先天性巨结肠肠壁NOS阳性神经元定性定量研究

目的:探讨一氧化氮合酶(NOS)阳性神经元与先天性巨结肠发生的关系。方法:对扩张段和移行段分别在双目解剖显微镜下做全层铺片,以NADPH-d酶组化法着色,光学显微镜下观察并以医学图像测算软件对神经元的面积、光密度等作量化处理。结果:扩张段神经节和神经纤维排列层次明显,色深量多,节内神经元色深,沿神经节周边及突起的基部排列。移行段神经节及神经纤维杂乱,量少色浅,节内神经元多浅染或未染,着色程度有较大差异。移行段近端、中段、远端神经元着色依次减弱。结论:NOS阳性神经元分布与代谢异常使肠壁舒张不能,部分导致了先天性巨结肠的发生。     

μ阿片受体在大鼠结肠肠肌丛的神经化学特性

目的:探索μ阿片受体(MOR)在大鼠结肠肠肌丛的分布及化学神经解剖学特性。方法:取大鼠结肠右曲和左曲之间肠管行全层铺片,剥离粘膜层、粘膜下层和环形肌层,保留纵行肌层。运用免疫荧光组织化学双重标记技术染色,共聚焦显微镜下观察MOR在大鼠结肠肠肌丛的分布及与一氧化氮合酶(NOS)、肠血管活血肽(VIP)、P物质(SP)或降钙素基因相关蛋白(CGRP)等在肠神经细胞的共存关系。结果:免疫荧光组织化学染色显示:在大鼠结肠肠肌层,MOR阳性细胞聚集形成神经节,其阳性突起穿行于神经节之间形成网格状神经丛。在肠肌丛神经细胞内,可见MOR分别与NOS、VIP、SP、CGRP共存。MOR阳性神经纤维和终末分布于NOS阳性神经细胞周围,也可见部分NOS、VIP阳性神经纤维和终末分布于MOR阳性神经细胞表面。有大量SP和CGRP阳性神经纤维穿行于MOR阳性神经细胞之间并包绕于MOR阳性神经细胞的胞体周围。结论:在结肠的肠肌丛,MOR与抑制性和感觉性神经递质在肠神经细胞有共存,且相互之间有纤维联系,推测MOR可能介导了阿片类调节肠神经细胞内抑制性神经递质的释放,并可能对肠感觉信息的传递有调节作用。     

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。     

先天性巨结肠中SMMHC、Smoothelin的表达及其临床意义

目的 探讨SMMHC、Smoothelin在先天性巨结肠神经节细胞、神经纤维及肠壁平滑肌中的表达及其在诊断中的应用价值。方法 采用免疫组化EnVision法检测46例先天性巨结肠中SMMHC、Smoothelin的表达，并分析其与神经节细胞及平滑肌细胞的关系。结果 SMMHC及Smoothelin在先天性巨结肠不同发育阶段的神经节细胞内的表达模式不同，在狭窄段、移行段及扩张段肠壁平滑肌内表达缺失，差异有统计学意义。46例标本中有42例狭窄段肠壁部分肌层SMMHC表达缺失(42/46,91.30%),37例狭窄段肠壁部分外纵肌层Smoothelin表达缺失(37/46,80.43%),SMMHC与Smoothelin在狭窄段肠壁平滑肌中的表达缺失率高于移行段、扩张段，差异有统计学意义(P<0.05),而SMMHC与Smoothelin表达差异无统计学意义(P>0.05);移行段与扩张段相比差异无统计学意义(P>0.05),且狭窄段肠壁平滑肌中SMMHC、Smoothelin的部分表达缺失与患儿性别、年龄差异均无统计学意义(P均>0.05)。结论 SMMHC、Smo...     

