| BackgroundDepression is a common mental disorder.The main manifestations are the higher cognitive dysfunction and mood depression of human beings,accompanied by the corresponding thinking language and psychomotor disorders.Depression is recurrent and chronic,has high suicide rate,and seriously endangers human physical and mental health.According to the relevant information of the WHO,upon the social development of China in 2030,depression will rank the first among all the high-burden diseases.Therefore,it is of great significance to analyze and study the relevant contents of depression,such as the clinical treatment and pathogenesis of depression.Since it is difficult to obtain tissue specimens and there are many limitations in studies of humans.With the continuous progress and development of science and technology,people have tried to use animal models to analyze the pathology,biochemistry and other aspects of this disorder,so as to clarify the root cause and pathogenesis of human depression.The development of brain imaging can further understand the pathogenesis of depression from brain structure,function and neurometabolism.The changes of brain structure and function were observed in vivo,repeatedly,noninvasively and dynamically by means of 7.0T small animal MRI,which has provided a lot of help to study the pathogenesis of depression to a certain extent and has been the core imaging technique to analyze brain function and brain tissue structure at this stage.Many scholars have found through studies that it is relevant to brain regions that perform cognitive and emotional functions and that such regions are mainly in the cortex and limbic system.In recent years,many scholars at home and abroad have showed that the morphology,structure,function and metabolism of brain regions of patients with resting-state or task-state depression with MRI from different perspectives and have obtained some significant results.There are few reports on the studies on depression brain imaging with 7.0T small animal MRI;and most of such studies are limited to a certain structure or function.Besides,the neural network of the brain is very complicated.Previous studies have showed that the pathogenesis of depression is related to the disorder of neuroendocrine and neuroregulatory functions.However,most of the studies in recent years have explored the pathogenesis of depression at the cellular molecular level and have gradually exceeded traditional neurobiochemical studies.Although such studies have revealed the pathogenesis of depression from several perspectives and have provided some theoretical basis,in the field of mental disorders,the pathogenesis of depression is complicated,such studies cannot comprehensively explain the various phenomena in the occurrence and development of depression;in addition,there are many signal pathways associated with the onset of depression;such signal pathways also have certain correlation.The changes in such signal transduction pathways and brain-derived neurotrophic factor?BDNF?have attracted more and more attention of scholars.Therefore,it is very necessary to combine MRI and histological detection to conduct a systematic study on the pathogenesis of depression.PurposeThe pathogenesis of depression is very complicated and is closely correlated to stress.The long-term chronic stress stimulation may cause the injury and apoptosis of the neurons in brain regions such as the hippocampus and prefrontal cortex;and the promotion of the repair and regeneration of such brain regions is also the key to the treatment of depression.This study established animal model of the chronic unpredictable mild stress and used 7.0T small animal MRI to observe the changes in the neurometabolites and fiber bundles in the brain of depression rats with hippocampus and prefrontal cortex as the regions of interest and by DTI and MRS sequential scanning;meanwhile,the detection and analysis of the protein expression of BDNF,P-ERK,P-CREB,ERK and CREB in brain tissues was carried out to observe the protein expression of key effectors in the BDNF,P-ERK and P-CREB in the hippocampus and prefrontal cortex in chronic stress depression rats.Next,the abnormal changes in MRI and those in the effectors on BDNF/ERK/CREB signal pathway were compared and analyzed to better understand and explain the pathogenesis of depression,provide new ideas for the treatment and prevention of depression and the R&D of antidepressants,and provide reliable basic experimental data for clinical practice.Methods1.The preparation of rat model and behavioral evaluation of depression:experimental animals were randomly divided into a control group and a model group.Animal model of chronic stress was established by chronic mild stress,including noise,frequent flashing lighting,sundries,fasting,water deprivation,clamping tail,45°tilt and mat odor,combined with isolated rearing.Each stress was randomly given once every week;7 days was a cycle;and there were 6 cycles in total.Six weeks later,the appearance and mental state of model rats were observed;the sucrose preference test?SPT?was conducted on the model;and behavioral evaluation was carried out on open field test?OFT?2.The chronic unpredictable mild stress?CUMS?was combined with isolated rearing model rats respectively underwent small animal MRI and image analysis:First,the CUMS models with isolated rearing were established successfully.Second,the image acquisition was carried out on the model group and the control group by means of the 7.0T MRI scanner?70/20 PharmaScan,Bruker Biospin GmbH,Germany?for small animals at the Binzhou Medical University.T2 weighted imaging?T2WI?acquisition was respectively conducted on horizontal,coronal and sagittal planes with TuberRARE sequence;1H MRS spectrum was acquired by PRESS sequence;DTIEPIseg30dir was selected for DTI image acquisition.Finally,image processing and analysis were carried out.?1?Diffusion tensor imaging?DTI?was