The efficacy of nebivolol on spontaneously hypertensive rats with overactive bladder – an experimental study

Abstract

Introduction Overactive bladder (OAB) is a disease that affects physical functioning and social life and significantly decreases quality of life. According to the International Continence Society definition, OAB consists of urinary urgency with or without urge incontinence, often accompanied by frequency and nocturia. OAB was estimated to affect 20% of the world population in 2018 [1]. Current guidelines recommend antimuscarinic drugs Andrzej F. Wróbel, Anna Stępniak, Małgorzata Bańczerowska-Górska, Klaudia Stangel-Wójcikiewicz, Piotr Czuczwar 2 Arch Med Sci as a mainstay of management of OAB [2]. Side effects of antimuscarinic agents occur in a high percentage of patients and may lead to withdrawal of treatment [3]. Adverse effects commonly include dry mouth and constipation [4]. Moreover, changes in blood pressure, pulse rate, or ECG may occur, and these effects may be important in patients with hypertension [4]. Therefore, a search for new drugs with fewer side effects is justified. In June 2012, the FDA approved mirabegron as a new therapeutic approach to OAB. Mirabegron is a selective agonist at β 3 -adrenergic receptor (β 3 -AR), which activates the β 3 -AR in the urinary bladder, resulting in its relaxation [5]. Although mirabegron is a well-tolerated alternative to antimuscarinic drugs, it also affects the cardiovascular system by stimulation of β 1 -adrenergic receptor (β 1 -AR) in the heart. It has been shown that mirabegron acts on both β 1 -AR and β 2 -adrenergic receptor (β 2 -AR), increasing heart rate and blood pressure; therefore, it is not recommended for patients with severe uncontrolled hypertension [6]. Nebivolol (NEBI) is a potential novel treatment option for patients with OAB and concomitant hypertension. It is a cardioselective, lipophilic, third-generation β-blocker, which blocks β 1 -ARs in the heart and has a nitric oxide (NO) potentiating and vasodilatatory effect. NEBI is approved for hypertension and heart failure treatment [7]. Moreover, similarly to mirabegron, NEBI is an agonist of β 3 -ARs. Activation of β 3 -AR results in relaxation of the bladder and prevention of frequent urination [8]. That is why this drug seems to be a reasonable treatment option for OAB patients, especially those with concomitant hypertension [9]. The aim of this study was to assess the impact of NEBI on OAB symptoms in spontaneously hypertensive rats (an animal model of OAB and hypertension). Additionally, we investigated the influence of NEBI on MAP, other cardiovascular parameters, and biochemical markers. Material and methods Study design In the experiments 60 female rats, weighing 200–225 g, were divided into four experimental groups of 15 animals each: 1) Wistar-Kyoto rats, receiving physiological saline for 14 days (WHY), which were used as normotensive age-matched controls; 2) Spontaneously hypertensive rats receiving phy siological saline for 14 days (SHR); 3) WHY plus nebivolol 0.05 mg/kg for 14 days (WHY + NEBI); 4) SHR plus nebivolol 0.05 mg/kg for 14 days (SHR + NEBI). The animals were kept in environmentally controlled rooms in standard cages with unlimited access to aliment. All animals were experimentally naive and were tested one time only. At the beginning of the experiment the bladder and arterial catheters were surgically inserted in all rats. Then NEBI (at a single daily dose of 0.05 mg/kg) or vehicle were administered intraarterially via a polyethylene catheter inserted into the carotid artery for 14 days. Cystometry and bladder blood flow (BBF) assessment were performed 30 minutes after the last injection. After finishing cystometric investigations the rats were put in metabolic cages (3700M071, Tecniplast) for 24 hours to assess urine production (UP), heart rate (HR), and mean arterial pressure (MAP). Then the bladders of experimental rats were removed, fixed in buffered 10% formalin, and processed for paraffin blocks to conduct biochemical analyses. Finally, the animals were sacrificed by decapitation (the brains were stored for future studies). A detailed description of the animal model used in this study and of all experimental procedures was presented in a previous study [10]. All experimental procedures were performed in accordance with the European Communities Council Directive of 22 September 2010 (2010/63/EU) and were approved by the Local Ethics Committee (324/2018). Surgical procedures The applied surgical procedures have been described in detail in a previous study [10]. All surgical procedures were performed under general anaesthesia with intraperitoneal injection of 15 mg/kg of xylazine (Sedazin, Biowet, Puławy, Poland) and 75 mg/kg of ketamine hydrochloride (Ketanest, Pfizer, Warsaw, Poland). The abdominal wall was opened through an approximately 10 mm vertical midline incision. Then, a double lumen catheter was inserted through the apex of the bladder dome and fixed with 6–0 absorbable