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06:58 min
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July 12th, 2021
DOI :
July 12th, 2021
•0:04
Introduction
1:06
Preparation for the Heat-Induced Seizure Assay
2:36
Heat-Induced Seizure Assay
4:36
Results: Screening for Occurrence of Heat-Induced Seizures in Mice with the Different Genetic Backgrounds
6:17
Conclusion
필기록
GEFS+mutations cause febrile seizures in patients. The ability to study hypothermia induced seizures in GEFS+mice allows us to examine the relationship between specific mutations and seizure profile. The digital temperature controller helps set the temperature gradient in the heating box in a reproducible manner.
Thus, the mouse body temperature can be increased at a slow and steady rate. This protocol can be used to identify potential therapies, such as drugs or dietary restrictions, that can be used to reduce or eliminate febrile seizures. To insert the rectal temperature probe into the mouse without injury, we'd recommend briefly anesthetizing the mouse, and lubricating the tip of the rectal probe.
Start by switching on the mouse heat chamber with the power on button and then press the heat on button. Using the keypad on the digital temperature controller, set the temperature of the heat chamber to 50 degrees Celsius. Line the floor of the heat chamber with cob bedding.
Mount a video recording camera in front of the heat chamber. Line a 140 millimeter diameter Petri dish with thick layers of tissue paper and place it on ice to serve as a cooling pad. For the screening assay, ensure the body weight of the mouse is 15 grams or more.
Before starting the procedure, confirm that the mouse is completely anesthetized with a toe pinch. Coat the metal tip of the rectal temperature probe with a lubricant and gently insert it into the mouse. Secure the rectal probe to the tail of the mouse with tape.
Place the mouse in a new recovery cage lined with cob bedding. Start a timer and observe the mouse for five minutes while monitoring the core body temperature, until the mouse completely recovers from anesthesia and the temperature stabilizes at 35 to 36 degrees Celsius. At the end of five minutes, note the body temperature of the mouse as the initial body temperature at zero minute.
Transfer the mouse quickly to the floor of the preheated mouse heat chamber to start the experiment trial. After the mouse is placed into the preheated mouse chamber, start the camera and the stopwatch, increase the temperature of the heat chamber at regular intervals, such that the body temperature of the mouse increases at a rate of 25, 2.5 degrees Celsius per minute. Begin recording the body temperature of the mouse at one minute intervals for the duration of the experiment.
At 9.5 minutes, set the temperature of the heat chamber to 55 degrees Celsius to stabilize the temperature of the heat chamber at 55 degrees Celsius by the 10th minute. Similarly achieve a stable temperature of the heat chamber to 60 degrees Celsius by the 20th minute. Each seizure screening trial lasts for 30 minutes.
If the mouse experiences a seizure, record the body temperature of the mouse during the seizure, as a seizure threshold temperature. Take note of the seizure behavior characteristics displayed by the mouse, then quickly pick up the mouse from the chamber and place it on the cooling pad. If a mouse does not experience heat induced seizures within the 30 minute observation period, or the body temperature of the mouse reaches 44 degrees Celsius, place the mouse on the cooling pad.
When the body temperature of the mouse comes down to 36 to 37 degrees Celsius, transfer the mouse to a recovery cage. To remove the rectal probe from the mouse, carefully cut the tape between the mouse tail and the rectal probe wire. Wipe and clean the metal tip of the rectal probe with 70%ethanol.
Observe the mouse until it recovers before returning it to the home cage. Keep monitoring the status of the mouse. Mark the end of the experiment trial.
Reset the temperature of the mouse heat chamber to 50 degrees Celsius and allow it to equilibrate before the next assay. In the heat seizure assay, the mean body temperature of the mice was recorded every minute using the heating protocol, and the rate of the body temperature change over time was evaluated. There was no difference in rate of body temperature change between heterozygous mutant and wild type mice, in the respective genetic backgrounds.
Experimental and control groups of mice displayed different behavior when exposed to a periodic increase in body temperature. All heterozygous mutant mice from 129X1 or B6NJ genetic backgrounds exhibited heat induced seizures. None of the wild type mice in the 129X1 enriched background experienced seizures, while only a third of the mice from the seizure sensitive B6NJ background showed seizures.
A mean seizure threshold temperature of 129X1 mutant mice was not significantly different from the mean seizure threshold temperature seen in B6NJ mice. One third of B6NJ wild type mice that did display heat induced seizures, had a seizure threshold temperature that was significantly higher than B6NJ heterozygous mutant mice. The severity of the seizures was measured using the modified Racine scale.
The maximum Racine score of heterozygous mutant mice in 129X1 enriched background was not significantly different from heterozygous mutant mice in B6NJ genetic background. While setting the heating protocol, please ensure that the mouse body temperature does not increase at a rate faster than 25 degrees Celsius per minute, or it can be harmful to the health of the animal. It would be very cool to combine the heating protocol with simultaneous EEG recordings, that would allow us to correlate different patterns of brain activity with different types of seizure behavior.
The goal of the method is to screen for hyperthermia or heat-induced seizures in mouse models. The protocol describes the use of a custom-built chamber with continuous monitoring of the body temperature to determine whether elevated body temperature leads to seizures.
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