Following injury to the peripheral nervous system (PNS), microglia in the spinal dorsal horn (SDH) become activated and contribute to the development of local neuro‐inflammation, which may regulate neuropathic pain processing. The molecular mechanisms that control microglial activation and its effects on neuropathic pain remain incompletely understood. We deleted the gene encoding the plasma membrane receptor, LDL Receptor‐related Protein‐1 (LRP1), conditionally in microglia using two distinct promoter‐Cre recombinase systems in mice. LRP1 deletion in microglia blocked development of tactile allodynia, a neuropathic pain‐related behavior, after partial sciatic nerve ligation (PNL). LRP1 deletion also substantially attenuated microglial activation and pro‐inflammatory cytokine expression in the SDH following PNL. Because LRP1 shedding from microglial plasma membranes generates a highly pro‐inflammatory soluble product, we demonstrated that factors which activate spinal cord microglia, including lipopolysaccharide (LPS) and colony‐stimulating factor‐1, promote LRP1 shedding. Proteinases known to mediate LRP1 shedding, including ADAM10 and ADAM17, were expressed at increased levels in the SDH after PNL. Furthermore, LRP1‐deficient microglia in cell culture expressed significantly decreased levels of interleukin‐1β and interleukin‐6 when treated with LPS. We conclude that in the SDH, microglial LRP1 plays an important role in establishing and/or amplifying local neuro‐inflammation and neuropathic pain following PNS injury. The responsible mechanism most likely involves proteolytic release of LRP1 from the plasma membrane to generate a soluble product that functions similarly to pro‐inflammatory cytokines in mediating crosstalk between cells in the SDH and in regulating neuropathic pain.

Sensory neurons in the PNS demonstrate substantial capacity for regeneration following injury. Recent studies have identified changes in the transcriptome of sensory neurons, which are instrumental for axon regeneration. The role of Schwann cells (SCs) in mediating these changes remains undefined. We tested the hypothesis that SCs regulate expression of genes in sensory neurons before and after PNS injury by comparing mice in which LDL Receptor-related Protein-1 (LRP1) is deleted in SCs (scLRP1 mice) with wild-type (scLRP1 ) littermates. LRP1 is an endocytic and cell-signaling receptor that is necessary for normal SC function and the SC response to nerve injury. scLRP1 mice represent a characterized model in which the SC response to nerve injury is abnormal. Adult DRG neurons, isolated from scLRP1 mice, with or without a conditioning nerve lesion, demonstrated increased neurite outgrowth when cultured ex vivo, compared with neurons from wild-type mice. Following sciatic nerve crush injury, nerve regeneration was accelerated in vivo in scLRP1 mice. These results were explained by transcriptional activation of RAGs in DRG neurons in scLRP1 mice prior to nerve injury. Although the presence of abnormal SCs in scLRP1 mice primed DRG neurons for repair, nerve regeneration in scLRP1 mice resulted in abnormalities in ultrastructure, principally in Remak bundles, and with the onset of neuropathic pain. These results demonstrate the importance of SCs in controlling RAG expression by neurons and the potential for this process to cause chronic pain when abnormal. The SC may represent an important target for preventing pain following PNS injury.

Abnormalities in interactions between sensory neurons and Schwann cells (SCs) may result in heightened pain processing and chronic pain states. We previously reported that SCs express the NMDA receptor (NMDA-R), which activates cell signaling in response to glutamate and specific protein ligands, such as tissue-type plasminogen activator. Herein, we genetically targetedgrin1encoding the essential GluN1 NMDA-R subunit, conditionally in SCs, to create a novel mouse model in which SCs are NMDA-R-deficient (GluN1– mice). These mice demonstrated increased sensitivity to light touch, pinprick, and thermal hyperalgesia in the absence of injury, without associated changes in motor function. Ultrastructural analysis of adult sciatic nerve in GluN1– mice revealed increases in the density of Aδ fibers and Remak bundles and a decrease in the density of Aβ fibers, without alteredg-ratios. Abnormalities in adult Remak bundle ultrastructure were also present including aberrant C-fiber ensheathment, distances between axons, and increased poly-axonal pockets. Developmental and post radial sorting defects contributed to altered nerve fiber densities in adult. Uninjured sciatic nerves in GluN1– mice did not demonstrate an increase in neuroinflammatory infiltrates. Transcriptome profiling of dorsal root ganglia (DRGs) revealed 138 differentially regulated genes in GluN1– mice. One third of the regulated genes are known to be involved in pain processing, includingsprr1a,npy,fgf3,atf3, andcckbr, which were significantly increased. The intraepidermal nerve fiber density (IENFD) was significantly decreased in the skin of GluN1– mice. Collectively, these findings demonstrate that SC NMDA-R is essential for normal PNS development and for preventing development of pain states.SIGNIFICANCE STATEMENTChronic unremitting pain is a prevalent medical condition; however, the molecular mechanisms that underlie heightened pain processing remain incompletely understood. Emerging data suggest that abnormalities in Schwann cells (SCs) may cause neuropathic pain. We established a novel mouse model for small fiber neuropathy (SFN) in whichgrin1, the gene that encodes the NMDA receptor (NMDA-R) GluN1 subunit, is deleted in SCs. These mice demonstrate hypersensitivity in pain processing in the absence of nerve injury. Changes in the density of intraepidermal small fibers, the ultrastructure of Remak bundles, and the transcriptome of dorsal root ganglia (DRGs) provide possible explanations for the increase in pain processing. Our results support the hypothesis that abnormalities in communication between sensory nerve fibers and SCs may result in pain states.

The goal of stem cell therapy for spinal cord injury (SCI) is to restore motor function without exacerbating pain. Induced pluripotent stem cells (iPSC) may be administered by autologous transplantation, avoiding immunologic challenges. Identifying strategies to optimize iPSC-derived neural progenitor cells (hiNPC) for cell transplantation is an important objective. Herein, we report a method that takes advantage of the growth factor-like and anti-inflammatory activities of the fibrinolysis protease, tissue plasminogen activator tPA, without effects on hemostasis. We demonstrate that conditioning hiNPC with enzymatically-inactive tissue-type plasminogen activator (EI-tPA), prior to grafting into a T3 lesion site in a clinically relevant severe SCI model, significantly improves motor outcomes. EI-tPA-primed hiNPC grafted into lesion sites survived, differentiated, acquired markers of motor neuron maturation, and extended βIII-tubulin-positive axons several spinal segments below the lesion. Importantly, only SCI rats that received EI-tPA primed hiNPC demonstrated significantly improved motor function, without exacerbating pain. When hiNPC were treated with EI-tPA in culture, NMDA-R-dependent cell signaling was initiated, expression of genes associated with stemness (Nestin, Sox2) was regulated, and thrombin-induced cell death was prevented. EI-tPA emerges as a novel agent capable of improving the efficacy of stem cell therapy in SCI.