103 Proc. West. Pharmacol. Soc. 41: 103-106 (1998) Intramuscular Administration of Cholera Toxin in Balb/c Mice Induces an Inflammatory Reaction that is Prevented by Indomethacin L. MORENO-FIERROS 1 , R. LÓPEZ-REVILLA 2 & L. VERDIN 1 1 UMF UNAM ENEP Iztacala , Tlalnepantla A.P. 314 Edo. de México, México; & 2 Departamento de Biología Celular CINVESTAV del IPN, A.P. 14-740 Mexico, D.F. A primary action of cholera toxin (CT) is at intestinal epithelium where CT induces stimulation of adenylate cyclase. This leads directly to increased fluid secretion and electrolyte transport [1,2], although 5-hydroxytryp- tamine (5-HT) [3,4], prostaglandins [5] and the function of neuronal structures [6] also have been implicated in the pathogenesis of cholera. The enteric submucosal neurons appear to be involved in the propagation of the local se- cretion induced by cholera toxin [6,7]. Some evidence suggests that CT provokes the release of 5-HT from en- terochromaffin cells into submucosal plexus [3]. Some reports have found that cholera toxin increases local prostaglandin synthesis and that prostaglandin synthetase inhibitors impair the secretory effects of cholera toxin [4,8]. However, the results of other studies do not support these cholera toxin effects [9,10]. Prostaglandin E 2 has been shown to be an important intermediate in the trans- duction mechanism, which leads to 5-HT induced intesti- nal secretion. Beubler et al. [4] performed in vivo experi- ments to investigate the influence of cholera toxin on fluid secretion in the rat jejunum, luminal release of 5-HT and prostaglandin E 2 , and formation of cyclic AMP. They also examined the effects of ketanserin, indomethacin, vera- pamil, and nifedipine on the named parameters. Their data show that CT increased fluid secretion and the release of 5-HT and prostaglandin E 2 and stimulated the formation of cyclic AMP. They also found that indomethacin and ketanserin shifted to the right the dose-response curve for CT-induced secretion. Hence these results support the concept that CT-induced secretion is caused in part by the release of 5-HT from enterochromaffin cells. 5-HT may then stimulate prostaglandin E 2 formation via 5-HT re- ceptors and activate neuronal structures [2]. However, in spite of promoting prostaglandin E2 formation CT, in contrast to Clostridium difficile toxin A, is considered a noninflammatory toxin [6]. The above evidence shows that the role of CT in pro- moting prostaglandin synthesis and inflammation is still controversial. Therefore, we have performed a histologi- cal study to determine first whether the intramuscular application of CT in mice provoked an inflammatory re- action, and secondly whether indomethacin inhibited the inflammatory reaction provoked by CT. MATERIALS AND METHODS: Cholera toxin and indomethacin were obtained from Sigma Co. Inbred 3 month-old male mice of the Balb/c strain were used. At least 5 mice per group were used. Treatment groups. (i) Cholera toxin: Two doses of CT were tested (10 and 20 μg), and groups of five mice were divided as follows: (a) a single intramus- cular dose of CT and killed 10 min, 3 h, or 7 days later; (b) two weekly doses of CT, and killed 15 days after the first dose. (ii) Cholera toxin and indomethacin: Mice received indomethacin (10 mg/kg; po) 3 h before receiving the first CT dose and every 12 h thereafter until being killed. CT was given by one of the following treatments: (a) A single intramuscular dose of CT (10 μg) and killed 10 min, 3 h, or 7 days later; (b) two weekly dose of CT (10 μg), and killed 15 days after the first dose. (iii)Control mice receiving 50 μl of phosphate-buffered saline (PBS) vehicle as follows: (a) a single intramuscular dose of PBS and killed 10 min, 3 h, or 7 days later; (b) two weekly dose of PBS, and killed 15 days after the first dose. Histological technique. Muscles were dissected and fixed in 4% paraformaldeyde in PBS for 24 h and processed by routine histological techniques, immersed in paraplast and sections of 5 μm prepared and hematoxylin-eosin stained. RESULTS: Intramuscular administration of cholera toxin provokes an inflammatory reaction that increases with time. Ten minutes after administration there is a light acute inflammatory infiltrate and polymorphonuclear cells are presented. These are mainly neutrophils and eosino- phils, located near blood vessels. Three hours after ad- ministration, a more abundant purulent inflammatory infiltrate is observed. The infiltrate is immersed in mus- cular fibers. Mainly neutrophils and some eosinophils are present. The presence of extravascular red blood cells indicates a moderate hemorrhage although no muscular damage is seen. Seven days after administration of 10 μg (im) of chol- era toxin, an inflammatory reaction from moderate to severe is observed. The presence of some macrophages, lymphocytes and monocytes can be observed (Fig. 1a). The presence of muscular fibers in regeneration indicates previous muscular damage. A similar effect is observed 14 days after the administration of cholera toxin, although a higher number of muscular fibers regenerating can be observed. The administration of 20 μg (im) of cholera toxin pro- voked a severe inflammatory infiltrate, both acute and chronic, with hemorrhage, seven days later (Fig. 1b). The