Vitamin C Mitigates Early Auditory Cortical Hyperexcitability and Antioxidant Imbalance Induced by Acute Intermittent Hypoxia

Vitamin C Mitigates Early Auditory Cortical Hyperexcitability and Antioxidant Imbalance Induced by Acute Intermittent Hypoxia | 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 Research Article Vitamin C Mitigates Early Auditory Cortical Hyperexcitability and Antioxidant Imbalance Induced by Acute Intermittent Hypoxia Yue Wu, Tao Li, Wanxin Cao, Rui Fan, Hailing Jiang, Jiachen Han, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8528320/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objectives To test whether brief intermittent hypoxia (IH) induces early hyperexcitability and antioxidant imbalance in the primary auditory cortex (Au1), and whether high-dose vitamin C (VC) pretreatment mitigates these effects. Methods Adult male Sprague–Dawley rats were assigned to four groups (n = 6/group): control, IH, IH plus intraperitoneal normal saline (IH + IPNS), and IH plus intraperitoneal VC (IH + IPVC). Rats underwent a 3-h IH protocol; VC (500 mg/kg, i.p.) or saline was administered 30 min before IH. Three hours after IH, in vivo Au1 multiunit recordings quantified spontaneous firing rate (SFR). We quantified catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities and assessed apoptosis using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Results Acute IH increased Au1 SFR versus control, and saline did not alter this response; VC pretreatment reduced SFR toward control levels. IH decreased CAT activity and increased SOD activity, with minimal change in GPx. VC increased CAT, SOD, and GPx activities relative to IH. No overt neuronal apoptosis was detected. Conclusions Brief OSA-relevant IH triggers an early stage of auditory cortical dysfunction with hyperexcitability and antioxidant imbalance. High-dose VC pretreatment attenuates hyperexcitability and enhances antioxidant enzyme activity, supporting antioxidant strategies as adjuncts for central auditory protection. intermittent hypoxia auditory cortex neuronal excitability vitamin C antioxidant enzymes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1.introduction OSA is a sleep-related breathing disorder characterized by recurrent partial or complete collapse of the upper airway during sleep, leading to apnea and hypoventilation. These events cause intermittent hypoxia, sleep fragmentation, and a wide range of cardiovascular and metabolic problems [ 1 ] . OSA results in multi-system damage and is linked to type 2 diabetes [ 2 ] , hypertension [ 3 ] , heart failure, cardiac arrhythmias [ 4 ] , stroke [ 5 ] . Besides its systemic effects, increasing evidence shows that chronic intermittent hypoxia can also harm the auditory system, especially causing peripheral hearing loss at high frequencies [ 6 ] . IH has also been reported to impair the central auditory system, with clinical signs like prolonged auditory brainstem response (ABR) latencies [ 7 ] , reduced speech recognition scores [ 8 ] , and declines in verbal memory and cognitive function [ 9 ] . These findings indicate that intermittent hypoxia may damage auditory-related central pathways. The auditory cortex is a central part of the speech-processing network, maintaining effective connections with frontal, temporal, and temporoparietal regions to support the tracking and understanding of continuous speech; the health of this network is closely tied to speech comprehension [ 10 ] .Our previous work demonstrated that short-term intermittent hypoxia induces peripheral hearing loss, shortens ABR wave III–V latencies, and increases spontaneous firing rates in the auditory cortex [ 11 ] . Collectively, clinical and experimental data support the view that IH is associated with structural and functional impairment of the auditory cortex. Currently, continuous positive airway pressure (CPAP) is the mainstay of treatment for OSA. Previous studies have shown that adequately titrated CPAP can partially improve cochlear function and low-to-mid frequency hearing thresholds in patients with OSA [ 12 ] . However, there is still insufficient evidence that CPAP exerts a beneficial effect on OSA-related central auditory damage [ 13 , 14 ] . This highlights the need to explore adjunctive strategies targeting central auditory injury in OSA. VC is a classical water-soluble antioxidant with potent ROS-scavenging capacity. In the pathogenesis of OSA, repetitive hypoxia–reoxygenation episodes generate large amounts of oxygen-free radicals in the central nervous system, triggering oxidative stress [ 15 ] . By eliminating free radicals, it appears to improve hearing outcomes in patients with sudden sensorineural hearing loss [ 16 ] . Besides, high-dose VC has been shown to attenuate oxidative stress, mitochondrial fragmentation, and apoptosis in the brain and to ameliorate functional deficits [ 17 ] . Therefore, vitamin C supplementation may confer a protective effect against intermittent hypoxia–induced auditory cortical dysfunction and potential injury by scavenging reactive oxygen species and alleviating oxidative stress. Therefore, the present study used a pre-IH high-dose VC protocol to conceptually model pre-sleep VC supplementation and to investigate its protective effects against short-term intermittent hypoxia–induced oxidative stress and functional impairment in the auditory cortex. 2. Materials and Methods 2.1 Animals Male Sprague–Dawley rats (300–350 g; purchased from VITAL RIVER, Beijing, China) were used in this study. Animals were housed under controlled conditions (22 ± 2°C, 50 ± 10% relative humidity, 12 h light/dark cycle) with free access to food and water. All procedures were approved by the Institutional Animal Care and Use Committee of Peking University Health Science Center and complied with relevant guidelines for the care and use of laboratory animals. Rats were randomly assigned to four groups: control, IH, IH + IPNS, and IH plus, IH + IPVC. Vitamin C (500 mg/kg) was dissolved in sterile saline and administered intraperitoneally (i.p.) 30 min before the beginning of the AIH exposure. Control animals received an equal volume of normal saline using the same schedule. Electrophysiological and biochemical assessments of the auditory cortex were completed within 3h after the end of the exposure protocol. 2.2 Intermittent hypoxia apparatus and protocol The IH apparatus and parameters were based on our previous work.[22] Briefly, the system consisted of an air pump, a unidirectional ventilation tube, and a 3D-printed mask fitted to the rat’s snout. When the pump was on, room air was delivered at 2 L/min, flushing the mask and removing CO₂. When the pump was off, the mask formed a relatively closed local environment, leading to a progressive decline of arterial oxygen saturation and thus producing acute IH exposure. 2.3 Surgery and in vivo electrophysiological recording Three hours after the acute IH protocol, in vivo electrophysiological recordings were performed under general anesthesia to assess neuronal activity in Au1. After exposure of the skull, a craniotomy was made over the auditory cortex (anteroposterior − 4.56 to − 5.04 mm; mediolateral 6.4 to 7.6 mm from bregma; dorsoventral 3.8 to 5.0 mm according to the rat brain atlas). The dura was carefully removed to expose the cortex.A 4 × 4 microelectrode array (custom-made; pre-sterilized with povidone–iodine; mean impedance ≈ 0.3 MΩ) was slowly advanced into Au1 using a micromanipulator in 0.05-mm steps. After each advancement, the electrode was left in place for ~ 20 min to allow neuronal firing to stabilize. During recording, a copper mesh shield was used to reduce electrical noise, and exposed brain tissue was covered with saline-soaked cotton to prevent drying. 2.4 Electrophysiological data acquisition and analysis Multiunit activity was recorded using an OmniPlex multichannel acquisition system (Plexon Inc., Dallas, TX, USA) at a sampling rate of 40 kHz. Raw signals were exported to Offline Sorter (Plexon) for preprocessing. A Bessel filter with a 250 Hz high-pass cutoff was applied to remove low-frequency noise, and channels with a signal-to-noise ratio < 3.0 were excluded.Spike sorting was performed using principal component analysis followed by k-means clustering, and units with abnormal waveforms or clear artifacts (e.g., refractory period violations with interspike intervals < 1–2 ms) were manually discarded. SFR of each isolated unit was calculated using NeuroExplorer (Nex Technologies, Colorado Springs, CO, USA). Mean SFR values were compared across groups to evaluate the effects of acute IH and VC treatment on neuronal excitability in Au1. 2.5 Auditory cortex tissue collection Rats were euthanized by cervical dislocation, and brains were rapidly removed and placed in ice-cold saline. The anteroposterior extent of Au1 (9–12 mm posterior to the olfactory bulb) was determined according to The Rat Brain in Stereotaxic Coordinates (5th ed.). Brains were then placed in a pre-cooled brain matrix with millimeter markings to obtain coronal slabs encompassing Au1, and Au1 was microdissected under a dissecting microscope based on cortical landmarks and immediately processed for antioxidant enzyme assays. 