Changes of serum levels of Caspase-3 after trauma and ototoxic damage of the cochlea in rabbits: An in vivo study

Pavlos Pavlidis; Haralampos Gouveris; Vasileios Nikolaidis; Gregor Alexander Schittek

doi:10.4103/indianjotol.indianjotol_29_22

ORIGINAL ARTICLE

Year : 2022 | Volume : 28 | Issue : 1 | Page : 52-56

Changes of serum levels of Caspase-3 after trauma and ototoxic damage of the cochlea in rabbits: An in vivo study

Pavlos Pavlidis¹, Haralampos Gouveris¹, Vasileios Nikolaidis², Gregor Alexander Schittek³
¹ ENT Clinic, University Hospital Mainz, Mainz, Germany
² 2nd ENT-Department of Aristotle University Thessaloniki, Thessaloniki, Greece
³ Department for Anaesthisiology and Intensive Care, Medical University of Graz, Graz, Austria

Date of Submission	13-Feb-2022
Date of Acceptance	25-Mar-2022
Date of Web Publication	25-Apr-2022

Correspondence Address:
Dr. Pavlos Pavlidis
ENT Clinic, University Hospital Mainz, Mainz
Germany

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/indianjotol.indianjotol_29_22

Abstract

Background: Caspase-3 is one of the most important enzymes for the regulation of apoptosis. Aims and Objectives: Aim of our study was to examine the changes in serum levels of this factor during apoptotic phenomena in the cochlea, caused by traumatic or ototoxic causes. Materials and Methods: A cohort of 24 rabbits was studied for this purpose for 31 days. Eight animals were implanted with a cochlear implant electrode (group A), 8 were treated with intramuscular amikacin for 14 days (group B) and another 8 were the control group (group C). In all groups DPOAEs and serum levels of caspase-3 were tested every second day. Results: Serum levels of caspase-3 rise immediately after implantation, while a 3-day latency in levels was seen in group B. Caspase-3-levels in both groups remained elevated until the 31st day of the experiment. Levels of caspase-3 showed a moderate negative correlation with DPAOE amplitudes. Conclusion: Caspase-3 rises after traumatic and ototoxic causes and moderately correlates with cochlear outer hair cell function in rabbits. Therefore, serum caspase-3 levels should be tested as a surrogate marker of structural integrity of the cochlea after trauma or administration of ototoxic drugs in humans as well.

Keywords: Apoptosis, biomarker, Caspases-3, cochlear implant, distortion products otoacoustic emissions, ototoxicity

How to cite this article:
Pavlidis P, Gouveris H, Nikolaidis V, Schittek GA. Changes of serum levels of Caspase-3 after trauma and ototoxic damage of the cochlea in rabbits: An in vivo study. Indian J Otol 2022;28:52-6

How to cite this URL:
Pavlidis P, Gouveris H, Nikolaidis V, Schittek GA. Changes of serum levels of Caspase-3 after trauma and ototoxic damage of the cochlea in rabbits: An in vivo study. Indian J Otol [serial online] 2022 [cited 2022 May 27];28:52-6. Available from: https://www.indianjotol.org/text.asp?2022/28/1/52/343764

Introduction

Outer ear hair cells (HCs) are the mechanosensory cells essential for hearing function. As mammalian HCs do not regenerate, damage, or loss of HCs leads to permanent hearing impairment. One common cause of HC death is exposure to aminoglycoside antibiotics.^[1],[2] As a result, up to 25% of patients treated with aminoglycosides develop irreversible sensorineural hearing loss.^[3],[4] Another cause of HC death is the trauma of cochlea.^{[1],[2],[3],[4]} Cell death with autophagy is typically characterized by engulfment of cellular contents in autophagosomes inside the cytoplasm.^[5],[6]

