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.

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.

BackgroundTraumatic spinal cord injury (SCI) causes acute neuronal death followed by delayed secondary neuronal damage. However, little is known about how microenvironment regulating cells such as microglia, astrocytes, and blood inflammatory cells behave in early SCI states and how they contribute to delayed neuronal death.MethodsWe analyzed the behavior of neurons and microenvironment regulating cells using a contusion-induced SCI model, examining early (3–6 h) to late times (14 d) after the injury.ResultsAt the penumbra region close to the damaged core (P1) neurons and astrocytes underwent death in a similar spatial and temporal pattern: both neurons and astrocytes died in the medial and ventral regions of the gray matter between 12 to 24 h after SCI. Furthermore, mRNA and protein levels of transporters of glutamate (GLT-1) and potassium (Kir4.1), functional markers of astrocytes, decreased at about the times that delayed neuronal death occurred. However, at P1 region, ramified Iba-1+ resident microglia died earlier (3 to 6 h) than neurons (12 to 24 h), and at the penumbra region farther from the damaged core (P2), neurons were healthy where microglia were morphologically activated. In addition, round Iba-1/CD45-double positive monocyte-like cells appeared after neurons had died, and expressed phagocytic markers, including mannose receptors, but rarely expressed proinflammatory mediators.ConclusionLoss of astrocyte function may be more critical for delayed neuronal death than microglial activation and monocyte infiltration.