Enhanced Neural Recovery and Reduction of Secondary Damage in Spinal Cord Injury through Modulation of Oxidative Stress and Neural Response

Enhanced Neural Recovery and Reduction of Secondary Damage in Spinal Cord Injury through Modulation of Oxidative Stress and Neural Response | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Enhanced Neural Recovery and Reduction of Secondary Damage in Spinal Cord Injury through Modulation of Oxidative Stress and Neural Response Jiwen Zhu, Zhenyu Liu, Qi Liu, Qinghua Xu, Chengbiao Ding, Zhu Chen, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4297802/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Aug, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Spinal cord injury (SCI) presents a critical medical challenge, marked by substantial neural damage and persistent functional deficits. This study investigates the therapeutic potential of cold atmospheric plasma (CAP) for SCI, utilizing a tailored dielectric barrier discharge (DBD) device to conduct comprehensive in vivo and in vitro analyses. The findings show that CAP treatment significantly improves functional recovery after SCI, reduces neuronal apoptosis, lowers inflammation, and increases axonal regeneration. These findings illustrate the efficacy of CAP in fostering a conducive environment for recovery by modulating inflammatory responses, enhancing neuronal survival, and encouraging regenerative processes. The underlying mechanism involves CAP's reduction of reactive oxygen species (ROS) levels, followed by the activation of antioxidant enzymes. These findings position CAP as a pioneering approach for spinal cord injury (SCI) treatment, presenting opportunities for improved neural recovery and establishing a new paradigm in SCI therapy. Physical sciences/Engineering/Biomedical engineering Biological sciences/Neuroscience/Diseases of the nervous system/Neurodegeneration Cold atmospheric plasma spinal cord injury neuroprotective ROS Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Spinal Cord Injury (SCI) is one of the most prevalent and debilitating conditions worldwide, primarily due to the limited regenerative capacity of nerves, often resulting in permanent functional impairments 1 . When SCI happens, the repair of nerve cells occurs at a slow pace, with little to no promotion of natural neuron regeneration. The sequelae of SCI are essentially incurable with current medical and rehabilitative approaches, and spontaneous recovery of bodily functions often plateaus within approximately 1.5 years post-SCI 2 . Current therapeutic strategies for SCI primarily focus on mitigating secondary complications and compensating for lost parts rather than targeting neural recovery or restoration of pre-injury capabilities 3 . Therefore, it is critical to develop novel treatments for early-stage spinal cord injuries. In recent years, numerous emerging neural protection and regeneration strategies have provided new directions for SCI treatment. These approaches aim to reduce neuronal death following central nervous system injuries, while also enhancing the intrinsic regenerative capabilities of post-mitotic neurons. Furthermore, improving the hostile extracellular environment within the central nervous system that inhibits neuronal growth is also an effective strategy. 4–8 . Plasma, the fourth state of matter following solid, liquid, and gas, consists of highly reactive physical and chemical substances 9 . In recent years, cold atmospheric plasma (CAP) has applied widely in biomedicine because it doesn’t cause thermal damage to biological tissues and has no toxic side effects. 10 . CAP comprises highly active substances such as highly reactive species (reactive oxygen and nitrogen species), electric fields, ultraviolet radiation, and charged particles, which enable it to modulate biological processes 11 .Dielectric barrier discharge (DBD) is a typical method for generating CAP. Under different working gases and operating parameters, DBD could yield diverse active species 12,13 , including nitric oxide (NO), superoxide (O 2 − ), hydrogen peroxide (H 2 O 2 ); singlet oxygen ( 1 O 2 ); ozone (O 3 ) and even hydroxyl radical (•OH) can and do play essential roles in biological systems. 14,15 More and more research shows that CAP is a novel and promising way to treat neurological diseases 16 . CAP primarily addresses neural regression diseases through two pathways: On one hand, CAP has demonstrated neuroprotective effects against adverse conditions such as neuronal damage induced by glucose deprivation and hypoxia 17–20 .It is evident that exposure to mild “mini-insults” causes injury tolerance, making neurons more resilient to damage in the future 21 . A recent study showed that CAP induces the production of reactive oxygen and nitrogen species (RONS), leading to a significant and transient increase in cellular glutathione (GSH) levels and the activation of erythroid 2-related factor 2 (Nrf2), thereby mitigating glutamate excitotoxicity 18 . Another study reported that atmospheric pressure plasma jets (APPJ) could reduce the damage of middle cerebral artery occlusion and improve neurological function. Plasma-induced NO generation could be potentially used as a cytoprotective agent 22 . On the other hand, previous studies have reported that CAP could notably stimulate the differentiation of neuronal stem cells and promote increased neuronal regeneration following trauma [ 23 – 24 ] . Moreover, CAP have been directly applied to living organisms, such as cancer, 23 rheumatoid arthritis 24,25 ,and ischaemic stroke 26 ,demonstrating the potential of CAP for in vivo treatment. The potential usage of CAP in neuronal and Central Nervous System(CNS) injury treatment has been relatively proven in vitro 27 . However, the application of CAP in vivo and its experimental efficacy in treating neurodegenerative diseases remains limited and warrants further investigation. Using a compression injury model, this study introduces CAP to the spinal cord injury site. Various methods were utilized to assess the therapeutic effects, axon regression, neuronal apoptosis and oxidative stress levels. The research findings can offer a novel treatment approach for clinically managing SCI and pave the way for innovative treatments in neurodegenerative disease management Results Physical characterization of dielectric barrier discharge devices The DBD device shown in Fig. 1 is specifically designed for conducting experiments on spinal cord injury both in vivo and in vitro settings. Figure 1 A depicts a tri-layer structure: a titanium (Ti) electrode on top, a polyvinyl chloride (PVC) middle dielectric layer, and a copper (Cu) electrode at the base. The device's dimensions (5mm in length and 4mm in width) are optimized for cell culture applications. Similarly, the DBD functions effectively with a 3 mm gap during animal experiments, ensuring practicality, precision and accuracy across multiple sets of experiments. To monitor the real-time operating status of the CAP treatment, a digital oscilloscope was linked to the positive and negative electrodes of the DBD apparatus (Fig. 1 E). The DBD device employed air as its working gas. When the DBD device is discharging, two electrical sensors were used to measure the DBD discharge voltage and current waveforms, which are shown in Fig. 1 C. The voltage waveform is a typical sine wave with a frequency of 13.7 kHz. The overall discharge power of the DBD is 38.8 W. The power measurement method comes from Manley's classical DBD discharge model and is based on the equivalent circuit method. The charge Q(t) can be obtained as the integral of the measured current waveform with a sampling capacitance (47 µF). By measuring the Lissajous figure (Fig. 1 B) of DBD discharge, the average power could be obtained through the graphic area. The specific calculation formula is as follows. $$\text{P}=\frac{1}{\text{T}}{\oint }_{T}\text{Q}\left(\text{V}\right)\text{d}\text{V}$$ 1 The T refers to the discharge cycle, and the V refers to the voltage. Therefore, we can derive further from the above formula, which is $$\text{P}=\text{f} \times \text{C}\times \text{s}$$ 2 The f (13.7 kHz) refers to the frequency of discharge, the C (0.47 µF) refers to the sampling capacitance value, and the s (2.88) refers to the area of the Lissajous figure. The power value is calculated to be about 38.8 W. Optical emission spectroscopy (OES) was recorded to identify the composition of reactive species generated by DBD, as shown in Fig. 1 D. The range from 338 to 405 nm corresponds to transitions in the N 2+ second positive system. Additionally, a distinct emission peak at 770.09 nm signifies the presence of atomic oxygen. These two species correspond to ionized nitrogen and oxygen, which are the most abundant ingredient in the working gas. These active species play a significant role in regulating cellular activities within biological organisms, emphasizing their importance in biomedical applications and further studies. CAP treatment improved functional recovery after SCI To evaluate the effects of cold atmospheric plasma on the mouse spinal cord, we established a compression model in C57 mice (n = 8), as shown in Fig. 2 A, and the timeline of the experimental design is depicted in Fig. 2 B. Basso Mouse Scale (BMS) scores were used to assess hindlimb performance. The BMS scores at 0, 1, 7, 10, 14, and 28 dpi (days post injury) represents different levels of functional recovery (Fig. 2 C). The sham group was 9 points, and the hind limb motor function was completely normal. After surgery, the SCI and CAP groups immediately reached a state of complete hindlimb paralysis post-surgery (BMS score = 0), which showed irreversible neurological damage. Compared with the SCI group, the motor function of the CAP group recovered more quickly and reached higher motor performance scores, notably by the 28th day post-intervention 28 . It is well known that functional recovery is correlated with tissue structure and pathophysiology. Tissue samples were collected at 7 and 28 dpi. Hematoxylin and eosin (H&E) staining was used to visualize pathological changes in the spinal cord. As shown in Fig. 2 D and 2 E, the structure of the sham group was completed and normal with abundant neurons. At the same time, the SCI and CAP groups experienced atrophy and loose structural integrity due to the formation of scars and cavities. Compared to the CAP group, the injury group demonstrates a larger area of damage, more extensive edema, cyst formation and hemorrhage, and less nerve fiber density. CAP treatment decreased neuronal apoptosis and inflammation response after SCI Some in vitro studies suggest that CAP can enhance neuroprotection. However, there is no corresponding in vivo evidence to support this claim. Therefore, we conducted a separate analysis to assess apoptosis, neuronal survival and inflammation response following CAP treatment. As illustrated in Fig. 3 A and 3 B, TUNEL staining revealed a significant increase in cell death in injured spinal cords compared to the sham control group. Notably, the CAP treatment group exhibited a significant reduction in the percentage of apoptotic neurons compared to the SCI group. The apoptosis index for the SCI group was significantly higher (15.07%) than that of the CAP group (0.65%). Nissl staining demonstrated a decrease in