Name: using a bipolar electrode to create a temporal lobe epilepsy mouse model by electrical kindling of the amygdala
Uploaded: 2022-06-29T14:30:00
Description: Watch this Scientific Journal Video about Using a Bipolar Electrode to Create a Temporal Lobe Epilepsy Mouse Model by Electrical Kindling of the Amygdala at JoVE.com

The research was supported by the National Natural Science Foundation of China (No. 82030037, 81871009) and Beijing Municipal Health Commission (11000022T000000444685). We thank TopEdit (www.topeditsci.com) for its linguistic assistance during the preparation of this manuscript.

This experiment was approved by the Experimental Animal Ethics Committee of Xuanwu Hospital, Capital Medical University. All mice were kept in the animal laboratory of Xuanwu Hospital, Capital Medical University. This protocol is divided into four parts. The first two parts introduce the method of building the electrode and the electric circuit using a slip ring to connect the electrodes and the EEG recording/stimulation equipment. The third part describes the operation method of electrode implantation, and the fourth pa...

<ol>
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Epilepsy is a group of diseases with multiple manifestations and diverse causes18; it should be noted that no single model can be used for all types of epilepsy, and researchers must select an appropriate model for their specific study. The present study introduces one of the most accessible methods of electrode fabrication. Various parts of this method can be adjusted to adapt to different experimental conditions.
This method utilizes electrodes with both stimulating a...

Temporal lobe epilepsy (TLE) is the most prevalent type of epilepsy and has a high risk of conversion into drug-resistant epilepsy. Surgery, such as selective amygdalohippocampectomy, is an effective treatment for TLE, and the epileptogenesis and ictogenesis of the disease are still under investigation1,2. Pathogenesis of TLE has been shown to occur not only in the hippocampus but also extensively in the amygdala3,4. For example, both amygdala sclerosis and amygdala enlargement have been frequently reported as the origins of TLE seizures5,6. The importance of the amygdala cannot be underestimated; an amygdala model is essential for the study of epileptogenesis, and a clear illustration of this model is urgently needed.
Several approaches have been proposed to induce seizures in animal models. In the past, convulsant drugs were injected intraperitoneally at an early stage7. Although this method was convenient, the location of epileptic foci was uncertain. With the development of stereotactic technology and a detailed animal brain atlas, intracranial drug injection was applied to solve the problem of localization8. However, a lack of intervention for severe seizures during the acute stage resulted in a high mortality rate, and chronic spontaneous seizures were accompanied by the problem of unstable interictal and seizure frequency9,10. Finally, the electrical kindling method was developed; this method periodically stimulates specific brain regions several times, allowing seizures to be induced with definite control of both the location and the onset time11.
An advantage of this method is that the intracranial implantation of electrodes is minimally invasive12. Furthermore, the severity of the seizure is controllable by the termination of the stimuli, reducing the mortality caused by the seizures. These changes solved the shortcomings of the previous approaches. Notably, this model can adequately mimic human seizures and is especially suitable for the study of status epilepticus (SE) because of its ability to induce SE quickly13. It can also be used for anti-epileptic drug screening14 and in studies on the mechanism of epilepsy. Finally, it is well known that the amygdala is closely associated with memory modulation, reward processing, and emotion15. Disorders of these mental functions are often encountered in epileptic patients and, thus, the amygdala epilepsy model may be a better choice for studying emotional problems in epilepsy16.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 488-conjugated Donkey anti-Rabbit IgG</td>
			<td>invitrogen</td>
			<td>A-21206</td>
			<td></td>
		</tr>
		<tr>
			<td>c-Fos antibody</td>
			<td></td>
			<td>ab222699</td>
			<td></td>
		</tr>
		<tr>
			<td>Cranial drill</td>
			<td>SANS</td>
			<td>SA302</td>
			<td></td>
		</tr>
		<tr>
			<td>dental cement</td>
			<td>NISSIN</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EEG recording and stimulation equipment</td>
			<td>Neuracle Technology (Changzhou) Co., Ltd</td>
			<td>NSHHFS-210803</td>
			<td></td>
		</tr>
		<tr>
			<td>lead-free tin wire</td>
			<td>BAKON</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pin header/Female header</td>
			<td>XIANMISI</td>
			<td></td>
			<td>spacing of 1.27 mm</td>
		</tr>
		<tr>
			<td>Silver wire</td>
			<td>A-M systems</td>
			<td>786000</td>
			<td></td>
		</tr>
		<tr>
			<td>Slip ring</td>
			<td>Senring Electronics Co.,Ltd</td>
			<td>SNM008-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten wire</td>
			<td>A-M systems</td>
			<td>796000</td>
			<td></td>
		</tr>
		<tr>
			<td>ultrafine multi-stand wire</td>
			<td>Shenzhen Chengxing wire and cable</td>
			<td>UL10064-FEP</td>
			<td></td>
		</tr>
		<tr>
			<td>welding equipment</td>
			<td>BAKON</td>
			<td>BK881</td>
			<td></td>
		</tr>
	</tbody>
</table>

using a bipolar electrode to create a temporal lobe epilepsy mouse model by electrical kindling of the amygdala

