A Conditioning Sciatic Nerve Lesion Triggers a Pro-regenerative State in Primary Sensory Neurons Also of Dorsal Root Ganglia Non-associated With the Damaged Nerve

The primary sensory neurons of dorsal root ganglia (DRG) are a very useful model to study the neuronal regenerative program that is a prerequisite for successful axon regeneration after peripheral nerve injury. Seven days after a unilateral sciatic nerve injury by compression or transection, we detected a bilateral increase in growth-associated protein-43 (GAP-43) and superior cervical ganglion-10 (SCG-10) mRNA and protein levels not only in DRG neurons of lumbar spinal cord segments (L4-L5) associated with injured nerve, but also in remote cervical segments (C6-C8). The increase in regeneration-associated proteins in the cervical DRG neurons was associated with the greater length of regenerated axons 1 day after ulnar nerve crush following prior sciatic nerve injury as compared to controls with only ulnar nerve crush. The increased axonal regeneration capacity of cervical DRG neurons after a prior conditioning sciatic nerve lesion was confirmed by neurite outgrowth assay of in vitro cultivated DRG neurons. Intrathecal injection of IL-6 or a JAK2 inhibitor (AG490) revealed a role for the IL-6 signaling pathway in activating the pro-regenerative state in remote DRG neurons. Our results suggest that the pro-regenerative state induced in the DRG neurons non-associated with the injured nerve reflects a systemic reaction of these neurons to unilateral sciatic nerve injury

Inflammatory Profiling of Schwann Cells in Contact with Growing Axons Distal to Nerve Injury

Activated Schwann cells distal to nerve injury upregulate inflammatory mediators, including cytokines. The goal of the present study was to investigate expression of proinflammatory (IL-1β, TNFα) and anti-inflammatory cytokines (IL-4, IL-10) in activated Schwann cells in relation to growing axons distal to crush injury of rat sciatic nerves. Seven days from sciatic nerve crush, transverse cryostat sections were cut 5 mm distal to lesion and incubated for double immunostaining to indicate Schwann cells (GFAP or S100b) and individual investigated cytokines or to demonstrate growing axons (GAP43). The Schwann cells of naïve sciatic nerves and those removed from sham-operated rats displayed similar weak immunoreactivity for the investigated cytokines. In contrast, increased intensity of cytokine immunofluorescence was found in Schwann cells distal to crush lesion. The cytokine-positive Schwann cells were found in close contact with growing axons detected by immunostaining for GAP43. The results of immunohistochemical analysis distal to nerve crush injury suggest that inflammatory profiling of Schwann cells including upregulation of both pro- and anti-inflammatory cytokines does not prevent growth of axons distal to nerve crush injury

CD200R1 agonist attenuates glial activation, inflammatory reactions, and hypersensitivity immediately after its intrathecal application in a rat neuropathic pain model

© 2016 Hernangómez et al.[Background]: Interaction of CD200 with its receptor CD200R has an immunoregulatory role and attenuates various types of neuroinflammatory diseases.[Methods]: Immunofluorescence staining, western blot analysis, and RT-PCR were used to investigate the modulatory effects of CD200 fusion protein (CD200Fc) on activation of microglia and astrocytes as well as synthesis of pro- (TNF, IL-1β, IL-6) and anti-inflammatory (IL-4, IL-10) cytokines in the L4-L5 spinal cord segments in relation to behavioral signs of neuropathic pain after unilateral sterile chronic constriction injury (sCCI) of the sciatic nerve. Withdrawal thresholds for mechanical hypersensitivity and latencies for thermal hypersensitivity were measured in hind paws 1 day before operation; 1, 3, and 7 days after sCCI operation; and then 5 and 24 h after intrathecal application of artificial cerebrospinal fluid or CD200Fc.[Results]: Seven days from sCCI operation and 5 h from intrathecal application, CD200Fc reduced mechanical and thermal hypersensitivity when compared with control animals. Simultaneously, CD200Fc attenuated activation of glial cells and decreased proinflammatory and increased anti-inflammatory cytokine messenger RNA (mRNA) levels. Administration of CD200Fc also diminished elevation of CD200 and CD200R proteins as a concomitant reaction of the modulatory system to increased neuroinflammatory reactions after nerve injury. The anti-inflammatory effect of CD200Fc dropped at 24 h after intrathecal application.[Conclusions]: Intrathecal administration of the CD200R1 agonist CD200Fc induces very rapid suppression of neuroinflammatory reactions associated with glial activation and neuropathic pain development. This may constitute a promising and novel therapeutic approach for the treatment of neuropathic pain.This work was supported by the projects CEITEC from European Regional Development Fund (CZ.1.05/1.1.00/02.0068) and Employment of Newly Graduated Doctors of Science for Scientific Excellence (CZ.1.07/2.3.00/30.0009)

