SP-immunoreactive Nerve Fibers Research Articles

Neural Regeneration/Remodeling in Engineered Coronal Pulp Tissue in the Rat Molar

IntroductionThis study aimed to examine the process of reinnervation during coronal pulp tissue regeneration in a rat model in which rat bone marrow mesenchymal stem cells were implanted in pulpotomized molars. MethodsThe maxillary first molars of Wistar rats were pulpotomized, and preformed biodegradable porous poly L-lactic acid scaffolds and hydrogel carrying rat bone marrow mesenchymal stem cells were implanted in the pulp chamber. After 3, 7, and 14 days, the implanted teeth were processed for histologic analysis; immunoperoxidase staining for protein gene product 9.5 (a general neuronal marker), calcitonin gene-related peptide (CGRP), or substance P (SP); and real-time polymerase chain reaction for nerve growth factor (NGF) and growth-associated protein 43 (GAP-43) messenger RNA (mRNA) expression. ResultsHistologic analysis of the implanted region revealed sparse cellular distribution at 3 days, pulplike tissue with a thin dentin bridge–like structure at 7 days, and dentin bridge–like mineralized tissue formation and resorption of most scaffolds at 14 days. Protein gene product 9.5 and CGRP-immunoreactive nerve fibers showed the lowest density at 3 days and significantly increased until 14 days when the CGRP-immunoreactive fibers reached normal levels. SP-immunoreactive nerve fibers showed the highest density at 7 days and decreased to normal levels at 14 days. NGF mRNA increased with time, whereas GAP-43 mRNA levels peaked at 3 days and subsequently dropped until 14 days. ConclusionsRegeneration/remodeling of SP-immunoreactive and CGRP-immunoreactive nerve fibers with increased mRNA expression of NGF and GAP-43 occurred in a rat model of coronal pulp tissue engineering with bone marrow mesenchymal stem cells.

Substance P and the neurokinin-1 receptor expression in dog ileum with and without inflammation

In the gastrointestinal tract, the tachykinin Substance P (SP) is involved in motility, fluid and electrolyte secretion, and blood flow and regulation of immunoinflammatory response. SP exerts its biological activity on target cells by interacting mainly with the neurokinin-1 receptor (NK1R). The present study aims to quantify the percentage of SP-immunoreactive (SP-IR) enteric neurons and the density of SP-IR nerve fibers in the ileum of control dogs (CTRL-dogs; n=7) vs dogs with spontaneous ileal inflammation (INF-dogs; n=8). In addition, the percentage of enteric neurons bearing NK1R, and nitrergic neurons (nNOS-IR) expressing NK1R immunoreactivity were evaluated in both groups. The percentages of SP-IR neurons were similar in CTRL- and INF-dogs, in either the myenteric (MP) (15±8% vs. 16±7%, respectively) and submucosal plexus (SMP) (26±7% vs. 24±14%, respectively). In INF-dogs, the density of SP-IR mucosal nerve fibers showed a trend to decrease (P=0.07). Myenteric neurons of CTRL- and INF-dogs expressed similar percentages of NK1R-immunoreactivity (39±5% vs. 38±20%, respectively). Submucosal NK1R-IR neurons were occasionally observed in a CTRL-dog. MP nitrergic neurons bearing NK1R showed a trend to decrease in INF-dogs vs. CTRL- dogs (41±22% vs. 65±10%, respectively; P=0.11). In INF-dogs, muscle cells and immune cells overexpressed NK1R immunoreactivity. These findings should be taken as a warning for possible intestinal motility disorders, which might occur during administration of NK1R-antagonist drugs. Conversely, the strong expression of NK1R immunoreactivity observed in muscle and mucosal immune cells of inflamed tissues may provide a rationale for the use of NK1R antagonist drugs in the treatment of intestinal inflammation.

