6-OHDA所致PD大鼠模型黑质多巴胺能神经元与胶质细胞的变化

目的 应用6-羟多巴(6.OHDA)建立帕金森病(PD)大鼠模型。方法将6-OHDA立体注入大鼠前脑内侧束,应用免疫组织化学法检测6-OHDA注射后第3、7及14天后多巴胺能神经元、小胶质细胞与星形胶质细胞数量及形态的变化。用显微镜专业摄像头采集图像,计算机图像分析软件测量平均灰度值。结果 损伤侧与对侧相比,TH阳性多巴胺能神经元数量减少,损伤侧平均灰度值增加;小胶质细胞与星形胶质细胞显著增生,二者损伤侧的平均灰度值下降,经配对t检验,各组内比较差异均具有显著性意义(P<0.01);增生活化的小胶质细胞与星形胶质细胞形态发生明显的改变。结论 6-OHDA能成功建立PD大鼠模型。     

激活小胶质细胞对帕金森病模型大鼠黑质多巴胺能神经元的影响

目的 探讨脂多糖(LPS)激活小胶质细胞(MG)对黑质损伤多巴胺(DA)能神经元细胞存活的影响.方法 采用立体定向技术向大鼠单侧黑质(SNpc)内注入LPS激活MG后,隔48h于原位注射高、低剂量鱼藤酮(BT)分别建立重度及轻度损伤模型;采用免疫组化法观察黑质酪氨酸羟化酶(TH)阳性细胞和离子钙结合转接分子1(Ib α...     

褪黑素对帕金森病模型大鼠代谢机能和DA能神经元的保护作用

目的我们前期研究证实6-羟基多巴胺(6-OHDA)明显诱导性帕金森病(PD)模型大鼠行为学障碍及其纹状体神经元的损伤。目前进一步借助褪黑素(MT)探查证实6-羟基多巴胺对PD模型大鼠纹状体代谢机能损害和黑质DA能神经元的保护作用。方法选取55只成年雄性SD大鼠,并随机分成正常对照组(包括正常对照和稀释剂对照)、6-OHDA处理组和6-OHDA+MT处理组,实验借助Micro PET-CT、免疫组织化学染色及Westernblot技术检测和比较实验大鼠纹状体代谢、TH阳性纤维密度及TH蛋白表达量的变化。所获得数据使用SPSS20.0统计学软件进行分析处理,P0.05为有统计学意义。结果 Micro PET-CT检测结果显示,6-OHDA组大鼠纹状体糖代谢机能(1.34±0.114)明显较对照组(4.36±0.114)低(P=0.00),而6-OHDA+MT处理组(4.14±0.114)与6-OHDA组相比糖代谢机能增高,差异也具有统计学意义(P=0.00)。纹状体内TH阳性纤维密度和TH蛋白表达水平在6-OHDA组(分别为0.324±0.018、0.434±0.338)均显著低于对照组(0.378±0.026、1.626±0.526)(P=0.00,P=0.00)和6-OHDA+MT处理组(0.37±0.010、1.136±0.494)(P=0.00,P=0.03),但对照组和6-OHDA+MT处理组之间差异无统计学意义(P=0.52,P=0.12)。结论褪黑素对PD模型大鼠纹状体代谢机能障碍和中脑黑质DA能神经元损伤具有一定的保护作用。     

6-羟多巴诱导大鼠黑质的持续胶质细胞反应

本研究将40μg6- 羟多巴注射到SD大鼠一侧纹状体制作Parkinson病动物模型,研究黑质反应性神经胶质增生在Par kinson病发病过程中的可能作用。筛选成功的模型大鼠,术后12周处死。应用免疫荧光双标记法检测模型大鼠黑质胶质细胞对多巴胺能神经元损伤的反应。结果显示:在注射后12周,损伤侧黑质仍然存在明显的星形胶质细胞反应和小胶质细胞激活。此外,小胶质小结和淋巴细胞浸润的存在提示在注射后12周的注射侧黑质内依然有多巴胺能神经元死亡。结论: 6 -羟多巴对大鼠黑质多巴胺能神经元的急性损伤可以通过胶质细胞反应从而对多巴胺能神经元产生长期的毒性作用。     