surviving neurons, with atrophied cytoplasm post-surgery. In contrast, the CAP treated group displayed neurons with a more regular shape, increased neurites, and deeper blue-stained Nissl bodies in histological analysis (Fig. 3 C). The Nissl and TUNEL results collectively support a higher preservation rate of intact neuronal structures in the CAP-treated group. To assess the impact of CAP on the inflammatory response following SCI, we conducted immunofluorescent staining and ELISA to measure the concentrations of inflammatory cytokines 29,30 . As shown in Fig. 3 D and 3 F, immunofluorescent results demonstrated a pronounced accumulation of inflammatory factors at the injury site. Using ELISA, we detected changes in the expression levels of TNF-α and IL-1β in Fig. 3 F and 3 G. After calibration, the average protein content for TNF-α and IL-1β in the sham group was measured at 69.91 pg/mL and 22.62 pg/mL, respectively. In the CAP group, TNF-α levels were 38.09 pg/mL, significantly lower than the 89.14 pg/mL observed in the SCI group (p > 0.01). Likewise, the ELISA analysis of IL-1β revealed substantially lower expression in the CAP group (76.29 pg/mL) compared to the SCI group (144.2 pg/mL) (p > 0.05). CAP treatment enhanced axonal regeneration and reduced glial scar formation A series of histological analyses were conducted to examine the effects of the CAP treatment on SCI. The immunofluorescent staining of spinal cord sagittal sections was further performed to investigate the axonal regeneration in Fig. 4 A and 4 B. The neurofilaments (NF200) were selected to indicate the axon growth at the lesion site (within 400µm). As was shown in Fig. 3 C, while there is no significant difference between the CAP group and the SCI group, images demonstrate that the CAP group exhibits better neural preservation and axon regression. Glial fibrillary acidic protein (GFAP), a specific marker of astrocytes negatively correlated with NF, was also used to evaluate spinal cord repair—the GFAP + area of the excessive representative. The GFAP + area of the representative sections displays pronounced glial scarring. Compared to the CAP group, the SCI group exhibited a significant enhancement in GFAP fluorescence signals near the injury epicentre, decreasing radially towards the injury periphery (Fig. 4 D). Furthermore, NF signals did not colocalise with the GFAP astroglial scar. The results demonstrate that the CAP group exhibits more excellent neural preservation and axon regression. CAP treatment increased cell viability and altered apoptosis of SH-SY5Y cells in response to glutamate-induced cytotoxicity In our study, SH-SY5H cells were exposed to glutamate concentrations of 100, 150, and 200 µM to induce neurotoxicity 31,32 . A significant viability reduction to 50% was observed at 150 µM after 24 hours, as shown in Fig. 5 A using a CCK8 assay. Therefore, 150 µM was selected for the excitotoxicity model using SH-SY5Y cells. Following CAP treatments for 30, 60, 120, and 240 seconds after 24 h, cell viability improved to 71.2%, 68.91%, 86.84%, and 88.74% respectively, as depicted in Fig. 5 B. Notably, durations of 120 and 240 seconds exhibited a marked increase in cell viability compared to the control group, with p-values < 0.01 and < 0.001, demonstrating CAP's neuroprotective potential against glutamate-induced cytotoxicity. The apoptosis status of SH-SY5Y Cells was quantitatively analyzed by flow cytometry. Related results were shown in Fig. 5 C and 5 D. Early apoptosis significantly decreased in the 240 s CAP-treated group (47.57%) compared to the Glu group (66.1%) (p < 0.05). Late apoptosis rates increased significantly in both the 60 s (22.97%) and 240 s (25.9%) groups compared to Glu group (11.6%) (p < 0.05 and p < 0.01). No significant differences were found in overall apoptosis rates or the proportion of normal cells between CAP-treated and glutamate groups. The results indicated that CAP treatment can modify apoptosis rates in cells subjected to glutamate-induced toxicity. Specifically, CAP treatment for 240 seconds significantly reduced early apoptosis compared to cells exposed only to glutamate, suggesting a protective effect against glutamate-induced damage. Conversely, CAP treatment increased late apoptosis rates, which could imply a shift in the cell death mode under prolonged exposure. CAP reduced oxidative stress level and triggered the self-antioxidant capability of tissues Oxidative stress is considered as a key factor in neurodegenerative diseases. Spinal cord injury results in heightened oxidative stress, attributed to an imbalance between reactive oxygen species and antioxidants within cells and tissues. To further investigate the effect of cold atmospheric plasma on intracellular ROS in SH-SY5Y, flow cytometry was conducted. As illustrated in Fig. 6 A and 6 B, the results showed a rightward shift in fluorescence intensity across the groups compared to the sham group. The MFI revealed significant statistical differences between the sham group (MFI = 4.837) and the Glu group (MFI = 92.37), 60s group (MFI = 22.03), and 240s group (MFI = 28.5) (P < 0.0001, P < 0.01, and P < 0.001, respectively). Additionally, significant differences were found between the Glu group and both the 60s and 240s groups (P < 0.0001). These findings suggest that direct cold atmospheric plasma treatment on glutamate-induced excitotoxicity cells effectively reduces intracellular ROS levels, thereby mitigating cell damage. Antioxidant capacity of tissues plays a crucial role in recovery from spinal cord injury 33 . This study measures the levels of glutathione peroxidase (GSH-XP) and superoxide dismutase (SOD) - key agents in scavenging reactive oxygen species, along with malondialdehyde (MDA), an indicator of oxidative damage in 28 days post CAP intervention. Notably, the results showed the CAP group exhibiting significantly higher antioxidant levels compared to both sham and SCI groups (Fig. 6 C-E), indicating an enhanced recovery mechanism. In Fig. 6 D, The SOD activity in Sham and CAP groups was relatively low, measured at 286.8 U/mgprot and 287.4 U/mgprot. At the same time, the SCI group exhibited a value of 341.8 U/mgprot. Similarly, the activity of glutathione GSH-PX in SCI (26.30 U/mgprot) was higher than in CAP (14.39 U/mgprot). It is plausible that CAP may have triggered the early activation of SOD and GSH-PX, resulting in increased enzymatic activities. By the 28th day, these activities have normalized or reverted to their baseline levels. This is confirmed by the reduced concentration in MDA from 3.94 nmol/mgprot for SCI group to 2.65 nmol/mgprot for the CAP group. In general, CAP treatment reduced oxidative stress and enhanced antioxidant defense mechanisms in spinal cord injury Discussion Increasing scientific evidence suggests the potential of CAP as an effective strategy for treating neurodegenerative diseases. This study demonstrates that CAP can promote functional recovery and reduce secondary damages such as oxidative stress, apoptosis, and inflammation in SCI models,. In addition, a novel neuroprotective mechanism was reported for the first time, to the best of our knowledge. The physical structure of the DBD, Tailored DBD structures designed for both in vitro and in vivo applications ensure precise and consistent application of CAP across different research setups. The in vivo findings provide compelling evidence of CAP's benefits on functional recovery post-SCI. The improved BMS scores in the CAP-treated group not only highlight the functional improvements but also clarify underlying mechanisms of neuroprotection and tissue repair. Histological analysis showed reduced neuronal apoptosis, decreased inflammation, and enhanced axonal regeneration, which were consistent with the observed functional outcomes. These results collectively suggest that CAP treatment may facilitate a favorable recovery environment by by modulating inflammatory responses, supporting neuronal survival, and promoting regenerative processes. Firstly, CAP treatment promotes axonal regeneration, as evidenced by positive NF immunofluorescent staining. CAP-induced spinal axonal regeneration is critical for recovery Secondly, unlike the SCI group, where astrocytes near the injury site show hypertrophy and increased intermediate filament protein expression, the CAP group exhibits a uniform astrocyte distribution with minimal scarring, indicating CAP's role in regulating astrocyte proliferation and scarring [ 31 ] . The last but not the least, the negative correlation between GFAP and NF indicates that excess astrocyte proliferation may inhibit axonal regeneration. CAP treatment balances it by fostering axonal growth and limiting astrocyte proliferation. Astrocytes is beneficial for healing initially. However, obstruction induced by fibrotic scarring may also occur the in cases of overreaction. The above discussion indicates astrocytes have a dual role in SCI 34–36 . CAP may preserve and stimulate neurons, consequently inhibiting the excessive proliferation of astrocytes and the formation of glial scars triggered by trauma-induced stress and promoting axon regression. The significant improvement in cell viability following CAP treatment, especially for durations of 120 and 240 seconds, underscores CAP's potential to mitigate glutamate-induced cytotoxicity. This observation implies a dose-responsive nature of CAP's neuroprotective capabilities and aligns with in vivo experiment outcomes. Additionally, the modulation of apoptosis by CAP—significantly decreasing early apoptosis while interestingly increasing late apoptosis rates—suggests a shift towards a more regulated form of cell death. Such regulation could help reduce inflammatory responses and promote tissue repair, further underscoring CAP's therapeutic potential. ROS levels and oxidative stress-related enzymes were further examined to investigate the mechanism of CAP treatment. CAP treatment effectively reduced intracellular ROS levels in SH-SY5Y cells, which is a critical aspect given the role of oxidative stress in SCI pathophysiology and neurodegenerative diseases. The enhancement of antioxidant defenses, as seen through increased activities of SOD and GSH-PX and reduced MDA levels, further indicates CAP's potential to restore oxidative balance and support tissue recovery. It is hypothesized that the reactive oxygen species in a low concentration could change redox homeostasis and increase the activation of antioxidant enzymes, which renders the cells resilient to an exogenous stressor and support cell proliferation and survival pathways 37–39 . In conclusion, This study effectively bridges the gap between in vitro and in vivo research, illuminating the significant therapeutic potential of cold atmospheric plasma for treating spinal cord injuries and neurodegenerative diseases (Fig. 7 ). Through a range of biological responses—from boosting cell viability and modulating apoptosis in SH-SY5Y cells facing glutamate-induced cytotoxicity to enhancing functional recovery and neuroprotection in a mouse model of spinal cord injury—our findings underscore CAP's capacity to positively influence cellular and tissue responses. This suggests a powerful therapeutic approach for neurodegenerative conditions and traumatic injuries. While further research is required to comprehensively understand the underlying mechanisms, our findings establish a strong foundation for the development of CAP-based therapeutic approaches in neuroscience. Methods 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% CO 2 humidified incubator at 37 °C. The suspension containing SH-SY5Y (cell concentration 1 × 10 6 /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) guidelines 40 . 