2.6 Assays of CAT, SOD, and GPx activities Auditory cortex tissue was homogenized in ice-cold buffer, and protein concentration was determined according to the manufacturer’s instructions. The activities of CAT(Solarbio, Beijing, China; cat. no. BC4785), SOD(Solarbio; cat. no. BC5160) and GPx( Solarbio; cat. no. BC1190) were measured using the corresponding commercial assay kits following the manufacturers’ protocols. Enzyme activities were normalized to protein content and expressed as units per milligram of protein. 2.7 Histological assessment and neuronal apoptosis In animals designated for electrode localization and apoptosis assessment, brains were fixed by transcardial perfusion with 4% paraformaldehyde under deep anesthesia. After routine dehydration and paraffin embedding, coronal sections (4–6 µm) were cut at the level of the auditory cortex. A subset of sections was stained with hematoxylin–eosin (HE). Under light microscopy, electrode tracks and local depressions or cavities were identified and matched to corresponding plates of the rat brain atlas, using the lateral sulcus and other anatomical landmarks, to verify that electrode tips were located within Au1. Another subset of sections was processed for TUNEL staining to assess neuronal apoptosis. TUNEL labeling was performed using the In Situ Cell Death Detection Kit, TMR red (Roche; cat. no. 12156792910), according to the manufacturer’s instructions. Positive controls were prepared using a TUNEL apoptosis positive control kit (Beyotime; cat. no. C1082), which includes DNase I pretreatment, whereas omission of terminal deoxynucleotidyl transferase (TdT) served as a negative control. Nuclei were counterstained with DAPI. Fluorescence images from the Au1 region were acquired under identical exposure settings across groups. Two observers blinded to group allocation independently counted TUNEL-positive cells and total nuclei. The apoptotic index was calculated as the percentage of TUNEL-positive cells among total nuclei (TUNEL-positive cells / total nuclei × 100%), and the mean value from multiple fields was used as the data point for each animal. 2.8 Statistical analysis The animal was the primary experimental unit (n = 6 per group). For electrophysiology, unit-level measures were summarized within each animal (e.g., mean/median SFR per rat) for primary group comparisons. Data are presented as mean ± standard error of the mean (SEM). Normality was assessed using the Shapiro–Wilk test, and homogeneity of variance was evaluated using Levene’s test. When both assumptions were satisfied, group comparisons were performed using one-way analysis of variance (one-way ANOVA) followed by Tukey’s honestly significant difference (Tukey HSD) post hoc test. When assumptions were violated, appropriate non-parametric tests were applied. A two-tailed p value < 0.05 was considered statistically significant. Statistical analyses were conducted using SPSS (IBM Corp., Armonk, NY, USA) or GraphPad Prism (GraphPad Software, San Diego, CA, USA). 3. Results 3.1 Short-term intermittent hypoxia increases neuronal firing in the auditory cortex, and high-dose vitamin C partially attenuates this effect As detailed in the Methods, rats were assigned to four experimental groups (control group, IH group, IH+IPNS group, and IH+IPVC group; n = 6 per group). Rats in the control group wore the mask apparatus without hypoxic exposure. Rats in the IH group underwent 3h of IH, with mean maximal and minimal arterial oxygen saturation values of 98.02 ± 0.24% and 81.45 ± 0.91%, respectively. In the IH+IPNS group, 0.9% saline was administered intraperitoneally 30 min before IH (at the same volume as in the IH+IPVC group), and maximal and minimal oxygen saturation values were 98.02 ± 0.39% and 82.73 ± 1.05%. In the IH+IPVC group, rats received 500 mg/kg VC intraperitoneally 30 min before IH, with maximal and minimal oxygen saturation values of 97.93 ± 0.28% and 82.02 ± 0.78%. Maximal and minimal oxygen saturation did not differ significantly among the IH, IH+IPNS and IH+IPVC groups, indicating a comparable hypoxic load. After IH exposure, the SFR of auditory cortical neurons was 1.23 ± 0.074 Hz in the control group, 2.64 ± 0.18 Hz in the IH group, 2.73 ± 0.16 Hz in the IH+IPNS group, and 1.93 ± 0.17 Hz in the IH+IPVC group. Shapiro–Wilk and Levene’s tests confirmed that the assumptions of normality and homogeneity of variance were met; therefore, one-way ANOVA was applied for group comparisons. One-way ANOVA revealed a significant group effect (F = 20.42, p = 2.66 × 10⁻⁶). Tukey post hoc analysis showed that SFR was significantly higher in the IH group than in the control group (1.23 ± 0.074 vs 2.64 ± 0.18 Hz, p = 1.6 × 10⁻⁵), with no difference between IH and IH+IPNS (2.64 ± 0.18 vs 2.73 ± 0.16 Hz, p = 0.97). SFR in the IH+IPVC group was significantly lower than in the IH+IPNS group (1.93 ± 0.17 vs 2.73 ± 0.16 Hz, p = 0.0077), but remained higher than in the control group (1.93 ± 0.17 vs 1.23 ± 0.074 Hz, p = 0.022)（ Figure 1 ）. These findings indicate that short-term IH induces marked hyperexcitability of auditory cortical neurons, and that high-dose VC partially attenuates this hyperexcitability. After completion of in vivo recordings, brain sections were stained with hematoxylin–eosin and compared with the corresponding plates in The Rat Brain in Stereotaxic Coordinates (5th ed.). Electrode tips were consistently located within Au1, confirming that the recorded signals originated from this region ( Figure 2 ). 3.2 Short-term intermittent hypoxia decreases CAT activity, increases SOD activity, and has limited effects on GPx in the auditory cortex, whereas high-dose VC enhances all three enzymes To assess oxidative stress in the auditory cortex, we measured the activities of CAT, SOD and GPx in all groups. Shapiro–Wilk and Levene’s tests indicated that the data met the assumptions of normality and homogeneity of variance; therefore, one-way ANOVA with Tukey HSD post hoc tests was used for group comparisons. SOD activity Mean SOD activities were 686.8 ± 27.14 U/g in the control group, 1433.67 ± 143.94 U/g in the IH group, 1343.07 ± 128.92 U/g in the IH+IPNS group and 2402.83 ± 133.34 U/g in the IH+IPVC group. ANOVA showed a significant group effect (F = 35.83, p = 3.09 × 10⁻⁸). SOD activity was higher in the IH group than in the control group (686.8 ± 27.14 vs 1433.67 ± 143.94 U/g, p = 0.0012), and did not differ between the IH and IH+IPNS groups (1433.67 ± 143.94 vs 1343.07 ± 128.92 U/g, p = 0.948). The IH+IPVC group showed a further increase compared with the IH+IPNS group (2402.83 ± 133.34 vs 1343.07 ± 128.92 U/g, p = 1.9 × 10⁻⁵), and SOD activity in IH+IPVC was also higher than in the control group (686.8 ± 27.14 vs 2402.83 ± 133.34 U/g, p= 1.16 × 10⁻⁸).( Figure 3 ) CAT activity Mean CAT activities (U/g tissue, mean ± SEM) were 197.66 ± 7.94 in the control group, 146.53 ± 3.68 in the IH group, 155.15 ± 7.41 in the IH+IPNS group and 234.68 ± 14.64 in the IH+IPVC group. One-way ANOVA revealed a significant group effect (F = 19.24, p = 4.12 × 10⁻⁶). CAT activity was lower in the IH group than in the control group (197.66 ± 7.94 vs 146.53 ± 3.68 U/g, p = 0.0047), with no difference between the IH and IH+IPNS groups (146.53 ± 3.68 vs 155.15 ± 7.41 U/g, p = 0.91). In contrast, CAT activity was higher in the IH+IPVC group than in the IH+IPNS group (155.15 ± 7.41 vs 234.68 ± 14.64 U/g, p = 3.6 × 10⁻⁵) and also exceeded control levels (197.66 ± 7.94 vs 234.68 ± 14.64 U/g, p = 0.048)（ Figure 4 ）. GPx activity Mean GPx activities were 251.13 ± 8.66 U/g in the control group, 280.14 ± 3.95 U/g in the IH group, 285.80 ± 17.39 U/g in the IH+IPNS group and 314.80 ± 12.99 U/g in the IH+IPVC group. ANOVA indicated a significant group effect (F = 4.85, p = 0.011). Post hoc tests showed no significant differences between the control and IH groups (251.13 ± 8.66 vs 280.14 ± 3.95 U/g, p = 0.33), between the IH and IH+IPNS groups (280.14 ± 3.95 vs 285.80 ± 17.39 U/g, p = 0.99), or between the IH+IPNS and IH+IPVC groups (285.80 ± 17.39 vs 314.80 ± 12.99 U/g, p = 0.33). However, GPx activity in the IH+IPVC group was higher than in the control group (251.13 ± 8.66 vs 314.80 ± 12.99 U/g, p = 0.0057)( Figure 5 ). Taken together, short-term IH in the auditory cortex is associated with decreased CAT activity, increased SOD activity and minimal changes in GPx, whereas high-dose VC elevates all three enzyme activities, indicating an overall enhancement of the antioxidant defense system. 