Fractal Dimension in Liver Histological Findings of Wistar Rats Experimentally Intoxicated with Venom of Crotalus durissus terrificus

Isabella Keyko Navarro Saneshigue dos Santos, Paulo Felipe Izique Goiozo, Adriana Falco de Brito, Gabriela Haro de Melo, Cristiane de Pauli Pereira, Rosa Maria Barilli Nogueira


Background: Accidents caused by venom of Crotalus durissus snakes, popularly known in Brazil as rattlesnake, are second in relation to the occurrence and first place in deaths in humans and animals, mainly due to the great neurotoxic, myotoxic, coagulant, nephrotoxic and hepatotoxic potential of their venom. The effects observed are due to the action of the main poison fractions and among them we can mention crotoxin (representing 50% of the total poison), crotamine, gyroxine and conxulxin. The present study had the objective of analyzing by histology and fractal dimension liver samples of Wistar rats experimentally poisoned with venom of the snake Crotalus durissus terrificus. The hypothesis is that the venom of Crotalus durissus terrificus is can induce hepatic damage at the dose recommended in this study, that its alterations can be quantified by the fractal dimension and that the antiofidic serum botropic crotalic be able to minimize the hepatic lesions induced by the venom.  

Materials, Methods & Results: Ninety rats were distributed into different groups and treated with: control group (GC, n = 30) 0.9% sodium chloride solution; venom group (GV, n = 30) crotalic venom at the dose of 1 mg/kg; (GVS, n = 30) crotalic venom at the dose of 1 mg/Kg and antiofidic serum 6 h after the application of the venom at the dose recommended by the manufacturer. Liver samples were collected at 2 h (M1), 8 h (M2) and 24 h (M3) after venom administration and submitted to histological analysis and fractal dimension (DF) using the ImageJ® software and box-counting method. Procedures for collecting, processing and analyzing samples were standardized. For statistical analyzes, after the normality was verified by the Shapiro-Wilk test and homogeneity by the Bartlett test, the data were submitted to analysis of variance (ANOVA) with Duncan test contrast with a significance level of 5%. No significant lesions were observed in GC, however necrosis, cytoplasmic and nuclear vacuolization and absence of inflammatory infiltrate were observed in 8 h (M2) and 24 h (M3) in GV. In addition to the lesions found in the GV, mononuclear inflammatory infiltrate was evident always (2 h, 8 h and 24 h) in the GVS. The lesions of necrosis, cytoplasmic and nuclear vacuolization, considered of greater severity were visualized in M3 (24 h) in both GV and GVS. There was an increase in DF for the same changes in GV and GVS over time, but with no difference between them, but with a significant difference compared to CG. In this study, lesions evidenced in the liver were not minimized by the application of antiofidic serum at the recommended dose and during the time of observation of the animals.

 Discussion: This study agrees with other authors about the hepatotoxicity of crotalic venom in relation to histological findings and the results indicate an increase in DF for the findings of vacuolization and necrosis, proving to be an efficient method for the quantitative evaluation of morphological changes induced by venom without observer interference. In addition, non-protection of the liver by antiofidic serum was evident. It is concluded that Crotalus durissus terrificus venom causes important hepatotoxic effects in the first 24 h after experimental intoxication in rats; DF is effective in the quantitative morphological evaluation of liver changes and was characterized mainly by vacuolization and necrosis; the antiofidic serum did not protect the liver from lesions induced by crotalic venom according to the dose of venom and antiofidic serum and time of observation recommended in this study.

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