The etiology of overactive bladder (OAB) is highly complex. Previous studies suggest that altered activity in brain regions may be one of the pathogenic mechanisms underlying OAB [1]. However, these functional neuroimaging findings require further validation through animal experiments to strengthen the robustness of evidence and provide an experimental foundation for screening potential novel therapeutic targets in future research. By establishing an OAB mouse model and employing immunofluorescence staining and neural circuit tracing techniques, we identified brain regions potentially associated with the regulation of bladder function. This provides a novel potential therapeutic target for future OAB treatment.

OAB models were established via intravesical infusion of either 1% v/v acetic acid [2] or 100 μM PGE2 [3]. Two hours post-stimulation, brains were harvested and processed for c-Fos immunostaining (anti-c-Fos antibody, ab208942, Abcam) to identify activated neural populations.To map neural circuits controlling bladder function, we performed multipoint intramural injections of PRV-CAG-EGFP (1 μL in total, distributed across anterior/posterior/lateral walls and bladder dome) in C57BL/6 mouse bladders. Following viral expression, brain tissues were cryosectioned and analyzed by fluorescence microscopy.

In OAB models induced by intravesical 1% acetic acid or 100 μM PGE2, c-Fos immunostaining revealed functional activation in the pontine micturition center (PMC), locus coeruleus (LC), periaqueductal gray (PAG), paraventricular thalamic nucleus (PVT), primary somatosensory/motor cortices (S1/M1), bed nucleus of the stria terminalis (BNST) and prefrontal cortex. Pseudorabies virus  retrograde tracing identified structural connectivity between the bladder and discrete CNS regions, including the PMC, LC, PAG, red nucleus, lateral hypothalamus (LH), paraventricular nucleus (PVN), S1, and M1.

Combining c-Fos immunostaining in OAB models with retrograde viral tracing results, we demonstrate that the PMC, LC, PAG, S1, and M1 not only maintain direct structural connections with the bladder but also exhibit significant neural activation during OAB states.   Some regions (e.g. the prefrontal cortex, BNST) were not identified by retrograde viral tracing, which is potentially due to technical limitations in transsynaptic propagation efficiency. Overall, our result confirmed the abnormal activation of PMC, LC, PAG, S1, and M1 regions in OAB animal models, and these regions may form interconnected neural circuits that potentially serve as a fundamental mechanism for regulating bladder function.

Brain regions such as PMC, LC, PAG, S1, and M1 were abnormally activated in OAB mice, while retrograde viral tracing confirmed these regions were anatomically connected to the bladder. These results implicated a potential neural circuit for the regulation of micturition.

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