3.3 Acute intermittent hypoxia does not induce overt neuronal apoptosis in the auditory cortex In the same Au1 region used for electrophysiological recordings, five non-overlapping fields (≈400 × 400 µm) were randomly selected per animal, yielding approximately 1,000 nuclei per group. Across the control, IH, IH+IPNS and IH+IPVC groups, only sporadic TUNEL-positive cells were observed. Overall TUNEL signal remained at a low abundance close to background, and no clear trend of group differences was detected (Figure 6) . In contrast, the positive control with DNase pretreatment showed strong, diffuse nuclear TUNEL staining, whereas the negative control (omission of TdT) exhibited only background fluorescence, confirming the specificity and reliability of the TUNEL assay (Figure 7). Under the present experimental conditions (3h acute IH), neurons in the auditory cortex thus displayed altered excitability and antioxidant enzyme activity, but no apparent apoptotic cell loss. Discussion OSA is a sleep-related breathing disorder characterized by recurrent partial or complete upper airway collapse during sleep, leading to intermittent hypoxia, sleep fragmentation and multi-system dysfunction [ 1 ] . Increasing attention has been paid to the auditory consequences of OSA, as clinical studies have reported elevated hearing thresholds [ 6 ] , prolonged ABR latencies [ 7 ] and reduced speech recognition scores [ 8 ] , all of which are closely associated with intermittent hypoxia. Although CPAP may partially improve low-to-mid frequency hearing thresholds [ 12 ] , its efficacy in restoring central auditory function, particularly at the cortical level, appears limited [ 13 , 14 , 18 ] . Given that the auditory cortex is a hub of the speech processing network and its connectivity with frontal and temporo-parietal regions is critical for speech comprehension [ 10 ] , adjunctive strategies targeting hypoxia-induced damage in central auditory structures may have clinical value. In the present study, we used an acute 3h IH rat model to characterize early responses of the auditory cortex to IH at two levels: neuronal spontaneous firing and antioxidant enzyme activity. We further evaluated the effects of high-dose VC pretreatment. Our data show that short-term IH markedly increases SFR of auditory cortical neurons. Under comparable hypoxic load, intraperitoneal administration of high-dose VC partially attenuates this hyperexcitability and concomitantly elevates SOD, CAT and GPx activities, without inducing overt neuronal apoptosis. These findings suggest that at an early stage of IH, the auditory cortex primarily undergoes functional plasticity and reversible oxidative changes, indicating a potential window of opportunity for intervention. With regard to excitability, 3h of IH was sufficient to induce a robust increase in SFR in Au1, indicating a transient shift in the excitation-inhibition balance. Previous work has shown that excessive activation of NMDA receptors can markedly enhance ROS production via a nitric oxide (NO)–NADPH oxidase (NOX) pathway, and that genetic deletion or pharmacological inhibition of NOX abolishes this ROS elevation, supporting excitatory transmission–driven NOX activation as a major source of cortical ROS [ 19 , 20 ] .These observations support excitatory transmission-driven NOX activation as a major source of cortical ROS. In our model, high-dose VC pretreatment reduced the IH-induced increase in SFR without altering the severity of hypoxia and at the same time enhanced antioxidant enzyme activities. Although direct measurements of ROS were not obtained, this constellation of findings is consistent with a contribution of oxidative stress to IH-induced cortical hyperexcitability and suggests that antioxidant support can partially buffer this effect. We next examined the activities of three key antioxidant enzymes in the auditory cortex: SOD, CAT and GPx. SOD catalyzes the dismutation of superoxide anions (O₂⁻·) to H₂O₂, whereas CAT and GPx are involved in subsequent H₂O₂ detoxification. CAT is well suited for high-throughput decomposition of H₂O₂, while GPx/Prx pathways provide high-affinity, lower-capacity clearance [ 21 , 22 ] .In the present study, acute IH was accompanied by a decrease in CAT activity, a marked increase in SOD activity and only a small, non-significant change in GPx. Taken together, these enzyme profiles indicate an early, unbalanced adjustment of the antioxidant system in the auditory cortex, in line with observations from other hypoxia models [ 23 , 24 ] . At the mechanistic level, IH constitutes a repeated hypoxia–reoxygenation stress that can amplify ROS generation in the brain. Evidence from hypoxia-related models suggests that ROS accumulation can be driven by NADPH oxidase (NOX2)–dependent mechanisms and accompanied by mitochondrial redox disturbances, together increasing the burden of superoxide and downstream oxidants [ 25 , 26 ] . In response to oxidative pressure, the Nrf2–ARE pathway is typically engaged as a core antioxidant defense program, inducing the expression of SOD and other antioxidant components [ 27 ] . As a result, elevated SOD activity accelerates the dismutation of O₂⁻· to H₂O₂, which can shift redox stress from superoxide toward peroxide handling demands. Importantly, H₂O₂ is not merely a by-product of oxidative stress; it can influence neuronal physiology by modulating excitatory neurotransmission. Prior studies have shown that NMDA receptor activation contributes to H₂O₂-related pathophysiology and that NMDA receptor function is sensitive to redox conditions [ 28 , 29 ] . Collectively, these findings support a mechanistic framework in which IH-driven ROS production triggers an Nrf2-associated antioxidant response that upregulates SOD, while the resulting increase in H₂O₂ places greater reliance on downstream peroxide-buffering systems. In our acute IH model, the combination of increased neuronal firing and a SOD↑/CAT↓ pattern suggests that ROS generation and antioxidant compensation are temporally mismatched. One plausible upstream driver is activity-dependent ROS production: NMDA receptor activation can rapidly enhance free radical formation through a nitric oxide–NOX2 mechanism, providing a direct link between excitatory load and superoxide generation during early IH [ 30 ] . The rise in SOD activity would then be expected to accelerate conversion of O₂⁻· to H₂O₂, increasing reliance on downstream H₂O₂-detoxifying capacity. However, CAT activity may decline in the acute phase because enzyme capacity can be functionally compromised before transcriptional replenishment occurs, and because maintenance of peroxisomal antioxidant competence depends on intact PPARγ–peroxisome signaling; impairment of this axis has been shown to disrupt peroxisome functionality and weaken related redox homeostasis [ 31 ] . Together, these considerations support a conservative working model in which early IH produces a rapid, excitation-coupled ROS surge (NMDA–NOX2), while CAT-dependent buffering lags or is functionally constrained, yielding transient H₂O₂ pressure that may contribute to cortical hyperexcitability. This interpretation remains hypothetical and should be tested by time-resolved ROS measurements and targeted assays of NOX2 and peroxisomal/PPARγ markers. In our model, GPx activity in Au1 did not change significantly after 3h of IH, in line with findings by Weis et al. in acute hypoxia [ 32 ] . Considering the kinetics of H₂O₂ clearance in the brain, early-stage IH may rely predominantly on CAT-mediated high-throughput elimination, whereas GPx/Prx pathways become more relevant under sustained or chronic oxidative load [ 33 ] . Transcription, translation and substrate supply for GPx are also subject to temporal delay. These factors together may account for the observation that, at the 3h time point, SOD is already upregulated and CAT is reduced, whereas GPx remains relatively stable, consistent with the time course of acute IH. Notably, we did not detect significant neuronal apoptosis in Au1 after 3h of IH, with only sporadic TUNEL-positive cells and no group differences. Gozal et al. reported that IH-induced structural damage in the cortex and hippocampus typically peaks around 48 h [ 34 ] , suggesting that neuronal loss is more characteristic of subacute or chronic exposure. Our findings are consistent with this temporal profile: at an early stage of IH, the auditory cortex exhibits altered excitability and non-symmetric antioxidant adjustments without overt structural degeneration. This supports a sequence in which functional plasticity and redox imbalance precede more irreversible cellular injury. In summary, our data indicate that in an acute IH rat model, 3h of hypoxia is sufficient to induce hyperexcitability of auditory cortical neurons and to trigger a distinctive antioxidant response characterized by increased SOD, decreased CAT, and largely unchanged GPx activity. High-dose VC pretreatment, administered shortly before IH, partially normalizes the abnormal increase in SFR and simultaneously enhances CAT, SOD and GPx activities, without provoking detectable apoptosis. These findings suggest