Origins and projections of nerve fibres in rat pyloric sphincter

Nerve fibres play an important role in the regulation of gastric emptying. The aims of this study were to clarify the distribution, projections and origin of neuronal type nitric oxide synthase (NOS)-, tyrosine hydroxylase (TH)-, vesicular acetylcholine transporter (VAchT)- and peptide-containing nerve fibres of the rat pyloric sphincter. Extrinsic and local denervations of the sphincter were performed in order to reveal the origin and projections of the various nerve fibre populations. Pylorus from control and denervated animals were processed for the immunocytochemical demonstration of cholecystokinin (CCK), enkephalin, gastrin-releasing peptide (GRP), somatostatin, calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), pituitary adenylate cyclase-activating peptide (PACAP), substance P (SP), vasoactive intestinal peptide (VIP), galanin, NOS, VAchT and TH. VAchT, TH, nNOS, and all of the peptides investigated were found in nerve fibres innervating the pyloric sphincter, and coexistence of several putative neurotransmitters were revealed. Extrinsic denervation caused a total loss of NPY/TH-, SP/CGRP- and SP/CGRP/VIP/NOS/PACAP-containing nerve fibres. Local denervation immediately proximal to the sphincter markedly reduced the numbers of VIP/NOS/galanin- and VIP/NOS/galanin/PACAP +/- NPY-containing fibres within the sphincter suggesting an origin of these fibres in myenteric ganglia in the antral region; denervation at the level of the oxyntic-pyloric border had no effect. Local denervation immediately distal to the sphincter caused a marked decrease in VAchT-, SP/enkephalin-, enkephalin-, somatostatin-, CCK- and GRP-containing fibres within the sphincter suggesting that these emanate from the duodenum. The latter procedure also reduced the number of SP/CGRP-containing fibres of extrinsic origin within the pyloric sphincter.     

Decrease in nerve fibre density in human sigmoid colon circular muscle occurs with growth but not aging

Background Studies in animals suggest that enteric neurons decrease in density or number with increasing age. Neurons containing nitric oxide (NO), vasoactive intestinal peptide (VIP) and Substance P (SP) have been implicated. In human large intestine, NO‐utilizing neurons decrease during childhood or early adulthood but it is not known if the innervation of the muscle changes. This study examined the density of nerve fibres containing these transmitters in sigmoid colon circular muscle from children and adults. Methods Fluorescence immunohistochemistry using antibodies to neuronal NO synthase (nNOS), VIP and SP was performed on sigmoid colon from 18 adults with colorectal cancer, two children with familial adenomatous polyposis, and normal colon from nine children with Hirschsprung’s disease. The percentage area of immunoreactive (IR) nerve fibres containing each transmitter in circular muscle was quantified in confocal images. Key Results In the adult sigmoid colon circular muscle, the percentage area of nerve fibres containing nNOS>VIP>SP (6 : 2 : 1). Paediatric groups had significantly higher percentage area of nerve fibres containing nNOS, VIP or SP‐IR than adults, with the decrease in nerve fibre density occurring from birth to 30 years. Circular muscle thickness increased between 12 and 30 years. Total nerve fibre area remained constant, while the muscle increased in thickness. Conclusions & Inferences In human sigmoid colon circular muscle, there are reductions in nNOS‐, VIP‐ and SP‐IR nerve fibre density with growth from newborn to late adolescence but little further change with aging. The reduction in nerve density is due to an increase in circular muscle thickness rather than a loss of nerve fibres.     

Immunohistochemical characterization of the innervation of human colonic mesenteric and submucosal blood vessels

Abstract The aim was to characterize quantitatively the classes of nerves innervating human mesenteric and submucosal vessels. Specimens of uninvolved normal human mesentery and colon were obtained with prior informed consent from patients undergoing elective surgery for bowel carcinoma. Mesenteric and submucosal vessels were processed for double‐labelling immunohistochemical localization of tyrosine hydroxylase (TH), neuropeptide Y (NPY), calcitonin gene‐related peptide (CGRP), substance P (SP), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), somatostatin (SOM), vesicular acetylcholine transporter (VAChT) and enkephelin (ENK), each compared to the pan‐neuronal marker protein gene product 9.5. Branching patterns of individual nerve fibres were investigated using in vitro anterograde tracing. Sympathetic neurons containing TH and NPY were the largest population, accounting for more than 85% on all vessels. Extrinsic sensory axons, containing SP but not CGRP comprised a second major population on mesenteric vessels: these axons generally lacked TH, NPY and VAChT. On submucosal, but not mesenteric vessels, an additional population of SOM‐immunoreactive fibres was present: these axons did not co‐localize with TH. Major similarities and differences with enteric vessel innervation in laboratory animals were identified. Sympathetic neurons comprise the largest input. Extrinsic sensory neurons in humans largely lack CGRP but contain SP. Submucosal vessels receive an additional source of innervation not present in mesenteric vessels, which contain SOM, but are rarely cholinergic. These results have significant implications for understanding the control of blood flow to the human gut.     

