内脏高敏感大鼠肠道和脊髓5-羟色胺能神经元及神经纤维分布

目的　研究内脏高敏感大鼠肠道及脊髓内的 5 羟色胺 (5 HT)能神经元及神经纤维的分布 ,肥大细胞脱颗粒对 5 HT能神经元及神经纤维的影响。方法　内脏高敏感大鼠分为两组 (A组和B组 ) ,B组为事先给予促肥大细胞脱颗粒释放剂com pound 4 8 80的内脏高敏感大鼠。对内脏高敏感大鼠和正常对照组 (C组 )结肠和脊髓进行 5 HT免疫组化IHN Envision染色法并作定量分析。结果　A组和C组比较肠道 5 HT免疫阳性神经纤维在黏膜下层阳性指数 (PI)明显增加 (P <0 0 1,0 6 34± 0 2 75vs 0 2 4 5± 0 131) ,肌间神经丛内和脊髓后角 5 H…     

L-精氨酸甲酯对严重烫伤大鼠回肠NOS阳性神经元的影响

目的 了解一氧化氮合酶抑制剂L-精氨酸甲酯(L-NAME)对大鼠严重烫伤后回肠肌间神经丛内NOS阳性神经元的影响。方法50只SD大鼠随机分为正常组、烫伤组和烫伤+L-NAME组,应用NADPH-d酶组织化学染色观察回肠肌间神经内NOS阳性神经元数量和阳性反应面积。结果大鼠体表烫伤后3d,回肠NOS阳性神经元数目明显增加,染色呈强阳性,阳性反应面积增加。L-NAME可降低回肠肌问神经丛内NOS阳性神经元数目,阳性反应面积减少,染色较淡。结论烫伤可引起大鼠回肠内NOS活性增加,L-NAME可能通过降低NOS活性,从而减轻烫伤引起的神经元损伤。     

P物质、血管活性肠肽和乙酰胆碱能神经在大鼠肠道内的分布及其关系

应用乙酰胆碱酯酶(AChE)组织化学和PAP免疫组织化学方法,比较观察P物质(SP)、血管活性肠肽(VIP)和AChE三种阳性神经元在大鼠十二指肠、空肠、回肠、结肠和直肠内的分布特征及其相互关系。结果显示:SP、VIP、AChE阳性神经神经元和纤维均分布于肠壁各层,从十二指肠、空肠到回肠逐渐增多,但从结肠到直肠则逐渐减少;AChE阳性神经元或纤维在肠壁各层最丰富,其中VIP以粘膜层和粘膜下神经丛较丰富,SP以肠肌丛较丰富;三者的分布密度为AChE>VIP>SP。AChE、SP和VIP阳性神经元胞体及神经纤维在不同肠段的分布密度有明显差异(P<0.05),提示可能与不同肠段肠动力调节功能有关。     

MN9202对缺血后大鼠回肠肌间神经丛一氧化氮合酶阳性神经元的影响

目的　实验用 β NADPH脱氢酶组织化学研究MN92 0 2对缺血后大鼠回肠肌间神经丛一氧化氮合酶 (NOS)阳性神经元的影响。方法　用无创伤动脉夹夹闭大鼠肠系膜上动脉使回肠缺血。 1h后松夹复流 ,分别于缺血前 15min和再灌前 1min腹腔注射MN92 0 2 (1μg/kg)。缺血对照组腹腔注射等量生理盐水。动物存活 1d后取回肠进行β NADPH组化反应。 结果　肠缺血后NOS阳性神经元明显增多 (P <0 0 5 ) ,在缺前后应用MN92 0 2可使肌间神经丛内NOS阳性神经元较缺血对照组显著减少 (P <0 0 5 )。结论　MN92 0 2减少大鼠减少大鼠肠缺血后NOS阳性神经元 ,其NO量相应减少 ,可能是其对大鼠肠缺血再灌注损伤的保护作用机制之一     

犬心脏表面主要神经丛降钙素基因相关肽和P物质的免疫组织化学研究

目的 探明犬心脏表面神经丛的化学特性。方法 免疫组织化学ABC法。结果 在犬心脏表面各神经丛均见降钙素基因相关肽(CGRP)免疫反应阳性神经元，而SP免疫阳性神经元仅在心房背侧神经丛(DAP)、房间隔神经丛(IAP)和主动脉．肺动脉间神经丛(A—PP)内见到。CGRP—IR和SP—IR神经元形态、大小相似。心房表面神经丛内的CGRP—IR和SP—IR神经元都较心室表面神经丛者多。在心脏表面各脂肪垫及心肌间隙等处见到多量CGRP-IR、SP—IR神经纤维，多靠近血管或附于血管壁，在一些部位可见这两种肽能神经纤维似与心肌细胞接触。结论犬心脏表面神经丛内存在CGRP和SP；其在心脏内执行的功能可能有联系或相似之处，但也有不同；两种肽能神经对心房和心室的支配不对称，提示CGRP和SP可能直接参与心肌细胞和心脏血管活动的调控。     