that, during early IH, the auditory cortex remains in a “modifiable window” dominated by functional plasticity and reversible oxidative stress, and that VC-based antioxidant support may mitigate IH-related central auditory injury. Although extrapolation to clinical practice requires caution, the results provide experimental support for simple adjunctive strategies, such as evening VC supplementation, to be considered alongside CPAP in protecting central auditory structures in OSA. This study has several limitations. First, it is an exploratory basic study employing an acute 3h IH paradigm and a single observation time point, which does not fully capture the chronic and fluctuating nature of IH in OSA. Only male rats were used, and we tested a single VC dose and pretreatment schedule, without systematic evaluation of sex differences, dose–response relationships or different timing of administration. In addition, we focused on neuronal SFR and antioxidant enzyme activity as functional endpoints, without directly probing upstream molecular pathways or long-term behavioral and cognitive outcomes. Future studies using more clinically relevant chronic IH models, multiple time points, graded dosing regimens and combined molecular and behavioral readouts will be important to validate and extend the mechanistic inferences proposed here, and to better define the translational potential of antioxidant interventions for central auditory protection in OSA. Declarations Acknowledgement We thank the Animal Experimental Center of Peking University Third Hospital for assistance with animal housing and care. Declaration of interest statement No potential conflict of interest was reported by the author(s). Funding Declaration This work was supported by the Clinical Cohort Construction Project of Peking University Third Hospital (BYSYDL2025039). AI Use Statement Generative AI tools were used for language editing only. Specifically, Chat-GPT-5.2 was used to improve English grammar, clarity, and readability. No generative AI tools were used for study design, data collection, data analysis, or the generation of results, figures, or references. The authors take full responsibility for the content of the manuscript. Author Contribution Author contributions: Y.W. and T.L. contributed equally to this work. Y.W. and T.L. conceived and designed the study, conducted the experiments, analyzed the data, and drafted the manuscript. W.C., R.F., H.J., and J.H. assisted with data acquisition and interpretation and contributed to manuscript revision. F.M. and Y.Y. supervised the study and critically revised the manuscript. All authors reviewed and approved the final manuscript. References Gottlieb D J, Punjabi N M. Diagnosis and Management of Obstructive Sleep Apnea: A Review [J]. Jama, 2020, 323(14): 1389-400. 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Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: Oxidative eustress [J]. Redox biology, 2017, 11: 613-9. Gozal E, Row B W, Schurr A, Gozal D. Developmental differences in cortical and hippocampal vulnerability to intermittent hypoxia in the rat [J]. Neuroscience letters, 2001, 305(3): 197-201. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. 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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-8528320","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":575821887,"identity":"150654bc-e7ae-4294-8a75-4b6074bbc5aa","order_by":0,"name":"Yue Wu","email":"","orcid":"","institution":"Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Wu","suffix":""},{"id":575821892,"identity":"9faabcf3-963e-4764-a7de-f9b5e04e9d02","order_by":1,"name":"Tao Li","email":"","orcid":"","institution":"Peking University Third 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1","display":"","copyAsset":false,"role":"figure","size":33056,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of VC on SFR of neurons in the primary auditory cortex under acute intermittent hypoxia. Summary of SFR in each group (control group: n = 6 rats, 65 neurons; IH group: n = 6 rats, 62 neurons; IH+IPNS group: n = 6 rats, 66 neurons; IH+IPVC group: n = 6 rats, 70 neurons; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ****p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8528320/v1/b6d499dcd9a6235ec2072e7b.png"},{"id":100583616,"identity":"8d9048f0-0144-48b2-8eda-ecdc44e80686","added_by":"auto","created_at":"2026-01-19 11:33:01","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":383942,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative HE-stained section showing the electrode insertion site. The blue arrow indicates a local indentation corresponding to the electrode track in Au1.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8528320/v1/ab1802f2c9b413f9bf0c2814.jpeg"},{"id":100583632,"identity":"9551b275-f0b4-417b-96e5-61bca74e026e","added_by":"auto","created_at":"2026-01-19 11:33:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":46100,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of VC on SOD activity in the auditory cortex under acute IH. Summary of SOD activity in each group (control group: n = 6 rats; IH group: n = 6 rats; IH+IPNS group: n = 6 rats; IH+IPVC group: n = 6 rats,**p \u0026lt; 0.01, ****p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8528320/v1/fe211a46ab3310d671e9f7e7.png"},{"id":100583620,"identity":"be916490-7acf-4be5-b3c8-769a2154103d","added_by":"auto","created_at":"2026-01-19 11:33:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":34309,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of VC on CAT activity in the auditory cortex under acute IH. Summary of CAT activity in each group (control group: n = 6 rats,; IH group: n = 6 rats; IH+IPNS group: n = 6 rats; IH+IPVC group: n = 6 rats,*p \u0026lt; 0.05, **p \u0026lt; 0.01, ****p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8528320/v1/12189f413efc64fc5424e0f4.png"},{"id":100583493,"identity":"79adb718-560a-4430-956b-8cd5333d6aa2","added_by":"auto","created_at":"2026-01-19 11:32:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":26424,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of VC on GPx activity in the auditory cortex under acute IH. Summary of GPx activity in each group (control group: n = 6 rats; IH group: n = 6 rats; IH+IPNS group: n = 6 rats; IH+IPVC group: n = 6 rats,**p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8528320/v1/eb23d495335873e5de615743.png"},{"id":100583624,"identity":"ba937bab-9b67-4794-97d1-d23057547a44","added_by":"auto","created_at":"2026-01-19 11:33:06","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":390079,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative TUNEL immunofluorescence images in Au1. DAPI (blue) labels nuclei and TUNEL (red) labels apoptotic cells. In the control, IH, IH+IPNS and IH+IPVC groups, only rare or no obvious nuclear TUNEL-positive signals are observed. Scale bar = 200 µm.(control group: n = 6 rats; IH group: n = 6 rats; IH+IPNS group: n = 6 rats; IH+IPVC group: n = 6 rats.)\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8528320/v1/f234b81e16401ce9b758c58a.png"},{"id":100583595,"identity":"69b7b0e9-d537-4e83-ac92-0a6e6c38907e","added_by":"auto","created_at":"2026-01-19 11:32:53","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":215726,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative TUNEL staining in Au1 showing positive and negative controls. Staining is as in Figure 5. The positive control（PC） shows strong, diffuse nuclear TUNEL positivity, whereas the negative control（NC） displays only background fluorescence, confirming the specificity of the TUNEL assay. Scale bar = 200 µm.