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ANESTHETIC PHARMACOLOGY

The Effect of Isoflurane, Halothane and Pentobarbital on Noise-Induced Hearing Loss in Mice

Jong Woo Chung, MD^*, Joong Ho Ahn, MD^*, Jong Yang Kim, MD^*, Hyun Jung Lee, MS, Hun Hee Kang, MS, Yoon Kyung Lee, MD, Joung Uk Kim, MD, and Seung-Woo Koo, MD

From the Department of *Otolaryngology and Anesthesiology and Pain Medicine, Asan Medical Center, College of Medicine, University of Ulsan, Seoul, Korea; and Asan Institute for Life Science, Seoul, Korea.

Address correspondence and reprint requests to Kim, Joung Uk, MD, Department of Anesthesiology and Pain Medicine, Asan Medical Center, College of Medicine, University of Ulsan, 388-1, Poongnap-Dong, Songpa-Gu, Seoul 138-736, Korea. Address e-mail to jukim{at}amc.seoul.kr.

	Abstract

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BACKGROUND: Ear surgery using mastoid drills can lead to noise-induced hearing loss (NIHL). We investigated whether inhaled anesthetics or pentobarbital could have protective effects on NIHL in mice.

METHODS: Mice were exposed to broad band white noise for 3 h per day for 3 consecutive days, with or without anesthesia, using halothane, isoflurane, or pentobarbital. The hearing level of each mouse was analyzed before exposure, and 1 day, 1, 2, and 3 Wk, and 1 mo after noise exposure by measuring auditory brainstem response thresholds. At 1 Wk after noise exposure, the organ of Corti was stained with a fluorescent isothiocyanate-conjugated phalloidin probe and a TUNEL kit.

RESULTS: In the unanesthetized control group, the hearing threshold increased to 77.5 ± 8.0 dB hearing level (HL) after noise stimulation. In the pentobarbital, isoflurane, and halothane groups, hearing threshold increased to 62.5 ± 6.3 dB HL, 45.5 ± 9.8 dB HL, and 39.3 ± 6.2 dB HL, respectively, with all anesthetized groups of mice showing significantly preserved hearing compared with the control group (P < 0.05). But, in mice anesthetized with pentobarbital, hearing loss was more severe than in those treated with the inhaled anesthetics (P < 0.05). Hair cell survival was reduced in unanesthetized control mice and somewhat reduced in pentobarbital-treated mice, but largely unaffected in mice treated with inhaled anesthetics.

CONCLUSIONS: These findings indicate that, while halothane, isoflurane and pentobarbital could protect mice against NIHL and hair cell damage, inhaled anesthetics were more effective.

	Introduction

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Noise-induced hearing loss (NIHL) became widespread as a consequence of the Industrial Revolution, with the first account of deafness in boilermakers appearing in 1886 (1). Exposure to very high sound levels for a short period of time or prolonged exposure to intense sound can damage the hair cells of the cochlea, resulting in permanent hearing loss due to hair cell death in the organ of Corti, the auditory organ. Because hair cells cannot regenerate in the mammalian cochlea, the loss of each cell, whether due to noise or ototoxic drugs, is irreversible and cumulative. At present, there is no medical treatment for noise-induced sensorineural hearing loss, making prevention the best treatment of NIHL.

Several mechanisms have been proposed to account for hearing loss after intense sound, including direct mechanical trauma to the organ of Corti caused by unusually intense noise (2) and metabolic damage in the inner ear caused by the formation of reactive oxygen species (ROS)3 or the involvement of nitric oxide (NO)4 or glutamate receptors (5). Moreover, as sound intensity increases, cochlear bloodflow decreases, resulting in endolymph oxygenation and glucose uptake and leading to a greater potential for ROS damage (6).

The possibility that instrument-generated noise can induce cochlear damage has been raised since the mastoid drill was first used in ear surgery. Sound levels above 115 dB sound pressure level (SPL) cause sensorineural damage if sustained for more than 15 min (7). Mastoid drilling, however, can reach an intensity of 125 dB SPL, with a constant mean above 100 dB SPL (8). Noise from aspiration also appears to be significant, adding 10–31 dB to the noise from drilling alone (7). Drilling-induced vibration of the temporal bone may also contribute to cochlear damage (9). Middle ear surgery, however, is associated with a lower incidence of NIHL than expected, ranging from 1.2% to 4.5% of patients (10), suggesting that anesthetics may have some protective effect against NIHL. We previously showed that isoflurane has a protective effect against NIHL in mice (11). We have expanded this observation by assaying whether other anesthetics also have such effects. Specifically, we compared the effects of halothane, isoflurane, and pentobarbital on NIHL, as measured by changes in hearing thresholds and hair-cell damage.

