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。     

脑室内注射BDNF抗体对大鼠海马NOS表达的影响

探讨脑室内注射脑源性神经营养因子 (BDNF)抗体阻断内源性BDNF对大鼠海马一氧化氮合酶 (NOS)阳性神经元的影响。脑室内注射BDNF抗体一周后 ,采用Morris水迷宫进行行为检测 ;并用NADPH 黄递酶组化染色方法观察海马NOS阳性神经元数目的变化。与对照组相比 ,实验组大鼠空间学习和记忆能力明显下降 (P <0 0 1) ;实验组大鼠海马CA1区NOS阳性神经元数目 (38 37± 5 2 3)明显少于对照组 (4 9 5 3± 5 74 ) (P <0 0 1) ;实验组DG区NOS阳性神经元数目 (4 8 77± 5 5 1)明显少于对照组 (6 0 4 0± 7 39) (P <0 0 1)。脑室内注射BDNF抗体可导致大鼠空间学习记忆能力下降 ,海马NOS阳性神经元数目减少 ,提示BDNF对学习和记忆的影响可能与海马NOS阳性神经元数目的变化有关     

便秘型肠易激综合征大鼠胃排空及胃内肌间神经丛神经元的变化

目的通过评价便秘型肠易激综合征(IBS-C)模型大鼠胃排空率和胃肌间神经丛神经元的变化探讨肠神经系统在功能性胃肠病重叠综合征发病机制中的作用。方法冰水灌胃法制作IBS-C模型大鼠(n=7),设对照组;采用酚红排空定量法测定胃排空率;取远端胃组织制作石蜡切片标本,采用抗神经核抗体(anti-Hu)标记神经元总数,Hu/乙酰胆碱转移酶(ChAT)、Hu/一氧化氮合酶(NOS)抗体行免疫组织荧光双染色,观察、计算特异性阳性神经元占神经元总数百分比。结果冰水灌胃组大鼠胃排空率显著高于对照组(P<0.05)。IBS-C模型大鼠ChAT阳性神经元显著高于对照组(79.0%±2.4%vs 68.3%±1.0%,P<0.05),NOS阳性神经元显著低于对照组(18.9%±5.0%vs 33.1%±4.5%,P<0.01)。结论冰水灌胃法制作的IBS-C模型大鼠胃排空及胃肌间神经丛神经元均存在改变,表明肠神经系统的变化可能是功能性胃肠病重叠综合征发病机制之一。     

β-淀粉样蛋白诱导大鼠行为学改变及星形胶质细胞变化

为了探讨不同剂量β-淀粉样蛋白 2 5 -3 5片段 ( Aβ2 5- 35)诱导老年性痴呆 ( AD)大鼠动物模型中星形胶质细胞变化与大鼠行为学改变之间的关系 ,在大鼠双侧海马内注射不同剂量 Aβ2 5- 35( 2 .5、5、10和 2 0 μg)制作大鼠 AD模型 ,注射一周后采用胶质原纤维酸性蛋白 ( GFAP)免疫组化染色、NOS组化染色及 NOS组化和 GFAP双重染色分析大鼠海马 GFAP与 NOS的表达。结果发现海马内注射 Aβ2 5- 35剂量≥ 10 μg时 ,模型组大鼠出现学习记忆减退 ,海马星形胶质细胞增生、肥大 ,数目明显多于对照组 ( P<0 .0 5 ) ;海马 NOS阳性神经元数较对照组显著减少 ( P<0 .0 5 ) ;并出现 NOS阳性星形胶质细胞。结论 :星形胶质细胞参与Aβ神经毒性导致 NOS阳性神经元损伤 ,间接导致大鼠学习记忆能力下降 ,在 AD模型大鼠早期学习记忆功能减退中起重要作用     

