INTRODUCTION
Sensorineural hearing loss (SNHL) arises from various etiologies, including aging, noise exposure, genetic factors, infections, tumors, trauma, ototoxic drugs, autoimmune diseases, and Meniere’s disease [1-3]. Hearing loss is an independent risk factor for dementia, particularly among older adults [4-6]. Furthermore, hearing impairments are associated with depression, especially among women, and adversely affect socioeconomic status in terms of income, employment, and education [7,8]. Despite the rising incidence of hearing loss, no Food and Drug Administration–approved drug treatment currently exists. For sudden SNHL, the primary treatment options—oral steroids or intratympanic steroid injections—aim to produce an anti-inflammatory effect [9-11].
Auditory rehabilitation methods, such as hearing aids and cochlear implantation (CI), are also widely used for patients with SNHL. CI is increasingly utilized in cases of single-sided deafness and tinnitus, where it significantly suppresses symptoms. These broadened indications highlight the recognition of CI benefits beyond traditional severe-to-profound bilateral hearing loss [12,13].
In CI surgery, preserving residual hearing is critical, particularly for patients with functional low-frequency hearing, as an electro-acoustic stimulation (EAS) system can deliver a more natural hearing experience by combining high-frequency electrical stimulation with low-frequency acoustic amplification [14-16]. Various strategies have been developed to enhance hearing preservation. Soft surgical techniques—such as slow electrode insertion with steroid use—aim to minimize cochlear trauma and reduce inflammation. In addition, advances in thin, flexible electrode designs reduce insertion force and, consequently, the risk of cochlear damage. Together, these approaches significantly improve postoperative preservation of residual hearing [17-19].
For pharmacological interventions in hearing preservation during CI surgery, anti-inflammatory steroids have been administered using various methods, including osmotic pumps. Optimal hearing preservation can be achieved by delivering pharmacological agents at precise concentrations and dosages using osmotic pump techniques [20,21]. Moreover, animal studies of dexamethasone-eluting electrodes have shown promising results for preserving residual hearing after CI, although evidence from human studies remains limited [22,23]. Inflammation can lead to undesirable tissue growth near the electrode, making it a key target for pharmacological intervention. Recent advances have resulted in several drugs that effectively control such tissue proliferation. One of these is sarpogrelate, which is reported to modulate vascular sclerosis and blood viscosity [24]. Additionally, sarpogrelate has demonstrated anti-inflammatory effects in diabetic nephropathy model mice [25].
In this study, an animal model simulating the damaging effects of CI was employed using a dummy electrode. To protect the animals’ residual hearing, sarpogrelate was administered via an osmotic pump. The effects of sarpogrelate on residual hearing preservation were then investigated through functional and histological assessments.
MATERIALS AND METHODS
All procedures involving animals were approved by the Institutional Animal Care and Use Committee of Ajou University Graduate School of Medicine, Suwon, Republic of Korea (AUMC-IACUC; No. 2018-0005).
Cell culture and viability
House Ear Institute-Organ of Corti 1 (HEI-OC1) cells were cultured at 37 °C under 5% CO2 in high-glucose Dulbecco’s Modified Eagle’s Medium (11965092, Gibco; Thermo Fisher Scientific Inc.). A water-soluble tetrazolium salt (WST-1; Cayman Chemical) assay was used to analyze cell viability. HEI-OC1 cells were cultured in 96-well plates at a density of 5×103 cells/well for 24 hours. Subsequently, the cells were treated with lipopolysaccharide (LPS; 1,000 ng/mL) and sarpogrelate at various concentrations (0, 0.1, 1, 5, 10, and 25 μM) and were incubated for 24 hours (Fig. 1A). Cell viability was assessed by incubating the plate for 2 hours with WST-1, following the manufacturer’s instructions. Absorbance was recorded at 450 nm using an iMark Microplate Reader (Bio-Rad), and the results were normalized to those of control cells. The data were normalized to those of the untreated control group. Each assay was conducted in triplicate.
