*2.2. BTX E*ff*ects on Peripheral Sensory Nerves*

Chronic pain persisting from months to years can reduce people's quality of life and become a major health-care burden. The pathophysiology of chronic pain can be classified as inflammation, neuropathic pain, or dysfunction [13]. Jankovic et al. described the first clinical application of BTX on cervical focal dystonia and hemifacial spasms. They observed an improvement in the pain of these patients, supporting an antinociceptive or afferent-mediated activity by BTX-A [14]. This finding initiated an era of research on the analgesic effects of BTX-A. Figure 1 shows the proposed mechanism of BTX-A effects on peripheral sensory system.

**Figure 1.** Mechanism of Intravesical BTX-A Effects on Peripheral Nervous System. (**A**) Afferent nerves innervate bladder sensation and carry information toward the central nervous system (CNS). Bladder-stretching is detected by the afferent nerve endings that extend into detrusor smooth muscles. The afferent nerve terminals extend into urothelium and sub-urothelial interstitium. These nerve terminals are sensitized when bacteria invade urothelium or high potassium ion penetrate after the urothelial barrier breaks down. (**B**) When bladder distention, the stretching urothelium releases neurotransmitters, including ATP, NGF, acetylcholine, and NO to activate afferent nerves. BTX-A blocks the afferent input by normalizing the balance of NO and ATP (blocks ATP and enhances NO). Besides, BTX-A dampens NGF, leads to attenuation of afferent excitability. (**C**) Immune cells including mast cells, macrophages and neutrophils were recruited by the cytokines released during bacterial infection or tissue damage. Histamine, interleukins, neuropeptides and more cytokines are subsequently released, which activates the bladder afferents.

#### 2.2.1. Bladder Stretch (Spasm)-Related Visceral Pain

One possible pathomechanism of visceral pain has been proposed recently: tension-sensitive nerve terminals in the smooth muscle of hollow organs may respond to luminal distension or stretching [15,16]. In the bladder, BTX-A acts in detrusor muscle relaxation by inhibiting acetylcholine (Ach) release from parasympathetic nerve endings [17] (Figure 1, part B).

The transient receptor potential (TRP) superfamily of cationic ion channels is involved in many cellular functions and such channels are highly expressed in afferent neurons of the urinary bladder [18]. Members of the TRP channel superfamily include TRP vanilloid 1 (TRPV1), TRPV4, and TRP Ankyrin 1 (TRPA1), involved in the mechanosensory pathway of urothelial cells. Activation of such ion channels releases adenosine triphosphate (ATP), prostaglandin E2 (PGE2), and substance P, and causes visceral pain [18–20]. Therefore, it is inferred that the function of muscle paralysis by BTX-A is to help decrease

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bladder tension, reduce bladder spasms, downregulate such TRP channels, and consequently relieve bladder pain.

Chuang et al. had observed that intravesical BTX-A administration could significantly prolong the inter-contraction interval (ICI) of the bladder and produce analgesia against acetic acid-induced bladder pain in rats by inhibiting calcitonin gene-related peptide (CGRP) release from afferent nerve terminals [21].

When the bladder is distended, ligand-gated ion channel P2X purinoceptors 3 (P2X3) receptors on nerve endings in the bladder urothelium are activated by released ATP and evoke a neural discharge [22]. In an in vitro study, P2X3 subunits expressed by cultured IC bladder urothelial cells were upregulated during stretching; augmented ATP signaling in the bladder may explain IC symptoms [23]. Hanna-Mitchell et al. [24] and Collins et al. [25] demonstrated that the intravesical administration of BTX-A is effective in reducing stretch-induced ATP release in rats and mice models.
