大鼠束缚后脑内小胶质细胞的反应

目的　 探讨大鼠束缚后脑内小胶质细胞的反应及其时程变化。 方法　 将大鼠束缚于小的塑料桶内 1,3或 6h ,于解束后 30min被处死 ,脑组织进行抗OX4 2 (小胶质细胞的特异性标记物 )免疫组织化学染色。结果 　正常大鼠脑内的小胶质细胞一般为静息状态 (静息型 ) ,其特点是OX4 2为阴性或浅染 ,在切片上不易发现或细胞形态不清晰。束缚 1h后 ,小胶质细胞为轻度反应 ,OX4 2浅染 ,细胞形态隐约可见 ,仅于下丘脑的视上核、室旁核和弓状核有散在的表达。束缚 3h后 ,小胶质细胞的反应达到高峰 ,由静息状态变为早期反应状态 (早期反应型 ) ,OX4 2深染 ,细胞形态清楚 ,突起上可见到小棘。早期反应型小胶质细胞广泛分布于前脑 :扣带回、新皮质浅层、隔外侧核、海马CA3、齿状回、杏仁中央核 ;间脑 :下丘脑视前区、视上核、室旁核、第三脑室周区、弓状核、丘脑室旁核、外侧膝状体、内侧膝状体 ;脑干 :中脑的上丘视性层、中脑导水管周围灰质、下丘的皮质部 ;脑桥的外侧臂旁核、蓝斑、A5区 ;延髓的延髓内脏带 (MVZ)和三叉神经脊束核等处 ;束缚 6h后 ,小胶质细胞反应减弱 ,OX4 2浅染 ,分布范围减少 ,主要出现于扣带回、隔外侧核、海马、视上核、室旁核、中脑导水管周围灰质、脑桥的臂旁外侧核、蓝斑、延髓的延髓内脏带 (M     

经侧脑室注射脂多糖诱导大鼠黑质部位小胶质细胞激活及多巴胺能神经元损伤的研究

目的观察经脑室注射脂多糖(LPS)后大鼠的黑质部小胶质细胞激活及多巴胺(DA)能神经元的变化,探讨脑内炎性反应在黑质DA能神经元慢性变性过程中的作用。方法健康雄性SD大鼠30只,随机分为生理盐水(NS)对照组和LPS组,分别向大鼠右侧脑室注射20μL NS或50μg LPS,40周后用免疫组织化学方法检测大鼠黑质小胶质细胞是否激活、激活的程度(OX-42及OX-6抗体水平),以及酪氨酸羟化酶(TH)阳性神经元的形态和数量。以Fluoro-Jade B(FJB)染色法检测黑质部位神经元变性情况。结果 (1)NS对照组大鼠黑质部位OX-42阳性小胶质细胞呈静息状态,染色浅。LPS组大鼠黑质部OX-42阳性小胶质细胞呈部分激活状态,染色深。两组大鼠黑质部位均未发现OX-6阳性小胶质细胞。(2)NS对照组大鼠黑质部位有大量深染的TH阳性神经元。LPS组大鼠黑质部位TH阳性染色神经元数目(99.11±20.31)比NS对照组(189.52±12.12)减少47.7%(P<0.01)。(3)两组大鼠黑质部位均未见FJB阳性染色神经元。结论经侧脑室单次注射LPS可能造成大鼠黑质部位小胶质细胞长期慢性激活及DA能神经元慢性迟发性功能性损伤。     

黑质内注射脂多糖对不同年龄大鼠DA能神经元和小胶质细胞活性的影响

目的 探讨青年和老年大鼠黑质DA能(DA)神经元对脂多糖(LPS)所诱导的损伤作用的敏感性差异和对小胶质细胞活性的影响.方法 采用立体定向技术向大鼠单侧黑质内注入LPS建立PD大鼠模型;采用免疫组化法观察黑质酪氨酸羟化酶(TH)阳性细胞和OX6阳性小胶质细胞变化;采用Fluoro-Jade B染色法检测黑质的变性神经元.结果 黑质内注射LPS后,老年组大鼠黑质区TH阳性神经元数量较青年组明显减少(P<0.01);老年组大鼠黑质区Fluoro-Jade B阳性神经元明显多于青年组(P<0.01);青年组大鼠黑质部位的OX6阳性小胶质细胞主要是处在激活期的;而老年组大鼠黑质部位的OX6阳性小胶质细胞主要是活化期的(阿米巴样或巨噬细胞样).结论 老年大鼠的黑质DA能神经元对于LPS所诱导的损伤作用较青年大鼠更为敏感.     

