肠神经系统发育研究的新进展

肠神经系统 ( Enteric nervous system ENS)是自主神经系统的第三个组成部分 ,由神经元和神经胶质聚集而成。在胚胎时期 ,由迷走神经和骶神经水平的神经嵴细胞沿肠壁移行、分化而成。在这个过程中受肠壁微环境因素与细胞决定因子相互作用的影响 ,如层粘蛋白 ,内皮素 3及内皮素 B受体等。先天性巨结肠是肠神经系统发育不良的最常见疾病 ,近年来研究较多的 NO是介导胃肠道松弛反应的 NANC神经系统的主要递质 ,在先天性巨结肠病人中表现明显异常。随着生物学技术的发展 ,已发现与先天性巨结肠相关的基因有 :RET原癌基因、END3基因和编码 EDNRB基因。     

腹泻型肠易激综合征患者血清抗肠神经元抗体免疫反应特点及其临床意义

目的 通过检测腹泻型肠易激综合征(IBS-D)患者血清中抗肠神经元抗体(AENA),分析与症状的相关性,探讨AENA在IBS发病中的可能作用。方法 纳入符合罗马Ⅲ诊断标准IBS-D患者和健康对照者,采集血清,以豚鼠黏膜下神经丛为底物,间接免疫荧光法检测血清AENA,盲法判断免疫反应染色结果;比较AENA阳性和阴性/弱阳性IBS-D患者临床症状的差别。结果 1)127例IBS-D患者AENA阳性率为85.8%;86名健康对照者阳性率为7.0%。109例AENA阳性的IBS-D患者血清分别为强阳性23.6%、阳性43.3%、弱阳性18.9%,表现为单纯胞质染色、单纯胞核染色、胞质和胞核染色、核膜染色、胞质和核膜染色;6名AENA阳性的健康对照血清均为单纯胞质染色。2)AENA强阳性的IBS-D患者,其肠道症状重于抗体阴性和弱阳性患者,表现为肠道症状计分高分(10分者,58.8%比38.1%)、平素腹痛频发(91.7%比60.0%)、排便前腹痛/腹部不适严重的患者比例数高(24.7%比9.5%);AENA阳性IBS-D患者排便急迫感更常见(87.3%比57.1%)。结论 AENA在IBS发病中可能起一定作用,其有望成为IBS-D的生物学标志。     

豚鼠肠粘膜下神经丛淋巴管旁神经元的烯醇化酶免疫组织化学观察

作者曾报道豚鼠肠粘膜下神经丛内,可能有一种特殊类型的淋巴管旁神经元。为了对上述的研究提供进一步的证据,本文应用显示神经元特异性烯醇化酶(NSE)的免疫组织化学方法,对这种类型的神经元进行了观察。胃肠神经丛内所有的神经元都呈NSE阳性反应,小肠和结肠粘膜下丛内的淋巴管旁神经元,也显同样的阳性反应。平滑肌和结缔组织细胞均为阴性。大多数淋巴管旁神经元具有典型的神经元形态特征;有些细胞虽不具有明显的神经元形态特点,但它们都显相同的NSE阳性反应。本文为我们前文报道的淋巴管旁神经元提供了更可信的证据。     

人胚胎小肠EC细胞的免疫组化研究

本文用免疫组化双PAP法研究人胚胎小肠EC细胞的形态发生。结果表明,EC细胞于第9周出现,并随胎龄成比例地增加,以十二指肠上皮内密度最高,回肠最低,三肠段细胞数量变化曲线基本平行且趋于直线.EC细胞的形态多种多样,其细胞突起可伸至固有层。上皮外未发现EC细胞,作者赞同肠道内分泌细胞的内胚层起源学说。     

