Effects of platelet-rich plasma in experimental acute acoustic trauma

Abstract

Objectives

Acoustic trauma (AT) is a condition for which no standard treatment currently exists. Platelet-rich plasma (PRP) is a neuroregenerative agent. This prospective experimental study was performed to evaluate the effectiveness of PRP in rats subjected to AT.

Methods

Baseline auditory brainstem response (ABR) and distortion-product otoacoustic emissions (DPOAE) were measured prior to AT induction and again 3 days afterward. Following this, intratympanic PRP was administered to the treatment group (n = 10), while the control group (n = 10) received intratympanic saline. After 3 weeks, ABR and DPOAE tests were repeated and compared with the initial results.

Results

In the PRP group, significant improvements in DPOAE thresholds at 25,000 Hz and 32,000 Hz were observed between the post-trauma and post-treatment stages. No significant differences were found between baseline and post-treatment measurements. ABR thresholds in the PRP group increased significantly following trauma (P = .007) but showed no significant difference compared with baseline after treatment (P = .615). A statistically significant improvement was observed between post-trauma and post-treatment thresholds (P = .013).

Conclusion

PRP appears to contribute positively to auditory recovery following AT, likely due to its neuroregenerative properties. Nevertheless, further randomised, larger-scale studies are required to substantiate these preliminary findings.

Introduction

Noise is defined as an amplified or disruptive sound that can negatively impact human health. Among various types of hearing loss, age-related hearing loss is the most prevalent, followed closely by noise-induced hearing loss (NIHL). Hearing impairment resulting from acoustic overstimulation is typically classified into two categories: acoustic trauma (AT), which arises from brief exposure to extremely loud sound leading to sudden hearing loss, and NIHL, which develops through prolonged exposure to lower-level noise over time. ¹

The pathological process that takes place in the cochlea after AT begins through two pathways: mechanical trauma and metabolic damage. While mechanical trauma causes greater injury and loss of stereocilia, metabolic damage is primarily responsible for the loss of outer hair cells. Metabolic damage begins with vasoconstriction, triggered by activation of the sympathetic nervous system in response to noise, which reduces cochlear blood flow. It is believed that the resulting hypoxia and oxidative stress play fundamental roles in the cochlear damage that leads to NIHL. ²

In NIHL, the principal site of anatomical damage is at the level of the mechanical sensory receptors in the end organ of the auditory system. This results in significant damage to both the inner and outer hair cells of the cochlea. In the early stages, the outer hair cells are primarily affected.^3,4

To treat AT, intratympanic and systemic administration of steroids, hyperbaric oxygen, antioxidant agents, vasodilators, and vitamin complexes have been used. Although many agents and methods have been trialled for the prophylaxis and treatment of AT, no treatment modality has demonstrated proven efficacy or become routinely used.

Platelet-rich plasma (PRP), which is effective in wound healing, cell regeneration, and proliferation, has been applied in various areas of otolaryngology practice. PRP is derived from whole blood through centrifugation and contains higher concentrations of platelets and growth factors than whole blood. These properties make PRP a promising candidate for applications in tissue repair, neuroregeneration, and osteogenesis. Based on this, the present study considered that PRP may be beneficial in the treatment of AT because of its positive effects on cell healing and neuroregeneration.

Given these potential regenerative effects, PRP may hold promise as a therapeutic agent for AT. To our knowledge, its role in treating AT has not been conclusively determined. This study was performed to evaluate the potential efficacy of PRP in AT by analysing objective electrophysiological parameters in an animal model.

