| In the forensic practice,some cases with ones suffered from mental disorders or even death,induced by major mental incidents could be often observed.For instance,ones experienced traumatic events such as others hurt,natural or man-made disasters were contributed to some kinds of mental disorders.Ones suffered from intense mental stressors such as telecommunication fraud could lead to sudden death.And ones with restrictive position resulted in sudden death.Carefully analyzing above mentioned cases,one significant feature could be found,namely there is clear mental and physical stress.Although China's forensic appraisal standards for various types of injuries have been relatively perfect,the assessment and identification of various types of mental disorders resulting from intense mental stress stimulation are still blank.The mechanism and pathophysiological processes of injury and death induced by stress are still unclear.Due to the lack of scientific basis,it is difficult to objectively identify such cases,which result in adverse social impact.Therefore,studying the mechanism of injury or death induced by stress is a major problem that needs to be resolved in forensic science.The central nervous system is the most related to stress.Revealing the pathological changes in the central nervous system is a major scientific issue that needs to be resolved first.Epidemiological surveys showed that 51.2%-60.7% of people experienced at least one major traumatic event during their lifetime,indicating that psychological trauma is a part of life experience.About 7% could result in psychogeny,which seriously affect the quality of life and social functions.Previous studies indicated that soldiers undergoing Afghan warfare had higher levels of activation of the amygdala and insula in emotional stimulation processing.Compared with brain function and structuralneural network connections before and after the soldiers' departure from Afghanistan,they found that the the function of amygdala and medial frontal cortex had significant changes.The analysis was performed after three years of the 911 terrorist attacks and found that the volume of the amygdala,hippocampus,anterior cingulate and prefrontal cortex of witnesses closed to the World Trade Center were significantly reduced.Further investigating revealed that the decrease of size in amygdala was significantly positively correlated with recent life events.A study of survivors in the 2008 Wenchuan earthquake found that the excitement between the frontal lobe,marginal lobe,and striatum system was increased and the functional connectivity was reduced after 25 days of the earthquake.These suggested that severe and sustained stress could result in significant changes in the structure and function of the amygdala.Therefore,it is an important scientific issue to study the pathological mechanism of amygdala after stress stimulation.As a key component of the limbic system,the amygdala plays an indispensable role in the formation and expression of emotions.The basolateral of amygdala(BLA)has been confirmed to play a critical role in emotion-related learning and memory.Meanwhile,it has been widely studied as a key structure in the neural circuits that regulate anxiety-related states and behaviors.The amygdala was significant inhibitory in the resting state,and its spontaneous discharge activity was obviously weaker than that of the surrounding brain regions.But the amygdale of ones with stress disorder was over-exciting.The turbulence in balance of excitation and inhibition(E/I)was associated with the changes of amygdala,and also an underlying cause of anxiety disorders.The ?-aminobutyric acid(GABA),the most inhibitory neurotransmitter in BLA region,has not been fully elucidated on the mechanism of E/I imbalance induced by stress.Therefore,the present study was to investigate the pathological changes of the amygdala caused by stress and the mechanism of E/I in the neuronal damage of amygdala.Our previous studies indicated that long-term of stress could lead to pathological changes in rat hypothalamic neurons,and endoplasmic reticulumstress-related proteins were involved in the hypothalamic neurons damage.Endoplasmic reticulum stress(ERS)is a self-protective mechanism.While excessive long-term ERS could lead to pathological changes in cells.Studies have shown that ERS plays an important role in neuron damage and pathogenesis caused by neurodegenerative diseases.Factors such as hypoxia,ischemia,and energy metabolism could all introduce of ERS.When ERS occurs,the endoplasmic reticulum-glucose-regulated protein 78(GRP78)binds to unfolded or misfolded protein,which dissociates the proteins of PKRlike endoplasmic reticulum kinase(PERK),inositol requirement 1(IRE1),and artificial transcription factor 6(ATF6).Thus,the three signaling pathways are activated,promoting the correct proteins folding and protecting the cells.However,persistent and excessive ERS impairs the function of endoplasmic reticulum,which promotes the activation of CHOP and increases the expression of CHOP through the activation of PERK and ATF6.The high expression of CHOP will peak ERS and induce cell death.Caspase12 is a key molecule in inducing apoptosis during ERS.It is activated only during ERS while inactivated