Comparison of Actions of Ketamine and Telazol on Cochlear Function in a Rodent Model of Noise-Induced Hearing Loss

Abstract

Research has shown that anesthesia used in rodent models studying trauma-related changes in the peripheral auditory system can impact the results of standard functional tests like the auditory brainstem response (ABR). The anesthetic agents may also confound the effects of potential therapeutics under evaluation in the preclinical models. Ketamine, a N-methyl-D-aspartate (NMDA) receptor antagonist, is a commonly employed anesthetic in rodent models. Studies have shown that ketamine, unlike other anesthetics, exerts minimal effects on ABR measurements. Tiletamine, a compound chemically akin to ketamine, is also an NMDA antagonist. Tiletamine combined with zolazepam (Telazol) may be a substitute for ketamine given its less severe side-effects and long-acting capacity. In this study, we serially compare cochlear function in rats exposed to hazardous noise to induce noise-induced hearing loss (NIHL) under the effects of either ketamine or telazol. Awake male Brown Norway rats were exposed to octave band noise (4–8 kHz) at 110 dB SPL for 1 hour. Cochlear function was assessed over multiple time points using either intramuscular injection of ketamine (44mg/kg) and xylazine (5 mg/kg) or intraperitoneally injected telazol (20 mg/kg) and xylazine (5 mg/kg). Changes in ABR threshold, latency, and amplitude were compared to baseline (pre-NIHL) over 28 days. Functional results demonstrated that both ketamine- and telazol-anesthetized animals experience permanent changes in thresholds following noise. While both amplitude and latency were affected by noise, there were no significant differences in the changes between ketamine and telazol groups. Our findings suggest that telazol behaves similarly to ketamine and could be an alternative in rodent model experiments for the evaluations of hearing sensitivity following noise trauma.

1. INTRODUCTION

Hearing loss is a critical public health concern, affecting millions of individuals worldwide[1–3]. Projections indicate that by 2050, nearly 2.5 billion people will experience some degree of hearing impairment, representing an excess medical costs ranging from $3.3 to $12.8 billion in the United States [4, 5]. Despite its widespread prevalence and profound impact, there are currently no FDA-approved therapeutics, underscoring the urgent need for effective interventions [6]. This urgency drives ongoing extensive research efforts to elucidate the pathophysiology mechanisms of hearing loss and evaluate the efficacy of various therapeutic strategies [7–9].

Research efforts have largely focused on various hearing loss trauma models, with noise-induced hearing loss (NIHL) being one of the most extensively utilized due to its relevance as the most preventable cause of auditory damage [10–13]. A crucial factor in these studies is the choice of anesthesia, as it can significantly influence both the cellular, molecular and physiological responses of the subjects as well as the outcomes of auditory assessments [14–19]. For example, anesthetic agents that block N-methyl-D-aspartate receptors (NMDAR) may inadvertently alter hearing sensitivity profiles by mitigating glutamate excitotoxicity and protecting against cellular damage[20, 21]. Thus, selecting an appropriate anesthetic is essential for minimizing confounding variables and ensuring the reliability of experimental results.

Ketamine hydrochloride, a widely utilized anesthetic in rodent models, is a short-acting NMDAR antagonist commonly administered in combination with xylazine (K/X) due to its minimal cardiac and respiratory side effects [22–24]. Current evidence suggests that ketamine exerts negligible effects on auditory function in non-traumatized rodents as reflected by minimal alterations in auditory brainstem responses (ABR) [15, 25–28] and distortion product otoacoustic emissions (DPOAE) [26, 29]. However, ketamine has also demonstrated selective neuroprotective properties in various trauma models. For instance, in a NIHL model, the combination of ketamine and xylazine has been shown to reduce temporary threshold shifts (TTS) at specific frequencies, potentially due to the diminished sympathetic activity during anesthesia [30]. Moreover, ketamine has been associated with significant alterations in middle latency auditory response amplitude, latency, and waveform morphology in guinea pigs [31]. Beyond auditory contexts, the use of ketamine as an anesthetic has shown substantial neuroprotective effects in other scenarios including the reduction of glutamate-induced cell death, mitigation of neuronal excitation, and attenuation of inflammation in models of neurotrauma, such as neonatal pain [32], spinal cord ischemia (Lips et al., 2014), and traumatic brain injury [33].