人胎食管NOS阳性神经元发育的研究

目的：探讨人胎食管壁内NOS阳性神经元的发育。方法：用NADPH－d组织化学法对人胎食管NOS阳性神经元的分化,迁移和发育进行了观察。结果：第4－5月龄时,肌间神经节处的圆形细胞出现一氧化氮合酶阳性反应,并分化演变成梭形的NOS阳性神经细胞,呈条索状排列沿内环肌与外纵肌层之间迁移,有的穿过内环肌层进入粘膜下层,第6个月龄时,迁移到靶细胞附近的NOS阳性神经元胞体明显增大,突起伸长,在肌层和粘膜下层出现NOS阳性神经纤维分布。结论：人胎食管NOS阳性神经元来源于胚胎早期神经节处的圆形细胞,通过分化,迁移,生长发育形成成熟的NOS阳性神经元。     

扬子鳄食管颈段一氧化氮合酶(NOS)阳性神经元及神经纤维的分布

本实验采用还原型尼克酰胺腺嘌呤二核苷酸脱氢酶(NADPH-d)方法,观察扬子鳄食管颈段NOS阳性神经元和神经纤维的分布.结果食管颈段的粘膜下层和肌层分布着较丰富的NOS阳性神经纤维,一些神经纤维交织成丛状,其间散在分布着NOS阳性神经元胞体.粘膜下一些NO S阳性神经丛环绕血管壁走行.     

NO合酶阳性神经元在便秘型肠易激综合征大鼠结肠的表达

目的:探索一氧化氮合酶(NOS)阳性神经元在便秘型肠易激综合征(C-IBS)大鼠结肠的分布特点及变化。方法:冷水持续灌胃15 d制作C-IBS大鼠模型。通过对大鼠大便性状和结肠内膨胀刺激后腹肌反应的观察及结肠HE染色对模型进行评价。免疫荧光组织化学和Western Blot观察NOS阳性神经元在结肠肠肌丛的分布及改变。结果:造模15 d后,模型组大鼠大便呈腊肠状的碎块,结肠的腹肌反应阈值明显降低,HE染色显示结肠粘膜层完整。免疫荧光组织化学显示,C-IBS大鼠结肠肠肌从神经元NOS表达较对照组明显增高。Western Blot结果显示,C-IBS大鼠结肠NOS的表达明显增加。结论:NO在C-IBS大鼠结肠神经元内表达增加,此异常可能与C-IBS大鼠结肠运动功能紊乱相关。     

人胎小肠氮能神经元发育的研究

本研究用 NADPH-d组织化学法对人胎小肠氮能神经元的发育进行了观察。结果显示 :5个月胎龄时 ,肌间神经节处的圆形细胞中部分细胞分化成氮能神经细胞 ,细胞核不染色 ,胞质极少 ,呈浅蓝色 ,有的神经细胞有短小的突起。 6个月胎龄时 ,氮能神经元胞体明显增大 ,核大 ,胞质少 ,染色增深 ,在外纵肌层与内环肌层之间形成肌间神经节。由神经元胞体发出的神经纤维在肌间形成网状神经丛。同时 ,在内环肌层肌纤维之间出现氮能神经纤维分布。有的神经纤维穿过粘膜下层分布到粘膜肠腺基部。7个月胎龄时 ,肌间神经节细胞数目增多 ,胞质由少到多 ,染色强度增加 ,肌层神经纤维分布密度增加 ,在粘膜下层有时可见有 1～ 2个氮能神经元细胞体或小的神经节分布。8～ 10个月胎龄时 ,肌间神经节细胞和粘膜下层氮能神经元胞体及神经纤维呈深蓝色 ,内环肌层神经纤维分布密度增加。以上结果提示 :人胎小肠氮能神经元是由胚胎早期肌间神经节处的圆形细胞 ,通过细胞分化、增殖、生长发育而形成的     

