The diagnosis of OAB is symptom-based and involves assessing the conscious perception of urinary urgency. Knowing whether an animal is experiencing urgency, even if pseudoaffective changes in behavior may suggest it, is impossible. Animals cannot relate their symptoms to investigators, and consequently, the creation of an animal model of OAB has not been technically possible. Nevertheless several animal model of OAB, involving partial bladder outlet obstruction, spontaneous hypertension, hyperlipidemia, various neurological insults, and some gene knock-outs, have been suggested and used to study the mechanisms and treatment of OAB .
In this study, we used a partial bladder outlet obstruction animal model. This model has advantages in the replication of many of the structural and physiological bladder changes seen in human bladder outlet obstruction and is known to be a reliable model with good etiological validity . The main findings of the present study were: (1) bladder weights increased after PUO, but there was a no apparent histological change in urothelial layer; (2) DO, induced by PUO, was identified by CMG; (3) BDNF and NRG-1 expression levels were significantly higher in the OAB group than in the control group and significantly lower in the OAB + 5-HMT group than in the OAB group.
In this work, detrusor muscle hypertrophy was histologically observed in the OAB and OAB + 5-HMT groups. Although some reports have suggested that detrusor wall thickeness is a potential marker of OAB , other studies have suggested that detrusor wall thickness is not a diagnostic tool for OAB. Nevertheless, detrusor hypertrophy is associated with detrusor overactivity and it is important hallmark of reproducing OAB in animal model. Instead of measuring detrusor wall thickness, we evaluated bladder weight because the histology of the bladder tissue was inconsistent among the groups. Based on these results, we suggest that the increased bladder weight represents detrusor muscle hypertrophy because the detrusor muscle composes most of the bladder wall and 5-HMT is effective in decreasing detrusor hypertrophy.
DO is a urodynamic observation characterized by involuntary contractions during filling phase of cystometry . However, DO is not an essential characteristic of OAB, as indicated by a 2006 study that found only 64% of diagnosed OAB patients, according to the new International Continence Society (ICS) definition, had urodynamically demonstrable DO . The study also showed that more than 30% of DO patients did not have OAB. Nonetheless, DO has been used as the core surrogate for urgency in basic OAB research as urinary urgency cannot be ascertained in animal models of OAB . During CMG in the present study, MIs and MVs were significantly decreased and involuntary contractions were present during the MIs in the OAB group. The decreased MIs and MVs were thought to correspond to the clinical the frequent and involuntary contractions during MI.
Although the exact mechanism of OAB is not completely understood, OAB may be related to changes in or dysfunction of the muscarinic receptors of the detrusor muscle; other mechanisms may also be involved that include other receptor systems [14, 15]. These changes result in a predisposition towards unstable bladder contractions or overactivity of the detrusor muscle. Thus, the major mechanism of anticholinergic drugs, widely used in treatment of OAB, is their antagonism of the effect of acetylcholine on muscarinic receptors in the cholinergically innervated bladder detrusor muscle. The result is a decreased number of detrusor smooth muscle contractions in the bladder and a reduced intensity of urgency symptoms [16, 17]. In this study, we used 5-HMT, the active metabolite of fesoterodine, for treating experimental OAB; fesoterodine is a widely used muscarinic receptor antagonist for treating OAB, in humans . In our study, the bladder weights, MIs and MVs were improved in the OAB + 5-HMT group, compared with the OAB group, suggesting that the changes may be due to the anti-cholinergic effect of 5-HMT.
In this study, we also isolated the urothelium from the bladder and estimated BDNF expression as a control marker of NRG-1 for evaluating NRG-1’s potential as an OAB biomarker. OAB has been suggested to be related to neurotransmitters and other receptor systems in the urothelium. Furthermore, these sensor molecules and malfunctioning of the urothelium may be linked to the development of OAB [19–22]. The urothelium is increasingly recognized as a responsive structure capable of detecting physiological and chemical stimuli, and of releasing several signaling molecules and various trophic factors after physical or chemical stimulation. Thus, the functioning of the urothelium is closely related to the functioning of the nervous system, and control the urothelium signaling pathway may be a new therapeutic target for treatment of OAB.
