Chronic spinal cord injury (SCI) rostral to the lumbosacral level causes detrusor overactivity (DO) during the storage phase, which is mediated by spinal reflexes triggered by hyperexcitable C-fibre afferent pathways. In normal condition, Aδ-fibre bladder afferents are involved in micturition reflexes, while in chronic SCI condition, excitability of C-fibre bladder afferents is increased, therefore inducing neurogenic DO evident as non-voiding bladder contractions (NVCs) before micturition in rodent models of SCI [1]. It is also known that the second messenger signalling pathways activated by nerve growth factor (NGF) utilize p38 Mitogen-Activated Protein Kinase (p38 MAPK) [2], which is a serine-threonine kinase that is activated by phosphorylation and mediates cellular responses to a variety of chemical and physical insults [3]. However, it has not been clarified whether p38 MAPK activation contributes to the changes in electrophysiological properties of bladder afferent pathways following SCI.  Therefore, in the present study, we examined effects of a p38 MAPK inhibitor on DO and electrophysiological properties of capsaicin sensitive bladder afferent neurons with SCI mice.

Female C57BL/6 mice (8-10 weeks old, 18-22 g) were used. These mice were divided into 3 groups: spinal intact group (SI, n=22), spinal cord injury group (SCI, n=20), and SCI group treated with p38 MAPK inhibitor group (SCI + p38 MAPK inhibitor, n=15). In the mice SCI and SCI + p38 MAPK inhibitor groups, the Th8/9 spinal cord was completely transected under isoflurane anesthesia. In SI group, sham operation was performed. After spinal cord transection, their bladders were manually squeezed to eliminate urine once daily for 4 weeks until cystometric and electrophysiological evaluation. Two weeks after the spinal cord transection, an intrathecal catheter was positioned at the level of the L6-S1 spinal cord via an incision in the dura at the L1 vertebra under isoflurane anesthesia. An osmotic pump was connected to the intrathecal catheter and replaced subcutaneous space in the back. SB203580 (p38 MAPK inhibitor) (1 mg/ml) was continuously supplied intrathecally at infusion rate of 0.51 μl/hr for 2 weeks. Instead of SB203580, artificial cerebrospinal fluid was supplied in separate groups of SI and SCI mice as controls. Awake cystometry was performed to evaluate the bladder activity. To label the population of DRG neurons innervating the bladder, a retrograde fluorescent tracer Fast Blue (FB; 1.8% weight per volume) was injected into the bladder wall under isoflurane anesthesia.  Seven days after the FB injection (=4 weeks of SCI), DRG neurons were enzymatically dissociated and incubated at 37.0 °C in 5% CO2 per 95% air overnight before patch clamp experiments. Whole cell recordings were performed to evaluate the characteristics of action potentials and isolated voltage-gated potassium (Kv) currents in capsaicin-sensitive, FB-labeled C-fibre bladder afferent neurons. Two major types of Kv currents expressed in small-sized DRG neurons, namely slow decaying A-type K+ (slow KA) and sustained delayed rectifier-type K+ (sustained KDR) currents were evaluated. In these neurons; slow KA currents are activated by depolarizing voltage steps from hyperpolarized membrane potentials and inactivated when the membrane potential is maintained at a depolarized level more than -40 mV, therefore, slow KA currents are estimated by the difference in these currents activated by depolarizing voltage pulses from a holding potential (HP) of -40 mV and from a HP of -120 mV.

In cystometry, NVCs during bladder filling were significantly reduced in SCI + p38 MAPK inhibitor group compared to SCI group (Figure). In patch-clamp recordings, the threshold for eliciting action potentials was significantly reduced in SCI group compared to SI group. Also, the number of action potentials during 800 ms membrane depolarization in SCI group was significantly increased compared to SI group. Densities of slow KA and sustained KDR currents evoked by depolarization to 0 mV in capsaicin sensitive bladder afferent neurons in SCI group were significantly lower than those measured in SI groups. SB203580 treatment significantly reversed SCI-induced changes of the threshold, the number of action potentials and the density of slow KA current, but not KDR (Table).

We indicated that (1) SCI mice exhibit DO evident as NVCs, (2) capsaicin-sensitive bladder afferent neurons from SCI mice showed hyperexcitability, evident as decreased spike thresholds and increased firing rate of action potentials compared to neurons from SI mice, (2) slow KA and sustained KDR current densities of capsaicin-sensitive bladder afferent neurons from SCI mice were decreased compared to those from SI mice, and (3) p38 MAPK inhibitor treatment for two weeks significantly reversed SCI-induced DO and changes of spike thresholds, firing rate and slow KA current density. These results indicate that p38 MAPK that is activated via NGF-Trk A pathways has an essential pathophysiological role in SCI-induced hyperexcitability of capsaicin-sensitive bladder afferent neurons that underlie SCI-induced DO.

We functionally and electrophysiologically demonstrated that the p38 MAPK signalling pathway significantly contributes to C-fibre dependent DO after SCI and is a potential target for the treatment of SCI-induced lower urinary tract dysfunction.

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