Materials and methods

This study was conducted following approval from the Animal Research Ethics Committee and adhered to the ethical standards outlined in the Declaration of Helsinki. All procedures were carried out in the animal research laboratory at Erciyes University. Although none of the authors are currently affiliated with Erciyes University, the study was carried out at the animal research laboratory at Erciyes University which is the only certified animal laboratory in our city. We were granted legal and scientific access to the facility under the ethical approval provided by the Erciyes University Animal Experiments Local Ethics Committee (Approval No: 22/005). This procedure is in accordance with national and institutional regulations and is commonly applied in similar preclinical studies. Efforts were made to minimise the number of animals used and to reduce their suffering in accordance with the Guide for the Care and Use of Laboratory Animals, 8th Edition. ⁵ The reporting of this study conforms to the ARRIVE 2.0 guidelines. ⁶

A total of 24 healthy Wistar albino rats, each weighing 250–300 g and aged between 4 and 8 months, were used in the study. The sample size was determined through power analysis (Supplementary Figure 1). Rats with any apparent health problems were excluded from the experiment. The animals were housed under standard laboratory conditions (21°C, 12-hour light/dark cycle) and had free access to food and water. Rats with similar physical characteristics were randomly allocated to the experimental groups. PRP was prepared from four donor rats that were not included in the experimental groups. Baseline auditory brainstem response (ABR) and distortion-product otoacoustic emissions (DPOAE) were recorded. These measurements were repeated 3 days after AT was induced. Subsequently, intratympanic PRP was administered to the treatment group (n = 10), while the control group (n = 10) received intratympanic saline. ABR and DPOAE evaluations were repeated 3 weeks after treatment and compared with the baseline results. The investigators were blinded during analysis. The animals were sedated prior to euthanasia. Following sedation, they were euthanized via cervical dislocation. ⁷ Death was confirmed by the cessation of respiration and heartbeat after cervical dislocation.

AT procedure

AT was induced in the rats using a MATLAB programme that generates a single variance sound unit. A 1–12-kHz noise was recorded onto a computer-based WAV file and amplified using a sound amplifier. Continuous noise, measured at 110 dB, was played to the animals for 12 hours, with sound levels monitored using a decibel meter.

PRP preparation and application

Initially, all rats included in the study were sedated using intramuscular ketamine hydrochloride (15 mg/kg) and xylazine hydrochloride (6 mg/kg). Four rats were used solely for PRP preparation and were not included in any experimental group. We did not use each rat's own blood to prepare PRP because the volume required could have been lethal. Moreover, because platelets do not possess antigenic properties, using PRP prepared from the blood of other rats would not pose a risk to the recipient rats. Therefore, blood was obtained from four donor rats, which were excluded from the main study, and used for PRP preparation (Figure 1).

Figure 1.

Intracardiac blood collection for PRP preparation and the PRP obtained using a gel tube.

From each of these four rats, 4 mL of intracardiac blood was drawn and placed into PRP preparation kits containing a separating gel. This gel allowed the PRP layer to form clearly after centrifugation, enabling easy collection of pure and concentrated PRP while unwanted blood components remained beneath the cell separation layer. The transfer device and Luer-lock syringe system included in the kit made PRP collection straightforward and secure. After blood collection, the samples were centrifuged at 4000 rpm for 10 minutes to separate the blood into layers of red blood cells, a buffy coat of leukocytes, and plasma. The PRP was then collected using a transfer holder. All cell collection procedures were carried out in a sterile environment, ensuring the tubes remained sealed throughout (Figure 1).

The prepared PRP was applied bilaterally to the ears of the rats in the treatment group under an operating microscope. Saline was administered intratympanically to the control group.

DPOAE test procedure

DPOAE measurements were performed using the Otodynamic ILO-288 Echoport system (Otodynamics Ltd, London, UK). All tests were conducted in a quiet room. A 1-cm plastic adapter was inserted into the external auditory canal (Figure 2). The primary tone levels were set at 80 dB SPL for both f1 and f2, with an f1/f2 ratio of 1.22 to ensure optimal response. Recordings were taken at frequencies of 1001, 1501, 2002, 3003, 4004, 6006, and 7996 Hz.

Figure 2.

DPOAE probe and ABR electrode placement and test procedure.

ABR test procedure

ABR recordings were obtained under anaesthesia using an Interacoustics system (Denmark). Subdermal silver electrodes (Technomed Europe, Netherlands) were placed at the vertex (active), the ipsilateral mastoid (reference), and the contralateral mastoid (ground) (Figure 2). ABR measurements were performed prior to noise exposure, 3 days after AT, and again 3 weeks following intratympanic treatment. These recordings were compared with baseline values to assess hearing threshold shifts.

