Pro-inflammatory response and hemostatic disorder induced by venom of the coral snake Micrurus tener tener IN C57BL/6 mice

Abstract

Micrurus venoms are known to induce mainly neurotoxicity in victims. However, other manifestations, including hemorrhage, edema, myotoxicity, complement activation, and hemostatic activity have been reported. In order to develop a more complete pharmacological profile of these venoms, inflammatory responses and hemostasis were evaluated in C57BL/6 mice treated with a sub-lethal dose of M. t. tener (Mtt) venom (8 μg/mouse), inoculated intraperitoneally. The venom induced moderate bleeding into the abdominal cavity and lungs, as well as infiltration of leukocytes into the liver. After 30 min, the release of pro-inflammatory mediators (TNF-α, IL-6, and NO) were observed, being most evident at 4 h. There was a decrease in hemoglobin and hematocrit levels at 72 h, a prolongation in coagulation times (PT and aPTT), a decrease in the fibrinogen concentration and an increase in fibrinolytic activity. In this animal model, it was proposed that Mtt venom induces inflammation with the release of mediators such as TNF-α, in response to the toxins. These mediators may activate hemostatic mechanisms, producing systemic fibrinolysis and hemorrhage. These findings suggest alternative treatments in Micrurus envenomations in which neurotoxic manifestations do not predominate.

Keywords: Edema; Hemostasis; Inflammation; Micrurus tener tener; Snake venom.

Fig. 1.. Hemorrhage and cell infiltrates in lungs and livers of mice inoculated with Mtt venom.

C57BL/6 male mice inoculated i.p. with sterile saline solution (Control) or Mtt (8 μg/mouse) (Experimental). Lung (A–B) and liver (C–D) samples obtained 72 h post-inoculation, were fixed in 30% formaldehyde, stained with hematoxylin and eosin and observed under light microscopy at 400× magnification. Sections: A-C: Control; B-D: Experimental; E: Infiltration of mononuclear cells in hepatic tissue. Results represent 3 independent experiments and are expressed as the number of infiltrating cells/field (mean ± SD from 3 different zones in 10 mice). * p < 0.001 vs control. E: Erythrocytes. MC: Infiltrating mononuclear cells.

Fig. 2.. Edematogenic effect of Mtt venom in C57BL/6 mouse paws.

A) Mice were inoculated i.d. in the hind foot pad, with 8 or 50 μg of Mtt or Bi venom/ mouse, respectively, or sterile saline solution (control), and then sacrificed at different times (0–480 min). Percentage of edema = weight venom-injected limb/weight control limb. Results represent 3 independent experiments in triplicate (mean ± SD). B) Mice were inoculated i.d. in the posterior limb foot pad with 2 different doses of venom (Mtt, Cdc or Bi), or sterile saline (control), then sacrificed at 240 min. Percentage of edema = weight venom-injected limb/weight control limb. Results represent 3 independent experiments in triplicate (mean ± SD). * p < 0.05 Cdc vs. Mtt (8 μg) and Cdc vs Bi (20 μg). Mtt: Micrurus tener tener venom; Bi: Bothrops isabelae venom; Cdc: Crotalus durissus cumanensis venom.

Fig. 3.. Pro-inflammatory mediators in serum from mice inoculated with Mtt venom.

C57BL/6 male mice were inoculated i.p. with 200 μL sterile saline solution (Negative Control), 10 μg LPS/200 μL sterile saline solution (Positive Control) or 8 μg Mtt/200 μL sterile saline solution (Experimental). After 30, 60, 120, 240 and 480 min, serum TNF-α (A) and IL-6 (B) were determined by ELISA, and serum NO (C) by the Griess method. Results represent 3 independent experiments in triplicate (mean ± SD). * p < 0.05 vs. negative control; ° p < 0.001 vs. negative control.