Capsaicin-sensitive extrinsic afferents are involved in acid-induced activation of distinct myenteric neurons in the rat stomach

Challenge of the rat gastric mucosa with 0.5 mol L(-1) HCl activates nitrergic neurons in the myenteric plexus as visualized by c-Fos immunohistochemistry. In the present study, we characterized the activated neurons more extensively by their chemical coding and investigated whether a neural pathway that involves capsaicin-sensitive extrinsic afferents and/or cholinergic neurons transmitting via nicotinic receptors contributes to the activation of myenteric neurons. In multiple labelling experiments, c-Fos was examined for co-localization with nitric oxide synthase (NOS), vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), enkephalin (ENK), gastrin-releasing peptide (GRP), substance P (SP), calbindin D-28k (CALB) and neurofilament 145 (NF 145). All c-Fos-positive neurons were immunoreactive for NOS, VIP, NPY and NF 145, but not for SP, ENK, GRP and CALB. Nerve fibres co-expressing NOS, VIP and NPY were predominantly found in the external muscle layer and in the muscularis mucosae but rarely in the mucosa. Pre-treatment with capsaicin or hexamethonium or a combination of both pre-treatments reduced HCl-induced c-Fos expression by 54, 66 and 63%, respectively. Acid challenge of the stomach, therefore, leads to activation of presumably inhibitory motor neurons responsible for muscle relaxation. Activation of these neurons is partly mediated by capsaicin-sensitive afferents and involves ganglionic transmission via nicotinic receptors.     

An immunohistochemical study of neuropeptides and neuron-specific proteins present in the small intestine of the black-capped capuchin (Cebus appela)

Abstract Antisera against neuron specific enolase (NSE), calbindin and the neuropeptides substance P (SP), vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), enkephalin (ENK) and somntostatin (SOM) were used to demonstrate the presence of nerve fibres and cell bodies in the small intestine of the black-capped capuchin, Cebus appela. NSE-immunoreactive (IR) nerve fibres werefoundin all layers of the intestinal wall, except the longitudinal muscle, and immuno-reactive nerve cell bodies were found in both myenteric and submucous plexuses. The submucous plexus was composed of external and internal ganglionated plexuses. Endocrine cells in the epithelium were reactive for NSE. In ganglia of the myenteric plexus, SP-, VIP-, ENK-, SOM-, NPY- and calbindin-IR varicose and non-varicose fibres were found around reactive and unreactive cell bodies. Calbindin, ENK-, SP-, SOM- and VIP-IR, but not NPY-IR, nerve cell bodies were observed. The primary and secondary plexuses contained fibres of each type examined. The tertiary plexus was rich in SP-IR fibres, although ENK-, NPY-, SOM- and VIP-IR fibres were also observed. The deep muscular plexus had mainly ENK- and SP-IR fibres and few VIP-IR fibres, while in the remainder of the circular muscle VIP-IR fibres predominated. There were few SP-IR and no ENK-IR fibres in this layer. The two ganglionated plexuses of the submucosa contained ENK-, NPY-, SOM-, SP- and VIP-IR fibres, but calbindin-IR fibres were not found. SP- and VIP-IR cell bodies were found in both plexuses. Blood vessels had SP-, VIP- and NPY-IR fibres around them. SP-, VIP- and a few SOM-IR pbres innervated the mucosa, while endocrine cells were found with SP-, SOM- and NPY-IR. The present study demonstrated that the distribution of the neuropeptides SP- and VIP-IR is similar in the small intestine of the black-capped capuchin to that in the dog, guinea pig, human and rat. SP- and VIP-IR possibly mark neurons of the same function in each species. Distributions of ENK-, NPY-, SOM- and calbindin-IR differ substantially between species.     

Peptidergic innervation in the cerebral blood vessels of the guinea pig: an immunohistochemical study.

