Hypothesis / aims of study
Neurogenic lower urinary tract dysfunction after spinal cord injury (SCI) features detrusor overactivity (DO) and detrusor-sphincter dyssynergia (DSD), which results in inefficient voiding with high post-void residual volume. Previous studies have shown that nitric oxide (NO)-mediated pathways are involved in the control of lower urinary tract (LUT) function in multiple ways; including urethral and bladder neck smooth muscle relaxation, increased blood flow due to vascular smooth muscle relaxation and inhibition of bladder afferent activity . Also, phosphodiesterase type 5 (PDE5) inhibitors, which increase cellular levels of cyclic guanosine monophosphate (cGMP), are used for the treatment of male LUT symptoms with benign prostatic hyperplasia. NO is known to promote the production of cGMP from GTP via activation of soluble guanylate cyclase (sGC); therefore, this study examined the effects of sGC activation, which can increase cGMP production independent of NO inside the cell, on bladder and urethral dysfunctions in SCI mice.
Study design, materials and methods
Female C57BL/6N mice (9 weeks old) were used and divided into three groups: Group A of spinal cord intact mice; Group B of SCI mice treated with vehicle, and Group C of SCI mice treated with a sGC activator (BAY 60-2770). The mice in the SCI groups underwent Th8-9 spinal cord transection followed by oral administration of a vehicle or BAY 60-2770 (10 mg/kg/day) by oral gavage once a day in the morning during 2 to 4 weeks after spinal cord transection. The bladder of SCI mice was emptied via abdominal compression once daily after spinal cord transection. We evaluated the urodynamic parameters using awake cystometry (CMG) and external urethral sphincter (EUS)-electromyograms (EMGs), and the mRNA levels of mechanosensory channels, NO-related, ischemia, and inflammatory markers in L6-S1 dorsal root ganglia (DRG), urethra and bladder tissues at 4 weeks after SCI. In single-filling CMG, we measured the time to voiding, the number of non-voiding contractions (NVCs) per minute, postvoid residual volume (PVR), bladder capacity, bladder compliance, and voiding efficiency. NVC was defined as an increase in intravesical pressure more than 8 cmH2O above the baseline. In CMG and EUS-EMG recordings, we measured voiding contraction time, duration of notch-like reductions in intravesical pressure on CMG traces, and duration of reduced EMG activity. The voiding contraction time was measured as a duration between the rise of intravesical pressure beyond the threshold pressure and the point at which intravesical pressure returned to the level of threshold pressure. Reduced EMG activity was measured when EMG activity was reduced to the baseline level between tonic firings of EUS-EMG activity during voiding bladder contraction . In a separate group of animals, DRG, urethra and bladder tissues were harvested at 4 weeks of SCI to evaluate the mRNA levels various markers using real-time PCR, which included mechanosensory channels markers such as TRPA1, TRPV1, acid-sensing ion channel 1-3 (ASIC1-3), and piezo-type mechanosensitive ion channel component 2 (Piezo2), NO-related markers such as neuronal & endothelial nitric oxide synthase (nNOS & eNOS), and soluble guanylate cyclase alpha 1 (sGCα1), ischemia markers such as hypoxia-inducible factor 1-alpha (HIF-1α) and vascular endothelial growth factor (VEGF), and inflammatory markers such as transforming growth factor beta 1 (TGF-β1). All values are expressed as means ± standard deviations. We used the Mann Whitney U test to evaluate statistical differences between groups. A P-value < 0.05 was considered statistically significant.
In single-filling CMG, time to voiding, NVCs per minute, PVR and bladder capacity were significantly larger in Group B than in Group C (time to voiding: 44.4 ± 12.3 vs 33.5 ± 9.2 sec, NVCs: 0.9 ± 0.3 vs 0.5 ± 0.1 number/min, PVR: 0.4 ± 0.1 vs 0.3 ± 0.1 mL, bladder capacity: 0.5 ± 0.1 vs 0.3 ± 0.1 mL). Voiding efficiency was significantly higher in Group C than in Group B (13.0 ± 4.0 vs 6.5 ± 1.5 %) (Fig. 1A, B). In CMG and EUS-EMG recordings, voiding contraction time was significantly smaller in Group C than in Group B (20.2 ± 1.9 vs 25.1 ± 3.5 sec). Duration of notch-like reductions in intravesical pressure on CMG traces and reduced EMG activity time were significantly longer in Group C than in Group B (notch-like reductions: 1.7 ± 0.5 vs 1.0 ± 0.3 sec, reduced EMG activity: 1.5 ± 0.2 vs 0.9 ± 0.1 sec) (Fig. 1C, D). mRNA expressions of TRPA1, TRPV1, ASIC1, ASIC2, ASIC3, and Piezo2 in L6-S1 DRG were significantly higher in Group B than in Groups A and C (Fig. 2A). mRNA expressions of nNOS, eNOS, and sGCα1 in the urethra were significantly lower in Group B than in Groups A and C (Fig. 2B). mRNA expressions of HIF-1α, VEGF, and TGF-β1 in the bladder were significantly higher in Group B than in Groups A and C (Fig. 2C).
Interpretation of results
This is the first report of examining the effects of sGC activation on lower urinary tract dysfunction in SCI mice. In this study, the sGC activator treatment, which can directly increase cGMP production independent of NO, improved DO evident as reduced NVCs as well as DSD as evidenced by an increase in reduced EMG activity time in 4-weeks SCI mice. Also, the duration of notch-like reduction and reduced EMG activity were increased in the sGC-treated group, indicating the improvement of the EUS synergic relaxation during voiding, resulting in better voiding efficiency after the sGC treatment. Molecular studies also showed that the sGC treatment increased mRNA expressions of nNOS, eNOS, and sGCα1 in SCI mice, suggesting that the NO-GC-cGMP function, which is impaired after SCI, is recovered after sGC activation. Previous studies in rodent SCI models suggested that the afferent limb of micturition reflexes inducing NVCs and voiding bladder contractions are controlled by C-fiber and Aδ-fiber afferent pathways, respectively, after SCI . Also, it has been shown that ASICs and Piezo2 act as mechanosensitive channels in afferent pathways and that ASIC1-3 receptors are expressed in TRPV1 expressing, unmyelinated C-fiber neurons as well as in mechanosensitive, myelinated A-fiber neurons, whereas Piezo2 is expressed in mechanosensitive, myelinated A-fiber neurons. In this study TRPA1, TRPV1, ASIC1-3, Piezo2 transcripts in L6-S1 DRG were upregulated in SCI mice, and then reduced by the sGC treatment. Thus, the sGC treatment is likely to suppress excessive activities of Aδ- and C-fiber afferent pathways innervating the LUT. Moreover, the sGC treatment seems to be effective in reducing ischemia and inflammatory changes in the bladder, evident as decreased expressions of HIF-1α, VEGF, and TGF-β1 after the treatment.
BAY 60-2770, a soluble guanylate cyclase activator, reduced the number of NVCs, increased voiding efficiency and the mRNA expressions of NO-related markers, and decreased the mRNA expressions of C-fiber afferent markers, mechanosensitive channels, ischemia and inflammatory markers in SCI mice. Thus, sGC activation could be an effective modality for the treatment of SCI-related neurogenic LUT dysfunction such as DO and DSD.