姜黄素对帕金森病小鼠模型黑质多巴胺能神经元损伤的保护作用

目的研究姜黄素对由MPTP诱发的帕金森病小鼠模型的脑保护作用及其可能机制。方法应用免疫组织化学染色法和蛋白质印迹法(Western blotting)分别观察姜黄素干预前后帕金森病小鼠中脑黑质-纹状体系统中酪氨酸羟化酶、胶质纤维酸性蛋白阳性神经元数目的变化,以及酪氨酸羟化酶、诱导型一氧化氮合酶和胶质纤维酸性蛋白表达水平的变化。结果MPTP组小鼠中脑黑质酪氨酸羟化酶和胶质纤维酸性蛋白阳性神经元数目明显减少,与正常对照组及治疗组相比差异有统计学意义(均P<0.05)。经不同剂量(5mg/kg、50mg/kg和150mg/kg)的姜黄素干预治疗后,小鼠中脑纹状体中的酪氨酸羟化酶蛋白表达水平(相对灰度值)明显升高,而黑质中诱导型一氧化氮合酶和胶质纤维酸性蛋白表达水平明显降低,与MPTP组比较差异均有统计学意义(P<0.05);MPTP组与溶剂对照组(MPTP DMSO)之间差异无统计学意义(P>0.05)。结论姜黄素可以有效地拮抗MPTP诱导的帕金森病小鼠模型黑质多巴胺能神经元的丢失,其机制可能与姜黄素降低黑质多巴胺能神经元活性氧含量以及抑制炎症反应等作用有关。     

白藜芦醇通过SIRT1/AMPK信号通路减轻MPTP诱导的小鼠多巴胺能神经元丢失

目的观察白藜芦醇(RV)对帕金森病(PD)小鼠的神经保护作用,并探索其发挥神经保护作用的可能机制。方法将48只小鼠随机分为CON组、MPTP组、RV+MPTP组和EX527+RV+MPTP组,每组12只。腹腔注射MPTP建立PD小鼠模型,并给予RV灌胃、SIRT1特异性抑制剂EX257腹腔注射处理,利用免疫荧光、western blot等检测相关蛋白表达。结果给予RV后,与MPTP组相比,RV+MPTP组SIRT1蛋白表达显著增加(P0.001),p-AMPK蛋白水平显著升高(P0.01),Cleaved caspase 3蛋白水平显著下降(P0.01),小鼠黑质区TH阳性神经元丢失率明显降低,纹状体组织中TH蛋白表达显著增加(P0.01)。给予EX527后,阻断了RV的上述作用,p-AMPK蛋白水平显著降低(P0.01),Cleaved caspase 3显著升高(P0.01),小鼠黑质区TH阳性神经元丢失率明显升高,纹状体组织中TH蛋白表达显著下降(P0.001)。结论 RV可能通过激活SIRT1/AMPK信号通路,SIRT1与AMPK相互调控,减少MPTP小鼠黑质区多巴胺能神经元丢失。     

帕金森病模型鼠黑质α-突触核蛋白表达升高并集聚

目的观察低剂量1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)对小鼠黑质多巴胺能神经元α-突触核蛋白表达的影响。方法C57BL小鼠腹腔注射MPTP20mg/kg体重,每周注射1次连续3周,观察小鼠行为改变,应用逆转录-聚合酶链反应(RT-PCR)、Western印迹法、免疫组化染色检测α-突触核蛋白表达,荧光双标染色观察多巴胺能神经元内α-突触核蛋白聚集。结果与注射生理盐水组相比,注射MPTP组小鼠出现暂时性躯干震颤、竖毛、动作减少等;α-突触核蛋白基因表达百分比(79·5%±0·8%,生理盐水组为19·2%±3·3%,P<0·05)及蛋白表达百分比(82·2%±4·1%,生理盐水组为18·5%±1·0%,P<0·05)均明显增加;黑质部酪氨酸羟化酶(TH)阳性细胞数减少,α-突触核蛋白阳性细胞数增加;α-突触核蛋白与硫磺素S、TH与硫磺素S复合定位染色的阳性细胞数均升高。结论低剂量MPTP可诱导小鼠行为改变,黑质多巴胺能神经元α-突触核蛋白表达升高,并出现蛋白聚集。     

