The Roles And Mechanisms Of Glial Cells In Spinal Cord And Dorsal Root Ganglion Of Mice In Regulating Burn Injury-induced Pain

Burn injury is one of the common clinical severe traumas,which induced skin injury,inflammation,and nerve terminal injury can cause a severe pain response.However,due to the pathogenesis of burn injury-induced pain（BIP）remains unclear,the clinical treatment of BIP faces great challenges.In recent years,the role of the network between glial cells and neurons has received extensive attention in the development and maintenance of chronic pain.However,the regulation effect of glial cells on BIP remains unknown.Therefore,we established a mouse skin second-degree burn injury model（65℃,15 s）and discussed the roles and regulatory mechanisms of glial cells in the occurrence and development of BIP at central（spinal cord）and peripheral（dorsal root ganglion,DRG）levels,to provide theoretical support for the development of effective analgesia.The main research work in this paper includes two parts:Part I Roles and molecular mechanisms of spinal microglia and astrocytes in the occurrence and development of BIP in miceThe activation of spinal microglia and astrocytes varies in time and space,which plays a regulating role in different periods of pathological pain.In addition,the mutual regulation between microglia and astrocytes may further affect synaptic transmission and promotes the formation of chronic pain.Therefore,we studied the regulatory effect and functional linkage of spinal microglia and astrocytes in the second-degree burn injury model.Burn injury mice developed significant mechanical allodynia and thermal hyperalgesia for at least 2 weeks.Immunofluorescence results showed that microglial cells in the spinal dorsal horn（SDH）rapidly increased and peaked at 5 days post-burn.In contrast,the activation of astrocytes was significantly increased at 10 days post-burn.Intrathecally injected with the microglia inhibitor Minocycline（100 nmol）elicited powerful anti-nociceptive effects in the early-phase（day 3 and day 5 post-burn）of BIP,but the astrocyte toxin L-α-AA（50 nmol）potently reversed the mechanical allodynia and thermal hyperalgesia in the late-phase（day 10 and day 12 post-burn）of BIP.These results indicate that spinal microglia and astrocytes mainly contributed to the formation and maintenance of BIP,respectively.Numerous studies have shown that mitogen-activated protein kinases（MAPKs）signaling pathways play an important role in intracellular signaling in glial cells.Our present results demonstrated that the phosphorylation of p38 MAPKs in mice SDH microglia was significantly up-regulated on 5 days post-burn.In addition,the transcription and protein levels of TNF-αin microglia were significantly upregulated,and the selective p38 MAPKs inhibitor SB203580（30 nmol）significantly reversed the synthesis and release of TNF-α.Intrathecal injection of the selective p38 MAPKs inhibitor SB203580（30 nmol）or the TNF-αneutralizing antibody（1μg）completely reversed the mechanical allodynia and thermal hyperalgesia in the early-phase of BIP.It’s worth noting that intraperitoneal injection of thalidomide potently reduced burn injury-induced hypersensitivity and the activation of the astrocytes in the ipsilateral SDH.On the contrary,repeated intrathecal injection of TNF-αsignificantly increased the immunofluorescence intensity of GFAP in the SDH of na?ve mice,indicating that microglia-derived TNF-αserves as an activator of astrocytes in this BIP model.The expression of p-JNK MAPKs in the spinal cord was significantly upregulated in the late-phase of BIP,which was in alignment with the variation tendency of the activation of astrocytes.Meanwhile,the transcription of Cxcl1 was significantly increased on day 12 after burn injury.In addition,intrathecal the JNK inhibitor SP600125（50 nmol）or the selective CXCR2 antagonist SB225002（20μg）exhibited potent anti-allodynic and anti-hyperalgesic effects in the late-phase of BIP.From this,the astrocytic JNK MAPKs/CXCL1 signaling pathway might be involved in the regulation of the late-phase of BIP.The results of RT-PCR showed that the Cxcl1 m RNA levels of the primary astrocytes and mice spinal cord were substantially increased after TNF-αtreatment,and the JNK inhibitor SP600125 reversed TNF-α-induced Cxcl1m RNA upregulation.In conclusion,the