Regeneration of peripheral neurons after a nerve crush is enhanced if the axons have received a prior injury, a phenomenon known as the conditioning lesion response (CLR). Neurons in the mouse L3-L5 dorsal root ganglia (DRGs) that project into the sciatic nerve are commonly used to examine the CLR. These ganglia contain a diverse population of somatosensory neurons, which exhibit dramatic heterogeneity in their response to a CL. As reported previously, the nociceptors [i.e., isolectin B4 binding (IB4+) and calcitonin gene-related peptide positive (CGRP+) neurons] do not show enhanced axonal growth in culture after an in vivo CL, whereas the remaining neurons on average do. We asked whether the failure to produce a CLR was an inherent property of certain neurons. Two difficulties in interpreting previous findings are that only about half of the neurons in these ganglia project into the sciatic nerve and that both IB4 binding and CGRP immunoreactivity decrease after axotomy. Therefore, a possibility not considered previously is that neurons that are still CGRP+ or IB4+ after sciatic nerve transection have not been axotomized. Genetic mutants that express a reporter in CGRP neurons such that its expression is unchanged after axotomy demonstrate that the absence of a CLR is an inherent property of these neurons; however, expression of a reporter in the MrgD (Mas-related gene D)/IB4 neurons demonstrate that these neurons in fact do exhibit a CLR. Among the CGRP-/IB4- neurons, two populations found to exhibit a CLR are tropomyosin receptor kinase C+ (TrkC+) and MrgD+ neurons.

BACKGROUND:Since the 1990s, evidence has accumulated that macrophages promote peripheral nerve regeneration and are required for enhancing regeneration in the conditioning lesion (CL) response. After a sciatic nerve injury, macrophages accumulate in the injury site, the nerve distal to that site, and the axotomized dorsal root ganglia (DRGs). In the peripheral nervous system, as in other tissues, the macrophage response is derived from both resident macrophages and recruited monocyte-derived macrophages (MDMs). Unresolved questions are: at which sites do macrophages enhance nerve regeneration, and is a particular population needed.METHODS:Ccr2 knock-out (KO) and Ccr2gfp/gfp knock-in/KO mice were used to prevent MDM recruitment. Using these strains in a sciatic CL paradigm, we examined the necessity of MDMs and residents for CL-enhanced regeneration in vivo and characterized injury-induced nerve inflammation. CL paradigm variants, including the addition of pharmacological macrophage depletion methods, tested the role of various macrophage populations in initiating or sustaining the CL response. In vivo regeneration, measured from bilateral proximal test lesions (TLs) after 2 d, and macrophages were quantified by immunofluorescent staining.RESULTS:Peripheral CL-enhanced regeneration was equivalent between crush and transection CLs and was sustained for 28 days in both Ccr2 KO and WT mice despite MDM depletion. Similarly, the central CL response measured in dorsal roots was unchanged in Ccr2 KO mice. Macrophages at both the TL and CL, but not between them, stained for the pro-regenerative marker, arginase 1. TL macrophages were primarily CCR2-dependent MDMs and nearly absent in Ccr2 KO and Ccr2gfp/gfp KO mice. However, there were only slightly fewer Arg1+ macrophages in CCR2 null CLs than controls due to resident macrophage compensation. Zymosan injection into an intact WT sciatic nerve recruited Arg1+ macrophages but did not enhance regeneration. Finally, clodronate injection into Ccr2gfp KO CLs dramatically reduced CL macrophages. Combined with the Ccr2gfp KO background, depleting MDMs and TL macrophages, and a transection CL, physically removing the distal nerve environment, nearly all macrophages in the nerve were removed, yet CL-enhanced regeneration was not impaired.CONCLUSIONS:Macrophages in the sciatic nerve are neither necessary nor sufficient to produce a CL response.

Neutrophils are a vital part of the innate immune system. Many of their functions eliminate bacteria & viruses, like neutrophil extracellular traps (NETs), which trap bacteria, enhancing macrophage phagocytosis. It was surprising when it was demonstrated that neutrophils are a part of Wallerian degeneration, a process that is essential for nerve regeneration after a nerve injury. It is not known what signals attract neutrophils into the nerve and how they aid Wallerian degeneration. Neutrophils accumulate in the distal nerve within one day after an injury and are found in the nerve from one to three days. We demonstrate that CXCR2 mediates the trafficking of neutrophils into the distal nerve, and without CXCR2 Wallerian degeneration, as indicated by luxol fast blue staining, was reduced seven days after a sciatic nerve crush or transection injury. NETs were detected in the distal nerve after a sciatic nerve transection. NET formation has been shown to require protein arginine deiminase 4 (PAD4), which citrullinates histone 3. Inhibiting PAD4 reduced NET formation significantly in the distal nerve at two days and myelin clearance at seven days indicating that NETs aid myelin clearance. These results demonstrate another function for NETs other than clearing pathogens. Neutrophils have been detected after injuries to the central nervous system and diseases in humans and animal models. Our results demonstrate neutrophils aid myelin clearance, suggesting a role for their presence in central nervous system injuries and diseases.