Apoptosis is regulated by a family of enzymes called caspases. In humans, thirteen members of this family have been isolated and identified.^{[4],[5],[6],[7],[8]} Several signaling cascades are activated following an insult to the cochlea; these pathways can be pro-inflammatory, pro-death, and even pro-survival. The signaling cascades that occur on a cellular and a molecular level are highly complex and, in many ways, entwine; there is significant cross-communication between pathways, and it is the culmination of all of these activities that tilt the pendulum of cell survival and cell death in one direction or the other.^{[7],[8],[10],[12]} These enzymes are divided into initiators, effectors, and inflammatory. To the first group belong Caspases 8, 9, and 10 and to the second Caspases 3, 6, and 7.^{[7],[8],[9],[10],[11],[12]}

The primary goal of the present study was to examine the role of Caspase-3 as a surrogate marker of structural integrity after chemical or mechanical damage to the cochlea in a rabbit animal model. The secondary aim of the study was to correlate any functional changes in Distortion Products Otoacoustic Emissions (DPOAEs) to elevated titers of Caspase-3, which activates the apoptotic phenomena after cochlea-implantation and therapy with ototoxic antibiotics (amikacin). The third goal was to examine the time course of any changes in the concentration of Caspase-3 after cochlear implantation and amikacin administration.

Materials and Methods

The whole experimental protocol was organized according to the STROBE-Guidelines. After approval by the Animal Care and Use Committee of the National Veterinary Institute (Nr 186/27052005) and the Ethical Committee of the School of Medicine, Aristotle University of Thessaloniki, this study was conducted in accordance with the European Communities Council Directive of November 24, 1986 (86/609/EEC). All relevant protocols regarding care of animals during experimentation have been applied and observed. Every conceivable care has been taken to minimize pain and discomfort to the animals throughout the study.

Animals

Twenty-four rabbits, 1-month-old, New Zeeland type rabbits, weighting 450–650 g, were divided into three Groups of 8 members each (Group A, Group B and Group C). The animals were studied prospectively for 31 days. None of the rabbits had been used in another study and all had been grown under similar conditions. All experiments were done in the same room under low-noise conditions and constant room temperature and environment. The rabbits were also checked for parasitological diseases of their ears, before the beginning of the experiment and during the study. The parasitological control showed no signs of disease. Initially, the cochlear activity of the animals was measured using DPOAEs in a sound-proof booth. There were no animals with abnormal pretreatment cochlear activity as recorded by DPOAE.

The first 8 rabbits (rabbits Nr. 1–8-Group A) were implanted in one of their ears a cochlear-implant electrode (Med-el, Combi-40). The animals of this group were anesthetized and for this purpose, they were injected intramuscularly with 5 mg/Kg of Xylazine (Xylapan) and 35 mg/kg of Ketamine (Ketalar). Eight animals were injected intramuscularly with 15 mg/kg/24 h (once per day) of amikacin (rabbits 9–16-Group B). The dosage approved for veterinary purposes is 8–16 mg/kg/24 h for intramuscular use. The medication was administered every 24 h for 14 days. The remaining 8 were used as the control group (Group C).

We consider as “day 0” the day on which the electrode was implanted and the day on which the administration of amikacin began. The cochlear outer hair function of all rabbits was examined every 2 days with DPOAEs for the next 10 days after the implantation. The measurements were conducted in a soundproof cabin and the animals were not anesthetized to avoid any side-effect.^{[8],[15],[21],[24]} The 2f1/f2 (65/55 Hz) protocol for the DPOAEs was used.

No rabbit was decapitated after the end of the experiment. No rabbit has been reused in another experiment.

Blood samples

Blood samples were received on days 1, 3, 5, 7, 10, 14, and 31 of the experiment. We used Caspase-3 Colorimetric Assay Kit (Assay Designs Company, USA). The procedure was based on the centrifugation of rabbits' blood cells and serum. The outcome of the centrifugation was examined for possible action of the protease with the addition of a specific for the caspases peptide which combines with p-nitroaniline (pNA). The decomposition of the peptide by Caspases-3 releases the molecule of pNA which was quantitative analyzed with spectrometry at 405 nm wavelength. Increased levels of Caspases-3 are considered these in which the value is >400 U/ml.