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 level 41 . 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). Declarations Acknowledgments Funding: ITER Project of the Ministry of Science and Technology [grant numbers 2022YFE03080001] The Fundamental Research Funds for the Central Universities [grant numbers YD9110002012] The Fundamental Research Funds for the Central Universities [grant numbers USTC20210079] The joint Laboratory of Plasma Application Technology Funding [grant numbers JL06120001H]. The National Natural Science Foundation Incubation Program of the Second Affiliated Hospital of Anhui Medical University [grant numbers 2020GMFY06] The 2022 Natural Science Foundation of Anhui Province (C.B.D) [grant numbers 2208085MH254]. Author Contributions: Conceptualization: Zhengwei Wu Methodology: Zhu Chen, Jun Li, Chengbiao Ding Investigation: Jiwen Zhu, Zhenyu Liu, Qi Liu Visualization: Jiwen Zhu, Zhenyu Liu Supervision: Zhengwei Wu, Zhu Chen, Jun Li, Chengbiao Ding Writing—original draft: Jiwen Zhu, Qi Liu Writing—review & editing: Jiwen Zhu, Qinghua Xu Conflicts of Interest All other authors declare they have no competing interests. Data availability statement Data available on request / reasonable request. The author Jiwen Zhu will provide the data generated from this study upon direct email request to [email protected] . References Khachatryan, Z., Haunschild, J., von Aspern, K., Borger, M. A. & Etz, C. D. Ischemic Spinal Cord Injury-Experimental Evidence and Evolution of Protective Measures. Annals of Thoracic Surgery 113 , 1692-1702 (2022). https://doi.org:10.1016/j.athoracsur.2020.12.028 Alizadeh, A., Dyck, S. M. & Karimi-Abdolrezaee, S. Traumatic Spinal Cord Injury: An Overview of Pathophysiology, Models and Acute Injury Mechanisms. Frontiers in Neurology 10 , 282 (2019). https://doi.org:10.3389/fneur.2019.00282 Ahuja, C. S. et al. Traumatic spinal cord injury. Nature Reviews Disease Primers 3 , 17018 (2017). https://doi.org:10.1038/nrdp.2017.18 Saremi, J. et al. Advanced approaches to regenerate spinal cord injury: The development of cell and tissue engineering therapy and combinational treatments. Biomedicine & Pharmacotherapy 146 , 112-529 (2022). https://doi.org:10.1016/j.biopha.2021.112529 Cunningham, C. J. et al. The potential of gene therapies for spinal cord injury repair: a systematic review and meta-analysis of pre-clinical studies. Neural Regeneration Research 18 , 299-305 (2023). https://doi.org:10.4103/1673-5374.347941 Kumar, A., Kumar, N., Pathak, Z. & Kumar, H. Extra Cellular Matrix Remodeling: An Adjunctive Target for Spinal Cord Injury and Intervertebral Disc Degeneration. Neurospine 19 , 632-645 (2022). https://doi.org:10.14245/ns.2244366.183 Kathe, C. et al. The neurons that restore walking after paralysis. Nature 611 , 540-547 (2022). https://doi.org:10.1038/s41586-022-05385-7 Squair, J. W. et al. Recovery of walking after paralysis by regenerating characterized neurons to their natural target region. Science 381 , 1338-1345 (2023). https://doi.org:doi:10.1126/science.adi6412 Bruggeman, P. J., Iza, F. & Brandenburg, R. Foundations of atmospheric pressure non-equilibrium plasmas. Plasma Sources Science and Technology 26 , 123002 (2017). https://doi.org:10.1088/1361-6595/aa97af Sakudo, A., Yagyu, Y. & Onodera, T. Disinfection and Sterilization Using Plasma Technology: Fundamentals and Future Perspectives for Biological Applications. Int J Mol Sci 20 , 5216 (2019). https://doi.org:10.3390/ijms20205216 Laroussi, M., Lu, X. & Keidar, M. Perspective: The physics, diagnostics, and applications of atmospheric pressure low temperature plasma sources used in plasma medicine. Journal of Applied Physics 122 , 020901 (2017). https://doi.org:10.1063/1.4993710 Yan, X., Meng, Z., Ouyang, J., Qiao, Y. & Yuan, F. New Application of an Atmospheric Pressure Plasma Jet as a Neuro-protective Agent Against Glucose Deprivation-induced Injury of SH-SY5Y Cells. Journal of Visualized Experiments , 56323 (2017). https://doi.org:10.3791/56323 Weltmann, K. D. & Von Woedtke, T. Plasma medicine—current state of research and medical application. Plasma Physics and Controlled Fusion 59 , 014031 (2017). https://doi.org:10.1088/0741-3335/59/1/014031 Lu, X. et al. Reactive species in non-equilibrium atmospheric-pressure plasmas: Generation, transport, and biological effects. Physics Reports-Review Section of Physics Letters 630 , 1-84 (2016). https://doi.org:10.1016/j.physrep.2016.03.003 Di Meo, S., Reed, T. T., Venditti, P. & Victor, V. M. Role of ROS and RNS Sources in Physiological and Pathological Conditions. Oxid Med Cell Longev 2016 , 1245049 (2016). https://doi.org:10.1155/2016/1245049 Mitra, S., Kaushik, N., Moon, I. S., Choi, E. H. & Kaushik, N. K. Utility of Reactive Species Generation in Plasma Medicine for Neuronal Development. Biomedicines 8 , 348 (2020). Yan, X. et al. Cytoprotective effects of atmospheric-pressure plasmas against hypoxia-induced neuronal injuries. Journal of Physics D: Applied Physics 51 , 085401 (2018). https://doi.org:10.1088/1361-6463/aaa867 Tian, M. et al. Cold Atmospheric Plasma Elicits Neuroprotection Against Glutamate Excitotoxicity by Activating Cellular Antioxidant Defense. Plasma Chemistry and Plasma Processing 41 , 945-954 (2021). https://doi.org:10.1007/s11090-021-10172-9 Yan, X. et al. Atmospheric pressure plasma treatments protect neural cells from ischemic stroke‐relevant injuries by targeting mitochondria. Plasma Processes and Polymers 17 , 2000063 (2020). https://doi.org:10.1002/ppap.202000063 Steinbacher, P. & Eckl, P. Impact of oxidative stress on exercising skeletal muscle. Biomolecules 5 , 356-377 (2015). https://doi.org:10.3390/biom5020356 Coimbra-Costa, D. et al. Intermittent Hypobaric Hypoxic Preconditioning Provides Neuroprotection by Increasing Antioxidant Activity, Erythropoietin Expression and Preventing Apoptosis and Astrogliosis in the Brain of Adult Rats Exposed to Acute Severe Hypoxia. Int J Mol Sci 22 , 5272 (2021). https://doi.org:10.3390/ijms22105272 Chen, Y. et al. Application of an atmospheric pressure plasma jet in a rat model of ischaemic stroke: Design, optimisation, and characteristics. High Voltage 8 , 315-325 (2023). https://doi.org:10.1049/hve2.12267 Graves, D. B. Reactive Species from Cold Atmospheric Plasma: Implications for Cancer Therapy. Plasma Processes and Polymers 11 , 1120-1127 (2014). https://doi.org:10.1002/ppap.201400068 Ding, C. et al. Cold air plasma: A potential strategy for inducing apoptosis of rheumatoid arthritis fibroblast‐like synoviocytes. High Voltage 7 , 106-116 (2022). https://doi.org:10.1049/hve2.12132 Ding, C. et al. Cold air plasma improving rheumatoid arthritis via mitochondrial apoptosis pathway. Bioengineering & Translational Medicine 8 , 10366 (2022). https://doi.org:10.1002/btm2.10366 Chen, Y. et al. Inhalation of Atmospheric-Pressure Gas Plasma Attenuates Brain Infarction in Rats With Experimental Ischemic Stroke. Frontiers in Neuroscience 16 , 875053 (2022). https://doi.org:10.3389/fnins.2022.875053 Xiong, Z. L. Cold Atmospheric Plasmas: A Novel and Promising Way to Treat Neurological Diseases. Trends in Biotechnology 36 , 582-583 (2018). https://doi.org:10.1016/j.tibtech.2018.04.003 Zhou, X. et al. Microglia and macrophages promote corralling, wound compaction and recovery after spinal cord injury via Plexin-B2. Nat Neurosci 23 , 337-350 (2020). https://doi.org:10.1038/s41593-020-0597-7 Anjum, A. et al. Spinal Cord Injury: Pathophysiology, Multimolecular Interactions, and Underlying Recovery Mechanisms. International Journal of Molecular Sciences 21 , 7533 (2020). https://doi.org:10.3390/ijms21207533 Hellenbrand, D. J. et al. Inflammation after spinal cord injury: a review of the critical timeline of signaling cues and cellular infiltration. Journal of Neuroinflammation 18 , 284 (2021). https://doi.org:10.1186/s12974-021-02337-2 Zhao, R., Wu, X., Bi, X. Y., Yang, H. & Zhang, Q. Baicalin attenuates blood-spinal cord barrier disruption and apoptosis through PI3K/Akt signaling pathway after spinal cord injury. Neural Regen Res 17 , 1080-1087 (2022). https://doi.org:10.4103/1673-5374.324857 Sugiyama, K. & Tanaka, K. Spinal cord-specific deletion of the glutamate transporter GLT1 causes motor neuron death in mice. Biochemical and Biophysical Research Communications 497 , 689-693 (2018). https://doi.org:https://doi.org/10.1016/j.bbrc.2018.02.132 Singh, A., Kukreti, R., Saso, L. & Kukreti, S. Oxidative Stress: A Key Modulator in Neurodegenerative Diseases. Molecules 24 , 1583 (2019). https://doi.org:10.3390/molecules24081583 Bradbury, E. J. & Burnside, E. R. Moving beyond the glial scar for spinal cord repair. Nature Communications 10 , 3879 (2019). https://doi.org:10.1038/s41467-019-11707-7 Zamanian, J. L. et al. Genomic Analysis of Reactive Astrogliosis. The Journal of Neuroscience 32 , 6391-6410 (2012). https://doi.org:10.1523/jneurosci.6221-11.2012 Ayazi, M., Zivkovic, S., Hammel, G., Stefanovic, B. & Ren, Y. Fibrotic Scar in CNS Injuries: From the Cellular Origins of Fibroblasts to the Molecular Processes of Fibrotic Scar Formation. Cells 11 , 2371 (2022). https://doi.org:10.3390/cells11152371 Schottlender, N., Gottfried, I. & Ashery, U. Hyperbaric Oxygen Treatment: Effects on Mitochondrial Function and Oxidative Stress. Biomolecules 11 , 1827 (2021). Chen, X., Guo, C. & Kong, J. Oxidative stress in neurodegenerative diseases. Neural Regen Res 7 , 376-385 (2012). https://doi.org:10.3969/j.issn.1673-5374.2012.05.009 Lennicke, C. & Cochemé, H. M. Redox metabolism: ROS as specific molecular regulators of cell signaling and function. Mol Cell 81 , 3691-3707 (2021). https://doi.org:10.1016/j.molcel.2021.08.018 du Sert, N. P. et al. Reporting animal research: Explanation and elaboration for the ARRIVE guidelines 2.0. PLOS Biology 18 , e3000411 (2020). https://doi.org:10.1371/journal.pbio.3000411 Au - McDonough, A., Au - Monterrubio, A., Au - Ariza, J. & Au - Martínez-Cerdeño, V. Calibrated Forceps Model of Spinal Cord Compression Injury. JoVE , e52318 (2015). https://doi.org:doi:10.3791/52318 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 16 Aug, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 12 Jun, 2024 Reviews received at journal 12 Jun, 2024 Reviews received at journal 05 Jun, 2024 Reviewers agreed at journal 04 Jun, 2024 Reviewers agreed at journal 03 Jun, 2024 Reviewers invited by journal 03 Jun, 2024 Editor assigned by journal 03 Jun, 2024 Editor invited by journal 26 Apr, 2024 Submission checks completed at journal 26 Apr, 2024 First submitted to journal 20 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4297802","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":298115510,"identity":"85c98654-ec80-4f95-af83-61bf863e049a","order_by":0,"name":"Jiwen Zhu","email":"","orcid":"","institution":"Institute of Advanced Technology, University of Science and Technology of China, Hefei, Anhui","correspondingAuthor":false,"prefix":"","firstName":"Jiwen","middleName":"","lastName":"Zhu","suffix":""},{"id":298115514,"identity":"88392833-bb38-44bf-97fb-c3aed57ce66b","order_by":1,"name":"Zhenyu