Immunocytochemical localization of progesterone receptor in the guinea pig central nervous system

The distribution of neurons containing progesterone receptors in the brain of guinea pigs ovariectomized and primed by estradiol was investigated immunohistochemically using monoclonal antibodies to the progesterone receptor. We found that the picric acid paraformaldehyde perfusion provided satisfactory conditions of fixation for visualizing the progesterone receptor in sections of frozen tissue. Among the different techniques of immunocytochemical detection used, the indirect antibody peroxidase-antiperoxidase method gave the best results. The immunostained neurons were mainly located in two specific regions of the hypothalamus: the preoptic area and the mediobasal hypothalamus. Within the preoptic region, the majority of immunoreactive cells were present in the nucleus preopticus periventricularis particularly at the level of the pars suprachiasmatica. Within the mediobasal hypothalamus, immunostained neurons were found in the nucleus periventricularis, arcuatus, ventromedialis and premamillaris. Differences in the intensity of immunoreactivity appeared from one region to another. A marked cellular heterogeneity was observed: in each neuroanatomical structure, labeled cells alternated with non-labeled cells. The receptor, even in absence of progesterone, was localized in the nucleus. The nucleolus was not stained and only neurons were labeled. There was no progesterone receptor immunoreactivity in other regions of the brain, especially in the amygdala, hippocampus and cortex where biochemical studies have shown the presence of a non-estrogen regulated protein binding the progestin [3H]R 5020. Control experiments with antibody pretreated with purified progesterone receptor or with mouse receptor-unrelated monoclonal antibody did not show fluorescent or immunoreactive cells.     

Localization of ATP-gated P2X2 receptor immunoreactivity in the rat hypothalamus

Previous pharmacological studies have indicated that ATP receptors may be involved in the regulation of physiological functions in hypothalamus. In the present study, the distribution of P2X₂ receptor in the rat hypothalamus was studied with immunohistochemistry. It was shown that P2X₂ immunoreactivity-positive neurons and nerve fibres were localized in many hypothalamic nuclei. Intense labelling of both neuronal cell bodies and nerve fibres was observed in the paraventricular nucleus, arcuate nucleus, retrochiasmatic area, periventricular nucleus, and the ventral part of tuber cinereum area. In supraoptic, circular, and ventral tuberomammillary nuclei the neuronal cell bodies were strongly positive, but few nerve fibres were positive. Axons with strong P2X₂ immunoreactivity were found in the organum vasculosum of the lamina terminalis and median eminence. Some scattered positive neurons and nerve fibres were found in many hypothalamic nuclei including preoptic nucleus. The results of the present study demonstrated the existence of P2X receptors in hypothalamus, as a basis for detailed studies of the roles of P2X receptors in the regulation of hypothalamic functions.     

从成年大鼠中枢神经系统不同区域分离NG2蛋白聚糖阳性神经祖细胞

目的探索从成年大鼠中枢神经系统(CNS)不同区域是否能分离培养出NG2蛋白聚糖阳性神经祖细胞(NG2细胞)。方法从成年雌性大鼠解剖出CNS的9个不同区域,分别经木瓜蛋白酶消化和Optiprep不连续梯度离心,从离心产生的组织细胞密集带,用含B27添加剂和碱性成纤维细胞生长因子2(FGF2)的NeurobasalA培养液,分离培养出增殖性细胞,再以免疫荧光双重染色法鉴定细胞性质。结果应用上述方法,可从成年大鼠CNS的9个不同区域分离出具神经干细胞(NSCs)潜能的NG2细胞。结论成年大鼠CNS的非神经发生区域同样存在NSCs样细胞,且可通过适当方法体外培养。     

