Distribution and chemical coding of urinary bladder apex-projecting neurons in aorticorenal and testicular ganglia of the male pig.

Combined retrograde tracing and double-labelling immunofluorescence were used to investigate the distribution and chemical coding of neurons in aorticoerenal (ARG) and testicular (TG) ganglia supplying the urinary bladder apex (UBA) in the juvenile male pig (n=4, 12 kg. body weight). Retrograde fluorescent tracer Fast Blue (FB) was injected into the wall of the bladder apex under pentobarbital anesthesia. After three weeks all the pigs were deeply anesthetized and transcardially perfused with 4% buffered paraformaldehyde. TG and ARG were collected and processed for double-labelling immunofluorescence. The presence of tyrosine hydroxylase (TH) or dopamine beta-hydroxylase (DBH), neuropeptide Y (NPY), somatostatin (SOM), galanin (GAL), nitric oxide synthase (NOS) and vesicular acetylcholine transporter (VAChT) were investigated. The cryostat sections were examined with a Zeiss LSM 710 confocal microscope equipped with adequate filter blocks. The TG and ARG were found to contain many FB-positive neurons projecting to the UBA (UBA-PN). The UBA-PN were distributed in both TG and ARG. The majority were found in the left ganglia, mostly in TG. Immunohistochemistry disclosed that the vast majority of UBA-PN were noradrenergic (TH- and/or DBH-positive). Many noradrenergic neurons also contained immunoreactivity to NPY, SOM or GAL. Most of the UBA-PN were supplied with varicose VAChT-, or NOS- IR (immunoreactive) nerve fibres. This study has revealed a relatively large population of differently coded ARG and TG neurons projecting to the porcine urinary bladder. As judged from their neurochemical organization these nerve cells constitute an important element of the complex neuro-endocrine system involved in the regulation of the porcine urogenital organ function.

Enteric nervous system in the European beaver (Castor fiber) pylorus - an immunohistochemical study.

European beaver (Castor fiber), the largest rodent species inhabiting a wide area of Eurasia, feeds mainly on dry parts of plants, bark or wood. Such kind of nourishment needs to be properly digested in each part of the gastrointestinal tract. The time of stomach digestion, which directly influences all the following steps of the digestion process, is precisely controlled by the pylorus and its innervation. However, virtually no data is available on the organization of the enteric nervous system in most of the wild animal species, including beavers. On the other hand, a pecu- liar diet consumed by beavers, suggests that the arrangement of their stomach intramural nerve elements can be atypical. Therefore, the present study investigated the distribution and chemical coding of neurons and nerve fibers in the pylorus of the European beaver. The experiment was performed on stomachs obtained from a group of 6 beavers caught in Northeastern region of Poland (due to beaver overpopulation). Pyloric wall tissue cryosections were double immunostained with a mixture of antibodies against pan-neuronal marker PGP 9.5 (to visualize enteric neurons) and ChAT (cholinergic marker), nNOS (nitrergic marker), SP, CGRP, Gal (peptidergic markers). Confocal microscopy analysis revealed that the majority of enteric nerve cells were clustered forming submucosal and myenteric ganglia and all the studied substances were expressed (in various amounts) in these neurons. We conclude, that the anatomical arrangement and chemical coding of intramural nerve elements in the beaver pylorus resemble those found in other mammalian species.

Galaninergic intramural nerve and tissue reaction to antral ulcerations.

