C. Bolden1,2, M. Skibber3, S. D. Olson4, B. S. Gill1, C. S. Cox3 1University Of Texas Health Science Center At Houston,Surgery,Houston, TX, USA 2University Of Texas Health Science Center At Houston,Center For Translational Injury,Houston, TX, USA 3University Of Texas Health Science Center At Houston,Pediatric Surgery,Houston, TX, USA 4University Of Texas Health Science Center At Houston,Program In Regenerative Medicine,Houston, TX, USA
Introduction: Traumatic Brain Injury (TBI) is one of the leading causes of death and disability in the United States. TBI is a chronic disorder resulting from a heterogenous mechanical insult to the brain. This mechanical insult initiates a biochemical cascade of pathophysiological complications such as edema and neuroinflammation which severely impacts patient outcomes. Current experimental in vitro models attempt to mimic the complex pathophysiology of TBI, but often neglect the hemodynamic parameters that recapitulate in vivo TBI leading to a poor Blood Brain Barrier (BBB) phenotype. The development of a suitable in vitro TBI model will mimic pathogenesis by sequentially imposing the mechanical, metabolic, and inflammatory insults in a functional BBB in a high-throughput platform. This platform will allow for the independent modulation of the brain and blood compartments’ hydraulic forces, perfusion rates, and biochemical environments that have been neglected in previous models. Based on these parameters, we engineered a modular, dynamic platform around a Matrigel? coated Transwell? membrane. Our novel TBI platform utilized key BBB cellular components such as brain microvascular endothelial cells, astrocytes and mesenchymal stem cells (pericytes) arranged into either a direct contact coculture or triculture system.
Methods: Cellular layers were exposed to either a combination of Wall Shear Stress (WSS) (average of 4 dyne/cm2), and/or biochemical insult of tumor necrosis factor-alpha (TNF-a, 5 ng/ml) or Cytochalasin-D (cD, 2.5 ug/ml). To mimic conditions of TBI, cellular layers were also exposed to an oxygen-glucose depleted environment followed by reperfusion (OGD/R). Transendothelial electrical resistance (TEER), FITC-Dextran extravasation (1, 7, & 15 kDa), and tight junction immunofluorescence were used as validation markers of a BBB phenotype.
Results: Our results indicate that shear stress resulted in the enhancement of and maintenance of BBB phenotype through enhanced TEER in comparison to static condition in both the coculture and triculture BBB system. The triculture system achieved BBB characteristics including TEER up to 510 ? cm2 and a permeability coefficient (Papp) of 2.5 x 10-7 in 10 kDa FITC-dextran. Treatment with inflammatory insults TNF-a and cD caused a decrease in TEER over time, confirming the BBB phenotype of paracellular tightness. Glucose starvation with oxygen deprivation caused a decrease in TEER of the coculture system at the 2 hr timepoint and eventually recovered 4 hr after this metabolic insult.
Conclusion:Our findings suggest that our in vitro TBI platform is capable of initiating and/or enhancing the BBB phenotype over time to relevant in vivo characteristics. The goal in the development of our novel in vitro TBI platform is the potential refinement of current in vivo experiments, as well as the enhanced translation of preclinical results into clinically meaningful neuroprotective strategies for the future.