The distribution of peptidergic nerve fibers containing substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), and neuropeptide Y (NPY) in the cerebral arteries and veins of the guinea pig was studied using immunohistochemical techniques. The ultrastructure of these immunoreactive nerve terminals was also compared. The cerebral arteries were innervated by abundant peptidergic nerve fibers with characteristic running patterns, i.e., SP fibers in a meshwork, VIP and NPY fibers in a spiral fashion. Only CGRP fibers showed both meshwork and spiral patterns. In the cerebral veins, the abundant SP fibers innervated the cortical veins, deep cerebral veins, and dural sinuses. However, CGRP, VIP, and NPY fibers in extremely low density were noted merely in the cortical veins. Electron microscopic observations demonstrated that SP-immunoreactive nerve terminals existed apart from the arterial smooth muscle cells, while VIP- and NPY-immunoreactive nerve terminals adjoined them. As for CGRP nerve terminals, some existed close to the arterial smooth muscle cells, and others were found some distance from them. These morphological characteristics observed by light and electron microscopy suggest that SP fibers are not related directly to the vasomotor function, but VIP and NPY fibers are, and that CGRP fibers have a more complicated function. The distribution patterns of the peptidergic nerve fibers are consistent with the suggestion that vasomotor peptidergic fibers may function actively on cerebral arteries and passively on cerebral veins and that SP fibers regarded as sensory fibers may provide information regarding cerebral vascular conditions, innervating every part of both cerebral arteries and veins.     

Neurones in the chicken ureter are innervated by substance P- and calcitonin gene-related peptide-containing nerve fibres: Immunohistochemical and electrophysiological evidence

Numerous ganglia or single neurones immunoreactive to protein gene-product 9.5 (PGP) were demonstrated in the chicken ureter. Ganglia were observed in the main nerve trunks accompanying the ureter (400–2,000 cells), in the adventitia (1–45 cells; density; 79 ± 12 ganglia/cm²; mean ± S.E.M.), in the circular muscle (1–9 cells; 76 ± 10 ganglia/cm²) and in the longitudinal muscle (1–8 cells; 232 ± 41 ganglia/cm²). Most of the PGP-positive neurones in the nerve trunk ganglia (∼66%) and in the smooth muscle layers (85%) were encircled by a dense plexus of varicose nerve fibres containing both substance P (SP) and calcitonin gene-related peptide (CGRP). SP-positive somata were rarely observed. Immunogold electron microscopy revealed that SP- and CGRP-immunoreactivity were colocalised in the same dense core vesicles. A strong reduction of SP-positive nerve fibres was observed in organ cultures of the ureter, indicating their extrinsic origin. The fibres might originate from the dorsal root ganglia, where SP and CGRP were colocalised in 20–30% of the neurones. The sensitivity of ureteric neurones to SP and CGRP was investigated in recordings obtained from mechanosensitive nerve fibres with cell bodies located in or adjacent to the ureter (U-G units). The majority (71%) of the U-G units was excited by local application of SP in a dose-dependent manner. The SP-sensitive U-G neurones had higher mechanical thresholds (29 ± 5 mmHg) as opposed to the SP-insensitive ones (10 ± 3 mmHg). Repeated applications of high doses of SP to the U-G units resulted in desensitisation and reduced the response to mechanical stimuli. None of the U-G units responded to local application of CGRP, but all U-G units were excited by acetylcholine. The data support the hypothesis that SP-containing primary afferents are involved in the modulation of the activity of ureteric neurons in the chicken. J. Comp. Neurol. 380:105–118, 1997. © 1997 Wiley-Liss. Inc.     

Neuropeptides in idiopathic chronic constipation (slow transit constipation)

Tissue specimens from the large bowel of 18 patients with long-standing slow transit constipation were investigated to determine the distribution and density of several neuropeptides and amines in the enteric nerve system, and also of endocrine cells in comparison to normal individuals. CGRP (calcitonin gene-related peptide), galanin, glucagon, GRP (gastrin-releasing peptide), metenkephalin, motilin, neuropeptide Y (NPY), PACAP, peptide YY (PYY), serotonin, somatostatin, substance P and VIP were studied by immunohistochemistry. Tissue concentrations of VIP, substance P and galanin were also measured by radioimmunoassay. Significantly increased VIP, SP and galanin contents were found in specimens from the ascending colon. Levels of VIP and galanin were also increased in the transverse colon. Immunohistochemistry revealed only marginal changes with an increased density of PACAP nerve fibres in the smooth muscle and of VIP and PACAP nerves in the myenteric plexus of the transverse colon. In the descending colon substance P and NPY immunoreactivity were also increased in the myenteric plexus while the density of VIP nerve fibres was reduced in the mucosa/submucosa. The frequency of PYY-containing cells and the 5-HT-containing cells in the ascending colon was significantly increased in the constipated patients.     

