Mechanisms Of Microvascular Injury Following Mild Traumatic Brain Injury And Exploration Of Specific Biomarkers For Brain Pericytes

Background Traumatic brain injury(TBI)is a devastating neurological disease and a major cause of death and disability worldwide.It not only can cause acute brain injury,but also act as an important risk factor for a variety of chronic neurological disorders.Thus,in-depth investigating its pathophysiological mechanism and developing novel effective therapeutics is particularly important for prevention and treatment of this disease.Mechanistically,TBI can induce a series of neuropathological changes,including excitotoxicity,oxidative stress,inflammation,cell death,cerebral microvascular injury and blood-brain barrier(BBB)disruption,among others.These secondary injury responses interact highly with each other,which ultimately aggravates the complexity ofTBI pathology.In particular,brain microvascular damage and BBB dysfunction are considered to be important pathological mechanisms ofTBI,which can lead to plasma protein extravasation,tissue ischemia and hypoxia,accumulation of metabolic waste in the brain,glial cell activation,and neuronal cell injury and death.It has an important impact on the prognosis ofTBI patients.However,the pathological process and underlying mechanisms have not been fully elucidated.Based on the severity,TBI can be classified as mild,moderate and severeTBI.Among them,nearly 85% of cases are mildTBI(mTBI),and its importance in acute brain injury and chronic neurodegeneration is gradually being recognized.Accumulating clinical and preclinical data indicate that mTBI was closely related to the occurrence of neurodegenerative diseases such as Alzheimer’s disease,Parkinson’s disease and chronic traumatic encephalopathy.Currently,mTBI-induced neuropathological mechanism has not been fully elucidated,and the pathophysiological significance of brain microvascular damage and BBB disruption after mTBI has not been adequately addressed.This is partially due to the complex pathological mechanism of mTBI itself,as well as the sophisticated structure and function of cerebral microvasculature and BBB.The ultramicroscopic analysis of brain capillaries and BBB demonstrated that it is composed of endothelial cells,cell junction proteins,basement membranes,mural cells(including vascular smooth muscle cells(VSMC)and pericytes),and astrocytic endfeet.Noteworthily,as an important component of brain capillaries and BBB,pericytes are essential for maintaining their normal structure and function.Research evidence has demonstrated that the loss of pericytes is involved in the pathological process of acute brain injury and chronic neurodegeneration.Unfortunately,due to the lack of specific cell markers,current research aiming to elucidate the role of pericytes in the pathophysiological state of central nervous system diseases is still challenging.Thus,in this study,we aimed to explore the pathological changes of cerebral microvascular injury and BBB disruption after mTBI in a controlled cortical impact(CCI)mice model.Besides,based on single-cell sequencing analysis,transgenic animal generation as well as other experimental technologies,we explored and verified that Atp13a5 is a specific biomarker for cerebral vascular pericytes,which provide potential new research directions as well as tools for further investigating the mechanism of cerebral microvascular injury and BBB disruption in neurological diseases includingTBI.1.Pathological changes of cerebral microvascular injury and BBB disruption after mTBIPurpose This experiment aimed to detect and evaluate the cerebral microvascular injury and BBB breakdown after mTBI,investigate its pathological characteristics in the acute and subacute stages of mTBI,which would bring new perspectives for further exploration of the role and underlying mechanisms of brain microvascular injury and BBB disruption after mTBI.Method Three separate experiments were conducted in this part.(1)Build a controlled cortical impact(CCI)method to make a closed mTBI mouse model,and compared with severeTBI(severeTBI,sTBI).By using neurological evaluation(m NSS,Rotarod and Foot-fault test),Tunel staining,histochemical staining,brain tissue water content measurement and other technical methods to evaluate the acute/subacute cerebral microvascular injury and BBB disruption after mTBI.(2)Using histochemical staining and imaging technology(i.e.,Laser Speckle Contrast Imaging,LSCI)to evaluate the pathological changes of BBB’s important components(including pericytes and basement membrane)after mTBI,as well as changes in the perivascular space and microvascular blood supply after injury.(3)By measuring the content of Evans blue and Alexa Fluor 555-cadaverine in the brain tissue of mTBI and analyzing the activation of GFAP+ astrocytes and Iba-1+ microglia in the cortex and hippocampus via histochemical staining;Also,the relationship between BBB dysfunction and gliosis after mTBI was explored.Result(1)Compared with sTBI,mTBI caused transient neurological dysfunction and motor impairments in mice,which nearly recovered 3 days after injury;Pathological examination showed that mTBI did not cause visible cortical lesion 1 day after injury.Brain water content was not significantly changed 1 day after injury.No obvious cell death of Neu N+ neuronal death was detected 3 days after injury.Histochemical staining suggested that mTBI caused lighter axon damage and less activation of Iba-1+ microglia and GFAP+ astrocytes at day 3 after injury,compared to sTBI.