婴儿结肠肌间神经丛及神经节细胞的形态学观察

目的用镀银染色铺片法对婴儿结肠肌间神经丛进行了形态学观察,为结肠的ENS发生以及生理、病理学研究就及肠神经异常性疾病诊治提供形态学依据。方法大体解剖新鲜婴尸,取结肠各段应用镀银染色铺片法观察,测量神经节分布密度及体积。结果婴儿结肠各部神经节形态分布无显著差异,婴儿结肠肌间神经丛由神经纤维束和神经节组成,呈与肠管共同始终的连续网格状结构,神经节细胞形态呈高度的异质性,未见巨细胞,神经细胞平均密度为327.32个/cm2。结论婴儿结肠肌间神经丛的形态和其他实验动物及成人基本相似,但从神经细胞的形态来看,则存在明显的异质性。婴儿结肠肌间神经元数低于成人和其他实验动物。婴儿结肠肌间神经元形态学改变与肠神经异常性疾病具有紧密联系。     

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。     

Total colonic aganglionosis: case report, practical diagnostic approach and pitfalls

Hirschsprung disease remains a challenging diagnosis for many pathologists. The disease is characterized by a lack of ganglion cells in the myenteric and submucosal plexus, associated with increased numbers of acetylcholinesterase-positive nerve fibers. Hypertrophic nerve fibers are present in most but not all patients. Total colonic aganglionosis (TCA) is an uncommon form of Hirschsprung disease with clinical, histologic, and genetic differences and is even more difficult to diagnose and manage. This case illustrates some of the difficulties frequently faced by the pathologists dealing with total colonic aganglionosis. Suction rectal biopsy specimens often lack significant nerve hypertrophy and positive acetylcholinesterase staining, which aid in the diagnosis. Pathologists have to depend mainly on the lack of ganglion cells in adequate submucosa to establish the diagnosis. Transition zone is often long in total colonic aganglionosis and interpretation of frozen sections can be difficult. The presence of several uniformly distributed clusters of mature ganglion cells and lack of nerve hypertrophy are required to avoid connections at the transition zone.     

Ultrastructural localization of acetylcholinesterase activity in Hirschsprung's disease

Localization of acetylcholinesterase activity in congenital megacolon was studied by light and electron microscopy. Acetylcholinesterase activity was strongly positive at the light microscopic level in the Auerbach's plexus of the normal segment and in proliferated nerve fibers of the aganglionic segment. The reaction product observed by electron microscopy was deposited in and between the plasma membranes of the ganglion cells, nerve fibers, and their terminals. The product was also observed in the rough endoplasmic reticulum, nuclear envelope, and Golgi apparatus of the ganglion cells. Acetylcholinesterase activity in the aganglionic segment was observed in and between the plasma membranes of nerve fibers and nerve terminals, which terminated in proximity to smooth muscle cells. Reaction deposits were also observed in the interspace between nerve terminals and smooth muscle cells, suggesting direct innervation of smooth muscles by extrinsic nerve fibers.     

Glomerular handling of immune complex in the acute phase of active in situ immune complex glomerulonephritis employing cationized ferritin in rats

The distribution and co-localization of nitric oxide synthase (NOS) and vasoactive intestinal polypeptide (VIP) were examined by means of immunohistochemistry and NADPH diaphorase (NADPH-d) histochemistry in the gut of patients with Hirschsprung's disease. In the normoganglionic segment, many nitrergic nerve cells were localized in Auerbach's plexus and nerve fibres were observed preferentially in the circular muscle. The submucosal nitrergic nerve cells were mainly situated in Schabadasch's plexus with occasional cells demonstrable in Meissner's plexus. NOS and VIP were co-localized in most ganglion cells of Auerbach's plexus. In the oligoganglionic segment, a marked reduction of NOS- and VIP- positive nerve cells and fibres was noticed in both the myenteric and submucosal plexuses, and nitrergic fibres had disappeared in the inner layer of the circular muscle. In the aganglionic segment, NOS and VIP were revealed only in extrinsic nerve fasciculi and rami and co-localized in a few fibres. From these observations, the inner layer of the circular muscle of the oligoganglionic segment and the whole of the muscularis propria of the aganglionic segment were considered to be totally lacking in nitrergic innervation. Nitrergic nerves of the human colon comprise both intrinsic and extrinsic elements and the majority of intrinsic nitrergic nerve cells contain VIP. Very low numbers of extrinsic nitrergic fibres contain VIP.     