BACKGROUND: Well-developed galaninergic gastric intramural nerve system is known to regulate multiple stomach functions in physiological and pathological conditions. Stomach ulcer, a disorder commonly occurring in humans and animals, is accompanied by inflammatory reaction. Inflammation can cause intramural neurons to change their neurochemical profile. Galanin and its receptors are involved in inflammation of many organs, however, their direct participation in stomach reaction to ulcer is not known. Therefore, the aim of the study was to investigate adaptive changes in the chemical coding of galaninergic intramural neurons and mRNA expression encoding Gal, GalR1, GalR2, GalR3 receptors in the region of the porcine stomach directly adjacent to the ulcer location. METHODS: The experiment was performed on 24 pigs, divided into control and experimental groups. In 12 experimental animals, stomach antrum ulcers were experimentally induced by submucosal injection of acetic acid solution. Stomach wall directly adjacent to the ulcer was examined by: (1) double immunohistochemistry-to verify the changes in the number of galaninergic neurons (submucosal, myenteric) and fibers; (2) real-time PCR to verify changes in mRNA expression encoding galanin, GalR1, GalR2, GalR3 receptors. KEY RESULTS: In the experimental animals, the number of Gal-immunoreactive submucosal perikarya was increased, while the number of galaninergic myenteric neurons and fibers (in all the stomach wall layers) remained unchanged. The expression of mRNA encoding all galanin receptors was increased. CONCLUSIONS & INTERFERENCES: The results obtained unveiled the participation of galanin and galanin receptors in the stomach tissue response to antral ulcerations.

Immunohistochemical Characterization of Sympathetic Chain Ganglia (SChG) Neurons Supplying the Porcine mammary Gland.

The aim of this study was to investigate the chemical coding of mammary gland-projecting SChG neurons using double-labelling immunohistochemistry. Earlier observation showed that after injection of the retrograde tracer fast blue (FB) into the second, right thoracic mamma, FB+ mammary gland-projecting neurons were found in Th1-3, Th9-14 and L1-4 right SChG. The greatest number of FB+ nerve cell bodies was observed in Th10 (approx. 843) and Th11 (approx. 567). Neurons projecting to the last right abdominal mamma were found in L1-4 SChG. The greatest number of FB+ neurons was observed in L2 (approx. 1200). Immunohistochemistry revealed that the vast majority of FB+ mammary-projecting neurons contained immunoreactivities to TH (96.97%) and/or DßH (95.92%). Many TH/DßH-positive neurons stained for SOM (41.5%) or NPY (33.2%), and less numerous nerve cells expressed VIP (16.9%). This observation strongly corresponds to the results of previous studies concerning the immunohistochemical characterization of nerve fibres supplying the porcine mammary gland.

Vagal projections to the pylorus in the domestic pig (Sus scrofa domestica).

The goal of the present study was to examine the precise localization of the brainstem motor and primary sensory (nodose ganglion) vagal perikarya supplying the pylorus in the domestic pig. Using the Fast Blue retrograde tracing technique it has been established that all the vagal motor neurons projecting to the pylorus (about 337 ± 59 cells per animal) were localized bilaterally in the dorsal motor nucleus of the vagus nerve (DMX, 171 - left; 167 - right) and all other regions of the porcine brainstem were devoid of labeled neurons. The vagal perikarya supplying the porcine pylorus were dispersed throughout the whole rostro-caudal extent of the DMX and no somatotopic organization of these neurons was observed. The labeled neurons occurred individually or in groups up to five cell bodies per nuclear transverse cross section area (in the middle part of the nucleus). An immunocytochemical staining procedure disclosed that all Fast Blue labeled motor neurons were choline acetyltransferase (ChAT) immunoreactive, however some differences in immunofluorescence intensity occurred. The primary sensory vagal neurons were observed within the left (215±37 cells/animal) and right (148±21 cells/animal) nodose ganglion. The traced neurons were dispersed throughout the ganglia and no characteristic arrangement of these neurons was observed. The present experiment precisely indicates the sources of origin of the vagal motor and primary sensory neurons supplying the pyloric region in the pig, the animal of an increasing significance in biomedical research.

Nitric oxide in the bovine oviduct: influence on contractile activity and nitric oxide synthase isoforms localization.