局灶性脑缺血大鼠顶叶皮质一氧化氮合酶表达与CT灌注成像

目的:观察重度局灶性脑缺血状态下大鼠顶叶皮质一氧化氮合酶(NOS)表达过程,探讨NOS阳性神经元表达与脑血流量的关系.方法:SD大鼠随机分为5组即正常对照组,假手术组,脑缺血1、6、24 h组.线栓法建立大鼠脑缺血模型,多排螺旋CT灌注成像测量脑血流量,采用还原型尼克酰胺腺嘌呤二核苷酸黄递酶(NADPH-d)法,观察脑血流量低于20%正常值时顶叶皮质NOS表达.结果:与对照组比较,缺血1 h组顶叶皮质NOS阳性神经元数量明显增多;与对照组及缺血1 h组比较,缺血6 h组顶叶皮质NOS阳性神经元数量明显下降;与对照组及缺血1 h组比较,缺血24 h组顶叶皮质NOS阳性神经元数量进一步减少并几乎消失,与脑缺血6 h组比较,无显著差异.当血流量仅轻度下降时,顶叶皮质NOS阳性神经元表达并无明显变化.结论:缺血侧脑血流量低于正常值20%时,顶叶皮质NOS阳性神经元在缺血1 h时显著增加,缺血6、 24 h急剧减少;缺血后NOS阳性神经元表达,不仅与缺血持续时间有关,还与缺血程度有关.     

大鼠中缝背核一氧化氮合酶神经元投射纤维在大脑皮质微血管的分布

目的：研究通过损毁脑干中缝背核(DR)，探讨中缝背核NOS阳性神经元是否投射分布于大脑皮质微血管。方法：将16只SD雄性成年大鼠分为实验组与对照组。对实验组大鼠中缝背核微量注射喹啉酸，饲养1w，灌注固定，然后将大脑及脑干作冠状冰冻切片，NADPH—d组化染色。结果：实验组大鼠的中缝背核被有效损毁，其NOS阳性神经元的数量减少了59．1％(P＜0．001)。额、顶叶皮质NOS阳性纤维终末减少了32．1％(P＜0．05)，其中附着于皮质微血管的NOS阳性纤维终未了减少了37．8％(P＜0．01)。而枕额叶皮质NOS阳性纤维终末也减少了32．8％(P＜0．05)，其中附着于皮质微血管的阳性纤维终末减少了39．4％(P＜0．01)。结论：位于中缝背核的NOS阳性神经元投射分布于大脑皮质微血管，可能参与大脑皮质血流量的调节。     

胰岛素对糖尿病大鼠海马一氧化氮合酶阳性神经元变化的影响

观察实验性糖尿病大鼠海马一氧化氮合酶（NOS）阳性神经元变化及胰岛的治疗作用。给成年SD大鼠腹腔注射链脲佐菌素制备糖尿病大鼠模型，每日给予长效胰岛素2－3U使血糖低于10mmol/L，于第3月及第6月末以NADPH－d组化法显示海马NOS阳性神经元。并做Morris水迷宫行为学测试。海马NOS阳性神经元密度变化如下：糖尿病组齿状回3月时显著减少（P＜0．01），6月时更明显（P＜0．01）；CA1区6月时显著降低（P＜0．01）；治疗组齿状回3月时恢复正常，而6月时低于正常（P＜0．01）但高于糖尿病组3月时（P＜0．05），CA1区3月，6月均恢复正常。行为学测试中各组大鼠逃避潜伏期变化与齿状回NOS阳性神经元密度变化一致。以上变化可能与糖尿病学习记忆功能损伤有关。胰岛素治疗可延缓其发生。     

2型糖尿病模型db/db小鼠海马NOS阳性神经元变化

目的　观察人类 2型糖尿病模型———C5 7BL/KsJdb/db(db/db)小鼠海马NOS阳性神经元变化。方法　糖尿病组 :6周龄C5 7BL/KsJ(db +db +)小鼠 5只 ,尾静脉空腹血糖高于 11.1mmol/L且肥胖。对照组 :非糖尿病小鼠C5 7BL/KsJ(?+) 5只 ,尾静脉空腹血糖低于 6 .0mmol/L体重正常 ,于 30周龄 (成模第 6月末 )时 ,灌注固定取脑 ,以NADPH d组化法显示海马NOS阳性神经元。结果　与正常对照组相比 ,糖尿病组小鼠海马齿状回NOS阳性神经元密度显著减少 (P <0 0 1)。结论　糖尿病时NOS阳性神经元数量减少 ,NO的合成降低表明NO可能参与糖尿病中枢神经系统功能障碍     

