Animals and Treatments
All animal experiments were carried out in compliance with the ARRIVE guidelines and were approved by the Animal Care and Use Committee of Aviation General Hospital of China Medical University Laboratory (HK2022-03). Four to five week-old male ICR mice (Vital River, Beijing, China) weighing 25-27 g were maintained in cages under a 12 h light/12 h dark cycle, with room temperature kept at 23 ± 2 ℃ and kept humidity at 50 ± 5 %. Standard rodent chow and water were provided ad libitum. Investigators assigned randomly animals to four groups (n = 15 per group): the control (Ctrl) group, sleep deprivation (SD) group, low dose (LD) (11.5 μg/ml, i. p.) group, and a high dose (HD) (23 μg/ml, i. p.) group. Except for the control group, all groups had successive 21-day periods of sleep deprivation after 7 days of adaptive feeding (from 10 p.m. to 4 p.m. per day, 4 h for rest). The mice were administered ozone, once a day, from day 14 to day 21 at 4:30 p.m., and the Ctrl and SD groups received physiological saline (0.9% NaCl, 10 ml/kg, i. p.) simultaneously.
Preparation of Ozone Water
We used ozone therapy device to produce ozone water (KASTNER, German). This device can generate OW from O2 and water while while also can control the concentration of the dissolved O3. In every trial, OW was administered within 10 min after produced.
Establishment of the Chronic REM Sleep Deprivation Model
The chronic REM sleep deprivation model was established by a modified multi-platform water environment method [45]. Fifteen interconnected platforms (platforms were of 1cm diameter each) are put in the water tank (90 cm × 60 cm × 40 cm). The water reached a depth of 1.5 cm below the platform's base and was maintained at a temperature of 25 ± 2 ◦C. Therefore, when the animal entered the REM sleep state, it would fall into the water and wake up. The animals had unrestricted access to food and water. Mice of the Ctrl group were placed on larger platforms (7 cm in diameter) surrounded by water with their cage mates. Prior to the chronic REM sleep deprivation, the mice were placed on the platform to adapt to the condition for 1 week (30 minutes per day).
Morris Water Maze Test
MWM is a classic experiment used to assess rodent spatial memory ability [46]. The MWM consists of a black tank which was filled with enough fresh water (22-25 °C). Animals were taught to swim to a platform that was submerged 0.5 cm beneath the water. The spatial acquisition trial and the probing trial are the two components of the MWM trials. For the spatial acquisition experiment, four days of learning and followed by tests of learning. In brief, mice were randomly assigned to one of four different starting positions for four training trials per day. Each mouse was given 60 s to look for the platform in each trial. If the mouse was unable to locate the platform, the experimenter directed it to climb the platform and become acquainted with its surroundings for 30 s. The probe trial was held on the fifth day following the last day of training (Fig. 1). The hidden platform was removed before the probe trial. Mice were placed in the maze at 180° from their original platform position and permitted to swim in the tank for 60 s.
Open Field Test
This test was used to assess the locomotor activity and anxiety-like behavior in rodents [47]. Mice (n=6 per group) were placed in the open field apparatus (cubic black box of 42 cm × 42 cm × 25 cm in size) and allowed to move freely for 5 min. After each test, the apparatus was carefully cleaned with alcohol.
Novel Object Recognition Test
The NOR is a behavioral method that assesses the rodent's recognition and memory ability by utilizing the rodent's inherent tendency to approach and explore novel objects [48]. Two identical objects were presented, and the mice (n=6 per group) were free to explore for 5 min. Next, one of the objects is replaced by a novel one and the experiment is repeated. To eliminate potential odors, alcohol should be sprayed on the test equipment every two test intervals. The discrimination index was calculated as the percentage of time spent in exploring the novel object divided by total amount of time in exploring both objects.
Nissl Staining
Nissl staining revealed changes in the expression of hippocampal neuronal cells in mice. The dehydration and paraffin embedding methods described above were used. Brain slices were placed horizontally with drops of Nissl staining solution (0.5 % toluidine blue) for 10 - 20 minutes before being washed and dehydrated. Finally, xylene was used to clean the slides before the cover was slipped. The number of neurons in the CA1 and CA3 regions were counted by using an ImageJ analysis system (version 1.43, National Institutes of Health, USA).
