Sys micro4 controller

Ischemic damage was induced by injecting the vasoactive peptide endothelin-1 either onto the proximal branches of the middle cerebral artery along the lateral aspect of the frontal cortex or into the subcortical territory within and surrounding the internal capsule as previously described^{11 (link)–12 (link), 25 (link)}. The hemisphere contralateral to each animal’s preferred reaching paw was targeted for injury. Animals were anesthetized with ketamine hydrochloride (70 mg/kg; i.p.) and xylazine (5 mg/kg; i.p.) with supplemental isofluorane (0.15%) and ketamine (20 mg/kg; i.p.). Under sterile conditions, a midline incision was made and burr holes created over the injections sites. Endothelin-1 (0.2 μg/μL American Peptide, Sunnyvale) was delivered by the Nanolitre injection system (World Precision Instruments, Sarasota, Fl) using a SYS-Micro 4 Controller (World Precision Instruments, Sarasota, Fl). Stereotaxic coordinates of the injection site for MCAo were: anteroposterior, +0.9 mm; mediolateral, −5.2mm; and dorsoventral, −8.6 mm with respect to bregma^{25 (link)} whereas for SCII were: anteroposterior, −3.0 mm; mediolateral, −3.0 mm; and dorsoventral, −7.0 mm with respect to bregma^{11 (link)–12 (link)}. Doses of endothelin-1 were 3 μl for MCAo (240 pMol dissolved in 0.9% sterile saline) and1 μl for capsular injury (80 pMol dissolved in 0.9% sterile saline).

We bilaterally injected AAV8-hSynp-hM4Di-mCherry [Exp. 5 and Exp.6] (UNC vector core) into AIV: AP, +2.8; ML, ±4.9 (10°angle); DV, –6.2 mm from Bregma. We injected 0.75 µl over 5 min and left the needle in place for 5 min. We used 10 µl Nanofil syringes (World Precision Instruments) with 33-gauge needles, attached to an UltraMicroPump (UMP3) with SYS-Micro4 Controller (World Precision Instruments).

Virus particles (AAV8-U6-miRNA-Scramble-CMV-GFP or AAV8-U6-miR29b-CMV-GFP, SignaGen) were delivered to the anticipated ischemic territory (striatum) and penumbra (somatosensory cortex) in wild-type C57BL/6 animals, age 3.5–4 weeks, at a total of 6 injection sites with the following coordinates: Lateral (right) 3.5–4.0mm, Caudal 0.0–0.3 mm from bregma, at depths 0.8, 1.2, 2.0 mm, and Lateral (right) 4.0–4.5 mm, Rostral 0.0–0.5 mm from bregma, at depths 0.8, 1.2, and 2.0mm. A 10ul Hamilton syringe (Model 1701 RN Neuros Syringe 33G, tip: point 4, 60 degree cut) connected to a microinjector (UltraMicroPump UMP3, SYS-Micro4Controller, World Precision Instruments) was used to deliver a total of 1200ul in each animal (200ul per injection site) at a rate of 40 nanoliters/min. MCAo was performed ~4wks after AAV injection. A total of 8 animals were injected with AAV-miR29b and 6 with AAV-Scramble for were assayed by MRI/MRS at indicated stroke time points. After obtaining MRS measurements, 3 AAV-miR29b, and 3 AAV-Scramble injected animals were perfused for immunohistochemistry, remaining animals were used for cortical RNA and protein analysis.

The sequences for the 18-mer endcapped phosphorothioate ODNs for GR and MR, verified by BLAST search and based on previous literature (Engelmann et al., 1998 (link); Sakai et al., 2000 (link); Wang et al., 2004 (link); Wang et al., 2005 (link); Ferrari et al., 2013 (link)), used in this study were: GR antisense ODN (ASO): 5′-TGG AGT CCA TTG GCA AAT-3′, GR random sequence ODN (RSO): 5′-TGA AGT TCA GTG TCA ACT-3′; MR ASO: 5′-TTC CAT GTC TAG GCC TTC-3′, MR RSO: 5′-CAT TTT GAA GGT TCC GGT-3′. All ODN were synthesized by Eurofins MWG Operon (Huntsville, AL, USA) and supplied as dried, salt-free stocks that were resuspended in artificial cerebrospinal fluid (150 mM NaCl, 3.0 mM KCl, 1.4 mM CaCl₂, 0.8 mM MgCl₂, 0.8 mM Na₂HPO₄, 0.2 mM NaH₂PO₄, pH 7.4) to a concentration of 6 μg/μl. For daily infusions, rats were anesthetized with 2% isoflurane inhalation, before using a 33-gauge injector, 0.5 mm longer than the guide cannula, to deliver 0.5 μl of ODN to each CeA (1 μl/rat/day) at a rate of 0.1 μl/min via a Hamilton microsyringe attached to an UltraMicroPump with a SYS-Micro4 controller (World Precision Instruments, Sarasota, FL, USA). Injectors were left in place for an additional 2 min to allow for diffusion of the ODN solution before being withdrawn.