during mitochondrial or death receptor apoptosis.Caspase12 recombines from an inactive zymogen,segments and activates the caspase 9zymogen,which in turn initiates the classic apoptotic pathway of caspase 3and ultimately leads to cell death.Therefore,caspase12 is considered as a marker in ERS and plays an important role in pro-apoptotic pathways.Therefore,the present study was to investigate whether ERS was involved in the neurons injury in the BLA induced by stress.Among animal models of stress,restraint stress can cause animals with strong psychological stress by restricting the position in order to not move freely.At present,it has been recognized as an ideal model of replicating psychological stress.For the present study,we successfully established rat models with restraining and forced ice-water swimming,and systematically observed the changes of neurobehavioral,neuroendocrine,neuropathological,and ERS-related proteins expressions in the BLA region.Besides,we also investigated the relationship between pathological changes and neuroendo-crine and psychiatry,and the mechanism of changes in the BLA neurons induced by stress.All these were meaningful for assessing the stress-induced mental illness and providing theoretical support for possible future forensics identification.Part one Effect of stress on the BLA neuronsObjective: Models of restraining and forced ice-water swimming were successfully established.We observed changes of the behavioral and related neurotransmitters in stress rats.Using HE staining,thionin staining,FluoroJade B fluorescence staining and immunohistochemical staining,we investigated the pathological changes of the BLA neurons.Methods:1 Stress rat models were established by restricting and forced ice-water swimming.Using spontaneous activity and elevated plus maze to investigated the behavioral changes induced by stress.2 Enzyme-linked immunosorbent assay was used to detect the content of catecholamine and ATP in serum and amygdaloid nucleus.Liquid chromatography-mass spectrometry was to investigate the contents of GABA and GLU and their derivatives.Meanwhile,the changes of neuroendocrine were observed.3 Using HE staining,thiofluorescein staining,Fluoro-Jade B fluorescence staining and immunohistochemical staining to investigate the pathological changes of the BLA neurons.Result:1.Spontaneous activity and elevated plus maze results: Compared with the control group,stress groups showed obvious anxiety(P<0.05).Besides,with prolonged duration of stress,the symptoms gradually worsen(P<0.01).2.Serum catecholamines levels in stress rats increased rapidly at 1 day of stress(P<0.01),then decreased gradually.At 21 days of stress,serum catecholamine levels were still significantly higher than those of the control group(P<0.05).The amygdala adrenalin and norepinephrine in stress ratsgradually increased after stress,and there was a significant difference from the group at 1 day of stress(P<0.05).Dopamine in the amygdale was significantly increased at 14 days of stress(P<0.01).The ATP content in the amygdala of stress rats gradually decreased after stress,and showed a significant difference at 3 days after stress compared to the control group(P<0.05).3.Compared with the control group,GLU levels in amygdala were significantly up-regulated after stress,peaked at 7days,and maintained a high level at 14 days and 21 days(P<0.01).The level of GABA in amygdala showed fluctuate,and significantly decreased at 7 days of stress(P<0.05).The ratio of GLU/GABA was significantly increased after 3 days of stress(P<0.01).4.HE staining showed that with the prolongation of stress time,the neurocyte edema gradually appeared in the amygdala BLA nerve cells,the tissue structure was loose,the nerve cells were constriction,the microglial cells proliferated,and the satellite phenomenon and neuronophagia.5.Thionine staining showed that with prolonged duration of stress,tissue structure of the BLA was not clear,Nissl bodies were irregular and disappeared,nerve cells were pycnosis and deep dyeing,and the satellite phenomenon was seen.6.Fluoro-Jade B staining showed that neurons of the BLA were degeneration and death.7.GAD65/67 immunohistochemical staining showed that long term stress could lead to a decrease in the proportion of GABA neurons in the BLA area of amygdala.Summary:1.Stress could cause the degeneration and death of neurons in the BLA.2.The stress-induced E/I imbalance in the amygdala may be related to the degeneration and death of GABA neurons.Part two The mechanism research of catecholamine-mediated neurons injury in amygdala of stress ratsObjective:Using administering tyrosine hydroxylase inhibitor intraperitoneally,we detected the changes of serum catecholamine levels,amygdala neurotransmitters,amino acid and ATP levels.HE staining,thionin staining,and Fluoro-Jade B fluorescent labeling were used to compare the pathological changes of BLA neurons with or without inhibitor.Besides,we investigated whether the pathological changes of the BLA neurons induced by catecholamine was associated with apoptosis proteins caspase12 and caspase3.Methods:1 Intraperitoneal injection of tyrosine hydroxylase inhibitor was performed before establishing models.Spontaneous activities and an elevated plus maze were used to