Animals

The experiment was conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals in China (No. 14924, 2001) and was approved by the Ethics Committee of Zunyi Medical University (ZU21–2112–001). C57BL/6 mice, aged 20–22 days and weighing 8–10 g (equivalent to human infancy [13]), were obtained from China Tianqin Biotechnology Co., Ltd. The animals were housed and maintained in a pathogen-free facility under controlled environmental conditions, including a room temperature of 22–25 ℃ and a 12-h light/dark cycle. Food and water were provided ad libitum. This study exclusively utilized male mice. Any changes in the color of the gingival mucosa to pale (from the normal pink), an increase in respiratory rate, or abnormal heart rate during drug administration may indicate hypoxia and should be addressed promptly.

Determination of the drug dose

Dose–response curves [14] were generated to determine the 50% effective dose (ED50) and 95% effective dose (ED95) for the loss righting reflex (LORR) in mice induced by remimazolam tosylate (Jiangsu Hengrui Pharmaceutical Co. Ltd., China) and midazolam (Enhua Pharmaceutical, China). The specific steps are as follows:

First, the initial dose estimation of the two sedatives was performed. In a previous study [15], when CNS 7056 was 15 mg/kg, the number of LORR/total mice was 0/8, whereas when CNS 7056 was 20 mg/kg, the number of mice with LRR/total mice was 2/8; thus, we used 20 mg/kg as the initial dose of remimazolam. When the midazolam concentration was 30 mg/kg, the number of mice with LRR/total mice was 0/8, whereas when the midazolam concentration was 40 mg/kg, the number of mice with LRR/total mice was 7/8; thus, we used 40 mg/kg as the initial dose of midazolam.

Second, the LORRs of the maximum dose (Dmax) and the minimum dose (Dmin) were determined. Dmax indicates that no LORR occurred within 10 min, and Dmin indicates that all LORRs did not occur within 10 min. The common ratio was calculated as follows: q = n-1 √ Dmin/Dmax (n = 6 groups); the dose of each subgroup was Dmax × q (k-1); and K was the group serial number (K = 1,2,3…..6). We refer to the formula of the resource equation to estimate the sample size. The formula of the resource equation is E = N-K = Kn-K = K(n-1), where n is the total number of experimental units, n is the sample size of each group, k is the number of treatment groups, and e is the difference between them [16]. From this, we can obtain the minimum and maximum number of animals in each group: Min n = 10/K + 1, Max n = 20/K + 1. In this study, there are three groups (that is, K = 3). The results were integrated according to the formula Max n = 20/K + 1, and 6 mice in each group were used to explore the equivalent dose.

Finally, based on the previously mentioned formula, the typical q-value ratios for remimazolam and midazolam were 0.86 and 0.80, respectively. In a random manner, the mice were allocated into 6 subgroups, with 6 mice in each group. The injection volume was standardized at 0.1 mL per 10 g of body weight. Ultimately, the number of mice exhibiting LORR was documented within 10 min following the intraperitoneal administration of sedatives.

Randomized grouping and anesthesia protocol

Eighty-four juvenile mice were randomly distributed among three groups, namely, the normal control group (Con), the remimazolam group (Remi), and the midazolam group (Mida), with each group comprising 28 mice. Remi and Mida received intraperitoneal injections of their respective ED95 doses. The injection volume for each group was 0.1 mL/10 g of body weight, and the mice received two injections daily with a 4-h interval for 7 days.

Behavioral tests

An automatic tracking system (any-MAZE behavior software) was used to monitor all the behavior tests.

Y-maze test

The Y-maze test was conducted following 24 h of repeated drug exposure to assess the short-term memory of the mice by measuring their preference for exploring new pathways [17]. The Y-maze was composed of a start arm (always open), a second arm (always open), and a novel arm (initially blocked in the first test but accessible in the second test). The angle between each arm was set at 120 degrees. During the training phase, the mice could move freely among the open arms for 5 min. After 1 h, the memory assessment was performed, and the mice were permitted to investigate all three arms freely for another 5 min. After each Y-maze session, the waste was cleaned, and 75% ethanol solution was applied to eliminate odors from the maze floor and inner walls. The quantity, duration, and distance of items explored in each arm were recorded. A reduction in the percentage of items in the novel arm indicated impairment in short-term memory.

Morris water maze (MWM) tests

The MWM test was performed on the second day of the Y-maze (n = 24 mice). The MWM test is a classical method that can be adopted for evaluating spatial learning and long-term memory, and the specific process has been previously described [18]. The maze test was classified into two distinct parts: the memory training test and the memory retention test.

Throughout the 6-day training phase, the mice were positioned into the pool and tasked with locating the platform within a 60-s time frame. After finding the hidden platform, the mice were allowed to stay there for 10 s. If the mouse did not find the platform within a minute, the mouse was directed to the platform and allowed to stay there for 10 s.

