Regional variation in the neurochemical coding of the myenteric plexus of the human colon and changes in patients with slow transit constipation

Abstract There are differences in the structure and function between regions of the colon. In patients with slow transit constipation the activity of all regions is markedly slowed. Counts of colonic neurones in slow transit constipation have been semiquantitative and led to varying results. We have applied new methods of quantification of markers in whole mounts of the colonic myenteric plexus to compare density of innervation between regions and between normal patients and those undergoing resection for severe constipation. Whole mounts of colonic myenteric plexus were made from specimens removed for cancer treatment (controls) and cases of severe constipation. All neurones were labelled by anti‐human neuronal protein antibodies. Neurones synthesizing acetyl choline were labelled for choline acetyltransferase (ChAT) and those for nitric oxide by antisera to nitric oxide synthase (NOS). Four populations of neurones were distinguished and quantified according to the two selective markers, ChAT and NOS. In the normal major populations were NOS alone (51% of ascending colon neurones and 44% of descending colon neurones) and ChAT alone (41% ascending colon, 48% descending colon). Nitric oxide synthase/ChAT and NOS‐/ChAT‐comprised only small populations. In all regions in severe constipation, the percentage of NOS‐only colonic myenteric neurones was raised (54% ascending colon, 49% descending colon) and ChAT only was reduced (36% ascending colon, 42% descending colon). The other populations were not changed. Accurate quantification of neuronal populations in whole mounts of human colon reveals inter‐regional differences in innervation and marked changes in innervation in cases of very severe constipation.     

Correlation of morphology, electrophysiology and chemistry of neurons in the myenteric plexus of the guinea-pig distal colon.

Intracellular recordings were made from myenteric neurons of the guinea-pig distal colon to determine their electrical behaviour in response to intracellular current injection and stimulation of synaptic inputs. The recording microelectrode contained the intracellular marker biocytin, which was injected into impaled neurons so that electrophysiology, shape and immunohistochemistry could be correlated. Myenteric neurons in the distal colon were divided into four morphological groups based on their shapes and projections. One group (29 of the 78 that were characterized electrophysiologically, morphologically and immunohistochemically) was the multiaxonal Dogiel type II neurons, the majority (25/29) of which were calbindin immunoreactive. Each of these neurons had an inflection on the falling phase of the action potential that, in 24/29 neurons, was followed by a late afterhyperpolarizing potential (AHP). Slow excitatory postsynaptic potentials were recorded in 20 of 29 Dogiel type II neurons in response to high frequency internodal strand stimulation and two neurons responded with slow inhibitory postsynaptic potentials. Low amplitude fast excitatory postsynaptic potentials occurred in 3 of 29 Dogiel type II neurons. Neurons of the other three groups were all uniaxonal: neurons with Dogiel type I morphology, filamentous ascending interneurons and small filamentous neurons with local projections to the longitudinal or circular muscle or to the tertiary plexus. Dogiel type I neurons were often immunoreactive for nitric oxide synthase or calretinin, as were some small filamentous neurons, while all filamentous ascending interneurons tested were calretinin immunoreactive. All uniaxonal neurons exhibited prominent fast excitatory postsynaptic potentials and did not have a late AHP following a single action potential, that is, all uniaxonal neurons displayed S type electrophysiological characteristics. However, in 6/19 Dogiel type I neurons and 2/8 filamentous ascending interneurons, a prolonged hyperpolarizing potential ensued when more than one action potential was evoked. Slow depolarizing postsynaptic potentials were observed in 20/29 Dogiel type I neurons, 6/8 filamentous ascending interneurons and 8/12 small filamentous neurons. Six of 29 Dogiel type I neurons displayed slow inhibitory postsynaptic potentials, as did 2/8 filamentous ascending interneurons and 4/12 small filamentous neurons. These results indicate that myenteric neurons in the distal colon of the guinea-pig are electrophysiologically similar to myenteric neurons in the ileum, duodenum and proximal colon. Also, the correlation of AH electrophysiological characteristics with Dogiel type II morphology and S electrophysiological characteristics with uniaxonal morphology is preserved in this region. However, filamentous ascending interneurons have not been encountered in other regions of the gastrointestinal tract and there are differences between the synaptic properties of neurons in this region compared to other regions studied, including the presence of slow depolarizing postsynaptic potentials that appear to involve conductance increases and frequent slow inhibitory postsynaptic potentials.     

