Substance P-immunoreactive Nerve Fibers Research Articles

Substance P combined with epidermal stem cells promotes wound healing and nerve regeneration in diabetes mellitus.

Exogenous substance P accelerates wound healing in diabetes, but the mechanism remains poorly understood. Here, we established a rat model by intraperitoneally injecting streptozotocin. Four wounds (1.8 cm diameter) were drilled using a self-made punch onto the back, bilateral to the vertebral column, and then treated using amniotic membrane with epidermal stem cells and/or substance P around and in the middle of the wounds. With the combined treatment the wound-healing rate was 100% at 14 days. With prolonged time, type I collagen content gradually increased, yet type III collagen content gradually diminished. Abundant protein gene product 9.5- and substance P-immunoreactive nerve fibers regenerated. Partial nerve fiber endings extended to the epidermis. The therapeutic effects of combined substance P and epidermal stem cells were better than with amniotic membrane and either factor alone. Our results suggest that the combination of substance P and epidermal stem cells effectively contributes to nerve regeneration and wound healing in diabetic rats.

TRPM8 has a key role in experimental colitis-induced visceral hyperalgesia in mice.

Transient receptor potential channel melastatin 8 (TRPM8) is activated by cold temperatures and cooling agents (menthol and icilin). Recent studies showed TRPM8 is expressed in visceral organs and peripheral sensory pathways. However, the role of TRPM8 in visceral hyperalgesia is poorly understood in pathological states such as inflammatory bowel disease. Hence, we investigated the distribution of TRPM8 and its involvement in visceral hyperalgesia in experimental colitis mice. TRPM8 immunoreactivity was detected using immunohistochemical staining with fluorescein-conjugated tyramide amplification. Visceral hyperalgesia was measured by the intracolonic administration of TRPM8 agonist, WS-12, in control and dextran sodium sulfate (DSS)-induced colitis mice. TRPM8 immunoreactivity in the distal colon was much higher than in the transverse and proximal colon under physiological conditions. TRPM8 immunoreactivity markedly increased in the distal colon mucosa of DSS-induced colitis mice compared with control mice. The number of TRPM8 nerve fibers in mucosa of DSS- or 2,4,6-trinitrobenzene sulfonic acid-induced colitis model mice drastically increased compared with control mice. TRPM8 immunoreactivities colocalized with the calcitonin gene-related peptide- and substance P-immunoreactive nerve fibers in the mucosa. Intracolonic administration of WS-12 induced behavioral visceral pain-like responses. The numbers of these responses in the colitis model mice were 3 times higher than in control mice, and were decreased by pretreatment with the TRPM8 channel blocker AMTB. Increased expression of TRPM8 may contribute to the visceral hyperalgesia of experimental colitis.

Neuropeptide Analysis of Oral Mucosa in Diabetic Rats

Objective: Increasing evidence indicates that different neuropeptide-containing nerve elements are involved in the immune system and influence the inflammation of the gastrointestinal tract. The aim of this study was to investigate the morphological localization and distribution of the different immunoreactive (IR) nerve fibers and immunocompetent cells in the oral mucosa (e.g. tongue, gingiva) and compare the results with data received from streptozotocin (STZ)-induced diabetic rats. Materials and Methods: The different nerve elements and immunocytes were detected by ABC immunohistochemistry. Results: The IR nerve fibers were found in the tunica propria of oral mucosa with different densities. These IR nerve fibers were mainly located beneath the epithelial lining, around the blood vessels and glands, and some of them were also located in the taste buds. After 2 weeks of STZ treatment the total number of IR nerve fibers, especially the SP and neuropeptide Y (NPY) IR ones, was significantly increased (p < 0.05), as was also the number of immunocytes (lymphocytes, plasma cells, mast cells). Some of these cells also showed immunoreactivity for substance P (SP) and NPY. In several cases the SP IR nerve fibers were found in close proximity to the immunocytes. Electron microscopic investigation also revealed the close association between the IR nerve fibers and immunocompetent cells where the gap was 1 µm or even less. Conclusions: The close anatomical associations suggest communication between nerve fibers and immune cells which can be crucial for maintaining mucosal homeostasis and for ensuring an appropriate response to injury.

