FUNCTIONAL AND HISTOLOGICAL CHANGES IN RAT URINARY BLADDER FOLLOWING SHORT AND LONG TERM KETAMINE EXPOSURE

Abstract

Introduction: Due partly to their dissociative properties, ketamine and related new psychoactive substances (NPS) are widely used as recreational drugs. This has led to the discovery of a link between the chronic use of ketamine and methoxetamine and the development of cystitis, which is typically associated with an array of lower urinary tract symptoms indicative of an overactive bladder. Interestingly, however, in vitro organ bath experiments demonstrate that acute exposure to ketamine causes relaxation in human bladder. Ketamine has been shown to dose dependently inhibit Ca2+ influx through L-type calcium channels in various different tissues and species, and so this may contribute to the relaxant effects observed following acute exposure. Here, the effects of short term (20 minutes) and longer term (72 hours) ketamine exposure on carbachol-induced rat urinary bladder contractility are assessed in an in vitro organ bath assay. The mechanisms behind the functional changes observed were also investigated by looking at the effects of ketamine following blockade of L-type calcium channels; and through immunohistological assessment of L-type calcium channels. Methods: Rat bladder was dissected into longitudinal strips and suspended in organ baths containing 15mL Krebs-Heneselit solution, supplied with 95% O2 5% CO2, and maintained at 37°C. Strips were contracted with increasing concentrations of carbachol and then incubated with ketamine (up to 3mM) for 20 minutes within the organ bath, or 72 hours in an incubator, before carrying out another carbachol dose response. For histological assessment, rat bladder was dissected in half longitudinally and cultured at 37°C with 3mM ketamine for 72 hours, with the other half of the bladder acting as a time matched control. Bladders were then fixed in 10% formalin for 24 hours, dehydrated through graded alcohols and cleared with xylene, embedded in paraffin wax, sectioned at 5uM thickness and stained for immunohistochemical visualisation of L-type calcium channels. Results: Acute exposure to ketamine (≥300µM up to 3mM) decreased the contractile response of bladder strips to carbachol — a response that is predominantly L-type calcium channel dependent. In contrast, longer term 3mM ketamine exposure significantly increased contractility of bladder, compared to control tissues. The longer term effects of organ culture and ketamine on L-type calcium channels will be presented. Conclusions: This is the first study to demonstrate in vitro rat bladder overactivity following ketamine exposure. Further results will provide insight into the mechanisms behind the functional and histological bladder changes induced by ketamine.