Summary Purpose The role of granule cell axon (mossy fiber) sprouting in temporal lobe epileptogenesis is unclear and controversial. Rapamycin suppresses mossy fiber sprouting, but its reported effects on seizure frequency are mixed. The present study used high-dose rapamycin to more completely block mossy fiber sprouting and measure the effect on seizure frequency. Methods Mice were treated with pilocarpine to induce status epilepticus. Beginning 24 h later and continuing for 2 months, vehicle or rapamycin (10 mg/kg/d) was administered. Starting 1 month after status epilepticus, mice were video-monitored 9 h/d every day for 1 month to measure the frequency of spontaneous motor seizures. At the end of seizure monitoring, a subset of mice was prepared for anatomical analysis. Mossy fiber sprouting was measured as the proportion of the granule cell layer and molecular layer that displayed black labeling in Timm-stained sections. Key findings Extensive mossy fiber sprouting developed in mice that experienced status epilepticus and were treated with vehicle. In rapamycin-treated mice, mossy fiber sprouting was blocked almost to the level of naïve controls. Seizure frequency was similar in vehicle- and rapamycin-treated mice. Significance These findings suggest mossy fiber sprouting is not necessary for epileptogenesis in the mouse pilocarpine model. They also reveal that rapamycin does not have anti-seizure or anti-epileptogenic effects in this model.

Objectives/Hypothesis: The goal of this study was to objectively examine vocal fold (VF) motion dynamics after iatrogenic recurrent laryngeal nerve (RLN) injury in a mouse surgical model. Furthermore, we sought to identify a method of inducing injury with a consistent recovery pattern from which we can begin to evaluate spontaneous recovery and test therapeutic interventions.Study Design: Animal model. Methods: The right RLN in C57BL/6J mice was crushed for 30 seconds using an aneurysm clip with 1.3-N closing force. Transoral laryngoscopy enabled visualization of VF movement prior to surgery, immediately post-crush, and at two endpoints: 3 days (n = 5) and 2 weeks (n = 5). VF motion was quantified with our custom motion-analysis software. At each endpoint, RLN samples were collected for transmission electron microscopy for correlation with VF motion dynamics.Results: Our VF tracking software permitted automated quantification of several measures of VF dynamics, such as range and frequency of motion. By 2 weeks post-injury, the frequency of VF movement on the right (injured) side equaled the left, yet range of motion only partially recovered. These objective outcome measures enabled detection of VF dysfunction that persisted at 2 weeks post-crush. Transmission electron microscopy images revealed RLN degeneration 3 days post-crush and partial regeneration at 2 weeks, consistent with functional results obtained with automated VF tracking.Conclusions: Our motion-analysis software provides novel objective, quantitative, and repeatable metrics to detect and describe subtle VF dysfunction in mice that corresponds with underlying RLN degeneration and recovery. Adaptation of our tracking software for use with human patients is underway.

Pilocarpine-treated mice are an increasingly used model of temporal lobe epilepsy. However, outcomes of treatment can be disappointing, because many mice die or fail to develop status epilepticus. To improve animal welfare and outcomes of future experiments we analyzed results of previous pilocarpine treatments to identify factors that correlate with development of status epilepticus and survival. All treatments were performed by one investigator with mice of the FVB background strain. Results from 2413 mice were evaluated for effects of sex, age, body weight, and latency between administration of atropine methyl bromide and pilocarpine. All parameters correlated with effects on outcomes. Best results were obtained from male mice, 6–7 weeks old, and 21–25 g, with pilocarpine administered 18–30 min after atropine methyl bromide. In that group only 23% failed to develop status epilepticus, and 64% developed status epilepticus and survived. Those results are substantially better than that of the total sample in which 31% failed to develop status epilepticus and only 34% developed status epilepticus and survived.