GDNFR-α在成年大鼠脊髓和背根节的分布的免疫组织化学研究

为了探讨胶质细胞源性神经营养因子对脊髓运动神经元和感觉神经元的作用 ,本实验采用抗胶质细胞源性神经营养因子受体 -α多克隆抗体免疫组织化学方法 ,观察了胶质细胞源性神经营养因子受体 -α在成年大鼠腰段脊髓和背根节的分布。结果发现 :胶质细胞源性神经营养因子受体 -α免疫反应出现于成年大鼠腰段脊髓神经元和背根节神经元中。提示 :胶质细胞源性神经营养因子受体 -α可能对脊髓运动神经元、背根节神经元具有一定的调节作用     

大麻素Ⅱ型受体(CB2)在人皮肤及皮肤恶性肿瘤的组织分布

目的:研究大麻CB2受体在人皮肤及皮肤恶性肿瘤的组织分布.方法:用兔抗人CB2受体抗体做免疫组化,检测人类皮肤及皮肤恶性肿瘤组织CB2受体表达分布情况.结果:CB2受体在人类皮肤及皮肤恶性肿瘤均有表达分布,且皮肤恶性肿瘤的CB2受体过度表达.结论:大麻素受体CB2的表达与皮肤恶性黑素瘤、基底细胞癌、鳞状细胞癌关系密切.     

内源性大麻素含量升高可激活脊髓背角星形胶质细胞并导致机械性触诱发痛

目的:观察丁高大鼠鞘内大麻素水平后实验动物的疼痛行为学变化及脊髓背角星形胶质细胞的激活状态,探讨内源性大麻素参与触诱发痛的可能机制。方法:成功建立大鼠鞘内置管模型后,分别鞘内注射外源性大麻素2-AG、大麻素受体激动剂CP55940、大麻素水解酶抑制剂JZL195。使用Von-frey纤维丝观察给药后不同时间点大鼠机械性缩足反射阈值的变化,运用共聚焦显影观察脊髓背角星形胶质细胞的激活状态。结果:(1)鞘内注射2-AG、CP55940、JZLl95后1 d即产生明显的触诱发痛(P0.01)并至少持续到给药后第21 d(P0.001);(2)鞘内注射2-AG、CP55940和JZLl95后第5 d即有明显的星形胶质细胞激活(P0.01),并可持续到给药后第21 d(P0.01)。结论:升高鞘内大麻素水平可能通过激活星形胶质细胞导致触诱发痛。     

NG2细胞的研究进展

NG2细胞,是在发育和成年中枢神经系统的灰质和白质束中发现的,因表达NG2蛋白多糖而得名.NG2细胞先前被假定代表少突胶质细胞前体细胞,后来使用转基因小鼠的研究表明,NG2细胞在体内不仅能够产生少突胶质细胞,还能产生原浆性星形胶质细胞,以及某些情况下的神经元[1].NG2细胞是中枢神经系统中不同于神经元、成熟少突胶质细胞、星形胶质细胞和小胶质细胞的一类细胞亚群.此外,在发育和成年中枢神经系统的多个区域,发现NG2细胞和神经元之间存在独特的突触联系,暗示NG2细胞除了可能作为分化成多种细胞的可塑性前体细胞群,还可能会和神经元联系从而形成一个独特的神经胶质网络[2].进行综述对NG2细胞的近年研究,以及其功能特性和在神经网络中所扮演的角色.     

