This study has revealed that a functional ‘detour’ can be established around a Hx, from the lesioned ventrolateral white matter above to ipsilateral motoneurons below, using a novel combination treatment with the following components: an antibody to the major inhibitory molecule Nogo-A, the neurotrophin NT-3, and the NR2d regulatory subunits to enable NT-3-induced plasticity. Combining neurotrophins with other agents to improve their effectiveness in restoring function is consistent with the results of recent studies adopting this approach (Lu et al., 2004
; Nothias et al., 2005
; Arvanian et al., 2006b
; Chen et al., 2008
; Massey et al., 2008
). Furthermore, the VLF contains reticulospinal and long propriospinal fibers (Reed et al., 2008
) known to participate in the recovery of locomotor function in rats following thoracic injuries (Basso et al., 2002
; Schucht et al., 2002
; Arvanian et al., 2009
). Therefore, development of a treatment aimed at the restoration of VLF projections is an important strategy in promoting recovery of function after thoracic injuries.
Electrophysiological experiments revealed the appearance of novel long-latency responses in L5 motoneurons from the ipsilateral VLF rostral to Hx in rats receiving the combination treatment (). The fact that these responses were preserved after re-transection of the spinal cord through the pre-existing lesion strongly suggests that these novel responses were not due to axons regenerating through the lesion, but were the result of the establishment of novel functional connections around the Hx. Although the increase in branching from white-matter fibers observed in the tracing studies is suggestive of new connections being responsible for the detour, we cannot rule out a contribution from already existing subliminal connections or ‘silent’ synapses (Kerchner & Nicoll, 2008
) that were strengthened and became visible after the treatment (Wall, 1988
Previous double-labeling studies in intact adult rats using tracers injected into the lumbar cord and VLF have identified a population of cervical neurons that cross to the contralateral VLF and recross to terminate in the ipsilateral upper lumbar cord (Reed et al., 2008
). However, the absence of electrophysiological responses observed in control hemisected preparations suggests very little functional connectivity to L5 motoneurons mediated by propriospinal fibers crossing above and below the Hx (). The propriospinal fibers studied here apparently did not sprout below the lesion spontaneously, as indicated by the virtual absence of midline-recrossing fibers below the Hx in the absence of treatments. However, after the full combination treatment the number of fibers recrossing caudal to Hx increased substantially ().
The mild behavioral effects of the combination treatment occurred on a background of robust spontaneous recovery of locomotor function observed after thoracic Hx in rodents (Courtine et al., 2008
; Arvanian et al., 2009
); this spontaneous recovery makes further improvements difficult to detect. More challenging tests such as narrowing beam, horizontal ladder and the swimming symmetry revealed minor yet significant improvement of motor function in rats with the full combination treatment (), with no change in nociceptive function (). More robust recovery may depend on strengthening the synaptic connectivity from the descending fiber systems on the hemisected side to neurons responsible for the detour.
Together these studies suggest that the combination treatment produced larger effects electrophysiologically, anatomically and behaviorally than components tested separately or in pairs. In the electrophysiology where all possible combinations were tested with corresponding controls, the ability of these agents to produce a detour is clear. In the case of the anatomy and behavior, the full combination treatment elicited more sprouting or functional recovery than any of the treatments tested. However, because not all combinations were studied anatomically and behaviorally, and because the behavioral recovery may not parallel the electrophysiological recovery, we remain cautious about making conclusions concerning the ability of these treatments to promote recovery of behavior. Another caveat is the possibility of small differences in tissue sparing among the different preparations (). However, the uniform differences in plasticity at all levels between the treatment groups, and the uniformity of the lesion size, make it very unlikely that systematic differences in tissue sparing were a major factor determining the findings reported here.
How does the combination treatment produce the detour? The requirement for Nogo-A specific antibody and NT-3 and NR2d suggests that detour formation required sprouting or growth of axons as well as an increase in synaptic efficacy. Although our present results do not reveal the location of the novel connections, we believe that they are distributed throughout the cord but are probably most numerous or strongest close to the lesion site where the concentration of the exogenous agents is highest.
