2.11. Mouse Ear Anti-Inflammatory Activity

Anti-inflammatory activity was evaluated using the mouse ear model, as described previously [7,18]. Under anesthesia (pentobarbital 25 mg/kg animal weight), acute inflammation was induced by topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA; phorbol myristate acetate; Sigma Cat. P8139; 2.5 µg in 10 µL acetone) to both surfaces of the right ear of CD1 mice. Ten microliters (10 µL) vehicle were topically applied to each surface of the left ear. GAGs extract (125 µg/20 µL water/per ear, each surface) or the anti-inflammatory dexamethasone (DXA) were applied (250 µg/10 µL water/per ear, each surface) 15 min after treatment with TPA. Six hours later, the mice were re-anesthetized, and an electronic micrometer used to assess ear edema and thickness. The animals were then euthanized by cervical dislocation, and each ear removed and weighed (total ear weight). A sterile biopsy punch (Integra Miltex^®, Integra LifeSciences Corp, PA.USA.) was used to remove two representative samples (5 mm diameter discs) from each ear, and the discs weighed. Inflammation was quantified as the difference between the average weight of the right ear discs (TPA alone or TPA+dexamethasone or TPA+extract) and that of the left ear discs (vehicle: acetone). Samples were taken from the ear discs for analysis of gene expression (immersed in 400 µL RNAlater [Ambion] and stored at −80 °C) and for histological evaluation (fixed in 10% formalin).