Serpinin in the Skin.

The serpinins are relatively novel peptides generated by proteolytic processing of chromogranin A and they are comprised of free serpinin, serpinin-RRG and pGlu-serpinin. In this study, the presence and source of these peptides were studied in the skin. By Western blot analysis, a 40 kDa and a 50 kDa protein containing the sequence of serpinin were detected in the trigeminal ganglion and dorsal root ganglia in rats but none in the skin. RP-HPLC followed by EIA revealed that the three serpinins are present in similar, moderate amounts in rat dorsal root ganglia, whereas in the rat skin, free serpinin represents the predominant molecular form. There were abundant serpinin-positive cells in rat dorsal root ganglia and colocalization with substance P was evident. However, much more widespread distribution of the serpinins was found in dorsal root ganglia when compared with substance P. In the skin, serpinin immunoreactivity was found in sensory nerves and showed colocalization with substance P; as well, some was present in autonomic nerves. Thus, although not exclusively, there is evidence that serpinin is a constituent of the sensory innervation of the skin. The serpinins are biologically highly active and might therefore be of functional significance in the skin.

The influence of castration on intramural neurons of the urinary bladder trigone in male pigs.

The present study investigated the influence of castration performed at neonatal age on neuronal elements in the intramural ganglia of the urinary bladder trigone (UBT) in male pigs using double-labeling immunohistochemistry. The ganglia were examined in intact (IP) 7-day-old (castration day) pigs, and at 3 and 6 months after surgery. In IP and control (3- and 6-month-old noncastrated pigs) groups, virtually, all neurons were adrenergic (68%) or cholinergic (32%) in nature. Many of them (32%, 51%, and 81%, respectively; 56%, 75%, and 85% adrenergic; and 32%, 52%, and 65% cholinergic, respectively) stained for the androgen receptor (AR), and only a small number of nerve cells were caspase-3 (CASP-3)-positive. In 3- and 6-month-old castrated pigs, an excessive loss (87.6% and 87.5%, respectively) of neurons and intraganglionic nerve fibers was observed. The majority of the surviving adrenergic (61% and 72%, respectively) and many cholinergic (41% and 31%, respectively) neurons expressed CASP-3 and were also AR-positive (61% and 66%, and 40% and 36%, respectively). This study revealed for the first time the excessive loss of intramural UBT neurons following castration, which could have resulted from apoptosis induced by androgen deprivation.

Changes in the Neurochemical Coding of the Anterior Pelvic Ganglion Neurons Supplying the Male Pig Urinary Bladder Trigone after One-Sided Axotomy of Their Nerve Fibers.

The present study investigated the effect of unilateral axotomy of urinary bladder trigone (UBT)-projecting nerve fibers from the right anterior pelvic ganglion (APG) on changes in the chemical coding of their neuronal bodies. The study was performed using male pigs with immunohistochemistry and quantitative real-time PCR (qPCR). The animals were divided into a control (C), a morphological (MG) or a molecular biology group (MBG). APG neurons supplying UBT were revealed using the retrograde tracing technique with Fast Blue (FB). Unilateral axotomy resulted in an over 50% decrease in the number of FB+ neurons in both APG ganglia. Immunohistochemistry revealed significant changes in the chemical coding of FB+ cells only in the right ganglion: decreased expression of dopamine-B-hydroxylase (DBH)/tyrosine hydroxylase (TH) and up-regulation of the vesicular acetylcholine transporter (VAChT)/choline acetyltransferase (ChAT), galanin (GAL), vasoactive intestinal polypeptide (VIP) and brain nitric oxide synthase (bNOS). The qPCR results partly corresponded with immunofluorescence findings. In the APGs, genes for VAChT and ChAT, TH and DBH, VIP, and NOS were distinctly down-regulated, while the expression of GAL was up-regulated. Such data may be the basis for further studies concerning the plasticity of these ganglia under experimental or pathological conditions.

Inferior vagal ganglion galaninergic response to gastric ulcers.