神经生长因子通路调节大鼠结肠的肠神经系统可塑性

目的探讨神经生长因子（NGF）是否可以诱导新生期母婴分离（NMS）模型下的肠神经系统（ENS）可塑性。方法雄性SD大鼠出生后行NMS。每天NMS前10min腹腔注射K252a（非特异性神经生长因子受体TrK拮抗剂）阻断NGF信号,对正常新生鼠每天注射NGF模拟NMS诱导的肠神经可塑性。8周后腹壁撤退反射检测内脏痛觉过敏。通过铺片技术和免疫荧光技术。比较各组近端结肠神经节（HuD阳性细胞）大小和数目以及胶质细胞的变化。检测肌间神经丛和粘膜下神经丛肠神经递质类型（ChAT,VIP,nNOS,Cab,TrKA,P75阳性细胞）,分析神经递质的可塑性变化。结果新生期应激可致成年鼠内脏敏感性增高,NGF可诱导内脏敏感性增高,K252a能使之部分缓解。NGF可以诱导部分神经结构重排、近端肌间神经丛ChAT的增高,所有结果经统计学分析,差异有统计学意义。结论早期生活事件是引起成年后肠神经系统可塑性改变的重要原因。这种可塑性变化可能是依赖NGF通路调节。     

大鼠心内血管活性肠肽神经元──PAP法研究

应用PAP免疫组织化学技术对大鼠心内血管活性肠肽神经元进行了研究、结果表明：在大鼠心脏心房后壁和心传导系统中的心内神经节内，存在着血管活性肠肽免疫活性神经细胞，这些细胞多为梭形或圆形，大小中等，并对它的功能进行了讨论。     

力竭运动大鼠心脏心钠素表达的免疫组化研究

目的：探讨力竭运动对大鼠心脏心钠素表达的影响。材料和方法：４月龄雄性大鼠３０只,体重２２０－３６０ｇ。随机分为（１）正常对照组（Ａ组）、（２）有训练游泳力竭组（Ｂ）、（３）无训练游泳力竭组（Ｃ组）。每组各１０只。灌注后取出整心,用免疫组化ＡＢＣ法及图像分析技术,显示和检测大鼠心脏心钠素的表达和含量。结果：心钠素免疫阳性反应为：右心耳〉右心房〉左心房〉右心室近心房处。与Ａ组相比,Ｂ组大鼠心房心钠素表     

Immunohistochemical localization of protein-O-carboxylmethyltransferase in rat brain neurons

The distribution of the enzyme protein-O-carboxylmethyltransferase (EC 2.1.1.24) has been investigated in the rat brain using both immunohistochemical and biochemical techniques. The enzyme, which carboxylmethylates free aspartic and glutamic acid residues of protein substrates, was localized in neurons, but not other cell types throughout the brain. The highest immunoreactivity was detected throughout the cortex, followed by the hippocampus, the corpus striatum, the thalamus and the amygdala. Immunoreactive cells were detected in other brain regions but were not as prominent as those regions listed above. The distribution of immunoreactivity in the hippocampus was most striking, with considerable labelling of the pyramidal and granule cells in all regions. Numerous pyramidal cells were labelled in the cerebral cortex, with some ascending processes exhibiting immunoreactivity. The corpus striatum was uniformly labelled, suggesting that the enzyme was not localized to any specific neurotransmitter system. The antisera employed in this study was generated against purified bovine brain protein-O-carboxylmethyltransferase and Western immunoblot analysis showed cross reactivity against both rat brain and human erythrocyte forms of the enzyme. Enzyme activity and methyl acceptor protein capacity were examined in 1.5 mm coronal sections of rat brain. The regions with highest enzyme activities were found in cross-sections containing cortex and corpus striatum or cortex and hippocampus. The lowest enzyme activities were noted in slices of brainstem and cerebellum, areas exhibiting low amounts of immunoreactive protein-O-carboxylmethyltransferase. Methyl acceptor protein capacity was highest in slices of cortex and corpus striatum, cortex and hippocampus and was lowest in slices of brainstem and cerebellum. These results demonstrate that protein-O-carboxylmethyltransferase has an unique neuronal pattern of distribution in the rodent central nervous system, and suggest that the carboxylmethylation of proteins may be of functional significance in these neurons.     

