Outer Submucous Research Articles

The T2 Toxin Produced by Fusarium spp. Impacts Porcine Duodenal Nitric Oxide Synthase (nNOS)-Positive Nervous Structures-The Preliminary Study.

T2 toxin synthetized by Fusarium spp. negatively affects various internal organs and systems, including the digestive tract and the immune, endocrine, and nervous systems. However, knowledge about the effects of T2 on the enteric nervous system (ENS) is still incomplete. Therefore, during the present experiment, the influence of T2 toxin with a dose of 12 µg/kg body weight (b.w.)/per day on the number of enteric nervous structures immunoreactive to neuronal isoform nitric oxide synthase (nNOS—used here as a marker of nitrergic neurons) in the porcine duodenum was studied using the double immunofluorescence method. Under physiological conditions, nNOS-positive neurons amounted to 38.28 ± 1.147%, 38.39 ± 1.244%, and 35.34 ± 1.151 of all enteric neurons in the myenteric (MP), outer submucous (OSP), and inner submucous (ISP) plexuses, respectively. After administration of T2 toxin, an increase in the number of these neurons was observed in all types of the enteric plexuses and nNOS-positive cells reached 46.20 ± 1.453% in the MP, 45.39 ± 0.488% in the OSP, and 44.07 ± 0.308% in the ISP. However, in the present study, the influence of T2 toxin on the intramucosal and intramuscular nNOS-positive nerves was not observed. The results obtained in the present study indicate that even low doses of T2 toxin are not neutral for living organisms because they may change the neurochemical characterization of the enteric neurons.

The influence of experimental inflammation and axotomy on leucine enkephalin (leuENK) distribution in intramural nervous structures of the porcine descending colon

BackgroundThe enteric nervous system (ENS), located in the intestinal wall and characterized by considerable independence from the central nervous system, consists of millions of cells. Enteric neurons control the majority of functions of the gastrointestinal tract using a wide range of substances, which are neuromediators and/or neuromodulators. One of them is leucine–enkephalin (leuENK), which belongs to the endogenous opioid family. It is known that opioids in the gastrointestinal tract have various functions, including visceral pain conduction, intestinal motility and secretion and immune processes, but many aspects of distribution and function of leuENK in the ENS, especially during pathological states, remain unknown.ResultsDuring this experiment, the distribution of leuENK – like immunoreactive (leuENK-LI) nervous structures using the immunofluorescence technique were studied in the porcine colon in physiological conditions, during chemically-induced inflammation and after axotomy. The study included the circular muscle layer, myenteric (MP), outer submucous (OSP) and inner submucous plexus (ISP) and the mucosal layer. In control animals, the number of leuENK-LI neurons amounted to 4.86 ± 0.17%, 2.86 ± 0.28% and 1.07 ± 0.08% in the MP, OSP and ISP, respectively. Generally, both pathological stimuli caused an increase in the number of detected leuENK-LI cells, but the intensity of the observed changes depended on the factor studied and part of the ENS. The percentage of leuENK-LI perikarya amounted to 11.48 ± 0.96%, 8.71 ± 0.13% and 9.40 ± 0.76% during colitis, and 6.90 ± 0.52% 8.46 ± 12% and 4.48 ± 0.44% after axotomy in MP, OSP and ISP, respectively. Both processes also resulted in an increase in the number of leuENK-LI nerves in the circular muscle layer, whereas changes were less visible in the mucosa during inflammation and axotomy did not change the number of leuENK-LI mucosal fibers.ConclusionsLeuENK in the ENS takes part in intestinal regulatory processes not only in physiological conditions, but also under pathological factors. The observed changes are probably connected with the participation of leuENK in sensory and motor innervation and the neuroprotective effects of this substance. Differences in the number of leuENK-LI neurons during inflammation and after axotomy may suggest that the exact functions of leuENK probably depend on the type of pathological factor acting on the intestine.

Zinc Transporter 3 (ZnT3) in the Enteric Nervous System of the Porcine Ileum in Physiological Conditions and during Experimental Inflammation.