大鼠黑质损毁后多巴胺能神经元形态及纹状体c-fos表达的变化

目的:观察毁损黑质后,多巴胺(DA)能神经元形态学变化和纹状体内相关神经元c-fos表达情况,探讨c-fos表达与毁损程度的关系。方法:利用6羟多巴胺(6OHDA)特异毁损大鼠黑质DA能神经元,采用阿朴吗啡(APO)诱导旋转实验观察术后1、7、14和21d行为学变化;利用HE染色、Nissl染色、免疫组织化学方法和电镜,观察各时间点黑质DA能神经元形态学变化和纹状体c-fos表达。结果:毁损侧DA能神经元逐渐减少,超微结构损伤逐渐加重;DA神经元丢失比例≥80%时,纹状体毁损侧c-fos表达上调,APO诱导的旋转实验>7r/min。结论:黑质DA能神经元丢失是毁损大鼠行为改变的病理学基础;cfos的表达与DA能神经元的毁损程度、行为改变有一定的关系。     

Mrh-aFGF对帕金森病大鼠黑质神经元的保护

目的 探讨重组人改构体酸性成纤维细胞生长因子(Mrh-aFGF) 对帕金森病(PD) 大鼠黑质神经元病变的影响。 方法 SD大鼠72只,随机分为4组：对照组、PD组、生理盐水（NS）处理组、　Mrh-aFGF 处理组,每组18只。 6-OHDA 分别注入左侧黑质和腹侧被盖区后建立PD 大鼠模型,侧脑室内注射Mrh-aFGF,用阿扑吗啡诱导旋转行为;Nissl 染色法观察大鼠黑质神经元病理学改变;电子显微镜观察黑质神经元超微结构的变化。 结果 对照组均未出现旋转行为;PD组术后旋转速度逐渐加快;NS 处理组旋转行为未见明显改善;Mrh-aFGF 处理组旋转速度明显减慢(P<0.05);PD组及NS 处理组大鼠损毁侧黑质神经元数目较健侧显著减少(P<0.05);Mrh-aFGF 处理组术后1周、2周、4周损毁侧神经元数目均较PD组及NS 处理组明显增加(P<0.05);PD组大鼠黑质神经元超微结构明显受损,出现核固缩,线粒体肿胀、嵴消失,粗面内质网扩张、脱颗粒,以及突触前后膜肿胀,突触间隙消失,Mrh-aFGF处理组黑质神经元超微结构有明显改善。 结论 Mrh-aFGF能改善PD大鼠的旋转行为,减少PD大鼠黑质神经元的丢失,并改善其黑质神经元的超微结构。     

6-OHDA制备的帕金森病大鼠黑质多巴胺能神经元的形态观察

帕金森病(Parkinson's disease,PD)主要病理改变是中脑黑质多巴胺(DA)能神经元变性、死亡、缺失,黑质-纹状体DA能系统功能减退及嗜酸性包涵体(Lewy小体)形成。PD在动物中没有自发的倾向,进行PD实验研究需要建立适当的动物模型。应用神经毒素6-羟基多巴胺(6-OHDA)直接注入黑质纹     

蛋白激酶Cδ在6-OHDA诱导大鼠多巴胺能神经元损伤过程中的作用

目的:探究蛋白激酶Cδ(PKCδ)对6-羟基多巴胺(6-OHDA)诱导的帕金森病(PD)模型大鼠多巴胺能神经元损伤的影响及其机制.方法:50只2月龄雄性SD大鼠,随机分为对照组(control)和6-羟基多巴胺注射组(6-OHDA).通过大鼠右侧纹状体注射6-OHDA溶液构建PD模型.运用旋转实验和圆筒实验检测平均旋转...     

