Diazepam

In deeply anesthetized rats (intraperitoneal (ip) injected 40 mg/kg thiopental (Rotexmedica, Germany) and 10 mg/kg diazepam (Apaurin; Krka, Slovenia)), we induced abdominal compartment syndrome by intraperitoneal insufflation of ordinary air controlled by a manual and digital manometer with a data logger connected to a computer (DD890, Dostmann Electronic GmbH, Germany) and maintained high abdominal pressure at 25 mmHg for 120 min before sacrifice, with a pressure measurement interval of 1 s. High abdominal pressure at 25, 30, 40, or 50 mmHg was maintained until sacrifice at 60 min (25 mmHg), 30 min (30 mmHg, 40 mmHg), or 15 min (50 mmHg). Rats received BPC 157 (10 µg or 10 ng/kg subcutaneously) or saline (5 ml) at 10 min abdominal compartment syndrome-time. Alternatively, using esketamine anesthesia (40 mg/kg esketamine (Rotexmedica, Germany) and 10 mg/kg diazepam (Apaurin; Krka, Slovenia) intraperitoneally), we induced abdominal compartment syndrome as described before and maintained high abdominal pressure at 25 mmHg for 120 min before sacrifice. Medication (BPC 157 (10 µg or 10 ng/kg sc) or saline (5 ml)) was given after 10 min of high abdominal pressure.
Recordings of brain swelling were performed in rats before sacrifice after complete calvariectomy was performed (Gojkovic et al., 2021a (link); Knezevic et al., 2021a (link); Knezevic et al., 2021a (link); Knezevic et al., 2021b (link)). Briefly, six burr holes were drilled in three horizontal lines, all of them medially to the superior temporal lines and temporalis muscle attachments. The two rostral burr holes were placed just basal from the posterior interocular line, the two basal burr holes were placed just rostral to the lambdoid suture (and transverse sinuses) on both sides, respectively, and the two middle burr holes were placed in line between the basal and rostral burr holes.
Rats were laparatomized before sacrifice for the corresponding presentation of the peripheral vessels (azygos vein, superior mesenteric vein, portal vein, inferior caval vein, and abdominal aorta). The recording was performed with a camera attached to a VMS-004 Discovery Deluxe USB microscope (Veho, United States) at the end of the experiment and assessed as before (Gojkovic et al., 2021a (link); Knezevic et al., 2021a (link); Knezevic et al., 2021a (link); Knezevic et al., 2021b (link); Strbe et al., 2021 (link)).

Video recordings from SHD and RLD groups were used to score behavioral seizures on modified Racine scale at 5 min epochs as previously described [33] (link). The persons scoring the episodes were unaware of the treatment groups. In each epoch, the highest seizure stage reached was considered. For example in a 5 min epoch, if stage-1 and -2 seizures occurred during the first 3 min followed by a 2 min stage-3 seizure, then the highest stage considered for behavioral scoring was CMS stage-3. This way, the seizure severity was calculated as cumulative seizure severity score (mean ± standard error) for the duration of SE based on the total amount of time spent in CMS from the time they reached first CMS to the time they received diazepam. The more severe the seizures during the SE, the higher the score. In the present study the SE refers to the 2 h duration starting from the first onset of CMS (stage≥3 for SHD group or stage-5 for RLD group) after administering KA to the time-point they received diazepam. Student's unpaired t-test was used to compare the mortality rate, duration of CMS and stage-5 seizures alone between the SHD and RLD groups using GraphPad Prism software. To compare the mortality rate between SHD and RLD groups, the mice that died were taken as “0” and survived as “1”. To compare the number of animals that reached stage-5 seizures in SHD and RLD groups, the mice that did not reach stage-5 was taken as “0” and the mice that reached were taken as “1”. The spike rate between SHD and RLD groups were compared using two-way ANOVA.

Sample size and power calculations were performed using G*Power 3.1, an open-source statistical power analysis program (Faul et al., 2007 (link)). The sample size was chosen based on previous experiments in our lab investigating the effects of the sedative diazepam on sleep (McKillop et al., 2021 (link)), which indicated an effect size of Cohen’s d=0.90 for the key outcome parameter NREM sleep time. We therefore decided that our study should be sufficiently powered to detect effects of sizes of d=1 designed between saline and individual CNO treatment conditions with a power of 0.9 at the given α-error probability of 0.05. The estimated sample size from this calculation was 12–13 animals per group. Based on experiences from previous EEG studies in mice, we aimed to account for an attrition rate of approximately 20% and decided to include 16 animals in this study.