Microcirculatory Dysfunction and Oxidative Stress in Sudden Sensorineural Hearing Loss: Insights From a Case-Control and Experimental Study

Introduction

Sudden sensorineural hearing loss (SSNHL) is an acute otological emergency characterized by the rapid onset of sensorineural hearing impairment, conventionally defined as a hearing loss of at least 30 dB across 3 consecutive frequencies occurring within a 72-hour period [1]. SSNHL has an estimated annual incidence of 5 to 20 cases per 100 000 individuals and imposes a substantial burden on the quality of life of affected patients, frequently leading to communication difficulties, social isolation, and psychological distress [2]. Despite the clinical urgency of SSNHL, its underlying etiology remains incompletely understood. Multiple pathophysiological mechanisms have been proposed, including viral infection, autoimmune dysregulation, metabolic abnormalities, and vascular insufficiency [3,4]. Among these, disturbances in inner ear microcirculation and heightened oxidative stress have received increasing attention as potentially interrelated contributors to cochlear dysfunction in SSNHL, although their combined impact and mechanistic interplay remain insufficiently characterized [5,6]. The inner ear, and particularly the cochlea, is highly metabolically active and depends on a continuous and tightly regulated blood supply, as it lacks substantial collateral circulation [7]. Disruption of microvascular perfusion can therefore predispose cochlear tissues to hypoxic stress. Alterations in perfusion-related parameters – such as reduced cerebral blood flow (CBF), decreased cerebral blood volume (CBV), and prolonged mean transit time (MTT) – have been proposed as indirect indicators of impaired microcirculatory function that may compromise oxygen and nutrient delivery to the cochlea [8]. In parallel, oxidative stress is a well-recognized mediator of cochlear injury. Excessive generation of reactive oxygen species, reflected by increased lipid peroxidation products such as malondialdehyde (MDA), together with attenuation of endogenous antioxidant defenses including superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), can promote cellular apoptosis, inflammatory signaling, and structural damage within the inner ear [9,10]. Although hypoperfusion-related stress and oxidative imbalance have each been implicated in SSNHL, most previous studies have examined these processes in isolation. As a result, the extent to which microcirculatory dysfunction and oxidative stress interact and jointly contribute to hearing impairment remains unclear, limiting the development of mechanism-oriented therapeutic strategies [11,12]. The present study was designed to address this gap by adopting an integrated translational framework that combines clinical observations with experimental validation. Specifically, we assessed perfusion-related surrogate parameters using high-resolution magnetic resonance perfusion imaging and peripheral microcirculatory assessment, together with systemic and cochlear oxidative stress markers quantified by biochemical assays. In parallel, a rat model of transient ischemia–reperfusion was used to examine cochlear oxidative injury and apoptotic changes under controlled conditions. By integrating human case–control data with complementary animal experiments, this approach allows for a more comprehensive evaluation of how impaired microcirculatory function may be linked to oxidative stress–mediated cochlear damage, while avoiding assumptions of direct cochlear blood flow measurement. Accordingly, the aim of this study is to elucidate the respective and combined roles of microcirculatory dysfunction and oxidative stress in the pathogenesis of SSNHL. By examining perfusion-related parameters, oxidative stress markers, and functional hearing outcomes across clinical and experimental settings, we seek to clarify their mechanistic associations and relative contributions to hearing impairment. Ultimately, this integrated analysis may help identify early pathophysiological indicators and inform the rational development of therapeutic strategies – such as perfusion-modulating and antioxidant interventions – within the narrow therapeutic window characteristic of SSNHL.

Material and Methods

STUDY DESIGN AND PARTICIPANTS:

This prospective case–control study was conducted over a 2-year period from March 2022 to March 2024 at Nanjing Drum Tower Hospital, a tertiary academic medical center, and the Affiliated Jiangning Hospital of Nanjing Medical University, both of which provide specialized otolaryngology services. We enrolled a total of 100 patients diagnosed with SSNHL and 100 age- and sex-matched healthy controls to ensure group comparability and minimize confounding effects.

