| Objective: In criminal cases,many factors,such as physical injury and mental stress,can lead to sudden disturbance of consciousness and mental injury of party,even death.For example,when the party suffer from sustained binding or limiting position,it may happen mental disorders,damage to vital organs and even death.The injury in these cases is insufficient to cause these serious consequences,even there are no obvious visible injuries.As a result,the cases can not be resolved due to uncertain or disputed death reasons,which will result in adverse social effects.These cases were characterized by varying degrees of mental stress.Research results confirm that psychological stress can cause mental disorders,but the underlying mechanism remains unknown.It has become an urgent scientific problem in the field of forensic science.Stress is a systemic non-specific neuroendocrine response,when the body suffers from some stressors.In normal cases,moderate stress plays defensive effects.But too much or too long stress can lead to a variety of organ dysfunction and metabolic disorders.The reserves and defense mechanisms of body are gradually consumed,resulting in a serious disorder of internal environment,even MODS and death.It is one of the main neuroendocrine response of stress that HPA axis excites and GC increases.Normal concentration of GC plays an important role in maintaining the body's internal environmental of the steady state and promoting the body's growth and development.However,the GC will damage the central nervous system,when GC concentrations continue to rise or stressor exists for too long.Studies have found that high concentration of GC can cause degeneration and damage of hippocampal neurons,when subjected to excessive stress and excessive activation of the HPA axis.The damage effect fo GC on central nervous system injury is likely to be an important mechanism by which stress induce mental disorder.Amygdala is one of the the most important nucleus of the limbic system.Its fiber connections and functions are extremely broad.It is the nerve center which produces,and identifies,as well as regulates emotion and controls fear and memories.The amygdala is sensitive to stress reaction,but also plays an important role in formation and storage of the emotional memory.Research reported that the amygdala damage plays an important role in the pathogenesis of PTSD.Therefore,investigating the damage effect of GC on amygdala plays an significant scientific role in revealing the mechanism of mental damage caused by stress.The present study aimed to explore the damage effect of GC on the amygdala through primary cultured amygdaloid neurons,which will provide scientific basis for clarifying the GC's role in stress-induced damage and revealing the molecular mechanism of restraint stress leading to the amygdala damage.Methods:1 Newborn SD rats less than 24 hours were sacrificed after sterilized by 75% alcohol and the bilateral amygdaloid tissues were collected on ice plate to prepare monoplast suspension.Then the cells were seeded on tissue culture plates which were coated with Poly-L-Lysine(PLL).2 After matured,the amygdaloid primary neurons were identified by immunefluorescence techniques with antibody against microtubule associated protein-2(MAP2),which is neural specific marker,and were detected the existence of glucocorticoid receptor(GR)by immunefluorescence techniques with antibody against GR.3 In order to clarify the GC can lead to apoptosis of primary cultured amygdaloid neurons,give different concentrations of Dexamethasone to activate 48 hours.The cells were randomly divided into five groups: Control?10-9M?10-8M?10-7M?10-6M.Detect the rate of apoptosis of every group by Flow cytometry Annexin-FITC/PI technique after 48 hours.4 In order to further confirm whether GC has cause the apoptosis of amygdala neurons and whether the GC receptor was involved,use GC receptor antagonist Mifepristone and Dexamethasone(10-6M)to activate the neurons 48 hours together.The cells were randomly divided into four groups: Control?DEX?DEX+ MIF?MIF.Detect the rate of apoptosis of every group by TUNEL technique.Detect the expressions of BAX mRNA of every group by Real-time PCR technique.5 The data were presented as the “mean ± standard deviation(Mean±SD)” and were analyzed with analysis of one-way variance(AVOVA)followed by Least Significant Difference(LSD)test for multiple comparisons with SPSS 21.0 statistical program.Values of P<0.05 were considered to be statistically significant.Results:1 Results of amygdala neurons in primary culture: When cultured for 7-10 days,the primary cultured amygdaloid neurons had full cell body which is round or oval with strong third dimension,surrounded by a Halo.And the dendrites which connected with each other formed a network-like structure.The cultured neurons were matured at present and could be used for subsequent experiments.The primary culture techniques of this experiment were improved on the basis of traditional techniques for primary culture of neurons.Operate at low temperature all the time,centrifuge for several times to remove impurities and update laboratory equipment.Thus,the survival rate and the quality of primary cultured neurons were improved largely.Meanwhile,the growth environment and the growth rate of the neurons were all better.Ductility performance of the dendrites were more prominent,which would be more convenient to observe and to post-process under the microscope.2 The amygdaloid primary neurons were identified with antibody against MAP2,which is neural specific marker.Results showed that the purity of neurons in the present study was more than 90% and the neurons can be used for subsequent experiments.It was confirmed that there exists a wealth of GC receptor(GR)on the membrane of amygdaloid neurons by immunefluorescence techniques with antibody against GR.3 Results of the apoptosis rate detected by Flow cytometry Annexin-FITC/PI technique: Compared with the control group,the percentage of apoptosis cells gradually increased significantly when DEX((10-8M-10-6M)treatment.While the apoptosis rate of the 10-9M group had no significant difference compared with the control group,prompted that a certain concentration of GC can lead to apoptosis of neurons in the amygdala.4 The TUNEL test showed that the negative cells expression of the DEX group increased significantly,compared with the control group.While the negative cells expression of the DEX+MIF group decreased significantly,compared with the DEX group.There was no difference between the MIF group and the control group.The results confirmed GC receptor could inhibit the apoptosis of amygdaloid neurons.5 Results of the expressions of BAX mRNA detected by Real-time PCR technique: Compared with the control group,the expressions of BAX mRNA of the DEX group increased significantly.While the expressions of BAX mRNA of the DEX+MIF group decreased significantly,compared with the DEX group.Also there was no difference between the MIF group and the control group.Consequently,it was further confirmed that GC could induce the apoptosis of amygdaloid neurons through the receptor mechanism.Conclusion:The primary neurons of amygdala were successfully cultured in vitro for the first time in the present study.The effects of GC on apoptosis of amygdaloid neurons were observed at the cellular level.And through the observation on pathological changes of neurons and changes of the expression of BAX mRNA,the conclusion can be reached as follows: GC can independently induce the apoptosis of primary cultured neurons in the amygdala by combining with GC receptor. |
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