Immunohistochemical localization of gastrin/CCK-B receptors in the dog and guinea-pig stomach

Gastrin/CCK-B receptors are involved in the regulation of several types of cells of the gastric mucosa, including the parietal cells, the ECL cells and the D cells. In this study, we aimed at localizing such receptors in the gastric mucosa. For this purpose, we prepared monospecific antibodies against two sequences of the canine gastrin/CCK-B receptor. Sections of formalin-fixed, paraffin-embedded corpus and antrum from dog and guinea-pig were immunostained with these antibodies. In parallel, sections were stained with antibodies against somatostatin. Staining with gastrin/CCK-B receptor antibodies was observed in a few, small epithelial cells in the bottom part of the corpus mucosa. Immunoreactive cells of the antral mucosa were structurally similar, but more frequent. The same cells also stained with somatostatin antibodies. In addition, one of the gastrin/CCK-B antibodies reacted with canine submucosal smooth muscle cells. No staining was observed in sections exposed to antibodies that were pre-absorbed with the corresponding antigen. We conclude that gastrin/CCK-B receptors are present in D cells of the gastric mucosa and in submucosal smooth muscle cells.     

Immunohistochemical study of arginase in cancer of the stomach

High levels of arginase have been detected in gastric adenocarcinoma. To examine the hypothesis that this is due to macrophage infiltration into the tumour, we localized the cellular distribution of arginase by immunohistochemical staining. We examined gastric adenocarcinomas and their corresponding normal tissues (n=45), leiomyomas (n=2), leiomyosarcomas (n=3), human gastric adenocarcinoma cell lines (n=3), and benign gastric ulcers (n=4) by the avidin-biotin-peroxidase complex technique. Macrophages with strong arginase immunoreactivity were observed infiltrating both gastric normal and cancer tissues. No arginase immunoreactivity was observed in normal mucosal gland, muscular and serosal tissues or benign gastric ulcers. The immunoreactivity of arginase was positive but heterogeneous in most specimens of gastric adenocarcinoma (62.2%) and was absent from gastric intestinal metaplasia, leiomyomas and leiomyosarcomas. Among the 28 neoplasms with arginase immunoreactivity, scattered immunoreactivity was also noted in adjacent dysplastic glands in 12 (42.8%) specimens. Arginase immunoreactivity was observed in all three gastric cancer cell lines. Arginase is present in the cytoplasm but not in the nucleus. These data suggest that the high arginase levels in adenocarcinoma cancer tissues originate largely from cancer cells.     

Immunohistochemical localization of galanin receptors (GAL-R1, GAL-R2, and GAL-R3) on myenteric neurons from the sheep and dog stomach

Galanin exerts its biological activities (inhibitory or excitatory) via three different G protein-coupled receptors. In the present study, double immunocytochemical labeling was used to localize GAL-R1, GAL-R2 and GAL-R3 on PGP 9.5-positive myenteric neurons from the dog and sheep stomach/forestomachs. In both species, the occurrence of galanin in neurons and nerve fibers of gastric ganglia was also studied. Myenteric ganglia of the dog stomach were supplied with numerous, mainly varicose, galanin-immunoreactive (IR) nerve terminals whereas the frequency of galanin-positive nerve fibers in myenteric ganglia of the ovine stomach and forestomachs was moderate. The number of PGP 9.5-IR/galanin-IR myenteric neurons was significantly lower in the dog stomach (12.3+/-1.3%) as compared to the sheep rumen (20.1+/-0.7%), omasum (19.5+/-2.9%), abomasum (23.8+/-1.2%) but not reticulum (8.1+/-0.8%). In the canine stomach the frequencies of GAL-R1, GAL-R2 and GAL-R3 expressing myenteric neurons were statistically equivalent (4.4+/-0.9%, 3.5+/-0.7% and 3.1+/-0.5%, respectively). Immunoreactivity to GAL-R1 was absent in myenteric ganglia from the ovine rumen, reticulum as well as omasum. GAL-R1 was localized on 0.5+/-0.3% of myenteric perikarya from the abomasum. GAL-R2 bearing myenteric neurons were localized in the ovine rumen (0.6+/-0.3%), reticulum (0.5+/-0.3%), omasum (1.0+/-0.2%) and abomasum (1.1+/-0.3%). The percentages of PGP 9.5-IR/GAL-R3-IR neurons were 0.8+/-0.2% in the rumen, 0.6+/-0.3% in the reticulum, 0.7+/-0.2% in the omasum and 0.9+/-0.3% in the abomasum. In all compartments of the sheep stomach, the proportions of GAL-R1, GAL-R2 and GAL-R3 expressing neurons were significantly lower when compared to analogous neuronal subpopulations present in the dog. It is suggested that, although endogenous galanin may potentially inhibit or stimulate the activity of sparse gastric enteric neurons, its general role in indirect mediation of gastric motility and/or secretion seems to be of minor importance.     

