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

Summary

This paper demonstrates the performance of acupuncture, epilepsy models, and the analysis of sleep in rodents. The acupuncture procedure and the identification of acupoints are described. Pilocarpine or pentylenetetrazol (PTZ) is used to induce epilepsy. Electrocorticogram (ECoG), electromyogram (EMG), brain temperature, and locomotor activity recordings are employed for sleep analysis.

Abstract

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little research to reveal the possible mechanisms behind these effects. To investigate the effect of acupuncture on epilepsy and sleep, several issues need to be addressed. The first is to identify the acupoints, which correspond between humans, rats, and mice. Furthermore, the depth of insertion of the acupuncture needle, the degree of needle twist in manual needle acupuncture, and the stimulation parameters for electroacupuncture (EA) need to be determined. To evaluate the effects of acupuncture on epilepsy and sleep, a feasible model of epilepsy in rodents is required. We administer pilocarpine into the left central nucleus of the amygdala (CeA) to simulate focal temporal lobe epilepsy (TLE) in rats. Intraperitoneal (IP) injection of pilocarpine induces generalized epilepsy and status epilepticus (SE) in rats. Five IP injections of pentylenetetrazol (PTZ) with a one-day interval between each injection successfully induces spontaneous generalized epilepsy in mice. Recordings of electrocorticograms (ECoGs), electromyograms (EMGs), brain temperature, and locomotor activity are used for sleep analysis in rats, while ECoGs, EMGs, and locomotor activity are employed for sleep analysis in mice. ECoG electrodes are implanted into the frontal, parietal, and contralateral occipital cortices, and a thermistor is implanted above the cerebral cortex by stereotactic surgery. EMG electrodes are implanted into the neck muscles, and an infrared detector determines locomotor activity. The criteria for categorizing vigilance stages, including wakefulness, rapid eye movement (REM) sleep, and non-REM (NREM) sleep are based on information from ECoGs, EMGs, brain temperature, and locomotor activity. Detailed classification criteria are stated in the text.

Introduction

Epilepsy is a common neurological disorder in which recurrent seizures occur throughout a patient's lifespan. Most epileptic recurrences can be well-controlled by anti-epileptic drugs (AEDs). However, about 30% of epileptic patients develop refractory epilepsy¹. Epilepsy causes sleep disturbances, which can further exacerbate recurrence. Evidence demonstrates that epilepsy may either disrupt sleep at night or may cause excessive daytime sleepiness^2,3. Our previous studies further indicate that epilepsy occurring at zeitgeber time (ZT) 0, i.e., the beginning of the light period in the light:dark cycle, decreases sleep; this is mediated by corticotropin-releasing hormone (CRH), a homeostatic factor. Epilepsy at ZT13 (the beginning of the dark period) enhances the expression of another homeostatic factor, interleukin-1 (IL-1), which increases sleep. Sleep circadian rhythms are altered when epilepsy occurs at ZT6, the middle of the light period^4,5. On the other hand, sleep problems further exacerbate the progression and recurrence of epilepsy⁶. Based on the aforementioned evidence, we try to reveal an optimal therapeutic method to simultaneously control epilepsy and prevent sleep disruptions in epilepsy patients. We previously found that electroacupuncture (EA) with a 10-Hz stimulation frequency, in which a certain amount of current is delivered into the acupoint through a stainless-steel needle, successfully suppresses electrocorticogram (ECoG) epileptic activities and epilepsy-induced sleep disturbances⁷. EA with a 100-Hz stimulation frequency further deteriorates epileptic activities and sleep disruptions in rats^8,9. This successful experiment depends on three factors: firstly, a feasible epileptic animal model; secondly, a method for sleep recording and analysis in rodents; and thirdly, the accurate performance of acupuncture and the accuracy of the acupoint locations.

Epilepsy has been categorized into two major types: focal epilepsy and generalized epilepsy. We are interested in focal temporal lobe epilepsy (TLE), generalized epilepsy, status epilepticus (SE), and the recurrence of spontaneous generalized epilepsy. Therefore, different manipulations are applied to create suitable epileptic models for our experiments. To establish focal TLE, a low dose of pilocarpine is administered into the left central nucleus of the amygdala (CeA). To verify this model, six ECoG electrodes are implanted on the frontal (F1 & F2), parietal (P1 & P2), and occipital (O1 & O2) lobes in both the left and right hemispheres, and another two reference electrodes (R1 & R2) are placed over the cerebellum in both hemispheres. An additional microinjection guide cannula is surgically implanted into the left CeA (AP, 2.8 mm from bregma; ML, 4.2 mm; DV, 7.8 mm relative to bregma). The coordinates are adapted from the Paxinos and Watson rat atlas¹⁰. If the focal TLE is successfully induced, only the recording from the electrode on left parietal cortex (P1), which is near the left CeA, should acquire the dominant epileptiform ECoGs, with no significant epileptiform ECoGs recorded from the other ECoG electrodes. Intraperitoneal (IP) injections of pilocarpine into rats induce generalized epilepsy and SE, but this can be fatal. Five IP injections of pentylenetetrazol (PTZ) with a one-day interval between each injection successfully induce spontaneous generalized epilepsy in mice and also ensure the mice's survival. Two wire ECoG electrodes are implanted into the frontal and parietal cortices in the mice to receive ECoG signals and to verify spontaneously recurrent epilepsy.

