Neurogenic lower urinary tract dysfunction (NLUTD) due to spinal cord injury (SCI) is a condition that affects both storage and voiding function. In human SCI patients, the normal micturition reflex is initially eliminated in the acute phase and then slowly recovers by time.  In chronic phase, the main functional problems inducing storage and voiding dysfunctions include detrusor overactivity (DO) and detrusor sphincter dyssynergia (DSD) that causes disruption of coordinating activity of the bladder and the external urethral sphincter (EUS), resulting in inefficient voiding with increased residual urine.  Animal models of SCI have been used to study these conditions; however, the adequate timing of evaluation of NLUTD after SCI in mouse models has not been well explored. Thus, this study evaluated the time-course changes of bladder and EUS activity as well as mechanosensitive channels in L6-S1 dorsal root ganglia (DRG), which contain bladder and EUS afferent neurons.

All experiments were conducted in accordance with institutional guidelines and was approved by the institutional animal care and use committee. Fifteen 8 to 9 weeks old female C57BL/6N mice were used and divided into 3 groups; (1) 2-weeks post-SCI, (2) 4-weeks post-SCI, and (3) 6-weeks post-SCI groups. All mice underwent complete transection of the Th8/9 spinal cord under isoflurane anesthesia. Thereafter, the bladder was emptied by perineal stimulation and bladder compression daily until evaluation. At the time of evaluation at 2-, 4- and 6-weeks after SCI, the mice in each group underwent single-filling cystometry (CMG) and EUS electromyogram (EMG) recordings under an awake condition in the method previously described (ref). The lower abdomen was opened, and the bladder dome was punctured with an 18-gauge needle for insertion of a PE-50 tube with the end flared by heat, which was then tied by a 6-0 silk thread suture. The abdomen wound was then closed, and the mice were gently restrained in a cage. First, urodynamic parameters of CMG were evaluated without EUS-EMG recordings in an awake condition.  After recovery from anesthesia, the PE-50 tube was connected to a three-way stopcock which was connected to a pressure transducer and to a syringe pump. CMG was recorded during saline filling into the bladder at a rate of 0.01ml/min. After the bladder activity became stable, the bladder was emptied and was evaluated for 3 times after bladder emptying each time. In the single-filling CMG recordings, the number of non-voiding contractions (NVC), which was determined as bladder pressure elevation of 8mmHg or higher above the baseline bladder pressure was measured.  Also, post-void residual volume (RU) was measured by withdrawing saline from the bladder by gravity, and voiding efficiency (VE) was calculated. After single-filling CMG measurement, coordinating activities of the bladder and EUS during the voiding phase were evaluated using simultaneous recordings of CMG and EUS-EMG. The SCI mice were again anesthetized, and epoxy-coated stainless-steel wire electrodes were placed into the EUS percutaneously using a 30-gauge needle, and the EUS-EMG activity was recorded under an awake condition after recovery from anesthesia. In the EUS-EMG recordings, voiding contraction time, reduced EMG activity time and the ratio of reduced EMG activity time to voiding contraction time were calculated to evaluate DSD. After evaluation, L6-S1 DRG were harvested to measure mRNA expression of TRP channels and mechanosensitive ion channels such as ASICs and Piezo2 by RT-PCR. For the molecular study, spinal intact mice were added as controls.

In SCI mice, non-voiding contractions (NVC) during bladder filling were confirmed at 2 weeks post-SCI and did not increase over time to 6 weeks (Fig. 1A). In EUS-EMG measurements, DSD was observed at 2 weeks, but periodic EMG reductions during bladder contraction, resulting in urination, were not observed in most 2-weeks SCI mice, thereby leading to urinary retention (Fig. 1A).  At 4 weeks, SCI mice showed increases of EMG activity reduction time with increased VE.  At 6 weeks, SCI mice exhibited further increases in bladder capacity, residual volume and EMG reduction time compared to 2-weeks or 4-weeks SCI groups without significant changes in VE compared to 4 weeks SCI mice (Fig. 1A).  RT-PCR of L6-S1 DRG showed increased mRNA levels of TRPV1 and ASIC1-3 in SCI mice compared to spinal intact mice, with a later decrease of ASIC2 and 3 at 6 weeks compared to 4-weeks SCI mice whereas Piezo2 showed a slow increase at 6 weeks of SCI compared to 4-weeks SCI mice (Fig. 1B).

These results indicate that; (1) DO evident as NVC is established in the early phase (2weeks) whereas DSD is completed later at 4weeks with a slight improvement evident as increased EMG reduction time at 6 weeks post-SCI, and (2) ASIC2/3 and Piezo2 mechanosensitive channels expressed in L6-S1 DRG, which contain bladder and EUS afferent neurons, could be involved in the progression of DSD in early (2-4 weeks) and late phases (4-6 weeks) of SCI, respectively.

Differences in time-dependent progression of NLUTD such as DO and DSD are identified in the mouse model of SCI.  Also, different types of mechanosensitive channels such as ASICs and Piezo2 could contribute at different time points to the establishment of DSD after SCI.  These data would help us to understand the progression mechanisms of SCI-induced NLUTD.

Kadekawa K et al. The role of capsaicin-sensitive C-fiber afferent pathways in the control of micturition in spinal-intact and spinal cord-injured mice. Am J Physio Renal Physiol (2017); 313: F796-F804