Paper Title
The Influence of Lipopolysaccharides (LPS) from Salmonella Enteritidis on Nitrergic Neurons in The Enteric Nervous System (ENS) of the Porcine Duodenum

Abstract
Lipopolysaccharide (LPS, bacterial endotoxin) is a component of the cellular membrane of Gram-negative bacteria, which is known as an important pathological factor. In spite of many previous studies describing multidirectional negative effects of LPS on the living organism, the knowledge concerning the influence of LPS on the enteric nervous system (ENS) is extremely scarce. The ENS is located in the wall of the gastrointestinal (GI) tract and in large mammals consists of millions of neurons grouped into three types of ganglionated plexuses: myenteric plexus (MP), which is situated between the longitudinal and circular muscle layers, the outer submucous plexus (OSP), near the internal side of the circular muscle layer and the inner submucous plexus (ISP), locatedbetween the muscularis mucosa and lamina propria. Enteric neurons show wide variations in terms of their neurochemical characterization. Till now tens of neuronal active substances have been noted in enteric neurons. One of them is gaseous neurotransmitter - nitric oxide (NO), which is knownas an important inhibitory factor within the GI tract, which takes part also in pathological processes. the aim of this study was to describe the influence of LPS on distribution of nitrergic neurons in the ENS of porcine duodenum. Fragments of duodenumwere collected from five control pigs (C-group) and from five animals, which seven days earlier received intravenously 5 µg/kg body weight solution of LPS from Salmonella Enteritidis. Tissues were subjected to standard double-labelling immunofluorescence technique using commercial antisera against protein gene-product 9.5 (PGP 9.5, used here as a pan-neuronal marker) and neuronal isoform of nitric oxide synthase (nNOS – used here as a marker of nitrergic neurons). To evaluate the percentage of nNOS – like immunoreactive (nNOS-LI) neurons in relation to all enteric neuronal cells, at least 500 cells labeled with PGP-9.5 (treated as 100%) in the particular type of enteric plexuses from each animal were examined for the presence of nNOS. LPS administration caused clear, statistically significant (p≤0.05) increase in the percentage of nNOS-LI neurons in all types of enteric ganglia. In control animals the percentage of neuronal cells immunoreactive to nNOS amounted to 14.4 ± 0.62% of all PGP 9.5 – positive cells in the MP, 19.42 ± 0.92% in the OSP and 10.58 ± 0.63% in the ISP. After LPS administration these values achieved 21.26 ± 0.81%, 24.44 ± 0.8% and 14.62 ± 0.61% in the MP, OSP and ISP, respectively. The obtained results show that nitric oxide is an important neuronal factor in the ENS of the porcine duodenum, not only under physiological conditions, but also under influence of LPS. Observed changes in the percentage of nNOS-LI enteric neurons suggest participation of nitric oxide in adaptive and/or neuroprotective processes within nervous structures supplying the duodenum. However, the explanation of exact roles of nitrergic neurons during bacterial LPS intoxication requires further investigations. Supported by KNOW (Leading National Research Centre) Scientific Consortium “Healthy Animal – Safe Food”, decision of Ministry of Science and Higher Education No. 05-1/KNOW2/2015