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CHAPTER 3 connective tissues and bathed in paraffin oil. Silver wire bipolar hook electrodes were used to stimulate sural nerves and a single hook silver wire electrode was used to record from sciatic nerves to ensure complete recruitment of all sural nerve fibres upon electrical stimulation (square wave pulse, 0.5-1.1 mA, 0.05 ms). A platinum wire electrode was used to record from a single midline position on the brainstem at a location that was established to provide evoked potentials of equal magnitude and latency from left and right sural nerve stimulation. 33 individual evoked potentials were recorded and averaged from the sciatic nerve and the brainstem in response to repeated sural nerve stimulations. Signals recorded from the brainstem were then processed offline (MATLAB, MathWorks). The averaged signal was band-pass filtered (500 Hz – 3350 Hz) and response magnitudes calculated from the integral of rectified signals (integral limits: 5.00 ms before and 8.75 ms after the primary peak) after subtraction from baseline levels obtained prior to the stimulus. Latency was measured from the filtered signal where it first exceeded 3 standard deviations (confidence interval 99.7%) of background levels. 3.3.7 Locomotor assessment Prior to surgery, animals were trained to run along an 80 cm custom build transparent walking- track with mirrors that reflected left and right sides and underneath of the animal. This enabled exquisite locomotor detail from all sides of interest to be video captured simultaneously from a single viewpoint. 2 h following surgery, initial recordings of animals running 3 consecutive times down the walking-track were acquired with a digital camera (Sony, NEX-VG20EH) at 50 frames per second, which provided adequate data for detailed gait analysis. Recordings were repeated every 24 hrs post-surgery for 7 consecutive days. Each video file was coded and assessed blind by one assessor. The BBB locomotor scale (Basso et al., 1995) for the left and right hind-limbs was used to generate locomotor scores from video files assessed in slow motion. 3.3.8 Immunohistochemistry and TUNEL Animals from both groups (SCI, n = 15; SCI+670, n = 15) were divided into 3 recovery time points and sacrificed at 1, 3 and 7 days post-injury. At the end of designated recovery periods, animals were transcardially perfused with saline and 4% buffered paraformaldehyde (w/v). Harvested spinal cords were cryoprotected in 30% sucrose (w/v), cryosectioned at 20 μm in the longitudinal plane using a Leica CM1850 cryostat, and dorsal sections labelled with primary antibodies (1:200) against rat CD68 (ED-1 clone, MAB1435, Millipore), and Arginase-1 (AB60176, Abcam) or CD80 (AB53003, Abcam) to quantify microglia/macrophages (ED1+) and polarized subtypes M1 (CD80+ED1+) and M2 (Arginase1+ED1+) respectively. Tissue was subsequently incubated with the appropriate secondary antibodies (1:1000, Invitrogen, Alexa 594 conjugated chicken anti-goat #A21468, Alexa 488 conjugated goat anti-mouse #A31619, Alexa 594 conjugated goat anti-mouse #A31623, Alexa 488 conjugated donkey anti-rabbit #A21206). Slides were then incubated in Hoechst solution (2 μg/ml Sigma-Aldrich). Standard immunohistochemical controls were included. 61PDF Image | Effects of Red Light Treatment on Spinal Cord Injury
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