Liu","email":"","orcid":"","institution":"Department of Rehabilitation Medicine, The Second Affiliated Hospital of Anhui Medical University, Hefei, Anhui","correspondingAuthor":false,"prefix":"","firstName":"Zhenyu","middleName":"","lastName":"Liu","suffix":""},{"id":298115518,"identity":"3f2fb06f-401a-4717-85d0-6a003665a006","order_by":2,"name":"Qi Liu","email":"","orcid":"","institution":"School of Nuclear Science and Technology, University of Science and Technology of China, Hefei, Anhui","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Liu","suffix":""},{"id":298115522,"identity":"c0d3b4bd-d125-4b10-8d4b-8eba27401d00","order_by":3,"name":"Qinghua Xu","email":"","orcid":"","institution":"Anhui Provincial Center for Disease Control and Prevention, Public Health Research Institute of Anhui Province","correspondingAuthor":false,"prefix":"","firstName":"Qinghua","middleName":"","lastName":"Xu","suffix":""},{"id":298115526,"identity":"fad19710-cdfe-47bc-8e8d-90eff8f33b02","order_by":4,"name":"Chengbiao Ding","email":"","orcid":"","institution":"Department of Rehabilitation Medicine, The Second Affiliated Hospital of Anhui Medical University, Hefei, Anhui","correspondingAuthor":false,"prefix":"","firstName":"Chengbiao","middleName":"","lastName":"Ding","suffix":""},{"id":298115528,"identity":"80e1eb52-a4c1-47a5-a1aa-588ad6450e5e","order_by":5,"name":"Zhu Chen","email":"","orcid":"","institution":"the First Affiliated Hospital of USTC, University of Science and Technology of China","correspondingAuthor":false,"prefix":"","firstName":"Zhu","middleName":"","lastName":"Chen","suffix":""},{"id":298115529,"identity":"aaaaa76c-56d0-4c09-8dc4-c6fea120e249","order_by":6,"name":"Jun Li","email":"","orcid":"","institution":"Department of Spinal and Neural Function Reconstruction, China Rehabilitation Research Center","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Li","suffix":""},{"id":298115530,"identity":"c6646bfb-8882-4d91-8c46-a99fca277ba6","order_by":7,"name":"Zhengwei Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAr0lEQVRIiWNgGAWjYLCCD1BagmgdjDOgqonXwsxDkhaDG+nPpG3b6ur4G5gP3uZhsMsjQktCmnRu22EJiQNsydY8DMnFBLWY3Ug4dju37YCEAQOPmTQPw4HEBsJaEttuW7bVAbXwfyNWSzLbbcY2ZpAtbMRpsT/zjP1nz7nDkjMOsxlbzjFIJqxFsj39scGPsjp+/vbmhzfeVNgR1oIAzCDCgHj1o2AUjIJRMArwAACcVjUQHFFs+wAAAABJRU5ErkJggg==","orcid":"","institution":"School of Nuclear Science and Technology, University of Science and Technology of China, Hefei, Anhui","correspondingAuthor":true,"prefix":"","firstName":"Zhengwei","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2024-04-20 14:03:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4297802/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4297802/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-69861-y","type":"published","date":"2024-08-16T15:57:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":55798423,"identity":"72efd0c6-1af8-43a3-be91-ea2027f0a1f1","added_by":"auto","created_at":"2024-05-03 11:53:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":71324,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDielectric barrier discharge device. (A) \u003c/strong\u003e2D diagram of dielectric barrier discharge device.\u003cstrong\u003e (B)\u003c/strong\u003eLissajous figure of DBD discharge. \u003cstrong\u003e(C)\u003c/strong\u003eDBD discharge voltage and current waveform. \u003cstrong\u003e(D) \u003c/strong\u003eOptical emission spectroscopy spectrum of dielectric barrier discharge plasma at wavelengths ranging from 300 to 1000 nm. \u003cstrong\u003e(E)\u003c/strong\u003e Schematic illustration of the DBD device in operation.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4297802/v1/65cc8b9cb58fed7dfc1a1c02.png"},{"id":55798421,"identity":"c912011b-6336-4f00-b858-8b10a6ec1fad","added_by":"auto","created_at":"2024-05-03 11:53:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":146497,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluating neuroprotective effects of CAP treatment \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. (A) \u003c/strong\u003eTraumatic spinal cord injury in mice, laminectomy was performed at the T9-10 vertebrate. Forceps were applied to a 5-second complete spinal cord compression at the T10 level. \u003cstrong\u003e(B)\u003c/strong\u003e Schematic diagram of the experimental design. \u003cstrong\u003e(C)\u003c/strong\u003e Basso Mouse Scale (BMS) motor recovery scale was measured at 0, 1, 7, 14, 21, 28 dpi (n=8). \u003cstrong\u003e(D)\u003c/strong\u003e Representative images of H\u0026amp;E staining of the injury site in 10× and magnified images in the black box regions in 40× in 7 dpi (n=8). Scale bar = 200μm and 50μm. \u003cstrong\u003e(E)\u003c/strong\u003e H\u0026amp;E staining in 10× and magnified images in the black box regions in 40× in 28 dpi (n=8). Scale bar = 200μm and 50μm. Data are expressed as the mean ± SEM. *P \u0026lt; 0.05, **P \u0026lt; 0.01 vs. SCI group.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4297802/v1/c82150f8dabfc9ad57b794d8.png"},{"id":55798419,"identity":"f053ff07-d8bc-4794-af47-40a528ed07a5","added_by":"auto","created_at":"2024-05-03 11:53:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":159290,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssessment of apoptosis and inflammation response properties \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eWhite stars indicate the lesion site.\u003cstrong\u003e (A)\u003c/strong\u003e Tunel immunofluorescence staining in 28dpi (n=3). Scale bar = 50μm. (B) Quantitative analysis of the cell apoptosis ratio. \u003cstrong\u003e(C)\u003c/strong\u003e Nissl staining in 28 dpi (n=3). Scale bar = 50μm. \u003cstrong\u003e(D)\u003c/strong\u003e TNF-α immunofluorescence staining in 28dpi (n=3). Scale bar = 50μm.\u003cstrong\u003e (E)\u003c/strong\u003e IL-1β immunofluorescence staining in 28dpi (n=3). Scale bar = 50μm. \u003cstrong\u003e(F) \u003c/strong\u003eELISA assay of inflammatory cytokines collected at tissue supernatants for TNF-α (n=3). (\u003cstrong\u003eG)\u003c/strong\u003e ELISA assay of inflammatory cytokines collected at tissue supernatants for IL-1β (n=3). Data are expressed as the mean ± SEM, *P \u0026lt; 0.05, **P \u0026lt; 0.01 vs SCI group.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4297802/v1/024bca44b3bd962663b556a8.png"},{"id":55798418,"identity":"05e405b5-bf5d-44a3-a89b-bfea34cc9717","added_by":"auto","created_at":"2024-05-03 11:53:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":187537,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssessment of axon regression and glial scar \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003eWhite stars indicate the lesion site. \u003cstrong\u003e(A)\u003c/strong\u003eNeurofilaments marker (NF) and Astrocytes marker (GFAP) double immunofluorescence staining images (n=5). Scale bar = 200μm. \u003cstrong\u003e(B)\u003c/strong\u003e Magnified images for NF and GFAP within 400μm from the injury site (n=5). Scale bar = 50μm. \u003cstrong\u003e(C) \u003c/strong\u003eQuantification of the density of NF+. \u003cstrong\u003e(D) \u003c/strong\u003eQuantification of the density of GFAP+. Data are expressed as the mean ± SEM, *P \u0026lt; 0.05, **P \u0026lt; 0.01 vs SCI group.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4297802/v1/d73f01c45048f15331ab1b05.png"},{"id":55798420,"identity":"9e5d0528-18b4-47a9-9f16-cee9a5533db7","added_by":"auto","created_at":"2024-05-03 11:53:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":89567,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluating neuroprotective effects of CAP treatment \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Schematic illustration of the DBD device in operation. \u003cstrong\u003e(B)\u003c/strong\u003e Cell viability after 24-hour exposure to varying concentrations of glutamate. \u003cstrong\u003e(C)\u003c/strong\u003e Cell survival rates following different durations of DBD treatment.\u003cstrong\u003e (D)\u003c/strong\u003e Effects of different CAP treatment durations on apoptosis \u003cem\u003ein vitro \u003c/em\u003eafter 24 h. Data are expressed as the mean ± SEM *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001, vs Glu group.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4297802/v1/1eb652208d649035d9bb6653.png"},{"id":55798981,"identity":"e7f55722-c2c5-470b-be16-92dc471524b9","added_by":"auto","created_at":"2024-05-03 12:01:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":35917,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntracellular ROS content \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and oxidative stress indicators level (n=3) \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e (A)\u003c/strong\u003e Effects of different CAP treatment durations on ROS levels \u003cem\u003ein vitro\u003c/em\u003e.\u003cstrong\u003e (B) \u003c/strong\u003eQuantitative analysis graph of ROS fluorescence intensity. \u003cstrong\u003e(C) \u003c/strong\u003eAntioxidant enzymes collected at tissue supernatants for GSH-PX (n=3). \u003cstrong\u003e(D)\u003c/strong\u003eAntioxidant enzymes collected at tissue supernatants for SOD (n=4). \u003cstrong\u003e(E) \u003c/strong\u003e\u0026nbsp;Oxidative stress indexes at tissue supernatants for MDA (n=3). Data are expressed as the mean ± SEM, P \u0026lt; 0.05, ****P \u0026lt; 0.0001 vs SCI group\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4297802/v1/2178bd480c3f11b437351fde.png"},{"id":55798982,"identity":"7bad64d7-808b-48b3-b299-653c58c78ed1","added_by":"auto","created_at":"2024-05-03 12:01:50","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":78373,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of the mechanism of CAP's therapeutic effect on the spinal cord.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4297802/v1/8a30d4b936c3224c83a076a2.png"},{"id":63071013,"identity":"6674c218-dd83-40c3-8382-dc796de1d44d","added_by":"auto","created_at":"2024-08-22 20:02:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1514268,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4297802/v1/4a7b224c-8aa5-46fd-8f3e-3239c9b98234.