LPS激发大鼠延髓内脏带星形胶质细胞GFAP和神经元FOS表达水平的变化

观察腹腔注射细菌内毒素（LPS）后,大鼠延髓内脏带（MVZ）星形胶质细胞胶质原纤维酸性蛋白（GFAP）及神经元FOS表达水平随时间变化的规律及其相互关系。大鼠经LPS腹腔注射后1,3,6,12h分别行固定取材制片。每个时间点的切片分为3组,分别进行抗FOS、抗GFAP免疫组织化学染色及抗FOS／GFAP／酪氨酸羟化酶（TH）三重免疫组织化学染色。结果表明：（1）FOS反应在LPS注射后3h达到高峰,阳性产物主要分布于MVZ内。（2）GFAP反应在注射后1h即达到高峰,表现为胶质细胞肥大,数量增多。其分布于FOS基本相同。（3）三重染色观察到GFAP与FOS的多种聚集方式（FOS／GFAP／TH,FOS／GFAP,GFAP／TH）,FOS阳性神经元周围GFAP免疫反应产物更密集。提示星形胶质细胞对LPS起反应,其反应高峰的出现先于神经元。     

大鼠延髓内脏带与下丘脑室旁核、视上核往返投射通路参与高渗调节反应的研究

目的探讨延髓内脏带(MVZ)与下丘脑室旁核(PVN)和视上核(SON)之间是否存在往返渗透压投射通路。方法通过给予大鼠饮用3%氯化钠的方法制作高渗刺激模型,并用WGA-HRP逆行追踪、抗Fos、抗酪氨酸羟化酶(TH)或加压素(VP)及胶质纤维酸性蛋白(GFAP)免疫组织化学相结合的四重标记方法,观察MVZ、PVN和SON中WGA-HRP、Fos、TH、VP和GFAP阳性分布及表达状况。结果高渗刺激后MVZ、PVN和SON内Fos阳性细胞明显增多;GFAP阳性结构也明显增多,其分布与Fos阳性细胞分布基本一致,表现为胞体肥大、突起粗长。星形胶质细胞(AST)紧密包绕在神经元周围形成神经元-AST复合体(N-ASC)。结论神经元和AST以N-ASC的形式共同参与渗透压调节反应,体内存在MVZ和SON或PVN之间往返的渗透压调节通路。     

延髓内脏带至杏仁核投射通路参与迷走神经刺激抑制癫痫发作的研究

目的 观察迷走神经→延髓内脏带(MVZ)→杏仁中央核的儿茶酚胺能通路是否参与了迷走神经刺激(vagus nerve stimulation,VNS)抑制癫痫的调节；是否存在由迷走神经→延髓内脏带→海马的直接投射参与抑痫。方法 将逆行追踪剂WGA—HRP注入大鼠—侧杏仁中央核或腹侧海马，48h后，给予迷走神经刺激，观察MVZ内WGA—HRP逆行标记的细胞、Fos蛋白、TH阳性神经元的表达及分布。结果 杏仁核注射组大鼠MVZ内可见HPR／Fos／TH二重标记的细胞；海马注射组MVZ内未见HRP逆标神经元，但HRP逆行标记与Fos阳性双重标记细胞出现存隔区和下丘脑室旁该。结论 提示迷走神经→延髓内脏带→杏仁中央核的投射通路直接参与VNS抑痫过程，而且与儿茶酚胺能神经元有关；迷走神经→延髓内脏带→隔区、下丘脑室旁核中继至海马的间接通路也参与了抑痫。     

