All experimental procedures were approved by the Keio University Institutional Animal Care and Use Committee (authorizations A2021-006 and A2022-257). All procedures were performed in accordance with university guidelines and Animal Research: Reporting of In Vivo Experiments (ARRIVE) reporting guidelines for the care and use of laboratory animals.

Fifty C57BL/6 mice (25 males and 25 females, 8 weeks old) were purchased from CLEA Japan Inc. (Tokyo, Japan). They were housed for acclimatization under a 12-h dark–light cycle in an air-conditioned room (temperature of 23.0 °C ± 1.0 °C and humidity of 55% ± 7%) with unrestricted access to water and food for > 1 week before conducting the following experiments.

Of the 25 male and 25 female mice, the following animals were excluded from the analysis: Male mice, 2 due to death during exposure to cigarette smoke and 1 due to insufficient sample volume for blood gas analysis in the smoking group, 2 due to poor blood gas, and 1 due to a failure of femoral artery catheterization in the sham group; female mice: 2 due to death during smoke exposure, 1 due to poor blood gas, and 1 due to insufficient sample volume for blood gas analysis in the smoking group, 3 due to poor blood gas, 1 due to insufficient sample volume for blood gas analysis, and 1 due to ear bleeding in the sham group. Thus, 7–10 mice from each group (male-smoking, male-sham, female-smoking, and female-sham) were analyzed.

The mice were randomly assigned to either smoking or sham groups. Mice in the smoking group were restrained in an immobilization tube and exposed to cigarette smoke from commercially available filtered cigarettes (Marlboro, 12 mg tar/1.0 mg nicotine, Philip Morris, Richmond, VA) through nasal inhalation for 1 h, as previously reported. Following dilution with compressed air, the mass concentration of total particulate matter was 1,202 ± 196 mg/m3 [13]. Mice in the sham group were restrained in the same setting as the smoking group for immobilization and exposed to room air. One hour was allowed for recovery after exposure to cigarette smoke or room air so that the physiological state could remain unchanged during the recordings.

The following parameters were recorded: direct current (DC) potential, regional cerebral blood flow (rCBF), systemic arterial blood pressure (ABP), and heart rate (HR). The rectal temperature was maintained at 37 °C with a heating pad and thermocontroller (BWT-100, Bio Research Center Co. Ltd., Nagoya, Japan) as described previously [14] (Fig. 1-(a), (b)). In brief, one hour after smoking or being restrained, a catheter was inserted into the femoral artery to monitor blood pressure under isoflurane anesthesia (2%–2.3%). The mouse was secured to a head-holder (SGM-4, Narishige Scientific Instrument Laboratory, Tokyo, Japan). Prior to CSD evaluation, arterial blood was drawn through the catheter for blood gas analysis with RapidLab 348EX (Siemens AG., Munich, Germany). DC potentials were monitored using three electrodes (EEG-5002Ag; tip diameter = 200 µm; Bio Research Center) that were fixed in the bilateral parietal (± 2 mm lateral and 2 mm caudal to the bregma) and right frontal (2 mm lateral and 1 mm rostral to the bregma) openings in the skull. Over the parietal bone, rCBF was monitored bilaterally using laser Doppler flowmeters (ALF21, Advance Co. Ltd., Tokyo, Japan). Continuous recordings of DC potential, rCBF, ABP, and HR were stored on a multichannel recorder (PowerLab 8/35; ADInstruments Pty Ltd., Sydney, Australia). The LabChart software (ADInstruments Pty Ltd.) was used for offline analysis (Fig. 1-(b)). We considered it a CSD occurrence when we observed unilateral CBF changes that were characteristic of CSD and were synchronized with the deflection of DC potentials. DC potentials were recorded with monopolar electrodes, with a reference electrode placed subcutaneously in the dorsum.

a Experimental protocol and CSD setup. Fifty C57BL/6 mice (25 males and 25 females) were divided either into the smoking group to receive cigarette smoke exposure for one hour (1 h) or the sham group to receive room air for 1 h in a tube for immobilization. After 1 h of recovery, followed by preparation, the minimum KCl concentration to evoke CSD (threshold of CSD induction) and frequency of CSD induced by a 1 M KCl cotton ball were measured in 1 h. The propagation velocity was calculated from the time differences and distances between two direct current (DC) electrodes. b Representative raw tracing of recorded CSD. DC, direct current; rCBF, regional cerebral blood flow

After reducing the isoflurane concentration to 1.5% and confirming that no CSD occurred for a duration exceeding 30 min, 5 µL of 0.025 M potassium chloride (KCl) solution was introduced into the left posterior hole; the KCl concentration was increased in a stepwise manner by 0.025 M at intervals of at least 5 min. The concentration at which CSD first manifested was designated as the threshold for CSD [14, 15]. CSD induction was validated by the presence of characteristic DC potential deflection and unilateral rCBF fluctuation. The frequency of CSD induced in the right hemisphere was assessed by calculating the number of CSD events that occurred within a 60-min period. The 1-M KCl cotton ball was replaced every 15 min. The propagation velocity of the first-manifested CSD was calculated using the distance and time difference between the two electrodes. CSD measurement was performed in part by a blinded investigator.

We analyzed the threshold to induce CSD, the frequency of CSD at 1 M KCl, and the propagation velocity of CSD (Fig. 1). Animals that were deceased or exhibited poor physiological status, or in which technical errors occurred during the procedure were excluded. In general, we used the Wilcoxon rank sum test. The Wilcoxon rank sum test with Bonferroni correction was used for the statistical analysis of the effect on the CSD threshold. Statistical analysis was performed using the SAS version 9.4 software (SAS Institute, Cary, NC), and graphs were created using the Prism version 9.2.0 software (GraphPad Software, La Jolla, CA).