Golgi-Cox Staining
Golgi-Cox staining of experimental samples to clearly observe the dendritic spines on neurons (n=3 per group). Briefly, under deep anesthesia with 2 % pentobarbital sodium (50 mg/kg), mice were transcardially perfused with 0.9% saline. Then the whole brains of the mice were immersed in Golgi-Cox solution in the dark for 14 days. Every 48 hours, the staining solution was changed. After that, the distilled water were used to wash tissues (three times), immersed overnight in 80% glacial acetic acid, washed again in distilled water, then put in 30% sucrose. Finally, the dried tissue slides (100 μm) were treated with concentrated ammonia (15 mins), distilled water (washed 1 min), acidic firm film fixing solution (15 mins), distilled water (3 mins), dried, and sealed with glycerin gelatin. The Panoramic 250 (3D Histech) digital section scanner was used to capture images, which were then processed with ImageJ software for spine density analysis.
Western Blotting
We used cold PBS to wash tissues twice or three times with before being homogenized in 1% PMSF RIPA lysis buffer (Servicebio, Wuhan, China). Total proteins were quantified using the BCA protein assay kit (Servicebio, Wuhan, China). After activation with methanol, samples (n=3) were separated by SDS PAGE and electro-transferred onto PVDF (0.45 um) membranes. Following that, the membranes were blocked for 1 hour at room temperature. Then, the membranes were incubated with the following primary antibodies overnight at 4 °C: PSD-95 (1:1000, P78352, Cell Signaling Technology, USA), SYN (1:1000, P08247, Cell Signaling Technology, USA), Sema3A (1:1000, ab199475, Abcam, UK), CRMP2(1:10000, ab129082, Abcam, UK), phosphor-Thr514-CRMP2 (1:1000, ab62478, Abcam, UK), Acet-α-Tubulin (1:1000, sc-23950, Santa Cruz Biotechnology, USA) and β-Actin (1:1000, GB11001, Servicebio, China). TBST was applied to wash the membranes five times, each for five minutes. After that, the membranes were incubated in blocking buffer with HRP conjugated Goat Anti-Rabbit IgG (1:3000, GB23303, Servicebio, China) for 1 h before being washed with TBST. For capturing images, the enhanced chemical lighting (ECL) method was applied. The optical density value of the target band was quantified with the ImageJ (n=3 per group).
Immunohistochemistry
After deparaffinizing and rehydrating the paraffin section, the tissue sections were put in a microwave oven which contain with citric acid antigen repair buffer (pH 6.0) for antigen repair. Subsequently, the tissues were incubated in 3 % hydrogen peroxide to block endogenous peroxidase and sealed with 3% BSA. Then incubated with primary antibodies: Sema3A (1:8000, ab199475, Abcam, UK), PlexinA1(1:200, orb6757, Biorbyt, UK), CRMP2(1:1000, ab129082, Abcam, UK) at 4 ◦C overnight. The biotinylated HRP goat anti-rabbit (IgG) secondary antibody was added and incubated for 50 minutes at room temperature following washed with PBS. The immune reaction was visualized by incubating with DAB for 5 min. The Image-Pro-Plus software (Media Cybernetics, Silver Spring, USA) was used to calculate the average optical density value.
ATP Synthase Activity
The Mitochondrial Complex V Kit (AIDISHENG, Jiangsu, China) was applied for measuring ATP synthase activity after isolating mitochondria from the hippocampus following the manufacturer's instructions. ATP synthesis results in production of ATP which is accompanied with the reduction of NAD+ to NADH that is then monitored as an increase in absorbance at 340nm. The OD340 was continuously monitored using a Spectra Max M5 microplate reader (Molecular Devices, USA).
Statistical Analysis
All of the data were presented as the mean ± SD. All statistical analyses were carried out using SPSS 26.0 software (IBM Corporation, Armonk, NY, USA). All charts are created by GraphPad Prism 8.4.3 software (GraphPad Software, La Jolla, CA). The difference between two groups was compared using the unpaired t-test. Multigroup comparisons were carried out using one-way ANOVA followed by Tukey post-hoc test. Values of P < 0.05 were considered statistically significant. All experiments were repeated at least 3 times.