Animals and auditory brainstem response
Five-week-old male albino guinea pigs were used in the present study. All animals were maintained in an environment with controlled temperature (24 °C) and humidity (40%–45%). The lighting was cycled every 12 hours to maintain the circadian rhythm. This study was approved by the Institutional Animal Care and Use Committee. Guinea pigs were anesthetized via intramuscular injection with a mixture of Zoletil 50 (tiletamine-zolazepam, 40 mg/kg; Virbac Animal Health) and Rompun (xylazine, 10 mg/kg; Bayer Health Care). Needle electrodes were positioned subcutaneously at the vertex and beneath the pinnae of both ears to measure hearing thresholds. The thresholds were recorded at frequencies of 8, 16, and 32 kHz using tone-burst stimuli with sound levels ranging from 10 to 90 dB in a soundproof chamber. Auditory brainstem responses (ABRs) were measured before and 1, 7, and 30 days after dummy electrode insertion surgery.
Dummy electrode and pump insertion surgery
Twenty-five animals were used in this study. The animals were divided into three groups: control (dummy electrode insertion only, n=12), SPG-1004 (dummy electrode plus low-capacity pump with sarpogrelate, n=7), and SPG-2004 (dummy electrode plus high-capacity pump with sarpogrelate, n=6). Pumps (models 1004 and 2004; Alzet Osmotic Pump, Alza Corp.) were used to deliver sarpogrelate. The low-capacity pump (model 1004) administered 100 μL of sarpogrelate over a 4-week period, corresponding to a delivery rate of 0.11 μL/hr. Conversely, the high-capacity pump (model 2004) dispensed 200 μL of sarpogrelate over the same duration, achieving a delivery rate of 0.25 μL/hr.
A retroauricular incision was made through the bullae under aseptic conditions, to visualize the round window of the cochlea. The round window niche, which obscured visualization of the round window membrane, was partially removed. After incising the round window membrane, an electrode with a pump or a dummy electrode (manufactured by the Daegu Gyeongbuk Institute of Science and Technology; shaft diameter: 0.64 mm, length: 5 mm, tip diameter: 0.43 mm), composed of silicone rubber, was slowly inserted into the round window. In the control group, only the dummy electrode was positioned, and the round window was sealed with a soft tissue graft and muscle. Muscle closure and skin sutures were then performed. In the experimental group that underwent surgery with the dummy electrode and pump, the pump was positioned in the subcutaneous layer of the animal’s dorsal area. A microcatheter connected to the pump was inserted through the dorsal subcutaneous tunnel into the bullotomy site via dissection. The catheter tip was positioned to reach the incised round window membrane. To prevent movement, the bulla dead space was filled with soft tissue, such as muscle. Additionally, to minimize movement, the catheter was secured to the muscle tissue outside the bulla using a tagging suture. The drug was administered from the pump through the catheter and electrode, then delivered into the cochlea via the round window, reaching the scala tympani where the electrode is located.
Reverse transcription polymerase chain reaction
Animals were sacrificed 7 and 30 days after electrode insertion, and the cochleae were harvested for reverse transcription polymerase chain reaction (RT-PCR). Total RNA was extracted from cochlea using RNAiso Plus (9108, TaKaRa). For the synthesis of cDNA, 1.0 μg of RNA was reverse transcribed using the PrimeScript 1st Strand cDNA Synthesis Kit (6110A, TaKaRa). The synthesized cDNA was amplified using a CFX96 Real-Time PCR Cycler (Bio-Rad) with specific primers and SYBR Green PCR Master Mix (NanoHelix Co., Ltd.). The PCR protocol involved an initial activation at 95 °C for 15 minutes, followed by 35 cycles of denaturation at 95 °C for 20 seconds, and annealing/extension at 60 °C for 30 seconds. The primer sequences used for RT-PCR are listed in Table 1.
Hematoxylin and eosin staining
The harvested cochleae were first fixed in 4% paraformaldehyde and then stored overnight at 4 °C. Subsequently, they were washed with phosphate-buffered saline and decalcified in Calci-Clear Rapid Decalcifying Solution (HS-105; National Diagnostics) for 3 days. After decalcification, the cochlea were embedded in paraffin using an automated tissue processor. The paraffin blocks were sectioned into 3.5-μm-thick slices, which were subsequently deparaffinized with xylene. Sections were rehydrated in ethanol washes, and the slides were stained with hematoxylin and eosin (H&E) for histological analysis. The slides were observed under a microscope (Olympus Optical BX51), and the tissue reaction within the scala tympani was quantified using ImageJ software (National Institutes of Health).