DPOAEs

Prior to testing, acoustic calibration was done. Following the appropriate configuration of stimuli waveform, DPOAEs (GSI 60, Grason-Stadler) were measured in diagnostic mode by giving nonlinear clicks. The ratio of f2/f1 was kept at 1.22. Stimuli intensities were L1 and L2 for f1 and f2 frequencies, respectively, and L1–L2 was kept at 10 dB SPL (L1 = 65 dB SPL, L2 = 55 dB SPL), as it has been proposed in previous studies.^{[21],[22],[23]} DPOAEs were created by presenting two stimuli to the external ear canal from two different speakers (for f1 and f2, respectively), and emissions were recorded by a microphone in the probe placed in the canal. Continuous measurements were done for f2 frequencies (3–8 kHz), without removing the probe from the ear. DPOAEs were assessed on the same day with the blood samples. To avoid any inconvenience for the rabbits, the measurement of DPOAEs took always place prior to the blood reception. The normative data for the DPOAEs used in the study were the same with those applied in humans.^[24]

Statistics

IBM SPSS Statistics 26 (IBM Chicago, USA) was used for statistical analysis. Dependent and independent variables of the experiment were DPOAEs and time, respectively.^[13] DPOAEs for 3–8 kHz frequencies were recorded. The mean DPOAE value of all frequencies was used for analysis. All values were compared using Wilcoxon signed-rank test. Nonparametric correlations were performed with Spearman's rho. For all tests, two-sided exact P values below 0.05 were deemed to indicate statistical significance.

Results

Rabbits of Group A showed increased levels of Caspases-3 from day 1 after implantation (1^st day after “day 0”). Rabbits of Group B (daily injected with amikacin) showed increased levels of Caspases-3 from the 3^rd day. While Caspase-3 serum levels are not significantly higher in Group A during the first 10 days, this occurs later. Detailed levels of each group in comparison to the control group are shown in [Figure 1].

Figure 1: Caspase-3 levels (Y-axis) of Group A (Trauma), Group B (Ototoxic) and Group C (Control) on the 1^st, 3^rd, 5^th, 7^th, 10^th, and 31^st day (X-axis) after initiation of the experiment

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Disturbance of the cochlear activity lasted longer in rabbits of Group B, which received amikacin. The abnormal cochlear activity was observed from day 7 onward. The average signal-to-noise level was 13 dB [Figure 2]. No changes in the cochlear activity were detected for the animals in Group C and the nonoperated ear of Group A rabbits. Interestingly, measurements in the operated ear (Group A) on the last day of the experiment were better, compared to those measured in the animals of the control group (P = 0.019).

Figure 2: Average distortion products otoacoustic emissions (frequency 1687) of Group A (Trauma), Group B (Ototoxic), and Group C (Control) on the 1^st, 3^rd, 5^th, 7^th, 10^th, and 31^st day (X-axis) after initiation of the experiment. Because the cochlea electrode was implanted only on one side we added the results of the nonoperated ear in this figure

Click here to view

Another interesting finding was the difference in the DPOAEs measurements in the animals of Group C and those of Group A and B. It seems that any intervention, pharmaceutical or surgical, has an immediate impact on hearing. The degree of the disturbances varies, according to the case.

When levels of Caspase-3 and DPAOEs measurements are correlated with Spearman's rho a moderate negative correlation could be found (r = −0.557, P < 0.001). This means when levels of Caspase-3 are rising, DPAOE decrease.

Discussion

The purpose of our study is to examine changes in serum levels of Caspase-3 during apoptotic phenomena in the cochlea, caused by traumatic or ototoxic causes. In fact, increased levels of Caspase-3 can be detected as early as 1 day after the insertion of the electrode. On the other hand, levels of Caspase-3 seem to increase on the 3^rd day after the beginning of intramuscular amikacin administration, but they remain elevated for weeks after the end of the therapy. The results of the present study agree with those of previous ones.^[16],[17]

Both cochlear implantation trauma and ototoxic-induced hearing loss involve a physical disruption of the organ of Corti and may involve several mechanisms of cell death at the molecular level, i.e., necrosis, necrosis-like programmed cell death, and apoptosis (reference). Caspases are a family of cysteine proteases that play a critical role in mammalian apoptosis, activation of cytokines, and induction of inflammation.^[1],[3],[6] To date, 11 human and 10 murine Caspases (Casp) have been identified.^[1],[2] They can be subdivided into two main groups: upstream or initiator caspases (Casp 1, 2, 4, 5, 8, 9, 10, 12, 13) and downstream or effector caspases (Casp 3, 6, 7).^{[1],[2],[3],[17],[18]} Upstream caspases are responsible for caspase activation or regulation of inflammatory processes and they have long N-terminal prodomains.^{[16],[19],[20],[21]}

