Tibial fracture surgery in elderly mice caused postoperative neurocognitive disorder via SOX2OT lncRNA in the hippocampus

Animals

Male C57BL/6J mice (12 months old) weighing 30–35 g were purchased from the Air Force Military Medical University. All mice experiments were carried out in accordance with the guidelines for experimental animals of the National Institutes of Health and approved by the Animal Use and Care Committee of the Air Force Military Medical University (IACUC-20200650). All experimental mice were housed under a 12-/12-h light/dark cycle at 20–24 °C and 50–60% humidity with free access to food and water. In part I of the experiment, the mice were randomly divided into either a sham (n = 20) or surgery (n = 20) group.

In Part II, a mouse model with SOX2OT knockdown was established via stereotactic injection of lentivirus containing SOX2OT shRNA into the hippocampus. The tibial fracture model was established after a week, and the mice were divided into two groups: Lv-neg (stereotactic injection of empty lentivirus vectors; n = 10) and Lv-SOX2OT (stereotactic injection of Lv-SOX2OT; n = 10).

Tibial fracture model

Tibial fracture surgery was performed, as reported previously [20,21,22]. Briefly, isoflurane (1.5–2.5%) and oxygen-mixed gas were administered via inhalation as anesthesia before tibial fracture surgery. Only the left hind limb was prepared for surgery. A 0.4-mm stainless steel needle was inserted into the intramedullary canal, the periosteum was separated, and osteotomy was performed. Finally, 0.2% ropivacaine was injected subcutaneously before closing the incision. The skin was then sutured with 4/0 nylon. Aseptic conditions were maintained throughout the procedure. The sham group was subjected to all surgical procedures except bone fracture and pin insertion.

HT22 cell culture

HT22, a hippocampal neuronal cell line, was provided by Dr. Xiuquan Wu, from the Department of Neurosurgery, Air Force Military Medical University. All cells were cultured in complete medium at 37 °C in the presence of 5% CO2 under saturated humidity. HT22 cells were treated with lipopolysaccharide (LPS) to produce inflammatory factors to simulate the inflammatory reaction of the body. The cells were divided into four groups as follows: (1) the Lv-neg group (cells were transfected with empty lentivirus vectors for 48 h and replaced with fresh culture medium), (2) Lv-neg + LPS group (cells were treated with medium containing LPS [100 ng/mL] for 24 h after transfection of empty lentivirus vectors for 48 h), (3) Lv-SOX2OT group (cells were transfected with Sox2ot lentivirus for 48 h and then cultured in fresh medium for 24 h), and (4) Lv-SOX2OT + LPS group (cells were transfected with Sox2ot for 48 h and then treated with culture medium containing LPS [100 ng/mL] for 24 h).

Apoptosis assay

According to a previous study [23], we used flow cytometry (Coulter XL, Beckman USA) to detect the apoptosis rate of HT22 cells with a PE Annexin V apoptosis detection kit (559763; BD Biosciences). Briefly, after being digested with trypsin, the cells were collected in a 15 mL tube and centrifuged at 900 r/min for 5 min and the supernatant discarded. The cells were then resuspended in 200 μL of combined buffer, collected in a flow tube, 5 μL of Annexin V PE was added into the flow tube, incubated at 24 °C for 10 min, and then washed once with 200 μL combined buffer. The cells were then centrifuged at 900 r/min for 5 min, and the supernatant was discarded. The cells were then resuspended with 190 μL of combined buffer, 10 μL of 7ADD was added into the flow tube, mixed, and subjected to flow cytometer. EXPO32-ADC, the system software of flow cytometry, was used for apoptosis analysis.

Mitochondrial membrane potential analysis

Based on pre-established research methods [23] and the manufacturer's protocol, we used a JC-1 mitochondrial membrane potential determination kit (C2006; Beyotime Biotechnology, Shanghai, China) to measure the mitochondrial membrane potential.

Detection of ROS in mitochondria and HT22 cells

Mitochondrial reactive oxygen species and total oxidative activity were analyzed using the mitochondrial fluorescent probe MitoSox (M36008; Thermo Fisher, USA) and a fluorescent intracellular reactive oxygen species kit (S0033; Beyotime Biotech, China), respectively, according to a previously reported methodology [24]. Images were acquired using a confocal laser-scanning microscope (FV 1000; Olympus, Japan).

Estimation of mitochondrial morphology in HT22 cells

To observe the morphology of mitochondria, we used the MitoTracker probe (100 mM, 37 °C, 10 min) (M7510; Thermo Fisher Scientific, USA) to stain the cells. The image was then acquired using a confocal laser-scanning microscope (Olympus). We used Image-pro plus software to measure and analyze the number and volume of mitochondria per five separate regions of per sample, according to a previous study [25].