Zinc transporter 3 (ZnT3) is a member of the solute-linked carrier 30 (SLC 30) zinc transporter family. It is closely linked to the nervous system, where it takes part in the transport of zinc ions from the cytoplasm to the synaptic vesicles. ZnT3 has also been observed in the enteric nervous system (ENS), but its reactions in response to pathological factors remain unknown. This study, based on the triple immunofluorescence technique, describes changes in ZnT3-like immunoreactive (ZnT3-LI) enteric neurons in the porcine ileum, caused by chemically-induced inflammation. The inflammatory process led to a clear increase in the percentage of neurons immunoreactive to ZnT3 in all “kinds” of intramural enteric plexuses, i.e., myenteric (MP), outer submucous (OSP) and inner submucous (ISP) plexuses. Moreover, a wide range of other active substances was noted in ZnT3-LI neurons under physiological and pathological conditions, and changes in neurochemical characterisation of ZnT3+ cells in response to inflammation depended on the “kind” of enteric plexus. The obtained results show that ZnT3 is present in the ENS in a relatively numerous and diversified neuronal population, not only in physiological conditions, but also during inflammation. The reasons for the observed changes are not clear; they may be connected with the functions of zinc ions and their homeostasis disturbances in pathological processes. On the other hand, they may be due to adaptive and/or neuroprotective processes within the pathologically altered gastrointestinal tract.

Changes in expression of calbindin 28 kDa in the small intestine of red kidney bean (Phaseolus vulgaris) lectin-treated suckling piglets

Calbindin (CB) is a calcium binding protein playing a role in calcium uptake and anti-apoptotic cellular protection. The expression of CB was immunohistochemically studied in the small intestine of normal and red bean kidney lectin-treated suckling piglets. In the duodenum and jejunum (but not ileum) of lectin-treated animals overexpression of CB was noted in chromogranin A-immunoreactive (CgA-IR) neuroendocrine (NE) cells. In both control and experimental group a small population of CB-IR NE cells exhibited the presence of somatostatin (but not serotonin, histamine or CRF). After the lectin treatment, an increased (however not statistically significant) immunoreactivity to CB was found in a small subpopulation of neurons of outer submucous (but not inner submucous and myenteric) plexus. It is suggested that there is a functional interaction between lectin administration and CB-expression in the porcine small intestine. Future studies will be needed to clarify this processes.

Substance P as a neuronal factor in the enteric nervous system of the porcine descending colon in physiological conditions and during selected pathogenic processes

The present investigation pertains to changes in substance P-like immunoreactive (SP-LI) nerve structures of the enteric nervous system (ENS) in the porcine descending colon, caused by chemically-induced inflammation and nerve injury (axotomy). The distribution pattern of SP-LI structures was studied using the double immunofluorescence technique in the myenteric (MP), outer submucous (OSP) and inner submucous (ISP) plexuses, as well as in the circular muscle and mucosal layers. Under physiological conditions, SP-LI neurons have been shown to constitute 4.13 ± 0.24%, 3.36 ± 0.26%, and 7.92 ± 0.16% in the MP, OSP, and ISP, respectively. Changes in SP-immunoreactivity depended on the pathological factor studied. The numbers of the SP-LI perikarya amounted to 7.89 ± 0.34, 5.56 ± 0.30, and 19.96 ± 0.57 in chemically-induced colitis, and 4.28 ± 0.13%, 7.18 ± 20%, and 11.62 ± 0.48% after axotomy in MP, OSP, and ISP, respectively. The both studied processes generally resulted in an increase in the number of SP-LI nerve fibers in the circular muscle and mucosal layers. The obtained results suggest that SP-LI nerve structures of the ENS may participate in various pathological processes in the porcine descending colon and exact functions of SP probably depend on the type of the pathological factor.

Inflammation- and axotomy-induced changes in cocaine- and amphetamine-regulated transcript peptide-like immunoreactive (CART-LI) nervous structures in the porcine descending colon

This study reports on changes in CART-like immunoreactive (CART-LI) nerve structures in the porcine descending colon during chemically driven inflammation and after axotomy. The distribution pattern of CART-LI nerve structures was studied using doublelabeling immunofluorescence technique in the circular muscle layer, myenteric (MP), outer submucous (OSP) and inner submucous plexuses (ISP) and also in the mucosal layer of the porcine descending colon in physiological conditions as well as under pathological factors. In the control animals, CART-LI perikarya have been shown to constitute 5.11% +/- 0.64, 4.03% +/- 1.17 and 0.05% +/- 0.04 in MP, OSP and ISP, respectively. Changes in CART-immunoreactivity depended on the pathological factor and the part of the enteric nervous system (ENS) studied. Numbers of CART-LI perikarya amounted to 2.77% +/- 0.64, 2.60% +/- 0.36 and 0.26% +/- 0.19 during chemically-induced colitis and 3.04% +/- 0.88, 2.46% +/- 0.8 and 0.43% +/- 0.09 after axotomy in MP, OSP and ISP, respectively. Both studied pathological processes also caused an increase in the number of CART-LI nerve fibers in the circular muscle as well as in the mucosal layer.

