局部亚低温对大鼠大脑中动脉闭塞后神经元凋亡的影响

目的研究局部亚低温对脑缺血后半胱天冬氨酸酶-3（caspase-3）、细胞凋亡诱导因子（AIF）和微管相关蛋白-2（MAP-2）表达的影响。并探讨亚低温脑保护的机制。方法采用改良的Longa法制备永久性大鼠大脑中动脉闭塞（MCAO）模型。低温组于缺血后立即给予贴敷式局部亚低温（33℃）治疗2h。于缺血后2、6、12、24h及3d、1周和2周取脑。进行HE染色，并采用SABC法进行免疫组化染色，检测大鼠脑缺血区caspase-3、AIF和MAP-2的表达。结果脑缺血后2h，脑梗死病灶周围区即出现caspase-3和AIF免疫阳性细胞，24h达高峰。之后开始逐渐减低，但在2周时仍可见少量免疫阳性细胞。caspase-3在缺血中心区和周围区均有表达，而AIF主要表达于脑梗死周围区。与常温组相比，低温组caspase-3和AIF表达显著减低，而MAP-2显著增加。结论亚低温能抑制caspase-3和AIF表达，促进MAP_2表达。并可抑制caspase介导的细胞凋亡，亦可抑制不依赖于caspase的AIF介导的细胞凋亡。     

亚低温治疗对大鼠脑梗死后Bax和Bcl-2表达的影响

目的探讨亚低温对大鼠急性脑梗死Bax、Bcl-2表达的影响。方法清洁级（SPF）雄性健康SD大鼠,采用线栓法建立局灶性脑梗死模型,分别于缺血3、6、12 h给予亚低温治疗,缺血24 h取材观察。实验分常温组（大鼠10只）、亚低温3、6、12 h组（每组大鼠各10只）,假手术组（大鼠6只）,用TTC染色测定脑梗死体积,用免疫组化的方法检测Bax、Bcl-2的表达水平。结果与常温组比较,亚低温组脑梗死体积明显减少（P〈0.01）、Bcl-2表达上调、Bax表达下调（P〈0.05）。亚低温组脑梗死体积3 h〈6 h〈12 h组,Bcl-2表达3 h〉6 h〉12 h、Bax表达3 h〈6 h〈12 h（P〈0.05）。结论亚低温治疗可能通过抑制Bax和Bcl-2表达,从而抑制神经元凋亡、降低脑梗死体积,脑缺血后越早期给予亚低温治疗效果越好。     

大鼠脑缺血再灌注后Caspase-3、Bcl-2和Bax的表达

目的探讨大鼠脑缺血再灌注后caspase-3、Bcl-2和Bax在脑皮质神经元中的表达。方法将动物随机分为假手术组及缺血组,参照zea longa线栓法建立大鼠左侧大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)局灶性脑缺血再灌注模型,各组大鼠分别在左侧MCAO2h再灌注不同时间点断头取脑,脑皮质神经元中caspase-3、Bcl-2和Bax的表达通过免疫组化法来测定。结果缺血组大鼠脑皮质caspase-3的表达较假手术组显著增强(P<0.01),缺血组大鼠脑皮质Bcl-2的表达较假手术组显著增强(P<0.01),缺血组大鼠脑皮质Bax的表达较假手术组显著增强(P<0.01)。结论短暂性脑缺血再灌注上调脑皮质神经元中caspase-3和Bax的表达促细胞凋亡,上调脑皮质神经元中Bcl-2的表达抗细胞凋亡。     

亚低温疗法联合依达拉奉对大鼠脑缺血再灌注损伤的保护作用

目的探讨亚低温疗法联合依达拉奉对大鼠脑缺血再灌注损伤后的保护作用。方法采用线栓法建立局灶性脑缺血再灌注模型,缺血6h后再灌注,并分为假手术组、模型组、依达拉奉组、亚低温联合依达拉奉组,再灌注24h后进行神经功能缺损评分,采用TTC染色测定脑梗死体积和用免疫组化的方法检测Bax和Bcl-2的阳性细胞数。结果与模型组比较,依达拉奉未能减少脑梗死体积,但能通过下调Bax及上调Bcl-2减少细胞的凋亡,对脑细胞有保护作用;与模型组、依达拉奉组比较,亚低温联合依达拉奉组能通过上调Bcl-2及下调Bax明显降低大鼠神经缺陷评分及减少脑梗死体积。结论亚低温疗法联合依达拉奉对大鼠脑缺血再灌注损伤具有很好的保护作用。     

