Sławomir Gonkowski

Inflammation- and axotomy-induced changes in galanin-like immunoreactive (GAL-LI) nerve structures in the porcine descending colon

This study reports on changes caused by chemically driven inflammation and axotomy in galanin-like immunoreactive (GAL-LI) nerve structures in the porcine descending colon. The distribution pattern of GAL-LI structures was studied using the immunofluorescence technique in the circular muscle layer, the myenteric (MP), outer submucous (OSP) and inner submucous plexuses (ISP), and also in the mucosal layer. Under physiological conditions GAL-LI perikarya were shown to constitute 3.68 +/- 0.32%, 7.02 +/- 0.93% and 10.99 +/- 0.71% in MP, OSP and ISP, respectively. Both colitis and axotomy caused an increase in GAL-like immunoreactivity, which was different in particular parts of the bowel segment studied. The numbers of GAL-LI perikarya increased to 14.16 +/- 0.49%, 16.78 +/- 1.09% and 37.46 +/- 1.18% during colitis and 7.92 +/- 0.72%, 10.44 +/- 0.71% and 16.20 +/- 0.96% after axotomy in MP, OSP and ISP, respectively. Both these processes caused an increase in the number of GAL-LI nerve fibres in the circular muscle and mucosal layers as well as the appearance of a population of GAL-LI cells in the mucosa.

Distribution of cocaine- and amphetamine-regulated transcript-like immunoreactive (CART-LI) nerve structures in the porcine large intestine

The aim of the present study was to investigate the number of cocaine- and amphetamine-regulated transcript-like immunoreactive (CART-LI) nerve structures in the large intestine of juvenile pigs. The distribution pattern of CART-LI structures was studied by immunohistochemistry in the circular muscle layer, myenteric (MP), outer submucous (OSP) and inner submucous plexus (ISP) as well as in the mucosal layer of six regions of the large bowel: caecum, centripetal and centrifugal turns of the proximal colon, transverse colon, descending colon and rectum. CART-LI neural structures were observed in all gut fragments studied. CART-LI nerve fibres were numerous within the circular muscle layer and in the MP of all the regions studied, while they were moderate or few in number in other layers of the intestinal wall. The numbers of CART-LI neurons within the MP amounted to 2.02% in the caecum to 7.92% in the rectum, within the OSP from 2.73% in the centrifugal turns of the proximal colon to 5.70% in the rectum, and within the ISP from 2.23% in the transverse colon to 5.32% in the centrifugal turns of the proximal colon. The present study reports for the first time a detailed description of the CART distribution pattern within the enteric nervous system (ENS) of the porcine large intestine.

Characterisation of cocaine- and amphetamine- regulated transcript-like immunoreactive (CART-LI) enteric neurons in the porcine small intestine

The aim of this study was to investigate the distribution and the number of cocaine- and amphetamine-regulated transcript-like immunoreactive (CART-LI) neurons and the co-localisation of CART with substance P (SP), somatostatin (SOM), nitric oxide synthase (NOS) and vasoactive intestinal polypeptide (VIP) within the enteric nervous system (ENS) in the porcine small intestine. Accordingly, the myenteric plexus (MP), outer submucous plexus (OSP) and inner submucous plexus (ISP) of the small intestine (duodenum, jejunum and ileum) were studied by double-labelling immunofluorescence technique. CART-LI neurons were observed in all gut fragments and all types of intramural plexuses studied and amounted from 0.2 ± 0.1% in the ISP of ileum to 22.4 ± 2.4% in the MP of this segment. The co-localisation of CART and NOS or/and VIP was observed depending on the segment of the gut and the complexity of the intramural plexus. On the other hand, during this study the co-localisation of CART and SOM or/and SP was not observed. The present study, for the first time, presents a detailed description of the CART distribution pattern and co-localisation with other neuromodulators within the ENS of the porcine small intestine.

Axotomy-induced changes in the chemical coding pattern of colon projecting calbindin -positive neurons in the inferior mesenteric ganglia of the pig.

