J. Kaleczyc

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

Abstract. 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.

The Distribution and Chemical Coding of Intramural Neurons Supplying the Porcine Stomach – the Study on Normal Pigs and on Animals Suffering from Swine Dysentery

The present study was designed to investigate the expression of biologically active substances by intramural neurons supplying the stomach in normal (control) pigs and in pigs suffering from dysentery. Eight juvenile female pigs were used. Both dysenteric (n = 4; inoculated with Brachyspira hyodysenteriae) and control (n = 4) animals were deeply anaesthetized, transcardially perfused with buffered paraformalehyde, and tissue samples comprising all layers of the wall of the ventricular fundus were collected. The cryostat sections were processed for double‐labelling immunofluorescence to study the distribution of the intramural nerve structures (visualized with antibodies against protein gene‐product 9.5) and their chemical coding using antibodies against vesicular acetylcholine (ACh) transporter (VAChT), nitric oxide synthase (NOS), galanin (GAL), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), Leu5‐enkephalin (LENK), substance P (SP) and calcitonin gene‐related peptide (CGRP). In both inner and outer submucosal plexuses of the control pigs, the majority of neurons were SP (55% and 58%, respectively)‐ or VAChT (54%)‐positive. Many neurons stained also for CGRP (43 and 45%) or GAL (20% and 18%) and solitary perikarya were NOS‐, SOM‐ or VIP‐positive. The myenteric plexus neurons stained for NOS (20%), VAChT (15%), GAL (10%), VIP (7%), SP (6%) or CGRP (solitary neurons), but they were SOM‐negative. No intramural neurons immunoreactive to LENK were found. The most remarkable difference in the chemical coding of enteric neurons between the control and dysenteric pigs was a very increased number of GAL‐ and VAChT‐positive nerve cells (up to 61% and 85%, respectively) in submucosal plexuses of the infected animals. The present results suggest that GAL and ACh have a specific role in local neural circuits of the inflamed porcine stomach in the course of swine dysentery.

Has Active Immunization Against Gonadotrophin‐Releasing Hormone any Effect on Testis Innervation in the Pig? – An Immunohistochemical Study

The innervation of porcine testes was studied in intact animals and in boars undergoing active immunization against gonadotrophin‐releasing hormone (GnRH) by means of immunohistochemistry using antibodies to tyrosine hydroxylase (TH), dopamine β‐hydroxylase (DβH), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), synaptosome‐associated protein of 25 kDa (SNAP‐25) and protein gene product 9.5 (PGP 9.5). Moreover, the distribution of luteinizing hormone (LH) receptors in clusters of Leydig cells was also investigated. To identify these cells easily, either the NADPH‐diaphorase histochemical technique or the Mayer counter‐staining procedure was applied. Differences in the distribution pattern and relative density of particular subsets of intratesticular nerve fibres were observed in immunized boars as compared to those found in the intact animals. In the testes of non‐treated animals, only single TH‐immunoreactive (TH‐IR) nerve fibres were observed. However, many DβH‐IR nerve terminals surrounded blood vessels in the tunica albuginea and parenchyma. Very scarce VIP‐IR nerves occurred only in the tunica albuginea, mainly in close vicinity to blood vessels. Immunoreactivity to NPY occurred in single nerve fibres. Immunoreactivity to SNAP‐25 and PGP 9.5 was found in single nerve fibres distributed mainly in the tunica albuginea. The interstitial cells were heavily stained for LH‐receptors and NADPH‐diaphorase. In the testes of immunized animals, only single TH‐IR nerve fibres, scattered mainly in the tunica albuginea, were observed. Some TH‐IR nerve terminals were also encountered in the parenchyma of the organ, where they were always associated with blood vessels. DβH‐IR nerve fibres formed a dense network distributed throughout the testis in association with the capsule, vasculature and interstitium. Some fibres were observed to run between seminiferous tubules. VIP‐IR nerve fibres were located in the neighbourhood of blood vessels in the tunica albuginea and parenchyma. Only single VIP‐IR nerves were found between seminiferous tubules. Numerous NPY‐IR nerve fibres occurred in the tunica albuginea and parenchyma of the organ. SNAP‐25‐IR and PGP 9.5‐IR nerve terminals formed a dense network distributed throughout the testis and many fibres were observed between seminiferous tubules. Interstitial cells were very weakly stained for LH receptors or NADPH‐diaphorase.