延迟亚低温治疗对大鼠永久性大脑中动脉阻塞后凋亡相关蛋白表达的影响

目的 研究延迟亚低温治疗对大鼠永久性脑缺血的神经保护作用以及作用机制.方法 线栓法制备永久性大脑中动脉阻塞(pMCAO)模型,随机分为3组,每组10只.亚低温治疗组(HT组)分为HT 2h组和HT 6h组,前者在pMCAO后2h给予亚低温(33±0.5℃)治疗22h,后者在pMCAO后6h给予亚低温(33±0.5℃)治疗18h.缺血对照组(NT组)在pMCAO后放置于室温(25℃).制备pMCAO模型过程中,应用激光多普勒血流监测仪监测局部大脑中动脉供血区脑血流量.持续监测直肠温度.各组大鼠均于pMCAO后24h灌注取脑制备冰冻切片,进行TUNEL染色以及Bcl-2、Bax、Caspase-3免疫组织化学染色.结果 NT组有3只大鼠死亡,亚低温治疗组无大鼠死亡.与NT组比较,HT组皮层缺血半暗带区域凋亡细胞减少,Bcl-2蛋白表达增多,Bax蛋白表达减少,Bcl-2/Bax比值升高,Caspase-3蛋白表达显著减少(P<0.05).与HT 6h组比较,HT 2h组皮层缺血半暗带区域凋亡细胞减少(P<0.05),但Bcl-2、Bax、Caspase-3蛋白表达以及Bcl-2/Bax比值均无显著性差异(P>0.05).结论 永久性脑缺血后延迟6h给予亚低温治疗18h仍然可以减少缺血后神经细胞凋亡.延迟亚低温治疗通过抑制Bcl-2基因家族蛋白介导的线粒体依赖性Caspase激活途径,抑制Caspase介导的细胞凋亡途径,从而发挥神经保护的作用.在脑缺血的治疗过程中,要尽早给予亚低温治疗,以保护更多的神经元和更好地促进神经功能的恢复.     

依达拉奉对局灶性脑缺血再灌注损伤大鼠caspase-3、Bcl-2表达的影响

目的 对局灶性脑缺血再灌注损伤大鼠,给予自由基清除剂依达拉奉后,观察再灌注不同时间点脑组织caspase-3和Bcl-2蛋白表达情况,探讨依达拉奉对脑缺血再灌注损伤的保护作用.方法 制作局灶性脑缺血再灌注模型.随机分为正常组、假手术组、脑缺血组、依达拉奉组.假手术组于术后,脑缺血组和依达拉奉组于缺血1h后再灌注2h、6h、12h、24h、48h不同时间点,依达拉奉组于再灌注后30min腹腔内及皮下各注射依达拉奉1 次(3mg/kg.wt),30min 后重复1次.按时间点处死大鼠,灌注固定、取脑,行免疫组化染色,观察和计数不同脑区的caspase-3和Bcl-2蛋白表达阳性细胞数.结果 脑缺血组再灌注后2h在大脑额、顶叶皮质和海马均可见到caspase-3和Bcl-2阳性细胞,caspase-3表达高峰在12h;Bcl-2表达高峰在6h.依达拉奉组各时间点caspase-3阳性细胞较脑缺血组明显减少,而Bcl-2阳性细胞数明显增加 (均为P＜0.05).结论 依达拉奉可抑制caspase-3,提高Bcl-2的蛋白表达,对脑缺血再灌注损害有明显的保护作用.     

全身亚低温对全脑缺血再灌注大鼠脑保护作用与Bcl-2、Bax表达变化的关系

目的　研究 Bcl- 2、Bax及海马锥体细胞形态在全脑缺血再灌注后的表达及全身亚低温对其表达变化的影响 ,探讨亚低温脑保护作用的机制。方法　采用四血管阻断法制作大鼠全脑缺血再灌注模型 ,分别进行 Bcl-2、Bax免疫组织化学及 H- E染色。结果　与常温组相比 ,全身亚低温组死亡细胞数明显减少 (P<0 .0 1) ;Bcl- 2蛋白免疫反应强度峰值增高 ,持续时间延长 ;Bax蛋白免疫反应强度峰值减低 ,持续时间缩短。结论　 33℃全身亚低温并持续 4 h,对缺血性锥体细胞损害有较好的保护作用。增强 Bcl- 2蛋白的表达 ,延长其表达持续时间 ,而减弱 Bax表达 ,同时缩短其表达持续时间 ,可能是亚低温保护缺血性脑组织损害的分子机制之一。     