Statistical analysis

All statistical analyses were performed using IBM SPSS Statistics version 20.0 (IBM Corp., Armonk, NY, USA). Prior to analysis, data were assessed for normality using the Shapiro–Wilk test. Data that met the assumption of a normal distribution were analysed using parametric tests, while non-normally distributed data were analysed using appropriate non-parametric methods. Variance homogeneity across groups was tested using Levene's test. Where variances were found to be unequal, appropriate corrections (e.g. Welch's analysis of variance or adjusted degrees of freedom in t-tests) were applied. The variation within each group is presented as mean ± standard deviation or median with interquartile range, depending on the data distribution. Statistical comparisons between two groups were made using the unpaired two-tailed Student's t-test (for parametric data) or the Mann–Whitney U test (for non-parametric data). For comparisons involving more than two groups, one-way analysis of variance followed by Tukey's post hoc test was used if the data were normally distributed, or the Kruskal–Wallis test followed by Dunn's multiple-comparisons test for non-parametric data. All tests were two-tailed, and a P-value of <.05 was considered statistically significant. When multiple comparisons were made, the Bonferroni correction was applied to control for type I error. Effect sizes were calculated for major comparisons to provide a measure of practical significance. The Mann–Whitney U test, cross-tabulation, and t-test were used to compare the outcomes in each group, with P-values of <.05 indicating statistical significance.

Results

Baseline ABR and DPOAE measurements were taken in both groups before the induction of AT (ABR1 and OAE1). Measurements were repeated 3 days after AT (ABR2 and OAE2) and again 3 weeks after the intratympanic injections (ABR3 and OAE3).

DPOAE

There were no significant differences in baseline DPOAE thresholds between the groups across all frequencies (P = .08, .392, .621, .675, and 1.00, respectively).

In the control group, no significant differences were observed at 25,000 Hz and 32,000 Hz across day 0, post-AT, and post-intratympanic injection measurements. However, significant differences were noted at 8000 Hz, 12,000 Hz, and 17,000 Hz across these time points. At these frequencies, significant threshold shifts were found between day 0 and post-AT measurements, but no differences were noted between post-AT and post-intratympanic injections.

In the PRP group, no significant differences were found at 17,000 Hz across day 0, post-AT, and post-intratympanic injection measurements. Significant differences were found at 8000, 12,000, 25,000, and 32,000 Hz across the same time points. At 8000 and 12,000 Hz, significant differences were detected between day 0 and post-AT, with no further differences between the post-AT and post-intratympanic injection periods. At 25,000 Hz and 32,000 Hz, significant differences were observed both between day 0 and post-AT and between post-AT and post-intratympanic injections. No significant differences were found between day 0 and the post-intratympanic injection period (Table 1).

Table 1.

Comparison of OAE results of groups.

	Grup 1± SD	Grup 2± SD	P
OAE1_8	10.56 ± 5.63	13.82 ± 2.52	.007*
OAE2_8	−0.22 ± 3.34	−2.00 ± 3.79	.295
OAE3_8	4.33 ± 3.42	2.82 ± 3.21	.295
OAE1_12	14.56 ± 1.33	14.91 ± 2.46	.412
OAE2_12	3.33 ± 4.38	2.64 ± 4.24	.656
OAE3_12	8.00 ± 4.30	2.64 ± 5.88	.031*
OAE1_17	4.78 ± 5.06	5.64 ± 6.42	.656
OAE2_17	3.11 ± 4.04	3.27 ± 5.25	.941
OAE3_17	5.56 ± 3.04	−0.18 ± 6.32	.02*
OAE1_25	11.78 ± 4.54	12.82 ± 4.19	.710
OAE2_25	16.44 ± 2.40	12.82 ± 2.13	.002*
OAE3_25	15.78 ± 2.16	15.55 ± 2.80	1.00
OAE1_32	7.78 ± 4.94	8.00 ± 7.65	1.00
OAE2_32	2.00 ± 3.60	5.55 ± 3.41	.056
OAE3_32	4.56 ± 4.21	1.27 ± 5.21	.295
ABR_1	41.67 ± 7.95	44.55 ± 6.10	.412
ABR_2	67.22 ± 9.39	66.36 ± 15.98	.656
ABR_3	65.00 ± 11.45	45.00 ± 9.48	.001*

Mann–Whitney U test.