Galanin is a neuropeptide widely expressed in central and peripheral nerves and is known to be engaged in neuronal responses to pathological changes. Stomach ulcerations are one of the most common gastrointestinal disorders. Impaired stomach function in peptic ulcer disease suggests changes in autonomic nerve reflexes controlled by the inferior vagal ganglion, resulting in stomach dysfunction. In this paper, changes in the galaninergic response of inferior vagal neurons to gastric ulceration in a pig model of the disease were analyzed based on the authors' previous studies. The study was performed on 24 animals (12 control and 12 experimental). Gastric ulcers were induced by submucosal injections of 40% acetic acid solution into stomach submucosa and bilateral inferior vagal ganglia were collected one week afterwards. The number of galanin-immunoreactive perikarya in each ganglion was counted to determine fold-changes between both groups of animals and Q-PCR was applied to verify the changes in relative expression level of mRNA encoding both galanin and its receptor subtypes: GalR1, GalR2, GalR3. The results revealed a 2.72-fold increase in the number of galanin-immunoreactive perikarya compared with the controls. Q-PCR revealed that all studied genes were expressed in examined ganglia in both groups of animals. Statistical analysis revealed a 4.63-fold increase in galanin and a 1.45-fold increase in GalR3 mRNA as compared with the controls. No differences were observed between the groups for GalR1 or GalR2. The current study confirmed changes in the galaninergic inferior vagal ganglion response to stomach ulcerations and demonstrated, for the first time, the expression of mRNA encoding all galanin receptor subtypes in the porcine inferior vagal ganglia.

Effect of castration on pelvic neurons in the male pig.

The present study investigated the influence of castration performed at neonatal age on neuronal elements in the anterior pelvic ganglion of the male pig with immunohistochemistry and quantitative real-time PCR (qPCR). The ganglia were examined 3 and 6 months after surgery. In 3-month-old castrated pigs (3MCP) 74% of adrenergic and 31% of cholinergic neurons stained for caspase-3 (CASP-3), and much greater numbers of perikarya than in the control animals expressed CGRP, galanin (GAL) and VIP (peptides known to have neuroprotective properties). In 6-months-old castrated pigs (6MCP), an excessive loss (90%) of neurons and intraganglionic nerve fibres was found. The survived adrenergic and cholinergic neurons also expressed CASP-3, CGRP, GAL or VIP. The qPCR results corresponded with immunofluorescence findings. In 3MCP, genes for CASP-3 and CGRP were up-regulated, while the expression of those for DßH, VAChT, GAL, VIP and SP displayed statistically insignificant variations. In 6MCP, distinctly up-regulated were genes for CGRP, GAL, VIP, SP, DßH and VAChT, while the expression of casp3 gene was down-regulated. The study revealed for the first time the excessive loss of pelvic neurons following castration, and a realistic assumption is proposed, that the neurons died due to apoptosis triggered by androgen deprivation.

Neuropeptides and lymphocyte populations in the porcine ileum and ileocecal lymph nodes during postnatal life.

The maturation-related changes in the concentrations of galanin (Gal), vasoactive intestinal polypeptide (VIP), substance P (SP) and somatostatin (Som), as well as in subpopulations of lymphocytes expressing antigens CD2 (lymphocytes T), CD4 (T helper), CD8 (T cytotoxic), CD21 (B lymphocytes), CD5-/CD8+ (NK cells) and TCRgamma/delta (gut mucosal/intraepitelial cells) were studied in the ileal Peyer's patches and ileo-cecal lymph nodes in female pigs aged 3 days, 2 weeks, 4 weeks and 4 months. As regards neuropeptide concentrations statistically significant changes in the ileum and lymph nodes were found only in case of Gal and VIP. The concentrations of neuropeptides were significantly higher only in new-born animals. As regards the changes in subpopulations of lymphocytes, statistically significant changes were noticed only in 4-months old animals and were dealing only with CD2+ and TCRgamma/delta cells in the ileum as well as CD4+, CD8+, CD21+ and TCRgamma/delta in lymph nodes. The highest number of CD8+, CD21+ and TCRgamma/delta lymphocytes occurred in 4-months old animals.

Immunohistochemical characteristics and distribution of neurons in the paravertebral, prevertebral and pelvic ganglia supplying the urinary bladder in the male pig.

The distribution and chemical coding of neurons supplying urinary bladder in the male pig were studied in the sympathetic chain ganglia, inferior mesenteric ganglia and anterior pelvic ganglia. The combined retrograde tracing and immunohistochemistry for tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), neuropeptide Y (NPY), somatostatin(SOM), galanin (GAL), vasoactive intestinal polypeptide(VIP), nitric oxide synthase (NOS), calcitonin gene related peptide (CGRP), substance P (SP), choline acetyltransferase (ChAT) and vesicular acetylcholine transporter(VAChT) were applied in the experiment. Bladder projecting neurons were found in all the ganglia studied. The majority of sympathetic ganglia neurons (inferior mesenteric ganglia and sympathetic chain ganglia) expressed immunoreactivity(IR) to DBH. In sympathetic chain ganglia these neurons simultaneously expressed NPY, GAL or VAChT,while in inferior mesenteric ganglia they contained NPY, SOM and/or GAL. A small number of these bladder projecting neurons was VAChT-IR and some contained NPY. In the pelvic ganglia bladder-projecting neurons formed two populations: DBH- and VAChT-IR. Some of DBH-IR neurons contained IR to NPY, SOM or GAL, while VAChTIR neurons were NPY-, SOM- or NOS-IR. The results indicate that sympathetic ganglia contain mainly adrenergic neurons,while pelvic ganglia contain both adrenergic and cholinergic neurons. All these neurons contain typical combinations of neuropeptides.

