Regulation of bladder dynamic elasticity: a novel method to increase bladder capacity and reduce pressure using pulsatile external compressive exercises in a porcine model

Abstract

Purpose:

Dynamic elasticity is a biomechanical property of the bladder in which muscle compliance can be acutely adjusted through passive stretches and reversed with active contractions. The aim of this study was to determine if manipulating dynamic elasticity using external compression could be used as a novel method to acutely increase bladder capacity and reduce bladder pressure in a porcine model.

Methods:

Ex vivo experiment: Bladders underwent continuous or pulsatile compression after establishing a reference pressure at bladder capacity. Bladders were then filled back to the reference pressure to determine if capacity could be acutely increased. In vivo experiments: Bladders underwent five cycles of pulsatile external compression with ultrasound confirmation. Pre- and post-compression pressures were measured, and pressure was measured again 10 minutes post-compression.

Results:

Ex vivo experiment: Pulsatile compression demonstrated increased bladder capacity by 16% (p=0.01). Continuous compression demonstrated increased capacity by 9% (p<0.03). Comparison of pulsatile to continuous compression showed that the pulsatile method was superior (p=0.03). In vivo experiments: Pulsatile external compression reduced bladder pressure by 19% (p <0.00001) with a return to baseline 10 minutes post compression.

Conclusions:

These results suggest that regulation of bladder dynamic elasticity achieved with external compression can acutely decrease bladder pressure and increase bladder capacity. Pulsatile compression was found to be more effective as compared to continuous compression. These results highlight the clinical potential for use of non-invasive pulsatile compression as a therapeutic technique to increase bladder capacity, decrease bladder pressure, and reduce the symptoms of urinary urgency.

INTRODUCTION

Historically bladder compliance was viewed as a static property, and changes to bladder compliance were thought to be a result of irreversible long-term processes which led to alterations in the structure of the bladder wall[1], [2]. More recently, studies involving isolated mammalian bladder strips have shown that bladder compliance can acutely change depending on the strain and activation history of the muscle[3]. It is postulated that cycling actin-myosin cross bridges are acutely broken by passive stretches and that these cross-bridges are restored with active contractions[3], [4]. In bladder strip studies, this process has been termed adjustable passive stiffness and in human comparative-fill urodynamic studies involving individuals with and without OAB, the process has been identified and called dynamic elasticity[5], [6]. These results challenge the traditional notion that bladder compliance is static in nature and offer a unique insight into biomechanical mechanisms to acutely regulate compliance during filling and contraction.

Dynamic elasticity is a property of bladder biomechanics and other smooth muscles in which there is an acute and reversible increase in compliance after repeated stretching[6], [7]^,[8]. The ability to acutely increase compliance has been observed in latex balloons with repeat passive filling, explaining why stretching a balloon makes it easier to inflate. However, in latex balloons, the property is irreversible[8]. In contrast, increases in compliance are reversible in mouse, rabbit, and human bladders and can be restored by active voiding[9]–[11]. Dynamic elasticity was demonstrated in individuals with overactive bladder, but not detrusor overactivity, during repeat passive filling and emptying during clinical Urodynamics studies by Colhoun et al and Cullingsworth et al[5], [6]. However, the clinical utility of this method was limited by the invasive use of catheters and the non-physiologic nature of repeat passive filling. More recently, a non-invasive method to demonstrate dynamic elasticity using repeat external compression was developed in ex vivo porcine bladders[8].

The aim of this study was to further evaluate the property of dynamic elasticity in ex vivo and in-vivo porcine bladders. Specifically, the aim was to determine if a non-invasive method of dynamic elasticity regulation using pulsatile external compression can be used to increase bladder capacity and reduce bladder pressure. In doing so, the hope was to generate a robust biomechanical model which may provide targets for the development of novel treatments for individuals with overactive bladder and other forms of voiding dysfunction.

