Effects of Ketamine and Related New Psychoactive Substances in Rat Urinary Bladder

Abstract

Introduction: In 2007, Shahani et al. first described the clinical entity of ketamine-induced cystitis (KC) — a condition seen in recreational users of the drug, characterised by the development of various lower urinary tract (LUT) symptoms indicative of bladder overactivity. Histopathologically, individuals with KC typically present with thinning or denudation of the urothelium and inflammatory cell infiltration, and in severe cases, complete or partial cystectomy are the only recourse. Despite being investigated for more than 15 years, the aetiology surrounding KC remains incompletely understood, and there is currently no known treatment. The development of KC has also been reported in individuals using therapeutic ketamine, and so, when considering ketamine’s expanding clinical application, uncovering its mechanism of action in KC is becoming increasingly important. The aim of this thesis was to provide further insight into this phenomenon, with the ultimate hope of developing treatments and/or prevention of KC. Also explored were the effects of ketamine-related new psychoactive substances (NPS), which, despite a lack of information regarding their pharmacological effects or potential toxicity, are generally regarded as safer alternatives to ketamine. Methods: For functional assessment, rat bladder strips were suspended in organ baths containing oxygenated Krebs-Henseleit solution and maintained at 37°C. The effects of acute (20-minute) exposure to ketamine and ketamine-related compounds (up to 3mM) were investigated on contractility evoked by carbachol (CCH), high potassium, and electrical field stimulation (EFS). Longer-term (3-day) exposure to ketamine was examined by contracting tissues with either CCH or high potassium, incubating for 3 days in tissue culture media containing 3mM ketamine, and reassessing the contractile response. For histological and immunohistochemical assessment, rat bladder was dissected in half longitudinally and cultured at 37°C with ketamine or related compounds (up to 3mM). Bladders were then fixed in 10% formalin, processed, and embedded in paraffin wax. Tissue sections were stained with haematoxylin and eosin for visualisation of nuclei and cytoplasm, or stained with antibody for visualisation of various proteins of interest. Results: Ketamine and all ketamine-related compounds tested inhibited rat bladder strip contractility following acute exposure. In contrast, 3-day ketamine exposure enhanced bladder contractility to CCH, but not high potassium, which is a novel finding. Ketamine and nifedipine (an L-type calcium channel antagonist) concentration-dependently attenuated EFS-induced contractility potentiated by Bay K8644 (an L-type calcium channel agonist), and Bay K8644 concentration-dependently enhanced EFS-evoked contractility inhibited by ketamine or nifedipine. Histological examination revealed a loss of mucosal cells following 3 days culture with ketamine, (S)-ketamine, norketamine, or ketamine-related NPS (3mM). Three-day exposure to ketamine and (S)-ketamine also induced a loss of urothelial umbrella cells, which has not previously been reported in in vitro rat bladder. Conclusion: The evidence presented here suggests that acute inhibition of rat bladder contractility by ketamine and related compounds is mediated through L-type calcium channel blockade. The mechanism behind increased rat bladder strip contractility to CCH following 3-day ketamine exposure remains elusive; however, it seems likely that this effect is not L-type calcium channel-mediated, and may be due to loss of a diffusible inhibitory factor released from the urothelium following muscarinic receptor activation. It is suggested that, acutely, direct contact of ketamine and its metabolites with the bladder reduces the magnitude of bladder contractility, leading to bladder relaxation or poor bladder efficiency, ultimately resulting in an increased contact time of ketamine and its metabolites with the urothelium. Over time, urothelial damage (which can at least be partly explained by loss of umbrella cells) and reduced expression of tight junction-associated proteins, presumably induced by ketamine and/or norketamine, leads to defective barrier function of the urothelium. Increased urothelial permeability can lead to leakage of irritative agents and urinary potassium to the underlying tissue layers, which could cause an inflammatory response and also directly depolarise nerves and smooth muscle, potentially contributing to the LUT symptoms of KC. Overall, the mechanisms mediating KC appear complex, and are likely the result of the interplay of various ketamine-related effects. The novel ex vivo tissue culture assay described here shows clear functional and histopathological effects of ketamine, and so could form the basis of a functional or histopathological screening assay that is sensitive to ketamine toxicity. The evidence presented in this thesis provides further avenues to explore in the hope of fully understanding the aetiology of KC.