Nitric oxide synthase, choline acetyltransferase, catecholamine enzymes and neuropeptides and their colocalization in the anterior pelvic ganglion, the inferior mesenteric ganglion and the hypogastric nerve of the male guinea pig

By the indirect immunofluorescence method, the distribution of nitric oxide synthase (NOS)-like immunoreactivity (LI) and its possible colocalization with neuropeptide immunoreactivities, with two enzymes for the catecholamine synthesis pathway, tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH), as well as the enzyme for the acetylcholine synthesis pathway, choline acetyltransferase (ChAT) were studied in the anterior pelvic ganglion (APG), the inferior mesenteric ganglion (IMG) and the hypogastric nerve in the male guinea pig. The analyses were performed on tissues from intact animals, as well as after compression/ligation or cut of the hypogastric nerve. In some cases the colonic nerves were also cut. Analysis of the APG showed two main neuronal cell populations, one group containing NOS localized in the caudal part of the APG and one TH-positive group lacking NOS in its cranial part. The majority of the NOS-positive neurons contained ChAT-LI. Some NOS-positive cells did not contain detectable ChAT, but all ChAT-positive cells contained NOS. NOS neurons often contained peptides, including vasoactive intestinal peptide (VIP), neuropeptide tyrosine (NPY), somatostatin (SOM) and/or calcitonin gene-related peptide (CGRP). Some NOS cells expressed DBH, but never TH. The second cell group, characterized by absence of NOS, contained TH, mostly DBH and NPY and occasionally SOM and CGRP. Some TH-positive neurons lacked DBH. In the IMG, the NOS-LI was principally in nerve fibers, which were of two types, one consisting of strongly immunoreactive, coarse, varicose fibers with a patchy distribution, the other one forming fine, varicose, weakly immunoreactive fibers with a more general distribution. In the coarse networks, NOS-LI coexisted with VIP- and DYN-LI and the fibers surrounded mainly the SOM-containing noradrenergic principal ganglion cells. A network of ChAT-positive, often NOS-containing nerve fibers, surrounded the principal neurons. Occasional neuronal cell bodies in the IMG contained both NOS- and ChAT-LI. Accumulation of NOS was observed, both caudal and cranial, to a crush of the hypogastric nerve. VIP accumulated mainly on the caudal side and often coexisted with NOS. NPY accumulated on both sides of the crush, but mainly on the cranial side, and ENK was exclusively on the cranial side. Neither peptide coexisted with NOS. Both substance P (SP) and CGRP showed the strongest accumulation on the cranial side, possibly partly colocalized with NOS. It is concluded that the APG in the male guinea-pig consists of two major complementary neuron populations, the cholinergic neurons always containing NOS and the noradrenergic neurons containing TH and DBH. Some NOS neurons lacked ChAT and could represent truly non-adrenergic, non-cholinergic neurons. In addition, there may be a small dopaminergic neuron population, that is containing TH but lacking DBH. The cholinergic NOS neurons contain varying combinations of peptides. The noradrenergic population often contained NPY and occasionally SOM and CGRP. It is suggested that NO may interact with a number of other messenger molecules to play a role both within the APG and IMG and also in the projection areas of the APG.     

Origin of peptide-containing fibers in the inferior mesenteric ganglion of the guinea-pig: Immunohistochemical studies with antisera to substance P,enkephalin, vasoactive intestinal polypeptide,cholecystokinin and bombesin

