Macrophages have been implicated in peripheral nerve regeneration for some time, supposedly through their involvement in Wallerian degeneration, the process by which the distal nerve degenerates after axotomy and is cleared by phagocytosis. Thus, in several studies in which macrophage accumulation in the distal nerve was reduced and Wallerian degeneration inhibited, regeneration was delayed. However, this interpretation ignores the more recent findings that macrophages also accumulate around axotomized cell bodies. The function of macrophage action at this second site has not been clear. In two mutant strains of mice, the slow Wallerian degeneration (Wld s ) mouse and the chemokine receptor CCR2 knock-out mouse, we report that macrophage accumulation after axotomy was abolished in both the dorsal root ganglion (DRG) and the distal sciatic nerve. To measure neurite outgrowth, DRG neurons were given a conditioning lesion, and outgrowth was measured in vitro 7 d later in the absence of the distal nerve segment. The increased growth normally seen after a conditioning lesion did not occur or was reduced in Wld s or CCR2 Ϫ/Ϫ mice. In the superior cervical ganglion (SCG), particularly in Wld s mice, macrophage accumulation was reduced but not abolished after axotomy. In SCG neurons from Wld s mice, the conditioning lesion response was unchanged; however, in CCR2 Ϫ/Ϫ mice in which the effect on macrophage accumulation was greater, SCG neurite outgrowth was significantly reduced. These results indicate that macrophages affect neurite outgrowth by acting at the level of peripheral ganglia in addition to any effects they might produce by facilitation of Wallerian degeneration.

Peripheral nerves regenerate following injury due to the effective activation of the intrinsic growth capacity of the neurons and the formation of a permissive pathway for outgrowth due to Wallerian degeneration. Wallerian degeneration and subsequent regeneration are significantly influenced by various immune cells and the cytokines they secrete. Although macrophages have long been known to play a vital role in the degenerative process, recent work has pointed to their importance in influencing the regenerative capacity of peripheral neurons. In this review, we focus on the various immune cells, cytokines, and chemokines that make regeneration possible in the peripheral nervous system, with specific attention placed on the role macrophages play in this process.

Neuroinflammation plays a critical role in the regeneration of peripheral nerves following axotomy. An injury to the sciatic nerve leads to significant macrophage accumulation in the L5 DRG, an effect not seen when the dorsal root is injured. We recently demonstrated that this accumulation around axotomized cell bodies is necessary for a peripheral conditioning lesion response to occur. Here we asked whether overexpression of the monocyte chemokine CCL2 specifically in DRG neurons of uninjured mice is sufficient to cause macrophage accumulation and to enhance regeneration or whether other injury-derived signals are required. AAV5-EF1α-CCL2 was injected intrathecally, and this injection led to a time-dependent increase in CCL2 mRNA expression and macrophage accumulation in L5 DRG, with a maximal response at 3 wk post-injection. These changes led to a conditioning-like increase in neurite outgrowth in DRG explant and dissociated cell cultures. This increase in regeneration was dependent upon CCL2 acting through its primary receptor CCR2. When CCL2 was overexpressed in CCR2 −/− mice, macrophage accumulation and enhanced regeneration were not observed. To address the mechanism by which CCL2 overexpression enhances regeneration, we tested for elevated expression of regeneration-associated genes in these animals. Surprisingly, we found that CCL2 overexpression led to a selective increase in LIF mRNA and neuronal phosphorylated STAT3 (pSTAT3) in L5 DRGs, with no change in expression seen in other RAGs such as GAP-43. Blockade of STAT3 phosphorylation by each of two different inhibitors prevented the increase in neurite outgrowth. Thus, CCL2 overexpression is sufficient to induce macrophage accumulation in uninjured L5 DRGs and increase the regenerative capacity of DRG neurons via a STAT3-dependent mechanism.

