Amplified Sensory Symptoms after Inflammation

It has been observed that women with a history of recurrent urinary tract infection in childhood are more prone to be diagnosed with IC/BPS later in life [60]. This phenomenon may be due to a protective hypersensitive response during the remission process after a preceding bladder infection or inflammation. Basic studies have demonstrated that bladder insults in neonatal rats lead to a hypersensitive response to inflammation stimuli when tested in adults [61], and strengthens the spontaneous bladder distension-evoked activity of spinal visceral nociceptive neurons [62]. Altered spinal cord circuits regulate this situation because neonatal inflammation can prompt a downregulation of GABA (Aα-1) receptor microRNA and altered opioid peptide content in the dorsal horn [63,64].

#### *2.3. BTX E*ff*ects in Bladder Urothelium and Lamina Propria*

The bladder sensory system is complex and encompasses not only local afferent nerves, but also the bladder urothelium and lamina propria (LP), thus including the entire bladder mucosa. The urothelium was previously viewed as merely a passive blood-urine permeability barrier; however, it now apparently plays an active role in the bladder's sensory system by having certain "neuronal-like properties" [65]. In vitro studies have shown that some neurotransmitters, including NO, ATP, Ach, and prostaglandins, are released from the urothelium after the application of chemical or physical

stressors [66]. BTX-A is able to bind to the toxin's receptor, SV2, within bladder urothelium and suppress hypotonic-evoked ATP release from rat urothelial cultures [24]. The LP lies between the urothelium and detrusor muscle, and contains mainly connective tissue, lymphatics, and abundant vasculatures [67]. The LP consists of afferent and efferent nerve endings, and acts as a "communication center" to integrate signals of the urothelium and local afferent nerve terminals [68]. Two specific kinds of cells, telocytes (Tc) and myofibroblasts (Myo), constitute a three-dimensional (3D) network structure in the LP that acts as a mass of stretch-receptors capable of perceiving physical and chemical stimuli and consequently behaving as a "functional syncytium" [69]. The Myo/Tc 3D network contributes to bladder compliance, avoids organ deformity and expresses muscarinic, vanilloid, and purinergic receptors that recognize signals from the urothelium and afferent nerve terminals to propagate information through this network to the bladder detrusor [69]. BTX-A was proposed to induce phenotypic changes in the Myo/Tc network, including the inhibition of expression of purinergic and SP receptors, and a reduction in the expression of contractile and gap junction proteins [70].

#### Nerve Sprouting and Exhaustion of BTX efficacy

The progressive loss of BTX-A efficacy can be seen during the treatment. When BTX-A was injected into a striated muscle, the efficacy persisted till antibodies against BTX-A were formed [71]. In the bladder, however, the phenomenon of losing BTX-A effectiveness may not work the same [68].

While BTX-A injection blocks nerve terminals, new nerve endings sprout to restore synaptic activity. Haferkamp et al. [72] biopsied the urothelium and LP of NDO patients, before and after the first BTX-A injection, and found axonal degeneration, nerve sprouting, and Schwann cell activation. In order to transduce signals correctly within the bladder sensory system, an appropriate distance between cells is necessary. Since sprouting is likely to be disorganized, the integration of signaling inside the LP system may be disturbed [71]. The excitation of new sprouting afferent nerve endings contributes to chronic neurogenic inflammation. Inflammation also activates the sensory nerve endings of the LP and causes the release of neuropeptides (SP, ATP, CGRP, neuropeptide Y) that mediate multidirectional interactions in Myo/Tc multicellular networks, and acts on endothelial, smooth muscle, and immune cells, and even back on nerve endings. These effects cause a positive feedback loop and turn into a vicious cycle [73]. The exhaustion of BTX efficacy is observed in NDO patients and may be due to the growth of afferent sprouts after repeated injections, which produce a chain reaction over time by maintaining and amplifying neurogenic inflammation [68].

