Mouse Model of Sciatic Nerve Crush Injury.

Sciatic nerve crush injury was performed as previously described with pressure-gauge-tethered forceps.²¹ Briefly, after IP ketamine (100 mg/kg)/xylazine (10 mg/kg) anesthesia, the right hindlimb was shaved and prepared with alcohol swabs and povidone–iodine (Betadine). Under a binocular microscope (Model PZMIII, World Precision Instruments), a lateral skin incision (~2.5 cm) was made along the length of the femur and the sciatic nerve (SN) was bluntly exposed through the iliotibial band. Crush injury was performed ~3 mm proximal to the SN trifurcation using calibrated forceps (3.3 mm tip width; 18–1107, Miltex Instruments, York, PA) for a 30 s duration at a pressure of 4.4 MPa. Skin was closed via surgical staples and post-operative slow release buprenorphine (0.05 mg/kg) was given subcutaneously as an analgesic. The experimental animals (n = 5/group) were randomly assigned to Sham (normal saline, 0.1 mL/mouse, IP), SN crush injury with saline (normal saline, 0.1 mL/mouse, IP), SN crush injury with systemic 4-AP (4-AP, 2 mg/kg, IP), SN crush injury with PLGA–PEG vehicle (PLGA–PEG, ~20 μL on sciatic nerve injury site), and SN crush injury with (4-AP)–PLGA–PEG ((4-AP)–PLGA–PEG, 1.4 mg/kg, ~20 μL on sciatic nerve injury site) groups. Systemic 4-AP was given IP once daily for 28 days. Local administration groups received (4-AP)–PLGA–PEG immediately after crush injury. Post-injury functional recovery was assessed by walking track analysis (WTA), sensory nerve testing (SNT), and grip strength testing on days 1, 3, 7, 14, 21, and 28. The animals were euthanized on post-injury day 28 to harvest sciatic nerves for histological analysis.