Experimental envenomation with Crotalus durissus terrificus venom in dogs treated with antiophidic serum - part II: laboratory aspects, electrocardiogram and histopathology

The present work shows laboratory aspects, electrocardiogram and histopathology results during experimental envenomation by Crotalus durissus terrificus in dogs treated with antiophidic serum. Twenty-one dogs were divided into three groups of seven animals each. Group I received 1mg/kg venom (sc); Group II received 1mg/kg venom (sc), 50mg antiophidic serum (iv) and fluid therapy including 0.9% NaCl solution (iv); and Group III received 1mg/kg venom (sc), 50mg antiophidic serum (iv) and fluid therapy including 0.9% NaCl solution containing sodium bicarbonate diluted to the dose of 4mEq/kg. Urinalysis showed brown urine, proteinuria, occult blood and myoglobinuria. Respiratory acidosis and hypotension were also observed. At the venom inoculation site, there was discreet edema, popliteal lymph node response, musculature presenting whitish areas and necrotic myositis with myoregenerative activity. There was not evidence of electrocardiographical and biochemical alterations.

Ophidic accidents can be classified as light, moderate and severe, and the only effective treatment to neutralize the action of crotalic venom is intravenous administration of antiophidic serum. Acute renal failure with tubular necrosis can be the main complication of a crotalic accident (18,26).  Laboratory studies included biochemical tests for urea and creatinine, (blood samples were collected by jugular vein puncture using a 30X08mm hypodermic needle and a 10ml syringe), blood gas analysis (blood samples were collected by femoral artery puncture using a 20X5.5mm hypodermic needle and a 1ml syringe containing heparin), urinalysis (urine was collected by cystocentesis using a 25X07mm hypodermic needle and a 10ml syringe), systolic blood pressure (Doppler), histopathology and electrocardiogram. For variables whose results were given by scores, groups were compared at every moment using Kruskal-Wallis non-parametric test, and the effect of moments on each group was compared using Friedman non-parametric test (19,29). A significance level of p<0.05 was adopted.

Urinalysis
Animals of Group III presented increased urine pH due to the alkalization process of the used urine.
Brown color and positive myoglobin were observed at M1 in all three groups, at M2 in Groups I and II and at M3 in Group I ( Figure 1A).
Alteration in the urine color is due to the venom systemic myotoxic activity, denominated rhabdomyolysis and characterized by myoglobinuria (1-5, 12, 18).
Proteinuria was also observed (500mg/dl) in Groups II and III at M1 and M2, and in Group I at M1, M2, M3 and M4.
Occult blood was observed at every moment starting from M1 in all groups.
According to Rosenfeld et al. (26), occult blood in the urine is due to the hemolytic action of the crotalic venom. Nogueira and Sakate (20) and Collicchio et al. (12) also reported occult blood in the urine.

Biochemistry and blood gas analysis
Urea and creatinine levels were within normal values for the studied species at every moment for all three groups.
Respiratory acidosis was observed, with increased pCO 2 and decreased blood pH values at M1 in all groups, which kept up to M2 for animals of Group I (28).

Systolic blood pressure and electrocardiogram
Systolic blood pressure significantly decreased (p<0.05) in animals of Groups I and III at M2, relative to the control moment, and in animals of all three groups at M3.
Other authors have reported hypotension and shock in an accident involving humans

Histopathology
At the inoculation site, discreet edema, as well as delimited focal and multifocal areas of whitish coloration, was observed due to degeneration and necrosis ( Figure 1B).
Alterations in biceps femoris and semitendinous muscles were characterized by moderate to severe necrotic myositis. In the adjacent musculature, there was no alteration ( Figure 1C) and the complete muscle, including form, color and texture, was different from those reported by other authors such as Azevedo-Marques et al.
(2) and Koscinczuk et al. (16). The absence of alterations in other muscles might be attributed to the evaluation moments and the inoculated venom dose.
Inflammatory process, varying from focal to diffuse and multifocal, was noticed in animals of all three groups. With regard to fiber hyalinization, Group I showed diffuse hyalinization accompanied by necrotic areas; Group II presented marked hyalinization and Group III showed one animal with rare fiber hyalinization, another animal with marked hyalinization and a third animal with diffuse hyalinization.
In Group I, diffuse fiber necrosis prevailed, whereas in Groups II and III, focal fiber necrosis and multifocal necrosis were observed ( Figure 1D).
Groups I and III showed edema and marked hemorrhage and Group II presented edema and light hemorrhage ( Figures 1E and 1F).
Myoregenerative activity was discreet and noticeable in animals of all three groups.
In 50% animals of Groups I and II, popliteal lymph nodes response was observed.
The present histological findings agree with those described by Dal Pai & Neto (13) and Salvini et al. (27).  The levels of urea and creatinine were within normality for the studied species at all moments for all three groups. The findings show that there was not renal compromising because of the employed venom dose.
Respiratory acidosis in blood gas analysis and dyspnea were observed six hours after venom inoculation in all three groups, which kept only up to 48h after envenomation in animals of Group I. Systolic blood pressure decreased at M3 (9h AV) in animals of all three groups. These effects are attributed to the neurotoxic action of this venom and to the shock developed.
No alterations were observed in the electrocardiogram for all three groups.
At the inoculation site there was discreet edema besides well-marked focal and multifocal areas of whitish coloration due to degeneration and necrosis, popliteal lymph node response and discreet myoregenerative activity. These effects are due to the venom myotoxic action and rhabdomyolysis.
The employed antiophidic serum dose was effective in neutralizing the venom, all animals survived and recovered after one week.