For the human study, 142 potentially eligible patients with suspected SSNHL were screened; 42 were excluded due to chronic ear diseases (n=15), systemic conditions known to affect microcirculation (n=20), or pregnancy/breastfeeding (n=7), resulting in 100 enrolled patients with SSNHL. In parallel, 135 individuals undergoing routine health examinations were screened as potential controls; 35 were excluded because of a history of hearing-related disorders (n=25) or failure to meet age- and sex-matching criteria (n=10), yielding 100 healthy controls. The recruitment, exclusion, and enrollment process for both human participants and experimental animals is summarized in a flow diagram (Figure 1).

Sample size estimation was performed using G*Power software based on CBF as the primary outcome. Assuming an effect size of Cohen’s d=0.5, a 2-sided α of 0.05, and a statistical power of 0.80, the minimum required sample size was 64 participants per group. To account for variability in secondary outcomes, including oxidative stress markers, and to enhance the robustness of multivariable analyses, the sample size was increased to 100 participants per group.

All SSNHL cases were diagnosed by experienced otolaryngologists in accordance with the American Academy of Otolaryngology–Head and Neck Surgery (AAO-HNS) criteria, defined as a sensorineural hearing loss of at least 30 dB occurring within 72 hours [1]. Unilateral and bilateral cases were included; bilateral cases constituted a minority and were analyzed together with unilateral cases under a unified SSNHL classification. Healthy controls were consecutively recruited from individuals undergoing routine health check-ups at the same institutions and were drawn from the same source population, with no history of hearing loss or otologic disease. This case–control design was selected to enable systematic comparison of perfusion-related parameters, oxidative stress markers, and hearing outcomes between patients with SSNHL and healthy individuals.

INCLUSION AND EXCLUSION CRITERIA:

Inclusion criteria were as follows: (1) adult male and female participants aged 18 to 65 years; (2) for the SSNHL group, presentation with unilateral or bilateral sensorineural hearing loss within 72 hours, with a hearing impairment ≥30 dB meeting AAO-HNS diagnostic criteria; and (3) for the control group, absence of hearing loss or any history of hearing-related disease.

Exclusion criteria included (1) a history of chronic otologic disease, ear trauma, Ménière’s disease, or autoimmune disorders; (2) severe cardiovascular disease, diabetes mellitus, renal insufficiency, or other systemic conditions known to affect microcirculation; and (3) pregnancy or breastfeeding.

Controls were matched to SSNHL patients by age (±5 years) and sex and were consecutively recruited to minimize selection bias, ensuring a representative comparison group free of conditions that could confound microcirculatory or oxidative stress measurements.

EXPERIMENTAL ANIMALS AND ISCHEMIA–REPERFUSION MODEL:

To complement the clinical observations and explore mechanistic associations under controlled conditions, an experimental animal model was used. A total of 20 male Sprague-Dawley rats (8 weeks old, 200–250 g) were included. Initially, 30 rats were screened; 10 were excluded due to health concerns (n=6) or body weight outside the predefined range (n=4). Animals were housed under standardized conditions (22±2 °C, 55±5% humidity, 12-h light/dark cycle) with free access to food and water.

Rats were randomly assigned to either a microcirculation disorder group (n=10) or a sham-operated control group (n=10). Animals in the experimental group were anesthetized with isoflurane (2% maintenance) and subjected to bilateral common carotid artery clamping for 10 minutes, followed by clamp release to induce transient ischemia–reperfusion injury. Control animals underwent identical anesthesia and surgical exposure without carotid occlusion and received an equivalent volume of saline [6].

This bilateral carotid artery ischemia–reperfusion model was selected as an established experimental proxy for acute cochlear hypoperfusion and oxidative stress. Although it represents a global rather than unilateral ischemic insult, transient carotid occlusion produces rapid and reproducible reductions in inner ear perfusion due to the cochlea’s dependence on the labyrinthine artery and its limited collateral supply. The model therefore provides a controlled framework for examining oxidative stress, apoptotic injury, and functional hearing changes relevant to SSNHL pathophysiology.

