Expression of c-Fos protein and nitricoxide synthase in neurons of cerebral cortex from fetal rats in hypoxia and protective role of Angelica sinensis

BACKGROUND： Both c-Fos protein and nitricoxide synthase （NOS） have been used as general indexes in relative research about neurons, but it is lack of reports that c-Fos protein and NOS are applied synchronously to study the neurons of hypoxic fetal rats in uterus. OBJECTIVE： To study the effect of hypoxia in uterus on the expression of c-Fos protein and NOS in neurons of cerebral cortex from fetal rats and whether Angelica sinensis has the protective effect on these neurons in hypoxia. DESIGN： Randomized control experiment.SETTING ： Department of Histology and Embryology, Luzhou Medical College.MATERIALS ： Twelve adult female Wistar rats in oestrum and 1 male Wistar rat with bodymass from 220 to 250 g were chosen. Parenteral solution of Angelica sinensis mainly contained angelica sinensis, 10 mL/ampoule, was provided by Department of Agent of the Second Hospital Affiliated to Hubei Medical University （batch number： 01062310）. METHODS ： This experiment was completed in the Department of Histology and Embryology of Luzhou Medical College from September 2003 to June 2004. ①Twelve adult female Wistar rats in oestrum and 1 male Wistar rat were housed in one rearing cage. Vaginal embolus was performed on conceive female rat at 8： 00 am next day. On the 15^th conceiving day, all conceiving rats were divided randomly into three groups： control group, hypoxia group and Angelica group with 4 in each group. Rats in hypoxia group and Angelica group were modeled with hypotonic hypoxia in uterus. Angelica group： Rats were injected with 8 mL/kg Angelica sinensis injection through caudal veins before hypoxia. Hypoxia group： Rats were injected with the same volume of saline. Control group： Rats were not modeled and fed with normal way. ② Twenty embryos of rats were chosen randomly from each group and then routinely embedded in paraffin. Paraffin sections were cut from the brain of embryos to anterior fontanelle. Double-label staining was used to detect the expression of nNOS and c-Fos in neurons of cerebral cortex from embryos of rats. OLYMPUS Bx-50 microscope was used to observe sections and DP12 digit camera was also used under 400 times to detect types of cells. Under microscope, the number of c-Fos, NOS, c-Fos/NOS positive neurons in cerebral cortex from embryos of rats were counted in 2 fields with magnification of 400 in one section per animal. ③ The data in experiments were analyzed by one-way analysis of variance （ANOVA） followed by q test. MAIN OUTCOME MEASURES： ① Results of immunohistochemical double-label staining of c-Fos/NOS from cerebral cortex; ② Comparison of amount immunohistochemical double-label staining of c-Fos/NOS positive cells from cerebral cortex. RESULTS：① The positive NOS cells and c-Fos/NOS cells in the three groups were mainly distributed in cerebral cortex, but positive c-Fos neurons were not observed. ② Positive NOS cells and c-Fos/NOS cells in hypoxia group were more than those in control group （76.55±12.02, 50.45±10.39; 33.35±7.42, 26.35±6.67, P 〈 0.05）, but those in Angelica group were less than those in hypoxia group （51.70±9.82, 35.65±8.37, P 〈 0.05）. CONCLUSION： Hypoxia can stimulate the increase of expression of c-Fos protein and NOS in neurons of cerebral cortex. However, Angelica sinensis can decrease this expression so as to play a protective role in cerebral neurons of hypoxic fetal rats.

Effect of polygonatum polysaccharide on the hypoxia-induced apoptosis and necrosis in in vitro cultured cerebral cortical neurons from neonatal rats