METHODS AND MATERIALS

Ex Vivo Bladder Experiments:

Harvest and Preparation. Bladders from young adult pigs of both genders were harvested from local abattoirs immediately after slaughter, following previously published protocols[12], [13]. Briefly, the relevant genitourinary anatomy and associated vasculature were isolated en bloc, and the aorta was then flushed with heparinized Krebs-Henseleit (KH) buffer. The tissues were then stored in a container with cold 3‐(N-morpholino) propanesulfonic acid (MOPS) buffer on ice and transported to the lab. All specimens were used within 48 hours of procurement. In the lab, bilateral superior vesical arteries were cannulated with 8Fr polyethene vascular catheters and the ureters ligated. The urethra was cannulated with a 16 Fr. Foley catheter used for bladder filling, and a 7 Fr T-DOC® pressure sensing catheter connected to an Aquarius TT Urodynamics system (Laborie, Mississauga, Canada). The bladders were then placed in a humidified chamber with a 250-W incandescent bulb (03917 R40, Globe Electric, Montreal, QC, Canada) to maintain physiologic conditions.

Perfusion and Data Acquisition.

Bilateral vesical arteries were perfused with KH buffer infused with 95%/5% O₂/CO₂, at a physiologic rate of 4 ml/min[12]. The urodynamics catheter permitted simultaneous monitoring of bladder pressure and bladder filling at 50 mL/min with NPSS. A syringe perfusion pump (PHD 4400 Programmable; Harvard Apparatus, Holliston, MA) allowed for administration of potassium enriched agents at 10 mL/min to induce active voiding. In a series of initial comparative-fill urodynamics experiments, bladders underwent four fill-empty cycles to a volume of 250 ml, followed by either active or passive voiding to establish dynamic elasticity, quantified as the change in pressure over the change in percent capacity (ΔP_ves/Δ%Capacity) (Fig. 1a). Subsequent experiments were performed using external compression protocols to establish dynamic elasticity as follows: Two control fills were used to establish the mean reference pressure (P_ref) at bladder capacity[8]. This was accomplished by filling the bladders at 50ml/min to a capacity of 300ml followed by potassium induced active voiding. Subsequent fills were used to perform either continuous compression (Fig. 2a, top inset) or pulsatile compression (Fig. 2a, bottom inset) at ~50% capacity. Pulsatile compression was performed for four cycles consisting of 30 seconds of compression to P_ref followed by 5 seconds of rest and continuous compression to P_ref was performed for one minute. Following completion of pulsatile or continuous compression, bladder filling was performed until P_ref was reached. After each fill, bladders were voided by potassium-induced contraction, and voided volumes recorded.

Figure 1.

a. Comparative-fill urpodynamics protocol. Fill1 indicates the fill used to collect pre-strain softening data. Fill2 indicates fill used to collect data after a passive empty and Fill3 shows the fill used to collect data after an active void.

b. (A) Average elasticity loss seen in Fill2 due to strain softening in Fill1 and (B) average elasticity gain seen during Fill3 after active voiding following Fill2.

Figure 2:

a Ex vivo compression protocols. (A) Continuous compression was performed for one minute to Pref followed by active voiding. (B) Pulsatile compression was performed for four cycles of 30 s of compression to Pref followed by 5 s of rest, followed by active voiding. b (A) Bladder capacity prior to compressive exercises, (B) following pulsatile compressive exercises and (C) after repeat filling of bladder after active voiding following completion of compressive exercises. The asterisk indicating statistical significance between bladder capacity of the control compared to after pulsatile compressive exercises. c (A) Bladder pressure readings prior to bladder filling, (B) after bladder filling, and (C) after completion of pulsatile or continuous compression exercises. The asterisk indicating statistical significance between bladder pressure before and after both compressive and pulsatile exercises when compared to bladder pressure prior to completion of exercises

In-Vivo Bladder Experiments:

Male juvenile Yorkshire pigs were obtained from commercial sources, sedated, and placed under general endotracheal anesthesia as part of an ongoing hemorrhagic shock study[14]. A lower midline incision was created to access the bladder. Once exposed, a 0.5 cm cystotomy was created and 16Fr foley catheter was inserted into the bladder and secured with a purse-string suture. The bladder was then emptied. The urethra was ligated distally to prevent leakage. The catheter was connected to a pressure transducer, and the bladder was then rapidly filled with 250 ml of saline through the catheter. A compression protocol was performed prior to initiation of hemorrhagic shock. After the bladder was filled with 250 ml of saline, baseline bladder pressure was recorded. Five cycles of external compression were then performed with 1 minute of compression followed by 30 seconds of rest (Fig 3a). An ultrasound was performed to ensure compression to 50% of bladder anterior-posterior diameter with each compression cycle. The bladder pressure was recorded immediately after completion of the external compression protocol and again 10 minutes post-compression.