Sample size for the animal experiment was determined based on prior ischemia–reperfusion studies demonstrating medium-to-large effect sizes for oxidative stress markers and auditory brainstem response (ABR) thresholds. Assuming an effect size of 1.0, α=0.05, and power of approximately 0.80, a minimum of 8 animals per group was required; 10 animals per group were included to account for potential intraoperative variability while adhering to the principle of reduction. Hearing function was assessed using ABR testing before and after ischemia–reperfusion injury.

HEMODYNAMIC MEASUREMENTS:

In the quantitative assessment of perfusion-related parameters, high-resolution magnetic resonance perfusion imaging was performed within 72 hours of hospital admission using a 3.0-Tesla MRI scanner (Philips Achieva, Philips Healthcare, Netherlands). Dynamic contrast-enhanced MRI was used to derive perfusion-related surrogate parameters, including CBF, CBV, and MTT. Imaging parameters, contrast administration, calibration procedures, image processing, and inter-observer analysis were conducted as previously described, with regions of interest manually delineated around cochlear and vestibular structures. Measurements from 2 independent radiologists were averaged, with discrepancies greater than 10% resolved by consensus.

In the peripheral microcirculatory assessment, peripheral microcirculatory perfusion was evaluated using laser Doppler flowmetry at the earlobe, using a PeriFlux System 5000 (Perimed AB, Sweden). Measurements were conducted under controlled environmental conditions, calibrated prior to each session, and repeated 3 times per participant, with mean values used for analysis.

DETECTION OF OXIDATIVE STRESS MARKERS:

Serum levels of MDA, SOD, and GSH-Px were quantified using enzyme-linked immunosorbent assays in fasting blood samples. Cochlear tissue from experimental animals was processed 24 hours after reperfusion for parallel assessment of oxidative stress markers and apoptotic injury. Apoptosis was evaluated using TUNEL staining, with quantitative analysis performed by 2 blinded pathologists according to predefined criteria.

HUMAN AUDIOMETRY:

Pure tone audiometry was performed in all participants across standard frequencies (0.5–4 kHz), and thresholds were averaged to calculate pure tone audiometry threshold values.

ANIMAL AUDITORY FUNCTION:

ABR testing was conducted at 4, 8, 16, and 32 kHz before and after ischemia–reperfusion injury to assess functional hearing changes in rats.

STATISTICAL ANALYSIS:

Statistical analyses were performed using SPSS version 26.0. Continuous variables were assessed for normality using the Shapiro-Wilk test and are presented as mean±standard deviation. Group comparisons were conducted using independent samples t tests, paired t tests, or one-way ANOVA with Bonferroni correction, as appropriate. Pearson correlation and multiple linear regression analyses were applied to examine associations among perfusion-related parameters, oxidative stress markers, and hearing outcomes. A 2-sided P value <0.05 was considered statistically significant.

ETHICS AND COMPLIANCE:

Ethics approval for the human study was obtained from the institutional ethics committee (approval No. 2020-03-080-K01), and written informed consent was obtained from all participants. The animal experiments were approved by the institutional animal ethics committee (approval No. AECNJU2022-0402-AN). Both approvals pertain to the same overarching project investigating microcirculatory dysfunction and oxidative stress in SSNHL and were conducted in accordance with established ethical guidelines.

CONSENT TO PARTICIPATE:

Written informed consent was obtained from all human participants prior to their inclusion in the study.

CONSENT TO PUBLISH:

All participants consented to the publication of anonymized data related to this study.

Results

HEMODYNAMIC MEASUREMENT RESULTS:

Perfusion-related surrogate parameters differed significantly between the SSNHL group and healthy controls. Specifically, the SSNHL group demonstrated lower CBF (38.52±5.34 vs 45.67±4.76 mL/100 g/min, P=0.003) and CBV (3.21±0.65 vs 3.78±0.72 mL/100 g, P=0.018), together with prolonged MTT (5.43±1.12 vs 4.96±0.98 s, P=0.021) (Table 1). These findings indicate an association between SSNHL and altered perfusion-related parameters derived from contrast-enhanced perfusion imaging, consistent with a microcirculatory dysfunction phenotype. Baseline characteristics of the SSNHL and control groups are summarized in Table 2. The groups were comparable with respect to age and sex by design, and no significant between-group differences were observed for body mass index or smoking status (all P>0.05), supporting the validity of subsequent group comparisons. Figure 2 presents the distribution of perfusion-related parameters across groups. In Figure 2A and 2B, box plots illustrate lower CBF and CBV values in the SSNHL group relative to controls, with visible between-individual variability in both cohorts. These graphical findings are consistent with the summary statistics reported in Table 1. Peripheral microcirculation assessed by laser Doppler flowmetry at the earlobe also differed between groups. Earlobe perfusion was lower in SSNHL patients than in controls (15.72±3.48 vs 18.34±3.10 perfusion units), with a mean difference of −2.62 (95% CI: −4.66 to −0.58; P=0.012) (Table 3). Together, these results support an association between SSNHL and altered peripheral microcirculatory perfusion, complementing the imaging-derived perfusion-related parameters.

