Fig 1.
Diagram presenting the method of tissue sampling in the tracing (T), histochemical (H) and molecular (M) animal subgroups. The markings are applied in the picture presenting the interior surface of the "experimental animal" stomach which was cut along the greater curvature. Ulcers are indicated by red circles. Photomicrograph presenting the transverse section of the deeply penetrating stomach ulcer labelled with HE technique is shown in the red frame. Tissues containing Fast Blue (FB) traced perikarya were collected from the gastric antrum (blue frame) of the tracing subgroup pigs (T) and were cut into 20 μm thick cryostat consecutive microscopic sections. Tissues of the pyloric orifice wall (green frame) were collected from the histochemical subgroup of animals (H) and were cut into 20 μm thick cryostat microscopic sections. Tissues for Real-Time PCR were cut out bilaterally (about 0.5 cm from the torus pylori) from the pyloric orifice wall (violet circles with letter M) from the animals of the molecular subgroup (M). The circular-shaped samples, having a diameter of 1 cm, were cut transversally to the stomach wall by the use of a round cutter and comprised all layers of the pyloric orifice wall (violet frame).
Table 1.
Sequences of primers used in Real-Time PCR.
Fig 2.
Localization of the myenteric plexus ganglia (containing Gal-immunoreactive perikarya) within the deep layers of the pyloric circular muscles.
Set of microphotographs at different magnifications of the pyloric wall cross-section comprising myenteric plexus ganglion with PGP 9.5/Gal-immunoreactive neurons. The section was taken from the experimental pig of the subgroup H and double-immunolabeled with antibodies against PGP 9.5 (b, c) and galanin (b’, c’) [pictures (a, a’, b”, c”) present overlap of both fluorescence channels]. Low magnification picture (a, red frame) shows a myenteric plexus ganglion localized deep within the pyloric circular muscle layers [cm]. Medium magnification picture (a’) of the selected area [dotted line boarder from the picture (a)] presents Gal-immunofluorescent perikarya (arrows). High magnification pictures (b, b’, b’, c, c’, c”) show medium (b, b’, b”) and large (c, c’, c”) in a diameter PGP9.5/Gal-immunoreactive perikarya (arrows). The characteristic pattern of Gal-immunoreactivity observed in the neurons (b’, c’) blurred the outlines of the perikarya, precluding accurate measurements of the cell bodies using red channel [Gal immunostaining]. Thus, all the measurements of Gal-immunoreactive nerve cells were performed using the green channel [PGP 9.5 staining (b, c)]. Scale bars are included in the pictures.
Fig 3.
Typical characteristics (shapes, immunofluorescence) of Gal-immunoreactive perikarya.
Set of photomicrographs showing shapes and patterns of immunofluorescence observed in the typical Gal-positive (single arrows) myenteric (a, a’, b’ b’, c, c’, c”) and submucosal (d, d’, e, e’) perikarya of the pyloric orifice wall in the control (a, a’, d, d’) and experimental (b, b’, c, c’, c”, e, e’) pigs of the subgroup H. In both groups of the animals most of the myenteric neurocytes immunoreactive to galanin (arrows) were round (a, c’) or oval (b, c’) and expressed medium to strong immunoreactivity. In the experimental animals (b, b’, c, c’, c”) some of Gal-positive perikarya (double arrows) seemed to express weak immunofluorescence and/or were larger in a diameter. Most of the submucosal neurocytes immunoreactive to Gal (arrows) in the control (d, d’) and experimental (e, e’) animals were oval and measured about 19.45 ± 0.65 x 11.33 ± 0.38 μm. Scale bars are included in the pictures.
Fig 4.
Double immunolabeled (PGP 9.5 and Gal) perikarya of the pyloric orifice wall.
Set of microphotographs showing sections of the pyloric orifice wall taken from the control and experimental pigs of the subgroup H and double-immunolabeled with antibodies against PGP 9.5 (a, b, c, d) and galanin (a’, b’, c’, d’). Some of the myenteric plexus perikarya (arrows) of the control (a, a’, a”) and experimental (b, b’, b”) animals simultaneously co-expressed immunoreactivity to PGP 9.5 (a, b) and galanin (a’, b’). The number of PGP 9.5+/Gal+ neurons was higher in the experimental animals and the difference was statistically significant. Some of the submucosal neurons (arrows) of the control (c, c’, c”) and experimental (d, d’, d”) animals simultaneously co-expressed immunoreactivity to PGP 9.5 (c, d) and galanin (c’, d’), and these percentages did not differ significantly between both groups of animals. Pictures (a”, b”, c”, d”) show the overlap of both fluorescence channels (PGP 9.5—green, Gal—red). Scale bars are included in the pictures.
Fig 5.
Percentages of myenteric and submucosal PGP+/Gal+ perikarya in the pyloric wall tissue.
Graph showing percentages of the myenteric and submucosal PGP+/Gal+ perikarya in the pyloric wall samples collected from the control and experimental animals of the subgroup H. Statistically significant differences between the control and experimental animals are marked by asterisks, ** P ≤ 0.005.
Fig 6.
Differences between the percentages of myenteric and submucosal PGP+/Gal+ perikarya in the pyloric wall tissue.
Graph presenting differences between the percentages of myenteric and submucosal PGP+/Gal+ perikarya in the control and experimental pigs of the subgroup H. The number of galaninergic submucosal neurons was significantly higher in both groups of animals. Statistically significant differences between numbers of myenteric and submucosal neurons are marked by asterisks, **** P ≤ 0.0001.
Fig 7.
FB-positive neurons of the stomach antrum double-immunostained for PGP 9.5 and Gal.
Set of microphotographs showing stomach antrum sections with FB-positive neurons (a, e) from the animals of subgroup T and double-immunostained with antibodies against PGP 9.5 (b, f) and galanin (c, g). Some of FB-positive perikarya (solid arrows) in the control (a) and experimental (e) animals simultaneously co-expressed immunoreactivity to PGP 9.5 (b, f) and galanin (c, g), while the other traced neuronal somata (empty arrows) were devoid of galanin immunoreactivity. Differences in the number of FB+/PGP 9.5+/Gal+ neurons did not differ significantly between both groups of the animals. Pictures (d, h) show the overlap of all three fluorescence channels (FB—blue, PGP 9.5—green, Gal—red). Scale bars are included in the pictures.
Fig 8.
Percentages of FB+/PGP+/Gal+ perikarya in the myenteric plexus of the stomach antrum.
Graph presenting percentages of FB-positive neurons supplying the pyloric sphincter in the control and experimental animals of the subgroup T which simultaneously co-expressed immunoreactivity to PGP 9.5 and Gal. The differences between the control and experimental animals were statistically insignificant.
Fig 9.
Expression of mRNA encoding Gal, GalR1, GalR2, GalR3 in the pyloric wall tissue.
Expression of Gal (a), GalR1 (b), GalR2 (c) and GalR3 (d) mRNA in the pyloric wall tissue collected from the control and experimental animals of subgroup M. Levels of Gal, GalR1, GalR2, GalR3 mRNA were measured by Real-Time PCR. The data obtained from each sample were normalized to GAPDH. Relative quantities (RQ) of mRNA were analysed using the comparative Ct method. Each cDNA sample was amplified in triplicate and all data are expressed as the mean ± S.E.M., **** P ≤ 0.0001; ** P ≤ 0.005 (vs the control animals).