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8528320/v1/c209750f95a9f7aa2e8109b3.jpeg"},{"id":100583658,"identity":"0eda2afc-532e-4f58-81fc-b3d95e1d8a36","added_by":"auto","created_at":"2026-01-19 11:33:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1841299,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8528320/v1/7afcd2a2-9cc6-40e7-a75a-72959159891d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Vitamin C Mitigates Early Auditory Cortical Hyperexcitability and Antioxidant Imbalance Induced by Acute Intermittent Hypoxia","fulltext":[{"header":"1.introduction","content":"\u003cp\u003eOSA is a sleep-related breathing disorder characterized by recurrent partial or complete collapse of the upper airway during sleep, leading to apnea and hypoventilation. These events cause intermittent hypoxia, sleep fragmentation, and a wide range of cardiovascular and metabolic problems\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. OSA results in multi-system damage and is linked to type 2 diabetes\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e, hypertension\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e, heart failure, cardiac arrhythmias\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e, stroke\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Besides its systemic effects, increasing evidence shows that chronic intermittent hypoxia can also harm the auditory system, especially causing peripheral hearing loss at high frequencies\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. IH has also been reported to impair the central auditory system, with clinical signs like prolonged auditory brainstem response (ABR) latencies\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e, reduced speech recognition scores\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e, and declines in verbal memory and cognitive function\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. These findings indicate that intermittent hypoxia may damage auditory-related central pathways. The auditory cortex is a central part of the speech-processing network, maintaining effective connections with frontal, temporal, and temporoparietal regions to support the tracking and understanding of continuous speech; the health of this network is closely tied to speech comprehension\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e.Our previous work demonstrated that short-term intermittent hypoxia induces peripheral hearing loss, shortens ABR wave III\u0026ndash;V latencies, and increases spontaneous firing rates in the auditory cortex\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Collectively, clinical and experimental data support the view that IH is associated with structural and functional impairment of the auditory cortex.\u003c/p\u003e \u003cp\u003eCurrently, continuous positive airway pressure (CPAP) is the mainstay of treatment for OSA. Previous studies have shown that adequately titrated CPAP can partially improve cochlear function and low-to-mid frequency hearing thresholds in patients with OSA\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. However, there is still insufficient evidence that CPAP exerts a beneficial effect on OSA-related central auditory damage\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. This highlights the need to explore adjunctive strategies targeting central auditory injury in OSA.\u003c/p\u003e \u003cp\u003eVC is a classical water-soluble antioxidant with potent ROS-scavenging capacity. In the pathogenesis of OSA, repetitive hypoxia\u0026ndash;reoxygenation episodes generate large amounts of oxygen-free radicals in the central nervous system, triggering oxidative stress\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. By eliminating free radicals, it appears to improve hearing outcomes in patients with sudden sensorineural hearing loss\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Besides, high-dose VC has been shown to attenuate oxidative stress, mitochondrial fragmentation, and apoptosis in the brain and to ameliorate functional deficits\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Therefore, vitamin C supplementation may confer a protective effect against intermittent hypoxia\u0026ndash;induced auditory cortical dysfunction and potential injury by scavenging reactive oxygen species and alleviating oxidative stress.\u003c/p\u003e \u003cp\u003eTherefore, the present study used a pre-IH high-dose VC protocol to conceptually model pre-sleep VC supplementation and to investigate its protective effects against short-term intermittent hypoxia\u0026ndash;induced oxidative stress and functional impairment in the auditory cortex.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animals\u003c/h2\u003e \u003cp\u003eMale Sprague\u0026ndash;Dawley rats (300\u0026ndash;350 g; purchased from VITAL RIVER, Beijing, China) were used in this study. Animals were housed under controlled conditions (22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 50\u0026thinsp;\u0026plusmn;\u0026thinsp;10% relative humidity, 12 h light/dark cycle) with free access to food and water. All procedures were approved by the Institutional Animal Care and Use Committee of Peking University Health Science Center and complied with relevant guidelines for the care and use of laboratory animals.\u003c/p\u003e \u003cp\u003eRats were randomly assigned to four groups: control, IH, IH\u0026thinsp;+\u0026thinsp;IPNS, and IH plus, IH\u0026thinsp;+\u0026thinsp;IPVC. Vitamin C (500 mg/kg) was dissolved in sterile saline and administered intraperitoneally (i.p.) 30 min before the beginning of the AIH exposure. Control animals received an equal volume of normal saline using the same schedule.\u003c/p\u003e \u003cp\u003eElectrophysiological and biochemical assessments of the auditory cortex were completed within 3h after the end of the exposure protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Intermittent hypoxia apparatus and protocol\u003c/h2\u003e \u003cp\u003eThe IH apparatus and parameters were based on our previous work.[22] Briefly, the system consisted of an air pump, a unidirectional ventilation tube, and a 3D-printed mask fitted to the rat\u0026rsquo;s snout. When the pump was on, room air was delivered at 2 L/min, flushing the mask and removing CO₂. When the pump was off, the mask formed a relatively closed local environment, leading to a progressive decline of arterial oxygen saturation and thus producing acute IH exposure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Surgery and in vivo electrophysiological recording\u003c/h2\u003e \u003cp\u003eThree hours after the acute IH protocol, in vivo electrophysiological recordings were performed under general anesthesia to assess neuronal activity in Au1. After exposure of the skull, a craniotomy was made over the auditory cortex (anteroposterior \u0026minus;\u0026thinsp;4.56 to \u0026minus;\u0026thinsp;5.04 mm; mediolateral 6.4 to 7.6 mm from bregma; dorsoventral 3.8 to 5.0 mm according to the rat brain atlas). The dura was carefully removed to expose the cortex.A 4 \u0026times; 4 microelectrode array (custom-made; pre-sterilized with povidone\u0026ndash;iodine; mean impedance\u0026thinsp;\u0026asymp;\u0026thinsp;0.3 MΩ) was slowly advanced into Au1 using a micromanipulator in 0.05-mm steps. After each advancement, the electrode was left in place for ~\u0026thinsp;20 min to allow neuronal firing to stabilize. During recording, a copper mesh shield was used to reduce electrical noise, and exposed brain tissue was covered with saline-soaked cotton to prevent drying.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Electrophysiological data acquisition and analysis\u003c/h2\u003e \u003cp\u003eMultiunit activity was recorded using an OmniPlex multichannel acquisition system (Plexon Inc., Dallas, TX, USA) at a sampling rate of 40 kHz. Raw signals were exported to Offline Sorter (Plexon) for preprocessing. A Bessel filter with a 250 Hz high-pass cutoff was applied to remove low-frequency noise, and channels with a signal-to-noise ratio\u0026thinsp;\u0026lt;\u0026thinsp;3.0 were excluded.Spike sorting was performed using principal component analysis followed by k-means clustering, and units with abnormal waveforms or clear artifacts (e.g., refractory period violations with interspike intervals\u0026thinsp;\u0026lt;\u0026thinsp;1\u0026ndash;2 ms) were manually discarded.\u003c/p\u003e \u003cp\u003eSFR of each isolated unit was calculated using NeuroExplorer (Nex Technologies, Colorado Springs, CO, USA). Mean SFR values were compared across groups to evaluate the effects of acute IH and VC treatment on neuronal excitability in Au1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Auditory cortex tissue collection\u003c/h2\u003e \u003cp\u003eRats were euthanized by cervical dislocation, and brains were rapidly removed and placed in ice-cold saline. The anteroposterior extent of Au1 (9\u0026ndash;12 mm posterior to the olfactory bulb) was determined according to \u003cem\u003eThe Rat Brain in Stereotaxic Coordinates\u003c/em\u003e (5th ed.). Brains were then placed in a pre-cooled brain matrix with millimeter markings to obtain coronal slabs encompassing Au1, and Au1 was microdissected under a dissecting microscope based on cortical landmarks and immediately processed for antioxidant enzyme assays.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Assays of CAT, SOD, and GPx activities\u003c/h2\u003e \u003cp\u003eAuditory cortex tissue was homogenized in ice-cold buffer, and protein concentration was determined according to the manufacturer\u0026rsquo;s instructions. The activities of CAT(Solarbio, Beijing, China; cat. no. BC4785), SOD(Solarbio; cat. no. BC5160) and GPx( Solarbio; cat. no. BC1190) were measured using the corresponding commercial assay kits following the manufacturers\u0026rsquo; protocols. Enzyme activities were normalized to protein content and expressed as units per milligram of protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Histological assessment and neuronal apoptosis\u003c/h2\u003e \u003cp\u003eIn animals designated for electrode localization and apoptosis assessment, brains were fixed by transcardial perfusion with 4% paraformaldehyde under deep anesthesia. After routine dehydration and paraffin embedding, coronal sections (4\u0026ndash;6 \u0026micro;m) were cut at the level of the auditory cortex.