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.