SD: standard deviation.

*P < .05.

ABR

There were no significant differences in the baseline ABR thresholds between the groups (P = .371). Similarly, no significant difference was observed between the groups regarding ABR thresholds following the AT procedure (P = .615). However, a significant difference was found between the ABR thresholds of the two groups 3 weeks after the intratympanic injections (P = .002).

In the control group, significant differences were observed in ABR thresholds between day 0, post-AT, and post-intratympanic injection measurements (P = .003). ABR thresholds were significantly elevated both post-AT and post-intratympanic injections compared with day 0 (P = .008 and P = .015, respectively). No statistically significant difference was found between post-AT (day 3) and post-intratympanic injection (week 3) measurements (P = .599).

In the PRP group, significant differences were also noted in ABR thresholds between day 0, post-AT, and post-intratympanic injections (P = .007). ABR thresholds were significantly higher post-AT than on day 0 (P = .007). However, no significant difference was found between baseline ABR measurements and those taken after intratympanic PRP treatment (P = .615). A significant difference in ABR thresholds was observed between post-AT and post-intratympanic injection measurements (P = .013) (Tables 2 and 3).

Table 2.

Group 1 ABR values.

	N	Mean	Std. Deviation	P
ABR_1	10	41.67	7.906	<.001
ABR_2	10	67.22	9.391
ABR_3	10	65.00	11.456

Table 3.

Group 2 ABR values.

	N	Mean	Std. Deviation	P
ABR_1	10	44.55	6.105
ABR_2	10	66.36	15.983	.002
ABR_3	10	45.00	9.487

Discussion

Various animal species have been used in experimental studies of AT. In this study, we chose rat models, which are frequently used in hearing research because of their wide frequency range and sensitivity to AT.

Moreover, in experimental studies on AT, the methods used to induce AT vary and cannot be fully standardised. The noise intensity applied to create AT has ranged between 90 dB and 160 dB, while the exposure duration has varied between 15 minutes and 24 hours. In our study, we selected a noise level of 110 dB and a 12-hour exposure duration, which have been commonly used in previous research. We confirmed the induction of AT using ABR and DPOAE results.

Although hearing loss caused by AT is very common and numerous agents have been explored for its treatment and prevention, no proven or routinely used agent or method has yet been established.

In the treatment of AT, systemic and intratympanic steroids have been the most commonly investigated approaches. Steroids are frequently used to treat a range of inner ear disorders, including sudden sensorineural hearing loss, NIHL, and hearing loss caused by ototoxic drugs. In a study involving 263 military personnel with AT, Zloczower et al. ⁸ compared changes in bone and air conduction thresholds at 2–8 kHz in individuals who received steroid treatment (prednisone, 1 mg/kg, with a daily limit of 60 mg). Mutlu et al. ⁹ evaluated the protective effects of synthetic adrenocorticotropic hormone analogues and systemic steroids on rats exposed to NIHL. They reported that steroids were effective for the treatment of NIHL, and adrenocorticotropic hormone analogues also demonstrated promising therapeutic potential. ⁹ Gumrukcu et al. ¹⁰ investigated the impact of intratympanic steroid therapy on hearing in rats by assessing otoacoustic emissions and found that dexamethasone had a beneficial effect on NIHL. By contrast, Mamelle et al. ¹¹ examined hearing outcomes following the administration of a hyaluronic acid gel containing liposome-encapsulated dexamethasone into the middle ear after AT. They found that local delivery of dexamethasone 48 hours after mild AT did not enhance hearing recovery, despite the use of a sustained-release gel specifically designed for inner ear treatment. ¹¹ The main therapeutic effect of corticosteroids is thought to stem from their ability to reduce autoimmune inflammation. In the present study, we used PRP, which also possesses anti-inflammatory properties, and found that it contributed to improved hearing recovery.

