Animal groups
The animal protocol of the present study was approved by the Institutional Animal Care and Use Committee at Tongji Medical College, Huazhong University of Science and Technology. All animal experiments were performed in compliance with animal care guidelines. Thirteen- to eighteen-month-old male Sprague Dawley (SD) rats were commercially obtained from Biont (Wuhan, China). To verify our hypothesis, this study conducted four animal experiments. First, we used 3% sevoflurane in 100% oxygen to induce the SIN model, and the SD rats were randomly divided into two groups, the control and sevoflurane (Sevo) groups. SD rats in the Sevo group were exposed to 3% sevoflurane in 100% oxygen for 4 h, and rats in the control group were exposed to 100% oxygen for 4 h.
In the second experiment, rats were fed AIN-76A chow supplemented with PLX3397, a selective CSF1R (Colony-stimulating factor 1 receptor) inhibitor that has been demonstrated to readily cross the blood-brain barrier (BBB) and deplete microglia by oral delivery in rat chows [12, 13]. Vehicle chow (AIN-76A chow) was used to feed rats in the control group. Then, the rats used for SIN establishment by sevoflurane exposure were assigned to two groups: the Sevo-AIN76A and Sevo-PLX3397 groups.
In the third experiment, rats were intraperitoneally injected with meloxicam, a nonsteroidal anti-inflammatory drug (NSAID) that has been shown to effectively reduce hippocampal inflammation [14]. Intraperitoneal injection of normal saline (NS) was used as a carrier control. Then, following SIN establishment by sevoflurane exposure, these rats were assigned to two groups: the Sevo-NS and Sevo-meloxicam groups.
In the fourth experiment, rats received intracerebroventricular (i.c.v) injection of 10 μl C1q neutralizing antibody (clone JL-1, GTX54404, GeneTex) or IgG2b in PBS, which were administered at 1 h after anesthesia end. According to previous work, the i.c.v. injection site was located as: AP (anteroposterior) = −1.2 mm, ML (mediolateral) = 1.8 mm, and DV (dorsoventral) = 4.0 mm [15]. Then, following SIN establishment by sevoflurane exposure, these rats were assigned to two groups: the Sevo-IgG and Sevo-C1q groups.
The SIN rat model
Focusing on examining the SIN, we used a long time and 3% sevoflurane in 100% oxygen for rat anesthesia, which has been reported to effectively trigger cognitive impairment in previous studies [2, 16, 17]. SD rats were exposed to 3% sevoflurane in 100% oxygen for 4 h in an anesthetizing box in which the concentration of sevoflurane and carbon dioxide and the temperature were detected through an anesthesia monitor. After sevoflurane exposure, the rats were immediately transferred to a resuscitation chamber filled with fresh oxygen. The air temperature was set in the range of 23–25 °C. Rats in the control group were exposed to the carrier gas (100% oxygen) for 4 h.
PLX3397 formulation in rat chow
PLX3397 (TOPSCIENCE, China) was purchased to eliminate microglia in the brain. Following the methods of a previous study, PLX3397 was formulated in AIN-76A chow at a concentration of 300 mg/kg chow [18]. PLX3397-supplemented chow was produced by Jiangsu Xietong, Inc. (Nanjing, China). Based on published studies, rats were fed either AIN-76A or AIN-76A chow supplemented with PLX3397 (300 mg/kg) for 21 days [18, 19]. Then, after feeding for 21 days, the number of microglia and the expression of iba1 (a microglial marker) were measured to estimate the efficacy of microglia elimination.
Meloxicam injection
Meloxicam injection (Kangdien, China) was used to inhibit neuroinflammation in the SIN rats. A dose of 2.5 mg/kg was proven to show a protective anti-inflammatory effect [20]. Meloxicam injection was administered three times: at the start of anesthesia, at the end of anesthesia, and 1 h later, based on a previous study [20]. Inflammatory cytokines were detected to evaluate the anti-inflammatory effect.
Tests of cognition
The Morris water maze (MWM) test, a widely used measure of learning and memory, was used to evaluate cognitive impairment, as described in a published study [21]. During neurobehavioral experiments in this study, experimenters were all blind to group assignment and outcome assignment. Briefly, SD rats underwent 5 days of training, including three periods (swimming training with a visible platform, swimming training with a hidden platform, and a probe trial). On day 1, the rats received swimming training four times a day with a visible platform in one quadrant of the pool. From days 2 to 5, the rats received swimming training four times a day with a hidden platform in the same quadrant of the pool. During the training period, if the rats found the platform within 60 s, they were allowed to stand on the platform for 5 s. Rats that failed to find the platform within 60 s were placed on the platform for 20 s. In the probe trial, the platform was removed from the pool. Then, the time spent in the platform quadrant and the number of platform quadrant crossings were measured for 60 s. The probe trial was conducted on the first and third days after anesthesia. Since the MWM test showed no difference on day 3 after sevoflurane exposure, we ended the MMW test on the third day after anesthesia.