人胎幽门括约肌内NOS阳性神经元发育的研究

用ＮＡＤＰＨ－ｄ组织化学法对第３个月龄至足月（１０个月胎龄）人胎幽门括约肌内ＮＯＳ阳性神经元的发育进行了观察。结果显示：第５个月胎龄时,肌间神经节处的圆形细胞中部分细胞出现较弱的ＮＯＳ阳性反应。第６个月胎龄时,该处圆形细胞ＮＯＳ阳性反应增强,并分化形成梭形ＮＯＳ阳性神经细胞,部分细胞呈条索状排列向内环肌层方向延伸,有的到达粘膜下层。第７个月胎龄时,肌间神经节细胞胞体明显增大,细胞数增加,胞质增多,     

Presence and regional variation in peptide-containing nerves in the human ureter.

The occurrence, distribution and regional variation of neurones immunoreactive for the neuropeptides, vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), enkephalin (ENK), calcitonin gene-related peptide (CGRP), and substance P (SP) were investigated in human ureters by indirect immunohistochemistry. In addition, immunoreactivities to tyrosine hydroxylase (TH), a marker of noradrenergic neurones and to protein gene product (PGP) 9.5, a general marker of neurones, were also studied. Neurones displaying PGP-, NPY-, VIP- and TH-like immunoreactivity (-LIR) provided a rich innervation to the smooth muscle and blood vessels of the ureter, where they formed dense muscular and perivascular nerve plexuses. In contrast, there was only a moderate to sparse innervation by SP and CGRP-LIR neurones, most of which were distributed to blood vessels and to the sub mucosal layer, and only rarely to smooth muscle bundles. No ENK-LIR was detected in this study. Nerve fibre bundle densities were estimated for each of the localized neurochemicals according to a method described. NPY-LIR nerve fibre bundles were found to account for 80% of the total nerve fibre bundles (i.e. PGP-LIR) in the ureter. On the other hand, TH-LIR and VIP-LIR nerve fibre bundles each accounted for 50% of the total ureteral innervation, whereas SP- and CGRP-LIR nerve fibre bundles each comprised 20% of the total innervation. The abundance and pattern of tissues innervated by these immunoreactive neurones is consistent with the view that some of these neuropeptide substances co-exist with other peptide substances and/or with other known neurotransmitters, such as noradrenaline or acetylcholine. A gradient of innervation was found to exist for all the neurochemicals demonstrated in the ureter, whereby the lower ureter receives a greater density of innervation than the upper ureter. This finding suggests the human ureter is primarily innervated by fibres arising from or via the lower pelvis, i.e. the pelvic plexus. It also supports the view that the lower ureter may perform an important physiological role, such as coordinating the tone of this region during bladder filling and emptying.     

Distribution of calcitonin gene-related peptide, somatostatin, substance P and vasoactive intestinal polypeptide in experimental colitis in rats

Immunohistochemistry was used to examine the distribution of calcitonin gene-related peptide (CGRP), substance P, somatostatin and vasoactive intestinal polypeptide (VIP) in experimental colitis induced with trinitrobenzene sulphonic acid (TNBS) in rats. CGRP immunoreactivity was observed throughout the colonic wall. A significant reduction of CGRP-immunoreactive (IR) nerve fibres was observed in the mucosa after the induction of colitis. After TNBS treatment substance P immunoreactivity was reduced throughout the colon; however, after 7 days there was a marked re-innervation of the circular muscle. Somatostatin immunoreactivity was distributed sparsely within the colonic wall, and was comparatively less affected by colitis. VIP immuno- reactivity was abundantly distributed in the colonic wall and underwent an immediate reduction in the mucosa after TNBS treatment. After 2 days, there was a consistent and progressive increase in the number and density of VIP-IR nerve fibres in the inflamed colon, particularly the circular muscle. This change was associated with a proliferation of nerve fibres within the muscle layers. It was concluded that the early decrease in these neuropeptides was consistent with release from peripheral nerve terminals or the loss of nerves during the initial stages of colonic inflammation, which may be an essential condition for the development of colitis in this model. The observation that the intensity and density of substance P and VIP-IR nerve fibres increased in the circular muscle 7 days after the induction of colitis suggests their possible involvement in tissue repair.     