Although there are several drugs that can injure auditory HCs, the most commonly encountered ototoxic drugs are aminoglycosides and cisplatin.^[22],[23] In brief, aminoglycoside antibiotics, such as gentamicin, amikacin, kanamycin, and neomycin, can initiate apoptotic cell death in auditory HCs and vestibular HCs.^[24] In particular, the outer HCs of the basal turn are the most vulnerable to aminoglycoside ototoxicity.^{[24],[25],[26]} The most predominant form of cell death is apoptosis, however, necrotic features are also seen following exposure to aminoglycosides.^[25] Aminoglycosides can induce mitochondrial apoptotic cell death and DNA fragmentation through oxidative stress, and Caspase 3 activity.^{[26],[27],[28]} Evidence for the role of caspase-8 and extrinsic apoptosis is aminoglycoside ototoxicity is not strong.^[28],[29]

Likened to cellular suicide, apoptosis is an actively driven process with distinct morphological and biochemical features.^[25],[27] Cell shrinkage, nuclear and cytoplasmic condensation, chromatin fragmentation, and cysteine-aspartate protease (caspase) activation are all hallmarks of apoptotic cell death.^[28] It is the activation of caspases that ultimately results in apoptotic cellular degradation. All cells contain caspases that are present constitutively but synthesized in an inactive precursor form.^[8],[20]

Rabbits were used for convenience reasons since they were easier to examine during measurements and it was easier to estimate the exact drug dosage. DPOAEs have been used by other researchers as well for the study of hearing loss on rabbits.^[14],[15] Sensitivity of the recording device and the noise level are also important factors that affect the detection threshold for DPOAEs.^[26],[27] Although the adjustments should be individualized, in practice, this was not possible since the animals were rabbits. The only data available in rabbits is the range of the hearing frequencies. Therefore, we compared the frequencies and the intensity of the distortion product of every measurement with those which were taken prior to the administration. DPOAEs were measured every 2 days to get an accurate estimation of the possible changes caused by apoptotic phenomena.

The results of our experiment are indicative of the different mechanisms which cause the cochlear lesions. In the animals of Group A, the levels of Caspases-3 rise immediately after implantation. However, in the animals of Group B the rise occurs later.

We should mention the methodological problems which would appear if our study was based only on the measurement of Caspases 3.^{[30],[31],[32],[33],[34]} Drug toxicity in the rabbits of this Group B is not located only in cochlea but in other organs such as the vestibular organ or the kidneys. For this reason, the rise of caspase levels could be attributed to cell damage of other tissues. We avoided to sacrifice animals, as it was done in previous studies, because we wanted to investigate the time course of the respective damage in the animals.^[35] This was resolved with the use of DPOAEs-measurements on the “healthy” ear.

The main limitation of our exploratory study is the absence of correlation of our findings to any structural changes, given that we have chosen not to sacrifice the laboratory animals. The reader could also think that the number of the rabbits in each group, though calculated according to the STROLE-Guidelines, is not great enough to lead to findings that could be generalized and applies on human beings.

Based on these very promising results, the next logical step will be to test the use of Caspase-3 as a surrogate marker of cochlear damage in humans.

Conclusion

Caspase-3 rises after both traumatic and ototoxic causes in a rabbit animal model. It has the potential to become a surrogate marker of the structural integrity (or degree of structural damage) of the cochlea after trauma or administration of ototoxic drugs.

Ethics approval

Ethics approval has been obtained (Auth: Nr 168-23.05.2003).

Acknowledgments

The authors would like to thank Mrs. A. Tsipropoulou for her help by typing and checking the text.

Financial support and sponsorship

This study was supported by General Hospital Veria, Veria, Greece.

Conflicts of interest

There are no conflicts of interest.

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