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.

Neurochemical Characterization of Zinc Transporter 3-Like Immunoreactive (ZnT3+) Neurons in the Intramural Ganglia of the Porcine Duodenum

The SLC30 family of divalent cation transporters is thought to be involved in the transport of zinc in a variety of cellular pathways. Zinc transporter 3 (ZnT3) is involved in the transport of zinc into synaptic vesicles or intracellular organelles. As the presence of ZnT3 immunoreactive neurons has recently been reported in both the central and peripheral nervous systems of the rat, the present study was aimed at disclosing the presence of a zinc-enriched neuron enteric population in the porcine duodenum to establish a preliminary insight into their neurochemical coding. Double- and triple-immunofluorescence labeling of the porcine duodenum for ZnT3 with the pan-neuronal marker (PGP 9.5), substance P, somatostatin, vasoactive intestinal peptide (VIP), nitric oxide synthase (NOS), leu-enkephalin, vesicular acetylcholine transporter (VAChT), neuropeptide Y, galanin (GAL), and calcitonin gene-related peptide were performed. Immunohistochemistry revealed that approximately 35, 43, and 48 % of all PGP9.5-postive neurons in the myenteric (MP), outer submucous (OSP), and inner submucous (ISP) plexuses, respectively, of the porcine duodenum were simultaneously ZnT3+. In the present study, ZnT3+ neurons coexpressed a broad spectrum of active substances, but co-localization patterns unique to the plexus were studied. In the ISP, all ZnT3+ neurons were VAChT positive, and the largest populations among these cells formed ZnT3+/VAChT+/GAL+ and ZnT3+/VAChT+/VIP+ cells. In the OSP and MP, the numbers of ZnT3+/VAChT+ neurons were two times smaller, and substantial subpopulations of ZnT3+ neurons in both these plexuses formed ZnT3+/NOS+ cells. The large population of ZnT3+ neurons in the porcine duodenum and a broad spectrum of active substances which co-localize with this peptide suggest that ZnT3 takes part in the regulation of various processes in the gut both in normal physiology and during pathological processes.

Changes in Cocaine- and Amphetamine-Regulated Transcript-Like Immunoreactive (CART-LI) Nerve Structures of the Porcine Descending Colon During Proliferative Enteropathy

Abstract The distribution pattern of CART- like immunoreactive (CART-LI) nerve structures was studied by a double immunofluorescence technique in the circular muscle layer, myenteric (MP), outer submucous (OSP), and inner submucous plexuses (ISP), as well as in the mucosal layer of porcine descending colon under physiological conditions and during proliferative enteropathy (PE). In control animals, CART-LI perikarya have been shown to constitute 3.18 ±0.51%, 3.44 ±0.6%, and 3.33 ±0.72% in MP, OSP, and ISP, respectively. PE caused a decrease in the number of CART - LI neurons in MP and ISP. In OSP, the observed changes were not statistically significant. During PE, the number of CART-LI perikarya amounted to 2.18 ±0.27%, 3.07 ±0.06%, and 0.07 ±0.02% within MP, OSP, and ISP, respectively. Moreover, PE caused an increase in the number of CART-LI nerve fibers in the colonic circular muscle and mucosal layers as well as in MP. This study describes for the first time changes in CART-LI nerve structures of the porcine descending colon during Lawsonia intracellularis infection, and suggests the participation of this neuropeptide in the regulation of gut functions, not only under physiological conditions, but also during pathological processes.

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.

