While the deleterious effects of reactive astrocytes and their associated glial scar after CNS injury are well established (Sofroniew, 2009), their beneficial roles have only been evidenced relatively recently (White & Jakeman, 2008). Using several conditional knock-out mice targeting STAT3 signalling in reactive astrocytes, we and others have previously observed that the compaction of inflammatory cells by migrating reactive astrocytes is associated with enhanced locomotor recovery after SCI (Herrmann et al, 2008; Okada et al, 2006). Here, we report that the pharmacological inhibition of GSK-3 using the novel, highly potent agent Ro3303544 successfully stimulated the migration of astrocytes both in vitro and in vivo and promoted functional recovery after SCI.
Mechanism of increased migration by the sustained inhibition of GSK-3
The historical model for describing the mesenchymal migration mode (DiMilla et al, 1991; Lauffenburger & Horwitz, 1996; Palecek et al, 1997) implies that cell migration speed depends on the strength of cell adhesion to the substratum. In agreement with the reduced surface expression of β1-integrin, the effect of Ro3303544 on migration was observed to depend on the concentration of the laminin coating. The apparent discrepancy between the administration period of Ro3303544 in vivo (the first 5 days after injury) and the observation that the compaction of inflammatory cells increased compared to control at 14, but not 7 DPI suggests that the pro-migratory effect of Ro3303544 in vivo indeed depends on the spontaneous upregulation of ECM proteins following SCI, which we have demonstrated occurs at 10 DPI. Given the complexity of the lesion environment, as well as the number of molecules that potentially modulate the migration of reactive astrocytes, it seems plausible that the actual mechanism for the in vivo enhancement of migration by Ro3303544 is more complex than our proposed model.
Mechanism of improved functional outcome by GSK-3 inhibition
The beneficial effect of GSK-3 inhibition in SCI using less potent and specific reagents has been previously reported (Cuzzocrea et al, 2006; Dill et al, 2008). This effect may involve reduced apoptosis and the direct promotion of axon outgrowth. While the direct stimulation of axon growth upon GSK-3 inhibition is still a matter of controversy in the literature (Alabed et al, 2010), we observed that Ro3303544 promoted the neurite outgrowth of embryonic hippocampal neurons in vitro, thereby demonstrating its lack of toxicity. However, to selectively evaluate the effects of enhanced astrocyte migration in vivo, Ro3303544 administration was restricted to the first 5 days after injury. This protocol allowed us to distinguish the observed results from possible direct axon growth-promoting effects of the drug, because axonal growth is a delayed event.
The observed decrease of astrocyte-devoid spaces filled with CD11b-positive inflammatory cells and the consistent reduction in CSPG- (Fitch & Silver, 1997) and collagen IV-positive areas demonstrated that GSK-3 inhibition at the acute phase of SCI accelerated the compaction of the lesion. We propose that this progressive seclusion of inflammatory cells by reactive astrocytes significantly contributes to the beneficial effect of GSK-3 inhibition after SCI. Although the local inflammatory reaction triggered by SCI is known to be capable of enhancing repair, the involvement of inflammatory cells in secondary neuronal damage such as demyelination is uncontested (Alexander & Popovich, 2009). The beneficial effect of walling off inflammatory cells by scar-forming reactive astrocytes is well established in innate (Bush et al, 1999; Faulkner et al, 2004; Herrmann et al, 2008; Myer et al, 2006; Okada et al, 2006) and adaptive inflammation (Voskuhl et al, 2009).
The major defects observed after ganciclovir-targeted death revealed the contribution of the dividing reactive astrocyte pool to the process of walling off leukocytes after brain and spinal cord injuries (Bush et al, 1999; Faulkner et al, 2004; Myer et al, 2006; Voskuhl et al, 2009). The mitogenic effect of Ro3303544 observed in vivo in brain progenitors (Adachi et al, 2007) and in vitro in astrocytes led us to investigate whether proliferation was involved in its in vivo effect. In vivo BrdU incorporation experiments suggest that the contribution of proliferation to the effect of Ro3303544 in vivo is not significant.
What is the mechanism whereby the accelerated compaction of inflammatory cells improves functional recovery? Although lesions ultimately demonstrated similar compaction levels at the last time-point examined (42 DPI), locomotor function was permanently superior in the Ro3303544 group compared to control. This observation highlights the critical role of this sub-acute period after the lesion in the recovery process, as previously suggested (Okada et al, 2006). The finding also strongly suggests that white matter sparing is crucial to recovery, as we and others have previously observed when reactive astrocytes wall off inflammatory cells (Faulkner et al, 2004; Herrmann et al, 2008; Okada et al, 2006). Whether remyelination also contributes to the increased myelin staining is difficult to analyse experimentally, as is the conflicting literature concerning the role of β-catenin signalling in remyelination (Azim & Butt, 2011; Fancy et al, 2009).
Given the wide actions of GSK-3 and Wnt/β-catenin, and the role of GSK-3 in inflammation (Jope et al, 2007), additional direct effects of Ro3303544 on inflammatory or immune cells are likely. Nevertheless, the normal infiltration of CD11b cells observed at 7 DPI, the peak of their invasion (Beck et al, 2010), suggests that Ro3303544 does not affect the recruitment of inflammatory cells.
A major question arising from this study is whether our current observation is relevant to other animal models or human SCI. In rat and human, cystic cavity formation is a common complication of brain and spinal cord damage. Unfortunately, these cystic cavities are not observed in most mouse strains, including the C57 BL6/J mice used for this study. In vitro studies have suggested that the development of these cavities is closely related to the relationship between inflammatory cells and reactive astrocytes (Fitch et al, 1999). While it is speculated that the physical contraction of the fibrous scar may also contribute to cavity formation in some previous reports (Klapka & Muller, 2006), two recent studies using different experimental approaches have observed that reduction of the scarring was associated with reduced cystic cavities in rat (Iannotti et al, 2006; Xia et al, 2008). However, further studies in relevant models are needed to examine whether the stimulation of reactive astrocyte migration through GSK-3 inhibition does indeed limit the development of these cystic cavities.
In conclusion, our findings reveal a novel beneficial effect of GSK-3 inhibition for SCI and suggest that the pharmacological stimulation of reactive astrocyte migration holds promise as a new therapeutic strategy for the treatment of SCI.