Targeting Trauma‐Induced Blood Brain Barrier Disruption: an Innovative Approach for Treating Concussion

1.7 million concussions occur each year in the United States. Repetitive concussions have been linked to chronic neurodegenerative changes in athletes and soldiers several years after injury. How these concussions cause lasting brain damage is not well understood. Emerging evidence has revealed that mild traumatic brain injury can cause blood brain barrier (BBB) disruption. Protein kinase C (PKC) activity is increased, which disrupts the tight junction proteins that help form the rigid barrier between endothelial cells. This acute disruption leaves the brain susceptible to peripheral inflammatory cytokines and toxic byproducts of red blood cell breakdown. Over time these toxic byproducts can contribute to a chronic neuroinflammatory environment. We propose that targeting PKC activity post‐injury in our clinically relevant rodent traumatic brain injury model will preserve tight junction protein integrity, and prevent BBB disruption. A compression wave model was used to produce concussive injury in young‐adult male Sprague Dawley rats. 40 rats were split into 4 groups: control with saline injection, bryostatin injection (2.5 μg/kg i.p.) only, single blast only (50PSI), and single blast with bryostatin injection (2.5 μg/kg i.p.). In a subset of rats, blood was drawn from the tail vein at 30 min, 6h, and 24h post traumatic brain injury with terminal blood collected at 72h to be used for rtPCR. Half the rats were sacrificed at 72 hours and prefrontal cortex tissue prepared for western blot analysis and immunohistochemistry. The other half of the rats were injected with the tracers Rhodamine 123 and Texas Red to exquisitely assess the integrity of the BBB at 6h and 24h. The brains were removed and analyzed by immunohistochemistry. A 2.546 fold increase in Texas Red permeation in the contralateral cortex was seen 6 hours following concussive injury (p<0.05) compared to control indicating significant BBB disruption. The increase was associated with an upregulation of PKCα (q=5.043, p<0.01) and PKCδ (q=8.049, p<0.001). The disrupted BBB was accompanied by increased iron uptake (19%) into surrounding neurons that was released from damaged red blood cells. Bryostatin had a profound neuroprotective effect by reducing the harmful PKCα (q=4.559, p<0.01) and increasing the beneficial PKCɛ (q=7.509, p<0.001). The protective PKC regulation resulted in increased integrity at the BBB with significant reduction of Texas Red permeation and concurrent upregulation of tight junction proteins occludin (q=14, p<0.001) and ZO‐1 (q=11.45, p<0.001). Importantly, elevated plus maze testing at 48h revealed a significant increase in exploratory behavior into the open arms following traumatic brain injury compared to controls (p<0.01). Ongoing investigation is looking into the protective role of bryostatin on behavior. The neuroprotection at the BBB may be mediated by targeting upstream micro RNAs. BBB specific micro RNAs are being examined in serum samples from rats exposed to injury and human serum samples from traumatic brain injury patients. It is clear that vascular disruption plays an important role in concussion and warrants continued investigation. We have shown the significant protective benefit of modulating PKC activity at the BBB post‐injury. This protective benefit is likely to have a long‐term beneficial effect on behavior and potentially reduce chronic neurodegeneration. Support or Funding Information American Foundation of Pharmaceutical Education American Medical Association Foundation American Association of Pharmaceutical Scientists West Virginia University Foundation Neurosurgery Research and Education FoundationElevated plus maze showing increased exploratory behavior following compression wave injury 48 hours post‐injury.Texas Red staining showing increased BBB disruption 6 hours post‐injury.Astrocyte activation 72 hours post‐injury.