triggering and monitoring heat-induced seizures in a transgenic mouse model

Triggering and Monitoring Heat-Induced Seizures in a Transgenic Mouse Model

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 5 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 5 minutes, note the body temperature of the mouse as the initial body temperature at 0 minute. Transfer the mouse quickly to the floor of the preheated mouse heat chamber to start the experiment&#39;s 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 0.25 to 0.5 degrees Celsius per minute. Begin recording the body temperature of the mouse at 1-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. 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.

triggering-monitoring-heat-induced-seizures-transgenic-mouse

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JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Epilepsy and Seizure Disorders

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation for the heat-induced seizure assay
<ol>
	<li>Switch on the Power On button on the heat chamber, followed by the Heat On button.</li>
	<li>Set the temperature of the heat chamber at 50 &#176;C using the keypad on the digital temperature controller.</li>
	<li>Wait for a minimum of 1 h to preheat the chamber to 50 &#176;C before introducing the first mouse into the chamber. Preheating ensures uniform heating inside the chamber.</li>
	<li>Line the floor of the mouse heat chamber with cob bedding.</li>
	<li>Mount a video recording camera in front of the heat chamber to record each heat-induced seizure assay trial.</li>
	<li>Line a 140 mm diameter Petri dish with thick layers of tissue paper and place it on ice to serve as a cooling pad. 
	&#8203;NOTE: At the end of the assay, individual mice will be transferred to the prechilled cooling pad to help bring down their elevated body temperature.</li>
</ol>
2. Preparing the mouse for heat-induced seizure assay
<ol>
	<li>Select 10 adult mice (P30-P40), 5 that carry the epilepsy-causing mutation, and 5 of the wild-type litter mates for heat-induced seizure screening assay. 
	NOTE: Wild-type mice, not harboring any epilepsy-causing mutation, do not exhibit heat-induced seizures at temperatures below 44 &#176;C and serve as the control group.</li>
	<li>Weigh each mouse to be used for the screening assay and record its body weight. Only mice weighing 15 g or more should be used for the assay.</li>
	<li>Screen one mouse at a time in the mouse heat chamber.</li>
	<li>Briefly anesthetize the mouse for 10-15 s using a few drops of isoflurane at the bottom of a bell jar.</li>
	<li>Take the animal out of the bell jar and place it on a paper towel.</li>
	<li>Ensure that the mouse is completely anesthetized by checking that the mouse is unresponsive to a noxious toe pinch.</li>
	<li>Coat the metal tip of the rectal temperature probe with a lubricant (such as petroleum jelly) and gently insert it into the mouse at a depth of less than or equal to 2 cm.</li>
	<li>Secure the rectal probe to the mouse&#39;s tail with tape so the probe does not come out during the assay. 
	NOTE: Alternatively, place the animal in a mouse restrainer cone and insert the rectal temperature probe. Secure it by taping it to the tail.</li>
	<li>Ensure the rectal probe is connected to a multimeter that displays the internal body temperature of the mouse.</li>
	<li>Place the animal in a fresh cage lined with cob bedding, i.e., the recovery cage.</li>
	<li>Start a timer and wait for 5 min. Observe the mouse until it has completely recovered from anesthesia and the mouse is active and grooming.
	<ol>
		<li>Simultaneously, monitor the core body temperature of the mouse till it stabilizes at 35-36 &#176;C.</li>
	</ol>
	</li>
	<li>At the end of 5 min, note the body temperature of the mouse. This is the initial body temperature at time &#34;0&#34; min. 
	NOTE: If the core body temperature of the mouse is below 35 &#176;C, wait for additional time for the animal to recover from anesthesia-induced hypothermia.</li>
	<li>Quickly transfer the individual mouse into the preheated mouse chamber. This marks the START of the experiment trial. Only one mouse is screened at a given time.</li>
</ol>
3. Heat-induced seizure assay
<ol>
	<li>After gently placing the mouse on the floor of the pre-heated mouse heat chamber, close the plexiglass door and start the camera for video recording the experiment.</li>
	<li>Start the stopwatch. Record the body temperature of the mouse from the rectal thermometer at 1 min intervals for the duration of the experiment.</li>