suture. In the same session a cannula was inserted into the carotid artery and filled with 40 IU/ml heparinised saline in order to ease drug administration directly to the bloodstream and to measure cardiovascular system parameters: HR and MAP. Drugs Nebivolol hydrochloride (NEBI): rel-(aR,a′R,2R,2′S) -a,a′-[iminobis(methylene)]bis[6-fluoro-3,4-dihydro-2H-1-benzopyran-2-methanol] hydrochloride) was dissolved in saline and administered intra – arterially via a polyethylene catheter inserted into the carotid artery in a single daily dose of 0.05 mg/kg. NEBI dosage was chosen according to literature data [8] and confirmed in a pilot study. Cystometry and bladder blood flow Conscious cystometry was performed via a bladder catheter connected to a microinjection The efficacy of nebivolol on spontaneously hypertensive rats with overactive bladder – an experimental study Arch Med Sci 3 pump (CMA 100) and to a pressure transducer (FT03, Grass Technologies). During the examination the bladder was filled with physiological saline at a constant rate of 0.05 ml/min. Micturition volumes were measured using a fluid collector attached to a force displacement transducer (FT03C, Grass Technologies). Cystometric profiles and micturition volumes were recorded continuously on a Grass polygraph and were determined graphically. The measurements in each rat represent average values of five bladder micturition cycles after obtaining repetitive voiding. Data on five reproducible micturition cycles for each animal were analysed, and a mean ± standard error of the mean (SEM) in each experimental group was calculated. The following cystometric parameters were assessed: 1) basal pressure (BP, cm H2O), 2) threshold pressure (TP, cm H 2 O), 3) micturition voiding pressure (MVP, cm H 2 O), 4) voided volume (VV, ml), 5) post – void residual (PVR, ml) – bladder capacity minus voided volume/fluid remaining in the bladder at the end of micturition, 6) volume threshold (VT, ml), 7) voiding efficiency (VE, %), 8) intercontraction interval (ICI, s), 9) bladder contraction duration (BCD, s), 10) relaxation time (RT, s), 11) bladder compliance (BC, ml/cm H 2 O) – calculated as the bladder capacity divided by the difference in the pressure threshold and baseline pressure using the formula [(VV + PVR)/(TP – BP)], 12) detrusor overactivity index (DOI, cm H 2 O/ml) – depicted as the quotient of the sum of amplitudes of all detrusor contractions during the filling phase and functional bladder capacity, 13) nonvoiding contraction amplitude (ANVC, cm H 2 O), 14) nonvoiding contraction frequency (FNVC, times/filling phase), 15) volume threshold to elicit NVC (VTNVC, %). The BBF was assessed by laser Doppler blood perfusion imager (PeriScan PIM III, Perimed) and displayed as changes in frequency with a colour scale. BBF was measured five times for each rat’s bladder immediately after bladder emptying. UP and cardiovascular parameters After cystometry the rats were observed in metabolic cages (3700M071, Tecniplast, Buguggiate, Italy) for 24 hours to investigate the effects of the examined substances on UP, HR, and MAP. Biochemical analyses Levels of the following biomarkers were measured in the urinary bladder tissue collected from the tested animals: interleukin 1-β (IL-1β; ELISA Kit for IL1b, Cloud-Clone, Katy, TX), interleukin 6 (IL-6; Rat IL6 ELISA Kit, LifeSpan BioSciences, Seattle, WA), tumour necrosis factor α (TNF-α; ELISA Kit for TNF Alpha, LifeSpan BioSciences), nerve growth factor (NGF; Rat NGF ELISA Kit, LifeSpan BioSciences), and brain-derived neurotrophic factor (BDNF; Emax ImmunoAssay System, Promega, Madison, WI). Statistical analysis Raw data were evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test (GraphPad Software, San Diego, CA). The obtained results were presented as mean ±SEM. P values < 0.05 were considered statistically significant. Results Cystometry and bladder blood flow Comparative analysis of WHY and SHR cystometrograms showed statistically significant differences. An increase in FNVC, ANVC, DOI, TP, and BP was observed, while a decrease in VTNVC, BC, ICI, VT, and VV was demonstrated. There were no statistically significant differences in BCD, RT, MVP, and PVRVE between these groups (Figures 1–3). The administration of NEBI in a 0.05 mg/kg/ day dose did not cause any significant cystometric changes in WHY. On the contrary, in SHR NEBI normalised the changes in cystometric parameters characteristic for DO: the mean values of assessed parameters in SHR + NEBI were not significantly different in comparison to those in WHY. NEBI decreased FNVC, ANVC, DOI, TP, and BP and simultaneously increased VTNVC, BC, ICI, VT, and VV to similar values as those observed in WHY. No significant differences in BCD, RT, MVP, PVR, and VE were observed between any of the studied groups (Figures 1–3). The basic BBF level was lower in SHR than in WHY. The administration of NEBI had no influence on BBF in WHY, but significantly increased BBF in SHR (Figure 4 D). UP and cardiovascular parameters Mean