丁苯酞对血管性痴呆大鼠认知功能、海马神经元结构及nNOS的影响

目的研究丁苯酞对血管性痴呆（VD）大鼠认知功能、海马神经元结构、及神经元型一氧化氮合酶（nNOS）的影响。方法采用结扎双侧颈总动脉方法制备慢性前脑缺血动物模型,100只老龄大鼠随机分5组,应用水迷宫、透射电镜及免疫组化方法对各组大鼠学习记忆、神经元结构、nNOS表达进行观察。结果与假手术组比较,大鼠学习记忆能力在造模后差异有显著性意义（P〈0.05）;大鼠海马区神经元在造模后变性水肿明显,与模型组比较,丁苯酞不同剂量治疗1月后,大鼠学习记忆能力明显改善（P〈0.05）,神经元变性水肿减轻,nNOS阳性神经元表达减少（P〈0.05）。结论丁苯酞能显著改善VD大鼠学习记忆能力;减轻神经元变性水肿;抑制nNOS阳性神经元表达。     

丁苯酞对大鼠脑缺血再灌注损伤后HSP10表达的影响

目的研究丁苯酞干预对大鼠脑缺血再灌注损伤后不同时点脑组织中热休克蛋白10(HSP10)表达的影响,并探讨丁苯酞对缺血性脑血管病保护作用的机制。方法采用夹闭双侧颈总动脉的方法制作大鼠前脑缺血再灌注模型;H&E染色和NSE免疫组化染色神经元,观察脑组织的形态变化和记数各组神经元数目;免疫组织化学检测对照组、缺血再灌注组及丁苯酞干预组大鼠脑组织HSP10的表达水平变化。结果脑缺血再灌注后丁苯酞干预组及缺血再灌注组HSP10表达水平于再灌注后6h开始上调,3d达到高峰,丁苯酞干预组各时间点HSP10阳性表达指数均高于缺血再灌注组,丁苯酞干预组脑组织的损伤程度明显轻于脑缺血再灌注组(P<0.05)。结论丁苯酞可能通过上调大鼠缺血再灌注损伤后脑组织HSP10的表达,从而抑制前脑缺血再灌注损伤后细胞凋亡,减少神经元死亡,减轻缺血再灌注后脑组织的损伤。     

金纳多对血管性痴呆大鼠认知功能及生长抑素表达的影响

目的研究金纳多对血管性痴呆(vascular dementia,VD)大鼠认知功能及生长抑素(SS)表达的影响。方法采用结扎双侧颈总动脉方法制备慢性前脑缺血动物模型。将100只老龄大鼠随机分为假手术组、模型组和金纳多组。应用水迷宫及免疫组化方法对各组大鼠学习记忆及SS表达进行观察。结果模型组与假手术组学习记忆能力差异有统计学意义(P<0.05);与模型组比较,金纳多治疗30 d后大鼠学习记忆能力明显改善(P<0.05),SS阳性神经元表达增加(P<0.05)。结论金纳多可增加SS阳性神经元表达,改善VD大鼠学习记忆能力。     

丁苯酞对帕金森病大鼠模型的疗效研究

目的研究丁苯酞对帕金森病(PD)大鼠行为学、中脑氧化应激水平及黑质多巴胺能神经元的影响并探讨其可能的疗效。方法采用鱼藤酮立体定向注射方法制作偏侧PD大鼠模型。将造模成功的大鼠随机分为按体质量丁苯酞20、40、80 mg/kg 3个治疗组及对照组,观察各组大鼠经丁苯酞干预前后旋转行为情况,采用分光光度计检测中脑还原型谷胱甘肽(GSH)、丙二醛(MDA)水平及超氧化物歧化酶(SOD)活性,另用免疫组化染色测定黑质酪氨酸羟化酶(TH)阳性细胞数量。结果 (1)与治疗前相比,按体质量丁苯酞80 mg/kg治疗组治疗后旋转圈数明显减少(P0.05);(2)与对照组损毁侧相比,各治疗组损毁侧中脑GSH水平增高,MDA水平降低,SOD活性增强(P0.05,P0.01);(3)与对照组损毁侧相比,按体质量丁苯酞80 mg/kg治疗组损毁侧黑质TH阳性细胞计数增多(P0.05)。结论丁苯酞可显著改善PD大鼠旋转行为,增强抗氧化能力,提高黑质多巴胺能神经元残存率,提示其可能具有治疗和保护作用。     

