Handling-Induced Seizures in a Mouse Model

Abstract

Source: Batot, G., et al. A Model for Epilepsy of Infectious Etiology using Theiler's Murine Encephalomyelitis Virus. J. Vis. Exp. (2022).

This video demonstrates the induction of handling-induced seizures in a mouse infected with Theiler's Murine Encephalomyelitis Virus (TMEV). The infected mouse, which has hyperexcitable neurons, is exposed to external physical stimuli. This exposure causes stress, leading to abnormal electrical activity in the neurons, which results in seizures.

Protocol

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

1. Virus inoculation

1. Sterilization of the injection syringe
   1. Remove the cap of the insulin syringe. Add polyethylene tubing as a collar around the needle to ensure an adequate injection depth of 2.5 mm. Submerge the syringe in ethanol for 30 min. Place the syringe under UV light for 30 min.
   2. Put the cap back onto the needle. Wrap the syringe in a sterilizing pouch and tape it closed. Label with the date of preparation.
2. Virus injection
   1. Get aliquots of Daniel's strain of TMEV from the −80 °C freezer. Thaw the virus and keep it on ice. Avoid thawing and re-freezing the virus. Load the syringe with the virus suspension (3 x 10⁵ plaque-forming unit [PFU] diluted in 20 µL of phosphate-buffered saline [PBS] or Dulbecco's modified Eagle medium [DMEM] culture medium).
      CAUTION: The virus is infectious to mice but not to humans.
   2. Clean the bench with a disinfectant and work under a fume absorber. Virus inoculation is not sterile but is as clean as possible.
   3. Transfer the mouse into the anesthesia induction chamber and use 2% isoflurane in oxygen to induce anesthesia. Reaching surgical tolerance takes a few minutes; adjust the concentration based on the animal's breathing and depth of anesthesia.
   4. Transfer the anesthetized mouse from the anesthesia box under the hood. Check surgical tolerance, e.g., by toe pinch. The whole procedure is performed in less than 30 s, so the animal does not need to be inhaling isoflurane during the injection. Add eye ointment to prevent drying of the cornea.
   5. Clean the head of the animal with an alcohol pad. Tilt the mouse's head slightly to the left so that the injection site is pointing upwards.
   6. Pull the skin a little backward, insert the needle into the head, and inject 20 µL intracortically to a depth of 2.5 mm in the temporal region of the right hemisphere (posterior and medial to the right eye).
   7. Perform the injection unilaterally in the same hemisphere in all animals. Use the eye and ear as landmarks for the location of the parietal cortex. The placement has been previously verified histologically; see Figure 1. Injection coordinates according to stereotaxic injection in relation to bregma are −2.0 (AP); +3.0 (ML); −1.5 (DV).
   8. Leave the syringe in place for 5-15 s. Write down if there is any leakage from injection or if air bubbles are seen - in that case, prepare a new syringe. When carefully pulling the syringe out, rotate it slightly. Apply eye ointment to prevent dryness while under anesthesia.
   9. OPTIONAL: Code the animal on the tail or the ear depending on local procedures (optional but easy as the animal is unconscious).
   10. Transfer the animal to a new cage, which is placed half-on/half-off a heating pad (35-40 °C) during recovery from the anesthesia. Do not leave the animals unattended until they have regained sufficient consciousness to maintain sternal recumbency.
       NOTE: Animals can be group-housed again after recovery (infected animals are not mixed with mock-infected animals). Infected mice should not be housed in the same room as non-infected mice.
   11. Keep track of the weight of the mice for the first 7 days post-infection as they lose weight after infection and might require additional feeding.

2. Seizure monitoring of handling-induced seizures

1. Perform this procedure by an experimenter blinded to the treatment. Bring all the cages to the bench. Observe the animals for seizures 2x daily during the light phase.
2. Score the seizure activity by a modified Racine scale: 0 = no change in behavior, 1 = mouth and facial movements, 2 = head nodding, 3 = unilateral forelimb clonus, 4 = bilateral forelimb clonus with rearing, 5 = generalized tonic-clonic activity with loss of postural tone, sometimes jumping, 6 = prolonged and excessive jumping and hyperactivity. Report the number and intensity of seizures.
3. Slide a pen across the cage to make some noise.
4. Transfer each animal to another box and back.
5. Gently shake the cage using a back-and-forth motion, taking care not to shake the cage so vigorously that animals will hit the sides or the top of the cage and are in danger of sustaining physical injury.
6. Monitor all animals in the cage for seizures. At any time, if a mouse has a seizure, transfer it back to the home cage and write down the level of the seizure without further seizure stimulation by noise or handling.
7. For animals that have not seized spontaneously or after gentle cage shaking, trigger seizures by more intense handling: Carefully turn over the mouse by flipping it at its tail from left to right.
   NOTE: Animals with seizures are hyperexcitable and can be jumpy.
8. Observe each animal for seizure behavior again. Repeat the process for subsequent cages.

Results

Figure 1: Schematic of TMEV injection procedure. From left to right: For a right parietal cortex injection, the needle is injected slightly lateral of an imaginary line between the eye and opposite ear. The collar for depth control is indicated in yell...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Animal balance	Ohaus (Parsippany, NJ, U.S.A.)	STX2202	0.01 g–2200 g
BD Lo-Dose U-100 Insulin Syringes	BD (Mississauga, ON, Canada)	BD329461	Lo-Dose sterile syringes with permanent BD Micro-Fine IV needle - 1 mL
Daniel's strain of TMEV	kindly provided by Robert Fujinami (University of Utah)	-	3 x 105 plaque-forming units aliquot(s)
Disinfectant, e.g. VennoVet 1 super	Menno Chemie Vertriebsgesellschaft GmbH, Germany	-	Recommended by campus veterinarians with less than or equal to 5% alcohol
Fisherbrand medium sterile Alcohol prep pad C7	Thermo Fisher Scientific (Waltham, MA, U.S.A.)	22-363-750
Fluriso	VETone (Boise, ID, U.S.A)	502017	Isoflurane 250 mL, 2%–5%
Fume absorber	Labconco (Kansas City, MO, U.S.A.)	-	-
General Protection Disposable SMS White Lab Coats	Thermo Fisher Scientific (Waltham, MA, U.S.A.)	17-100-810A
Ice bucket	-	-	any
Mouse cage	-	-	any mouse cage holding at least 5 mice
PrecisionGlide needles	BD (Mississauga, ON, Canada)	329652	BD Slip Tip with PrecisionGlide Needle Insulin Syringes - 26 G x 3/8 - 0.45 mm x 10 mm
Self-Sealing Sterilizing Pouch	Fisher Scientific (Hampton, NY, U.S.A.)	NC9241087	12.6 x 25.5 cm
Small glass flask	-	-	any, volume 25 mL
sterile PBS	Thermo Fisher Scientific (Waltham, MA, U.S.A.)	10010056
Syringe Luer-Lok	BD (Mississauga, ON, Canada)	309628	1 mL syringe only
Ultrasonic Cleaner, Heater/Mechanical Timer	Cole-Parmer (Vernon Hills, IL, U.S.A.)	EW-08895-23	Bath sonicator - 0.5 gal, 115 V