BDNF/TrkB Signaling Was Involved In The Mechanism Of Depression Facilitating Neuropathic Pain Mediated By ACC-SDH Circuitry

BackgroundIn recent years,the incidence of mental disorders such as depression has increased year by year.Depressed patients often suffer from decreased pain tolerance,and are prone to suffer from hyperalgesia and refractory pain when combined with pain-related diseases.Pain can further aggravate the degree of depression and develop into a vicious cycle of mutual promotion of depression and pain.Depression-pain comorbidity can lead to a decline in the quality of life of patients and impairment of work ability,increase the consumption of medical resources,and increase the social burden.Therefore,it is of great significance to study the pathogenesis of depression-pain comorbidity.ObjectiveThe objective of this study is to investigate the role of brain-derived neurotrophic factor（BDNF）/tyrosine kinase receptor B（Trk B）signaling in the mechanism of depression facilitating neuropathic pain mediated by anterior cingulate cortex（ACC）-spinal dorsal horn（SDH）circuitry,based on a rat model of depression combined with neuropathic pain,using chemogenetic methods,calcium imaging and other techniques.It might provide new ideas and potential targets for the treatment of depression-pain comorbidity.Methods（1）Specific pathogen-free（SPF）male SD rats,5 weeks old,weighing 140g to 160g were selected as the research subjects.A rat model of depression was constructed by chronic unpredictable mild stress（CUMS）treatment,and combined with chronic sciatic nerve compression injury（CCI）treatment on the 21^stday of CUMS.Rats were randomly divided into four experimental groups:Sham+Control,CCI+Control,Sham+CUMS and CCI+CUMS.The depressive behavior were measured by sugar water preference test and forced swimming test.The pain behavior,including Paw withdrawal mechanical threshold（PWMT）and Paw withdrawal thermal latency（PWTL）,were measured by Von Frey and Plantar Test respectively.The expression level and distribution of c-Fos protein in ACC and SDH were detected by western blot（WB）and immunofluorescence staining（IF）.（2）In rats treated with CUMS combined with CCI,Cre recombinase-dependent AAV-DIO-h M4Di and chemogenetic methods were used to selectively inhibit the activity of descending projection neurons of the ACC from day 0 of CCI to day 14 of CCI.Rats were randomly divided into two experimental groups,AAV-h M4Di group and Mock group.Protein expression levels of c-Fos in ACC and SDH were detected by WB.Activity of ACC neurons was detected using calcium imaging techniques.The changes in depression behavior and pain behavior were observed after the activity of ACC-SDH projection neurons was inhibited.（3）In rats treated with CUMS combined with CCI,Cre recombinase-dependent AAV-DIO-sh BDNF was used to selectively knock down BDNF in ACC-SDH projection neurons.Rats were randomly divided into two experimental groups,sh BDNF group and Scramble group.The protein and m RNA level as well as the distribution of BDNF in ACC and SDH were detected by western blot（WB）,q PCR and immunofluorescence staining（IF）.（4）In rats treated with CUMS combined with CCI,the Trk B receptor selective antagonist ANA-12 was intrathecally injected to block the BDNF/Trk B pathway at the spinal cord level.Rats were randomly divided into two experimental groups,ANA-12 group and Vehicle group.The changes of depression behavior and pain behavior were observed.（5）Using RNA-sequencing technology,we analyzed the gene expression changes at the spinal cord level after chemogenetic inhibition of ACC-SDH projection neurons in rats treated with CUMS combined with CCI,and screened possible downstream molecular pathways.Based on the RNA-sequencing results,the Lamb1 protein was further quantitatively detected and cell localization analysis by WB and IF.Golgi staining was used to reflect the dendritic growth changes of SDH neurons,and to explore the downstream mechanism of depression-facilitated neuropathic pain.Results（1）After 21 days of CUMS treatment,a rat model of depression was successfully established.CUMS-induced depression exacerbates CCI-induced hyperalgesia.The PWMT of the CCI+CUMS group was significantly lower than that of the CCI+Control group from the 3rd day of CCI,and the difference between the two groups reached the maximum at the 7th day of CCI（2.178 vs 6.778,p=0.0001）.The change trend of PWTL was consistent with PWMT.On the 21st day of CUMS（ie,CCI day 0）,the c-Fos protein level in the ACC of the Sham+CUMS group was significantly higher than that of the Sham+Control group（p=0.0286）,while there was no difference in the c-Fos protein level in the SDH between the two groups.These results suggest that in the absence of CCI stimulation,CUMS-induced depression can lead to ACC neuron activation,but not SDH directly.On the 7th day of CCI,the c-Fos protein level in ACC of CCI+CUMS group and Sham+CUMS group was significantly higher than that of CCI+Control group（p=0.0103）and Sham+Control group（p=0.0003）,respectively.The CCI+Control group was slightly higher than the Sham+Control group（p=0.0429）,but there was no significant difference between the CCI+CUMS group and the Sham+CUMS group.The results of immunofluorescence staining were consistent with the trend of WB.The comparison of the c-Fos protein levels in SDH on the 7th day of CCI showed that the c-Fos protein levels in the CCI+Control group and CCI+CUMS group were significantly higher than those in the Sham+Control group（p=0.0223）and Sham+CUMS group（p=0.0003）.The c-Fos protein level in the CCI+CUMS group was higher than that in the CCI+Control group（p=0.0433）,but there was no difference between the Sham+CUMS group and the Sham+Control group.This suggests that CUMS-induced depression can enhance the activation of SDH neurons after CCI-induced neuropathic pain,but for the Sham group without neurological damage,CUMS-induced depression has no significant effect on the activation of SDH neurons.The results of immunofluorescence staining were consistent with the trend of WB.