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhanced Neural Recovery and Reduction of Secondary Damage in Spinal Cord Injury through Modulation of Oxidative Stress and Neural Response","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSpinal Cord Injury (SCI) is one of the most prevalent and debilitating conditions worldwide, primarily due to the limited regenerative capacity of nerves, often resulting in permanent functional impairments\u003csup\u003e1\u003c/sup\u003e. When SCI happens, the repair of nerve cells occurs at a slow pace, with little to no promotion of natural neuron regeneration. The sequelae of SCI are essentially incurable with current medical and rehabilitative approaches, and spontaneous recovery of bodily functions often plateaus within approximately 1.5 years post-SCI\u003csup\u003e2\u003c/sup\u003e. Current therapeutic strategies for SCI primarily focus on mitigating secondary complications and compensating for lost parts rather than targeting neural recovery or restoration of pre-injury capabilities\u003csup\u003e3\u003c/sup\u003e. Therefore, it is critical to develop novel treatments for early-stage spinal cord injuries. In recent years, numerous emerging neural protection and regeneration strategies have provided new directions for SCI treatment. These approaches aim to reduce neuronal death following central nervous system injuries, while also enhancing the intrinsic regenerative capabilities of post-mitotic neurons. Furthermore, improving the hostile extracellular environment within the central nervous system that inhibits neuronal growth is also an effective strategy. \u003csup\u003e4\u0026ndash;8\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePlasma, the fourth state of matter following solid, liquid, and gas, consists of highly reactive physical and chemical substances\u003csup\u003e9\u003c/sup\u003e. In recent years, cold atmospheric plasma (CAP) has applied widely in biomedicine because it doesn\u0026rsquo;t cause thermal damage to biological tissues and has no toxic side effects. \u003csup\u003e10\u003c/sup\u003e. CAP comprises highly active substances such as highly reactive species (reactive oxygen and nitrogen species), electric fields, ultraviolet radiation, and charged particles, which enable it to modulate biological processes\u003csup\u003e11\u003c/sup\u003e .Dielectric barrier discharge (DBD) is a typical method for generating CAP. Under different working gases and operating parameters, DBD could yield diverse active species\u003csup\u003e12,13\u003c/sup\u003e, including nitric oxide (NO), superoxide (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e), hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e); singlet oxygen (\u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e); ozone (O\u003csub\u003e3\u003c/sub\u003e) and even hydroxyl radical (\u0026bull;OH) can and do play essential roles in biological systems.\u003csup\u003e14,15\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eMore and more research shows that CAP is a novel and promising way to treat neurological diseases\u003csup\u003e16\u003c/sup\u003e. CAP primarily addresses neural regression diseases through two pathways: On one hand, CAP has demonstrated neuroprotective effects against adverse conditions such as neuronal damage induced by glucose deprivation and hypoxia \u003csup\u003e17\u0026ndash;20\u003c/sup\u003e .It is evident that exposure to mild \u0026ldquo;mini-insults\u0026rdquo; causes injury tolerance, making neurons more resilient to damage in the future\u003csup\u003e21\u003c/sup\u003e. A recent study showed that CAP induces the production of reactive oxygen and nitrogen species (RONS), leading to a significant and transient increase in cellular glutathione (GSH) levels and the activation of erythroid 2-related factor 2 (Nrf2), thereby mitigating glutamate excitotoxicity\u003csup\u003e18\u003c/sup\u003e. Another study reported that atmospheric pressure plasma jets (APPJ) could reduce the damage of middle cerebral artery occlusion and improve neurological function. Plasma-induced NO generation could be potentially used as a cytoprotective agent\u003csup\u003e22\u003c/sup\u003e. On the other hand, previous studies have reported that CAP could notably stimulate the differentiation of neuronal stem cells and promote increased neuronal regeneration following trauma\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Moreover, CAP have been directly applied to living organisms, such as cancer,\u003csup\u003e23\u003c/sup\u003e rheumatoid arthritis\u003csup\u003e24,25\u003c/sup\u003e ,and ischaemic stroke\u003csup\u003e26\u003c/sup\u003e ,demonstrating the potential of CAP for \u003cem\u003ein vivo\u003c/em\u003e treatment.\u003c/p\u003e \u003cp\u003eThe potential usage of CAP in neuronal and Central Nervous System(CNS) injury treatment has been relatively proven \u003cem\u003ein vitro\u003c/em\u003e\u003csup\u003e27\u003c/sup\u003e. However, the application of CAP \u003cem\u003ein vivo\u003c/em\u003e and its experimental efficacy in treating neurodegenerative diseases remains limited and warrants further investigation. Using a compression injury model, this study introduces CAP to the spinal cord injury site. Various methods were utilized to assess the therapeutic effects, axon regression, neuronal apoptosis and oxidative stress levels. The research findings can offer a novel treatment approach for clinically managing SCI and pave the way for innovative treatments in neurodegenerative disease management\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePhysical characterization of dielectric barrier discharge devices\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe DBD device shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e is specifically designed for conducting experiments on spinal cord injury both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e settings. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA depicts a tri-layer structure: a titanium (Ti) electrode on top, a polyvinyl chloride (PVC) middle dielectric layer, and a copper (Cu) electrode at the base. The device's dimensions (5mm in length and 4mm in width) are optimized for cell culture applications. Similarly, the DBD functions effectively with a 3 mm gap during animal experiments, ensuring practicality, precision and accuracy across multiple sets of experiments. To monitor the real-time operating status of the CAP treatment, a digital oscilloscope was linked to the positive and negative electrodes of the DBD apparatus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eThe DBD device employed air as its working gas. When the DBD device is discharging, two electrical sensors were used to measure the DBD discharge voltage and current waveforms, which are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC. The voltage waveform is a typical sine wave with a frequency of 13.7 kHz. The overall discharge power of the DBD is 38.8 W. The power measurement method comes from Manley's classical DBD discharge model and is based on the equivalent circuit method. The charge Q(t) can be obtained as the integral of the measured current waveform with a sampling capacitance (47 \u0026micro;F). By measuring the Lissajous figure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) of DBD discharge, the average power could be obtained through the graphic area. The specific calculation formula is as follows.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Equ1\" class=\"Equation\"\u003e \u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\text{P}=\\frac{1}{\\text{T}}{\\oint }_{T}\\text{Q}\\left(\\text{V}\\right)\\text{d}\\text{V}$$\u003c/div\u003e \u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe T refers to the discharge cycle, and the V refers to the voltage. Therefore, we can derive further from the above formula, which is\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Equ2\" class=\"Equation\"\u003e \u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\text{P}=\\text{f} \\times \\text{C}\\times \\text{s}$$\u003c/div\u003e \u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe f (13.7 kHz) refers to the frequency of discharge, the C (0.47 \u0026micro;F) refers to the sampling capacitance value, and the s (2.88) refers to the area of the Lissajous figure. The power value is calculated to be about 38.8 W.\u003c/p\u003e \u003cp\u003eOptical emission spectroscopy (OES) was recorded to identify the composition of reactive species generated by DBD, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD. The range from 338 to 405 nm corresponds to transitions in the N\u003csup\u003e2+\u003c/sup\u003e second positive system. Additionally, a distinct emission peak at 770.09 nm signifies the presence of atomic oxygen. These two species correspond to ionized nitrogen and oxygen, which are the most abundant ingredient in the working gas. These active species play a significant role in regulating cellular activities within biological organisms, emphasizing their importance in biomedical applications and further studies.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCAP treatment improved functional recovery after SCI\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo evaluate the effects of cold atmospheric plasma on the mouse spinal cord, we established a compression model in C57 mice (n\u0026thinsp;=\u0026thinsp;8), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, and the timeline of the experimental design is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB. Basso Mouse Scale (BMS) scores were used to assess hindlimb performance. The BMS scores at 0, 1, 7, 10, 14, and 28 dpi (days post injury) represents different levels of functional recovery (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The sham group was 9 points, and the hind limb motor function was completely normal. After surgery, the SCI and CAP groups immediately reached a state of complete hindlimb paralysis post-surgery (BMS score\u0026thinsp;=\u0026thinsp;0), which showed irreversible neurological damage. Compared with the SCI group, the motor function of the CAP group recovered more quickly and reached higher motor performance scores, notably by the 28th day post-intervention\u003csup\u003e28\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIt is well known that functional recovery is correlated with tissue structure and pathophysiology. Tissue samples were collected at 7 and 28 dpi. Hematoxylin and eosin (H\u0026amp;E) staining was used to visualize pathological changes in the spinal cord. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, the structure of the sham group was completed and normal with abundant neurons. At the same time, the SCI and CAP groups experienced atrophy and loose structural integrity due to the formation of scars and cavities. Compared to the CAP group, the injury group demonstrates a larger area of damage, more extensive edema, cyst formation and hemorrhage, and less nerve fiber density.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCAP treatment decreased neuronal apoptosis and inflammation response after SCI\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSome \u003cem\u003ein vitro\u003c/em\u003e studies suggest that CAP can enhance neuroprotection. However, there is no corresponding \u003cem\u003ein vivo\u003c/em\u003e evidence to support this claim. Therefore, we conducted a separate analysis to assess apoptosis, neuronal survival and inflammation response following CAP treatment. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, TUNEL staining revealed a significant increase in cell death in injured spinal cords compared to the sham control group. Notably, the CAP treatment group exhibited a significant reduction in the percentage of apoptotic neurons compared to the SCI group. The apoptosis index for the SCI group was significantly higher (15.07%) than that of the CAP group (0.65%). Nissl staining demonstrated a decrease in surviving neurons, with atrophied cytoplasm post-surgery. In contrast, the CAP treated group displayed neurons with a more regular shape, increased neurites, and deeper blue-stained Nissl bodies in histological analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The Nissl and TUNEL results collectively support a higher preservation rate of intact neuronal structures in the CAP-treated group.