Michal Zalecki

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

The present study examines the response of colon-projecting neurons localized in the inferior mesenteric ganglia (IMG) to axotomy in the pig animal model. In all animals (n = 8), a median laparotomy was performed under anesthesia and the retrograde tracer Fast Blue was injected into the descending colon wall. In experimental animals (n = 4), the descending colon was exposed and the bilateral caudal colonic nerves were identified and severed. All animals were euthanized and the inferior mesenteric ganglia were 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, vesicular acetylcholine transporter, or galanin. Immunohistochemistry revealed significant changes in the chemical coding pattern of injured inferior mesenteric ganglion neurons. In control animals, Fast Blue-positive neurons were immunoreactive to TH, NPY, SOM, VIP, NOS, LENK, and CB. In the experimental group, the numbers of TH-, NPY-, and SOM-expressing neurons were reduced, whereas the number of neurons immunoreactive to LENK was increased. Our data indicate that the colon-projecting neurons of the porcine IMG react to the axotomy in a similar, but not an identical manner in a comparison to other species, especially rodents. Further studies are needed to elucidate the detailed factors/mechanisms involved in the response to nerve injury.

Localization and neurochemical characteristics of the extrinsic sympathetic neurons projecting to the pylorus in the domestic pig

The pylorus, an important part of the digestive tract controlling the flow of chyme between the stomach and the duodenum, is widely innervated by intrinsic and extrinsic nerves. To determine the locations of postganglionic sympathetic perikarya that innervate the pylorus of the domestic pig, a retrograde tracing method with application of Fast Blue tracer was used. All positive neuronal cell bodies (ca. 1750) were found in the celiac-cranial mesenteric ganglion complex (CSMG), however, the coeliac poles of this complex provided the major input to the pylorus. Afterwards, the immunohistochemical staining procedure was applied to determine biologically active substances expressed in the FB-labeled perikarya. Approximately 77% of the FB-positive cell bodies contained tyrosine hydroxylase (TH), 87% dopamine β-hydroxylase (DβH), 40% neuropeptide Y (NPY), 12% somatostatin (SOM) and 7% galanin (GAL). The presence of all these substances in the ganglion tissue was confirmed by RT-PCR technique. Double immunocytochemistry revealed that all of the TH-positive perikarya contained DβH, about 40% NPY, 12% SOM and 8% GAL. Additionally, all above-cited immunohistochemical markers as well as VIP, PACAP, ChAT, LEU, MET, SP and nNOS were observed within nerve fibers associated with the FB-positive perikarya. Immunocytochemical labeling of the pyloric wall tissue disclosed that TH+, DβH+ and NPY+ nerve fibers innervated ganglia of the myenteric and submucosal plexuses, blood vessels, both muscular layers and the muscularis mucosae; nerve fibers immunoreactive to GAL mostly innervated both muscular layers, while SOM+ nerve fibers were observed within the myenteric plexus. Presented study revealed sources of origin and immunohistochemical characteristics of the sympathetic postganglionic perikarya innervating the porcine pylorus.

The Influence of Antral Ulcers on Intramural Gastric Nerve Projections Supplying the Pyloric Sphincter in the Pig ( Sus scrofa domestica )—Neuronal Tracing Studies

Background Gastric ulcerations in the region of antrum pylori represent a serious medical problem in humans and animals. Such localization of ulcers can influence the intrinsic descending nerve supply to the pyloric sphincter. The pyloric function is precisely regulated by intrinsic and extrinsic nerves. Impaired neural regulation could result in pyloric sphincter dysfunction and gastric emptying malfunction. The aim of the study was to determine the effect of gastric antral ulcerations on the density and distribution of intramural gastric descending neurons supplying the pyloric sphincter in pigs. Methodology/Principal Findings The experiment was performed on 2 groups of pigs: healthy gilts (n=6) and gilts with experimentally induced peptic ulcers in the region of antrum pylori (n=6). Gastric neurons supplying pyloric sphincter were labeled using the retrograde neuronal tracing technique (20μl of Fast Blue tracer injected into the pyloric sphincter muscle). After a week survival period the animals were sacrificed and the stomachs were collected. Then, the stomach wall was cross-cut into 0.5cm thick sections taken in specified intervals (section I - 1.5cm; section II - 3.5cm; section III - 5.5cm; section IV – 7.5cm) starting from the sphincter. Consecutive microscopic slices prepared from each section were analyzed under fluorescent microscope to count traced neurons. Obtained data were statistically analyzed. The total number of FB-positive perikarya observed within all studied sections significantly decreased from 903.3 ± 130.7 in control to 243.8 ± 67.3 in experimental animals. In healthy pigs 76.1 ± 6.7% of labeled neurons were observed within the section I, 23.53 ± 6.5% in section II and only occasional cells in section III. In experimental animals, as many as 93.8 ± 2.1% of labeled cells were observed within the section I and only 6.2 ± 2.2% in section II, while section III was devoid of such neurons. There were no traced perikarya in section IV observed in both groups of pigs. Conclusions/Significance Obtained results revealed for the first time significant impact of antral ulcerations on intramural descending nerve pathways supplying the pyloric sphincter in pigs, animals of increasing value in biomedical research and great economic importance.

