Abstracts

Rationale: Malformations of cortical development (MCD) are common causes of neurological deficits, including seizures and intellectual disability. Hemimegencephaly (HME), an MCD subtype, is characterized by a single enlarged hemisphere in which multiple histological and electrophysiological abnormalities are observed; including phosphorylated ribosomal S6 protein positive (PS6+) dysmorphic neurons, an increase in activated microglia and epilepsy. A subset of HME cases are associated with de novo somatic gain-of-function mutations in AKT3. To generate an accurate model of AKT3 HME in rats, we hypothesized that expression of constitutively active AKT3 in one cortex leads to increased cortical size, abnormal cell morphology, inflammatory cell activation and seizures. Methods: Sprague-Dawley rats (F; 65-75d; 200-224g) were anesthetized with ketamine/xylazine. A cranial window was created over the left primary somatosensory forelimb cortex. Eight µl of AAV2 virus containing constitutively active AKT3 plasmid or control plasmid was injected into the cortex. Incisions were closed and animals allowed to recover. Animals were monitored for behavioral seizures using the Racine scale (RS) for up to 6 weeks post-op. To collect brain specimens, animals were anesthetized and perfused with 4% PFA. Specimens were post-fixed in 4% PFA for 24hrs, immersed in 30% sucrose solution, embedded in OTC and sliced on a cryostat at 30 µm. Sections were probed with anti-PS6 (ser 235/236), -hemmaglutinin (HA), -MAP2, and -IBA-1 antibodies and mounted on slides using Fluoromount-g with DAPI. Sections were imaged on a fluorescent microscope. Results: Behavioral seizures began two weeks post-surgery as continuous spasms in the forelimb contralateral to the injected hemisphere (RS-1). Seizures progressed into frequent tonic-clonic seizures (RS-5) lasting approximately 45 sec (n=8) over the following 2-4 weeks. No control animals experiences seizures (n=6). AKT3 injected hemispheres (n=8) had, on average, a 42% greater slice area than the contralateral hemisphere (p AKT3 injected cortices vs. contralateral or control cortices. PS6 immunoreactivity extended throughout the hemisphere beyond the injection site (marked by HA staining). PS6+ neurons in injected cortices had larger cell bodies than did neurons in contralateral (p AKT3 injected vs. contralateral or control cortices. Conclusions: We demonstrate increased hemisphere size, increased cell body size, inflammatory cell activation and severe behavioral seizures after constitutively active AKT3 injection into adult rat brains. These finding recapitulate what is observed in patients with AKT3-associated HME and provide a novel model of HME that provides the groundwork for further HME studies. Funding: