Dielectric barrier discharge device
The DBD device with dimensions of 5mm in length and 4mm in width, is specifically designed for cell culture and spinal cord animal model applications. Additionally, a working distance of 3 mm is set to accommodate animal model studies. In the device's discharge circuit, a current probe is strategically placed in series to precisely measure high-frequency currents. The emission spectra of the DBD devices, spanning a spectral range from 300 nm to 1000 nm, were recorded using an ocean spectrometer. For these measurements, the fiber optic probe was positioned at a vertical height of 10 mm above the discharge area, ensuring accurate spectral data collection.
Cell culture and intervention
SH-SY5Y cells were obtained from iCell and cultivated using DMEM medium (Gibco, Grand Island, NY, USA) containing fetal bovine serum (10%), penicillin (100 units/mL) and streptomycin (100 μg/mL) in a 5% CO2 humidified incubator at 37 °C. The suspension containing SH-SY5Y (cell concentration 1 × 106/mL) was added to a 96‐well culture plate at 100 μL per well. A stable neurotoxicity model was established by treating the cells with 150 μM glutamate for 24 hours, which reduced cell viability to 50% of the control. Culturing continued for 24 h. The culture medium was removed before the intervention. The 96‐well culture plate was placed under the DBD equipment with a distance of 5 mm, and then treated for 0, 60, 120 and 240 s. Post-treatment, cells were further cultured for 24 hours for analysis.
Cell count kit 8 determination
To measure cell viability, the Cell Counting Kit-8 (CCK-8) was used. Firstly, SH-SY5Y cells were washed with PBS solution after 24 hours of cold atmospheric plasma treatment. Then, add 100 μL of DMEM medium to each well followed by 10 μL of 10% CCK-8 solution. Incubate the plates in a 37 ℃ water bath for 1.5 hours. After incubation, measure the absorbance (OD values) of each well at 450nm wavelength using a microplate reader.
Determination of apoptosis
The Annexin V FITC Apoptosis Kit is used to detect apoptosis in SH-SY5Y cells. Adherent cells were digested with trypsin without EDTA, and 1 × 106/mL of cells were collected for each sample. SH-SY5Y cells were washed with PBS solution and incubated with 5 μL of Annexin V-FITC and 5 μL of PI for 15–20 min under dark conditions. The percentage of early apoptotic and late apoptotic cells were analyzed by flow cytometry (Beckman Coulter, CytoFLEX LX).
Intracellular ROS content
To measure the ROS content in SH-SY5Y cells, the mean fluorescence intensity (MFI) of DCFH-DA was utilized. The procedure began with trypsin digestion of adherent cells, followed by centrifugation at 1500 rpm and 4 ° C for 10 minutes. After discarding the supernatant, the cells were resuspended in 1 mL of complete culture medium, incorporating the DCFH-DA solution at a final concentration of 10 μmol/L. The cell suspension was then incubated at 37 ° C in the dark for 20 minutes, with intermittent mixing. Subsequent to washing with PBS thrice, the MFI of SH-SY5Y cells from each group was assessed through flow cytometry.
Animals
Female C57BL/6 J mice (WT, 20-25g) were purchased obtained from the Animal Ethics Committee of Anhui Medical University and approved for the study by Animal Ethics Committee of Anhui Medical University (Approval No. 20200851). For each experiment, mice were carefully matched for age and weight. They were housed in a controlled environment with regulated temperature and humidity, following a 12-hour day/night cycle, and provided ad libitum access to food and water.All the experimental protocols were approved by the Animal Ethics Committee of Anhui Medical University. All experimental procedures were planned and reported in compliance with the guidelines outlined in the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines40.
Ethical statement
Animal Ethics Committee of Anhui Medical University provided official approval for the studies presented in this manuscript (Approval No. 20200851). All animals received humane care. The study followed the guidelines for the ethical and humane use of laboratory animals, and all animal procedures were approved by the animal Ethics Committee of Anhui Medical University. All methods reported in this manuscript are in accordance with ARRIVE guidelines(http://www.nc3rs.org.uk/page.asp?id=1357).
Spinal cord injury model and intervention
Before SCI induction, the mice were randomly divided into sham, SCI, and CAP. The total number of animals used in this study was 25. The mice were anesthetised with a 2% intraperitoneal injection of pentobarbital sodium. The Tenth Thoracic vertebra (T10) was identified using anatomical landmarks, and the lamina was removed. We used Dumont #5 forceps to apply a 5-second complete spinal cord compression at the T10 level41. The forceps' arms were positioned within the epidural space on adjacent sides, ensuring their tips contacted the vertebral canal floor for consistent injury reproduction. Successful establishment of the SCI model was confirmed by rapid dural sac congestion, edema, and the onset of hindlimb tremors in mouse. In the CAP group, DBD treatment was administered for 120 seconds, while the sham group mouse underwent laminectomy without spinal cord injury. The incision was sutured and disinfected with iodine, followed by three days of consecutive intramuscular injections of penicillin (0.8 units/g). After surgery, mice's bladders were manually pressed three times a day to assist with urination until their urination reflex recovered.