抗帕颗粒对帕金森病模型小鼠酪氨酸羟化酶神经元的影响

目的 探讨抗帕颗粒对1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)帕金森病(PD)模型小鼠黑质纹状体酪氨酸羟化酶(TH)阳性神经元的影响。方法 90只健康雄性C57BL/6小鼠,鼠龄8～12周,并随机分为3组,即正常对照组30只、PD模型对照组30只、PD模型干预组30只; MPTP腹腔注射(40 mg·kg^-1·d^-1×7 d)制备小鼠PD模型; 正常对照组及PD模型对照组予生理盐水1 mL·d^-1灌胃,PD模型干预组给予抗帕颗粒40 mg·kg^-1·d^-1灌胃,连续喂养4个月; 黑质纹状体切片、HE染色、免疫组织化学染色TH神经元及Western blotting检测TH蛋白的表达量。结果 ①正常对照组30只(30/30只)最终均存活,PD模型对照组4个月存活27只(27/30只),PD模型干预组4个月存活28只(28/30只); ②PD模型对照组、PD模型干预组小鼠每次注射MPTP后先有短暂兴奋[持续(7.61±2.17)min],表现为四处窜跳,随即出现全身中重度震颤,皮毛及尾巴时有竖立,活动减少,持续(24.23±3.89)min后震颤消失,随后出现活动减少; ③HE染色显示正常对照组大量褐色TH阳性细胞,PD模型对照组TH阳性细胞数明显减少,PD模型干预组TH阳性细胞数有所增加; ④免疫组织化学染色后经Imagepro-Plus 5.1系统分析,正常对照组TH阳性细胞面积为64 145 μm²,高倍镜下可见大量胞质为褐色颗粒的TH阳性细胞; PD模型对照组TH阳性细胞染色面积为40 012 μm²,高倍镜下见TH细胞数明显减少; PD模型干预组TH阳性细胞染色面积为60 952 μm²,高倍镜下见TH阳性细胞数较PD模型对照组增加; ⑤Western blotting检测显示正常对照组TH蛋白表达量与PD模型对照组和PD模型干预组比较均有明显差异(P<0.001),PD模型对照组TH蛋白表达量与PD模型干预组比较也有明显差异(P<0.05)。结论 抗帕颗粒可使PD小鼠黑质纹状体中多巴胺能神经元一定程度地减少丢失,对多巴胺能神经元的数量、形态及功能具有一定的保护作用。     

丁基苯酞对帕金森模型小鼠中脑黑质多巴胺能神经元数及TH、TNF-α蛋白表达的影响

目的研究丁基苯酞(dl-3n-butylphthalide,NBP)对由MPTP诱导的C57BL/6小鼠帕金森模型中脑黑质多巴胺能神经元数及TH、TNF-α蛋白表达的影响,进一步探讨其保护机制。方法 24只C57BL/6小鼠,随机分成3组:正常对照组,MPTP组,NBP治疗组。MPTP腹腔注射法制备帕金森模型,免疫组织化学法观察中脑黑质TH阳性神经元细胞数,蛋白质印迹法观察中脑黑质TH、TNF-α蛋白含量的变化。结果 (1)与正常对照组比较,MPTP组可见帕金森病小鼠中脑黑质TH阳性神经元明显减少(P0.01);与MPTP组比较,NBP治疗组帕金森病小鼠中脑黑质TH阳性神经元数目明显增加(P0.01);(2)与正常对照组比较,MPTP组帕金森病模型小鼠中脑黑质TH蛋白表达减少(P0.01),而TNF-α蛋白表达增加(P0.05);(3)与MPTP组比较,NBP治疗组帕金森病模型小鼠中脑黑质TH蛋白表达明显增加(P0.01),而TNF-α蛋白表达减少(P0.05)。结论丁基苯酞可能通过提高中脑黑质中TH的含量及减少TNF-α炎性介质表达发挥对MPTP所致C57BL/6小鼠帕金森模型的神经元保护作用。     

