The pathophysiology of overactive bladder (OAB) is multifactorial, and one of the pathophysiologic factors is the hyperexcitability of bladder afferent pathways. Furthermore, afferent hyperexcitability may induce neural remodeling in the central nervous system (CNS). Although many OAB patients have been treated with anticholinergics and/or β3-adrenoceptor agonists, there has not been any clear pathophysiologic evidence explaining how long OAB treatments should be continued. We, therefore, investigated the CNS effect of mirabegron, a β3-agonist, using an OAB animal model and compared the therapeutic effect between treatment cessation and treatment continuation groups.

Thirty-two 8 week old female C57BL/6 mice were divided into 4 groups: (1) Sham group (N=8), (2) OAB group (N=8), (3) Treatment cessation group (N=8), and (4) Treatment continuation group (N=8). For producing an OAB model, 3-times weekly intravesical instillations of 0.05ml KCl (75mg/ml) solution mixed with 5mg/ml of hyaluronidase was conducted for one hour. Mirabegron was systemically and continuously administered using an osmotic pump (daily 2mg/kg, s.c.) from the last intravesical instillation of KCl/hyaluronidase. The osmotic pump lasts for 20 days in the continuation group but was removed on day 10 in the cessation group. After awake cystometry (CMG), RT-PCR was conducted to examine the changes in mRNA expression levels of muscarinic, β-adrenergic, and purinergic receptors in the bladder mucosa. The expression levels of CCL2 and CCR2, which are reportedly associated with central sensitization [1, 2], in L6-S1 dorsal root ganglia (DRG) were also examined. Using immunofluorescent staining methods, the immunoreactivity of CX3CR1 (microglial marker) and GFAP (astrocyte marker) in the L6 spinal cord were assessed.

In CMG results, the OAB group exhibited reductions in intercontraction intervals (ICI), voided volume, bladder capacity, voiding efficiency, and non-voiding contractions (NVCs) compared to the sham group. The treatment continuation group showed improved parameters of ICI, NVCs, voided volume, bladder capacity, and voiding efficiency compared to the OAB group. Compared with the treatment cessation group, the treatment continuation group showed better improvements in NVCs and voiding efficiency (Fig.1).
The OAB group demonstrated elevated mRNA expression levels of M2, M3, β2, β3, P2X2, P2X3, P2X4, and P2X7 receptors in the bladder as well as CCL2 and CCR2 in L6-S1 DRG vs. the sham group. In the treatment continuation group, β2, β3, P2X2, P2X3, P2X4, P2X7, CCL2, and CCR2 were reduced vs. the OAB group. The treatment continuation group showed greater reductions in β3 and CCL2 levels vs. the cessation group (Fig.2 A). 
CX3CR1 and GFAP were highly expressed in the dorsal horn of L6 spinal cord of the OAB group vs. the sham group in immunofluorescent staining. The treatment continuation group exhibited lower expression of CX3CR1 and GFAP vs. the OAB group. Comparing with the treatment cessation group, the treatment continuation group showed lower CX3CR1 levels (Fig.2 B and C).

The increased expressions of muscarinic, β-adrenergic, and purinergic receptors in the bladder mucosa, and the upregulated CCL2 and CCR2 of L6-S1 DRG reflect bladder overactivity and afferent hyperexcitability, respectively, induced by the repeated bladder sensitization. Moreover, these changes resulted in CNS neural remodeling, evidenced by overexpression of CX3CR1 and GFAP in the OAB model. Glial activation is also known to play an important role in central sensitization. Therefore, our results represent the strong possibility that central sensitization and long-term afferent potentiation may be involved in the OAB mechanisms. There has been no consensus about the duration of OAB medication yet. According to our results, untreated OAB may sustain harmful signals from the bladder to CNS and may exacerbate the disease progression.
Continuous treatment of mirabegron improved bladder overactivity and afferent hyperexcitability as evidenced by reduced ICI and NVCs as well as decreased expressions of β-adrenergic and purinergic receptors, and decreased CCL2 and CCR2. Furthermore, continuous treatment interfered with central sensitization evidenced by decreased immunoreactivity of CX3CR1 and GFAP in L6 spinal cord. Some of these parameters were not entirely recovered in the treatment cessation group compared with the treatment continuation group.

Central sensitization may be an important pathophysiological mechanism of OAB.
Continuous treatment of OAB with mirabegron seems to prevent the process of central sensitization via improvement of CNS neural remodeling. Therefore, continuous OAB medication may be desirable for the long-term disease control.

Ma SB, Xian H, Wu WB et al. CCL2 facilitates spinal synaptic transmission and pain via interaction with presynaptic CCR2 in spinal nociceptor terminals. Mol Brain 2020;13:161.
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