3-硝基丙酸对沙土鼠海马CA1区锥体细胞超微结构的影响

目的观察小剂量3-硝基丙酸(≤20mg/kg)对沙土鼠海马CA1区锥体细胞超微结构的影响.方法将沙土鼠腹腔注射不同剂量3-硝基丙酸或双蒸水后3d,取海马CA1 区用戊二醛和锇酸固定,电镜下观察CA1区锥体细胞层神经元细胞核和细胞器超微结构形态变化,用体视学方法计算每张照片线粒体和粗面内质网体面积变化.结果实验组与对照组比较细胞核无明显变化,线粒体和粗面内质网轻度肿胀,体密度高于对照组.结论小剂量3-硝基丙酸可引起神经元缺氧代偿性反应,无致死性损害.     

3-硝基丙酸对沙土鼠海马CA1区锥体细胞超微结构的影响

目的　观察小剂量3-硝基丙酸(≤2 0mg/kg)对沙土鼠海马CA₁区锥体细胞超微结构的影响。方法　将沙土鼠腹腔注射不同剂量3-硝基丙酸或双蒸水后3d ,取海马CA₁区用戊二醛和锇酸固定,电镜下观察CA₁区锥体细胞层神经元细胞核和细胞器超微结构形态变化,用体视学方法计算每张照片线粒体和粗面内质网体面积变化。结果　实验组与对照组比较细胞核无明显变化,线粒体和粗面内质网轻度肿胀,体密度高于对照组。结论　小剂量3-硝基丙酸可引起神经元缺氧代偿性反应,无致死性损害。     

鼠脑缺血再灌注后星形胶质细胞的变化及补阳还五汤对其的影响

目的 探讨沙土鼠脑缺血再灌注后星形胶质细胞（AS）的变化及补阳还五汤对其的影响。方法 采用沙土鼠双侧颈动脉夹闭脑缺血模型，于脑缺血15min、再灌注24h和48h后，运用免疫组化技术观察星型胶质纤维酸性蛋白（GFAP）的动态表达。结果 缺血再灌注24h后，GFAP免疫阳性反应达高峰，补阳还五汤可使GFAP免疫反应减轻；缺血再灌注48h后GFAP表达减弱，补阳还五汤可使其增强。结论 补阳还五汤对脑缺血再灌注后星形胶质细胞的调节作用可能与脑缺血损伤后神经功能的恢复有关。     

亚低温对沙土鼠海马CA1区缺血后细胞凋亡的影响

目的 探讨亚低温对沙土鼠短暂性脑缺血后海马CA1区细胞凋亡的影响。方法 阻断沙土鼠双侧颈总动脉15分钟造成前脑缺血模型。实验动物随机分为假手术组、缺血再灌注组、亚低温治疗组。用光镜观察海马CA1区的神经元死亡过程．采用原位末端标记(TUNEL)法检测凋亡的神经元。结果 脑缺血后．海马CA1区锥体神经元于再灌注后2～7天死亡,再灌注第3天神经元开始凋亡．第5天达高峰。亚低温治疗抑制了缺血后海马CA1区神经元的死亡及细胞凋亡。结论 亚低温治疗对脑缺血后的神经元具有保护作用．并可抑制神经细胞凋亡的发生。     

3-硝基丙酸的神经毒理

３－硝基丙酸的神经毒理何凤生付以同（中国预防医学科学院劳动卫生与职业病研究所，北京１０００５０）３－硝基丙酸（３－ＮＰＡ）是引致我国北方流行的变质甘蔗中毒的一种霉菌毒素．儿童吃后数小时内即可引起急性非炎性脑病，出现抽搐与昏迷；重者还可留有迟发性肌张力...     

