2010 Copyright @ by the Shock Society. Unauthorized reproduction of this article is prohibited. MECHANISMS OF THE BENEFICIAL EFFECT OF HYPERTONIC SALINE SOLUTION IN ACUTE PANCREATITIS Ana Maria Mendonc ¸a Coelho,* Jose ´ Jukemura,* Sandra N. Sampietre,* Joilson O. Martins, † Nilza A. T. Molan,* Rosely A. Patzina, ‡ Bjo ¨ rn Lindkvist, § Sonia Jancar, † Jose ´ Eduardo M. Cunha,* Luiz A. Carneiro D’Albuquerque,* and Marcel Cerqueira Cesar Machado* *Department of Gastroenterology (LIM/37), Medical School, † Department of Immunology, Institute of Biomedical Sciences, and ‡ Department of Pathology, Medical School, University of Sao Paulo, Sao Paulo, Brazil; and § Department of Clinical Sciences, Malmo ¨ University Hospital, Lund University, Malmo ¨ , Sweden Received 18 Feb 2010; first review completed 3 Mar 2010; accepted in final form 4 Mar 2010 ABSTRACT—Administration of hypertonic saline (HS) solution to rats with acute pancreatitis (AP) decreases mortality and systemic inflammation. We hypothesized that these effects are related not only to systemic inflammatory reduction, but also to a reduction of the pancreatic lesion. Acute pancreatitis was induced in Wistar rats by injection of 2.5% sodium taurocholate. Animals were divided in groups: without AP, not treated AP, AP treated with NaCl 0.9%, and AP treated with NaCl 7.5%. Trypsinogen activation peptides and amylase activity were increased in ascitic fluid and serum and were not affected by treatment with HS. Pancreatic inflammation was evaluated by increased myeloperoxidase activity, malondialdehyde formation, and histopathology for severity of pancreatic lesions. The HS did not affect these parameters. Expression of cyclooxygenase 2 and inducible nitric oxide synthase was markedly increased in the pancreas of the AP group and was reduced by treatment with HS. This treatment also reduced the levels of TNF-! and IL-6 but not of IL-10 in the pancreatic tissue. These results show that HS modulates cytokine production and expression of enzymes responsible for inflammatory mediator production in the pancreas without affecting the severity of the pancreatic lesions. KEYWORDS—Acute pancreatitis, hypertonic saline, pancreatic lesions, inflammation, cytokines INTRODUCTION Clinical management of severe acute pancreatitis (AP) re- mains a difficult problem mainly in cases with intense systemic inflammatory response syndrome usually associated with a high mortality rate (1, 2). Local alterations in AP are characterized mainly by increased capillary permeability, tissue infiltration of inflammatory cells, and release of cytokines that increase tissue damage (3, 4). Systemic alterations involve severe hemodynamic alterations, acute lung injury secondary to AP, increased plasma levels of inflammatory cytokines, bowel damage, and increased bacterial translocation. It has been previously demonstrated that administration of hypertonic saline (HS) during hemorrhagic shock corrects the hemodynamic alterations and decreases capillary and sinusoi- dal luminal narrowing in resuscitation of hemorrhagic shock (5, 6). Hypertonic saline resuscitation of hemorrhagic shock also reduces neutrophil rolling and adherence into endothelial cells, therefore diminishing vascular lesions (7, 8). Treatment with HS was shown to be also beneficial in exper- imental AP (9Y11). Studies from our group have shown that administration of HS to rats with AP induced by taurocholic acid avoided mortality, reduced hemodynamic alterations, and was associated with a 10-fold decrease of IL-6 and IL-10 serum levels. Pancreatic infection and pulmonary and liver alterations were also reduced (12). We hypothesized that these effects are related not only to systemic inflammatory reduction, but also to a reduction of the pancreatic lesion. In the present study, we investigated the effects of HS ad- ministration in the same experimental model of AP focusing on the pancreatic inflammation. We found that the severity of pancreatic lesions was not affected by HS treatment, whereas inflammation markers (iNOS, cyclooxygenase 2 [COX-2], TNF-!, and IL-6) in pancreatic tissue were all reduced by this treatment. The volume of ascitic fluid and the level of TNF-! in ascitic fluid were also reduced by HS. MATERIALS AND METHODS Animals Adult male Wistar rats weighing 230 to 270 g were housed in individual cages and kept under standard conditions (12 h of light/dark cycle and tem- peratures between 22-C and 28-C) with free access to a standard rat chow and water. The experimental protocol was approved by the Ethics Committee for Animal Research from the Medical School of Sa ˜o Paulo University. All Animals received care in accordance with the Guide for the Care and Use of Laboratory Animals. Reagents Ketamine chloride (Ketalar; Parke Davis, Sa ˜o Paulo, Brazil); TNF-!, IL-6, and IL-10 assay kits (Biosource International, Camarillo, Calif ); TAP radio- immunoassay kits; Bicinchoninic Acid Protein Assay kit (Pierce, Rockford, Ill); ECL chemiluminescence kit (Amersham Biosciences Corp, Piscataway, NY); Mini-Gel System and Trans-Blot SD-Semidry Transfer Cells (Bio-Rad Laboratories, Hercules, Calif); rabbit polyclonal antibodies to COX-2 or rabbit antiserum to iNOS (1:500; Cayman Chemical, Ann Arbor, Mich); Rainbow protein molecular weight markers (Amresco Inc, Solon, Ohio); immunocomplexed peroxidase-labeled antibodies. The other chemical reagents were obtained from Sigma Chemical Co (St Louis, Mo). 502 SHOCK, Vol. 34, No. 5, pp. 502Y507, 2010 Address reprint requests to Marcel Cerqueira Cesar Machado, MD, PhD, Department of Gastroenterology, University of Sao Paulo, R. Peixoto Gomide, 515 13 andar, Sa ˜o Paulo, Brazil. E-mail: amcoelho@usp.br. This research was supported by the Fundac ¸a ˜o de Amparo a ` Pesquisa do Estado de Sa ˜o Paulo and by the Conselho Nacional de Desenvolvimento Cientı ´fico e Tecnolo ´gico, Brazil. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work. DOI: 10.1097/SHK.0b013e3181defaa1 Copyright Ó 2010 by the Shock Society