5-HT及其2A受体在大鼠丘脑前核的表达

目的研究5-羟色胺(5-HT)及其5-羟色胺2A受体(5-HT2AR)在大鼠丘脑前核的表达,探讨两者参与学习记忆的形态学依据。方法免疫组织化学ABC法观察5-HT及5-HT2AR在丘脑前核内的表达情况。包埋前免疫电镜技术观察丘脑前核群的5-HT能投射纤维终末。结果免疫组化结果显示:在大鼠丘脑前核群的前、中、后部均可见阳性的5-HT能神经元及大量串珠状的投射纤维终末,其中背侧核(AD)的神经元着色较深,胞体较大,纤维密集,平均光密度值(A值)与腹侧核(AV)的比较差异显著(P0.05);5-HT阳性反应产物主要定位于胞浆内,胞核不着色。包埋前免疫电镜显示:阳性5-HT能轴突终末与树突形成非对称性的轴-树突触。在AD、AV内可见黄色的5-HT2AR阳性神经元,其中AD的神经元胞体较大,着色较AV深,阳性产物灰度值二者比较差异显著(P0.05);阳性产物主要定位于神经元胞膜,胞核不着色。结论 5-HT和5-HT2AR在大鼠丘脑前核表达,在AD、AV的表达强度不同。     

炎性胃痛幽门括约肌内VIP和CGRP能神经的变化

目的：研究大鼠幽门括约肌内肠神经系统的分布与胃肠运动功能的相互关系。方法：30只大鼠，设对照组，实验组，采用免疫组织化学技术，显示括约肌内VIP－和CGRP－ir神经元成分，结果：（1）正常幽门括约肌肌层可见个别的CGRP－和VIP－ir神经元胞体，阳性纤维丰富，于肌纤维增厚的括约肌部位神经元成分特别丰富，（2）甲醛致胃炎性痛鼠，肌间神经丛VIP神经元胞体和肌层内神经纤维的活性（图像分析，平均光密度）降低，甲醛组比对照组均显著降低（P＜0．01），但CGRP的甲醛组较对照组升高（P＜0．01），结论：肠神经系统中这两种神经元成分参与幽门括约肌活动与胃肠运动的调控机制。     

Nogo-A在大鼠胃肠神经丛中的表达

目的：探讨Nogo-A蛋白在成年大鼠胃肠神经丛中的表达。方法：利用免疫组织化学法,观察Nogo-A蛋白在大鼠胃肠神经丛中的表达,并用RT—PCR检测Nogo-AmRNA在胃肠组织中的表达情况。结果：在成年大鼠胃肠神经丛,包括黏膜下神经丛、肌间神经丛、浆膜下神经丛内的神经元出现Nogo-A免疫反应阳性,胞质和胞核都可见棕褐色颗粒;胃肠组织的RT—PCR结果显示Nogo-AmRNA为阳性。结论：Nogo-A可广泛表达于各器官系统的神经元中,提示Nogo-A的功能可能与神经元的某个共同的生理活动有关。     

大鼠回肠肌间神经丛内一种特殊类型的神经元

实验用乙酰胆碱酯酶和一氧化氮合成酶组织化学方法,对１０只大鼠回肠肌间神经丛进行研究。发现肌间神经丛的一些神经元,沿毛细血管周围分布,有的甚至与毛细血管壁相紧贴,将其称为毛细血管周神经元。这些神经元胞体呈梭形,梨形或多角形,均呈一氧化氮合成酶阳性反应,而乙酰胆碱酯酶反应阴性。这种神经元类似于中枢神经系统的接触脑脊液神经元,它们可能具有神经分泌或感受血液中某些化学成分成分变化之功能。     

Effect of heroin dependence on 5-hydroxytryptamine, somatostatin and substance P immunoreactive cells in the rat ileum

目的:探讨海洛因依赖对大鼠回肠5-羟色胺(5-HT)、生长抑素(SS)和P物质(SP)免疫反应(IR)细胞的影响.方法:选取成年SD大鼠,分为正常对照组、盐水对照组和海洛因依赖组,建立大鼠海洛因依赖模型,取回肠组织用免疫组织化学SABC法及图像分析法进行研究.结果:与正常及盐水对照组比较,海洛因依赖组大鼠回肠5-HT免疫反应细胞增多,SS及SP免疫反应细胞数无明显变化.5-HT、SS-、SP-IR细胞免疫显色增强,图像分析显示海洛因依赖期间大鼠回肠内5-HT、SS及SP-IR细胞的平均灰度值均低于正常及盐水对照组.结论:海洛因依赖期间,回肠5-HT、SS和SP-IR细胞的5-HT、SS和SP表达增加.     