儿童抗MOG抗体及抗NMDAR抗体双阳性中枢神经系统脱髓鞘1例报道并文献复习

目的 探讨缺血性脑卒中(Cerebral ischemic stroke,CIS)患者发生睡眠呼吸紊乱(Sleep-disordered breathing,SDB)的影响因素。方法 纳入2018年2月-2019年4月本院神经内科收治的288例首次发病的CIS患者,于发病后的第7 d左右采用Apnea Link^TM睡眠测试装置整夜连续进行呼吸睡眠检测,发生SDB的患者[呼吸暂停-低通气指数(Apnoea-hypopnoea index,AHI)≥10]纳入研究组,未发生SDB的患者纳入对照组,考察CIS患者中SDB的发生率,采用单因素分析的方法比较发生SDB的可疑危险因素,对于单因素分析有意义的自变量,采用Logistic回归逐步向前法进一步识别SDB的独立影响因素。结果 CIS患者中SDB的发病率为63.1%。多因素Logistic回归分析显示,年龄≥65岁[OR(95%CI)=2.078(1.317～3.28),P=0.002]、脑干病变[OR(95%CI)=2.306(1.418～3.75),P=0.001]、NIHSS得分[OR(95%CI)=2.368(1.34～4.185),P=0.003]、mRS得分[OR(95%CI)=2.033(1.146～3.606),P=0.015]和发生吞咽困难[OR(95%CI)=2.392(1.031～5.545),P=0.042]是脑卒中患者发生SDB的独立危险因素。结论 脑干损伤是脑卒中患者发生SDB的主要危险因素,而SDB加重了急性期缺血性脑卒中患者的神经系统症状,老年缺血性脑卒中患者是SDB的易感人群。对脑干损伤的老年患者,要重视早期监测呼吸睡眠功能,发生SDB迹象时要及时采取干预措施。     

Characterisation of central adenosine A1 receptors and adenosine transporters in mice lacking the adenosine A2a receptor

The present study was designed to assess whether adenosine A_2a receptor knockout mice exhibit altered purine utilisation in brain nuclei. Specifically, the properties of adenosine transporters and adenosine A₁ receptors were characterised in brain membranes and on slide-mounted sections. The B_MAX for [³H]nitrobenzylthioinosine ([³H]NBTI) binding (adenosine transporter density) was significantly reduced in brainstem membranes of homozygotes (560±52 fmol/mg protein, n=5, P<0.05, Kruskal–Wallis ANOVA) compared to wildtype (1239±213 fmol/mg protein) and heterozygous mice (1300±558 fmol/mg protein). Quantitative autoradiography data indicated that [³H]NBTI binding in the medulla oblongata of heterozygous mice was seen to decrease significantly (P<0.05) in the subpostremal nucleus tractus solitarius (NTS), medial NTS, inferior olive and area postrema (AP). On the other hand, in the homozygous mice a decrease was seen in the medial NTS and AP. In the pons, [³H]1,3-dipropyl-8-cyclopentylxanthine ([³H]DPCPX) (adenosine A₁ receptor density) binding increased significantly (P<0.05, Kruskal–Wallis ANOVA) in the lateral parabrachial nucleus, caudal pontine reticular nucleus and locus coeruleus of homozygotes compared to wildtype. In higher brain centres, [³H]NBTI binding was reduced in the paraventricular thalamic nucleus of both heterozygous and homozygous mice, whereas [³H]DPCPX binding was reduced in the hippocampus and lateral hypothalamus of heterozygotes. In homozygotes, [³H]DPCPX binding in the hippocampus increased compared to wildtype mice. The present study indicates that deletion of the A_2a receptor may have contributed to region-specific compensatory changes in purine utilisation in brain nuclei associated with autonomic, neuroendocrine and behavioural regulation.     

Distribution of hippocampal cholinergic neurostimulating peptide (HCNP) immunoreactivity in the central nervous system of the rat

Hippocampal cholinergic neurostimulating peptide (HCNP), an undecapeptide isolated from the hippocampal tissue of young rats, enhances the cholinergic development in explant cultures of medial septal nuclei. This report concerns the distribution of HCNP immunoreactivity in the central nervous system (CNS) of 11- and 28-day-old Wistar rats; two affinity-purified anti HCNP antibodies were used. Immunoblot analyses of extracts of different regions of the brain revealed a single 23 kDa band that corresponded to the presumed HCNP precursor protein. Immunostaining of the various CNS structures of the 28-day-old rats was more intense than in those of 11-day-old animals. HCNP immunoreactivity was detected in neurons as well as in glia cells, particularly oligodendroglia. The perikarya of neurons in the cerebral cortex, hippocampus, limbic cortex, caudate, putamen, arcuate nucleus of hypothalamus, trigeminal subnuclei, rostroventrolateral reticular nucleus and dorsal horn of the spinal cord were positively stained. In addition, nerve fibers and terminals in the hypothalamic subnuclei, zona incerta, thalamic subnucleus, caudate, putamen, locus coeruleus, trigeminal subnuclei, dorsal motor nucleus of the vagus, dorsal horn of the spinal cord and intermediolateral column also displayed HCNP immunoreactivity. These observations would suggest that HCNP and its related molecules may have multifunctional roles in the CNS.     