	METHODS

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Four-week-old BALB/c mice, with normal Preyer's reflex and a normal hearing threshold, were purchased from Orient Charles River Technology (Seoul, Korea). The mice were housed in cages and maintained in environmentally controlled rooms with a 12-h light/dark cycle, with food and water available ad libitum. All animal experiments were performed with the approval of the Animal Care Committee of University of Ulsan College of Medicine. The care and use of the animals reported in this study were in accordance with the guidelines of the Laboratory Animal Unit of the Asan Institute for Life Sciences.

Forty mice were exposed to white noise (300–10,000 Hz, 122 dB peak equivalent SPL) in a noise booth for 3 h per day for three consecutive days, with 3 to 5 mice exposed at the same time. White noise was generated by a personal computer and amplifier (R-399, Interm, Seoul, Korea) and delivered through a speaker (290–8L, Altec Lansing, OK) inside the noise booth. Their location inside the noise booth was changed daily so that each animal was exposed to the same level of noise. Equipotent doses of the three anesthetics were determined by the preliminary studies that demonstrated that about 50% of animals would move in response to a tail-clamp 1 h after drug administration. Inhaled anesthetic concentrations from the noise booth were monitored by a Datex Model 222 Anesthetic Agent Analyzer^TM. Ten mice each were anesthetized with inspired 1.0–1.2 Vol% for halothane or inspired 1.5–1.7 Vol% for isoflurane, 10 mice were anesthetized with an intraperitoneal (i.p.) injection of 80 mg/kg pentobarbital sodium, with an additional 40 mg/kg i.p. every hour during noise exposure, and 10 control mice were not treated. Mice of all groups received supplemental oxygen (4 L/min) during noise exposure.

The body temperature of the mice was monitored with a rectal temperature probe connected to a continuous monitoring device (Datex-Ohmeda, S/5, Bradford, UK) to eliminate the effect of whole body cooling or heating. The mice were maintained at a rectal temperature of 35–37°C with circulating-water mattress cooling (Blanketrol II, Cincinnati Sub-Zero CO, Cincinnati, OH) and a heat lamp.

The hearing level of each mouse was analyzed before exposure, and 1 day, 1, 2, and 3 Wk, and 1 mo after noise exposure by measuring auditory brainstem response thresholds, using the Traveler Express apparatus (Bio-logic System, Mundelein, IL). Mice were anesthetized by an IM injection of ketamine hydrochloride (30 mg/kg) and xylazine (2 mg/kg), and each ear was stimulated with an ear probe sealed into the ear canal. The auditory brainstem response in response to click stimuli was recorded, and thresholds were obtained for each ear. Hearing thresholds are shown as mean ± sd.

In a separate experiment, 16 mice in 4 groups (control, isoflurane, halothane, and pentobarbital group, each group n = 4) were exposed to white noise as described above. One week after noise exposure, mice were anesthetized with ketamine hydrochloride and xylazine as above, and both cochlea were removed. The stapes were removed and a small hole was made in the cochlear apex with a fine pick. Fixative (4% formalin and 1% glutaraldehyde in 0.1 M sodium phosphate buffer) was infused through this hole and the cochlea was immersed in fixative for 48 hours at 4°C. After killing the animal, fixation of both cochleas was completed within 10 min. The cochlea were washed with phosphate-buffered saline, the outer bony wall and tectorial membrane were gently removed and the organ of Corti was harvested with fine forceps beginning from the apex.

The organ of Corti was stained with a fluorescent isothiocyanate-conjugated phalloidin probe (Sigma, St Louis, MO), washed and examined at high power magnification (x400) using a fluorescence microscope. The number of fluorescent isothiocyanate-positive cells was counted, and the relative survival ratio (%) of hair cells was calculated by dividing the number of surviving hair cells by the total number of hair cells in each field of view and plotted relative to the percent distance from the apex. Under these magnification conditions, each field of view encompassed a length of 0.32 mm, with 16 consecutive lengths being 5.12 mm long, or about 85% of the entire 6.04 mm length of the organ of Corti (12).

Fixed cochlea, obtained as described above, were incubated in 5.5% EDTA in phosphate-buffered saline for 3 d, dehydrated, and embedded in paraffin, and the paraffin blocks were sectioned at 4 µm thickness along the mid-modiolar axis. The sections were stained with a terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) kit (Fluorescein In Situ Cell Death Detection kit; Boehringer Mannheim, Mannheim, Germany), mounted in Vectashield containing 4`,6-diamidino-2-phenylindole nuclear counterstain (Vector Laboratories, Burlingame, CA), and viewed under a microscope (400x) using epifluorescence.