observe the behavioral changes of stress.2 Enzyme-linked immunosorbent assay was used to detect the content of catecholamine and ATP in rat serum and amygdaloid nucleus.The contents of GABA and GLU and their derivatives were detected by liquid chromatography-mass spectrometry.3 HE staining,thionin staining and Fluoro-Jade B fluorescence staining were used to observe the pathological changes of the BLA nucleus.4 Immunohistochemistry was used to observe the expression of GAD65/67(GABAergic neuron specific marker),caspase12 and caspase3 in the BLA.5 Western blot was used to detect the expression of caspase12 and caspase3 in the amygdala.6 Immuofluorescence multiple staining was used to detect the number of GAD65/67 positive cells co-expressing caspase12/ caspase3 in GABAergic neurons of the BLA.Results:1.Spontaneous activity and elevated plus maze results: compared with the stress group,the stress+AMPT group showed a significant decrease in anxiety(P<0.01),but there was still difference when compared with the control group and the AMPT group(P<0.05).2.Catecholamine in serum of the stress+AMPT group was significant lower than the stress group rats(P<0.01),while there was insignificant difference when compared with the control group.Compared with the control group,catecholamine in the amygdale was significantly decreased(P<0.05),while norepinephrine in the amygdala was obviously increased(P<0.01).Compared with the stress group,the ATP content of the amygdala was significantly increased in the stress+AMPT group,but there was still decrease compared to the control group and the AMPT group(P<0.05).3.Compared with the stress group,GLU content was significantly decreased in the stress+AMPT group(P<0.01),while GABA content was significantly increased(P<0.05).The GLU/GABA ratio tended to be more stable.4.HE staining showed that cell pyknosis in the stress+AMPT group was significantly decreased when compared with the stressed group.5.Thionine staining indicated that cells degeneration in the BLA neurons of the stress+AMPT group was significantly decreased compared to the stress group.6.Fluoro-Jade B staining indicated that the number of neuronal degeneration in the BLA neurons of the stress+AMPT group was less than that in the stress group(P<0.05).However,it was still an increase compared to the control group and the AMPT group(P<0.05).7.Immunohistochemical staining showed that the expression of apoptosis proteins caspase12 and caspsae3 in the amygdala stress+AMPT group were significantly decreased compared to the stress group(P<0.01),while they were obviously increased when compared with the control group and the AMPT group(P<0.01).8.Western blot analysis showed that compared with the stress group,the expression of caspase12 and caspsae3 in the stress+AMPT group were significant decreased(P<0.05),while they were obviously increased when compared with the control group and the AMPT group(P<0.05),which consistent with the result of immunofluorescence.9.Multilamellar immunofluorescence staining: compared with the stress group,the number of caspase12,caspsae3 colocalized with GAD65/67 was slightly decreased in the stress+AMPT group(P<0.05).10.GAD65/67 immunohistochemical staining showed that the number of GABAergic neurons of the BLA in the stress+AMPT group was significantly increased when compared to the stress group(P<0.05).Summary:1.Tyrosine hydroxylase inhibitor could reduce the catecholamine levels in serum and amygdala of stress rats,and improve the E/I ratio,which suggested that it could effectively alleviate anxiety in stress rats.2.Tyrosine hydroxylase inhibitor could reduce the expression of apoptosis proteins caspase12 and caspsae3,which improved the survival rate of GABAergic neurons.Part three The mechanism research on the injury of GABAergic neurons in the BLA of stress rats induced by endoplasmic reticulum PERK pathwayObjective:The present study was to investigate the changes of ERS proteins GRP78,p-PERK,CHOP,caspase12 and caspase3 as well as the relationship between dynamic changes of these proteins and neuron injury.Using administering the PERK pathway-related inhibitor salubrinal,the study was to detect the changes of behavioral and neuroendocrine and morphological changes of the GABAergic neurons of the BLA in stress rats with or without inhibitors,which finally indicated that the role of PERK pathway in the injury of GABAergic neurons of the BLA.Methods:1 Intraperitoneal injection of salubrinal was performed before establishing models.Spontaneous activities and an elevated plus maze were used to observe the behavioral changes of stress.2 Enzyme-linked immunosorbent assay was used to detect the content ofcatecholamine and ATP in rat serum and amygdaloid nucleus.The contents of GABA and GLU and their derivatives were detected by liquid chromatography-mass spectrometry.3 HE staining,thionin staining and Fluoro-Jade B fluorescence staining were used to observe the pathological changes of the BLA.4 Immunohistochemistry was used to observe the expression of GRP78,p-PERK,CHOP,caspase12 and caspase3 in the BLA neurons.The number of GABAergic neurons was counted by labeling and counting GAD65/67 positive cells in the BLA.5 Western blot was used to detect the expression of GRP78,p-PERK,CHOP,caspase12 and