Hearing is essential for animals as it allows them to interact with their environment in a variety of ways. Thus, the decline or loss of hearing function can be fatal. Deaf animals can survive; however, their utility is decreased. This is particularly true for working dogs. In pet-family relationships as well as between animals, communication is impeded and vehicles or predatory animals can be dangerous (34).

Sound waves are mechanical vibrations transmitted through a medium such as air or water. In the air, they are composed of rapid oscillations of atmospheric pressure. Upon entering the outer and middle ear, these sound waves are converted into electrical impulses in the inner ear and are transmitted to the auditory nerve, the brainstem, and finally to the cerebral auditory cortex (16,22,34).

The use of an auditory evoked response (AER), which has replaced behavioral testing as a method of evaluating hearing in dogs, is growing in popularity. It is relatively easy, non-invasive, and, transportable with a short inspection time. It allows for objectivity, sensitivity, and is a comprehensive index. In addition, it is more cost effective compared to other hearing tests and provides a specific anatomic evaluation. In general, it is not significantly affected by the level of consciousness or drug use. However, despite these advantages, inaccuracies could be produced depending on the level of expertise of the examiner (37).

AER is composed of many waves (negative and positive peaks), and each wave represents neuronal activities in one or more of the brain structures. There are specific recording types depending on the average length of time after stimulation. In humans, AER is divided into early (0-10 ms), middle (10-50 ms), late (50-250 ms), and long (more than 250 ms) waves. In general, components of the successive types of AER represent the activity of the neural generators at progressively higher levels in the neuroaxis. In the early latency component, the generators are located mostly within the brainstem. This series of waves is called the brainstem auditory evoked response (BAER). The BAER measures pulses ascending through the initial component of the auditory pathway. This response consists of seven waveforms, five of which are clinically significant (25,37). Each positive peak begins within 1.0-1.5 ms after the auditory stimulation, and the subsequent waveform is formed at an interval of less than 1 ms. Each positive peak is labeled with either a Roman or Arabic number or a latency value according to its polarity designation. Each peak is characterized by latency and amplitude, and these values are clinically significant (28). Waves I, II, and V have large amplitudes and III, IV, and VII have small amplitudes. Wave VII is not visible, and wave IV can be seen as a single wave merging with waves III or V (37).

Each wave is known to occur at a specific anatomical location. Waves I and II occur in the vestibulocochlear nerves, wave III inside the cochlear nucleus or in nearby neurons, and wave V in the contralateral caudal colliculus of the brainstem (36). According to previous studies, all waveforms were thought to be associated with one anatomical location. However, it is now known that each wave, except for waves I and II, originates from one or more anatomical structures (37). Based on this, the location of a lesion as well as the hearing threshold can be estimated using a BAER test (25,28).

Electrophysiological auditory tests are often conducted under deep anesthesia (35). In human medicine, sedation is commonly used in infants and toddlers when they are unable to sleep or remain still during the test (27). However, in veterinary medicine, general anesthesia is essential when invasive surgery is required or when an animal’s movements must be restricted to minimize artifacts during an examination (35). As BAER is not significantly affected by anesthesia, more data can be collected in a shorter time when the animal is chemically restrained (28).

Various studies have been conducted in humans, but comparative studies of BAER regarding sedation, injectable anesthetics, inhalational anesthetics, or depth of anesthesia are lacking for animals. Additionally, there are differences in the results from studies on various animals (29,35). Therefore, this study aimed to identify the differences in BAER test measurements under three conditions: 1. when medetomidine was used for sedation, 2. when general anesthesia was administered with propofol, a commonly used injectable anesthetic, and 3. when isoflurane, an inhalational anesthetic, was used after propofol administration.

Subjects

This study was conducted on 4 healthy adult Beagles that were approximately 4 years old, medium-sized, and weighed between 10-13 kg. Prior to the study, all dogs fasted for a period of 12 h. No dog took any drugs that could have caused ototoxicity, and topical agents were not used. In addition, an otoscopic examination, an otic swab examination, a complete physical examination, hematology and complete biochemistry tests, and radiography were conducted to ensure that the dogs did not have any underlying disease or structural deformities that could have affected their auditory function. Ethical approval for this study was obtained from the Institutional Animal Care and Use Committees of Gyeongsang National University (approval no. GNU-201106-D0081).

