Studies evaluating the mechanism of the detrusor muscle pathophysiology have led to recognizing myosin II inhibition as a potential place of action in reducing bladder muscle contractions. Blebbistatin (BLEB), a 1-phenyl-2-pyrrolidinone derivative, is able to penetrate specific cells and modulate the protein function. BLEB is currently being assessed with regard to its potential biological utility, i.e. the ability to control its concentration in certain assessed material, the simplicity of its use in in-vivo and in-vitro studies and its use in combined drug therapy. 
The aim of the study was to determine the effectiveness of the blebbistatin (BLEB), on detrusor overactivity (DO) in the animal model, and, due to the potential urothelial permeability, to evaluate the potential degenerative impact of the BLEB on the urothelium.

A total of 60 female Wistar rats were used and randomly assigned to one of the following four treatment groups of 15 animals each: 1. Control (CON) 2. Retinyl acetate (RA) 3. Blebbistatin (BLEB) 4. Retinyl acetate plus blebbistatin (RA + BLEB). The following drugs were used:  Retinyl acetate with a mixture of Polysorbate 80 and saline; (±)-Blebbistatin: a small cell permeable selective inhibitor of the myosin-ADP-Pi complex blocking the myosin II in an actin-detached state, was dissolved in DMSO. Animals were catheterised and drug instillation was performed followed by the cystometry and urothelium thickness measurement. Cystometric evaluation was performed 3 days after surgical procedures in conscious unrestrained animals. The bladder catheter was attached and connected to a microinjection pump (CMA 100, CMA Microdialysis AB, Kista, Sweden) and to a pressure transducer (FT03, Grass Technologies, West Warwick, RI, U.S.A.)  using a three-way stopcock.  The rat bladder was first infused with 125 nmol of the BLEB or vehicle for 30 min. Conscious cystometry was then performed by slowly filling the bladder with physiological saline at a constant rate 0.05 mL/min to provoke repetitive voiding.  The following cystometric parameters were recorded: volume threshold (VT, ml), micturition voiding pressure (MVP, cm H2O), basal pressure (BP, cm H2O), nonvoiding contractions amplitude (ANVC, cm H2O), detrusor overactivity index (DOI, cm H2O/ml),  bladder compliance (BC, ml/cm H2O), threshold pressure (TP, cm H2O), voided volume (VV, ml), post-void residual (PVR, ml), , voiding efficiency (VE, %), intercontraction interval (ICI, s), bladder contraction duration (BCD, s), volume threshold to elicit NVC (VTNVC, %), nonvoiding contractions frequency (FNVC, times/filling phase), and relaxation time (RT, s). After the cystometric assessment, bladder edema and urothelium thickness were measured.
The obtained data were assessed by the one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test (Statistica, v. 10, StatSoft, Inc., Tulsa, OK, U.S.A.). All results are presented as the means ± standard error of the mean (SEM). Herein, p < 0.05 was considered as a statistically significant difference.

Administration of BLEB alone did not cause any statistically significant differences in measured parameters when compared with the control group. However, instillation of RA solution into the urinary bladder led to changes in cystometric parameters characteristic of DO. Increases in BP [F(2,42) = 8.856; p < 0.007], TP [F(2,42) = 4.862; p < 0.028], DOI [F(2,42) = 18.75; p < 0.0001], ANVC [F(2,42) = 8.138; p < 0.0007] and FNVC [F(2,42) = 8.138; p < 0.0001] were recorded, while decreases were noted in VV [F(2,42) = 7.812; p < 0.001], VT [F(2,42) = 5.933; p < 0.006], ICI [F(2,42) = 10.29; p < 0.004], BC [F(2,42) = 5.085; p < 0.008] and VTNVC [F(2,42) = 18.49; p < 0.0001]. Treatment with RA did not lead to any changes in MVP, PVR, VE, BCD or RT. (Fig. 1, Fig. 2)
An administration of BLEB to rats previously treated with RA resulted in decreases in BP [F(2,42) = 5.106; p < 0.029], TP [F(2,42) = 6.890; p < 0.002], DOI [F(2,42) = 14.64; p < 0.0006], ANVC [F(2,42) = 10.16; p < 0.0036]  and FNVC [F(2,42) = 35.09; p < 0.0001], while increases were observed in VV [F(2,42) = 9.298; p < 0.0001], VT [F(2,42) = 5.264; p < 0.015], ICI [F(2,42) = 7.981; p < 0.011], BC [F(2,42) = 9.300; p < 0.0004] and VTNVC [F(2,42) = 15.91; p < 0.0023]. After BLEB treatment, no statistically significant changes were noted in MVP, PVR, VE, BCD or RT.(Fig. 1, Fig. 2)
One-way ANOVA demonstrated no statistically significant changes in Evans Blue extravasation into bladder tissue and urothelium thickness between the study groups.

The current research provides new data on the possible utility of blebbistatin in the pharmacotherapy of detrusor overactivity. 
Three main findings concerning the action of blebbistatin should be particularly underlined: 
1. It did not influence the cystometric results obtained in the healthy rats treated with BLEB
2. It revealed a beneficial effect on the cystometric parameters which are specific for the detrusor overactivity.
3. It was shown not to induce degenerative effect on the urothelium after local administration.  

This is the first study showing the efficacy of BLEB in an animal model of DO. Further studies in human patients with DO/OAB are warranted to confirm these pre-clinical results.

Blebbistatin did not influence the cystometric results obtained in the healthy rats treated with the BLEB, it revealed a beneficial effect on the cystometric parameters specific for the DO and it was shown not to induce degenerative effect on the urothelium after local administration.