Topically applied semaphorin 3A ointment inhibits scratching behavior and improves skin inflammation in NC/Nga mice with atopic dermatitis

Epidermal hyperinnervation in atopic dermatitis (AD) is activated directly by various external stimuli, causing enhanced itching. Nerve density is regulated by the nerve repulsion factor semaphorin 3A (Sema3A), along with nerve elongation factors. To investigate the effects of Sema3A ointment in the NC/Nga mouse model of AD. An AD-like phenotype was induced by repeated application of Dermatophagoides farinae body (Dfb) ointment to the dorsal skin of NC/Nga mice. Vaseline, heparinoid, betamethasone, tacrolimus and recombinant Sema3A ointments were applied to the lesional skin once a day for 4 days. Transepidermal water loss (TEWL) was measured before and after each treatment. We also scored the degree of dermatitis and recorded videos to observe scratching behavior. Subsequently, we collected skin samples from these mice for histological analyses. Topical application of Sema3A, betamethasone and tacrolimus ointments significantly inhibited scratching behavior and improved dermatitis scores in Dfb-treated mice compared with control mice, whereas vaseline and heparinoid had no effects. A significant improvement of TEWL was observed only in Sema3A ointment-treated mice. Moreover, Sema3A ointment reduced the densities of PGP9.5- and substance P-immunoreactive nerve fibers in the epidermis and the numbers of inflammatory cells, such as CD4 immunoreactive T cells and eosinophils, and improved acanthosis in the Dfb-treated mice compared with controls. Sem3A ointment may have therapeutic efficacy in patients with pruritus and dermatitis of AD.

TRPV1 expressing extrinsic primary sensory neurons play a protective role in mouse oxazolone-induced colitis

TRPV1 expressing sensory neurons which have been considered to be largely associated with neurogenic inflammation were chemically denervated by capsaicin treatment in neonatal mice. However, neonatal capsaicin treatment aggravated mouse oxazolone-induced colitis, and did not affect the expression of calcitonin gene-related peptide (CGRP)- or substance P-immunoreactive nerve fibers in the colon. Meanwhile, the capsaicin-induced contraction was absent in the colon of neonatal capsaicin treatment mouse. These results suggest a protective role of TRPV1 expressing extrinsic sensory neurons in oxazolone-induced colitis and the involvement of some neurotransmitter other than CGRP and substance P in the pathogenesis of the colitis.

An immunohistochemical study of endocrine cells in the iodoacetamine-induced rat's gastritis

The gastric epithelium is a complex stucture formed by tubular gastric glands. The gastric glands contain a wide variety of cell types according to the secretion of hydrochloric acid, proteases, mucus, pepsinogen and hormones. Endocrine and local paracrine mediators are of crucial importance for maintaining structural and functional integrity of the epithelium. Therefore our aim was to investigate the precise localization and density of the endocrine cells and their contacts in iodoacetamine-induced rat's gastritis. Methods: somatostatin (SOM), calcitonin gene-related peptide CGRP), substance P (SP), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) and galanin immunoreactive (IR) endocrine cells and nerve elements were detected by ABC immunohistochemistry. Results: The quantitative analysis of the density of the different neuropeptide containing endocrine cells and nerve fibres showed that the (IR) nerve fibres were found with different density. The number of SOM and CGRP IR endocrine cells increased significantly in the gastritis. Both endocrine cells possessed long cytoplasmic extensions and no cell-to-cell contact between parietal and endocrine cells was found. The number of SP, VIP, NPY IR nerve fibres also increased significantly in the gastritis, however, the others showed no alteration. The number of the immunocompetent cells was also increased (being lymphocytes, plasma cells and mast cells) and 16,8% of the immunocytes were IR for SP and 9, 4% of them were IR for NPY. In several cases close contacts were detected between IR nerve fibres and the immunocytes. Conclusions: The increased number of SOM and CGRP endocrine cells and SP IR nerve fibres might play a crucial role in the regulation of gastric acid secretion, including gastric ulcer disease and autoimmune gastritis. The main inhibitor of acid secretion is somatostatin. CGRP activates the mucin biosynthesis, both SOM and CGRP have a preventive action on gastric mucosal injury. The changes in the density of nerve fibres and immunocytes might be the consequences of the inflammation.