褪黑素减弱帕金森病模型大鼠纹状体神经元的ROS反应

目的 帕金森病(PD)纹状体神经元损伤是否牵涉到氧化应激因素一直备受关注,本文结合行为学分析探讨褪黑素(MT)对纹状体不同神经元活性氧自由基(ROS)反应的影响.方法 随机将24只清洁级SD大鼠分为对照组、6OHDA组和6OHDA+MT处理组,实验借助行为学、组化、免疫组化技术对PD大鼠模型予以探察.结果 ①如同本实验室前期研究,6-羟基多巴胺(6OHDA)致使实验大鼠的学习记忆及肌张力改变,MT的使用明显改善其变化.②二氢乙锭(DHE)组化染色结果显示,作为氧化应激反应产物的ROS主要定位于纹状体投射神经元(NeuN+),同时可见一些单染色的ROS+以及NeuN+神经元.6OHDA组纹状体的ROS出现在NeuN+神经元的百分率(ROS-NeuN/ROS)以及NeuN+神经元呈现ROS+反应百分率(NeuN-ROS/NeuN)[分别为(80.34±1.75)％、(89.08±2.86)％]皆高于其对照组[分别为(68.67±2.57)％、(61.24±1.52)％](P＜0.05),而使用MT明显遏制由6OHDA诱导升高的百分率[6OHDA+MT组分别为(71.78±3.19)％、(64.64±2.97)％](P＜0.05)o③ROS-Parv双标记实验显示,作为纹状体主要类型的小清蛋白(Parv)中间神经元呈现ROS阳性反应,其百分率在60HDA组(91.62±8.63)％高于它的对照组(68.66±7.52)％(P＜0.05),MT对6OHDA诱导升高的百分率显示抑制效应(71.83±1.45)％(P＜0.05).结论 本文结果提示氧化应激因素可能牵涉到PD纹状体神经元损伤的病理过程,而MT对其具有保护作用.     

重复注射6-羟多巴胺建立帕金森病动物模型

目的探讨通过大鼠中脑内重复注射 6-羟多巴胺 (6-hydroxydopamine,6-OHDA)建立高效、稳定、可靠的帕金森病动物模型。方法将 60 只雄性 SD 大鼠随机分为一次打击组和二次打击组,经腹腔注射 4%水合氯醛(40 mg / 100g)麻醉,脑立体定位仪固定,于左侧黑质致密部(SNC)和中脑腹侧被盖区(VTA)分别注射 6-羟多巴胺 (6-OHDA,2 g / L,2μl),二次打击组一周后在相同位置重复注射同剂量的 6-OHDA,建立帕金森模型,观察其行为改变,通过 HE、TH 和 DIG-dUTP 染色观察其细胞形态的改变及凋亡情况。结果经过二次打击的大鼠,模型成功率(旋转周数 >7 r / min)为 86.7%,明显多于一次打击组的 33.3%;HE 染色显示,二次打击组凋亡细胞的阳性率(37.12%)明显多于一次打击组(21.25%);DIG-dUTP 染色显示,一次打击组大鼠左侧中脑黑质区神经细胞肿胀的数量多 ,凋亡数量少,凋亡细胞阳性率为 20.73%,二次打击组细胞凋亡数显著增多,凋亡细胞阳性率达 36.03%;TH 染色显示,二次打击组 TH 阳性细胞数明显减少,TH 细胞阳性率(18.61%)显著低于一次打击组(36.55%)。结论通过二次打击建立帕金森病动物模型成功率高于一次打击。     

