*2.5. Hot Plate Test*

The hot plate test was performed on day 0 and 3, 5, 7, 14, and 21 days post-injection. The hot-plate latency was evaluated using a metal surface maintained at 53.6 ◦C (Ugo Basile, Milan, Italy). The rat was monitored and the licking of a hind paw was acquired as the end point. Maximal latency accepted was 45 s [42].

#### *2.6. The Tail-Flick Warm Water Test*

The tail-flick warm water test was performed on day 0 and 3, 5, 7, 14, and 21 days post-injection. The warm water tail-flick test was employed to evaluate pain threshold. 4 cm of the rat-tail was located in 50 ± 0.5 ◦C warm water and the time between tail input and retraction was noted (three tests were conducted and the average in units of seconds was recorded). The latency was assessed with a sensitivity of 0.01 s. A maximum tail-flick latency of 10 s was employed to minimize tissue damage to the tail [42].

### *2.7. Forced Swimming Test (FST)*

The forced swimming test (FST) was performed on day 0 and 3, 5, 7, 14, and 21 days post-injection according to the original method by Porsolt et al. [43] and modified by Detke and Lucki [44]. Each rat was individually placed in a plexiglass cylinder for 5 min. It was considered immobile when it remained floating in the water making only essential movements to keep its head above water. The total duration of immobility was recorded as immobility time (sec/5 min).

## *2.8. Estimation of Lipid Peroxidation*

Twenty-one days after reserpine injection brain tissues were harvested and the malondialdehyde content, an indicator of lipid peroxidation, was measured in the form of thiobarbituric acid-reactive substances by the method of Wills [45]. Briefly, 0.5 mL of cytosolic fraction of brains and 0.5 mL of Tris-HCl were incubated at 37 ◦C for 2 h. After incubation 1 mL of 10% trichloroacetic acid was added and centrifuged at 1000× *g* for 10 min. Then 1 mL of 0.67% thiobarbituric acid was added to 1 mL of supernatant and the tubes were kept in boiling water for 10 min. After cooling, 1 mL double-distilled water was added and absorbance was measured at 532 nm. Thiobarbituric acid-reactive substances were quantified using an extinction coefficient of 1.56 × 105 M−<sup>1</sup> cm<sup>−</sup><sup>1</sup> and expressed as nmol of malondialdehyde per mg protein.

#### *2.9. Estimation of Non Protein Thiols*

Twenty-one days after reserpine injection brain tissues were harvested and non protein thiols were calculated by the method of Jollow [46]. Briefly, 1.0 mL of cytosolic fraction of brain tissues were precipitated with 1.0 mL of sulphosalicylic acid (4%). The samples were kept at 4 ◦C for at least 1 h and then subjected to centrifugation at 1200× *g* for 15 min at 4 ◦C. The assay mixture contained 0.1 mL supernatant, 2.7 mL phosphate buffer (0.1 M, pH 7.4) and 0.2 mL 5,5-dithiobis- (2-nitrobenzoic acid) (Ellman's reagent, 0.1 mM, pH 8.0) in a total volume of 3.0 mL. Samples was read at 412 nm and the reduced glutathione levels were reported as mmol/mg protein.

## *2.10. Estimation of Superoxide Dismutase*

Twenty-one days after reserpine injection brain tissues were harvested and superoxide dismutase activity was assayed by the method of Kono [47]. The assay system consisted of 0.1 mM EDTA, 50 mM sodium carbonate, and 96 mM of nitro-blue tetrazolium (NBT). In a cuvette, 2 mL of the above mixture was taken and 0.05 mL of cytosolic fraction of brains and 0.05 mL of hydroxylamine hydrochloride (adjusted to pH 6.0 with NaOH) were added to it. The auto-oxidation of hydroxylamine was observed by measuring the change in optical density at 560 nm for 2 min at 30-/60-s intervals.
