A Methodological Study On The Extraction Of Brain-derived Microparticles

Objective: In recent years,microparticles(MPs)have gradually become a research hotspot in the fields of biology and medicine.At present,there are many different methods for extracting MP in various related experiments.There are many studies which have shown that traumatic brain injury(TBI)will produce brain-derived microparticles(BDMP),and these brain-derived microparticles have a very important impact on the pathophysiological changes of patients with traumatic brain injury.In this experiment,we first establish a model of moderate traumatic brain injury in mice,and then we use the three different methods of grinding,papain digestion,and pipetting to extract the brain-derived source of traumatic TBI from mice.Then we examined and compared the differences in extraction efficiency,procoagulant activity,microscopic morphology,particle size and distribution of BDMP from mice extracted by these three different methods.We try to discuss the characteristics of the particles extracted by different methods through analysis and comparison,so as to provide a methodological reference and direction for the subsequent research on the particles.Methods:(1)We established a model of moderate traumatic brain injury in mice by using a hydraulic shock(FPI)instrument;(2)we extracted brain-derived microparticles from mice,using a grinding method,and performing tissue homogenization and step-by-step Centrifugation;(3)we extract brain-derived microparticles from mice,make papain digestion,and perform tissue homogenization and stepwise centrifugation;(4)we extract brain-derived microparticles from mice to make a blow,and Perform tissue homogenization and stepwise centrifugation;(5)We use flow cytometry to detect the amount of BDMP extracted by different methods,and then compare the extraction efficiency of the three methods;(6)We used flow cytometry to detect the positive expression rates of GFAP,NSE,AnnexinV and Mito-Tracker Green in each group of BDMP;(7)we used coagulometer to detect prothrombin time(PT),and then Compare the procoagulant effects of BDMP extracted by three different methods;(8)We used transmission electron microscopy to observe the microscopic morphology and size of BDMP extracted by three methods;(9)We used NanoSight analyzer to detect and compare the size and distribution of the BDMPs.Results:(1)There is a statistical difference in the extraction efficiency of BDMP of these three different extraction methods we used.And among them,the extraction method and the enzymatic digestion method are close to the extraction efficiency of BDMP,and the extraction efficiency of BDMP is the lowest;(2)The number of Mito-Tracker Green,NSE,and GFAP-positive BDMPs extracted by these three different methods were different;(3)The procoagulant activity of BDMP in each group was different.Among them,the clotting activity of BDMP extracted by enzymatic digestion was significantly higher than that of BDMP extracted by the method of extraction and grinding and blowing,while the difference between the BDMPs by the grinding method and blowing method was small.(4)There was no significant difference in the morphological structure and particle size of BDMP extracted by different methods observed by transmission electron microscopy;(5)The difference in particle size distribution of the above 6 groups of BDMP detected by NanoSight analyzer was small.BDMP of the grinding method was significantly smaller than the BDMP of the enzymatic digestion method and the blow method.Conclusion: The experimental study showed that there were some differences in the efficiency of extraction,the procoagulant function of the produced microparticles and the micromorphological characteristics of the mouse BDMP extracted by different methods.The method of uniformly grinding and extracting particles is the most widely used,and it is more efficient than the method of enzymatic digestion and mechanical blowing to extract BDMP.The average particle size of the particles extracted by the grinding method is smaller than that of the other two methods.In this experiment,there were some differences in the cell source and procoagulant activity of the six groups of BDMP.This experiment can provide a reference and new ideas for the future experimental research to take different methods to extract particles according to different needs.