便秘型肠易激综合征大鼠胃排空及胃内肠神经系统的变化

 目的 观察便秘型肠易激综合征（IBS-C）模型大鼠胃排空及胃肌间神经丛神经元总数、乙酰胆碱转移酶（ChAT）阳性神经元、一氧化氮合酶（NOS）阳性神经用。方法 冰水灌胃法制作IBS-C模型大鼠（n＝7），设对照组；采用酚红排空定量法测定胃排空率；取远端胃组织制作石蜡切片标本，采用抗神经核抗体（anti-Hu）标记神经元总数， Hu/ChAT、Hu/NOS抗体行免疫组织荧光双染色，观察、计算特异性阳性神经元占神经元总数百分比。结果 冰水灌胃组大鼠胃排空率显著高于对照组（P<0.05）。IBS-C模型大鼠ChAT阳性神经元显著高于对照组（79.0% ± 2.4%比68.3 % ± 1.0%，P<0.05），NOS阳性神经元显著低于对照组（18.9% ± 5.0%比33.1% ± 4.5%，P<0.01）。结论 冰水灌胃法制作的IBS-C模型大鼠胃排空及胃肌间神经丛神经元均存在改变，表明肠神经系统的变化可能是功能性胃肠病重叠综合征发病机制之一。     

去卵巢后雌性大鼠下丘脑视前区NOS阳性神经元形态学和突触素表达的变化

目的：为解释围绝经期的精神、神经症状提供实验依据。方法：采用免疫细胞化学方法显示去卵巢雌性大鼠下丘脑视前内侧区(MPA)和视前外侧区(LPA)内NOS阳性神经元，形态学改变以及突触素表达。结果：(1)去卵巢组和对照组大鼠的视前内、外区内可见大量NOS阳性神经元；(2)去卵巢动物的下丘脑内NOS阳性神经元突起长度比对照组的明显变短，分支明显变少；(3)去卵巢组动物下丘脑内突触素表达比对照组的明显减少。结论：大鼠去卵巢后，由于雌激素分泌减少，导致了NOS阳性神经元的突起减少和突触减少，结果NO介导的神经元信号传导失常，这些变化可能是引发围绝经期的精神、神经症状的原因之一。     

一氧化氮合酶阳性神经元在大鼠胃肌间神经丛中的分布

目的：了解一氧化氮合酶阳性神经元在大鼠胃肌间神经丛中的分布规律。方法：采用还原性辅酶Ⅱ黄递酶组化法和消化道铺片技术观察了胃各部肌间神经丛中神经元的形态和分布。结果：一氧化氮合酶阳性神经元广泛分布于大鼠胃肌间神经丛中,其形态基本类似,以圆形和卵圆形为主。在胃不同区域神经元的分布密度、胞体大小和染色强度存在较大差异。从胃底、胃体至胃窦神经元密度逐渐增加,各处相关显著（Ｐ〈０．０１）。胃底部的神经元胞体     

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.     

Alpha-synuclein-immunopositive myenteric neurons and vagal preganglionic terminals: Autonomic pathway implicated in Parkinson's disease?

