Drug delivery model to inform investigations into bladder pathophysiology: potential role for suburothelial pericyte cells

Pathologies affecting the bladder, such as overactive bladder, interstitial cystitis/bladder pain syndrome and ketamine-induced cystitis have a dramatic effect on quality of life. Unfortunately, the pathological mechanisms are poorly understood and therefore treatment is mostly empirical, often ineffective or with intolerable side effects. Intravesical drug delivery is a therapeutic strategy that provides direct delivery of drugs into the bladder, minimizing systemic side effects associated with oral therapies. However, there is limited knowledge regarding drug distribution across the different bladder layers when an effect beyond the superficial layer is desired. In a proof of concept study, an ex vivo porcine whole bladder model was used to investigate the bladder wall distribution of lidocaine after intravesical instillation with clinically relevant solutions. It was demonstrated that concentrations within all layers of the bladder are dependent on the pH and concentration of the solution administered. Alkalinised lidocaine resulted in higher concentrations within the bladder and was less affected by urine dilution but was also associated with severe urothelial changes. In a subsequent study, the aim was to estimate the concentrations of ketamine accumulated in the urine following recreational use and investigate if urinary ketamine penetrates the bladder wall to achieve harmful concentrations, which ultimately lead to the development of ketamine-induced cystitis. Higher concentration and longer exposure to ketamine were associated with urothelial damage, supporting the hypothesis that urinary ketamine has a direct toxic effect. It has also been proposed that the pathophysiological mechanism involves microvascular changes induced by ketamine. To investigate this further, a viable murine bladder tissue model was developed to study microvascular blood flow regulation, with a particular focus on the role of pericyte cells. Using this model, it was observed that ketamine evoked pericyte-mediated constriction of the suburothelial capillaries of the bladder, which may translate in vivo into reduction of blood flow to the tissue and eventually lead to bladder dysfunction. Overall, this thesis highlights that changes to the barrier function and microvascular blood flow can have an important role in bladder pathophysiology

Murine Live Bladder Tissue Model to Study the Microvasculature Properties and Function In Situ

Regulation of microvascular blood flow in the bladder has historically been associated with regulation by pre-capillary arterioles and more recently to post-capillary venules. Although pericytes are known to regulate capillary vessel diameter in various capillary beds (e.g. brain, retina, heart and kidney), their role in regulating bladder capillary diameter remains controversial. We have developed a mouse full-thickness live bladder tissue model in which the microvasculature of the mucosa can be visualised in situ, enabling the regulation of suburothelial capillary diameter to be investigated. Bladder tissue viability was first investigated by fluorescence imaging of tissue labelled with Hoechst and propidium iodide to determine the live to dead cell ratio. Data indicated that tissue was viable in situ for up to 5 hours (>60%; P>0.05; n = 3 per time point). Real time DIC images of live tissue sections were collected for all experiments in which live tissue was exposed to vasoactive agents to investigate the pericyte-mediated regulation of capillary diameter. Images were analysed off line and vessel diameter was measured at both pericyte and non-pericyte sites along a capillary for each experiment. Angiotensin II (100 nM) and endothelin I (1 nM) evoked a pericyte-mediated vasoconstriction of suburothelial capillaries at pericyte sites (13.3 ± 5.8%, 22.0 ± 9.7%) that was significantly greater (p<0.05, n = 6 per drug) than that measured at non-pericyte sites (3.2 ± 2.7%, 6.0 ± 3.4%). Prostaglandin E2 (30 μM) evoked a pericyte-mediated dilation of capillaries (12.1 ± 2.6%) that was significantly greater than that measured at non-pericyte sites (2.3 ± 2.3%, p = 0.03, n = 3). In this study we therefore demonstrate that suburothelial capillary pericytes act to regulate capillary diameter in response to agents previously reported to regulate capillary beds of other organs. This indicates that in addition to smooth muscle-mediated regulation of upstream vessels in the bladder, spatial regulation of capillary diameter downstream of these vessels also occurs in the bladder