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

The Influence of Antral Ulcers on Intramural Gastric Nerve Projections Supplying the Pyloric Sphincter in the Pig (Sus scrofa domestica)-Neuronal Tracing Studies.

Gastric ulcerations in the region of antrum pylori represent a serious medical problem in humans and animals. Such localization of ulcers can influence the intrinsic descending nerve supply to the pyloric sphincter. The pyloric function is precisely regulated by intrinsic and extrinsic nerves. Impaired neural regulation could result in pyloric sphincter dysfunction and gastric emptying malfunction. The aim of the study was to determine the effect of gastric antral ulcerations on the density and distribution of intramural gastric descending neurons supplying the pyloric sphincter in pigs.The experiment was performed on 2 groups of pigs: healthy gilts (n=6) and gilts with experimentally induced peptic ulcers in the region of antrum pylori (n=6). Gastric neurons supplying pyloric sphincter were labeled using the retrograde neuronal tracing technique (20μl of Fast Blue tracer injected into the pyloric sphincter muscle). After a week survival period the animals were sacrificed and the stomachs were collected. Then, the stomach wall was cross-cut into 0.5cm thick sections taken in specified intervals (section I - 1.5cm; section II - 3.5cm; section III - 5.5cm; section IV - 7.5cm) starting from the sphincter. Consecutive microscopic slices prepared from each section were analyzed under fluorescent microscope to count traced neurons. Obtained data were statistically analyzed. The total number of FB-positive perikarya observed within all studied sections significantly decreased from 903.3 ± 130.7 in control to 243.8 ± 67.3 in experimental animals. In healthy pigs 76.1 ± 6.7% of labeled neurons were observed within the section I, 23.53 ± 6.5% in section II and only occasional cells in section III. In experimental animals, as many as 93.8 ± 2.1% of labeled cells were observed within the section I and only 6.2 ± 2.2% in section II, while section III was devoid of such neurons. There were no traced perikarya in section IV observed in both groups of pigs.Obtained results revealed for the first time significant impact of antral ulcerations on intramural descending nerve pathways supplying the pyloric sphincter in pigs, animals of increasing value in biomedical research and great economic importance

Tissue sampling and cell measurements.

<p>(<b>a</b>) Picture presenting the interior surface of the "experimental animal" stomach, exposed by cutting along the greater curvature of the stomach. Ulcers are indicated by red arrows. (<b>b</b>) Microphotograph showing Haematoxylin and Eosin staining of the gastric ulcer margin. The gastric mucosa shows a defect (red ellipse with the inscription “Ulcer” and arrows) that extends into the deep muscular layers. (<b>c</b>) Diagram showing the method of tissue sampling. The tracer injection site is indicated by the violet arrows. The red vertical stripes symbolize the subsequent 0.5 cm thick transverse sections taken in specific intervals (section I—1.5 cm; section II—3.5cm; section III—5.5 cm; section IV—7.5cm) starting from the pyloric sphincter (section 0—blue vertical stripe). Subsequently, each of these sections (I, II, III, IV) was cut into 20 μm thick cryostat consecutive microscopic slices for future processing. The red circle symbolizes the localization of ulcer induced in experimental animals. (<b>d, e, f</b>) Photomicrographs showing: (<b>d</b>) bundles of PGP-immunoreactive fibers (arrowheads) and perikarya (arrows). Double arrow points to the perikaryon which was fast blue (FB) positive [the perimeter (l) and area (A) of the cell body are included]; (<b>e</b>) fast blue (FB) positive cell body (double arrow) with lines indicating its dimensions; (<b>f</b>) the image formed by merging both channels (green: PGP 9.5 and blue: FB).</p

Traced neurons observed in studied sections.