\u003c/p\u003e \u003cp\u003eA subset of sections was stained with hematoxylin\u0026ndash;eosin (HE). Under light microscopy, electrode tracks and local depressions or cavities were identified and matched to corresponding plates of the rat brain atlas, using the lateral sulcus and other anatomical landmarks, to verify that electrode tips were located within Au1.\u003c/p\u003e \u003cp\u003eAnother subset of sections was processed for TUNEL staining to assess neuronal apoptosis. TUNEL labeling was performed using the In Situ Cell Death Detection Kit, TMR red (Roche; cat. no. 12156792910), according to the manufacturer\u0026rsquo;s instructions. Positive controls were prepared using a TUNEL apoptosis positive control kit (Beyotime; cat. no. C1082), which includes DNase I pretreatment, whereas omission of terminal deoxynucleotidyl transferase (TdT) served as a negative control. Nuclei were counterstained with DAPI. Fluorescence images from the Au1 region were acquired under identical exposure settings across groups. Two observers blinded to group allocation independently counted TUNEL-positive cells and total nuclei. The apoptotic index was calculated as the percentage of TUNEL-positive cells among total nuclei (TUNEL-positive cells / total nuclei \u0026times; 100%), and the mean value from multiple fields was used as the data point for each animal.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe animal was the primary experimental unit (n\u0026thinsp;=\u0026thinsp;6 per group). For electrophysiology, unit-level measures were summarized within each animal (e.g., mean/median SFR per rat) for primary group comparisons. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Normality was assessed using the Shapiro\u0026ndash;Wilk test, and homogeneity of variance was evaluated using Levene\u0026rsquo;s test. When both assumptions were satisfied, group comparisons were performed using one-way analysis of variance (one-way ANOVA) followed by Tukey\u0026rsquo;s honestly significant difference (Tukey HSD) post hoc test. When assumptions were violated, appropriate non-parametric tests were applied. A two-tailed p value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Statistical analyses were conducted using SPSS (IBM Corp., Armonk, NY, USA) or GraphPad Prism (GraphPad Software, San Diego, CA, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Short-term intermittent hypoxia increases neuronal firing in the auditory cortex, and high-dose vitamin C partially attenuates this effect\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs detailed in the Methods, rats were assigned to four experimental groups (control group, IH group, IH+IPNS group, and IH+IPVC group; n = 6 per group). Rats in the control group wore the mask apparatus without hypoxic exposure. Rats in the IH group underwent 3h of IH, with mean maximal and minimal arterial oxygen saturation values of 98.02 \u0026plusmn; 0.24% and 81.45 \u0026plusmn; 0.91%, respectively. In the IH+IPNS group, 0.9% saline was administered intraperitoneally 30 min before IH (at the same volume as in the IH+IPVC group), and maximal and minimal oxygen saturation values were 98.02 \u0026plusmn; 0.39% and 82.73 \u0026plusmn; 1.05%. In the IH+IPVC group, rats received 500 mg/kg VC intraperitoneally 30 min before IH, with maximal and minimal oxygen saturation values of 97.93 \u0026plusmn; 0.28% and 82.02 \u0026plusmn; 0.78%. Maximal and minimal oxygen saturation did not differ significantly among the IH, IH+IPNS and IH+IPVC groups, indicating a comparable hypoxic load.\u003c/p\u003e\n\u003cp\u003eAfter IH exposure, the SFR of auditory cortical neurons was 1.23 \u0026plusmn; 0.074 Hz in the control group, 2.64 \u0026plusmn; 0.18 Hz in the IH group, 2.73 \u0026plusmn; 0.16 Hz in the IH+IPNS group, and 1.93 \u0026plusmn; 0.17 Hz in the IH+IPVC group. Shapiro\u0026ndash;Wilk and Levene\u0026rsquo;s tests confirmed that the assumptions of normality and homogeneity of variance were met; therefore, one-way ANOVA was applied for group comparisons. One-way ANOVA revealed a significant group effect (F = 20.42, p = 2.66 \u0026times; 10⁻⁶). Tukey post hoc analysis showed that SFR was significantly higher in the IH group than in the control group (1.23 \u0026plusmn; 0.074 vs 2.64 \u0026plusmn; 0.18 Hz, p = 1.6 \u0026times; 10⁻⁵), with no difference between IH and IH+IPNS (2.64 \u0026plusmn; 0.18 vs 2.73 \u0026plusmn; 0.16 Hz, p = 0.97). SFR in the IH+IPVC group was significantly lower than in the IH+IPNS group (1.93 \u0026plusmn; 0.17 vs 2.73 \u0026plusmn; 0.16 Hz, p = 0.0077), but remained higher than in the control group (1.93 \u0026plusmn; 0.17 vs 1.23 \u0026plusmn; 0.074 Hz, p = 0.022)（\u003cstrong\u003eFigure 1\u003c/strong\u003e）. These findings indicate that short-term IH induces marked hyperexcitability of auditory cortical neurons, and that high-dose VC partially attenuates this hyperexcitability.\u003c/p\u003e\n\u003cp\u003eAfter completion of in vivo recordings, brain sections were stained with hematoxylin\u0026ndash;eosin and compared with the corresponding plates in \u003cem\u003eThe Rat Brain in Stereotaxic Coordinates\u003c/em\u003e (5th ed.). Electrode tips were consistently located within Au1, confirming that the recorded signals originated from this region (\u003cstrong\u003eFigure 2\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Short-term intermittent hypoxia decreases CAT activity, increases SOD activity, and has limited effects on GPx in the auditory cortex, whereas high-dose VC enhances all three enzymes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess oxidative stress in the auditory cortex, we measured the activities of CAT, SOD and GPx in all groups. Shapiro\u0026ndash;Wilk and Levene\u0026rsquo;s tests indicated that the data met the assumptions of normality and homogeneity of variance; therefore, one-way ANOVA with Tukey HSD post hoc tests was used for group comparisons.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSOD activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMean SOD activities were 686.8 \u0026plusmn; 27.14 U/g in the control group, 1433.67 \u0026plusmn; 143.94 U/g in the IH group, 1343.07 \u0026plusmn; 128.92 U/g in the IH+IPNS group and 2402.83 \u0026plusmn; 133.34 U/g in the IH+IPVC group. ANOVA showed a significant group effect (F = 35.83, p = 3.09 \u0026times; 10⁻⁸). SOD activity was higher in the IH group than in the control group (686.8\u0026nbsp;\u0026plusmn;\u0026nbsp;27.14 vs 1433.67\u0026nbsp;\u0026plusmn;\u0026nbsp;143.94 U/g, p = 0.0012), and did not differ between the IH and IH+IPNS groups (1433.67\u0026nbsp;\u0026plusmn;\u0026nbsp;143.94 vs 1343.07\u0026nbsp;\u0026plusmn;\u0026nbsp;128.92 U/g, p = 0.948). The IH+IPVC group showed a further increase compared with the IH+IPNS group (2402.83 \u0026plusmn; 133.34 vs 1343.07 \u0026plusmn; 128.92 U/g, p = 1.9 \u0026times; 10⁻⁵), and SOD activity in IH+IPVC was also higher than in the control group (686.8\u0026nbsp;\u0026plusmn;\u0026nbsp;27.14 vs 2402.83\u0026nbsp;\u0026plusmn;\u0026nbsp;133.34 U/g, p= 1.16\u0026nbsp;\u0026times;\u0026nbsp;10⁻⁸).(\u003cstrong\u003eFigure 3\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCAT activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMean CAT activities (U/g tissue, mean \u0026plusmn; SEM) were 197.66 \u0026plusmn; 7.94 in the control group, 146.53 \u0026plusmn; 3.68 in the IH group, 155.15 \u0026plusmn; 7.41 in the IH+IPNS group and 234.68 \u0026plusmn; 14.64 in the IH+IPVC group. One-way ANOVA revealed a significant group effect (F = 19.24, p = 4.12 \u0026times; 10⁻⁶). CAT activity was lower in the IH group than in the control group (197.66 \u0026plusmn; 7.94 vs 146.53 \u0026plusmn; 3.68 U/g, p = 0.0047), with no difference between the IH and IH+IPNS groups (146.53 \u0026plusmn; 3.68 vs 155.15 \u0026plusmn; 7.41 U/g, p = 0.91). In contrast, CAT activity was higher in the IH+IPVC group than in the IH+IPNS group (155.15 \u0026plusmn; 7.41 vs 234.68 \u0026plusmn; 14.64 U/g, p = 3.6 \u0026times; 10⁻⁵) and also exceeded control levels (197.66 \u0026plusmn; 7.94 vs 234.68 \u0026plusmn; 14.64 U/g, p = 0.048)（\u003cstrong\u003eFigure 4\u003c/strong\u003e）.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGPx activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMean GPx activities were 251.13 \u0026plusmn; 8.66 U/g in the control group, 280.14 \u0026plusmn; 3.95 U/g in the IH group, 285.80 \u0026plusmn; 17.39 U/g in the IH+IPNS group and 314.80 \u0026plusmn; 12.99 U/g in the IH+IPVC group. ANOVA indicated a significant group effect (F = 4.85, p = 0.011). Post hoc tests showed no significant differences between the control and IH groups (251.13 \u0026plusmn; 8.66 vs 280.14 \u0026plusmn; 3.95 U/g, p = 0.33), between the IH and IH+IPNS groups (280.14 \u0026plusmn; 3.95 vs 285.80 \u0026plusmn; 17.39 U/g, p = 0.99), or between the IH+IPNS and IH+IPVC groups (285.80 \u0026plusmn; 17.39 vs 314.80 \u0026plusmn; 12.99 U/g, p = 0.33). However, GPx activity in the IH+IPVC group was higher than in the control group (251.13 \u0026plusmn; 8.66 vs 314.80 \u0026plusmn; 12.99 U/g, p = 0.0057)(\u003cstrong\u003eFigure 5\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eTaken together, short-term IH in the auditory cortex is associated with decreased CAT activity, increased SOD activity and minimal changes in GPx, whereas high-dose VC elevates all three enzyme activities, indicating an overall enhancement of the antioxidant defense system.