Ultimately, while most auditory assessments indicate that ketamine does not significantly impact cochlear function, its neuroprotective potential in other specific contexts may introduce confounding variables when evaluating trauma-induced damage, particularly in studies focused on auditory or neurotrauma outcomes. As an alternative, some research groups have chosen telazol, a 1:1 combination of tiletamine and zolazepam, as their anesthetic of choice [34]. Tiletamine, an NMDA receptor antagonist, has a chemical structure closely resembling that of ketamine, which explains their similar mechanisms of action [35, 36]. Nonetheless, tiletamine is often preferred due to its rapid induction capability, lower risk of cardiovascular and respiratory depression [37–39] and anxiolytic effects in rodents, a characteristic not commonly associated with ketamine[40]. Importantly, despite these advantages, the effects of telazol in cochlear injury models have not been thoroughly investigated. Given its potential to reduce dosing frequency, minimize the number of injections, and improve overall health outcomes, it is essential to explore how telazol may influence functional outcomes in longitudinal NIHL rodent studies.

In this study, we compare the effects of two commonly used anesthetics, ketamine-xylazine (K/X) and telazol-xylazine (T/X), in rats subjected to an NIHL model. Our objective is to determine how these anesthetics affect auditory function, as measured by survival analysis and auditory brainstem responses (ABR).

2. METHODS

2.1. Noise Chamber & Exposure

A custom noise chamber was utilized with the aim of mimicking translational noise exposure. The system consists of four speakers and an animal holder system placed on a motorized stage within a soundproof chamber. Four speakers were mounted above the animal holder system for equal distribution of noise throughout the chamber (Pyle-Pro A-B Box PDBT45, Pyle Electronics, [Brooklyn, NY]). The containment cells within the holder system were vented and covered with a wire mesh cover to secure the animals during exposure (RestorEar Devices LLC [Bozeman, MT]). The holder was placed atop a rotating stage (0.25 Hz) to ensure uniform exposure profiles independent of animal location within the cage. A mini sound meter with Bluetooth was placed inside the rotating cage to ensure that sound levels were within +/− 2 dB SPL (Uni-T, UT353 Mini Sound Level Meter, [Bellingham, WA]).

Normal hearing Male Brown Norway rats (n=10) were acquired to perform this study (Charles River Laboratories). The use of animals was approved by the University of Miami’s The Institutional Animal Care and Use Committee (protocol 21–129) and was in compliance with USDA and NIH Guidelines for the Care and Use of Laboratory Animals. Prior to any intervention or functional testing, animals were randomly assigned to one of two anesthetic groups, under which cochlear function was assessed: (1) intramuscular ketamine (44 mg/kg) and xylazine (5 mg/kg) (K/X; n=5), or (2) intraperitoneal telazol (20 mg/kg) and xylazine (5 mg/kg) (T/X; n=5). Following baseline functional measurements, rats were exposed to 110 dB SPL octave band noise (4 – 8 kHz) for one hour. The noise parameters were specifically selected to induce permanent threshold shifts (PTS) [41, 42].

2.2. Auditory Brainstem Responses

Auditory brainstem responses (ABRs) were recorded before noise exposure (i.e., baseline) and at days 1, 3, 7, 14, and 28 post-trauma to quantify shifts in hearing thresholds in anesthetized animals. These measurements have been described previously by our lab [43–46]. Briefly, during the data collection period, rats were placed on heating pads to maintain physiological conditions. Using subcutaneous needle electrodes, two needles were placed behind both ears, an EEG electrode was placed toward the nasion and grounded at the hind leg. ABRs were recorded using a pre-amplifier and a data acquisition system (Intelligent Hearing Systems, IHS [Miami, FL]). The ABR system included a soundproof chamber containing the anesthetized rats as acoustic stimuli were administered through earphones inserted via the external ear canal. Hearing sensitivity was assessed at six different pure frequencies – 2, 4, 8, 16, 24, and 32 kHz – with sound intensities beginning at 90 dB SPL and decreasing in increments of 5 dB SPL. Thresholds were defined as the minimum, distinct ABR response as determined by a visible node-to-peak of wave I (pertaining to cochlear nerve afferents). ABRs were also assessed for latency and amplitude changes at wave I. All ABR results are reported as an average between the left and right ears (mean ± SEM). Designated anesthetics were administered exclusively during serial ABR collection. Neither group received any alternative treatments or interventions for the duration of this study.