Statistical analyses
Significant differences between two groups were analyzed using an independent t-test, and more than two groups were compared using one-way analysis of variance, with Tukey’s post-hoc test. Data are presented as means±standard errors of the mean. The data were analyzed using SPSS Statistics for Windows version 23.0 (IBM Corp.). Statistical significance was set at P-value <0.05.
RESULTS
Sarpogrelate effects on cell viability in LPS-treated HEI-OC1 cells
To assess the impact of sarpogrelate on HEI-OC1 cell viability, cells were treated with various concentrations of sarpogrelate (0.1, 1, 5, 10, and 25 μM) in the absence of LPS. As shown in Fig. 1B, no significant reduction in cell viability was observed at any concentration, indicating that sarpogrelate had no adverse effects even at higher doses.
To mimic the inflammation that develops after CI, HEI-OC1 cells were pretreated with sarpogrelate (0, 0.1, 1, 5, 10, and 25 μM) for 1 hour, followed by treatment with LPS (1,000 ng/mL). Cell viability was assessed after 24 hours (Fig. 1A). LPS treatment significantly reduced cell viability compared with the control group (P<0.001). While low doses of sarpogrelate had little effect, pretreatment with higher concentrations resulted in a dose-dependent recovery of cell viability. In particular, 25 μM sarpogrelate significantly improved cell viability relative to LPS treatment alone, restoring viability to levels comparable to the untreated control group. These results suggest that sarpogrelate effectively mitigates LPS-induced cytotoxicity in HEI-OC1 cells in a dose-dependent manner (Fig. 1C).
Audiologic outcomes of animals treated with sarpogrelate delivered by osmotic pump after electrode insertion
To evaluate audiological outcomes, ABR thresholds at 8, 16, and 32 kHz were measured preoperatively and on days 1, 7, and 30 after electrode insertion via a round window approach (Fig. 2A and B). Hearing threshold shifts were compared among the control group (dummy electrode insertion only) and groups receiving electrode insertion combined with either low-capacity (model 1004) or high-capacity (model 2004) pumps positioned subcutaneously (Fig. 2C-G).
One day after electrode insertion, no significant differences in ABR thresholds were observed among groups at 8, 16, or 32 kHz (Fig. 3A). The threshold shifts were comparable across the control, SPG-1004, and SPG-2004 groups, indicating that sarpogrelate administration had no immediate effect on hearing preservation. At 1 week, similar findings were observed; threshold shifts remained stable across all groups (Fig. 3B). The hearing thresholds remained relatively stable across all groups. In the control group, threshold shifts increased over time, indicating worsening hearing loss. Specifically, at 1 day the shifts were 20.0±18.83 (8 kHz), 33.75±22.88 (16 kHz), and 33.33±22.60 (32 kHz); by 1 week they had increased to 26.25±22.97 (8 kHz), 35.42±17.64 (16 kHz), and 36.67±18.87 (32 kHz); and by 1 month they reached 39.38±19.90 (8 kHz), 48.13±15.57 (16 kHz), and 50.0±16.90 (32 kHz).
In contrast, sarpogrelate administered via an osmotic pump resulted in a decreasing trend in threshold shifts over time. Notably, in the SPG-1004 group a significant reduction in threshold shift at 8 kHz was observed between 1 day and 1 month post-surgery (P=0.039). Both the SPG-1004 and SPG-2004 groups exhibited significantly smaller threshold shifts than the control group, indicating a protective effect on hearing over time. At 1 month, the SPG-1004 group had an 8 kHz threshold shift of 11.0±10.25 dB compared with 39.38±19.90 dB in the control group (P=0.003). Although the SPG-2004 group showed a reduction (P=0.073), the difference did not reach statistical significance. At 16 kHz, both the SPG-1004 (13.0±13.04 dB, P=0.002) and SPG-2004 (16.25±14.93 dB, P=0.008) groups demonstrated significantly lower shifts compared to the control group (48.12± 15.57 dB). At 32 kHz, the control group shift was 50.0±16.90 dB, whereas the SPG-1004 group shift was significantly lower at 18.0±12.55 dB (P=0.003); the SPG-2004 group again showed a reduction (20.0±21.60 dB, P=0.073) that did not achieve statistical significance (Fig. 3C). These results indicate that by 1 month post-surgery both sarpogrelate-treated groups demonstrated significant hearing preservation across all frequencies compared to the control group, with the pump volume and delivery rate having minimal impact on the overall outcome.