Statistical analysis of data was performed using ANOVA for repeated measures followed by Bonferroni or Kruskal–Wallis test using SPSS windows (version 12.0). Data were expressed mean ± sd. Changes within or between groups were considered statistically significant when P < 0.05.

	RESULTS

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At 1 d before noise exposure, the hearing threshold was in the normal range (15–25 dB hearing level (HL)) in all four groups of mice. In the control group, the hearing threshold increased to 77.5 ± 8.0 dB HL 1 day after noise exposure. In the pentobarbital, isoflurane, and halothane groups, hearing threshold increased to 62.5 ± 6.3 dB HL, 45.5 ± 9.8 dB HL, and 39.3 ± 6.2 dB HL, respectively, with all three anesthetized groups of mice showing significantly preserved hearing compared with the control group (P < 0.05). But, in mice anesthetized with pentobarbital, hearing loss was more severe than in mice treated with inhaled anesthetics (P < 0.05). Hearing recovered gradually in three groups of anesthetized mice, but did not change in the control group up to 4 wk after noise exposure. At 4 wk after noise exposure, hearing threshold was 76.3 ± 5.8, 50.0 ± 3.5, 28.5 ± 10.8, 30.0 ± 3.7 dB HL in the control, pentobarbital, isoflurane, and halothane groups, respectively (P < 0.05) (Fig. 1).

View larger version (12K): [in this window] [in a new window]   Figure 1. Changes in hearing threshold measured using auditory brainstem responses (ABR) in mice anesthetized with halothane, isoflurane, or pentobarbital, compared with unanesthetized (control) mice (n = 10 each). Mice were anesthetized and exposed to white noise for 3 h per day on three consecutive days as described in the Methods section. Data are presented as mean ± sd. *P < 0.05: control versus other groups. P < 0.05: pentobarbital versus inhalation groups.

One week after noise exposure, viable stereocillia were quantitatively analyzed using cytocochleograms. In the control group (n = 4 mice), the survival rate for each row of hair cells gradually decreased from the apical to the basal turn of the cochlea. At the basal turn (80% of the total length from the apex), the survival rates of the outer and inner hair cells in the isoflurane (n = 4 mice), halothane (n = 4 mice), and pentobarbital group (n = 4 mice) were higher than those of the control group (Fig. 2). The total survival rate of all hair cells was a 65.1 ± 2.0% in the control group, 96.6 ± 0.5% in the isoflurane group, 88.4 ± 0.5% in the halothane group, and 72.6 ± 0.4% in the pentobarbital group (data not shown). The survival rate of total hair cells among the groups was significantly different (P < 0.05).

View larger version (20K): [in this window] [in a new window]   Figure 2. Number of damaged hair cells in the organ of Corti of mice anesthetized with halothane, isoflurane, or pentobarbital, compared with unanesthetized (control) mice. The complete organ of Corti was stained with FITC-phalloidin and was examined at high power magnification (x400). The FITC-positive cells were counted, and the relative survival ratio (%) was calculated by dividing the number of surviving hair cells by the total number of hair cells in each region of examination.

When we performed TUNEL staining for the detection of cell death in the inner ear of control mice, we observed many TUNEL-positive nuclei in the inner and outer hair cells, the stria vascularis, and the spiral ganglion. By contrast, there were fewer TUNEL-positive nuclei in the isoflurane and halothane groups than the pentobarbital group (Fig. 3).

View larger version (25K): [in this window] [in a new window]   Figure 3. Effects of anesthetics on noise-induced cell death. Paraffin blocks of the cochlea of control mice (A) and mice anesthetized with isoflurane (B), halothane (C), or pentobarbital (D) were sectioned at 4 µm thickness along the mid-modiolar axis. The sections were stained with a terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) kit, mounted in Vectashield containing 4`,6-diamidino- 2-phenylindole (DAPI) nuclear counter- stain, and viewed under a microscope (x400) using epifluorescence.

	DISCUSSION

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We have shown here that exposure to intense noise (122 dB SPL) resulted in elevated hearing thresholds (more than 60 dB HL) and 30%–40% hair cell loss in mice, but this damage was attenuated by anesthesia with halothane, isoflurane, or pentobarbital during noise exposure. We also found that attenuation of this damage was less in mice anesthetized with pentobarbital than in those anesthetized with isoflurane or halothane, indicating that pentobarbital has a smaller protective effect than the inhaled anesthetics against NIHL in mice. The other finding was that exposure of mice to three consecutive days of noise induced fragmentation of nuclei in inner ear cells. Mice exposed to noise had many TUNEL-positive cells, suggesting that noise exposure induces cell death of inner ear cells. This noise-induced cell death was more attenuated in the isoflurane and halothane groups than in the pentobarbital group.