The role of inflammation in nervous system injury and disease is attracting increased attention. Much of that research has focused on microglia in the central nervous system (CNS) and macrophages in the peripheral nervous system (PNS). Much less attention has been paid to the roles played by neutrophils. Neutrophils are part of the granulocyte subtype of myeloid cells. These cells, like macrophages, originate and differentiate in the bone marrow from which they enter the circulation. After tissue damage or infection, neutrophils are the first immune cells to infiltrate into tissues and are directed there by specific chemokines, which act on chemokine receptors on neutrophils. We have reviewed here the basic biology of these cells, including their differentiation, the types of granules they contain, the chemokines that act on them, the subpopulations of neutrophils that exist, and their functions. We also discuss tools available for identification and further study of neutrophils. We then turn to a review of what is known about the role of neutrophils in CNS and PNS diseases and injury, including stroke, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, spinal cord and traumatic brain injuries, CNS and PNS axon regeneration, and neuropathic pain. While in the past studies have focused on neutrophils deleterious effects, we will highlight new findings about their benefits. Studies on their actions should lead to identification of ways to modify neutrophil effects to improve health.

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 Ccl2 fl/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–RFP fl/fl mice. CCL2–CCR2 signaling was further examined using global Ccl2 KOs and Ccr2 gfp 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, Ccr2 gfp knock-in/knock-out animals were used to differentiate resident and recruited macrophages in the injured tissues. C cr2 gfp/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.

Injury to the sciatic nerve leads to degeneration and debris clearance in the area distal to the injury site, a process known as Wallerian degeneration. Immune cell infiltration into the distal sciatic nerve plays a major role in the degenerative process and subsequent regeneration of the injured motor and sensory axons. While macrophages have been implicated as the major phagocytic immune cell participating in Wallerian degeneration, recent work has found that neutrophils, a class of short-lived, fast responding white blood cells, also significantly contribute to the clearance of axonal and myelin debris. Detection of specific myeloid subtypes can be difficult as many cell-surface markers are often expressed on both neutrophils and monocytes/macrophages. Here we describe two methods for detecting neutrophils in the axotomized sciatic nerve of mice using immunohistochemistry and flow cytometry. For immunohistochemistry on fixed frozen tissue sections, myeloperoxidase and DAPI are used to specifically label neutrophils while a combination of Ly6G and CD11b are used to assess the neutrophil population of unfixed sciatic nerves using flow cytometry.

Neuroinflammation 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.

Wallerian degeneration (WD) is considered an essential preparatory stage to the process of axonal regeneration. In the peripheral nervous system, infiltrating monocyte-derived macrophages, which use the chemokine receptor CCR2 to gain entry to injured tissues from the bloodstream, are purportedly necessary for efficient WD. However, our laboratory has previously reported that myelin clearance in the injured sciatic nerve proceeds unhindered in the Ccr2−/− mouse model. Here, we extensively characterize WD in male Ccr2−/− mice and identify a compensatory mechanism of WD that is facilitated primarily by neutrophils. In response to the loss of CCR2, injured Ccr2−/− sciatic nerves demonstrate prolonged expression of neutrophil chemokines, a concomitant extended increase in the accumulation of neutrophils in the nerve, and elevated phagocytosis by neutrophils. Neutrophil depletion substantially inhibits myelin clearance after nerve injury in both male WT and Ccr2−/− mice, highlighting a novel role for these cells in peripheral nerve degeneration that spans genotypes. SIGNIFICANCE STATEMENT The accepted view in the basic and clinical neurosciences is that the clearance of axonal and myelin debris after a nerve injury is directed primarily by inflammatory CCR2+ macrophages. However, we demonstrate that this clearance is nearly identical in WT and Ccr2−/− mice, and that neutrophils replace CCR2+ macrophages as the primary phagocytic cell. We find that neutrophils play a major role in myelin clearance not only in Ccr2−/− mice but also in WT mice, highlighting their necessity during nerve degeneration in the peripheral nervous system. These degeneration studies may propel improvements in nerve regeneration and draw critical parallels to mechanisms of nerve degeneration and regeneration in the CNS and in the context of peripheral neuropathies.