OXIDATIVE STRESS MARKER DETECTION RESULTS:

Systemic oxidative stress markers differed significantly between SSNHL patients and healthy controls. As shown in Figure 3A–3C, the SSNHL group exhibited higher MDA levels and lower antioxidant enzyme activities (SOD and GSH-Px) compared with controls (all P<0.05). These findings are consistent with increased oxidative stress burden and reduced antioxidant capacity in SSNHL. In the animal experiment, cochlear tissue oxidative stress markers showed a parallel pattern. Figure 3D–3F demonstrates increased MDA and reduced SOD and GSH-Px in the ischemia–reperfusion (microcirculation disorder) group relative to sham controls (P<0.05). Apoptotic injury in cochlear tissue, assessed by TUNEL staining, is quantified in Figure 3G. The ischemia–reperfusion group exhibited a significantly higher apoptotic cell burden than did controls (P=0.002), indicating increased cochlear cell apoptosis under ischemia–reperfusion conditions.

HEARING ASSESSMENT RESULTS:

Functional hearing outcomes differed between groups in both the clinical and experimental settings. In humans, pure tone audiometry thresholds were significantly elevated across tested frequencies in SSNHL patients compared with controls (Figure 4A; P<0.05). In the animal model, ABR thresholds were significantly higher at 4, 8, 16, and 32 kHz in the ischemia–reperfusion group compared with sham controls (Figure 4B; P<0.05). These findings indicate that both clinical SSNHL and experimentally induced ischemia–reperfusion are associated with measurable impairments in hearing function.

CORRELATION AND REGRESSION ANALYSES:

Correlation analyses demonstrated significant associations between perfusion-related parameters and oxidative stress markers (Table 4). CBF was negatively correlated with MDA (r=−0.45, P=0.002) and positively correlated with SOD (r=0.38, P=0.004) and GSH-Px (r=0.35, P=0.008). CBV showed similar correlations with oxidative stress markers (all P < 0.05). MTT was positively correlated with MDA (r=0.49, P=0.001) and negatively correlated with SOD (P=0.018) and GSH-Px (P=0.011), suggesting that less favorable perfusion-related profiles are associated with a higher oxidative stress burden. Multiple linear regression analysis (Table 5) evaluated independent associations of perfusion-related parameters and oxidative stress markers with hearing thresholds. CBF (β=−0.42, P=0.003) and CBV (β=−0.25, P=0.027) were independently associated with lower hearing thresholds, whereas MTT (β=0.51, P=0.001) and MDA (β=0.36, P=0.009) were independently associated with higher hearing thresholds. Antioxidant markers SOD (β=−0.29, P=0.015) and GSH-Px (β=−0.22, P=0.045) were also inversely associated with hearing thresholds. The model explained 68% of variance in hearing thresholds (R2=0.68), indicating that these parameters collectively account for a substantial proportion of between-individual differences in hearing impairment within the study cohort.

