A Study On The Alteration Of Gray Matter Structure And The Abnormality Of Iron Metabolism In The Post Traumatic Stress Disorder

PART Ⅰ.Alteration of gray matter structure in burn patients with post traumatic stress disorder:a functional MRI studyBackgrounds and objective:Post-traumatic stress disorder(PTSD)is a disabling neuropsychiatric disorder characterized by intrusion of the event,avoidance of reminding it,alterations in cognition and mood and hyperarousal based on the Diagnostic and Statistical Manual of Mental Disorders(Fifth Edition,DSM-Ⅴ).The overall lifetime prevalence rate of PTSD is approximately 13% of women and 6% of men in the United States,making it one of the most common psychiatric disorders.Despite high morbidity associated with PTSD,the pathophysiology of PTSD remains largely unclear.At present,a large number of clinical studies of functional magnetic resonance imaging proved that the hippocampus,anterior cingulate gyrus,amygdala,prefrontal cortex and other areas play an important role in the pathogenesis of PTSD and other brain areas including post cingulate gyrus,left temporal pole,middle temporal gyrus,cerebellum,and visual cortex(lingual gyrus,angular gyrus),premotor cortex,parietal cortex,striatum involving in different types of trauma patients with PTSD,which reflected the neural heterogeneity and complex of pathogenesis involving in PTSD.The nature and severity of traumatic events is a prerequisite for the occurrence of the disease.Different types of traumatic events have different effects on the structure and function of the human brain,and the pathogenesis of PTSD is not consistent.At present,functional magnetic resonance researches are mainly focused on the PTSD disease caused by major natural disasters,war,rape,abuse in children and other types of stress,but less is known about the alteration of brain structure in PTSD after burn.It is very meaningful to understand the pathophysiological basis of PTSD after burn,not only for the patients with burn,or even for the recovery of the survivors of war or major disasters.Methods:Twenty post traumatic stress disorder patients after burn and twenty healthy controls(matched with age,sex and extent of education)underwent functional MRI.The imaging data were analyzed and compared with DPARSFv2.3 and REST1.8 analysis system.Main Results:By using the VBM method,We found that:Compared with the normal control group,PTSD patients after burn demonstrated alteration of cerebral structure,including :Decreased cortical thickness on the right side of frontal cortex,left insula(temporal island junction),left paracentral lobule,left postcentral gyrus,left cuneus.The right precuneus,left supramarginal gyrus,right angular gyrus region increased grey matter volume.Main Conclusion:Neuronal damage may occurred in the early stage of the PTSD patients and dysfunction in orbitofrontal cortex,insula cortex which involving in the limbic system,and the precuneus involving in the DMN might play a critical role in pathophysiology of PTSD.PART Ⅱ.Experimental study on SPS model and model validationBackgrounds and objective:Previous study have proved that neuronal damage may occurred in the early stage of the PTSD patients,but it is not very clear about the pathogenesis caused by stress in PTSD.Due to the lack of noninvasive methods for PTSD patients to study the molecular pathology,most of the experiments were conducted in animal models.Among various animal models of PTSD,single prolonging stress model(SPS)nowadays has been extensively applied in the investigation of PTSD,which was validated to mimic the pathophysiological abnormalitiesand behavioral characteristics of PTSD,such as enhanced anxiety-like behavior,elevated glucocorticoid negative feedback,and exaggerated acoustic startle response.Furthermore,studies have shown that the SPS model was well-established for investigation of abnormal metabolism of the nervous system.In present study,we intend to use behavioral and hematological indices of the tests to verify the validity of the SPS model,and lay an experimental foundation for further research on iron metabolism.Methods:1.The SPS rats were bound for two hours and forced swimming was performed for 20 minutes.Next,rats had a rest for 15 minutes and were then anesthetized with ether to a deep coma.2.Behavioral changes in the model group were detected by Elevated Plus Maze Test and Open Field Test.3.Changes of serum cortisol concentration were detected by the Enzyme-linked Immunosorbent Assay.4.Changes of glucocorticoid receptor(GR)in hippocampus were detected by Immunohistochemistry(IHC).5.Cell morphological changes in different brain regions were observed by Hematoxylin & eosin(H&E)staining.Main results1.SPS exposure induced anxiety-like symptomsCompared with the control group,SPS paradigm dramatically decreased the percentage of open arm time by 49%,corresponding percentage of distance was decreased by 30.2%,and the percentage of entries was reduced by 36.5%.The open field test showed a significant decrease in central time(decreased by 