	<li>At regular intervals, increase the temperature of the mouse heat chamber such that the body temperature of the mouse increases at a rate of 0.25-0.5 &#176;C /min. 
	NOTE: Rapid increases in body temperature can lead to heat stroke or death and should be avoided.</li>
	<li>Following this protocol, increase the temperature of the mouse heat chamber by 5 &#176;C every 10 min as shown in Figure 1A.</li>
	<li>At 9.5 min, set the temperature of the heat chamber to 55 &#176;C, to stabilize the temperature of the heat chamber to 55 &#176;C by the 10th min as shown on the digital temperature display.</li>
	<li>Similarly, increase the temperature to 60 &#176;C at 19.5 min to stabilize the temperature of the heat chamber to 60 &#176;C by the 20th min. Each seizure screening trial lasts for 30 min.</li>
	<li>If the mouse has a seizure (vocalizes, shows head nodding, forelimb clonus, hindlimb extension, falls on its side, or experiences generalized tonic/clonic convulsions), record the following information.
	<ol>
		<li>Record the body temperature of the mouse during the seizure (seizure threshold temperature) from the rectal temperature thermometer.</li>
		<li>Record the seizure behavior characteristics such as head nodding, forelimb clonus, hind limb extension, falling on the side, and/or generalized tonic/clonic seizures (GTCS) displayed by the mouse.</li>
	</ol>
	</li>
	<li>Quickly but gently pick up the mouse from the chamber and place it on the cooling pad prepared in step 1.6. 
	NOTE: If a mouse is experiencing Racine scale 5 seizures and exhibiting uncontrolled jumping, it can be difficult to pick up the animal from the heat chamber and transferred to the cooling pad outside. However, a typical heat-induced seizure lasts between 30 s to 60 s.Thus, the mouse should be taken out of the heat-chamber and put on the cooling pad within 60 s of start of heat-induced seizure episode.</li>
	<li>Wait for the mouse body temperature to come down to 36-37 &#176;C, before transferring it to a recovery cage. Only one mouse is placed in a recovery cage at a time. 
	NOTE: Do not mix mice that are yet to be used for heat-induced screening with the mouse that has already experienced the heat-induced seizure experiment trial.</li>
	<li>Gently and carefully, cut the tape between the mouse tail and rectal probe wire with a pair of scissors to remove the rectal probe from the mouse.</li>
	<li>Wipe clean the metal tip of the rectal probe with 70% alcohol and soft tissue wipes to keep it ready for the next trial.</li>
	<li>Continue to observe the mouse in the recovery cage until it resumes normal activity (walking, grooming, etc.), before returning the mouse to its home cage. This marks the END of the experiment trial for this mouse.</li>
	<li>Record the animal status after the assay-alive and recovered from the test session or dead. High intensity seizures involving uncontrolled jumping and generalized tonic/clonic seizures can sometimes result in the death of the mouse.</li>
	<li>If a mouse does not experience heat-induced seizures within the 30 min observation period or the body temperature of the mouse reaches 44 &#176;C, remove the mouse from the heat chamber and place on the cooling pad till the body temperature of the mouse returns to 36-37 &#176;C.</li>
	<li>Reset the temperature of the mouse heat chamber to 50 &#176;C and allow it to equilibrate till the display temperature on the digital temperature controller shows 50 &#176;C.</li>
	<li>Change cob bedding between individual mouse trials.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Axial fan</td>
			<td>Farnam</td>
			<td>AF20-200-120-xx10-3.1</td>
			<td>Farnam custom products -Axial Fan Heater with Fan</td>
		</tr>
		<tr>
			<td>Digital temperature controller</td>
			<td>Inkbird</td>
			<td>ITC-100RH</td>
			<td>Inkbird digital PID temperature controller ITC-100RH with K thermocouple</td>
		</tr>
		<tr>
			<td>Mouse rectal temperature probe</td>
			<td>ThermoWorks, Braintree Scientific, Inc</td>
			<td>RET-3</td>
			<td>Mouse rectal temperature probe with thermometer</td>
		</tr>
	</tbody>
</table>

Source: Das, A., et al. <a href="https://app.jove.com/v/62846">A Behavioral Screen for Heat-Induced Seizures in Mouse Models of Epilepsy.</a> J. Vis. Exp. (2021)
This video demonstrates the procedure to induce heat-induced seizures in a transgenic mouse with epilepsy-related gene mutations by gradually increasing body temperature in a controlled chamber. This protocol is used to study seizure mechanisms and potential therapies for epilepsy.

