2.1 Ethics statement and animal care
This study was conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of Okayama University Faculty of Medicine. The protocol was specifically approved by the Institutional Animal Care and Use Committee of Okayama University Faculty of Medicine (Protocol# OKU-2019651) and was carried out in accordance with the ARRIVE guidelines (https://arriveguidelines.org). Adult male Sprague-Dawley rats (Shimizu Laboratory Supplies Co., Ltd., Japan) weighing 250 to 300 g at the beginning of the study served as subjects for all experiments. Animal housing consisted of individual cages in a temperature- and humidity-controlled room that was maintained on a 12 h light/dark cycle with ad libitum access to food and water. Considering homeostatic control, all experiments were conducted during the daytime, and KA was administered between 8:00 A.M. and 11:00 A.M. Care was taken to record each animal at the same time of day. In addition, if the body weight decreased by more than 20% during the study, the experiment was terminated, and the animals were euthanized. In case of prolonged epileptic seizures, diazepam (8 mg/kg) was administered intraperitoneally for its anti-seizure effect, in the interest of animal welfare.
2.1.1 Experiment 1 Evaluation of a rat model of status epilepticus by behavioral test, immunohistochemical investigation and electroencephalograms
The male Sprague-Dawley rats (n = 7 in each group) was received implantation of the device and spinal cord stimulation at day 0. Stimulation continued for 9 consecutive days, with battery replacement every 3 days. The animals were injected with KA (10 mg/kg) on day 2. The behaviors of rats were video monitored for 6 h after KA injection and seizure severity was evaluated by the Racine scale 22. The animals were returned to the vivarium after the seizure evaluation period. Seven days after KA injection, rats underwent euthanasia and brains were harvested for immunohistochemical examination to evaluate glial cells: Ionized calcium binding adapter protein 1 (Iba-1), glial fibrillary acidic protein (GFAP) and 4',6-diamidino-2-phenylindole (DAPI) staining). In another cohort, we also measured electroencephalograms (EEGs) for 6 h after the administration of KA (n = 3 in each group).
2.1.2 Experiment 2 Evaluation of molecular mechanisms with qRT-PCR
We performed qRT-PCR (n = 5 in control and 300 Hz group) to investigate the mechanism after confirming the anti-seizure effects for the high-frequency group (Fig. 1A). In the qRT-PCR group, the hippocampus was isolated from excised brains and evaluated for C-C motif chemokine ligand 2 (CCL2), C-C motif chemokine receptor 2 (CCR2), interleukin-6 (IL-6), IL-1β, tumor necrosis factor-α (TNF-α), Janus kinase 2 (JAK2), and signal transducer and activator of transcription 3 (STAT3), respectively.
2.2 Surgical procedure of SCS therapy
SCS surgery was performed as described previously 23. The rats were anesthetized with isoflurane and placed in a stereotaxic instrument (Narishige, Japan). Following xylocaine injection to the midline of the head, animals underwent skin incision from the midline of the head to the back, and the spinal muscles were then carefully dissected so they were exposed and a C3 laminectomy was performed (Fig. 1B). We implanted a silver bipolar ball electrode (anode electrode) with a diameter of 2 mm epidurally on the dorsal surface of the spinal cord and fixed it to the muscle using 5 − 0 silk thread (Fig. 1C). We then drilled through the right occipital bone using a small hand drill and implanted a cathode electrode (grounding electrode) on the epidural space, with the lead tunneled subcutaneously to the back of the rats. Finally, the rats received a stimulation device that was fixed on their back using 1 − 0 silk thread at four fixing holes and encased in a protective jacket (Fig. 1D, E). The device is small, and the stimulation was performed continuously up to sacrifice. In the stimulation group, stimulation began immediately after surgery.
2.3 Small mobile device for continuous electrical stimulation
We developed an electrical stimulation device called SAS-200 (Unique Medical Co., Ltd., Japan) that offered convenient adjustment of stimulation conditions via Bluetooth and allowed free movement of the rats owing to its small size 23, 24 (Fig. 1D). The SAS-200, which was attached to the back of the rats and was connected to the SCS electrode, delivered the stimulation. This stimulation required no anesthesia, thereby allowing rats to move around freely and making continuous stimulation possible. Additionally, the stimulation conditions could be easily adjusted wirelessly. The SAS-200 measured 20 mm × 40 mm × 20 mm, with a net weight of 26 g (including the battery). The stimulation parameters were as follows: pulse width, 100 µs; frequency, 2, 50, and 300 Hz; intensities, correspond to 80% of motor thresholds. It consisted of a control panel, a rechargeable lithium-ion battery, and an aluminum case. A standard Windows PC with a specific application controlled these stimulation conditions, namely, the beginning, duration, and particular conditions. A battery change involved simply removing the screws and replacing the depleted battery with a fully charged battery.