Anesthetic protocol

No premedication was used with any subject. The first measurement was carried out for approximately 10 min after 8 mg/kg propofol (Provive; Baxter) was slowly injected intravenously while spontaneous breathing was maintained. After the test, the dogs were intubated using a 5.5 mm or 6 mm internal diameter cuffed endotracheal tube. Respiratory anesthesia was maintained by setting the inspiratory concentration of isoflurane (Ifran; Hana pharm) at 2%. At the same time, 100% O2 was supplied at 2 L/min, and end tidal CO2 was maintained at 30-40 mmHg with a respiratory rate of 6-10 respirations/min. The second measurement was carried out using the same method. After one week, 0.015 mg/kg medetomidine (Sedator; Dechra) was injected intravenously, and the third measurement was carried out with the animals in a sedated state. At the end of the test, an intramuscular injection of 0.05 mg/kg of atipamezole hydrochloride (Atipam; Dechra) was administered to the animal to reverse the effect of the medetomidine hydrochloride sedation. Anesthetic depth was clinically evaluated by checking the palpebral reflex, jaw tone, and muscle relaxation. Electrocardiography, pulse quality, and capnography were monitored, and body temperature was maintained at 37-39°C using a heating pad during the study.

BAER test and equipment

BAER tests were conducted using the Neuropack M1, MEB-9200 electrodiagnostic system (Nihon Kohden, Tokyo, Japan) auditory brainstem response program according to the manufacturer’s manual. During the BAER test, the main unit was connected to an electrode junction box, JB-902BK (Nihon Kohden; Tokyo, Japan). The electrode junction box was connected to four monopolar needle electrodes, NM-710T (Nihon Kohden; Tokyo, Japan), with a length of 13 mm and a diameter of 0.25 mm. An insert earphone YE-103J (Nihon Kohden; Tokyo, Japan) was used as the transducer.

The BAER test was conducted in the electrogram room at the Gyeongsang National University Animal Medical Center. Interference was minimized to maintain a quiet environment. The animal was kept in sternal recumbency, and the head was slightly elevated using a folded towel. The four monopolar needle electrodes were fully inserted into the vertex, forehead, and subcutis just anterior to the tragus on each ear, respectively (Fig. 1). An impedance check was conducted prior to each test to confirm the correct insertion of the electrode. Auditory stimuli were set at a click rate of 0.1 ms, and the intensity was decreased by 10 dB hearing level (HL) from the 90 dB of normal HL to 60 dB HL. To avoid any crossover recordings, the ear that was not being examined was provided white noise that was 40 dB lower than the sound provided to the ear being tested. Each waveform was obtained at an average of 200-500 click stimulations with a 0.1 ms interval. Electrical activity was amplified to 100-2,000 Hz, and an alternating current filter was used. Latency and amplitude were manually marked after the test by the same examiner.

Figure 1. Animal posture and location of needle electrodes in a dog injected with propofol (A) and in a dog inhaling isoflurane after intubation (B).

Statistics

Statistical analysis was performed using the Statistical Package for the Social Sciences for Windows (SPSS) 25 software (IBM Corp., Armonk, NY, USA); a Kruskal-Wallis test was used, with a significance threshold of p < 0.05.

Altogether, eight ears from four Beagles were examined. The first measurement was carried out after propofol injection, the second measurement after propofol injection and maintenance of anesthesia with isoflurane, and the third measurement after medetomidine injection.

Two dogs showed paradoxical movements such as paddling and myoclonus that appeared immediately before isoflurane inhalation after propofol injection and intubation. This was temporarily relieved by increasing the respiratory minute volume ventilation with manual hyperventilation using isoflurane.

For the BAER recordings, the waveform, latency, and amplitudes for each peak were evaluated. In all dogs, four peaks (waves I, II, III, and V) appeared at 90 dB, 80 dB, 70 dB, and 60 dB. Latency is the time to each positive peak and is expressed in ms, and amplitude refers to the voltage difference between the negative peaks following each positive peak and is expressed in uV. A significant negative peak (trough) was confirmed after wave V. The mean and standard deviation graphs for the latencies of waves I, II, III, and V, interpeak wave latencies (IPLs) of I-III , III-V, and I-V, and the amplitudes of waves I, II, III, and V in dogs are presented in Figs. 2-4.

Figure 2. Mean wave latencies of waves I, II, III, and V for propofol (●), propofol-isoflurane (□) and medetomidine (▲). Vertical bars represent mean ± SD.

Figure 3. Mean wave amplitudes for waves I, II, III, and V for propofol (●), propofol-isoflurane (□) and medetomidine (▲) in each decibel. Vertical bars represent mean ± SD.

Figure 4. Mean wave IPLs for waves I, II, III, and V for propofol (●), propofol-isoflurane (□) and medetomidine (▲) for each decibel. Vertical bars represent mean ± SD.

When comparing the shape of the waveforms visually, no significant difference between the three groups consisting of the same subjects was observed. There was no statistically significant difference in latency and amplitude of the four waves and IPLs of I-III, III-V, and I-V between three protocols.

