Effect of Allopurinol on Autonomic Dysfunction in Rat Jejunal Segments Exposed to Cold Ischemic Preservation for Transplantation M.O. Taha, M.M. Fraga, D.J. Fagundes, A. Jurkiewicz, and A. Caricati-Neto T HE CONTRACTILE FUNCTION of the small bowel is mainly regulated by acetylcholine (ACh) released from enteric cholinergic nerves, which acts at nicotinic cholinergic receptors to stimulate ACh release and at muscarinic cholinergic receptors to induce smooth muscle contraction. 1 These regulatory actions of ACh are highly dependent on the structural and functional integrity of enteric cholinergic nerves. 2 Histological studies of small bowel after cold ischemic preservation (CIP) for organ transplantation have shown severe lesions of the enteric autonomic nerves. 3–5 Func- tional studies have revealed a dramatic reduction in intes- tinal contractile function mediated by these nerves. 6 These autonomic dysfunctions that reduce contractile functions of small bowel exposed to CIP apear to result from the actions of cytotoxic substances generated under ischemic condi- tions. 7 Ischemia injury is one of the leading causes of loss of transplanted organs. Oxidative damage is thought to play an important role in ischemia injury, including the outcome of organ transplantation. Xanthine oxidase has been widely reported to be an important source of cytotoxic free radicals under ischemic conditions. 7 Various pharmacological agents have been evaluated. To reduce free radical levels under ischemic conditions. 8 –10 Some studies suggest that allopurinol, an analogue of hypoxanthine, is a specific potent inhibitor because it acts as a substrate for xanthine oxidase, thereby reducing free radical generation under ischemic conditions. 8 –12 To evaluate the long-term effects of allopurinol on autonomic dysfunction in small bowel exposed to CIP, for we studied the contractile functions mediated by enteric cholinergic nerves in rat jejunal seg- ments exposed for 12, 18, or 24 hours to CIP (4°C) in Ringer’s lactate solution containing 0.5 mmol/L, allopuri- nol. METHODS Animals Male Wistar rats weighting 240 to 300 g (16 to 20 weeks old) obtained from animal care UNIFESP/EPM were maintained with day–night (12-hour light/dark) cycles and given free access to tap water and pellet chow. The animals were deprived of food for 12 hours before the experiment but were allowed water ad lib. The study design was approved by the Animal Ethics Committee of the UNIFESP-EPM. Biological Preparation Rats sacrificed sulfuric ether inhalation underwent midline lapa- ratomy for surgical removal of the small bowel. The jejunum was isolated, washed intraluminally, and cleaned of surrounding tis- sues. 13 Jejunal segments (2 cm) were exposed to CIP (4°C in Ringer’s lactate solution) without (-) or with (+) addition of 0.5 mmol/L -1 allopurinol (AL). After preservation for 12, 18, or 24 hours, AL- and AL+ jejunal segments were mounted in parallel under 1 g tension in isolated organ baths containing 10 mL of aerated nutrient solution containing (in mmol/L): NaCl 138, KCl 5.7, CaCl 2 1.8, NaH 2 PO 4 0.36, NaHCO 3 15, and dextrose 5.5 (37°C, pH 7.4). 13 Longitudinal muscle contractions were induced by stimulation of the enteric autonomic nerves (electrical transmural stimulation, ETS) or nicotinic (nicotine, NIC) or muscarinic (Car- bachol, CCh) cholinergic receptors. For ETS, jejunal segments were mounted between two parallel platinum electrodes connected to an electrical stimulator (Grass S88) for delivery of electrical pulses (10 to 30 Hz, 1 ms duration and 60 V). The effects of nicotinic (hexametonium, HEX) and muscarinic (atropine, ATR) antagonists were studied on these contractions. Contractile re- sponses were recorded by means of force-displacement transducers connected via a bridge amplifier to a computerized recording system (PowerLab, ADInstruments, USA). The maximal contrac- tions (C max ) produced by ETS, NIC, and CCh in AL- and AL+ jejunal segments were analyzed by and one-way analysis of variance (ANOVA) and Student t test. A P value of .05 was considered significant. From the Departments of Surgery (M.O.T., M.M.F., D.J.F.) and Pharmacology (A.J., A.C.N.), Universidade Federal de Sa ˜ o Paulo (UNIFESP), Escola Paulista de Medicina (EPM), Sa ˜ o Paulo, Brazil. This work was supported by grants from the Fundo de Auxilio dos Docentes e Alunos (FADA)—UNIFESP/EPM, Centro de Estudos da Disciplina de Te ´ cnica Operato ´ ria e Cirurgia Experi- mental (CEDITEC)—UNIFESP-EPM, Conselho Nacional de Des- envolvimento Cientifico e Tecnolo ´ gico (CNPq), and Fundo de Amparo a ` Pesquisa do Estado de Sa ˜ o Paulo (FAPESP). Address reprint requests to Murched Omar Taha, PhD, Sur- gery Department, UNIFESP/EPM, Rua Botucatu, 740, CEP 04023-900, Sa ˜ o Paulo-SP, Brazil. E-mail: taha@uol.com.br © 2004 by Elsevier Inc. All rights reserved. 0041-1345/04/$–see front matter 360 Park Avenue South, New York, NY 10010-1710 doi:10.1016/j.transproceed.2004.01.095 Transplantation Proceedings, 36, 293–295 (2004) 293