A Model for Epilepsy of Infectious Etiology using Theiler's Murine Encephalomyelitis Virus

The TMEV model is one of the first experimental platforms to allow for the investigation of the mechanisms of seizure development as a consequence of CNS infection. CNS infections significantly increase the risk for epilepsy. These model helps to identify potential therapeutic targets and compounds for patients at risk.

Experimenters should be trained in working with mice. The virus injection can be practiced very well by injecting dye into dead animals to achieve sufficient precision and routine. Demonstrating the procedure will be Laura Bell, a graduate student from my laboratory.

To begin, remove the cap of the insulin syringe and add polyethylene tubing as a collar around the needle to ensure an adequate injection depth of 2.5 millimeters. Then submerge the syringe in ethanol for 30 minutes and place the syringe under UV light for 30 minutes. Next, put the cap back onto the needle and wrap the syringe in a sterilizing pouch.

Tape the pouch to close it and label with the date of preparation. To perform virus injection, get aliquots of Daniels strain of TMEV from the minus 80 degrees Celsius freezer. Thaw the virus and keep it on ice.

Then load the syringe with the virus suspension. Afterward, clean the bench with disinfectant and work under a fume absorber. Transfer the anesthetized mouse from the anesthesia box under the hood and check the surgical tolerance by performing a toe pinch.

Then clean the head of the animal with an alcohol pad and tilt the mouse's head slightly to the left so that the injection site is pointing upwards. Pull the skin a little backward and insert the needle into the head. Next, inject 20 microliters intracordically to a depth of 2.5 millimeters in the temporal region of the right hemisphere.

Leave the syringe in place for five to 15 seconds and check for any leakage from the injection or if air bubbles are seen, then prepare a new syringe. When carefully pulling the syringe out, rotate it slightly. Then transfer the animal to a new cage, which is placed half-on, half-off a heating pad during recovery from the anesthesia.

And do not leave the animals unattended until they have regained sufficient consciousness to maintain sternal recumbency. First, bring all the cages to the bench and observe the animals for seizures twice daily during the light phase. Score the seizure activity by a modified racing scale and report the number and intensity of seizures.

Next, slide a pen across the cage to make some noise and transfer each animal to another box and back. For animals that have not seized spontaneously or after gentle cage shaking, trigger seizures by more intense handling while carefully turning over the mouse by flipping it at its tail from left to right. Observe each animal for seizure behavior again and repeat the process for subsequent cages.

Behavioral seizures were recorded if they occurred during drug injections or during the subsequent seizure handling monitoring sessions. The observed data were presented as a heat map created by picturing the seizure stages in different colors. By recording EEG, various epileptic events can be identified in the chronic phase, months after infection with TMEV.

Quantification of the hippocampal degeneration and epileptic TMEV-infected mice shows a significant decrease in hippocampal area and a corresponding increase in the ventricular area of TMEV mice as compared to controls. Immunohistological analysis can deliver information on various parameters such as the presence of inflammatory cells. In this case, sections containing the hippocampus were stained with antibodies to label T lymphocytes.

Mock-infected animals show no T-cell infiltration, whereas moderate infiltration was seen in the majority of TMEV-infected mice. Some of the infected mice show severe infiltration of T lymphocytes. Not all of the animals have seizures during each observation period.

Also, the highest incidence of seizures occurs on days five and six after inoculation. We use the TMEV model to test for efficacy of novel anti-seizure medications against infection-induced seizures. Assays investigating comorbid behavioral changes and electrophysiological changes can also be evaluated.

The TMEV model has provided the epilepsy research community with the first CNS infection-induced model of temporal lobe epilepsy, allowing for the investigation of underlying mechanisms of epileptogenesis following infection.