Polysomnography (PSG) is a comprehensive method to record physiological changes that occur during sleep, and it can objectively classify sleep into different stages of non-rapid eye movement (NREM) and rapid eye movement (REM) sleep. PSG records parameters of body functions, including brain waves (electroencephalogram, EEG), eye movements (electrooculogram, EOG), skeletal muscle tones (electromyogram, EMG), heart rhythms (electrocardiogram, ECG), and blood oxygen levels and respiratory parameters. In rats, we record ECoGs, EMGs, cortical temperature, and locomotor activity to classify vigilance states into wakefulness, NREM sleep, and REM sleep. Sleep analysis in mice is conducted using ECoGs, EMGs, and locomotor activity results. Rats are surgically implanted with three ECoG screw electrodes at the frontal, parietal, and contralateral cerebellar cortices by stereotaxic surgery. Post-acquisition determination of the vigilance states (wakefulness, NREM sleep, and REM sleep) is conducted according to the parameters acquired from the ECoGs, EMG, brain temperature, and locomotor activity. Detailed criteria for categorizing the animal's behavior in both rats and mice are described in the protocol.

Both rats and mice need to be anesthetized with a low dose of zoletil (25 mg/kg), which is half the dosage of anesthetics normally administered during stereotaxic surgery, before performing manual acupuncture or EA. This dosage allows animals to wake up 30 to 35 min after the injection. Either manual acupuncture or EA is performed at the beginning of the dark period, with a constant time period of 30 min, and each animal is consecutively treated for two to three days. Stimulating EA currents are delivered into a particular acupoint through a stainless-steel needle that is inserted into the acupoint. The stimulus current is a train of biphasic square pulses, in which the pulse duration is 150 ms and the stimulation intensity is 1 mA. If a dry needle is used for manual acupuncture, the needle inserted into the acupoints is twitched 10 times every 5 min. The difficult part of manual acupuncture or EA is to localize the acupoints in rodents. The location of acupoints in rats or mice is similar to their anatomical location in humans. For example, the bilateral Fengchi acupoints are located 3 mm away from the posterior median line on the neck, between the two ears, which is similar to its anatomical location in humans¹¹. Furthermore, the acupoints with low impedance on the skin can be further confirmed. Sham acupuncture or sham EA manipulation is necessary for acupuncture or EA experiments. Sham acupuncture or sham EA should be performed at a non-acupoint located close to the acupoint, such as near the axilla¹².

To successfully investigate the effects of acupuncture or EA on epilepsy and epilepsy-induced sleep disruptions, the following factors must be in place: a feasible epileptic animal model, the precise analysis of epileptiform ECoGs and the recurrence of epilepsy, a method to classify vigilance states, and the accurate performance of acupuncture or EA in rodents.

Protocol

All experimental protocols are approved by the Institutional Animal Care and Use Committee (IACUC) of National Taiwan University.

