Abstract
Toll-like receptor 4 (TLR4) is important for the pathogenesis of inflammatory reactions and the promotion of pain processing after ischemia/reperfusion (IR) in spinal cord. Recently, C-X-C chemokine ligand 12 (CXCL12) and its receptor, C-X-C chemokine receptor 4 (CXCR4), were demonstrated to be simultaneously critical for inflammatory reactions, thereby facilitating glial activation. However, whether CXCL12/CXCR4 expression can contribute to IR-induced inflammatory pain via spinal TLR4 remained unclear. A rat model was established by 8 min of aortic arch occlusion. The effects of CXCL12/CXCR4 expression and TLR4 activation on inflammatory hyperalgesia were investigated by pretreatments with CXCL12-neutralizing antibody, CXCR4 antagonist (AMD3100) and TLR4 antagonist (TAK-242) for 5 consecutive days before surgery. The results indicated that IR induced significant and sustained inflammatory pain, observed as decreases in paw withdrawal threshold (PWT) and paw withdrawal latency (PWL), throughout the post-injury period. The increased levels of TLR4 and proinflammatory chemokine CXCL12, as well as its receptor, CXCR4, were closely correlated with the PWT and PWL trends. Double immunostaining further suggested that TLR4, which is mainly expressed on astrocytes and microglia, was closely co-localized with CXCL12 and CXCR4 in spinal dorsal horn. As expected, intrathecal pretreatment with the TLR4 antagonist, TAK-242 markedly ameliorated pain by inhibiting astrocytic and microglial activation, as shown by decreases in TLR4 immunoreactivity and the percentage of double-labeled cells. These protective effects were likely due in part to the reduced production of the downstream cytokines IL-1β and TNF-α, as well as for the recruitment of CXCL12 and CXCR4. Additionally, intrathecal pretreatment with CXCL12-neutralizing antibody and AMD3100 resulted in similar analgesic and anti-inflammatory effects as those receiving TAK-242 pretreatment. These results suggest that intrathecal blockade of CXCL12/CXCR4 expression may attenuate IR-induced pain sensation and the release of inflammatory cytokines by limiting glial TLR4 activation in spinal cord.
Highlights
Spinal cord ischemia-reperfusion (IR) injury is a devastating, incapacitating complication that often inflicts persistent inflammatory pain and affects a considerable proportion of the human population worldwide [1]
Pain is processed in neural networks, increasing evidence suggests that neuron-glial interactions, such as those of microglia and astrocytes, and Toll-like receptor (TLR)-mediated glial neuroinflammation in the spinal cord play important roles in the promotion and maintenance of pain [2,3,4]
Compared with rats in the IR group, paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were markedly increased at all the observed time points in rats that were intrathecally injected with CXCL12-neutralizing antibody (I/R + N group), CXCR4-specific antagonist (I/R + A group) or Toll-like receptor 4 (TLR4) inhibitor (I/R + T group) (P < 0.05)
Summary
Spinal cord ischemia-reperfusion (IR) injury is a devastating, incapacitating complication that often inflicts persistent inflammatory pain and affects a considerable proportion of the human population worldwide [1]. Pain is processed in neural networks, increasing evidence suggests that neuron-glial interactions, such as those of microglia and astrocytes, and Toll-like receptor (TLR)-mediated glial neuroinflammation in the spinal cord play important roles in the promotion and maintenance of pain [2,3,4]. As shown in our previous studies, IR-activated microglia, which exert their effects via TLR4 and induce further release of the proinflammatory chemokine IL-1β, can contribute to a secondary cascade of inflammatory responses as well as to decreased pain thresholds [8]. A recent study showed that in a model of chronic pancreatitis-induced mechanical allodynia, TLR4 was greatly increased in astrocytes of the spinal dorsal horn. Our previous study suggested that neutralizing TLR4 function significantly attenuated the mechanical allodynia after spinal cord IR [8]. Finding and inhibiting the mediators that evoke and amplify TLR4 expression might lead to relatively broad therapies for interrupting the inflammatory feedback and improving the inflammatory pain of patients with IR injury
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