Other agents investigated for the treatment of AT include antioxidants such as N-acetyl cysteine, carnitine, edaravone, coenzyme Q, and vitamins A, C, and E.^12–16 PRP also possesses antioxidative effects, which may have contributed to its healing effect on AT in this study.

In addition, various neuroregenerative agents have been explored as potential treatments for AT. Wan et al. applied neurotrophin-3 following AT and observed enhanced recovery of cochlear function along with the regeneration of ribbon synapses. ¹⁷ Similarly, Inaoka et al. administered hepatocyte growth factor locally after AT and demonstrated that this treatment significantly reduced noise-induced ABR threshold shifts and prevented the loss of outer hair cells in the basal region of the cochlea. ¹⁸ Lin et al. also reported positive outcomes using insulin-like growth factor-1 for AT therapy. ¹⁹ In the present study, we propose that PRP, known for its neuroregenerative properties in various tissues, may likewise offer therapeutic benefits following AT.

PRP contains hyperphysiological levels of growth factors due to its high platelet concentration and is known for its beneficial effects on healing. In the field of otology, its neuroregenerative effects have previously been investigated, and in the present study, we explored its effect on AT based on these positive findings. Stolle et al. ²⁰ conducted culture studies using the spiral ganglia of newborn rats and observed that PRP significantly increased neuronal survival and neuronal growth. Yurtsever et al. ²¹ examined the impact of intratympanic PRP treatment on cisplatin-induced ototoxicity in rats and reported that both histopathological findings and ABR results were better in the PRP group following intratympanic application (Table 4). Additionally, studies have shown that intratympanic application of PRP is beneficial in cases of sudden idiopathic hearing loss. ²²

Table 4.

A comparison between the study investigating the effect of PRP on ototoxicity and our own findings.

Features	Yurtsever et al.	Our study
Study design	Controlled experimental study on animals	Controlled experimental study on animals
Model	Cisplatin-induced ototoxicity model in rats	Acoustic trauma model in rats
Sample size	Eight female rats (4 control, 4 PRP)	24 female rats (10 control, 10 PRP, 4 for PRP preparation)
PRP protocol	Intratympanic PRP injection, single dose	Intratympanic PRP injection, single dose
Evaluation methods	Histopathology, auditory brainstem response (ABR)	Auditory brainstem response (ABR), distortion-product otoacoustic emissions (DPOAE)
Follow-up duration	3 weeks	3 weeks
Findings	PRP reduced damage to outer hair cells and desquamation and erosion in vessels in the stria vascularis, improvement in ABR thresholds	Improvement in ABR and DPOAE thresholds
Conclusion	PRP may exert a protective effect against ototoxicity	PRP may have a potential therapeutic effect in cases of acoustic trauma

📌 “Although both studies indicate the potential beneficial effects of PRP on hearing, Yurtsever et al. demonstrated this effect using an ototoxicity model, while our study shows its possible efficacy in an acoustic trauma model.”

In our study, PRP had to be administered to the rats under general anaesthesia, and because we needed to sacrifice four rats each time to prepare PRP, we were limited to a single-dose application. Although we consider this a limitation of our study, there are animal studies in the literature that have also used PRP in a single dose – likely for similar reasons – and have reported positive effects on nerve healing.^21,23,24

In the present study, intratympanic application of PRP after AT appeared to have a positive effect on hearing outcomes, even with a single dose. It is possible that this positive effect could be enhanced with repeated administrations.

The limitations of our study include the necessity of applying only a single dose of PRP. While studies, including ours, have reported the effectiveness of a single dose, repeated applications may lead to increased therapeutic efficacy. Other limitations were the inability to perform long-term follow-up measurements and the absence of histopathological confirmation of the functional outcomes.

Conclusion

In our study, hearing function was observed to be better in the group of rats exposed to AT and treated with intratympanic PRP than in the control group. These findings suggest that intratympanic PRP may have potential therapeutic value in cases of AT. However, larger prospective, randomised studies are required to confirm its beneficial effects.