Histological analysis following sarpogrelate administration
For histological analysis, cochlear tissues were harvested 1 month after the experimental period and subjected to H&E staining, comparing samples based on drug administration. Extensive inflammation and fibrosis were observed in the tympanic cavity region of the basal turn in samples obtained 1 month after electrode insertion (Fig. 4A-C). The control group (dummy electrode only) exhibited 38.0%±9.6% inflammatory cell infiltration and fibrosis, compared with 22.4%±19.0% in the SPG-1004 group (not statistically significant) and 3.2%±2.1% in the SPG-2004 group (P<0.01) (Fig. 4D). These results indicate that the experimental groups, particularly the SPG-2004 group, exhibited significantly reduced tissue reactions relative to controls. In comparison to the ABR outcomes, the concentration-dependent effect of sarpogrelate was even more pronounced in reducing tissue reactions.
Effects of sarpogrelate on inflammatory gene expression
To elucidate the mechanisms underlying these effects, the expression of several inflammation-related genes—Il1b, Il6, Nos, and Tnfa—was evaluated. Baseline values were established using a normal-hearing group that did not undergo surgery. One week after pump administration, Il1b expression in the control group was elevated by 5.88-fold relative to the normal ear, whereas this increase was attenuated to 1.9-fold in the SPG-2004 group. After 1 month, Il1b expression in the control group increased to 9.81-fold, while the SPG-1004 and SPG-2004 groups showed significantly lower levels (3.50-fold and 3.07-fold, respectively), indicating notable suppression of Il1b upregulation (Fig. 5A). Therefore, a higher drug dose was initially effective; although similar effects were observed later. For Il6, expression increased by 3.74-fold in the control group relative to the normal ear; however, at 1 week no significant reduction was observed in the SPG-1004 (1.13-fold) or SPG-2004 (2.81-fold) groups. At 1 month, Il6 expression in the control group further increased to 4.97-fold, while both treatment groups exhibited significant reductions (1.09-fold for SPG-1004 and 1.24-fold for SPG-2004) (Fig. 5B). For Nos, expression was elevated by 2.71-fold in the control group at 1 week, but this increase was significantly attenuated in both the SPG-1004 (1.04-fold) and SPG-2004 (1.15-fold) groups; at 1 month, the reduction persisted in the SPG-1004 group (1.81-fold), whereas the SPG-2004 group showed a partial rebound (2.66-fold) (Fig. 5C). Regarding Tnfa, expression increased 4.12-fold in the control group at 1 week, with no significant suppression in the SPG-1004 (2.95-fold) or SPG-2004 (1.92-fold) groups. However, by 1 month, Tnfa levels in the control group surged to 9.65-fold, while both treatment groups exhibited significant reductions (1.53-fold for SPG-1004 and 2.54-fold for SPG-2004), indicating effective downregulation in response to treatment (Fig. 5D). Over time, the control group showed progressive increases in inflammation-related gene expression, while sarpogrelate administration mitigated this response.
DISCUSSION
Although CI can restore hearing in patients with hearing loss, preserving residual hearing is critical for ensuring optimal benefit from EAS. Previous animal and clinical studies have identified factors affecting residual hearing after CI, including surgical technique, steroid use, and the type (length and shape) of the implanted electrode [26-29]. Various pharmacological approaches have been explored, with systemic steroid administration before and after surgery and intraoperative steroid irrigation being the most established [30].
Several studies have reported tissue reactions in the cochlea following CI. Nadol and Eddington [31] described robust fibrous and bony tissue responses at the cochleostomy site and within the cochlea in 21 human specimens, with inflammatory responses—including infiltration of mononuclear leukocytes, histiocytes, and foreign-body giant cells—observed in 12 specimens. Chronic local inflammation accompanied by fibrosis and new bone growth after CI may result from delayed hypersensitivity or a localized tissue response to the electrode array [32]. These changes are significant because they can affect postoperative performance. In later stages, the host response to the electrode array’s biomaterials triggers a foreign-body reaction characterized by intense inflammation, macrophage activation, and fibroblast migration, eventually forming a fibrous capsule around the implant [32,33]. Intracochlear fibrosis following CI can even contribute to soft failure mechanisms leading to device malfunction [34].