Ultrastructural identification of vasoactive intestinal polypeptide- and neuropeptide Y-containing nerve fibres in the vas deferens of the guinea-pig

Vasoactive intestinal polypeptide (VIP)-like and neuropeptide Y (NPY)-like immunoreactive nerve fibres were identified by electron microscopic immunohistochemistry in the guinea-pig vas deferens. Labelled nerve fibres were observed in all layers of the wall. However, the NPY-like immunoreactive nerve fibres were most numerous in the muscle layer, and only a few labelled nerve processes were found in the tunica mucosa. In contrast, many of the VIP-like immunoreactive nerve fibres were found in close relation to the blood vessels, especially in the tunica mucosa. In the muscle layer, most immunoreactive nerve fibres were found in close relation to the smooth muscle cells; in these situations the separation between axon and muscle cell membranes was nearly always less than 100 nm, and in many cases as little as 20 nm. The ratio of large to small vesicles in the VIP-like immunopositive axons was 1:3.1, while in the NPY-like immunoreactive nerve terminals the ratio was 1:6.2. These ultrastructural observations confirm that VIP- and NPY-like immunoreactive nerve fibres exist in vas deferens and provide a morphological basis for the possibility that these fibres may participate in the regulation of smooth muscle activity and may influence blood flow in the vas deferens.     

Distribution of calcitonin-gene-related peptide, neuropeptide-Y, vasoactive intestinal polypeptide, cholecystokinin-8, substance P and islet peptides in the pancreas of normal and diabetic rats

Neuropeptides and peptides are particularly important in the co-ordination of pancreatic exocrine and endocrine secretions. In diabetes mellitus, pancreatic endocrine secretion is particularly impaired. This study investigates whether there is a change in the pattern of distribution of neuropeptides including calcitonin-gene-related peptide (CGRP), neuropeptide-Y (NPY), vasoactive intestinal polypeptide (VIP), cholecystokinin-octapeptide (CCK-8), substance P (SP), and islet peptides including insulin (INS), glucagon (GLU), somatostatin (SOM) and pancreatic polypeptide (PP) in the pancreas of streptozotocin (STZ)-diabetic rats. After the onset of diabetes, the pattern of distribution of INS, GLU, SOM and PP cells was deranged. CGRP was demonstrated in ganglion cells of both normal and diabetic pancreas. CGRP was also localized in nerve fibres innervating the blood vessels of both normal and diabetic pancreas. The pancreata of both normal and diabetic rats contained numerous NPY-immunopositive varicose nerve fibres in the wall of blood vessels. In normal pancreatic tissue, VIP-immunopositive nerve fibres were observed in all areas of the pancreas. After the onset of diabetes, VIP-positive nerve fibres were still discernible in the interacinar regions of the pancreas. CCK-8 was identified in nerve fibres innervating both the normal and diabetic rat pancreata. These CCK-8-immunopositive nerves were varicose in nature and distributed in the wall of blood vessels. SP was demonstrated in neurons located in the interlobular areas of normal tissue and in fine varicose nerve fibres of the interacinar region of STZ-induced diabetic pancreas. In conclusion, CGRP, NPY, VIP, CCK-8 and SP are well distributed in both normal and diabetic pancreas.     

Vagal mechanisms controlling serotonin release from the gastrointestinal tract and pyloric motor function

This chapter focuses on investigations of two mechanisms controlled by the vagal nerve; serotonin (5-HT) release from the small intestine and pyloric motor function. Morphological, physiological, pharmacological and biochemical methods were combined in these studies. 5-HT is mainly stored in enterochromaffin cells (EC), but is present also in mast cells and nerve terminals of the gut, as observed by immunocytochemistry. Vagal nerve stimulation causes a release of 5-HT from EC to the portal circulation and to the gut lumen. Morphological evidence for the endoluminal release of 5-HT was obtained by autoradiography and immunofluorescence. The 5-HT release from EC is mediated by a beta-adrenoceptor mechanism via sympathetic adrenergic fibers in the vagal nerve, originating from sympathetic ganglia, e.g. the superior cervical ganglion. This vagal adrenergic pathway was studied by fluorescence microscopy and retrograde tracing of horseradish peroxidase. The vagal peptidergic (nonadrenergic, noncholinergic) control of pyloric motor function was studied in chloralosed cats by means of an in vivo model, where changes of an applied flow of body-warm saline through the pylorus were recorded. Also, gastric volume changes were monitored. By means of immunofluorescence the presence of VIP-, enkephalin (ENK)- and substance P (SP)-like immunoreactivity was demonstrated in pyloric neurons and in the vagal nerve. Physiological evidence for a vagal VIPergic relaxatory mechanism was obtained, while ENK-neurons seem to mediate the vagally induced pyloric contraction, prevented by naloxone pretreatment. SP may mediate part of the vagally induced pyloric and gastric contraction, the latter probably via axon collaterals on final cholinergic neurons. ENKergic and SPergic vagal contractile mechanisms seem to be additive for the pylorus.     

