Effect of Preservation Conditions on Autonomic Transmission in Rat Small Bowel M.O. Taha, M.M. Fraga, D.J. Fagundes, C.P. Bandeira, A. Caricati-Neto, and A. Jurkiewicz I N small bowel, acetylcholine (ACh) released from intra- mural cholinergic nerves acts at nicotinic cholinergic receptors to cause excitation of enteric nerves and at muscarinic cholinergic receptors to cause contractions of intestinal smooth muscle. 1 Thus, contractile function of small bowel regulated by ACh is highly dependent on structural and functional integrity of enteric nerves. 2 His- tological studies performed on small bowel submitted to cold ischemic preservation prior to transplantation suggest that neurological lesions following prolonged ischemia se- verely compromises contractile functions of the intestine. 3–5 However, autonomic dysfunction has not been studied in small bowel grafts preserved for transplantation. In order to investigate autonomic transmission in small bowel grafts preserved for transplantation, the contractile effects mediated by muscarinic and nicotinic cholinergic receptors in longitudinal muscle of rat jejunum submitted to cold ischemic preservation (Ringer-Lactate solution at 4°C) for long-term (12, 18, and 24 hours) were studied by means of stimulation with electrical or cholinergic agonists (carbachol and nicotine). METHODS Animals The experiments were conducted with female Wistar rats, weighing 200 to 240 g (12 to 16 weeks old). The day-night cycle was constant, with 12 hours of light and dark. The animals had free access to tap water and pelletted chow. The animals were deprived of food for 12 hours before the experiment but were allowed water ad libitum. The study design was approved by the Animal Ethics Committee of the UNIFESP. Biological Preparation The rats were killed by sulfuric ether inhalation for removal of small bowel. The jejunum was isolated, dissected free of surround- ing tissues, and intraluminally washed. Jejunal segments (2 cm) were preserved in Ringer’s lactate solution at 4°C for 12 (P12), 18 (P18), or 24 hours (P24). Jejunum segments after preservation for 12 (n = 8), 18 (n = 8) or 24 (n = 8) hours were mounted, in parallel with freshly harvested segments (n = 24), in an isolated organ bath, containing 10 mL of nutrient solution with the following composi- tion (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, 7.4 and bubbled with air). Longitudinal muscle contractions induced by electrical or drug stimulation were recorded by means of isometric transducers HP FTA10 coupled to a HP 7754A polygraph. Electrical stimulation (ES) was applied by means of platinum electrodes coupled to an electrical stimulator Grass S88 (10 to 30 Hz, 1 ms, 60 to 80 V). Jejunal segments were also stimulated by the cholinergic agonists carbachol (CCh) or nicotine (NIC) and by a purinergic agonist (ATP). Effects of ES and agonists were studied in absence or presence of the cholinergic antagonists atropine (ATR; 10 -6 mol/L) or hexametonium (HEX; 10 -5 mol/L). The relaxing effects of adrenaline (ADR; 10 -3 mol/L) were studied in preserved and control segments. Maximal effects (E max ) produced by ES or drugs were determined in preserved and control segments. Statistical analyses of E max were performed by analysis of variance. RESULTS In preserved and control jejunum, contractile response induced by ES with 10, 20, and 30 Hz were biphasic and composed of an initial rapid component followed by a sustained component (Fig 1). In relation to the control, the response induced by ES was significantly lower in segments preserved for 12, 18, 24 hours (Fig 1; Table 1). These contractions were blocked by ATR (10 -6 mol/L) in control and in preserved preparations (Fig 2). Contractions induced by CCh (10 -3 mol/L) or NIC (10 -3 mol/L) were lower in segments preserved for 12, 18, or 24 hours than in control segments (Fig 3 and 4; Table 1). Contractions induced by CCh were blocked by ATR (10 -6 mol/L; (Fig 5) while contractions induced by NIC were blocked by HEX (10 -6 mol/L, not shown). The relaxing effects of ADR (10 -3 mol/L) were not different in pre- served and control segments (Fig 5). From the Departments of Surgery (M.O.T., M.M.F., D.J.F., C.P.B) and Pharmacology A.C.N., A.J.), Universidade Federal de Sa ˜ o Paulo (UNIFESP), Escola Paulista de Medicina,Sa ˜ o Paulo- SP, Brazil. This was supported by Fundac ¸a ˜ o de Amparo a ` Pesquisa do Estado de Sa ˜ o Paulo (FAPESP) and Centro de Estudos da Disciplina de Te ´ cnica Operato ´ ria e Cirurgia Experimental (CEDITEC)—UNIFESP/EPM. Address reprint requests to Murched O. Taha, PhD, UNIFESP/ EPM, Te ´ cnica Operato ´ ria e Cirurgia Experimental, Rua Botucatu, 740, CEP 04023-900, Sa ˜ o Paulo-SP, Brazil. © 2002 by Elsevier Science Inc. 0041-1345/02/$–see front matter 655 Avenue of the Americas, New York, NY 10010 PII S0041-1345(02)02698-2 Transplantation Proceedings, 34, 1021–1024 (2002) 1021