An immunohistochemical study of the distribution of enteric GABA-containing neurons in the rat and guinea-pig intestine

gamma-Aminobutyric acid (GABA) antiserum was applied to sections of rat and guinea-pig intestine which were subsequently processed to reveal any immunoreactivity using either fluorescence or peroxidase techniques. Immunopositive fibres were demonstrated in stomach, duodenum, ileum and colon of rat and guinea-pig intestine. Myenteric ganglia and nerve bundles in the circular muscle contained immunopositive nerve fibres, while the longitudinal muscle, submucosa and mucosa were only rarely innervated. In favourable sections, immunopositive fibres could be seen running from the myenteric plexus into the circular muscle, thus suggesting that the GABA-immunopositive nerves in the circular muscle originate from neurons in the myenteric plexus. In both rat and guinea-pig, immunoreactive nerve cell bodies were most numerous in the myenteric plexus of the colon. In the rat, immunopositive fibres in the circular muscle were most abundant in the ileum, whereas in the guinea-pig it was the colon circular muscle that was most richly innervated. The results demonstrate that neurons which show GABA immunoreactivity are present along the length of the gastrointestinal tract. Their distribution in both myenteric ganglia and circular muscle is heterogeneous both within and between the two species studied. It is probable that this heterogeneity reflects the diversity and specificity of function of this class of enteric neurons.     

Interactions of Serotoninergic,Cholinergic, and Tachykinin‐Containing Nerve Elements in the Rabbit Small Intestine

This report presents novel results on the effects of serotonin (5‐HT) on longitudinal muscle contractions in the rabbit ileum and the interactions of serotonin with some neuronal elements of the myenteric plexus. We showed previously that serotonin‐triggered contractions involved two mechanisms in the rabbit ileum: neuronal excitation (via 5‐HT₂ receptors in the neurons) and direct muscular stimulation (via 5‐HT₄ receptors in the muscle). Here, we focus on the neuronal 5‐HT₂ receptor pathway and report further pharmacological and immunocytochemical data clarifying the details of the mechanisms. We observed that antagonists for neurokinin (NK1 and NK2) receptors partially blocked the serotonin response, but NK3 receptor antagonists had no effect. Pretreatment by atropine (ATR) eliminated the NK1 receptor antagonist resistant contractions. In contrast, the NK1 antagonist did not depress the ATR‐resistant contraction when ATR was added first. 5‐HT₂ receptor agonist‐induced contractions were partially suppressed by ATR, hexamethonium, and NK1 or NK2 receptor antagonists. In conclusion, serotonin acting through 5‐HT₂ receptors could stimulate interneurons and excitatory motor neurons. Immunocytochemical staining revealed an extensive tachykinin‐immunoreactive (IR) network in the myenteric plexus. Approximately 52% of all myenteric neurons were labeled. 5‐HT‐IR fibers could be detected around both choline acetyltransferase‐ and tachykinin‐IR cells, suggesting functional relationships between them. Consistent with our pharmacological observations, we found that immunopositive nerve elements for 5‐HT_2A receptor and double‐labeled immunostaining revealed a remarkable overlap between tachykinin‐IR neurons and 5‐HT_2A‐IR elements. Anat Rec, 2009. © 2009 Wiley‐Liss, Inc.     

Distribution of enteric nerve cell bodies and axons showing immunoreactivity for vasoactive intestinal polypeptide in the guinea-pig intestine