The influence of ileitis on the neurochemistry of the caudal mesenteric ganglion in the pig

Background  Some literature data suggest that there is a regulatory neuronal circuit between the small and the large bowel. To verify this hypothesis the present study investigated: (i) the distribution, chemical coding and routing of caudal mesenteric ganglion (CaMG) neurons participating in an intestinointestinal reflex pathway involving ileal descending neurons and viscerofugal colonic neurons and (ii) possible changes in the neuroarchitecture of this pathway evoked by chemically induced ileitis in juvenile pigs (n = 16).

Zebrafish: an animal model for research in veterinary medicine

The zebrafish (Danio rerio) has become known as an excellent model organism for studies of vertebrate biology, vertebrate genetics, embryonal development, diseases and drug screening. Nevertheless, there is still lack of detailed reports about usage of the zebrafish as a model in veterinary medicine. Comparing to other vertebrates, they can lay hundreds of eggs at weekly intervals, externally fertilized zebrafish embryos are accessible to observation and manipulation at all stages of their development, which makes possible to simplify the research techniques such as fate mapping, fluorescent tracer time-lapse lineage analysis and single cell transplantation. Although zebrafish are only 2.5 cm long, they are easy to maintain. Intraperitoneal and intracerebroventricular injections, blood sampling and measurement of food intake are possible to be carry out in adult zebrafish. Danio rerio is a useful animal model for neurobiology, developmental biology, drug research, virology, microbiology and genetics. A lot of diseases, for which the zebrafish is a perfect model organism, affect aquatic animals. For a part of them, like those caused by Mycobacterium marinum or Pseudoloma neutrophila, Danio rerio is a natural host, but the zebrafish is also susceptible to the most of fish diseases including Itch, Spring viraemia of carp and Infectious spleen and kidney necrosis. The zebrafish is commonly used in research of bacterial virulence. The zebrafish embryo allows for rapid, non-invasive and real time analysis of bacterial infections in a vertebrate host. Plenty of common pathogens can be examined using zebrafish model: Streptococcus iniae, Vibrio anguillarum or Listeria monocytogenes. The steps are taken to use the zebrafish also in fungal research, especially that dealing with Candida albicans and Cryptococcus neoformans. Although, the zebrafish is used commonly as an animal model to study diseases caused by external agents, it is also useful in studies of metabolic disorders including fatty liver disease and diabetes. The zebrafish is also a valuable tool as a model in behavioral studies connected with feeding, predator evasion, habituation and memory or lateralized control of behavior. The aim of the present article is to familiarize the reader with the possibilities of Danio rerio as an experimental model for veterinary medicine.

Immunohistochemical Characterization of Neurones in the Hypoglossal Nucleus of the Pig

With 4 figures and 1 table

Sources of Sensory Innervation of the Hip Joint Capsule in the Rabbit – A Retrograde Tracing Study

The aim of the study was to investigate the sensory innervation of the hip joint capsule in the rabbit. Individual animals were injected with retrograde fluorescent tracer Fast Blue (FB) into the lateral aspect of the left hip joint capsule (group LAT, n = 5) or into the medial aspect of the hip joint capsule (group MED, n = 5), respectively. FB‐positive (FB+) neurons were found within ipsilateral lumbar (L) and sacral (S) dorsal root ganglia (DRG) from L7 to S2 (group LAT) and from L6 to S4 (group MED). They were round or oval in shape with a diameter of 20–90 μm. The neurons were evenly distributed throughout the ganglia. The average number of FB+ neurons was 16 ± 2.8 and 27.6 ± 3.5 in rabbits from LAT and MED, respectively. The largest average number of FB+ neurons in animals of group LAT was found within the S1 DRG (8 ± 1.7), while S2 ganglion contained the smallest number of the neurons (3.6 ± 1). In the L7 DRG, the average number of FB+ neurons was 6.2 ± 1.6. In rabbits of MED group, the largest number of FB+ neurons was found within the S1 DRG (13.4 ± 4), while the smallest one was found within the S3 ganglion (1.4 ± 0.4). In L6, L7, S2 and S4 ganglia, the number of retrogradely labelled neurons amounted to 1.6 ± 0.5, 4 ± 1.5, 4.4 ± 1.5 and 2.8 ± 1.7, respectively. The data obtained can be very useful for further investigations regarding the efficacy of denervation in the therapy of hip joint disorders in rabbits.

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.

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.

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.