The neurochemical changes in the innervation of human colonic mesenteric and submucosal blood vessels in Ulcerative colitis and Crohn's disease

Neurogenic inflammation involves vasodilation, oedema and sensory nerve hypersensitivity. Extrinsic sensory nerves to the intestinal wall mediate these effects and functional subsets of these extrinsic nerves can be characterized by immunohistochemical profiles. In this study such profiles were examined in samples from patients with inflammatory bowel disease (IBD), in particular ulcerative colitis (UC) and Crohn's disease (CD). Healthy margins from cancer patients were compared to specimens from IBD patients. All nerve fibres were labelled by PGP 9.5. Double and triple labelling with TH, NPY, SP, SOM, NOS, VIP, VAChT, CGRP, TRPv1 were performed. Perivascular nerve fibres in the mesentery, and submucosa, were examined. The percentage of all labelled nerve fibres was calculated with a transect method. Total number of varicosities on mesenteric vessels increased in IBD but decreased around submucosal vessels. The percentage of nerve fibres around submucosal arteries labelled by SP increased from 11% in controls to 20% (UC) and 24% (CD) and mesenteric artery nerve fibres were unchanged. Nerve fibres labelled by SOM were markedly reduced surrounding submucosal arteries, from 22% to 1% (UC) and 2% (CD), but not perivascular mesenteric nerve fibres. 87 to 93% of SP immunoreactive nerve fibres were also reactive for TRvP1. TRPv1 labelling without SP was 12%in controls and increased to 40% in CD submucosal specimens. There is an increase in SP and TRPv1, and a reduction in SOM immunoreactive nerve fibres in IBD. Changes in the perivascular functional nerve subclasses may underlie the hyperaemia, and ulceration, characteristic of IBD. Furthermore, pain may relate to underlying neural changes.

Neuroimmunomodulation in human autoimmune liver disease

Bidirectional interaction between immune and nervous systems is considered an important biological process in health and disease. However, little is known about the mechanisms involved in their interaction in the human liver. This study examines the distribution of intrahepatic NPY, SP immunoreactive (IR) nerve fibers and their antomical relationship with immunocells containing tumor necrosis factor-α (TNF-α) and nuclear factor κB (NF-κB) in patients with autoimmune hepatitis. Liver specimens were obtained from control liver and autoimmune hepatitis patients. The immunoreactivity was determined by immunohisto- and immunocytochemistry and confocal laser microscopy. In hepatitis, the number of NPY-IR and SP-IR nerve fibers increased significantly. These IR nerve fibers were in very close contact with the lymphocytes. In healthy controls, no NPY-IR, SP-IR or NF-κB IR lymphocytes and only a few TNF-α positive cells, were observed. In hepatitis, some of the lymphocytes showed immunoreactivity for SP and NPY in the portal area. Fluorescent double-labeled immunostaining revealed that in these cells NPY did not colocalize with TNF-α or NF-κB. However, some of the SP fluorescence-positive immune cells exhibited immunostaining for p65 of NF-κB, where their labeling was detected in the nuclei. Under the electronmicroscope, these cells could be identified (lymphocytes, plasmacells and mast cells). The gap between the IR nerve fibers and immunocells was 1 μm or even less. Overexpression of SP in lymphocytes may amplify local inflammation, while NPY may contribute to liver homeostasis in hepatitis. Neural immunomodulation (SP antagonists and NPY) might be a novel therapeutic concept in the management of liver inflammation.

Immunohistochemical evidence for the presence of a vasoactive intestinal peptide, neuropeptide Y and substance P in rat adrenal medulla after heat stress

Immunohistochemistry revealed the presence of VIP-, NPY- and SP-immunoreactivity in the rat adrenal medulla. VIP- and NPY-immunoreactivity was detected in chromaffin and ganglion cells and in nerve fibers, but SP-immunoreactivity was found only in chromaffin cells. After acute heat stress, VIP- and NPY- immunoreactivities in cells and nerve fibers were reduced, probably as a result of the release of these peptides with catecholamines. The absence of SP-immunoreactive ganglion cells in the adrenal medulla suggests that the SP-immunoreactive nerve fibers are extrinsic in origin.

