extracellular acetylcholine (ACh) levels in the retrosplenial cortex induced by administration of dizocilpine (0.5 mg/kg, i.p.). These results suggest that LY379268 has a protective effect on the neurotoxicity in the rat retrosplenial cortex after administration of NMDA receptor antagonists such as dizocilpine.     

胰蛋白酶抑制剂对实验性缺血性脑水肿的作用

目的观察各种胰蛋白酶抑制剂对大鼠缺血性脑水肿的作用。方法大鼠脑缺血模型采用线栓法，脑含水量测定采用称重法，常规病理学检查。结果５５ｍｇ／ｋｇ和１６５ｍｇ／ｋｇ胰蛋白酶抑制剂（ＳＢＴＩ）对ＬＭＣＡＯ３ｈ大鼠脑含水量无显著影响（Ｐ均＞０．０５）；６６０００ＫＩＵ／ｋｇ特血乐、５００００Ｕ／ｋｇ尿胰蛋白酶抑制剂（ＵＴＩ）可显著降低脑水肿大鼠脑含水量（Ｐ均＜０．０５），并可减轻缺血区神经元变性及间质水肿程度。结论６６０００ＫＩＵ／ｋｇ特血乐、５００００Ｕ／ｋｇＵＴＩ具有减轻实验性缺血性脑水肿的作用。     

Delayed administration of interleukin-1 receptor antagonist reduces ischemic brain damage and inflammation in comorbid rats

Many neuroprotective agents have been effective in experimental stroke, yet few have translated into clinical application. One reason for this may be failure to consider clinical comorbidities/risk factors in experimental models. We have shown that a naturally occurring interleukin-1 receptor antagonist (IL-1Ra) is protective against ischemic brain damage in healthy animals. However, protective effects of IL-1Ra have not been determined in comorbid animals. Thus, we tested whether IL-1Ra protects against brain injury induced by experimental ischemia in aged JCR-LA (corpulent) rats, which have clinically relevant risk factors. Male, aged, lean, and corpulent rats exposed to transient (90 minutes) occlusion of the middle cerebral artery (tMCAO) were administered two doses of IL-1Ra (25 mg/kg, subcutaneously) during reperfusion. Brain injury and neuroinflammatory changes were assessed 24 hours after tMCAO. Our results show that IL-1Ra administered at reperfusion significantly reduced infarct volume measured by magnetic resonance imaging (50%, primary outcome) and blood–brain barrier disruption in these comorbid animals. Interleukin-1Ra also reduced microglial activation, neutrophil infiltration, and cytokines levels in the brain. These data are the first to indicate that IL-1Ra protects against ischemic brain injury when administered via a clinically relevant route and time window in animals with multiple risk factors for stroke.     

血脑屏障内皮细胞与细胞间粘附分子-1在鼠脑缺血性脑水肿发生机制中的作用

目的研究缺血性脑水肿病理过程中血脑屏障内皮细胞及其细胞间粘附分子-1(intercellularadhesionmolecule-1,ICAM-1)的表达与血脑屏障通透性的关系.方法用电镜技术和免疫组织化学方法观察脑缺血过程中大鼠血脑屏障内皮细胞超微结构及其ICAM-1的表达和对镧离子的通透性改变.结果脑缺血后1h,缺血区脑组织血脑屏障内皮细胞未见ICAM-1表达,但电镜下可见内皮细胞轻度肿胀.3h,ICAM-1开始表达,缺血区脑组织轻度水肿,内皮细胞肿胀.至12h,ICAM-1表达达到高峰,脑组织严重水肿,内皮细胞、神经细胞及胶质细胞明显肿胀;内皮细胞胞饮小泡包含有被吞噬的镧离子,有些镧离子进入紧密连接间隙.72h,ICAM-1表达已明显减少,脑组织水肿仍较重,内皮细胞严重破坏,大量镧离子漏出血管外,进入脑实质.结论脑缺血后血脑屏障的破坏与ICAM-1的表达密切相关,提示ICAM-1参与血脑屏障内皮细胞的破坏过程,而且这些改变在缺血性脑水肿的病理过程中起重要作用.     