Detection of mitochondrial oxygen consumption rate

We used the XF24 extracellular flux analyzer (Agilent Seahorse Bioscience, USA) to determine the oxygen consumption rate (OCR) of HT22 cells [26]. The same numbers of HT22 cells (approximately 0.8 × 103 cells per well) were cultured in Seahorse 24-well plates before OCR detection. HT22 cells were successively cultured in oligomycin to inhibit ATP synthase (1 μmol/L), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone to promote the uncoupling of electron (FCCP; 0.5 μmol/L), and antimycin A to inhibitor the electron transfer (1 μmol/L). Thereafter, the baseline and maximum OCRs were calculated with these data. All OCR measurements were normalized to hemocytometry cell counts.

RNA immunoprecipitation

All operations were conducted according to the instructions in the kit (#EZ-Magna RIP kit 17–701). Briefly, HT22 cells were collected and incubated with 5 µg of SOX2 (ab133337; Abcam)-specific antibody or normal IgG antibody for 2 h at 4 °C after adding an equal precipitation volume of RNA immunoprecipitation (RIP) complete lysate. The cell lysate was then added to the samples and spun overnight at 4 °C to produce beads, which were then washed six times with RIPA buffer. Finally, proteinase K treatment was used to release RNA from the bound protein. RNA was isolated using phenol:chloroform:isoamyl alcohol and precipitated using ethanol for the subsequent RT-PCR analysis.

Luciferase reporter assay

To explore the SOX2-dependent transcription of Drp1, the 2 kb 5ʹ promoter (− 2000/0, see Additional file 1) sequence upstream of the mouse Drp1 transcription start site was cloned into a PGL3-basic vector upstream of the firefly luciferase gene. The plasmid was generated by Wuhan GeneCreate Biological Engineering Co., Ltd. Briefly, HEK293 cells were cultured and inoculated in 24-well plates. After 12 h of growth, the cells were transfected with either Drp1 promoter alone, co-transfected with the internal reference plasmid (Renilla luciferase reporter plasmid, pRL-TK), co-transfected with SOX2 small interfering RNA (siRNA), or empty virus for 24 h. After removing the transfection medium, the cells were supplemented with the medium containing 6 μM curcumin (Sigma-Aldrich, St. Louis, MO, USA), which was dissolved in 100% dimethyl sulfoxide. Forty-eight hours after transfection, the Dual-Luciferase Reporter Assay System (Promega) was used to measure luciferase activity. The pRL-TK vector was introduced to eliminate the intergroup error caused by cell transfection and other factors.

Lentiviral infection

Lentiviral-SOX2OT is the SOX2OT silencing vector. The small volume infection method with a 1:2 ratio was used to replace the cell culture medium with serum-free culture medium, according to manufacturer’s instructions. After adding lentivirus infection to the serum-free medium for 4 h, the fresh medium was replenished. After continuously infecting the cells for 12 h, replace the fresh medium once, and then replace the fresh medium every 1–2 days according to the cell growth. After 48 h of transfection, cell fluorescence expression was observed, and the expression of LncRNA was detected using qRT-PCR.

Western blotting

Mouse hippocampus tissue and HT22 cells were analyzed using western blotting, as described previously [27]. The cells were incubated overnight with the primary antibodies against Sox2 (ab171380, ab92494; Abcam), Drp1 (ab184248; Abcam), Fis1 (ab156865; Abcam), Opa1 (ab157457; Abcam), Mfn1 (ab221661; Abcam), Bcl-2 associated X (Bax, ab182733; Abcam), B-cell lymphoma-2 (Bcl-2, ab32124; Abcam), and β-actin (ab8227, Abcam) (all at 1:1000 dilution), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz, CA; 1:5000) at 24 °C for 2 h. The bicinchoninic acid (BCA) method was used to quantify the protein concentration, the value of each well was read by an enzyme-labeling instrument, and the concentration and sample loading amount of each group was calculated. Target proteins were visualized using chemiluminescence (Bio-Rad, Hercules, CA, USA). Band intensities were analyzed using ImageJ. β-actin was used as the loading control.

Quantitative real-time PCR

RNA extraction and quantitative RT-PCR were performed according to the manufacturer’s protocol (Invitrogen, USA), as described previously [27]. PCR primers are summarized in Table 1. The final results were normalized to the expression levels of β-actin and expressed as fold change compared with the control.

Table 1 Primers used in qRT-PCR analysisNeurobehavioral tests

Open field (OFT), novel object recognition (NOR), and fear conditioning (FCT) tests were performed on day 3 post-operation. The same behavioral tests were used for both the sham and surgery groups.

OFT was conducted to evaluate anxiety and locomotor activity in the experimental mice [28]. Mice from the sham and surgery groups were placed in a 40 cm × 40 cm × 40 cm plastic box and allowed to move freely in the box for 5 min before testing. The total distance traveled and amount of time spent by each mouse in the central square were recorded by an observer who was blinded to animal grouping.

NOR was used to test the recognition and nonspatial memory abilities of the mice [29]. Briefly, the mice were allowed to explore two identical objects (A and B) that were placed 10 cm away from the wall of the box during training. The recognition test phase was performed 24 h after the end of the familiarization phase. Object B was replaced with a new object C, and the mice were allowed to explore freely for 5 min. The duration of exploration for each object was recorded. The total sniffing time of novel object was recorded for further analyses. The new object preference index was calculated as follows:

$$T_}} /\left( }} + T_}} } \right) \, \times 00\% ,$$

where TA and TC are the time spent exploring objects A and C, respectively.

