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Primary blast injury is caused by the direct impact of an overpressurization wave on the body. Due to limitations of current models, we have developed a novel approach to study primary blast-induced traumatic brain injury. Specifically, we employ a bioengineered 3D brain-like human tissue culture system composed of collagen-infused silk protein donut-like hydrogels embedded with human IPSC-derived neurons, human astrocytes, and a human microglial cell line. We have utilized this system within an advanced blast simulator (ABS) to expose the 3D brain cultures to a blast wave that can be precisely controlled. These 3D cultures are enclosed in a 3D-printed surrogate skull-like material containing media which are then placed in a holder apparatus inside the ABS. This allows for exposure to the blast wave alone without any secondary injury occurring. We show that blast induces an increase in lactate dehydrogenase activity and glutamate release from the cultures, indicating cellular injury. Additionally, we observe a significant increase in axonal varicosities after blast. These varicosities can be stained with antibodies recognizing amyloid precursor protein. The presence of amyloid precursor protein deposits may indicate a blast-induced axonal transport deficit. After blast injury, we find a transient release of the known TBI biomarkers, UCHL1 and NF-H at 6 h and a delayed increase in S100B at 24 and 48 h. This in vitro model will enable us to gain a better understanding of clinically relevant pathological changes that occur following primary blast and can also be utilized for discovery and characterization of biomarkers.

Traumatic Brain injury-induced disturbances in mitochondrial fission-and-fusion dynamics have been linked to the onset and propagation of neuroinflammation and neurodegeneration. However, cell-type-specific contributions and crosstalk between neurons, microglia, and astrocytes in mitochondria-driven neurodegeneration after brain injury remain undefined. We developed a human three-dimensional in vitro triculture tissue model of a contusion injury, composed of neurons, microglia, and astrocytes, and examined the contributions of mitochondrial dysregulation to neuroinflammation and progression of injury-induced neurodegeneration. Pharmacological studies presented here suggest that fragmented mitochondria released by microglia are a key contributor to secondary neuronal damage progression after contusion injury, a pathway that requires astrocyte-microglia crosstalk. Controlling mitochondrial dysfunction thus offers an exciting option for the development of therapies for TBI patients.

Total body irradiation (TBI) can result in death associated with hematopoietic insufficiency. Although radiation causes apoptosis of white blood cells, red blood cells (RBC) undergo hemolysis due to hemoglobin denaturation. RBC lysis post-irradiation results in the release of iron into the plasma, producing a secondary toxic event. We investigated radiation-induced iron in the spleens of mice following TBI and the effects of the radiation mitigator captopril. RBC and hematocrit were reduced ~7 days (nadir ~14 days) post-TBI. Prussian blue staining revealed increased splenic Fe3+ and altered expression of iron binding and transport proteins, determined by qPCR, western blotting, and immunohistochemistry. Captopril did not affect iron deposition in the spleen or modulate iron-binding proteins. Caspase-3 was activated after ~7–14 days, indicating apoptosis had occurred. We also identified markers of iron-dependent apoptosis known as ferroptosis. The p21/Waf1 accelerated senescence marker was not upregulated. Macrophage inflammation is an effect of TBI. We investigated the effects of radiation and Fe3+ on the J774A.1 murine macrophage cell line. Radiation induced p21/Waf1 and ferritin, but not caspase-3, after ~24 h. Radiation ± iron upregulated several markers of pro-inflammatory M1 polarization; radiation with iron also upregulated a marker of anti-inflammatory M2 polarization. Our data indicate that following TBI, iron accumulates in the spleen where it regulates iron-binding proteins and triggers apoptosis and possible ferroptosis.