NeuN is not a reliable marker of dopamine neurons in rat substantia nigra

Quantification of neuronal cell number is a key endpoint in the characterization of neurodegenerative disease models and neuroprotective regimens. Immunohistochemistry for phenotypic markers, followed by unbiased stereology is often used to quantify the relevant neuronal population. To control for loss of phenotypic markers in the absence of cell death, or to determine if other types of neurons are lost, a general neuronal marker is often desired. Vertebrate neuron-specific nuclear protein (NeuN) is reportedly expressed in most mammalian neurons. In Parkinson's disease models, NeuN has been widely used to determine if there is actual nigral dopamine neuron loss or simply loss of tyrosine hydroxylase expression, a prominent phenotypic marker. To date, the qualitative value of NeuN expression as such a marker in the substantia nigra has not been assessed. Midbrain tissue sections from control rats were stained for NeuN and tyrosine hydroxylase and assessed by light or confocal microscopy. Here we report that NeuN expression level in the rat substantia nigra was highly variable, with many faintly stained cells that would not be meet stereological scoring criteria. Additionally, dopamine neurons with little or no NeuN expression were readily identified. Subcellular compartmentalization of NeuN expression was also variable, with many cells dorsal and ventral to the nigra exhibiting expression in both the nucleus and cytoplasm. NeuN expression also appeared to be much higher in non-dopamine neurons within the ventral midbrain. This characterization of nigral NeuN expression suggests that it is not useful as a quantitative general neuronal marker in the substantia nigra.     

Cholinergic stimulation of substantia nigra: abolition of carbachol-induced eating by unilateral 6-hydroxydopamine lesion of nigrostriatal dopamine neurones

Summary Microinjection of cholinergic agonists into the substantia nigra is known to elicit increases in eating, drinking and sexual behaviour under appropriate circumstances. It has been suggested that these effects are dependent on stimulation of nigrostriatal dopamine-containing neurones in the substantia nigra pars compacta, but no direct evidence has confirmed this. The present experiment was therefore undertaken to determine whether unilateral lesions of nigrostriatal dopamine neurones made by 6-hydroxydopamine would attenuate or abolish eating in satiated rats elicited by intranigral microinjection of the muscarinic agonist carbachol. Two groups of rats were tested: a 6-hydroxydopamine- and a sham-lesion group. Before lesions were made intranigral microinjection of 0.5 g/0.5 l carbachol stimulated significantly more eating than control microinjections in both groups. After 6-hydroxydopamine lesions, microinjection of carbachol elicited no more eating than vehicle alone. Rats given sham lesions (ascorbate-saline vehicle only) showed increased feeding to intra-nigral carbachol before and after sham-lesioning. Post-mortem analysis by HPLC was used to determine the concentration of dopamine, DOPAC, HVA, serotonin and 5-HIAA in the lesioned and nonlesioned hemispheres of both 6-hydroxydopamine- and sham-lesioned rats. In caudate-putamen there were significant reductions in the concentration of DA (to 50.03% of the level in control sides), DOPAC (to 49.34%) and HVA (to 63.98%) in the 6-hydroxydopamine-lesioned but not sham-lesioned rats. The concentration of dopamine, DOPAC and HVA were not affected in the nucleus accumbens. The turnover of dopamine (assessed by calculating the ratio of dopamine to DOPAC) in the caudateputamen but not nucleus accumbens was also altered by the 6-hydroxydopamine lesions. The concentration and turnover of serotonin was not affected in either the caudate-putamen or nucleus accumbens in either group of rats. These data show that loss of dopamine from the caudate-putamen but not nucleus accumbens is sufficient to abolish completely the eating stimulated by intranigral carbachol.     