BACKGROUND： Cardiocerebrovascular diseases induced cerebral circulation insufficiency and senile vascular dementia can result in ischemic/hypoxic apoptosis of central neurons, which we should pay more attention to and prevent and treat as early as possible. Traditional Chinese medicine possesses the unique advantage in this field. Polygonatum, a Chinese herb for invigorating qi, may play a role against the hypoxic apoptosis of brain neurons. OBJECTIVE ： To observe the protective effect of polygonatum polysaccharide on hypoxia-induced apoptosis and necrosis in cerebral cortical neurons cultured in vitro. DESIGN： A comparative experiment.SETTING： Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine. MATERIALS： The experiment was carried out in the Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine from November 2003 to April 2005. Totally 218 Wistar rats （male or female） of clean degree within 24 hours after birth were purchased from the animal center of Jiangxi Medical College （certification number was 021-97-03）. METHODS：① Preparation of cerebral cortical neurons of rats： The cerebral cortical tissues were isolated from the Wistar rats within 24 hours after birth, and prepared to single cell suspension, and the cerebral cortical neurons of neonatal rats were in vitro cultured in serum free medium with Neurobasal plus B27 Supplement. ② Observation on the non-toxic dosage of polygonatum polysaccharide on neurons： After the neurons were cultured for 4 days, polygonatum polysaccharide of different dosages （1-20 g/L） was added for continuous culture for 48 hours, the toxicity and non-toxic dosage of polygonatum polysaccharide on neurons were observed and detected with trypan blue staining. ③Grouping： After hypoxia/reoxygenation, the cultured neurons were divided into normal control group, positive apoptotic group and polygonatum polysaccharide group. In the normal control group, the neurons were cultured at 37℃ in CO2 with the volume fraction of 0.05 under saturated humidity for 6 days. In the apoptotic positive group, the neurons were cultured with hypoxia for 12 hours after 4-day culture, and followed by reoxygenation for 48 hours. In the polygonatum polysaccharide group, polygonatum polysaccharide with the terminal concentration of 0.5, 1 and 1.5 g/L was added to some neurons at 10 hours before the hypoxia culture, and then the neurons were cultured with hypoxia for 12 hours, followed by reoxygenation for 48 hours; polygonatum polysaccharide with the terminal concentration of 0.5, 1 and 1.5 g/L was added to the other neurons at 12 hours after hypoxia followed by reoxygenation for 48 hours.④ The Hoechst33342 fluorescence staining, Annexin V/PI flow cytometer, appearance of DNA agarose gel electrophoresis gradient strap and immunohistochemical staining were used to observe the expressions of Bcl-2, Bax and Caspase-3 apoptotic and anti-apoptotic proteins and the ratio of Bcl-2/Bax, and observe the effect of polygonatum polysaccharide against the hypoxic apoptosis of cerebral cortical neurons of neonatal rats. MAIN OUTCOME MEASURES： ① Toxicity and non-toxic dosage of polygonatum polysaccharide on neurons;② Apoptotic rate of neurons detected with Hoechst33342 fluorescence staining;③ Early apoptotic rate and necrotic rate of neurons detected with Annexin V/PI flow cytometer; ④DNA agarose gel electrophoresis ladder-like strap appeared or not;⑤ Expressions of Bcl-2, Bax and Caspase-3 apoptotic and anti-apoptotic proteins and the ratio of Bcl-2/Bax. RESULTS：① Polygonatum polysaccharide within 6 g/L had no cytotoxicity on the normal cultured cerebral cortical neurons （P 〉 0.05）. ②The apoptotic rates of neurons detected with Hoechst33342 fluorescence staining had significant differences between the polygonatum polysaccharide groups and positive apoptosis group added to neurons at 10 hours before the hypoxia culture [（13.00±4.52）%,（12.72±2.15）%, （11.80±1.18）%,（38.03±1.05）%, P 〈 0.01], and had no significant differences between the polygonatum polysaccharide groups and positive apoptosis group added to neurons at 12 hours after the hypoxia culture （36.77±1.45）%, （36.60±1.61）%, （36.37±2.02）%, （38.03±1.05）%, P 〉 0.05].③ Annexin V/PI flow cytometer detected that the anti-necrotic effect was enhanced with the increased concentration of polygonatum polysaccharide within 0.5-1.5 g/L （P 〈 0.01）. Polygonatum polysaccharide of 0.5-1.5 g/L added before hypoxia could significantly decrease the apoptotic rate of neurons induced by hypoxia/reoxygenation （P 〈 0.01）. ④ No DNA agarose gel electrophoresis ladder-like strap appeared in the groups with polygonatum polysaccharide of 0.5-1.5 g/L added at 10 hours before hypoxia;⑤ After Polygonatum polysaccharide of 0.5-1.5 g/L was added before hypoxia, the expression of Bcl-2 protein of hypoxic neurons was increased （P 〈 0.01）, and those of Bax protein and Caspase-3 protein were reduced （P 〈 0.01）, and the ratio of Bcl-2/Bax was increased （P 〈 0.01）. CONCLUSION： Polygonatum polysaccharide within 6 g/L has no cytotoxicity on the normal cultured cerebral cortical neurons. Polygonatum polysaccharide of 0.5-1.5 g/L added before hypoxia plays a role agains necrosis of neurons induced by hypoxia. Polygonatum polysaccharide of 0.5-1.5 g/L can significantly reduce the apoptosis of neurons induced by hypoxia through up-regulating the expression of Bcl-2 protein, down-regulating the expressions of Bax protein and Caspase-3 protein, and increasing the ratio of Bcl-2/Bax.