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Acute intermittent hypoxia does not induce overt neuronal apoptosis in the auditory cortex\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the same Au1 region used for electrophysiological recordings, five non-overlapping fields (\u0026asymp;400 \u0026times; 400 \u0026micro;m) were randomly selected per animal, yielding approximately 1,000 nuclei per group. Across the control, IH, IH+IPNS and IH+IPVC groups, only sporadic TUNEL-positive cells were observed. Overall TUNEL signal remained at a low abundance close to background, and no clear trend of group differences was detected \u003cstrong\u003e(Figure 6)\u003c/strong\u003e. In contrast, the positive control with DNase pretreatment showed strong, diffuse nuclear TUNEL staining, whereas the negative control (omission of TdT) exhibited only background fluorescence, confirming the specificity and reliability of the TUNEL assay \u003cstrong\u003e(Figure 7).\u003c/strong\u003e Under the present experimental conditions (3h acute IH), neurons in the auditory cortex thus displayed altered excitability and antioxidant enzyme activity, but no apparent \u0026nbsp;apoptotic cell loss.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOSA is a sleep-related breathing disorder characterized by recurrent partial or complete upper airway collapse during sleep, leading to intermittent hypoxia, sleep fragmentation and multi-system dysfunction\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Increasing attention has been paid to the auditory consequences of OSA, as clinical studies have reported elevated hearing thresholds\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e, prolonged ABR latencies\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e and reduced speech recognition scores\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e, all of which are closely associated with intermittent hypoxia. Although CPAP may partially improve low-to-mid frequency hearing thresholds\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e, its efficacy in restoring central auditory function, particularly at the cortical level, appears limited\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Given that the auditory cortex is a hub of the speech processing network and its connectivity with frontal and temporo-parietal regions is critical for speech comprehension\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e, adjunctive strategies targeting hypoxia-induced damage in central auditory structures may have clinical value.\u003c/p\u003e \u003cp\u003eIn the present study, we used an acute 3h IH rat model to characterize early responses of the auditory cortex to IH at two levels: neuronal spontaneous firing and antioxidant enzyme activity. We further evaluated the effects of high-dose VC pretreatment. Our data show that short-term IH markedly increases SFR of auditory cortical neurons. Under comparable hypoxic load, intraperitoneal administration of high-dose VC partially attenuates this hyperexcitability and concomitantly elevates SOD, CAT and GPx activities, without inducing overt neuronal apoptosis. These findings suggest that at an early stage of IH, the auditory cortex primarily undergoes functional plasticity and reversible oxidative changes, indicating a potential window of opportunity for intervention.\u003c/p\u003e \u003cp\u003eWith regard to excitability, 3h of IH was sufficient to induce a robust increase in SFR in Au1, indicating a transient shift in the excitation-inhibition balance. Previous work has shown that excessive activation of NMDA receptors can markedly enhance ROS production via a nitric oxide (NO)\u0026ndash;NADPH oxidase (NOX) pathway, and that genetic deletion or pharmacological inhibition of NOX abolishes this ROS elevation, supporting excitatory transmission\u0026ndash;driven NOX activation as a major source of cortical ROS\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e.These observations support excitatory transmission-driven NOX activation as a major source of cortical ROS. In our model, high-dose VC pretreatment reduced the IH-induced increase in SFR without altering the severity of hypoxia and at the same time enhanced antioxidant enzyme activities. Although direct measurements of ROS were not obtained, this constellation of findings is consistent with a contribution of oxidative stress to IH-induced cortical hyperexcitability and suggests that antioxidant support can partially buffer this effect.\u003c/p\u003e \u003cp\u003eWe next examined the activities of three key antioxidant enzymes in the auditory cortex: SOD, CAT and GPx. SOD catalyzes the dismutation of superoxide anions (O₂⁻\u0026middot;) to H₂O₂, whereas CAT and GPx are involved in subsequent H₂O₂ detoxification. CAT is well suited for high-throughput decomposition of H₂O₂, while GPx/Prx pathways provide high-affinity, lower-capacity clearance\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e.In the present study, acute IH was accompanied by a decrease in CAT activity, a marked increase in SOD activity and only a small, non-significant change in GPx. Taken together, these enzyme profiles indicate an early, unbalanced adjustment of the antioxidant system in the auditory cortex, in line with observations from other hypoxia models\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAt the mechanistic level, IH constitutes a repeated hypoxia\u0026ndash;reoxygenation stress that can amplify ROS generation in the brain. Evidence from hypoxia-related models suggests that ROS accumulation can be driven by NADPH oxidase (NOX2)\u0026ndash;dependent mechanisms and accompanied by mitochondrial redox disturbances, together increasing the burden of superoxide and downstream oxidants\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. In response to oxidative pressure, the Nrf2\u0026ndash;ARE pathway is typically engaged as a core antioxidant defense program, inducing the expression of SOD and other antioxidant components\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. As a result, elevated SOD activity accelerates the dismutation of O₂⁻\u0026middot; to H₂O₂, which can shift redox stress from superoxide toward peroxide handling demands. Importantly, H₂O₂ is not merely a by-product of oxidative stress; it can influence neuronal physiology by modulating excitatory neurotransmission. Prior studies have shown that NMDA receptor activation contributes to H₂O₂-related pathophysiology and that NMDA receptor function is sensitive to redox conditions\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Collectively, these findings support a mechanistic framework in which IH-driven ROS production triggers an Nrf2-associated antioxidant response that upregulates SOD, while the resulting increase in H₂O₂ places greater reliance on downstream peroxide-buffering systems.\u003c/p\u003e \u003cp\u003eIn our acute IH model, the combination of increased neuronal firing and a SOD\u0026uarr;/CAT\u0026darr; pattern suggests that ROS generation and antioxidant compensation are temporally mismatched. One plausible upstream driver is activity-dependent ROS production: NMDA receptor activation can rapidly enhance free radical formation through a nitric oxide\u0026ndash;NOX2 mechanism, providing a direct link between excitatory load and superoxide generation during early IH\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. The rise in SOD activity would then be expected to accelerate conversion of O₂⁻\u0026middot; to H₂O₂, increasing reliance on downstream H₂O₂-detoxifying capacity. However, CAT activity may decline in the acute phase because enzyme capacity can be functionally compromised before transcriptional replenishment occurs, and because maintenance of peroxisomal antioxidant competence depends on intact PPARγ\u0026ndash;peroxisome signaling; impairment of this axis has been shown to disrupt peroxisome functionality and weaken related redox homeostasis\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Together, these considerations support a conservative working model in which early IH produces a rapid, excitation-coupled ROS surge (NMDA\u0026ndash;NOX2), while CAT-dependent buffering lags or is functionally constrained, yielding transient H₂O₂ pressure that may contribute to cortical hyperexcitability. This interpretation remains hypothetical and should be tested by time-resolved ROS measurements and targeted assays of NOX2 and peroxisomal/PPARγ markers.