Nerve Fibres in the Parathyroid Gland of the Golden Hamster (Mesocricetus auratus): Immunohistochemical and Ultrastructural Investigations

We investigated the morphology and the distribution of the nerve fibres in golden hamster (Mesocricetus auratus) parathyroid glands using antibodies to calcitonin gene-related peptide (CGRP) and substance P, and electron microscopy. CGRP-immunoreactive nerve fibres were densely distributed in the interstitial tissues and the capsules of the hamster parathyroid glands. Some nerve fibres were detected in close proximity of the parathyroid chief cells. The distribution pattern for substance P-immunoreactive nerve fibres was roughly the same as for CGRP-immunoreactive fibres. Ultrastructurally, we found numerous nerve fibres joining the blood vessels. Axon bundles were located adjacent to the smooth muscle cells of the arterioles. The axons formed structurally specialized neuromuscular junctions with the vascular smooth muscle cells. Some axons were in close vicinity to the parathyroid chief cells. These findings indicate that the hamster parathyroid gland contain CGRP and substance P, which may regulate the blood flow and the secretory activity of the gland.

Change of substance P containing neural pathway in rat model of irritable bowel syndrome

AIM: To investigate the expression of substance P (SP) and c-fos in the enteric nervous system (ENS) and central nervous system (CNS) of a rat model of constipation-pre- dominant irritable bowel syndrome (C-IBS), and to explore the possible role of SP-immunoreactive nerve fibers in IBS. METHODS: The rat model was established by intragastrical injection with ice-cold water (0-4 ). The expressions of SP and c-fos in the ENS and CNS of model group (n = 10) and control group (n = 10) were detected by immunohis- tochemistry, and analyzed semi-quantitatively by comput- erized color image analyzer. The statistical differences of the opacity density (OD) and immunoreactive areas (IA) between the two groups were examined by t-test. RESULTS: The expressions of SP in ileocecus, colonic myenteric plexus, spinal dorsal horn, hypothalamus, and the fore part of gyrus cinguli were significantly higher in model group than those in the corresponding regions of control group (OD: 176.6 vs 155.5, 172.3 vs 152.0, 182.1 vs 160.2, 128.3 vs 117.9, 127.9 vs 114.5, P <0.05; IA: 169318 vs 137655, 144728 vs 114403, 145117 vs 117612 except in ileocecus and colonic myenteric plexus, P <0.05). The expression of c-fos in the above tissues was significantly higher in model group than that in the corresponding regions in control group (OD: 120.9 vs 109.0, 101.3 vs 92.2, 125.4 vs 88.7, 115.5 vs 88.6, 120.6 vs 105.1, P<0.05; IA: 191 712 vs 154 363, 73 597 vs 41 179, 46 258 vs 33 238, 302 369 vs 204 563, 708 506 vs 409 856, P <0.05). There was a close correlation between the expression of SP and c-fos in the same region (r = 0.594-0.721, P <0.05). CONCLUSION: The abnormal SP immunoreactive nerve fibers may be involved in the pathogenesis of IBS. SP containing neural pathway may play an important role in the regulation of the gastrointestinal function.

Changes in nasal mucosal innervation in horses with grass sickness.

