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 mediate....

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....

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