PS激发大鼠延髓内脏带星形胶质细胞GFAP和神经元FOS表达水平的变化

观察腹腔注射细菌内毒素 (LPS)后 ,大鼠延髓内脏带 (MVZ)星形胶质细胞胶质原纤维酸性蛋白 (GFAP)及神经元FOS表达水平随时间变化的规律及其相互关系。大鼠经LPS腹腔注射后 1,3,6,12h分别行固定取材制片。每个时间点的切片分为 3组 ,分别进行抗FOS、抗GFAP免疫组织化学染色及抗FOS/GFAP/酪氨酸羟化酶 (TH)三重免疫组织化学染色。结果表明 :①FOS反应在LPS注射后 3h达到高峰 ,阳性产物主要分布于MVZ内。②GFAP反应在注射后 1h即达到高峰 ,表现为胶质细胞肥大、数量增多。其分布与FOS基本相同。③三重染色观察到GFAP与FOS的多种聚集方式 (FOS/GFAP/TH ,FOS/GFAP ,GFAP/TH) ,FOS阳性神经元周围GFAP免疫反应产物更密集。提示星形胶质细胞对LPS起反应 ,其反应高峰的出现先于神经元     

红藻氨酸致痫大鼠海马Fos和GFAP的共同表达

目的 研究红藻氨酸（kainic acid,KA)诱导大鼠癫痫发作后海马（hippocampus,HI)内神经元和星形胶质细胞的时空效应性反应变化。方法 大鼠侧脑室内注射KA，用抗即刻早期基因Fos蛋白和抗胶质原纤维酸性蛋白（GFAP）的双重免疫荧光组织化学方法结合激光共聚焦显微镜技术，显示痫性发作后HI同一部位内反应性神经元与星形胶质细胞的分布。结果 KA诱导大鼠癫痫发作，HI内的Fos阳性神经元和GFAP阳性星形胶质细胞明显增多。两分布范围基本一致，且癫痫诱发30min后GFAP开始增多，1h达高峰；1h后Fos阳性产物开始增多；2h达高峰；部分Fos阳性神经元周围有GFAP免疫反应产物包绕，显示反应性神经元（Fos阳性）与反应性星形胶质细胞（GFAP阳性）之间关系密切。结论 HI内的神经元和星形胶质细胞与癫痫发作直接相关且存在相互关系。可能共同参与癫痫的发生及其调节。     

模拟失重大鼠延髓内脏带向下丘脑室旁核投射的儿茶酚胺能神经元表达Fos

确定延髓内脏带向下丘脑室旁核（PVN）投射通路是否参与对模拟失重的反应,用HRP逆行追踪结合抗Fos和抗栈氨酸羟化酶（TH）的免疫组织化学三重标记技术。观察4周模拟失重大鼠延髓内脏带向PVN投射的儿茶酚胺能神元Fos表达情况。发现有7种不同的标记细胞：HRP,Fos,TH单标细胞;Fos/HRP,Fos/TH,HRP/TH双标细胞;Fos/HRP/TH三标细胞,主要分布于延髓内脏带即延髓中尾段的孤束核和腹外侧区以及两者之间的网状结构。向PVN投射的神经元中有15.3%为Fos阳性细胞,即对失重起反应,而这些神经元中有62.6%为儿茶酚胺能神经元。结果显示,延髓内脏带投射至PVN的儿茶酚胺能神经元有些参与对失重的心血管反应。     

血栓素A_2受体在正常大鼠脑内的分布定位

目的探讨血栓素A2受体(TP)在正常大鼠脑内的表达分布特点。方法正常成年SD大鼠脑组织冰冻切片,TP免疫荧光染色,TP/神经核蛋白(Neu N),TP/胶质纤维酸性蛋白(GFAP),TP/谷氨酸脱羧酶67(GAD67)免疫荧光双标染色,观察TP在脑内分布表达情况。结果 TP免疫阳性产物主要分布在扣带回皮质、皮层Ⅲ~V层、下丘和小脑的浦肯野细胞层;免疫荧光双标结果显示TP与神经元胞核特异性标记物Neu N共存,但不与星形胶质细胞标记物GFAP共存;同时,所有TP阳性神经元表达γ氨基丁酸(GABA)能神经元标记物GAD67。结论 TP表达于大鼠扣带回皮质、皮层Ⅲ~V层、下丘脑和小脑的浦肯野细胞层,主要分布于GABA能神经元,提示TP可能参与了大鼠大脑GABA能神经元的功能调节和病变过程。     

