Single-Cell Transcriptomic Analysis of Rat Urethral Tissue Following Simulated Childbirth Injury and Microenergy Acoustic Pulse Therapy

Microenergy acoustic pulse (MAP) therapy has been associated with functional and structural improvement after childbirth-related urethral injury, but its multicellular effects remain incompletely defined. We explored treatment- and injury-associated differences in the urethral microenvironment using single-cell RNA sequencing. Eight female Sprague–Dawley rats were assigned to sham, vaginal balloon dilation plus ovariectomy (VBDO), VBDO plus beta-aminopropionitrile (BAPN), or VBDO plus BAPN and MAP (MAP) groups, with one 24-week-old and one 48-week-old rat per group. Urethral tissues were analyzed for cell composition, differential gene expression, Gene Ontology enrichment, stem/progenitor features, senescence-associated secretory phenotype activity, and CellChat-inferred intercellular communication. A balanced single-cell dataset comprising 23,128 cells was used for downstream comparative analyses and resolved 19 annotated cell-type/state categories. Across the injury- and age-associated experimental conditions, epithelial, vascular, stromal, immune, and muscle compartments showed distinct transcriptional patterns, whereas MAP-treated samples showed a distinct cellular distribution; a separate histological cohort showed higher urethral muscle content. MAP-treated samples also showed differences in CellChat-inferred signaling activity in selected extracellular-matrix, adhesion, vascular, developmental, and neural/neuromuscular pathways, including fibronectin 1 (FN1), collagen (COLLAGEN), angiopoietin (ANGPT), vascular endothelial growth factor (VEGF), Wingless/Integrated (WNT), Notch (NOTCH), reelin (RELN), Slit guidance ligand (SLIT), neural cell adhesion molecule (NCAM), and neurexin (NRXN). Exploratory enrichment analysis identified treatment-associated differences in translation, oxidative metabolism, stress adaptation, synaptic-organization processes, progenitor-associated transcriptional states, and senescence-related programs. The treatment-split marker manifold approximation and projection (UMAPs) are descriptive and do not provide evidence of increased muscle-cell number; the reported difference in muscle-associated staining content derives from a separate animal-level histological analysis. Overall, the findings are descriptive and hypothesis-generating and require validation in independently replicated cohorts.