Nanopump

Focal demyelination was induced via stereotactic injection of L-α-Lysophosphatidylcholine (LPC, L4129, Sigma-Aldrich) into the corpus callosum of 12–14 week old C57BL/6 male mice (n=2–3 per time point). Anesthetized animals received 2μL of 1% LPC through a burr hole in the skull and at stereotactic coordinates 1.2 mm posterior, 0.5 mm lateral, 1.4 mm deep to the bregma over 4 minutes using a 30 gauge needle attached to a Hamilton syringe and driven by a Nano pump (KD Scientific Inc., Holliston, MA). The needle was additionally left on site for additional 4 minutes to avoid backflow. Similar stereotactic injection of PBS was followed to create a surgical control (PBS control). LPC causes reversible focal demyelination without axonal loss. The time course of de- and remyelination is well established and reproducible with demyelinated lesions appearing as early as 3 days post injection (3dpi). Two time points with early (14 dpi) and late (28 dpi) remyelination were analyzed. Mice were subsequently perfused with 4% PFA, the brain tissue was harvested, cryoprotected in 30% sucrose and frozen embedded in OCT compound.

Rats were anesthetized with ketamine (75 mg/kg) and xylazine (10 mg/kg). The skull was exposed and holes were drilled in the skull bilaterally above the PL. A Hamilton syringe with a 33 gauge needle, mounted onto a nanopump (KD Scientific, Holliston, MA), was stereotactically inserted into the PL (14 degree angle towards midline, 2.8 mm anterior to Bregma, 1.45 mm lateral from midline, 3.7 mm ventral). Recombinant CTb conjugated with Alexa Fluor-555 (0.5% in PBS, 0.5 µL per side; Invitrogen, Waltham, MA) was microinjected at a rate of 0.1 µL/min. The needle was left in place an additional 5 min following microinjection to ensure complete diffusion of the tracer, and then slowly retracted. The scalp was sutured. One week after injection, the rats were transcardially perfused with 4% paraformaldehyde in PBS pH7.4, and their brains were post-fixed in this solution overnight at 4°C. 40 µm brain sections were collected by a vibratome and mounted with Prolong Diamond antifade mountant with DAPI (Invitrogen, Waltham, MA). Images were collected by the Olympus VS120 virtual slide microscope (Olympus, Tokyo, Japan).