（2）Chemogenetic inhibition of ACC-SDH projection neurons did not affect depressive-like behaviors in CCI+CUMS rats,but improved pain behaviors.From CCI 3 days to CCI 14 days,the PWMT of the AAV-h M4Di group was higher than that of the Mock group,and the difference between the two groups reached the maximum at CCI 7 days（6.778 vs3.222,p=0.0012）.The PWTL trend is consistent with the PWMT.On the7th day of CCI,the fluorescence calcium signal intensityΔF/F（%）of AAV-h M4Di group after pain stimulation was lower than that of Mock group（p=0.0079）,and the level of c-Fos protein in ACC and SDH of AAV-h M4Di group was lower.in the Mock group.These results suggest that after chemogenetic inhibition of activation descending projection neurons in the ACC,the activation of SDH neurons at the spinal cord level was also inhibited.（3）BNDF levels in the ACC of CUMS-treated rats increased over time after CCI.The level of BDNF protein in SDH of CUMS-treated rats on day 7 of CCI was higher than that of Control group（p=0.0286）.Chemogenetic inhibition of ACC-SDH projection neurons reversed the upregulation of BDNF in ACC and SDH in CCI+CUMS rats.After sh BDNF knockdown,the transcription and protein levels of BDNF were down-regulated in ACC of CCI+CUMS rats,while the transcription level of BDNF in SDH was unchanged but the protein level was down-regulated.Immunofluorescence staining revealed co-localization of BDNF with tool virus reporter fluorescence in ACC and SDH.The above results suggest that BDNF expressed by descending projection neurons in the ACC has a material and structural basis for transmission to SDH via ACC-SDH projection fibers.After selective knockdown of BDNF in ACC-SDH projection neurons by sh BDNF,depressive behaviors were not changed in CCI+CUMS rats,but pain behaviors were improved.The PWMT in the sh BDNF group was higher than that in the Scramble group after CCI,and the difference between the two groups reached the maximum on the 7th day of CCI（7.111 vs 2.889,p=0.0002）.The changing trend of PWTL is consistent with that of PWMT.（4）Intrathecal injection of ANA-12 had no effect on depression-like behavior in CUMS-CCI-treated rats,but improved pain behavior.The PWMT in the ANA-12 group was higher than that in the Vehicle group,and the difference between the two groups reached the maximum at 7days of CCI（6.889 vs 3.111,p=0.0003）.The change trend of PWTL was consistent with PWMT.The above results suggest that BDNF is involved in the facilitation of CUMS-induced depression on CCI-induced neuropathic pain at the spinal level through the BDNF/Trk B pathway.（5）The results of RNA-sequencing indicated that the expression of dendrite-related genes in the AAV-h M4Di group was up-regulated.As an important molecule regulating extracellular matrix,Lamb1 has the highest up-regulation fold.Further quantitative detection of Lamb1protein level found that Lamb1 protein level was down-regulated in SDH of CCI+CUMS rats.In contrast,after chemogenetic inhibition of ACC-SDH projection neurons,the downregulation of Lamb1 protein levels in SDH was reversed.The results of immunofluorescence staining in the spinal cord showed that 79.59%of Lamb1 co-localized with Neu N,13.62%of Lamb1 and GFAP co-localized,and 5.82%of Lamb1 co-localized with Iba1.Lamb1 is mainly expressed in neurons.The results of Golgi staining showed that the number of dendrites and the density of dendritic spines in the CCI+CUMS group were significantly higher than those in the CCI+Control group,and the number of dendritic spines in the CCI+CUMS group was significantly higher than those in the CCI+Control group.Dendritic spine density was not significantly different.The above results suggest that CUMS-induced depression can promote active dendritic growth in SDH neurons after CCI,and the density of mature dendritic spines is up-regulated.However,the number of dendrites and the density of mature dendritic spines in the AAV-h M4Di group were significantly lower than those in the Mock group,and there was no significant difference in the density of dendritic spines in the immature morphology.It is suggested that inhibition of ACC-SDH projection neurons by chemogenetic methods reversed the effect of CUMS-induced depression on dendritic growth of SDH neurons after CCI.ConclusionCUMS-induced depression can lead to activation of rat ACC neurons and up-regulation of BDNF protein expression.BDNF is transmitted to SDH via ACC-SDH projection fibers,activates the BDNF/Trk B pathway at the spinal cord level,promotes the growth of SDH neuron dendrites,and participates in the mechanism of hyperalgesia.Lamb1 participates in the mechanism of depression facilitating neuropathic pain by regulating synaptic plasticity of SDH neurons.