2.1 Animals preparation
This study was performed in line with the principles of the Declaration of Helsinki. All experimental procedures involving animals were approved by the Ethics Committee of Suzhou Xiangcheng People's Hospital (Suzhou, Jiangsu Province, China). Approval document number (No.2020007). Healthy and clean male SD rats, weighing 300-400g and aged 12–16 weeks, were provided by the Experimental Animal Center of Xuzhou Medical University one week before surgery. The feeding temperature was 25 ℃, humidity was 50%, day and night alternated 12 hours /12 hours, and food and water were freely consumed.
2.2 Conditional fear memory test system
The conditioned fear experiment was performed using the infrared video fear system, which consists of two parts: two soundproof boxes A and B with different internal colors to block communication between the tested animal and the outside world, and A ventilation fan to provide ventilation and background noise. Soundproof box each has an experiment box (transparent plexiglass), the experiment box side wall has a computer control sound. At the bottom of the soundproof box A is A conductive stainless steel fence floor. Each metal fence is connected to A current generator. Soundproof box B has a fast detachable hard PVC floor at the bottom. Soundproof box B is equipped with a near-infrared video camera and video analysis system, which can take video images without visible light, and can automatically recognize the frozen behavior of rats in fear, and record the time of the frozen behavior.
2.3 Conditioned fear memory training
The rats were placed in speaker box A before administration and allowed to move freely for 90s. Conditional stimulation: a sound with a frequency of 2.2 kHz, a volume of 96 dB and a duration of 30 s; At the last 2 s of the sound playback, an unconditional stimulus was given: 0.8 mA current was injected into the fence for 2 s, and 30 s after the shock was completed, the experimental rats were taken out and injected intraperitoneally with solvent or corresponding drugs.
2.4 Conditioned fear memory test
Rats were put into soundproof box B for detection of conditioned fear memory effect. The rats were placed into soundbox B and allowed to move freely for 90 s. Then, they were given 3 sounds with a frequency of 2.2 kHz, a volume of 96 dB and a duration of 30 s, with 20 s interval between each sound. Nir camera recorded the time of the rat's rigid behavior in three sounds for a total of 90 s. Freeze is a fearful rodent behavior characterized by a lack of movement other than breathing. The level of fear memory was measured by the percentage of time of rigid behavior in rats.
2.5 Study design
Effects of different doses of dexmedetomidine on propofol enhancement of conditioned fear memory in rats. 100 SD rats were divided into 5 groups by random number table method (n = 20): Control group (group C), propofol group 1(group P1), propofol + dexmedetomidine low-dose group (10 µg/kg, P + DEX10 group), propofol + dexmedetomidine medium-dose group (20 µg/kg, P + DEX20 group) and propofol + dexmedetomidine high-dose group (40µg/kg, P + DEX40 group). Immediately after conditioned fear memory training, rats in 5 groups were intraperitoneally given 1 mL /kg of solvent (sesame oil, Batch:No.20152548, Sigma-Aldrich®) or propofol (Batch:No.5C201526, Sigma-Aldrich®), P + DEX10 group, P + DEX20 group and P + DEX40 group were intraperitoneally given 10µg/kg, 20 µg/kg and 40µg/kg dexmedetomidine (Batch:No.20062331, Yangzijiang Pharmaceutical Group Co., LTD.), 48h later, the freezing time of rats in each group was recorded and the proportion was calculated; Pulse oxygen saturation (SpO2) of rats under anesthesia was monitored by small animal pulse oximetry monitor and the number of cases with SpO2 < 90 was recorded. The lowest value was taken for analysis during the monitoring of SpO2 in each rat.
Effects of dexmedetomidine on the effect of propofol on conditioned fear memory in rats during different administration time windows: Another 100 SD rats were divided into 5 groups by random number table method (n = 20) : Propofol group 2(P2 group), propofol + dexmedetomidine T0 group (given simultaneously with propofol, P + DEXT 0min group), propofol + dexmedetomidine T30 group (given 30 min after propofol, P + DEXT 30min group), propofol + dexmedetomidine T60 group (given 60 min after propofol, P + DEXT 60min group) and propofol + dexmedetomidine T90 group (90 min after propofol administration, P + DEXT 90min group). Immediately after conditioned fear memory training, rats in 5 groups were intraperitoneally given 1mL/kg propofol, P + DEXT 0min, P + DEXT 30min, P + DEXT 60min and P + DEXT 90min groups were intraperitoneally given dexmedetomidine 20 µg/kg at 0, 30, 60 and 90 min after propofol administration, respectively. 48 h later, the freezing time of rats in each group was recorded and the proportion was calculated.
2.6 Tissue preparation
Rats were anesthetized with 10% chloral hydrate (0.3 ml/ 100 g, i.p.) and perfused transcardially with 200 ml of 0.9% saline followed by 300 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brain tissues were removed and postfixed in 4% paraformaldehyde overnight and cryoprotected in 30% sucrose. Themicron-thick frozen sections from the rat brains were cut using a freezing microtome and serially collected throughout the hippocampus. Free-floating tissue sections were rinsed three times with PBS-T. The tissue sections were incubated with 10% normal donkey serum in PBS-T for 2 h.
2.7 Immunofluorescent staining
The tissue sections were incubated for 48 h at 4°C with rabbit anti-c-fos antibody (1:500, Cell Signaling Technology, Inc., Beverly, MA), mouse glutamic acid decarboxylase67 (GAD67) monoclonal antibody (1:500, Abcam, Cambridge, UK) or mouse mono clonal anti-CaMKⅡ antibody (1:500, Abcam, Cambridge, UK). After three 5-min rinses in PBS, the sections were incubated with donkey anti-rabbit IgG conjugated to Alexa Fluor® 488 and donkey anti-mouse IgG conjugated to Alexa Fluor® 594 (1:500, Life Technologies, Carlsbad, CA, USA) in the dark for 2h at 37°C. Fluorescence intensity was visualized under a confocal microscope (FV1000, Olympus Corp., Tokyo, Japan). The intensity on four slides (three to four sections per slide) was averaged for each animal and then normalized by that of the control group.
2.8 Statistical analysis
SPSS 25.0 software or GraphPad Prism 5.0 for Windows was used for analysis. The measurement data of normal distribution was expressed as mean ± standard deviation, one-way ANOVA was used for comparison between groups, and χ2 test was used for comparison of count data, P < 0.05 was considered statistically significant.