亚低温对大鼠急性脑缺血再灌注损伤后脑组织半胱氨酸蛋白酶-12表达及神经元凋亡的影响

目的 观察亚低温对大鼠急性脑缺血再灌注损伤后脑组织半胱氨酸蛋白酶( caspase) - 12表达及神经元凋亡的影响.方法 56只大鼠随机分为正常组、假手术组、缺血组和亚低温组;后两组再分为再灌注3h、6h、12h、24h、72 h及7d6个亚组.应用线栓法制作大鼠大脑中动脉栓塞局灶性脑缺血(2 h)再灌注模型;亚低温组于缺血30 min实施病灶侧头部亚低温4h.各组大鼠在相应时点处死前行神经功能缺损评分(NDS);脑组织切片行HE、免疫组化及原位末端标记法(TUNEL)染色观察病理改变、caspase-12表达及神经元凋亡.结果 正常组及假手术组脑组织均未见明显病理改变及caspase-12和TUNEL阳性细胞.缺血组可见明显的脑梗死灶,大量神经元坏死及消失;亚低温组梗死灶较小,可见神经元固缩.与缺血组比较,亚低温组各时间点亚组NDS明显降低,脑组织caspase-12及TUNEL阳性细胞数明显减少(均P＜0.05).结论 急性脑缺血再灌注损伤后亚低温治疗可抑制脑组织caspase-12表达,减少神经元凋亡及神经功能的损害.     

亚低温对大鼠局灶性脑缺血再灌注后MMP-9表达和细胞凋亡的影响

目的 通过研究亚低温对大鼠局灶性脑缺血再灌注后基质金属蛋白酶-9(MMP-9)表达和细胞凋亡的影响,探讨亚低温脑保护的可能机制.方法 将雄性SD大鼠39只分为假手术组、常温缺血组和缺血期亚低温组.制作大脑中动脉阻塞(MCAO)模型,缺血2h再灌注48h,HE染色观察各组大鼠脑组织形态学改变;采用TTC染色法观察梗死体积;TUNEL法检测细胞凋亡;免疫组化法检测MMP-9表达.结果 亚低温减轻脑缺血组织病理学损伤,明显缩小脑梗死体积(P<0.05).常温下缺血侧脑组织可见大量TUNEL阳性细胞和MMP-9免疫阳性细胞,主要位于皮质缺血半暗带区.亚低温减少脑缺血后TUNEL阳性细胞数目(P<0.05),明显下调MMP-9蛋白表达(P<0.05).结论 亚低温可能通过下调脑缺血再灌注后MMP-9表达,抑制细胞凋亡,从而发挥确实的脑保护作用.     

大鼠脑缺血预处理再灌注模型Bcl-2和Bax的表达

目的研究大鼠脑缺血预处理再灌注模型Bcl-2和Bax的表达。方法采用线栓法建立大鼠脑缺血预处理再灌注模型,缺血预处理后分别再灌注6h、12h、1d、2d、3d、5d和7d,免疫组化染色观察Bcl-2和Bax蛋白表达。结果Bcl-2阳性细胞数于缺血再灌注后第1d明显增加,第3d达到高峰,并维持到第7d,均明显高于对照组(均P<0·01)。不同缺血再灌注时间Bax阳性细胞数与对照组比较差异无显著性(均P>0·05)。结论上调Bcl-2表达可能是缺血预处理产生脑保护作用的机制之一。     

Immunohistochemical study of the distribution of sodium-dependent vitamin C transporters in adult rat brain

Sodium-dependent vitamin C transporters (SVCTs) is known to transport the reduced form of ascorbic acid into the cell, whereas the oxidized form of vitamin C (VC) is moved through a facilitative sugar transporter, such as glucose transporter (GLUT). With regard to the distribution of SVCT1 and -2 within the various organs, they were reported to be expressed in different types of cells. Especially in the central nervous system, only SVCT2 mRNA was expressed mainly in neurons and some types of neuroglial cells. However, data on the expression of SVCT proteins in the brain are scant. Therefore, we tried to develop comprehensive data on the distribution of SVCT proteins in adult rat brain by using immunohistochemical techniques for the first time. In our study, SVCT2 immunoreactivities (IRs) were intensely localized in the neurons of cerebral cortex, hippocampus, and Purkinje cells of cerebellum, and much weaker SVCT2 IRs were found in the other brain regions. Judging from double-immunohistochemical data, most of the cells expressing SVCT2 IRs were likely to be neurons or microglia, even though the cells in choroids plexus or ependymal cells around the ventricles also exhibited SVCT2 IRs. Complete mapping of the distribution of SVCT2 IRs was available by using a semiquantitative method. The subcellular localization of SVCT proteins is necessary for understanding the exact role of the protein, so the current overall mapping of SVCT IRs in the rat brain could be the basis for further studies on related subjects.     