2.3. Statistical Analysis

GraphPad Prism was used to generate figures and perform statistical analysis. The Kaplan-Meier survival analysis was performed to determine the probability of an animal dying over the course of this longitudinal study. ABR data thresholds, latencies, and frequencies were reported as averages ± standard error of the mean (SEM). A paired Student’s t-test was performed for each group at baseline (pre-NIHL) to compare ABR threshold between left and right ears to confirm absence of significantly different baseline differences. Further, this test was repeated at day 1 post-noise exposure to ensure both ears were equally affected by the noise trauma, validating averaging of ear thresholds for functional analysis. In all presented data sets, Mixed-effects model (REML) (post-hoc Bonferroni multiple comparisons test) with the Geisser-Greenhouse correction was applied. Any significant differences identified between groups were analyzed assuming for p-value threshold of less than 0.05.

3. RESULTS

3.1. Ketamine- and Telazol-anesthetized groups show equivalent survival rates

To assess the safety of using telazol as an alternative anesthetic to ketamine, a survival analysis was performed to determine the percent survival after several data collection periods over 28 days. As shown in Figure 1, the survival rate remained above 50% in both groups, with no identifiable median survival. An early death was recorded in the ketamine group, which was unrelated to the group assignment. However, for the purposes of this study, it is important to report and include in this case for comparative purposes. The Kaplan-Meier survival analysis highlighted that there was no significant finding for probability of survival with respect to days elapsed after noise trauma (Log-rank test, p = 0.32).

Fig. 1. Telazol and Ketamine anesthetized animals survival analysis.

The percentage for the probability of survival between ketamine- (red) and telazol-anesthetized (black) groups was determined using the Kaplan-Meier survival analysis. There was no significant difference in the probability of animal mortality between the two groups (p = 0.32). The survival rate remained above 50% in both groups.

3.2. Telazol- and Ketamine-anesthetized groups have similar auditory thresholds after intense exposure

In order to determine functional differences between anesthesia groups, we performed ABRs at five different time points post-noise trauma. At day 1 post-noise, the average ABR thresholds for the ketamine and telazol groups at the representative frequency of 8 kHz were 84.50 (± 2.42) dB SPL and 79.50 (± 3.74) dB SPL (mean ± SEM), respectively. By day 28, the ketamine group’s average threshold at 8 kHz was 59.38 (± 5.90) dB SPL and the telazol group’s average threshold were 67.50 (± 5.92) dB SPL, demonstrating a small recovery in both groups. Table 1 shows the average threshold at all frequencies tested. As shown in Figure 2A1–A6, both groups exhibited similar tonotopic changes in ABRs following noise trauma. No significant differences were observed in average ABR thresholds at any of the frequencies tested at any timepoint (Mixed-effects model with Geisser-Greenhouse correction and post-hoc Bonferroni test, p > 0.05) (Figure 2). When comparing changes in threshold shift over time, telazol and ketamine again had comparable averages at day 28 (Telazol: 21.42 ± 6.06 dB; Ketamine: 13.61 ± 6.76 dB) and no significant differences were observed between groups (Mixed-effects model with Geisser-Greenhouse correction and post-hoc Bonferroni test, p > 0.05). The trends in our data (Figure 2A1–A6) demonstrate that both anesthesia groups were equally affected by noise and followed identical patterns in recovery approaching day 28. Neither anesthesia group fully returned to baseline, though this was expected given the noise profile used during exposure.

Table 1. Mean ABR Thresholds for Ketamine (K)- and Telazol (T)- anesthetized animals.

Data is provided for each frequency stimulus tested at days 1 and 28 post-noise (also see Figure 2).