Changes in the somatostatin (SOM)-like immunoreactivity within nervous structures of the porcine descending colon under various pathological factors

This study reports on changes in the somatostatin-like immunoreactive (SOM-LI) nerve structures of the enteric nervous system (ENS) in the porcine descending colon, caused by chemically driven inflammation, proliferative enteropathy (PE), which is a “natural” inflammation with proliferative changes and nerve injury (axotomy). The distribution pattern of SOM-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 SOM-LI perikarya have been shown to constitute 1.97 ± 0.36%, 2.06 ± 0.33% and 4.23 ± 0.40% in the MP, OSP and ISP, respectively. Changes in SOM-immunoreactivity depended on the pathological factor and the part of the ENS studied. Numbers of the SOM-LI perikarya amounted 1.81 ± 0.30, 1.97 ± 0.24 and 11.15 ± 0.95 during chemically induced colitis and 3.21 ± 0.37%, 4.33 ± 0.33% and 4.42 ± 0.32% after axotomy in MP, OSP and ISP, respectively. Moreover during PE SOM-positive cell bodies were not observed at all in MP, whereas within OSP and ISP the number of SOM-LI perikarya amounted to 3.34 ± 0.36 and 10.92 ± 059, respectively. All processes studied resulted in a decrease in the number of SOM-LI nerve fibers in the mucosal layer, whereas within the circular muscle layer chemically induced inflammation and axotomy caused an increase in the number of the SOM-LI nerve fibers contrary to PE, which reduced the number of such fibers. The obtained results suggest that SOM-LI nerve structures of the ENS may participate in various pathological states within the porcine descending colon and their functions probably depend on the type of pathological factor.

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.

Proliferative enteropathy (PE): Induced changes in galanin-like immunoreactivity in the enteric nervous system of the porcine distal colon

The aim of this study was to investigate the changes of galanin (GAL) like immonoreactivity in the porcine descending colon during proliferative entheropathy (PE). Accordingly, the distribution pattern of GAL - like immunoreactive (GAL-LI) nerve structures was studied by the immunofluorescence technique in the circular muscle layer, myenteric (MP), outer submucous (OSP) and inner submucous plexuses (ISP), as well as in the mucosal layer of the porcine descending colon under physiological conditions and during PE. In control animals GAL-LI perikarya have been shown to constitute 4.03 ± 0.1%, 6.67 ± 0.3% and 11.20 ± 0.5% in MP, OSP and ISP, respectively. PE caused changes in the GAL - like immunoreactivity, which differed in particular parts of the studied bowel segment. During PE the number of GAL-LI perikarya amounted to 2.90±0.5%, 8.42±1.0% and 21.72±1,4% within the MP, OSP and ISP, respectively. Moreover PE caused an increase in the number of GAL-LI nerve fibers in the colonic circular muscle and mucosal layers, as well as in all intramural plexuses, especially in ISP, where nearly every ganglion contained a very dense meshwork of the GAL-positive nerve fibers under the studied pathological factor. This study for the first time reports on changes in GAL-LI nerve structures of the porcine descending colon during Lawsonia intracellularis infection.

Nitric oxide synthase expression in enteric neurons during development in the pig duodenum

The expression of the constitutive neural isoform of nitric oxide synthase (bNOS) is dynamic and thus forms an ideal parameter to evaluate whether development and region affect the enteric nervous system. By applying NADPH-diaphorase histochemistry on whole-mount preparations of the myenteric and submucosal plexuses and by using the 'unbiased counting frame', a qualitative and quantitative description of bNOS-expression in enteric neurons in the pig duodenum in various developmental stage and region was obtained. Examinations were carried out on the oral and aboral duodenum of fetal pigs from the second half of gestation, of 1-2-day-old pigs and of 6-8-week-old pigs. In the pig duodenum, three enteric plexuses were readily distinguished: the inner submucous, the outer submucous and the myenteric plexuses. All three plexuses already harboured, to different degrees, bNOS-expressing neurons at midgestation. Although the enteric nervous system was present at midgestation, the enteric neurons had not yet reached their adult phenotype and morphology. During gestation, the number of inner submucous bNOS-expressing neurons increased approximately 50-fold, whereas after birth that number fell to about 10% of the prenatal value. During further postnatal development it returned to prenatal values. In addition, the number of bNOS-expressing myenteric neurons doubled postnatally. These changes favour a role for NO in mediating the development of enteric neurons and point to a greater necessity for inhibitory innervation in the adult pig as compared with the fetal pig. Furthermore, the number of bNOS-expressing outer submucosal and myenteric neurons was significantly higher in the oral duodenal segment compared with the aboral duodenal segment. This regional difference suggests that the oral duodenal segment is more prominently involved in the regulation of NO-mediated gastrointestinal processes than the aboral one. The developmentally and regionally dependent bNOS-expression can be explained by shifts and differences in the balanced system of hormones, presynaptic input and target-derived signals that affects neurotransmitter expression.