Figure 1.
Modifications of the Yasargil aneurysm clip used for SCC, and tissue appearance following SCC.
(A) Before (left) and after (right) trimming of the clip blades down to 150 µm. The stopper (St) was fashioned from a slice of polypropylene tubing. (B) When fully released, the clip maintains an interblade distance of 230 µm. (C) Image sequence from surgery showing the laminectomy (“Before”), the clip-driven compression, and the subsequent hemorrhagic contusion and edema (“After”). (D) Representative spinal cords dissected at different times after injury. The level of the compression site is indicated by the black arrowhead. The third panel is a high magnification view of the boxed area in the second panel. Scale bars: 1 mm.
Table 1.
Summary of the number of mice used for each procedure performed at different timepoints after injury (SCC, SCT) or surgery (sham).
Figure 2.
Cellular damage after SCC assessed by immunohistochemistry.
(A and B) Immunohistochemistry for NeuN 1 day (A) and 8 days (B) after SCC, seen in transverse sections rostral to, at, and caudal to the lesion. At both timepoints NeuN immunostaining is greatly diminished within the compressed region. (C and D) Immunohistochemistry for 200 kD neurofilaments 1 day (C) and 8 days (D) after SCC, seen in horizontal sections. At 1 day after injury (C) many axons (arrowheads) still traverse the compressed region (arrows), although some fascicles appear to be interrupted. At 8 days after injury (D), there is white matter continuity across the lesion site, with a relatively even density of axons. Abbreviations: cc = central canal, DH = dorsal horn, VH = ventral horn. Scale bars: 200 µm.
Figure 3.
Ultrastructure of SCC and sham control spinal cords 1 day after injury.
(A) Axon profiles in the ventrolateral white matter in sham control at P2. Note the presence of filament-like structures within the axon cytoplasm (inset). (B) Ventrolateral white matter in a SCC spinal cord 1 day post-injury. Note the paucity of axons and loosely organized membrane structures (inset). (C) Gray matter in sham control spinal cord at P2. Dendrites, axon terminals, synapses (inset), and subcellular structures such as mitochondria, synaptic vesicles and postsynaptic densities (arrowheads) can be observed. (D) Gray matter in a SCC spinal cord 1 day post-injury. Degenerated structures (inset) are present among less compromised but nevertheless disorganized and discontinuous structures such as dendrites, axon terminals and synapses (double arrowheads). (E) A damaged neuron soma in a SCC spinal cord presents cytoplasmic vacuoles (single arrowheads and inset) and an indented nucleus likely to be in an early stage of nuclear fragmentation (double arrowheads). (F) Cell debris from fragmented plasma membrane and endomembranes. Scale bars: 350 nm (A-D), 760 nm (E) and 390 nm (F). Abbreviations: A = axon; At = axon terminal; D = dendrite; DS = degenerated structure; m = mitochondria; Nu = nucleus.
Figure 4.
Loss of spinal neurons with descending axons traversing the injury following SCC.
(A) Schematic representation of interneurons, propriospinal neurons and projection neurons in the spinal cord based on [34]. The compression was performed in the lower thoracic cord (T10–12, grey area). RDA was applied one segment below the compressed region to label ascending (a) and descending (d) ipsilaterally (I) and contralaterally (C) projecting neurons (INs). (B and C) Representative confocal images of one side of the ventral region of transections containing RDA-labeled dCINs (B) and aCINs (C) in one sham and one SCC mouse 1 day (B1–2, C1–2) and 14 days (B3–4, C3–4) after surgery/injury. Very few are labeled in SCC (arrows, B2) compared to sham controls (B3). The inset in B1 shows the contour of the entire section with the imaged area indicated by the stippled red square. (D) Counts of labeled neuronal profiles of the indicated projection types within a 1 mm stretch of spinal cord immediately rostral to the compressed region (or the equivalent in sham animals) and immediately caudal to the RDA application site (see also Fig. S1). Bars represent averages of total counts taken from every other section in the relevant 1 mm length of spinal cord, with number of animals indicated in the first graph. Error bars represent standard deviations. Differences in counts of dIINs and dCINs between sham control and SCC animals are significant at p<0.025 at 1 day after surgery/injury and at p<0.01 at 14 days after surgery/injury (Mann-Whitney U-test, U = 0 in both cases). Scale bars: 100 µm. Additional abbreviations: Vr = ventral root.
Figure 5.
Recovery of hindlimb motility following spinal cord injury.
