Down-Regulation of CXCL12/CXCR4 Expression Alleviates Ischemia-Reperfusion-Induced Inflammatory Pain via Inhibiting Glial TLR4 Activation in the Spinal Cord

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.

Elevation of the Chemokine Pair CXCL10/CXCR3 Initiates Sequential Glial Activation and Crosstalk During the Development of Bimodal Inflammatory Pain after Spinal Cord Ischemia Reperfusion

“…IR-induced pain was established in the rats through an 8-min occlusion of the aortic arch [1]. Briefly, all the rats were anesthetized with 2% sevoflurane and placed in a lateral position to expose the aortic arch through a cervicothoracic approach.…”

“…Mechanical allodynia was measured by recording the PWTs using von Frey filaments (37450, Ugo Basile, Dublin, Ireland), as previously described [1]. The tests were performed by another observer who was blinded to the experimental protocols.…”

“…Thermal allodynia was measured 15 min after the PWT measurements by recording the PWL using a hot plate (35150, Ugo Basile, Dublin, Ireland) [1]. The PWL was determined as the average from triplicate measurements of the time that the rat stayed on a hot plate at 50°C before it initially clearly withdrew its paws.…”

“…Because CXCR3 expression was closely related to glial activation, we further evaluated the statuses of microglia and astrocytes via immunofluorescence staining because this method intuitively displays the extent of activation through different fluorescence intensities [1]. Microglia, which are immunoreactive for Iba-1, were highly activated and showed increased fluorescence intensities at 12 h and 48 h post-IR.…”

“…It usually inflicts severe and persistent IR-induced pain, which is characterized by symptoms of mechanical and cold allodynia but not involve direct nerve injury [1,2]. The pathophysiological mechanisms are multifactorial and have not been completely elucidated.…”