Establishing a Pentylenetetrazole-Induced Epileptic Seizure Model in Mice

Abstract

Source: Shimada, T., et al. Pentylenetetrazole-Induced Kindling Mouse Model. J. Vis. Exp. (2018).

This video demonstrates the process of inducing epilepsy in mice using pentylenetetrazole (PTZ) injections, a GABA-A receptor antagonist. Repeated intraperitoneal administration of PTZ progressively lowers the seizure threshold by inhibiting chloride ion influx, disrupting inhibitory neurotransmission, and leading to hyperexcitability in neurons that trigger seizures. This process results in kindling and the development of spontaneous seizures, mimicking chronic epilepsy.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of Pentylenetetrazole (PTZ)

1. Dissolve 2 mg/mL PTZ in sterile 0.9% (w/v) NaCl. Prepare PTZ on the day of use.

2. Injection of PTZ

1. Perform all experiments between 9:00 a.m. and 12:00 p.m. (noon).
2. Measure the animal's body weight.
3. Place the animal in an observation chamber for habituation.
4. During the habituation period (3 min), calculate the volume of PTZ solution for the injection based on the body weight of the animal and the previously determined injection dose.
   NOTE: For example, when 35 mg/kg PTZ is used, 0.875 mg of PTZ is required for a mouse weighing 25 g (35 mg/kg x 0.025 kg = 0.875 mg). Therefore, 437.5 μL of a 2 mg/mL PTZ solution should be injected (0.875 mg / 2 mg/mL = 0.4375 mL). The injection dose of PTZ depends on the mouse genotype and strain. For wild-type C57BL/6 mice, a dose of 30-35 mg PTZ/kg body weight is recommended for the first trial. Sensitivity to PTZ depends on the mouse strain used. The injection dose varies according to the purpose of the experiment.
5. Inject PTZ intraperitoneally with a 1-mL syringe attached to a 27-gauge needle into the left or right quadrant of the abdomen of the animal. Avoid injecting at the midline.
   1. Avoid repeated injections at the same position.
6. Observe animal behaviors for 30 min after PTZ administration (Figure 1A) and classify and score the abnormal behavior as shown below. If possible, keep the animals under observation for 24 h, or at least an additional 6-10 h post-injection, especially once the seizure severity score reaches 3 or above.
   1. Note any mild seizures or behavioral changes in the animals beyond the 30-min observation period. This prolonged observation period may address a particularly important and complete effect of a subconvulsive dose of PTZ in the generation of chronic unprovoked seizures in epilepsy.
   2. In addition, measure the seizure duration of each observed seizure as changes in seizure duration relate to the seizure severity. Moreover, the latency to the first seizure after the PTZ injection is another important measure to collect. Monitoring the seizure frequency, duration, and latency is critical for any post-seizure molecular studies.
7. Inject PTZ every other day (Figure 1A). The number of injections depends on the objective of the experiment. Representative examples are as follows.
   1. To generate completely kindled animals, once an animal experiences a seizure with a score of 5 (tonic-clonic seizure), finish the injection within an additional three administrations. In this case, increase the injection dose if the seizure score does not increase for three consecutive administrations. Each animal can receive a different number and dose of PTZ injections.
   2. Fix the number and dose of PTZ injection in every condition to evaluate the vulnerability to PTZ, including in assessments of anti-epilepsy drugs and studies of the phenotype of genetically modified mice. Give all animals the same number of PTZ injections; a total of 8 to 12 injections is recommended. If an animal dies, the seizure score should be denoted as 6.
   3. Determine the injection dose necessary to maintain a high seizure score (4 or 5) for 10 or more injections to study the histopathological changes that occur epileptic seizures. In this case, the total injection number should range from 25 to 30. Decrease the injection dose if the seizure score reaches 5. If the seizure score decreases to 3 or lower, increase the injection dose.

Results

Figure 1: Brief description of the protocol. (A) Schematic illustration of PTZ-mediated kindling. (B) Illustrations of representative animal behaviors for respective seizure scores.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Pentylenetetrazole	Sigma-Aldrich	P6500
Sodium chloride	MANAC	7647-14-5
Mouse	CLEA Japan		C57Bl/6NJcl, postnatal 8 week, male
Syringe (1mL)	Terumo	SS-01T
Needle(27G x 3/4") (0.40 x 19 mm)	Terumo	NN-2719S
Weighing scale	Mettler	PE2000	This item is a discontinued product. Almost equivalent to FX-2000i with FXi-12-JA from A&D company.