Equine grass sickness is a dysautonomia characterised by widespread destruction of autonomic ganglia, resulting in the clinical signs of dysphagia, constipation, profuse sweating, tachycardia, rhinitis sicca and high mortality rate. Rhinitis sicca is a common finding in horses with the chronic form and we have postulated that alterations in autonomic innervation of the nasal mucosa might underlie this clinical presentation. In this study, the expression and distribution of nerve fibres immunoreactive for calcitonin gene-related peptide (CGRP), substance P (SP), the general neuronal marker protein gene-product 9.5 (PGP 9.5; ubiquitin) and the intermediate neurofilaments (PAN-N; neurorfilaments L, M and H) in the nasal mucosa of normal horses (n = 10) and horses with EGS (n = 18; acute n = 8, subacute n = 3, chronic n = 7) was assessed. Innervation density and distribution was investigated in the different groups using standard immunohistochemical techniques. Significant differences were noted when comparing the density and distribution of nerve fibres immunoreactive for PGP 95 and PAN-N, with PGP 95 consistently giving better staining in all groups and at all sites in the nasal mucosa. An apparent increase in the density of innervation was noted for acute vs. normal cases. A significant reduction in the density of innervation was noted only with PAN-N when comparing normal horses and acute cases with the chronic group (P < 0.05). CGRP and SP immunoreactive nerve fibres were typically most abundant in the epithelial and subepithelial layers, but the quality of staining and nerve fibre density was greater for SP, achieving significant difference in several comparisons. The density of innervation for SP was significantly reduced in the chronic group compared to the normal and acute groups (P < 0.01). A significant decrease was noted for CGRP only for the acute and chronic groups (P<0.05). These results demonstrate a reduction in the expression of the sensory neuropeptides in nasal mucosal innervation as a consequence of equine dysautonomia, and may underlie the clinical presentation of rhinitis sicca noted with this disease. Nasal biopsy may be of use in antemortem diagnosis of grass sickness and identification of mucosal denervation; and might also be useful in the treatment of rhinitis in EGS cases.

Do Cardiac Neurons Play a Role in the Intrinsic Control of Heart Rate in the Rat?

Three experiments were performed to see whether cardiac neurons contribute to the intrinsic control of heart rate in right atria of adult rats. The intrinsic heart rate response (IRR) was examined by raising right atrial pressure from 2 to 8 mmHg for 3 min. In isolated preparations of the right atrium, the IRR was not significantly altered by the addition of either 1 microM atropine (n =6; control +19+/- 3 min(-1) ; atropine+18+/-3 min(-1); (mean /+/-S.E.M.)) or 1 microM propranolol (n = 5; control +22+/- 4 min(-1); ; propranolol +21+/-3 min(-1); ). Tetrodotoxin (0.5 microm) had no effect on the IRR (n = 6; control +37+/-5 min(-1); tetrodotoxin 38+/-5 min(-1); ). In another experiment, 2-day-old rat pups were injected with capsaicin (50 mg kg(-1); treated) or with vehicle(control). There was no difference in the IRR of right atrial preparations taken from control and treated animals after they reached adulthood (control (n = 7) and treated (n = 8): +30+/- 4 and +32+/- 4 min(-1)). The influence of right atrial pressure on the efficacy of vagal stimulation was examined. The rate response to vagal stimulation was reduced similarly in control and treated preparations when pressure was elevated from 2 to 4 mmHg (control and treated: -34+/- 5% and -33+/- 3%). The effectiveness of the capsaicin treatment was confirmed by the depletion of substance P-immunoreactive nerve fibres in cardiac tissues. Together, these results strongly suggest that cardiac neurons are not involved in intrinsic heart rate control.

Morphological examination of the termination pattern of substance P-immunoreactive nerve fibers in human antral mucosa.