巢蛋白在成年C57小鼠和SD大鼠中枢神经系统中的分布及比较

目的 观察巢蛋白(nestin)在成年C57小鼠和SD大鼠中枢神经系统中的分布，探讨并比较神经干细胞在两种啮齿类动物中分布的异同。方法 采用免疫组织化学ABC法和间接免疫荧光法，显示含神经干细胞特征性的标志物巢蛋白的阳性结构在上述两种啮齿类动物中枢神经系统中的分布。结果 在成年C57小鼠中枢神经系统中，巢蛋白在整个脑室系统周围均有表达，免疫阳性结构位于室管膜细胞胞体及其突起，并呈现由吻端向尾端渐次减少的趋势。在成年SD大鼠的脑室系统中，巢蛋白主要分布在侧脑室外侧壁及其周围脑区。此外，在两者的齿状回、穹窿下器、最后区等脑结构中也有巢蛋白的表达。成年C57小鼠中枢神经系统中巢蛋白的表达，无论在免疫阳性结构的数量上还是在染色强度上都明显多于和强于成年SD大鼠。结论 巢蛋白在成年C57小鼠中枢神经系统中的广泛脑区均有表达，而在成年SD大鼠中仅见于有限的脑区，提示成年C57小鼠中枢神经系统可能具有较强的神经可塑性和损伤修复能力。     

GFR-α1在Parkinson病大鼠黑质区的表达

本文观察了胶质细胞源性神经营养因子受体-α1(GFR-α1)在Parkinson病(PD)大鼠黑质区的表达变化。分别向大鼠左侧尾壳核和黑质致密部、中脑腹侧被盖区内注射6-羟基多巴胺(6-OHDA)建立部分损伤模型和完全损伤模型后,取大鼠黑质区脑组织作冰冻切片,行GFR-α1和TH免疫组织化学染色,用图像分析系统进行细胞计数。结果表明,在部分损伤组和完全损伤组中,GFR-α1在黑质区神经元及胶质细胞中均有表达,且部分损伤组GFR-α1阳性细胞的分布、数量随受损时程的不同出现差异。TH阳性神经元形态学改变,数量的变化与GFR-α1阳性细胞的分布、数量有着密切的关系。以上结果提示,GFR-α1的表达可能与脑内受损部位细胞的营养、修复、再生有关。     

体内和体外条件下Nogo-A在大鼠小脑颗粒神经元上表达差异的研究

Nogo-A是网膜家族蛋白的成员之一,在抑制成年哺乳动物中枢神经系统损伤后轴突再生的过程中发挥着重要作用。Nogo-A表达于寡突胶质细胞和多种神经元,但在成年动物的小脑颗粒神经元中却未检测到。为探讨Nogo-A在小脑颗粒神经元上的表达情况及其影响因素,本实验应用免疫荧光组织化学染色法研究了Nogo-A蛋白在新生大鼠脑切片上和不同体外培养条件下小脑颗粒神经元中的表达。结果显示:在体条件下Nogo-A蛋白在新生大鼠的小脑颗粒神经元上的表达逐渐减少,至新生14d时检测不到;而在体外培养的来源于新生7d大鼠的小脑颗粒神经元中Nogo-A蛋白持续表达,可维持到14d;与胶质细胞共培养,或加入胶质细胞培养上清的小脑颗粒神经元仍然表达Nogo-A蛋白。本研究结果表明,体内和体外两种条件下Nogo-A蛋白在小脑颗粒神经元上的表达存在差异,新生期Nogo-A在小脑颗粒神经元上的表达下调可能与Purkinje细胞有关,提示Nogo-A在生后发育过程中可能与神经元的迁移或突触的形成密切相关。     

大麻素抑制背根节神经元ATP诱发的[Ca~(2+)]i升高

目的:观察大麻素对背根节神经元ATP诱发的[Ca~(2+)]i升高的影响及机制。方法:培养SD大鼠背根节神经元,采用激光共聚焦技术检测培养神经元[Ca~(2+)]i的变化。结果:ATP(100μmol/L)经P2X受体介导可导致培养的背根节神经元[Ca~(2+)]i增高(P0.05);大麻素受体激动剂CP55940预孵育10 min可剂量依赖性地抑制背根节神经元ATP所致的[Ca~(2+)]i升高(P0.05);CB1受体(cannabinoid receptor 1,B1R)的拮抗剂AM251(10μmol/L)、CB2受体(Cannabinoid receptor 2,CB2R)的拮抗剂AM630(10μmol/L)均可显著降低CP55940(1μmol/L)的抑制效应(P0.05);腺苷酸环化酶激动剂Forskolin(10μmol/L)可逆转CP55940对ATP的抑制作用(P0.05)。结论:CP55940可显著抑制背根节神经元ATP诱发的[Ca~(2+)]i升高,CP55940的抑制效应可能是由CB1、CB2受体介导抑制背根节神经元PKA活性所致。     