The oviducts of 64 Holstein cows in luteal (early I, early II and late) and follicular phases were evaluated to determine the protein expression and mRNA transcription of different nitric oxide synthase isoforms (eNOS, iNOS, nNOS) as well as the effect of nitric oxide (NO) on spontaneous contractility in vitro. The expression patterns of nitric oxide synthase (NOS) isoforms in isthmus and ampulla (n = 6 for each phase) were determined by immunohistochemistry, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. In the contractility studies, longitudinal and circular isolated strips of isthmus and ampulla (n = 10 for each phase) of oviducts located ipsilateral to the luteal structure or preovulatory follicle were treated as follows: a) L-arginine, an endogenous NO donor (10(-8) to 10(-3)m), b) N(ω)-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor (10(-5)m) and L-arginine (10(-3)m), c) methylene blue (MB), an inhibitor of soluble guanylate (10(-5)m) and L-arginine (10(-3)m) and d) sodium nitroprusside (SNP), an exogenous NO donor (10(-8) to 10(-4)m). Immunohistochemical evaluation revealed that endothelial NOS (eNOS) expression detected in epithelial layer of isthmus and ampulla was strong in early I luteal phase, moderate in follicular phase and weak in other phases. Neuronal NOS (nNOS) immunoreactivity was strong in isthmus and moderate in ampulla, and staining of nerve fibers was observed mostly in early I luteal and follicular phases. All eNOS, nNOS and inducible NOS (iNOS) isoforms were detected by RT-PCR. eNOS and iNOS proteins were evident, whereas nNOS was undetectable by Western blot analysis in the tissue examined. L-arginine applied alone or after L-NAME did not alter or increase the contractile tension of the strips in most tissues examined. However, L-arginine applied after MB increased contractile tension in the strips of ampulla and longitudinal isthmus from early I luteal phase and circular isthmus from follicular phase but decreased it in isthmus from early II luteal phase. SNP differentially modulated oviductal contraction depending on the type of muscular strips and period examined. These results showed the estrous phase-dependent changes related to endogenous NO system which might be of physiological importance to the oviduct for secretory and ciliary functions involved in gametes and embryo(s) transportation.

Evidence for coexistence of choline acetyltransferase (ChAT)- and calcitonin gene-related peptide (CGRP)-immunoreactivity in the thoracolumbar and sacral spinal cord neurons of the pig.

Previous immunocytochemical studies provided conflicting data concerning occurrence of the CGRP-immunoreactive neuronal cell bodies in the porcine spinal cord. In the present study, we have investigated expression of the CGRP and its possible coexpression with ChAT in the gray matter of the thoracic, lumbar and sacral spinal cord of the pig. Our study revealed a large number of CGRP-immunoreactive cells in the motor nucleus of the ventral horn, and less and singe perikarya intermediolateral and intermediomedial nuclei, respectively. Double staining immunocytochemistry, depending on the cross-section level, disclosed the highest ChAT/CGRP colocalization subsequently in the motor nucleus of the ventral horn, then in the intermediolateral and intermediomedial nucleus. Our data provide morphological evidence confirming expression of CGRP in the porcine spinal nuclei while its coexpression in cholinergic neurons suggests that CGRP may play a role in modulation of the spinal cholinergic transmission.

Re-examination of the topographical localization of facial nucleus in the pig.

Previous publications have provided different descriptions of the topographical organization of the facial nucleus of the pig. Since swine is used in biomedical research due to its embryological, anatomical and physiological similarities to human, we have reinvestigated the anatomical organization of the facial nucleus with application of fluorescent retrograde tracer Fast Blue, antibody to choline acetyltransferase and acetylcholinesterase histochemistry. Our findings demonstrate that in the porcine medulla facial motoneurons constitute a large cellular group occupying the ventro-lateral medulla. The neuronal group is interposed rostro-caudally between the superior and inferior olive, and located ventro-medially to the spinal nucleus of the trigeminal nerve. The present results clarify the anatomical description of this important brain stem nucleus in the pig.