The present study examines the response of colon-projecting neurons localized in the inferior mesenteric ganglia (IMG) to axotomy in the pig animal model. In all animals (n = 8), a median laparotomy was performed under anesthesia and the retrograde tracer Fast Blue was injected into the descending colon wall. In experimental animals (n = 4), the descending colon was exposed and the bilateral caudal colonic nerves were identified and severed. All animals were euthanized and the inferior mesenteric ganglia were harvested and processed for double-labeling immunofluorescence for calbindin-D28k (CB) in combination with either tyrosine hydroxylase (TH), neuropeptide Y (NPY), somatostatin (SOM), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), Leu-enkephalin (LENK), substance P, vesicular acetylcholine transporter, or galanin. Immunohistochemistry revealed significant changes in the chemical coding pattern of injured inferior mesenteric ganglion neurons. In control animals, Fast Blue-positive neurons were immunoreactive to TH, NPY, SOM, VIP, NOS, LENK, and CB. In the experimental group, the numbers of TH-, NPY-, and SOM-expressing neurons were reduced, whereas the number of neurons immunoreactive to LENK was increased. Our data indicate that the colon-projecting neurons of the porcine IMG react to the axotomy in a similar, but not an identical manner in a comparison to other species, especially rodents. Further studies are needed to elucidate the detailed factors/mechanisms involved in the response to nerve injury.

T2 Toxin-Induced Changes in Cocaine- and Amphetamine-Regulated Transcript (CART)-Like Immunoreactivity in the Enteric Nervous System Within Selected Fragments of the Porcine Digestive Tract

T-2 toxin is a mycotoxin produced by some Fusarium species, which may affect the synthesis of DNA and RNA and causes various pathological processes. Till now, the influence of T-2 toxin on the enteric nervous system (ENS) located in the wall of gastrointestinal tract has not been studied. On the other hand, cocaine- and amphetamine-regulated transcript (CART) is one of enteric neuronal factors, whose exact functions in the intestines still remain not fully explained. The present study describes the influence of low doses of T-2 toxin on CART-positive neuronal structures in porcine stomach, duodenum, and descending colon. Distribution of CART was studied using the double immunofluorescence technique in the plexuses of the ENS, as well as in nerve fibers within the circular muscle and mucosal layers of porcine gastrointestinal tract. Generally, after T-2 toxin administration the greater number of CART-LI structures were studied, but intensity of changes depended on part of the ENS and digestive tract fragment studied. The obtained results show that even low doses of T-2 toxin may change the expression of CART in the ENS.

The Influence of Low Doses of Zearalenone and T-2 Toxin on Calcitonin Gene Related Peptide-Like Immunoreactive (CGRP-LI) Neurons in the ENS of the Porcine Descending Colon

The enteric nervous system (ENS) can undergo adaptive and reparative changes in response to physiological and pathological stimuli. These manifest primarily as alterations in the levels of active substances expressed by the enteric neuron. While it is known that mycotoxins can affect the function of the central and peripheral nervous systems, knowledge about their influence on the ENS is limited. Therefore, the aim of the present study was to investigate the influence of low doses of zearalenone (ZEN) and T-2 toxin on calcitonin gene related peptide-like immunoreactive (CGRP-LI) neurons in the ENS of the porcine descending colon using a double immunofluorescence technique. Both mycotoxins led to an increase in the percentage of CGRP-LI neurons in all types of enteric plexuses and changed the degree of co-localization of CGRP with other neuronal active substances, such as substance P, galanin, nitric oxide synthase, and cocaine- and amphetamine-regulated transcript peptide. The obtained results demonstrate that even low doses of ZEN and T-2 can affect living organisms and cause changes in the neurochemical profile of enteric neurons.

Proliferative Enteropathy (PE)—Induced Changes in the Calbindin-Immunoreactive (CB-IR) Neurons of Inferior Mesenteric Ganglion Supplying the Descending Colon in the Pig

A subpopulation of the pig inferior mesenteric ganglia (IMG) neurons projecting to the colon exhibit calbindin-like immunoreactivity. It is not known if there are any changes in the chemical coding patterns of these neurons during porcine proliferative enteropathy (PE). To answer this question, juvenile Large White Polish pigs with clinically diagnosed Lawsonia intracellularis infection (PE; n = 3) and a group of uninfected controls (C; n = 3) were compared. The retrograde tracer fast blue (FB) was injected into the descending colons of all animals and then tissue comprising IMGs from both groups was processed for double-labeling immunofluorescence with calbindin-D28k (CB) in combination with either tyrosine hydroxylase (TH), neuropeptide Y (NPY), somatostatin (SOM), vasoactive intestinal polypeptide (VIP), nitric oxide synthase, Leu-enkephalin, substance P, vesicular acetylcholine transporter, galanin, or pituitary adenylate cyclase-activating polypeptide. Immunohistochemistry revealed changes in the chemical coding pattern of calbindin-immunoreactive neurons in the inferior mesenteric ganglia of the pig. In control animals, FB/CB-positive neurons were immunoreactive to TH, NPY, SOM, and VIP. In the experimental group, TH-expressing neurons were unaffected, NPY-expressing neurons were increased, whereas the number of neurons immunoreactive to SOM or VIP was reduced. Changes in chemical coding of CB neurons during PE may play an important role in adaptation of these IMG cells under pathological conditions.

