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 reduce the threshold of both clonic and GTC seizures, increase the rate of kindling and produce cellular pathology. Here, we present a detailed description of seizures during and after kindling.Methods: C3HeB/FeJ male 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. The mice were immediately removed from the chamber once a GTC seizure was confirmed and allowed to recover. After the kindling period, the mice were left untreated for a 28-36 day period and then retested with flurothyl.Results: Initially, we attempted an 8-day kindling protocol, but stopped at 7 days because the survival rate fell precipitously between day 5 (90%) and day 7 (29%). During this time the latency to clonus decreased ~35% reaching a plateau on day 5 in all mice. Interestingly, the number of clonic seizures before the GTC seizure increased on days 2-4 and then decreased on days 5-7. Concomitantly, the GTC seizure latency initially increased ~20% before decreasing ~20% by day 5 and 50% by day 7. Upon retesting, latency to clonus remained reduced by ~20%, mice only experienced one clonic seizure before GTC and the latency to GTC remained ~30% reduced. The 7-day kindling protocol mortality was high, therefore we determined whether a 5-day protocol would suffice. The 5-day protocol had a 90% survival rate and all seizure parameters were similar to the 7-day protocol. Upon retest, latency to clonus, the number of clonic seizures and GTC remained reduced.Conclusions: Allowing GTC seizures to develop in the flurothyl kindling model shortens kindling time to five days and provides a greater number of metrics that may increase the sensitivity for compound evaluation. Ongoing experiments will determine possible pathological consequences of this kindling protocol.