Stereotaxic apparatus

For stereotaxic surgery, animals were anesthetized with a combination of ketamine and xylazine (80 and 5 mg/kg, ip; respectively) and placed in a stoelting stereotaxic apparatus (stoelting, USA) in the flat skull position. The small central incision was made to make the skull appear. A 23 gauge sterile cannula was inserted into the injection site as a guide cannula for subsequent insertion of the injection tube into the striatum. The coordinates for this position, with reference to the atlas of Paxinos &Watson,¹⁹ were: anteroposterior from bregma (AP)= 0.4 mm, mediolateral from the midline (ML)= 2.8 mm and dorsoventral from the skull (DV)= -5 mm. Subsequently, 6-OHDA (10 μg/ rat in 2 μl saline containing 0.2% ascorbic acid) was infused by an infusion pump at the flow rate of 0.2 μl/min into the right striatum. Lesioned rats were subjected to the designed protocols after a 3-week recovery period. All of these procedures were performed for sham-operated animals, but they only received intra-striatal of 2 μl vehicle of 6-OHDA (0.9% saline containing 0.2% (w/v) ascorbic acid).

The mice were anesthetized with pentobarbital sodium (80 mg/kg, Sigma-Aldrich) and fixed to a stereotaxic apparatus (Stoelting). The guide cannula (RWD Life Science) was unilaterally implanted into the right GP [(Anterior–Posterior (AP): −0.3 mm, Medial–Lateral (ML): +1.9 mm, Dorsal-Ventral (DV): 2.6 mm) (according to the mouse brain in Stereotaxic Coordinates; Paxinos and Franklin, 2001 )]. Four skull screw holes were drilled, and tightly fitting screws were driven through the skull until the surface of the dura was reached. Both the cannula and the stainless steel anchoring screws were fixed to the skull with dental cement. After the surgical procedures, the animals were allowed to recover in individual chambers for at least 7 days. During experimentation, each animal was transferred to a chamber and connected to an optical fiber.

In Experiment 1, EEG after ROSC was monitored, as reported previously [19 ]. Briefly, before intubation and cannulation procedures, animals underwent implantation of EEG electrodes bilaterally under isoflurane, using a stereotaxic apparatus (Stoelting, Chicago, IL, USA). Each animal had five screw electrodes (Plastics One, Roanoke, VA, USA) cortically implanted 2 mm lateral and 2 mm anterior or posterior to the bregma. In addition, a ground electrode was placed at 3 mm lateral on the right and 9 mm anterior to the bregma in the midline. EEGs were recorded using an Intan RHS Stim/Recording 16 channel recording controller (Intan Technologies, Los Angeles, CA, USA) during the baseline, asphyxial CA, resuscitation, and after ROSC. Raw EEG signals were used to determine the electrical activity. In addition, the durations between ROSC and onset of both EEG amplitude activity (defined as >5% of the basal value with no EEG activity), as well as continuous background EEG as markers of brain’s electrical recovery after CA/CPR, were also recorded [20 (link)]. Continuous background EEG activity was defended as continuous EEG activity/burst without low amplitude activity (suppression, <10 µV). EEG analysis was performed offline using MATLAB 7.0 (MathWorks, Inc., Natick, MA, USA) after the experiment was completed.

Rats were anesthetized by injection of ketamine (100 mg/kg) and xylazine (5 mg/kg), intraperitoneally. Then, 4 μL of 6-OHDA dissolved in isotonic saline containing 0.2 mg/mL of ascorbic acid was administrated into 4 sites in the right striatum using stereotaxic apparatus (Stoelting, USA) by a 10-μL Hamilton syringe. Coordinates for 6-OHDA injections were AP: 1.5, L: −2.5, DV: −6, and AP: 0.8, L: −3, DV: −6, and AP: 0.1, L: −3.2, DV: −6, and AP: −0.5, L: −3.6, DV: −6. AP and L were evaluated from bregma and DV was assessed from the surface of skull according to the atlas of Paxinos and Watson (2007) . In the end of surgery, the needle of the Hamilton syringe was left in the brain for an additional 5 minutes and, then, withdrawn at a rate of 1 mm/min.

Cryoanesthetized P1–3 rat pups were injected with a total amount of 1 μl of AAV1-hSynap-eGFP-WPRE-bGH (titer 10¹¹ copies/ml; Hutson et al., 2012 (link)) into four sites of right sensorimotor cortex (0.25 μl/site; coordinates from Bregma: 1.5 mm ML: 0.7 mm AP; 1 mm ML: 0.3 mm AP; 1 mm ML: 0.0 mm AP; 2 mm ML: 0.5 mm AP). Briefly, animals were fixed on a stereotaxic apparatus (Cunningham, Stoelting Co., Wood Dale, IL, USA) previously cooled with dry ice and viral injections were performed by using a pneumatic ejection pump (PDES-02TX, npi) attached with a pulled glass micropipette (30 μm tip). Pups were then placed under a heating lamp until they were fully awake and transferred to the home cage with their mothers.