When experiencing an aversive stimulus, mice adopt a typical “freezing” posture, which is memorized via contextual clues that are related to a previously learned fear-inducing stimulus–response pairing [30]. In this study, sound was used as a conditioning stimulus, and an electric shock to the foot acted as an unconditional stimulus. Sound (2000 Hz and 90 db for 30 s) and electrical (1 mA for 2 s) stimuli administered during the training phase indicated that the mice would associate the link between the environment and electric shock following training. A contextual test was performed in a similar context chamber for 5 min without any sound or electric shock stimulation. During the cued test, the cue (90 db sound stimuli) was applied in a plastic box with black and white stripes different from those of the pre-operation training. The cued test was performed after completion of the contextual test.

After the behavioral test, the mice were euthanized, their brain tissue was removed immediately and placed on ice, and the hippocampal tissue was isolated and cryopreserved at − 80 °C for lncRNA sequencing (Shanghai Jingneng Company).

Cell Counting Kit-8 (CCK-8) and lactate dehydrogenase analysis

CCK-8 (Dojindo ck04) and lactate dehydrogenase (LDH) (c0017; Beyotime) analyses were performed after the cells were subjected to lentiviral infection. Cell viability was analyzed using the CCK-8 kit according to the manufacturer’s instructions. After incubating the cells for 1–4 h, color changes were observed, the absorbance at 450 nm was measured using a microplate reader. The cell survival rate was calculated according to the absorbance value [31]. Prism 8.0 software was used to analyze data. The amount of LDH released in each group was measured following the instructions on the LDH assay kit. The cells were divided into different groups and then cultured again. LDH release was detected at 24, 48, and 72 h. the absorbance at 490 nm was measured using a microplate reader. LDH enzyme activity was calculate according to the absorbance value [32]. The time at which neuronal injury occurred was determined for the subsequent experiment.

Transmission electron microscopy (TEM)

According to previous research [33], the hippocampus sample from the brain was obtained, fixed with 2.5% glutaraldehyde (pH 7.4, 4 °C) for 24 h, and then fixed with 1% osmium tetroxide in deionized water for 4 h. All sections were observed using a TEM (JEM-1230; JEOL Co., Ltd., Tokyo, Japan). We generated a semi-quantitative score for the morphological damage of mitochondria [34]: 0 = no damage, normal morphology, electronic dense structure; 1 = mild injury: the number of cristae decreased, slightly swollen, and the electronic dense appearance disappeared; 2 = severe injury: (near) the cristae completely disappeared, and the mitochondria were severely swollen; 3 = complete destruction: outer membrane rupture, mitochondrial structure collapse. Image J software was used to score all mitochondria in five separate regions of each sample, and the average score of each region was plotted.

Small interfering RNA (siRNA) transfection

SOX2 siRNA was provided by Hanbio Company, Shanghai, China. Diluted siRNA and RNA transfection partners (Lipofectamine; Hanbio Company Shanghai, China) were added to six-well (1500 µL/well) plates when HT22 cells reached 60–70% confluency, and siRNA transfection was performed in Opti-MEM medium. After 4 h, fresh medium was added to each well. Transfection efficiency was evaluated by measuring the expression level of SOX2 protein.

Lentivirus stereotactic injection

Mice were anesthetized via the intraperitoneal injection of ketamine (0.1 mg/kg) before the skin was prepared and fixed on the brain stereotactic injection instrument to ensure the symmetrical placement of ear rods. After complete disinfection, the skin of the skull top was cut open on both sides and the bregma and lambda points were fully exposed on the skull top plane. Craniotomies were performed at ± 1.85 mm anterior to the bregma and − 1.94 mm lateral to the midline. Coordinates were determined according to the mouse brain atlas (x =  ± 1.85 mm, y = − 1.94 mm, z = − 2 mm) and holes were drilled into the skull with a dental drill. Two sites in the CA1 region of the hippocampus were selected for injecting the virus, and 1 µL of virus was injected to each site over 30 min. The needle was left in place for 10 min. After the localization injection, the skin on top of the skull was disinfected and sutured. The mice were returned to the cage to continue feeding when fully awake.

LncRNA sequencing

LncRNA sequencing analyses were conducted to compare the hippocampal tissues of mice from the PND and control groups. Shanghai Jingneng Co., Ltd. Gene Ontology (GO) analysis was used to analyze the main functional differentially expressed genes, and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was used to analyze the signal pathway of differential gene enrichment (P < 0.05) (see Additional file 2).

Statistical analysis

All values are presented as mean ± standard error. T-test was used for the analysis of two groups of data, multiple groups of data analysis was performed using ANOVA followed by Tukey's post-hoc test with GraphPad Prism 8.0. Statistical significance was set at P < 0.05.

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