\u003c/p\u003e \u003cp\u003eTo assess the impact of CAP on the inflammatory response following SCI, we conducted immunofluorescent staining and ELISA to measure the concentrations of inflammatory cytokines\u003csup\u003e29,30\u003c/sup\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, immunofluorescent results demonstrated a pronounced accumulation of inflammatory factors at the injury site. Using ELISA, we detected changes in the expression levels of TNF-α and IL-1β in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG. After calibration, the average protein content for TNF-α and IL-1β in the sham group was measured at 69.91 pg/mL and 22.62 pg/mL, respectively. In the CAP group, TNF-α levels were 38.09 pg/mL, significantly lower than the 89.14 pg/mL observed in the SCI group (p\u0026thinsp;\u0026gt;\u0026thinsp;0.01). Likewise, the ELISA analysis of IL-1β revealed substantially lower expression in the CAP group (76.29 pg/mL) compared to the SCI group (144.2 pg/mL) (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCAP treatment enhanced axonal regeneration and reduced glial scar formation\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eA series of histological analyses were conducted to examine the effects of the CAP treatment on SCI. The immunofluorescent staining of spinal cord sagittal sections was further performed to investigate the axonal regeneration in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB. The neurofilaments (NF200) were selected to indicate the axon growth at the lesion site (within 400\u0026micro;m). As was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, while there is no significant difference between the CAP group and the SCI group, images demonstrate that the CAP group exhibits better neural preservation and axon regression.\u003c/p\u003e \u003cp\u003eGlial fibrillary acidic protein (GFAP), a specific marker of astrocytes negatively correlated with NF, was also used to evaluate spinal cord repair\u0026mdash;the GFAP\u0026thinsp;+\u0026thinsp;area of the excessive representative. The GFAP\u0026thinsp;+\u0026thinsp;area of the representative sections displays pronounced glial scarring. Compared to the CAP group, the SCI group exhibited a significant enhancement in GFAP fluorescence signals near the injury epicentre, decreasing radially towards the injury periphery (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Furthermore, NF signals did not colocalise with the GFAP astroglial scar. The results demonstrate that the CAP group exhibits more excellent neural preservation and axon regression.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e \u003cb\u003eCAP treatment increased cell viability and altered apoptosis of SH-SY5Y cells in response to glutamate-induced cytotoxicity\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn our study, SH-SY5H cells were exposed to glutamate concentrations of 100, 150, and 200 \u0026micro;M to induce neurotoxicity\u003csup\u003e31,32\u003c/sup\u003e. A significant viability reduction to 50% was observed at 150 \u0026micro;M after 24 hours, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA using a CCK8 assay. Therefore, 150 \u0026micro;M was selected for the excitotoxicity model using SH-SY5Y cells. Following CAP treatments for 30, 60, 120, and 240 seconds after 24 h, cell viability improved to 71.2%, 68.91%, 86.84%, and 88.74% respectively, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB. Notably, durations of 120 and 240 seconds exhibited a marked increase in cell viability compared to the control group, with p-values\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and \u0026lt;\u0026thinsp;0.001, demonstrating CAP's neuroprotective potential against glutamate-induced cytotoxicity.\u003c/p\u003e \u003cp\u003eThe apoptosis status of SH-SY5Y Cells was quantitatively analyzed by flow cytometry. Related results were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD. Early apoptosis significantly decreased in the 240 s CAP-treated group (47.57%) compared to the Glu group (66.1%) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Late apoptosis rates increased significantly in both the 60 s (22.97%) and 240 s (25.9%) groups compared to Glu group (11.6%) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). No significant differences were found in overall apoptosis rates or the proportion of normal cells between CAP-treated and glutamate groups. The results indicated that CAP treatment can modify apoptosis rates in cells subjected to glutamate-induced toxicity. Specifically, CAP treatment for 240 seconds significantly reduced early apoptosis compared to cells exposed only to glutamate, suggesting a protective effect against glutamate-induced damage. Conversely, CAP treatment increased late apoptosis rates, which could imply a shift in the cell death mode under prolonged exposure.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCAP reduced oxidative stress level and triggered the self-antioxidant capability of tissues\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eOxidative stress is considered as a key factor in neurodegenerative diseases. Spinal cord injury results in heightened oxidative stress, attributed to an imbalance between reactive oxygen species and antioxidants within cells and tissues. To further investigate the effect of cold atmospheric plasma on intracellular ROS in SH-SY5Y, flow cytometry was conducted. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, the results showed a rightward shift in fluorescence intensity across the groups compared to the sham group. The MFI revealed significant statistical differences between the sham group (MFI\u0026thinsp;=\u0026thinsp;4.837) and the Glu group (MFI\u0026thinsp;=\u0026thinsp;92.37), 60s group (MFI\u0026thinsp;=\u0026thinsp;22.03), and 240s group (MFI\u0026thinsp;=\u0026thinsp;28.5) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively). Additionally, significant differences were found between the Glu group and both the 60s and 240s groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). These findings suggest that direct cold atmospheric plasma treatment on glutamate-induced excitotoxicity cells effectively reduces intracellular ROS levels, thereby mitigating cell damage.\u003c/p\u003e \u003cp\u003eAntioxidant capacity of tissues plays a crucial role in recovery from spinal cord injury\u003csup\u003e33\u003c/sup\u003e. This study measures the levels of glutathione peroxidase (GSH-XP) and superoxide dismutase (SOD) - key agents in scavenging reactive oxygen species, along with malondialdehyde (MDA), an indicator of oxidative damage in 28 days post CAP intervention. Notably, the results showed the CAP group exhibiting significantly higher antioxidant levels compared to both sham and SCI groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-E), indicating an enhanced recovery mechanism. In Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, The SOD activity in Sham and CAP groups was relatively low, measured at 286.8 U/mgprot and 287.4 U/mgprot. At the same time, the SCI group exhibited a value of 341.8 U/mgprot. Similarly, the activity of glutathione GSH-PX in SCI (26.30 U/mgprot) was higher than in CAP (14.39 U/mgprot). It is plausible that CAP may have triggered the early activation of SOD and GSH-PX, resulting in increased enzymatic activities. By the 28th day, these activities have normalized or reverted to their baseline levels. This is confirmed by the reduced concentration in MDA from 3.94 nmol/mgprot for SCI group to 2.65 nmol/mgprot for the CAP group. In general, CAP treatment reduced oxidative stress and enhanced antioxidant defense mechanisms in spinal cord injury\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIncreasing scientific evidence suggests the potential of CAP as an effective strategy for treating neurodegenerative diseases. This study demonstrates that CAP can promote functional recovery and reduce secondary damages such as oxidative stress, apoptosis, and inflammation in SCI models,. In addition, a novel neuroprotective mechanism was reported for the first time, to the best of our knowledge.\u003c/p\u003e \u003cp\u003eThe physical structure of the DBD, Tailored DBD structures designed for both in vitro and in vivo applications ensure precise and consistent application of CAP across different research setups.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e findings provide compelling evidence of CAP's benefits on functional recovery post-SCI. The improved BMS scores in the CAP-treated group not only highlight the functional improvements but also clarify underlying mechanisms of neuroprotection and tissue repair. Histological analysis showed reduced neuronal apoptosis, decreased inflammation, and enhanced axonal regeneration, which were consistent with the observed functional outcomes. These results collectively suggest that CAP treatment may facilitate a favorable recovery environment by by modulating inflammatory responses, supporting neuronal survival, and promoting regenerative processes. Firstly, CAP treatment promotes axonal regeneration, as evidenced by positive NF immunofluorescent staining. CAP-induced spinal axonal regeneration is critical for recovery Secondly, unlike the SCI group, where astrocytes near the injury site show hypertrophy and increased intermediate filament protein expression, the CAP group exhibits a uniform astrocyte distribution with minimal scarring, indicating CAP's role in regulating astrocyte proliferation and scarring\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. The last but not the least, the negative correlation between GFAP and NF indicates that excess astrocyte proliferation may inhibit axonal regeneration. CAP treatment balances it by fostering axonal growth and limiting astrocyte proliferation. Astrocytes is beneficial for healing initially. However, obstruction induced by fibrotic scarring may also occur the in cases of overreaction. The above discussion indicates astrocytes have a dual role in SCI \u003csup\u003e34\u0026ndash;36\u003c/sup\u003e. CAP may preserve and stimulate neurons, consequently inhibiting the excessive proliferation of astrocytes and the formation of glial scars triggered by trauma-induced stress and promoting axon regression.\u003c/p\u003e \u003cp\u003eThe significant improvement in cell viability following CAP treatment, especially for durations of 120 and 240 seconds, underscores CAP's potential to mitigate glutamate-induced cytotoxicity. This observation implies a dose-responsive nature of CAP's neuroprotective capabilities and aligns with \u003cem\u003ein vivo\u003c/em\u003e experiment outcomes. Additionally, the modulation of apoptosis by CAP\u0026mdash;significantly decreasing early apoptosis while interestingly increasing late apoptosis rates\u0026mdash;suggests a shift towards a more regulated form of cell death. Such regulation could help reduce inflammatory responses and promote tissue repair, further underscoring CAP's therapeutic potential.