	AVERAGE THRESHOLD (dB SPL)
Frequency	Day 1		Day 28
	K	T	K	T
2	77.50 (8.37)	66.50 (5.40)	43.85 (5.15)	51.50 (8.61)
4	77.50 (3.48)	71.50 (6.64)	43.13 (6.07)	60.50 (9.23)
8	84.50 (2.42)	79.50 (3.74)	59.38 (5.90)	67.50 (5.96)
16	79.50 (2.55)	75.50 (3.82)	48.13 (4.25)	58.50 (5.22)
24	67.00 (3.20)	69.00 (4.37)	36.25 (5.64)	43.00 (7.68)
32	65.00 (5.12)	69.50 (5.61)	40.00 (5.20)	37.50 (5.48)

Fig. 2. There are no functional ABR differences in rats anesthetized with Ketamine versus Telazol following awake noise exposure.

A1–6) Following awake noise exposure in rats, ABRs were collected at days 1, 3, 7, 14, and 28 to determine threshold changes for ketamine- (red) and telazol-anesthetized (black) animals across a representative tonotopic mapping of the cochlea. Both telazol and ketamine anesthetized group thresholds were significantly affected by noise and followed similar patterns in recovery approaching day 28. No significant differences (NS) were determined by the two anesthesia groups at any timepoint across any of the frequencies tested (p > 0.05). B1–4) Latency and amplitude of ABR wave I was assessed to determine if there were any permanent changes following noise, and to see if there were differences between anesthesia groups. There were no differences in shifts across frequencies between ketamine and telazol animals (p > 0.05).

3.3. Telazol and Ketamine exert similar effects on ABR changes, amplitude and latency

Amplitude and latencies of the ABR wave I peaks were analyzed to further investigate how the two different anesthetic agents might affect the robustness of the cochlear nerve response. As displayed in Table 2 and 3, we observed no significant differences between either group for amplitude or latency at any timepoint and frequency (Mixed-effects model with Geisser-Greenhouse correction and post-hoc Bonferroni test, p > 0.05). When analyzing wave I latency and amplitude shifts from baseline to day 28, there were complimentary trends in the recovery pattern between telazol and ketamine though there was no statistical significance between groups (Mixed-effects model with Geisser-Greenhouse correction and post-hoc Bonferroni test, p > 0.05) (Figure 2B1–B4).

Table 2. Mean latency values of ABR Wave I response for Ketamine (K)- and Telazol (T)- anesthetized animals.

Data is provided for each frequency stimulus tested and all time points (days 1, 3, 7, 14, and 28 post-intervention).

	AVERAGE LATENCY OF WAVE I (MEAN (ms) ± SEM)
Frequency	Baseline		Day 1		Day 3		Day 7		Day 14		Day 28
	K	T	K	T	K	T	K	T	K	T	K	T
2	1.41 (0.14)	1.28 (0.07)	2.20 (0.40)	2.03 (0.16)	1.88 (0.38)	1.82 (0.21)	1.49 (0.14)	1.85 (0.21)	1.59 (0.38)	1.35 (0.41)	1.58 (0.21)	1.44 (0.25)
4	1.55 (0.17)	1.51 (0.09)	1.99 (0.13)	1.77 (0.16)	1.96 (0.23)	1.45 (0.17)	1.73 (0.10)	1.40 (0.22)	1.54 (0.22)	1.36 (0.15)	1.56 (0.14)	1.75 (0.40)
8	1.43 (0.20)	1.40 (0.08)	2.39 (0.19)	1.77 (0.27)	1.97 (0.06)	1.40 (0.22)	2.00 (0.05)	1.64 (0.24)	1.74 (0.17)	1.60 (0.09)	1.63 (0.11)	1.46 (0.30)
16	1.43 (0.08)	1.43 (0.03)	1.98 (0.27)	1.47 (0.04)	1.58 (0.04)	1.39 (0.11)	1.56 (0.05)	1.37 (0.05)	1.43 (0.19)	1.34 (0.08)	1.54 (0.03)	1.40 (0.07)
24	1.38 (0.04)	1.29 (0.03)	1.39 (0.14)	1.36 (0.05)	1.36 (0.05)	1.27 (0.06)	1.39 (0.06)	1.40 (0.08)	1.36 (0.09)	1.21 (0.07)	1.34 (0.03)	1.33 (0.07)
32	1.31 (0.05)	1.23 (0.02)	1.47 (0.08)	1.32 (0.01)	1.54 (0.26)	1.30 (0.06)	1.42 (0.05)	1.33 (0.11)	1.33 (0.10)	1.31 (0.02)	1.37 (0.08)	1.21 (0.12)

Table 3. Mean amplitude values of ABR Wave I response for Ketamine (K)- and Telazol (T)- anesthetized animals.

