The animal model used in this study, does not completely represent patients with OAB, but it has been demonstrated in several studies as the best animal model of OAB [14, 15]. The methods used here are based on previous finding, as discussed below. Hedlund et al. reported on CB1 and CB2 receptors, and showed that CB1 and CB2 agonists inhibit nerve-mediated contractions of the mouse bladder [16]. Representative CB1 agonists include, CP55244, WIN55,212–2, delta-9-tetrahydrocannabinol (THC), arachidonyl-2′-chloroethylamide (ACEA), and nabilone. CB2 agonists, CP55940, CB65. In this study, we used WIN55,212–2 and, CB65 as CB1 and CB2 agonists, respectively. Systemically administered cannabinoids can act at multiple sites, including the bladder and many sites in the central nerve system. Intravesical activation of CB1 does block Nerve Growth Factor-induced increased bladder activity [17]. In addition, according to Tyagi et al., CB1 and CB2 receptors have higher expression rates in the bladder urothelium, and bladder strips incubated for 15 min CB agonists show a decreased detrusor muscle contraction amplitude [8]. Therefore, we thought that intravesically instilling these drugs into the urothelium of bladder could affect detrusor contraction.

We interpret the experimental results of this study as follows. First, the CMG findings of a higher CP and shorter CI in the BOO group than in the control group provided clear evidence that DO was present in the BOO group. The results also showed that DO was significantly improved by the inhibitory effects of CB1 and CB2 receptor agonists. Second, the expression of the CB1 receptor was significantly increased in the urothelium and detrusor muscle in rats with BOO-associated DO, but no significant change in the expression of the CB2 receptor was observed. These findings suggest a key role for CB1 and CB2 receptors, particularly the CB1 receptor, in the regulation of bladder function in rats with BOO-associated DO. Many previous studies have shown that cannabinoids affect bladder DO in patients with OAB symptoms and CMG parameters reflecting sensory function, and they reduce the sensory activity of isolated tissues, and induce antihyperalgesia in animal studies of bladder inflammation [16]. Endocannabinoids relax the detrusor muscle during filling of the bladder by binding to CB1 and CB2 receptors, possibly modulating sensory afferent signals [18]. However, there are also contradictory results from previous studies of the expression and function of CB receptors, thus, the mechanism of this effect remains unclear.

In our study, both Western blotting and immunofluorescence showed that in BOO-associated DO, CB1 receptor expression in the urothelium and detrusor muscle was significantly increased, while CB2 receptor expression was not significantly changed. These results are consistent with those of Tyagi et al. [8], who suggested that both CB1 and CB2 receptors are expressed in the urothelium and detrusor muscle and that CB1 receptor expression was significantly higher than that of CB2 receptor. They also suggested that CB2 receptor expression did not increase at the onset of DO or an inflammatory response because CB2 receptors are mainly expressed in peripheral immune cells; our results support their hypothesis. In a study using CB1 receptor-knockout mice, Füllhase et al. reported that the pathophysiologic role of CB1 receptors, but not that of CB2 receptors, is related to central and peripheral nervous control of micturition [19, 20].

In contrast to these studies, there are reports that it is not CB1 receptors, but rather CB2 receptors that play an important role. For example, Gratzke et al. [21] reported that increased CB2 receptor expression, but not increased CB1 receptor expression, was present in the urothelium and sensory nerves, and that a CB2 agonist improved the micturition interval and threshold, suggesting that the CB2 receptor mediate afferent signals in the bladder. Another study reported that the CB2 receptor was upregulated in a rat model of bladder inflammation [22]. Lastly, it was recently reported by Bakali et al. [23, 24] that in both rat and human bladders, CB1 receptors were localized in the urothelium, detrusor muscle, and nerve fiber structures of the subendothelium, and have both pre- and post-synaptic inhibitory effects, whereas CB2 receptors were localized in the same sites in the human bladder, but only in the detrusor muscle in rat bladders, suggesting that CB2 receptors only have postsynaptic inhibitory effects.

The differences between our study and previous studies could be due to the different methodologies. In our study, we used intravenous ketamine and xylazine to anesthetize rats, whereas Gratzke et al. [21] used intraperitoneal administration of isoflurane and urethane and performed CMG on conscious rats. It has been reported that ketamine inhibits the micturition reflex [25].

This study had several limitations. First, we used one dose and type of agonist for CB1 and CB2 receptors, however, these doses and types may not produce the maximal effect on the bladder of rats. Definitive characterization of CB receptor expression in tissues depends on the availability of selective agonists and antagonists. Further studies are warranted to examine these receptors in the urothelium and detrusor muscle and to develop drugs targeting these receptors. Such studies would be helpful for regulating DO caused by BOO and to control OAB symptoms. Second, we did not measure residual urine volume or functional bladder capacity in the rat model used here. CB1 and CB2 agonists may reduce the resistance provided by external sphincter, thereby indirectly reducing bladder CP. Therefore, we can not exclude the possibility that reduction in CP was because of a decrease in urethral resistance after the administration of CB1 and CB2 agonists. Third, although we attempted to measure non-voiding contractions, we were unable to do so; in this study, CP and CI were measured at voiding. It is also possible that our conclusions are not valid for technical reasons. We considered that the changes in CP and CI suggested the presence of DO, and may therefore provide information regarding changes in bladder status related to DO. Fourthly, both immunofluorescence staining and Western blotting showed that CB2 receptor density was not significantly higher in the BOO group than in the control group. The cause to explain this discrepancy is not precisely known, but it is presumed that CB2 receptors are involved in the mechanisms discussed in this study but to a lesser degree than CB1 receptors. The factors underlying these differences were not revealed by the methods used here. However, there was a statistically significant difference in the control, CB1 agonist, and CB2 agonist group for CMG parameters relative to the BOO group (p < 0.05).

In addition, although treatment with CB1 and CB2 agonists can have beneficial effects on DO, they can also cause complications or side effects during long-term administration. However, our study did not reveal any serious complications. Complications may have been avoided because agonists were administered intravesically, which is a local injection rather than a systemic injection. A potential problem is that it may be difficult to determine the most appropriate method of administration and dosage of CB agonist in future clinical studies of human patients.

Ultimately, CB1 receptor expression was upregulated in the bladder in BOO-associated DO. A possible explanation for this may be feedback activation via decreased levels of endocannabinoid ligands. The concentration of endocannabinoids in the bladder, which have inhibitory effects, may decrease during BOO-associated DO.