MORPHOLOGICAL STUDY OF FETAL NEOCORTICAL TRANSPLANT GRAFTED TO THE CEREBRAL CORRESPONDING AREA IN YOUNG RATS DIFFERENTIATION OF IMMATURE NEURONS AND RECIPROCAL CONNECTIONS OF FIBERS BETWEEN GRAFT-HOST BRAIN

The fetal neocortical transplant (E15-17 days gestation) of Wistar rat was grafted to the corresponding neocortical region (frontal-parietal lobe) of the same strain in young rats (4-5 weeks old). On the 7th, 15th, 30th, 60th, 150th day after transplantation, the sections cut through the middle area of graft-ost brain were examined by HE, Nissl, Glees stain, immunohistochemical technique for GFAP and NF, Nissl, Glees stain, immunohistochemical technique for GFAP and NF, acetylcholinesterase (AChE) histochemistry as well as horseradish peroxidase (HRP) retrograde tracing with light microscope. Some of the sections were also examined with TEM. The result showed that most immature neurons within the graft can survive, grow, differentiate and mature, and are similar to the structure of the neocortical neurons of host brain. This study also provides patterns of integration of the interface between graft-host brain varying with the proliferation of reactive astrocyte as well as graft-host reciprocal connection of fibers.

Effect of Batroxobin on Expression of Neural Cell Adhesion Molecule in Temporal Infarction Rats and Spatial Learning and Memory Disorder

The effect of Batroxobin expression of neural cell adhesion molecule (NCAM) in left temporal ischemic rats with spatial memory disorder was investigated by means of Morri's water maze and immunohistochemical methods. The results showed that the mean reaction time and distance of temporal ischemic rats for searching a goal were significantly longer than those of sham-operated rats and at the same time NCAM expression of left temporal ischemic region was significantly increased. However, the mean reaction time and distance of Batroxobin-treated rats were shorter and they used normal strategies more often and earlier than those of ischemic rats. The number of NCAM immune reactive cells of Batroxobin-treated rats was more than that of ischemic group. In conclusion, Batroxobin can improve spatial memory disorder of temporal ischemic rats and the regulation of the expression of NCAM is probably related to the neuroprotective mechanism.

Inosine inhibits apoptosis and cytochrome C mRNA expression in rat neurons after cerebral ischemia/reperfusion

BACKGROUND: It has been demonstrated that adenosine can induce glial cell to release cytochrome C, enhance expression of apoptotic gene bax, inhibit anti-apoptotic gene bcl-2, and activate caspase-3 to apoptosis; Whereas inosine can inhibit neuronal apoptosis which is similar to bcl-2. OBJECTIVE: To observe the effects of inosine on neuronal apoptosis and expression of cytochrome C mRNA in rats after focal cerebral ischemia/reperfusion, and analyze the pathway of its neuroprotective effect. DESIGN: A randomised controlled animal trial. SETTINGS: Department of Neurology, Rongcheng Second People's Hospital; Department of Neurology, Affiliated Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. MATERIALS: Sixty-eight rats, weighing 230-280 g and clean grade, were used. TdT-mediated dUTP-biotin nick end labeling (TUNEL) and cytochrome C mRNA in situ hybridization kits and DAB staining kit were purchased from Wuhan Boster Biological Co., Ltd.; Inosine injection [200 mg (2 mL) each] from Qingdao First Pharmaceutical Factory. METHODS: The experiment was accomplished in the animal experimental center in Tongji Medical College of Huazhong University of Science and Technology from December 2003 to June 2005. ① Sixty-four rats were made into focal ischemia by middle cerebral artery occlusion (MCAO) with a nylon monofilament suture. The successfully induced rats were assigned to inosine group (n =32) and model group (n =32) at random. Rats in the inosine group were intraperitoneally administrated with inosine in dose of 100 mg/kg preoperatively, twice a day, 7 days in all. The rats in the control group were injected with the same dose of saline solution by the similar way preoperatively. Each group was randomized into ischemia /reperfusion 2, 6, 12, 24 hours, 2, 3, 7 and 14 days subgroups consisted of 4 rats. The other 4 rats were taken as the sham-operated group, the rats were given the same treatment except for not introduced the filament into the external carotid artery stump, and brain tissue was removed at 2 hours of reperfusion. ② In situ hybridization was performed to examine the expression of cytochrome C mRNA while TUNEL staining was made to characterize apoptosis. ③ The t test was used to compare the difference of measurement data. MAIN OUTCOME MEASURES: ① Neuronal apoptosis in the different regions of the ischemic brain tissue; ② Expression of cytochrome C mRNA in the different regions at different time points after MCAO. RESULTS: All the 68 rats were involved in the analysis of results. ① Neuronal apoptosis: A small number of TUNEL-positive cells were detected in the sham-operated brain and non-ischemic brain. The number of apoptotic cells in the ischemic cortex peaked at 24 hours of reperfusion [(72.00±1.98) cells] and that in the striatum peaked at 2 days [(94.75±3.57) cells], then decreased to the level of sham-operated group at 14 days. Inosine could reduce apoptotic cells from 12 hours to 7 days of reperfusion as compared with the model group (t =6.19-26.67, P < 0.01). ② Cytochrome C mRNA expression: There was weak expression of cytochrome C mRNA in both sham-operated brain and contralateral brain. Cytochrome C was detected at 2 hours of reperfusion in ischemic brain [(25.75±3.50), (39.75±2.49) cells], and strongly increased to a peak at 12 hours and 24 hours of reperfusion in cortex and striatum [(122.50±6.69), (119.25±5.12) cells], respectively. Furthermore, inosine could significantly decrease cytochrome C expression in cortex at 12 hours to 14 days of reperfusion after ischemic reperfusion and that in striatum at 12 hours to 3 days (t =8.67-43.26, P < 0.01). CONCLUSION: Inosine can exert a neuroprotective effect by inhibiting apoptosis and cytochrome C mRNA expression.

