Abstracts

Rationale: Flurothyl (2,2,2-trifluroethyl ether) is a volatile compound that induces electro-clinical seizures, progressing from myoclonic jerks to limb clonus to generalized tonic-clonic (GTC) seizures to death, dependent on the duration of inhalation. Flurothyl is eliminated by exhalation; therefore, simply removing the animal from flurothyl exposure terminates seizure progression. A current method of flurothyl kindling involves 8 trials over 8 consecutive days, each time terminating flurothyl exposure once a clonic seizure develops. This treatment results in sustained reduction of clonic seizure threshold, increases brain BDNF levels and neurogenesis, but does not cause cell death, astrogliosis or spontaneous recurrent seizures. We hypothesized that allowing progression to GTC seizures during kindling would increase the rate of kindling and produce cellular pathology. Here, we present a detailed description of the effects of a 5-day flurothyl kindling on seizure threshold, hippocampal cellular pathology and development of spontaneous epileptiform activity in both male and female mice. Methods: C3HeB/FeJ male and female mice were placed in a glass chamber (2.7L) and 10% flurothyl was infused onto suspended filter paper at a constant rate (50 nl/min) using an automated syringe pump. The latency to first clonic seizure, the number of clonic seizures and the latency to GTC seizure was recorded. After daily exposures over five consecutive days, the mice were left untreated for a 28-36 day period and then retested with flurothyl. Hippocampal cellular pathology was determined using immunohistochemistry techniques using hematoxylin and eosin staining and antibodies for GFAP (astrocytes), zinc transporter 3 (ZnT3, mossy fibers) and Prox-1 (dentate granule cells). A separate cohort of mice were implanted with subdural electrodes one week post-kindling and video-EEG recorded periodically over six months. Results: Overall female mice had higher seizure thresholds and lower mortality rates than male mice. However, both sexes demonstrated a 25-40% reduction in clonic and GTC seizure latency over the kindling period that was sustained at retest. Both sexes also displayed similar pathologies with cellular hypertrophy in CA1 stratum pyramidale, astrogliosis and reactive astrocytes, aberrant ZnT3 staining, increased dentate granule cells and ectopic granule cells in the hilar region. No region examined exhibited cell death. Video-EEG monitoring and analysis is ongoing, however, at one month post-kindling 50% of mice had putative interictal spikes that have increased in frequency at two months post-kindling. Conclusions: Allowing GTC seizures to develop in the flurothyl kindling model reduces kindling time to five days, produces distinct and consistent cellular pathology and may produce spontaneous epileptiform activity. Funding: This work is supported by a CURE award and NIH R01 NS085389 grant