Authors :
Presenting Author: Nicolas Simmons, MS – University of Virginia
Jaideep Kapur, MBBS, PhD – University of Virginia
John Williamson, - – University of Virginia
Chia-Yi Kuan, MD, PhD – University of Virginia
Rationale:
Post-stroke seizures (PSS) are common in severe stroke with austere outcomes. Yet, there is insufficient evidence to support the routine use of anti-epileptic drugs in acute stroke and no specific therapy due to poor understanding of its pathological mechanisms. The current preclinical stroke models do not reliably induce PSS, which has further hindered the development of prophylactic PSS treatment. The goal of this study is to optimize a hypoxia-ischemia (HI)-based murine stroke model that has been reported to produce PSS and respond to thrombolytic treatment (Am J Pathol 2006; PMID: 16877357). Subsequently, we aim to test whether PAR1 activation due to thrombin extravasation is an important mechanism of PSS.
Methods:
Four-to-six-month-old C57BL/6 mice were used to compare three versions of HI model. All three versions involve unilateral common carotid artery ligation (UCCAO) followed by 30 min hypoxia (7.5% O2/92.5% N2) but differ in the interval between the two insults and in the method to deliver hypoxia. In the first (original) model, hypoxia was delivered through a nose cone immediately after UCCAO, with the mouse was anesthetized by ~1.5% isoflurane and core temperature kept at 37.5 ± 0.5 °C. The second (separated HI) model, there is a 18 h interval between UCCAO and nose cone-delivered hypoxia with ~1.5% isoflurane and the core temperature controlled at 37.5 ± 0.5 °C. In the third version, a hypoxic chamber was used to deliver hypoxia immediately after UCCAO, while neither anesthesia or temperature control was used. Video-electroencephalogram (EEG) was recorded pre/post-ligation, during hypoxia, and for 24 h post-HI. C-Fos labeling was used to assess neuronal hyper-excitation. The infarct size was measured by Triphenyltetrazolium chloride (TTC) staining at 24 h post-HI.Results:
More than 80 C57BL/6 mice were used to compare the three models. The separated HI model produces the highest seizure incidence (92%) and fewest deaths (7%), while the mortality rate was too high in the original model and the incidence of PSS too low in the chamber model. Post-HI mice presented ictal-like EEG patterns, including spike-wave and post-ictal suppression, and convulsive seizures (CS) such as rapid running, jumping, barrel rolling, and falling. Induction of c-Fos were found in mice with CS, and biochemical analysis indicated a rapid onset of thrombin extravasation in the post-HI cerebral cortex. Severe PSS correlated with the infarct sizes.Conclusions:
The separated HI model induces a high incidence of PSS represented by convulsive behaviors, ictal-like EEG signals and peri-infarct c-Fos induction within 24 h after stroke-onset. Severe PSS in the separate HI model also predicts large infarction as seen in clinical studies. The rapid onset of thrombin extravasation in the optimized HI model suggests its causal role in PSS. We are testing this hypothesis using anti-thrombin and anti-PAR1 intervention in the optimized HI model, positive outcomes of which may have clinical implications.Funding: --