1. Stereotaxic Surgery for Implanting ECoG Electrodes, EMG Electrodes, Brain Thermistor, and Injection Guide Cannula

1. For rats (250 - 350 g, 6- to 8-week-old Sprague-Dawley rats)
   1. Anesthetize the rats by IP injection with 50 mg/kg zoletil. Confirm the proper depth of anesthesia by observing a lack of response after pinching the hind paw. Apply eye ointment, shave the fur, and sterilize the skin with povidone-iodine solution and 75% ethanol. Inject an antibiotic (penicillin G) to prevent infection.
   2. Prepare scalpels, scissors, hemostats, gauzes, and cautery machine for surgery. Sterilize surgical gear and gauzes by an autoclave and the cautery by 75% ethanol.
   3. Place an ear bar into the ear canal and mount the rat to the stereotaxis.
   4. Using a scalpel, make an approximate 2-cm midline incision on the skull along a line between two the ears, moving caudally. Clip the skin flaps with hemostats to expose the skull and remove the tissue over the skull using a scalpel.
   5. Drill eight holes (F1, F2, P1, P2, O1, O2, R1, and R2), each approximately 0.7 mm in diameter, on the skull with a rotary tool. Screw eight ECoG electrodes on the frontal, parietal, and occipital lobes and the cerebellum in both the left and right hemispheres. These electrodes are used for focal epilepsy detection.
      1. Use the following coordinates for the recording electrodes: frontal (F1 and F2): +2.0 mm anterior to bregma and +2.5 mm from the midline, parietal (P1 and P2): -2.0 mm anterior to bregma and +3.0 mm from the midline, and occipital (O1 and O2): -5.5 mm anterior to bregma and +3.0 mm from the midline.
      2. Place two reference electrodes (R1 and R2) over the cerebellum (-11.0 mm anterior to bregma and +4.0 mm from the midline).
   6. In a separate groups of rats, drill three holes and place two screw EEG electrodes over the right frontal (F2) and parietal lobes (P2) of the cortices with the same coordinates as described in step 1.1.5.1. Place a third EEG electrode over the left cerebellum (R1), which serves to ground the animal and reduce signal artifacts. These electrodes are used for confirming generalized epilepsy and analyzing vigilance stages.
   7. Separate the neck skin and muscle and insert two EMG electrodes into the neck muscle.
   8. Drill another hole on the skull and place a microinjection guide cannula into the left CeA (AP, 2.8 mm from bregma; ML, 4.2 mm; DV, 7.8 mm relative to bregma) in rats. The coordinates are adapted from the Paxinos and Watson rat atlas¹⁰.
   9. Drill a bigger hole (with a diameter of 1.6 mm) on the skull and insert a calibrated 30-kV thermistor on the surface of the parietal cortex, which will be cemented later, to monitor the cortical temperature in rats.
   10. Use gauze and cautery to stop bleeding when it occurs.
   11. Route the insulated leads from the ECoG electrodes and EMG electrodes to a pedestal and connect the thermistor to the tether. Cement the pedestal and guide cannula to the skull with dental acrylic.
   12. Treat the incision topically with polysporin (bacitracin zinc/polymyxin B sulfate) to prevent infection. Give the animals both ibuprofen and penicillin G in water for one week after surgery.
2. For mice (20 - 30 g, 6- to 8-week-old C57BL/C mice)
   1. Anesthetize the mice by IP injection with 50 mg/kg zoletil and confirm the proper depth of anesthesia by observing a lack of response after pinching the hind paw. Apply eye ointment. After shaving the fur, sterilize the skin with povidone-iodine solution and 75% ethanol. Inject an antibiotic (penicillin G) to prevent infection.
   2. Place an ear bar into the ear canal and mount the mouse to the stereotaxis.
   3. Using a scalpel, make an approximate 1.5-cm midline incision on the skull along a line between the two ears, moving caudally. Clip the skin flaps with hemostats to expose the skull and remove the tissue over the skull with a scalpel.
   4. Poke two holes on the skull with surgical scissors and place two wire ECoG electrodes on the right frontal lobe (F2: +2.0 mm to bregma and +1.5 to the midline) and left parietal lobe (P1: -3.0 mm to bregma and -2.5 mm to the midline).
   5. Separate the neck skin and muscle and insert two EMG electrodes into the neck muscle.
   6. Connect the insulated leads from the wire ECoG electrodes and EMG electrodes to the female terminals and to a 2.54-mm connector, and then cement to the skull with dental acrylic.
   7. Treat the incision topically with polysporin (bacitracin zinc/polymyxin B sulfate) to prevent infection. Give the animals both ibuprofen and penicillin G in water for one week after surgery.
3. Allow all animals to recover for seven days prior to the initiation of the experiments.
4. House rats or mice separately, in individual recording cages, in the isolated room where the temperature is maintained at 23 ± 1 °C and the light:dark (L:D) rhythm is controlled in a 12:12-h cycle (40 W x 4 tubes illumination). Provide food and water ad libitum.
5. Connect the ECoG, EMG, and thermistor through a tether to the amplifiers one week after surgery to start the recordings.

2. Establishment of Focal TLE Epilepsy, SE, and Spontaneously Recurrent Epilepsy

1. For rats
   1. Administer 0.5 µL of pilocarpine (2.4 mg/µL) into the left CeA through the injection guide cannula using a microinjection pump. The injection rate should be set at 0.2 µL/min to induce focal TLE.
   2. IP inject 300 mg/kg of pilocarpine to induce generalized epilepsy with recurrent SE.
2. For mice
   1. IP administer PTZ (0.035 mg/g mouse bodyweight) at a particular ZT point every other day. Five consecutive injections will cause the development of spontaneously and recurrently generalized epilepsy.
3. For both rats and mice
   1. Amplify the ECoG signals by 5,000 and filter the analog bandpass between 0.1 and 40 Hz.
   2. Use an A/D converting board to convert the analog ECoGs signals to digital signals with a 128-Hz sampling rate.
   3. Use a software for visual scoring and analyze the onset and the duration of epilepsy. Measure the time scale in order to represent the duration.
   4. Define ECoG epilepsy by the appearance of epileptic spikes with amplitudes greater than 2 mV and with durations of more than 30 s¹³.