Age-related changes in substance P-immunoreactive nerve structures of the rat recto-anal region

Abstract The recto-anal region is innervated by extrinsic and intrinsic nerves and a number of neuropeptides including substance P (SP) have been suggested to participate in the regulation of intestinal movements. We examined the age-related changes in the distribution of SP-immunoreactive nerve structures in the distal part of the rat large intestine. Using immunohistochemistry, the presence of SP was studied in fresh tissues from Wistar rats at different ages taken at three sampling sites, the distal rectum, anal canal and internal anal sphincter. In the 15-day old rats the myenteric plexus of the distal rectum and anal canal was well outlined by numerous SP-immunoreactive varicose nerve fibres encircling immunonegative perikarya. In the circular muscle layer, nerve fibres and small nerve bundles ran parallel to the muscle cells, while in the longitudinal muscle layer, only occasional nerve fibres were seen. At the level of the internal anal sphincter, no myenteric ganglia were present. Here, thin varicose fibers ran parallel to the smooth muscle cells. In the 3-month old rats, a larger number of intensely staining SP-immunoreactive nerve fibres were found and in the circular muscle layer, thicker nerve strands were observed. In the 26-month old rats, the density and staining intensity of SP-immunopositive nerve fibres in the myenteric plexus was lower than in the 3-month-old rats. Similar changes in the SP-immunostained fibres in the internal anal sphincter were observed. Degenerative alterations in SP-containing fibres during aging appear to play a role in ano-rectal motility and sphincter control.

Fall in density, but not number of myenteric neurons and circular muscle nerve fibres in guinea‐pig colon with ageing

In guinea-pig ileum, ageing has been associated with a decrease in enteric neurons. This study examined guinea-pig colon and measured changes in gut dimensions, neuron size, density and ganglionic area. Changes in motor nerve fibres in the circular muscle were also measured. Myenteric neurons in whole-mount preparations of mid-colon from 2-week, 6-month, and 2-year-old guinea-pigs were labelled immunohistochemically with the neuronal marker human neuronal protein HuC/HuD, and numbers of neurons mm(-2), neuronal size, ganglionic area mm(-2), gut length, circumference and muscle thickness were measured. Corrected numbers of neurons mm(-2) and ganglionic area mm(-2) accounting for growth of the colon were calculated. Additionally, nerve fibres in circular muscle cross-sections were labelled with antibodies against nitric oxide synthase (NOS) and substance P (SP) and the density of nerve fibres in circular muscle was measured. The numbers of neurons mm(-2) decreased by 56% (from 2 weeks to 2 years) with no change in neuron size. Total neuron numbers decreased by 19% (P = 0.14) when adjusted for changes in length and circumference with age. The percentage area of NOS- and SP-immunoreactive (IR) nerve fibres in the circular muscle decreased (P < 0.001), but the total area of NOS and SP-IR nerve fibres increased (P < 0.01) due to an age-related increase in muscle thickness. The density of myenteric neurons in guinea-pig mid-colon halved from 2 weeks to 2 years, but when the increase in colon dimensions was considered, the number of neurons decreased by only 19%. The percentage area of motor nerve fibres in the circular muscle decreased with no change in total volume of nerve fibres.

Substance P Immunoreactivity in the Rat Adrenal Gland during Postnatal Development

Substance P (SP)-immunoreactive nerve fibers in the adrenal medulla was very few during postnatal day 0-5, indicating that its synthesis in the neurons and the transport to nerve endings was incomplete by the end of this period. Since the number of SP-immunoreactive nerve fibers gradually increased during postnatal week 1-2, it was hypothesized that SP was not fully transported to nerve endings until postnatal week 1-2. At postnatal week 3, numerous SP-immunoreactive nerve fibers contacted some noradrenaline (NA) cells but not adrenaline (A) cells in the medulla. From postnatal week 3 onward, the abundance and expression patterns of SP-immunoreactive nerve fibers in the medulla were similar to those in adults. At postnatal week 3, the innervation with SP-immunoreactive nerve fibers was completed indicating the possibility that SP affected on the secretory activity of NA cells but not of A cells in the medulla. The medullary SP-immunoreactive nerve fibers contacting the chromaffin cells possessed a few dense-cored vesicles in their endings at postnatal week 8. Very few SP-immunoreactive chromaffin cells were found in the medulla from postnatal day 0 onward, and SP immunoreactivity was primarily observed in granular cores of the cells suggesting that SP and catecholamine synthesized in the chromaffin cells were released from the granules by adequate stimuli. Very few or a few SP-immunoreactive nerve fibers, acting as a vasomotor effect were found around blood vessels in the superficial cortex from postnatal day 0 onward.