The amygdala is one of the most common origins of seizures, and the amygdala mouse model is essential for the illustration of epilepsy. However, few studies have described the experimental protocol in detail. This paper illustrates the whole process of amygdala electrical kindling epilepsy model making, with the introduction of a method of bipolar electrode fabrication. This electrode can both stimulate and record, reducing brain injury caused by implanting separate electrodes for stimulation and recording. For long-term electroencephalogram (EEG) recording purposes, slip rings were used to eliminate the record interruption caused by cable tangles and falling off.
After periodic stimulation (60 Hz, 1 s every 15 min) of the basolateral amygdala (AP: 1.67 mm, L: 2.7 mm, V: 4.9 mm) for 19.83 &plusmn; 5.742 times, full kindling was observed in six mice (defined as induction of three continuous grade V episodes classified by Racine's scale). An intracranial EEG was recorded throughout the entire kindling process, and an epileptic discharge in the amygdala lasting 20-70 s was observed after kindling. Therefore, this is a robust protocol for modeling epilepsy originating from the amygdala, and the method is suitable for revealing the role of the amygdala in temporal lobe epilepsy. This research contributes to future studies on the mechanisms of mesial temporal lobe epilepsy and novel antiepileptogenic drugs.

The electrode and circuit enable the EEG to be recorded and function as a stimulation (Figure 1); this setup avoids the complexity of implanting recording and stimulating electrodes separately and minimizes damage to the brain tissue. The application of slip rings allows electrode connection with all types of devices.
We performed electrode implantation surgery on six healthy adult male C57BL/6 mice, and electrical stimulation was performed 2 weeks after surgery. ...

Watch this Scientific Journal Video about Using a Bipolar Electrode to Create a Temporal Lobe Epilepsy Mouse Model by Electrical Kindling of the Amygdala at JoVE.com

Using a Bipolar Electrode to Create a Temporal Lobe Epilepsy Mouse Model by Electrical Kindling of the Amygdala

The amygdala plays a key role in temporal lobe epilepsy, which originates in and propagates from this structure. This article provides a detailed description of the fabrication of deep brain electrodes with both recording and stimulating functions. It introduces a model of medial temporal lobe epilepsy originating from the amygdala.

The amygdala is one of the most common origins of seizures, and the amygdala mouse model is essential for the illustration of epilepsy. However, few ...

