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CXCL12 and CXCR4 proteins and mRNAs were monitored in the dorsal root ganglia (DRGs) of lumbar (L4–L5) and cervical (C7–C8) spinal segments of naïve rats, rats subjected to sham operation, and those undergoing unilateral complete sciatic nerve transection (CSNT) on post-operation day 7 (POD7). Immunohistochemical, Western blot, and RT-PCR analyses revealed bilaterally increased levels of CXCR4 protein and mRNA in both lumbar and cervical DRG neurons after CSNT. Similarly, CXCL12 protein levels increased, and CXCL12 mRNA was upregulated primarily in lumbar DRGs ipsilateral to the nerve lesion. Intrathecal application of the CXCR4 inhibitor AMD3100 following CSNT reduced CXCL12 and CXCR4 protein levels in cervical DRG neurons, as well as the length of afferent axons regenerated distal to the ulnar nerve crush. Furthermore, treatment with the CXCR4 inhibitor decreased levels of activated Signal Transducer and Activator of Transcription 3 (STAT3), a critical transforming factor in the neuronal regeneration program. Administration of IL-6 increased CXCR4 levels, whereas the JAK2-dependent STAT3 phosphorylation inhibitor (AG490) conversely decreased CXCR4 levels. This indicates a link between the CXCL12/CXCR4 signaling axis and IL-6-induced activation of STAT3 in the sciatic nerve injury-induced pro-regenerative state of cervical DRG neurons. The role of CXCR4 signaling in the axon-promoting state of DRG neurons was confirmed through in vitro cultivation of primary sensory neurons in a medium supplemented with CXCL12, with or without AMD3100. The potential involvement of conditioned cervical DRG neurons in the induction of neuropathic pain is discussed. [ABSTRACT FROM AUTHOR]

To assess the potential role of IL-6 in sciatic nerve injury-induced activation of a pro-regenerative state in remote dorsal root ganglia (DRG) neurons, we compared protein levels of SCG-10 and activated STAT3, as well as axon regeneration in IL-6 knockout (IL-6ko) mice and their wild-type (WT) counterparts. Unilateral sciatic nerve compression and transection upregulated SCG-10 protein levels and activated STAT3 in DRG neurons not only in lumbar but also in cervical segments of WT mice. A pro-regenerative state induced by prior sciatic nerve lesion in cervical DRG neurons of WT mice was also shown by testing for axon regeneration in crushed ulnar nerve. DRG neurons from IL-6ko mice also displayed bilaterally increased levels of SCG-10 and STAT3 in both lumbar and cervical segments after sciatic nerve lesions. However, levels of SCG-10 protein in lumbar and cervical DRG of IL-6ko mice were significantly lower than those of their WT counterparts. Sciatic nerve injury induced a lower level of SCG-10 in cervical DRG of IL-6ko than WT mice, and this correlates with significantly shorter regeneration of axons distal to the crushed ulnar nerve. These results suggest that IL-6 contributes, at the very least, to initiation of the neuronal regeneration program in remote DRG neurons after unilateral sciatic nerve injury. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The anatomical position of the subarachnoid space (SAS) in relation to dorsal root ganglia (DRG) and penetration of tracer from the SAS into DRG were investigated. We used intrathecal injection of methylene blue to visualize the anatomical position of the SAS in relation to DRG and immunostaining of dipeptidyl peptidase IV (DPP-IV) for detecting arachnoid limiting the SAS. Intrathecal administration of fluorescent-conjugated dextran (fluoro-emerald; FE) was used to demonstrate direct communication between the SAS and DRG. Intrathecal injection of methylene blue and DPP-IV immunostaining revealed that SAS delimited by the arachnoid was extended up to the capsule of DRG in a fold-like recess that may reach approximately half of the DRG length. The arachnoid was found in direct contact to the neuronal body-rich area in the angle between dorsal root and DRG as well as between spinal nerve roots at DRG. Particles of FE were found in the cells of DRG capsule, satellite glial cells, interstitial space, as well as in small and medium-sized neurons after intrathecal injection. Penetration of FE from the SAS into the DRG induced an immune reaction expressed by colocalization of FE and immunofluorescence indicating antigen-presenting cells (MHC-II+), activated (ED1+) and resident (ED2+) macrophages, and activation of satellite glial cells (GFAP+). Penetration of lumbar-injected FE into the cervical DRG was greater than that into the lumbar DRG after intrathecal injection of FE into the cisterna magna. Our results demonstrate direct communication between DRG and cerebrospinal fluid in the SAS that can create another pathway for possible propagation of inflammatory and signaling molecules from DRG primary affected by peripheral nerve injury into DRG of remote spinal segments. [ABSTRACT FROM AUTHOR]

One of the changes brought about by Wallerian degeneration distal to nerve injury is disintegration of axonal mitochondria and consequent leakage of mitochondrial DNA (mtDNA)—the natural ligand for the toll-like receptor 9 (TLR9). RT-PCR and immunohistochemical or Western blot analyses were used to detect TLR9 mRNA and protein respectively in the lumbar (L4-L5) and cervical (C7-C8) dorsal root ganglia (DRG) ipsilateral and contralateral to a sterile unilateral sciatic nerve compression or transection. The unilateral sciatic nerve lesions led to bilateral increases in levels of both TLR9 mRNA and protein not only in the lumbar but also in the remote cervical DRG compared with naive or sham-operated controls. This upregulation of TLR9 was linked to activation of the Nuclear Factor kappa B (NFκB) and nuclear translocation of the Signal Transducer and Activator of Transcription 3 (STAT3), implying innate neuronal immune reaction and a pro-regenerative state in uninjured primary sensory neurons of the cervical DRG. The relationship of TLR9 to the induction of a pro-regenerative state in the cervical DRG neurons was confirmed by the shorter lengths of regenerated axons distal to ulnar nerve crush following a previous sciatic nerve lesion and intrathecal chloroquine injection compared with control rats. The results suggest that a systemic innate immune reaction not only triggers the regenerative state of axotomized DRG neurons but also induces a pro-regenerative state further along the neural axis after unilateral nerve injury. [ABSTRACT FROM AUTHOR]