Mouse brain matrix

Fresh brains were dissected from adult wild-type male animals and 1 mm coronal slices were collected using a mouse brain matrix (Zivic Instruments). SCN samples were collected with a 1 mm in diameter tissue punch, frozen in liquid nitrogen, and stored at −80°C until further processing. Tissue punches were homogenized by sonication in 50 μl of 0.4 N perchloric acid solution. The homogenate was centrifuged at 15,000g for 12 min at 4°C. 25 μl of the resulting supernatant was loaded into an auto sampler connected to a high-performance liquid chromatography instrument with an electrochemical detector (Decade, Antec Leyden B.V., Zoeterwoude, The Netherlands) to measure the levels of dopamine. Retention time for DA was determined through comparison with DA standards.

Infarct size was determined 3–5 days after stroke by staining with a 2% triphenyl tetrazolium chloride (TTC; gr/mL, made in PBS; Sigma-Aldrich, St Louis, MO) solution for 40 minutes at 37°C. The brain was then cut in 1 mm sections using a mouse Brain Matrix (Zivic Instruments, Pittsburgh, PA) and imaged using a stereomicroscope. The same preparation was used to quantify hydrogel volume one day after injection. For immunohistochemical studies mice were euthanized at 2 weeks or 6 weeks after cell or gels or cells in gels injections and perfused with 4% paraformaldehyde (Fisher Scientific, Pittsburgh, PA; in PBS, pH: 7.4) and cryoprotected in sucrose for 48 hours. Brains were cut on cryostat at 16 μm intervals and mounted on gelatin-coated Superfrost^® slides (Fisher Scientific).

Locus coeruleus adra2a mRNA levels were determined from adra2a KD and scramble AAV-injected mice by real time (quantitative, q)-PCR (n=4), using a Taqman® RNA-to-CTTM1-Step kit (Applied Biosystems, Foster City, USA). Briefly, brains were removed and 1-mm tissue punches were collected using 1-mm interval mouse brain matrix (Zivic Instruments, Pittsburgh, PA, USA) and 1-mm core diameter hollow needles. Total RNA was extracted from frozen tissues using TRIzol. qPCR was performed on an ABI StepOne Plus Real Time PCR system (Applied Biosystems, Foster City, USA) using the adra2a primers (Applied Biosystems, Mm00845983_s1, described previously⁴³ . Data were evaluated with SDS 2.1 software, using the Comparative CT method (ΔΔCT) to measure gene expression. Relative expression of the adra2a mRNA was determined by comparing AAV-shRNA mRNA levels in the knockdown group to those in AAV-scramble, mice and normalized to expression of tyrosine hydroxylase (TH) mRNA (TH primers were:Applied Biosystems, Mm00447557_m1).

At 7 days post-TETS exposure, a subset of mice imaged in this study was anesthetized with 4–5% isoflurane in medical-grade oxygen, perfused with ~25 mL of 4% (w/v) paraformaldehyde (PFA; Sigma; St. Louis, MO, USA) in phosphate-buffered saline (PBS; 3.6 mM Na₂HPO₄, 1.4 mM NaH₂PO₄, 150 mM NaCl; pH 7.2) using a Peri-Star Pro peristaltic pump (5 mL/min). Brains were removed, and the freshly perfused whole brains were immediately placed into 20 mL glass scintillation vials containing 10 mL of 4% (w/v) PFA in PBS (pH 7.2). After 24 h, whole brains were transferred into 30% (w/v) sucrose (Fisher; Houston, TX, USA) in PBS (3.6 mM Na₂HPO₄, 1.4 mM NaH₂PO₄, 150 mM NaCl; pH 7.2) for an additional 48 h or until the tissue sank completely to the bottom of the vial. Fixed whole brains were serially sectioned into 2-mm thick coronal slices using a mouse brain matrix (Zivic Instruments, #5325; Pittsburgh, PA, USA) and embedded by flash freezing in optimal cutting temperature medium (OCT; Fisher HealthCare; Waltham, MA, USA). Blocked brain sections were stored at −80 °C until cryosectioned using a Microm HM550 cryostat (Thermo Fisher Scientific, Waltham, MA, USA) into 10-μm thick slices on Superfrost plus microscope slides (Fisher HealthCare). Slides were stored at −80 °C prior to staining or immunohistochemistry.