Role Of VTA-vHIP In Modulating Chronic Pain Comorbid With Anxio-depressive Disorders:Conjoint AES-SDM Meta-analysis And Collateral Projections

Chronic pain has been one of the most important diseases threatening human health.Anxio-depressive disorder induced by persistent chronic pain is one of the most important types in chronic pain-related psychological disorders,the prevalence of which is more than 40%.Thus,persistent chronic pain usually induces the anxio-depressive disorders,which in turn will further aggravate the symptoms of chronic pain,forming a vicious cycle.However,the underlying mechanism of chronic pain comorbid with anxio-depressive disorders is not well understood in clinical now,causing the challenges in clinical interventions.Recently,magnetic resonance imaging(MRI)has been widely used to explore the potential central nervous mechanism in kinds of diseases because of the noninvasive advantage.Previous neuroimaging studies showed that chronic pain and anxio-depressive disorders patients had significant structural and functional alterations compared to healthy populations.In animal studies,researchers observed that activations of the dopaminergic(DAergic)neuropathway from the ventral tegmental area(VTA)to the nucleus accumbens(NAc)could reverse the chronic pain-related negative emotional disorders.Additionally,hippocampal neurogenesis was also regarded as an important link between the chronic pain and depression.Taken together,mesolimbic DA system may play an important role in chronic pain-related anxio-depressive disorders.But the smaller sample sizes,controversial findings in current neuroimaging studies,as well as poor interact proof between clinical and preclinical studies limited the further exploration in this disease.Furthermore,the findings of our team also demonstrated that the DAergic neuropathway from the VTA to the ventral hippocampus(vHIP)may be also the potential mechanism in chronic pain-related anxio-depressive disorders.However,whether the VTA DAergic neurons send collateral projections to both the vHIP and NAc,and whether these collateral VTA neurons are involved in chronic pain and associated negative emotions information transmission have not been reported now.Thus,AES-SDM meta-analysis and collateral projections were conjoined to perform the study,which included three parts as follows.Part 1:Gray matter volume alterations in chronic pain patients comorbid with anxio-depressive disorders:An AES-SDM meta-analysis of voxel-based morphometry studiesObjective:Chronic pain is a complex emotional experience.Persistent chronic pain is usually accompanied by various negative emotions,including anxiety and depression.Accumulating previous neuroimaging evidence indicates that the structural alterations in specific brain areas are closely related to the chronic pain and anxio-depressive disorders comorbidity.However,the neuroimaging findings on the brain structural alterations in chronic pain patients with anxio-depressive disorders(CP-AD)are still controversial.Therefore,it is necessary to perform a meta-analysis to explore the consistent and significant structural alterations for further diagnosis and intervention.Methods:Systematic search of PubMed,MEDLINE,Web of Science,and Cochrane Library databases updated to July 13,2021,obtaining 1722 studies.In addition,four studies were manually searched in references.The altered GMV between CP-D and HCs in VBM studies was included in this meta-analysis.A total of 18 studies(20 datasets)and 1320 participants(520 patients and 800 healthy controls(HCs))were included.Significant coordinate information(x,y,z)reported in standard space and the effect size(t-value or z-score)were extracted and analyzed by anisotropic effect size-signed differential mapping(AES-SDM)5.15 software.Results:According to the main analysis,CP-AD showed significant and consistent increased GMV in the left hippocampus(HIP.L)and decreased GMV in the left superior frontal gyrus,medial(SFGmedial.L,BA 10)compared to HCs.Subgroup analysis showed significant decreased GMV in the right superior frontal gyrus,medial orbital(PFCventmed.R,BA 10)in chronic neuropathic pain with anxio-depressive disorders(CNP-AD),as well as significant increased GMV in the right parahippocampal gyrus(PHG.R,BA 35),HIP.L(BA 20),and right middle frontal gyrus(MFG.R)in chronic musculoskeletal pain with anxio-depressive disorders(CMP-AD).Furthermore,meta-regression showed the positive relationship between the decreased GMV in the SFGmedial.L and the percentage of female patients(r=0.3450,P<0.0005).Conclusion:This study suggested that the GMV alterations in specific brain regions,especially in the HIP.L and SFGmedial.L,may be important biomarkers for patients with chronic pain and anxio-depressive disorders comorbidity.Sexual difference may be a potential risk for the brain structural alterations in CP-AD.Part 2:Collateral projections from the ventral tegmental area to the ventral hippocampus and nucleus accumbens in mice with acute painObjective:Based on the first part of the study,CP-AD showed significant brain structural alteration in the HIP.L,suggesting the potential mechanism of limbic system in chronic pain and depression comorbidity.Previous studies have demonstrated that the ventral tegmental area(VTA),ventral hippocampus(vHIP),and nucleus accumbens(NAc)were important brain structures in the mesolimbic dopaminergic(DAergic)neurocircuits in modulating chronic pain and associated negative emotions.However,whether the VTA DAergic neurons send collateral projections to both the vHIP and NAc,and whether the collateral VTA neurons are involved in the nociceptive information transmission are not reported.This study aimed to explore this question by collateral projections in acute pain mice model.Methods:Eighteen mice were randomly divided into saline control(n=9)and formalin acute pain groups(n=9).Fluoro-gold(FG)and tetramethylrhodamine(TMR)were injected into the vHIP and the NAc,respectively.A double retrograde tracing method of FG and TMR,as well as the immunofluorescent triple-labeled staining for TH or FOS induced by pain stimulations were conducted to provide morphological evidence.Results:(1)FG/TMR double-labeled VTA neurons were observed in both saline control and formalin acute pain groups,which was characterized by bilateral distribution and ipsilateral dominance.These retrogradely labeled VTA neurons can be fusiform,triangular or rhombus,with diameter nearly from 10～20 μm.