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

Summary

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.

Abstract

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 ± 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.

Introduction

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 investigation^1,2. Pathogenesis of TLE has been shown to occur not only in the hippocampus but also extensively in the amygdala^3,4. For example, both amygdala sclerosis and amygdala enlargement have been frequently reported as the origins of TLE seizures^5,6. 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 frequency^9,10. 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.

Protocol

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 part presents the EEG recording and stimulation parameters used for the amygdala epilepsy model.

1. Fabrication of electrodes

1. Keep the following previously prepared materials ready: two 3 cm long pieces of Teflon-coated tungsten wire (bare diameter: 76.2 µm), one piece of silver wire (bare diameter: 127 µm) of the same length, and one set of 2 x 2 gauged row pins.
2. Use a lighter to burn one end of each tungsten wire to remove 5 mm of the insulation coating.
   NOTE: The tungsten wire with the insulation removed turns black; this part of the tungsten wire is referred to as the upper end.
3. Peel a section of ultrafine multi-strand wire and wrap it from the bottom to the upper end where it starts to get dark, continuing to the top. Combine this superfine wire (has a soft texture) with the tungsten wire by pinching one end and gently twisting the other end, which enables the two materials to be easily entwined together.
4. Gently pull to ensure that they are tightly wrapped and cut off excess superfine wire. Try to keep the tungsten wire straight throughout the process.
5. Fix the row 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 °C; melt and smear some lead-free tin wire with the torch tip.
6. Overlap the upper end of the tungsten wire with one needle of the row pins and use the solder on the torch to bond the tungsten wire to the pin.
   NOTE: It would be very difficult to weld the tungsten wire with the pins directly without the help of the superfine wire.
7. Weld another tungsten wire and another silver wire to the row pin in the same way so that each wire corresponds to a needle (see Figure 1,i).
8. 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.
   NOTE: Although there are three wires, if two of them are insulated, the three wires will not be in series; a tube can also be added to the silver wire.
9. 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, using a good thermal conductivity clamp with slightly more force.
10. Turn on the air duct and heat until a temperature of 320 °C is reached. Blow the heat-shrinkable tube for several seconds until it is tightened (see Figure 1,ii).
11. If the needles separate from the plastic body during the welding process, splice the welding part and the plastic body with a hot melt adhesive (see Figure 1,iii). Be careful not to smear it on the interface as this would affect the interface insertion.
12. Hold the two tungsten wires and twist them together, keeping their ends apart (see Figure 1,iv). Trim the twisted tungsten wires to approximately 10 mm in length so that the separation at the ends does not exceed 0.5 mm.
    ​NOTE: This step can also be performed before electrode implantation to allow flexible adjustment of electrode length.
13. Heat the glue gun and apply the glue evenly around the electrode.
14. Check the electrodes with a multimeter: place one bar of the multimeter on the unwelded side of the row pins, and gently touch 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.

2. Slip ring connection and circuit description

NOTE: When the electrodes on the mice are plugged into an EEG device via cables in a free-moving condition, the cables can become tangled as the mice move and turn around. This causes the cables to become shorter, eventually hindering the mice from moving or causing the cables to fall off their heads. In the method described here, a four-channel slip ring is introduced to prevent the cables from falling off. The four channels are represented in four colors in Figure 1B.

1. Peel off 5 mm of the insulation skin at each end to expose the metal wire inside.
2. Add a section of heat-shrinkable tube to each stator wire.
3. Weld each wire with the EEG device connector plug.
4. Shrink the heat-shrinkable tube with hot air.
5. Add a section of heat-shrinkable tube to each rotor wire.
6. Screw the conducting parts of the red and orange wires together and weld them to a joint in the header to fit the row pin.
7. Weld the other two wires on the header to each joint.
   ​NOTE: The brown channel that corresponds to the silver wire is connected to the EEG device for grounding. The red and orange channels receive signals from the same tungsten wire, and the orange channel serves as a reference for the EEG device. The signals in the red channel are meaningless, but they must coexist with the black channel to form a current stimulus. The signals in the black channel are the real electrical signals in the brain. Different circuits can be designed with multi-channel slip rings to suit different devices.