硫辛酸对帕金森病小鼠模型自噬蛋白表达水平的影响

目的 探讨硫辛酸(lipoic acid,LA)对MPTP诱导的C57BL/6帕金森病(Parkinson's disease,PD)小鼠模型黑质及纹状体TH、parkin、PINK1、DJ-1自噬相关蛋白表达水平的影响。方法 将60只C57BL/6小鼠随机分为PD模型组、正常组、治疗组、预保护组,采用背部皮下注射MPTP制作PD模型(每组各15只),行为学评价造模,Western Blotting法检测黑质、纹状体TH、parkin、PINK1、DJ-1蛋白的表达水平,免疫组化法观察黑质及纹状体阳性神经元数,免疫荧光检测PINK1的表达水平。结果(1)与正常组比较,PD模型组小鼠黑质、纹状体内TH、Parkin、PINK1、DJ-1的表达水平均明显降低(P<0.01);(2)与PD模型组比较,预保护组黑质、纹状体内TH、PINK1、Parkin、DJ-1表达水平均明显增高(P<0.05);(3)与治疗组比较,预保护组小鼠黑质、纹状体内TH、PINK1、Parkin表达水平略增高,预保护组DJ-1表达水平略降低,但两组比较无统计学差异(P>0.05)。结论 线粒体自噬参与PD小鼠的发病过程; 硫辛酸可能通过上调PD小鼠自噬相关蛋白的表达水平而发挥对多巴胺神经元的保护作用,从而为PD的防治提供新的思路。     

DNA聚合酶β抑制剂齐墩果酸对帕金森病小鼠模型的保护作用

目的 观察DNA聚合酶β抑制剂齐墩果酸对帕金森病小鼠模型的保护作用.方法 利用MPTP腹腔注射法制作小鼠帕金森病模型,在MPTP注射前后分别给予DNA聚合酶β抑制剂齐墩果酸干预,用免疫组织化学染色法检测小鼠中脑酪氨酸羟化酶（TH）阳性神经元,免疫印迹法检测中脑腹侧TH蛋白和活化的Caspase-3表达水平,HPLC法检测纹状体中多巴胺及其代谢产物的水平.结果 小鼠腹腔注射MPTP后出现行为学异常,中脑黑质多巴胺能神经元损伤,中脑腹侧TH表达降低,活化的caspase-3水平增高,同时纹状体多巴胺及其代谢产物3,4二羟基苯乙酸（DOPAC）水平降低.齐墩果酸干预能够显著改善小鼠的行为学评分,增加黑质多巴胺能神经元数量,抑制caspase-3活化,并增加纹状体多巴胺和DOPAC水平.结论 齐墩果酸可能对帕金森病小鼠多巴胺能神经元具有保护作用.     

尼莫地平对帕金森病模型小鼠黑质iNOS和COX-2表达的抑制作用

目的研究尼莫地平对1-甲基-4-苯基-1、2、3、6四氢吡啶(MPTP)所诱导的帕金森病(PD)模型小鼠黑质多巴胺能神经元的保护作用。方法雄性健康C57BL/6N小鼠随机分为对照组,腹腔注射生理盐水;模型组,腹腔注射MPTP(25 mg/kg);尼莫地平治疗组,每次注射MPTP前3 h灌胃给予尼莫地平(15 mg/kg)。观察各组小鼠行为学变化,免疫组织化学和免疫蛋白印迹法观察中脑黑质酪氨酸羟化酶(TH)、诱导型一氧化氮合酶(induciblenitricoxidesynthase,i NOS)和环氧化酶-2(Cyclooxygenase,COX-2)的表达变化。结果与对照组小鼠比较,MPTP模型组小鼠出现典型的PD症状,中脑黑质TH阳性神经元大量丢失,i NOS和COX-2阳性细胞显著增多,蛋白水平明显升高;经尼莫地平处理后,上述情况得到明显改善。结论在此PD小鼠模型中,尼莫地平通过抑制炎症因子i NOS和COX-2的表达,可对多巴胺能神经元起到一定的保护作用。     