Motor Function Assessment
The Basso Mouse Scale (BMS) motor recovery scale was used to assess the recovery of mice hind limb motor functions after injury. The scores were performed on 1, 3, 7, 14, 21 and 28 days after SCI. Different groups of mice were allowed to walk freely in an open field. Two independent observers blinded to the experimental groups evaluated the motor behavior within 5 mins for mouse.
Histological procedures
Upon completion of the respective treatments, the mice were euthanized with an overdose of 2% pentobarbital sodium. Some of their spinal cord samples (0.5 cm) were collected following perfusion with 4% paraformaldehyde via cardiac perfusion. Meanwhile, other portions of the mouse were subjected to partial cryopreservation for subsequent ELISA and ROS analysis.
H&E and Nissl staining
Dehydrated spinal cord samples were embedded in paraffin, and 5 μm thick sections were prepared from the paraffin blocks. After deparaffinization and dehydration, the cells were subjected to Hematoxylin–eosin (H&E) and Nissl staining using respective staining kits. All sections were observed and photographed under a bright-field optical microscope.
Immunofluorescent staining
Spinal cord sections were frozen and sectioned. The sections underwent a series of preparatory steps beginning with three 10-minute xylene washes, followed by dehydration through three 5-minute pure ethanol washes, and then rinsed in distilled water. Subsequently, the sections were incubated in PBS (pH 7.4) for 5 minutes and agitated on a decoloring shaker three times for 5 minutes each. To block non-specific binding, 3% BSA was applied and left at room temperature for 30 minutes. Primary antibodies for neurofilaments (NF) and Glial fibrillary acidic protein (GFAP) were then added, and the slides were incubated overnight at 4°C in a humidified chamber. Following another series of PBS washes, corresponding secondary antibodies were applied and incubated at room temperature for 50 minutes in the dark. This was followed by a 10-minute incubation with 4',6-diamidino-2-phenylindole (DAPI) solution in the dark. After washing in PBS and on a decoloring shaker, an autofluorescence quencher was applied for 5 minutes, and the slides were then rinsed under running water for 10 minutes. Slides were mounted with anti-fade mounting medium for microscopic examination. Fluorescent microscopy was employed for the detection and image collection, focusing on Regions of Interest (ROIs) within a 3.5 mm segment of the spinal cord centered on the injury site for quantification.
Tunel staining
The frozen sections were thawed at room temperature for 2 hours and rinsed thrice with PBS. Next, 0.3% Triton X-100 and 0.1% citric acid sodium were added and incubated with the sections for 5 minutes. Subsequently, the TUNEL reaction mixture was added and set in a dark, humid environment at 37 °C for 60 minutes. After three PBS rinses, DAPI was added and set for 15 minutes. Finally, the sections were examined under a fluorescent microscope.
ELISA analysis
For the enzyme-linked immunosorbent assay (ELISA), dilute the antibody in carbonate buffer to 1-10 μg/mL and add 100 μL to each ELISA plate well; incubate overnight at 4°C. The next day, empty the wells, wash thrice with wash buffer for 3 minutes each, then block with 200 mL of blocking solution at 37 °C for 1-2 hours. Wash the plate 3-5 times manually or using a plate washer. Add 100 μL of test samples, controls, and standards to the wells and incubate at 37 °C for 1-2 hours. Following a rewash, add 100 μL of biotinylated antibody, incubate for 1 hour, wash, then add enzyme conjugate and incubate in the dark for 30 minutes. Develop with TMB substrate until colour develops, stop the reaction with 2M sulfuric acid, and read OD at 450 nm within 10 minutes. The IL-1β and TNF-α were determined by correspondingELISA kits following the manual instruction.
Oxidative stress level in vivo
After sacrificing the mouse on the 28th day, spinal tissue was collected and mixed with nine times the volume of normal saline at a weight-to-volume ratio of 1:9. This preparation yielded a 10% tissue homogenate, which was then subjected to centrifugation at 3000 rpm for 10 minutes using a commercial kit from Nanjing Jiancheng Bioengineering Institute, China. The superoxide dismutase (SOD), malondialdehyde (MDA)and glutathione peroxidase (GSH-PX) levels in the tissue homogenate were determined following the manufacturer's protocol.
Statistical analysis
All the experimental results were analyzed by GraphPad Prism 9.0 and Origin 2023. Data are expressed as the mean ± SEM of at least three independent experiments. One-way ANOVA conduct statistical analysis among groups. p < 0.05 was taken as statistically significant (*p < 0.05, **p < 0.01, *** p < 0.001 and **** p < 0.0001).