Considering the influence of brain trauma after intracerebroventricular injection on rats’ motor ability as well as water in MWM causing wound infection, we used contextual fear conditioning (CFC) to test memory ability. The CFC chamber was composed of a white plastic chamber with a stainless steel grid floor. Each rat was taken from its home cage, and each animal was allowed to explore for 3 min for habituation. Each rat was conditioned with A 30-s, 80-dB, 4500-Hz tone co-terminated with a 2-s, 1.5-mA footshock, with an inter-trial interval of 1 min, for 4 min. Next 2 days, each rat was placed back in the CFC chamber for 3 min. The freezing behaviors of rats were recorded by Xeye software (Zhongshi Technology, China). The memory performance was estimated by the percentage of freezing time.
Exploratory activity and anxiety-like behavior
Exploratory activity and anxiety-like behavior were evaluated by the open field test (OFT) and elevated plus maze (EPM) test [22]. In the OFT, an open field apparatus was used, and the central zone was a square 20 cm away from the wall of the apparatus. Rats were placed in the central zone and allowed autonomous exploration. The time spent in the central zone and the number of crossings in the central zone were recorded for 5 min. In the EPM test, an apparatus mainly consists of two closed arms and two opened arms. The closed arms in the apparatus were enclosed by a black wall. Each rat was placed in the central region of the EPM apparatus facing the opened arm. The time of staying in different arms and the number of entering different arms were measured for 5 min. Since the cognitive tests showed no difference on day 3 after anesthesia, we focused only on the behavioral change on day 1 after sevoflurane exposure. The OFT and EPM tests were performed on the first day after anesthesia.
Electrode placement and hippocampal local field potential (LFP) recordings
The rats were administrated with continuous 3% sevoflurane on an animal anesthesia mask and then fixed onto a stereotaxic frame (RWD, China). After disinfecting the scalp and applying additional analgesia using bupivacaine, a longitudinal incision along the midline was made to expose the bregma. One PFA-coated LFP electrode (stainless steel wire) was positioned in the left hippocampus (AP −4.5 mm; ML −3.0 mm; DV −4.0 mm), and a reference electrode (silver wire) was placed over the left cerebellum. Subsequently, all electrodes were skull-secured with dental cement. Before the LFP recordings, at least 7 days were allowed for the animals to recover from the surgery.
Rats were individually positioned in a custom-made 20-L airtight chamber. An inlet and an outlet were placed on the opposite sidewalls for gas delivery and discharge. EEG signals were continuously sampled (1 kHz/s) and bandpass-filtered at 0.1–500 Hz using PowerLab 8/35 (ADInstruments, Australia). Moreover, LabChart 8.0 software (ADInstruments, Australia) was used for data recording and analysis. The LFP was sequentially recorded in the air for 30 min as a control and in sevoflurane (3%) for 30 min as a test. Recordings ended when the rats emerged from anesthesia. Only the data recorded during relatively quiet conditions were used for analysis to avoid interference caused by vigorous exercise.
Burst suppression identification and analysis
The raw data were first bandpass-filtered at 0–45 Hz and exported into MATLAB R2016b (Mathworks, MA, USA) for processing, as described in our previous study [23]. In the present study, a suppression signal is defined as an amplitude limited within ± 15 μV for at least 200 ms, and bursts are defined as epochs between suppression events. Suppression and burst are, respectively, given a value of 1 and 0 to produce a binary time series. The burst suppression ratio (BSR) was calculated as the percentage of suppression time of each 1 min binary series [12].
Brain tissue collection
Since the cognitive tests showed a significant difference on the first day after anesthesia, rats were deeply anesthetized with sevoflurane on the first day after the SIN model establishment for collecting tissues. In brief, after opening up the skull, brain tissue was collected for molecular biology experiments, and a glass dissecting needle was employed to peel off the brain tissue. We used a saline flush and paraformaldehyde fixation, and brain slices were prepared for staining.