Discussion

The present study demonstrates that SSNHL is associated with significant alterations in perfusion-related surrogate parameters and oxidative stress markers, providing convergent evidence from clinical observations and experimental validation. In the clinical cohort, reduced CBF and CBV together with prolonged MTT were observed in patients with SSNHL compared with healthy controls, indicating an unfavorable perfusion-related profile. Given the high metabolic demand of the cochlea and its limited collateral circulation, such alterations are plausibly linked to impaired oxygen and nutrient delivery, thereby increasing susceptibility to tissue stress and dysfunction [8,12]. Rather than implying direct cochlear blood flow measurement, the observed changes in imaging-derived parameters should be interpreted as indirect indicators of microcirculatory impairment. These alterations were accompanied by functional consequences, as reflected by elevated hearing thresholds in patients with SSNHL. Collectively, these findings support an association between perfusion-related dysfunction and hearing impairment, consistent with prior reports suggesting a vascular contribution to SSNHL pathophysiology [13,14]. In parallel, pronounced oxidative stress was evident in SSNHL. Elevated serum MDA levels together with reduced SOD and GSH-Px activities indicate an imbalance between pro-oxidant and antioxidant processes. Lipid peroxidation products such as MDA reflect excessive reactive oxygen species generation, which can disrupt cellular membranes and impair cochlear cell integrity [9,10]. The observed correlations between perfusion-related parameters and oxidative stress markers further suggest that compromised microcirculatory function is linked to an increased oxidative burden, rather than acting as an isolated mechanism. This relationship is biologically plausible, as hypoxia and ischemia–reperfusion are well-established triggers of oxidative stress across multiple tissues, including the inner ear [15]. The experimental ischemia–reperfusion model provided mechanistic support for these clinical associations. In rats, transient carotid artery occlusion resulted in increased cochlear oxidative stress, reduced antioxidant enzyme activity, and a significant rise in apoptotic cell burden. These changes were accompanied by elevated ABR thresholds, indicating functional hearing impairment. Although this model represents a global ischemic insult rather than the predominantly unilateral presentation of SSNHL in humans, it offers a controlled framework to examine oxidative injury and cochlear vulnerability under conditions of acute hypoperfusion. The concordance between human and animal findings strengthens the inference that oxidative stress and microcirculatory dysfunction are interrelated components of SSNHL-associated cochlear damage. Regression analyses further indicated that perfusion-related parameters and oxidative stress markers were independently associated with hearing thresholds, collectively explaining a substantial proportion of inter-individual variability. Importantly, these findings should be interpreted as associative rather than predictive or causal, given the case–control design. The unexplained variance highlights the likely contribution of additional factors, including genetic susceptibility, inflammatory pathways, and environmental influences, which were not captured in the present study [16,17]. From a clinical perspective, these results have potential therapeutic implications. The coexistence of perfusion-related alterations and oxidative stress suggests that combined strategies aimed at improving microcirculatory function and enhancing antioxidant defenses may be beneficial, particularly if implemented early within the narrow therapeutic window of SSNHL. Pharmacological agents that modulate microcirculation or reduce oxidative injury have been proposed in prior studies, and the present findings provide mechanistic support for their further investigation [18,19]. However, the present data do not support direct treatment recommendations and should be viewed as hypothesis-generating.

Several limitations should be acknowledged. First, although the sample size was increased beyond the minimum requirement for the primary perfusion-related outcome, it was not specifically optimized for all secondary analyses. Second, despite matching for age and sex, residual confounding by unmeasured variables cannot be excluded. Third, while the ischemia–reperfusion animal model corroborates key clinical observations, differences in cochlear physiology and the predominantly unilateral nature of human SSNHL may limit direct translational extrapolation. Fourth, the study population was restricted to adults aged 18 to 65 years and was predominantly Chinese, which can limit generalizability to other age groups or ethnic populations. Future studies incorporating longitudinal designs, broader populations, and complementary animal models are warranted to refine translational relevance.

Conclusions

This study demonstrates that sudden sensorineural hearing loss is associated with unfavorable perfusion-related surrogate parameters and increased oxidative stress, accompanied by reduced antioxidant defense capacity, as observed in both the clinical and experimental settings. The concordance between human data and animal model findings supports a close relationship between microcirculatory dysfunction, oxidative imbalance, and cochlear injury in SSNHL. Although causal inference cannot be established, these results suggest that perfusion-related impairment and oxidative stress are interrelated pathophysiological features of SSNHL. Further studies are warranted to determine whether therapeutic strategies targeting microcirculation and oxidative stress may have clinical relevance within the narrow therapeutic window of this condition.