Figure 3:

a In-vivo compression protocols. Bladders were filled manually to a volume of 250 cc. Pulsatile compression was performed for five cycles with one minute of compression to 50% of bladder height followed by 30 s of rest (black arrows). (A) Baseline bladder pressure, (B) post-compression bladder pressure, and (C) bladder pressure 10 min after completion of compression were obtained with pressure sensing catheter. b In-vivo porcine bladder model of stress strain relaxation using pulsatile external compression. (A) Bladder pressure readings prior to prior to compression, (B) immediately after completion of external compression protocol, and (C) ten minutes post-compression were obtained. The asterisk indicating statistical significance between bladder pressure before and after completion of the pulsatile compression protocol

Statistical Analysis:

To determine the minimum number of bladders needed for this study, a power analysis was performed, which determined that a sample size of 6 bladders was needed to identify a statistical difference when comparing paired data with a power level of 0.8 and an alpha value of 0.05. Comparisons were performed using paired Student’s t-tests with statistical significance set to p < 0.05. Continuous variables are reported as mean ± SE.

RESULTS

Ex Vivo Dynamic Elasticity Quantification:

In N=6 bladders, comparative fill urodynamics showed that dynamic elasticity decreased by 37.8% ± 8.1% (p< 0.05) after passive voiding and returned to baseline values after active voiding with dynamic elasticity increased by 41.3% ± 9.9% (p<0.05) (Fig. 1b).

Ex Vivo External Compression:

In N=8 bladders, there was a 15.7% ± 4.7% increase in bladder capacity with pulsatile compression (p=0.01) (Fig. 2b). This was significantly different from continuous compression which showed an increase in bladder capacity of 9.4% ± 3.8% (p=0.03). A subset of five bladder were evaluated with both pulsatile and continuous compression. There was a 46.6% ± 2.4% decrease in bladder pressure with pulsatile compression, and a decrease of 39.4% ± 6.3% with continuous compression which were both significantly different from baseline bladder pressure (p<0.05) (Fig. 2c). However, there was no significant difference when comparing pulsatile to continuous compression (p=0.15).

In Vivo External Compression:

In N=8 bladders, pressure was reduced by 17.3% ± 1.8% (p <0.001) after pulsatile external compression (Fig. 3b). Ten minutes after completion of the external bladder compression protocol, bladder pressure returned to the baseline with no statistically significant difference (Fig. 3b) (p=0.97).

DISCUSSION

Key findings of this study were that in both the ex vivo and in vivo porcine bladder models, pulsatile external compression significantly reduced bladder pressure. In addition, in the ex vivo model, pulsatile external compression significantly increased bladder capacity, and was superior to continuous compression. Changes in dynamic elasticity affect bladder wall tension during filling and consequently affect the stimulation of tension-sensitive nerves responsible for the sensation of bladder fullness[15], [16]. We previously published an equilibrium model in which acute bladder elasticity is regulated by competing passive forces (i.e. external compressions) and active micromotions. Increases in active micromotions could alter the equilibrium and lead to OAB [6]. Therefore, by reducing bladder pressure and wall tension, pulsatile external compression could potentially provide a non-invasive way to reduce bladder sensation and provide symptomatic relief in patients with OAB.

This study helps establish the in vitro and in vivo whole porcine bladder models for the study of biomechanical bladder properties. Other groups, including our own have previously used whole perfused bladders. Anele et al, and Vince et al created a working perfused ex vivo pig bladder models with similar techniques to our current study and showed that alterations in blood flow can lead to direct changes in bladder wall contractility[12], [13]. At least two other groups are using this model. Lentle et al demonstrated that contractile tone may be related to the propagating areas of contraction identified using spatiotemporal mapping[17]. In addition, Fry et al have used this model extensively to evaluate bladder contractility as a translational model of human urodynamics[18]. However, to our knowledge this is the only study performing in vivo urodynamics on anesthetized pigs using commercially available urodynamics equipment and showing that bladder pressure can be reduced by a non-invasive external compression protocol. Indeed, pulsatile external compression is a non-invasive technique that could be performed by a patient at almost any time.