Immunohistochemical characteristics and distribution of sensory dorsal root Ganglia neurons supplying the urinary bladder in the male pig.

The study determined the distribution and immunohistochemical coding of the sensory neurons innervating the male pig urinary bladder. Retrograde tracer Fast Blue was injected bilaterally into the bladder trigone, base or dome. The presence of neuropeptide Y (NPY), somatostatin (SOM), galanin (GAL), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), calcitonin gene-related peptide (CGRP) and substance P (SP) were studied with immunofluorescence. Fast Blue-positive neurons were localized bilaterally in dorsal root ganglia from L1 to L6 and from S3 to S4 with specific differences regarding the injection site. The number of Fast Blue-positive neurons was higher in the right ganglia. Immunohistochemistry revealed that sensory neurons innervating the urinary bladder trigone, base and dome displayed immunoreactivities to CGRP, SP, NOS, GAL and SOM. Differences in the neuropeptide content were observed between the Fast Blue-positive neurons in lumbar and sacral ganglia. Taken together, these data indicate that the lumbar and sacral pathways probably play different roles in sensory transmission from the urinary bladder trigone, base and dome.

Plasticity of neuropeptidergic neoplasm cells in the primary and metastatic Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a rare and highly aggressive cutaneous carcinoma with characteristics of neuroendocrine tumor. We performed immunohistochemical analysis to demonstrate the presence of various neuropeptides within cells of MCC resected from a 75-year old woman. The cells of primary tumor of cheek were compared with the cells of regional right submandibular metastatic tumor which was found eight months later. A double- staining IHC for the pan-neuronal marker, PGP 9.5, and selected neuropeptides in the tissue material obtained from both locations was performed. Single multipolar cells in the main mass of primary tumor stained positively for PGP 9.5 and such neuropeptides as GAL, VIP, PACAP, NPY and CGRP. Moreover, we demonstrated for the first time the presence of neuropeptides in metastatic MCC cells. In the metastatic tumor, cells showing the co-localization of PGP-9.5 and neuropeptides were more numerous, mostly of oval shape, and significantly smaller than in the primary tumor. Thus, the progression of MCC may be associated with the acquisition by its cells of new morphological and biological features.

Characteristic of galaninergic components of the enteric nervous system in the cancer invasion of human large intestine.

BACKGROUND: This study presents changes that take place in galanin (GAL)-containing components of the enteric nervous system in patients with colorectal carcinoma. The main goal of our study was to investigate the morphological and biochemical characteristics of the GAL-ergic neurons and nerve fibers in the cancer-affected part of the colon and to compare results to the unchanged part of the intestine. MATERIAL AND METHODS: The study was performed on tissue samples collected from 15 patients (9 women and 6 men). The material was collected during surgery, i.e., resection of the anterior sigmoid colon and anterior amputation of the rectum, from patients in good general condition, without any other significant disease. Immunohistochemistry and ELISA were used to study, respectively, the distribution of neurons and nerve fibers containing GAL and concentration of this neuropeptide in the human large intestine during the colorectal cancer infiltration. RESULTS: Morphological examinations have revealed that a statistically significantly higher percentage of GAL+ neurons (46%) was observed in the pathologically changed myenteric plexuses as compared to the unchanged part of the intestine (35%). No changes were observed in the density of GAL+ nerve fibers in the submucosal and myenteric plexuses. Biochemical examinations performed with the ELISA revealed that the average GAL content in the cancer tissues was 9.38 ng/g versus 12.27 ng/g in the morphologically unchanged tissues (the difference was statistically significant). CONCLUSIONS: The results indicate the continuous presence of GAL-ergic innervation in the immediate vicinity of the cancer invasion, which may be attributed to increased contraction of the affected part of intestine.

Inflammation-induced changes in the chemical coding pattern of colon-projecting neurons in the inferior mesenteric ganglia of the pig.