BNIP3L介导的线粒体自噬在镉致大鼠大脑皮质神经元凋亡中的作用

为探究线粒体自噬受体Bcl-2/腺病毒E1B 19 kDa相互作用蛋白3样(BNIP3L)介导的线粒体自噬对镉致大鼠大脑皮质神经元凋亡的调控作用,利用RNA干扰技术建立大鼠大脑皮质神经元BNIP3L基因沉默模型,并经10μmol/L镉处理12 h,Western blot检测BNIP3L、帕金森氏蛋白2(Parkin)和线粒体凋亡通路相关蛋白cleaved caspase-9、cleaved caspase-3的表达水平,免疫荧光染色检测BNIP3L与线粒体标志蛋白TOMM20共定位,透射电镜检测细胞内线粒体自噬体数目,FITC Annexin V染色检测细胞凋亡水平。结果:大鼠大脑皮质神经元经镉处理12 h后,BNIP3L蛋白表达水平极显著升高(P<0.01),BNIP3L与TOMM20共定位增加,沉默BNIP3L极显著抑制镉致Parkin线粒体转位(P<0.01),抑制镉致细胞内线粒体自噬体形成,极显著促进镉致caspase-9、caspase-3激活和神经元凋亡(P<0.01)。结果表明,BNIP3L介导的线粒体自噬可抑制镉致大鼠大脑皮质神经元凋亡。

Role of chloride channels in nitric oxide-induced rat hippocampal neuronal apoptosis in vitro

BACKGROUND：Chloride channels participate in non-neuronal apoptosis.However,it remains unclear whether chloride channels are involved in ischemic neuronal apoptosis.OBJECTIVE：To explore the effects of 4-acetamido-4＇-isothiocyanatostilbene-2,2＇-disulfonic acid （SITS） and 4,4＇-diisothiocyanostilbene-2,2＇-disulfonic acid （DIDS）,two chloride channel blockers,on the hippocampal neuronal apoptosis induced by 3-morpholinosydnonimine （SIN-1） based on the nitric oxide toxicity theory of neuronal apoptosis following ischemic brain injury.DESIGN,TIME AND SETTING：Comparative observation and in vitro experiments were performed at the laboratory of Zhuhai Campus of Zunyi Medical College from January to May 2009.MATERIALS：SIN-1,SITS,and DIDS were purchased from Sigma,USA.METHODS：Hippocampal neurons from Sprague-Dawley rats,aged 1 day,were cultured In vitro for 12 days and randomly assigned to control,SIN-1,or chloride channel blocker groups.SIN-1 group neurons were induced by SIN-1 for 18 hours to establish a model of ischemic neuronal apoptosis.Neurons in chloride channel blocker groups were treated with SITS or DIDS plus SIN-1 for 18 hours.The controls were cultured in DMEM/Ham＇s F12 complete medium alone.MAIN OUTCOME MEASURES：The apoptotic neurons and nuclear appearance were detected by Hoechst 33258 fluorescence staining; neuronal viability was quantitatively determined by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide analysis.Caspase-3 activity was analyzed by Western blot.RESULTS：SIN-1 （1 mmol/L） dramatically induced apoptosis （50%-60%）.SITS and DIDS inhibited nitric oxide-induced neuronal injury in a dose-dependent manner,suppressed caspase-3 activation,reduced neuronal apoptosis,and improved neuronal survival.CONCLUSION：Chloride channel blockers can protect against neuronal injury induced by NO.Chloride channels might be involved in neuronal apoptosis following cerebral ischemia.