The platform was removed on the 7th day. All the mice were then directed to enter the water, and their swimming path, duration spent in the initial platform quadrant, and frequency of crossing the original platform within 60 s were recorded. This observation assessed alterations in the mice's spatial orientation abilities and memory.

After behavioral evaluation, each group of mice (n = 18 mice) was classified into three groups (for western blot analysis, TUNEL detection, and electrophysiology; n = 6 in each subgroup). They were anesthetized with 3% sevoflurane (Lunan Better Pharmaceutical Co., Ltd., China).

Western blot analysis

The heart was perfused with 200 mL of normal saline. Whole brain tissue was quickly dissected and collected on ice. Hippocampal tissue was isolated and preserved at – 80 °C.

Protein extraction was performed with a BCA kit (C503021-0500, Sangon Biotech, China) to determine protein concentrations. Following established protocols, western blotting was conducted [19]. SDS–PAGE sample loading buffer-5 × (C510003–0050; Sangon Biotech, China) was used to denature the proteins. The extracts were resolved via SDS-PAGE and then subjected to western blot analysis. In our study, the following antibodies were utilized: a rabbit polyclonal antibody against caspase-3 (AF6311; Affinity Biosciences, USA; RRID: AB_2835170), a rabbit polyclonal antibody against BDNF (DF6387; Affinity Biosciences, USA; RRID: AB_2838350), a rabbit polyclonal antibody against PSD95 (AF5283; Affinity Biosciences, USA; RRID: AB_2827690), and a mouse monoclonal antibody against beta-actin (T0022; Affinity Biosciences, USA; RRID: AB_2839417). The bands were visualized with Western Bright ECL reagent (Advansta, United States) and imaged with a Fusion IPP image analysis system (Media Cybernetics, USA).

TUNEL detection

After anesthetizing and perfecting the heart with cold PBS and 4% paraformaldehyde (PFA), intact brain tissue was acquired and fixed in 4% PFA at 4 °C for 48 h. Then, the brain tissue was incubated in 30% sucrose consecutively at 4 °C until it completely sank. Dehydrated brains were embedded in optimal cutting temperature (OCT) compound and frozen at – 80 °C. Using a freezing cryostat (CM1950, Leica, Germany), the tissues were sliced into 30-µm serial coronal sections. These sections were then classified into three equal groups and preserved in 0.01 M PBS at 4 °C. Apoptosis in hippocampal neurons was assessed following the instructions provided with a tuned cell apoptosis detection kit (Beyotime Biotechnology, c1088). The cells were sealed with a sealing solution containing DAPI and visualized via an inverted fluorescence microscope (BX51W1–IR7). ImageJ software was used to compute the gray values, to determine the staining intensity and number of nuclei in the observed region.

Measurement of long-term potentiation (LTP) of the Schaffer collateral

Previous studies were performed [20]. Mouse brain tissue was immersed in artificial cerebrospinal fluid (ACSF) at a temperature of 0–4 °C for 5 min. Subsequently, the relevant coronal tissue block of the target nucleus was retained following the stereotactic map of the mouse brain. Brain slices (400 µm) were prepared with an automatic oscillating microtome, and the experiment started after the samples had incubated for 1 h.

The stimulation electrode was positioned on the side branch of the CA3 → CA1 Schaffer pathway, and the concentric stimulation electrode was linked to the Master 8 stimulator via the ISO flex isolator. For recording, a glass microelectrode filled with 4 mol/L NaCl was employed, which was inserted into the pyramidal cell layer of the CA1 region and connected to an EPC10 HEKA patch clamp amplifier. The basic stimulus intensity was set at 40% of the maximum response. The average fEPSP slope over a 20-min period was recorded as the baseline value. Three sets of 200 Hz, 0.1 mA high-frequency stimulation (HFS) were subsequently administered at intervals of 200 ms to induce fEPSP, and the outcomes were monitored over a 90-min duration. If the slope of the fEPSP was increased by 30% and maintained for at least 30 min, LTP induction was considered successful; otherwise, it was eliminated.

Statistical analysis

All the data were subjected to one-way analysis of variance (ANOVA) via IBM SPSS Statistics 19.0 (IBM Corp., Armonk, NY, United States). The estimation of ED50 and ED95 was conducted via probit analysis. The results are shown as the mean ± SD. The results of the Y-maze test, western blot analysis, immunofluorescence, and electrophysiology were analyzed via one-way ANOVA among groups, and a homogeneity of variance test was conducted before analysis. To compare the mean values among multiple groups, a pairwise least significant difference (LSD) test was used in this study. The Morris water maze test results were analyzed via repeated-measures analysis of variance mixed model and two-way repeated-measures analysis of variance (group × days), and those that did not satisfy Mauchly’s spherical test results were corrected via the Greenhouse–Geisser method. When p was less than 0.05, statistical significance was identified.