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1

Study of neuropeptide functions using zebrafish as a model organism

100%

Podlasz P.

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nr Suppl.1

XX

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.

2

Axotomy does not induce apoptosis in some sympathetic neurons innervating the porcine aterus

51%

Wasowicz K. , Podlasz P. , Bukowski R.

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tom 65

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nr 3

3

Adrenergic and cholinergic innervation of pulmonary tissue in the pig

51%

Wojtarowicz A. , Podlasz P. , Czaja K.

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2003

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tom 62

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nr 3

215-218

EN

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.

4

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

51%

Obremski K. , Wojtacha P. , Podlasz P. , Żmigrodzka M.

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5

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

51%

Chmielewska M. , Losiewicz K. , Podlasz P. , Wasowicz K.

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nr 3

EN

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.

6

Immunohistochemical characteristics of neurons in the ganglia of the greater splanchnic nerve of the pig

51%

Nourinezhad J. , Podlasz P. , Wasowicz K.

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nr 4

7

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

45%

Czaja K. , Wasowicz K. , Klimczuk M. , Podlasz P. , Lakomy M.

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nr 3

205-211

EN

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⁺.

8

Zebrafish: an animal model for research in veterinary medicine

38%

Nowik N. , Podlasz P. , Jakimiuk A. , Kasica N. , Sienkiewicz W. , Kaleczyc J.

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nr 3

9

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

32%

Wasowicz K. , Podlasz P. , Chmielewska M. , Losiewicz K. , Kaleczyc J. , Zmudzki J. , Zalecki M. , Pidsudko Z. , Lakomy M.

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tom 58

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nr 1

EN

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.

10

Preliminary study on adverse effects of phenanthrene and its methyl and phenyl derivatives in larval zebrafish, Danio rerio

32%

Wolińska L. , Brzuzan P. , Woźny M. , Góra M. , Łuczyński M. K. , Podlasz P. , Kolwicz S. , Piasecka A.

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tom Vol. 7, No. 1

26-33

EN

Toxic effects of polycyclic aromatic hydrocarbons (PAHs) have been extensively studied in fish, although knowledge concerning biological activities of phenanthrene and its derivatives remains still incomplete. The aim of this work was to evaluate lethal and sublethal effects of benzo(a)pyrene, phenanthrene and phenanthrene derivatives (1-methylphenanthrene, 4-methylphenanthrene, 1-phenylphenanthrene and 4-phenylphenanthrene) on zebrafish (Danio rerio) larvae. We conducted acute toxicity test, using 96h static renewal exposure to a series of the PAH concentrations (0.05, 0.50, 5.00, 50.00µmol*l-1), to determine the No Effect Concentration (NEC) value for each compound examined. The mean NEC estimates obtained in the study were 5.16۪.45µmol*l-1 (B[a]P), 4.88۪.13µmol*l-1 (Ph), 40.24䔰.93µmol*l-1 (1P-Ph), 47.92ۭ.61µmol*l-1 (1M-Ph), 24.31۱.33µmol*l-1 (4P-Ph) and 3.11۫.01µmol*l-1 (4M-Ph) and suggested the following order of PAH toxicities on Danio rerio larvae: 4M-Ph>Ph˜B[a]P>4PPhᲙP-Ph>1M-Ph. To gain insight into possible molecular mechanisms of apparent toxicity of phenanthrene derivatives on zebrafish larvae, we examined mRNA expression of cyp1a, cyp1b1, and vtg genes in the larvae exposed for 48h to a PAH concentration of 0.50µmol*l-1. Whereas the larvae exposed to each tested PAH displayed many developmental abnormalities (i.e. pericardial and yolk sac edema, dorsal curvature, or tail malformations), no significant upregulation of cyp1a and cyp1b1 mRNA was observed, except for zebrafish exposed to B[a]P. However, significant reduction of vtg mRNA was observed in the larvae exposed to phenanthrene and its 4P- derivative. The results may contribute to the development of a new knowledge about effects of structurally diverse phenanthrene derivatives on vertebrate organisms.

11

The effect of T-2 toxin on percentages of CD4plus, CD8plus, CD4plusCD8plus and CD21plus lymphocytes, and mRNA expression levels of selected cytokines in porcine ileal Peyer’s patches

26%

Obremski K. , Podlasz P. , Zmigrodzka M. , Winnicka A. , Wozny M. , Brzuzan P. , Jakimiuk E. , Wojtacha P. , Gajecka M. , Zielonka L. , Gajecki M.

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nr 2

EN

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).

12

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

26%

Obremski K. , Podlasz P. , Żmigrodzka M. , Winnicka A. , Woźny M. , Brzuzan P. , Jakimiuk E. , Wojtacha P. , Gajęcka M. , Zielonka Ł. , Gajęcki M.

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nr 2