氯胺酮对沙土鼠脑缺血再灌注损伤后细胞凋亡的影响

目的：研究氯胺酮对沙土鼠脑缺血再灌注损伤后细胞凋亡状态及相关基因bax、bcl—2表达的影响。方法：沙土鼠全脑缺血模型，缺血10min，再灌注24h。分为假手术组、缺血再灌注组和氯胺酮组。采用TUNEL法原位标记DNA片段检测凋亡细胞，采用SP法进行bax、bcl—2的免疫组织化学染色。结果：缺血再灌注组及氯胺酮组脑组织中TUNEL及bax阳性细胞数明显多于假手术组(P＜0．01)；氯胺酮组阳性细胞数目明显低于缺血再灌注组(P＜0．01)。bcl—2阳性细胞数3组间比较无明显差异。结论：氯胺酮减少沙土鼠脑缺血再灌注损伤后细胞的凋亡，bax/bcl—2比值较再灌注组相对降低可能为此作用的机理之一。     

3-硝基丙酸叔丁酯的制备

3-硝基丙酸叔丁酯(1)可用于合成caspase二肽抑制剂[1,2],这类抑制剂能有效抑制细胞凋亡,对与细胞过度凋亡相关的神经退行性疾病、以及延长待移植器官保存时间等作用明显.本研究参考相关文献[3,4],用丙烯醛(2)硝化得到3-硝基丙醛(3),不经分离,直接氧化得到3-硝基丙酸(4),简化了操作,两步收率51%(文献[4]:60%).4再用异丁烯法[5]制得1(图1).操作简便,总收率30%.     

布比卡因抑制谷氨酸诱导大鼠海马星形胶质细胞内钙升高的机制

本文以原代培养的大鼠海马星形胶质细胞为研究对象,探讨布比卡因(bupivacaine)抑制谷氨酸诱导的海马星形胶质细胞内钙升高的机制。运用免疫荧光技术确定代谢型谷氨酸受体(mGluR5受体)在神经元和星形胶质细胞上的表达情况;利用钙离子成像技术,观察布比卡因和mGluR5受体拮抗剂2-甲基-6-(苯乙炔)吡啶盐酸盐[2-methyl-6-(2-phenylethynyl)-pyridine, MPEP]对谷氨酸诱导的大鼠海马神经元和星形胶质细胞胞内游离钙(intracellular free Ca²⁺concentration,[Ca²⁺]_i)变化的影响;观察布比卡因对mGluRs (mGluR1/5)受体激动剂(RS)-3,5-二羟基苯基甘氨酸[(RS)-3,5-dihydroxyphenylglycine, DHPG]及mGluR5受体特异性激动剂2-氯-5羟苯基甘氨酸钠盐[(RS)-2-chloro-5-hydroxyphenylglycine sodium salt, CHPG]诱导的大鼠海马神经元和星形胶质细胞...     

全脑缺血诱导沙土鼠纹状体,海马和皮层氨基酸水平的改变(英文)

目的:研究全脑缺血时沙土鼠海马、纹状体和皮层谷氨酸、天冬氨酸、γ-氨基丁酸(GABA)、谷氨酰胺、甘氨酸和牛磺酸含量的变化及氯胺酮对上述氨基酸含量的影响.方法:采用结扎双侧颈总动脉的方法制备沙土鼠全脑缺血模型,应用HPLC和荧光检测器联用测定氨基酸的含量.结果:全脑缺血显著增加沙土鼠海马,纹状体和皮层的谷氨酸,天冬氨酸,谷氨酰胺,GABA,甘氨酸和牛磺酸含量;氯胺酮(120 mg/kg,ip)预处理能完全逆转缺血诱导的谷氨酸、天冬氨酸、甘氨酸和谷氨酰胺释放的增加,但不能完全逆转缺血诱导的GABA和牛磺酸释放增加.结论:脑缺血诱发的神经元损伤可能与其增加谷氨酸、天冬氨酸、甘氨酸、谷氨酰胺含量有关,而抑制性氨基酸GABA和牛磺酸释放增加则可能是机体一种重要的自身脑保护机制.氯胺酮逆转脑缺血诱导的兴奋性氨基酸释放增加可能是其抗兴奋性神经毒的生化基础.     

