Kainic acid injection protocols, video-EEG monitoring and signal analysis

We used two experimental models to: i) elicit convulsive (intra-peritoneal kainic acid) or non-convulsive (intra-hippocampal kainic acid) status epilepticus (SE); ii) generate a steady yield of spontaneously seizing mice (intra-hippocampal kainic acid).

Mice were anesthetized with chloral hydrate and placed in a stereotaxic frame. A 10μL micro-syringe (stainless steel 33G needle) was filled with a 20 mM solution of kainic acid (KA; Sigma-Aldrich) in 0.9% sterile NaCl, and positioned in the right dorsal hippocampus (AP = −2, ML = −1.5, DV = −2 mm). Mice were injected (over a 1 minute) with 50 nL of KA solution (1 nmol), using a micro-pump operating the micro-syringe. After injection, the cannula was left in place for an additional 2 min to avoid reflux. Sham mice were injected with 50 nL of 0.9% sterile NaCl. Mice were implanted with a bipolar electrode in correspondence of the injection site. Mice were also equipped with three monopolar electrodes placed on the right fronto-parietal cortex, while the reference was placed on the cerebellum (cortical EEG traces not shown). Electrodes were secured on the skull using dental acrylic cement. After surgery the animals were kept under observation for 8h to assess the behavioral changes corresponding to SE ^28–30. Generally, i.h KA elicited non-convulsive SE, with mild asymmetric clonic movements of the forelimbs along with deviations of the head, rotations, and periods of stillness ³¹. In some cases, bilateral clonic seizures associated with rearing were observed ^{30, 31}. Video-EEG recording was performed using a Pinnacle Technology wired system (Lawrence, KS, USA). Signals acquisition (Sirenia®, Pinnacle Technology Inc.) was filtered at 40 Hz, and sampling set at 200–400 Hz. Video was synchronized to the EEG. Mice were monitored daily starting from 1 week post-SE and for the 5 consecutive weeks (at least 3 hours/session) to detect spontaneous recurrent seizure (SRS). The occurrence of SRS activity was characterized using EEG analysis (pClamp, Sirenia) and criteria derived from previously published guidelines ^28–30. Briefly: i) rhythmic sharp waves were selected when their amplitude was > 2 × baseline; ii) spike activity of > 5 sec duration was considered a seizure; iii) two distinct seizures were separated by > 1 sec one from another. Figure 2 provides quantifications of seizure activity and the number of seizures calculated according to the above criteria and normalized by EEG recording time (hour). Scoring in Figure 2H–H1 is proportional to the duration of the longest hippocampal discharges detected for each mouse during 3 consecutive EEG sessions performed between weeks 1 and 5. The latter method was previously described by ²⁹ and it reflects the level of maturation of an epileptic network and epileptogenesis.

A–A1) RT-qPCR transcripts of Cyp were upregulated at SRS. B–B1) Cyp quantification expressed as % immunoreactivity area. n = 4–5 mice/condition (duplicate or triplicate). Data are shown as box plot with individual values (mean ± SD). The non-parametric Mann-Whitney test was used, p<0.05. C–D) Examples of Cyp2e1 and Cyp3a staining in liver samples obtained from control and SRS mice. Note the increased signals at SRS (arrowheads). Scale bars 1 mm and 50 μm. E–E1) Examples of intra-hippocampal EEG traces relative to low (green) and high (blue) seizing mice as detected 1 week after i.h. KA. F) At SRS mice displayed an overall increased seizure frequency and severity. G–G1) Quantification of seizure duration and number of seizures in the cohort used. Data are shown as box plot with individual values (mean ± SD). Parametric t-test was used in D-D1, p<0.05. H–H2) Cyp hepatic protein levels at SRS positively correlates with EEG seizure scores (n = 9 mice).

Mice were anesthetized using ketamine/xylazine and placed in a stereotaxic frame. Intra-hippocampal bipolar electrodes were implanted (AP = −2.5, ML = −3.4, DV = −2.7). See above for surgery details. Behavior was scored according to the Racine scale during SE as induced by i.p. kainic acid injection (KA, 25 mg/kg; stock solution 5 mg/ml KA in PBS, pH = 7 adjusted with NaOH 0.2M). Animals were selected when a generalized tonic-clonic SE associated with a loss of balance was observed (Stage VI Racine). We chose animals experiencing at least 3 tonic-clonic convulsive SE (lasting at least 30–45 seconds over a period of 90 min).

The following groups of animals were generated using intra-hippocampal kainic acid injections: sham control, 24h–72h, 1 week (n= 5–6 / group) and 6 weeks post-KA (n = 9). Two separate series of animals were used to perform histology, western blots and RT-qPCR (PFA fixed vs. snap frozen), leading to a total of n = 20–24 × 2 mice (up to 1 week) and 9 × 2 mice (6 weeks). Intra-peritoneal kainic acid injections were also performed to obtain: sham control, 24h–72h, 1 week (n= 5–6 mice / group). Two series: n= 20–24 × 2 mice. Samples obtained from 24h and 72h groups were pulled together to increase the number of animals. Mice were processed as follows: i) intracardiac perfusion with 4% PFA + overnight post-fixation for brain and liver histology. Livers were transferred to 30% EtOH; ii) intracardiac perfusion with PBS and liver and hippocampi were snap frozen for western blot and RT-qPCR analysis; iii) blood samples were obtained from the left cardiac ventricles prior to PBS/PFA perfusions. Models efficiency: i) i.h KA non-convulsive SE induction rate 100%, mortality 2 out of 52 (~4%) occurring during SE; ii) i.p KA convulsive SE induction rate 75%, mortality 9 out 34 (~25%) during SE or 24 hours after.