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Neuropeptides constitute a large class of signaling molecules in the nervous system of many groups of animals. These peptides are extracellular chemical messengers that act as circulating neurohormones, local co‑transmitters, or neuromodulators. Neuropeptides have been well conserved during the course of evolution, indicating their major role as regulators of physiological processes. In our lab, we study neuropeptide functioning using zebrafish as a model organism. One of the more interesting neuropeptides we study is galanin. Galanin is a 29‑30 amino acid neuropeptide widely expressed in the central and peripheral nervous systems in vertebrates. It co‑exists within neurons with several small-molecule classical neurotransmitters and exerts strong inhibitory actions on synaptic transmission by reducing the release of neurotransmitters. Galanin has been implicated in several higher order physiological functions such as nociception, cognition, feeding, mood, and neuroendocrine regulation, which may be relevant to disease states and clinical therapy. Galanin is a highly inducible neuropeptide, showing distinct up-regulation after pathological disturbance within the nervous system. A significant increase in galanin expression is observed after peripheral nerve injury, inflammation, in the basal forebrain in Alzheimer’s disease, multiple sclerosis, and during neuronal development. These early studies suggested that increased galanin concentration might have a trophic influence on nerve repair. Galanin has also been observed in a variety of tumors, where it may affect growth and apoptosis. The above information indicates that galanin is a multifaceted peptide. Although it has been 35 years since its discovery, many of its functions are not fully elucidated. We believe that so far applied research models have some limitations which prevent full understanding of the function of galanin. Therefore, we decided to study the function of galanin using another, not used so far, model – zebrafish. Our previous studies revealed that the zebrafish galanin gene is very similar to that of other studied species. The structure of the zebrafish galanin gene is identical to the gene present in mammals. This strong evolutionary conservation may suggest important and similar roles of galanin in all vertebrates. We already studied the expression of galanin during development and in adult zebrafish. In addition, using antisense oligonucleotides (morpholino), we examined the role of galanin in the development of the nervous system. We also confirmed that galanin regulates blood glucose levels. Our studies in transgenic line with inducible overexpression of galanin revealed that galanin reduced the incidence of seizure‑like behavior episodes and their intensity. Recently we generated a CRISPR-cas9 zebrafish mutant that is demonstrated to lack the expression of galanin. We use it, among others, to study the role of galanin in the neuroendocrine system, innate immunity, bacterial infection, and regeneration of the nervous system.
The expression of 3 types of peroxide dismutase (SOD1, SOD2 and SOD3) was studied with Real-Time PCR in the colonic wall of domestic pig suffering from swine dysentery. The expression of enzymes was studied separately in the mucosa and the muscular membrane. It was found that in the mucosa the expression of SOD1 (cytoplasmic) did not change, while the levels of expression of mitochondrial SOD2 and extracellular SOD3 were raised in inflamed colon. More dramatic changes were seen in the muscular mebrane where expression of SOD1 rose twice, this of SOD2 rose ca. 5-fold and the expression of SOD3 rose dramatically, even 30-fold.The obtained data are contradictory to findings in other types of colonic inflammation, which were studied either in the whole colonic wall, or in mucosa alone. The results show a very strong reaction of antioxidant systems in the muscular membrane in the enteritis.
Both the adrenergic and the cholinergic components of the autonomic nervous system (ANS) have been found to be an important source of nerve fibres supplying the lungs. On the other hand, data regarding the innervation of the pulmonary tissue in breeding animals are surprisingly scarce. Therefore, in the present study noradrenergic and acetylcholinesterase-positive (AChE-positive) innervation of the lungs of sexually immature pigs was studied using histochemical methods. Studies were performed on six juvenile female pigs (aged 9 weeks, body weight 15–20 kg). Samples of the tissue were collected from the caudal lobe of the right lung. 10µm cryostat sections were processed for the sucrosepotassium phosphate-glyoxylic acid technique to determine the occurrence and distribution of noradrenergic nerve fibres, while AChE-positive nerves were detected according to the acetylcholinesterase histochemistry. The present results revealed a dense network of noradrenergic nerve fibres localised mainly in the muscular membrane surrounding the epithelium of the bronchuli while AChE- -positive nerve terminals supplied functional capillary vessels localised in the inter-alveolar septum and mucous membrane of the bronchi and bronchuli. The results of the present study confirm those of physiological experiments reporting the influence of the adrenergic and cholinergic components of the autonomic nervous system on the lung functions of pigs.
Immunohistochemical characteristics of neurones innervating the porcine uterus located in paracervical ganglia were studied with a combination of retrograde fluorescent tracing and immunofluorescence. Retrograde fluorescent tracer Fast Blue (FB) was injected into the uterine horn and uterine cervix. The presence of biologically active substances, tyrosine hydroxylase (TH), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), galanin (GAL), Met-enkephalin-Arg-GlyLeu (MEAGL) and calcitonin gene-related peptide (CGRP) was studied in FBpositive neurones localised in paracervical ganglia. FB-positive neurones containing TH, NPY, VIP and MEAGL were numerous, while those containing CGRP were scarce. The results pointed to some species-related differences in immunohistochemical coding of neurones of paracervical ganglion responsible for uterus innervation.