<p>(<b>a, b, c, d</b>) Microphotographs presenting the Fast Blue positive neurons (arrows) in section I (<b>a, b</b>), II (<b>c</b>) and III (<b>d</b>) obtained from the stomachs of control animals. In section I the traced neurons occurred mostly in groups of 4–7 neurons (<b>a</b>), although there were more numerous clusters, up to 12 cells observed (<b>b</b>). In section II the traced cells were observed in groups up to 5 neurons (<b>c</b>), while in section III only occasional cells were noticed (<b>d</b>). There were no traced neurons observed within section IV. (<b>e, f, g</b>) Photomicrographs showing the Fast Blue positive neurons (arrows) in section I (<b>e, f</b>), and II (<b>g</b>) obtained from the stomachs of experimental animals. In section I the majority of labeled cells was scattered within the myenteric ganglia singly or in pairs (<b>e</b>), however, occasional groups up to 4 labeled neurons were additionally noticed (<b>f</b>). In section II the traced cells were observed mostly singly (<b>g</b>). There were no labeled neurons observed within section III nor IV. (<b>h, i, j</b>) Higher magnification microphotographs showing the Fast Blue labeled perikarya (arrows) of different sizes and shapes: (<b>h</b>) medium-sized oval (single arrow) and round (double arrow), (<b>i</b>) small-sized round and (<b>j</b>) large-sized triangular in shape labeled perikarya.</p

Graph presenting percentage of FB-positive neurons in studied sections.

<p>Percentage of FB-positive neurons observed in subsequent sections (I, II, III, IV) in control (green) and experimental (orange) animals. Observed intergroup differences were statistically significant (P<0.05) in section I and II (asterisk).</p

Histogram showing the maximum number of traced neurons in studied sections.

<p>Histogram showing the maximum number of traced neurons in studied sections.</p

Cocaine and Amphetamine Regulated Transcript (CART) Expression Changes in the Stomach Wall Affected by Experimentally Induced Gastric Ulcerations

Cocaine- and amphetamine-regulated transcript (CART) is a peptide suggested to play a role in gastrointestinal tract tissue reaction to pathology. Gastric ulceration is a common disorder affecting huge number of people, and additionally, it contributes to the loss of pig livestock production. Importantly, ulceration as a focal disruption affecting deeper layers of the stomach wall differs from other gastrointestinal pathologies and should be studied individually. The pig’s gastrointestinal tract, due to its many similarities to the human counterpart, provides a valuable experimental model for studying digestive system pathologies. To date, the role of CART in gastric ulceration and the expression of the gene encoding CART in porcine gastrointestinal tube are completely unknown. Therefore, we aimed to verify the changes in the CART expression by Q-PCR (gene encoding CART in the tissue) and double immunofluorescence staining combined with confocal microscopy (CART immunofluorescence in enteric nervous system) in the porcine stomach tissues adjacent to gastric ulcerations. Surprisingly, we found that gastric ulcer caused a significant decrease in the expression of CART-encoding gene and huge reduction in the percentage of CART-immunofluorescent myenteric perikarya and neuronal fibers located within the circular muscle layer. Our results indicate a unique CART-dependent gastric response to ulcer disease

Cocaine and Amphetamine Regulated Transcript (CART) Expression Changes in the Stomach Wall Affected by Experimentally Induced Gastric Ulcerations

Cocaine- and amphetamine-regulated transcript (CART) is a peptide suggested to play a role in gastrointestinal tract tissue reaction to pathology. Gastric ulceration is a common disorder affecting huge number of people, and additionally, it contributes to the loss of pig livestock production. Importantly, ulceration as a focal disruption affecting deeper layers of the stomach wall differs from other gastrointestinal pathologies and should be studied individually. The pig’s gastrointestinal tract, due to its many similarities to the human counterpart, provides a valuable experimental model for studying digestive system pathologies. To date, the role of CART in gastric ulceration and the expression of the gene encoding CART in porcine gastrointestinal tube are completely unknown. Therefore, we aimed to verify the changes in the CART expression by Q-PCR (gene encoding CART in the tissue) and double immunofluorescence staining combined with confocal microscopy (CART immunofluorescence in enteric nervous system) in the porcine stomach tissues adjacent to gastric ulcerations. Surprisingly, we found that gastric ulcer caused a significant decrease in the expression of CART-encoding gene and huge reduction in the percentage of CART-immunofluorescent myenteric perikarya and neuronal fibers located within the circular muscle layer. Our results indicate a unique CART-dependent gastric response to ulcer disease.</jats:p