Protective effects of mesenchymal stromal cell-derived secretome on dermonecrosis induced in rabbits by Loxosceles intermedia spider venom

Abstract

Background: Loxoscelism refers to a set of clinical manifestations caused by the bite of spiders from the Loxosceles genus. The classic clinical symptoms are characterized by an intense inflammatory reaction at the bite site followed by local necrosis and can be classified as cutaneous loxoscelism. This cutaneous form presents difficult healing, and the proposed treatments are not specific or effective. This study aimed to evaluate the protective effect of mesenchymal stromal cells-derived secretome on dermonecrosis induced by Loxosceles intermedia spider venom in rabbits.

Methods: Sixteen rabbits were distributed into four groups (n = 4). Except for group 1 (G1), which received only PBS, the other three groups (G2, G3, and G4) were initially challenged with 10 μg of L. intermedia venom, diluted in 100 μL of NaCl 0.9%, by intradermic injection in the interscapular region. Thirty minutes after the challenge all groups were treated with secretome, except for group 2. Group 1 (G1-control group) received intradermal injection (ID) of 60 μg of secretome in 0.15 M PBS; Group 2 (G2) received 0.9% NaCl via ID; Group 3 (G3) received 60 μg of secretome, via ID and Group 4 (G4), received 60 μg of secretome by intravenous route. Rabbits were evaluated daily and after 15 days were euthanized, necropsied and skin samples around the necrotic lesions were collected for histological analysis.

Results: Rabbits of G1 did not present edema, erythema, hemorrhagic halo, or necrosis. In animals from G2, G3, and G4, edema appeared after 6h. However, minor edema was observed in the animals of G2 and G3. Hemorrhagic halo was observed in animals, six hours and three days after, on G2, G3, and G4. Macroscopically, in G4, only one animal out of four had a lesion that evolved into a dermonecrotic wound. No changes were observed in the skin of the animals of G1, by microscopic evaluation. All animals challenged with L. intermedia venom showed similar alterations, such as necrosis and heterophilic infiltration. However, animals from G4 showed fibroblast activation, early development of connective tissue, neovascularization, and tissue re-epithelialization, indicating a more prominent healing process.

Conclusion: These results suggest that secretome from mesenchymal stromal cells cultured in a xeno-free and human component-free culture media can be promising to treat dermonecrosis caused after Loxosceles spiders bite envenoming.

Keywords: Loxosceles intermedia; Loxoscelism; Regenerative therapy; Secretome.

Figure 1.. Evolution of rabbit dermonecrotic wound after challenged with Loxosceles intermedia venom and treatment with 0.9% NaCl (G2); after injection of Loxosceles intermedia venom and treatment with ID secretome (G3); and after injection of L. intermedia venom and treatment with IV secretome (G4). Group 1 (G1) was challenged with PBS and treated with secretome, and no macroscopic changes were observed.

Figure 2.. (A) Edema mean values (cm) of rabbits challenged with PBS and treated with secretome (G1) and challenged with L. intermedia venom and treated with 0.9% NaCl (G2), with secretome intradermally (G3) and intravenous (G4), at time 1 (six hours), third, ninth, and fifteenth day after challenge and treatments. (B) Global mean value ± standard deviation of edema of rabbits challenged with PBS and treated with secretome (G1) and challenged with L. intermedia venom and treated with 0.9% NaCl (G2), with secretome intradermally (G3) and intravenous (G4), after challenge and treatments. (C) Hemorrhagic halo values (cm) of rabbits challenged with PBS and treated with secretome (G1) and challenged with L. intermedia venom and treated with 0.9% NaCl (G2), with secretome intradermally (G3) and intravenous (G4), at time 1 (six hours), third, ninth, and fifteenth day after challenge and treatments. (D) Global mean value ± standard deviation of hemorrhagic halo of rabbits challenged with PBS and treated with secretome (G1) and challenged with L. intermedia venom and treated with 0.9% NaCl (G2), with secretome intradermally (G3) and intravenous (G4), after challenge and treatments.

Figure 3.. Histological image of skin from rabbits inoculated with L. intermedia venom after HE staining. (A) Rabbit of G1. Normal skin is seen, with no histological changes in the skin layers. (B) Rabbit of G2. Note that in the epidermis, superficial, deep dermis, extending to the hypodermis, there are multifocal to coalescent areas of necrosis (n), and tissue loss with accentuated heterophilic inflammatory infiltrate (i). (C) Rabbit of G2. Multifocal areas of vitreous basophilic material deposition are observed, with evidence of dystrophic mineralization (dm) in the final assessment. (D) Rabbit of G3. In greater magnification, intense heterophilic inflammatory infiltrate (i), necrosis (n), and angiogenesis (an) are seen. (E) Rabbit of G4. Collagen fibers (c) and angiogenesis (an) are visualized, characterizing the granulation tissue. (F) Rabbit of G4. Note granulation tissue (g), necrosis (n), heterophilic inflammatory infiltrate (i) and angiogenesis (an).