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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.

There is a growing body of evidence that cytokines contribute to both induction and maintenance of neuropathic pain derived from changes in dorsal root ganglia (DRG), including the activity of the primary sensory neurons and their satellite glial cells (SGC). We used immunofluorescence and in situ hybridization methods to provide evidence that chronic constriction injury (CCI) of the sciatic nerve induces synthesis of interleukin-6 (IL-6) in SGC, elevation of IL-6 receptor (IL-6R) and activation of signal transducer and activator of transcription 3 (STAT3) signalling. Unilateral CCI of the rat sciatic nerve induced mechanoallodynia and thermal hyperalgesia in ipsilateral hind paws, but contralateral paws exhibited only temporal changes of sensitivity. We demonstrated that IL-6 mRNA and protein, which were expressed at very low levels in naïve DRG, were bilaterally increased not only in L4-L5 DRG neurons but also in SGC activated by unilateral CCI. Besides IL-6, substantial increase of IL-6R and pSTAT3 expression occurred in SGC following CCI, however, IL-6R associated protein, gp130 levels did not change. The results may suggest that unilateral CCI of the sciatic nerve induces bilateral activation of SGC in L4-L5 DRG to transduce IL-6 signalling during neuroinflammation.

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]

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 rostralventromedial 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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]