丁苯酞对缺血大鼠大脑皮质中钙通道Cav1.3表达的影响

目的探讨丁苯酞对脑缺血大鼠大脑皮质中钙通道Cav1.3表达的影响及其脑保护作用的机制。方法雄性Wistar大鼠36只,随机分为假手术组、缺血对照组和丁苯酞治疗组,每组12只。采用免疫组织化学方法和蛋白质印迹法分别检测各组大鼠大脑皮质中钙通道Cav1.3表达的情况。采用TUNEL法观察大鼠大脑皮质内细胞凋亡的情况。结果与缺血对照组比较,丁苯酞治疗组大鼠大脑皮质中钙通道Cav1.3表达有所增加(P0.05),但仍低于假手术组(P0.05),大脑皮质中凋亡神经元数量丁苯酞治疗组明显少于缺血对照组但多于假手术组(P0.05)。结论丁苯酞可以抑制缺血造成的脑皮质中钙通道Cav1.3表达的减少,减少缺血后大脑皮质中的神经元凋亡。     

血管性痴呆大鼠认知功能及nNOS的表达

目的 探讨血管性痴呆(VD)大鼠认知功能、海马神经元结构及nNOS的表达.方法 采用双侧颈总动脉结扎法制备慢性前脑缺血动物模型,40只老龄大鼠随机分为假手术组(S)、模型组(M).应用水迷宫、透射电镜及免疫组化方法对2组大鼠学习记忆、神经元结构、nNOS表达进行观察.结果 与假手术组比较,大鼠水迷宫学习记忆能力在造模2个月后差异有统计学意义(P＜0.05),大鼠海马神经元在造模后变性水肿明显,大鼠海马及颞叶皮层nNOS在造模2个月后表达增加 (P＜0.05).结论 海马及颞叶皮层nNOS表达增加,神经元变性,可能导致血管性痴呆大鼠学习记忆障碍.     

丁苯酞对大鼠脑缺血再灌注损伤后的Fas／FasL蛋白表达的影响

目的研究大鼠脑缺血再灌注损伤后Fas和FasI。蛋白的表达和丁笨酞保护作用的机制。方法线栓法制备大鼠大脑中动脉缺血再灌注模型,SD大鼠随机分为正常对照组、假手术组、缺血再灌注组和丁苯酞组;应用尼氏染色显示存活神经元,末端转移酶介导的缺口末端标记（TUNEI。）方法检测程序化死亡细胞;免疫组织化学法观察脑缺血再灌注后6h、1d、3d、7d四个时间点脑组织中Fas和FasI．蛋白的表达变化。结果丁苯酞组皮层存活神经元数量多于缺血再灌注组,TUNEI．阳性细胞数减少,丁苯酞组在再灌注后各时间点的Fas和FasL阳性细胞数较缺血再灌注组少,差异均有统计学意义。结论丁苯酞可能通过抑制Fas／FasL蛋白的表达,从而减少神经元凋亡,减轻缺血再灌注后脑组织的损伤。     

丁苯酞氯化钠注射液对大鼠延髓缺血后神经元凋亡、NGF、BDNF表达的影响

目的探索丁苯酞氯化钠注射液对大鼠延髓缺血后脑神经元凋亡、神经生长因子(NGF)、脑源性神经生长因子(BDNF)表达的影响。方法将Wistar大鼠分为假手术组、丁苯酞氯化钠注射液治疗组(以下简称治疗组)、缺血对照组。采用电凝双侧椎动脉和结扎右侧颈总动脉的方法制造大鼠延髓缺血模型。分别给予治疗组和缺血对照组丁苯酞氯化钠注射液及等计量生理盐水腹腔注射。大鼠于相应时段灌注取材,每组标本分别采用尼氏染色、Tunel染色,免疫组织化学方法显示脑组织中的神经元、凋亡神经元数量、BDNF和NGF。光镜下观察脑组织中神经元数量、神经元凋亡的数量。同时观察NGF和BDNF的变化,利用灰度值进行分析。结果神经元凋亡数量及NGF、BDNF灰度值治疗组均低于缺血对照组。结论 NGF、BDNF水平与神经元数量密切相关,丁苯酞可上调大鼠缺血脑组织中NGF、BDNF的表达,减轻脑组织损伤。     