Immunohistochemical and enzyme-histochemical study on the accessory olfactory bulb of the dog

The accessory olfactory bulb (AOB) is a primary center of the vomeronasal system. In the dog, the position and morphology of the AOB remained vague for a long time. Recently, the morphological characteristics of the dog AOB were demonstrated by means of lectin-histochemical, histological, and immunohistochemical staining, although the distribution of each kind of neuron, especially granule cells, remains controversial in the dog AOB. In the present study, we examined the distribution of neuronal elements in the dog AOB by means of immunohistochemical and enzyme-histochemical staining. Horizontal paraffin or frozen sections of the dog AOB were immunostained with antisera against protein gene product 9.5 (PGP 9.5), brain nitric oxide synthase (NOS), glutamic acid decarboxylase (GAD), tyrosine hydroxylase (TH), substance P (SP), and vasoactive intestinal polypeptide (VIP) by avidin-biotin peroxidase complex method. In addition, frozen sections were stained enzyme-histochemically for NADPH-diaphorase. In the dog AOB, vomeronasal nerve fibers, glomeruli, and mitral/ tufted cells were PGP 9.5-immunopositive. Mitral/ tufted cells were observed in the glomerular layer (GL) and the neuronal cell layer (NCL). In the NCL, a small number of NOS-, GAD-, and SP-immunopositive and NADPH-diaphorase positive granule cells were observed. In the GL, GAD-, TH-, and VIP-immunopositive periglomerular cells were observed. In the GL and the NCL, TH-, and VIP-immunopositive short axon cells were also observed. In addition to these neurons, TH- and SP-immunopositive afferent fibers were observed in the GL and the NCL. We could distinctly demonstrate the distribution of neuronal elements in the dog AOB. Since only a small number of granule cells were present in the dog AOB, the dog AOB did not display such a well-developed GCL as observed in the other mammals. Anat. Rec. 252:393–402, 1998. © 1998 Wiley-Liss, Inc.     

Immunohistochemical study of C cell follicles in dog thyroid glands

In dog thyroid glands there are C cell follicles which are lined solely by C cells and which accumulate a colloidlike substance in the luminal cavities. In order to clarify the properties of the colloidlike substance secreted by C cells, the C cell follicles were stained with PAS reaction and immunoperoxidase method using anticalcitonin, anti-C-thyroglobulin, and anti-19S-thyroglobulin antisera, respectively. The colloidlike substance was PAS positive and revealed the strong immunoreaction for C-thyroglobulin but a faint reaction for calcitonin. It was nonreactive with anti-19-thyroglobulin antiserum. These results confirm that C cells synthesize the glycoprotein immunoreactive to anti-C-thyroglobulin antiserum in addition to calcitonin and can store it in the follicular lumens.     

