Figure 2

Acute MMP-9/2 inhibition attenuates CCI-induced allodynia and neuroinflammation. (A) Gelatin zymography of sciatic nerve extracts (70 μg total protein/lane) obtained at 3 h, 6 h, 1 day, 3 days, 5 days, and 7 days post-CCI. The arrows point to the MMP-9 and MMP-2 species. (B) Taqman qRT-PCR for MMP-9 in the sham (Sh) and CCI sciatic nerves (day 1). The mean relative mRNA ± SEM of n = 6/group normalized to GAPDH compared to naïve (N) nerve (**, P < 0.01). (C) Immunostaining of MMPs (2’2-diaminobenzidine, brown) in the sciatic nerve at day 1 and week 1 post-CCI. Methylene Blue, counterstain. MMP-9 is in the Schwann cells (asterisks) and axoplasm (arrowheads). MMP-2 is at the blood-nerve barrier (arrow), surrounding the vessel (V) endothelium, perivascular areas and at the Schwann cell plasma and basement membranes. Representative of n = 3/group. Scale bar, 40 μm. (D) Top panel, MMP-9 (red, left; green, right) co-localizes (yellow) with the S100-positive Schwann cells (left) at day 1 and with the CD68-positive macrophages (right) at week 1 post-CCI. Bottom panel, MMP-2 (red) localizes along the blood-nerve barrier and vWF-positive endothelial cells (green) at both time-points. Representative of n = 3/group. Scale bar, 40 μm. (E) von Frey testing after CCI and administration of SB-3CT (10 mg/kg) or the vehicle (10% DMSO) i.p., twice, at days 0 and 1 post-CCI. Decline in the withdrawal threshold in the ipsilateral (ipsi) to CCI hind paw corresponds to allodynia. SB-3CT-treated rats displayed no sensitivity to stimuli below 10–15 grams, comparable to that of contralateral (contra), uninjured hind paws. The mean withdrawal threshold (gram force; g) ± SEM of n = 6/group (**, P < 0.01; *, P < 0.05). (F) Taqman qRT-PCR for TNF-α and IL-17A analyzed at the completion of E (day 9 post-CCI). The mean relative mRNA ± SEM of n = 5/group normalized to GAPDH compared to the contralateral nerve (*, P < 0.05).