NMDA receptors on motoneurons become functional in the prenatal period (Ziskind-Conhaim, 1990
; Kalb & Hockfield, 1992
), but they suffer a decline in function during the second postnatal week due to Mg2+
blockade (Arvanian et al., 2004
). We previously found that restoring NMDA receptor function by adding back the NR2d subunit of the NMDA receptor using an HSV viral construct enabled NT-3 to induce NMDA receptor-dependent potentiation of VLF synaptic transmission (Arvanian et al., 2004
). When combined with NT-3, the NR2d subunit induced the appearance of synaptic responses in motoneurons from damaged VLF axons (Arvanian et al., 2006c
). These results suggest that activity of NMDA receptors in the target neurons might be an essential factor required for growing axons to establish glutamatergic synaptic contacts upon them. Although our studies were in motoneurons, it seems likely that novel connections on interneurons are important for establishing the connections to motoneurons described here.
Our current results demonstrate that combinatorial treatment with NT-3 and NR2d resulted in VLF connections in approximately 33% of the motoneurons in injured adult rats. Similar treatments with NT-3 and NR2d in contused or staggered double-hemisected neonatal rats resulted in recovery of some connectivity to virtually all motoneurons (Arvanian et al., 2006c
). One possible explanation for the limited efficacy of the treatment with NT-3 and NR2d in adult rats is the age-related development of myelin-associated neurite growth inhibition in the spinal cord. The localization of Nogo-A in oligodendrocytes, where expression starts at a relatively late developmental stage (Huber et al., 2002
; Taketomi et al., 2002
), fits well with its role as an age-dependent myelin-associated inhibitor of regenerative fiber growth in adult mammals. Here we demonstrate that additive treatment with NT-3 and HSV–NR2d in adult rats was sufficient to form connections via conduction around the Hx only when combined with anti-Nogo-A antibody. Considering that HSV-1-mediated NR2d expression lasts 1–2 weeks and Nogo-Ab delivery lasts 2 weeks, we hypothesize that they play a role in initiating the establishment of polysynaptic connections observed 7–12 weeks post-injury.
The establishment of connections to motoneurons via the detour is supported by anatomical experiments that indicate an increase in the number of branches given off by propriospinal or supraspinal axons (either ascending or descending) in the contralateral white matter caudal to Hx. Growth of these branches to the ipsilesional side of the cord could provide access to the stimulating electrode above the lesion; similarly, the recrossing between the hemisection and L5 could provide access to ipsilesional motoneurons, perhaps via strengthening of polysynaptic connections. Branches of fibers ipsilateral to the Hx may also send branches to contact propriospinal neurons on the contralateral side which then recross below the Hx to contact L5 motoneurons either directly or via relays from short propriospinal interneurons (Courtine et al., 2008
; Etlin et al., 2010
). Axotomized fibers descending from supraspinal centers (Reed et al., 2008
) including the corticospinal tract and serotonergic raphe spinal fibers known to be influenced by anti-Nogo (Liebscher et al., 2005
; Müllner et al., 2008
) could also play a role in re-establishing the connectivity observed in these experiments.
A further consideration is the recent finding that thoracic Hx can reduce conduction through the uninjured contralateral white matter beginning 1–2 weeks after Hx as well as a decline in conduction velocity for axon segments across from the Hx (Arvanian et al., 2009
). These changes were associated with decreased excitability in these axons (manifested by an increased rheobase), partial demyelination of the VLF and rubrospinal tract axons contralateral to the Hx (Hunanyan et al., 2011
) and accumulation of chondroitin sulfate proteoglycans (CSPGs) in tissue surrounding the Hx (Hunanyan et al., 2010
). Such changes undoubtedly contributed to the absence of any response through the region of injury in the controls; the treatments given at the time of the injury could have either prevented this decline in conduction or reversed it. In this context, NT-3 has been found to induce oligodendrocyte proliferation and myelination of regenerating axons in the contused adult rat spinal cord (McTigue et al., 1998
), and the presence of Nogo has complex effects on oligodendrocyte differentiation which could affect myelination and impulse conduction (Pernet et al., 2008
). The effects of NT-3 and anti-Nogo on myelination of regenerating fibers and conduction through the region contralateral to Hx, as well as the combination of this treatment with intraspinal digestion of CSPGs (Fawcett, 2009
), remain to be determined in the current Hx model.
In conclusion, these results demonstrate that combination treatments using anti-Nogo, NT-3 and the NR2d subunit promote the establishment of a synaptic detour around a Hx. This pathway involves sprouting of white-matter fibers to the opposite side and may contribute to behavioral improvement. Future experiments should explore this combination approach to studying other spinal injuries, e.g. contusion, to determine whether recovery of function is improved under these experimental conditions.