Galanin regulates blood glucose level in the zebrafish: a morphological and functional study.

The present study has demonstrated the galaninergic innervation of the endocrine pancreas including sources of the galaninergic nerve fibers, and the influence of galanin receptor agonists on blood glucose level in the zebrafish. For the first time, a very abundant galaninergic innervation of the endocrine pancreas during development is shown, from the second day post-fertilization to adulthood. The fibers originated from ganglia consisting of galanin-IR, non-adrenergic (non-sensory) neurons located rostrally to the pancreatic tissue. The ganglia were found on the dorsal side of the initial part of the anterior intestinal segment, close to the intestinal branch of the vagus nerve. The galanin-IR neurons did not show immunoreactivity for applied antibodies against tyrosine hydroxylase, choline acetyltransferase, and vesicular acetylcholine transporter. Intraperitoneal injections of galanin analog NAX 5055 resulted in a statistically significant increase in the blood glucose level. Injections of another galanin receptor agonist, galnon, also caused a rise in blood glucose level; however, it was not statistically significant. The present findings suggest that, like in mammals, in the zebrafish galanin is involved in the regulation of blood glucose level. However, further studies are needed to elucidate the exact mechanism of the galanin action.

The influence of experimental administration of low zearalenone doses on the expression of Th1 and Th2 cytokines and on selected subpopulations of lymphocytes in intestinal lymph nodes.

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.

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.

The expression of mitochondrial, cytoplasmic and extracellular superoxide dismutase in the colonic wall of pigs suffering from swine dysenteria.

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.

The effect of T-2 toxin on percentages of CD4+, CD8+, CD4+ CD8+ and CD21+ lymphocytes, and mRNA expression levels of selected cytokines in porcine ileal Peyer's patches.

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 microg 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-gamma, 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-gamma 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).

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.

Distribution and immunohistochemical characterisation of paracervical neurons innervating the oviduct in the pig.

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 beta-hydroxylase (D beta H), 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 microns-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+/D beta H+, TH+/NPY+, TH+/NOS+, TH+/NOS-, SP-/NOS+, SP+/CGRP+.

Vagal projections to the pylorus in the domestic pig (Sus scrofa domestica).

The goal of the present study was to examine the precise localization of the brainstem motor and primary sensory (nodose ganglion) vagal perikarya supplying the pylorus in the domestic pig. Using the Fast Blue retrograde tracing technique it has been established that all the vagal motor neurons projecting to the pylorus (about 337 ± 59 cells per animal) were localized bilaterally in the dorsal motor nucleus of the vagus nerve (DMX, 171 - left; 167 - right) and all other regions of the porcine brainstem were devoid of labeled neurons. The vagal perikarya supplying the porcine pylorus were dispersed throughout the whole rostro-caudal extent of the DMX and no somatotopic organization of these neurons was observed. The labeled neurons occurred individually or in groups up to five cell bodies per nuclear transverse cross section area (in the middle part of the nucleus). An immunocytochemical staining procedure disclosed that all Fast Blue labeled motor neurons were choline acetyltransferase (ChAT) immunoreactive, however some differences in immunofluorescence intensity occurred. The primary sensory vagal neurons were observed within the left (215±37 cells/animal) and right (148±21 cells/animal) nodose ganglion. The traced neurons were dispersed throughout the ganglia and no characteristic arrangement of these neurons was observed. The present experiment precisely indicates the sources of origin of the vagal motor and primary sensory neurons supplying the pyloric region in the pig, the animal of an increasing significance in biomedical research.

Galanin gene expression and effects of its knock-down on the development of the nervous system in larval zebrafish.

Despite the known importance of galanin in the nervous system of vertebrates, the galanin gene structure and expression and the consequences of galanin deficiency in developing zebrafish are unknown. We cloned the galanin gene and analyzed its expression by using in situ hybridization, PCR, and immunocytochemistry throughout the early development of zebrafish until the end of the first week of life. The single zebrafish galanin gene encoded for a single amidated galanin peptide and a galanin message-associated peptide. Two forms resulting from alternative processing were identified. Galanin mRNA was maternally expressed and found in developing fish throughout early development. In situ hybridization showed the first positive neurons in three groups in the brain at 28 hours postfertilization. At 2 days postfertilization, three prosencephalic neuron groups were seen in the preoptic area and in rostral and caudal periventricular hypothalamus. In addition, two other groups of weakly stained neurons were visible, one in the midbrain and another in the hindbrain. Translation inhibition of galanin mRNA with morpholino oligonucleotides caused complete disappearance of galanin immunoreactivity in the brain until 7 dpf and did not induce known cascades of nonspecific pathways or morphological abnormalities. A minor disturbance of sensory ganglia was found. Galanin knockdown did not alter the expression of tyrosine hydroxylases 1 and 2, choline acetyltransferase, histidine decarboxylase, or orexin mRNA. The results suggest that galanin does not regulate the development of these key markers of specific neurons, although galanin-expressing fibers were in a close spatial proximity to several neurons of these neuronal populations.

Characterization of autonomic nerve markers and lymphocyte subsets in the ileal Peyer's patch of pigs infected experimentally with Brachyspira hyodysenteriae.

The aim of the present study was to investigate potential interrelationships between immune and neural elements of Peyer's patches in normal pigs (n=8) and in pigs infected experimentally with Brachyspira hyodysenteriae and suffering from swine dysentery (n=8). Assessment of tissue concentration of neuropeptides by enzyme linked immunosorbent assay revealed increased levels of galanin (GAL) and substance P (SP) in samples from the infected animals. In contrast, concentrations of vasoactive intestinal polypeptide (VIP) and somatostatin (SOM) were similar in both groups. Immunohistochemistry demonstrated reactivity of nerve fibres with antibodies specific for dopamine ß hydroxylase, vesicular acetylcholine transporter, SOM, GAL, VIP and SP in the interfollicular region and peripheral areas of the Peyer's patch lymphoid follicles. In the dysenteric pigs, the GAL-positive nerve fibres were more numerous and more intensely labelled than those in the normal animals. Flow cytometry revealed a decreased percentage of CD21(+) lymphocytes and lymphocytes expressing T-cell receptor (TCR)-γ, with or without CD8 (TCR-γ(+)CD8(-) and TCR-γ(+)CD8(+)), in the dysenteric pigs as compared with the normal animals. Percentages of other lymphocyte subsets (CD2(+), CD4(+), CD5(+), CD8(+), CD5(-)CD8(+)) were comparable between the groups. Immunohistochemical investigations generally correlated with results obtained by flow cytometry related to lymphocyte subpopulations. Swine dysentery can therefore affect neuroimmunomodulatory processes in the ileal Peyer's patch, in addition to the large intestine. GAL and SP may play a specific role in this neuroimmune cross-talk.