Oral Neuroimmune Network and Mast Cells

Inflammation is a critical process in the oral cavity, especially in gingival inflammation and pulpitis, as seen in periodontitis and decayed tooth structure. Nevertheless, the cellular process involved in oral inflammation is not well delineated. Recent evidence from other organs as well as the mouth suggests that neurogenic inflammation involving mast cells (MCs) may be a critical factor. MCs, best known for their role in allergic reactions, are also involved in immunity and inflammation. They are located at strategic points close to small blood vessels and nerve fibers often containing substance P (SP). The pain models of reversible or irreversible pulpitis simply suggest the complexity of neural-inflammatory interactions within the dental pulp. In the pulp and periapical area, neuropeptides and cytokines modulate vascular responses, increase permeability and leukocyte migration. SP-immunoreactive nerve fibers and TNF-positive MCs were found localized around blood vessels in all samples of periapical granulomas. By generating a profound number of potent mediators, MCs may serve as a link between the immune, endocrine and nervous systems in pulp inflammation. This role has been strengthened by recent evidence of MCs involvement in inflammatory conditions.

Changes of substance P-immunoreactive nerve fiber innervation density in the sublesional bones in young growing rats at an early stage after spinal cord injury

Spinal cord injury (SCI) causes osteoporosis (OP), and the neuropeptide substance P (SP) may play important roles in the pathogenesis of OP after SCI. Our study confirmed SCI-induced sublesional bone loss in young rats at an early stage is associated with a significant increase of SP-immunoreactive nerve fiber innervation density. Spinal cord injury (SCI) causes osteoporosis (OP), and neuropeptides may play important roles in the pathogenesis of OP after SCI. However, few data exist concerning the relationship between neural factors and OP following SCI. One hundred and eight SCI and hindlimb cast immobilization (HCI) rats were studied for skeletal innervation of substance P (SP) and neurofilament 200 (NF200) with immunocytochemistry. Bone and serum SP levels were also assessed using enzyme immunoassay. Developing bone loss was successfully induced by SCI at 3 wks and by HCI at 6 wks. We observed a significant increase of SP-immunoreactive (IR) nerve fibers and decrease of NF200-IR nerve fibers in the tibiae of SCI rats compared with HCI and control (CON) rats at all time points. SP in the proximal tibiae in SCI rats was significantly higher than that in HCI and CON rats at all time points, but no difference was found in the serum. SCI-induced sublesional bone loss in young rats at an early stage is associated with a significant increase of nerve fiber innervation density of SP-IR and decrease of NF200-IR. We speculated that neural factors may play an important role in pathogenesis of OP after SCI.

Rapid changes in substance P signaling and neutral endopeptidase induced by skin-scratching stimulation in mice

Skin-scratching is a commonly seen behavior in patients with pruritus which sometimes exacerbates original lesions. Substance P (SP) signaling may play a predominant role in the pathophysiology induced by skin-scratching, however, it has not been well-elucidated. To clarify changes in SP, its receptor NK-1R and a degradating enzyme neutral endopeptidase (NEP) induced by skin-scratching stimulation in mice. After skin-scratching stimulation was given to mice, changes in SP signaling were investigated as follows. Mast cell degranulation was examined with toluidine blue staining. SP-immunoreactive nerve fibers and the expressions of NK-1R and NEP were examined with immunofluorescence. Protein contents of SP and the enzymatic activity of NEP were examined with an ELISA and a colorimetric assay, respectively. After skin-scratching stimulation, mast cells significantly degranulated within several minutes. SP-immunoreactive nerve fibers disappeared immediately from sensory nerve fibers, indicating the quick secretion and the depletion of SP. Both protein contents of SP and NEP activity in skin decreased dramatically soon after skin-scratching stimulation and thereafter they returned to the basal level within a week. The expression of NK-1R was significantly upregulated in epidermal basal keratinocytes after several days, in which NEP and NK-1R were well-coexpressed. Blocking NK-1R by an NK-1R antagonist suppressed scratching-induced decreases in SP-immunoreactive nerve fibers and in NEP activity. The present study clarified changing patterns of factors involved in SP signaling and NEP induced by skin-scratching stimulation. These findings provide basic and useful information to understand the pathophysiology of scratching-associated pruritic skin diseases.

