脊髓损伤
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Figure 3 ｜ Remodeling of excitatory Gi and Gi+LPGi fibers of the ReST in response to cervical spinal cord injury.
The reticulospinal tract is not only important to control locomotion in healthy mice but also following spinal cord injury as it allows the re-establishment of walking (Filli et al., 2014; Z?rner et al., 2014; Engmann et al., 2020). To determine the remodeling of excitatory and inhibitory reticulospinal fiber within the Gi and LPGi nuclei, we used respectively vGlut2-cre and vGat-cre mice. We labeled these mice with cre-dependent AAVs expressing mCherry injected either in the gigantocellular nucleus (Gi; vGlut: n = 9 controls and n = 8 injured; vGat: n = 6 controls and n = 4 injured) or in the gigantocellular and the lateral paragigantocellular nucleus (Gi + LPGi; vGlut n = 5 controls and injured; vGat n = 4 controls and injured) ipsilateral to the lesion. We chose to analyze LPGi plasticity together with the Gi due to the difficulty of targeting the LPGi alone as it is a very small nucleus. We first verified the accuracy of all our injections (Additional Figure 4) before analyzing the distribution of the fibers originating from the Gi or the Gi + LPGi in the cervical spinal cord (segment C5/6 above the lesion). To determine whether excitatory and inhibitory fibers originating from the Gi and the Gi + LPGi remodel following unilateral spinal cord lesion, we analyzed the dense topographical innervation and distribution of fibers in the cervical segments C5–C6 above the lesion using heatmaps of the axonal grey matter expression in vGlut2-cre and vGatcre mice. We first verified that our heatmaps analysis reflects the true fiber innervation and distribution in the grey matter by performing correlation of the collateral length and fiber count to the density of transduced axons in the spinal cord quantified in the heat maps (Additional Figure 5). We then proceeded to map regional densities of either excitatory or inhibitory fibers coming from the Gi or the Gi + LPGi 21 days following cervical spinal cord injury and represented them as heatmaps (as described above; Figure 3 for excitatory profiles and Figure 4 for inhibitory profiles). Here we selected 6 regions of interest: dorsal horn, intermediate laminae and ventral horn ipsi- and contra-lateral to the lesion (Figures 3 and 4). For excitatory fibers, we mapped first the regional densities from injection in the Gi in vGlut2- cre mice (Figure 3A). We found that following the spinal cord injury the excitatory Gi contralateral innervation of the spinal cord was slightly, albeit significantly, increased in the ventral horn at 21 days post-injury and that this increase was specifically significant in laminae VI and VII–IX (Figure 3B). Interestingly, the ipsilateral excitatory innervation from the Gi levels was not significantly changed (Figure 3B) and the contralateral innervation in the dorsal and intermediate horns was also not significantly affected (Additional Figure 6A). The fold change heatmap of the Gi when injured and control mice were compared demonstrated a slight but significant general increase in density between the ipsilateral and contralateral sides in the dorsal horn and intermediate lamina, and a significant regional increase in lamina V was found (Figure 3C). This underscores that subtle plastic changes of Gi excitatory fibers mostly occur in the contralateral ventral horn 21 days post-injury. We then focused on the injections that targeted not only the Gi but also the LPGi to determine whether the additional labeling of the lateral paragigantocellular nucleus would reveal additional plasticity (Figure 3D). Labeling of both Gi and LPGi nuclei demonstrate that there is an overall decrease in ipsilateral innervation balanced by an increase in contralateral innervation (Figure 3D and E). By concentrating on the contralateral side, we noticed that the increase was particularly significant in laminae V and VII– IX (Figure 3E) which is reminiscent of the plasticity seen when the Gi alone was labeled although it appears increased here. Similarly, to the Gi labeling, we did not notice any significant changes in ipsilateral and contralateral innervation of the dorsal and intermediate horns (Additional Figure 5B). The fold change heatmap of the Gi + LPGi injection demonstrated significantly more differences between the ipsilateral and contralateral sides with a significant increase in the intermediate and ventral horns that was particularly noticeable in laminae VII–IX (Figure 3F).
Figure 4 ｜ Remodeling of inhibitory Gi and Gi+LPGi fibers of the ReST in response to cervical spinal cord injury.
We then turned our attention to inhibitory fibers located in the Gi and LPGi. To label those fibers we used vGat-cre mice (Figure 4A) and we either labeled the fibers originating from the Gi (Figure 4A–C and Additional Figure 5C) or the Gi and LPGi (Figure 4D–F and Additional Figure 5D). In contrast to excitatory fibers, we could not detect any changes in heatmaps neither between control and injured mice nor between the ipsilateral and contralateral spinal cord sides for any injections considered. This suggests that unlike excitatory fibers, inhibitory fibers from the Gi and LPGi do not remodel following spinal cord injury (Figure 4). Finally, to ascertain that vGlut and vGat neurons behave differently in terms of their plastic abilities following SCI and that the lack of significance in the vGat group is due to a lack of plasticity, we compared the fold changes of vGlut and vGat fibers to each other in the previously defined regions (Additional Figure 7). In accordance with our previous data showing no changes in distribution in the ipsilateral spinal cord, we did not see any significance ispilaterally (Additional Figure 7A and B). In the contralateral hemisphere, we could detect significance in the Gi and GiLPGi in particular in the intermediate laminae and ventral horn (specifically also lamina VII/IX) respectively (Additional Figure 7C and D) suggesting that vGlut and vGat neurons differ indeed in their post-injury plastic abilities.