RNA sequencing (RNA-seq)
Hippocampal tissues were collected and immediately sent to BGI Genomics for RNA-seq processing (Beijing Genomics Institute, BGI, China). The cDNA library establishment was in compliance with the BGI standard procedures. Briefly, the fragment was end-repaired, dA-tailed, adaptor-ligated, and then subjected to a 4-cycle PCR program. The libraries were sequenced based on the BGI protocols for RNA-seq on the Illumina HiSeq 2500 platform using the 50 bp pair-end sequencing strategy. Transcript expression levels were evaluated by using fragments per kilobase per million reads (FPKM). In terms of functional enrichment analysis, all DEGs (differentially expressed genes) were mapped to terms in the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) databases. Dor. Tom from BGI (a web-based multiomics visualization tool: https://biosys.bgi.com/#/report/mrna/en/help) was used to analyze and visualize the results of RNA-seq.
Real-time polymerase chain reaction (RT-PCR)
Following the manufacturer’s instructions, an EntiLink™ 1st Strand cDNA Synthesis Kit (ELK Biotechnology, China) was used to prepare the cDNA of the hippocampus. RT-PCR was implemented by using an EnTurbo™ SYBR Green PCR SuperMix kit (ELK Biotechnology, China) and a StepOne™ Real-Time PCR machine (Thermo Fisher Scientific, USA). The reaction system was prepared according to the instructions of the EnTurbo™ SYBR Green PCR SuperMix kit. The cycling parameters were set at 95 °C for 3 min, then 40 cycles at 95 °C for 10 s, followed by 58 °C for 30 s and 72 °C for 30 s. All gene primer pairs are reported in Additional file 1: Table. S1.
Enzyme-linked immunosorbent assay (ELISA)
Proinflammatory cytokines (TNF-α, IL-6, and IL-1β) and C1q in the hippocampus were measured with ELISA kits (Bioswamp, China). According to the manufacturer’s instructions, an ELISA was employed to detect the TNF-α, IL-6, IL-1β, and C1q levels in the protein supernatant. A microplate reader (Thermo Fisher Scientific, USA) with absorption at 450 nm was employed to measure the absorbance values of the samples, and the proinflammatory cytokine concentration was detected based on the standard curve produced.
Western blotting
Western blotting was performed as described in our previous work in detail [24, 25]. The hippocampus was minced into homogenates in a lysis buffer with protease and phosphatase inhibitors. The supernatants were collected after the homogenates were centrifuged at 4 °C at 12,000 rpm for 15 min. A BCA protein assay Kit (Biosharp, China) was used to detect the protein concentration. Then, the supernatants were mixed with 5×SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) loading buffer (Biosharp, China). Hippocampal proteins were separated by 8–10% SDS-PAGE and transferred to PVDF membranes (Millipore, USA). Then, the PVDF membranes were immersed in the indicated primary antibodies overnight. The primary antibodies were as follows: anti-iba1 (1/1000, ab178846, Abcam, UK), anti-PSD95 (1/1000, 36233S, Cell Signaling Technology, USA), anti-Synapsin-1 (SYN1) (1/1000, 5297S, Cell Signaling Technology, USA), anti-Synaptophysin (SYP) (1/1000, 36406S, Cell Signaling Technology, USA), anti-C1qa (1/1000, sc-58920, Santa Cruz Biotechnology, USA), anti-CD68 (1/1000, ab125212, Abcam, UK), anti-Phospho-NF-κB p65 (1/1000, 3039S, Cell Signaling Technology, USA), anti-Caspase-3 (1/1000, 9662S, Cell Signaling Technology, USA), anti-bcl-2 (1/1000, ab196495, Abcam, USA), anti-bax (1/1000, 2772S, Cell Signaling Technology, USA), and anti-β-actin (1/5000, ab170325, Abcam, UK). Afterward, the PVDF membranes were incubated with the appropriate horseradish peroxidase-labeled secondary antibodies, including anti-rabbit (1/10,000, Abcam, UK), anti-rat (1/10,000, Abcam, UK), and anti-mouse (1/10,000, Abcam, UK) antibodies. Then, ECL-A buffer and ECL-B buffer (Biosharp, China) were used for imaging. β-actin antibody was used as a loading control. The western blotting bands were quantified by using ImageJ software.