Immunohistochemical distribution of cocaine and amphetamine regulatory peptide-like immunoreactive (CART-LI) nerve fibers in the circular muscle layer and their relationship to other peptides in the human caecum.

Motor activity of the gastrointestinal tract is extensively controlled by the enteric nervous system (ENS). Numerous neurotransmitters and neuromodulators are responsible for this regulation. One of them is cocaine- and amphetamine-regulated transcript peptide (CART). So far, there are few reports available concerning the distribution, functions, and co-localization of CART in the human gastrointestinal tract. The aim of the present investigation was to study the distribution and degree of co-localization of CART with substances taking part in conducting sensory stimuli, such as: substance P (SP), neurokinin A (NKA), calcitonin gene related peptide (CGRP) and Leu 5 enkephalin (L-ENK) in the circular muscle layer of the human caecum. CART-like immunoreactive (CART-LI) nerve fibers formed a very dense meshwork in the circular muscle layer of the caecum in all patients studied. Moreover, all neuronal substances tested during the present investigation were observed in CART-LI processes, but the degree of co-localization depended on the type of substance. The highest number of CART-positive nerves also contained L-ENK. A slightly lower level of co-localization was observed in the case of CART and SP or NKA, while only single nerve fibers were simultaneously CART- and CGRP-positive.

Chemical coding of zinc-enriched neurons in the intramural ganglia of the porcine jejunum

Zinc ions in the synaptic vesicles of zinc-enriched neurons (ZEN) seem to have an important role in normal physiological and pathophysiological processes in target organ innervation. The factor directly responsible for the transport of zinc ions into synaptic vesicles is zinc transporter 3 (ZnT3), a member of the divalent cation zinc transporters and an excellent marker of ZEN neurons. As data concerning the existence of ZEN neurons in the small intestine is lacking, this study was designed to disclose the presence and neurochemical coding of such neurons in the porcine jejunum. Cryostat sections (10 mμ thick) of porcine jejunum were processed for routine double- and triple-immunofluorescence labeling for ZnT3 in various combinations with immunolabeling for other neurochemicals including pan-neuronal marker (PGP9.5), substance P (SP), somatostatin (SOM), vasoactive intestinal peptide (VIP), nitric oxide synthase (NOS), leu-enkephalin (LENK), vesicular acetylcholine transporter (VAChT), neuropeptide Y (NPY), galanin (GAL), and calcitonin-gene related peptide (CGRP). Immunohistochemistry revealed that approximately 39%, 49%, and 45% of all PGP9.5- positive neurons in the jejunal myenteric (MP), outer submucous (OSP), and inner submucous (ISP) plexuses, respectively, were simultaneously ZnT3+. The majority of ZnT3+ neurons in all plexuses were also VAChT-positive. Both VAChT-positive and VAChT-negative ZnT3+ neurons co-expressed a variety of active substances with diverse patterns of co-localization depending on the plexus studied. In the MP, the largest populations among both VAChT-positive and VAChT-negative ZnT3+ neurons were NOS-positive cells. In the OSP and ISP, substantial subpopulations of ZnT3+ neurons were VAChT-positive cells co-expressing SOM and GAL, respectively. The broad-spectrum of active substances that co-localize with the ZnT3+ neurons in the porcine jejunum suggests that ZnT3 takes part in the regulation of various processes in the gut, both in normal physiological and during pathophysiological processes.

Co-localization of zinc transporter 3 (ZnT3) with sensory neuromediators and/or neuromodulators in the enteric nervous system of the porcine esophagus

Zinc transporter 3 (ZnT3) is one of the zinc transporters family. It is closely connected to the nervous system, where enables the transport of zinc ions from the cytoplasm to synaptic vesicles. This substance has been described within the central and peripheral nervous system, especially in the enteric nervous system (ENS). The aim of the present study was to describe the co-localization of ZnT3 with selected neuromediators and/or neuromodulators participating in sensory stimuli conduction in neurons of the ENS within the porcine esophagus. Co-localization of ZnT3 with substance P (SP), leucine enkephalin (LENK) and calcitonin gene-related peptide (CGRP) was studied using standard double-immunofluorescence technique. The obtained results show that ZnT3, SP and/or LENK may occur in the same enteric neurons, and the degree of co-localization of these substances clearly depends on the fragment of esophagus studied and the type of enteric ganglia. In contrast, the co-localization of ZnT3 with CGRP was not observed during the present investigation. The obtained results suggest that ZnT3 in the ENS may be involved in the conduction of sensory and/or pain stimuli.