Sarpogrelate, the drug employed in this study, is a selective 5-hydroxytryptamine 2A receptor antagonist with antiplatelet, antithrombotic, and antiatherosclerotic properties. Its effects have primarily been studied in peripheral arterial diseases, where it has been effective in reducing fibrosis and improving blood flow [35,36]. Because fibrosis is largely driven by the transforming growth factor-beta (TGF-β) signaling pathway, sarpogrelate’s ability to inhibit TGF-β expression or activity may reduce fibroblast activation and subsequent fibrosis [35]. Its role in modulating inflammatory responses has also been investigated; for example, in a cirrhotic animal model, sarpogrelate treatment reduced inflammatory cell infiltration compared to controls [37].
The inflammation induced by LPS and CI arises from distinct mechanisms. CI triggers a foreign-body reaction driven by inflammation and tissue remodeling, involving upregulation of cochlear inflammatory genes (Cxcl1, IL-1β, TNF-α, Tnfrsf1a/b) as well as key tissue remodeling genes such as TGF-β, MMP2, and MMP9 [38]. In studies modeling LPS-induced inflammation in HEI-OC1 cells, LPS not only upregulated TLR4 expression but also promoted the production of pro-inflammatory cytokines such as TNF-α, interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1) [39,40]. Many of these cytokines are also elevated during the foreign-body reaction, indicating a partial overlap in the inflammatory pathways. To further investigate these mechanisms, an in vitro model employing LPS—a well-established inducer of inflammation—was used. Additionally, LPS stimulation commonly leads to prostaglandin production (e.g., PGD2 and PGE2), which can induce macrophage migration and activation; this inflammatory response was inhibited by sarpogrelate [25]. Nevertheless, studies on sarpogrelate’s effects on the inner ear are limited. When Choo et al. [41] investigated its potential benefits in aging mice, no significant improvements in hearing outcomes were observed. In our study, pretreatment of HEI-OC1 cells with sarpogrelate, followed by LPS-induced inflammation, resulted in a significant, concentration-dependent increase in cell viability compared to untreated cells, thereby confirming its anti-inflammatory effect.
To determine whether these effects extended to both fibrosis and inflammation after CI surgery, an osmotic pump was employed for drug delivery. Osmotic pumps can administer pharmacological agents at varying concentrations and durations. Since the inflammatory process initiated by electrode placement within the cochlear scala tympani progresses gradually over time [32], drug delivery during CI primarily influences the initial phase of inflammation, with diminished effects in later stages. Previous studies in CI animal models have shown that continuous topical steroid delivery via an osmotic pump can preserve hearing [20,21]. In our study, sarpogrelate not only preserved hearing thresholds but also yielded positive histological outcomes.
The inflammatory response to the electrode is most pronounced at the basal turn of the cochlea near the cochleostomy site—likely due to surgical trauma during insertion triggering a cascade of inflammatory effects—although similar histologic changes have been observed in the scala vestibuli [32]. The extent of this response varies among patients, with some experiencing only mild fibrosis and others developing significant foreign body granulomas and new bone formation [34]. Factors such as electrode insertion depth, surgical approach (cochleostomy versus various round window techniques), insertion technique, and electrode type may all contribute to these differences [42]. In this study, the catheter was positioned near the round window membrane to administer the drug via a pump, and this placement may have contributed to increased fibrotic and inflammatory changes. Our previous study demonstrated that PBS infusion via a pump elevated IL-6 and TNF levels, supporting the notion that the pump system itself can influence the inflammatory response [43]. Furthermore, the anti-inflammatory effects of sarpogrelate observed in diabetic nephropathy were more pronounced in diabetic mice compared to normal mice [25], suggesting that its impact on CI in diabetic conditions merits further investigation.
