8 to 10 week-old male C57BL/6 mice ordered from Charles River Laboratories (Wilmington, MA) were used. The mice were housed in cages (five in each) and had access to chow and water ad libitum. The chow was either Picolab® Rodent Diet 20 (5053) or Rodent Laboratory Diet (5001) from LabDiet® (St. Louis, MO), containing both of which contain omega-3 to omega-6 PUFA ratio of about 0.15.
Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were purchased from Cayman Chemical as solutions in ethanol. For these experiments, after evaporating the ethanol solvent under a gentle nitrogen stream, DHA and EPA were delivered in a soybean oil control vehicle (Crisco Pure Vegetable Oil) containing the anti-oxidant vitamin E. Soybean oil was chosen as the control vehicle for its low omega-3 to omega-6 PUFA ratio of 0.14, which is similar to the mice’s background diet. Acetylsalicylic acid (ASA) was purchased from Sigma. All treatments were delivered daily to the mice via oral gavage, on the day before surgery through post-operative day 12. Mice in the appropriate treatment groups received approximately 200 mg/kg of DHA/EPA and 30 mg/kg of ASA per treatment. Dosing estimates were based on, the average mass of an adult C57BL/6 mouse of 26 g. Control groups received soybean oil over the same time span. DHA and EPA were delivered together in the soybean oil carrier. ASA was delivered in water.
STNI surgery was performed under 2–3% isofluorane anesthesia, as described by Shields. The left hind limb was immobilized in a lateral position. After skin incision at the mid-thigh level and dissection through the underlying muscle, the sciatic nerve trifurcation was exposed. The common peroneal and sural nerve branches were tightly ligated with 6–0 silk sutures and then severed. Throughout the procedure, the tibial nerve was preserved by carefully avoiding any stretch or nerve contact. For sham surgeries, the sciatic nerve trifurcation was exposed without imposing any nerve injury.
Mice were habituated to the testing environment for 2 days prior to baseline testing and for at least 30 min on each subsequent test day. The mice were placed in plastic boxes on an elevated wire-mesh apparatus. Mechanical allodynia was assessed by stimulating the left hind paw with Von Frey filaments of logarithmically increasing stiffness (0.04–2.00 g, Stoelting Co, Wood Dale, IL), applied perpendicularly to the plantar surface. Specifically, the hindpaw was stimulated in the distribution of the tibial nerve, in the center of the plantar surface. The 50% paw withdrawal thresholds were determined using the up-down method of Dixon. Testing was performed by a blinded researcher at baseline, post-operative day (POD) 3 and every following third day, finishing on POD 21.
Statistical analysis of mouse behavioral data was performed in GraphPad. Paw withdrawal thresholds were normalized to baseline and are presented as mean percentage of baseline with standard error of the mean. Missing data was imputed with the mean of the shared treatment group’s paw withdrawal threshold for the time point. The paw withdrawal thresholds of the treatment groups were compared using ANOVA and ad hoc Tukey tests corrected for multiple comparisons.