Stereotaxic surgeries

In the first stereotaxic surgery, the rats received 3 unilateral 6-OHDA micro injections (each 3μg) into the right dorsal striatum as described previously [54]. In the second surgery two weeks later, an infusion catheter was implanted into the right dorsal striatum in order to make site specific infusions of the compounds used in the study (Fig. 4B; for detailed information see below).

BT44 (0.3μg/24h) and GDNF (3μg/24h) alleviate amphetamine-induced rotational asymmetry in 6-OHDA lesioned rats. Design of the in vivo experiment with 6-OHDA lesioned hemiparkinsonian rats (A). Unilateral striatal 6-OHDA injections, striatal drug delivery with osmotic pumps, time points for amphetamine-induced turning behavior (Amph I-III) and cylinder (Cylinder I and II) tests, and perfusion time points are depicted on the timeline. Schematic illustration of 6-OHDA injection sites and treatment infusion site in the dorsal striatum (B). All rats received 3 deposits of 6-OHDA (3μg/deposit) along the rostrocaudal axis of the right striatum. The syringes indicate the 6-OHDA injection sites and the vertical cannula the treatment infusion site. Amphetamine-induced rotational asymmetry (cumulative data for 120 min) at 2 weeks (C), 6 weeks (D), and 12 weeks (E) after 6-OHDA lesion. Rotation rate per 5 min at 12 weeks post lesion (F). PBS, phosphate buffered saline; PG, propylene glycol. ^*p < 0.05, ^***p < 0.001 vs. PG; ^#p < 0.05, ^# # #p < 0.001 vs. PBS; ^†††p < 0.001 vs. GDNF, Tukey HSD after one-way ANOVA (D, E). ^**p < 0.01 BT44 0.3μg/24 h vs. PG; ^#p < 0.05 BT44 0.3μg/24 h vs. PBS, Tukey HSD after RM ANOVA (F). Mean±SEM, n = 8–11 per group.

All rats received a unilateral injection of 6-OHDA (6-hydroxydopamine hydrochloride, Sigma-Aldrich) into 3 different sites in the right dorsal striatum (A/P + 1.6, L/M –2.8, D/V –6.0; A/P 0.0, L/M –4.1, D/V –5.5; and A/P –1.2, L/M –4.5, D/V –5.5, mm relative to the bregma, according to the rat brain atlas [55]). The dose of 6-OHDA injected to each site was 3μg (calculated as free base) in 1.5μl of ice-cold, de-oxygenated, saline with 0.02% ascorbic acid (the concentration of solution was 2μg/μl). All surgical procedures were performed under general isoflurane (Vetflurane^® 1000 mg/g, Virbac, France) anesthesia (4.5% during induction and 2-3% during maintenance). Rats were placed in a stereotaxic frame (Stoelting, USA) and a small amount of lidocaine-adrenalin-solution (10 mg/ml; Orion Pharma, Finland) was injected under the scalp for local anesthesia and to prevent bleeding. The skull was exposed and burr holes were made using a high-speed drill. The 6-OHDA solution was injected at the flow rate of 0.5μl/min using an electronic injector (Quintessential stereotactic injector, Stoelting) and a 10μl -needle (NanoFil 33G, World Precision Instruments, USA). The needle was lowered into the brain at a 10° angle to avoid lateral ventricles. At the completion of each injection, the needle was kept in place for 5 min to minimize backflow of the solution. Desipramine (15 mg/kg i.p.; calculated as free base; Sigma-Aldrich) was administrated 30 min before the 6-OHDA injections to prevent the uptake of 6-OHDA into noradrenergic and serotonergic nerve terminals. One group of animals (lesion control group, n = 4) was sacrificed 2 weeks after the lesion. This group served as a reference showing the extent of dopamine neuron degeneration at the time of the treatment infusion initiation.

Rats were assigned into equal treatment groups according to the first amphetamine-induced rotation rate at 12 days after the 6-OHDA lesion (see below in the Materials and Methods and Fig. 4A and C). Subsequently, osmotic infusion pumps were implanted in the second stereotaxic surgery and treatment infusions were started 2 weeks after the 6-OHDA injections. The rats were anesthetized and the skull was exposed. A brain infusion cannula (Alzet Brain infusion kit no. 2, Durect, USA) was implanted to coordinates relative to the bregma A/P + 0.2; L/M –3.0; D/V –5.0 mm and secured to the skull with 3 stainless steel screws and polycarboxylate cement (Aqualox, VOCO, Germany). The tip of the cannula was placed in the right dorsal striatum, in the middle of the 6-OHDA injection sites (Fig. 4B). The cannula was connected via a 3 cm-long catheter tubing to an osmotic infusion pump (Alzet osmotic pump model 2002, Durect) which was placed into a subcutaneous pocket between the scapulae. The pump constantly delivered BT44 0.1μg/24 h (n = 10), BT44 0.3μg/24 h (n = 10), GDNF 3μg/24 h (n = 11, positive control), PG (n = 11, vehicle control) or PBS (n = 9, vehicle control) at a flow rate of 0.5μl/h for 14 days. At the end of the treatment infusions, 14 days after the implantation of the pumps, the rats were anesthetized and fixed in the stereotaxic frame for the third time. The osmotic pumps, infusion cannulas, and screws were removed, and the incision was cleaned, disinfected, and sutured.

Before every stereotaxic surgery the rats rec-eived buprenorphine 0.05 mg/kg s.c. (Temgesic^® 0.3 mg/ml, Indivior UK Limited, United Kingdom) for analgesia. Carprofen 5 mg/kg (Rimadyl Vet^® 50 mg/ml, Zoetis, USA) was injected s.c. immediately after the surgeries to relieve postoperative pain. Additional doses of buprenorphine and carprofen were given 1 day after the surgeries.