The distribution of cholecystokinin-8 in the central nervous system of turtles: An immunohistochemical and biochemical study

Immunohistochemical techniques, radioimmunoassay (RIA) and high performance liquid chromatography (HPLC) were used to: (1) determine the regional distribution and amounts of cholecystokinin-8 (CCK8)-like immunoreactivity in the turtle central nervous system, and (2) chemically characterize the CCK8-like material present in the turtle central nervous system. High levels of CCK8-like immunoreactivity were found in the turtle central nervous system, with the highest levels being present in the hypothalamus and neurohypophysis. Moderate levels of the CCK8-like material were found in all other regions of the turtle nervous system except the cerebellum, the olfactory bulbs and the dorsal ventricular ridge of the telencephalon, which contained low levels. The bulk (87%) of the CCK8-like material in turtle central nervous system co-eluted with CCK8-sulfate in gradient elution HPLC. The distribution of CCK8-like immunoreactivity (CCK8LI) observed using immunohistochemistry was consistent with the results of the RIA studies. Numerous CCK8LI-containing neurons and fibers were observed in the hypothalamus and neurohypophysis. Neurons and fibers containing CCK8 were, however, more sparsely distributed outside the hypothalamus. The immunohistochemical data provided evidence for the existence of two major CCK8-containing pathways in turtles that have been previously described in mammals: a pathway from the supraoptic and paraventricular magnocellular nuclei to the external zone of the median eminence and neurohypophysis and a pathway from dorsal root ganglia to the dorsal horn of the spinal cord. Overall, the present results, in conjunction with several previous studies, indicate that CCK8 has had a relatively stable evolutionary history as a CNS neuropeptide among land vertebrates. The molecular structure of CCK8 appears to have been largely (if not entirely) conserved, as has its concentration in many brain regions. A noteworthy exception to such conservatism in the localization of CCK8 is that the concentration of CCK8 in the telencephalon, particularly in the telencephalic cortex, is much lower in turtles than in mammals. The present results therefore suggest that CCK8 may not have become a prominent peptide in the telencephalic cortex (or its anatomical equivalents) until the evolution of neocortex in the mammalian lineage.     

Autoradiographic analysis of mu1, mu2, and delta opioid binding in the central nervous system of C57BL/6BY and CXBK (opioid receptor-deficient) mice

The recent development of in vitro autoradiography techniques has enabled investigators to determine the distribution and relative levels of multiple ligand binding sites in discrete anatomical areas. In this study we used semi-quantitative in vitro autoradiography to compare the levels of binding to central mu₁, mu₂, and delta opioids sites in two strains of mice, C57BL/6BY and CXBK. The CXBK strain is known to be deficient in whole brain opioid binding sites and to be less sensitive than the C57 strain to the analgesic and locomotor stimulatory effects of opiates and opioids. Delta sites were visualized using [³H][d-Ala²-d-Leu⁵]-enkephalin (DADL) plus a low concentration of morphine, total mu sites (mu₁ and mu₂) were visualized using [³H]dihydromorphine (DHM), and mu₂ sites were visualized using [³H]DHM plus a low concentration of DADL. Binding to mu₁ sites was determined by subtracting mu₂ binding from total mu binding. We found that the two strains did not consistently differ in the levels of delta sites; in some areas the CXBKs had lower levels but in many areas they had levels equal to or greater than those for the C57s. The CXBK strain, however, either had less or the same amount of mu binding as the C57 strain in all areas studied. The CXBK strain was especially deficient in mu₁ binding, particularly in areas involved in pain processing.     

1,25-Dihydroxyvitamin D3 receptors in the central nervous system of the rat embryo

We have mapped areas within the central nervous system (CNS) of the developing fetal rat which immunostain for the 1,25-dihydroxyvitamin D₃ receptor (VDR). The VDR was detected from days 12 to 21 of gestation throughout the CNS; immunostaining was particularly intense in the neuroepithelium and within the differentiating fields of various areas of the brain. Cells within the spinal cord, dorsal root, and other ganglia exhibited positive staining for the VDR. The intensity of staining for the VDR diminished or disappeared in the neuroepithelium throughout the CNS during the later days of development, while in the differentiating fields single VDR immunoreactive cells were observed. The presence of the VDR in the CNS was confirmed by in situ hybridization and RNA-based polymerase chain reaction methods with di-deoxy sequencing of the resultant DNA product. These results support the hypothesis that 1,25-dihydroxyvitamin D₃, through interactions with the VDR, may play a role in the development of the CNS.     