The aim of this study was to characterize the immune response taking place in ileocecal lymph nodes (ICLN) in control (n=15) and zearalenone (ZEN)-treated (n=15) pigs. The experiment was carried out over 42 days; a dose of 0.1 mg kg⁻¹ feed day⁻¹ of ZEN was administered to the animals. The dose used in the experiment was at a level where no adverse effects are observed (NOAEL) in the ovaries, uterus and vagina. ICLN samples for analysis were collected on the 14th, 28th and 42nd day of the experiment. The analysis of cytokine concentration in the tissues showed that pigs treated with ZEN had an increased level of cytokines produced by helper Th1 lymphocytes (IL-2, IL-12 and IFN- γ) on the 28th day of the experiment. The level of cytokines produced by helper Th2 lymphocytes (IL-4 and IL-10) was characterized by a statistically non-significant upward trend, as compared with the control group. Flow cytometry showed a linear decrease in the percentage of CD21+ B, CD2+ T and CD4+CD8- T cells and an increase in the percentage of CD8+CD4- and TCRγδ + T cells in pigs treated with ZEN. Both ZEN and α-ZEL (α-zearalenone) concentrations increased over time in the liver, but only ZEN concentration increased in ICLN. The results obtained demonstrate that a NOAEL concentration of ZEN shifts the immune response in pig ICLN towards Th1/Th17, probably with a simultaneous activation of M1 macrophages. Moreover, we observed an increase in humoral cytokine secretion; this can be explained by a negative feedback loop and a phenotypic switch of macrophages from M1 to M2, as well as a switch of immune response from Th1 to Th2 type. ZEN can therefore influence the process of cytokine secretion and the percentage of lymphocytes in ileocecal lymph nodes.
The present study was aimed at disclosing the distribution of paracervical neurons projecting to the ampulla and isthmus of the porcine oviduct and the pattern(s) of co-existence of tyrosine hydroxylase (TH), dopamine β-hydroxylase (DβH), neuropeptide Y (NPY), substance P (SP), calcitonin gene-related peptide (CGRP) and nitric oxide synthase (NOS) within these nerve cell bodies. The fluorescent retrograde tracer Fast Blue (FB) was injected into the wall of the ampullar (n = 3) and isthmal (n = 3) part of the organ in six sexually immature female pigs. After a survival period of three weeks paracervical ganglia (PCG) were collected. 10 µm-thick cryostat sections of the ganglia were examined for the presence of FB-positive (FB⁺) nerve cells under the fluorescent microscope. Tracered neurons were counted in every third section and processed for double-labelling immunofluorescence according to the method of Wessendorf and Elde. 78.6% of FB⁺ neurons were projecting to the isthmus while 21.4% of the studied population innervated the ampulla of the oviduct. Double-labelling immunofluorescence revealed the existence of the following different chemically coded subpopulations of the studied perikarya: TH⁺/DβH⁺, TH⁺/NPY⁺, TH⁺/NOS⁺, TH⁺/ NOS⁻, SP⁻/NOS⁺, SP⁺/CGRP⁺.
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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.
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.
The immune system is one of the main toxicity targets of the T-2 toxin. In view of scant research data demonstrating the effect of T-2 on cellular and humoral responses in gut-associated lymphoid tissue (GALT), this study set out to investigate the effects of chronic exposure to low doses of the T-2 toxin (200 μg T-2 toxin kg⁻¹ feed) on percentages of CD4⁺ and CD8⁺ T lymphocytes, CD4⁺/CD8⁺ double-positive T lymphocytes, CD21⁺ B cells, and IL-2, IFN-γ, IL-4 and IL-10 mRNA expression levels in porcine ileal Peyer’s patches. The investigated material comprised ileum sections sampled from piglets (aged 8-10 weeks, body weight of 15-18 kg) on days 14, 28 and 42 of the experiment. After 42 days of exposure to T-2, a significant drop in the quantity of the IL-10 product was observed (R=0.94; S.E. 0.49-0.79; p<0.001). A gradual decrease in the amount of IL-4 and IFN-γ cytokine transcripts was found throughout the experiment, but the reported trend was not significant. On experimental days 14 and 42, a significant increase in the percentage of CD8⁺ T lymphocytes was observed in comparison with the control (p=0.04 and p=0.05, respectively), whereas on day 28, a significant decrease in the percentage of the above subpopulation was noted (p=0.00). The percentage of CD21⁺ B cells in the experimental group decreased steadily in comparison with the control, and the observed drop was significant on days 28 and 42 (p=0.06 and p=0.00, respectively). On days 14 and 28, the percentages of CD4⁺ and CD8⁺ T lymphocytes were lower in the experimental animals than in the control group, and the drop reported on day 28 was statistically significant (p=0.03).
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