Differential distribution of nerve fibers immunoreactive for substance P and calcitonin gene-related peptide in the superficial and deep muscle layers of the dorsum of the rat

We compared the distribution of substance P (SP) and calcitonin gene-related peptide (CGRP) fibers of the superficial muscle layer (trapezius muscle), median muscle layer (rhomboideus muscle), and deep muscle layer (longissimus and spinalis muscles) of the dorsum of the rat. SP- and CGRP-immunoreactive fibers were seen along the walls of various types of blood vessels and within nerve bundles in skeletal muscles of all layers. Coexistence of SP and CGRP was evident in nerve fibers along the blood vessel walls. The total number of CGRP varicosities per millimeter square of muscle surface area was evaluated quantitatively, and CGRP varicosities were found to be significantly more numerous in the superficial muscle layer than in the deeper ones. After capsaicin treatment, most of the SP and CGRP fibers along the blood vessel walls were eliminated. These results suggest that sensory nerve fibers containing SP and CGRP are distributed more abundantly in the superficial muscle layer than in the deeper ones and that they might be involved in the regulation of local blood flow. The finding of SP- and CGRP-immunoreactive nerve fibers along the blood vessel walls connecting the trapezius muscle and the hypodermis raises the possibility that sensory stimuli to the skin affect the local blood flow of superficial muscle through collaterals of cutaneous fibers.     

Substance P and vasoactive intestinal peptide are reduced in right transverse colon in pediatric slow‐transit constipation

Background Slow‐transit constipation (STC) is recognized in children but the etiology is unknown. Abnormalities in substance P (SP), vasoactive intestinal peptide (VIP) and nitric oxide (NO) have been implicated. The density of nerve fibers in circular muscle containing these transmitters was examined in colon from children with STC and compared to other pediatric and adult samples. Methods Fluorescence immunohistochemistry using antibodies to NO synthase (NOS), VIP and SP was performed on colonic biopsies (transverse and sigmoid colon) from 33 adults with colorectal cancer, 11 children with normal colonic transit and anorectal retention (NAR) and 51 with chronic constipation and slow motility in the proximal colon (STC). The percentage area of nerve fibers in circular muscle containing each transmitter was quantified in confocal images. Key Results In colon circular muscle, the percentage area of nerve fibers containing NOS > VIP > SP (6 : 2 : 1). Pediatric groups had a higher density of nerve fibers than adults. In pediatric samples, there were no regional differences in NOS and VIP, while SP nerve fiber density was higher in sigmoid than proximal colon. STC children had lower SP and VIP nerve fiber density in the proximal colon than NAR children. Twenty‐three percent of STC children had low SP nerve fiber density. Conclusions & Inferences There are age‐related reductions in nerve fiber density in human colon circular muscle. NOS and VIP do not show regional variations, while SP nerve fiber density is higher in distal colon. 1/3 of pediatric STC patients have low SP or VIP nerve fiber density in proximal colon.     

An increase in the expression of neuropeptidergic vasodilator, but not vasoconstrictor, cerebrovascular nerves in aging rats

Perivascular nerve fibres containing noradrenaline (NA), serotonin (5-HT), substance P (SP), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) and calcitonin gene-related peptide (CGRP) were localized in whole-mount stretch preparations of the arteries of the rat circle of Willis using fluorescence and immunohistochemical techniques. Changes in the pattern and density of these perivascular nerves were studied from birth to 27 months of age. All perivascular nerve types reached a peak density of innervation at 1 month of age. This was followed by a general fall in the density of fluorescent nerve fibres. However, with aging, there was a decrease in the expression of vasoconstrictor neurotransmitters (NA and 5-HT) in cerebrovascular nerves, whereas the expression of vasodilator neurotransmitter (VIP and CGRP) in perivascular nerve fibres supplying the rat cerebral arteries was strikingly increased in old age. The density of NPY- and SP-containing nerve fibres was not significantly altered in old age. These changes are discussed in relation to the increased incidence of cerebrovascular disorders in the elderly.     

