Experimental animals
Experiments were performed on 31 male Wistar rats (two to three months old) with an initial body weight of 180–220 g. Animals were kept in standard conditions (constant temperature of 23°C, relative humidity of 60%, 12 h dark/light cycles; standard diet and tap water were given ad libitum) and were acclimatized to the laboratory conditions for at least seven days before the experiment. All experiments were carried out following the National Institutes of Health guidelines for the care and use of laboratory animals and the European Council Directive on 24 November 1986 for Care and Use of Laboratory Animals (86/609/EEC). The experiments were approved by the Odesa National Medical University Bioethics Committee (UBC) (approval No. 3 dated 14/03/2018) before the study.
Epilepsy model
Kindled seizures were induced as described previously [9, 26]. PTZ (Sigma Aldrich, St. Louis, MO, USA) was administered intraperitoneally (i.p.) at a daily dose of 35.0 mg/kg for 21 days.
Myoclonus precipitation is associated with typical absence seizure manifestations [27–29]. Meanwhile, typical absences in genetic epilepsy are a feeble level of myoclonus that does not encompass the whole body repeatedly. That is why we addressed severe whole-body myoclonus in the third stage of kindling development. Hence, the seizure responses were observed over a 30-minute cutoff period and were classified as initially described by Racine: zero: no response; one: vibrissae twitching, mouth, ear, and facial twitching; two: convulsive waves
through the body; three: whole-body myoclonic jerks, rearing; four: tonic-clonic convulsions with rearings and falling; five: repeated seizures as at stage four, loss of postural control [21, 22].
Criteria for inclusion of rats into observation
The early kindled stage was modeled in rats demonstrating minor absence manifestations, including myoclonus of muscle groups of extremities and body (one-two scored the severity of seizures). Rats with whole body myoclonus or rearings (three-scored severity) until the 11th PTZ administration composed the group treated with PTZ for the induction of fully kindled seizures.
Groups of animals
Sham-stimulated rats, which were PTZ-administered for three weeks (35.0 mg/kg, i.p. ), served as a control group. Thus, seven rats composed a control group for the early kindling group, and seven rats formed the control group of fully kindled animals (14 rats in total) (Fig. 1).
Experimental groups subjected to ES trials included eight rats with early kindling and nine with fully kindled seizures (17 rats).
Overall, at the stages of operations, kindling development, and performing simulations, 12 rats were excluded due to the absence of correspondence to criteria for inclusion – 5; not stable electrodes fixation – 2, death – 2, the incorrect location of electrodes – 3 rats.
Surgery
In animals, anesthesia was performed with i.p. ketamine administration (100 mg/kg, i.p., Farmak, Ukraine). Rats were placed in a stereotactic apparatus, “SEZh-5” (Kyiv, Ukraine). A 0.5% Novocain solution infiltration (Darnitsa, Ukraine) was used for local anesthesia, and the head was shaved and cleaned with iodine before incision. EEG acquisition and stimulation electrodes were placed in relation to the bregma after skin dissection (2 cm along a middle sagittal axis) and the removal of all soft tissues from the skull surface. A 1.5-2.0 mm burr hole for EEG acquisition and a 3.0–4.0 mm hole for stimulation electrodes were drilled through the cranium with a standard dental portable drill (Colt 1, Charkov, Ukraine).
The nichrome monopolar electrodes were implanted in the ventral hippocampus (AP=-4.3; ML = 4.5; DV=-8.0) and frontal cortex (AP = 1.7; ML = 2.0; DV=-1.0) of both hemispheres, according to the rat brain atlas [30]. The custom-made nichrome electrodes had a diameter of 0.15 mm and an interelectrode distance of 0.25 to 0.30 mm, except for the tip, which was insulated with 25 µm polyesterimide. They were implanted at the left LCN (AP=-10.8; ML = 3.5; DV = 6.0). The reference electrode was located in the nasal bones. The electrodes were fixed on the cranial surface with dental cement. A total of 5.0 ml of 0.9% NaCl solution was heated to 35°C and injected i.p. at the end of surgery to prevent dehydration. Penicillin potassium salt (100,000 IU/kg, intramuscular injection) was administered every 12 h for 48 h postsurgery to prevent infection. The animals were allowed to recover for 10 to 14 days after surgery before observation.
Electrical stimulation
ES was conducted daily from 11.00–12.00 AM for five days using the ESU-2 electrical stimulator (universal electrical stimulator, FSU), with a 100 Hz impulse frequency, 0.25 ms monophasic pulse duration, 50–100 mcA intensity, and 4.5-5.0 s ES duration manually controlled (Fig. 1). Sham stimulations were conducted by connecting the animal's electrodes to the stimulator without delivering an electrical current.
EEG data acquisition and analysis
The analog data were acquired using a computer electroencephalograph DX-5000 (Kharkiv, Ukraine), and the data were digitized at a 256 Hz sampling rate. The time constant was 0.1, and the low-pass filter was set at 70 Hz. Shallow frequency and high amplitude (excessive) waves on EEG synchronously appearing in all leads coinciding with behavioral movements were treated as artifacts and excluded from the analysis. The polygraph recordings were analyzed offline visually, and epochs containing artifacts were eliminated.
Histological light microscopic examination
At the end of the administration, the animals were euthanized by an overdose (100 mg/kg) of sodium pentobarbital. Brain specimens from sham-stimulated animals – early kindled (7 animals) and fully kindled (7 animals) seizures (Fig. 1) – were taken for histological examination. Saline-treated false-operated rats (n = 6) were used as a control (not shown in Fig. 1).
Hematoxylin and eosin (H&E) study specimens for light microscopy were fixed in 10% buffered formalin and processed to prepare 5-µm-thick sagittal paraffin sections for Harris H&E staining [31].
Light microscopic specimens were examined under a Nikon Eclipse E-400 light microscope (Nikon Corporation, Japan) equipped with a digital imaging system and image analysis software (ImageJ, free access).
The cerebellum was dissected starting 2.0-2.4 laterally from the sagittal line, and tissue samples were taken, including anterior lobules IV and V, simple lobule, crus one, crus two ansiform lobule, and paramedian lobule [30, Outline resource 1]. Purkinje cells (PC), which were identified by the cell body, were counted in lateral parts of cerebellar lobules for each slice. Altogether, no fewer than 200 cells were counted for each sagittal cerebellar section at 200× magnification, and then the mean value for each section was estimated. The density of PC was calculated as the mean cell count per millimeter length of cerebellar tissue [32, 33].
The sections were also assessed for microscopic alterations pertaining to neuronal damage, such as distorted morphology of cells or pyknotic nuclei.
Visual control of electrode locations
All experimental animals were euthanized with Nembutal (100.0 mg/kg, i.p.). Upon completion of the experiment, the visual quality control of the electrode placement was ex tempore on the gently removed tissue (no transcardial perfusion). For this purpose, electrocoagulation was performed in the electrode placement area, applying a direct current with an amplitude of 5.0 mA over 30 s and using the electrodes as an anode [9].
Statistics
Values were compared using one-way ANOVA and Newman-Keuls test for the latency of seizures; Kruskal-Wallis followed with a post hoc test for seizure severity; and "z" criteria for comparing two proportions. The results are presented as M ± S.D. The Shapiro-Wilk test for normality was used for the latent period. P values < 0.05 were considered significant. Only observations falling between the median ± 3.0 S.D. of the sample were included in the dataset to avoid the influence of outliers.