Although the central branches of the dorsal root ganglion (DRG) sensory neurons do not spontaneously regenerate, a conditioning peripheral injury can promote their regeneration. A potential role of macrophages in axonal regeneration was proposed, but it has not been critically addressed whether macrophages play an essential role in the conditioning injury model. After sciatic nerve injury (SNI) in rats, the number of macrophages in DRGs gradually increased by day 7. The increase persisted up to 28 d and was accompanied by upregulation of inflammatory mediators, including oncomodulin. A macrophage deactivator, minocycline, reduced the macrophage number and expressions of the inflammatory mediators. Molecular signatures of conditioning effects were abrogated by minocycline, and enhanced regenerative capacity was substantially attenuated both in vitro and in vivo. Delayed minocycline infusion abrogated the SNI-induced long-lasting heightened neurite outgrowth potential, indicating a role for macrophages in the maintenance of regenerative capacity. Intraganglionic cAMP injection also resulted in an increase in macrophages, and minocycline abolished the cAMP effect on neurite outgrowth. However, conditioned media (CM) from macrophages treated with cAMP did not exhibit neurite growth-promoting activity. In contrast, CM from neuron-macrophage cocultures treated with cAMP promoted neurite outgrowth greatly, highlighting a requirement for neuron-macrophage interactions for the induction of a proregenerative macrophage phenotype. The growth-promoting activity in the CM was profoundly attenuated by an oncomodulin neutralizing antibody. These results suggest that the neuron-macrophage interactions involved in eliciting a proregenerative phenotype in macrophages may be a novel target to induce long-lasting regenerative processes after axonal injuries in the CNS.

CNS neurons in adult mammals do not spontaneously regenerate axons after spinal cord injury. Preconditioning peripheral nerve injury allows the dorsal root ganglia (DRG) sensory axons to regenerate beyond the injury site by promoting expression of regenerationassociated genes. We have previously shown that peripheral nerve injury increases the number of macrophages in the DRGs and that the activated macrophages are critical to the enhancement of intrinsic regeneration capacity. The present study identifies a novel chemokine signal mediated by CCL2 that links regenerating neurons with proregenerative macrophage activation. Neutralization of CCL2 abolished the neurite outgrowth activity of conditioned medium obtained from neuron-macrophage cocultures treated with cAMP. The neuron-macrophage interactions that produced outgrowth-promoting conditioned medium required CCL2 in neurons and CCR2/CCR4 in macrophages. The conditioning effects were abolished in CCL2-deficient mice at 3 and 7 d after sciatic nerve injury, but CCL2 was dispensable for the initial growth response and upregulation of GAP-43 at the 1 d time point. Intraganglionic injection of CCL2 mimicked conditioning injury by mobilizing M2-like macrophages. Finally, overexpression of CCL2 in DRGs promoted sensory axon regeneration in a rat spinal cord injury model without harmful side effects. Our data suggest that CCL2-mediated neuron-macrophage interaction plays a critical role for amplification and maintenance of enhanced regenerative capacity by preconditioning peripheral nerve injury. Manipulation of chemokine signaling mediating neuron-macrophage interactions may represent a novel therapeutic approach to promote axon regeneration after CNS injury.

The cystic cavity that develops following injuries to brain or spinal cord is a major obstacle for tissue repair in central nervous system (CNS). Here we report that injection of imidazole-poly(organophosphazenes) (I-5), a hydrogel with thermosensitive sol–gel transition behavior, almost completely eliminates cystic cavities in a clinically relevant rat spinal cord injury model. Cystic cavities are bridged by fibronectin-rich extracellular matrix. The fibrotic extracellular matrix remodeling is mediated by matrix metalloproteinase-9 expressed in macrophages within the fibrotic extracellular matrix. A poly(organophosphazenes) hydrogel lacking the imidazole moiety, which physically interacts with macrophages via histamine receptors, exhibits substantially diminished bridging effects. I-5 injection improves coordinated locomotion, and this functional recovery is accompanied by preservation of myelinated white matter and motor neurons and an increase in axonal reinnervation of the lumbar motor neurons. Our study demonstrates that dynamic interactions between inflammatory cells and injectable biomaterials can induce beneficial extracellular matrix remodeling to stimulate tissue repair following CNS injuries.

Combining cell transplantation with activity-based rehabilitation is a promising therapeutic approach for spinal cord repair. The present study was designed to investigate potential interactions between the transplantation (TP) of neural stem cells (NSCs) obtained at embryonic day 14 and treadmill training (TMT) in promoting locomotor recovery and structural repair in rat contusive injury model. Combination of TMT with NSC TP at 1 week after injury synergistically improved locomotor function. We report here that combining TMT increased the survival of grafted NSCs by Ͼ3-fold and Ͼ5-fold at 3 and 9 weeks after injury, respectively. The number of surviving NSCs was significantly correlated with the extent of locomotor recovery. NSCs grafted into the injured spinal cord were under cellular stresses induced by reactive nitrogen or oxygen species, which were markedly attenuated by TMT. TMT increased the concentration of insulin-like growth factor-1 (IGF-1) in the CSF. Intrathecal infusion of neutralizing IGF-1 antibodies, but not antibodies against either BDNF or Neurotrophin-3 (NT-3), abolished the enhanced survival of NSC grafts by TMT. The combination of TP and TMT also resulted in tissue sparing, increased myelination, and restoration of serotonergic fiber innervation to the lumbar spinal cord to a larger extent than that induced by either TP or TMT alone. Therefore, we have discovered unanticipated beneficial effects of TMT in modulating the survival of grafted NSCs via IGF-1. Our study identifies a novel neurobiological basis for complementing NSC-based spinal cord repair with activity-based neurorehabilitative approaches.

Traumatic injuries to the spinal cord lead to severe and permanent neurological deficits. Although no effective therapeutic option is currently available, recent animal studies have shown that cellular transplantation strategies hold promise to enhance functional recovery after spinal cord injury (SCI). This review is to analyze the experiments where transplantation of stem/progenitor cells produced successful functional outcome in animal models of SCI. There is no consensus yet on what kind of stem/progenitor cells is an ideal source for cellular grafts. Three kinds of stem/progenitor cells have been utilized in cell therapy in animal models of SCI: embryonic stem cells, bone marrow mesenchymal stem cells, and neural stem cells. Neural stem cells or fate-restricted neuronal or glial progenitor cells were preferably used because they have clear capacity to become neurons or glial cells after transplantation into the injured spinal cord. At least a part of functional deficits after SCI is attributable to chronic progressive demyelination. Therefore, several studies transplanted glial-restricted progenitors or oligodendrocyte precursors to target the demyelination process. Directed differentiation of stem/progenitor cells to oligodendrocyte lineage prior to transplantation or modulation of microenvironment in the injured spinal cord to promote oligodendroglial differentiation seems to be an effective strategy to increase the extent of remyelination. Transplanted stem/progenitor cells can also contribute to promoting axonal regeneration by functioning as cellular scaffolds for growing axons. Combinatorial approaches using polymer scaffolds to fill the lesion cavity or introducing regeneration-promoting genes will greatly increase the efficacy of cellular transplantation strategies for SCI.