Substance P as an Immunomodulatory Neuropeptide in a Mouse Model for Autoimmune Hair Loss (Alopecia Areata)

Alopecia areata (AA) is an autoimmune disorder of the hair follicle characterized by inflammatory cell infiltrates around actively growing (anagen) hair follicles. Substance P (SP) plays a critical role in the cutaneous neuroimmune network and influences immune cell functions through the neurokinin-1 receptor (NK-1R). To better understand the role of SP as an immunomodulatory neuropeptide in AA, we studied its expression and effects on immune cells in a C3H/HeJ mouse model for AA. During early stages of AA development, the number of SP-immunoreactive nerve fibers in skin is increased, compared to non-affected mice. However, during advanced stages of AA, the number of SP-immunoreactive nerves and SP protein levels in skin are decreased, whereas the expression of the SP-degrading enzyme neutral endopeptidase (NEP) is increased, compared to control skin. In AA, NK-1R is expressed on CD8+ lymphocytes and macrophages accumulating around affected hair follicles. Additional SP supply to the skin of AA-affected mice leads to a significant increase of mast cell degranulation and to accelerated hair follicle regression (catagen), accompanied by an increase of CD8+ cells-expressing granzyme B. These data suggest that SP, NEP, and NK-1R serve as important regulators in the molecular signaling network modulating inflammatory response in autoimmune hair loss.

A co-localization study on the ovine pancreas innervation

The expression of DbetaH and several neuropeptides was investigated in neuronal elements of the ovine pancreas using double immunocytochemical stainings. Immunoreactivities to DbetaH, NPY, VIP and SP were seen to various extents in nerve terminals associated with the acini, islets, ducts, blood vessels, interlobular connective tissue as well as in the neurons of intrapancreatic ganglia. The expression of CGRP was limited to nerve fibers lying in the connective tissue septa, amongst the acini and in close vicinity to the pancreatic blood vessels. Single GRP-positive nerve endings were located around the acini, ducts and in the interlobular connective tissue. With the exception of the ductal system in a co-localization of NPY with DbetaH was frequently found in all regions of the pancreas. Moderately numerous blood vessel-associated VIP-positive nerve fibers as well as the vast majority of VIP-containing intrapancreatic neurons were found to co-express DbetaH. Single SP-immunoreactive (IR) nerve fibers of the exocrine pancreas and interlobular connective tissue as well as SP-positive intrapancreatic neurons additionally showed the presence of DbetaH. The co-localization of VIP and NPY was found in nerve terminals located around the blood vessels and acini, in the connective tissue septa as well as in numerous pancreatic neuronal perikarya. Rare nerve terminals located between the acini and around small blood vessels as well as several neurons of intrapancreatic ganglia were VIP-IR/ SP-IR. Simultaneous expression of SP and CGRP was found in nerve fibers supplying large pancreatic arteries and veins, interlobular connective tissue and, occasionally, around the acini. Throughout the pancreas the population of CGRP-positive nerve endings showed lack of VIP and NPY. In a moderate number of GRP-containing nerve fibers, a co-expression of NPY was noted. Nerve terminals containing both GRP and VIP were detected sporadically, whereas none of the GRP-positive nerve terminals showed expression of SP. We conclude that the presented noradrenergic as well as peptidergic innervation patterns of the ovine pancreas are species-dependent. On the basis of the occurrence of DbetaH, NPY, VIP and SP (alone or in combination) in pancreatic neuronal elements we can suggest that these substances presumably act as regulators of the endocrine and/or exocrine pancreas. Involvement of CGRP and GRP in the ovine pancreas physiology seems to be of minor importance. The co-localization study indicated that the pancreas of the sheep is innervated from several sources including intrinsic as well as extrinsic ganglia.

Neuroimmune Link in the Mucosa of Chronic Gastritis with Helicobacter pylori Infection

It is suggested that different neuropeptides regulate gastric mucosal integrity and participate in the development of chronic gastritis. The aim of this study was to examine the roles and changes of immunoreactive (IR) nerves and immunocompetent cells in human gastritis. Immunohistochemical, immunocytochemical, and confocal laser microscopic methods were used. All investigated nerve fibers were found in different quantities in the mucosa of both control and gastritis samples. The number of SP, NPY, and VIP IR nerve fibers increased significantly (P < 0.05) in gastritis. No IR immunocompetent cells (lymphocytes, plasma cells, mast cells) were found in the control, however, some showed NPY (16.8%) and SP (9.4%) immunoreactivity in chronic gastritis. The distance between nerve fibers and immunocompetent cells was 200 nm to 1 microm. In conclusion, the increased number of SP, NPY, and VIP IR nerves and IR immunocytes suggests that they participate in development of neurogenic inflammation, repairing processes of chronic gastritis.

