Abstracts

Rationale: Drug discovery efforts to prevent posttraumatic epilepsy (PTE) have been hindered by the lack of an animal model platform that reliably results in high rates of PTE, especially following non-penetrating closed-head injuries; the most common form of traumatic brain injury. This is due, at least in part, to the fact that existing models do not reliably recapitulate the clinical PTE risk factors observed in human subjects, namely repeated injury, and intracranial hemorrhage. The mechanisms of hemorrhage-related hyperexcitability are not well-studied but may be mediated via neuroinflammation. We therefore investigated the effects of promoting hemorrhage and repeated injuries on epileptogenesis and inflammatory responses.


Methods: Because rodents have a fourfold shorter clotting time compared to humans, traumatic hemorrhage was promoted with pre-impact administration of the anticoagulant heparin in a repeated closed-head controlled cortical impact (CCI) model (HTBI group). The TBI group was subjected to CCIs without heparin, and controls were sham treated. Continuous epidural cortical electroencephalography (EEG) was obtained for one month after the last injury (n=3 per group). The activity of the nucleotide-binding oligomerization domain (NOD)-like receptor 3 (NLRP3)-caspase 1 inflammasome in the brain was serially monitored in vivo in transgenic caspase-1 reporter mice (n=3-4 per group).


Results: Compared to TBI, the HTBI group had more visible subdural bleeding post-impact and significantly higher EEG spiking rates. None of the controls had spikes or seizures. Compared to TBI, HTBI had a significantly higher caspase-1 activation for at least 10 days post-injury, and both were significantly higher than controls (p< 0.05). Ex vivo brain slice imaging also revealed significant caspase-1 activation in response to heme iron (a biproduct hemorrhage), and these responses were attenuated by MCC950, an NLRP3-specific inhibitor and Tak242, a Toll-like receptor 4 (TLR4) inhibitor.


Conclusions: Our data suggest that combining traumatic bleeding with repeated injuries potentiates neuroinflammation and hyperexcitability, and thus may help in establishing a reliable animal model platform for drug discovery research. Given the known long latency of PTE, ongoing work and more prolonged EEG recordings by our team aim at confirming the role of hemorrhage, and specifically heme iron, in promoting epileptogenesis and potentially underlying neuroinflammatory mechanisms.


Funding: This research project was funded by Indiana University School of Medicine to MO, and Indiana State Department of Health and Department of Veterans Affairs I01RX004297 to FW.