Immunoreactivity for vasoactive intestinal polypeptide has been localized in neurons in the guinea-pig ileum, colon and stomach. In the ileum, 2.5% of the nerve cell bodies of the myenteric plexus and 45% of those of the submucous plexus showed vasoactive intestinal polypeptide-like immunoreactivity. Varicose axons containing vasoactive intestinal polypeptide ramified amongst the nerve cell bodies of both plexuses and in some cases formed rings of varicosities around non-reactive nerve cells. Axons were traced from the myenteric plexus to the circular muscle and deep muscular plexus. There were numerous positive axons running in fine strands within the circular muscle, parallel to the muscle bundles. Axons containing vasoactive intestinal polypeptide were associated with mucosal blood vessels, but few supplied the vascular network of the submucosa; some immunoreactive axons also contributed to the periglandular plexus of the mucosa. There were no changes in the distribution of axons in the ileum after extrinsic denervation.The results are discussed in relation to the possible functional roles of neurons that contain vasoactive intestinal polypeptide in the intestine: the distribution of such nerve cells in the myenteric plexus and of axons in the circular muscle and sphincters is consistent with this polypeptide being a transmitter of enteric inhibitory neurons; it is also possible that vasoactive intestinal polypeptide is the enteric vasodilator transmitter.     

Distribution of enteric neurons showing immunoreactivity for substance P in the guinea-pig ileum

Substance P-like immunoreactivity has been localized in whole mount preparations of the isolated layers of the guinea-pig ileum. Axons containing substance P formed dense networks around the nerve cells and ran in the primary, secondary and tertiary nerve bundles of the myenteric plexus. 3.6% of the nerve cell bodies of the myenteric plexus and 11.3% of the cell bodies in the submucous plexus showed immunoreactivity for substance P. Axons ran in fine nerve bundles parallel to the longitudinal muscle, between this muscle and the myenteric plexus. Axons containing substance P also ran in small nerve trunks parallel to the circular muscle throughout its thickness and in the deep muscular plexus at the base of this muscle coat. In the submucosa, these axons ramified amongst ganglion cells of the plexus and ran in the internodal strands. In addition they formed a perivascular network around submucous arteries and contributed to the paravascular nerves following these arteries. Axons containing substance P formed a delicate plexus in the mucosa. After extrinsic denervation the nerves containing substance P that were associated with submucous arteries, and some in the submucous plexus, disappeared. The nerves in the other areas were not detectably different from normal.Comparison with the distribution of somatostatin, enkephalin and vasoactive intestinal polypeptide indicated the neurons containing substance P constitute a separate population within the enteric nervous system.     

Light and electron microscopic studies of pituitary adenylate cyclase-activating peptide (PACAP) – immunoreactive neurons in the enteric nervous system of rat small and large intestine

 Pituitary adenylate cyclase-activating peptide (PACAP)-immunoreactive (IR) neurons in the myenteric and submucosal plexus of the rat small and large intestine were examined by immunostaining with purified polyclonal antiserum against PACAP (1–15), using both light and electron microscopy. Many PACAP-IR neuronal cell bodies and fibers were found in the myenteric and submucosal plexus. Many of the PACAP-IR fibers originated from the cell bodies of the myenteric and submucosal ganglia. The ganglia were also innervated by PACAP-IR fibers. PACAP-IR fibers penetrated both the circular and longitudinal muscle layers, confirming the previous observations indicating that PACAP neurons act as motor neurons. Ultrastructural study demonstrated that PACAP-IR nerve terminals formed synaptic contacts with PACAP-IR nerve cell bodies or dendritic processes. This observation suggests that PACAP-IR neurons innervate other PACAP-IR neurons, and that PACAP neurons work as interneurons in the enteric nervous system. PACAP-IR nerve cells received not only PACAP-positive nerve terminal input also PACAP-negative nerve terminal input. It also suggests that PACAP neurons are regulated not only by PACAP-IR enteric neurons, but also by neurons originating elsewhere. Our observations support the view that PACAP-IR neurons are involved in the control of gut motility. Accepted: 20 April 1998     

Serotonin in the rabbit ileum: localization,uptake, and effect on motility

Repeated experiments to localise serotonin in the myenteric plexus of rabbit ileum failed. After preincubation in serotonin (10(-5) M), an extensive varicose fibre system was detected by immunocytochemical methods. Stained fibres left the myenteric plexus and ran to the muscle layers. Labelled cell bodies could not be found, even after pretreatment with colchicine or pargyline. Application of reserpine (10(-5) M) and fluoxetine (10(-5) M) prevented serotonin uptake. Antisera against tryptophan hydroxylase revealed a rich fibre system, including those processes that entered the tertiary plexus. These fibres were able to accumulate serotonin, but again the cell bodies could not be detected. Serotonin caused concentration-dependent contraction in the longitudinal muscle layer of the rabbit ileum. Pretreatment with tetrodotoxin strongly reduced the effect of serotonin. Preapplication of atropine caused a slight decrease of response evoked by serotonin. Combined administration of tetrodotoxin and atropine significantly reduced the responses to serotonin, but did not abolish them. At the same time, agonists of 5-HT(2) and 5-HT(4) receptors caused concentration-dependent contractions. Our studies show that: 1). Without pretreatment, serotonin cannot be detected in the myenteric plexus of rabbit ileum. 2). An extensive uptake system works in this plexus. If released from myenteric nerve fibres, serotonin may evoke contractions in indirect and direct ways. 3). There may be an extrinsic serotoninergic innervation from the mesenteric ganglia. 4). Serotonin exerts its effect through 5-HT(2) and 5-HT(4) receptors on smooth muscle cells and nerve elements.     