PPARα促进自噬流抑制脑缺血后星形胶质细胞的过度激活

目的阐明过氧化物酶体增殖物激活受体α(PPARα)在脑缺血后星形胶质细胞活化中的作用及机制。方法大脑中动脉闭塞(MCAO)建立小鼠脑缺血损伤模型,缺氧缺糖再灌注(OGD/R)建立小鼠原代培养星形胶质细胞活化模型。应用PPARαnull小鼠及PPARα激动剂,观察PPARα功能缺失或者激活后对星形胶质细胞活化的影响。进一步通过观察自噬经典标记物LC3Ⅱ/LC3Ⅰ和P62表达变化,联合应用巴弗洛霉素或雷帕霉素观察自噬流的变化,应用自噬双标腺病毒(mRFP-GFP-LC3)检测自噬体和自噬溶酶体的变化,应用透射电子显微镜观察超微结构等方法,研究PPARα功能缺失或者激活后对星形胶质细胞自噬的影响。结果 PPARαnull小鼠或原代培养的PPARαnull星形胶质细胞在脑缺血损伤或OGD/R处理后,星形胶质细胞活化标记物GFAP,Neurocan等的表达较野生型对照组相比明显增加。脑缺血损伤或OGD/R处理后活化星形胶质细胞中存在自噬流障碍,自噬双标腺病毒检测及透射电子显微镜的结果均发现活化的星形胶质细胞中存在大量自噬小体的堆积,PPARαnull星形胶质细胞较野生对照组相比堆积更加明显,而PPARα激动剂可显著改善自噬流障碍,抑制星形胶质细胞的过度活化。结论 PPARα可促进脑缺血后星形胶质细胞的自噬流,抑制星形胶质细胞的过度活化。     

缺血预适应对沙土鼠脑缺血再灌注后纹状体多巴胺及其代谢产物的影响

目的　研究缺血预适应对沙士鼠脑缺再灌注后纹状体中多巴胺(DA)及其代谢产物的影响,以探讨其神经保护作用的可能机制。方法　应用沙土鼠脑缺血再灌注模型,脑缺血时间为 5min。96只沙土鼠随机分为假手术组 (S)、缺血组(I)、单纯预适应组 (Po)、缺血预适应组 (Ip)。在再灌注 0(即缺血末)、5、30、60min时,断头、分离纹状体,用高效液相-电化学检测器 (HPLC ECD)分别测定各组沙土鼠纹状体DA、3, 4双羟苯乙酸 (DOPAC)和高香草酸 (HVA)的含量。结果　与假手术组相比,短暂缺血组在缺血再灌注期间纹状体中DA、DOPAC和HVA含量均无显著性变化 (P>0 05)。再灌注期间,预适应、缺血再灌注组DA含量均显著低于假手术组,但在再灌注 0min和 30min时,预适应组DA含量及显著高于缺血再灌注组,分别增加了 28% (P<0 01)和 22% (P<0 05)。与假手术组相比,预适应组的DOPAC和HVA含量在再灌注期间均无显著变化 (P>0 05 );缺血再灌注组DOPAC含量在再灌注 5min时有明显增加;HVA含量在再灌注 30min时增加至峰值。结论　缺血预适应能部分抑制沙土鼠脑缺血再灌注期间纹状体DA的降低,显著减少DA氧化代谢产物的产生,从而在脑缺血再灌注损伤中起保护作用。     

血清尿酸水平与急性缺血性脑卒中患者脑微出血的相关性研究

目的 探讨血清尿酸水平与急性缺血性脑卒中患者脑微出血之间的关系.方法 回顾性分析连续性收治的212例急性脑卒中患者的人口学特征,临床和实验室及影像学数据,CMBs的诊断采用MRI T2加权梯度回波序列检查.应用Logistic回归模型分析测定尿酸和脑微出血的独立相关性.结果 94例(44.3%)患者发现存在CMBs.与未出现CMBs患者相比,发生CMBs的患者平均年龄更大、尿酸值更高,合并高血压病、既往脑卒中病史、WMLs分级及高总胆固醇几率更高,差异均有统计学意义(P<0.05).与尿酸水平第1分位数组相比,尿酸水平第4分位数组的患者合并高血压、高胆固醇血症以及心脏疾病及发生CMBs的几率更大、体重指数更高(P<0.05).单因素分析结果表明,高血压、高胆固醇血症、脑卒中病史、SBP、DBP、WMLs分级和尿酸与CMBs的发生有关(P<0.05).多变量分析显示:尿酸、WMLs分级、脑卒中病史、高胆固醇血症和高血压均为CMBs发生的独立预测因素,并且尿酸水平的OR值显著高于其他指标(P<0.05).结论 血清尿酸水平是急性缺血性脑卒中患者CMBs的独立相关因素.     