粒细胞集落刺激因子对百草枯干预小鼠黑质纹状体通路的影响

目的探讨粒细胞集落刺激因子(G-CSF)对百草枯干预小鼠脑内黑质纹状体通路的影响。方法 C57BL/6小鼠随机分为百草枯组、G-CSF干预组和对照组(均n=8)。高效液相色谱分析测定纹状体多巴胺含量,同时采用免疫组化ABC法观察黑质部酪氨酸羟化酶(TH)阳性表达神经元。结果腹腔注射百草枯20 mg·kg~(-1)·d~(-1),连续5 d后,小鼠自发性活动明显减少,纹状体多巴胺含量明显减少,黑质部TH阳性表达明显减少,与对照组比较差异有统计学意义(P0.05);给予G-CSF干预后纹状体多巴胺含量和黑质部TH阳性表达明显增加,与百草枯组比较差异有统计学意义(P0.05)。结论 G-CSF干预后小鼠纹状体的多巴胺含量升高,黑质部TH阳性表达增加,提示G-CSF对小鼠黑质纹状体通路多巴胺能神经元可能具有一定的保护作用。     

晚期糖基化终末产物受体对帕金森病模型小鼠脑内酪氨酸羟化酶表达的影响

目的探讨晚期糖基化终末产物受体(RAGE)对1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)诱导的帕金森病(PD)模型小鼠脑中酪氨酸羟化酶(TH)表达量的影响。方法采用12周龄的C57BL/6雄性小鼠依据不同处理方法分为6组(均n=10):对照组;MPTP组;空载病毒阴性对照(RAGE-NC)组;目的基因阴性对照(siRNA-RAGE)组;RAGE-NC+MPTP组;siRNA-RAGE+MPTP组。携带空载siRNA的慢病毒(RAGE-NC)与携带抑制RAGE表达的目的 siRNA-RAGE慢病毒经脑立体定位仪定向注射于小鼠两侧黑质,根据分组情况给予腹腔注射MPTP 30mg·kg~(-1)或等量生理盐水(每周2次×5周)。5周后予小鼠断头取脑,利用免疫组织荧光染色法检测各组小鼠脑组织黑质中TH数量变化情况,采用Western blot法分别检测各组RAGE、caspase-3、TH蛋白表达水平。结果与RAGENC+MPTP组比较,siRNA-RAGE+MPTP组RAGE蛋白表达减少(0.782 8±0.139 6 vs 1.039 0±0.146 4,P0.01),caspase-3蛋白表达减少(0.864 4±0.105 3 vs 1.240 0±0.080 7,P0.001),TH蛋白表达量显著升高(1.114 0±0.201 1vs 0.771 1±0.211 3,P0.05),差异有统计学意义。结论抑制RAGE表达可抑制凋亡反应,提高PD多巴胺能神经元模型中TH蛋白表达水平,有潜在的神经保护作用。     

Characteristics of the sympathetic innervation of the nictitating membrane and of the vasculature of the nose and tongue of the cat

Summary Vasomotor responses from the nasal mucosa and tongue, and contractions of the nictitating membrane, were recorded on stimulation of the cervical sympathetic or internal carotid nerves.Preganglionic sympathetic nerve fibres which elicited a membrane response possessed a lower threshold than those which evoked nasal vasoconstriction, while the latter displayed a lower threshold than fibres which evoked tongue vasoconstriction. The sympathetic vasodilator fibres to the tongue, whose activity was revealed after-receptor blockade, had a similar threshold to the vasoconstrictor fibres.Membrane contraction, nasal vasoconstriction and occasionally tongue vasoconstriction could be evoked by stimulating the internal carotid nerve. The postganglionic fibres innervating the nasal mucosa had a similar threshold to those of the nictitating membrane, which may indicate that there are small myelinated fibres innervating the mucosa.The preganglionic compound nerve action potential had four major components, S₁–S₄. S₁, S₂ and usually S₃ fibres were associated with membrane contraction; S₂, S₃ and sometimes S₁ fibres were associated with nasal vasoconstriction; and S₃, usually S₂ and occasionally S₁ fibres were associated with vasoconstriction in the tongue. It is concluded that each of these three groups of nerve fibres, but not S₄ fibres, may include fibres associated functionally with the three effectors.There was a considerable difference between the relative amplitude of the responses of the three effectors elicited by stimulation of the cervical sympathetic nerve at frequencies between 0.2 and 2 Hz. Vasoconstrictor responses were relatively larger than membrane contractions suggesting differences in the mechanisms of neurotransmission at the neuroeffector junctions.     