present study for the first time elaborates that the SDH microglia p38 MAPKs/TNF-αpathway and astrocytes JNK MAPKs/CXCL1 pathway attribute to the early-phase and late-phase BIP,respectively.In addition,spinal microglia-derived TNF-αis a critical mediator in burn injury-induced astrogliosis.PartⅡEffects and molecular mechanisms of DRG sensory neuron-SGCs crosstalk on the mice BIP modelTransient receptor potential vanillic acid 1（TRPV1）in DRG sensory neurons is a heat sensor（>43℃）,which contributes to the development of burn injury-induced hypersensitivity.In addition,the crosstalk between DRG neurons and SGCs participates in the formation of peripheral sensitization of pathological pain.Therefore,the present research further investigates the effect and molecular mechanisms of the bidirectional regulation between DRG sensory neurons and SGCs in BIP.In comparison to the sham group,the transcription of Trpv1 in mice DRG was significantly increased on day 5 after burn injury.Calcium imaging results showed that TRPV1-mediated Ca²⁺influx in burn injury mice DRG neurons was significantly stronger than that in sham group.In addition,burn injury also promoted the expression of calcitonin gene-related peptide（CGRP）in the DRG of BIP mice.And the TRPV1inhibitor AMG9810 reversed the upregulation of CGRP after burn injury,implying that the release of CGRP was regulated by TRPV1 in DRG neurons after burn injury.Consistent with these results,intraplantar injection of capsaicin,an agonist of TRPV1,significantly promoted CGRP expression in DRG neurons.The behavioral tests indicated that continuous intrathecal injection of the TRPV1 inhibitor AMG9810（45μg）or the CGRP receptor antagonist BIBN 4096BS（3 mg/kg）completely reversed the burn injury-induced mechanical allodynia.Furthermore,immunofluorescence results showed that SGCs markedly activated in the DRG for at least 12 days.It’s worth noting that continuous subcutaneous injection of CGRP receptor antagonist BIBN 4096BS（3 mg/kg）potently reduced burn injury-induced proliferation of SGCs.In contrast,intrathecal injection of CGRP（2μg）significantly decreased the mechanical thresholds and increased the immunofluorescence intensity of GFAP in the DRG of na?ve mice,indicating that DRG neurons-derived CGRP contributed to the activation of SGCs after burn injury.Gap junctions are involved in the cellular communication between SGCs.The expression of gap junction and the transcription of Cxcl1 and Ccl2 significantly increased in the SGCs after burn injury.The gap junction inhibitor carbenoxolone（50 mg/kg）reduced the coupling activation of SGCs and down-regulated Cxcl1 and Ccl2 m RNA levels after burn injury.In addition,carbenoxolone exhibited potent antinociceptive effects in the burn injury mice.These results demonstrated that the gap junction play important regulatory roles in the coupling activation of SGCs,the expression of chemokines,and the formation of allodynia.The results of electrophysiological recordings revealed that CXCL1（4 n M）significantly increased the firing rate of action potentials of primary DRG neurons.In addition,intrathecal injection of CXCL1（100 ng）significantly promoted the release of CGRP and decreased the mechanical thresholds in the DRG of the na?ve mice,indicating SGCs-derived CXCL1 could facilitate the release of CGRP,the excitability of DRG sensory neurons and the formation of peripheral sensitization after burn injury.The above results demonstrated that the neurotransmitter CGRP released by DRG neurons contributed to the activation of local SGCs after burn injury.Gap junction further promoted coupling activation of SGCs.In addition,SGCs-derived chemokines further enhanced the excitability of sensory neurons,which results in the formation of mechanical allodynia in mice after burn injury.In conclusion,we have systematically investigated the functional roles and regulatory mechanisms of microglia,astrocytes,and SGCs in the spinal cord and peripheral（DRG）levels in a mouse model of second-degree BIP using behavioral evaluation,molecular biology,and electrophysiological patch-clamp recording.These new findings expand the regulation theory of glial cells in BIP and raise the theoretical basis for the subsequent clinical treatment and new drug development of BIP targeting glial cells and their downstream signaling pathways.