Cadmium Activates Reactive Oxygen Species-dependent AKT/mT OR and Mitochondrial Apoptotic Pathways in Neuronal Cells

Objective To examine the role of Cd-induced reactive oxygen species（ROS） generation in the apoptosis of neuronal cells. Methods Neuronal cells（primary rat cerebral cortical neurons and PC12 cells） were incubated with or without Cd post-pretreatment with rapamycin（Rap） or N-acetyl-L-cysteine（NAC）. Cell viability was determined by MTT assay, apoptosis was examined using flow cytometry and fluorescence microscopy, and the activation of phosphoinositide 3’-kinase/protein kinase B（Akt）/mammalian target of rapamycin（m TOR） and mitochondrial apoptotic pathways were measured by western blotting or immunofluorescence assays. Results Cd-induced activation of Akt/m TOR signaling, including Akt, m TOR, p70 S6 kinase（p70 S6K）, and eukaryotic initiation factor 4E binding protein 1（4E-BP1）. Rap, an m TOR inhibitor and NAC, a ROS scavenger, blocked Cd-induced activation of Akt/m TOR signaling and apoptosis of neuronal cells. Furthermore, NAC blocked the decrease of B-cell lymphoma 2/Bcl-2 associated X protein（Bcl-2/Bax） ratio, release of cytochrome c, cleavage of caspase-3 and poly（ADP-ribose） polymerase（PARP）, and nuclear translocation of apoptosis-inducing factor（AIF） and endonuclease G（Endo G）. Conclusion Cd-induced ROS generation activates Akt/m TOR and mitochondrial pathways, leading to apoptosis of neuronal cells. Our findings suggest that m TOR inhibitors or antioxidants have potential for preventing Cd-induced neurodegenerative diseases.

抑制环氧合酶⁃2表达对大鼠脑缺血/再灌注损伤后神经元凋亡的影响

目的探讨大鼠脑缺血/再灌注(CI/R)损伤后环氧合酶-2(COX-2)引起神经元凋亡的作用机制。方法45只雄性SD大鼠,随机数字法分为3组:假手术组(sham)、模型组(CI/R)和COX-2抑制剂组(NS-398)。阻塞大脑中动脉建立模型,缺血开始时,NS-398组经腹腔注射NS-398(20 mg/kg),假手术组和模型组注射等量的DMSO。缺血2 h时对大鼠进行神经功能学评分,再灌注24 h时,2,3,5-氯化三苯四氮唑(TTC)染色检测大鼠脑梗死体积,同时取缺血侧额顶叶皮质半暗带区脑组织,Nissl染色检测神经元的损伤,TUNEL法检测神经元的凋亡,Western blotting检测COX-2、Bax和Bcl-2蛋白的表达水平。结果CI/R组大鼠神经功能学评分、脑梗死灶体积、凋亡指数、COX-2和Bax蛋白表达水平均高于假手术组(P<0.05),而Bcl-2蛋白表达水平和神经元数目均低于假手术组(P<0.05);NS-398组大鼠神经功能学评分、脑梗死灶体积、凋亡指数、COX-2和Bax蛋白表达水平均低于CI/R组(P<0.05),同时,Bcl-2蛋白表达水平和神经元数目均高于CI/R组(P<0.05)。结论COX-2可能通过调控Bcl-2和Bax的表达促进脑缺血/再灌注损伤后神经元的凋亡。

巴豆霜干预脑缺血再灌注损伤大鼠皮质区JNK/p38 MAPK及神经元凋亡的机制

背景:巴豆霜能够激活ERK通路、对神经元细胞具有抗凋亡作用,是否具有抑制JNK、p38通路的激活而发挥抗凋亡的协同效应尚不清楚。目的:探讨巴豆霜对脑缺血再灌注损伤大鼠缺血侧皮质区神经元损伤和凋亡的影响及机制。方法:①将90只SD大鼠随机分为假手术组,模型组,巴豆霜低、中、高剂量组,尼莫地平组,每组15只,除假手术组外,剩余各组均采取线栓法制备大脑中动脉栓塞大鼠模型,巴豆霜各组大鼠分别按剂量20,40,60 mg/kg灌胃;假手术组和模型组大鼠给予等量生理盐水,1次/d,连续给药7 d。运用神经功能缺损评分、TTC染色、脑组织含水量、苏木精-伊红染色及尼氏染色筛选出最佳浓度即巴豆霜高剂量。②将120只SD大鼠随机分为假手术组、模型组、巴豆霜组、JNK抑制剂组、巴豆霜+JNK抑制剂组、p38 MAPK抑制剂组、巴豆霜+p38 MAPK抑制剂组和尼莫地平组,每组15只,除假手术组外,剩余各组大鼠均制备大脑中动脉栓塞大鼠模型,在造模之前30 min,将10μL JNK抑制剂SP600125或10μL p38 MAPK抑制剂SB203580分别注射到大鼠侧脑室,巴豆霜各组大鼠灌胃60 mg/kg巴豆霜,7 d后,采用Western Blot、TUNEL染色及流式细胞术检测各组大鼠脑组织JNK/p38 MAPK信号通路及凋亡相关蛋白水平及细胞凋亡情况。结果与结论:①与假手术组相比,模型组神经功能缺损评分、脑含水量、脑梗死体积、细胞凋亡率显著升高(P<0.05),神经细胞呈散乱分布;与模型组相比,巴豆霜中、高剂量组和尼莫地平组大鼠神经功能缺损评分、脑组织含水量、脑梗死体积显著降低(P<0.05),神经细胞病理形态明显改善;②与JNK抑制剂组相比,巴豆霜+抑制剂组大鼠脑组织p-JNK/JNK、p-p38/p38、Bax表达显著降低(P<0.05),细胞凋亡率显著降低(P<0.05),而Bcl-2表达显著升高(P<0.05);③结果提示,巴豆霜可能通过抑制JNK/p38 MAPK信号通路的激活、减少神经元凋亡等途径,达到对脑缺血再灌注损伤大鼠的神经保护作用。

