In Vitro Traumatic Brain Injury Model of Mouse Brain Microvascular Endothelial Cells
Authors: Lotze Felicia P. , Salzman Michele M. , Pille Johannes A. , Balzer Claudius , Hees Josephine E. , Cleveland William J. , Riess Matthias L. 
Published: June 2021

INTRODUCTIONTraumatic brain injury (TBI) continues to be a leading cause of death and disability, not only in the US but worldwide. Approaches to mimic the injury mechanisms underlying TBI (e.g., stretch, shear and compression) include various models; in‐vivo models better simulate clinical effects, while in‐vitro models focus on the biomechanics of TBI. The effect of the injury in‐vitro can be examined without influence of systemic confounders, and the experiments are well‐controlled, reproducible and isolated from environmental impacts. Therefore, our objective was to establish an in‐vitro TBI model using mouse brain microvascular endothelial cells (MBEC), focusing on compression injury, to further understand underlying pathomechanisms and to test potential treatments. We compared 1) normoxic cells ± compression vs hypoxic cells ± compression and 2) varied hypoxia exposure times.METHODSMBEC cultures were grown to confluency and placed into either normoxic (complete media; 5% CO2, 95% air [21% O2]; 37°C) or hypoxic (glucose‐, serum‐free media; 0.01% O2, 5% CO2, N2 balance; 37°C) conditions. Hypoxia times of 5h, 7.5h, 10h, 12.5h vs 15h were compared. In addition, during the first hour of normoxia/hypoxia, compression (1kg/0.16cm²) was added. After 2h of reperfusion in normoxic conditions, following normoxia/hypoxia, samples were assayed for cell number, cytotoxicity (lactate dehydrogenase [LDH] release), and metabolic activity. Statistics: Data expressed as mean ± SEM. Kruskal‐Wallis one‐way analysis of variance (ANOVA) on Ranks, Dunn's Method; p <0.05, * vs normoxia (each timepoint), Ϯ vs hypoxia (prior timepoint).RESULTSCompared to normoxic conditions, a significant decrease in cell number and metabolic activity, as well as an increase in LDH release, was seen after exposing cells to hypoxia at all durations; except for 12.5h hypoxia, where there was no significant difference in metabolic activity. Compression added significant damage to hypoxia‐exposed MBEC by further decreasing cell number and metabolic activity. However, the effect of compression during hypoxia decreased with increasing hypoxia time. Furthermore, there were significant differences in cell number among the different hypoxia times. Surprisingly, there was only a significant difference for LDH release at 7.5h vs 10h and 12.5h vs 15h of hypoxia and for metabolic activity at 5h vs 7.5h and 12.5h vs 15h of hypoxia.CONCLUSIONOur data show that 5h hypoxia with compression is sufficient to cause significant injury in MBEC cultures. Extending hypoxia time leads to an even greater increase in damage; however, with increasing hypoxia time, the effect of compression is reduced. Therefore, 5h hypoxia with 1h compression is a suitable in‐vitro MBEC TBI model for testing potential treatments.Support or Funding InformationThis work was supported by institutional funds, NIH grant (5R01 HL123227), and a Merit Review Award (I01 BX003482) from the U.S. Department of Veterans Affairs Biomedical Laboratory R&D Service.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.