(2)Cresyl violet staining showed that the perivascular space was enlarged in mTBI mouse brain tissue slices;Evans blue staining showed that the accumulation of Evans blue mainly occurred in the superficial area of the injured side of the cortex after injury;The staining of endogenous Ig G and Fibrinogen as well as the exogenous tracer 555-cadaverine showed that the extravasation of the above-mentioned substances was still significantly higher than that of the control group at day 3 after injury.Histochemical staining suggested the number and coverage of CD13+ pericytes in the cortical brain tissue were significantly reduced after mTBI compared to the contralateral side;The expression of Collagen IV and Laminin α2 in the cortical brain tissue of the mTBI-injured side was significantly higher than that of the contralateral side,while the expression of Laminin α4 was significantly reduced.LSCI imaging showed mTBI can cause a ~50% reduction in cerebral blood flow in the cortex of the injured side 3 days after injury.(3)555-cadaverine staining showed that it accumulates in the cortical brain tissue of the injured side most obviously 1 day after injury,and then gradually decreases after mTBI;It can also be detected at 1,3,and 8 days in the hippocampus after injury,which was most obvious on day 3 after injury;Immunohistochemical staining showed that the number of GFAP+ astrocytes in the injured side cortex and hippocampus brain tissue was significantly increased on the 3rd and 8th day after mTBI;Iba-1+ microglia were increased significantly on day 1,3,8 after injury.Conclusion This study demonstrated that mTBI can cause brain microvascular injury,BBB disruption,pericytes loss,basement membrane changes,and decrease in cerebral blood flow in mice,and ultimately participate in the pathological process after injury.2.Analysis and verification of Atp13a5 as a specific biomarker for cerebral vascular pericytesPurpose This experiment aimed to explore and verify potential biomarkers specifically expressed by cerebrovascular pericytes based on single-cell RNA sequencing(sc RNA-seq)data analysis;Besides,the expression and distribution of Atp13a5 m RNA in different brain regions and from different ages were explored.Furthermore,transgenic animals were generated to verify and analyze Atp13a5 as a specific marker of cerebrovascular pericytes in the mouse brain.Investigating such specific biomarkers for brain pericytes contributes to developing new research tools to further explore the role of cerebrovascular pericytes and BBB pathological changes in the pathogenesis of central nervous system diseases includingTBI.Method(1)Analysis of brain vascular sc RNA-seq data sets,compare the transcriptome information of different cell subgroups in the brain and explore the potential specific expression genes of cerebrovascular pericytes.(2)Based on the analysis results of sc RNA-seq data,combined with in situ hybridization and histochemical staining techniques,analyze the expression of Atp13a5 m RNA in the perivascular cells of the brain and peripheral organs(such as heart,liver,and kidney).Explore its expression in brain pericytes as well as its developmental expression patterns.(3)Based on the generation of Atp13a5-2A-Cre ER-IRES-td Tomato knockin transgenic mice,combined with PCR and immunohistochemical staining techniques,to explore the expression of Atp13a5 in cerebral pericytes as well as its relationship with other brain cells.Result(1)Sc RNA-seq data analysis showed that unlike Vtn and Kcnj8,the two pan-pericyte markers,Atp13a5 is specifically and highly expressed in brain pericytes.(2)Chromogenic in situ hybridization demonstrated that Atp13a5 m RNA is widely expressed in different brain regions,including cortex,hippocampus,striatum,thalamus,midbrain,pons and cerebellum.Fluorescence in situ hybridization indicated that Atp13a5 m RNA is specifically co-expressed on CD13+ pericytes in cortical brain tissue,but not on Lectin+ endothelial cells,and it is also not expressed in peripheral organs(such as heart,liver,and kidney).(3)Sc RNA-seq data analysis showed that Atp13a5 m RNA is expressed in perinatal pericytes,and it increases from childhood to adulthood and maintains a stable expression level after that.Fluorescence in situ hybridization indicated that Atp13a5 m RNA was almost undetectable in E15 brain slices,and the expression gradually increased from P0,reaching a stable level at P10,and there was no significant difference in expression from 8W and 18 M.(4)In 8-week-old Atp13a5-2A-Cre ER-IRES-td Tomato knockin mice,the td Tomato signal can be detected on the entire coronary brain slice;Histochemical staining showed that the td Tomato signal is distributed along the Lectin+ capillary endothelium,but not the VCAM1+ venous or SMA+ arterial smooth muscle cells.In addition,the td Tomato signal is expressed in CD13+ cerebrovascular pericytes with a coincidence of 97.3±1.4%.Also,td Tomato signal is not expressed on Oligo+ oligodendrocytes,Neu N+ neurons,Iba-1+ microglia and GFAP+ astrocytes.Conclusion This study demonstrated that Atp13a5 is a specific biomarker of brain pericytes.It has potential application as a research tool to study the role of cerebrovascular pericytes and BBB pathological changes in the pathogenesis of central nervous system diseases includingTBI.