61.3%),and accompanying distance was decreased by 66.1% in the SPS group.2.SPS exposure increased the level of the serum cortisolOur results suggested that the level of serum cortisol was markedly higher in the SPS group,which reflected the state of stress in the rats.The level of the cortisol in the SPS group was approximately 2.1-fold of the control.3.SPS exposure increased the GR expression in the hippocampusResults of IHC analysis of the GR in the hippocampus showed positive immunoreactivity was localized in the cytoplasm of pyramidal neurons and Purkinje neurons.Glucocorticoid receptor(GR)immunoreactivity in the SPS-exposed group was increased by 22.0%.Compared with the control group,the SPS group showed more injured cells in the hippocampus,prefrontal cortex and striatum,which showed heterogeneous and hyperchromatic neurons.Main conclusion1.Our results showed that the rats had higher degree of anxiety and fear in both behavioral tests,which was consistent with a previous study.2.Cortisol is the most representative changed hormone in response to stress.In our study,the serum cortisol level was markedly increased and GR immunoreactivity was increased in the hippocampus,which was in agreement with previous findings.All these data suggest that changes in the expression of GR may represent the primary level of dysfunction in glucocorticoid signaling,combined with altered response to challenging conditions as well as higher anxious and fear.3.Heterogeneous and hyperchromatic neurons in the three brain regions were more than those in the control group,which indicated that neurons were damaged.With behavioral evidence,our data showed that the rats were in the morbid state were affected by severe stress and can simulate the conditions of PTSD patients to some extent.4.SPS exposure induce morphology changes of cells in the brainPART Ⅲ.Deposition of iron-induced neuronal damage in the SPS modelBackgrounds and objective:Maintenance of functional activity of central nervous system requires the participation of many metal ions and the function of iron is the most prominent.Although the activity mediated by iron is very important,cells require highly sophisticated and precisely regulatory mechanisms due to highly oxidative activity of iron resulting in cytotoxic effects.In recent years,many studies have shown that redistribution and deposition of iron in the brain in some neurodegenerative diseases,such as Alzheimer’s disease(AD),Parkinson’s disease(PD),Huntington’s disease and multiple sclerosis.Recently,iron accumulation was observed prior to plaque formation in an animal model of AD,moreover,gene mutations were discovered in neuroferritinopathy.These studies suggest that elevated iron level could be one of the initial factors contributing to neuronal death.A key component of iron regulatory system in the brain is transferrin receptor1(Tf R1)and ferritin(Fn).Studies have shown that iron can cross neuronal cellular membranes primarily through the transferrin receptor-mediated endocytotic mechanism.Tf R1 is mainly distributed in the neurons of the hippocampus and cortex,and uniquely functions in neural development.Some researchers have found that the high density of Tf R1 in the hippocampus and basal ganglia region indicated sensitivity to degeneration in AD.Tf R1 expression has been reported to change in animal models of PD and is involved in cellular apoptosis.Fn is a main intracellular protein storing excess iron within its shell,which is considered a pro-oxidant as well as an antioxidant.Alterations in the level of Fn have been reported in the brain of subjects with AD and PD,and is associated with neurodegeneration.Decreased Fn in these diseases following loss of iron storage capacity increases bioavailability of potentially toxic free iron.Recently,Fn in mitochondrion was observed to be increased and acted as an important protective protein in AD.During oxidative stress,Fn is the main source of iron and reactive oxygen species;however,whether iron,Tf R1 and Fn are involved in the progression of PTSD remains unclear.Some studies have recently shown a disorder of iron metabolism in the serum and brains of rats with psychological stress.Notably,the iron level has been shown to be significantly higher in areas of the brain that are very similar to the brain regions affected by PTSD,such as the prefrontal cortex,hippocampus and striatum.Therefore,we hypothesized that abnormal metabolism of iron in the brain may exist in rats after strong traumatic stress.To address this question,we assessed iron level and iron-related proteins by using single prolonged stress(SPS)-induced paradigm,which is a reliable model and has been extensively applied in the investigation of PTSD.Methods:1.The SPS rats were bound for two hours and forced swimming was performed for 20 minutes.Next,rats had a rest for 15 minutes and were then anesthetized with ether to a deep coma.2.Iron concentration was detected by Inductively Coupled Plasma Optical Emission Spectrometer(ICP-OES).3.The expression of the Tf R1 and Fn was