Search

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.

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.

The cellular distribution and changes in CX3CL1/fractalkine and its receptor CX3CR1 protein levels in the trigeminal subnucleus caudalis (TSC) of rats with unilateral infraorbital nerve ligation (IONL) were investigated on postoperation days 1, 3, 7, and 14 (POD1, POD3, POD7, and POD14, respectively) and compared with those of sham-operated and naïve controls. Behavioral tests revealed a significant increase in tactile hypersensitivity bilaterally in the vibrissal pads of both sham- and IONL-operated animals from POD1 to POD7, with a trend towards normalization in sham controls at POD14. Image analysis revealed increased CX3CL1 immunofluorescence (IF) intensities bilaterally in the TSC neurons of both sham- and IONL-operated rats at all survival periods. Reactive astrocytes in the ipsilateral TSC also displayed CX3CL1-IF from POD3 to POD14. At POD1 and POD3, microglial cells showed high levels of CX3CR1-IF, which decreased by POD7 and POD14. Conversely, CX3CR1 was increased in TSC neurons and reactive astrocytes at POD7 and POD14, which coincided with high levels of CX3CL1-IF and ADAM17-IF. This indicates that CX3CL1/CX3CR1 may be involved in reciprocal signaling between TSC neurons and reactive astrocytes. The level of CatS-IF in microglial cells suggests that soluble CX3CL1 may be involved in neuron–microglial cell signaling at POD3 and POD7, while ADAM17 allows this release at all studied time points. These results indicate an extended CX3CL1/CX3CR1 signaling axis and its role in the crosstalk between TSC neurons and glial cells during the development of trigeminal neuropathic pain. [ABSTRACT FROM AUTHOR]

Glial cells activated by peripheral nerve injury contribute to the induction and maintenance of neuropathic pain by releasing neuromodulating cytokines and chemokines. We investigated the activation of microglia and astrocytes as well as the cellular distribution of the chemokine CCL2 and its receptor CCR2 in the trigeminal subnucleus caudalis (TSC) ipsilateral and contralateral to infraorbital nerve ligature (IONL). The left infraorbital nerve was ligated under aseptic conditions, and sham controls were operated without nerve ligature. Tactile hypersensitivity was significantly increased bilaterally in vibrissal pads of both sham- and IONL-operated animals from day 1 to 7 and tended to normalize in sham controls surviving for 14 days. Activated microglial cells significantly increased bilaterally in the TSC of both sham- and IONL-operated animals with a marked but gradual increase in the ipsilateral TSC from 1 to 7 days followed by a decrease by day 14. In contrast, robust activation of astrocytes was found bilaterally in the TSC of IONL-operated rats from 3 to 14 days with a transient activation in the ipsilateral TSC of sham-operated animals. Cellular distribution of CCL2 varied with survival time. CCL2 immunofluorescence was detected in neurons within 3 days and in astrocytes at later time points. In contrast, CCR2 was found only in astrocytes at all time points with CCR2 intensity being dominant in the ipsilateral TSC. In summary, our results reveal bilateral activation of microglial cells and astrocytes as well as changes in the cellular distribution of CCL2 and its receptor CCR2 in the TSC during the development and maintenance of orofacial neuropathic pain. [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]

Background: IL-6 is a typical injury-induced mediator. Together with its receptors, IL-6 contributes to both induction and maintenance of neuropathic pain deriving from changes in activity of primary sensory neurons in dorsal root ganglia (DRG). We used in situ hybridization to provide evidence of IL-6 and IL-6 receptors (IL-6R and gp130) synthesis in DRG along the neuraxis after unilateral chronic constriction injury (CCI) of the sciatic nerve as an experimental model of neuropathic pain. Results: All rats operated upon to create unilateral CCI displayed mechanical allodynia and thermal hyperalgesia in ipsilateral hind paws. Contralateral hind paws and forepaws of both sides exhibited only temporal and nonsignificant changes of sensitivity. Very low levels of IL-6 and IL-6R mRNAs were detected in naïve DRG. IL-6 mRNA was bilaterally increased not only in DRG neurons but also in satellite glial cells (SGC) activated by unilateral CCI. In addition to IL-6 mRNA, substantial increase of IL-6R mRNA expression occurred in DRG neurons and SGC following CCI, while the level of gp130 mRNA remained similar to that of DRG from naïve rats. Conclusions: Here we evidence for the first time increased synthesis of IL-6 and IL-6R in remote cervical DRG nonassociated with the nerve injury. Our results suggest that unilateral CCI of the sciatic nerve induced not only bilateral elevation of IL-6 and IL-6R mRNAs in L4-L5 DRG but also their propagation along the neuraxis to remote cervical DRG as a general neuroinflammatory reaction of the nervous system to local nerve injury without correlation with signs of neuropathic pain. Possible functional involvement of IL-6 signaling is discussed. [ABSTRACT FROM AUTHOR]