尼莫地平对脑缺血早期神经元型一氧化氮合酶的影响

目的观察局灶性脑缺血大鼠早期缺血部位神经元型一氧化氮合酶(neuronal nitric oxide synthase,nNOS)的变化,以及钙拮抗剂尼莫地平对其的影响。方法以线栓法制作大鼠大脑中动脉闭塞模型为基础,选取雄性SD大鼠共75只,随机分成假手术组(n=25)、模型对照组(n=25)和尼莫地平处理组(n=25)。以NDAPH-d组织化学方法观测各组大鼠脑缺血后30min、2h和6h纹状体中nNOS阳性细胞的变化;利用生物化学法和Hoechst凋亡染色分别检测各组大鼠脑缺血30min纹状体nNOS活性和6h半暗区细胞凋亡。结果脑缺血30min,与假手术组相比,模型对照组大鼠缺血侧纹状体中nNOS活性和nNOS阳性细胞数均升高(P<0.01),而尼莫地平组无明显变化(P>0.05);随脑缺血时间延长,模型对照组和尼莫地平组nNOS阳性细胞数都下降(P<0.05);脑缺血6h,尼莫地平组半暗区的凋亡细胞少于模型对照组(P<0.01)。结论钙拮抗剂尼莫地平可抑制脑缺血早期的nNOS上升,并减轻随后的脑组织损伤。     

醒脑静联合丁苯肽对脑缺血再灌注损伤后细胞凋亡的影响

目的 观察醒脑静、丁苯酞及二者联合分别对大鼠脑缺血再灌注损伤后神经细胞凋亡及Bcl-2和Bax表达的影响。方法 60只雄性wistar大鼠(250±20)g采用改良线栓法制作脑缺血再灌注损伤模型(Middle cerebral artery occlusion,MCAO),随机分为4组,即模型组、醒脑静组、丁苯酞组、醒脑静联合丁苯酞(联合用药)组,每组又分为6、24、72 h三个亚组; 通过原位末端转移酶标记技术(TUNEL)检测神经细胞凋亡情况,采用免疫组化法观察大鼠脑缺血再灌注各个时间点Bcl-2、Bax的表达水平。结果(1)模型组手术对侧大脑半球偶见凋亡细胞,病灶区可见大量神经细胞凋亡。丁苯酞用药组、醒脑静用药组凋亡细胞数明显减少,醒脑静联合丁苯酞组凋亡细胞数最少(P<0.05);(2)丁苯酞组及联合用药组Bcl-2阳性表达水平较模型组均有提高,联合用药组Bcl-2阳性表达水平在各时间点均最高(P<0.05); 丁苯酞组及联合用药组Bax阳性表达水平较模型组均有降低,联合用药组Bax阳性表达水平最低(P<0.05)。结论(1)醒脑静、丁苯酞及二者联合均可能通过抑制脑缺血再灌注损伤后神经细胞凋亡来实现神经细胞保护作用,其中二者联合效果最佳;(2)丁苯酞可能通过增加脑缺血再灌注损伤大鼠Bcl-2表达,减少Bax表达的方式来减少神经细胞凋亡,从而减轻脑缺血再灌注损伤;(3)醒脑静本身不能对Bcl-2、Bax的表达水平产生影响,但其可能通过增强丁苯酞作用的方式影响Bcl-2、Bax的表达,从而减轻脑缺血再灌注损伤。     

Colocalization of somatostatin, neuropeptide Y, neuronal nitric oxide synthase and NADPH-diaphorase in striatal interneurons in rats