NG2胶质细胞在中枢神经系统病变中作用的研究进展

NG2胶质细胞是中枢神经系统中除星形胶质细胞、小胶质细胞和少突胶质细胞外的第四类胶质细胞,均匀地分布在整个脑区。当中枢神经系统发生损伤时,NG2胶质细胞能通过改变其细胞形态、增殖和分化对多种损伤类型作出反应。弄清NG2胶质细胞对不同损伤的反应机制,有助于更好地发挥其在损伤修复和髓鞘再生中的作用。本文着重讨论了NG2胶质细胞在几种损伤类型中的反应。     

AM1241抑制ADPβS诱发培养大鼠脊髓背角小胶质细胞P2Y_(12)和P2Y_(13)受体表达及炎症因子释放

目的:离体条件下,观察大麻素CB2受体激动剂AM1241对嘌呤P2Y受体激动剂ADPβS诱发的脊髓背角小胶质细胞P2Y_(12)和P2Y_(13)受体mRNA表达以及炎症因子IL-1β、IL-6和TNF-α释放的影响。方法:培养纯化新生SD大鼠(3 d)脊髓背角小胶质细胞,荧光定量PCR技术检测AM1241(10~(-5)mol/L,1 h)对ADPβS(10~(-5)mol/L,3 h)诱发的背角小胶质细胞P2Y_(12)和P2Y_(13)受体mRNA表达的影响;ELISA技术检测AM1241(10~(-5)mol/L,1 h)对ADPβS(10~(-5)mol/L,3 h)诱发的小胶质细胞IL-1β、IL-6和TNF-α释放的影响。结果:与正常对照组相比,ADPβS(10~(-5)mol/L,3 h)可以刺激脊髓背角小胶质细胞P2Y_(12)和P2Y_(13)受体在mRNA水平表达上调(P0.05),IL-1β、IL-6和TNF-α释放相应增加(P0.05)。AM1241(10~(-5)mol/L,1 h)几乎完全阻断ADPβS刺激小胶质细胞P2Y_(12)和P2Y_(13)受体mRNA表达上调以及IL-1β、IL-6和TNF-α释放的效应(P0.05);AM1241的这种抑制效应可以被CB2受体拮抗剂AM630(10~(-5)mol/L,1 h)反转(P0.05)。结论:大麻素CB2受体激活可以抑制ADPβS诱发的脊髓背角小胶质细胞P2Y_(12)和P2Y_(13)受体mRNA水平表达上调以及IL-1β、IL-6和TNF-α释放。     

Cannabinoid receptors in microglia of the central nervous system: immune functional relevance

Microglia, resident macrophages of the brain, function as immune effector and accessory cells. Paradoxically, they not only play a role in host defense and tissue repair but also have been implicated in a variety of neuropathological processes. Microglia, in addition to exhibiting phenotypic markers for macrophages, express CB1 and CB2 cannabinoid receptors. Recent studies suggest the existence of a third, yet-to-be cloned, non-CB1, non-CB2 cannabinoid receptor. These receptors appear to be functionally relevant within defined windows of microglial activation state and have been implicated as linked to cannabinoid modulation of chemokine and cytokine expression. The recognition that microglia express cannabinoid receptors and that their activation results in modulation of select cellular activities suggests that they may be amenable to therapeutic manipulation for ablating untoward inflammatory responses in the central nervous system.     