Tentative identification of a second central nervous system myelin membrane autoantigen (M2) by a biochemical comparison with the basic protein (BP)

Two central nervous system myelin autoantigens, M2 and basic protein (BP), were examined, using complement-fixing antibodies against each autoantigen as markers on myelin. M2 activity was very labile and very insoluble, PB activity was very resistant. Trypsin reduced both activities an this reduction was greater after phospholipase treatment. Both activities were slightly solubilized in 8 M urea. It is known that BP is not present on the surface of myelin and is considered a peripheral membrane protein. M2 appears to be a surface and integral membrane protein, and as such resembles Folch Pi proteolipid protein. The relationship between M2 and BP requires further study.     

Prostaglandin E2 (PGE2)-evolted chloride secretion in guinea-pig colon is mediated by nerve-dependent and nerve-independent mechanisms

Abstract Conventional flux chamber and intracellular recording methods were used to investigate the effect of prostaglandin E₂ on ion transport and on electrical behaviour of submucosal neurones in guinea-pig colon. In flux chamber experiments, prostaglandin E₂ evoked a dose-dependent increase in short-circuit current. The response was reduced by serosal addition of bumetanide, tetrodotoxin or atropine, but not hexamethonium or piroxicam. This indicates that the response to prostaglandin E₂ was mediated in part by activation of chloride secretion via submucosal neurons. Application of prostaglandin E₂ to submucosal neurones evoked a depolarization of the membrane potential associated with an enhanced spike discharge which was frequently triggered by fEPSPs. The depolarizing response to prostaglandin E₂ was not affected by tetrodotoxin, indicative of a direct effect of prostaglandin E₂ on the impaled neurones. However, the increased spike activity was synaptically driven suggesting an additional activation of other cells. Prostaglandin E₂ had no excitatory or inhibitory effect on cholinergic fast excitatory postsynaptic potentials. The study suggests that prostaglandin E₂ may function as a neuromodulator to evoke nerve-mediated chloride secretion through activation of submucosal neurones. The results further indicate that prostaglandin E₂ may influence mucosal function by altering electrical behaviour of submucosal neurones leading to spread of excitation throughout the plexus.     

原发性中枢神经系统淋巴瘤的诊治

目的 探讨原发性中枢神经系统细胞瘤的临床诊断和治疗.方法 回顾性分析我院2001年12月至2005年5月间经立体定向活检或手术病理证实的12例原发性中枢神经系统细胞瘤临床表现特点及治疗结果.结果 12例病人中手术切除7例,立体定向活检5例.本组12例共21个肿瘤,其中7例(58.3%)为多发性肿瘤,肿瘤多位于额部、颞顶部及基底节区,11例(91.7%)病人肿瘤主体均位于小脑幕上.组织病理学检查发现12例病人均为B细胞来源.4例病人辅以放疗,8例病人辅以放疗和化疗,生存时间为4-37个月,中位生存时间为16.3个月.结论 原发性中枢神经系统细胞瘤术前诊断困难,预后差,诊断主要靠病理.该疾病多采用综合治疗,术后辅以放、化疗.近年的大剂量甲氨蝶呤化疗受到关注.     

Coxsackie B antigen in the central nervous system of a patient with fatal acute encephalitis: immunohistochemical studies of formalin-fixed paraffin-embedded tissue

Summary A case of fatal acute encephalitis due to Coxsackie B₁ virus is described. Confirmation of Coxsackie B virus as the etiological agent of encephalitis was based on identification of the virus antigen in formalin-fixed paraffin-embedded tissue sections. In the past, the diagnosis was obtained by serological studies of peripheral blood and viral isolation. This is the first report in which indirect immunofluorescent and immunoper-oxidase methods using rabbit antiserum raised against Coxsackie B types 1–6 was utilized in determining the etiology of encephalitis. It must be emphasized that these methods can be used both on biopsy or autopsy specimens, even retrospectively.Presented in part at the sixty-third Annual Meeting of the American Association of Neuropathologists, Seattle, Washington, June 11–14, 1987     