Age-related changes in the expression of cytokeratin and vimentin in human choroid plexus

Using immunohistochemical methods the expression of intermediate filaments in fetal and adult human choroid plexus epithelium was investigated. The results indicate that the full expression of cytokeratin and vimentin was seen at the 22nd gestational week corresponding to the onset of liquor production. In neural tube and crest cells of human embryos no cytokeratins were detectable, whereas vimentin could be demonstrated in early stages.     

Age-related changes in rat colon mechanics

To determine whether myogenic factors are responsible for slowed colonic transit in senescent rats, maximum shortening velocity (V₀), compliance of the series elastic component (SEC), measurements of passive force in calcium-depleted tissue and peak isometric force (F₀) were examined in proximal and distal colonic circular smooth muscle from 6- and 30-monthold Fischer rats (n = 5). After mucosa was removed, measurements were made on strips stimulated with 80 m>m KCl in a 2.5 m>m Ca²⁺ Kreb's solution. Muscle strips were quick released at peak isometric force (F₀) to afterloads of 60% of F₀. The changes in muscle length from zero to 40 msec and 1 to 2 sec after release during isotonic contraction were used to calculate the SEC and V₀ as a fraction of total muscle length. Passive force (F_p) was measured in 2.5 m>m Ca²⁺ Kreb's solution and in a zero Ca²⁺, 0.1 m>m EGTA solution to determine the contribution of contractile and passive elements to passive force. The results of these studies indicate there is no difference in the V₀ (L₀/sec) of adult (8.4 ± 1.5) and aged (7.5 ± 2.0) animals (P ± 0.05). Peak force (F₀) in the distal colon of the aged rats was greater than adult rats (1.23 ± 0.1 vs 0.85 ± 0.01 kg/cm², P = 0.05). The stiffness of the parallel elastic component and the length-tension relationship were similar in adult and aged animals. Negligible decreases in F_p were observed in zero calcium medium. However, basal contractile tone was elevated in aged animals (P = 0.05). These studies indicate basic differences in aged colonic circular muscle that may contribute to altered bowel transit and function during ageing.     

Regeneration of myenteric plexus in the mouse colon after experimental denervation with benzalkonium chloride

Recent reports suggest a far greater plasticity in nerve tissue than previously believed. As the digestive tract is exposed to a variety of insults, this question is relevant to enteric nerves, but little is known about their ability to recover from damage. To address this problem, we ablated the myenteric plexus of the mouse colon with the detergent benzalkonium chloride (BAC) and followed the ensuing morphologic changes for up to 60 days by using light- and electron microscopy. We found that, 2 days after BAC application, the treated area was essentially devoid of intact nerve elements. From day 7, new nerve fibers were observed within the denervated region. This growth progressed until, at days 30-60, newly grown nerve fibers were present in most of this region, and the pattern of muscle innervation was similar to the normal one. At least part of these fibers originated at neurons within intact ganglia surrounding the denervated region. The cross-sectional area of neurons near the denervated region at day 14 was 52% greater than controls. Glial cells were closely associated with the regenerating nerve fibers. From day 14 onward, we observed undifferentiated cells and differentiating neurons in ganglia surrounding the denervated region, and by day 30, new neurons were present in the myenteric region, along with regenerating nerve fibers. We conclude that the myenteric plexus is endowed with a considerable ability of regeneration and plasticity. The results provide evidence for the presence of stem cells and for an adult neurogenesis in this plexus.     

Effect of aminoguanidine treatment on diabetes-induced changes in the myenteric plexus of rat ileum