ROS has been thought to be a major contributor to noise-induced hair cell damage. In a previous study, we proposed that isoflurane could protect against hearing loss by reducing the generation or action of ROS (11). Halothane and barbiturates have also been shown to have ROS scavenging activity. For example, halothane has been shown to attenuate the toxic effects of ROS on left ventricular pressure development in isolated hearts (13) and to decrease hydroxyl radical generation in ischemic canine hearts (14). Pentobarbital, phenobarbital, methohexital, and thiopental dose-dependently inhibited formation of 2,3-dihydroxybenzoic acid iron-stimulated lipid peroxidation (15).

Other proposed protective mechanisms of isoflurane and halothane include antagonistic effects on N-methyl-d-aspartate (NMDA) receptors and NO. For example, halothane has been shown to depress glutamate-mediated excitatory postsynaptic potentials and to inhibit cGMP production associated with NMDA receptor stimulation (16). This effect is thought to alter glutamate activity by presynaptically altering Ca²⁺ levels. Halothane has also been found to inhibit receptor/calcium-activated NO synthase (NOS) action (17). Furthermore, halothane and isoflurane have been shown to inhibit brain NOS activity in the in vitro system (18). Pentobarbital also has been observed to have a protective mechanism, including the attenuation of NMDA-mediated glutamate excitotoxicity (19). However, pentobarbital has no effect on NOS activity (18).

Other possible mechanisms underlying the protective effects of isoflurane and halothane include vasodilation caused by the activation of K_ATP channels, or by their effect on intracellular Ca²⁺ homeostasis in vascular smooth muscle. Both increased metabolic activity and reperfusion after noise-induced vasoconstriction may intensify ROS production. Intense noise has been found to decrease cochlear bloodflow, leading to rebound reperfusion (20). In addition, noise has been shown to increase the metabolic rate of the cochlea. During acoustic over-stimulation, this upregulation of metabolism leads to perturbations in cochlear homeostasis. The ability of halothane to stimulate the central circulation (21) suggests that this anesthetic may exert its protective effects by preserving bloodflow. Barbiturates, however, have been found to induce a proportional decrease in cerebral bloodflow. Moreover, pentobarbital anesthesia elicited relatively minor effects on cardiac output and regional bloodflow distribution (22). The different vasodilatory and NO inhibitory activities of the inhaled anesthetics and pentobarbital may explain the difference in their protective effects against NIHL.

The protective effects of the anesthetics against noise may be due to their ability to induce unconsciousness. For example, in a group of eight soldiers exposed to noise for more than 2 h, three were unconscious, but the remaining five were able to seek some form of protection. However, labyrinthine damage was greater in the conscious group (23). But, in another case, permanent sensorineural hearing loss was caused by sleeping with an ear against a train window (24). There are no other data available concerning the effects of sleeping on NIHL and, thus, further study is needed to investigate such effects.

In conclusion, we have shown that isoflurane, halothane, and pentobarbital protected against NIHL as well as hair cell damage in mice, with the inhaled anesthetics having a greater protective effect than pentobarbital. Although this animal study has many limitations, our results could help explain the low incidence of NIHL in noisy surgical fields. And it is possible that total IV anesthesia will cause different results compared with inhaled anesthesia. Identification of the mechanisms underlying these effects and the differences among the anesthetics awaits further study.

	Footnotes

 
Supported by Asan Institute for Life Sciences, Seoul, Korea, grant (2004-177).