After different denervation procedures the guinea-pig inferior mesenteric ganglion was analysed by immunohistochemistry using antisera to substance P, enkephalin, vasoactive intestinal polypeptide, cholecystokinin and bombesin. The results demonstrate that each of the nerve trunks connected to the ganglion carries specific peptidergic pathways. Thus, the lumbar splachnic nerves contain substance P-immunoreactive primary afferent neurons, which to a large extent traverse the ganglion, and enkephalin-immunoreactive preganglionic neurons; the colonie nerves carry vasoactive intestinal polypeptide-, cholecystokinin- and bombesin-immunoreactive fibers from the distal colon to the ganglion; the hypogastric nerves contain vasoactive intestinal polypeptide-positive fibers from the pelvic plexus; and the intermesenteric nerve contains vasoactive intestinal polypeptide, cholecystokinin, substance P and enkephalin from divergent sources. By studying accumulations of peptides in ligated lumbar splanchnic, intermesenteric, hypogastric and colonic nerves the existence of these major peptidergic pathways was confirmed and evidence was obtained for additional, not so prominent, peptidergic projections. The results are discussed in view of earlier morphological and physiological studies.     

Target specific organization and neuron types of the dog pelvic ganglia: a retrograde-tracing and immunohistochemical study

The major pelvic ganglion in both the rat and guinea pig has been extensively studied because of its anatomical simplicity. To clarify the target specific neural pathway in the diffusely distributed pelvic ganglia of larger animals, the pelvic plexus of the female dog was investigated by retrograde tracing and immunohistochemistry. The whole mount staining of the pelvic plexus with acetylcholinesterase histochemistry revealed 70-100 ganglia of varying sizes. Neurons retrogradely labeled from the rectum were mainly found in ganglia located in the dorso-caudal part of the plexus. The majority of these were non-catecholaminergic, immunoreactive for either calbindin (Calb) or neuropeptide Y (NPY), and characteristically associated with baskets of enkephalin (ENK)-immunoreactive varicose fibers. Neurons projecting to the utero-vaginal walls were distributed in ganglia located in the ventro-caudal part of the plexus. These mainly consisted of two major neuron groups: catecholaminergic Calb-immunoreactive neurons, and non-catecholaminergic neurons containing nitric oxide synthase (NOS) and/or vasoactive intestinal peptide (VIP), which were preferentially associated with a network of ENK-immunoreactive varicose fibers. Neurons retrogradely labeled from the urinary bladder mainly occurred in ganglia located around the junction between the ureter and the bladder. These consisted of catecholaminergic Calb neurons and noncatecholaminergic neurons containing Calb or NOS. Only a few ENK-immunoreactive fibers were found within the clusters of catecholaminergic neurons. These results indicate that organ specific neurons are located in separate ganglia and have both a distinctive composition of neuron types as well as different innervation by preganglionic fibers.     

Parasympathetic innervation of cutaneous blood vessels by vasoactive intestinal polypeptide-immunoreactive and acetylcholinesterase-positive nerves: histochemical and experimental study on rat lower lip

The distribution and origin of perivascular acetylcholinesterase-active and vasoactive intestinal polypeptide-immunoreactive nerve fibers were studied in the rat lower lip by means of acetylcholinesterase histochemistry and vasoactive intestinal polypeptide immunohistochemistry. The perivascular nerve fibers stained intensely with both histochemical techniques and were widely distributed on small arteries and arterioles of the lower lip, especially in the transitional zone between the hairy skin and the mucous membrane. The distributions of the two types of fibers were very similar and most of them showed overlapping coloration, on consecutive staining for vasoactive intestinal polypeptide and acetylcholinesterase. Both acetylcholinesterase-positive and vasoactive intestinal polypeptide-immunoreactive fibers were completely lost on removal of the otic ganglion, while they were not affected by sympathetic ganglion removal or sensory nerve sectioning. In the otic ganglion, most cells exhibited acetylcholinesterase activity, and about 60% of the cells showed light to heavy vasoactive intestinal polypeptide immunoreactivity. These findings indicate that vessels in the rat lip are innervated by parasympathetic fibers originating from the otic ganglion and support the view that vasoactive intestinal polypeptide is present in cholinergic neurons. This may suggest the possible control by the parasympathetic nervous system of cutaneous blood vessels through vasoactive intestinal polypeptide-containing cholinergic neurons, in general or at least in the facial area.