The Effect Of Brain Derived Microparticle On Cerebral Vasospasm And Involved Mechanism

Objective Cerebral vasospasm（CVS）is one of the serious complications caused by traumatic brain injury（TBI）.It is also one of the factors that cause ischemic cerebral infarction,long-term neurological damage and even mortality after TBI.But,the definite pathogenesis of CVS induced by TBI remains to be unclear.Our previous research found that brain derived microparticles（BDMP）rised sharply in blood circulation after TBI,and showed a dynamic change.Moreover,BDMP caused CVS and reduced cerebral blood flow after injected into the tail vein of mice.In vitro,we also found that BDMP contracted the isolated carotid artery of mice,and increase the cytoplasmic concentration of free calcium ion in smooth muscle cells（SMC）and endothelial cells.However,the mechanisms of ion channels that BDMP promotes contraction of carotid artery and SMC is still unintelligible.Previous studies have confirmed that large-conductance calcium-activated potassium channels（BKca channel）play a key role in regulating cerebral vascular tone,so this study focused on the role of BKca channel in vasoconstriction induced by BDMP.Otherwise,using calcium channel blockers to explain the source of calcium ion helping vascular SMC contract which was causeded by BDMP.Through this experiment,a new perspective for the pathophysiological mechanism of cerebral vasospasm after TBI may be provided.Further,this finding enriched the theoretical basis pertaining to clinical diagnosis and treatment of cerebral vasospasm.At the same time,in order to stabilize the experimental basis,this study explored the preservation method of BDMP.Methods（1）Extract BDMP in vitro and stored at different temperature for different time.Afterwards,characterise BDMP by flow cytometry,nanoparticle tracking analysis,and transmission electron microscope and explore stable BDMP storage methods to provide a reliable and effective source of BDMP for subsequent experiments;（2）Explore the relationship of concentration-tension response induced by BDMP in isolated carotid artery;（3）Use T-type and L-type calcium channel blockers to observe their effects on isolated carotid arteries pre-incubated by BDMP;（4）Observe the relaxation effect of BKca channel selective opener NS1619 on vasoconstriction induced by BDMP in isolated carotid artery,and determine the concentration-dose relationship;（5）Using ryanodine receptor blocker,observe the relationship between vascular contraction induced by BDMP and sarcoplasmic reticulum mediated calcium release;（6）By flow cytometry,detect the inhibitory effect of NS1619 on the increase of free Ca²⁺in cerebral arteries smooth muscle cells caused by BDMP;（7）Observe the effect of NS1619 on the morphological change of smooth muscle cell induced by BDMP using hopping probe ion conductance microscropy（HPICM）.Results（1）The complete membrane structure of BDMP can be seen under the TEM,but destroyed after cryopreservation.The content of Annexin-V positive BDMP definited by flow cytometry and the concentration of BDMP determined by NTA have changed significantly underwent cryopreservation;（2）When the concentration of BDMP is（0.1、0.5、1.0、5.0）×10^4/ul,the tension of isolated carotid artery caused by BDMP respectively is 11.4%±1.9%,31.0%±4.2%,50.8%±6.3%,258.5%±21.7%（the tension normalised by 60m MKCl）;（3）200u M NS1619 reduced the tension of isolated carotid artery pre-contracted by BDMP（-29.5%±10.12%）and statistical differences existed（P=0.002）compared with control（-0.50%±3.11%）;The relaxation effect of NS1619 exhibited concentration-dependent.When NS1619 is800u M,the vasodilation of isolated blood vessels achieved-89.75%±4.19%.（4）Blocking T-VDCC,vascular tone decreased by-2.25%±1.89%,there was no statistically significant difference（P=0.60）compared with PBS control（-3.25%±3.10%）;Blocking L-VDCC,the vascular tone decreased by-41.75%±8.22%（P<0.01）and statistical differences existed;（5）In the absence of exogenous Ca²⁺,BDMP can also cause a significant increase in vascular tone（3.04±0.35m N）,which is statistically different from the PBS control（0.23±0.08m N）（P<0.01）;And there was a statistically significant difference（P=0.004）compared with increased vascular tone（4.87±0.72m N）caused by BDMP in calcium solution;（6）In a solution lacking of Ca²⁺,Ry R blocker reduced vascular tone caused by BDMP（5.71±0.69m N）to3.76±0.38m N significantly and statistical differences existed（P=0.003）.Similarly,IP3 receptor blocker also reduced vascular tone caused by BDMP（5.71±0.69m N）to3.49±0.33m N significantly and statistical differences existed（P=0.001）.（7）Flow cytometry showed that the percentage of cytosolic free Ca²⁺positive SMC（21.34%±1.97%）caused by BDMP was significantly higher than that PBS control（3.68%±1.10%）（P<0.01）;After pre-incubation with NS1619,the percentage of free Ca²⁺positive SMC（8.50%±1.33%）was significantly lower than that of the BDMP group（P<0.01）;In a solution lacking of Ca²⁺,the proportion of free Ca²⁺positive SMC（16.34%±1.48%）after BDMP treatment was significantly higher than that of PBS control（5.02%±1.33%）（P<0.01）,but it is low significantly compared with BDMP treatment（23.68%±2.50%）in Ca²⁺system（P<0.01）;（8）HPICM found that the height of SMC（8.30±0.27um）was significantly higher than that of the control（6.69±0.59um）（P=0.013）after BDMP treatment.And NS1619 incubation did not change its height（10.04±0.47um）compared with BDMP treatment（P=0.005）.Conclusion（1）Cryopreservation affected the morphology,concentration and particle size distribution of BDMP;（2）Contraction of isolated carotid artery caused by BDMP may be related to its blocking of BKca channels.BDMP can increase the concentration of free Ca²⁺in the cytoplasm.Elevated Ca²⁺originated partly from the outside of the cell and partly from the sarcoplasmic reticulum of SMC.Namely,BDMP can opened the calcium bank of SMC and lead to extracellular Ca²⁺inflow into the cytoplasm,thereby promoting vascular spasm.Briefly a new ion channel mechanism about CVS caused by BDMP after TBI was revealed,and this may provid an enlightenment for the diagnosis and treatment of CVS related to TBI.