#### *2.4. BTX E*ff*ects in Central Nervous System*

So far, the analgesic properties of BTX-A have been widely investigated in a variety of pain models. During investigations the following interesting observations were noted: (1) the effects induced by BTX-A administration are observed distantly from the site of injection; and (2) BTX-A affects not only the peripheral but also the central nervous system (CNS) [74]. In the experiments with radiolabeled BTX-A, the retrograde transport of BTX-A to the CNS has been recognized for decades [75]. However, the neurotoxin was thought to be possibly inactivated when reaching the CNS due to the slow rate of retrograde axonal transport [76]. Restani et al. observed that BTX-A was internalized by spinal cord motor neurons and underwent fast axonal retrograde transport by directly monitoring endocytosis and axonal transport of the neurotoxin [77].

The long-distance retrograde effects of BTX-A were thoroughly reported by Antonicci et al. [78]. They were the first to show that BTX-A applied in the peripheral nerves affected central circuits via retrograde transport and transcytosis [78]. The hypothesis of retrograde action of BTX-A in pain pathway is illustrated in Figure 2. In a rat bladder model, the concentrations of radiolabeled BTX-A increased over time in both L6-S1 dorsal root ganglia and L6-S1 spinal cord segments after injections it into the bladders of rats [79]. Because of its retrograde axonal transport to the CNS, the BTX-A neurotoxin may gain access to second-order neurons to affect these.

Interestingly, recent studies described how the unilateral injection of BTX-A can bilaterally reduce pain. These analgesic effects were shown in rat models of paclitaxel-induced peripheral neuropathy, carrageenan-induced hyperalgesia, and acidic saline-induced mirror pain [80–82]. Favre-Guilmard et al. [82] designed a carrageenan-induced pain model by subplantarly injecting BTX-A into experimental rats. Dramatic anti-hyperalgesia effects in uninjected contralateral hind paws of the rats were found in this study, which cannot be explained by the peripheral mechanism of BTX-A. These results suggested that BTX-A might have a central effect via the retrograde axonal transport system, which is also presumed to be the mechanism by which BTX-A acts to induce central neuropathic pain [83].

Intrathecal BTX-A administration by Coelho et al. [84] unlocks a brand new field of investigation into a deeper understanding of the actions of BTX-A [85]. This brilliant study, using animal models of severe bladder pain, described the administration of BTX-A via an alternative intrathecal route, which effectively functioned while undesirable side effects were avoided, including decreased detrusor pressure and increased post-void residual in bladder injections. This intrathecal route of administration was further investigated for intractable or refractory patterns of pain [6].

A recent breakthrough study using concurrent functional magnetic resonance imaging (fMRI) and urodynamic studies in female patients with multiple sclerosis and neurogenic overactive bladders reported that an intradetrusor injection of BTX-A increased the activity of most brain regions (cingulate body, prefrontal cortex, insula, and pontine micturition center) involved in the sensation and process of urinary urgency [86]. This was a pilot study to evaluate the possible effects of BTX-A at the level of the brain where sensory awareness is located. However, to date and to the best of our knowledge, no available evidence exists which directly demonstrates the effects of BTX-A on ascending bladder pain via a CNS system.

#### **3. Conclusions**

BTX-A is a promising option for treating bladder pain. Although the mechanisms involved are complicated, recent research efforts using a growing body of diverse expertise have been fruitful, especially in understanding the molecular architecture of the neurotoxin, as well as in the use of bioengineered animal models and in gaining electrophysiological-based insights. The analgesics effects of BTX-A are thought to be mainly mediated by muscle relaxation as well as the blockage of neurotransmitters and inflammatory substances. Recently, a hypothesis of BTX-A affecting the CNS via retrograde transportation to target neurotransmission in pain sensory circuits has been developed but is still very controversial when applied to humans [87]. Further research on the central action of BTX-A is important and will provide crucial information to better understand the pathophysiology of bladder pain. To date, BTX-A has only been approved by the U.S. Food and Drug Administration for NDO and OAB refractory to first-line therapy. This review comprehensively includes current molecular evidence of the effects of BTX-A on bladder pain. Further basic studies and clinical trials with a large number of patients are required in order to provide much more robust evidence-based support in using BTX-A to treat bladder pain.
