The Involvement Of Peripheral PKG-I-Mediated Ascending Pain Sensitization And Lateral Hypothalamus-vlPAG Descending Inhibitory Pathway In Regulation Of Chronic Pain

Background Patients with chronic pain often suffer severe pain sensation accompanied with pain-related negative emotions,which seriously endangers the quality of life in patients.In recent years,it is still intractable for clinicians to treat refractory pain although the research of chronic pain has made great progress.Therefore,it has become frontier topics on neuroscience and medicine to study and reveal the physiology and pathogenesis underlying it,and also to explore new targets,strategies and approaches for the treatment of chronic pain.The perception of pain is initiated by activation of peripheral nociceptors,and the nociceptive sensory information is then processed to spinal cord and relayed to the brain.via the ascending pathway.Accumulated studies have documented the key significance of nociceptor hyperexcitability,spinal synaptic plasticity in sensitization of ascending pain pathway.There also exists an endogenous top-down inhibition pathway besides bottom-up pathway of pain sensation.In pathological state,it is an effective way that facilitating or enhancing the endogenous inhibition pathway could resist ascending pain sensitization,which help produce analgesic effect.Therefore,it could provide novel strategies and theoretical foundation to explore the molecular mechanism of bottom-up sensitization for developing novel strategies with peripheral nociceptors as targets,and to explore the composition pattern and characters of endogenous inhibition pathway,revealing the key molecular mechanisms of the plasticity change of the pathway.Aims 1.Our first aim is to investigate the mechanisms of PKG-I localized in nociceptors involved in neuropathic pain with comorbidity through ascending pain pathway.2.Our second aim is to explore the mechanisms of LH^PV-PAG circuit involved in regulating chronic pain through top-down inhibition pathway.Methods 1.Ongoing primary afferent inputs are critical for maintaining exaggerated pain response in peripheral neuropathy.The spared nerve injury（SNI）model was carried out in wild-type mice.The modeled animals were divided into two groups,and the ipsilateral sciatic nerve was injected with saline（control group）and lidocaine（experimental group）separately.Behavior test was performed to observe the pain threshold of bilateral hindpaws in two groups of animals.2.PKG-I in nociceptors is involved in pain hyperalgesia and pain-related aversive emotions induced by peripheral nerve injury.Western blotting was used to detect the expression of PKG-I in DRG after peripheral nerve injury.Genetic and pharmacological methods combined with behavior test were used to determine the effect of PKG-I on hyperalgesia and negative emotion after spared nerve injury.3.PKG-I localized in primary afferent nociceptors,which influences central sensitization by enhancing cortical plasticity in the ACC via regulation of BDNF function after nerve injury.Immunofluorescence staining was used to observe expression of c-Fos and p-ERK in ACC.The brain slice whole-cell patch clamp recording and western blotting were used to clarify the central sensitization and molecular mechanism underlying it.4.Parvalbumin-positive(LH^PV)neurons in lateral hypothalamus regulate chronic inflammatory pain.Chronic inflammatory pain was produced by injection of CFA into the unilateral hindpaw.Behavior test was performed to observe the pain threshold while activation or inhibition of LH^PV neurons by optogenetics and chemical genetics.5.LH^PV-vlPAG pathway regulates chronic inflammatory pain.Stereotactic injection of brain combined with immunofluorescence was performed to observe the projection of lateral hypothalamus.Behavioral test was performed to observe the involvement of the pathway in chronic pain while activation or inhibition of LH^PV-vlPAG pathway.6.LH^PV-vlPAG pathway regulates chronic pain by activating the descending inhibition pathway.Spinal cord fiber photometry recording combined with the optogenetics was performed to specifically activate the LH^PV-vlPAG neural circuit and observe the change of calcium signal of spinal cord neurons.Optogenetics combined with the pharmacological method was to observe the effect of blocking the PAG-RVM inhibition pathway on specifically activating LH^PV-vlPAG pathway induced antinociceptive behavior.7.The intrinsic properties and plastic changes of LH^PV neurons was identified.The character of PV neurons was determined by situ hybridization and patch clamp.The plasticity change and ih current of LH^PV neurons were recorded by patch clamp.Results1.At 7 d following SNI,the mice developed significant mechanical and thermal hyperalgesia of bilateral hindpaw.After ipsilateral sciatic nerve administration,the pain threshold of mice in experimental group increased significantly while the control group did not change.We infer from the above that ongoing primary afferent inputs are critical for maintaining primary and secondary pain hypersensitivity after peripheral neuropathy.2.After SNI 7d,the expression level of PKG-I in DRG changed significantly and showed lumbar segment dependent characteristic.PKG-I in L3 DRG was significantly up-regulated after SNI,and there was no change in L4 DRG,but significantly down-regulated in L5 DRG,suggesting that PKG-I in DRGs may play an important role in the occurrence of neuropathic pain.Deficiency of PKG-I in nociceptors significantly inhibited SNI-induced neuropathic pain