Innervation pattern of polycystic ovaries in the women.

The aim of the present study was to determine the changes in both the distribution pattern and density of nerve fibers containing dopamine β-hydroxylase (DβH), vesicular acetylcholine transporter (VAChT), neuronal nitric oxide synthase (nNOS), substance P (SP), calcitonin gene related peptide (CGRP), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), somatostatin (SOM), galanin (GAL) and pituitary adenylate cyclase-activating polypeptide (PACAP) in the human polycystic ovaries. In the polycystic ovaries, when compared to the immunoreactions pattern observed in the control gonads, following changes were revealed: (1) an increase in the number of DβH-, VAChT-, VIP- or GAL-immunoreactive (IR) nerve fibers within the stroma as well as in the number of DβH-IR fibers near primordial follicles and medullar veins and venules; (2) a reduction in the number of nerve fibers containing nNOS, CGRP, SOM, PACAP within the stroma and in the numbers of CGRP-IR fibers around arteries; (3) an appearance of SP- and GAL-IR fibers around medullar and cortical arteries, arterioles, veins and venules, with except of GAL-IR fibers supplying medullar veins; and (4) the lack of nNOS-IR nerve fibers near primordial follicles and VIP-IR nerves around medullar arteries and arterioles. In conclusion, our results suggest that the changes in the innervation pattern of the polycystic ovaries in human may play an important role in the pathogenesis and/or course of this disorder.

The Influence of Gastric Antral Ulcerations on the Expression of Galanin and GalR1, GalR2, GalR3 Receptors in the Pylorus with Regard to Gastric Intrinsic Innervation of the Pyloric Sphincter.

Gastric antrum ulcerations are common disorders occurring in humans and animals. Such localization of ulcers disturbs the gastric emptying process, which is precisely controlled by the pylorus. Galanin (Gal) and its receptors are commonly accepted to participate in the regulation of inflammatory processes and neuronal plasticity. Their role in the regulation of gastrointestinal motility is also widely described. However, there is lack of data considering antral ulcerations in relation to changes in the expression of Gal and GalR1, GalR2, GalR3 receptors in the pyloric wall tissue and galaninergic intramural innervation of the pylorus. Two groups of pigs were used in the study: healthy gilts and gilts with experimentally induced antral ulcers. By double immunocytochemistry percentages of myenteric and submucosal neurons expressing Gal-immunoreactivity were determined in the pyloric wall tissue and in the population of gastric descending neurons supplying the pyloric sphincter (labelled by retrograde Fast Blue neuronal tracer). The percentage of Gal-immunoreactive neurons increased only in the myenteric plexus of the pyloric wall (from 16.14±2.06% in control to 25.5±2.07% in experimental animals), while no significant differences in other neuronal populations were observed between animals of both groups. Real-Time PCR revealed the increased expression of mRNA encoding Gal and GalR1 receptor in the pyloric wall tissue of the experimental animals, while the expression(s) of GalR2 and GalR3 were not significantly changed. The results obtained suggest the involvement of Gal, GalR1 and galaninergic pyloric myenteric neurons in the response of pyloric wall structures to antral ulcerations.