\u003c/p\u003e \u003cp\u003eROS levels and oxidative stress-related enzymes were further examined to investigate the mechanism of CAP treatment. CAP treatment effectively reduced intracellular ROS levels in SH-SY5Y cells, which is a critical aspect given the role of oxidative stress in SCI pathophysiology and neurodegenerative diseases. The enhancement of antioxidant defenses, as seen through increased activities of SOD and GSH-PX and reduced MDA levels, further indicates CAP's potential to restore oxidative balance and support tissue recovery. It is hypothesized that the reactive oxygen species in a low concentration could change redox homeostasis and increase the activation of antioxidant enzymes, which renders the cells resilient to an exogenous stressor and support cell proliferation and survival pathways\u003csup\u003e37\u0026ndash;39\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn conclusion, This study effectively bridges the gap between \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e research, illuminating the significant therapeutic potential of cold atmospheric plasma for treating spinal cord injuries and neurodegenerative diseases (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Through a range of biological responses\u0026mdash;from boosting cell viability and modulating apoptosis in SH-SY5Y cells facing glutamate-induced cytotoxicity to enhancing functional recovery and neuroprotection in a mouse model of spinal cord injury\u0026mdash;our findings underscore CAP's capacity to positively influence cellular and tissue responses. This suggests a powerful therapeutic approach for neurodegenerative conditions and traumatic injuries. While further research is required to comprehensively understand the underlying mechanisms, our findings establish a strong foundation for the development of CAP-based therapeutic approaches in neuroscience.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eDielectric barrier discharge device\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture and intervention\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSH-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% CO\u003csub\u003e2\u003c/sub\u003e humidified incubator at 37 °C. The suspension containing SH-SY5Y (cell concentration 1 × 10\u003csup\u003e6\u003c/sup\u003e/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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell count kit 8 determination\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of apoptosis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntracellular ROS content\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFemale 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.\u0026nbsp;All experimental procedures were planned and reported in compliance with the guidelines outlined in the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines\u003csup\u003e40\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal Ethics Committee of Anhui Medical University\u0026nbsp;provided official approval for the studies presented in this manuscript (Approval No. 20200851).\u0026nbsp;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).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpinal cord injury model and intervention\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBefore 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 level\u003csup\u003e41\u003c/sup\u003e. 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMotor Function Assessment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistological procedures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUpon 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH\u0026amp;E and Nissl staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDehydrated 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\u0026amp;E) and Nissl staining using respective staining kits. All sections were observed and photographed under a bright-field optical microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescent staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpinal 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTunel staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eELISA analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOxidative stress level\u003cem\u003e\u0026nbsp;in vivo\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll 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 \u0026lt; 0.05 was taken as statistically significant (*p \u0026lt; 0.05, **p \u0026lt; 0.01, *** p \u0026lt; 0.001 and **** p \u0026lt; 0.0001).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eITER Project of the Ministry of Science and Technology [grant numbers 2022YFE03080001]\u003c/p\u003e\n\u003cp\u003eThe Fundamental Research Funds for the Central Universities [grant numbers YD9110002012]\u003c/p\u003e\n\u003cp\u003eThe Fundamental Research Funds for the Central Universities [grant numbers USTC20210079]\u003c/p\u003e\n\u003cp\u003eThe joint Laboratory of Plasma Application Technology Funding [grant numbers JL06120001H].\u003c/p\u003e\n\u003cp\u003eThe National Natural Science Foundation Incubation Program of the Second Affiliated Hospital of Anhui Medical University [grant numbers 2020GMFY06]\u003c/p\u003e\n\u003cp\u003eThe 2022 Natural Science Foundation of Anhui Province (C.B.D) [grant numbers 2208085MH254].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: Zhengwei Wu\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Methodology: Zhu Chen, Jun Li, Chengbiao Ding\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Investigation: Jiwen Zhu, Zhenyu Liu, Qi Liu\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Visualization: Jiwen Zhu, Zhenyu Liu\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Supervision: Zhengwei Wu, Zhu Chen, Jun Li, Chengbiao Ding\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Writing—original draft: Jiwen Zhu, Qi Liu\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Writing—review \u0026amp; editing: Jiwen Zhu, Qinghua Xu\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll other authors declare they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData available on request / reasonable request.\u0026nbsp;The author Jiwen Zhu will provide the data generated from this study upon direct email request to\u0026nbsp;[email protected].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eKhachatryan, Z., Haunschild, J., von Aspern, K., Borger, M. A. \u0026amp; Etz, C. D. Ischemic Spinal Cord Injury-Experimental Evidence and Evolution of Protective Measures. \u003cem\u003eAnnals of Thoracic Surgery\u003c/em\u003e \u003cstrong\u003e113\u003c/strong\u003e, 1692-1702 (2022). https://doi.org:10.1016/j.athoracsur.2020.12.028\u003c/li\u003e\n \u003cli\u003eAlizadeh, A., Dyck, S. M. \u0026amp; Karimi-Abdolrezaee, S. Traumatic Spinal Cord Injury: An Overview of Pathophysiology, Models and Acute Injury Mechanisms. \u003cem\u003eFrontiers in Neurology\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 282 (2019). https://doi.org:10.3389/fneur.2019.00282\u003c/li\u003e\n \u003cli\u003eAhuja, C. S.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Traumatic spinal cord injury. \u003cem\u003eNature Reviews Disease Primers\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 17018 (2017). https://doi.org:10.1038/nrdp.2017.18\u003c/li\u003e\n \u003cli\u003eSaremi, J.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Advanced approaches to regenerate spinal cord injury: The development of cell and tissue engineering therapy and combinational treatments. \u003cem\u003eBiomedicine \u0026amp; Pharmacotherapy\u003c/em\u003e \u003cstrong\u003e146\u003c/strong\u003e, 112-529 (2022). https://doi.org:10.1016/j.biopha.2021.112529\u003c/li\u003e\n \u003cli\u003eCunningham, C. J.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e The potential of gene therapies for spinal cord injury repair: a systematic review and meta-analysis of pre-clinical studies. \u003cem\u003eNeural Regeneration Research\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 299-305 (2023). https://doi.org:10.4103/1673-5374.347941\u003c/li\u003e\n \u003cli\u003eKumar, A., Kumar, N., Pathak, Z. \u0026amp; Kumar, H. Extra Cellular Matrix Remodeling: An Adjunctive Target for Spinal Cord Injury and Intervertebral Disc Degeneration. \u003cem\u003eNeurospine\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 632-645 (2022). https://doi.org:10.14245/ns.2244366.183\u003c/li\u003e\n \u003cli\u003eKathe, C.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e The neurons that restore walking after paralysis. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e611\u003c/strong\u003e, 540-547 (2022). https://doi.org:10.1038/s41586-022-05385-7\u003c/li\u003e\n \u003cli\u003eSquair, J. W.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Recovery of walking after paralysis by regenerating characterized neurons to their natural target region. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e381\u003c/strong\u003e, 1338-1345 (2023). https://doi.org:doi:10.1126/science.adi6412\u003c/li\u003e\n \u003cli\u003eBruggeman, P. J., Iza, F. \u0026amp; Brandenburg, R. Foundations of atmospheric pressure non-equilibrium plasmas. \u003cem\u003ePlasma Sources Science and Technology\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 123002 (2017). https://doi.org:10.1088/1361-6595/aa97af\u003c/li\u003e\n \u003cli\u003eSakudo, A., Yagyu, Y. \u0026amp; Onodera, T. Disinfection and Sterilization Using Plasma Technology: Fundamentals and Future Perspectives for Biological Applications. \u003cem\u003eInt J Mol Sci\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 5216 (2019). https://doi.org:10.3390/ijms20205216\u003c/li\u003e\n \u003cli\u003eLaroussi, M., Lu, X. \u0026amp; Keidar, M. Perspective: The physics, diagnostics, and applications of atmospheric pressure low temperature plasma sources used in plasma medicine. \u003cem\u003eJournal of Applied Physics\u003c/em\u003e \u003cstrong\u003e122\u003c/strong\u003e, 020901 (2017). https://doi.org:10.1063/1.4993710\u003c/li\u003e\n \u003cli\u003eYan, X., Meng, Z., Ouyang, J., Qiao, Y. \u0026amp; Yuan, F. New Application of an Atmospheric Pressure Plasma Jet as a Neuro-protective Agent Against Glucose Deprivation-induced Injury of SH-SY5Y Cells. \u003cem\u003eJournal of Visualized Experiments\u003c/em\u003e, 56323 (2017). https://doi.org:10.3791/56323\u003c/li\u003e\n \u003cli\u003eWeltmann, K. D. \u0026amp; Von Woedtke, T. Plasma medicine\u0026mdash;current state of research and medical application. \u003cem\u003ePlasma Physics and Controlled Fusion\u003c/em\u003e \u003cstrong\u003e59\u003c/strong\u003e, 014031 (2017). https://doi.org:10.1088/0741-3335/59/1/014031\u003c/li\u003e\n \u003cli\u003eLu, X.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Reactive species in non-equilibrium atmospheric-pressure plasmas: Generation, transport, and biological effects. \u003cem\u003ePhysics Reports-Review Section of Physics Letters\u003c/em\u003e \u003cstrong\u003e630\u003c/strong\u003e, 1-84 (2016). https://doi.org:10.1016/j.physrep.2016.03.003\u003c/li\u003e\n \u003cli\u003eDi Meo, S., Reed, T. T., Venditti, P. \u0026amp; Victor, V. M. Role of ROS and RNS Sources in Physiological and Pathological Conditions. \u003cem\u003eOxid Med Cell Longev\u003c/em\u003e \u003cstrong\u003e2016\u003c/strong\u003e, 1245049 (2016). https://doi.org:10.1155/2016/1245049\u003c/li\u003e\n \u003cli\u003eMitra, S., Kaushik, N., Moon, I. S., Choi, E. H. \u0026amp; Kaushik, N. K. Utility of Reactive Species Generation in Plasma Medicine for Neuronal Development. \u003cem\u003eBiomedicines\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 348 (2020).\u003c/li\u003e\n \u003cli\u003eYan, X.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Cytoprotective effects of atmospheric-pressure plasmas against hypoxia-induced neuronal injuries. \u003cem\u003eJournal of Physics D: Applied Physics\u003c/em\u003e \u003cstrong\u003e51\u003c/strong\u003e, 085401 (2018). https://doi.org:10.1088/1361-6463/aaa867\u003c/li\u003e\n \u003cli\u003eTian, M.