| BackgroundChronic stress has been recognized as a major risk factor for serious mental illness,including depression and post-traumatic stress disorder(PTSD),however,the underlying mechanism has not been illuminated yet.In addition,there are resilient individuals who experience the same stress while successfully maintaining normal psychological and physical functioning,which may involve an active stress coping mechanism.Therefore,understanding individual differences in neurobiological mechanism of susceptibility and resilience is of vital importance for illustrating pathogenesis of stress-induced disorders as well as for developing novel or more effective treatments.Hippocampus(Hi)plays a vital regulatory role in both stress-related psychiatric disorders and stress susceptibility,while its structure and function are affected by stress.Due to this dual role of Hi as well as the paucity of longitudinal studies,the cause-andeffect relationship between psychiatric symptoms and hippocampal changes is still obscure.On the other hand,Hi is structurally and functionally distinct along its dorsoventral axis as well as in its bilateral sides.As a consequence,taking the whole structure as one analytic subject may mask some stress effects.Accordingly,the left and right,dorsal(dHi)and ventral hippocampus(vHi)should be investigated as separate brain regions.Proteomics can reflect the comprehensive interactions between genes and the environment,and is more closely related to the pathophysiological mechanism of stressrelated psychiatric disorders.Isobaric tags for relative and absolute quantitation(iTRAQ)enable to identify differential protein expression and functional patterns with highthroughput,accuracy and sensitivity,which provides a solid foundation for screening protein biomarkers of stress-related psychiatric disorders.As for the sample,Hi is of higher tissue specificity,while blood samples are more easily accessible which makes it applied in longitudinal research and clinical transformation applications.The organic combination of the two can more fully reflect the stress-related mental disorders protein level.In this study,we aim to reveal the causal relationship between Hi and stress-related psychiatric disorders or stress susceptibility from a perspective of morphology and protein expression levels,to find out whether there is stress-induced lateralized effect on hippocampal subregions,as well as to preliminary screen potential proteomic biomarkers of stress susceptibility and resilience.Therefore,based on a chronic social defeat stress(CSDS)paradigm,bilateral dorsal and ventral hippocampal volumes and diffusion properties of experimental mice were monitored longitudinally with a repeated MRIbased morphometry and diffusion tensor imaging(DTI),respectively.Moreover,we used an iTRAQ method to screen susceptibility-related differentially expressed proteins in the left and right Hi as well as in the pre-and post-stress serum.Methods1.The chosen aggressors were then placed on the left side of adapted hamster cages(which were divided into two parts by a transparent perforated plexiglass)overnight before the formal initiation of the defeat sessions.For 10 consecutive days,each C57 BL/6J(C57)mouse(n=17)was first placed into a new aggressor's home cage compartment for a 10-minute social defeat session,followed by placement in the right side of the same cage for the remainder of the 24 hours for continuous non-physical social stress exposure.On the other hand,the control C57 mice(n=7)were housed with other C57 partners in the same adapted hamster cage,with one mouse on each side.The control mice were rotated to a new cage at the same time on a daily basis without any physical contact.Pre-and post-stress open field test,sucrose preference test,social interaction test,forced swimming test as well as body weight were conducted to evaluate their stress-induced behavioral and physical changes.2.We performed 7.0 T MRI scans before and after the CSDS paradigm to get longitudinal data on hippocampal volume and microstructural parameters(FA,MD,RD,and AD).According to the mouse atlas,bilateral dorsal(dHi)and ventral hippocampus(vHi)were manually delineated for independent regions of interest(ROI)analysis.3.The iTRAQ method was used to quantify the protein expression in bilateral Hi of stress susceptible or resilient mice.Differential expressed proteins were screened out and their enrichment in cellular component,molecular function,biological process and protein pathway were analyzed,to further screening of potential hippocampal protein biomarkers.4.The iTRAQ method was also performed on pre-or post-stress serum samples to screen out proteins differential expressed among groups on cross section analysis or proteins that significantly changed before and after stress in one group.Similarly,GO and protein pathway enrichment was analyzed to get a general map of mechanisms underlying stress as well as to get preliminary potential serum biomarkers.Results1.After the CSDS paradigm,stressed mice were divided into stress susceptible(SS,n=10)and resilient(SR,n=7)subgroups according to their social interaction ratios(SIR).Their immobility time in forced swimming test in the two stress groups increased significantly,but there was no significant change in anxiety-like behavior,motility,body weight,and sucrose preference.2.No significant difference in pre-stress volumes was found in either side of Hi.Only the SR group had a remarkable increase in right hippocampal volume,which occurred specifically in the right vHi and was positively related to the SIR.3.A significantly lower FA was found in the right vHi of the SS mice prior to the CSDS paradigm than that found in the SR mice,suggesting that pre-existing microstructural abnormalities may result in stress susceptibility.MD and RD values were found to be significantly greater only in the right dHi of the SR group compared to those of the SS mice.Furthermore,a significant longitudinal decrease was only observed in the right dHi RD value of the SS mice.Moreover,SIR was positively related to post-stress left MD,right dHi MD,and right dHi RD values and the longitudinal right dHi MD percent change.Meanwhile,a negative relationship was detected between the SIR and bilateral mean of the post-stress left relative to right vHi FA value,highlighting the important role of right Hi in SR phenotype.4.For the hippocampal proteomics,there were more differentially expressed proteins in left Hi of the SR group than that of the SS group,which was the opposite in right Hi.Meanwhile,differentially expressed proteins that found in the left Hi demonstrated similar GO enrichment items in both stress groups,however,in the ri ght Hi,there are different enrichment characteristics in cell composition,molecular function,biological processes and protein pathways,suggesting that right Hi may play a more important regulatory role in stress phenotypes.Potential biomarkers were preliminarily screened,including MBP,NLRP-1,GTPase HRas,PrPC,annexin A7 in the left Hi,and PPP3 CB,14-3-3?,14-3-3?/?,CLIC6,haptoglobin,TTR in the right Hi,demonstrating potential stress susceptible mechanisms in myelination,neuronal apoptosis,energy metabolism,axon and synaptic plasticity,hormones,etc.Moreover,the left-right hippocampal laterality was more intense in the susceptible group,which was enriched in axon guidance and hippocampal signaling pathways.5.In serum,differentially expressed proteins of each group exhibited different GO enrichment characteristics both before and after stress.Differential proteins that were in common but of different direction of regulation in the two stress groups may work as effective biomarkers,including 14-3-3?/?,ApoB-100,LBP,IGKC,which were closely related to metabolism,immunity,etc.From the longitudinal perspective,there were more differentially expressed proteins in the SR group,while the was the SS group demonstrated a farther change pattern than the other two groups.Conclusions1.Longitudinal imaging studies of hippocampus have highlighted an important lateralized role of the right hippocampus,especially the right vHi,both before and after stress,which makes it a potential morphological biomarker for the stress-related psychiatric disorder and SR phenotype,and an effective target for intervention and stress resilience training.2.The right hippocampus demonstrated a more distinct pattern of differential expressed protein enrichment between the two phenotypes,suggesting a more important regulatory role of the right hippocampus.3.In addition,MBP,annexin A7,PPP3 CB,14-3-3?,14-3-3?/?,ApoB-100,LBP,IGKC were preliminary screened as protein biomarkers of stress susceptibility,suggesting the mechanisms of myelination,neuronal apoptosis,axonal and synaptic plasticity,energy metabolism,hormones as well as immunity deserve further investigation. |
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