3. Surgery for implantation

1. Animals
   1. Use 8-week-old C57BL/6 wild-type male mice, weighing 24-26 g, for surgeries.
   2. House them with a 12 h light-dark cycle (light time: 8:00-20:00) in a temperature-controlled environment (22 ± 1 °C) and provide water and feed ad libitum.
   3. Use an extra heat mat to keep the animals warm during the surgery.
   4. After the surgery, inject meloxicam subcutaneously (10 mg/kg) as the first administration of analgesics. Then, place the animals in separate cages to optimize recovery. Add meloxicam to the animal's diet for the first week after surgery.
   5. After the experiment, infuse the left ventricles of the mice with 4% paraformaldehyde under anesthesia, and collect the brain tissues for histological verification of the electrode target.
2. Weigh the mouse and anesthetize it by intraperitoneal injection of 1% pentobarbital solution. Sterilize all the surgical instruments and consumables to be used, including drill bits, electrodes, dental cement, etc., by autoclaving.
3. When the mouse is completely anesthetized, shave the hair from the eye to the ear area with a razor.
4. Fix the mouse on the stereotaxic frame. Put the front upper teeth into the incisor bar and insert both ear bars equally deeply into the ears. Apply erythromycin eye ointment to the eyes to prevent dryness and blindness caused by a bright light during surgery.
5. Disinfect the surgical area with three alternating swabs of iodophor and 75% alcohol in a circular motion. Then, make a sagittal incision forward from the middle of this incision, and cut off the skin on either side of the incision to create a triangular window.
6. 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 clearly seen.
7. Adjust the anterior and posterior heights so that the anterior and posterior fontanelle is in the horizontal position. Consider the position of the anterior fontanelle to be the origin of the axes.
8. Fix a stainless-steel screw to the left cerebellar skull, using a drill to create a flat surface. Ensure that the screw protrudes halfway out of the skull.
9. Ensure the coordinates for the amygdala kindling are −1.67 mm posterior, −2.7 mm lateral, and −4.9 mm ventral from the bregma. Adjust the stereotaxic device to locate this spot and mark it.
10. Drill a hole on the marked spot with a 0.5 mm diameter skull drill.
11. Fix the electrodes to the locating rod of the stereotaxic device, place the electrode vertically above the hole, and drop the position to −4.9 mm slowly. Wrap the silver wire around the screw three times, taking care not to shake the electrode body during operation.
12. Mix the dental cement and 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.
13. When the cement has hardened, release the electrode from the stereotaxic device. Subcutaneously administer meloxicam 10mg/kg to relieve discomfort caused by pain in animals. Administer meloxicam to animal food for analgesic effect in the first week after surgery. Remove the mouse and place it back in the cage, keeping it separate from the other mice.

4. Electrical kindling

1. Allow the mice to rest for at least 1 week after surgery before kindling to allow for postoperative recovery and to allow the inflammation to subside.
   NOTE: In general, mice that have not recovered adequately do not respond well to kindling.
2. Put the mouse in a customized box with slip-ring cables connecting the electrode on the mouse's head and the EEG device. Run the cable through a hole in the lid of the box, and adjust the length left in the box to allow the mouse to move freely.
3. Turn on the EEG device and check whether it is working properly. Set the stimulator to deliver 1 ms monophasic square-wave pulses at 60 Hz for a 1 s train duration.
4. Start with a current intensity of 50 µA for the first stimulation; monitor the EEG for after-discharge, which is characterized by high-frequency spikes. If no after-discharge is observed, add 25 µA to the next stimulus, and continue this process every 10 min until an after-discharge is observed and lasts 5 s.
   NOTE: If the experiment does not require a discharge, step 4.4 can be skipped; 300 µA is strong enough for kindling.
5. Stimulate the mouse with the current intensity determined in step 4.3 every 15 min, no more than 20 times a day.
6. Monitor the behavioral responses to the stimulus.
   NOTE: The occurrence of three consecutive grade V episodes is considered full kindling, combined with Racine rank standard¹⁷.

Results

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. ...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
Alexa Fluor 488-conjugated Donkey anti-Rabbit IgG	invitrogen	A-21206
c-Fos antibody		ab222699
Cranial drill	SANS	SA302
dental cement	NISSIN
EEG recording and stimulation equipment	Neuracle Technology (Changzhou) Co., Ltd	NSHHFS-210803
lead-free tin wire	BAKON
Pin header/Female header	XIANMISI		spacing of 1.27 mm
Silver wire	A-M systems	786000
Slip ring	Senring Electronics Co.,Ltd	SNM008-04
Tungsten wire	A-M systems	796000
ultrafine multi-stand wire	Shenzhen Chengxing wire and cable	UL10064-FEP
welding equipment	BAKON	BK881