In BAER related research in humans, data from multiple studies on various sedatives and anesthetics as well as the depth of the anesthesia have been established (10,14,26). In animals, sedation or anesthesia is required for a successful BAER testing process and for accurate results with minimal artifacts caused by muscle movements. Comparative studies have been conducted on various drugs in veterinary medicine; however, BAER studies based on sedatives and anesthetics for animals are lacking. In general, the results are not affected by drugs, but some studies have found significant differences (29,35).

This study compared BAER test measurements after administration of medetomidine (a sedative), propofol (an injectable anesthetic), and isoflurane (an inhalational anesthetic) after propofol administration, and all study conditions were identical. Since latency could increase at temperatures below 36°C, the animal’s body temperature was maintained at 37-39°C (37).

In general, as stimulus intensity increases, latencies decrease and amplitudes increase, but Interpeak latencies (IPL) do not significantly change (37). In normal adults and dogs, when stimulus intensity decreases, the degree of latency prolongation is more pronounced in wave I than in wave V, and I-V IPL shortens (37). The III-V IPL is independent of wave I, so it is less affected by changes in the stimulus intensity. In this study, an increase in stimulus intensity led to a decrease in latencies and an increase in amplitudes in all waves.

BAER tests generally include the assessment of morphology and repeatability of waveforms, latencies, interwave latencies, interaural comparisons, amplitudes, and interaural comparisons (25). BAER is used to diagnose or estimate the location of a lesion, and it is typically measured at intensities of 70 dB or above. This is because the characteristics of wave morphology are the most prominent at intensities of 70 dB or more, and it is possible to accurately evaluate the latencies and IPLs of the waves (37). When the stimulus intensity decreases to 50 dB and below, waves II, IV, VI, and VII disappear and only wave V remains. The lowest stimulus intensity at which wave V can be seen is called the hearing threshold (25,37). In this study, measurements were taken between 60 dB and 90 dB to obtain the correct form of all waves. However, because the measurements were not taken at a lower intensity, the hearing threshold could not be confirmed.

In humans, BAER test is useful in evaluating whether postoperative damage, which may affect hearing, has occurred in structures within or anatomically near the auditory pathway or in areas related to brainstem function (10). In addition, post-anesthetic hearing loss may occur due to various causes, such as effect of pressure in the middle ear or damage to its’ vascular structure, cerebrospinal fluid pressure changes, embolism, and/or ototoxic drugs (33). In this study, there was no difference in the results when the BAER test was re-administered one week after anesthesia using propofol and isoflurane. This confirmed that there was no hearing damage after the anesthesia.

In humans, various studies regarding BAER tests have established the appropriate sedatives and anesthetics. In general, volatile agents are known to increase latency in a dose-dependent manner. In addition, propofol increases latency and decreases amplitude, and isoflurane increases latency but does not affect amplitude (10,14,26).

In veterinary medicine, there are some studies comparing BAER results regarding sedatives and anesthetics in various animals, although the data has not been established as accurately as in humans. In dogs, acepromazine had no effect, but thiamylal sodium was reported to change the shape of the waveform (15,30). In addition, latencies of all waves, except for wave I, increased when methoxyflurane was used for anesthesia (20).

There have been studies comparing the results obtained from awakened gerbils and gerbils anesthetized with ketamine and xylazine. One study showed that there was no significant difference in the threshold and only small magnitude differences in latency and amplitude. This suggests that relatively accurate testing is possible when anesthetized with ketamine and xylazine (31). Another study reported that only wave V latency increased, which may be due to ketamine that blocks the N-methyl-D-aspartate receptor channel and reduces synaptic transmission (13,18). In addition, isoflurane, as opposed to ketamine and xylazine, was reported to elevate the auditory brainstem response threshold in rats. This probably occurs because isoflurane increases blood flow to the brainstem and allows tissue perfusion. It inhibits glutamic acid decarboxylase and decreases gamma amino butyric acid (GABA), an inhibitory neurotransmitter. This results in neural excitation (17,24).

A study in cats reported that xylazine and ketamine anesthesia increased latency in some waves compared to that with xylazine-alone anesthesia, but since the difference was not significant, combination anesthesia could be more useful (29). In addition, there was no significant difference when using sevoflurane, an inhalable anesthetic, or alfaxalone, a sedative (23). Another study revealed that there was no significant difference in wave morphology or latency when using sodium pentobarbital, ketamine, halothane, or chloralose, which were administered via various routes such as intraperitoneal injection, intramuscular injection, or inhalational anesthesia through a face mask (22). In conclusion, the basic waveform did not change even with a variety of parenteral and inhalational anesthetic agents (3).

A study in guinea pigs showed that isoflurane dose-dependently reduced the amplitude of ABR and increased latency. This effect was more evident at a 2% concentration and in peaks of waves IV and V, as opposed to that of earlier waves I and III (35).