天麻对帕金森病大鼠黑质凋亡细胞表达及酪氨酸羟化酶阳性神经元的影响

目的研究和探讨帕金森病（PD）的发病机理及天麻防治PD的作用机制。方法采用6-羟基多巴胺（6-OHDA）大鼠模型,通过免疫组化技术观察天麻对PD大鼠黑质酪氮酸羟化酶阳性（TH^＋）神经元及Caspase-3阳性（Caspase-3^＋）神经元表达水平的影响。结果PD大鼠脑组织黑质致密部（SNpc）及腹侧被盖区（VTA）TH^＋神经元表达水平组间比较差异有统计学意义（F值分别为13．29、5．04,P〈0．01）。与正常组大鼠脑组织SNpc和VTA区TH^＋神经元表达水平[（32．13±4．84）,（30．23±3．21）]比较,模型组大鼠TH^＋神经元表达水平[（18．89±3．73）和（24．20±6．35）]明显下降（P〈0．01）;经天麻和美多巴治疗后TH^＋神经元丢失减少,天麻小剂量组TH^＋神经元表达水平分别为（26．24±3．06）和（26．31±6．1）,中剂量组分别为（22．44±3．81）和（26．91±6．28）;大剂量绀分别为（20．95±4．71）和（27．3±7．1）;美多巴组分别为（27．89±4．56）和（25．974±5．14）;其中,天麻小剂量组和美多巴组SNpc区TH^＋神经元表达水平和模型组比较差异有统计学意义（P〈0．05）。PD大鼠脑组织SNpc及VTA区Caspase-3阳性细胞表达水平组间比较差异有统计学意义（F值分别为6．09、5．53,P〈0．01）。与正常组Caspase-3阳性细胞表达水平[（9．83±3．03）,（7．04±1．76）]比较,模型组火鼠Caspase-3阳性细胞表达水平[（14．53±2．33）和（12．84±2．58）]明显升高（P〈0．01）;经天麻和美多巴治疗后Caspase-3阳性细胞表达水平下降,天麻小剂量组分别为（10．68±1．83）和（7．72±1．92）,中剂量组分别为（11．29±2．8）和（10．38±3．79）;大剂量组分别为（11．89±2．97）和（11．37±1．86）;美多巴组分别为（9．99±3．3）和（10．69±3．11）;其中,美多巴组SNpc区和天麻小剂量组VTA区Caspase-3阳性细胞表达水平和模型组比较差异有统计学意义（P〈0．05）。天麻和美多巴治疗各组TH^＋神经元表达与Caspase-3^＋神经元表达呈相反趋势。结论细胞凋亡可能是PD大鼠中腑多巴胺神经元丢失的主要方式。天麻可通过调节Caspase-3减少细胞凋亡,其中,小剂量天麻对治疗PD具有较好的作用。本研究结果提示天麻可不同程度地保护多巴胺神经元。     

帕金森病大鼠黑质小胶质细胞及星形胶质细胞的变化

目的探讨帕金森病(PD)发病中黑质小胶质细胞和星形胶质细胞的变化。方法采用立体定向术将神经毒素6-羟基多巴胺(6-OHDA)注入大鼠右侧黑质和内侧前脑束内,制备大鼠PD模型。将制模成功的16只PD大鼠随机分为2周和8周模型组,另6只正常大鼠作为对照组。观察各组大鼠黑质致密带内多巴胺(DA)能神经元、OX-42(小胶质细胞的特异性标志物)及神经胶质纤维酸性蛋白(GFAP,星形胶质细胞的特异性标志物)阳性细胞的分布和形态变化。结果 2周和8周模型组损毁侧黑质致密部DA能神经元较健侧显著减少(P0.01),损毁侧OX-42阳性细胞的数量较健侧明显增加(P0.01),形态呈"阿米巴状"。损毁侧GFAP阳性细胞数量较健侧明显增加(P0.01),突起变短,染色加深。2周模型组和8周模型组DA能神经元及两种胶质细胞的变化情况相似。结论 PD大鼠模型中存在着小胶质细胞和星形胶质细胞的激活,且两种胶质细胞的活化程度在PD发病过程中的不同时间无明显差别。     

Hypoxic-ischemic injury decreases anxiety-like behavior in rats when associated with loss of tyrosine-hydroxylase immunoreactive neurons of the substantia nigra

Neonatal Sprague-Dawley rats were randomly divided into normal control, mild hypoxia-ischemia (HI), and severe HI groups (N = 10 in each group at each time) on postnatal day 7 (P7) to study the effect of mild and severe HI on anxiety-like behavior and the expression of tyrosine hydroxylase (TH) in the substantia nigra (SN). The mild and severe HI groups were exposed to hypoxia (8% O₂/92% N₂) for 90 and 150 min, respectively. The elevated plus-maze (EPM) test was performed to assess anxiety-like behavior by measuring time spent in the open arms (OAT) and OAT%, and immunohistochemistry was used to determine the expression of TH in the SN at P14, P21, and P28. OAT and OAT% in the EPM were significantly increased in both the mild (1.88-, 1.99-, and 2.04-fold, and 1.94-, 1.51-, and 1.46-fold) and severe HI groups (1.69-, 1.68-, and 1.87-fold, and 1.83-, 1.43-, and 1.39-fold, respectively; P < 0.05). The percent of TH-positive cells occupying the SN area was significantly and similarly decreased in both the mild (17.7, 40.2, and 47.2%) and severe HI groups (16.3, 32.2, and 43.8%, respectively; P < 0.05). The decrease in the number of TH-positive cells in the SN and the level of protein expression were closely associated (Pearson correlation analysis: r = 0.991, P = 0.000 in the mild HI group and r = 0.974, P = 0.000 in the severe HI group) with the impaired anxiety-like behaviors. We conclude that neonatal HI results in decreased anxiety-like behavior during the juvenile period of Sprague-Dawley rats, which is associated with the decreased activity of TH in the SN. The impairment of anxiety and the expression of TH are not likely to be dependent on the severity of HI.     