虎杖苷对β淀粉样蛋白诱导体外皮质神经元线粒体氧化应激和功能障碍的保护作用

目的探讨虎杖苷对β淀粉样蛋白25~35(Aβ_(25~35))诱导的原代培养大鼠皮质神经元损伤的保护作用及可能的作用机制。方法原代培养SD大鼠皮质神经元,分为对照组、虎杖苷组、Aβ_(25~35)组和联合组(Aβ_(25~35)+虎杖苷)。采用四甲基偶氮唑盐检测皮质神经元活力,2,7-二氯荧光素二乙酸酯探针或红色线粒体超氧化物荧光探针染色检测细胞内和线粒体活性氧水平,线粒体膜通透性转换孔(MPTP)试剂盒检测MPTP开放程度,蛋白免疫印记法检测细胞色素C及线粒体转录因子A(TFAM)表达。另外,测定细胞内电子传递链复合物(包括复合物Ⅰ、Ⅱ、Ⅲ、Ⅳ)活性和三磷酸腺苷(ATP)水平。采用高效液相色谱法测定线粒体中8-羟基脱氧鸟苷(8-OHdG)。结果与对照组比较,Aβ_(25~35)组皮质神经元细胞活力、线粒体荧光强度、线粒体呼吸链酶活性(复合物Ⅰ、Ⅱ、Ⅲ、Ⅳ)、细胞内ATP、线粒体内TFAM表达明显降低,差异有统计学意义(P<0.05,P<0.01);与Aβ_(25~35)组比较,虎杖苷组线粒体荧光强度、线粒体呼吸链酶活性(复合物Ⅰ、Ⅱ、Ⅲ、Ⅳ)、细胞内ATP、线粒体内TFAM表达明显增高,皮质神经元暴露3、6、12、24h细胞内和线粒体内活性氧明显降低(P<0.05,P<0.01),联合组细胞色素C细胞质/线粒体比值、线粒体内8-OHdG水平明显低于Aβ_(25~35)组[3.02±0.28 vs 5.73±0.45,P<0.05;(8.07±1.45)×10^(6)dG vs(16.07±2.29)×10^(6)dG,P<0.05]。结论虎杖苷可有效地保护皮质神经元免受Aβ_(25~35)诱导的损伤,至少部分作用是通过抑制线粒体氧化应激和改善线粒体功能实现。

缺血性脑卒中SD大鼠血清、脑脊液中NSE、MBP水平及其与神经功能缺损评分和脑梗死体积的相关性

目的探讨缺血性脑卒中SD大鼠血清、脑脊液中神经元特异性烯醇化酶(NSE)、髓鞘碱性蛋白(MBP)水平及其与神经功能缺损评分和脑梗死体积的相关性。方法购买SD大鼠进行大脑中动脉闭塞缺血模型造模手术,将术后成功造模的大鼠随机分为8组,每组8只,术后8 h进行神经功能缺损评分,术后在8 h、12 h、1 d、3 d、5 d、7 d及21 d时间段采集腹腔静脉血及抽取延髓脑脊液后,断头取脑,进行0.4%的2,3,5-氯化三苯基四氮唑大脑染色测定脑梗死体积,并检测NSE及MBP水平。采用Spearman相关对血清、脑脊液NSE、MBP水平与脑梗死体积和神经功能缺损评分的相关性进行分析。结果各组血清、脑脊液中NSE和MBP水平在术后8 h开始升高,3 d达峰值,7 d下降至与对照比较差异无统计意义(P>0.05);术后8 h至5 d,脑脊液NSE和MBP水平明显高于血清。血清、脑脊液NSE、MBP水平与脑梗死体积、神经功能缺损评分均呈正相关(P<0.05)。结论血清、脑脊液中NSE、MBP为脑梗死的新型标志物,通过动物实验可为临床患者标本采集推荐最佳时间窗口。

