Rat CSF sample collection

Rats were anesthetized using intraperitoneal administration of an injectable cocktail of medetomidine (0.5 mg/kg) and ketamine (75 mg/kg). Once the animal was deeply anesthetized, as indicated by the absence of withdrawal reflexes (tail and limbs) and the eye positioning reflex, the surgical area was shaved and the animal was secured in the stereotaxic frame with the head tilted at roughly 45°. The surgical area was then cleaned with Hibiscrub and a surgical drape was placed around the operating area with a hole to expose only the surgical area. A skin incision along the midline of the skull extending from between the eyes to 3-4 cm caudally to make sure the back of the neck is fully exposed. The fascia and the superficial and deep layers of the neck muscles were then dissected to expose the membrane of the dura mater at the atlanto-occipital joint between the occipital condyles and the rostral facets of atlas. The cisterna magna was then carefully pierced by a pulled glass pipette (1 cm long) connected to a 2.5 mL syringe through 30 cm of PE-50 tubing. A small volume of CSF entered the glass pipette through the capillary action and the flow was maintained by gently pulling the plunger. The CSF was collected into cryoprotective tubes and snap-frozen immediately in liquid nitrogen. Animals were then given a lethal dose of anesthesia, decapitated, and the brain was exposed and the areas of interest were dissected and snap-frozen in liquid nitrogen.