AMPA/kainate-related mechanisms contribute to convulsant and proconvulsant effects of 3-nitropropionic acid

The role of the glutamatergic system in the convulsant and proconvulsant action of a mitochondrial toxin, 3-nitropropionic acid, was studied in mice. The occurrence of 3-nitropropionic acid-induced seizures was inhibited by the -amino-2,3-dihydro-5-methyl-3-oxo-isoxazole-propionate (AMPA)/kainate receptor antagonists, 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione disodium (NBQX) and 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine HCl (GYKI 52466), with ED₅₀ of 14.1 (7.9–25.2) and 7.2 (5.3–9.6) mg/kg, respectively. The N-methyl- -aspartate (NMDA) receptor antagonists, dizocilpine (MK-801) and 3-(2-carboxypiperazine-4-yl)propenyl-1-phosphonic acid (CPPene), were ineffective. Moreover, 3-nitropropionic acid given in a subthreshold dose potently enhanced seizures generated by intracerebroventricular administration of AMPA and kainate, lowering their CD₅₀ from 0.98 (0.83–1.17) and 0.73 (0.64–0.83) to 0.55 (0.45–0.66) (P<0.001) and 0.58 (0.51–0.65) (P<0.05) nmol, respectively. In contrast, NMDA action was not changed by 3-nitropropionic acid application. We conclude that AMPA/kainate-mediated events are involved in proconvulsive and convulsive effects of 3-nitropropionic acid.     

电压依赖性氯通道在3-吗啉斯德酮亚胺诱导的大鼠海马神经元凋亡中的作用

目的探讨氯通道与缺血性脑损伤的关系。方法离体培养12d的SD大鼠海马神经元,分为4组:正常对照组、3-吗啉斯德酮亚胺(SIN-1)处理组(SIN-11.0mmol.L-1作用18h)、SIN-1+4-4-二异硫氰茋-2,2′-二磺酸组(DIDS,0.1mmol.L-1作用18h)及SIN-1+4-乙酰氨基-4′-异氰酸茋-2,2′-二磺酸组(SITS,0.5mmo.lL-1作用18h)。DNA荧光染色观察神经元形态及检测凋亡数目的变化;免疫荧光染色观察神经元两种电压门控氯通道(ClC-2/ClC-3)的表达;全细胞膜片钳技术记录神经元氯通道电流。结果Hoechst33258染色显示,正常对照组、SIN-1处理组、SIN-1+SITS组和SIN-1+DIDS组神经元凋亡百分数分别是:(18.61±0.59)%,(50.43±0.56)%,(23.37±0.52)%和(23.37±0.84)%;两种氯通道ClC-2/ClC-3在正常神经元上存在;SIN-1处理后的神经元氯通道电流较正常增加55%～56%;氯通道阻断剂SITS和DIDS分别能抑制氯通道电流的30%～40%和50%～60%。结论电压依赖性氯通道可能参与了SIN-1诱导的神经元凋亡,氯通道可能参与缺血性脑损伤过程。     

Effects of N-acetylcysteine and ebselen on arachidonic acid release from astrocytes and neurons cultured in normoxic or simulated ischemic conditions

Arachidonic acid (AA) is released from cells after nervous tissue injuries. We treated rat cortical neurons and astrocytes cultured under normoxic or simulated ischemic conditions with N-acetylcysteine (100 or 200 M) or ebselen (10 or 20 M). N-acetylcysteine decreased AA release in normoxic astrocytes, while ebselen decreased AA release from astrocytes in both conditions. N-acetylcysteine produced no changes in neuronal AA release. A low dose of ebselen significantly increased AA release from neurons in both conditions. The influence of N-acetylcysteine and ebselen on AA release might be implicated in their effects on astrocytes and neurons, however, the exact mechanisms have yet to be explained.     