detected by WB.4.Changes of the Tf R1 and Fn in hippocampus were detected by the Immunohistochemistry(IHC).5.The expression of Tf R1 and Fn m RNA were detected by Q-PCR.6.Cell morphological changes in hippocampus were observed by transmission electron microscopic observations.Main results1.SPS exposure increased the concentrations of iron in the brainIn order to test whether iron content changed with the stress or not,we examined iron content in the hippocampus,frontal cortex,and striatum by using the ICP-OES method.Our results demonstrated iron accumulated in the brain areas after acute stress.In the SPS group,the content of brain iron was increased by 3.1 fold in the hippocampus.Iron accumulation in the prefrontal cortex and striatum in the SPS paradigm was increased by 2.5-fold and 3.4-fold,respectively,compared with the control.2.SPS exposure caused region-specific changes in Tf R1 expressionOur results showed that differences were evident in the hippocampus and prefrontal cortex.Tf R1 immunoreactivity in the hippocampus was decreased by 38.6% in the SPS group.The expression of Tf R1 was increased by 38.9% in the prefrontal cortex.However,results of Western blot analysis showed no significant difference in the striatum and cerebellum compared with the controls.3.SPS exposure caused region-specific changes of Fn expressionWestern blot analysis of Fn expression showed a decreasing trend,similar to Tf R1 in the hippocampus,and the expression of Ferritin was decreased by 43.5% in the SPS group compared with control.Immunoreactivity of ferritin in the striatum was increased by 29.4% of the SPS-exposed rats.There was no obvious difference in the expression between the prefrontal cortex and cerebellum.4.SPS exposure decreased the Tf R1,Fn expression in the hippocampusIHC analysis of the Tf R1 and Fn showed similar results as Western blot analysis.Tf R1 was distributed mainly in large pyramidal neurons and located mainly on the membrane,as described previously.Tf R1 immunoreactivity in the SPS-exposed group was decreased by 26.1% as compared with the control.Fn was obviously located in the cytoplasm and nucleus of the pyramidal neurons in the hippocampus of the SPS group,but was decreased by 20.6% compared with the control group.The expression of the three proteins in the hippocampal neurons were significantly different.5.SPS exposure decreased the expression of Fn and Tf R1 m RNA in the hippocampusQ-PCR results showed that Fnm RNA level was significantly inhibited and decreased by 87.4% in the SPS group.Tf R1 m RNA level was significantly inhibited by 41.6%.6.SPS exposure induce morphology changes of cells in the brainThe neurons in the hippocampus,observed under the transmission electron microscope,showed marked swelling mitochondria,crista degranulation and cell apoptosis in the SPS group.Main conclusion1.By using the higher sensitivity ICP-OES method,our data showed iron accumulated evidently in the hippocampus,prefrontal cortex,striatum after the SPS stress and may do harm to the neurons in these area.In fact,we observed that heterogeneous and hyperchromatic neurons in the three brain regions were more than those in the control group,which indicated that neurons were damaged by accumulated iron.By using the transmission electron microscope,we found that hippocampal neurons showed marked mitochondrial swelling and cell apoptosis in the SPS group,which suggested that iron is an important factor in cellular injury.2.Our study demonstrated that Fn was decreased in the hippocampus.We hypothesized that decreased ferritin may result in subsequent cellular free iron accumulation which aggravated neuronal damage,eventually leading to apoptosis.There is significantly elevated ferritin in the striatum potentially leading to a net reduction in the level of free iron.A deficit in bioavailable iron would activate microglia to damage the neurons through inflammation.Our finding suggested the Tf R1 should be increased in the prefrontal cortex inducing excess iron endocytosis,which could lead to cytotoxicity.The heterophanybetween these areas may be a reflection of differences in the regional susceptibility of iron accumulation and the hippocampus may be the most vulnerable area affected by iron in the brain which was supported by a previous study.3.Our data showed that Tf R1 and Fnm RNA in the hippocampus were significantly decreased and demonstrated the stress affects iron metabolism at the gene level.In summary,we showed iron accumulation in the prefrontal cortex,striatum,hippocampus in the SPS rat model.The expression of iron related protein and m RNA also changed in these areas.This study indicated that iron may be involved in the pathology of PTSD,and could be nominated as a novel molecule involved in the pathology of PTSD and provide a potential target for therapeutic intervention of PTSD.Our conclusions may highlight the role of iron in some other psychiatric disorders such as depression and obsessive compulsive disorder which are affected by stress factors in the pathogenesis of these disorders.