Abstracts

Rationale: Astrocytes play important roles in normal brain function and neurological diseases. Astogliosis, astrocyte vacuolization, and death are often identified in pathological specimens from animal models and patients with epilepsy and other neurological disorders. However the rapid, dynamic structural changes in astrocyte following seizures are not well documented. Abnormalities in astrocytes have been implicated in promoting epileptogenesis via a diversity of mechanisms. Several studies suggest that the mTOR pathway is activated in astrocytes in temporal lobe epilepsy. The use of mTOR inhibitors, such as rapamycin, may represent a rational and efficacious strategy for preventing astrocyte injury in epilepsy. In this study, we first characterized the acute, dynamic changes in astrocytes following kainate-induced seizures utilizing in vivo multiphoton imaging. Then we tested the hypothesis that treatment with rapamycin initiated before or after status epilepticus has protective effects against seizure induced astrocyte injury. Methods: Adult male transgenic mice expressing green fluorescent protein (GFP) under a glial fibrillary acidic protein (GFAP) promoter were used. Four experimental groups were utilized: control, kainate (KA, 20 mg/kg, i.p.), rapamycin pretreatment (30 mg/kg, i.p, daily, 24, 48 hr before KA) and post treatment (30 mg/kg, i.p, immediately after status epilepticus, daily, up to 1 wk) groups (n=6 mice/group). In vivo multiphoton imaging was used to monitor morphologic changes in GFAP-GFP expressing astrocytes after KA-induced status epilepticus (30 min cumulative duration of electrographic seizures, terminated by isoflurane). A thinned cranial window was created over neocortex and screw electrodes were placed adjacent to the cranial window to record EEG. After pre-seizure images were obtained, time-lapse imaging of the same astrocyte was followed at 0 hr, 4 hr, 24hr, 3d and 1wk after status epilepticus. Post-hoc image analysis was performed to evaluate changes in astrocyte number, vacuolization, and morphological features. Results: Compared with control mice, KA induced seizures caused acute astrocyte injury including vacuolization in ~80% astrocytes followed by astrogliosis. Vacuolization of astrocytes occurred immediately after seizures and persisted for at least 3 days. One week after seizures, astrogliosis developed, characterized by a decrease in astrocyte size (P < 0.05) and loss of their classical busy appearance, with fine individual processes becoming more prominent and extensive and without significant change in astrocyte number. The soma size did not change over the 1 wk period while the ratio of the soma to astrocyte size increased at 1 wk after seizures due to the decrease in astrocyte size (p < 0.05). Rapamycin partially prevented acute KA-induced astrocyte vacuolization, which occurred in only ~5% of astrocytes in rapamycin pre-treated mice and in ~35% of astrocytes in rapamycin post-treated mice immediately after seizure (p < 0.05). Rapamycin pre-treatment and post-treatment prevented the KA-induced astrogliosis and changes in astrocyte size and the soma-to-astrocyte ratio over the 1wk period. Conclusions: KA-induced seizures cause acute, dynamic structural changes in astrocytes, including vacuolization, decreased astrocyte size, and astrogliosis. Both pre- and post-rapamycin treatment attenuate astrocyte vacuolization and astrogliosis, although pre-treatment with rapamycin has a better protective effect against vacuolization of astrocytes than post-treatment. mTOR inhibitors may represent a rational strategy for preventing astrocyte injury in epilepsy. Funding: R01 NS079321, R21NS091047