GFAP和Fos蛋白在戊四氮致痫大鼠前脑中的表达变化

目的 研究大鼠在戊四氮导致癫痫发作时前脑内星形胶质细胞和神经元的形态学反应及其相互关系。方法 应用免疫组织化学单标记法分别显示前脑内GFAP和Fos蛋白表达的时间规律，并用免疫组织化学双重标记显示GFAP和Fos蛋白表达的相互关系。结果 在戊四氮导致大鼠癫痫发作早期，前脑的星形胶质细胞被激活，细胞体积增大，突起粗大，GFAP表达阳性，随着存活时间的变化，星形胶质细胞的反应经历先逐渐升高后降低的过程。被激活的星形胶质细胞和神经元表达Fos蛋白阳性，也呈现逐渐升高又降低的变化；另外，GFAP阳性星形胶质细胞和Fos阳性神经元在前脑主要分布在大脑皮层、海马、杏仁核等部位，二者的分布特征基本一致。结论 星形胶质细胞可能和神经元一起参与了戊四氮所致癫痫发作的变化。     

Distribution and Temporal Regulation of the Immune Response in the Rat Brain to Intracerebroventricular Injection of Interferon-γ

The response to intracerebroventricular administration of interferon (IFN)-γ was examined in the adult Wistar rat brain: major histocompatibility complex (MHC) antigens class I and II, CD8 and CD4 antigens, and the macrophage/microglia antigen OX42 were analyzed in respect to saline-injected cases over 1 week. The glial cell type expressing MHC antigens was characterized with double labeling. IFN-γ was thus found to induce MHC class I and II expression in microglia, identified by tomato lectin histochemistry, and not in GFAP-immunostained astrocytes. MHC antigen-expressing microglia was detected in the periventricular parenchyma, several fields of the cerebral cortex, cerebellum, major fiber tracts, and brainstem superficial parenchyma. Different gradients of density and staining intensity of the MHC-immunopositive elements were observed in these regions, in which MHC class I antigens persisted up to 1 week, when MHC class II induction had declined. Quantitative analysis pointed out the proliferation of OX42-immunoreactive cells in periventricular and basal brain regions. CD8+ T cells were observed in periventricular regions, basal forebrain, and fiber tracts 3 days after IFN-γ injection and their density markedly increased by 7 days. CD4+ T cells were also seen and they were fewer than CD8+ ones. However, numerous CD4+ microglial cells were observed in periventricular and subpial regions, especially 1 week after IFN-γ injection. Our data indicate that this proinflammatory cytokine mediatesin vivomicroglia activation and T cell infiltration in the brain and that the cells involved in this immune response display a regional selectivity and a different temporal regulation of antigen expression.     

Noxious somatic stimulation-induced expression of Fos-like immunoreactivity in catecholaminergic neurons with habenular nucleus projection in the medullary visceral zone of rat

In order to study the expression of Fos protein in catecholaminergic neurons in the medullary visceral zone (MVZ), which project to the habenular nucleus (HB), a triple-labeling method combining wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) retrograde tracing with anti-Fos and anti-tyrosine hydroxylase (TH) immunohistochemical staining was used in the rat. WGA-HRP was stereotaxically injected into unilateral HB. Forty-eight hours later, 50 μl of 8% formalin was injected into the foot pad of the right front paw. Two hours after formalin injection, animals were anesthetized and perfused transaortically. Coronal sections (40 μm) were cut from the cervical segment of spinal cord, the medulla oblongata and WGA-HRP injected area with a cryostat. First, sections of the injected area and the medulla oblongata were histochemically processed to demonstrate the presence of retrogradely transported WGA-HRP using the chromogen tetramethylbenzidine (TMB). Then sections of the spinal cord and the medulla oblongata were immunostained with anti-Fos and anti-TH antibodies using the ABC method. Under the light microscope, seven types of variously labeled neurons could be identified in MVZ, namely Fos and TH-immunoreactive (Fos- or TH-IL) neurons, WGA-HRP labeled ones, Fos/HRP, Fos/TH and HRP/TH double-labeled and Fos/HRP/TH triple-labeled cells. The results suggest that some catecholaminergic neurons in MVZ could send projections to HB and this pathway may be involved to relay nociceptive information from spinal cord to brainstem and on to the forebrain.     

Sexual behavior increases c-fos expression in the forebrain of the male rat.

The ability of a wide variety of pharmacological and physiological stimuli to increase neuronal expression of Fos has led to the suggestion that it might serve as a marker of neuronal activation. Psychomotor stimulants increase the release of dopamine from the terminals of nigrostriatal and mesolimbic neurons and enhance Fos immunoreactivity in the striatum and nucleus accumbens (NAc). Because sexual behavior also increases dopamine release in these and other forebrain regions, the present study examined the effect of copulation on Fos immunoreactivity in the forebrain of intact, sexually active male rats. Sexual behavior produced a striking increase in Fos immunoreactivity in the medial preoptic area (MPOA), NAc, bed nucleus of the stria terminalis and piriform cortex. However, no increase in Fos immunoreactivity was observed in the striatum. These results are consistent with neurochemical, physiological, and behavioral data suggesting that the MPOA and NAc are important substrates of sexual behavior.     

Differential Spatial Patterns of Fos Induction Following Generalized Clonic and Generalized Tonic Seizures

The expression of generalized clonic and generalized tonic seizures has been suggested to result from the activation of different and independent neuronal circuits. Using the induction of the c-fosprotein (Fos) as a marker of neuronal activity, we identified brain structures that are differentially associated with the expression of electroconvulsive shock-induced generalized clonic and generalized tonic seizures. Expression of either seizure phenotype resulted in a similar bilaterally symmetrical increase in Fos immunoreactivity in many forebrain structures, including the bed nucleus of the stria terminalis, hippocampal dentate gyrus, amygdala, and piriform cortex, compared to controls. However, following tonic hindlimb extension (THE), the degree of labeling in specific thalamic, hypothalamic, and brain stem areas was significantly greater than that of either controls or animals exhibiting clonic seizures. While a greater number of neurons in the hypothalamus (e.g., ventromedial nucleus), subparafascicular thalamic nucleus, peripeduncular area, deep medial superior colliculus, dorsal and lateral central gray, and paralemniscal nuclei were robustly labeled following THE, noticeably fewer cells were immunoreactive following face and forelimb clonic seizure behaviors. These differences were also found to be independent of the stimulus magnitude. In animals stimulated with the same current intensity but expressing either of the two seizure phenotypes, the pattern of Fos induction was consistent with the seizure phenotype expressed. These results demonstrate that specific subsets of neurons are differentially activated following the expression of different generalized seizure behaviors and that activity in discrete mesencephalic and diencephalic structures is more frequently associated with the expression of generalized tonic seizures than with the expression of generalized clonic seizures.     

Intracerebroventricular injection of senktide-induced Fos expression in vasopressin-containing hypothalamic neurons in the rat

Intracerebroventricular injection of senktide, a selective agonist for neurokinin B receptor (NK3), induced Fos expression in many neurons of the rat hypothalamus. Fos-positive neurons were predominantly present in the supraoptic and paraventricular hypothalamic nuclei, and some of them were seen in the lateral preoptic area, lateral hypothalamic area, arcuate nucleus, perifornical region, posterior hypothalamic area, circular nucleus, and along relatively large blood vessels (lateral hypothalamic perivascular nucleus) in the anterior hypothalamus. A double labeling study was performed to examine if vasopressin-containing neurons in the hypothalamus could be activated by the treatment. Neurons with both Fos-like immunoreactivity (-LI) and vasopressin-LI were found in the paraventricular nucleus, supraoptic nucleus, circular nucleus and lateral hypothalamic perivascular nucleus. In the supraoptic nucleus, about 87% of vasopressin-containing neurons exhibited Fos-LI, which corresponded to about 64% of Fos-positive neurons in the nucleus. In the paraventricular nucleus, about 80% of vasopressin-like immunoreactive neurons exhibited Fos-LI, which constituted about 51% of the total population of Fos-positive neurons in the region. The results suggest that NK3 receptor may be involved in the modulation of release of vasopressin from the hypothalamus in the rat.     

