2.2.2. Inhibition of Nociceptive Neurotransmitter Release in Peripheral Endings

The antinociceptive effects of BTX-A were initially considered in simple muscle relaxation. Recent studies found muscle relaxation effects may not directly overlap with pain relief, which implies that the mechanism of action of BTX-A in pain relief is more complex than first thought and may have possible effects on sensory neurons [26,27]. Of note, the analgesic effects of BTX-A often persist longer than the neuroparalytic effects, which indicates that BTX-A affects pain fibers or sensory nerves.

Unmyelinated C-fibers and lightly myelinated Aδ-fibers are two types of nerve fibers that transmit sensory information from the bladder [28,29]. C-fibers are the primary nociceptive fibers that innervate the suburothelium of the bladder. Under normal conditions, C-fibers are silent but become activated in several pathological conditions, such as the alteration of potassium channels. Activated C-fibers result in increased excitability, the transmission of painful stimuli, and increased afferent drive that consequently contributes to detrusor hyperreflexia [29].

Nerve growth factor (NGF) influences C-fiber hyperexcitability in studies done by Vizzard et al. [30] and Seki et al. [31]. They observed increased NGF levels in the bladder, spinal cord, and lumbosacral dorsal root ganglia (DRG) in animals with spinal cord injury or chronic cystitis that exhibited bladder overactivity. Yoshimura et al. [32] set up a study to mimic this mechanism by intrathecally injecting NGF into female rats. Cystometrograms showed a reduction in ICI and voided volume, indicating bladder overactivity. Liu and Kuo [33] confirmed that intravesical BTX-A treatment can reduce NGF production to a normal level and control pain in IC/BPS patients.

C-fibers also release neuropeptides such as CGRP and substance P (SP), which are upregulated in patients with IC/BPS [34]. Numerous studies have shown that BTX-A blocks the release of nociceptive neurotransmitters from peripheral sensory nerves [35]. Durham et al. provided the first evidence that BTX-A directly decreases the release of CGRP from trigeminal neurons [36]. Welch et al. reported that BTX-A inhibits the release of CGRP, glutamate, and SP from cultured embryonic rat DRG [37]. BTX-A inhibition of the stimulated release of CGRP and SP from afferent nerve terminals has also been confirmed ex vivo by Lucioni et al. in rats with cyclophosphamide (CYP)-induced cystitis [38] and Rapp et al. in a capsaicin-evoked rat bladder model [39]. The antinociceptive effects of BTX-A in peripheral endings are depicted in Figure 2.

TRPV1 is a vanilloid receptor expressed in C-fibers that are involved in pain transmission after activation by heat, capsaicin, or resiniferatoxin [40]. A previous study showed that increased severity of inflammation correlated with a higher expression of TRPV1-immunoreactive nerve fibers and NGF levels in bladder biopsies from IC/BPS patients [41].

**Figure 2.** Illustration of Actions of BTX-A along the pain pathway. (1) BTX-A blocks the release of nociceptive neurotransmitters (SP, glutamate and CGRP) from afferent nerve endings and the translocation of TRPV receptor, leading to peripheral desensitization and results in attenuation of inflammation and pain. (2) Intravesical-injected BTX-A gains access to dorsal root ganglia (DRG) and spinal cord via retrograde axonal transport pathway. By blocking SP and CGRP there, BTX-A inhibits further central sensitization.

In addition to C-fibers, BTX-A may also affect Aδ-fibers. In an in vitro mouse preparation, BTX-A weakened both low- and high-threshold afferent units firing during bladder distension, suggesting that it acts on both Aδ- and C-fibers [25]. This same study revealed a five-fold increase in nitric oxide (NO) in urothelium after BTX-A administration. The increase in NO may have diminished afferent activity by acting as an inhibitory transmitter on myofibroblasts or the urothelium [42]. A balance in urothelial neurotransmitters between excitatory ATP and inhibitory NO has been proposed to modulate afferent activity and it would seem that BTX-A contributed to normalizing this balance [43].
