4.3. Chemical Denervation

In this study, 6-OHDA has been used for sympathetic denervation by others in various studies [6,45,46]. To achieve effective denervation, we tested different dosages of 6-OHDA including one-time 200 mg/kg, two-times 100 mg/kg, two-times 50 mg/kg, and three-times 80 mg/kg in our preliminary experiments (data not shown). We speculated that a higher dosage of 6-OHDA might be harmful to the animals; therefore, we gave lower doses of repetitive injections. Three-times 80 mg/kg 6-OHDA was the only dosage effective in depleting TH⁺ fibers. Further, these fibers were absent for at least 60 days after three-times 80 mg/kg 6-OHDA treatment. Therefore, we used this dosage for all the subsequent analyses. Mice were injected intraperitoneally with sterile 80 mg/kg 6-hydroxydopamine hydrobromide dissolved (Sigma Aldrich, Saint Louis, MO, USA) in 0.1% l-ascorbic acid-containing saline for 3 consecutive days. The vehicle group was injected with only sterile saline for 3 days. Two weeks after the last 6-OHDA injection, mice were sacrificed and checked for the absence of TH⁺ neurons by immunofluorescence staining.