大麻二酚对大鼠多重脑震荡炎症反应的抑制作用

目的探究大麻二酚(CBD)抑制大鼠多重脑震荡神经炎症、保护神经组织的机制。方法制备大鼠多重脑震荡模型(MCC),分别采用HE、免疫组化染色检测多重脑震荡后神经元和小胶质细胞的变化及大麻二酚的保护作用。应用Western blot检测不同脑区Iba-1(小胶质细胞标记物)及炎症因子IL-1β、TNFα的表达。结果HE染色显示,较之Sham组,大鼠MCC后神经元出现明显病理改变;CBD干预后神经元形态趋于正常,高剂量组更显著。免疫组化显示,与Sham组呈静息状态的小胶质细胞相比,MCC后数量增加呈激活状态,CBD干预后小胶质细胞向静息态恢复,高剂量组更明显。Western blot结果显示:大鼠MCC后Iba-1、IL-1β、TNFα蛋白表达显著升高(P<0.05);CBD干预可下调不同脑区Iba-1、IL-1β和TNFα的表达(P<0.05)。结论大麻二酚可减轻多重脑震荡大鼠皮质及海马的炎症反应,发挥神经保护作用。

脑震宁颗粒调节三重脑震荡大鼠线粒体能量代谢的机制研究

目的探究脑震宁颗粒调节三重脑震荡(MCC)模型大鼠海马组织线粒体能量代谢的作用机制。方法取SPF级Wistar大鼠,通过“自由落体撞击法”制备MCC模型。将造模成功的大鼠分为模型组、吡拉西坦组和脑震宁颗粒低、中、高剂量组,另设正常组,每组8只。各给药组大鼠按吡拉西坦组0.324 g/kg,脑震宁颗粒低、中、高剂量组2.25、4.5、9 g/kg的剂量灌服相应药物,正常组和模型组给予等体积生理盐水,每天1次,连续14 d。检测大鼠运动探索能力和学习记忆能力;检测大鼠海马组织三磷酸腺苷(ATP)含量;观察大鼠海马组织线粒体结构变化;检测大鼠海马组织线粒体动力蛋白相关蛋白1(Drp1)、线粒体裂变蛋白1(Fis1)、线粒体融合蛋白1(Mfn1)、视神经萎缩蛋白1(Opa1)的荧光强度;检测大鼠海马组织过氧化物酶体增殖物激活受体γ共激活因子1α(PGC-1α)、核呼吸因子1(NRF-1)、线粒体转录因子A(TFAM)、Wnt-3a、β-连环蛋白(β-catenin)的蛋白表达水平。结果与正常组比较,模型组大鼠的运动总路程、中央格进入次数、直立次数,新物体识别指数,海马组织线粒体ATP含量,Mfn1、Opa1的荧光强度及PGC-1α、NRF-1、TFAM、Wnt-3a、β-catenin蛋白表达水平均显著降低(P<0.01),静止时间和海马组织Drp1、Fis1的荧光强度均显著升高(P<0.01);透射电镜结果显示,海马组织线粒体明显肿胀,嵴大量断裂、减少,部分线粒体膜内髓样变。与模型组比较,各给药组大鼠以上指标水平/含量均有不同程度的逆转,大部分差异有统计学意义(P<0.05或P<0.01);透射电镜结果显示,海马组织线粒体肿胀程度减轻,嵴少量断裂、减少,粗面内质网局部区域模糊断裂。结论脑震宁颗粒可以提高MCC模型大鼠的运动探索和学习记忆能力,修复神经元损伤,发挥神经保护作用;其机制可能与激活Wnt/β-catenin信号通路,维持线粒体融合分裂平衡,促进线粒体生物合成相关。

大麻二酚对多重脑震荡大鼠NLRP3炎性小体表达的影响

目的:探究大麻二酚(CBD)对多重脑震荡(MCC)大鼠NOD样受体热蛋白结构域相关蛋白3(NLRP3)炎性小体表达的影响。方法:制备大鼠多重脑震荡模型,分为Sham组、MCC组、溶剂组(MCC+TW)、CBD-L组(10 mg/kg)及CBD-H组(40 mg/kg)。应用免疫荧光双标染色法观测脑内NLRP3与小胶质细胞的变化,并用Western Blot检测NLRP3炎性小体的表达变化。结果:免疫荧光双标染色显示,MCC后皮质区大量lectin阳性小胶质细胞激活,胞体增大,小胶质细胞中NLRP3的免疫荧光强度明显升高(P<0.05);给予CBD可下调激活的小胶质细胞内NLRP3的表达,且CBD-H组较CBD-L组效果更明显(P<0.05)。Western Blot显示,大鼠MCC后皮质、海马及基底节中NLRP3、半胱氨酸天冬氨酸蛋白酶-1(caspase-1)和凋亡相关斑点样蛋白(ASC)的表达水平显著升高(P<0.05),且皮质区升高最明显;CBD-L组和CBD-H组中上述蛋白表达下降(P<0.05)。结论:大麻二酚可抑制多重脑震荡大鼠脑内NLRP3炎性小体表达,发挥抗炎保护作用。