3. Classification of Vigilance States

1. For rats
   1. Determine the vigilance states by using the parameters acquired from ECoGs, EMG, brain temperatures, and locomotion within a 12-s episode of recording. Connect the ECoG, EMG, and thermistor through a tether to the amplifiers one week after surgery to start the recording. Score the states with a custom-made software according to steps 3.1.5 - 3.1.7.
   2. Measure locomotor activities using an infrared motion detector, integrate the signals every 1 s, and store the signals.
   3. Measure cortical temperature and store the signals.
   4. Classify vigilance states according to our previously defined criteria¹⁴.
   5. Score wakefulness by using the following characteristics: small-amplitude ECoGs with high-frequency spectra, higher delta power (0.5 - 4.0 Hz), and lower theta power (6.0 - 9.0 Hz); dominant locomotor activity; high EMG activity; and gradually increasing cortical temperature.
   6. Score NREM sleep by using the following characteristics: delta-wave dominant ECoGs with large amplitudes, declined EMG activity, reduced cortical temperature, and no locomotor activity.
   7. Score REM sleep by using the following characteristics: decreased amplitude of ECoGs with the dominant theta frequency, suddenly increased cortical temperature, minimal EMG activity, and low locomotor activity with body twitches.
2. For mice
   1. Repeat the classification of vigilance states in the mice as conducted in the rats, except there is no cortical temperature recorded from the thermistor.

4. Performance of Manual Acupuncture and EA in Rats

1. The rat is anesthetized for the EA protocol. 
2. Locate the acupoint by anatomy and confirm low skin impedance at the acupoint. The light is flashing when detect the low skin impedance. Note: Fengchi acupoints are located 3 mm away from the posterior median line between the two ears, on the neck.
3. Insert stainless-steel needles into the acupoints at a depth of 2 mm.
4. Twitch the inserted needles 10 times every 5 min.
5. Using a functional electrical stimulator, deliver a train of biphasic pulses (150-µs duration each) with an intensity of 1 mA to the acupoints through the needle.
6. Perform a sham acupuncture or sham EA as a control.

Results

There are different rat and mouse models to satisfy the needs of different epilepsy types. To induce focal TLE, 0.5 µL of pilocarpine (2.4 mg/µL) is administered into the left CeA. The predominant epileptiform ECoGs are acquired from the ECoG electrode on the parietal lobe of the left hemisphere (Figure 1A: b), and rare epileptic activities are picked up from the rest of the ECoG electrodes (Figure 1A: a, c, d

Discussion

Choosing a feasible epilepsy animal model is essential for each experimental purpose. One of our goals is to elucidate the effects of EA on epilepsy suppression. EA is an alternative medicine that may exhibit therapeutic effect in epilepsy and has been documented in ancient Chinese literature. However, there is a lack of scientific evidence to prove it. To determine the effects of EA on epilepsy, we primarily focused on the effects of EA on mild focal epilepsy, rather than on severe generalized seizure or SE. Our previou...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Drugs
Zoletil	Virbac		50 mg/kg i.p.
pilocarpine	Sigma-Aldrich	P6503	300 mg/kg i.p.; 1.2 mg microinjection
PTZ	Sigma-Aldrich	P6500	0.035 mg/mouse
polysporin	Pfizer
Surgery
ECoG electrode	Plastics One	E363/20	screw electrode for rats
Pedestal	Plastics One	MS363
Cannula	Plastics One	C315G/spc
Thermistor	Omega Engineering	44008
Dental acrylic	Tempron
Stereotaxic Instrument	Stoelting		Dural arms
Recording equipments
ECoG amplifier	Colbourn Instruments	V75-01
A/D Board	National Instruments	NI PCI-6033E
Infrared-based motion detectors	Biobserve GmbH	custom-made
ICELUS	G-System
AxoScope 10 Software	Molecular Devices
Acupuncture needs
Stainless needles	Shanghai Yanglong Medical Articles Co.	32 gauge x 1”
Functions Electrical Stimulator	I.T.O., Japan	Trio 300
AcuPen	Lhasa OMS	Pointer Excel II