Absorption of 3-nitropropanol and 3-nitropropionic acid from the digestive system of sheep

When 3-nitropropanol (NPOH) was injected into the rumen (30 mg/kg), abomasum (10 mg/kg) or small intestine (10 mg/kg) of sheep, it was rapidly absorbed and converted to 3-nitropropionic acid (NPA). The reticulo-rumen was the major site of absorption for the miserotoxin aglycone but the abomasum and the small intestine also had the capacity to absorb NPOH. When NPA was injected into different regions of the alimentary tract, the reticulo-rumen was also the major site of absorption. Absorption of NPA or NPOH from the small intestine was much more rapid than from the abomasum. Plasma levels of NPA and inorganic nitrite were higher after dosing with NPOH than with NPA indicating a more rapid rate of uptake of the aglycone.     

Functional neurotoxic effects in rats elicited by 3-nitropropionic acid in acute and subacute administration

Changes possibly induced by 3-NP in electrophysiological functional characteristics of the central nervous system are, in contrast to biochemical and morphological alterations, less well known. In this study, the usability of a standard neurophysiological investigation system to detect functional changes caused by 3-NP administration in rats was studied. In subacute treatment, 10 or 15 mg/kg 3-NP was given i.p. on five consecutive days to groups of 10 rats and the effects were checked 4 weeks later. Acutely treated rats received 20 mg/kg i.p. after several control records. For recording, the animals’ left hemisphere was exposed in urethane anesthesia. Silver electrodes were placed on the cortical sensory foci and tungsten needles in the subcortical (caudatum, globus pallidus) recording sites. Spontaneous electrical activity, as well as somatosensory, visual and auditory evoked potentials, were recorded. Following subacute treatment, the slowest (theta) and fastest (beta2 and gamma) frequencies of the spontaneous activity were changed, differently in the cortical versus subcortical sites. In the sensory evoked potentials after subacute treatment, an increase of the latency was seen in all sensory areas. In the acutely treated animals, the amplitude of the somatosensory evoked potential decreased after giving 3-NP. With double stimuli, the relation of the two responses was treatment- and interval-dependent. Understanding the mechanism of these effects may widen the knowledge base for using 3-NP in disease models.     

Ovariectomy aggravates convulsions and hippocampal γ-aminobutyric acid inhibition induced by cyclosporin A in rats

The possible cyclosporin A application for rheumatoid arthritis that develops preferentially in middle-aged women raises concerns about adverse effects of cyclosporin A, including neurotoxicity in patients with climacterium. The present study was aimed at elucidating the effect of cyclosporin A on the convulsive activity and γ-aminobutyric acid (GABA) neural activity of the hippocampus in ovariectomized rats, as a menopause/climacterium model. Ovariectomy markedly aggravated the effect of repeated administration of cyclosporin A (40 mg/kg, once a day for 5 or 6 days), convulsions and reduction of the basal GABA levels and aminooxyacetic acid-evoked GABA accumulation. These aggravations were blocked by estradiol replacement. The present findings demonstrated that ovariectomy increased the susceptibility to cyclosporin A-induced convulsions by accelerating an inhibitory action of cyclosporin A on GABA neural activity in the hippocampus, this being blocked by estrogen replacement. Menopause/climacterium is, therefore, included in the risk factors for cyclosporin A-induced neurotoxicity and this risk is lowered by estrogen replacement therapy.     

Autophagy,mitochondria and 3-nitropropionic acid joined in the same model

Huntington''s disease (HD) is a neurodegenerative disorder caused by a mutation in the gene encoding the huntingtin protein. Although the precise mechanism by which neuronal degeneration occurs is still unclear, several elements are important to its development: (1) altered gene expression and protein synthesis, (2) mitochondrial damage and (3) improper regulation of the autophagy programme. In this issue of British Journal of Pharmacology, Galindo and co-workers provide the first evidence for a role of the mitochondrial permeability transition pore (mPTP) in mitochondrial fragmentation and autophagy activation. In a model of cell death induced by 3-nitropropionic acid (3-NP) in human neural cells, the authors describe clear functions for mPTP and Bax, but not the mitochondrial fusion/fission machinery, mitochondrial fragmentation and autophagy (mitophagy). This commentary summarises the significance of this relationship and suggests several points for future development.