Pups were anesthetized with isoflurane (3–5%) in oxygen and air. Percutaneous intrathecal injections were made at the low lumbar level (intervertebral space L4–5 or L5–L6) with a 30-gauge needle perpendicular to the skin. Injectate volumes of 0.5 or 1.0 mcl per gram bodyweight were delivered using a hand-driven micro injector (P3 and P10) or a 50 mcl Hamilton syringe (P21). As previously described in adult rats, intrathecal placement was suggested by a lateral tail flick as the needle entered the subarachnoid space33 (link) and confirmed by the distribution of 5% methylene blue in the injectate (Fisher Scientific, Fair Lawn, NJ). Within 2 h of injection animals were given intraperitoneal 100mg/kg pentobarbital, exsanguinated by cardiac puncture, and the spinal cord dissected. Intrathecal injections were defined by staining that was limited to the spinal cord and cerebrospinal fluid without pooling of dye in the epidural space or within paravertebral tissues. In most cases the injection site through the dura could be visualized but there was no obvious damage to underlying structures. The spread of dye was assessed by microscopic visualization and expressed as the number of vertebral segments above the injection level.
To assess intrathecal injectate distribution in vivo, P3 and P10 rats received intrathecal injections of 50% SAIVI™ Alexa Fluor® 680 in bovine serum albumin (Invitrogen, Eugene, OR) diluted in sterile saline. Injected volumes were 0.5, 1.0 or 1.5 mcl/g bodyweight. Rats were anesthetized with isoflurane anesthesia (1–2% in air/oxygen) and body temperature was maintained with a thermostatically controlled heat pad, and images were obtained at 30-min intervals for 2 h by the Xenogen®IVIS 100, in-vivo imaging system (Caliper Life Sciences, Hopkinton, MA).