Experimental animal

Male Mongolian gerbils (6-months-old, 75–85 g of body weight) purchased from Orient Bio (Seongnam, Korea) were used in this study. The animals were maintained at 22 °C and 50% humidity, and were fed freely every 12 h of the day/night cycle. All experimental procedures were performed according to standard work guidelines with due approval (Approval No. GWNU-2017-18) obtained from the Institutional Animal Care and Use Committee of the Gangneung-Wonju National University.

In this study, gerbils (total n = 63) were used. The experimental groups were as follows. There are the Sham group (n = 7) and Induction of transient cerebral ischemia group (Isch group; Ischemia group) (n = 7). The experimental group corresponding to the application time of cathodal tDCS after induction of transient cerebral ischemia (at 0, 5, 10, and 60 min) is divided into 0, 5, 10, and 60 min group (n = 7 per group). The experimental group corresponding to the persistence of cathodal tDCS after induction of transient cerebral ischemia (after 5, 7, and 10 days)) is divided into 5,7 and 10 days group (n = 7 per group).

Induction of transient cerebral ischemia in Mongolian gerbils

Prior to undergoing surgery, the experimental animals were anesthetized with 2.5% isoflurane (HANA PHARM CO., Seoul, Korea) mixed with 34% oxygen and 66% nitrous oxide. A rectal thermometer (TR-100, YSU, USA), along with a heating pad maintained a stable temperature of 37 ± 0.3 °C, was used during the surgery. After exposing the common carotid artery of the experimental animals, it was retracted using 4-0 silk (AILEE, Busan, Korea). Subsequently, the common carotid artery was subjected to clip ligation for 5 min in order to occlude the blood flow. After 5 min, the aneurysm clip was removed, and reperfusion of blood flow was examined. Following the induction of ischemic stroke, the experimental animals were maintained at body temperature in a thermostat to facilitate their recovery. The sham group underwent the same surgical procedure without clip ligation of the common carotid artery. Induction of ischemia was according to the protocol by O’Neill et al. [13].

Electrode fixture implantation and application

Experimental animals were anesthetized with 2.5% isoflurane mixed with 34% oxygen and 66% nitrous oxide. A rectal thermometer (TR-100), along with a heating pad maintained a stable temperature of 37 ± 0.3 °C, was used during electrode implantation and application. An active electrode fixture (cathode) having a size of 5 mm was placed in the calvaria where the bregma is located. while a contrast electrode fixture (positive electrode) was placed on the thoracic skin. Subsequently, a current stimulator was attached to the electrode fixture, and the “20 min stimulation-20 min rest-20 min stimulation” protocol was implemented for the application of cathodal tDCS [22, 23] (Fig. 5). All tDCS applications were performed according to this protocol.

Fig. 5
figure 5

Protocol of cathodal tDCS application after induction of transient cerebral ischemia. After induction of transient cerebral ischemia for 5 min, “20 min stimulation—20 min rest—20 min stimulation” protocol applied in this experiment

Application of cathodal tDCS

Application time of cathodal tDCS after induction of transient cerebral ischemia (at 0, 5, 10, and 60 min)

After induction of transient cerebral ischemia, cathodal tDCS was applied to confirm the alterations within the neurons in the hippocampal CA1 regions, in accordance with the timing of cathodal tDCS application. The application of cathodal tDCS was performed at an intensity of 0.2 mA immediately following the induction of transient cerebral ischemia (0 min), as well as after intervals of 5 min, 10 min, and 60 min. After 5 days, the brain tissue was extracted and analyzed histologically (Fig. 1A).

Persistence of cathodal tDCS after induction of transient cerebral ischemia (after 5, 7, and 10 days)

Cathodal tDCS was applied immediately after the induction of transient cerebral ischemia. To confirm the persistence of this tDCS, brain tissue was extracted 5 days, 7 days, and 10 days after its application. This was followed by histological analysis of the brain tissue (Fig. 3A).

Tissue preparation

To perform histochemical analysis, the animals were first anesthetized. They were perfused intracardially with 0.9% saline and 4% paraformaldehyde in phosphate-buffered saline (pH 7.5). Brain tissues were subsequently extracted and post-fixed with the same fixative for 8 h. Thereafter, the brain tissues were treated with 30% sucrose solution at room temperature for 12 h. The treated tissues were cut to a thickness of 30 µm using a frozen section and then stored in a preservation solution at 4 °C for further studies.

CV staining

Following the induction of transient cerebral ischemia, CV staining of the surrounding nuclei of surviving neurons was performed to confirm the neuronal protective effect according to the stimulation intensity of cathodal tDCS. Brain tissue was mounted on slides coated with gelatin, followed by staining with 1.0% (w/v) CV acetate solution for 40 min. Subsequently, the stained brain tissues were dehydrated in 70% to 100% ethanol and clarified using xylene. They were then mounted using Canada balsam (Kato, Japan). The completed slides were observed under a Axio Imager A2 microscope (Carl Zeiss, Oberkochen, Germany).

F-J C staining

After induction of transient cerebral ischemia, neuronal cell death in the hippocampal CA1 region was confirmed via fluorescent staining of neurons using F-J C staining. The brain tissues were treated with basic alcohol for 5 min and washed with 70% alcohol and distilled water for 2 min. Subsequently, the brain tissues were treated with 0.06% potassium permanganate solution for 20 min and washed twice with distilled water for 2 min each. Staining of the tissues with 0.001% Fluoro-Jade C (Histochem, Jefferson, USA) solution (containing 0.1% acetic acid) was performed for 30 min. Thereafter, the stained brain tissues were washed thrice with distilled water for 1 min each. Finally, the stained brain tissues were dried for 60 min on a slide warmer and mounted using dibutylphthalate polystyrene xylene (DPX) (Sigma, St. Louis, MO, USA). The completed slides were observed under a Axio Imager A2 fluorescence microscope (Carl Zeiss, Oberkochen, Germany) at an excitation wavelength (Ex) of 385 nm and emission wavelength (Em) of 425 nm.

Statistical analysis

The most predominant sites of the stained brain tissues were photographed using a microscope equipped with a CCD camera. To quantitatively analyze of surviving neuron and apoptosis, 10 section per animal were selected with 120 µm interval. The neurons were obtained in a 250 X 250 µm square at the same area of each subregion and counted by averaging total numbers using an image J analyzing system.

The numerical data obtained in this study were expressed as their mean ± SEM. Differences between the two groups were statistically analyzed using one-way ANOVA and Tukey post-hoc test. Data were considered statistically significant if the p-value was less than 0.05.

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