6 hydroxydopamine

Sixty male rats were anesthetized and placed in a stereotaxic frame (Narishige, Tokyo, Japan). We established the 6-hydroxydopamine (12 μL) PD model using the two-point method (Teng et al., 2015). The coordinates of two points in the brain were determined according to the Rat Brain Stereotactic Atlas (Bao and Shu, 1991), as follows: (1) substantia nigra pars compacta: 4.8 mm behind the anterior fontanelle, 2.0 mm from the right sagittal suture, and 8.0 mm below the dura; (2) ventral tegmental area: 4.8 mm behind the anterior fontanelle, 1.2 mm from the right sagittal suture and 8.2 mm below the dura. We punctured the skull using a dental drill and injected 6 μg 6-hydroxydopamine (Sigma, St. Louis, MO, USA) into each region at a rate of 1 μL/min. The needle was retained in place for 10 minutes before being removed. The incision was closed and the animals allowed to recover. Rats were injected with apomorphine (0.5 mg/kg intraperitoneally; Sigma) on the 14^th day after modeling. Rats that constantly rotated to the left more than seven times per minute were regarded as successful models of PD (Li et al., 2010). In total, 36 rats were successful models and used in the subsequent experiments. The remaining 12 naïve male rats were used as controls and underwent the same procedures but were injected with 6 μL normal saline instead of 6-hydroxydopamine.

A rodent model of Parkinson’s disease was established by creating a unilateral lesion by stereotaxic injection of 6-hydroxydopamine (Sigma-Aldrich). The stereotaxic surgery was conducted as described by Gomes et al.29 (link) Wistar rats (200–240 g) were anesthetized with a solution of ketamine/xylazine (0.15 mL/100 g body weight of a solution containing ketamine 10% and 70 μL of xylazine 2%) and submitted to stereotaxic injections with 6-hydroxydopamine 16 μg in 3 μL (of saline containing 0.05% ascorbic acid) into the right medial forebrain bundle, according to the following coordinates: anteroposterior −4.4 mm, medial-lateral +1.2 mm, and dorsal-ventral −8.2 mm in relation to bregma.30 On day 28 after the stereotaxic procedure, the animals were injected with stem cells on the right side of the dorsolateral striatum, according to the following coordinates: anteroposterior +0.7 mm, medial-lateral +3.0 mm, and dorsal-ventral −4.5 mm in relation to bregma. The stem cells was slowly injected (0.2 μL per minute) unilaterally into the adult rat striatum using a 10 μL Hamilton syringe mounted on a stereotactic apparatus (David Kopf Instruments, Tujunga, CA, USA).

Polyclonal anti-β-END antibody was obtained from rabbit antisera and purified to a final concentration of 8 mg/mL using staphylococcal protein A-Sepharose chromatography. Thirty minutes before each EA intervention, rats in the anti-β-END + EA group were injected with 110 µL anti-β-END antibody at the site with the lowest mechanical pain threshold.
6-hydroxydopamine (Sigma-Aldrich) was dissolved in saline containing 0.2% (w/v) ascorbic acid (Sigma-Aldrich) at a concentration of 100 mg/mL. We performed local chemical sympathectomy 30 hours before the first EA intervention and 6 hours before each intervention by injecting 100 µL 6-OHDA solution into the lowest mechanical pain threshold site of 6-OHDA + EA group rats.