Regional differences in nitrergic neuronal density in the developing porcine urinary bladder

Nitric oxide (NO) is involved in normal bladder physiology by regulating local arteriolar tone and smooth muscle relaxation and modulating the production of extracellular matrix proteins in vitro. Little information is available regarding the nitrergic innervation of the bladder during development. In this study we investigated the changes in density and morphology of the intramural nitrergic neurons of the porcine urinary bladder during development using whole-mount preparation. Bladder specimens were obtained from porcine foetuses of gestational age 60 days (n=5) and 90 days (n=5) and from newborn piglets (n=5) after perfusion fixation. Bladders were divided into base, body, and dome. Whole-mount preparation using NADPH-diaphorase (NADPH-d) histochemistry was used to visualize nitrergic innervation of the urinary bladders and to measure density of NADPH-positive ganglia (including single neurons), number of NADPH-d positive neurons per ganglion, and size of individual neurons. One-way ANOVA and chi-square tests were used for statistical analysis with a p-value <0.05 considered statistically significant. NADPH-d positive ganglia were numerous in the muscular layer of all three age groups. At E60, ganglion density was significantly higher in the body (mean 880/cm(2)) than in the dome (397/cm(2)) or the base (676/cm(2)). The ganglion density significantly decreased with age. The number of NADPH-d positive neurons per ganglion increased significantly between E90 and birth (p<0.01). A marked increase in the size of individual neurons over time was also seen (p<0.001), predominantly due to an increase in cytoplasm. Our data on whole-mount preparations demonstrate that significant maturation in nitrergic neuronal density and morphology occurs in the porcine urinary bladder, at least until birth.