Data is provided for each frequency stimulus tested and all time points (days 1, 3, 7, 14, and 28 post-intervention).

	AVERAGE AMPLITUDE OF WAVE I (MEAN (μV) ± SEM)
Frequen cy	Baseline		Day 1		Day 3		Day 7		Day 14		Day 28
	K	T	K	T	K	T	K	T	K	T	K	T
2	0.85 (1.01)	1.15 (0.09)	0.31 (0.04)	0.31 (0.07)	0.30 (0.07)	0.45 (0.10)	0.33 (0.05)	0.72 (0.18)	0.65 (0.14)	0.49 (0.23)	0.43 (0.03)	0.37 (0.09)
4	1.01 (0.12)	1.12 (0.14)	0.18 (0.04)	0.35 (0.07)	0.30 (0.08)	0.62 (0.11)	0.38 (0.08)	0.73 (0.16)	0.55 (0.10)	0.68 (0.17)	0.42 (0.08)	0.32 (0.07)
8	0.51 (0.08)	0.67 (0.12)	0.23 (0.06)	0.23 (0.06)	0.13 (0.04)	0.39 (0.13)	0.22 (0.01)	0.59 (0.06)	0.22 (0.02)	0.30 (0.08)	0.31 (0.06)	0.23 (0.06)
16	0.59 (0.08)	0.68 (0.18)	0.19 (0.07)	0.29 (0.08)	0.17 (0.05)	0.24 (0.08)	0.22 (0.10)	0.43 (0.07)	0.31 (0.06)	0.27 (0.08)	0.30 (0.06)	0.42 (0.05)
24	0.76 (0.21)	1.15 (0.16)	0.32 (0.07)	0.46 (0.12)	0.49 (0.02)	0.68 (0.04)	0.63 (0.14)	0.94 (0.17)	0.86 (0.10)	0.76 (0.14)	0.68 (0.05)	0.66 (0.07)
32	0.68 (0.14)	0.81 (0.16)	0.34 (0.04)	0.53 (0.15)	0.41 (0.07)	0.49 (0.06)	0.46 (0.06)	0.94 (0.11)	0.61 (0.04)	0.56 (0.10)	0.55 (0.05)	0.61 (0.10)

4. DISCUSSION

The current study aimed to profile the differential effects of telazol and ketamine on hearing function after an awake-state acoustic overexposure period. Animal experiments are a crucial tool in hearing research, and in NIHL models specifically, characterizing changes in functional measurements must be carried out under anesthesia. However, anesthetic agents that demonstrate protective mechanisms could represent confounding effects when studying hearing sensitivity and potential therapeutics. As demonstrated by the work described herein, an effective and safe anesthetic alternative for small animals may be telazol [34].

The use of anesthesia in noise-induced hearing loss (NIHL) rodent models has been well documented and characterized. Ketamine, isoflurane, and pentobarbital are commonly used in preclinical studies, and their mechanisms of action and effects on the auditory system are known. For instance, noise-exposed cochleae that were perfused with NMDAR antagonists showed significantly decreased cell swelling [21, 47, 48]. When compared to other anesthetics, such as Isoflurane and Pentobarbital, K/X anesthetized rats did not exhibit altered auditory responses while others did [27, 29]. Contrastingly, when a glutamate transporter highly expressed in the cochlea was deficient, increased auditory threshold shifts were found [49]. Other studies have suggested that K/X protects against hearing damage when compared to the awake state due to its reduced sympathetic influence [30]. When analyzing this information, it is important to consider the distinction between noise administered to awake-state versus anesthetized animals as the presence of NDMAR antagonists during exposure does influence the effects described. Given our study employed an awake-state noise protocol, we intended to minimize anesthesia usage, thereby reducing the potential for additional confounding results.