used for morphological and structural analysis of brain tissue:the software Imaging and Processing 5.0 was used to analyze the FA and MD values of the bilateral hippocampus and prefrontal cortex in the model group and the control group;and images were reconstructed to make pseudocolor images and fiber tractography.?2?Magnetic resonance spectrum?MRS?was used for metabolic analysis of brain function:1H PRESS program detection was applied to scan the bilateral hippocampus and prefrontal cortex;the software Topspin 5.0 was used to form the spectral peak of NAA,Glu,Cho,MI and Cr;and the ratios of NAA/Cr,Glu/Cr,Cho/Cr and MI/Cr were measured on this basis after baseline correction.3.Immumohistochemical?IHC?staining was used to show the protein expression of BDNF,P-CREB and P-ERK in bilateral hippocampus and prefrontal cortex:After the perfusion of 100ml saline through the heart,the rats in the control group and the model group were beheaded to take the brain,which was stored in 10%formaldehyde solution.The following procedures were carried out:IHC staining,dehydration embedding,bleaching,dewaxing and hydration,washing,incubating primary antibody and incubating secondary antibody,redyeing,and sealing,were carried out.The color pathological image analysis system was used to measure the optical density of the positive area of the target protein.4.Western blot was used to detect the protein expression of BDNF,P-CREB and P-ERK in bilateral hippocampus and prefrontal cortex:The rats in the control group and the model group were killed to take the brain tissue.After protein extraction and protein regulation,glue making,sample loading,electrophoresis,membrane transfer,sealing,incubating primary antibody,washing membrane,incubating secondary antibody,and exposure were carried out according to the conventional experimental methods of Western Blot to obtain the target protein band for analysis and grayscale measurement.5.Imaging and histological comparison analysis:to observe analyze whether the abnormal changes in the MRS and DTI performance of the bilateral frontal cortex and hippocampus in the model group are consistent with the changes in the protein expression of the BDNF,P-CREB and P-ERK in the corresponding brain tissue.Results1.Results of the preparation of rat model and behavioral evaluation of depression:After modeling,the appearance and mental state of the rats in the model group were significantly different from those in the control group.The SPT showed that the consumption of sucrose by the rats in the model group decreased significantly?P<0.01?.The OFT showed that the central distance,central duration and movement speed of the rats in the model group were much shorter or lower than those of the rats in the control group.The model had been successfully copied.2.MRS results:compared with the control group,the ratios of NAA/Cr,Cho/Cr and Glu/Cr in the bilateral hippocampus and the left prefrontal cortex decreased significantly?P<0.05?;and the NAA/Cr in the right prefrontal cortex also decreased significantly?P<0.05?.There was no significant difference in the ratios of Cho/Cr and Glu/Cr in the left prefrontal cortex between the model group and the control group?P>0.05?.3.IHC results:compared with the control group,the protein BDNF,P-ERK and P-CREB in the bilateral hippocampus and the prefrontal cortex decreased significantly?P<0.05,P<0.01?.There was significant difference in the protein expression of ERK and CREB in the left hippocampus between the model group and the control group?P<0.05?.There was no significant difference in the ERK and CREB protein in the right hippocampus and the bilateral prefrontal cortex between the model group and the control group?P>0.05?.4.Western Blot results:compared with the control group,the protein expression of the BDNF,P-ERK and P-CREB in the bilateral hippocampus and the prefrontal cortex decreased significantly?P<0.05,P<0.01?.There was significant difference in the protein expression of ERK and CREB in the bilateral hippocampus and the prefrontal cortex between the model group and the control group?P<0.05,P<0.01?.5.the observation indexes of MRS and DTI were consistent with the expression of target protein in the BDNF/ERK/CREB signaling pathway in the corresponding brain tissues,among which Glu/Cr of LH was correlated with BDNF in depression rat model.Conclusions1.The CUMS model combined with isolated rearing can simulate the imbalance of human body state caused by long-term stres.The depression model was successfully copied.2.The hippocampus and prefrontal cortex are important brain regions for depression study.Small animal MRI showed that there are different degrees of changes in neurometabolite and damages to fiber bundle in hippocampus and prefrontal cortex.And there is an imbalance between the hemispheres of the brain.3.The BDNF/ERK/CREB signal pathways in the hippocampus and prefrontal cortex in rat models of depression were inhibited.This shows that the BDNF/ERK/CREB signal pathway plays an important role in the pathogenesis of depression and it is an important target for clinical antidepressant therapy.4.The results show that the brain function imaging technique of small animal MRI can observe the structural and functional changes in the brain of depression rats,and that the relevant examination indicators of MRI have consistent performance with the protein detection results in the signal pathways in the corresponding brain tissue.Therefore,it can be speculated that the changes in the MRS and DTI of model rats are correlated to the inhibition of the BDNF/ERK/CREB signal pathway.5.Glutaminoenergy system does play a key role in the pathogenesis of depression.This study found that Glu/Cr of MRS could reflect the correlation between glutaminoenergy system and BDNF in brain tissues,further confirming that MRS could play an important role in the diagnosis and monitoring of depression. |
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