Traumatic Brain Injury (TBI), the main contributor to morbidity and mortality worldwide, can disrupt the cell membrane integrity of the vascular endothelial system, endangering blood–brain barrier function and threatening cellular subsistence. Protection of the vascular endothelial system might enhance clinical outcomes after TBI. Poloxamer 188 (P188) has been shown to improve neuronal function after ischemia/reperfusion (I/R) injury as well as after TBI. We aimed to establish an in vitro compression-type TBI model, comparing mild-to-moderate and severe injury, to observe the direct effects of P188 on Mouse Brain Microvascular Endothelial Cells (MBEC). Confluent MBEC were exposed to normoxic or hypoxic conditions for either 5 or 15 h (hours). 1 h compression was added, and P188 was administered during 2 h reoxygenation. A direct effect of P188 on MBEC was tested by assessing cell number/viability, cytotoxicity/membrane damage, metabolic activity, and total nitric oxide production (tNOp). While P188 enhanced cell number/viability, metabolic activity, and tNOp, an increase in cytotoxicity/membrane damage after mild-to-moderate injury was prevented. In severely injured MBEC, P188 improved metabolic activity only. P188, present during reoxygenation, influenced MBEC function directly in simulated I/R and compression-type TBI.

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.

Introduction: Traumatic brain injury (TBI), a major cause of severe disability and death, can lead to disruption of the vascular endothelial system and cell membrane integrity, threatening the survival of multiple cell types. Reducing these could improve the clinical outcome of TBI. Poloxamer 188 (P188) has been shown to protect different cell types against ischemia/reperfusion (IR) injury. Hypothesis: P188 protects mouse brain microvascular endothelial cells (MBECs) against injury in an in-vitro compression-type TBI model. Methods: Confluent MBEC cultures were exposed to normoxic (complete media; 21% O 2 , 5% CO 2 , 74% N 2 ; 37°C) or hypoxic (glucose-, serum-free media; 0.01% O 2 , 5% CO 2 , N 2 balance; 37°C) conditions for 5 hrs, with compression (9.81 N / 0.16 cm 2 ) added during the first hour of normoxia/hypoxia. All MBECs then underwent 2 hrs of reoxygenation in normoxic conditions ± P188 (10 μM, 100 μM, 1 mM). Samples were assayed for cell number, cytotoxicity (lactate dehydrogenase [LDH] release), and metabolic activity. Statistics: Data are mean ± SEM. Kruskal-Wallis one-way analysis of variance on Ranks, Dunn’s Method; p <0.05, * vs normoxia, † vs hypoxia, ** vs normoxia + compression, †† vs hypoxia + compression; n = 11-19 experiments/group. Results: Compared to normoxic cells without compression, cell number and metabolic activity decreased and cytotoxicity increased in cells exposed to hypoxic conditions +/- compression followed by reoxygenation. In hypoxic cells, 1 mM P188 increased cell number and metabolic activity and decreased cytotoxicity, while 100 μM only increased metabolic activity and decreased cytotoxicity and 10 μM only increased metabolic activity. In hypoxic compressed cells, no concentration of P188 improved cell number, however, 10 μM and 100 μM P188 increased metabolic activity, while 1 mM increased metabolic activity and decreased cytotoxicity. There was no difference between normoxic compressed and non-compressed cells in any assay, although all concentrations of P188 tested increased metabolic activity in normoxic compressed cells. Conclusion: P188, present during reoxygenation, provides protection to MBECs exposed to simulated IR injury, as well as compression-type TBI.