Immunohistochemical Characterization of Sympathetic Chain Ganglia (SChG) Neurons Supplying the Porcine mammary Gland

The aim of this study was to investigate the chemical coding of mammary gland‐projecting SChG neurons using double‐labelling immunohistochemistry. Earlier observation showed that after injection of the retrograde tracer fast blue (FB) into the second, right thoracic mamma, FB+ mammary gland‐projecting neurons were found in Th1‐3, Th9‐14 and L1‐4 right SChG. The greatest number of FB+ nerve cell bodies was observed in Th10 (approx. 843) and Th11 (approx. 567). Neurons projecting to the last right abdominal mamma were found in L1‐4 SChG. The greatest number of FB+ neurons was observed in L2 (approx. 1200). Immunohistochemistry revealed that the vast majority of FB+ mammary‐projecting neurons contained immunoreactivities to TH (96.97%) and/or DßH (95.92%). Many TH/DßH‐positive neurons stained for SOM (41.5%) or NPY (33.2%), and less numerous nerve cells expressed VIP (16.9%). This observation strongly corresponds to the results of previous studies concerning the immunohistochemical characterization of nerve fibres supplying the porcine mammary gland.

Changes in the expression of galanin and galanin receptors in the wall of the colon in pigs experimentally infected with Brachyspira hyodysenteriae

Abstract The expression of galanin (GAL) and its three receptors (GalR1, GalR2, and GalR3) were studied with real-time PCR in the colonic wall of pigs suffering from experimental colitis caused by the infection with Brachyspira hyodysenteriae. The expression was studied in the muscular membrane, mucosa/submucosa layer, and in lymphocytes isolated from mucosa/submucosa. The expression levels were normalized to glyceraldehyde-6-phosphate dehydrogenase (GAPDH) expression and compared to expression levels in control animals. GAL expression was found in all three studied compartments of the colonic wall. A significant decrease in GAL expression level was found in the mucosa/submucosa and in isolated lymphocytes, whereas the decrease was much less profound in the muscular membrane. In the case of galanin receptors their expression was found in all studied compartments of the colonic wall, however at different levels, as compared to GAPDH expression. The decrease of galanin receptors expression was found in all studied compartments of the colonic wall of the sick animals.

Effect of seminal plasma zinc–binding proteins on motility and membrane integrity of canine spermatozoa stored at 5°C

Abstract Sperm surface binding sites for non-zinc-binding proteins (nZnBPs) and zinc-binding proteins (ZnBPs) were studied by the fluorescence technique with biotin-labelled proteins. The nZnBPs binding pattern was unspecific, no characteristic sites on plasmalemma were found. ZnBPs were attached mainly to the acrosomal region of sperm head and to the sperm flagellum. ZnBPs added to the incubation mixture of the canine spermatozoa allowed the preservation of higher values for total motility, progressive motility, curvilinear line velocity, straight line velocity, and beat cross frequency (P < 0.05), both at time 0 and after 1 h incubation at 5ºC. The addition of nZnBPs to the incubation mixture caused only weak positive effects when compared with control sample (PBS). A higher percentage of canine-ejaculated spermatozoa with intact membranes were observed when ejaculate was incubated with ZnBPs in comparison to control sample stored with PBS (P < 0.05) or nZnBPs (P < 0.05). Spermatozoa diluted with ZnBPs and nZnBPs exhibited a higher percentage of cells with active mitochondria when compared with control, both at time 0 and after 1 h; however, no statistical differences were observed. Our results emphasise the role of seminal plasma protein in securing the correct quantity and availability of zinc ions as a component regulating the motility of canine spermatozoa. The protective effect of ZnBPs against the cooling effect may be due to their ability of preventing sperm membrane damage.

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.

Galaninergic intramural nerve and tissue reaction to antral ulcerations

Well‐developed galaninergic gastric intramural nerve system is known to regulate multiple stomach functions in physiological and pathological conditions. Stomach ulcer, a disorder commonly occurring in humans and animals, is accompanied by inflammatory reaction. Inflammation can cause intramural neurons to change their neurochemical profile. Galanin and its receptors are involved in inflammation of many organs, however, their direct participation in stomach reaction to ulcer is not known. Therefore, the aim of the study was to investigate adaptive changes in the chemical coding of galaninergic intramural neurons and mRNA expression encoding Gal, GalR1, GalR2, GalR3 receptors in the region of the porcine stomach directly adjacent to the ulcer location.