The termination pattern of substance P (SP)-containing axons in human antral mucosa was examined using immunohistochemical techniques at the light and electron microscopic level. SP-immunoreactive (IR) axons were found to extend towards the pit region of the glands, where intraepithelial axons were observed. Electron microscopy showed immunostained axon profiles in close contact with the basement membrane of surface mucous cells. Membrane-to-membrane contacts between labeled axons and myofibroblast-like cells were identified, and SP-IR axons that were apposed to the epithelium were also in contact with subjacent myofibroblast-like cells. The anatomical relationship between SP-IR axons and the cells of the muscularis mucosae was investigated by light microscopy. Immunoreactivity for alpha-smooth muscle actin (alpha-sma) was used to visualize the smooth muscle cells, and the alpha-sma-IR cells were found to create a network that surrounded the gastric glands. Immunostained varicose axons ran alongside and in close apposition to the labeled muscle strands. Ultrastructural examination showed close contacts between SP-IR axon profiles and smooth muscle-like cells. In conclusion, SP-containing neurons may be important for sensory and secretomotor functions in the human antral mucosa.

Slow transit constipation in children.

Patients with chronic constipation that fails to respond to treatment remain a challenge for paediatricians and surgeons. Ongoing work in our institution suggests that a number of children with intractable symptoms have slow transit constipation, which has only been described recently in paediatrics. Common features of slow transit are: delayed passage of the first meconium stool beyond 24 h of age, symptoms of severe constipation within a year, or treatment-resistant 'encopresis' at 2-3 years, soft stools despite infrequent bowel actions, and delay in colonic transit on a transit study. A proportion of children with slow transit constipation have an abnormality of intestinal innervation associated with the dysfunctional colonic motility, recognized as intestinal neuronal dysplasia (IND). Intestinal neuronal dysplasia type B, the most common variant of IND, is defined on rectal biopsy by hyperplasia of the submucosal plexus. On laparoscopic colon muscle biopsy, many specimens show reduced numbers of excitatory substance P-immunoreactive nerve fibres in the circular muscle. Functional markers of the nerves allow new diagnostic criteria to be developed which may also allow a more rational approach to treatment. The aetiology remains obscure and the optimal management poorly defined, although subtotal colectomy, proximal colostomy or appendicostomy (for antegrade enemas) have been tried. Once the anatomy and physiology of the colon in children with slow colonic transit is better understood, we will have defined not only a new form of constipation, but also will be able to consider new therapies.

Immunohistochemical study of the distribution of adrenergic and peptidergic innervation in the equine uterus and the cervix.

Little is known about neurogenic regulation of uterine contractility in mares. The present study investigated the distribution of adrenergic and peptidergic nerves in the mare uterus. Samples from the uterine horn, body and cervix were collected from 18 cyclic mares for immunohistochemistry. The uterus was well supplied with adrenergic nerves. A large number of tyrosine hydroxylase- and dopamine beta-hydroxylase-immunoreactive nerve bundles and fibres were present in the myometrium and endometrium in all regions of the uterus and cervix. These adrenergic nerve bundles and fibres travelled parallel to the muscle layers and were often associated with blood vessels. The density of peptidergic nerves was less than that of adrenergic nerves, but the pattern of distribution was similar. Neuropeptide Y-immunoreactive nerve fibres were the most abundant, whereas vasoactive intestinal polypeptide- and calcitonin gene-related peptide-immunoreactive nerve fibres were less frequently seen. Substance P-immunoreactive nerve fibres were the most sparse. Peptidergic nerves were distributed among the smooth muscle layers and near endometrial glands and were often associated with blood vessels in all regions of the uterus. The density of peptidergic nerve fibres was similar in the uterine horn and body but was slightly denser in the cervix. These findings indicate that uterine innervation may have an important role in controlling reproductive functions in mares.