<img alt="Figure 1" src="/files/ftp_upload/23110/23110_Figure1.jpg" /> 
Figure 1: Mutant mice exhibit heat-induced seizures. (A) The heating protocol for behavioral screening of heat-induced seizures in mice. (B-C) Mean body temperature of mice across time in wild-type (Scn1a+/+ - black triangles) and heterozygous mutant (Scn1aKT/+ - orange circles) mice in two genetic backgrounds 129X1 and B6N, respectively. (<stro...

Source: Das, A., et al. A Behavioral Screen for Heat-Induced Seizures in Mouse Models of Epilepsy. J. Vis. Exp. (2021)
This video demonstrates the ...

Take an anesthetized transgenic adult mouse carrying a gene mutation that leads to dysfunctional sodium channels in the inhibitory interneurons.
Secure a lubricated rectal probe to monitor its body temperature.
Place the mouse in a recovery cage until its body temperature stabilizes.
Next, transfer the animal to a preheated heat chamber and begin video recording.
Increase the chamber&#39;s temperature at regular intervals while monitoring the gradual rise in the mouse&#39;s body temperature.
As the body temperature rises, the dysfunction in sodium channels worsens, reducing inhibitory neurotransmitter release.
This reduced release disrupts the balance between neuronal excitation and inhibition, resulting in neuronal hyperexcitability and seizures.
Record the body temperature during the seizure and note its behavioral characteristics.
Now, place the mouse on a cooling pad until its body temperature normalizes.
Finally, remove the rectal probe and allow the mouse to recover.

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视频: Triggering and Monitoring Heat-Induced Seizures in a Transgenic Mouse Model - 实验

Simultaneous Video-EEG-ECG Monitoring to Identify Neurocardiac Dysfunction in Mouse Models of Epilepsy

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can potentially increase risk of sudden unexpected death in epilepsy (SUDEP). Electroencephalography (EEG) and electrocardiography (ECG) are widely used clinical diagnostic tools to monitor for abnormal brain and cardiac rhythms in patients. Here, a technique to simultaneously record video, EEG, and ECG in mice to measure behavior, brain, and cardiac activities, respectively, is described. The technique described herein utilizes a tethered (i.e., wired) recording configuration in which the implanted electrode on the head of the mouse is hard-wired to the recording equipment. Compared to wireless telemetry recording systems, the tethered arrangement possesses several technical advantages such as a greater possible number of channels for recording EEG or other biopotentials; lower electrode costs; and greater frequency bandwidth (i.e., sampling rate) of recordings. The basics of this technique can also be easily modified to accommodate recording other biosignals, such as electromyography (EMG) or plethysmography for assessment of muscle and respiratory activity, respectively. In addition to describing how to perform the EEG-ECG recordings, we also detail methods to quantify the resulting data for seizures, EEG spectral power, cardiac function, and heart rate variability, which we demonstrate in an example experiment using a mouse with epilepsy due to Kcna1 gene deletion. Video-EEG-ECG monitoring in mouse models of epilepsy or other neurological disease provides a powerful tool to identify dysfunction at the level of the brain, heart, or brain-heart interactions.

Here, we present a protocol to record brain and heart bio signals in mice using simultaneous video, electroencephalography (EEG), and electrocardiography (ECG). We also describe methods to analyze the resulting EEG-ECG recordings for seizures, EEG spectral power, cardiac function, and heart rate variability.

同步视频脑电图-心电图监测对小鼠癫痫模型 Neurocardiac 功能障碍的鉴别

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can ...