and anxiety like behavior.More importantly,the foramen injection of KT5823 significantly inhibited the neuropathic pain and pain related anxiety behavior after spared nerve injury.Under normal physiological conditions,peripheral PKG-I has no effect on the basal pain threshold.These results strongly suggest that PKG-I localized in nociceptors plays an important role in the co-morbidity of chronic neuropathic pain and negative emotion.3.SNI induced a robust enhancement for c-Fos and p-ERK expression in the ACC neurons derived from PKG-Ifl/fl mice,which was almost eliminated by the deficiency of PKG-I in nociceptors.These data indicate that PKG-I in nociceptors contributes to the enhanced neuronal activity in the ACC after peripheral neuropathies.At 7 day after SNI,ACC neurons in wild-type mice showed lowered rheobase and displayed a dramatic augmentation in firing frequency.Compared with wild-type mice,deficiency PKG-I in nociceptors suppressed hyperexcitability and rheobase was increased after SNI.At 7d following SNI surgery,PKG-Ifl/fl mice displayed enhanced synaptic transmission.By contrast,deficiency of PKG-I in nociceptors prevented this potentiation of synaptic transmission.PPR also showed the same trend,Upon SNI surgery,there was a significant reduction in PPF in ACC neurons from PKG-Ifl/fl mice.In contrast,this drop in PPF did not come about in neurons from SNS-PKG-I-/-mice;At 7d following SNI surgery,SNI induced a robust increase in the frequency of m EPSCs,but no change in the amplitude in ACC neurons derived from PKG-Ifl/fl mice.This facilitation in m EPSCs frequency induced by nerve injury was eliminated in nociceptor-specific PKG-I-deficient mice.LTP was induced by conditioning stimulus which produced a significant,long-lasting potentiation of e EPSCs in slices of PKG-Ifl/fl mice compared with baseline responses.By contrast,this synaptic potentiation was abolished in slices from SNS-PKG-I-/-mice.Lysates from ACC in PKG-Ifl/fl mice revealed that BDNF expression was upregulated at 7d after SNI,and returned back to basal level at 28 d after injury.This upregulation was excluded with the deficiency of PKG-I in nociceptors.It can thus be concluded that peripheral PKG-I contributes to ACC synaptic potentiation and pain hypersensitivity as well as pain-related aversions via BDNF signaling pathway in ACC.4.Optogenetic or chemical genetic activation LH^PV neurons significantly alleviated the CFA induced hyperalgesia,and optogenetic or chemical genetic inhibition of PV neurons induced mice to produce hyperalgesia.It is suggested that LH^PV neurons are involved in regulation of chronic pain.5.LH^PV neurons projected to vlPAG,LHB and PF brain areas.The activation of LH^PV-vlPAG pathway by optogenetics significantly alleviated the inflammatory pain induced by CFA.Inhibition of it by optogenetics induced hyperalgesia in normal state.This suggests that LH^PV-vlPAG pathway may be involved in the regulation of chronic pain.6.The calcium signals of SDH neurons induced by peripheral stimulation was not changed in normal state,in contrast,the calcium signal of SDH neurons was significantly decreased while LH^PV-vlPAG pathway was activated by optogenetics in chronic pain state.We further observe the influence on calcium signals of SDH neuron upon inhibition of the pathway.Our data showed the calcium signals of SDH neurons induced by peripheral stimulation is significantly increased in normal state upon inhibition of LH^PV-vlPAG pathway,and it was not altered in chronic pain state.It can be concluded that LH^PV-vlPAG pathway could have an effect on noxious afferent input to SDH neurons.We further determined whether activation of LH^PV-vlPAG pathway induced antinociceptive response was influenced by block of PAG-RVM inhibition pathway.Our data indicated that the inhibition of hyperalgesia by activation of LH^PV-vlPAG was blocked while blocking PAG-RVM pathway through intrathecal administration of 5-HT antagonist,which suggested activation of LH^PV-vlPAG induced antinociception was dependent on PAG-RVM descending pathway.7.PV-Tom neurons in lateral hypothalamus colocalized with VGLUT2,and EPSC was produced by recording the PAG projected LH neurons,which combined to indicate that LH^PV neurons were glutamatergic.LH^PV neurons exerted fast spiking in normal state while the frequency of spiking was significantly decreased in chronic pain states,and amplitude of ih current was decreased compared with basal state.It is suggested that the decreased excitatory plasticity of PV neurons may be related to the structure and function of HCN channels in chronic pain.Conclusion 1.Using genetic and pharmacological methods,we clarify that PKG-I in nociceptors plays a key role in the occurrence and maintenance of chronic neuropathic pain and pain-related negative emotion.2.In chronic pain state,the continuous activation of PKG-I in nociceptors triggers hyperexcitation and enhanced synaptic plasticity of ACC neurons,which in turn induces chronic pain and negative emotions.Further mechanism studies show that the effects are accomplished by activating the BDNF signaling pathway in ACC.3.LH^PV neurons participate in the regulation of chronic pain.Optogenetic activation of LH^PV neurons significantly inhibits chronic inflammatory pain and inhibition of LH^PV neurons induces hyperalgesia.4.LH^PV-vlPAG pathway regulate the chronic inflammatory pain by activating the PAG-RVM-SDH inhibition pathway.Optogenetic activation of LH^PV-vlPAG pathway inhibits chronic inflammatory pain and inhibition of the pathway induces hyperalgesia.