The aim of this study was to investigate the ability of aminoguanidine (AG) to prevent diabetes-induced changes in nitric oxide synthase- (nNOS), vasoactive intestinal polypeptide- (VIP) and noradrenaline- (NA) containing nerves of the rat ileum using immunohistochemical and biochemical techniques. Diabetes was induced in adult male Wistar rats by a single intraperitoneal injection of streptozotocin (65 mg/kg). AG was administered in the drinking water to control (1.8 g/l) and diabetic (0.9 g/l) rats over a period of 8 weeks. Diabetes caused a significant increase in the thickness of nNOS-containing nerve fibres (p<0.001) in the circular muscle, in nNOS activity (p<0.05) and in the size distribution of nNOS-containing myenteric neurons (p<0.001). The thickness of VIP-containing nerve fibres was significantly greater (p<0.01) and there was a significant increase in varicosity size (p<0.01) and proportion of VIP-positive myenteric neurons (p<0.01) in diabetes. NA levels were significantly reduced (p<0.01) and the size of varicosities containing tyrosine hydroxylase (TH) was significantly increased (p<0.001) in diabetes. AG treatment completely or partially prevented the diabetes-induced increase in nNOS activity, in VIP-containing varicosity size, and in fibre width of both VIP- and nNOS-containing fibres in the circular muscle but had no effect on the diabetes-induced increase in nNOS-containing neuronal size or proportion of VIP-containing myenteric neurons. In contrast to VIP, AG treatment had no effect on the increase in TH-containing varicosity size in diabetes and also failed to prevent the decrease in NA levels induced by diabetes. These results indicate that AG treatment for neuropathy is not equally effective for all autonomic nerves supplying the ileum and that diabetes-induced changes in NA-containing nerves are particularly difficult to treat.     

The myenteric plexus of the rat colon after fecal stream diversion: a morpho-quantitative study

The myenteric plexus undergoes adaptive changes under several conditions. Mucosal and muscular alterations of the colon have been described after fecal stream diversion but studies concerning the myenteric plexus after this procedure are scarce. Therefore, 28 Wistar rats were submitted to fecal diversion and followed for different periods (30, 60, 120 and 180 days), in order to study the myenteric plexus of the excluded segments. Seven non-operated rats were employed as control. The myenteric plexus was subsequently evaluated with the NADH and NADPH histochemical techniques. The colonic area of excluded segments is significantly decreased. The density of NADH-stained neurons continuously increases during the entire postoperative period but does not match the extent of surface reduction. Neuronal area measurements suggest hypertrophy of the remaining neurons in the late postoperative period. Morphological alterations of myenteric ganglia and neurons were also evident. An important surface area reduction combined with slight density increase points toward significant neuronal loss after fecal diversion. While studies correlating neuronal loss and functional changes are still lacking, surgeons should bear in mind the modifications of the myenteric plexus when performing fecal stream diversion surgeries.     

小鼠胸腺形态结构的增龄性变化

背景：树突状细胞是体内最重要的抗原提呈细胞,在免疫调节中扮演着免疫应答和免疫耐受的双重角色,胸腺含有较多的树突状细胞。随着鼠龄增加,小鼠胸腺结构呈退化趋势,树突状细胞抗原提呈作用也在减弱。目的：观察不同年龄时期小鼠胸腺形态结构的变化。方法：分别观察出生后4周龄、20周龄及10月龄小鼠的胸腺结构;通过S100免疫组织化学标记,计算S100+胸腺树突状细胞在参照物(胸腺)中所占面积百分比;计算胸腺树突状细胞密度。结果与结论：随着年龄的增长,小鼠胸腺皮质变薄,皮髓质界限清楚。胸腺髓质由散在斑片状连成片。10月龄小鼠胸腺实质部分明显减少,而被充满脂肪细胞的间质所取代。20周龄小鼠的胸腺树突状细胞所占面积比和胸腺树突状细胞密度均比4周龄的增加,然而10月龄者则明显减少(P < 0.01)。10月龄小鼠胸腺皮髓质交界处有较多S100+巨噬细胞。结果提示,随着鼠龄增加,小鼠胸腺结构呈退化趋势;老年鼠胸腺树突状细胞抗原提呈作用的减弱与胸腺树突状细胞数量的减少有密切关系。关键词：胸腺树突状细胞;小鼠;胸腺;增龄;形态学     

