Manipulation of Epileptiform Electrocorticograms (ECoGs) and Sleep in Rats and Mice by Acupuncture

The overall goal of this procedure is to assess epilepsy, sleep and the effects of acupuncture in an animal model. This method can help answer key questions in neuroscience field about how to establish epilepsy, analyze sleep and perform acupuncture in a small animal model. The main advantages of this technique are that the lateral cortical and the lateral myogram electrodes in that semester can be implanted quickly and easily by stereotaxic surgery.

Demonstrating this procedure will be Yi-Jou Wu, a graduate student from my laboratories. After confirming the proper depth of sedation by a lack of response to a toe pinch, apply eye ointment to a six-to eight-week-old 250 to 350 gram Spregg Dolly rat from the first group. Use clippers to remove the hair from the top of the skull.

Disinfect the skin with sequential applications of povidone iodine solution and 75 percent ethanol, followed by antibiotic injection to prevent infection. Next, mount the animal onto a stereotaxis machine and then place an ear bar into the ear canal. Using a scalpel, make an approximately two centimeter midline caudal incision along the skull between the ears.

Then use hemostats to clip the skin flaps, exposing the skull, and use a scalpel to remove the tissue. When the skull is clean, use a rotary tool to drill eight approximately 0.7 millimeter diameter holes into the frontal, parietal, and occipital lobes and the cerebellum in both the left and right hemispheres of the exposed bone. Then screw eight electrocorticogram electrodes into the holes for focal epilepsy detection.

In a separate group of rats, drill three holes and place two screw electroencephalogram electrodes over the right frontal and parietal lobes of the cortices with the same coordinates as in the first group. Next, place a third electroencephalogram electrode over the left cerebellum to ground the animal and reduce the signal artifacts. Separate the neck skin and muscle and insert two electromyogram electrodes into the neck muscle.

Drill another hole on the skull and place a microinjection guide canula into the left central nucleus of the amygdala. Then, drill a 1.6 millimeter hole in the skull and insert a calibrated 30 kilovolt thermoster on the surface of the parietal cortex to monitor the cortical temperature. Route the insulated electrocortico and electromyogram electrode leads to a pedestal.

Using dental acrylic to cement the pedestal, guide the canula to the skull and then connect the thermoster to the tether. Then, treat the incision topically with polysporin to prevent infection and give the animals water supplemented with ibuprofin and penicillin G for one week after surgery. To induce focal temporal lobe epilepsy, use a microinjection pump to administer 0.5 microliters of pilocarpine into the left central nucleus of the amygdala through the injection guide canula at 0.2 microliters per minute.

To induce generalized epilepsy with recurrent status epilepticus, inject 300 milligrams per kilogram of pilocarpine IP.For acupuncture, first manually locate the acupoints. For example, the feng chi acupoints are located three millimeters away from the posterior median line between the ears on the neck. After confirming a low skin impedance at the acupoints of interest, insert stainless steel needles into the acupoints at a two millimeter depth.

Then, using a functional electrical stimulator, deliver a 10 hertz train of 150 millisecond byphasic pulses with a one milliamp intensity to the acupoints through the needle. Here, a predominant epileptiform electrocorticogram acquired from the electrocorticogram electrode on the parietal lobe of the left hemisphere is shown. After pilocarpine administration, rare epileptic activities are picked up from the rest of the electrocorticogram electrodes, indicating a successful focal temporal lobe epilepsy induction.

Within five minutes, severe cholinergic effects on behavior occur, gradually increasing in severity until an electrocorticogram generalized seizure occurs followed by a status epilepticus event. No epileptiform electrocorticograms are observed during the dark period after the first pentilene tetrasol injection, or during the second dark period on an off-day for injection until the fifth pentilene tetrasol injection, along with spontaneously and recurrently generalized epilepsy. Wakefulness exhibits a high amplitude electromyogram and an abundance of locomotor activity, with a gradual increase in the cortical temperature as the vigilant state transits from either non-REM or REM sleep to wakefulness.

Non-REM sleep is characterized by synchronized electrocorticograms with a high amplitude and low frequency, with a gradual electromyogram amplitude decline and no locomotor activity, as well as a decrease in the cortical temperature as the vigilant state transits from wakefulness into NREM sleep. During REM sleep, the electrocorticogram wave is desynchronized with a reduced amplitude. The predominant electroencaphalogram power density occurs within the theta frequency.

The electromyogram activity is the lowest, phasic body twitch is observed, and the cortical temperature rapidly increases. Administration of 100 hertz of electroacupuncture into the bilateral feng chi acupoints exacerbates the pilocarpine induced epileptiform electrocorticograms, while 10 hertz of electroacupuncture suppresses the epileptiform electrocorticograms. Further, pilocarpine administration into the left central nucleus of the amygdala also suppresses non-REM sleep during the light period of the 12-hour light/dark cycle.

100 hertz of electroacupuncture further deteriorates the pilocarpine induced suppression of non-REM sleep. In contrast, a 10 hertz electroacupuncture into the bilateral feng chi acupoints increases non-REM sleep per se during the dark period, and blocks the pilocarpine-induced reduction of non-REM sleep during the light period. Once mastered, this technique can be completed in one and a half hours if it is performed properly.

Before attempting this procedure, it is important to practice the skill of stereotaxic surgery until proficiency. After its development, this technique paves the way for the researchers in the field of neuroscience to explore the effects of acupuncture in epilepsy and sleep disturbance. After watching this video, you should have a good understanding of how to surgically implant electroencephalo and electromyogram electrodes, a guide canula, and a semester to assess sleep patterns and other effects of acupuncture on brain activity.