The neuropeptides somatostatin (SS), neuropeptide Y (NPY), the enzyme neuronal nitric oxide synthase (nNOS) and enzymatic activity for NADPH diaphorase (NADPHd) are extensively colocalized in striatal interneurons, which has led to the widespread tendency to operationally treat all four substances as being completely colocalized within a single class of striatal interneurons. We have explored the validity of this assumption in rat striatum using multiple-labeling methods. Conventional epi-illumination fluorescence microscopy was used to examine tissue triple labeled for SS, NPY and nNOS, or double-labeled for SS and nNOS or for SS and NPY. In tissue double-labeled for SS and nNOS, confocal laser scanning microscopy (CLSM) images of SS and nNOS labeling were compared to subsequent NADPHd labeling. We found that SS, NPY and nNOS co-occurred extensively, but a moderately abundant population of neurons containing SS and nNOS but not NPY was also observed, as were small populations of SS only and nNOS only neurons. About 80% of SS + neurons contained NPY, and no NPY neurons were devoid of SS or nNOS. All neurons containing nNOS in rat striatum were found to contain NADPHd. Combining our various quantitative observations, we found that of those striatal neurons containing any combination of SS, NPY, nNOS and NADPHd in rats, about 73% contained all four, 16% contained SS, nNOS and NADPHd, 5% contained SS only, and 6% contained only nNOS and NADPHd. These results indicate that while there is a large population of striatal neurons in which SS, NPY, nNOS and NADPHd are colocalized in rats, there may be smaller populations of striatal neurons devoid of NPY in which SS or nNOS/NADPHd are found individually or together.     

铝盐致大鼠学习记忆功能障碍的β—APP、SS作用机制研究

目的：观察β－淀粉样前体蛋白（APP）、生长抑素（SS）在铝盐致大鼠学习记忆功能障碍模型脑内海马区表达的改变,并探讨其作用机制。方法：采用免疫组织化学方法检测β-APP、SS在大鼠脑内海马区的表达,HE染色观察实验组与对照组海马区锥体细胸层神经元形态结构,结果：实验组大鼠学习记忆功能较对照组明显降低（P＜0．01）;实验组海马区β－APP表达较对照组明显增加（P＜0．01）;SS免疫反应性神经元数目却显著减少（P＜0．01）;β-APP阳性度与SS阳性度之间存在等级负相关（P＜0．05）,实验组与对照组海马区神经元形态结构均基本 正常,数目无明显差异,结论：铝盐致大鼠海学记忆障碍的病理机制可能与脑内β-APP、SS表达的改变相关。     

Responses of neuronal nitric oxide synthase expression in the brainstem to electroacupuncture Zusanli (ST 36) in rats

Recent studies have reported that l-arginine-derived nitric oxide (NO) in the gracile nucleus modifies the hypotensive responses to electroacupuncture (EA) stimulation of Zusanli (ST 36). The purpose of this study was to examine the influence of EA stimulation of ST 36 on neuronal NO synthase (nNOS) expression in the brainstem nuclei in rats. EA stimulation of ST 36 and a non-acupoint was performed using 3 Hz of stimulation for 10 s every 2 min for a period of 120 min in rats anesthetized with ketamine. Rats in the sham-treated group received surgery and EA needles were placed into the acupoints without performing the stimulation. After 2-h stimulation and sham treatment, animals were perfused with 4% paraformaldehyde. Sections of rat medulla were examined by immunolabeling with a polyclonal antibody directed against nNOS. The brainstem nuclei were also visualized by NADPH-diaphorase histochemistry, a marker of nNOS activity. nNOS expression and NADPH-diaphorase reactivity were quantified by using a microscope with reticule grid to count the number of positive cells over a nucleus. Unilateral EA stimulation of ST 36 in rats caused increases in nNOS immunostained cells in the rostral region of the ipsilateral gracile nucleus, but was not altered in the contralateral gracile nucleus compared with sham-treated rats (P < 0.05, n = 6-7). NADPH-diaphorase-positive cells were also increased in the ipsilateral gracile nucleus of rats with EA stimulation. nNOS immunostaining and NADPH-diaphorase-positive neurons were significantly increased in both ipsilateral and contralateral sides of the medial nucleus tractus solitarius (mNTS) in rats receiving EA ST 36 compared with sham-treated animals (P < 0.05). nNOS immunostaining and NADPH-diaphorase reactivity was neither altered in the gracile nucleus and mNTS of non-acupoint stimulated rats nor other brainstem nuclei in rats with EA ST 36. These results show that nNOS immunoreactivity and NADPH-diaphorase reactivity are consistently increased in the gracile nucleus and the mNTS by EA ST 36. We conclude that EA ST 36 induces nNOS expression in the gracile nucleus and mNTS, and enhanced nNOS-NO in the nuclei may modify central cardiovascular regulation, which contribute to hypotensive effects of acupuncture.     