Nerve fibre studies in skin biopsies in peripheral neuropathies. I. Immunohistochemical analysis of neuropeptides in diabetes mellitus

Standardised skin biopsies followed by immunohistochemical examination for the presence of terminal nerve fibres reacting for neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) were evaluated. Healthy subjects regularly displayed free nerve endings of both fibre types in the papillary and reticular dermis. Both fibre types were present close to blood vessels, while CGRP immunoreactive fibres were more often encountered near sweat gland acini compared to SP fibres. Diabetes mellitus complicated by polyneuropathy was accompanied by marked reduction of SP and CGRP reactive fibres in the dermis layers. Five type I diabetes patients without clinical or neurophysiological evidence of polyneuropathy also had reduced density of both fibre types, being significant for CGRP fibres when compared with controls. Skin biopsy with immunohistochemical staining for neuropeptides may represent a sensitive tool in evaluation of patients with peripheral neuropathies.     

Immunocytochemical localization of substance P,methionine-enkephalin and somatostatin in the cat intestinal wall

Summary The localization of substance P-(SP-), methionine-enkephalin (met-Enk-) and somatostatin (SOM-)like immunoreactivity was studied in the cat pyloric sphincter, ileum, ileocecal sphincter and proximal colon. The enteric plexuses in all regions examined contained SP-, met-Enk- and SOM-like immunoreactive varicose nerve fibres. A large number of especially SP-and met-Enk-containing varicosities were often seen to encircle the nerve cell bodies and processes in the two ganglionic plexuses.The SOM-like immunoreactive perikarya were the only peptide-containing nerve cells, preferentially located in the submucous ganglia. The predominant localization of the SOM-like immunoreactive neurons in the two enteric plexuses of the ileum was the most pronounced regional difference in the distribution pattern of the peptides. Among the layers of the cat intestinal wall the circular muscle contained the most peptide-immunoreactive fibres in contrast to the longitudinal muscle.Evidence was obtained for the occurrence of single peptide-immunoreactive varicose nerve fibres in muscularis mucosae as well as around the glands and the blood vessels. Immunoreactive endocrine cells occurred mainly in the ileum mucosa.     

Perivascular neuropeptides (NPY, VIP, CGRP and SP) in human brain vessels after subarachnoid haemorrhage

Introduction - Cerebral blood vessels are innervated by sympathetic nerve fibres storing neuropeptide Y (NPY), parasympathetic nerves storing acetylcholine, vasoactive intestinal peptide (VIP) and sensory afferent fibres containing calcitonin gene-related peptide (CGRP), substance P (SP) and neurokinin A. In experimental studies on subarachnoid haemorrhage (SAH) there are indications that perivascular peptides are involved. In the present study we have in man measured the levels of NPY, VIP, SP and CGRP in brain vessels of patients that have suffered a fatal SAH and compared this with the levels encountered in subjects that died of an extracerebral cause. Material and methods – Vessels from patients who have died from SAH or nonSAH were obtained during autopsy performed within 24 hrs after death. The peptides were extracted and fractionated with reversed phase liquid chromatography (HPLC). The levels of NPY, VIP, SP, and CGRP were measured with radioimmunoassay. Vasomotor responses of human cerebral arteries were Results – Human cerebral vessels contained NPY, VIP, CGRP and SP which eluted at the same positions as the authentic peptides. The level of CGRP was significantly lower (p < 0.01) in arteries removed from SAH patients as compared to control subjects. The level of SP was not changed, if anything it tended to be increased after SAH. The levels of NPY and VIP were not significantly altered after SAH. In isolated brain vessels α-CGRP was a potent vasodilator of arteries precontracted with whole blood, prostaglandin F_2α or endothelin. It had a poor effect on vessels precontracted with 60 mM potassium. Conclusion – The evidence suggest that the trigemino-cerebrovascular system, storing CGRP and SP, is to a differential degree involved in the pathophysiology of SAH in man and supports the hypothesis of an exhaustion of CGRP as one important factor in the development of late spasm occuring after SAH.