Supplementary Figure 6: Characterization of projection neurons with residual connection below the contusion in mice.

To visualize the interruption of the corticospinal tract in 3D, an AAV-Cre was co-injected with an AAV-flex-Synaptophysin-GFP into the motor cortex of Flex-tdTomato mice to label corticospinal tract fibers in the spinal cord. A large block of spinal cord containing the contusion was cleared using CLARITY. A representative 3D rendering of a CLARITY-processed spinal segment rostral to the contusion shows corticospinal tract fibers. Longitudinal photographs of spinal segments rostral and caudal to contusion show the synapses from corticospinal tract neurons. Scale bar 500 µm. (b) To verify the interruption of corticospinal tract projections independent of viral injections, spinal cord sections rostral (T6) and caudal (L1) to the contusion were stained with Protein Kinase C gamma (PKC-Gamma) antibodies, which label corticospinal tract axons. The bar plot reports the mean relative (arbitrary unit, a.u.) intensity of the PKC-Gamma signal in segments caudal and rostral to the contusion. Scale bars 500 µm, 50 µm insets. *P < 0.05. Paired Student’s t-test. (c) To quantify the interruption of corticospinal tract projections from the leg region of the motor cortex, fluorescence of the main corticospinal tract component in the dorsal column, the lateral component and the ventral component was measured in spinal cord sections rostral (T6) and caudal (L1) to the contusion. Scale bars 500 µm, 50 µm insets. Bar plots show the mean relative (arbitrary unit, a.u.) intensity of corticospinal tract axons projecting in the different regions of the white matter. (d) Diagram illustrating the injections of the retrograde tracer FastBlue in the lumbar spinal cord to identify projection neurons with residual connections below the contusion. The diagram illustrates the various regions where neurons were found and analysed. Bar plots report the number of neurons in these regions (n = 4 each for intact and contused mice). 3D cervical (e) and (f) thoracic spinal cord reconstructions in intact and contused mice, including quantifications. *P < 0.05; **P < 0.01. Mann-Whitney test. (g) The phenotype of brainstem neurons with residual projections below the contusion was studied with immunohistochemistry. Photographs show neurons retrogradely labelled from lumbar segments and the co-localisation with glutamate or serotonin (white arrows). Scale bars 25 µm. The polar plots display the mean percentage (+- s.e.m) of neurons co-localising with glutamate or serotonin with respect to the entire population of neurons retrogradely labelled from lumbar segments in intact mice (n = 4 mice).