Cannabinoid receptors and pain

Mammalian tissues contain at least two types of cannabinoid receptor, CB(1) and CB(2), both coupled to G proteins. CB(1) receptors are expressed mainly by neurones of the central and peripheral nervous system whereas CB(2) receptors occur centrally and peripherally in certain non-neuronal tissues, particularly in immune cells. The existence of endogenous ligands for cannabinoid receptors has also been demonstrated. The discovery of this 'endocannabinoid system' has prompted the development of a range of novel cannabinoid receptor agonists and antagonists, including several that show marked selectivity for CB(1) or CB(2) receptors. It has also been paralleled by a renewed interest in cannabinoid-induced antinociception. This review summarizes current knowledge about the ability of cannabinoids to produce antinociception in animal models of acute pain as well as about the ability of these drugs to suppress signs of tonic pain induced in animals by nerve damage or by the injection of an inflammatory agent. Particular attention is paid to the types of pain against which cannabinoids may be effective, the distribution pattern of cannabinoid receptors in central and peripheral pain pathways and the part that these receptors play in cannabinoid-induced antinociception. The possibility that antinociception can be mediated by cannabinoid receptors other than CB(1) and CB(2) receptors, for example CB(2)-like receptors, is also discussed as is the evidence firstly that one endogenous cannabinoid, anandamide, produces antinociception through mechanisms that differ from those of other types of cannabinoid, for example by acting on vanilloid receptors, and secondly that the endocannabinoid system has physiological and/or pathophysiological roles in the modulation of pain.     

Characterization of CB1 cannabinoid receptor immunoreactivity in postmortem human brain homogenates

The CB1 cannabinoid receptor (CB1) is the predominant type of cannabinoid receptor in the CNS, in which it displays a unique anatomical distribution and is present at higher densities than most other known seven transmembrane domain receptors. Nevertheless, as with almost all seven transmembrane domain receptors, the tertiary and quaternary structure of this receptor is still unknown. Studies of CB1 in rat cerebral tissue are scarce, and even less is known regarding the expression of CB1 in the human brain. Thus, the aim of the present work was to characterize CB1 expression in membranes from postmortem human brain using specific antisera raised against this protein. Western blot analysis of P1 and P2 fractions, and crude plasma membrane preparations from the prefrontal cortex showed that CB1 migrated as a 60 kDa monomer under reducing conditions. These data were confirmed by blotting experiments carried out with human U373MG astrocytoma cells as a positive control for CB1 expression and wild-type CHO cells as negative control. In addition, when proteins were solubilized in the absence of dithiothreitol, the anti-human CB1 antiserum detected a new band migrating at around 120 kDa corresponding in size to a putative CB1 dimer. This band was sensitive to reducing agents (50 mM dithiothreitol) and showed sodium dodecylsulphate stability, suggesting the existence of disulfide-linked CB1 dimers in the membrane preparations. Important differences in the anatomical distribution of CB1 were observed with regard to that described previously in monkey and rat; in the human brain, CB1 levels were higher in cortex and caudate than in the cerebellum.     

A light and electron microscopic study of the CB1 cannabinoid receptor in primate brain.

The immunohistochemical distribution and subcellular localization of the cannabinoid CB1 receptor was determined in the adult monkey using a polyclonal antiserum raised against the amino terminus of the rat CB1 receptor. At the level of light microscopy, our results generally parallel earlier studies investigating CB1 distribution in rodent brain with a few differences. In particular, high levels of receptor were found in the cortex, hippocampus, amygdala, cerebellum. However significant differences were also noted. The most striking differences were high levels of CB1 receptor in the monkey substantia nigra pars compacta, cerebellar Purkinje cells, and the principal cells of the hippocampus, while few receptors were found in the globus pallidus or substantia nigra pars reticulata. In contrast, in a previous study investigating the rat, using the same antibody, the opposite staining pattern was observed. At the electron microscopic level CB1 receptor was restricted to neurons. Here it was found both pre- and postsynaptically, particularly on dendritic spines and axon terminals. The CB1 receptor is widely distributed in higher brain regions in the monkey. While its distribution is similar to that in the rat, there are major differences, some of which may be significant when extrapolating the behavioral effects of cannabinoids observed in rodents to primates (e.g., humans). The ultrastructural localization of the CB1 receptor suggests that it modulates neuronal excitability by both pre- and postsynaptic mechanisms.     