大鼠实验性脑震荡的病变规律和神经细胞凋亡研究

采用Wistar大鼠复制脑震荡动物模型 ,于伤后 1、3、7、14及 30天活杀取脑组织 ,经光镜、电镜和原位末端标记等技术研究脑震荡的病变规律及神经细胞凋亡情况。结果显示 ,10 0g砝码于 1m处撞击大鼠头部可复制大鼠脑震荡的模型 ,其基本病变为脑血管扩张、淤血、出血、脑组织水肿 ,神经元变性、凋亡和坏死 ,尼氏体减少甚至消失。伤后 1～3天 ,脑组织呈点灶状坏死 ,周围组织疏松 ,单核及泡沫样细胞增多 ,神经元变性、凋亡和坏死。 7天脑水肿达高峰 ,海马锥体细胞减少 ,呈极度缺血性改变。 14～ 30天仍见血管扩张、淤血 ,可见出血 ,水肿好转。原位末端标记显示 ,伤后 1天见凋亡细胞增多 ,3天数量达高峰 ,伤后 30天凋亡的神经元持续存在。结果提示 ,脑震荡以血液循环障碍和实质细胞变性、凋亡和坏死为主要病理改变 ;神经细胞凋亡是脑震荡神经元迟发性损害的主要形式之一。

三重脑震荡鼠模型建立及组织病理学动态改变观察

目的建立三重脑震荡（MCC）大鼠模型，观察MCC鼠不同结构神经元及轴索的病理学变化，探索运动员多次脑震荡损伤机制。方法用金属单摆闭合性脑损伤打击装置复制大鼠MCC模型48只，随机分为1、2、4、8、16和24d组（n=8）。另设正常对照组（n=8）。定时戊巴比妥钠麻醉，4％多聚甲醛心腔灌注处死，取脑。用Nissl染色、Bielschowsky银染和Weil髓鞘染色显示神经元、轴索和髓鞘，对固缩变性神经元数目、轴索直径和髓鞘变性进行半定量分析。结果伤后：①1～24d皮层、海马和脑干网状结构神经元不同程度固缩变性，以2d组为重；②轴索不同程度的肿胀、变形、排列紊乱、收缩球形成；轴索直径平均值均大于对照组，以2、4d组最为明显（P〈0．05）；③髓鞘肿胀、淡染，灰度值增高，与对照组相比，1～16d组均有统计学意义（P〈0．05）。结论大鼠MCC后，脑神经元出现不同程度弥漫性的固缩变性，轴索肿胀、髓鞘淡染的病理改变。

川芎嗪对大鼠局灶性脑缺血损伤的神经保护作用

目的观察川芎嗪对局灶性脑缺血后脑损伤的保护作用。方法采用线栓法制作大鼠左侧大脑中动脉阻塞模型。氯化三苯基四氮唑(TTC)脑片染色测定脑梗死体积,干湿重法测定脑组织含水量,快速Golgi银染方法观察脑缺血周围区神经元的形态改变。结果川芎嗪能明显缩小脑梗死体积、降低脑组织含水量,随着川芎嗪剂量增大,作用更为明显,具有剂量依赖性。脑缺血后14 d Golgi银染显示,模型组在梗死周围区神经元明显减少,变性和正常神经元共存。变性神经元主要表现为突起断裂、增粗,突起有大的串珠,树突棘减少。川芎嗪组较模型组皮质梗死周围神经元变性较少。结论川芎嗪能缩小脑梗死体积、减轻脑水肿、保护缺血周围神经元,证实川芎嗪对脑缺血损伤有保护作用。

多重脑震荡大鼠脑神经元病理变化的研究

为探讨多重脑震荡(multiple cerebral concussion,MCC)后脑损伤的累加效应机制,本实验首次应用自制单摆式机械打击装置复制MCC大鼠模型,研究伤后大鼠脑神经元的组织病理学变化。将56只大鼠随机分为7组:对照组、伤后1、2、4、8、16和24d组(n=8),分别用Nissl染色显示神经元,并对变性神经元进行计数。结果显示:MCC后1d,大鼠大脑皮层、海马、齿状回及脑干网状结构变性神经元增加,伤后2d达到高峰,4～8d减轻,8～16d又有回升,24d基本恢复至正常水平。以上结果提示,多重脑震荡后,不同脑区的脑神经元可出现早期及迟发性的神经元变性,此病理变化可能与多次脑损伤的累积性加重有关。

胚鼠大脑皮质神经元的最佳分离和培养方法的探讨

用不同类型酶消化法及单纯机械法分离胚鼠大脑皮质 ,并分别采用两种不同的培养基进行神经元的体外培养以建立胚鼠脑皮质神经元的最佳分离和培养方法。结果表明 :采用 0 .0 5 %胰蛋白酶 +0 .0 5 % II型胶原酶消化 ,以神经元完全培养基作为培养基质可获得高纯度。