Efeitos do veneno de Loxosceles similis no modelo de implante subcutâneo de esponja em camundongos Swiss

Abstract

Envenomation by Loxosceles spider bite leads to a set of signs and symptoms, called loxoscelism, which in most cases manifests through the dermonecrotic frame. The development of a smaller size animal model, of easy handling and maintenance, and lower cost is needed to study the loxoscelism pathogenesis. The inflammatory effects of the Loxosceles similis crude venom was evaluated considering neutrophil and macrophage activation, vasodilatation, hyperhaemia, edema, hemorrhage and TNF- and VEGF production using the murine sponge implant model. Thirty two, male, Swiss mice (68 weeks old) were implanted subcutaneously with polyetherpolyurethane sponge discs. Fourteen days post implantation, animals were separated into two groups: (1) control group16 mice received 30 L of saline intra-implant; (2) treated group-sixteen mice injected with 0.5 g/30 L of L. similis crude venom intra-implant. The animals were euthanized with xylazine/ketamine after 1 and 4 h post-injection. Microscopically, implants of the treated groups presented an acute inflammation characterized by: neutrophilic infiltrate, edema, vasodilatation, hyperhaemia, and severe hemorrhage. Some vessels presented ruptured walls. Under morphometric analysis, vessel area was bigger in the treated groups compared with the control ones. The biochemical parameters, hemoglobin content, levels of the cytokines tumor necrosis factor (TNF) and vascular endothelial growth factor (VEGF), inflammatory enzyme activities n-acethyl--glucosaminidase (NAG) and myeloperoxidadase (MPO) were also significantly higher in the venom-treated groups. The effects of Loxosceles venom in the granulation tissue of the implant in mice were similar to those observed in cutaneous loxoscelism in other species (human and rabbits). Consequently, the murine sponge implant model provides a new method to investigate cellular/molecular mechanisms associated with cutaneous loxoscelism.Envenomation by the Loxosceles spider causes loxoscelism, a pattern of signs and symptoms that primarily manifests in the dermonecrotic form. Our studies have shown that a mouse subcutaneous sponge implantation model may be useful in evaluating the effects of Loxosceles similis venom. This model provides an ideal microenvironment in which to study loxoscelism; however, it is still important to evaluate its pathogenesis and to observe the effects of L. similis venom for longer time periods than those in previous studies of this model. The aims of this study are: (1) to histologically characterize the effects of L. similis crude venom in a subcutaneous sponge implant; (2) to quantify the mast cells present in the implant and to measure their degranulation activity; (3) to quantify collagen subtypes I and III; and (4) to verify, quantify, and evaluate the effects of apoptosis in the implant on the pathogenesis of loxoscelism at 1h, 4h, and 24 h after injecting the venom. Thirty Swiss mice (6-8 weeks, male) were subcutaneously implanted with polyester-polyurethane sponge discs. Fourteen days post-implantation, the animals were divided into six groups (5 animals per group): three control groups (C1h, C4h, and C24h), in which the mice received 30 l injections of intra-implant saline, and three treated groups (T1h, T4h, and T24h), in which the mice received 30 L (0.5 g) injections of L. similis crude venom at 1h, 4h, and 24 h intervals. After each time interval, the animals were euthanized, and the implants were harvested and processed for light and electron microscopic analyses. The following results were observed in the implants harvested from the treated groups: acute inflammation with marked edema, thrombus, and vasculitis, as well as increased levels of mast cells and mast cell degranulation, and apoptosis in giant cells. Furthermore, degradation of collagen types I and III was observed. An analysis of the ultrastructure revealed apoptosis in various cell types. The present results suggest that apoptosis in some cell types associated with an increase in mast cell degranulation and the degradation of collagen fibers are important in the pathogenesis of loxoscelism therefore may explain the difficulty in repairing the ulcer is commonly observed in severe cases of loxoscelism cutaneous in humans.