At 1 month, a significant reduction in ABR threshold shifts was observed in the sarpogrelate-treated groups compared to controls, with no notable differences related to pump volume. This finding suggests that sarpogrelate protects the cochlea against delayed inflammatory changes rather than the immediate postoperative inflammatory response. Similarly, Eshraghi et al. [44] showed in a noise-exposed guinea pig model that animals implanted with dexamethasone-eluting electrodes had consistently smaller threshold shifts than those without steroid exposure, with significant differences emerging from day 3 and becoming more pronounced by day 30.
This hearing preservation effect was further supported by the reduced expression of genes encoding inflammatory markers, which are typically elevated during CI procedures [45-47]. In our study, inflammation-related genes (Il1b, Il6, Nos, and Tnfa) increased significantly from 1 week to 1 month after electrode insertion in controls; however, sarpogrelate administration via a pump resulted in a more effective reduction in their expression, correlating with improved hearing preservation. Even in uncomplicated CI cases, the implant can induce a foreign body reaction in the inner ear, with both acute and delayed components. The delayed component—stemming from the reaction to the electrode array’s biomaterials—involves strong inflammatory responses, macrophage activation, and fibroblast migration, ultimately leading to fibrous capsule formation [33]. TGF-β, which is upregulated in fibrotic disease, regulates fibroblast growth factors (FGFs) and fibroblast growth factor receptors (FGFRs), influencing extracellular matrix remodeling by controlling bone resorption and formation through osteoblasts while also stimulating IL-6 and MMPs [48]. Therefore, IL-6 may also increase in a delayed phase, and the anti-fibrotic effect of sarpogrelate delivery may have contributed to its reduction. Additionally, TNFα binding to its receptors (TNFαR1a and TNFαR1b) can trigger signaling pathways that damage cochlear sensory cells [49]. After electrode insertion, inflammation- and fibrosis-associated genes such as Tnfa and Tgfb tend to increase over time [50]; electrode insertion trauma leads to significant upregulation of genes like Tnfa, Tnfr1a, and Tgfbr1 within 24 hours, with even greater increases after 7 days—a response mitigated by dexamethasone-eluting electrodes [44]. Our study suggests that managing delayed or chronic inflammatory and fibrotic responses with agents such as sarpogrelate is crucial for hearing preservation.
This delayed but significant effect underscores the potential utility of administering sarpogrelate via an osmotic pump as a long-term strategy for hearing preservation after CI. In our previous work, topical steroid delivery via an osmotic pump reduced scala tympani inflammation and produced significant hearing improvements compared with systemic dexamethasone [20,21]. The present results indicate that sarpogrelate not only reduces inflammatory and fibrotic responses but also improves hearing preservation, suggesting it may serve as a viable alternative to steroids in CI outcomes.
A limitation of this study is the difficulty in obtaining a large sample size in guinea pig experiments. Small sample sizes can reduce statistical power and limit the ability to account for confounding factors such as biological variability, surgical technique, or individual immune responses. Future studies should aim to increase sample sizes and incorporate additional experimental replicates, including evaluations of different drug concentrations delivered via a pump. Moreover, inherent differences between guinea pigs and humans—such as cochlear anatomy—must be considered, meaning these results represent only one factor among many influencing hearing preservation after CI.
In summary, this study revealed the beneficial effects of administering sarpogrelate in the inner ear via an osmotic pump during CI, which has not been reported previously. Nevertheless, although this study provides valuable insights into the effects of sarpogrelate on hearing preservation after CI, it has some limitations. The animal model used herein may not have fully replicated the complexities of human cochlear anatomy and immune responses. Further studies, including long-term human trials, are necessary to confirm these effects and to determine the optimal administration protocols, including the most effective dosage and delivery methods.
Sarpogrelate delivered via an osmotic pump effectively preserved residual hearing after simulated CI in an animal model, likely due to its anti-inflammatory and antifibrotic properties. This approach shows promise for improving postoperative outcomes in patients undergoing CI.
HIGHLIGHTS
▪ Sarpogrelate delivered via an osmotic pump significantly preserved residual hearing following cochlear implantation in guinea pigs.
▪ Sarpogrelate demonstrated anti-inflammatory and antifibrotic effects by reducing cochlear fibrosis and inflammatory cell infiltration in treated animals compared to controls.
▪ Continuous, controlled drug delivery via osmotic pumps enabled sustained release of sarpogrelate, resulting in effective hearing preservation over time post-surgery.
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