caspase3 in the amygdala.6 Immuofluorescence multiple staining was used to detect the number of GAD65/67 positive cells co-expressing GRP78/p-PERK/CHOP/caspase12/caspase3 in GABAergic neurons of the BLA.Results:1.Spontaneous activity and elevated plus maze results: compared with the stress group,the stress+SAL group showed a significant decrease in anxiety(P<0.05),but there was still difference when compared to the control group and the solvent group(P<0.05).2.Norepinephrine and adrenaline in the amygdala of the stress+SAL group was significantly decreased compared with the stress group(P<0.01),but was obviously increased when compared with the control group and the solvent group(P<0.05).The content of dopamine in the stress+SAL group was significantly decreased compared with the stress group(P<0.05).Compared with the stress group,the ATP content of the amygdala was significantly increased in the stress+SAL group(P<0.05),but there was still decrease compared to the control group and the solvent group(P<0.05).3.Compared with the stress group,GLU content was significantly decreased in the stress+SAL group(P<0.01).However,GABA content was significantly increased when compared with the stress group(P<0.01).The GLU/GABA ratio tended to be more stable.4.HE staining showed that cell pyknosis in the stress+SAL group was significant decrease compared to the stress group.5.Thionine staining indicated that cells degeneration in the BLA neurons of the stress+SAL group was significantly decreased compared to the stress group.6.Fluoro-Jade B staining indicated that the number of neuronal degeneration in the BLA neurons of the stress+SAL group was less than that in the stress group(P<0.01).However,it was still an increase compared to the control group and the solvent group(P<0.01).7.Immunohistochemical staining showed that the expression of GRP78,p-PERK and CHOP as well as the apoptotic proteins caspase12 and caspsae 3in the amygdala BLA neurons was siginificantly increased after stress compared to the control group.Immunohistochemical staining showed that the expression of CHOP,caspase12 and caspsae3 in the stress+SAL group were significantly decreased compared to the stress group(P<0.01),though they were obviously increased when compared with the control group and the solvent group(P<0.05).these results suggested that salubrinal could alleviate the injury of the BLA neurons.8.Western blot analysis showed that the ERS protein of GRP78,p-PERK,and CHOP as well as caspase12,caspsae3 was obviously increased(P<0.05),which consistent with the result of immuno-fluorescence.Western blot analysis showed that compared with the stress group,the expression of CHOP,caspase12 and caspsae3 in the stress+SAL group were significant decrease(P<0.05),while they were obvious increase when compared with the control group and the solvent group(P<0.05),which consistent with the result of immunofluorescence.9.Multilamellar immunofluorescence staining: with prolonged duration of stress,the number of GRP78,p-PERK,CHOP,caspase12,caspsae3 colocalized with GAD65/67 was showed a trend of increase first and then decrease slightly.Compared with the stress group,the number of CHOP,caspase12,caspsae3 colocalized with GAD65/67 was significant decrease in the stress+SAL group(P<0.01;P<0.05;P<0.01),while was obviously increased when compared with the control group(P<0.05).These results suggested that salubrinal could alleviate the injury of the BLA GABAergic neurons.10 GAD65/67 immunohistochemical staining showed that the number of GABAergic neurons in BLA was significantly decreased after 7 days of stress(P<0.05).Compared with the stress group,the number of GABAergic neurons was obviously increased in the stress+SAL group(P<0.05),which suggested that salubrinal could effectively improve the survival of the BLA GABAergic neurons.Summary:1.The ERS PERK pathway was involved in the degeneration and death of BLA neurons in stress rats.2.Salubrinal could reduce the injury of BLA neurons(including of GABAergic neurons),and improve the E/I ratio,which suggested that it could effectively alleviate anxiety in stress rats.Conclusions:In the study,we investigated the effect of stress on psychiatry,neuroendocrine,and GABAergic neuron-related proteins expression in the amygdala basolateral nucleus,based on successfully established stress rat models and the models of administering inhibitors,and got conclusions as follows:1.Restricting stress united with forced ice-water swimming can lead to degeneration of neurons in the basolateral nucleus(including GABA energy cells)of the amygdala,E/I imbalance,and anxiety like behavior.2.Catecholamine was associated with the degeneration and death of BLA neurons(including of GABAergic neurons)in stress rats.3.The ERS PERK pathway was involved in the degeneration and death of BLA neurons(including of GABAergic neurons)in stress rats.In conclusion,stress of restricting and forced ice-water swimming could continually activate the amygdala by inducing catecholamines,disturb the balance of E/I in amygdale,decrease ATP content and over-express the ERS proteins of the PERK pathway,which finally lead to the degeneration and death of BLA neurons(including of GABAergic neurons). |
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