We hypothesize that early post-traumatic events in the bladder following spinal cord injury (SCI) are critical in defining long-term bladder function and structure. This study aims to characterize the immediate molecular and functional changes in the bladder post-SCI and to identify potential early therapeutic windows before irreversible damage occurs.

Using a mouse model of SCI, we performed a longitudinal study over 8 weeks. We focused particularly on the early response phase (within 72 hours post-injury) and its progression over time. DNA damage was assessed using the Comet assay. Reactive oxygen species (ROS) levels were measured by flow cytometry. Immunofluorescence staining was used to assess the expression and localization of Pi16 (vascular leakage marker), CD31 (endothelial cells), LIVE/DEAD signals, and podoplanin (lymphatic endothelial marker). Histological evaluation was performed using H&E staining. We also calculated the bladder wet-to-dry weight ratio to quantify edema. Urodynamic recordings were conducted to evaluate bladder function. Inosine was administered post-SCI to evaluate its potential protective effect on bladder pathology.

Within the first 72 hours post-SCI, we observed a rapid increase in pro-inflammatory cytokines, accompanied by significant bladder edema and structural disorganization. Flow cytometry confirmed increased ROS levels in fibroblast and podoplanin positive cells (possibly SMCs), likely due to disrupted ATP synthesis from impaired contractility. The Comet assay revealed early DNA damage in bladder tissues. Pi16 expression was markedly downregulated, consistent with vascular and lymphatic leakage, and bladder edema was supported by an increased bladder-to-body weight ratio.
By one week post-SCI, Pi16 expression and edema resolved; however, this phase marked an onset of robust immune cell infiltration and activation, as shown by histological and immunofluorescent analysis. This second phase was characterized by structural remodeling of the bladder wall. Functional analysis via urodynamics revealed progressive bladder dysfunction, including detrusor overactivity and loss of coordinated voiding. Inosine treatment did not affect the acute phase but showed protective effects from the first week onward. Notably, it downregulated NF-κB activity and limited subsequent inflammatory remodeling.

SCI triggers a biphasic response in the bladder: an acute injury phase with oxidative stress, DNA damage, and vascular leakage, followed by a secondary immune-mediated remodeling phase. Pi16 serves as a useful early biomarker of vascular leakage and recovery. The protective effect of inosine is delayed, suggesting it acts on immune-driven processes rather than acute injury signals. Importantly, interventions targeting the early post-injury window may prevent the transition to chronic dysfunction.

Early events within 72 hours after SCI play a pivotal role in determining the trajectory of bladder injury and recovery. These findings highlight a critical therapeutic window to prevent long-term bladder dysfunction. Targeting oxidative stress and vascular instability may offer new strategies to preserve bladder function following SCI.