Noradrenergic innervation of serotoninergic neurons in the myenteric plexus

The monoaminergic innervation of the guinea pig small intestine was investigated to determine if there is an anatomical basis for the hypothesis that serotoninergic and noradrenergic neurons physiologically interact in the enteric nervous system. Initial rates of uptake of tritiated 5-hydroxytryptamine (3H-5-HT) or norepinephrine (3H-NE) by segments of guinea pig small intestine were measured in order to estimate the regional density of the serotoninergic and noradrenergic innervation. No change was found in the uptake of 3H-5-HT as a function of distance between duodenum and ileum, whereas the relative uptake of 3H-NE declined. The pattern of serotoninergic elements demonstrated radioautographically was compared with that obtained by visualizing 5-HT immunoreactivity. Both methods revealed that a small number of serotoninergic neurons, located in 35.3% +/- 1.5% of myenteric ganglia, give rise to many fibers that form thick bundles in interganglionic connectives. Moreover, there was a pronounced heterogeneity in the serotoninergic innervation of individual myenteric neurons and ganglia. In material fixed with aldehydes and postfixed with NaMnO4, noradrenergic axon terminals were identified by their characteristic small dense-cored vesicles. Following incubation with 3H-NE only terminals with small dense-cored vesicles were radioautographically labeled, confirming that these terminals are noradrenergic. When 3H-5-HT was substituted for 3H-NE, noradrenergic terminals were not labeled, showing that nonspecific uptake of 3H-5-HT into noradrenergic axons did not occur in the presence of 5-hydroxydopamine. The combination of aldehyde-NaMnO4 fixation with the radioautographic localization of 3H-5-HT thus permitted the simultaneous identification of serotoninergic and noradrenergic neural elements. Serotoninergic varicosities were found to differ from noradrenergic varicosities in the size, appearance, and packing density of their synaptic vesicles. In addition, recognizable but rudimentary pre- and postsynaptic membrane specializations were associated with serotoninergic but not noradrenergic varicosities. Most serotoninergic neuronal cell bodies were contacted both by serotoninergic synapses and noradrenergic varicosities. Similar appositions of noradrenergic varicosities with nonserotoninergic neurons appeared to be rare. In view of earlier observations that sympathetic nerves affect the release of 5-HT from stimulated enteric serotoninergic neurons, it seems likely that the noradrenergic appositions with serotoninergic neurons are the anatomical substrate for this effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intracellular calcium changes associated with cholinergic nicotinic receptor activation in cultured myenteric plexus neurones

Intracellular free calcium concentration ([Ca²⁺]_i) was measured in cultured explants of myenteric plexus neurones by using the fluorescent calcium indicator Indol in combination with patch-clamp techniques. The basal [Ca²⁺]_i was 94 nM and spontaneous oscillations in the internal free calcium concentration were recorded. These oscillations were associated with bursts of action potentials triggered by spontaneous nicotinic excitatory synaptic potentials. Under voltage clamp conditions, application of the selective nicotinic agonist m-hydroxyphenylpropyltri-methylammonium iodide (10 μM) induced an inward current and increased the intracellular free calcium concentration. We conclude that cholinergic synaptic excitatory activity provide a regular calcium entry in myenteric neurone and suggest that the nicotinic channel might be significantly permeable to calcium.     

Morphological changes of the myenteric plexus neurons in the bullfrog (Rana catesbeiana) duodenum during metamorphosis

Myenteric plexus neurons of the duodenum in the bullfrog Rana catesbeiana were examined during metamorphosis by the Gros-Bielschowsky silver impregnation method and electron microscopy. Larval type neurons with slender and curved cell soma were recognized in the duodenum of the premetamorphic tadpole. They degenerate and decrease in number during early metamorphic climax through shrinkage of the cell soma and autolysis of the cytoplasm. These larval type neurons reduce to debris and then disappear. Two new cell types (adult type neurons) subsequently appear. These new neurons develop and increase in number during late climax and after metamorphosis. Those that appear first are large type A neurons each with a prominent axon and they stain darkly with silver. They enlarge during late stages. Subsequently small type B neurons appear which stain weakly with silver. They increase the number of their dendrites, change their shape, but enlarge only slightly during late development. In summary, therefore, it is concluded that during metamorphosis, the larval myenteric plexus neurons in the bullfrog duodenum are replaced by two new populations of adult neurons.     