The protein alpha-synuclein is implicated in the development of Parkinson's disease. The molecule forms Lewy body aggregates that are hallmarks of the disease, has been associated with the spread of neuropathology from the peripheral to the CNS, and appears to be involved with the autonomic disorders responsible for the gastrointestinal (GI) symptoms of individuals afflicted with Parkinson's. To characterize the normative expression of alpha-synuclein in the innervation of the GI tract, we examined both the postganglionic neurons and the preganglionic projections by which the disease is postulated to retrogradely invade the CNS. Specifically, in Fischer 344 and Sprague–Dawley rats, immunohistochemistry in conjunction with injections of the tracer Dextran–Texas Red was used to determine, respectively, the expression of alpha-synuclein in the myenteric plexus and in the vagal terminals. Alpha-synuclein is expressed in a subpopulation of myenteric neurons, with the proportion of positive somata increasing from the stomach (3%) through duodenum (proximal, 6%; distal, 13%) to jejunum (22%). Alpha-synuclein is co-expressed with the nitrergic enzyme nitric oxide synthase (NOS) or the cholinergic markers calbindin and calretinin in regionally specific patterns: 90% of forestomach neurons positive for alpha-synuclein express NOS, whereas 92% of corpus-antrum neurons positive for alpha-synuclein express cholinergic markers. Vagal afferent endings in the myenteric plexus and the GI smooth muscle do not express alpha-synuclein, whereas, virtually all vagal preganglionic projections to the gut express alpha-synuclein, both in axons and in terminal varicosities in apposition with myenteric neurons. Vagotomy eliminates most, but not all, alpha-synuclein-positive neurites in the plexus. Some vagal preganglionic efferents expressing alpha-synuclein form varicose terminal rings around myenteric plexus neurons that are also positive for the protein, thus providing a candidate alpha-synuclein-expressing pathway for the retrograde transport of putative Parkinson's pathogens or toxins from the ENS to the CNS.     

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。     

The neurochemistry and innervation patterns of extrinsic sensory and sympathetic nerves in the myenteric plexus of the C57Bl6 mouse jejunum

In vitro anterograde tracing of axons in mesenteric nerve trunks using biotinamide in combination with immunohistochemical labelling was used to characterize the extrinsic nerve projections in the myenteric plexus of the mouse jejunum. Anterogradely-labelled spinal sensory fibres innervating the enteric nervous system were identified by their immunoreactivity for calcitonin gene-related peptide (CGRP), while sympathetic noradrenergic fibres were detected with tyrosine hydroxylase (TH), using confocal microscopy. The presence of these markers has been previously described in the spinal sensory and sympathetic fibres. Labelled extrinsic nerve fibres in the myenteric plexus were identified apposing enteric neurons that were immunoreactive for either calretinin (CalR), calbindin (CalB) or nitric oxide synthase (NOS). Of the total anterogradely labelled axons in the myenteric plexus, 20% were CGRP-immunoreactive. Labelled CGRP-immunoreactive varicosities were closely apposed to CalR-immunoreactive myenteric cells, many of which were Dogiel type I (40%; interneurons) or type II (20%; intrinsic sensory) neurons. Labelled CGRP-immunoreactive varicosities were also observed in close appositions to CalB-immunoreactive myenteric cell bodies, of which a small subset had type II morphology (18%; intrinsic sensory neurons). A further 43% of all biotinamide-filled fibres were immunoreactive for TH and these fibres were apposed to CalR-immunoreactive cell bodies (small-sized; excitatory motor neurons) and NOS-immunoreactive cell bodies (either type I or small neurons; inhibitory motor neurons and interneurons) in the myenteric plexus. The results provide a neurochemical and neuroanatomical basis for connections between dorsal root afferent neurons and myenteric neurons and suggest an anatomical substrate for the well-known modulation of enteric circuits from sympathetic nerves. No anterogradely-labelled fibres were stained for NOS-immunoreactivity, despite more than 60% of dorsal root ganglion (DRG) neurons retrogradely labelled from the jejunum showing NOS-immunoreactivity. This was due to a substantial, time-dependent, and apparently selective, loss of NOS from extrinsic axons under in vitro conditions. Lastly, a small population of non-immunoreactive biotinamide-filled fibres (<1%) gave rise to dense terminal structures around individual myenteric cell bodies lacking CalR, CalB or NOS. These specialized endings may represent vagal fibres or a subset of spinal sensory neurons that do not contain CGRP.     

Projections and chemical coding of neurons with immunoreactivity for nitric oxide synthase in the guinea-pig small intestine.

The distribution of nitric oxide synthase (NOS) immunoreactivity was investigated in the guinea-pig small intestine. There were many immunoreactive nerve cell bodies in the myenteric plexus but very few in submucous ganglia. NOS immunoreactivity was not found in non-neuronal cells except for rare mucosal endocrine cells. Abundant immunoreactive nerve fibres in both myenteric and submucous ganglia, and in the circular muscle, arose from myenteric nerve cells whose axons projected anally along the intestine. NOS immunoreactivity coexisted with VIP-immunoreactivity, but not with substance P immunoreactivity. We conclude that nitric oxide synthase is located in a sub-population of enteric neurons, amongst which are inhibitory motor neurons that supply the circular muscle layer.     

