Recovery of supraspinal control of stepping via indirect propriospinal relay connections after spinal cord injury

Abstract

Spinal cord injuries (SCIs) in humans and experimental animals are often associated with varying degrees of spontaneous functional recovery during the first months after injury. Such recovery is widely attributed to axons spared from injury that descend from the brain and bypass incomplete lesions, but its mechanisms are uncertain. To investigate the neural basis of spontaneous recovery, we used kinematic, physiological and anatomical analyses to evaluate mice with various combinations of spatially and temporally separated lateral hemisections with or without the excitotoxic ablation of intrinsic spinal cord neurons. We show that propriospinal relay connections that bypass one or more injury sites are able to mediate spontaneous functional recovery and supraspinal control of stepping, even when there has been essentially total and irreversible interruption of long descending supraspinal pathways in mice. Our findings show that pronounced functional recovery can occur after severe SCI without the maintenance or regeneration of direct projections from the brain past the lesion and can be mediated by the reorganization of descending and propriospinal connections. Targeting interventions toward augmenting the remodeling of relay connections may provide new therapeutic strategies to bypass lesions and restore function after SCI and in other conditions such as stroke and multiple sclerosis.

Figure 1

Recovery of supraspinal control of stepping after a lateral hemisection at T12. (a) Schematic of left hemisection at T12 (black). (b) Reflective markers overlying bony landmarks were video recorded (100 Hz). The limb axis from iliac crest to toe (dashed line) was used to characterize forward and backward hindlimb oscillation. (c) Schematic of left hemisection at T12 (black) combined with a delayed right hemisection at the same level after 10 weeks (red). (d) Representative stick diagrams of ipsilateral hindlimb movements during stance and swing before and 7–48 d after left T12 hemisection. (e) Bar graphs representing the average maximum (Max) backward (BW) and forward (FW) angular positions of the limb axis depicted in b (n = 8). (f) Averaged EMG activity (n = 15 steps) for the vastus lateralis (extensor) and tibialis anterior (flexor) muscles ipsilateral to left T12 hemisection over the duration of the step cycle. Horizontal bars represent stance (light shading) or paw dragging (dark shading) durations. (g) Bar graphs representing the average EMG burst amplitude (n = 4 mice; 10 bursts per mouse) for vastus lateralis (top) and tibialis anterior (bottom) muscles ipsilateral to the hemisection at T12. (h) Probability density distributions (n = 4 mice, 10 steps per animal) of normalized EMG amplitudes in the vastus lateralis and anterior muscles during treadmill stepping. The L-shaped pattern observed during stepping before lesion indicates reciprocal activation between the antagonist anterior and vastus lateralis motor pools. The D-shape during stepping at 14 d after a T12 hemisection indicates co-activation between anterior and vastus lateralis. Values are means ± s.e.m. *P < 0.01 and ** P < 0.001 indicate a statistically significant differences between before and after lesion values and between ipsilateral and contralateral hindlimbs, respectively.

Figure 2

Long-term loss of supraspinal but not propriospinal connections after a T12 lateral hemisection or after T12 (left) and delayed T7 (right) lateral hemisections. (a) Schematic of the areas evaluated in the brainstem and spinal cord after unilateral injection of retrograde tracer (Dextran–Alexa 568) at L1–L2 in uninjured mice and mice with an ipsilateral hemisection at T12. (b–d) Representative examples of retrograde tract tracing. (b) Retrogradely labeled neurons in contralateral (Contra) but not ipsilateral (Ipsi) red nucleus (n.) in an uninjured mouse. m, midline. Scale bar, 150 μm. (c) Tracer deposit in the unilateral injection site; arrows denote the location of retrogradely labeled neurons contralateral to the injection. Scale bar, 250 μm. (d) Differences in the number of retrogradely labeled neurons at T9 in uninjured mice or after the tracer was injected either immediately (acute) or at 10 weeks (chronic) after a T12 hemisection. Arrows indicate examples of tracer-labeled neurons. The areas delineated by the boxes are shown at a higher magnification in the lower panel for each condition. Scale bars, 150 μm or 50 μm for survey or detail images, respectively. (e) Bar graphs showing average counts (n = 8 mice per group except for the acute group, n = 6) of retrogradely labeled neurons in the spinal cord and brainstem nuclei. (f) Schematic of areas evaluated in the brainstem and spinal cord after bilateral injection of retrograde tracer (Dextran–Alexa 568) at L1–L2 in mice with T12 (left) and delayed (10 weeks) T7 (right) hemisections. (g) Bar graphs showing the average count (n = 6 mice per group) of retrogradely labeled neurons in the spinal cord of and brainstem nuclei of mice injected immediately after simultaneous bilateral hemisection (Simul). Values are means ± s.e.m. *P < 0.001, statistically significant difference compared to uninjured mice; ⁺P < 0.01, statistically significant difference from mice injected immediately after SCI (acute).

Figure 3

Recovery of supraspinal control of stepping after delayed but not simultaneous T12 (left) and T7 (right) lateral hemisections. (a) Schematic of different combinations of simultaneous or delayed (10 weeks) SCIs. (b) Bar graphs showing the average maximum backward and forward angular positions of the limb axis after different SCIs. (c) Representative EMG recordings from the left and right tibialis anterior during stepping before and 3 d after different SCIs. (d) Bar graphs showing the average EMG burst amplitude for left and right tibialis anterior muscles (n = 4 for simultaneous or delayed hemisections; n = 3 for complete delayed T7 transection). Values are normalized to prelesion EMG activity. Values represent means ± s.e.m. *P < 0.05 and **P < 0.01, statistically significant differencs between pre- and postlesion values and between ipsilateral and contralateral hindlimbs, respectively.

Figure 4

Excitotoxic ablation of T8–T10 neurons abolishes the recovered control of stepping after a T12 lateral hemisection and after a T12 lateral hemisection followed by a delayed T7 lateral hemisection. (a,b) Survey and detail images of neighboring spinal cord sections (T8) stained for cresyl violet to show neurons (a) or luxol fast blue to show myelin (b) in uninjured mice or in mice after NMDA infusion (arrowheads). (c) Bar graphs showing the average maximum backward and forward angular positions of the limb axis depicted in Figure 1b during stepping 2–5 d after NMDA injection. Values represent means ± s.e.m. *P < 0.001, statistically significant difference compared to uninjured mice. Scale bars, 170 μm and 35 μm for survey and detail images, respectively.