Most of these comparative studies in veterinary medicine used drugs that were frequently used in clinical practice earlier. There are only a few comparative studies on propofol or isoflurane, which are currently widely used.

In this study, BAER test was not performed in a conscious state; however, there was no difference in the results measured in the awaken state or the natural sleep state. In addition, there was no substantial impact on BAER latency or amplitude even in narcolepsy or metabolic coma (32,37). This suggested that consciousness did not affect BAER test results. Previous studies have demonstrated that sensory information processing can occur during surgical procedures, even in patients who are under general anesthesia (5,8). According to another study, it was not that sensory information in a loss of conscious (LOC) state did not stimulate the sensory cortex, but rather it activated the primary sensory cortex. However, it is not integrated into the sensory cortex hierarchy, and thus, it is simply not recognized (1). A study in coma patients found that sensory stimuli activates the primary sensory cortex even in a vegetative state; however it is not active enough to be recognized (2,4,11,12). Therefore, unconsciousness does not mean that hearing is lost, but rather it is simply not recognized. So it is thought that the degree of loss of consciousness when using sedatives or anesthetics does not significantly affect BAER test results. Based on previous studies, BAER tests can be conducted by selecting appropriate drugs without significantly considering the resulting depth of sedation or anesthesia.

Sedation is mainly used as a substitute for general anesthesia in minor procedures. Medetomidine, a potent alpha-2 adrenoceptor agonist, is widely used in veterinary medicine as a pre-anesthetic or sedative drug. It can be analgesic, and by stimulating central receptors, it causes major changes in the cardiovascular system. The dose range for sedation is 10-80 ug/g/kg for intravenous or intramuscular injection (19). Medetomidine can have a sedative effect with less respiratory depression than that with propofol, fentanyl, or midazolam (27).

General anesthesia refers to a state in which there is no cognition or response to stimuli due to the depression of the central nervous system activity. It can be achieved via inhalation or intravenous injection. Propofol, a widely used injectable anesthetic for inducing or maintaining anesthesia, acts on GABA receptors and can cause a rapid onset and smooth induction of anesthesia (9). In dogs, the recommended dose if pre-anesthetic is administered is 2-4 mg/kg (IV) and if alone is 6-8 mg/kg (IV). In this study, 8 mg/kg IV was administered as no pre-anesthetic was used (9). The side effects of propofol include excitatory symptoms such as paradoxical myoclonus, paddling, opisthotonos, and nystagmus (9). In this study, paradoxical movements appeared temporarily in two dogs, and rapid administration of an inhalational anesthetic relieved the symptoms. Such side effects could be prevented with use of pre-anesthetics.

Inhalational anesthetics are preferred to injectable anesthesia. With the former, the concentration can be controlled more precisely and a quick intervention can be initiated when the physiological state of an animal changes (21,35). Isoflurane is a widely used inhalational anesthetic agent in veterinary medicine. It is known to influence various neurotransmitter receptor systems such as the GABAergic, glycinergic, acetylcholinergic, serotoninergic, and glutamatergic systems (6,7,35). In this study, the test was performed while anesthesia was maintained at a 2% isoflurane concentration. Conducting BAER tests at various isoflurane concentrations will allow for comparison studies according to the dose of inhalational anesthesia.

Since medetomidine and propofol were injected intravenously, they were administered at room temperature. As a result, O2 could not be supplied, so this test could not be carried out under the same conditions as when isoflurane was administered. However, the effects of O2 administration in BAER tests remain unknown.

This study compared BAER test results obtained using various methods of behavioral restrictions, such as sedation, injectable anesthesia, and inhalational anesthesia. Four dogs were included in this study. In humans, the test can be performed even in a conscious state as movements can be controlled. However, sedation and anesthesia are required for infants to induce sleep or to restrict their movements during the test, and there are instances where general anesthesia is necessary. In animals, because accurate measurement is impossible in the awake state due to an animal’s uncooperative nature and/or muscle movements, sedation or anesthesia is required. There are many studies in humans regarding the type of anesthetic or the depth of anesthesia required for BAER tests. Studies in veterinary medicine on widely used drugs are scare. In conclusion, a sedative or anesthetic can be selected for a BAER test according to the patient’s condition and considering the effects and side effects of the drugs and route of administration. However, this study is not without limitations, which include the small sample size and that O2 was not supplied during medetomidine and propofol administration, which resulted in different conditions. Additionally, the duration of anesthesia was not considered. Our data will aid in the selection of drugs for sedation or anesthesia during the administration of BAER test. Overcoming the limitations of this study could lead to the further development of anesthetics and sedative drugs on BAER test in veterinary medicine.

The authors have no conflicting interests.