Immunohistochemical characteristics and distribution of neurons in the intramural ganglia supplying the urinary bladder in the male pig

This study investigated the distribution and chemical coding of neurons in intramural ganglia of the urinary bladder trigone (UBT-IG) and cervix (UBC-IG) in the male pig using combined retrograde tracing and double-labelling immunohistochemistry. Additionally, immunoblotting was used to confirm the presence of marker enzymes for main populations of autonomic neurons. Retrograde fluorescent tracer Fast Blue (FB) was injected into the wall of both the left and right side of the bladder trigone, cervix and apex during laparotomy performed under thiopental anaesthesia. Twelve gm-thick cryostat sections were processed for double-labelling immunofluorescence with antibodies against tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), neuropeptide Y (NPY), somatostatin (SUM), galanin (GAL), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), calcitonin gene-related peptide (CGRP), substance P (SP) and vesicular acetylcholine transporter (VAChT). UBT-IG and UBC-IG neurons in both parts of the organ formed characteristic clusters (from few to tens of neuronal cells) found under visceral peritoneum or in the outer muscular layer. Immunohistochemistry revealed several subpopulations in UBT-IG and UBC-IG neurons, namely noradrenergic (ca. 76% and 76%), cholinergic (ca. 22% and 20%), non-adrenergic/non-cholinergic nerve cells (ca. 1.5% and 3.8%), NPY- (ca. 66% and 58%), SOM- (ca. 39% and 39 %), VIP- (ca. 5% and 0%) and NOS- immunoreactive (IR) (ca. 1.5% and 3.8%), respectively. Immunoblotting using antibodies to TH and VAChT showed the presence of studied proteins as revealed by the presence of protein bands of the correct molecular weight. This study has revealed a relatively large population of differently coded UBT- and UBC- IG neurons, which constitute an important element of the complex neuroendocrine system involved in the regulation of the male urogenital organs function.