Projections of intestinal neurons showing immunoreactivity for vasoactive intestinal polypeptide are consistent with these neurons being the enteric inhibitory neurons.

Experiments were performed to determine if the distribution of vasoactive intestinal peptide(VIP)-like immunoreactivity in nerve cell bodies and axons of the myenteric plexus and circular muscle of the small intestine is consistent with VIP being the transmitter of enteric inhibitory neurons. Immunoreactivity for VIP was found in nerve cell bodies of the myenteric plexus and in axons within the myenteric plexus and circular muscle. When the axons in the myenteric plexus were interrupted, there was accumulation of material showing reactivity for VIP on the oral side, indicating that the neurons project in an anal direction. The VIP-like immunoreactivity in axons which supply the circular muscle disappeared after a myectomy in which the overlying myenteric plexus was removed, but remained intact when extrinsic nerves were served. The projections of VIP neurons from the myenteric plexus to the circular muscle correspond to the expected projections of enteric inhibitory neurons determined by functional studies.     

Neurons with 5-hydroxytryptamine-like immunoreactivity in the enteric nervous system: their visualization and reactions to drug treatment

Immunoreactive nerve cell bodies and fibres in the intestine have been examined using three antibody preparations raised against 5-hydroxytryptamine. Cross reactivity studies indicate that the substance localized was an hydroxylated indoleamine. In the guinea-pig small intestine, nerve cell bodies were located in the myenteric plexus and varicose fibres were found in the ganglia of the myenteric and submucous plexus. The nerve cell bodies had prominent short, broad processes and a single long process. Similar nerve cells and fibres were found in the guinea-pig stomach and large intestine and areas of intestine that were examined in mice, rabbits and rats. Properties of the neurons were examined in the small intestine of the guinea-pig. The immunoreactive material was depleted by treatment with reserpine, but not by guanethidine or 6-hydroxydopamine in dose sufficient to deplete noradrenaline stores in axons in the intestine. No depletion of 5-hydroxytryptamine by the neurotoxin 5, 7-dihydroxytryptamine was observed. After depletion by reserpine, immunoreactivity of the neurons could be restored by application in vitro of 5-hydroxytryptamine, 5,7-dihydroxytryptamine or 5-hydroxytryptophan. The restoration by 5-hydroxytryptophan was prevented by the inhibitor of L-aminoacid decarboxylase, benserazide. After reserpine treatment, immunoreactivity was not restored by tryptophan. Uptake of 5, 7-dihydroxytryptamine into the nerves was antagonized by fluoxetine. The distribution of neurons with 5-hydroxytryptamine-like immunoreactivity was compared with the distribution of enteric amine-handling neurons that take up and decarboxylate L-dopa. This comparison indicated that there are two classes of aromatic amine neuron in the guinea-pig small intestine, the enteric 5-HT neurons and enteric, non-5-HT, amine handling neurons.     

Cytoplasmic delayed neuronal death in the myenteric plexus of the rat small intestine after ischemia

The present study demonstrates light and electron microscopic changes in neurons in the myenteric plexus of the rat ileum following four-hour ischemia. Macroscopically, an intestinal constriction occurred at the damaged portion at three weeks after ischemia; the segment oral to the constriction markedly swelled at four weeks. In light microscopy, at three weeks after ischemia, the myenteric neurons appeared spongy or foamy, containing many vacuoles in their somatic cytoplasm. At four weeks, the neuronal cytoplasm and nerve fiber bundles had disintegrated to form vacant spaces in the myenteric plexus. The neuronal nucleus of the damaged plexus did not show positive nick-end labeling. In electron microscopy, neuronal cytoplasm revealed degenerative signs already at one week after ischemia: a distended endoplasmic reticulum and swollen mitochondria with fragmentary cristae. The nerve fibers also showed destruction of the mitochondria, and degenerative changes in the postsynaptic sites appeared earlier than the presynaptic terminals. The results suggest that intestinal ischemia causes delayed neuronal death, which differs from the apoptotic process previously demonstrated in the ischemia-damaged brain.