\u003c/p\u003e \u003cp\u003eIn our model, GPx activity in Au1 did not change significantly after 3h of IH, in line with findings by Weis et al. in acute hypoxia\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Considering the kinetics of H₂O₂ clearance in the brain, early-stage IH may rely predominantly on CAT-mediated high-throughput elimination, whereas GPx/Prx pathways become more relevant under sustained or chronic oxidative load\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Transcription, translation and substrate supply for GPx are also subject to temporal delay. These factors together may account for the observation that, at the 3h time point, SOD is already upregulated and CAT is reduced, whereas GPx remains relatively stable, consistent with the time course of acute IH.\u003c/p\u003e \u003cp\u003eNotably, we did not detect significant neuronal apoptosis in Au1 after 3h of IH, with only sporadic TUNEL-positive cells and no group differences. Gozal et al. reported that IH-induced structural damage in the cortex and hippocampus typically peaks around 48 h\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e, suggesting that neuronal loss is more characteristic of subacute or chronic exposure. Our findings are consistent with this temporal profile: at an early stage of IH, the auditory cortex exhibits altered excitability and non-symmetric antioxidant adjustments without overt structural degeneration. This supports a sequence in which functional plasticity and redox imbalance precede more irreversible cellular injury.\u003c/p\u003e \u003cp\u003eIn summary, our data indicate that in an acute IH rat model, 3h of hypoxia is sufficient to induce hyperexcitability of auditory cortical neurons and to trigger a distinctive antioxidant response characterized by increased SOD, decreased CAT, and largely unchanged GPx activity. High-dose VC pretreatment, administered shortly before IH, partially normalizes the abnormal increase in SFR and simultaneously enhances CAT, SOD and GPx activities, without provoking detectable apoptosis. These findings suggest that, during early IH, the auditory cortex remains in a \u0026ldquo;modifiable window\u0026rdquo; dominated by functional plasticity and reversible oxidative stress, and that VC-based antioxidant support may mitigate IH-related central auditory injury. Although extrapolation to clinical practice requires caution, the results provide experimental support for simple adjunctive strategies, such as evening VC supplementation, to be considered alongside CPAP in protecting central auditory structures in OSA.\u003c/p\u003e \u003cp\u003eThis study has several limitations. First, it is an exploratory basic study employing an acute 3h IH paradigm and a single observation time point, which does not fully capture the chronic and fluctuating nature of IH in OSA. Only male rats were used, and we tested a single VC dose and pretreatment schedule, without systematic evaluation of sex differences, dose\u0026ndash;response relationships or different timing of administration. In addition, we focused on neuronal SFR and antioxidant enzyme activity as functional endpoints, without directly probing upstream molecular pathways or long-term behavioral and cognitive outcomes. Future studies using more clinically relevant chronic IH models, multiple time points, graded dosing regimens and combined molecular and behavioral readouts will be important to validate and extend the mechanistic inferences proposed here, and to better define the translational potential of antioxidant interventions for central auditory protection in OSA.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Animal Experimental Center of Peking University Third Hospital for assistance with animal housing and care.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interest statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo potential conflict of interest was reported by the author(s).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Clinical Cohort Construction Project of Peking University Third Hospital (BYSYDL2025039).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAI Use Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenerative AI tools were used for language editing only. Specifically, Chat-GPT-5.2 was used to improve English grammar, clarity, and readability. No generative AI tools were used for study design, data collection, data analysis, or the generation of results, figures, or references. The authors take full responsibility for the content of the manuscript.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthor contributions: Y.W. and T.L. contributed equally to this work. Y.W. and T.L. conceived and designed the study, conducted the experiments, analyzed the data, and drafted the manuscript. W.C., R.F., H.J., and J.H. assisted with data acquisition and interpretation and contributed to manuscript revision. F.M. and Y.Y. supervised the study and critically revised the manuscript. All authors reviewed and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGottlieb D J, Punjabi N M. Diagnosis and Management of Obstructive Sleep Apnea: A Review [J]. 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Nature neuroscience, 2009, 12(7): 857-63.\u003c/li\u003e\n\u003cli\u003eDi Cesare Mannelli L, Zanardelli M, Micheli L, Ghelardini C. PPAR- \u0026gamma; impairment alters peroxisome functionality in primary astrocyte cell cultures [J]. BioMed research international, 2014, 2014: 546453.\u003c/li\u003e\n\u003cli\u003eWeis S N, Schunck R V, Pettenuzzo L F, Krolow R, Matt\u0026eacute; C, Manfredini V, Do Carmo R P M, Vargas C R, Dalmaz C, Wyse A T, Netto C A. Early biochemical effects after unilateral hypoxia-ischemia in the immature rat brain [J]. International journal of developmental neuroscience : the official journal of the International Society for Developmental Neuroscience, 2011, 29(2): 115-20.\u003c/li\u003e\n\u003cli\u003eSies H. Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: Oxidative eustress [J]. Redox biology, 2017, 11: 613-9.\u003c/li\u003e\n\u003cli\u003eGozal E, Row B W, Schurr A, Gozal D. Developmental differences in cortical and hippocampal vulnerability to intermittent hypoxia in the rat [J]. Neuroscience letters, 2001, 305(3): 197-201.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"intermittent hypoxia, auditory cortex, neuronal excitability, vitamin C, antioxidant enzymes","lastPublishedDoi":"10.21203/rs.3.rs-8528320/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8528320/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjectives\u003c/h2\u003e \u003cp\u003eTo test whether brief intermittent hypoxia (IH) induces early hyperexcitability and antioxidant imbalance in the primary auditory cortex (Au1), and whether high-dose vitamin C (VC) pretreatment mitigates these effects.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eAdult male Sprague\u0026ndash;Dawley rats were assigned to four groups (n\u0026thinsp;=\u0026thinsp;6/group): control, IH, IH plus intraperitoneal normal saline (IH\u0026thinsp;+\u0026thinsp;IPNS), and IH plus intraperitoneal VC (IH\u0026thinsp;+\u0026thinsp;IPVC). Rats underwent a 3-h IH protocol; VC (500 mg/kg, i.p.) or saline was administered 30 min before IH. Three hours after IH, in vivo Au1 multiunit recordings quantified spontaneous firing rate (SFR). We quantified catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities and assessed apoptosis using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAcute IH increased Au1 SFR versus control, and saline did not alter this response; VC pretreatment reduced SFR toward control levels. IH decreased CAT activity and increased SOD activity, with minimal change in GPx. VC increased CAT, SOD, and GPx activities relative to IH. No overt neuronal apoptosis was detected.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eBrief OSA-relevant IH triggers an early stage of auditory cortical dysfunction with hyperexcitability and antioxidant imbalance. High-dose VC pretreatment attenuates hyperexcitability and enhances antioxidant enzyme activity, supporting antioxidant strategies as adjuncts for central auditory protection.\u003c/p\u003e","manuscriptTitle":"Vitamin C Mitigates Early Auditory Cortical Hyperexcitability and Antioxidant Imbalance Induced by Acute Intermittent Hypoxia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-19 11:31:44","doi":"10.21203/rs.3.rs-8528320/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f91aec13-aa4c-46f7-8ff8-c748f70fc56d","owner":[],"postedDate":"January 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-22T07:55:29+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-19 11:31:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8528320","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8528320","identity":"rs-8528320","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}