Ketamine is widely used in rodent models of hearing loss and auditory research. Its derivatives have also been compared in cochlear function tests, providing similar hearing thresholds and shorter recovery times[50]. However, ketamine’s effects on auditory processing can vary between strains, with some showing lasting disruptions in sensory encoding [51]. When comparing anesthetics, K/X produces significantly lower ABR thresholds than isoflurane in rats, with differences exceeding 27 dB across frequencies [25]. Beyond auditory research, ketamine has gained attention as a rapid-acting antidepressant in rodent stress models, capable of rescuing stress-associated morphological and behavioral changes [52]. These studies highlight the diverse applications of ketamine in rodent research, from hearing assessments to psychiatric models, while emphasizing the importance of considering strain differences and anesthetic choices in experimental design. Ketamine, a noncompetitive NMDA receptor antagonist, also has multiple mechanisms of action that contribute to its anesthetic, analgesic, and antidepressant effects in rodents. While NMDA receptor antagonism is considered a central mechanism [53, 54] other pathways are also involved. These include interactions with AMPA receptors, mTOR signaling, monoaminergic systems, and various ion channels [53, 55]. The drug’s effects are dose-dependent, with subanesthetic doses producing antidepressant-like effects in stressed rodents [56]. Long-term ketamine use can lead to structural and functional brain impairments in both rodents and humans[55]. While understanding these complex mechanisms, it is important to characterize its effects on auditory functional assessments such as ABRs following stressors like noise or blast exposures which are used to research occupational and recreational hearing loss. As highlighted in previous NIHL therapeutic work in our lab, Rincon Sabatino et. al assessed the role of ketamine anesthesia in relation to noise exposure and recovery and determined that anesthetic agent alone did not have an observed significant protective effect, specifically with respect to ribbon synapses [46]. Additionally, as observed in histology analyses (data not included for brevity), no significant difference in inner or outer hair cell survival was observed between K/X and T/X anesthesia groups, complimenting previous research. Ultimately, it is important to explore and compare anesthesia effects which may offer reduced side effects.

Telazol, a combination of tiletamine and zolazepam, has shown promise as an anesthetic in rodent research. In gerbils, telazol was found suitable for major surgical procedures, though with prolonged recovery time [57]. In mice, telazol combined with xylazine resulted in short, repeatable induction times and superior anesthesia duration compared to other regimens [58]. Rat studies demonstrated that telazol produced dose-dependent increases in anesthesia duration and showed fewer adverse cardiovascular effects compared to ketamine-xylazine and pentobarbital, with doses of 40–50 mg/kg appearing optimal [38].While telazol offers advantages in rodent anesthesia, its application in ongoing research exploring drug delivery methods for hearing loss studies or on cochlear function after exposures to stressor such as noise remain to be explored.

In this study, we aimed to compare functional measurements in animals anesthetized with T/X following awake noise exposure to the known effects observed in animals anesthetized with K/X. We found that there were no significant differences between telazol and ketamine in functional ABR thresholds. This indicates that telazol, like ketamine, did not suppress the robustness of the ABRs nor did it elevate the observed thresholds following noise. In addition, consistent with previous studies on the effects of ketamine on ABRs, telazol did not appear to significantly alter changes in latency or amplitude. This suggests that neither anesthetic has a substantial influence on glutamatergic inner hair cells (IHCs) at a molecular level, unlike other anesthetics such as isoflurane, which have shown sensitivity to auditory system alterations.

Overall, our results indicate that telazol produces similar outcomes to ketamine and using either anesthetic, does not confound auditory functional data obtained for NIHL studies.

5. CONCLUSION

In all, no significant differences were found in functional and histological outcomes between groups subjected to the two distinct anesthetic agents. This suggests that, similarly to ketamine, telazol is a suitable agent for use in NIHL rodent models without introducing confounding effects on noise sensitivity profiles. For some studies, telazol may be preferable due to its reduced impact on the respiratory and cardiovascular system, as well as its shorter induction period.

Highlights:

• Application of anesthesia in preclinical models impacts auditory processing.

• Ketamine and Telazol show similar functional changes post-noise in rats.

• Telazol, with rapid induction and fewer side effects, may be superior to Ketamine.