Summary: Peripheral neuropathic pain (PNP) frequently occurs as a consequence of nerve injury and may differ depending upon the type of insult and the individual patient. Progress in our knowledge of PNP induction mechanisms depends upon the utilization of appropriate experimental models in rodents based on various types of peripheral nerve lesions. In this review, we draw attention to current knowledge on basic cellular and molecular events in various experimental models used to induce the PNP symptoms. Spontaneous ectopic activity of axotomized and non-axotomized primary sensory neurons, the bodies of which are located in the dorsal root ganglion (DRG), seems to be a key mechanism of PNP induction. The primary sensory neurons are directly affected by nerve injury or indirectly by activated satellite glial cells and adjoining immune cells that release a variety of molecules changing the microenvironment of the neurons. Recently, it has become clear that molecules produced during Wallerian degeneration play an important role not only in axon-promoting conditions distal to nerve injury but also in initiation of neuropathic pain. The molecules, transported by the blood, influence afferent neurons and their axons not only in DRG associated, but also those not directly associated with the injured nerve (i.e., in the contralateral DRG or at different spinal segments). Generally, all experimental PNP models based on a partial injury of peripheral nerve segments contain mechanisms initiated by signal molecules of Wallerian degeneration. [Copyright &y& Elsevier]

The bodies of primary sensory neurons and their satellite glial cells (SGCs) are limited by the basal laminae from extracellular matrix of the dorsal root ganglia (DRG). The basal laminae displayed uniform immunofluorescence staining for laminin-1 in the sections of rat intact (naive) DRG. A proximal or distal ligature of the spinal nerves resulted in a heterogeneous immunostaining for laminin-1 around neuron-SGC units in the sections of the corresponding DRG. The pattern of irregular laminin-1 immunofluorescence was more extensive in the ipsilateral than the contralateral DRG of the operated rats. The immunofluorescence for laminin-1 exactly coincided with binding of Concanavalin-A as well as immunostaining for type IV collagen in both naive DRG and DRG affected by nerve ligature. Nidogen immunostaining decreased or fully disappeared at the surface of the SGCs consistently with immunofluorescence staining for laminin-1, but retained or increased in the endothelial cells and ED-1 positive cells invaded the DRG affected by nerve ligature. The results indicate an alteration of the content of basal laminae surrounding the bodies of primary sensory neurons and their SGSs following nerve constriction injury. A modulation of the basal laminae may be related with other cellular and molecular alterations related with peripheral neuropathic pain, for example, expansion of sympathetic sprouts. [ABSTRACT FROM AUTHOR]

Summary: In our experimental paradigm we sutured the distal stump of a transected musculocutaneous nerve to an intact ulnar nerve of the rat in an end-to-side fashion. We demonstrated the formation of collateral sprouts from intact afferent and motor axons by application of one type of molecule conjugated by two different fluorophores (Fluoro-Ruby© and Fluoro-Emerald©). Fluoro-Ruby and Fluoro-Emerald were applied distal to end-to-side suture into fresh cut ends of the ulnar and musculocutaneous nerves, respectively. Formation of collateral sprouts was evidenced by findings of mixed (a yellow to orange color) fluorescence labeling of spinal motoneurons and dorsal root ganglia neurons. Colocalization of two different tracers retrogradely transported to the neurons was verified by the individual green and red fluorescence profiles analyzed by means of the computer-assisted image-analyzing system. Our results unequivocally demonstrate that a nerve stump attached to an intact nerve can induce collateral sprouting of both afferent and motor axons. [Copyright &y& Elsevier]