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Cold Atmospheric Plasma Elicits Neuroprotection Against Glutamate Excitotoxicity by Activating Cellular Antioxidant Defense. \u003cem\u003ePlasma Chemistry and Plasma Processing\u003c/em\u003e \u003cstrong\u003e41\u003c/strong\u003e, 945-954 (2021). https://doi.org:10.1007/s11090-021-10172-9\u003c/li\u003e\n \u003cli\u003eYan, X.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Atmospheric pressure plasma treatments protect neural cells from ischemic stroke‐relevant injuries by targeting mitochondria. \u003cem\u003ePlasma Processes and Polymers\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 2000063 (2020). https://doi.org:10.1002/ppap.202000063\u003c/li\u003e\n \u003cli\u003eSteinbacher, P. \u0026amp; Eckl, P. Impact of oxidative stress on exercising skeletal muscle. \u003cem\u003eBiomolecules\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 356-377 (2015). https://doi.org:10.3390/biom5020356\u003c/li\u003e\n \u003cli\u003eCoimbra-Costa, D.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Intermittent Hypobaric Hypoxic Preconditioning Provides Neuroprotection by Increasing Antioxidant Activity, Erythropoietin Expression and Preventing Apoptosis and Astrogliosis in the Brain of Adult Rats Exposed to Acute Severe Hypoxia. \u003cem\u003eInt J Mol Sci\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 5272 (2021). https://doi.org:10.3390/ijms22105272\u003c/li\u003e\n \u003cli\u003eChen, Y.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Application of an atmospheric pressure plasma jet in a rat model of ischaemic stroke: Design, optimisation, and characteristics. \u003cem\u003eHigh Voltage\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 315-325 (2023). https://doi.org:10.1049/hve2.12267\u003c/li\u003e\n \u003cli\u003eGraves, D. B. Reactive Species from Cold Atmospheric Plasma: Implications for Cancer Therapy. \u003cem\u003ePlasma Processes and Polymers\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 1120-1127 (2014). https://doi.org:10.1002/ppap.201400068\u003c/li\u003e\n \u003cli\u003eDing, C.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Cold air plasma: A potential strategy for inducing apoptosis of rheumatoid arthritis fibroblast‐like synoviocytes. \u003cem\u003eHigh Voltage\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 106-116 (2022). https://doi.org:10.1049/hve2.12132\u003c/li\u003e\n \u003cli\u003eDing, C.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Cold air plasma improving rheumatoid arthritis via mitochondrial apoptosis pathway. \u003cem\u003eBioengineering \u0026amp; Translational Medicine\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 10366 (2022). https://doi.org:10.1002/btm2.10366\u003c/li\u003e\n \u003cli\u003eChen, Y.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Inhalation of Atmospheric-Pressure Gas Plasma Attenuates Brain Infarction in Rats With Experimental Ischemic Stroke. \u003cem\u003eFrontiers in Neuroscience\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 875053 (2022). https://doi.org:10.3389/fnins.2022.875053\u003c/li\u003e\n \u003cli\u003eXiong, Z. L. Cold Atmospheric Plasmas: A Novel and Promising Way to Treat Neurological Diseases. \u003cem\u003eTrends in Biotechnology\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e, 582-583 (2018). https://doi.org:10.1016/j.tibtech.2018.04.003\u003c/li\u003e\n \u003cli\u003eZhou, X.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Microglia and macrophages promote corralling, wound compaction and recovery after spinal cord injury via Plexin-B2. \u003cem\u003eNat Neurosci\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 337-350 (2020). https://doi.org:10.1038/s41593-020-0597-7\u003c/li\u003e\n \u003cli\u003eAnjum, A.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Spinal Cord Injury: Pathophysiology, Multimolecular Interactions, and Underlying Recovery Mechanisms. \u003cem\u003eInternational Journal of Molecular Sciences\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 7533 (2020). https://doi.org:10.3390/ijms21207533\u003c/li\u003e\n \u003cli\u003eHellenbrand, D. J.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Inflammation after spinal cord injury: a review of the critical timeline of signaling cues and cellular infiltration. \u003cem\u003eJournal of Neuroinflammation\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 284 (2021). https://doi.org:10.1186/s12974-021-02337-2\u003c/li\u003e\n \u003cli\u003eZhao, R., Wu, X., Bi, X. Y., Yang, H. \u0026amp; Zhang, Q. Baicalin attenuates blood-spinal cord barrier disruption and apoptosis through PI3K/Akt signaling pathway after spinal cord injury. \u003cem\u003eNeural Regen Res\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 1080-1087 (2022). https://doi.org:10.4103/1673-5374.324857\u003c/li\u003e\n \u003cli\u003eSugiyama, K. \u0026amp; Tanaka, K. Spinal cord-specific deletion of the glutamate transporter GLT1 causes motor neuron death in mice. \u003cem\u003eBiochemical and Biophysical Research Communications\u003c/em\u003e \u003cstrong\u003e497\u003c/strong\u003e, 689-693 (2018). https://doi.org:https://doi.org/10.1016/j.bbrc.2018.02.132\u003c/li\u003e\n \u003cli\u003eSingh, A., Kukreti, R., Saso, L. \u0026amp; Kukreti, S. Oxidative Stress: A Key Modulator in Neurodegenerative Diseases. \u003cem\u003eMolecules\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 1583 (2019). https://doi.org:10.3390/molecules24081583\u003c/li\u003e\n \u003cli\u003eBradbury, E. J. \u0026amp; Burnside, E. R. Moving beyond the glial scar for spinal cord repair. \u003cem\u003eNature Communications\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 3879 (2019). https://doi.org:10.1038/s41467-019-11707-7\u003c/li\u003e\n \u003cli\u003eZamanian, J. L.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Genomic Analysis of Reactive Astrogliosis. \u003cem\u003eThe Journal of Neuroscience\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, 6391-6410 (2012). https://doi.org:10.1523/jneurosci.6221-11.2012\u003c/li\u003e\n \u003cli\u003eAyazi, M., Zivkovic, S., Hammel, G., Stefanovic, B. \u0026amp; Ren, Y. Fibrotic Scar in CNS Injuries: From the Cellular Origins of Fibroblasts to the Molecular Processes of Fibrotic Scar Formation. \u003cem\u003eCells\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 2371 (2022). https://doi.org:10.3390/cells11152371\u003c/li\u003e\n \u003cli\u003eSchottlender, N., Gottfried, I. \u0026amp; Ashery, U. Hyperbaric Oxygen Treatment: Effects on Mitochondrial Function and Oxidative Stress. \u003cem\u003eBiomolecules\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 1827 (2021).\u003c/li\u003e\n \u003cli\u003eChen, X., Guo, C. \u0026amp; Kong, J. Oxidative stress in neurodegenerative diseases. \u003cem\u003eNeural Regen Res\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 376-385 (2012). https://doi.org:10.3969/j.issn.1673-5374.2012.05.009\u003c/li\u003e\n \u003cli\u003eLennicke, C. \u0026amp; Cochem\u0026eacute;, H. M. Redox metabolism: ROS as specific molecular regulators of cell signaling and function. \u003cem\u003eMol Cell\u003c/em\u003e \u003cstrong\u003e81\u003c/strong\u003e, 3691-3707 (2021). https://doi.org:10.1016/j.molcel.2021.08.018\u003c/li\u003e\n \u003cli\u003edu Sert, N. P.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Reporting animal research: Explanation and elaboration for the ARRIVE guidelines 2.0. \u003cem\u003ePLOS Biology\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, e3000411 (2020). https://doi.org:10.1371/journal.pbio.3000411\u003c/li\u003e\n \u003cli\u003eAu - McDonough, A., Au - Monterrubio, A., Au - Ariza, J. \u0026amp; Au - Mart\u0026iacute;nez-Cerde\u0026ntilde;o, V. Calibrated Forceps Model of Spinal Cord Compression Injury. \u003cem\u003eJoVE\u003c/em\u003e, e52318 (2015). https://doi.org:doi:10.3791/52318\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Cold atmospheric plasma, spinal cord injury, neuroprotective, ROS","lastPublishedDoi":"10.21203/rs.3.rs-4297802/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4297802/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSpinal cord injury (SCI) presents a critical medical challenge, marked by substantial neural damage and persistent functional deficits. This study investigates the therapeutic potential of cold atmospheric plasma (CAP) for SCI, utilizing a tailored dielectric barrier discharge (DBD) device to conduct comprehensive \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro \u003c/em\u003eanalyses. The findings show that CAP treatment significantly improves functional recovery after SCI, reduces neuronal apoptosis, lowers inflammation, and increases axonal regeneration. These findings illustrate the efficacy of CAP in fostering a conducive environment for recovery by modulating inflammatory responses, enhancing neuronal survival, and encouraging regenerative processes. The underlying mechanism involves CAP's reduction of reactive oxygen species (ROS) levels, followed by \u0026nbsp;the activation of antioxidant enzymes. These findings position CAP as a pioneering approach for spinal cord injury (SCI) treatment, presenting opportunities for improved neural recovery and establishing a new paradigm in SCI therapy.\u003c/p\u003e","manuscriptTitle":"Enhanced Neural Recovery and Reduction of Secondary Damage in Spinal Cord Injury through Modulation of Oxidative Stress and Neural Response","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-03 11:53:45","doi":"10.21203/rs.3.rs-4297802/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-12T11:10:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-12T06:19:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-05T13:19:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"110490329256638357324509710030003067089","date":"2024-06-04T11:57:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"158808356414607258040836403091647404480","date":"2024-06-03T23:59:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-03T09:56:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-03T09:48:16+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-04-26T12:34:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-26T12:32:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-04-20T14:01:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c1d83464-e61a-4b15-b381-70ded200ee0c","owner":[],"postedDate":"May 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":31450073,"name":"Physical sciences/Engineering/Biomedical engineering"},{"id":31450074,"name":"Biological sciences/Neuroscience/Diseases of the nervous system/Neurodegeneration"}],"tags":[],"updatedAt":"2024-08-22T19:30:53+00:00","versionOfRecord":{"articleIdentity":"rs-4297802","link":"https://doi.org/10.1038/s41598-024-69861-y","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-08-16 15:57:00","publishedOnDateReadable":"August 16th, 2024"},"versionCreatedAt":"2024-05-03 11:53:45","video":"","vorDoi":"10.1038/s41598-024-69861-y","vorDoiUrl":"https://doi.org/10.1038/s41598-024-69861-y","workflowStages":[]},"version":"v1","identity":"rs-4297802","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4297802","identity":"rs-4297802","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}