The tissues were homogenized and centrifuged to obtain the supernatant. Nitrate (NO3 - ) was converted into nitrite (NO2 - ) after reacting with nitrate reductase.Total nitrite was measured using Griess reagent with 1:1 ratio and read at OD 540 nm. The sum of nitrite and nitrates obtained from this procedure represented the NO concentration in paw tissue (μM/g wet weight of tissue).