Correlation between the Appearance of Neuropeptides in the Rat Trigeminal Ganglion and Reinnervation of the Healing Root Socket after Tooth Extraction

The neuropeptide substance P (SP) modulates bone metabolism. This study examined the temporal appearance of the neuropeptides SP and brain-derived nerve growth factor (BDNF) and their receptors (neurokinin-1 receptor (NK1-R) and Trk B, respectively) in the rat trigeminal ganglion to investigate the role of neuropeptides in healing after tooth extraction. Rats were anesthetized and their upper right first molars were extracted; the rats were sacrificed 3 hours and 1–21 days after extraction. Their trigeminal ganglion and maxilla were removed, and cryosections were prepared and immunostained using specific antibodies against SP, BDNF, NK1-R, and Trk B. In the tooth sockets after extraction, new bone and a few SP-­immunoreactive nerve fibers were first seen at day 7, and bone completely filled the sockets at day 21. In the trigeminal ganglion, the proportions of NK1-R-, BDNF-, and Trk B-immuno­reactive neurons changed similarly, i.e., they initially decreased, increased rapidly to ­maximum levels by day 3, and then decreased gradually to control levels until 21 days. These findings suggest that the appearance of neuropeptides in the trigeminal ganglion, the reinnervation of SP-immunoreactive nerve fibers, and bone repair in the tooth socket during healing after extraction were correlated.

Relationships between neurokinin receptor-expressing interstitial cells of Cajal and tachykininergic nerves in the gut.

The so-called interstitial cells of Cajal (ICC) are distributed throughout the muscle coat of the alimentary tract with characteristic intramual location and species-variations in structure and staining. Several ICC sub-types have been identified: ICC-DMP, ICC-MP, ICC-IM, ICC-SM. Gut motility is regulated by ICC and each sub-type is responsible for the electrical activities typical of each gut region and/or muscle layer. The interstitial position of the ICC between nerve endings contain tachykinins. Three distinct tachykinin receptors (NK1r, NK2r and NK3r) have been demonstrated by molecular biology. Each of them binds with different affinities to a series of tachykinins (SP, NKA and NKB). In the ileum, SP-immunoreactive (SP-IR) nerve fibers form a rich plexus at the deep muscular plexus (DMP), distributed around SP-negative cells, and ICC-DMP intensely express the SP-preffered receptor NK1r; conversely a faint NK1r-IR is detected on the ICC-MP and mainly after receptor internalization was induced by agonists. ICC-IM are never stained in laboratory mammals, while those of the human, antrum are NK1r-IR. RT-PCR conducted on isolated idleal ICC-MP and gastric ICC-IM showed that these cells express NK1r and NK3r. Colonic ICC, except those in humans, do not express NK1r-IR, at least in resting conditions. Outside the gut, NK1r-IR cells were seen in the arterial wall and exocrine pancreas. In the mouse gut only, NK1r-IR is present in non-neuronal cells located within the intestinal villi, so-called myoid cells, which are c-kit-negative and α-smooth muscle actin-positive. Immunohistochemistry and functional studies confirmed that ICC receive input from SP-IR terminals, with differences between ICC sub-types. In the rat, very early after birth, NK1r is expressed by the ICC-DMP and SP by the related nerve varicosities. Studies on pathological conditions are few and those on mutant strains practically absent. It has only been reported that in the inflamed ileum of rats the NK1r-IR ICC-DMP disappear and that at the peak of inflammatory conditions ICC-MP are NK1r-IR. In ileum of mice with a mutation in the W locus, ICC-DMP were seen to express c-kit-IR but not NK1-IR, and SP-IR innervation seems unchanged. In summary, there are distinct ICC populations, each of them under a different tachkininergic control and, likely, having different functions. Further studies are recommended at the aim of understanding ICC involvement in modulating/transmitting tachykininergic inputs.

