Induction of Temporal Lobe Epilepsy in Mice with Pilocarpine

Abstract

In the pilocarpine model of temporal lobe epilepsy (TLE) in rodents, systemic injections of pilocarpine induce continuous, prolonged limbic seizures, a condition termed "Status Epilepticus" (SE).With appropriate doses, many inbred strains of mice show behavioral seizures within an hour after pilocarpine is injected. With the behavioral scoring system based on a modification of the original Racine scale, one can monitor the seizures behaviorally, as they develop into more prolonged seizures and SE. SE is typically associated with damage to subsets of hippocampal neurons and other structural changes in the hippocampus and generally subsides on its own. However, more precise control of the duration of SE is commonly achieved by injecting a benzodiazepine into the mouse 1 to 3 h after the onset of SE to suppress the seizures. Several days following pilocarpine-induced SE, electrographic and behavioral seizures begin to occur spontaneously. The goal of this protocol is to reliably generate mice that develop spontaneous recurrent seizures (SRS) and show the typical neuropathological changes in the brain characteristic of severe human mesial temporal lobe epilepsy (mTLE), without high mortality. To reduce mortality, multiple subthreshold injections of pilocarpine are administered, which increases the percentage of mice developing SE without concomitant mortality. Precise control of the duration of SE (1 or 3 h) is achieved by suppressing SE with the benzodiazepine Midazolam (Versed). We have found that this protocol is an efficient means for generating mice that subsequently develop characteristics of human mTLE including high-frequency interictal spike and wave activity and SRS. In addition, we and others have shown that this protocol produces mice that show excitotoxic cell death of subsets of hippocampal GABAergic interneurons, particularly in the dentate gyrus and compensatory sprouting of excitatory projections from dentate granule cells (mossy fiber sprouting). Aspects of this protocol have been described in several of our previous publications.

Pre‐synaptic and post‐synaptic A‐type K+ channels regulate glutamatergic transmission and switching of the network into epileptiform oscillations

“…These findings support the view that A-channel-tuned glutamatergic synaptic transmission plays a role in the generation of seizures. Intraperitoneal injection of NS-5806 also effectively reduced the severity of acute seizures induced by either pilocarpine (Arshad & Naegele, 2020;Turski et al, 1983) or pentylenetetrazole (PTZ, Lüttjohann et al, 2009) (Figure 1c,d), broadening the potential antiseizure scope of the A-channel activator. One of the common seizure models is 'kindling', in which initially innocent, repetitive, focal electrical stimulation of telencephalic structures can eventually lead to an epileptic brain (Morimoto et al, 2004).…”

“…After 30 min, the mice were then injected with 300 mg·kg −1 pilocarpine (50 mg·kg −1 , in 0.9% NaCl) to induce seizures. The stages of behavioural seizures were analysed by the following behavioural characteristics according to the literature (Arshad & Naegele, 2020; Turski et al, 1983): stage 0, normal behaviour; stage 1, frozen or motionless posture; stage 2, facial automatisms in the presence of stiffened tail; stage 3, partial clonus of forelimb or hindlimb; stage 4, rearing; stage 5, tonic–clonic seizure with loss of posture. When the stage‐3 behavioural seizure was reached (typically around ~50 min after injection of pilocarpine), the animal was then anaesthetised with isoflurane to temporarily stop seizures and received either vehicle or 5 mg·kg −1 NS‐5806 (1 mg·ml −1 , in a solvent mixture with a volume ratio of 1: 4: 5 for DMSO, polyethylene glycol and 0.9% NaCl).…”

Hippocampal transplants of fetal GABAergic progenitors regulate adult neurogenesis in mice with temporal lobe epilepsy

“…Two of the established methods to induce TLE in rodents are systemic pilocarpine and intrahippocampal kainic acid injections ( Curia et al, 2008 ; Lévesque and Avoli, 2013 ; Arshad and Naegele, 2020 ). For our experimental group, multiple, subthreshold systemic injections of pilocarpine were given to the mice to induce SE ( Arshad and Naegele, 2020 ). Prior studies established that 3 h of SE is sufficient to induce TLE in all animals.…”

“…Injections of systemic pilocarpine have become a common laboratory method for models of TLE in inbred strains of mice, due to the relative ease of inducing status epilepticus (SE) using repeated subthreshold injections of the drug. This method leads to the reliable development of spontaneous, recurrent seizures in mice ( Ahmed Juvale and Che Has, 2020 ; Arshad and Naegele, 2020 ; Arshad et al, 2021 ). Pilocarpine-induced SE also results in aberrant neurogenesis ( Parent et al, 1997 ; Parent et al, 2006 ; Jessberger and Parent, 2015 ; Danzer, 2018 ; Sasaki-Takahashi et al, 2020 ) and loss of GABAergic interneurons ( Thind et al, 2010 ; Buckmaster et al, 2017 ).…”

Hippocampal Transplants of Fetal GABAergic Progenitors Regulate Adult Neurogenesis in Mice with Temporal Lobe Epilepsy

“…To generate mice with chronic TLE, we injected adult male or female mice at approximately 5-6 weeks of age and weighing 18-22 g with 0.07 mL methyl scopolamine (i.p., 0.5 mg/mL diluted in sterile saline; Sigma). After 30 min, we injected pilocarpine-HCl (i.p., 280 mg/kg), as described previously (Arshad and Naegele, 2020). Mice that did not develop SE were excluded from further study.…”