Proteome analysis of isolated myenteric plexus reveals significant changes in protein expression during postnatal development

The enteric nervous system in vertebrates is the most complex part of the peripheral nervous system. Concerning chemical coding, ultrastructure and neuronal circuits, it is more similar to the central than to the peripheral nervous system. Its networks, the myenteric and submucous plexus are integrated in the gut wall. The enteric nervous system is a system of high plasticity, which not only changes during pre- and postnatal development, but also with disease or changing dietary habits. The Aim of this study was to elucidate changes in protein expression during the first two postnatal weeks in the rat myenteric plexus. Colonic and duodenal myenteric plexus from newborn (P1) and fourteen-day old (P14) Sprague-Dawley rats was isolated following a procedure that combines enzymatic digestion and mechanical agitation. The neuronal tissue was collected and processed for two-dimensional gel electrophoresis (2-DE). The obtained 2-D gels were stained with silver for image analysis or with colloidal Coomassie for subsequent protein identification. Gels from the various samples showed a high degree of consistence concerning protein-spots found in all preparations. Nevertheless, there was a number of proteins that were clearly detected in one sample but not, or only in significantly smaller amounts in the other. Several differentially expressed proteins in the postnatal myenteric plexus were identified with MALDI-TOF mass spectrometry. Especially stathmin, polyubiquitin and heterogeneous nuclear ribonucleoprotein seem to play an important role in pre- and postnatal development. 2-DE combined with mass spectrometry can help to identify pathological relevant proteins in the enteric nervous system, and so deliver a valuable tool for the early diagnosis of also central nervous system diseases by using biopsies from the gut.     

Neurological disorders of the myenteric plexus: a review

The myenteric plexus is the neuronal complex that regulates the motility of the gut; a brief review of its pathology is presented in this paper as well as a tentative etiopathogenetic classification. Disorders of gut innervation include congenital (e.g. Hirschsprung's disease) or acquired diseases; the latter can be idiopathic or related to a more general pathological involvement of the whole organism as in the case of bacterial toxins, diabetes mellitus, Riley-Day disease and primary orthostatic hypotension. In view of the fundamental similarity of the myenteric plexus to the central nervous system, the study of this organ can be useful both for diagnosis of degenerative diseases of the central nervous system (i.e. via rectal biopsy) and for gaining a better etiopathogenetic insight into peripheral and central nervous system disease.     

帕金森病大鼠胃肠功能障碍和肌间神经丛一氧化氮变化的研究

目的探讨6-羟基多巴胺(6-hydroxydopamine,6-OHDA)诱导的SD大鼠帕金森病(PD)模型胃肠功能障碍及肌间神经丛一氧化氮的变化。方法 60只SD大鼠随机分为对照组和6-OHDA组,每组30只,以6-OHDA诱导制备PD大鼠模型。4w后收集大鼠1h粪便排出量;计算粪便含水量;测定餐后2h大鼠胃内固体食物残留率。采用免疫组化法检测胃肠神经丛神经元型一氧化氮合酶(nNOS)表达变化;反转录聚合酶链式反应技术(RT-PCR)检测胃、结肠组织nNOS mRNA水平的变化。结果与对照组相比,6-OHDA组1h粪便湿重、干重、及含水量明显降低,胃内固体食物残留率明显增加(均P<0.01);胃窦和结肠肌间神经丛nNOS阳性区平均积分光密度明显降低(P<0.01);胃窦、结肠组织nNOS mRNA水平明显降低(P<0.01)。结论 PD大鼠胃肠功能障碍可能与胃肠神经系统nNOS水平降低有关。     

Age-related changes in the dynamics of human albino visual pathways

A deficiency of melanin in the retinal pigment epithelium, which regulates the development of neural retina, leads to chiasmal misrouting such that the uncrossed pathway (to the ipsilateral hemisphere) is reduced relative to the crossed pathway (to the contralateral hemisphere). This study examines age-related changes in the flash and pattern appearance visual evoked potentials (VEP) of human albinos. Scalp recorded cortical VEPs to flash (FVEP) and pattern appearance stimulation were recorded in 58 albino (8 months to 60 years) and 34 normal subjects (4-55 years). VEPs were analysed by amplitude and latency. The contralateral hemisphere FVEP amplitude decreased with age in albino subjects, as in both hemispheres in normals. However, the ipsilateral hemisphere FVEP amplitude was significantly lower in young albino subjects, initially giving a marked interhemispheric asymmetry, but this normalized with age. Significant interhemispheric FVEP latency asymmetries were not observed. The contralateral pattern appearance VEP latency in albino subjects decreased with age, as in both hemispheres in normals; the ipsilateral latency increased significantly with age. Significant interhemispheric pattern appearance VEP amplitude asymmetries were not observed. These novel and unexpected observations indicate significant age-related changes in the retinocortical pathways of the human albino. These changes have implications for our understanding of development and plasticity of the central visual pathways.