Neurochemical properties of the middle cervical ganglion in the sheep

The neurochemical properties of the ovine middle cervical ganglion (MCG) were studied using antibodies raised against tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DbetaH), neuropeptide Y (NPY), substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and galanin (GAL). Double-labelling immunocytochemistry revealed that the vast majority (95.5 +/- 0.8%) of postganglionic sympathetic MCG neurons expressed simultaneously both catecholamine-synthesizing enzymes (neurons were TH/DbetaH-positive). A large population of noradrenergic neurons exhibited immunoreactivity (IR) to NPY (62.2 +/- 2.2%), but single NPY-positive perikarya-lacking noradrenergic markers were also observed (2.0 +/- 0.3%). None of the examined MCG neuronal somata contained SP, CGRP, GAL or VIP. A moderate number of noradrenergic nerve fibres located amongst neuronal cell bodies was also found. In small number of these terminals the presence of NPYor GAL (but not CGRP or VIP) was detected. The ovine MCG was numerously innervated with SP-immunoreactive nerve fibres which sometimes formed basket-like formations around postganglionic neurons. The MCG exhibited a sparse CGRP-immunoreactive innervation and lacked VIP-positive nerve terminals. In many aspects the chemical coding of MCG postganglionic neurons and nerve terminals resembles that found in other mammalian cervico-thoracic paravertebral ganglia, but some important species-dependent differences exist. The functional implications of these differences remain to be elucidated.

Substance P and opioid peptidergic innervation of the anterior eye segment of the rat: an immunohistochemical study

Recently discovered endogenous opioid peptides such as nociceptin are known to modulate neurotransmitter release of primary afferent neurons (especially substance P, SP) and they have also been demonstrated in peripheral nerve fibres. The aim of this study was to investigate the opioid peptidergic innervation of the anterior eye segment and to compare it with the innervation pattern of SP in order to shed light on the functional relationship between these peptides. Anterior eye segments of 20 rat eyes were cut in a tangential plane and the sections stained with antibodies against SP, nociceptin, nocistatin, endomorphin 1 and 2, leu-enkephalin and met-enkephalin. Sections of the spinal cord or brain were used as positive controls. Numerous SP-immunoreactive nerve fibres were found in the conjunctiva, cornea, episclera, trabecular meshwork, iris and ciliary body. A weak staining for met-enkephalin and leu-enkephalin could only be found in the iris and anteriormost ciliary body. Nerve fibres immunoreactive for nociceptin, nocistatin, and endomorphin 1 or 2 could not be detected in any part of the anterior eye segment. It is tempting to speculate that the opioid peptidergic innervation of the anterior ciliary body may play a role in the modulation of intraocular inflammation.

Distribution of calcitonin gene-related peptide (CGRP), substance P (SP) and galanin (GAL) immunoreactive nerve fibers in the seminal vesicle and prostate of the male sheep

Double immunohistochemistry was used to determine the occurrence and distribution pattern of nerve fibres immunoreactive to calcitonin gene-related peptide (CGRP), substance P (SP) and galanin (GAL) in seminal vesicles and prostate of the male sheep. Numerous CGRP- and SP-immunoreactive (IR) nerve fibres were found in the mucosal layer and smooth musculature of the seminal vesicles and prostate. In both glands nerve terminals immunoreactive to CGRP were more numerous than SP-IR ones. The majority of CGRP-IR nerve fibers showed colocalization of this peptide and SP. In both layers of the seminal vesicle and prostate, rare nerve terminals immunoreactive to GAL were also found. Immunoreactivity to SP was also found in all GAL-IR nerve fibers. The presence of numerous CGRP- and SP-IR nerve fibers in the seminal vesicle and prostate of the male sheep suggests that these neuropeptides may be involved in the sensory transmission and/or control of smooth muscle contractility. On the other hand, a relatively low number of GAL-IR nerve fibers of the seminal vesicle and prostate suggest that this peptide may act as an anti-nociceptive agent. It cannot be excluded that, in the seminal vesicle, GAL may also be involved in the control of the smooth muscle fiber activity. The possible role of CGRP, SP and GAL in the regulation of functions of the accessory sexual glands needs to be determined in further physiological studies.

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.