**4. BoNT-A Treatment for DSD in Patients with Spinal Cord Injury**

DSD is characterized by involuntary contraction of the external urethral sphincter during a detrusor contraction and is caused by CNS injury between the sacral spinal cord and pontine micturition center [55]. High post-void urine amount, incomplete emptying, and increased intravesical pressure during voiding phase are noticed in the SCI patient with DSD. BoNT-A injection to the urethral sphincter has demonstrated sphincter relaxation by blocking Ach from the presynaptic vesicles at the neuromuscular junction [9,56,57]. Although DSD patient receiving BoNT-A injection over the urethral sphincter may experience incontinence, most of the patients were satisfied by the treatment effects of significant urethral pressure reduction, increased voiding volume, and decreased urinary incontinence episodes [56,57].

### **5. BoNT-A Injection for Dysfunctional Voiding (DV) or Bladder Neck Dysfunction (BND)**

DV is characterized by voiding intermittency or urine flow rate fluctuation due to involuntary contraction of the periurethral striated muscle during voiding in non-neurological deficit individuals [49]. As BoNT-A injection treatment has been successfully used in SCI patients with DSD, it has been developed for adults with non-neurogenic voiding dysfunction. Fowler's syndrome consists of challenging symptoms of poor external urethral sphincter relaxation without neurological or anatomical abnormality, which may cause voiding difficulty and even urinary retention [58]. Patients with non-neurogenic voiding dysfunction classically present with open bladder neck but poorly relaxed urethral sphincter and normal-to-high voiding pressure during micturition by urodynamic study and are mostly refractory to medical treatment [59]. Several recent clinical studies have shown BoNT-A injection to the external urethral sphincter could be a safe and efficient treatment method for refractory DV patients [60–62]. Post-treatment, improvements were noticed in several clinical aspects in those studies, such as maximal flow rate, voided urine amount, decreased detrusor voiding pressure, international prostate symptom score (IPSS), and quality of life index. However, only the total IPSS and voided volume improvements were significantly greater than those in the placebo group in a randomized controlled double-blind study [62].

Some DV patients without strong electromyographic activity may have similar symptoms of DV due to poor relaxation of the pelvic floor muscle and the urethral sphincter [63]. Poor bladder

neck relaxation in BND may lead to weak stream and increased residual urine amount after voiding [63]. Recent studies demonstrated that BoNT-A injections in the external urethral sphincter, pelvic floor muscle, or bladder neck may offer promising therapeutic effects for DV symptoms improvement [61,64,65]. Synchronous significant reduction of the bladder outlet resistance and the pelvic floor pressure were observed in a study after BoNT-A injection treatment to the pelvic floor muscle, which indicates a more complex mechanism in DV symptoms. Further evidence is necessary to explain the pathophysiology of BoNT-A action in DV or bladder neck dysfunction.

### **6. BoNT-A Injection for IC**/**BPS**

IC/BPS is a clinical syndrome described as having "An unpleasant sensation (pain, pressure, discomfort) perceived to be related to urinary bladder, associated with lower urinary tract symptoms of more than 6 weeks duration, in the absence of infection or other identifiable causes" [62]. Though the real pathophysiology of IC/BPS has remained unclear for decades, recent studies have progressed in molecular biology, which have focused on urothelial dysfunction and neurogenic inflammation and could explain some part of the disease [61–66]. Urothelial defect with surface glycosaminoglycan and associated dysregulation of urothelial permeability has been established as one of the pathogenesis of IC/BPS [65]. IC/BPS also had upregulated P2X3 receptors and with increasing ATP release in the urothelium [66]. TRPV1 is a capsaicin receptor that detects bladder pain. A recent clinical study found that the increased inflammation severity correlated with a high TRPV1-immunoreactive nerve fibers and nerve growth factor (NGF) in IC/BPS expression and the correlation is directly positive to the clinical symptoms [67]. Substance P, a neurotransmitter secreted from the sensory nerve and a key cytokine in inflammatory process and pain, was also found with increased expression in the bladder nerve fibers of IC/BPS patients [68]. NGF, a neuropeptide involved in the regulation of growth released by the mast cell in the inflammatory process, was found with increased expression in the bladder mucosa, urine, and serum of IC/BPS patients [69,70]. NGF is also believed to play a pivotal role in the pathogenesis of IC/BPS.

BoNT-A injections improved urothelial function in IC/BPS patients by P2X3 and TRPV1 receptor expression reduction in the urothelium, which may be the chronic pain control mechanism [71]. BoNT-A injections inhibit the sensory neurotransmitters that further reduce pain sensation [71]. Various clinical studies have shown decreased NGF mRNA expression in the bladder for those who responded to intravesical BoNT-A injection treatment [72,73]. Recent clinical evidence suggests that BoNT-A stops the inflammation process in the bladder [74,75]. Declined tryptase expression was discovered over the urothelium after repeated BoNT-A injection, which indicated the reduction of mast cell activity in the treated IC/BPS patients' bladder [74]. Furthermore, BoNT-A injection treatment was found with decreased vascular endothelial growth factor (VEGF) and attenuated vasculogenesis in the bladder of IC/BPS patients [75]. Apoptotic signaling also decreased in evidence after BoNT-A injection treatment [75].
