Dissection Of Spinal Mechanisms Underlying Mirror-image Pain

Mechanical allodynia（MA）,which includes gentle brushing-evoked dynamic and light pressure-evoked punctate forms,is a common and debilitating symptom experienced by millions of patients worldwide following peripheral inflammation or nerve injury.In both humans and animals,some local unilateral injuries lead to full-blown bilateral MA;however,most unilateral injuries in human patients and commonly used inflammatory and neuropathic pain models in animals usually manifest mechanical allodynia on the side ipsilateral to the injury（called mirror-image pain）,but not the contralateral uninjured side.For example,patients with unilateral carpal tunnel syndrome can experience bilateral MA,but only a minority of patients with complex regional pain syndrome do so.Unilateral injection of complete Freund’s adjuvant（CFA）or carrageenan can induce bilateral MA in rats,while nerve injury in most cases only evokes unilateral MA in mice.How can certain unilateral injuries produce bilateral MA?To date,how such laterality is controlled remains poorly understood.MA pathways pre-exist,but normally“gated”in the spinal dorsal horn.The question of the control of MA laterality can be addressed by examining the gate control mechanisms of the bilaterality of MA.According to the gate control theory of pain,pain transmission neurons（T neurons）in the spinal dorsal horn（SDH）receive inputs from low-threshold Aβmechanoreceptors;however,these inputs are normally gated by inhibitory neurons（INs）in the SDH.Enhanced excitatory outputs from T neurons or disinhibition of INs caused by peripheral inflammation or nerve injury can open the gate,allowing non-painful mechanical stimuli to generate the perception of pain.Several studies have addressed the identities of T neurons and INs involved in somatosensory MA that support the gate control theory of pain.Despite recent progress regarding the identities of T neurons and INs,one major unanswered question remains:what are the main forces that drive and influence the excitability of T neurons and INs,to subsequently affect the gate control of bilateral MA pathways in the SDH?Some studies suggest that bilateral MA is mediated by bilateral descending modulation of dorsal horn circuits,presumably by modulating the excitability of T neurons and INs in the SDH.Activated glial cells may also influence the excitability of T neurons and INs in the SDH following peripheral inflammation or nerve injury.Several groups have supported the critical role of activated astrocytes in the development of bilateral MA.However,the role of microglia in the control of MA laterality remains controversial.For instance,Schreiber et al.,reported that intrathecal administration of minocycline,a selective inhibitor of microglial activation,inhibits the development of carrageenan-induced contralateral MA in mice.In contrast,Choi et al.,reported that activated spinal microglia prevent the development of carrageenan-induced contralateral MA in rats.The first part of this thesis（Chapters 3 and 4）aimed to investigate the role of microglia in the control of MA in mice.To this end,（1）we screened behavioral phenotypes in 6 pain models of unilateral injury-induced bilateral dynamic and punctate MA to perform loss-of-function studies;（2）to determine whether the behavioral signs of bilateral MA are correlated with a bilateral change in the gate control within the SDH,in dorsal root ganglion（DRG）–dorsal root–sagittal spinal cord slice preparations,we recorded low-threshold Aβ-fiber stimulation-evoked inputs and outputs of SDH neurons in the 6 MA models;and（3）we used genetic strategies to deplete microglia in these transgenic mice.Surprisingly,central microglial depletion did not prevent the induction of bilateral dynamic or punctate MA.Moreover,consistent with the behavioral tests,microglial depletion did not prevent the opening of bilateral gates for Aβpathways in the superficial dorsal horn.These results suggest that microglia are not required for the initiation of bilateral MA in mice.The first part of this study challenges the role of microglia in the control of MA laterality in mice.Future studies are needed to further understand whether role of microglia in the control of MA laterality is etiology-or species-specific.The second part of this thesis（Chapter 5）went on to explore neural mechanisms underlying laterality control of mechanical allodynia.Chapter 5examined the role of spinal kappa opioid receptors（KORs）in the laterality control of MA in mice.To this end,（1）we performed whole-cell patch clamp recording of KOR⁺neurons in the spinal dorsal horn and found increased excitability of contralateral KOR⁺neurons upon hindpaw capsaicin injection;（2）i.t application of KOR antagonist nor-BNI before hindpaw capsaicin injection prevented the opening of contralateral Aβpathways,suggesting that spinal KORs are required for the induction of contralateral Aβpathways in hindpaw capsaicin model;and（3）consistent with i.t.injection of KOR antagonist,genetic knockout of KORs from dorsal horn neurons with Lbx1^Cre/Oprk1^fl/fl mice also prevented the opening of contralateral Aβpathways in hindpaw capsaicin model.These results demonstrate that spinal KORs could control the laterality of mechanical allodynia via modulating the excitability of KOR⁺neurons in the spinal dorsal horn.Overall,these data collectively show that microglia,no matter what kind of role they may play in nociception-induced mechanical allodynia,they are not required for the initiation of contralateral allodynia;in the meantime,KORs from dorsal horn neurons control the laterality of mechanical allodynia,possibility via increasing the excitability of these neurons and subsequently opened the gate for contralateral mechanical allodynia pathways.Therefore,KORs from dorsal horn neurons,but not microglia,are required for the development of mirror-image pain.In the future,more experiments are warranted to further dissect the underlying mechanisms for the laterality control of mirror-image pain by dorsal horn KORs.