Importance of endogenous nitric oxide synthase in the rat hypothalamus and amygdala in mediating the response to capsaicin

Although capsaicin has been shown to activate certain neuronal groups in the hypothalamus and amygdala, the neurotransmitters involved and the exact mechanism of action are not clearly understood at present. The aim of this study was to examine the hypothesis that the effect of capsaicin in the rat hypothalamus and amygdala primarily involves direct activation of the endogenous nitric oxide synthase (NOS) neurons responsible for the synthesis of nitric oxide (NO). Subcutaneous capsaicin injection in male rats, compared with vehicle, caused a significant increase in Fos expression in the paraventricular nucleus (PVN), supraoptic nucleus (SON), and medial and cortical amygdala. The expression of nicotinamide adenine dinucleotide phosphate diaphorase, a histochemical marker for NOS, was also increased in these brain areas in addition to the periventricular and lateral hypothalamic area and central amygdaloid nucleus. Also, capsaicin significantly increased the expression of neuronal NOS messenger RNA and protein in the PVN, SON, and medial amygdala as demonstrated by in situ hybridization and immunohistochemistry, respectively. A higher proportion of the NOS neurons in the PVN, periventricular region, SON and amygdala showed Fos expression in response to capsaicin than vehicle injection. There was little, if any, Fos activation in the NOS-positive neurons in the lateral hypothalamic area. The capsaicin-induced activation of the hypothalamic PVN and SON neurons and the medial amygdaloid nucleus was attenuated in the NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) -pretreated animals in comparison with the inactive enantiomer D-NAME. These observations indicate that activation of the endogenous NOS system and production of NO constitute a major pathway through which capsaicin exerts its effect within the hypothalamus and amygdala.     

Coitus-induced activation of c-fos and gonadotropin-releasing hormone in hypothalamic neurons in female rabbits

Copulation induces hypothalamic release of neuropeptides and catecholamines, especially gonadotropin-releasing hormone (GnRH) and norepinephrine, in female rabbits. The forebrain distribution of GnRH cells and the cellular events responsible for the coitally induced GnRH surge have not been identified. We characterized the expression of c-fos mRNA before (0 min) and up to 60 min after coitus in forebrain tissues of mated and nonmated females and compared these findings with those in which single- and double-labeled GnRH/Fos protein cells were identified by immunocytochemistry (ICC). Enhanced expression of fos-mRNA occurred 30 min after coitus, especially in the anteroventral periventricular nucleus (AVPV), the encapsulated portion of the bed nucleus of the stria terminalis (BNSTe) and the ventrolateral hypothalamus (VLH); this increased fos-mRNA activity remained elevated at 60 min in the AVPV and VLH, and was reflected by Fos protein expression 90 min postcoitus. Both ICC Fos-labeled and ICC GnRH-labeled cells were widely distributed throughout the forebrain with postcoital increased double-labeling in the preoptic-septal areas, the anterior-medial hypothalamus and the VLH. The increased number of dual-labeled and unchanged number of single-labeled GnRH cells after coitus suggest some GnRH neurons were non-detected before coitus. Many dual-labeled neurons were adjacent to Fos-labeled cells, suggesting enhanced interneuronal input to GnRH cells after coitus. Collectively, the results suggest that coitus activates hypothalamic GnRH neurons via several loci that include the AVPV, BNSTe and VLH. The distinct anatomical location of the AVPV, BNSTe and VLH further suggests that coital signals may reach the hypothalamus via separate neural pathways that are likely developed within the brainstem.