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. 2011 Jul 13;475(7355):196-200.
doi: 10.1038/nature10199.

Functional regeneration of respiratory pathways after spinal cord injury

Warren J Alilain  1 , Kevin P Horn, Hongmei Hu, Thomas E Dick, Jerry Silver
Affiliations

Functional regeneration of respiratory pathways after spinal cord injury

Warren J Alilain et al. Nature. .

Abstract

Spinal cord injuries often occur at the cervical level above the phrenic motor pools, which innervate the diaphragm. The effects of impaired breathing are a leading cause of death from spinal cord injuries, underscoring the importance of developing strategies to restore respiratory activity. Here we show that, after cervical spinal cord injury, the expression of chondroitin sulphate proteoglycans (CSPGs) associated with the perineuronal net (PNN) is upregulated around the phrenic motor neurons. Digestion of these potently inhibitory extracellular matrix molecules with chondroitinase ABC (denoted ChABC) could, by itself, promote the plasticity of tracts that were spared and restore limited activity to the paralysed diaphragm. However, when combined with a peripheral nerve autograft, ChABC treatment resulted in lengthy regeneration of serotonin-containing axons and other bulbospinal fibres and remarkable recovery of diaphragmatic function. After recovery and initial transection of the graft bridge, there was an unusual, overall increase in tonic electromyographic activity of the diaphragm, suggesting that considerable remodelling of the spinal cord circuitry occurs after regeneration. This increase was followed by complete elimination of the restored activity, proving that regeneration is crucial for the return of function. Overall, these experiments present a way to markedly restore the function of a single muscle after debilitating trauma to the central nervous system, through both promoting the plasticity of spared tracts and regenerating essential pathways.

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Figures

Figure 1
Figure 1. 7 days following C2 hemisection there is increased expression of the perineuronal net and inhibitory proteoglycans around phrenic motor neurons
a, Phrenic motor neurons labeled with dextran Texas red (DTR PMN; red) in uninjured animals are ensheathed by the perineuronal net, indicated by Wisteria Floribunda Agglutinin staining (WFA - green, first column), but stained poorly for CS56, a marker for CSPGs (green, second column). Following C2 hemisection and treatment with saline there is increased WFA and CSPG staining (green, second row). Seven days after ChABC treatment WFA and CS56 staining disappears (third row). Scale bar = 40 μm. b, 2B6 (green), a marker for digested CSPGs, is present following ChABC treatment. Scale bar = 40 μm. b’, A lower power montage shows the area of CSPG digestion by ChABC (the side of montage left of central canal (CC) is ipsilateral to lesion and ChABC treatment). Scale bar = 200 μm. c, Within the region of CSPG degradation there is an increase of serotonergic fibers (5HT – green) around PMNs. Scale bar = 40 μm. Pixel intensity analysis shows a doubling in the amount of 5HT in ChABC treated animals compared to controls (n = 6, inset). Error bar indicates standard error.
Figure 2
Figure 2. After C2 hemisection and implantation of a PN graft with ChABC treatment there is an increase to near-normal levels of diaphragmatic EMG activity over time
a, Prior to twelve weeks post C2 hemisection there is minimal activity in the hemidiaphragm ipsilateral to the lesion in animals that received ChABC treatment and application of an autologous peripheral nerve bridge. At twelve weeks there is substantial recovery of hemidiaphragmatic inspiratory activity. b, At 12 weeks recovery in animals that received a PNG and ChABC treatment was close to that of a non-lesioned animal and better than grafted animals that received only vehicle treatment. c) Animals that received the graft and ChABC had a higher percentage of recovery 12 weeks after hemisection compared to other groups. Semi-quantitative analysis showed that there was no difference in the frequency of breaths between groups. C2 hemisected animals with a PNG and ChABC treatment (purple bars) had a higher average peak amplitude of the raw inspiratory bursts compared to the other groups. Although burst duration could, on occasion, reach near normal levels (2a,2b), on balance there was no difference in the average duration of inspiratory bursts between animals that showed recovery. Darker colored bars are all animals in a group, while the lighter bars represent only animals that showed recovered activity. ** = significantly different compared to saline and ChABC treated animals, p < 0.02. * = significantly different compared to grafted animals with saline, p < 0.05. The error bar indicates standard error.
Figure 3
Figure 3. There is significant regeneration of axonal fibers in the PN graft and back into the CNS with ChABC treatment
a, a’, In animals that received ChABC there is alignment of astroglial processes from the spinal cord (SC), identified by GFAP labeling (red), with the TAU positive (green) axons at the graft/SC interface that have regenerated back into the CNS. In saline treated animals, it appears that the astrocytes form a barrier-like structure at the interface. Scale bar = 40 μm. b, Only a small portion of the regenerated axons in the graft are serotonergic (green). Scale bar = 200 μm. b, b’, c, In ChABC treated animals, serotonergic fibers (arrows) penetrated deep into the CNS (identified by GFAP, red) from the graft (arrowheads). In c, scale bar = 40 μm. d, Anterograde tracing from the medulla with dextran Texas red shows regenerated fibers in the graft and back into the gray matter of the spinal cord. e, BDA labeling and immunocytochemistry show close proximity of regenerated fibers with synapsin puncta. Scale bar = 40 μm.
Figure 4
Figure 4. Transection of the PN graft after recovery leads to increased tonic EMG activity of the diaphragm
a, 12 weeks after C2 hemisection and graft+ChABC, there is recovery ipsilateral to the lesion that is rhythmic and synchronous with the contralateral side. b, Immediately after transection of the graft there is a reduction in the recovered activity but an increase in tonic activity and compensatory changes on the contralateral side. c-e, In another recovered animal where the PNG was transected the tonic activity declined after one hour. f, During times of inspiration (indicated by upper trace of the right hemidiaphragm) there is an increase in the spiking frequency in the resulting tonic activity of the left hemidiaphragm. g, In a third animal, the tonic activity is gone 24 hours after graft transection. h and i, In two examples of animals with graft and ChABC treatment, there is an abundance of activity in the graft that can be augmented during and after respiratory challenge, suggesting regeneration of respiratory related axons. j, Phrenic nerve activity after similar challenge mirrors the patterned increased activity in the graft (h and i).
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References

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