As described in the Introduction, new, noninvasive tests to diagnose OAB and assess therapeutic outcomes are urgently needed and some studies have focused on the detection and clinical application of OAB biomarkers . In this study, we presumed that elevated bladder pressure, caused by PUO, stimulated the urothelium, and increased the release of neuronal molecules, such as BDNF and NRG-1. These neuronal molecules were also presumed to activate a signaling pathway for inducing detrusor muscle hypertrophy and overactivity. The expected result was an increase in the number of detrusor smooth muscle contractions and an increased intensity of urgency symptoms. To confirm this hyposthesis, we measured the expression of BDNF and NRG-1 in isolated urothelium tissues.
In humans, the BDNF protein is encoded by the BDNF gene . BDNF is a member of the neutrophin family of growth factors, which are related to nerve growth factor. BDNF is the most abundant neurotrophin in the human body and contributes to the differentiation, survival and normal function of sensory neurons [25–28]. Many studies have suggested that BDNF is a biomarker of OAB because urinary bladder synthesis of BDNF is strongly increased after chronic bladder inflammation or spinal cord injury [29–31]. BDNF sequestration improves bladder function in rats with chronic cystitis , and the BDNF/creatinine ratio is significantly higher in OAB patients compared to controls . Furthermore, urinary concentrations of BDNF are higher at baseline than after administration of botulinum toxin A to the bladder trigone in patients with bladder pain syndrome/interstitial cystitis . These positive correlations suggest the potential utility of BDNF as a biomarker of OAB.
In humans, NRG-1 protein is encoded by the NRG-1 gene [7, 35] and is a member of the epidermal growth factor (EGF) family known to activate proliferation, differentiation, and survival of many tissue types [7, 36–38]. Although the function and mechanism of NRG-1 has not yet been clearly established, at least 6 major types (having different N termini) of NRG-1 are known . Type I NRG-1 plays a particular role in synapse development, influencing the upregulation of acetylcholine receptor genes beneath the endplate after mammalian motor neurons have made synaptic contact with muscle fibers. ErbB4 is highly expressed during re-epithelialization of urothelium , and antiadrenergic NRG-1/ErbB signaling disappears when the muscarinic cholinergic receptor is blocked . In addition, antiadrenergic muscarinic cholinergic signaling is diminished in the absence of NRG-1 . Although the molecular mechanism underlying the cooperation between the NRG-1/ErbB system and the cholinergic system are still under investigation, NRG-1 may be related to the recycling and maintenance of urothelium and may act in a manner similar to a cholinergic receptor in the urothelium.
In this study, both BDNF and NRG-1 were expressed at significantly higher levels in the OAB group, compared with the control group, and were significantly reduced in the OAB + 5-HMT group compared to the OAB group. As a result, we suggest that the increased expression of NRG-1 induced upregulation of the acetylcholine receptors, including detrusor muscle hypertrophy and increasing the involuntary contraction of the detrusor smooth muscle. Furthermore, NRG-1 expression may be altered by the anti-cholinergic drug, 5-HMT, which affected not only the muscarinic receptor in the detruosor muscle but also the muscarinic receptor in the urothelium. Therefore, the urothelium may be a therapeutic target for treatment of OAB.
We note that our study has some limitations. In this study, we compared NRG-1 expression in control, OAB, and OAB + 5-HMT groups without investigating the function and detailed mechanism of NRG-1 action in the urothelium. Had we assessed the signaling NRG-1 pathway in the bladder urothelium, better information about the function and mechanism of NRG-1 could have been provided, along with additional evidence for its consideration as an OAB biomarker. However, NRG-1 has not been studied in urological organ or disease models and our study was designed to investigate the possibility NRG-1’s role as a potential OAB biomarker. To clarify NRG-1’s role as a potential OAB biomarker, further investigations regarding the relationship between NRG-1 and the bladder urothelium signaling pathways are needed.