Development of the projection from the nucleus of the brachium of the inferior colliculus to the superior colliculus in the ferret

Neurons in the deeper layers of the superior colliculus (SC) have spatially tuned receptive fields that are arranged to form a map of auditory space. The spatial tuning of these neurons emerges gradually in an experience-dependent manner after the onset of hearing, but the relative contributions of peripheral and central factors in this process of maturation are unknown. We have studied the postnatal development of the projection to the ferret SC from the nucleus of the brachium of the inferior colliculus (nBIC), its main source of auditory input, to determine whether the emergence of auditory map topography can be attributed to anatomical rewiring of this projection. The pattern of retrograde labeling produced by injections of fluorescent microspheres in the SC on postnatal day (P) 0 and just after the age of hearing onset (P29), showed that the nBIC-SC projection is topographically organized in the rostrocaudal axis, along which sound azimuth is represented, from birth. Injections of biotinylated dextran amine-fluorescein into the nBIC at different ages (P30, 60, and 90) labeled axons with numerous terminals and en passant boutons throughout the deeper layers of the SC. This labeling covered the entire mediolateral extent of the SC, but, in keeping with the pattern of retrograde labeling following microsphere injections in the SC, was more restricted rostrocaudally. No systematic changes were observed with age. The stability of the nBIC-SC projection over this period suggests that developmental changes in auditory spatial tuning involve other processes, rather than a gross refinement of the projection from the nBIC.     

Mechanisms of the suppression of the nociceptive reflex of the opening of the mouth during stimulation of the structures of the limbic brain

The comparative effectiveness of the inhibitory influence of tetanic stimulation of hypothalamus, amygdala and limbic cortex on EMG-response of m. digastricus evoked by electrical stimulation of tooth pulp nociceptive afferents was studied in cats anesthetized with a mixture of chloralose and nembutal. It was found that inhibition of the EMG-component of the jaw-opening reflex is most pronounced in case of stimulation of medial and lateral region of the hypothalamus, the inhibitory effect of central and medial nuclei of the amygdala is less pronounced and the effect of the limbic cortex is the weakest. It was shown that the mechanism of the antinociceptive effect of tetanic stimulation of the hypothalamus is not related to the concomitant increase of the blood pressure. After stabilization of the blood pressure the suppressive effect of the hypothalamus remains without changes, that points out to a direct, primary, not baro-afferent mechanism of the inhibition of the activity of nociceptive neurons of the trigeminal sensory nuclei. Noradrenaline, injected intravenously, induced a large increase of the blood pressure accompanied by a pronounced inhibition of the pain reflex. Angiotensin causes the same degree of blood pressure elevation without changes in the amplitude of the EMG-response of the pain reflex. Hypothalamic and noradrenergic mechanisms for control of pain sensitivity are discussed.     

Origins of the sympathetic innervation of the cervical end of the uterus in the rat

A retrograde neuronal tracer (Fast Blue) was injected in the cervical end of the uterine horn of virgin rats. The majority of the retrogradely labeled post-ganglionic sympathetic neurons were found in the sympathetic chain (74%). The superior mesenteric ganglia, inferior mesenteric ganglia and suprarenal ganglia accounted for 22, 3 and <1%, respectively. The distribution of neurons in the sympathetic chain labeled from the uterus resembles that described for other pelvic organs.     

