Abstracts

Rationale: Seizures and status epilepticus (SE) occur following traumatic brain injury, lobar hemorrhage, and subarachnoid hemorrhage at the cortex and cause secondary injury. The neurobiological mechanisms underlying acute seizures or SE following cortical insult are unknown.

Methods: We implanted cobalt (Co) in the right supplementary motor cortex (M2) and 16 hours later gave a homocysteine injection (845 mg/kg, i.p.) to C57bl/6J mice to induce SE and monitored it by video and EEG. The responses to diazepam doses: 10, 30, 100, and 300 mg/kg i.p.; 10 mins after the continuous generalized seizure activity began and pretreatment (30 mins prior to homocysteine) with thrombin inhibitor α-NAPAP (0.75 mg/kg i.p.) and PAR-1 blocker SCH79797 (30 (n = 3), 100 (n = 5) and 1000 (n = 5) µg/kg, i.p.) were evaluated. Blood-brain barrier damage and edema were assessed using Evans blue staining, western blotting, and MRI. Thrombin (1 U/mL in 5 (n = 3), 10 (n = 6), 20 μL (n = 3) of ACSF) and albumin (0.4 mM of BSA in 5 µL of ACSF, n = 5) were intracerebrally administered at the supplementary motor cortex area in C57bl/6 animals. In various conditions, we quantified GCaMP7 fluorescence in vitro in cortical slices to measure thrombin-evoked neuronal Ca2+ activity.

Results: SE started with 2 to 4 discrete focal motor seizures, which evolved into continuous generalized convulsive SE. At the end of SE, the animal became comatose, had burst-suppression patterns on EEG, and died within 24 h. Seizures were refractory to diazepam, but 300 mg/kg of diazepam rapidly ended SE at the expense of increased mortality (100%, post-SE median survival time: 5.05 hrs). Evans blue and western blotting revealed that thrombin leakage peaked during early focal intermittent seizures in the perilesional area. Intracerebral thrombin infusion (5 U/µL) caused acute seizures within 25.53 ± 5.6s after infusion, whereas  40% (2/5) of animals had seizures 9.165 ± 3.06 hours post-infusion of albumin. Treatment with a thrombin inhibitor, α-NAPAP improved survival rate (α-NAPAP: 80% vs. control: 45%) and reduced median SE duration (α-NAPAP: 76 mins vs. control: 150.5 mins, p = 0.016, Mann-Whitney test) in cobalt-homocysteine group. T2-weighted MRI images in α-NAPAP treated animals revealed smaller edema and slight or no ventricular effacement than vehicle-treated animals. Treatment with a thrombin receptor (PAR1) SCH79797 improved animals' survival rate (SCH: 100% vs. control: 45%). Thrombin (5 U/mL) evoked neuronal Ca²⁺ activity in vitro, which was partly partially reduced by NMDA-blocker APV (50 µM) and completely eliminated by SCH79797 (1 mM). In the presence of TTX, thrombin caused a tonic elevation of Ca²⁺ activity, which APV abolished.

Conclusions: Thrombin activation and extravasation mediate seizures and cerebral edema following cortical injury. Thrombin alters the functions of neuronal NMDA receptors and TTX-sensitive Na⁺ channels via PAR-1 receptors to cause elevated neuronal activity. These findings suggest that thrombin inhibitors could be a potential therapy in patients with seizures or SE following neocortical injury.

Funding: National Institute of Health (R37 NS119012, R01NS120945 RO1 NS040337), UVA Brain Institute