Role of Ca-independent phospholipase A2 and n−3 polyunsaturated fatty acid docosahexaenoic acid in prostanoid production in brain: perspectives for protection in neuroinflammation

Various diseases of the central nervous system are characterized by induction of inflammatory events, which involve formation of prostaglandins. Production of prostaglandins is regulated by activity of phospholipases A₂ and cyclooxygenases. These enzymes release the prostaglandin precursor, the n−6 polyunsaturated fatty acid, arachidonic acid and oxidize it into prostaglandin H₂. Docosahexaenoic acid, which belongs to the n−3 class of polyunsaturated fatty acids, was shown to reduce production of prostaglandins after in vivo and in vitro administration. Nevertheless, the fact that in brain tissue cellular phospholipids naturally have a uniquely high content of docosahexaenoic acid was ignored so far in studies of prostaglandin formation in brain tissue. We consider the following possibilities: docosahexaenoic acid might attenuate production of prostaglandins by direct inhibition of cyclooxygenases. Such inhibition was found with the isolated enzyme. Another possibility, which has been already shown is reduction of expression of inducible cyclooxygenase-2. Additionally, we propose that docosahexaenoic acid could influence intracellular Ca²⁺ signaling, which results in changes of activity of Ca²⁺-dependent phospholipase A₂, hence reducing the amount of arachidonic acid available for prostaglandin production. Astrocytes, the main type of glial cells in the brain control the release of arachidonic acid, docosahexaenoic acid and the formation of prostaglandins. Our recently obtained data revealed that the release of arachidonic and docosahexaenoic acids in astrocytes is controlled by different isoforms of phospholipase A₂, i.e. Ca²⁺-dependent phospholipase A₂ and Ca²⁺-independent phospholipase A₂, respectively. Moreover, the release of arachidonic and docosahexaenoic acids is differently regulated through Ca²⁺- and cAMP-dependent signal transduction pathways. Based on analysis of the current literature and our own data we put forward the hypothesis that Ca²⁺-independent phospholipase A₂ and docosahexaenoic acid are promising targets for treatment of inflammatory related disorders in brain. We suggest that Ca²⁺-independent phospholipase A₂ and docosahexaenoic acid might be crucially involved in brain-specific regulation of prostaglandins.     

Intracerebroventricular injection of prostaglandin E2 induces thermal hyperalgesia in rats: the possible involvement of EP3 receptors

To determine what types of prostanoid receptors are involved in the central effect of prostaglandin E₂ (PGE₂) on nociception, we administered PGE₂ and its agonists, i.e., 17-phenyl-ω-trinor PGE₂ (an EP₁ receptor agonist), butaprost (an EP₂ receptor agonists), 11-deoxy PGE₁ (an EP₂/EP₃ receptor agonist, EP₂ EP₃) and M&B28767 (an EP₃ receptor agonist) into the lateral cerebroventricle (LCV) of rats and observed the changes of paw-withdrawal latency on a hot plate. The LCV injection of PGE₂ (10 pg/kg-10 ng/kg), 11-deoxy PGE₁ (100 pg/kg-10 ng/kg) and M&B28767 (1 pg/kg-100 pg/kg) produced a significant reduction in the paw-withdrawal latency. The maximal reduction was observed 15 min after the LCV injection of these drugs. Neither 17-phenyl-ω-trinor PGE₂ (1 pg/kg-1 μg/kg) nor butaprost (1 pg/kg-100 μg/kg) induced any significant changes in the paw-withdrawal latency. The LCV injection of PGE₂ (1 μg/kg) and 17-phenyl-ω-trinor PGE₂ (50 μg/kg) increased the latency only 5 min after LCV injection. These findings indicate that the LCV injection of PGE₂ induces thermal hyperalgesia through EP₃ receptors and analgesia through EP₁ receptors by its central action in rats.     

Indomethacin blocks central nociceptive effects of PGF2α

Intrathecal administration of PGE₂ and PGF_2α and intradermal administration of PGE₂ but not PGF_2α, in low doses, produce hyperalgesia as measured by the Randall-Selitto paw-withdrawal test. Indomethacin (2 mg/kg, i.p.) prevented intrathecal PGF_2α, but not PGE₂-induced hyperalgesia. We propose that the central nociceptive effects of PGF_2α are mediated, indirectly, by effecting the cyclooxygenation of arachidonic acid in the central nervous system.