2.4 Behavioral tests
We used the Racine scale (stage 1, absence-like immobility; stage 2, hunching with facial automatism and/or abducted forelimbs, wet-dog shaking; stage 3, rearing with facial automatism and forelimb clonus; stage 4, repeated rearing with continuous forelimb clonus and falling; and stage 5, generalized tonic–clonic convulsions with lateral recumbence or jumping) based on previous literature 22. Video monitoring was performed for 6 h after the administration of KA, and the seizure frequency, the mean value of the Racine scale for each hour, and the time from administration of KA to convergence of seizures were evaluated.
2.5 Construction of electrode implantation and EEG recording
After the rats were implanted with a ground electrode on the epidural space, we drilled a hole on the right frontal bone (anode) and two holes on the left parietal bone (reference and ground) (Fig. 2A). Next, animals received three epidural electrodes, which were fixed using dental cement. We secured a protective cap over the bone and the cord was housed inside. Two days after the operation, the animals were anesthetized with isoflurane again, and the EEG-electrodes were connected to the recording system (MEB-2200 Neuropack®, NIHON KOHDEN) and filtered (high-pass filter cut-off 0.5 Hz, low-pass filter cut-off 100 Hz). The animals were injected with KA solution (10 mg/kg), video EEG monitoring was performed for 6 h (Fig. 2B-G), and we evaluated the number of isolated spikes and seizures (high-voltage sharp waves lasting more than 4 s) 25.
2.6 Immunohistochemical investigations and morphological analyses
Seven days after KA injection, each animal was anesthetized and perfused transcardially with 4% paraformaldehyde in 0.01 M phosphate-buffered saline (PBS). We then harvested the brains carefully, post-fixed them in the same fixative, and soaked them in 30% sucrose solution. The brains were coronally cut and then embedded in Tissue Tek O.C.T. compound (Sakura Finetek, Torrance, CA) and subsequently sectioned at 35 µm thickness using a Leica freezing microtome. The following primary antibodies were used for tissue staining: rabbit anti-Iba1 antibody (1:250; Wako Pure Chemical Industries, Osaka, Japan), rabbit anti-GFAP antibody (1:1000; Novus Biologicals, Littleton, CO). After rinsing in PBS, sections were incubated for 1 h in FITC-conjugated affinity-purified donkey anti-rabbit IgG (H + L) and 4,6-diamidino-2-phenylindole (DAPI; 2 drops/mL, R37606; Thermo Fisher, Waltham, MA) in a dark chamber. The sections were then extensively washed with PBS and coverslipped. The fluorescent immunoreactivities were visualized using an inverted fluorescence phase-contrast microscope BZ-X710 (Keyence, Osaka, Japan). The number of Iba-1 and GFAP cells in the CA1, CA3, and dentate gyrus was counted using six randomly selected regions (500 × 500 µm2, from 2.8–4.52 mm posterior from the bregma) in each rat.
2.7 qRT-PCR
The hippocampus was collected immediately after decapitation 4 h after KA administration in the control group and the 300 Hz group (n = 5). Tissue samples were placed in individual tubes containing the tissue storage reagent and were stored at -70˚C until RNA isolation. Syntheses of cDNA and qRT-PCR procedures were conducted as described previously 26,27. As an internal control, we used GAPDH mRNA. The primer sequences used were as follows: CCL2: forward, GAG TAG GCT GGA GAG CTA CAA GAG; reverse, AGG TAG TGG ATG CAT TAG CTT CAG. CCR2: forward, CTT GTG GCC CTT ATT TTC CA; reverse, AGA TGA GCC TCA CAG CCC TA. IL-1β: forward, AGG CTT CCT TGT GCA AGT GT; reverse, TGA GTG ACA CTG CCT TCC TG. IL-6: forward, CCG GAG AGG AGA CTT CAC AG; reverse ACA GTG CAT CGC TGT TC. TNF-α: forward, CCA ACA AGG AGA AGT TCC; reverse, CTC TGC TTG GTG GTT TGC TAC. JAK2: forward, CTG AAA TCC TTG CAG CAT GA; reverse, CTC CAT GCC CTT GCA TAT CT. STAT): forward, CAG CCA AAC TCC CAG ATC AT; reverse, TCT GCT TTC ACA GCC ATC AC.
2.8 Statistical analyses
We used GraphPad Prism ver.9.00 for Windows (GraphPad Software, San Diego, CA). Unpaired t-test or the Mann–Whitney U test was used for analyzing the qRT-PCR between the control group and the 300 Hz group. Single analysis of variance and the post hoc Tukey’s test were used for analyzing the number of Iba-1- and GFAP-positive cells in each seizure stage. Significant differences were preset as p < 0.05. Data are shown as means ± standard error (SE).