Sexual pheromones and the evolution of the reward system of the brain: the chemosensory function of the amygdala

The amygdala of all tetrapod vertebrates receives direct projections from the main and accessory olfactory bulbs, and the strong similarities in the organization of these projections suggest that they have undergone a very conservative evolution. However, current ideas about the function of the amygdala do not pay sufficient attention to its chemosensory role, but only view it as the core of the emotional brain. In this study, we propose that both roles of the amygdala are intimately linked since the amygdala is actually involved in mediating emotional responses to chemical signals. The amygdala is the only structure in the brain receiving pheromonal information directly from the accessory olfactory bulbs and we have shown in mice that males emit sexual pheromones that are innately attractive for females. In fact, sexual pheromones can be used as unconditioned stimuli to induce a conditioned attraction to previously neutral odorants as well as a conditioned place preference. Therefore, sexual pheromones should be regarded as natural reinforcers. Behavioural and pharmacological studies (reviewed here) have shown that the females' innate preference for sexual pheromones is not affected by lesions of the dopaminergic cells of the ventral tegmental area, and that the systemic administration of dopamine antagonists do not alter neither the attraction nor the reinforcing effects of these pheromones. Anatomical studies have shown that the vomeronasal amygdala gives rise to important projections to the olfactory tubercle and the islands of Calleja, suggesting that these amygdalo-striatal pathways might be involved in the reinforcing value of sexual pheromones.     

药物治疗与合并认知行为治疗对强迫症疗效的比较

目的探讨认知行为心理治疗(CBT)在强迫症(OCD)患者各亚型治疗中的有效性和规律性。方法本研究为临床对照研究。符合入组标准的强迫症患者按患者自愿原则分为两组,治疗观察3、6、12个月。疗效评定分别运用Yale-Brown强迫量表,自拟的自评好转程度量表和临床疗效评定。结果认知行为心理治疗合并药物治疗组31例,临床有效率70.9%,其中治愈率1.8%。单纯药物治疗组24例,临床有效率33.3%。Yale-Brown强迫量表和自评量表得分在6个月和12个月两组有显著差异(P<0.05)。其中强迫症亚型(怕脏型、反复检查型和反复担心型)的疗效比较,怕脏型在治疗3个月末两组间自评量表评分有显著性差异(P<0.05);反复担心型在治疗6个月末两组间Yale-Brown强迫量表总分有显著性差异(P<0.05);反复检查型两组间无统计学差异。结论认知行为心理治疗合并药物治疗强迫症的疗效明显优于单纯药物治疗。强迫症的亚型在治疗中的有效性次序为:反复担心型>怕脏型>反复检查型。     

Topographical organization of the motoneuron pools that innervate the muscles of the pinna of the cat

The topographical organization of the 22 motoneuron pools that innervate the pinna muscles of the cat was examined by injecting the B-subunit of cholera toxin conjugated to horseradish peroxidase into individual muscles. All 22 pools are found in the facial nucleus, organized as rostro-caudally oriented columns, and arranged according to the action of the muscles they innervate. Pools innervating muscles that pull the pinna dorsally are located in the dorsal two thirds of the medio-dorsal subdivision, and those innervating muscles that pull the pinna ventrally are located in the ventral one half of the nucleus. Motoneurons innervating muscles that pull the pinna cranially are located laterally, those that pull the pinna caudally are located medio-ventrally, and those that change the shape of the pinna are located along the entire dorso-ventral extent in the center of the medio-dorsal subdivision. This topographical layout is consistent with the somatotopic organization of the entire facial nucleus as demonstrated in a variety of species. © 1995 Wiley-Liss, Inc.     

Histochemical methods for the further characterisation of the tumourettes of the posterior lobe of the pituitary

Summary The granular cell tumourettes of the posterior lobe of the pituitary possess neuraminic acid containing carbohydrate. After removal of neuraminic acid with neuraminidase and exposure to FITC (Fluorescein isothiocyanate) labelled peanut agglutinin (Arachis hypogaea) (PNA), intracellular receptor structures could be demonstrated. The significance of the findings is discussed.