1 M Na2HPO4; pH 4.5) to yield a final concentration of 2.24 mM. The reaction was stopped by the addition of (100 μL) of 0.2 M glycine buffer (pH 10.6). Hydrolysis of the substrate was determined by measuring the absorption at 400 nm. Results were expressed as change in OD/g of wet tissue. The measurement of VEGF and TNF-α in the implants was carried out spinning (10,000 rpm Selleck Sirolimus for 30 min) 100 μL of the supernatant prepared for hemoglobin dosage (item 2.6). The analysis was made with Immunoassay Kits (R & D Systems, USA) following the manufacturer’s protocol. Briefly, dilutions of cell-free supernatants were added in duplicate
to ELISA plates coated with a specific murine monoclonal antibody against VEGF and TNF-α, followed by the addition of a secondary horseradish-peroxidase-conjugated polyclonal antibody (goat anti-mouse VEGF and goat anti-mouse TNFα). After washing to remove any unbound antibody-enzyme reagent, a substrate solution (50 μL of a 1:1 solution of hydrogen peroxide and tetramethylbenzidine (10 mg/mL) in DMSO) was added OSI-906 purchase to the wells. The color development was stopped, after 20 min of incubation, with 2N
sulphuric acid (50 mL) and the intensity of the color was measured at 540 nm on a spectro-photometer (E max, Molecular Devices). Standards were 0.5 log10 dilutions of recombinant murine chemokines from 7.5 to 1000 pg mL (100 μL). The results were expressed as picogram of cytokine/mg of wet tissue. Results are presented as mean ± standard deviation. Comparisons between two groups were carried out using Student’s t-test for unpaired data. Comparisons between three or more groups were carried out using one-way analysis of variance (ANOVA) and differences between groups were assessed using Newman–Keuls (parametric data). When the groups distribution showed no normal distribution (nonparametric) Kruscal Wallis test and Dunn post test were applied. A P < 0.05 was considered significant. At the 14th day post implantation, the
sponge discs became enveloped by a fibrous connective tissue (Fig. 1A) containing visible blood vessels. Intra-implant venom injection resulted in intense hemorrhage more pronounced at 4 h after injection (Fig. 1B). Hemorrhage and hyperhaemia were confirmed by the amount of hemoglobin extracted from the venom-treated implants (Fig. 1C). The treated group presented mean hemoglobin values of 4.1 ± 1.2 μg/mg Vasopressin Receptor wet tissue at one hour post-injection and 4.7 ± 0.9 μg/mg wet tissue at 4 h post-injection. These values were higher than those of the control groups (1.4 ± 0.14 μg/mg wet tissue at 1 hour; and 1.3 ± 0.3 μg/mg wet tissue at 4 h). Under light microscopy, the implant of the control group contained an organized granulation tissue composed by fibroblasts and blood vessels and an inflammatory infiltrate of neutrophils and macrophages (Fig. 2A). In the venom-treated group implant, an intense neutrophilic inflammatory infiltrate, vasodilatation, hyperhaemia and edema were present at both time points (Fig.