Up-regulation Of LCN2 In The Anterior Cingulate Cortex Mediates The Development Of Chronic Neuropathic Pain

Neuropathic pain is a severe chronic pain caused by peripheral or central nerve injury,often accompanied by sensory hypersensitivity and nociceptive abnormalities at the site of nerve injury.The mechanisms that cause chronic neuropathic pain are currently being studied,including two main mechanisms:peripheral sensitization and central sensitization.Central sensitization refers to the activation of peripheral receptors that transmit pain signals to the spinal cord or supraspinal centers after nerve injury,causing changes in synaptic or neuronal functional plasticity.In chronic neuropathic pain,central sensitization plays an important role,but the molecular mechanisms by which central sensitization occurs are still unknown.The theory of neuronal plasticity is thought to be one of the mechanisms of action causing central sensitization,which can be observed in many pain-related regions such as the spinal cord^[1,2],anterior cingulate cortex（ACC）,thalamus,insula,amygdala and somatosensory cortex.In particular,the anterior cingulate cortex,as an essential brain region for the upstream and downstream nociceptive transmission pathways in the spinal cord,has been shown to be activated in patients with chronic neuropathic pain by neuroimaging;and in animals with chronic neuropathic pain,excitatory neurons were hyperactivated and excitatory synaptic responses were enhanced in the ACC brain region,suggesting that the ACC may be a key brain region for chronic neuropathic pain.However,it is not clear how the ACC brain region is involved in the pathological neuronal plasticity link through molecular mechanisms.Lipocalin-2（LCN2）,is involved in the iron death pathway and in the development of several diseases.In chronic neuropathic pain,LCN2 can have some effect on macrophages in peripheral nerves,while in the spinal cord,LCN2 mainly acts on neurons and thus activates microglia,leading to the release of pro-inflammatory factors and producing neuropathic pain symptoms.However,it is still unclear whether LCN2 causes central sensitization by altering neuronal plasticity in the centrally relevant brain region.In this study,we first established a spared nerve injury（SNI）mouse model and used Von Frey mechanical pain and Hargreaves thermal pain assays to detect pain thresholds in mice;used electrophysiological recording,two-photon calcium imaging techniques in isolated brain slices to detect glutamatergic neuronal activity in mice;regulated neuronal activity by chemical genetic inhibition or activation;determined LCN2 by WB and immunofluorescence;silent inhibition of LCN2 expression in vivo by constructing a broad-spectrum LCN2-interfering virus;and upregulation or reduction of LCN2expression in the ACC by cannula administration,thus revealing the expression of LCN2in the ACC brain region under chronic pain conditions.It was found that:1.ACC glutamatergic neurons were increased in chronic neuropathic pain mice;2.Chemogenetic modulation of ACC glutamatergic neurons could alter pain-sensitive behavior in mice;3.LCN2 expression was increased in ACC brain regions of chronic neuropathic pain mice;4.The downregulation of LCN2 expression resulted in a decrease of ACC glutamatergic neurons activity and relieved pain 5.LCN2expression modulate pain-sensitivty in chronic neuropathic pain mice.In summary,this study reveals that downregulation of LCN2 expression during chronic pain development mediates overactive ACC glutamatergic neurons,thereby promoting the development of chronic pain.Inhibition of LCN2 expression in ACC by viral injection or pharmacological methods effectively alleviate mechanical and thermal pain nociceptive behavior in SNI 2W mice,suggesting that LCN2 may be a viable pharmacological target for the treatment of chronic pain.