	REFERENCES

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1. Barr T. Enquiry into the effects of loud sounds upon the hearing of boilermakers and others who work amid noisy surroundings. Trans Phil Soc Glasgow 1886;17:223–39.
2. Slepecky N. Overview of mechanical damage to the inner ear: noise as a tool to probe cochlear function. Hear Res 1986; 22:307–21.[Web of Science][Medline]
3. Yamasoba T, Schacht J, Shoji F, Miller JM. Attenuation of cochlear damage from noise trauma by an iron chelator, a free radical scavenger and glial cell line-derived neurotrophic factor in vivo. Brain Res 1999;815:317–25.[Web of Science][Medline]
4. Fessenden JD, Schacht J. The nitric oxide/cyclic GMP pathway: a potential major regulator of cochlear physiology. Hear Res 1998;118:168–76.[Web of Science][Medline]
5. Puel JL, Ruel J, Gervais d'Aldin C, Pujol R. Excitotoxicity and repair of cochlear synapses after noise-trauma induced hearing loss. Neuroreport 1998;9:2109–14.[Web of Science][Medline]
6. Wangmann P, Schacht J. Homeostatic mechanisms in the cochlea. In: Springer handbook of auditory research. Vol 8. New York: Springer-Verlag, 1996:130–85.
7. Parkin JL, Wood GS, Wood RD, McCandless GA. Drill- and suction-generated noise in mastoid surgery. Arch Otolaryngol 1980;106:92–6.[Abstract/Free Full Text]
8. Holmquist J, Oleander R, Hallen O. Preoperative drill-generated noise levels in ear surgery. Acta Otolaryngol 1979;87:458–60.[Medline]
9. Zou J, Bretlau P, Pyykkö I, et al. Sensorineural hearing loss after vibration: an animal model for evaluating prevention and treatment of inner ear hearing loss. Acta Otolaryngol 2001;121: 143–8.[Medline]
10. Palva T, Karja J, Palva A. High-tone sensorineural losses following chronic ear surgery. Arch Otolaryngol 1973;98:176–8.[Abstract/Free Full Text]
11. Kim JU, Lee HJ, Kang HH, et al. Protective effect of isoflurane anesthesia on noise-induced hearing loss in mice. Laryngoscope 2005;115:1996–9.[Web of Science][Medline]
12. Santi PA, Blair A, Bohne BA, et al. The digital cytocochleogram. Hear Res 2004;192:75–82.[Web of Science][Medline]
13. Tanguay M, Blaise G, Dumont L, et al. Beneficial effects of volatile anesthetics on decrease in coronary flow and myocardial contractility induced by oxygen-derived free radicals in isolated rabbit hearts. J Cardiovasc Pharmacol 1991;18:863–70.[Web of Science][Medline]
14. Glantz L, Ginosar Y, Chevion M, et al. Halothane prevents postischemic production of hydroxyl radicals in the canine heart. Anesthesiology 1997;86:440–7.[Web of Science][Medline]
15. Almaas R, Saugstad OD, Pleasure D, Rootwelt T. Effect of barbiturates on hydroxyl radicals, lipid peroxidation, and hypoxic cell death in human NT2-N neurons. Anesthesiology 2000;92:764–74.[Web of Science][Medline]
16. Zuo Z, De Vente J, Johns RA. Halothane and isoflurane dose-dependently inhibit the cyclic GMP increase caused by N-methyl-d-aspartate in rat cerebellum: novel localization and quantitation by in vitro autoradiography. Neuroscience 1996;74: 1069–75.[Web of Science][Medline]
17. Zuo Z, Tichotsky A, Johns RA. Halothane and isoflurane inhibit vasodilation due to constitutive but not inducible nitric oxide synthase. Implications for the site of anesthetic inhibition of the nitric oxide/guanylyl cyclase signaling pathway. Anesthesiology 1996;84:1156–65.[Web of Science][Medline]
18. Tobin JR, Martin LD, Breslow MJ, Traystman RJ. Selective anesthetic inhibition of brain nitric oxide synthase. Anesthesiology 1994;81:1264–9.[Web of Science][Medline]
19. Daniell LC. Effect of anesthetic and convulsant barbiturates on N-methyl-d-aspartate receptor-mediated calcium flux in brain membrane vesicles. Pharmacology 1994;49:296–307.[Web of Science][Medline]
20. Quirk WS, Shapiro BD, Miller JM, Nuttall AL. Noise-induced changes in red blood cell velocity in lateral wall vessels of rat cochlea. Hear Res 1991;52:217–23.[Web of Science][Medline]
21. Price HL. General anesthetics: the pharmacological basis of therapeutics. New York: Macmillan, 1975;75–102.
22. Manders WT, Vatner SF. Effects of sodium pentobarbital anesthesia on left ventricular function and distribution of cardiac output in dogs, with particular reference to the mechanism for tachycardia. Circ Res 1976;39:512–17.[Abstract/Free Full Text]
23. Rubinstein M, Pluznik N. Effect of anesthesia on susceptibility to acoustic trauma. Ann Otol Rhinol Laryngol 1976;85:276–80.[Web of Science][Medline]
24. Robertson A, Bingham B, McIlwraith G. Can permanent sensorineural hearing loss be caused by sleeping with an ear against a train window? J Laryngol Otol 2002;116:695–8.[Web of Science][Medline]

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