3.1.1. Inhibition of Detrusor Muscle Activity

BoNT-A consists of a 50 kDa light chain and a 100 kDa heavy chain connected by a weak disulfide bond [13]. In presynaptic nerve endings, the C-terminal of the heavy chain binds to the synaptic vesicle protein 2 on the neuronal cell membrane, enabling BoNT-A to be internalized within the nerve terminal by endocytosis [14]. Although the heavy chain facilitates the entrance of BoNT-A into neurons, the light chain is the biologically active moiety of BoNT-A. Synaptosome-associated protein 25 facilitates intracellular vesicle docking and membrane fusion, and is involved in the exocytotic release of neurotransmitters during synaptic transmission [15]. The light chain of BoNT-A cleaves apart synaptosome-associated protein 25 in presynaptic neurons, thereby inhibiting the release of the acetylcholine neurotransmitter by disrupting vesicular fusion with the neuronal cell membrane, resulting in flaccid muscle paralysis [11]. A previous study showed that BoNT-A could attenuate bladder contractility. In a clinical study of intravesical injection of BoNT-A 200 U in patients with neurogenic detrusor overactivity, the maximum detrusor pressures during filling cystometry were significantly reduced at the four-week follow-up [16]. Recently, abnormal high tension or spasm in smooth muscle has been suggested to be a potential mechanism for the development of chronic visceral organ pain [17]. Tension-sensitive nerve endings in the smooth muscle of visceral organs may respond to luminal distension or stretching. Mechanotransduction of low-threshold afferent nerves is associated with transient receptor potential vanilloid receptor 1 (TRPV1) activation and resulting visceral pain [17]. Although electrophysiological evidence of this phenomenon in the human bladder is lacking, patients with IC/BPS commonly exhibit a tender bladder with "spasm-like" sensations [18]. Relaxation of bladder-muscle tension might be the mechanism by which BoNT-A treats bladder pain

in patients with IC/BPS. Furthermore, although the core symptom of IC/BPS is bladder pain, most patients with IC/BPS also exhibit urinary frequency, urgency, and incontinence. Our previous study showed that detrusor overactivity could be detected in approximately 10% of patients with IC/BPS [19]. BoNT-A injection-induced inhibition of detrusor muscle overactivity could relieve frequency and urgency symptoms, thus improving the quality of life for patients with IC/BPS.

### 3.1.2. Sensory Modulation in the Urothelium

Generally, patients with botulism do not lose sensory nerve function. However, intravesical BoNT-A injection results in sensory modulation, both in pain reduction and urgency reduction in patients with IC/BPS or an overactive bladder [2,9]. Recent laboratory evidence revealed that BoNT-A injection indeed could alter the expression levels of sensory neurotransmitters and receptors in the bladder. Substance P and calcitonin gene-related peptide are small peptides that act as neurotransmitters in nociception [20,21]. In rats with cyclophosphamide-induced cystitis, the intravesical BoNT-A injection has been shown to significantly reduce the bladder expression levels of substance P and calcitonin gene-related peptide [22]. Glutamate is an excitatory neurotransmitter that plays a crucial role in central and peripheral pain pathways, along with its receptor [23]. BoNT-A injection induced significant downregulation of glutamate expression levels in human and rat skin [24,25]. In the bladder, adenosine 5'-triphosphate acts on the purinergic receptor, serving as the signal for the central nervous system to perceive bladder fullness [26]. In animal studies, BoNT-A intravesical injection was found to inhibit adenosine 5'-triphosphate release from rat bladder urothelium [27,28]. TRPV1 is a vanilloid receptor that is expressed in nociceptive afferent neurons, which can be activated by heat, protons, or vanilloid chemicals (e.g., capsaicin and resiniferatoxin) [29,30]. TRPV1 plays an important role in neural transmission in the pain pathway, and the upregulation of TRPV1 in certain diseases is accompanied by elevated pain [29,30]. In a clinical study of the use of intravesical BoNT-A injection in patients with an overactive bladder, a significant reduction of TRPV1-positive nerves was detected in bladder biopsy specimens [31]. Detrusor BoNT-A injections also led to significant reductions in the expression levels of the bladder muscarinic receptors M2 and M3, as well as the purinergic receptors P2X2 and P2X3, in patients with neurogenic detrusor overactivity [31,32]. Modulation of sensory neurotransmitters and their receptors in the bladder therefore constitutes an important mechanism by which BoNT-A can treat IC/BPS. In addition, the modulation of central nervous system sensory receptors in peripheral organs after BoNT-A injection has been observed in animal studies. The reduction of c-fos and calcitonin gene-related peptide expression levels in the dorsal horn of the spinal cord after pawl BoNT-A injection has been reported in association with the reduction of mechanical allodynia [33,34]. In a recent study, the injection of radiolabeled BoNT-A into the bladder caused retrograde transport to lumbosacral dorsal root ganglia [35]. Theoretically, intravesical injection of BoNT-A might change sensory receptor expression levels in the spinal cord, but further studies are needed to confirm this hypothesis. Although the Botulinum toxin does not directly block neurotransmitters released in the afferent nerve system, current clinical and laboratory evidence suggests that it indirectly modulates the sensory function in the bladder with pathological changes.
