FIGURES AND TABLES
FROM:
Tetracycline Protects against Dermonecrosis Induced by Loxosceles Spider Venom
Danielle Paixão-Cavalcante, Carmen W van den Berg, Rute M Gonçalves-de-Andrade, Matheus de F Fernandes-Pedrosa, Cinthya Kimori Okamoto and Denise V Tambourgi
BACK TO ARTICLE
Figure 1.
Effect of L. intermedia venom and SMase D treatment on rabbit fibroblasts. (a) Viability: rabbit fibroblast cell cultures were incubated with increasing concentrations of Loxosceles venom (
) or SMase D (
) and after 3 days of treatment, cell viability was analyzed by the Alamar Blue assay. (b) Effect of tetracycline, doxycycline, and minocycline on the loss of cell viability: rabbit fibroblast cell cultures were cultivated in 24-well plates in DMEM without FBS. At day zero, cells were treated with 15 
g/ml of SMase D and simultaneously incubated with different concentrations of tetracycline (), doxycycline (
), or minocycline (). After 3 days of treatment the cell viability was analyzed by the Alamar Blue assay. Results are representative for two independent experiments and are expressed as the mean of triplicates
SD. (c) DNA gel electrophoresis: rabbit fibroblasts, cultured in DMEM without FBS, were treated with 15 g/ml of venom (V) or SMase D (P2) for 24 hours at 37°C in the presence or absence of tetracycline (50 g/ml). Cells incubated with (c) PBS and H2O2 (100
M) were used as negative and positive controls, respectively. After incubation, cells were detached, washed in cold PBS, and the DNA was isolated using Trizol reagent and analyzed by agarose gel electrophoresis.
Figure 2.
Protective effect of tetracyclines on the development of dermonecrotic lesion. Adult rabbits were injected intradermally with 5 g of venom or SMase P2 from L. intermedia spider. After 6 hours treatments with (a and b) tetracycline or (c and d) doxycycline were initiated. The animals were treated topically (
) with a cream containing lanolin and 5% of doxycycline or tetracycline, or orally (
) with 15 mg/kg of the inhibitors, twice a day during 48 hours. Control animals were inoculated with venom or SMase D and not treated () or treated with lanolin cream alone (). Results are representative for four independent experiments and are expressed as the mean of triplicatesSD. The asterisks indicate values statistically different (P<0.05) from the controls, that is, non-treated, or lanolin cream-treated venom, or SMase D-injected animals.
Figure 3.
Histological analysis of the dermonecrotic lesion induced by L. intermedia venom and SMase D after MMP inhibitors treatment. Rabbits were injected with 5 g of L. intermedia venom and, after 6 hours of inoculation, treated twice a day during 48 hours with tetracycline or doxycycline, as described in Materials and Methods. Control sites were injected with an equal volume of PBS. Panels correspond to skin sections from rabbits injected with (panel 1: a–d) PBS; (panel 2: a–d) L. intermedia venom; (panel 3: a–d) L. intermedia venom and treated with cream of lanolin alone or (panel 4: a–d) containing tetracycline or (panel 6: a–d) doxycycline; (panel 5: a–d) L. intermedia venom and orally treated with tetracycline or (panel 7: a–d) doxycycline. Skin areas are (a) collagenous area, (b) muscle fibers, superficial dermis: detail of (c) hemorrhage, deep dermis: detail of (d) neutrophil infiltration. Bar=50 m.
Figure 4.
Determination of neutrophils number in the rabbit skin. Number of neutrophils in the histological skin sections obtained from rabbits injected with 5 g of L. intermedia venom and treated twice a day with tetracycline or doxycycline as described in Materials and Methods by (a) local or (b) oral routes. Control animals were inoculated with venom and not treated or treated with lanolin cream. Neutrophils quantification was performed in histological rabbit skin sections by counting the number of cells identified by morphological criteria. Results are representative of four different experiments and represented as meanSD. *
P<0.05.
Figure 5.
Loxosceles venom induced gelatinases expression in skin rabbit. Gelatinase activity was analyzed by zymography in skin samples obtained from animals injected with (a) PBS, (b) L. intermedia venom, and (c) topically treated with lanolin cream alone, or (d) containing tetracycline, or (e) doxycycline, or (f) orally treated with tetracycline, or (g) doxycycline. The arrows indicate gelatinase expression.
Full figure and legend (33K)