Cerebral hypoxia-ischemia and middle cerebral artery occlusion induce expression of the cannabinoid CB2 receptor in the brain

Until recently the cannabinoid CB2 receptor was believed to be absent from the central nervous system. In this study we have identified CB2 expressing cells that appear in the rat brain following stroke and hypoxic-ischemia. At 3 days following surgery CB2-positive macrophages, deriving from resident microglia and/or invading monocytes appear on the lesioned side of the brain. By day 7, a mixed population of CB2-positive cells is present. Microglia-derived macrophages are the key cells in the first stages of brain inflammation, and a pivotal step in the neurodegeneration that follows the acute stage of injury. Thus, CB2 may be important in the brain during injury, and in inflammatory neurodegenerative disorders. The presence of CB2-positive cells in the brain following stroke may provide a novel strategy for cannabinoid-mediated intervention into stroke induced neurodegeneration without the psychoactive effects of CB1 receptor stimulation.     

Functional expression of cell surface cannabinoid CB(1) receptors on presynaptic inhibitory terminals in cultured rat hippocampal neurons

At present, little is known about the mechanisms by which cannabinoids exert their effects on the central nervous system. In this study, fluorescence imaging and electrophysiological techniques were used to investigate the functional relationship between cell surface cannabinoid type 1 (CB(1)) receptors and GABAergic synaptic transmission in cultured hippocampal neurons. CB(1) receptors were labelled on living neurons using a polyclonal antibody directed against the N-terminal 77 amino acid residues of the rat cloned CB(1) receptor. Highly punctate CB(1) receptor labelling was observed on fine axons and at axonal growth cones, with little somatic labelling. The majority of these sites were associated with synaptic terminals, identified either with immunohistochemical markers or by using the styryl dye FM1-43 to label synaptic vesicles that had undergone active turnover. Dual labelling of neurons for CB(1) receptors with either the inhibitory neurotransmitter GABA or its synthesising enzyme glutamate decarboxylase, demonstrated a strong correspondence. The immunocytochemical data was supported by functional studies using whole-cell patch-clamp recordings of miniature inhibitory postsynaptic currents (mIPSCs). The cannabinoid agonist WIN55,212-2 (100nM) markedly inhibited (by 77+/-6.3%) the frequency of pharmacologically-isolated GABAergic mIPSCs. The effects of WIN55,212-2 were blocked in the presence of the selective CB(1) receptor antagonist SR141716A (100nM).In conclusion, the present data show that cell surface CB(1) receptors are expressed at presynaptic GABAergic terminals, where their activation inhibits GABA release. Their presence on growth cones could indicate a role in the targeting of inhibitory connections during development.     

Adenosine A2A receptor activation stimulates collagen production in sclerodermic dermal fibroblasts either directly and through a cross-talk with the cannabinoid system