Activity of neurons in the cat substantia nigra pars reticulata during drinking

Summary Extracellular activity of single neurons in the pars reticulata of the Substantia Nigra (SNpr) was recorded in cats during drinking. Two groups of cells were distinguished: I. Somatosensory cells which responded by a short decrease in firing rate to the arrival of water against the upper lip. We suggest that these stimulus-related responses reflect a complex process linked to preparation of buccolingual movements. II. Action-related cells which were de-activated at the beginning or during the entire drinking period but without modulation in relation to the individual movements of jaws and tongue. We suggest that de-activation of these cells during drinking operates as a gating mechanism which allows implementation of complex motor sequences by cortical and/or subcortical structures.     

Participation of the substantia nigra dopaminergic neurons in the occurrence of gastric mucosal erosions

Previous reports indicate that dopaminergic systems play an important role on gastric mucosal erosions. In the present study, the participation of intrinsic neurons of the substantia nigra (SN) and ventral tegmental area (VTA) on the occurrence of stomach ulceration was investigated. It was found that bilateral microinfusions of a neurotoxic dose (20 microg/microl) of N-methyl-D-aspartate (NMDA) into the SN, but not in the VTA, lead to gastric erosions 24 h after the surgery. A decrease in dopamine levels in the caudate 24 h after the microinfusion of NMDA into the SN was also observed. Destruction of SN cell bodies with 6-hydroxydopamine (6-OHDA) did not induce gastric ulceration or changes in dopamine levels in the caudate nucleus 24 h after the lesioning procedure. NMDA neurotoxicity is mediated by the acute excitatory or activational effects, in contrast to 6-OHDA, suggesting that the occurrence of gastric ulceration after the infusion of NMDA into the SN is not due to the cell death per se but is related to an overactivation of these cells that precede their death. Taken together, these results suggest that modulation of dopaminergic levels by neurons located within the SN may play an important role for the development of gastric erosions.     

帕金森病大鼠黑质区神经干细胞分化的观察

目的:探讨帕金森病(Parkinson's disease,PD)大鼠中脑黑质区神经干细胞(NSCs)的分化情况.方法:将6-OHDA注入纹状体内制作PD大鼠模型.随机将成功模型分为3 d、5 d、7 d、14 d、28 d组,每组6只;另设假手术及正常对照组各3只.向成功模型鼠腹腔注射5-溴脱氧尿苷(Brdu).用Brdu/NeuN、Brdu/GFAP、Brdu/TH免疫双标组织化学方法检测黑质区内源性NSCs向神经元、神经胶质细胞和多巴胺(DA)能神经元的分化情况.结果:PD模型7 d组,在黑质区Brdu /GFAP 、Brdu /NeuN 细胞开始出现,14 d组双标的阳性细胞数量逐渐增加,28 d达到高峰,14 d、28 d组与其它组比较差异显著(P＜0.05).在这些双标细胞中,Brdu /GFAP 细胞数量较多,Brdu /NeuN 细胞数量较少(P＜0.05),未发现Brdu /TH 细胞.结论:PD大鼠模型黑质区NSCs多数分化为神经胶质细胞,少数分化为神经元,在没有诱导和干预下,未见有向DA能神经元分化.