The Preliminary Process Of Mechanical Damage On Neurons Induced By Hematoma Through The Method Of Brain Slice

Hemorrhagic stroke is the most lethal and crippling cerebrovascular disease and with serious dysneuria among the survivors.Most papers researched the biochemistry damages in the past studies of hypertension cerebral hemorrhage,such as hypoxia,excitatory amino acid toxicity,free radical damage,red blood cell lysate and inflammatory reaction.Besides,there are various mechanical factors in the process of cerebral hemorrhage,including instantaneous blood flow impact stress,shear stress,and long-term compression induced by hematoma.However,whether these mechanical factors play the key role in the occurrence and development of cerebral hemorrhage is rarely studied.In this paper,the primary process of neuron and tissue injury induced by hematoma occupying is researched through the method of brain organic brain slice and with the auxiliary proof of neuron culture.It is a new perspective to study the occurrence and development of hemorrhagic stroke through researching the neuron damage mechanism based on biomechanics,which will provide a new thought for the clinic treatment and new drug discovery.The research and result can be divided into three aspects(1)A mechanical compress loading system for brain slice and neurons is constructed.The culture is consisted of MEM,EBSS balanced salt solution,penicillin-streptomycin,nystin,horse serum and glucose.According to the method of microporous filtering film,brain slices were cultured with Transwell,and then compressed by the mechanical compress loading system.Though the method of lactate dehydrogenase detecting,fluorescent quantitative PCR,and observation of inverted phase contrast microscope,the best condition for brain slice culturing and the best period for experiment are obtained.The culture medium is changed every 24 hours,and the brain slices compressed under the pressure of 40 kPa were cultured for 2 to 7 days.(2)The primary process of neuron damage induced by mechanical stress is researched at the level of brain slice in this research.1)It is obviously that the activity of neurons and glial cells reduced rapidly after being compressed from the immunofluorescence staining result of NSE,GFAP and ?-III.2)The content of PIEZO,TRPV4 genes are detected through the method of fluorescent quantitative PCR.The results indicated that the genes expression raised rapidly after being compressed,and then continued to increase significantly and reach a peak value after 12 hours culturing from the time removing stress.3)The expression of ion channel proteins PIEZO and TRPV4 are tested through westernblot.Compared with the gene expression,the protein expression increased after being compressed,but did not continue to raise,otherwise decreased.The increase of these ion channel proteins will induce the change of cation concentration in the microenvironment of neurons.4)So the concentration of Ca2+ is detected through Ca2+ fluorescent probe.Ca2+ ions were transferred to intracellular space after being compressed,which would increase the intracellular Ca2+ concentration.And the high density of intracellular Ca2+ can activate the expression of apoptosis promoting gene.5)The expression of apoptosis promoting gene BAX increased rapidly while apoptosis restraining gene Bcl-2 decreased significantly,and the tendency continued after mechanical stress being removed,which showed a similar result with the immunofluorescence experiments.(3)After brain slice experiment,verification at cell level is continued.Neurons were cultured and pressed to confirm the primary process of neuron damage.It showed a similar LDH detection result with brain slice.However,as a result of different microenvironment,the concentration of LDH in neuron culture raised directly,other than decreased after being compressed and followed by increase.It is obviously through the immunofluorescence staining that,besides the ion channel protein PIEZO1,PIEZO2 and TRPV4,the expression of skeleton structure ?-III microtube protein is also increased significantly after pression.And while cultured for 12 h after removing mechanical stress,the expression declined rapidly.?-III is a kind of microtube protein with the key function of maintaining neuron's shape,burdening the external mechanical stress.It is obviously that,the expression of mechanical-activate genes in neurons increase after being compressed,and induced the raise of relative protein expression,which results in the influx of Ca2+ and increase the intracellular Ca2+ concentration.The high concentration of intracellular Ca2+ will activates the expression of apoptosis promoting gene BAX and prevent the expression of apoptosis resistant gene Bcl-2,and ultimately accelerates the process of cell apoptosis.