Systemic Pilocarpine Treatment: Mouse Model of Temporal Lobe Epilepsy

Overview

This video presents the procedure for inducing status epilepticus (SE) in mice by systemic administration of pilocarpine to model temporal lobe epilepsy.

Protocol

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

1. SE Induction

1. Purchase 8-week-old male C57BL/6NHsd mice and weigh each mouse. Then, use a marker pen to mark the tails of all mice for their easy identification during SE induction.
2. Calculate the amount of scopolamine methyl bromide (scopolamine; 2 mg/kg), terbutaline hemisulfate (terbutaline; 2 mg/kg), and pilocarpine hydrochloride (pilocarpine; 280 mg/kg) based on the mouse weight and add saline (0.9% NaCl, 10 mL/kg) to make solutions.
   NOTE: For example, if the weight of the mouse is 25 g, the following amounts are applied: Scopolamine and terbutaline: 2 mg/kg * (25 g/1,000 g) = 0.05 mg, Saline: 10 mL/kg * (25 g/1,000 g) = 0.25 mL, Pilocarpine: 280 mg/kg * (25 g/1,000 g) = 7 mg, Saline: 10 mL/kg * (25 g/1,000 g) = 0.25 mL.
3. Load the scopolamine and terbutaline mixture in a 1 mL syringe with a 30 G needle. Inject the solution intraperitoneally into each mouse, and then return the mice to their cages. At 30 min after scopolamine and terbutaline administration, inject pilocarpine solution into each mouse (i.p.; 1 mL syringe; 30 G needle). Immediately after pilocarpine injection, place the mice in an incubator (28–30°C) for observation.
4. Carefully monitor the behavior of the mice until SE is induced. If limbic motor seizures that correspond to stage 3 or higher according to Racine's scale are detected, record the time and monitor the mice to determine whether the seizures occur more frequently. Once continuous motor seizures last more than 2 min, place the mouse in a new cage at room temperature and keep monitoring for 3 h to determine whether their convulsive seizures continue and SE is induced. Euthanize the mice that failed to enter SE at about 2 h after pilocarpine injection.
   NOTE: Racine's scale; stage 1, mouth and facial movement; stage 2, head nodding; stage 3, forelimb clonus; stage 4, rearing with forelimb clonus; stage 5, rearing and falling with forelimb clonus. If the mouse is not transferred to the cage at room temperature immediately after SE induction, the mouse can die due to hyperthermia.
5. Terminate behavioral acute seizures at 3 h after SE onset by injecting diazepam solution (i.p.; 10 mg/kg; 1 mL syringe; 30 G needle). Make the diazepam solution by diluting 5 mg/mL of diazepam in 10% polyoxyl 35 hydrogenated castor oil by adding saline (i.p.; 10 mL/kg; 1 mL syringe; 30 G needle). 
   NOTE: 10% polyoxyl 35 hydrogenated castor oil solution: 1 mL polyoxyl 35 hydrogenated castor oil solution + 9 mL saline. Store the solution at room temperature. For example, if the mouse body weight is 25 g, inject 250 µL of diazepam solution: 50 µL 5 mg/mL diazepam + 200 µL 10% polyoxyl 35 hydrogenated castor oil solution = 250 µL 1 mg/mL diazepam.
6. For sham mice, perform an i.p. injection of the scopolamine methyl bromide and terbutaline hemisulfate mixture (i.p.; both 2 mg/kg, 10 mL/kg; 1 mL syringe; 30 G needle). 30 min later, inject the saline intraperitoneally (i.p.; 10 mL/kg; 1 mL syringe; 30 G needle).
   NOTE: If the mouse body weight is 25 g, inject 250 µL of saline instead of pilocarpine.
7. After diazepam injection (i.p.; 1 mL syringe; 30 G needle), administer 1 mL of 5% dextrose per individual mouse (i.p.; 1 mL syringe; 26 G needle).
   NOTE: 1 mL of 5% dextrose injection provides energy source and hydration that can increase the survival rate.
8. During post-care in the incubator (28–30 °C), wipe off excessive secretions such as saliva, tears, and feces.
9. At day 1 after SE induction, weigh the mice and keep them in the incubator (28–30 °C) for one extra day. At day 2, after SE induction, weigh the mice and return them to their home cage. Provide moist chow to facilitate their recovery.
   NOTE: In this experiment, the mortality rate for C57BL/6NHsd after SE termination was 8.57% on average (3 out of 35 mice tested).
10. Measure daily body weight of the animals until 7 days post-pilocarpine and inject the mice with 5% dextrose (i.p.; 1 mL syringe; 26 G needle) when the body weight has not increased. Stop body weight monitoring when the mice start to regain body weight and consume moist chow without difficulty at 2 days after pilocarpine injection.
    NOTE: If the body weight of a mouse did not increase until 7 days post SE, exclude the mouse from the experiment and euthanize by carbon dioxide. Depending on the background of the mouse, death can occasionally occur; however, in the case of C57BL/6NHsd, less than 1% of mice died in these experiments. With survival of more than 7 days after SE induction, the mice rarely die during the latent period.

Materials

Name	Company	Catalog Number	Comments
C57BL/6	Envigo	C57BL/6NHsd
Scopolamine methyl nitrate	Sigma	S2250	Make 10X stock
Terbutaline hemisulfate salt	Sigma	T2528	Make 10X stock
Pilocarpine hydrochloride	Sigma	P6503
Diazepam	SAMJIN
Castor oil (Kolliphor EL)	Sigma	C5135	Polyoxyl 35 hydrogenated castor oil
Saline	Daihan pharm. Co.
5% Dextrose	Daihan pharm. Co.
1 mL syringe	Sung shim medial Co.  Ltd.
26 guage needle	Sung shim medial Co.  Ltd.		26 G * 13 mm (1/2")
30 guage needle	Sung shim medial Co.  Ltd.		30 G * 13 mm (1/2")