(2)After cell counting in the ipsilateral VTA,there were 61.6～62.2%FG-labeled and 67.4～72.7%TMR labeled neurons were FG/TMR double-labeled neurons in the saline control,as well as 67.5～67.7%FG-labeled and 74.3～80.2%TMR-labeled neurons were FG/TMR double-labeled neurons in the formalin acute pain group.(3)After performing immunofluorescent triple-labeled staining of FG/TMR/TH,findings showed that:In saline control group,FG/TMR/TH triple-labeled VTA neurons accounted for 48.8±7.7%in FG-labeled VTA neurons,56.9±5.7%in TMR-labeled VTA neurons,and 16.5 ± 3.5%in TH-labeled VTA neurons,respectively.In addition,FG/TMR/TH triple-labeled VTA neurons accounted for 78.2 ± 3.3%in FG/TMR double-labeled VTA neurons.In formalin acute pain group,FG/TMR/TH triple-labeled VTA neurons accounted for 48.7±5.5%in FG-labeled VTA neurons,57.6±4.6%in TMR-labeled VTA neurons,and 17.1±1.9%in TH-labeled VTA neurons,respectively.In addition,FG/TMR/TH triple-labeled VTA neurons accounted for 71.9±4.5%in FG/TMR double-labeled VTA neurons.(4)FG/TMR/FOS triple-labeled neurons were further explored and observed by confocal laser scanning microscopic imagines.Findings indicated that FOS-labeled neurons were mainly in bilateral VTA,decreasing from the rostral to the caudal part.Cell count showed that:In saline control group,FG/TMR/FOS triple-labeled VTA neurons accounted for 15.2±3.4%in FG-labeled VTA neurons,16.7±3.6%in TMR-labeled neurons,and 16.8 ± 2.9%in FOS-labeled neurons,respectively.In addition,FG/TMR/FOS triple-labeled VTA neurons accounted for 25.1 ± 7.3%in FG/TMR double-labeled VTA neurons.In formalin acute pain group,FG/TMR/FOS triple-labeled VTA neurons accounted for 21.7±2.4%in FG-labeled VTA neurons,24.0±4.4%in TMR-labeled neurons,and 16.2±3.1%in FOS-labeled neurons,respectively.In addition,FG/TMR/FOS triple-labeled VTA neurons accounted for 32.3 ± 5.4%in FG/TMR double-labeled VTA neurons.Conclusion:Most VTA neurons sending the collateral axons to both the vHIP and NAc in mice were DAergic(TH positive)neurons,some of which may be involved in the peripheral acute nociceptive information processing.Part 3:Some VTA neurons sending collateral projections to the ventral hippocampus and nucleus accumbens may be involved in the peripheral chronic pain and anxiety information transmissionObjective:Based on the second part study,it has demonstrated that collateral projections from the VTA to the vHIP and NAc were involved in the nociceptive information transmission.Thus,whether those collateral VTA neurons were also involved in chronic pain and associated anxio-depressive information transmission?This study further explored the relationship between the collateral projections(from the VTA to the vHIP and NAc)and chronic pain-associated anxiety,providing the direct behavioral and morphological evidence for chronic pain and anxiety information transmission.Methods:Sixteen mice were randomly divided into sham(n=7)and chronic pain(n=9)groups.Mechanical allodynia was tested at 1th,3th,7th,and 14th day after cuffing operation.Mice in cuff group were stereotactically injected with TMR into the left NAc and FG into the left vHIP at 7th day after cuffing operation.Open field test(OFT)and elevated plus-maze test(EPM)were performed to test the anxiety-like behavior at 14th after cuffing operation.Combining the immunofluorescence histochemical staining method,the FG/TMR double-labeled neurons,FG/TMR/FOS,and FG/TMR/TH triple-labeled VTA neurons were observed in mice with chronic pain and anxiety-like behavior.Results:(1)Compared with the sham group,mice in cuff group showed mechanical allodynia at 1th day after cuffing operation,which last until 14th day.And mice in cuff group showed anxiety-like behavior at 14th day.(2)The findings of immunofluorescent double-labeled staining indicated that some retrogradely labeled VTA neurons were FG/TMR double-labeled in mice with chronic pain and anxiety comorbidity.(3)The findings of immunofluorescent triple-labeled staining of FG/TMR/TH indicated that most FG/TMR double-labeled VTA neurons were TH-immunopositivity.Cell count showed that 46.2±2.0%FG-and 50.6±4.0%TMR-labeled neurons were FG/TMR double-labeled neurons,as well as 78.3 ± 6.9%FG-,70.2 ± 1.6%TMR-labeled,and 76.4±0.7%FG/TMR double-labeled VTA neurons expressed TH.(4)The findings of immunofluorescent triple-labeled staining of FG/TMR/FOS indicated that some FG/TMR double-labeled VTA neurons expressed FOS protein.Cell count showed that 50.9±7.8%FG-and 57.4±4.3%TMR-labeled VTA neurons were FG/TMR double-labeled neurons,as well as 41.5±4.0%FG-,40.6±7.1%TMR-,and 58.2±7.7%FG/TMR double-labeled VTA neurons expressed FOS protein.Conclusion:Mice VTA DAergic(TH positive)neurons can send collateral projections to the vHIP and NAc.Some collateral VTA neurons may be involved in chronic pain and associated anxiety information transmission.