The Mechanistic Investigation Of Brain-derived Microparticles On The Formation Of Traumatic Brain Injury Associated Cerebral Vasospasm

Objective Cerebral vasospasm after traumatic brain injury is one of the serious complications after TBI,and it is an important resson causing secondary brain injury such as cerebral ischemia,cerebral infarction.The patients have a poor prognosis,high disability rate and mortality rate.However,the exact pathogenesis is still not clear.Our group’s previous study confirmed that mice can release brain-derived microparticles into the peripheral circulating blood through the disrupted blood-brain barrier after TBI.A certain number of brain-derived microparticles injected into the tail vein of rats can cause mice to die.However,pathological autopsy did not find organic lesions in the dead mice.Therefore,we speculated that the cause of death is functional changes such as cerebral vasospasm.Based on previous studies,this study will continue to study the role of brain-derived microparticles in cerebral vasospasm and cerebral blood flow,and examine its effects on intracellular calcium concentration in vascular constituent cells such as smooth muscle cells and endothelial cells.The therapeutic effect of nimodipine,a calcium channel antagonist,on brain-derived microparticle-mediated cerebral vasospasm was examined.This study will clarify the early occurrence mechanism of cerebral vasospasm after TBI,enrich the theory of the physiological mechanism of cerebral vasospasm after TBI,and provide new ideas and theoretical basis for the diagnosis and treatment of cerebral vasospasm after TBI.Methods(1)Brain-derived microparticles were prepared in vitro and identified by transmission electron microscopy and particle size identification.The median lethal dose was determined by injecting BDMP into the tail vein in mouses.It can provide reliable and valid basic data for follow-up in vivo and in vitro studies on the pathophysiology of brain-derived microparticles.(2)Vascular ring tension test was used to detect the vasospasm effect of brain-derived microparticles and the antispasmodic effect of nimodipine in vitro.The role of brain-derived microparticles in cerebral blood flow perfusion was tested by laser speckle flow dynamic imaging in vivo.(3)The effects of brain-derived microparticles on the intracellular free calcium ion in cerebral artery smooth muscle cells and umbilical vein endothelial cells were determined by confocal fluorescence microscopy and flow cytometry in vitro(4)The role of brain-derived microparticles in the regulation of voltage-gated calcium channels was examined in vitro by patch-clamp technique trying to explore the mechanism of vasospasm caused by brain-derived microparticles.(5)The effects of brain-derived microparticles on the morphology of vascular constitutional cells such as cerebral artery smooth muscle cells and umbilical vein endothelial cells were observed using ion-conducting microscopy in vitro.(6)Confocal microscopy was used to observe the effects of brain-derived microparticles on the cytoskeleton of cerebral artery smooth muscle cells and umbilical vein endothelial cells in vitro.(7)Western blotting was used to determine the effect of brain-derived microparticles on the final stage of smooth muscle contraction.(8)The phospholipid composition of brain-derived microparticles was determined by mass spectrometry.the in vitro prepared phospholipids were removed and removed.The above-mentioned effects of brain-derived microparticles of phospholipids clarify the relationship between the cariogenic effects of brain-derived microparticles and their structural integrity.The above-mentioned effects of phospholipid microparticles prepared in vitro and brain-derived microparticles with phospholipid removal were explored to clarify the relationship between the vasospasm effect of brain-derived microparticles and their structural integrity.Results(1)A complete membrane structure can be seen under the transmission electron microscope in the prepared BDMPs.The diameter of BDMPs is between 100 and 1000 nm.Different clinical manifestations can be seen after injecting BDMPs into the tail vein.There was a positive correlation between the number of BDMP and mortality.(2)Brain-derived microparticles can cause contraction of carotid artery rings in vitro.It confirmed that brain-derived microparticles can cause a decrease in cerebral blood flow perfusion in vivo.Nimodipine can partially block these effects.(3)It confirmed that brain-derived microparticles can cause increased intracellular free calcium concentration in SMC and HUVEC.Nimodipine can partially block such effects.(4)It confirmed that brain-derived microparticles can cause height elevation of smooth muscle cells and endothelial cells through ionic conductivity microscopy.It confirmed that BDMPs can lead to changes in cytoskeletal proteins and cause contraction of smooth muscle cells and endothelial cells.(5)Brain-derived microparticles can promote the opening of voltage-gated calcium channels,and nimodipine can partially block this effect.(6)The above-described effect disappears when the phospholipid microparticles prepared in vitro and the brain-derived microparticles which phospholipids have been removed by SDS injected into the tail vein..Conclusion(1)Brain-derived microparticles released into circulating blood after TBI act on smooth muscle cells and endothelial cells to cause vasospasm,which in turn leads to a decrease in cerebral blood flow.Severe cases can cause death.(2)Brain-derived microparticles can increase the intracellular free calcium concentration of smooth muscle cells and endothelial cells,which in turn leads to changes in cell morphology and cytoskeleton.The main mechanism is that brain-derived microparticles promote opening the voltage-gated calcium channels.(3)The vasospasm effect of brain-derived microparticles depends on the structural integrity,and nimodipine can partially block its vasodilatory effects.This study revealed a new physiological mechanism of cerebral vasospasm after traumatic brain injury and explored a corresponding treatment method.