科研

JoVE Journal

遗传学

Inducing Post-Traumatic Epilepsy in a Mouse Model of Repetitive Diffuse Traumatic Brain Injury

Traumatic brain injury (TBI) is a leading cause of acquired epilepsy. TBI can result in a focal or diffuse brain injury. Focal injury is a result of direct mechanical forces, sometimes penetrating through the cranium, creating a direct lesion in the brain tissue. These are visible during brain imaging as areas with contusion, laceration, and hemorrhage. Focal lesions induce neuronal death and glial scar formation and are present in 20%&minus;25% of all people who incur a TBI. However, in the majority of TBI cases, injury is caused by acceleration-deceleration forces and subsequent tissue shearing, resulting in nonfocal, diffuse damage. A subpopulation of TBI patients continues to develop post-traumatic epilepsy (PTE) after a latency period of months or years. Currently, it is impossible to predict which patients will develop PTE, and seizures in PTE patients are challenging to control, necessitating further research. Until recently, the field was limited to only two animal/rodent models with validated spontaneous post-traumatic seizures, both presenting with large focal lesions with massive tissue loss in the cortex and sometimes subcortical structures. In contrast to these approaches, it was determined that diffuse TBI induced using a modified weight drop model is sufficient to initiate development of spontaneous convulsive and non-convulsive seizures, even in the absence of focal lesions or tissue loss. Similar to human patients with acquired post-traumatic epilepsy, this model presents with a latency period after injury before seizure onset. In this protocol, the community will be provided with a new model of post-traumatic epilepsy, detailing how to induce diffuse non-lesional TBI followed by continuous long-term video-electroencephalographic animal monitoring over the course of several months. This protocol will detail animal handling, the weight drop procedure, the electrode placement for two acquisition systems, and the frequent challenges encountered during each of the steps of surgery, postoperative monitoring, and data acquisition.

This systematic protocol describes a new animal model of post-traumatic epilepsy after repetitive mild traumatic brain injury. The first part details steps for traumatic brain injury induction using a modified weight drop model. The second part provides instructions on the surgical approach for single- and multi-channel electroencephalographic data acquisition systems.

在重复性扩散创伤性脑损伤小鼠模型中诱导创伤后癫痫

Traumatic brain injury (TBI) is a leading cause of acquired epilepsy. TBI can result in a focal or diffuse brain injury. Focal injury is a result of ...

行为学

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

One of the main causes of epilepsy is an infection of the central nervous system (CNS); approximately 8% of patients who survive such an infection develop epilepsy as a consequence, with rates being significantly higher in less economically developed countries. This work provides an overview of modeling epilepsy of infectious etiology and using it as a platform for novel antiseizure compound testing. A protocol of epilepsy induction by non-stereotactic intracerebral injection of Theiler's murine encephalomyelitis virus (TMEV) in C57BL/6 mice is presented, which replicates many of the early and chronic clinical symptoms of viral encephalitis and subsequent epilepsy in human patients. The clinical evaluation of mice during encephalitis to monitor seizure activity and detect the potential antiseizure effects of novel compounds is described. Furthermore, histopathological consequences of viral encephalitis and seizures such as hippocampal damage and neuroinflammation are shown, as well as long-term consequences such as spontaneous epileptic seizures. The TMEV model is one of the first translational, infection-driven, experimental platforms to allow for the investigation of the mechanisms of epilepsy development as a consequence of CNS infection. Thus, it also serves to identify potential therapeutic targets and compounds for patients at risk of developing epilepsy following a CNS infection.

Intracerebral infection with the Theiler's murine encephalomyelitis virus (TMEV) in C57BL/6 mice replicates many of the early and chronic clinical symptoms of viral encephalitis and subsequent epilepsy in human patients. This paper describes the virus infection, symptoms, and histopathology of the TMEV model.

基于泰勒鼠脑脊髓炎病毒的感染性病因癫痫模型

One of the main causes of epilepsy is an infection of the central nervous system (CNS); approximately 8% of patients who survive such an infection develop ...

Triggering and Monitoring Heat-Induced Seizures in a Transgenic Mouse Model

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Triggering and Monitoring Heat-Induced Seizures in a Transgenic Mouse Model