A new case of malonic aciduria with a presymptomatic diagnosis and an early treatment

Malonyl-CoA decarboxylase deficiency (MLYCD) is a rare autosomal recessive inborn error of metabolism presenting a variable clinical phenotype. We report an affected Italian male receiving an early diagnosis (8 days after birth) and a timely dietary therapy (high carbohydrate, low long chain fatty acid and medium chain triglyceride supplemented diet with l-carnitine supplementation). The boy was born at term and presented normal function of the heart (except for a tricuspid Ebstein-like dysplasia) and neurodevelopmental status. Genomic sequencing of MLYCD gene revealed two point mutations (c.672G>A, c.869C>T) not listed in the Human MLYCD Allelic Variant Database nor in Human Gene Mutation Database, responsible for a deleterious effect on protein structure and function according to a computational analysis (MuPro, SIFT, ConSEQ v1.1). At the age of 2 years he only showed a mild language and psychomotor delay, while heart functioning became normal. Brain MRI examination was normal. Thirty-five cases, including our patient, have been described to date. This is the first report concerning a malonic aciduria patient diagnosed on newborn screening and treated in a presymptomatic stage of the disease.     

Homozygous protein C deficiency combined with heterozygous dysplasminogenemia found in a 21-year-old thrombophilic male

A congenital, probably homozygous deficiency of protein C combined with a heterozygous molecular abnormality of plasminogen was found in a 21-year-old male who had been suffering from recurrent venous thrombosis since the age of 14. Although the homozygous deficiency of protein C has been reported to be closely associated with fatal purpura fulminans or severe and massive thrombotic diseases during the neonatal period, the patient had survived the neonatal period without any clinical manifestations relevant to thrombosis. The patient apparently inherited the genetic abnormality of protein C deficiency from both of his parents and that of abnormal plasminogen from his father.     

实验性自身免疫性脑脊髓炎模型的建立和长期观察研究

目的 建立实验性自身免疫性脑脊髓炎小鼠模型(EAE)并长期观察研究.方法 C57BL/6小鼠30只,随机分为EAE模型组、PBS对照组和正常对照组.应用神经功能评分进行临床评估,通过HE和髓鞘染色观察组织病理变化.结果 小鼠在诱导后的12±3d急性起病,16±2d内达到高峰,严重度评分为3.2±0.6分.半年观察期内复发1次,复发率为25%.光镜下可见EAE组以脊髓组织病变为主,表现为大量炎性细胞浸润和白质脱髓鞘.结论 采用MOG_(35-55)诱导C57BL/6小鼠建立的模型既往被认为是一种慢性迁延EAE模型,本研究通过长期观察发现其存在缓解复发现象.

Abstract：

Objective To establish a mice model of experimental autoimmune encephalomyelitis (EAE) and perform a long term study. Methods C57BL/6 mice were immunized with 300μg MOG_(35-55) in complete Freund' s adjuvant (CFA) to establish EAE model in EAE group (n = 10). Mice in adjuvant group( n = 10)were treated with CFA without MOG_(35-55) and control group( n = 10)were treated with normal saline. The pathologic changes of the central nervous system were studied by HE staining and myelin staining. Results The clinic symptoms of EAE were present in the 12±3th day post-immunization,and went to the peek in the 16±2 th day post-immunization. The severity score was 3.2±0.6. One relapse was observed in the term of 6 months, and the rate was 25%. Light microscopy showed there were abundant inflammatory cells infiltrated especially in spinal cord tissues in EAE mice,with evident demyelination in white matter. Conclusion The relapse of this EAE model was observed in the study, though it was believed to be a chronic persistent model without relapse.     

Frontotemporal lobar degeneration and amyotrophic lateral sclerosis: Molecular similarities and differences

In the last years, new disease proteins and genes have been identified in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS), leading to a dramatic shift in our understanding of the molecular mechanisms underlying both conditions. The vast majority of FTLD and ALS are characterized by the abnormal accumulation of TDP-43, including genetic forms associated with mutations in the genes C9ORF72, GRN, TARDBP and VCP. The overlap in pathology and of genetic factors, particularly C9ORF72 as common cause of ALS and FTLD, provides molecular evidence that both conditions represent a spectrum of diseases sharing similar pathomechanisms. Accumulation of the protein FUS defines another subset of FTLD and ALS. However, here some striking differences have been identified. All members of the FET family (FUS, EWS, TAF15) are co-accumulating with their nuclear import receptor Transportin in FTLD-FUS which is usually not associated with FUS mutations, whilst ALS-FUS is almost always associated with FUS mutations and reveals only FUS aggregates. Together with recent data demonstrating differences in the arginine methylation status of FUS in FTLD-FUS and ALS-FUS, these findings strongly imply at least partially distinct underlying disease mechanisms in these molecular subtypes of ALS and FTLD.     

