Tracing Axonal Plasticity in Spinal Cord

To detect the axonal plasticity induced by lithium treatment, we traced axons of the lateral spinothalamic tract. Animals were anesthetized at 42 dpi and laminectomy was performed at the L2 level to expose the dorsal segment of the spinal cord (Caglayan et al., 2019 (link)). Under visual inspection, 1 μl biotinylated dextran amine (BDA, 10,000 MW; D1956; Invitrogen, Waltham, MA, USA) diluted in 0.01 M PBS was bilaterally injected 0.3 mm lateral to the midline and 0.7 mm deep to the dorsal surface using a Hamilton syringe connected to a glass tip. The syringe tip remained inside the injection area for 1 min to prevent backflow. After 14 days, animals were sacrificed by transcardial perfusion with 4% PFA and coronal sections were collected. Then, sections were washed with PBS, blocked with 10% normal goat serum in PBS for 1 h at room temperature, incubated with Alexa Fluor 555 conjugated streptavidin (S21391; Invitrogen) for 90 min at room temperature and analyzed using confocal microscopy (LSM780, Carl Zeiss). The number of axons crossing the rostral and caudal spinal cord segments were blindly counted in the lateral spinothalamic tract of the ipsilesional and contralesional hemicord in ROI measuring 62,500 μm².