Immunohistochemical study of the distribution of adrenergic and peptidergic innervation in the equine uterus and the cervix

Little is known about neurogenic regulation of uterine contractility in mares. The present study investigated the distribution of adrenergic and peptidergic nerves in the mare uterus. Samples from the uterine horn, body and cervix were collected from 18 cyclic mares for immunohistochemistry. The uterus was well supplied with adrenergic nerves. A large number of tyrosine hydroxylase- and dopamine beta-hydroxylase-immunoreactive nerve bundles and fibres were present in the myometrium and endometrium in all regions of the uterus and cervix. These adrenergic nerve bundles and fibres travelled parallel to the muscle layers and were often associated with blood vessels. The density of peptidergic nerves was less than that of adrenergic nerves, but the pattern of distribution was similar. Neuropeptide Y-immunoreactive nerve fibres were the most abundant, whereas vasoactive intestinal polypeptide- and calcitonin gene-related peptide-immunoreactive nerve fibres were less frequently seen. Substance P-immunoreactive nerve fibres were the most sparse. Peptidergic nerves were distributed among the smooth muscle layers and near endometrial glands and were often associated with blood vessels in all regions of the uterus. The density of peptidergic nerve fibres was similar in the uterine horn and body but was slightly denser in the cervix. These findings indicate that uterine innervation may have an important role in controlling reproductive functions in mares.

A parasympathetic ganglion innervating the harderian gland and lacrimal gland of the musk shrew (Suncus murinus): fluorescent tracing and immunohistochemical studies.

A small ganglion, named the peri-trigeminal ganglion (PTG), was found in the ventromedial border of the rostral half of the trigeminal ganglion (TG) in the musk shrew (Suncus murinus). In frontal sections, the PTG was semicircular or elliptical in shape. Most of the neurons constituting this ganglion were round in shape and much smaller than those of the TG. The retrograde fluorescent tracer fluoro-gold was injected into various regions of the face in order to investigate innervation by the PTG neurons. When the tracer was injected subcutaneously around the external acoustic meatus and around the circumference of the orbit, a number of labeled neurons were seen not only in the TG but also in the PTG. After applying the tracer to the lacrimal gland (LG) and the harderian gland (HG), numerous labeled neurons were detected only in the PTG. A few labeled neurons were found in the PTG after injection into the palatoglossal arch. Immunohistochemically, most of the neurons constituting the PTG were positive for vasoactive intestinal polypeptide (VIP) antiserum. And a moderate number of somatostatin (SOM)-immunoreactive neurons and a small number of leucine-enkephalin (L-ENK)-immunoreactive neurons were detected. Numerous substance P-immunoreactive nerve fibers and varicosities were found in the PTG, and fewer L-ENK-, SOM- and VIP-immunoreactive fibers were observed. The present results suggest that the PTG is an autonomic ganglion that resembles in part the pterygopalatine ganglion in other species, and mainly innervates the HG and LG.

Activation of neurokinin 1 receptors on interstitial cells of Cajal of the guinea-pig small intestine by substance P

The aims of this work were to determine whether cells that are similar to the interstitial cells of Cajal (ICC) and have immunoreactivity for the neurokinin 1 (NK1) receptor are indeed ICC; to determine whether the agonist, substance P, binds to and activates the receptor on presumptive ICC; and to investigate the relationship between substance P-immunoreactive nerve fibres and ICC. ICC at the level of the myenteric plexus and in the deep muscular plexus in the duodenum and ileum of the guinea-pig were investigated. Immunoreactivities for the ICC marker, Kit, and the NK1 receptor were colocalised in ICC of the myenteric and deep muscular plexuses. In tissue fixed immediately after its removal from the animal, NK1 receptor-immunoreactive ICC were found at the level of the myenteric plexus in the duodenum, but not in the ileum, and in the deep muscular plexus in the duodenum and ileum. The majority of receptor immunoreactivity was on the cell surface. ICC were exposed to substance P (10(-7) M), initially at 4 degrees C for 1 h to allow the agonist to bind, followed by incubation at 37 degrees C to allow receptor internalisation to proceed. Exposure to substance P caused the NK1 receptor immunoreactivity to aggregate in clumps in the cytoplasm of ICC of the myenteric and deep muscular plexuses, including the ICC of the myenteric plexus of the ileum, where NK1 receptor immunoreactivity was not seen if tissue was not exposed to substance P. Substance P, to which the fluorescent label, cyanine 3.18 (Cy-3), was coupled, bound to the ICC. The Cy-3-substance P was internalised with the receptor following warming to 37 degrees C. Many, but not all, ICC were closely apposed by nerve fibres with immunoreactivity for substance P. It is concluded that the NK1 receptor immunoreactivity on ICC represents receptor that is functional in the sense that it binds the natural agonist substance P and undergoes agonist-induced internalisation. ICC are likely to receive excitatory innervation from the close approaches of tachykinin-containing nerve fibres.

