Intra-Amygdala Kainate Model (mouse)
The intra-amygdala kainate (IAK) microinjection mouse model of mesial temporal lobe epilepsy (MTLE) recapitulates many of the features of human MTLE including injury patterns in the hippocampus. Microinjection of the chemoconvulsant kainic acid (KA) into unilateral amygdala induces a prolong status epilepticus, following which the majority of mice develop spontaneous recurrent seizures with relatively stable average frequency after a short latent period. These features make this model well suited for screening novel therapies to treat pharmacoresistant epilepsy.
Experiments are performed on C57BL/6 male mice from Jackson Laboratories. Four to five-week-old mice are implanted under isoflurane anesthesia (2-5% in O2) with a 22-gauge guide cannula over the dura matter (from bregma: AP = -1.2 mm; ML = +3.3 mm) to deliver KA into the right basolateral amygdala, a Teflon insulated platinum iridium electrode (Plastics One Inc) in the right dorsal CA1 area of the hippocampus (from Bregma: AP = -2.3 mm; ML = 2.5 mm; DV = -1.8 mm) to record EEG, and a reference EEG electrode over the right hemisphere cerebellum. Animals are given buprenorphine for the treatment of pain 1 hour before surgery and a single dose of penicillin (60,000 units s.c.) after the surgery to prevent any infection. Mice are allowed to recover in their individual home cages for ≥ 3 days before the next experiment.
To inject KA, an injection cannula (Plastics One Inc) connected to a syringe pump is inserted into the guide cannula and slowly lowered into the basolateral amygdala at a depth of 3.7 mm below the dura and 0.39 µg of KA dissolved in 0.26 µL 0.9% saline is delivered at a rate of 50 nL/second. The injection cannula is left in place for an additional 2 minutes after KA microinjection to allow diffusion into the brain tissue and minimize movement of KA up the cannula track. The mice are continuously monitored by time synchronized video and EEG and status epilepticus (SE), as defined by continuous EEG spikes >1 Hz and concomitant stage 3-5 seizures, typically begins 10-30 minutes after KA injection. Forty minutes after the SE is confirmed, mice are detached from their tethered EEG cables, given lactated ringers, and returned to their individual home cages.
Three days after SE-induction, mice are placed in individual plexiglass cages and continuously monitored by video-EEG for two-four weeks to identify mice with recurrent spontaneous seizures. Only mice that have experienced at least one spontaneous seizure are enrolled in the screening study. Seizures are defined as high frequency (>5 Hz) and high amplitude (>2x baseline) poly-spike discharges of >10 seconds duration, and seizure termination is defined as a return of amplitude and frequency to baseline values with or without postictal depression. Behavioral seizures are scored as non-convulsive, focal (facial automatisms or unilateral forelimb clonus) or secondarily generalized (bilateral forelimb clonus, rearing, and/or falling). A cross-over experimental design is used to screen the drugs, i.e., after a 7-day baseline observation period, half of the mice receive test drug and the other half receive vehicle for 5 days, and following a 2-day washout period where no drug or vehicle is administered, each group is given the opposite treatment for an additional 5 days. Finally, all mice are monitored for seizures in absence of any treatment for an additional 7 days in order to determine any reversal of drug-induced effects. At the end of the study, the effect of the treatment on total number of seizures and the number of mice achieving seizure freedom is computed. Seizure freedom is defined as zero seizures occurring between the first and last dose of drug. The dose(s) of the test compound are determined based on the effective dose identified in the prior screening tests.