2.4. BDA Injection into Sensorimotor Cortex that Projects to Low Thoracic/Lumbar Spinal Cord

Injections of biotinylated dextran amine were spaced in virtually identical positions as the earlier AAV-CNTF^mCherry or AAV-GFP transduction injections in order to cover the same hindlimb projection fields in the sensorimotor cortex. For each of the 4 injections, 0.5–1 μl of 10% (w/v) BDA were injected using a nanojet device (World Precision Instruments) at a depth of 1 mm. The location of these injections is based on studies showing the location of CMN that project to the hindlimbs and forelimbs in Fischer rats [2] and confirmed by the examination of the label in appropriate thalamic motor nuclei. In some rats, gelfoam soaked in the BDA solution was placed over the exposed cortex prior to the closure of the craniotomy.

A variety of behavioural tests for injured rats were used to give a valid indication of functional recovery [24, 25, 32–35] including (i) “open-field” locomotion test (BBB scoring method [36] to assess spontaneous movements), (ii) ladder walking [37] to assess general hindlimb recovery, and (iii) our own novel computerized quantitative gait analysis method (Ratwalk® [38]) which allows objective analyses of a large number of locomotion parameters, such as interlimb coordination (step sequence), stride length, step length, and base of support (stance width) [38]. All functional (locomotor) behaviour was assessed weekly for up to day 56 after SCI. BBB scoring was analysed using at least 3 blinded raters both at the time of assessment and later in silica via slow motion/frame-by-frame replay of high definition (1080 p) digital recordings taken during the test. The BBB rating score is composed of 22 nonlinear operational definitions (0–21 scale) studying several aspects involved in the locomotion of quadrupedal animals such as weight support, plantar stepping, and forelimb/hindlimb coordination. Ladderwalk and Ratwalk assays were performed on animals once they had reached weight support on their hindlimbs, and involved comparison of preinjury performance with weekly assessments from day 14 post-SCI until day 56 (endpoint). Digital recordings of animals traversing 3 lengths per time point of Ladderwalk and Ratwalk (preinjury and weekly up to day 56 post-SCI) were also similarly assessed by at least 2 blinded raters in silica via slow motion/frame-by-frame replay of digital recordings taken during the tests for preinjury and up to day 56 post-SCI. Ladderwalk scores are an average of missteps over a 1 m horizontal ladder with unevenly spaced bars. Ratwalk data analysis involves frame by frame designation from digital recordings of left and right fore- and hindlimb placement on a 1 m glass platform in the Ratwalk apparatus in low light, using an average of 3 “runs” per time point [38].