The present study examines the chemical coding of the inferior mesenteric ganglia after chemically induced colitis in the pig animal model. In all animals (n = 6), a median laparotomy was performed under anesthesia, and the Fast Blue retrograde tracer was injected into the descending colon wall. In experimental animals (n = 3), the thick descending colon were injected with formalin solution to induce inflammation. The animals were euthanized and the inferior mesenteric ganglion was harvested and processed for double-labeling immunofluorescence for calbindin-D28k (CB) in combination with either tyrosine hydroxylase (TH), neuropeptide Y (NPY), somatostatin (SOM), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), Leu-enkephalin (LENK), substance P (SP), vesicular acetylcholine transporter (VAChT), or galanin (GAL). Immunohistochemistry revealed significant changes in the chemical coding pattern of inferior mesenteric ganglion neurons. In control animals, Fast Blue-positive neurons were immunoreactive to TH, NPY, SOM, VIP, LENK, CB, and NOS. In the experimental group, TH, NPY, SOM, VIP, and LENK expressing neurons were reduced, whereas the number of neurons immunoreactive to CB, NOS, and GAL were increased. The increase of so-called neuroprotective neuropeptides suggests that the changes in the chemical coding of inferior mesenteric ganglion neurons reflect adaption under pathological conditions to promote their own survival.

Distribution and chemical coding of neurons in intramural ganglia of the porcine urinary bladder trigone.

This study presents the distribution and chemical coding of neurons in the porcine intramural ganglia of the urinary bladder trigone (IG-UBT) demonstrated using combined retrograde tracing and double-labelling immunohistochemistry. Retrograde fluorescent tracer Fast Blue (FB) was injected into the wall of both the left and right side of the bladder trigone during laparotomy performed under pentobarbital anaesthesia. Ten-microm-thick cryostat sections were processed for double-labelling immunofluorescence with antibodies against tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), neuropeptide Y (NPY), somatostatin (SOM), galanin (GAL), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), calcitonin gene-related peptide (CGRP), substance P (SP), Leu5-enkephalin (LENK) and choline acetyltransferase (ChAT). IG-UBT neurons formed characteristic clusters (from a few to tens neuronal cells) found under visceral peritoneum or in the outer muscular layer. Immunohistochemistry revealed four main populations of IG-UBT neurons: SOM- (ca. 35%), SP- (ca. 32%), ChAT- and NPY- immunoreactive (-IR) (ca. 23%) as well as non-adrenergic non-cholinergic nerve cells (ca. 6%). This study has demonstrated a relatively large population of differently coded IG-UBT neurons, which constitute an important element of the complex neuro-endocrine system involved in the regulation of the porcine urogenital organ function.

The influence of inflammation on the expression of neuropeptides in the ileum-projecting primary sensory neurones in the pig.

In the present study the ELISA test was used to investigate the influence of chemically-induced ileitis on the dorsal root ganglia (DRG) neurons in the pig. The preliminary retrograde fluorescent tracing study revealed that ileum-projecting sensory neurones (IPN) are located in the thoracic ganglia (Th; Th8-Th13). The ileum wall in experimental (E) pigs was subjected to multiple injection with 4% paraformaldehyde to induce inflammation, while in the control (C) animals the organ was injected with 0.1 M phosphate buffer. Three days later the DRGs (Th8-Th13) collected from all the animals were evaluated for VIP, SP, CGRP, NPY, GAL and SOM content with an ELISA test. It was found that the inflammation increased clearly the tissue level of SP, GAL and SOM.

Distribution, immunohistochemical characteristics and nerve pathways of primary sensory neurons supplying the porcine vas deferens.

The present study investigated: (1) the distribution and chemical coding of primary sensory neurons supplying the vas deferens in juvenile pigs by the use of retrograde tracing combined with double-labelling immunofluorescence, (2) nerve pathways from dorsal root ganglia (DRG) to the vas deferens by means of denervation procedures involving transection of the hypogastric or pelvic nerve combined with a retrograde tracing method, and (3) possible interactions of the substance P (SP)/calcitonin gene-related peptide (CGRP)-immunoreactive varicose nerve fibres on vas deferens projecting neurons (VDPN) in the anterior pelvic ganglion (APG). The vast majority of VDPN were found mainly in the lumbar L2, L3 and sacral S2, S3 pairs of DRG and showed a clear ipsilaterally organized projection pattern. Immunohistochemistry revealed that most of these neurons contained SP and/or CGRP, occasionally coexpressed with galanin. Interestingly, pronounced differences in the expression of SP and/or CGRP were observed between the lumbar and sacral VDPN in that most of the lumbar but less than half of the sacral neurons stained for these peptides. Denervation experiments showed that the neurons located within the lumbar DRG project through the ipsilateral hypogastric nerve, whereas those found within the sacral DRG send their processes through the ipsilateral and contralateral pelvic nerve. In the nerve-lesioned animals, especially in those with the hypogastric nerve cut, a dramatic reduction in the number of SP and/or CGRP-containing nerve terminals surrounding the efferent VDPN within the APG was observed. This study has disclosed the distribution and, for the first time, chemical coding and nerve pathways of vas deferens-projecting primary sensory neurons in a mammalian species, the pig. The results obtained also provide some novel information about the possible morphological and functional relationship between vas deferens-projecting primary sensory and pelvic efferent nerve cells.