Tunneling Nanotubes Formation And Protective Role On Cerebral Ischemia And Reperfusion Models Of Neural Stem Cells

Background and objectivesStroke is one of the three diseases with the highest mortality and morbidity in the world,of which 87%are cerebral infarction.For acute cerebral infarction,the only drug treatment currently approved by the FDA is early thrombolysis.However,the thrombolysis time window is relatively strict,and more than 95%of patients cannot reach the hospital for thrombolytic therapy within the onset time window.Our country has slowly entered an aging society.The stroke mortality rate and disability rate have been increasing year by year.It has seriously lowered the quality of life and the average life expectancy of patients,and has brought a heavy burden on the national economy.Therefore,it is of great practical significance for researchers to explore new ischemic stroke treatment strategies that can cover more patients.In recent years,many neural stem cells have emerged in the study of the mechanisms of stroke treatment.This has provided new ideas and methods for solving the stroke problem of the world.In the study of acute stroke,the protective effect of neural stem cells?NSCs?on microvascular endothelial cells is not yet clear.Brain microvascular endothelial cells?BMECs?are not only lining cells of the cerebral microvascular lumen,but also secrete active factors to affect the functional status of neurons.BMECs injury is the basic cause of ischemia-reperfusion injury after acute cerebral infarction.Due to ischemia-reperfusion injury,BMECs edema and expose subcutaneous collagen.In the end,white blood cells and platelets adhere and microthrombus are formed.At the same time,the mitochondria of BMECs are damaged and are strongly activated to carry out oxidative activities,generating a large amount of oxygen free radicals and aggravating brain tissue damage.However,in the current study of neural stem cells in the treatment of cerebral infarction,most of the studies focused on the repair of damaged nerve tissue.However,the protective effect of NSCs on BMECs after cerebral ischemic reperfusion injury is unclear.To this end,this study conducted a series of experimental explorations on the mechanism of NSCs repairing ischemia-reperfusion injury of BMECs.This study confirmed that NSCs can protect endothelial cells from ischemia-reperfusion injury through direct contact.In the experiment,we successfully isolated and cultured NSCs and BMECs;in order to further simulate the pathophysiological conditions of cerebral microvascular endothelial cells in ischemia-reperfusion injury,we established a co-culture model of NSCs and BMECs,and confirmed that NSCs can Tunneling nanotubes?TNTs?,a type of bridge formed only between mammalian cells,is a special type of intercellular connection,consisting primarily of a-tubulin and f-actin aggregation.Long-distance mitochondrial transport.?1?The formation of TNT between BMECs and NSCs,the promotion of the formation of TNTs by the MSEC gene,and the inhibition of TNTs by Nocodazole?tubulin depolymerization agent?were identified in this study.?2?Because TNT lacks specific markers,NSCs were added with MSEC or Nocodazole pretreatment to affect TNT formation,and MCAO models were treated with stem cell transplantation.The changes of the expression of major oxidative stress factors SOD,H2O2,T-AOC,SOD,CAT,GSH-Px,and MDA were examined;the role of preconditioning factors in the mitochondrial apoptosis signaling pathway was analyzed,and its influence on MCAO model pathology,Functional roles and mechanisms,indirectly explain the role of TNT.This study mainly explored new mechanisms for the treatment of NSCs in stroke through ex vivo experiments and in vivo experiments,providing theoretical basis for the further benefit of human stem cell therapy.TNT is composed of a superficial cell membrane and an internal cytoskeleton.Its length and diameter can fluctuate within a wide range,and different types of cells and different culture conditions may cause differences in the degree.TNT can mediate the transport of substances and signal exchanges between cells.With its wide caliber and relatively long length,TNT has a unique advantage in transporting high molecular weight substances and even organelles.Methods?1?Primary separation and culture of neural stem cells,brain microvascular endothelial cells;?2?Identification of neural stem cells by Nestin antibody and identification of brain microvascular endothelial cells by?factor antibody;?3?Constructing neural stem cells and brain microvascular endothelial cells co-culture model;?4?CFSE labeling of brain microvascular endothelial cells;?5?Mitochondrial mitotracker tracing of neural stem cells;?6?Construction of MSEC lentivirus;?7?MSEC transfection of neural stem cells or Nocodazole pretreatment;?8?Extracting the common RNA of neural stem cells of each group,detecting the expression level of ki-67 by PCR;?9?Confocal laser scanning microscope observation of TNTs formation;?10?establishment of MCAO model;preparation of MSEC lentivirus infection,Nocodazole preconditioning of neural stem cells,empty virus transfected neural stem cells and PBS pretreated neural stem cells as a control group;?11?Neural stem cell transplantation in MCAO model;?12?frozen section staining,the transplanted cells were traced;electron microscopy to observe the structure of the blood-brain barrier;?13?Establishment of TTC stained cerebral infarction model and comparison of infarct size;?14?Nissl staining,observed morphological changes of brain tissue after neural stem cell transplantation,and counted neurons;?15?MNSS behavioral functional scores of MCAO model rats in each group;?16?Changes in expression of oxidative stress related factors in animal models were examined;?17?Western Blot detection of MCAO model intervention,the mitochondrial apoptotic pathway of key molecules Bcl-2,Bax and Cyt-c expression.Results?1?Primary cultured BMECs were positive for Factor VIII antigen and NSCs were positive for Nestin antigen.Prove cell division success;?2?The results showed that TNTs existed in BMECs and NSCs co-culture system;?3?Overexpression of MSEC gene can promote TNT formation,Nocodazole pretreatment of NSCs inhibit cells inter-TNT formation;?4?MSEC overexpression virus transfection and Noco pretreatment did not affect the proliferation activity of NSCs;?5?Mouse MCAO model was constructed,TTC staining showed the formation of cerebral infarction;?6?Nissl staining,neurological mNSS score results showed that:MSEC overexpression promotes neural stem cell protection of MCAO;Nocodazol preconditioning attenuates the protective effects of neural stem cells on MCAO;?7?Western blot results showed that:MSEC preconditioning of neural stem cells up-regulates anti-apoptotic protein Bcl-2,And decreased the expression of pro-apoptotic protein Bax and downstream pathway Cyt-c.The results of Nocodazole preconditioning were opposite.?8?In general,MSEC pretreatment of neural stem cells can promote the role of antioxidant factors,inhibition of H₂O₂,ROS,MDA and other oxidative factors;Nocodazole pretreatment of neural stem cells to obtain the opposite result.ConclusionIn this study,we successfully constructed a co-culture model of BMECs and NSCs and verified the formation of TNTs between BMECs and NSCs.MSEC gene can promote the formation of TNTs,Nocodazole pretreatment of neural stem cells will hinder the formation of TNTs.We found that the miRNA let-7b directly or indirectly target gene MSEC and reduce the formation of long dendritic cells,probably because of its down-regulation of MSEC expression,so that the cell's"budding"characteristics are inhibited.After NSCs was pretreatment with MSEC,the effect of mitochondrial apoptosis on MCAO mouse brain cells was weakened.The results of Nocodazole preconditioning were the opposite.MSEC pretreatment could attenuate the oxidative stress response.The result of Neural stem cells pretreated with Nocodazole is opposite.