Abstract Text: Traumatic brain injury (TBI) is a major health issue for the U.S. military. During the recent conflicts in Iraq and Afghanistan it is estimated that over 444,000 service members suffered a TBI; the vast majority of them (approximately 80%) were mild TBIs. Military personnel are exposed to multiple blasts both during training in garrison, and while deployed in theatre. While moderate and severe brain injuries are easier to diagnose, it is often difficult to determine if blast exposure has resulted in damage to the brain. There also could be a cumulative detrimental effect of multiple sub concussive blasts. Biomarkers released into the serum from blast-induced traumatic brain injury (bTBI) can be used to diagnose and assess injury severity, to monitor recovery, predict outcomes, and provide insight into the pathological changes. However, there are currently no serum biomarkers to diagnose bTBI. We have developed a bioengineered 3D brain-like human tissue culture system that provides a completely novel approach to the discovery of serum biomarkers for bTBI. The 3D brain-like tissue is composed of hybrid collagen-infused silk donut hydrogels embedded with human IPSC derived neurons, human astrocytes, and a human microglial cell line. The 3D brain-like tissues form mature neural networks by 5 weeks with axons extending across the central region of the donut. The unique advantages of our 3D TBI model includes complete control over cellular composition and microenvironment, with the ability to profile changes in released proteins and microRNAs over time and to perform multi-parameter imaging studies on blast-affected tissue. In addition, functional tissues are generated in much shorter time frames than animal studies and offer the reproducibility and scalability critical for larger scale target and drug screens. We have utilized the Advanced Blast Simulator (ABS) to produce a blast wave that can be precisely controlled. The 3D tissue cultures are enclosed in a custom designed 3D printed sterile surrogate skull-like material containing media. The skulls are placed in a holder apparatus, which allows for exposure to the blast wave without any other damage occurring. The enclosed cells exhibit functional responses to injury and allow us to examine cellular damage and biomarker release at multiple time points post-injury. High-speed video confirmed that this apparatus is stable during the progression of the blast wave. We have shown that blast induces an increase in lactate dehydrogenase activity and glutamate release indicating cellular injury and membrane disruption. Additionally, we observe a significant increase in the amount of axonal varicosities on Tuj1 stained axons after blast. These varicosities often contain strong staining of the amyloid precursor protein that may indicate a blast induced axonal transport deficit. Blast injury leads to an early transient release of known TBI biomarkers UCHL1 and NF-H at 6 hours and a delayed increase in S100B at 24 and 48 hours providing proof of principle, and suggesting that this system has validity to discover other biomarkers of injury. We have also isolated miRNAs and evaluated over 7,000 proteins from the media at different time points after injury and found a complex pattern of miRNAs and proteins released with temporal specificity. These studies will utilize a novel, clinically relevant human culture model to assess and identify protein and miRNA biomarkers released following bTBI that will guide subsequent analysis of human serum samples. The discovery and characterization of biomarkers following mild bTBI will allow for the objective detection of such injuries in military personnel, as well as providing useful information on long-term prognosis and potential outcomes of therapeutic interventions.
Keywords: Biomarkers, Blast, Primary blast injury, 3D cultures, Bioengineering