Immunohistochemical evidences for localization and production of D-serine in some neurons in the rat brain

D-Amino acids are thought not to occur in mammalian tissues. However, previous studies reported D-serine was present only in astrocytes in the rat brain. In the present study, it was indicated by a highly sensitive immunocytochemical method with a D-serine specific antibody that D-serine was contained not only in astrocytes but also in some neurons, such as pyramidal neurons in the cerebral cortex, and neurons in the nucleus of the trapezoid body. Some amacrine cells also showed strong immunoreactivity for D-serine in the eyes which were injected with colchicine into the corpus vitreum.     

Immunohistochemical study of neuropeptides in vagal and glossopharyngeal afferent neurons in the rat

The presence and distribution of multiple neuropeptides in vagal and glossopharyngeal afferent ganglia of the rat were studied using immunohistochemistry. Substance P-, calcitonin-gene related peptide-, cholecystokinin-, neurokinin A-, vasoactive intestinal polypeptide-, and somatostatin-immunoreactive neurons were detected in each visceral afferent ganglion. Neurotensin-immunoreactive cells were not observed. In the nodose ganglion (inferior ganglion of the vagus nerve) occasional immunoreactive cells were scattered throughout the main (caudal) portion of the ganglion with small clusters of cells seen in the rostral portion. The pattern of distribution of the various peptides in the nodose ganglion was similar, with the exception of vasoactive intestinal polypeptide-immunoreactive neurons which exhibited a more caudal distribution. The relative numbers of immunoreactive cells varied, with the greatest numbers being immunoreactive for substance P or vasoactive intestinal polypeptide, and the lowest numbers being immunoreactive for neurokinin A and somatostatin. A build-up of immunoreactivity for each of the peptides, except somatostatin and neurotensin, was detected in vagal nerve fibers of colchicine-injected ganglia. Numerous peptide-immunoreactive cells were also found in the petrosal (inferior ganglion of the glossopharyngeal nerve) and jugular (superior ganglion of the vagus nerve) ganglia. No specific intraganglionic distribution was noted although the relative numbers of cells which were immunoreactive for the different peptides varied considerably. Substance P and calcitonin-gene related peptide were found in large numbers of cells, cholecystokinin was seen in moderate numbers of cells, and neurokinin A, vasoactive intestinal polypeptide and somatostatin were seen in fewer cells. These data provide evidence for the presence and non-uniform distribution of multiple peptide neurotransmitters in vagal and glossopharyngeal afferent neurons. In general, relatively greater numbers of immunoreactive cells were located in the rostral compared with caudal nodose ganglion, and in the petrosal and jugular ganglia compared with the nodose ganglion. Thus, multiple neuropeptides may be involved as afferent neurotransmitters in the reflexes mediated by vagal and glossopharyngeal sensory nerves.     

Immunohistochemical localization of estrogen receptors within aromatase-immunoreactive neurons in the fetal and neonatal rat brain

We elucidated the anatomical relationship between estrogen receptors and aromatase, the enzyme converting androgens to estrogens, in the fetal and neonatal rat brain by means of double immunohistochemical labeling, using antibodies against rat estrogen receptors and human placental aromatase cytochrome P450. Numerous aromatase-immunoreactive neurons were found in the medial preoptic area, the bed nucleus of the stria terminalis, the medial amygdaloid nucleus and the ventromedial nucleus. Estrogen receptors were also abundant in these areas. Most of the aromatase-immunoreactive neurons showed immunoreactivity for estrogen receptors in the medial subdivision of the bed nucleus of the stria terminalis and in the posterodorsal division of the medial amygdaloid nucleus. There were also many double-labeled cells in the ventromedial nucleus. However, in the medial preoptic area the localization of aromatase-immunoreactive neurons was distinct from that of neurons containing estrogen receptors. These results suggested that estrogens, which are converted from androgens in aromatase-containing neurons, are involved in the sexual differentiation of the brain through estrogen receptors within aromatase-immunoreactive neurons in the bed nucleus of the stria terminalis, the medial amygdaloid nucleus and the ventromedial nucleus, but through estrogen receptors in aromatase-immunonegative neurons in the medial preoptic area.