Veno-occlusive disease prophylaxis with fresh frozen plasma and heparin in bone marrow transplantation

Fresh frozen plasma (FFP) contains natural anticoagulants, such as antithrombin (AT) and Protein C (Prot-C). We hypothesized that FFP given in addition to heparin, could potentially replace the consumption of endogenous anticoagulants occurring during conditioning and moreover, corrected AT levels could augment heparin's anticoagulant function. This could therefore result in an effective anti-VOD prophylaxis. In this study, we retrospectively analyzed the incidence of hepatic VOD in 403 consecutive bone marrow transplants (BMTs) comparing 2 prophylactic regimens and no prophylaxis. Patients received no prophylaxis (70/403), heparin-only (27/403) or heparin+2FFP daily during conditioning (306/403). VOD was significantly lower in the heparin+FFP group (5.9%) compared to heparin (20%) and no prophylaxis group (15.7%) [p<0.01]. Day 8 AT and Prot-C levels, were lower in the VOD- compared to the non-VOD group (AT: 69+/-26% vs. 89+/-19%, Prot-C:68+/-26% vs. 91+/-29%, respectively, p=0.001). In a multivariate logistic regression, risk factors for developing VOD were: the administration of >2 hepato-nephrotoxic drugs, previous history of hepatitis B or C and number of BMT. Multivariate analysis in a subset of 198 patients (all having recorded AT, Prot-C), demonstrated as VOD-related factors, the low day 8 Prot-C, number of BMT>1 and prior abdominal radiotherapy. Our study implies that FFP during conditioning, in addition to heparin, potentially has an anti-VOD prophylactic effect, presumably by minimizing the drop of natural anticoagulants around day 8. In order to evaluate if there truely is a beneficial effect of heparin+FFP in VOD prophylaxis, we have initiated a prospective randomized trial.     

<Emphasis Type="Italic">In vitro</Emphasis> study of haematoporphyrin monomethyl ether-mediated sonodynamic effects on C6 glioma cells

OBJECTIVES: To study the cytotoxicity induced by haematoporphyrin monomethyl ether (HMME)-mediated sonodynamic therapy (SDT) on C6 glioma cells. METHODS: The potent photosensitizer HMME was used as the sensitizer. Rat C6 glioma cells were incubated with HMME (10 mug/mL) in the dark for 2 h and then subjected to ultrasound treatment at 1.0 MHz and 0.5 W/cm(2) for 2 min. The growth inhibition rate at different time points after SDT was determined by MTT assay. The apoptotic rate and cell circle profiles were examined with flow cytometry. Fine structures were observed with transmission electron microscope (TEM). The sonodynamic effect on the glioma cells was also studied in the absence or presence of various reactive oxygen species (ROS) scavengers. RESULTS: The growth inhibition rate of C6 glioma cells after SDT significantly increased. SDT also increased the apoptosis and proliferation rate (APR). TEM examination showed the morphological features of apoptosis or necrosis. The addition of NaN(3) showed a strong protective effect again SDT. CONCLUSIONS: Our data indicated that SDT could kill C6 glioma cells in vitro and possibility through induction of apoptosis and necrosis. Singlet oxygen ((1)O(2)) may play an important role in SDT.     

PDGF-B链基因三链形成寡核苷酸对C6胶质瘤细胞增殖和凋亡的影响

目的观察PDGF—B链基因三链形成寡核苷酸(triplex—forming oligonucleotide，TFO)对C6胶质瘤细胞增殖和凋亡的影响。方法应用流式细胞技术观察TFO对C6胶质瘤细胞PDGF—B、PCNA表达的影响，应用流式细胞技术观察TFO对C6胶质瘤细胞凋亡的影响。结果TFO对C6胶质瘤细胞PDGF—B、PCNA的表达有明显抑制作用，而且抑制作用存在浓度依赖性。TFO有明显诱导C6胶质瘤细胞细胞凋亡作用，而且诱导作用存在浓度依赖性。结论TFO抑制C6胶质瘤细胞细胞增殖，同时诱导C6胶质瘤细胞细胞凋亡。