The pulsatile external compression exercises used in the current protocols demonstrated the ability to increase bladder capacity and reduce bladder pressure, both of which are therapeutic targets for overactive bladder. While the effects were temporary in the in vivo model, with a return to baseline 10 minutes after completion of external compression, as well as with active voiding in the ex vivo model, it is possible that pulsatile external compression in humans could be used to reduce urgency and potentially extend the interval between voids.

While response durability is an important question for future investigations with this model, repetitive exercises with pelvic floor physical therapy in humans have been shown to reduce the activation levels of circuits in the brain responsible for bladder sensation and the urge to void[19]–[21]. Other studies performed in rats and humans investigated the effects of pulsatile increases in bladder pressure using bladder pumping with CO2 gas in pathologic bladders with reduced compliance and capacity [22], [23]. The authors found that pulsatile bladder pumping disrupts bladder collagen fibers and increases bladder capacity and elasticity [22], [23] with durable symptom and urodynamic improvement at 4 weeks and subjective symptom improvement for up to 6 years through retreatment [22], [23]. These prior investigations likely improved poor bladder compliance through chronic structural remodeling. However, most patients with idiopathic OAB have normal bladder compliance. Our pulsatile compression protocol works by acutely improving compliance through passive bladder stretches and reversal after active contractions. Therefore, this acute process of dynamic elasticity could potentially benefit patients with OAB and would not be expected to benefit individuals with pathologic, poorly compliant bladders due to altered collagen/elastin deposition and interstitial fibrosis. This acute process is also completely different than structural remodeling and has been demonstrated in multiple studies of animal and human bladders [6], [17], [22], [23], [27], [28]. Additional benefits of our external compression protocol include the possibility of patient-driven treatments with repeat exercises without the need for catheter placement or gas infusion.

Currently the first line muscle-based therapy for overactive bladder includes pelvic floor physical therapy in addition to behavioral modifications[20], [21]. The premise of pelvic floor physical therapy is to improve the function, strength, and ability to voluntarily relax the pelvic floor with the goal of improving urge suppression and decreasing frequency[21]^,[19]. Activation of the medial pre-frontal cortex and the dorsal anterior cingulate areas of the brain trigger the sensation of bladder fullness and the urge to void[21]. Pelvic floor physical therapy has been show to decrease activity in these areas on MRI[21]. Second and third line muscle-based therapies for overactive bladder include use of anti-muscarinic medications, β₃ agonist therapy, and bladder botulinum injection therapy which work by decreasing overactivity, increasing muscle compliance, and reducing sensitivity to bladder filling[24], [25] However, all of these therapeutic options are limited by poor compliance due to side-effects, the invasive nature of the treatment, costs, and time consumption[19], [26]. In this regard, the potential benefit of pulsatile external compression as non-invasive, patient-controlled therapy is clear.

This study was limited by a small sample size; however, results across the various protocols were consistent. While porcine bladder function may not accurately represent human bladder function, many different mammalian models including human studies have demonstrated that dynamic elasticity, a biomechanical property of bladder muscle, can be acutely regulated through cycling actin-myosin crossbridge formation [6], [17], [22], [23], [27], [28]. In addition, the whole porcine bladder is becoming and established translational model to evaluate bladder biomechanics and contractility. Symptom evaluation and demonstration of clinical significance could also not be achieved with the porcine model, although the results are promising and provide an avenue for further investigation in humans and pathologic conditions such as overactive bladder.

CONCLUSION

This study showed that it is possible to regulate the property of dynamic elasticity through non-invasive external compression with ex vivo and in-vivo porcine models, which can increase bladder capacity and decrease bladder pressure. These results highlight the clinical potential for use of non-invasive pulsatile compression as a possible therapeutic technique to increase bladder capacity, decrease bladder pressure, and reduce the symptoms associated with overactive bladder. Future studies in humans are needed to determine the clinical significance and utility of these exercises.