Upregulated expression of neuronal nitric oxide synthase by insulin in both neurons and astrocytes

Both insulin and nitric oxide (NO) play important roles in the brain. However, there are no unequivocal evidences pointing to a direct effect of insulin on nitric oxide pathway in the brain. In the present study, the effects of insulin on the expression and activity of neuronal nitric oxide synthase (nNOS) were investigated in the cultured cerebellum cell line R2, cerebral cortical astrocytes, and neurons of rats by using flow cytometry, in situ hybridization, RT-PCR, and electron spin resonance (ESR) techniques. In astrocytes, the expression of nNOS was significantly stimulated by insulin in a concentration-dependent manner, with a maximal increase of about 47.6% compared with the control values (p<0.05, t test, n=5). Furthermore, in situ hybridization analysis showed that the expression of nNOS was also significantly increased by insulin (0.64 ng/ml, 6 h), reaching 134.2+/-9.6% of the control values (p<0.05, t test, n=3). In addition, by using nNOS specific primers, RT-PCR analysis also demonstrated the same effect of insulin (0.64 ng/ml, 6 h) on nNOS mRNA expression. Similarly, significant increase of the expression of nNOS protein and mRNA were also observed in both R2 cells and neurons of rats after incubation with insulin. In addition, significant increase of the activity of nNOS in R2 cells and astrocytes were also detected after incubation with insulin (0.64 ng/ml, 9 h) by using ESR technique. Overall, our results suggested that exogenous insulin could upregulate the expression and activity of nNOS in R2 cells, cerebral cortical astrocytes, and neurons of rats. The phenomena opened new insights for further investigation of the physical and pathological significances of insulin in the brain.     

睡眠剥夺大鼠大脑皮层和海马nNOS表达的改变在其学习记忆损伤中的作用

目的观察睡眠剥夺（SD）后,大鼠学习记忆能力及大脑皮层和海马神经元型一氧化氮合酶（nNOS）蛋白表达的变化。方法采用小平台水环境法建立大鼠SD模型。观察大鼠经过不同时间SD后,各组“Y”迷宫成绩及大脑皮层和海马nNOS阳性神经元的变化情况。结果（1）“Y”迷宫实验：3组SD组的迷宫成绩较正常笼养对照组（CC）分别下降了24．3％、17．6％和41．9％（P〈0．01）。（2）nNOS阳性神经元数目：在皮层,SDld组较CC组增加了8．7％（P〈0．01）,SD2d组和SD3d组较CC组则减少了9．0％和16．0％（P〈0．01）;在海马CA。区,3个SD组较CC组分别减少了9．4％、16．0％和22．0％（P〈0．01）。结论SD可能通过改变大鼠大脑皮层和海马CA．区nNOS蛋白的表达来损害大鼠学习记忆能力。     

星形胶质细胞源性因子对神经干细胞分化的实验研究

目的探讨星形胶质细胞源性因子对神经干细胞分化的影响。方法分离和培养新生大鼠脑组织的神经干细胞;采用差速贴壁法和振荡法分离纯化星形胶质细胞,用免疫细胞化学染色法,胶质纤维酸性蛋白(GFAP)标记星形胶质细胞,进行细胞的纯度鉴定;将星形胶质细胞和神经干细胞在互不接触的情况下进行共培养,免疫荧光法观察神经干细胞分化后神经元特异性烯醇化酶(NSE)、GFAP和酪氨酸羟化酶(TH)的表达。结果纯化的星形胶质细胞GFAP抗体标记阳性,细胞纯度达98%;星形胶质细胞与神经干细胞共培养时,神经干细胞贴壁分化加快,NSE阳性细胞及TH阳性细胞明显多于对照组(P<0·05)。结论星形胶质细胞源性因子可快速诱导神经干细胞向神经元细胞、包括多巴胺神经元细胞分化,提示星形胶质细胞支持神经元发生。     

Ontogenesis of neurons immunoreactive for nitric oxide synthase in rat forebrain and midbrain.