Activation of Astrocytes and Microglial Cells and CCL2/CCR2 Upregulation in the Dorsolateral and Ventrolateral Nuclei of Periaqueductal Gray and Rostral Ventromedial Medulla Following Different Types of Sciatic Nerve Injury

Peripheral nerve injuries (PNIs) may result in cellular and molecular changes in supraspinal structures possibly involved in neuropathic pain (NPP) maintenance. Activated glial cells in specific supraspinal subregions may affect the facilitatory role of descending pathways. Sterile chronic compression injury (sCCI) and complete sciatic nerve transection (CSNT) in rats were used as NPP models to study the activation of glial cells in the subregions of periaqueductal gray (PAG) and rostral ventromedial medulla (RVM). Molecular markers for activated astrocytes (glial fibrillary acidic protein, GFAP) and microglial cells (OX42) were assessed by quantitative immunohistochemistry and western blotting. The cellular distribution of CCL2/CCR2 was monitored using immunofluorescence. sCCI induced both mechanical and thermal hypersensitivity from day 1 up to 3 weeks post-injury. Unilateral sCCI or CSNT for 3 weeks induced significant activation of astrocytes bilaterally in both dorsolateral (dlPAG) and ventrolateral PAG (vlPAG) compared to naïve or sham-operated rats. More extensive astrocyte activation by CSNT compared to sCCI was induced bilaterally in dlPAG and ipsilaterally in vlPAG. Significantly more extensive activation of astrocytes was also found in RVM after CSNT than sCCI. The CD11b immunopositive region, indicating activated microglial cells, was remarkably larger in dlPAG and vlPAG of both sides from sCCI- and CSNT-operated rats compared to naïve or sham-operated controls. No significant differences in microglial activation were detected in dlPAG or vlPAG after CSNT compared to sCCI. Both nerve injury models induced no significant differences in microglial activation in the RVM. Neurons and activated GFAP+ astrocytes displayed CCL2-immunoreaction, while activated OX42+ microglial cells were CCR2-immunopositive in both PAG and RVM after sCCI and CSNT. Overall, while CSNT induced robust astrogliosis in both PAG and RVM, microglial cell activation was similar in the supraspinal structures in both injury nerve models. Activated astrocytes in PAG and RVM may sustain facilitation of the descending system maintaining NPP, while microglial activation may be associated with a reaction to long-lasting peripheral injury. Microglial activation via CCR2 may be due to neuronal and astrocytal release of CCL2 in PAG and RVM following injury

Activation of Astrocytes and Microglial Cells and CCL2/CCR2 Upregulation in the Dorsolateral and Ventrolateral Nuclei of Periaqueductal Gray and Rostral Ventromedial Medulla Following Different Types of Sciatic Nerve Injury

Peripheral nerve injuries (PNIs) may result in cellular and molecular changes in supraspinal structures possibly involved in neuropathic pain (NPP) maintenance. Activated glial cells in specific supraspinal subregions may affect the facilitatory role of descending pathways. Sterile chronic compression injury (sCCI) and complete sciatic nerve transection (CSNT) in rats were used as NPP models to study the activation of glial cells in the subregions of periaqueductal gray (PAG) and rostral ventromedial medulla (RVM). Molecular markers for activated astrocytes (glial fibrillary acidic protein, GFAP) and microglial cells (OX42) were assessed by quantitative immunohistochemistry and western blotting. The cellular distribution of CCL2/CCR2 was monitored using immunofluorescence. sCCI induced both mechanical and thermal hypersensitivity from day 1 up to 3 weeks post-injury. Unilateral sCCI or CSNT for 3 weeks induced significant activation of astrocytes bilaterally in both dorsolateral (dlPAG) and ventrolateral PAG (vlPAG) compared to naïve or sham-operated rats. More extensive astrocyte activation by CSNT compared to sCCI was induced bilaterally in dlPAG and ipsilaterally in vlPAG. Significantly more extensive activation of astrocytes was also found in RVM after CSNT than sCCI. The CD11b immunopositive region, indicating activated microglial cells, was remarkably larger in dlPAG and vlPAG of both sides from sCCI- and CSNT-operated rats compared to naïve or sham-operated controls. No significant differences in microglial activation were detected in dlPAG or vlPAG after CSNT compared to sCCI. Both nerve injury models induced no significant differences in microglial activation in the RVM. Neurons and activated GFAP+ astrocytes displayed CCL2-immunoreaction, while activated OX42+ microglial cells were CCR2-immunopositive in both PAG and RVM after sCCI and CSNT. Overall, while CSNT induced robust astrogliosis in both PAG and RVM, microglial cell activation was similar in the supraspinal structures in both injury nerve models. Activated astrocytes in PAG and RVM may sustain facilitation of the descending system maintaining NPP, while microglial activation may be associated with a reaction to long-lasting peripheral injury. Microglial activation via CCR2 may be due to neuronal and astrocytal release of CCL2 in PAG and RVM following injur