Differential distribution of substance P binding sites in guinea-pig sympathetic ganglia

We have used a combination of autoradiographic and immunohistochemical techniques to investigate the distribution of binding sites for substance P in relation to the distribution of substance P-immunoreactive nerve fibres and specific functional populations of neurons in the sympathetic ganglia of guinea-pigs. There was considerable heterogeneity in the density of binding sites for Bolton Hunter labelled 125 I−substance P (BHSP). Binding sites were more dense in the prevertebral ganglia, such as the coeliac and inferior mesenteric ganglia, than in the paravertebral ganglia, such as the superior cervical or lumbar chain ganglia. The binding sites tended to be clumped within the ganglia. Within the prevertebral ganglia, they were associated predominantly with neurons projecting to the enteric plexuses. Many of these neurons contained somatostatin immunoreactivity. In the lumbar sympathetic chain ganglia, there was a weak association of binding sites with neurons containing immunoreactivity to vasoactive intestinal peptide. Overall, the density of binding sites matched the density of nerve fibres containing immunoreactivity to substance P in different ganglia. However, within particular ganglia, there was little, if any, correlation between the distribution of binding sites and nerve fibres containing substance P. Most of the binding sites in the ganglia had the pharmacological characteristics of NK 1 receptors. Our results show that there is considerable heterogeneity in the expression of NK 1 receptors in the sympathetic ganglia of guinea-pigs. However, given the relatively poor spatial correlation between the distribution of binding sites and potential sites of substance P release from intraganglionic nerve fibres, we suggest that substance P may diffuse for relatively large distances through the ganglia, with actions only on those neurons selectively expressing NK 1 receptors.

Substance P in the Gastrointestinal Tract of Non-Obese Diabetic Mice

Background: Gut dysmotility occurs in most diabetics. Substance P is a neurotransmitter that plays an important role in regulating gastrointestinal motility. The present investigation was carried out to evaluate the possible role of this neurotransmitter in the pathogenesis of dysmotility in diabetics. Methods: Pre-diabetic and diabetic female non-obese diabetic (NOD) mice aged 22-24 weeks were studied. As controls, BALB/CJ mice of the same age and sex were used. Substance P concentrations in tissue extracts from the antrum, duodenum, and colon were determined with radioimmunoassay. Substance P-immunoreactive nerve elements and endocrine cells were identified by immunocytochemistry and quantified with computerized image analysis. Results: Substance P levels in the antrum of both pre-diabetic and diabetic NOD mice were significantly lower than those of controls. In the duodenum and colon substance P levels were higher than those of the controls in both pre-diabetics and diabetic NOD mice. The relative volume density of substance P-immunoreactive nerve fibres in the colon of diabetic NOD mice was significantly decreased. There was no statistically significant difference between pre-diabetic and diabetic NOD mice and controls with regard to the relative volume density of substance P immunoreactive nerve fibres in the antrum and duodenum. In the antrum the number of substance P-immunoreactive cells decreased significantly in both pre-diabetic and diabetic NOD mice. In the duodenum and colon the numbers of these cells in NOD mice did not differ from those of controls. Conclusions: The changes in substance P contents in various parts of the gastrointestinal tract of NOD mice seem to be primary to the onset of diabetes. The decreased antral substance P contents in NOD mice seems to be caused by structural change in the mucosal endocrine cells. In the small and large intestine the increase in substance P levels appears to be caused by change in the physiologic activities of the nerve element and/or endocrine cells rather than by structure changes. The abnormalities observed here in an animal model for diabetes type I might have relevance for the gastrointestinal dysmotility displayed in human diabetes.