Sennosides do not kill myenteric neurons in the colon of the rat or mouse

Effects of senna on the myenteric plexus of the colon were investigated in view of earlier reports that this anthraquinone cathartic depletes the plexus of its intrinsic neurons. Rats and mice were given purgative doses of sennosides in their drinking water for 4 and 5 months, respectively. Body growth was reduced, and the weight of the colon with its contents was increased relative to the weight of the whole body in the treated animals. The latter change was attributed to depressed propulsive motility of the large intestine. Total numbers of myenteric neurons were determined from whole-mount preparations stained with Cuprolinic Blue-magnesium chloride, which selectively coloured the neuronal somata. The number of neurons in the rat's colon was unaffected by treatment with senna, but the colons of the treated mice contained significantly more neurons than those of their controls.

Staining with antisera to 10 putative neurotransmitters or their associated enzymes revealed immuno-reactive somata and axons in the myenteric plexus. Treatment with senna was not associated with absence of neuronal somata or fibres stainable with any of the antisera in either species. Thus, there was no evidence of toxic destruction of any identifiable population of neurons that might have been too small to affect the total counts. We conclude that senna does not kill myenteric neurons in the colon of the rat or mouse.     

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.     

神经型一氧化氮合酶与血红素氧合酶-2在应激后大鼠结肠的表达

目的探讨神经型一氧化氮合酶（nNOS）与血红素氧合酶-2（HO-2）在应激后大鼠结肠的表达.方法采用水浸-束缚应激（WRS）动物模型,用免疫组织化学SABC法检测nNOS和HO-2在大鼠结肠中的表达,并通过图像分析系统进行定量测定.结果对照组大鼠nNOS主要表达于结肠黏膜下神经丛和肌间神经丛的神经元,HO-2主要表达于结肠黏膜固有层黏膜肌、肌层环行肌及黏膜下层的血管内皮和平滑肌.应激组黏膜下神经丛和肌间神经丛的nNOS阳性神经元的平均灰度值较对照组明显增加,阳性神经元的平均数高于对照组,且在黏膜上皮细胞、固有层淋巴细胞也有nNOS表达;应激组HO-2阳性黏膜肌的平均灰度值较对照组增加,环行肌阳性单位（PU）明显高于对照组,在部分大肠腺也有HO-2表达.与应激组比较,应激＋L-NAME组的nNOS阳性神经元的平均灰度值减少,阳性神经元的平均数下降,应激＋ZnPP组HO-2阳性黏膜肌平均灰度值减少,环行肌PU下降.结论一氧化氮（NO）和一氧化碳（CO）均是结肠重要的气体信号分子和神经递质,两者在应激所致的结肠功能失调中可能具有协同作用.     

An immunohistochemical study of the projections of somatostatin-containing neurons in the guinea-pig intestine

Somatostatin-like immunoreactivity was localized in nerves in whole mount preparations of the separated layers of the guinea-pig intestine. The directions in which the neurons project were determined by examining the accumulation of somatostatin-like immunoreactivity after axonal flow was interrupted. In some experiments this was done by crushing or cutting the nerves in isolated preparations which were then maintained in oxygenated Krebs solution for 3–5 h. In other experiments, the nerves were cut in vivo and the animals allowed to survive for 4–8 days before the intestine was examined.Somatostatin immunoreactive nerve cell bodies were found in both the myenteric plexus, where they represented 4.7% of the total population of neurons, and in the submucous plexus, where they formed 17.4% of the total population. The axons of the somatostatin-containing neurons in the submucosa are not polarized while those of the somatostatin-containing neurons in the myenteric plexus of the small intestine project in the anal direction for 8–12 mm to form pericellular baskets around other enteric neurons, some of which are reactive for somatostatin.It is postulated that somatostatin-containing neurons in the myenteric plexus are interneurons in a descending nerve pathway, possibly the one involved in the descending inhibitory reflex of peristalsis.