This protocol proposed a model of epilepsy with amygdala as its origin. It paves the way to the investigation of mesial temporal lobe structures. This model could be interpreted as the basis of the experiment.As this protocol is a low-cost and efficient method so it can be carried out quickly in most of the laboratories. Demonstrating the procedure will be Yongchang Lu, a postgraduate in our laboratory. Begin by gathering the pre-prepared components starting with two-centimeter long pieces of Teflon-coated tungsten wire with a bear diameter of 76.2 micrometers.One piece of silver wire with a bear diameter of 127 micrometers of the same length and one set of two by two gauged rope pins. Use a lighter to burn one end of each tungsten wire to remove five millimeters of the insulation coating. Peel a section of ultra fine multi-strand wire.Unwrap from the bottom to the upper end where it starts to get dark continuing to the top. Combine this super fine wire and the tungsten wire by pinching one end and gently twisting the other, enabling the two materials to be easily entwined together. Gently pull to ensure that the wires are tightly wrapped and cut off the excess super fine wire.Try to keep the tungsten wire straight throughout the process. Fix the rope pin to the clamp on the welding table with the longer side of the pins facing outward. Use the syringe needle to pick up some solder paste and apply it to the pins.Heat the welding torch to 320 degrees Celsius. Melt and smear some lead-free tin wire with the torch tip. Overlap the upper end of the tungsten wire with one needle of the rope pins and use the solder on the torch to bond the tungsten wire to the pin.Weld another tungsten wire and another silver wire to the rope pin in the same way so that each wire corresponds to a needle. Cut two heat-shrinkable tubes slightly longer than the upper end of the tungsten wire. Put them on the solder joint of two tungsten wires ensuring that the conductive part is fully covered in the tube so that the circuit of the two tungsten wires is not placed in series.Remove the electrode from the welding table clamp and hold the electrode gently with large pliers as it is easy for electrodes to lose their shape when heating the shrinkable tube. Use a good thermal conductivity clamp with slightly more force. Turn on the air duct and heat until a temperature of 320 degrees Celsius is reached.Blow the heat-shrinkable tube for several seconds until it is tightened. Reinforce the electrode with hot melt adhesive. Hold the two tungsten wires and twist them together, keeping their ends apart.Trim the twisted tungsten wires to approximately 10 millimeters in length so that the separation at the ends does not exceed 0.5 millimeters. Check the electrodes with a multi-meter by placing one bar of the multi-meter on the unwelded side of the rope pins and gently touching the end of tungsten wire or silver wire to the other bar. Checking whether the circuit is smooth.Ensure that the lines are not placed in series. Peel off five millimeters of the insulation skin at each end to expose the metal wire inside. Add a section of heat-shrinkable tubing to each starter wire.Weld each wire with the EEG device connector plug. Shrink the heat-shrinkable tube with hot air. Add a section of heat-shrinkable tube to each rotor wire.Screw the conducting parts of the red and orange wires together and weld them to a joint and the header to fit the rope pin. Weld the other two wires on the header to each joint. Weigh the mouse.When the mouse is fully anesthetized, shave hair from the eye to the ear area with a razor. Fix the mouse on the stereotaxic frame. Put the front upper teeth into the incisor bar and insert both ear bars to equal depths into the ears.Apply erythromycin eye ointment to the eyes to prevent dryness and blindness caused by bright light during surgery. Disinfect the surgical area at least three times with alternating swabs of iodophor and 75%alcohol. Make a longitudinal incision to fully expose the surgical area or make a triangular incision as long as it exposes the anterior and posterior fontanelles and electrode implanting sites.Roll a small piece of cotton into a ball and wet it with 3%hydrogen peroxide. Remove the soft tissue attached to the skull by gently rubbing the exposed area with a small cotton ball until the anterior and posterior fontanelle are seen. Adjust the anterior and posterior heights so that the anterior and posterior fontanelle is horizontal.Consider the position of the anterior fontanelle to be the origin of the axes. Fix a stainless steel screw to the left cerebellar skull. Set the coordinates for the amygdala kindling from the bregma and adjust the stereotaxic device to locate this spot and mark it.Drill a hole on the march spot with a 0.5 millimeter diameter skull drill. Fix the electrodes to the locating rod of the stereotaxic device. Place the electrode vertically above the hole and drop the position to minus 4.9 millimeters slowly.Wrap the silver wire around the screw thrice. Taking care not to shake the electrode body during operation. Mix dental cement and to gently apply it to the electrode and skull surface.When the dental cement hardens, modify the outside until the cement that encloses the fixed electrode turns into a cone. Then release the electrode from the stereotaxic device. Remove the mouse and place it back in the cage.Keeping it separate from the other mice. Put the mouse in a customized box with slippering cables connecting the electrode on the mouse's head and the EEG device. Run the cable through a hole in the box lid and adjust the length left in the box to allow the mouse to move freely.Turn on the EEG device and ensure it works correctly. Set the stimulator parameters to deliver one millisecond monophasic square wave pulses at 60 hertz for a one second train duration for 10 stimulation cycles. Start with a current intensity of 50 microamperes for the first stimulation.Monitor the EEG for after discharge characterized by high frequency spikes. If no after discharge is observed add 25 microamperes to the next stimulus and continue this process every 10 minutes until an after discharge is observed and lasts five seconds. Stimulate the mouse with the determined current intensity every 15 minutes, no more than 20 times a day.Monitor the behavioral responses to the stimulus. The electrode implantation surgery was performed on six healthy adult males C57 black 6 mice and electrical stimulation was performed two weeks post-surgery. The behavioral seizure level gradually increased with the number of stimuli increasing and the number of stimuli required for complete kindling was recorded.The EEG after discharges lasted five to 15 seconds. Then the intracranial spontaneous discharges intensified and behavioral symptoms began. Seizure duration was usually less than one minute which reduces the risk of death from severe convulsions resulting in apnea.The expression of c-Fos in the brain tissue was detected by immunohistochemistry two hours after complete kindling. The results show that the expression of c-Fos in the ipsilateral amygdala significantly increased verifying the feasibility of this model. The separation of tungsten wire ends should not exceed half millimeters otherwise it'll not get through the brain hole.The length of the electrode should not be too long otherwise it'll affect activity of mice.

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