Innervation of "painful" lumbar discs.

The authors investigated the innervation of discographically confirmed degenerated and "painful" human intervertebral discs. To determine the type and distribution patterns of nerve fibers present in degenerated human intervertebral discs. The innervation of intervertebral discs has previously been extensively described in fetal and adult animals as well as humans. However, little is yet known about the innervation of severely degenerated human lumbar discs. The question may be posed whether a disc that has been removed for low back pain possesses an increased innervation compared with normal discs. The presence of nerve fibers was investigated using acetylcholinesterase enzyme histochemistry, as well as neurofilament and substance P immunocytochemistry. From 10 degenerated and 2 control discs, the anterior segments were excised and their nerve distribution studied by examining sequential sections. In all specimens, nerve fibers of different diameters were found in the anterior longitudinal ligament and in the outer region of the disc. In 8 of 10 degenerated discs, fibers were also found in the inner parts of the disc. Substance P-immunoreactive nerve fibers were sporadically observed in the anterior longitudinal ligament and the outer zone of the anulus fibrosus. Findings indicate a more extensive disc innervation in the severely degenerated human lumbar disc compared with normal discs. The nociceptive properties of at least some of these nerves are highly suggested by their substance P immunoreactivity, which provides further evidence for the existence of a morphologic substrate of discogenic pain.

Functional relationships between sensory nerve fibers and mast cells of dura mater in normal and inflammatory conditions

In this study, we have characterized the phenotype of mast cells in rat dura mater and their topological and functional relationships with C-fibers in normal and inflammatory conditions. Three mast cell populations with different size, morphology and localization were characterized by their content of specific neutral serine proteases. They showed immunoreactivity corresponding to rat mast cell protease I, rat mast cell protease II, or both proteases. Using confocal microscopy, all three mast cell types were observed in close apposition (distance less than 100 nm) to calcitonin gene-related peptide- and substance P-immunoreactive nerve fibers in both controls and rats infected with the nematode Nippostrongylus brasiliensis. After nematode infection or neonatal treatment with capsaicin, a large increase in the number of rat mast cell protease II-immunoreactive mast cells was found within dura mater segments (+1478% and +596%, respectively), without concomitant changes of rat mast cell protease I- or rat mast cell protease I/II-immunoreactive mast cells. Under both these conditions, the increase in mast cell number was accompanied by a significant increase in rat mast cell protease II level within tissue extracts (+281% after nematode infection and +36% after capsaicin treatment). The functional interaction of mast cells with sensory nerve fibers in the dura mater was assessed by evaluating [ 3H]histamine synthesis after administration of l-[ 3H]histidine, an index of mast cell activity. The H 3 receptor agonist ( R)- α-methylhistamine (15 mg/kg, i.p.) had no effect, but administration of the H 3 receptor antagonist, thioperamide (10 mg/kg, i.p.), resulted in a significant increase of [ 3H]histamine synthesis (+62%). This effect was reduced in neonatal capsaicin-treated rats, but not completely suppressed (+35%), very likely because of partial denervation, as assessed by monitoring calcitonin gene-related peptide immunoreactivity. It is concluded that, in the dura mater, as in peripheral tissues, sensory nerve fibers and mast cells actively synthesizing and releasing histamine form a short inhibitory feedback loop involving prejunctional H 3 receptors that could regulate the release of pro-inflammatory mediators, thus limiting the extent of inflammatory reactions.