MPEP,a selective metabotropic glutamate receptor 5 antagonist,attenuates conditioned taste aversion in rats

Metabotropic glutamate receptors (mGluRs) have been implicated in several types of cognitive and associative learning. Although recent evidence indicates an influence of mGluRs in conditioned taste aversion (CTA), the subtype-specific involvement of mGluRs in this learning paradigm remained to be determined. The aim of this study was to examine the role of Group I mGluR subtypes in CTA using a selective mGluR5 antagonist (2-methyl-6-(phenylethynyl)-pyridine, MPEP) and a selective mGluR1 antagonist (1-aminoindan-1,5-dicarboxylic acid, AIDA). Male, water-deprived, Sprague-Dawley rats were injected i.p. with 6 or 12 mg/kg MPEP or saline. Twenty-five minutes later, all rats received 15-min access to a 0.1% saccharin solution (Sac) immediately followed by an injection of 0.15M LiCl at 1.33% body weight. The animals were tested with 15-min access to Sac on each of four test days. MPEP-treated animals consumed more Sac on the test trials than saline-treated rats. In another experiment, controlled access to Sac was used by infusing the solution on the conditioning trial. Consistent with the above results, MPEP attenuated the degree of CTA. Similar experiments using the mGluR1 antagonist AIDA, have found no effect on CTA learning. These results suggest that the two subtypes of Group I mGluRs are differentially involved in taste aversion learning.     

U0126对缺血性脑损伤大鼠脑内水通道蛋白-4表达的影响

目的研究水通道蛋白-4(aquaporin4,AQP4)在缺血性脑损伤大鼠脑内的表达,及丝裂原活化蛋白激酶(MAPKs)信号转导通路抑制剂U0126对其表达的影响。方法用线栓法建立缺血性脑损伤大鼠模型,测定脑组织含水量及伊文斯蓝含量,并采用免疫组织化学、Westernblot和逆转录-聚合酶链反应技术,检测AQP4的表达。测定预先经侧脑室给予U0126后MAPKs信号通路关键蛋白ERK1/2和ELK1磷酸化水平,同时观察U0126对脑水肿和AQP4表达的影响。结果正常组AQP4表达较低[蛋白(吸光度值,下同):123·1±1·0,mRNA(吸光度比值,下同):0·173±0·017],在缺血损伤后表达升高(蛋白:153·6±0·8,mRNA:0·400±0·015),脑组织含水量及伊文斯蓝含量增加,给予U0126后AQP4的表达和脑组织含水量降低(蛋白:149·0±1·1,mRNA:0·328±0·010,P<0·01),同时ERK1/2和ELK1磷酸化水平降低。结论缺血性损伤后AQP4表达上升,脑水肿明显,预先给予U0126可抑制AQP4的表达,减轻脑水肿。     

Effects of LF 16-0687 Ms,a bradykinin B(2) receptor antagonist,on brain edema formation and tissue damage in a rat model of temporary focal cerebral ischemia

Bradykinin, an endogenous nonapeptide produced by activation of the kallikrein–kinin system, promotes neuronal tissue damage as well as disturbances in blood–brain barrier function through activation of B₂ receptors. LF 16-0687 Ms, a non-peptide competitive bradykinin B₂ receptor antagonist, was recently found to decrease brain swelling in various models of traumatic brain injury. We have investigated the influence of LF 16-0687 Ms on the edema formation, neurological outcome, and infarct size in temporary focal cerebral ischemia in rats. Sprague–Dawley rats were subjected to MCA occlusion for 90 min by an intraluminal filament. Local CBF was bilaterally recorded by laser Doppler flowmetry. Study I: animals were assigned to one of three treatment arms (n=11 each): (a) vehicle, (b) LF 16-0687 Ms (12.0 mg/kg per day), or (c) LF 16-0687 Ms (36.0 mg/kg per day) given repetitively s.c. over 3 days. The neurological recovery was examined daily. The infarct volume was assessed histologically 7 days after ischemia. Study II: brain swelling and bilateral hemispheric water content were determined at 48 h post ischemia in eight rats, subjected to the low dose regimen as described above, and in eight vehicle-treated control animals. All treated animals showed tendency to exhibit improved neurological recovery throughout the observation period as compared to the vehicle-treated controls, while this improvement was only significant within the low dose group from postischemic days 3 to 4. Low dose LF 16-0687 Ms significantly attenuated the total and cortical infarct volume by 50 and 80%, respectively. Furthermore, postischemic swelling (−62%) and increase in water content of the infarcted brain hemisphere (−60.5%) was significantly inhibited. The present findings provide strong evidence for an involvement of bradykinin-mediated secondary brain damage following from focal cerebral ischemia. Accordingly, specific inhibition of bradykinin B₂ receptors by LF 16-0687 Ms attenuated postischemic brain swelling, improved the functional neurological recovery, and limited ischemic tissue damage, raising its potential for clinical evaluation in patients with acute stroke.