Immunofluorescence staining
Brain slices were blocked with 5% BSA (bovine serum albumin) in PBS (phosphate-buffered saline) supplemented with 0.3% Triton X-100 for 1 h. Then, the brain slices were incubated with the indicated primary antibodies, including iba1 (1/200, ab178846, Abcam, UK), PSD95 (1/200, 36233S, Cell Signaling Technology, USA), PSD95 (1/200, 3450S, Cell Signaling Technology, USA), SYN1 (1/200, 5297S, Cell Signaling Technology, USA), SYP (1/200, 36406S, Cell Signaling Technology, USA), C1qa (1/50, sc-58920, Santa Cruz Biotechnology, USA), C1q (1/100, GTX54404, GeneTex, USA), CD68 (1/100, ab125212, Abcam, UK), C-fos (1/100, AF5354, Affinity Biosciences, China), Phospho-NF-κB p65 (1/100, 3033T, Cell Signaling Technology, USA), and CX3CR1 (1/50, sc-377227, Santa Cruz Biotechnology, USA). Two primary antibodies from different species were coincubated with the same brain slice for bimolecular staining. After washing, the brain slices were immersed in the appropriate secondary antibodies, including goat anti-rabbit IgG antibody conjugated with Alexa Fluor 488 (1/1000, Abcam, UK), goat anti-rabbit IgG antibody conjugated with Alexa Fluor 594 (1/1000, Abcam, UK), goat anti-mouse IgG antibody conjugated with Alexa Fluor 488 (1/1000, Abcam, UK), goat anti-mouse IgG antibody conjugated with Alexa Fluor 594 (1/1000, Abcam, UK), and donkey anti-rat IgG antibody conjugated with Alexa Fluor 488 (1/1000, Abcam, UK) for 1 h at RT (Room temperature) in a dark room. Finally, the sections were placed in a mounting medium with DAPI-Aqueous, Fluoroshield (Abcam, UK) and then imaged under a fluorescence microscope or a confocal laser scanning microscope.
Hematoxylin and eosin (HE) staining
HE staining was employed to observe neuronal morphology and inflammatory cell infiltration. Brain slices were stained by using an H&E kit (Solarbio, China) according to the manufacturer’s instructions and then imaged under a microscope.
Nissl staining
Nissl staining was used to detect Nissl bodies in the neurons, which is considered an indicator of neural damage [25]. Nissl staining was performed in accordance with the instructions of the Nissl staining kit (Solarbio, China), and the brain slices were imaged under a microscope. Then, morphological alterations in the neurons and the number of Nissl bodies in the hippocampus were observed by a microscopy.
TUNEL assay
Neuronal apoptosis was evaluated by TUNEL assay, as described in our previous work [24]. The TUNEL assay was performed using an In Situ Cell Death Detection Kit (Roche, Switzerland) according to the manufacturer’s instructions. Fluorescence microscopy was employed to identify neuronal apoptosis in the whole brain slice.
Scanning electron microscope (SEM)
Briefly, rats were perfused with 0.1 M sodium cacodylate buffer containing 4% paraformaldehyde (PFA) and 2.5% glutaraldehyde, and immediately, 1-mm coronal sections were collected. The CA1 region of the hippocampus was microdissected and fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at 4 °C for 24 h. Then, hippocampal samples were sent to Jarvis Inc. (Wuhan, China) for staining and processing. The synaptic vesicles, synapses, and synaptic clefts were used as features to identify the synaptic structure.
Golgi staining
An FD Rapid GolgiStain™ Kit (FD NeuroTechnologies, Inc., USA) was employed to examine the neuromorphopathological alterations. Golgi staining was performed according to the instructions. The rat brains were immersed in Golgi solution for 14 days in a dark room. The solution was changed every 2 days for 14 days. Then, the brain samples were sliced into 100-μm coronal sections using a vibrating microtome. The staining steps followed the instructions.
Sholl analysis and quantitative morphology
We all know that cellular structure is fundamental to the execution of its functions. The Sholl plugin in ImageJ was used to describe and trace the morphologies [26, 27]. The Sholl plugin can perform analysis on grayscale images of isolated neurons or microglia. The Sholl plugin can skeletonize neurons or microglia. Then, it was used to analyze the color-coded dendrites and pseudopodia. Warner hues produced by the Sholl plugin indicated the number of intersections. The intersection mask was employed as the quantitative descriptor for comparisons between groups. The maximal intersection number was used to depict cell complexity and reflect the highest number of processes in the cell [28]. Solidity was defined as microglial cell area/convex area, which estimates the area of the cell and skeleton [29]. We used ImageJ to measure the morphological transformation in two-dimensional space.
Statistics
The mean ± SD (standard deviation) was used to describe normally distributed data. The differences between the two groups were analyzed using t-tests or rank-sum tests. Paired t-tests were used to compare the results on the first and third days of the MWM. With regard to the RNA-seq results, |log2(fold change)| > 0.2 and adjusted P < 0.05 were set to achieve more DEGs [30], which have a potential association with SIN. The fold change of each gene was calculated by comparing the standardized read counts of one group to another group (fold change = standardized read counts of one group/standardized read counts of another group). A P < 0.05 was considered a significant difference. All data analyses and statistics were performed with Stata 16.0 (Stata Corporation, USA), and statistical charts were produced by GraphPad Prism 8.0 (GraphPad Software Inc., USA) and Stata 16.0.