Systemic sclerosis (SSc) is a connective tissue disease characterised by exaggerated collagen deposition in the skin and visceral organs. Adenosine A2A receptor stimulation (A2Ar) promotes dermal fibrosis, while the cannabinoid system modulates fibrogenesis in vitro and in animal models of SSc. Moreover, evidence in central nervous system suggests that A2A and cannabinoid (CB1) receptors may physically and functionally interact. On this basis, we investigated A2Ar expression and function in modulating collagen biosynthesis from SSc dermal fibroblasts and analysed the cross-talk with cannabinoid receptors. In sclerodermic cells, A2Ar expression (RT-PCR, Western blotting) was evaluated together with the effects of A2A agonists and/or antagonists on collagen biosynthesis (EIA, Western blotting). Putative physical and functional interactions between the A2A and cannabinoid receptors were respectively assessed by co-immuno-precipitation and co-incubating the cells with the unselective cannabinoid agonist WIN55,212-2, and the selective A2A antagonist ZM-241385. In SSc fibroblasts, (1) the A2Ar is overexpressed and its occupancy with the selective agonist CGS-21680 increases collagen production, myofibroblast trans-differentiation, and ERK-1/2 phosphorylation; (2) the A2Ar forms an heteromer with the cannabinoid CB1 receptor; and (3) unselective cannabinoid receptor stimulation with a per se ineffective dose of WIN55,212-2, results in a marked anti-fibrotic effect after A2Ar blockage. In conclusion, A2Ar stimulation induces a pro-fibrotic phenotype in SSc dermal fibroblasts, either directly, and indirectly, by activating the CB1 cannabinoid receptor. These findings increase our knowledge of the pathophysiology of sclerodermic fibrosis also further suggesting a new therapeutic approach to the disease.     

Novel cannabinoid-sensitive receptor mediates inhibition of glutamatergic synaptic transmission in the hippocampus

Psychoactive effects of cannabinoids are thought to be mediated, at least in part, by suppression of both glutamate and GABA release via CB1 cannabinoid receptor. Two types of cannabinoid receptor (CB1 and CB2) have been cloned so far. The CB1 receptors are abundantly expressed in the nervous system, whereas CB2 receptors are limited to lymphoid organs (Matsuda et al., 1990; Munro et al., 1993). Immunocytochemical and electrophysiological studies revealed that in the hippocampus CB1 receptors are expressed on axon terminals of GABAergic inhibitory interneurons (Tsou et al., 1999; Katona et al., 1999) and activation of these receptors decreases GABA release (Hájos et al., 2000). Other physiological studies pointed out the involvement of CB1 receptors in the modulation of hippocampal glutamatergic synaptic transmission and long-term potentiation (Stella et al., 1997; Misner and Sullivan, 1999), but anatomical studies could not confirm the existence of CB1 receptors on glutamatergic terminals. Here we examined cannabinoid actions on both glutamatergic and GABAergic synaptic transmission in the hippocampus of wild type (CB1+/+) and CB1 receptor knockout mice (CB1-/-). The synthetic cannabinoid agonist WIN55,212-2 reduced the amplitudes of excitatory postsynaptic currents in both wild type and CB1-/- mice, while inhibitory postsynaptic currents were decreased only in wild type mice, but not in CB1-/- animals. Our findings are consistent with a CB1 cannabinoid receptor-dependent modulation of GABAergic postsynaptic currents, but a novel cannabinoid-sensitive receptor must be responsible for the inhibition of glutamatergic neurotransmission.     

Distribution of 1,25-dihydroxyvitamin D3 receptor immunoreactivity in the rat brain and spinal cord

A complete mapping study on the 1,25-dihydroxyvitamin D3 receptor immunoreactivity within the rat central nervous system was performed with a monoclonal and a polyclonal antibody. Specific immunostaining was observed within both nuclear and cytoplasmic compartments of a variety of cells in the cerebellum, mesopontine area, diencephalon, cortex, spinal cord, and limbic system. Both monoclonal and polyclonal antibodies provided similar staining patterns. The monoclonal antibody stained distinct domains within the nuclei of all and the cytoplasm of specific neuronal cell types, like motor neurons, Purkinje cells, and pyramidal cells of the cortex more clearly than the polyclonal antibody. The expression of vitamin D3 receptor in the rat central nervous system was confirmed by in situ hybridisation. The widespread distribution of vitamin D3 receptor in distinct portions of the sensory, motor, and limbic brain systems suggests multiple functional properties of 1,25-dihydroxyvitamin D3 in the central nervous system.