It is widely recognized that neurons in the peripheral nervous system (PNS) are capable of regeneration after injury. In contrast, those neurons that are entirely located within the CNS (i.e., the brain and spinal cord) largely do not regenerate. What is often not emphasized, however, is that the extent of PNS regeneration is limited and that this results in incomplete functional recovery (1). A great deal of attention has been focused recently on the cellular and molecular mechanisms underlying degeneration and regeneration in the PNS, with an eye toward enhancing this regeneration. Regeneration is not a neuron-autonomous process, but depends heavily on interactions between neurons, glial cells (i.e., Schwann cells in the nerve and satellite cells in peripheral ganglia), and immune cells, particularly macrophages (2, 3). Among the several reasons that have been postulated for the incomplete regeneration in the PNS, is the extremely slow rate of this regeneration (about 1–2 mm per day), leading perhaps to an eventual decrease in the intrinsic growth capacity of the injured axons and the limited period after injury during which Schwann cells actively promote axonal elongation (4). Once an axon is transected or crushed (referred to as axotomy), the distal segment fragments and then is cleared by phagocytic cells by a process called Wallerian degeneration. Advances in our understanding of the mechanism of Wallerian degeneration have come from genetic studies on the mutant gene WLDs and its normal counterpart NMNAT1 , both of which temporarily protect the axon from degeneration (5), and SARM1 , which is involved in triggering axon degeneration (6, 7). Schwann cells upregulate their expression and release of the macrophage chemokine CCL2, which draws in inflammatory monocytes that then differentiate into macrophages and are involved in phagocytosis of myelin and axonal debris (2). Although it was … [↵][1]1Email: rez{at}case.edu. [1]: #xref-corresp-1-1

Neuropathy is a major diabetic complication. While the mechanism of this neuropathy is not well understood, it is believed to result in part from deficient nerve regeneration. Work from our laboratory established that gp130 family of cytokines are induced in animals after axonal injury and are involved in the induction of regeneration-associated genes (RAGs) and in the conditioning lesion response. Here, we examine whether a reduction of cytokine signaling occurs in diabetes. Streptozotocin (STZ) was used to destroy pancreatic β cells, leading to chronic hyperglycemia. Mice were injected with either low doses of STZ (5×60mg/kg) or a single high dose (1×200mg/kg) and examined after three or one month, respectively. Both low and high dose STZ treatment resulted in sustained hyperglycemia and functional deficits associated with the presence of both sensory and autonomic neuropathy. Diabetic mice displayed significantly reduced intraepidermal nerve fiber density and sudomotor function. Furthermore, low and high dose diabetic mice showed significantly reduced tactile touch sensation measured with Von Frey monofilaments. To look at the regenerative and injury-induced responses in diabetic mice, neurons in both superior cervical ganglia (SCG) and the 4th and 5th lumbar dorsal root ganglia (DRG) were unilaterally axotomized. Both high and low dose diabetic mice displayed significantly less axonal regeneration in the sciatic nerve, when measured in vivo, 48h after crush injury. Significantly reduced induction of two gp130 cytokines, leukemia inhibitory factor and interleukin-6, occurred in diabetic animals in SCG 6h after injury compared to controls. Injury-induced expression of interleukin-6 was also found to be significantly reduced in the DRG at 6h after injury in low and high dose diabetic mice. These effects were accompanied by reduced phosphorylation of signal transducer and activator of transcription 3 (STAT3), a downstream effector of the gp130 signaling pathway. We also found decreased induction of several gp130-dependent RAGs, including galanin and vasoactive intestinal peptide. Together, these data suggest a novel mechanism for the decreased response of diabetic sympathetic and sensory neurons to injury.

Neurite outgrowth is key to the formation of functional circuits during neuronal development. Neurotrophins, including nerve growth factor (NGF), increase neurite outgrowth in part by altering the function and expression of Ca(2+)-permeable cation channels. Here we report that transient receptor potential vanilloid 2 (TRPV2) is an intracellular Ca(2+)-permeable TRPV channel upregulated by NGF via the mitogen-activated protein kinase (MAPK) signaling pathway to augment neurite outgrowth. TRPV2 colocalized with Rab7, a late endosome protein, in addition to TrkA and activated extracellular signal-regulated kinase (ERK) in neurites, indicating that the channel is closely associated with signaling endosomes. In line with these results, we showed that TRPV2 acts as an ERK substrate and identified the motifs necessary for phosphorylation of TRPV2 by ERK. Furthermore, neurite length, TRPV2 expression, and TRPV2-mediated Ca(2+) signals were reduced by mutagenesis of these key ERK phosphorylation sites. Based on these findings, we identified a previously uncharacterized mechanism by which ERK controls TRPV2-mediated Ca(2+) signals in developing neurons and further establish TRPV2 as a critical intracellular ion channel in neuronal function.

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 weeks 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.

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 (WD). WD 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.