We studied the immunohistochemical localization of neuronal nitric oxide synthase (nNOS) in the developing rat brain on embryonic days 13 (E13), 15 (E15), 17 (E17) and 19 (E19) and postnatal days 0 (P0), 7 (P7) and 14 (P14). A few neurons positive for nNOS were first detected at E15 in the hypothalamus and pons. At E17, many positive cells became detectable in the thalamus. At E19, the positive cells in these three regions were rapidly increased in number, and a few positive neurons were also observed in such regions as the cerebral cortex and striatum. Positive cells in the hypothalamus tended to locate ventrolaterally. Positive neurons, stained very intensely as in adult rats, were seen in the pedunculopontine tegmental nucleus, laterodorsal tegmental nucleus and parafascicular nucleus. Two weeks after birth, positive neurons of larger somata with many processes were distributed widely in the cerebral cortex and hippocampus. The present study indicates that, in the forebrain and midbrain, the distribution pattern of nNOS-containing neurons is fundamentally completed by E19.     

匹罗卡品致(癎)大鼠海马中表达生长抑素的中间神经元数目变化及其轴突出芽

目的 探讨表达生长抑素(SS)的中间神经元在颞叶癫(癎)的发生和自我修复中的作用.方法 建立匹罗卡品致(癎)大鼠模型.免疫组织化学方法检测各时间点海马不同区域SS中间神经元的数目变化及其轴突出芽情况;结合Fluoro-Jade B(FJB)行免疫荧光双标方法特异性检测大鼠癫(癎)持续状态(status epilepticus,SE)后7 d和60 d海马不同区域SS中间神经元及其轴、树突的变性情况.结果 实验组大鼠海马各区Ss阳性神经元数目均在SE后7 d减至最低(门区11.1±3.3,CA1区2.8±0.9,CA1区1.8±0.7,t=13.519、9.644、8.808,均P<0.01),慢性期开始部分恢复,SE后60 d时CA1区SS神经元数目(12.8±1.5)超过对照组(8.8±1.3,t=-4.506,P<0.01),但门区和CA3区SS神经元数目(分别为25.5±4.6和4.8±0.8)仍明显低于正常水平(t=4.691、3.953,P<0.01);sE后30 d大鼠海马回腔隙.分子层(lacunosum-moleculare,lm)和齿状回外分子层出现大量SS染色阳性纤维,60 d时海马CA1区全层均可见大量增多的SS阳性纤维.实验组中SE后7 d的海马CA1区、60 d的CA1、CA1区和门区均可观察到少部分被FJB染色的SS中间神经元胞体及其轴、树突.结论 SS中间神经元的缺失在颞叶癫(癎)的发生中起重要作用,其缺失部分是由于神经元的变性死亡所致;慢性期CA,区SS阳性纤维大量增多,可能在颞叶癫(癎)的发生和自我修复中起重要作用.     

The sensitivity of neurons with non-periodic activity to sympathetic stimulation in rat injured dorsal root ganglion

Objective The relationship between firing pattern and sensitivity of neurons was studied in chronically compressed dorsal root ganglion (DRG) neurons and the Hindmarsh-Rose (HR) neuronal model. Methods Spontaneous activities from single fibers of chronically compressed DRG neurons in rats were recorded, and divided into periodic and non-periodic firing patterns. The sensitivity of the two kinds of firing pattern neuron to sympathetic stimulation (SS) was compared. Result It was found that 27.3% of periodic firing neurons and 93.2% of non-periodic firing neurons responded to SS respectively (periodic vs non-periodic, P<0.01). The responses to SS with different stimulation time were greater non-periodic firing neurons than periodic firing neurons (P<0.01). The non-periodic firing neurons obviously responded to SS. After the firing pattern of these neurons transformed to periodic firing pattern, their responses to SS disappeared or decreased obviously. The HR neuronal model exhibited a significantly greater response to perturbation in non-periodic (chaotic) firing pattern than in periodic firing pattern. Conclusion The non-periodic firing neurons with deterministic chaos are more sensitive to external stimuli than the periodic firing neurons.