(A) Kinematic assessment of trajectories of forepaws (blue traces) and hindpaws (green traces) during air stepping test of sham control and SCC mice at 3 times post-injury (post-surgery for sham controls). Upper panels show actual trajectories, and lower panels show instantaneous velocities. (B) Distribution of hindlimb instantaneous velocities in sham control (black), SCC (red) and SCT (blue) mice during 20 s video sequences, expressed as the proportion of trajectories with different velocities. Grey zone at far right of each graph indicates the bin containing instantaneous velocities greater then 170 mm/sec. This bin was distinct in that the proportion of velocities over this value increased markedly both in sham control and SCC mice over time. (C) Mean hindlimb trajectory amplitudes in sham control, SCC and SCT mice at different post-injury times normalized to sham controls at 14 days post-surgery. Numbers in or over bars represent numbers of animals tested. Error bars represent standard deviations. Statistical significance of differences of means tested by Mann-Whitney U-test: p≤0.002 (***), p = 0.01 (**), p = 0.09 (Ø). Note that p value for difference between sham control and SCC mice reaches non-significance by 8 days post-surgery. (D) Proportions of instantaneous velocities >170 mm/s in sham control, SCC and SCT mice at different times post-surgery/injury. Blue asterisk represents a measurement of value zero, otherwise the lack of a bar indicates that no measurement was made (which was the case for 6 h post-injury for SCT mice). Numbers in or over bars represent numbers of animals tested. Error bars represent standard deviations. Statistical significance of differences of means tested by Mann-Whitney U-test: p≤0.003 (***), p≤0.025 (**), p>0.1 (Ø). Note that p value for difference between sham control and SCC mice reaches non-significance by 14 days post-surgery.
Figure 6.
Recovery of responses to general bulbospinal pathway stimulation following SCC, assessed with electrophysiology.
(A) Schematic of the ex vivo preparation of the brain stem and spinal cord showing the site of bulbospinal pathway stimulation in the ventral funiculus (VFstim), the compressed region (red zone) and the recording suction electrodes placed on cervical (above the injury) and lumbar (below the injury) ventral roots. (B and C) Ventral root responses during stimulus trains (grey box, 25 pulses over 5 s, individual stimulus pulses indicated above) in cervical (B1, C1) and lumbar (B2, C2) ventral roots in 6 h post-surgery sham control (B, black traces), 6 h post-injury SCC (B, red traces), 4 day post-injury sham control (C, black traces), and 4 day post-injury SCC mice (C, red traces). Upper traces show raw recordings, graphs show instantaneous spike firing rates. (D) Tight seal recordings from cervical (D1) and lumbar (D2,3) ventral roots following single pulse stimulation of the ventrolateral funiculus (D1, D2) or the lumbar dorsal root (D3) in sham control (black traces) and SCC mice 4 days after surgery/injury. Filled black arrowhead indicates the monosynaptic component of the response, open arrowheads indicate polysynaptic components. The red arrow in D2 shows the increased delay between monosynaptic and first polysynaptic component 4 days post-injury.
Figure 7.
Recovery of responses to general bulbospinal pathway stimulation following SCC, assessed with optical recording.
(A) Schematic of the ex vivo preparation of the brain stem and spinal cord showing the site of bulbospinal pathway stimulation in the ventral funiculus (VF Stim) and the CGDA labeling of the L5 ventral root, and a photograph of the CGDA labeled lumbar MNs in the medial motor column (MMC) and lateral motor column (LMC). (B) Low temporal resolution (4 Hz frame rate) optical recordings of calcium transients elicited in MMC and LMC MNs in response to VF stimulation (same parameters as in Fig. 6) 6 h and 4 days after surgery/injury in sham control mice (B1 and B2, black traces) and SCC mice (B1 and B2, red traces). Green traces show responses to stimulation of the L5 dorsal root in SCC. ΔF/F represents fluorescence change in % relative to baseline. (C) High temporal resolution (50 Hz frame rate) recordings from individual L5 MMC MNs in response to VF stimulation in sham control (C1) and SCC (C2) mice 4 days after surgery/injury. Each colored trace shows the calcium transients in one of the individual MNs outlined by a colored region of interest in the photographic images to the left. Black and grey traces (right column) represent the superimposed individual traces (grey) and the averaged response (black) from the same MNs, with the expanded traces (outlined in red) showing the responses to the last 10 pulses of the train. Red arrows in the expanded trace of C2 (4 days post-injury) indicate absent calcium transients in response to the 15th and the last pulses of the train.
Figure 8.
Reorganization of vestibulospinal connections to lumbar medial and lateral motor columns following SCC.
(A) Schematic of the ex vivo preparation of the brain stem and spinal cord showing the site of vestibular nerve stimulation (VIII N Stim) and the CGDA labeling of the L5 ventral root. (B) Low temporal resolution (4 Hz frame rate) optical recordings of calcium transients elicited in LMC MNs (black traces) and MMC MNs (red traces) in response to train stimulation of the vestibular nerve (same parameters as in Fig. 6) in one sham control mouse (B1) and two SCC mice (B2, B3) 4 days after surgery/injury. ΔF/F represents fluorescence change in % relative to baseline. The small green traces (DR stim) show responses to stimulation of the L5 dorsal root. Insets above the MMC traces show the apparent reorganized synaptic connectivity from lateral vestibulospinal tract (VST) to LMC and MMC.
Figure 9.
Integration of human fetal neural progenitor cells following transplantation into neonatal spinal cords with SCC injuries.
(A) Transverse section showing GFP-positive f-NPC-derived cells 29 days post-injury. (B, C) Higher magnification views of GFP-positive f-NPC-derived cells showing morphological differentiation including the extension of thin, ramified processes. Scale bars: 20 µm (A), 32.5 µm (B, C).