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.

Keywords: Epileptogenesis; Hippocampus; Neurodegeneration; Pilocarpine; Seizures.

Video 1.. Video-EEG of a mouse exhibiting Modified Racine Stage 3 seizures.

In this stage, pilocarpine-treated mice develop generalized tonic-clonic seizures associated with a stiff tail, involuntary movements of the forelimbs, and orofacial movements of mouth and whiskers (Video 1). The behavioral seizure begins at 14 s in the video.

Video 2.. Video-EEG of a mouse exhibiting Modified Racine Scale Stage 5 seizures.

The seizure begins at 22 s in this video and shows the characteristic behaviors of mice in Stage 5 seizures. These include rearing up onto hind legs and falling, as well as involuntary, bilateral, tonic-clonic movements of the limbs. In this video, the mouse briefly loses postural control and then regains it, which is also typical.

Video 3.. Video-EEG of a mouse exhibiting SE.

In this stage called SE, mice have experienced 3 or more stage 3-5 seizures and develop continuous seizures, or SE. In this stage, the mice become immobile and exhibit prominent head-tics.

Figure 1.. Flow chart diagram of experimental protocol for inducing status epilepticus

Figure 2.. Examples of spontaneous epileptiform activity in mice following pilocarpine-induced SE.

The EEGs were acquired using Sirenia Acquisition and Sirenia Seizure Detection software.

Figure 3.. The graphical user interface in the Sirenia seizure detection software.

The setting for detection of seizures is based on the EEG line length, with a threshold setting of 6000.000 len/sec. The mouse is shown in a video camera view that has been superimposed onto the screenshot of the graphical user interface.