BackgroundNeuroinflammation accompanies neural trauma and most neurological diseases. Axotomy in the peripheral nervous system (PNS) leads to dramatic changes in the injured neuron: the cell body expresses a distinct set of genes known as regeneration-associated genes, the distal axonal segment degenerates and its debris is cleared, and the axons in the proximal segment form growth cones and extend neurites. These processes are orchestrated in part by immune and other non-neuronal cells. Macrophages in ganglia play an integral role in supporting regeneration. Here, we explore further the molecular and cellular components of the injury-induced immune response within peripheral ganglia.MethodsAdult male wild-type (WT) and Ccr2−/− mice were subjected to a unilateral transection of the sciatic nerve and axotomy of the superior cervical ganglion (SCG). Antibody arrays were used to determine the expression of chemokines and cytokines in the dorsal root ganglion (DRG) and SCG. Flow cytometry and immunohistochemistry were utilized to identify the cellular composition of the injury-induced immune response within ganglia.ResultsChemokine expression in the ganglia differed 48 h after nerve injury with a large increase in macrophage inflammatory protein-1γ in the SCG but not in the DRG, while C-C class chemokine ligand 2 was highly expressed in both ganglia. Differences between WT and Ccr2−/− mice were also observed with increased C-C class chemokine ligand 6/C10 expression in the WT DRG compared to C-C class chemokine receptor 2 (CCR2)−/− DRG and increased CXCL5 expression in CCR2−/− SCG compared to WT. Diminished macrophage accumulation in the DRG and SCG of Ccr2−/− mice was found compared to WT ganglia 7 days after nerve injury. Interestingly, neutrophils were found in the SCG but not in the DRG. Cytokine expression, measured 7 days after injury, differed between ganglion type and genotype. Macrophage activation was assayed by colabeling ganglia with the anti-inflammatory marker CD206 and the macrophage marker CD68, and an almost complete colocalization of the two markers was found in both ganglia.ConclusionsThis study demonstrates both molecular and cellular differences in the nerve injury-induced immune response between DRG and SCG and between WT and Ccr2−/− mice.

Background Peripheral nerve injuries stimulate the regenerative capacity of injured neurons through a neuroimmune phenomenon termed the conditioning lesion (CL) response. This response depends on macrophage accumulation in affected dorsal root ganglia (DRGs) and peripheral nerves. The macrophage chemokine CCL2 is upregulated after injury and is allegedly required for stimulating macrophage recruitment and pro-regenerative signaling through its receptor, CCR2. In these tissues, CCL2 is putatively produced by neurons in the DRG and Schwann cells in the distal nerve. Methods Ccl2fl/fl mice were crossed with Advillin-Cre, P0-Cre, or both to create conditional Ccl2 knockouts (CKOs) in sensory neurons, Schwann cells, or both to hypothetically remove CCL2 and macrophages from DRGs, nerves or both. CCL2 was localized using Ccl2–RFPfl/fl mice. CCL2–CCR2 signaling was further examined using global Ccl2 KOs and Ccr2gfp knock-in/knock-outs. Unilateral sciatic nerve transection was used as the injury model, and at various timepoints, chemokine expression, macrophage accumulation and function, and in vivo regeneration were examined using qPCR, immunohistochemistry, and luxol fast blue staining. Results Surprisingly, in all CKOs, DRG Ccl2 gene expression was decreased, while nerve Ccl2 was not. CCL2–RFP reporter mice revealed CCL2 expression in several cell types beyond the expected neurons and Schwann cells. Furthermore, macrophage accumulation, myelin clearance, and in vivo regeneration were unaffected in all CKOs, suggesting CCL2 may not be necessary for the CL response. Indeed, Ccl2 global knockout mice showed normal macrophage accumulation, myelin clearance, and in vivo regeneration, indicating these responses do not require CCL2. CCR2 ligands, Ccl7 and Ccl12, were upregulated after nerve injury and perhaps could compensate for the absence of Ccl2. Finally, Ccr2gfp knock-in/knock-out animals were used to differentiate resident and recruited macrophages in the injured tissues. Ccr2gfp/gfp KOs showed a 50% decrease in macrophages in the distal nerve compared to controls with a relative increase in resident macrophages. In the DRG there was a small but insignificant decrease in macrophages. Conclusions CCL2 is not necessary for macrophage accumulation, myelin clearance, and axon regeneration in the peripheral nervous system. Without CCL2, other CCR2 chemokines, resident macrophage proliferation, and CCR2-independent monocyte recruitment can compensate and allow for normal macrophage accumulation.