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ORL 1991;53:331-334

Tinnitus-Induced Weight Loss in Rats An Animal Model for Tinnitus Research B. Kellerhals, R. Zogg University ENT Department, Inselspital, Bern, Switzerland

Key Words. Tinnitus • Noise • Ototoxicity • Quinine • Salicylate • Gentamycin Abstract. Growing rats were exposed to noise and/or tinnitus-producing drugs (sodium salicylate, quinine, genta­ mycin). Growth inhibition or weight loss was absent or significantly lower in the noise-only and salicylate-only/ quinine-only groups than in the groups exposed to both noise and drugs. In the gentamycin groups, initially the same trend was found, but general toxicity soon prevailed, leading to heavy weight losses in both groups. The findings clearly favor the hypothesis that tinnitus-induced stress may be the cause for this effect. The experimental set-up thus could serve as an animal model for tinnitus research. In humans, high doses of salicylate or quinine should be avoided in persons exposed to elevated noise levels.

For patients suffering from any kind of inner ear damage, tinnitus often poses heavier problems than the associated hearing loss. Subjective tinnitus is believed to be the result of aberrant neural activity within the audi­ tory pathways, activity that may be erroneously inter­ preted as sound by higher auditory centers. Tinnitus takes a variety of forms and has more than one cause, but the pathophysiological mechanisms of its origin are still unknown [1], Tinnitus research therefore urgently needs animal models for experimental investigations. Evans et al. [2] published experimental data in cats, showing a significant increase of the spontaneous discharge rate of single cochlear nerve fibers after intravenous salicylate administration. For the first time, tinnitus thus could be recorded objectively. Jastreboff and Sasaki [3] showed analogous reduction of the spontaneous interspike inter­ vals in single units of the guinea pig inferior colliculus after salicylate administration. Animal models for tinni­ tus thus do exist, but their highly intricate technology limits their practical use. Sodium salicylate is well known to produce reversible hearing loss and tinnitus. Only recently, Carson et al. [4] and Brennan and Jastreboff [5] observed strange deaths and weight loss in rats within a few days during exposure

to salicylate and noise, whereas salicylate only or noise only were tolerated without apparent impairment of the animals’ well being. No apparent cause for this mortality could be found. The incidence of weight loss and deaths was dose dependent. Histopathological examination of various organs and tissues did not reveal any significant pathologies, especially no bleedings from the gastrointes­ tinal tract nor from any other organs were found. All hematological parameters and blood chemistry were within normal limits. Both research groups considered it premature to speculate on the physiological basis of the mortality observed. The hypothesis arises that tinnitus-induced stress could be the deleterious cause for these observations. This hypothesis could gain further weight, if analogous weight loss and mortality occurred in animals exposed to noise and other tinnitus-producing agents such as qui­ nine or aminoglycoside antibiotics. The present study was designed to answer this question.

Materials and Methods A pilot study with 54 rats (noise only, salicylate only, quinine only, salicylate + noise, quinine + noise) lasting 31 days preceded the present experiments. It confirmed the occurrence of inexplicable growth inhibition or weight loss in animals under the combined

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Introduction

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dB

Table 1. Mean daily aberration (in grams) from the initial body weight during the experimental period ( ± SD) N1 8.9 ±6.4 SI -0 .2 ±3.4 SN1 - 1.0 ± 5.3

N2 S2 SN2

12.8 ± 5.6 13.8 ± 4.5 1.8 ± 8.3

N1+N2 S1+S2 SN1+SN2

10.9 ±6.1 6.8 ±8.2 0.4 ±6.8

Cl 10.9 ±2.3 CN1 4.7 ±6.3

C2 CN2

11.5 ± 4.5 -1.1 ±8.2

C1+C2 CN1+CN2

11.2 ± 3.4 1.8 ± 6.9

Gl 11.7 ±4.1 GN1 4.7 ±6.3

G2 —39.2 ± 11.6 GN2 -42.2 ±28.1

G1+G2 - 13.8 ± 27.9 GN1+GN2 - 18.8 ± 31.2

N = Noise only, S = salicylate only, C = quinine only, G = gen­ tamycin only, SN - salicylate + noise, CN = quinine + noise, GN = gentamycin + noise.

Table 2. Mean daily weight aberration from the initial body weight, statistical significances (Wilcoxon test) N1+N2 t S1+S2 S1+S2 * SN1+SN2 N1+N2 t SN1+SN2

not significant p = 0.05 p - 0.002

N1+N2 4 C1+C2 C1+C2 4 CN1+CN2 N l + N 2 ^ CN1+CN2

not significant p < 0.001 p < 0.01

N1 + N2 4 G1+G2 G1+G2 4 GN1+GN2 N1 + N2 4 GN1+GN2

p < 0.1 not significant p < 0.001

N = Noise only, S = salicylate only, C = quinine only, G = gentamycin only, SN = salicylate + noise, CN = quinine + noise, GN = gentamycin + noise.

influence of noise and tinnitus-inducing drugs, and it allowed deter­ mination of the optimal duration of the experimental periods. In the present study, 44 growing female Sprague-Dawley rats (mean 257 ± 12 g SD) were divided into seven groups as follows: Group 1, 8 rats: noise exposure and physiological saline s.c. (16 consecutive days, groups N1 (days 0-8) and N2 (days 8-16). Group 2, 6 rats: salicylate only (days 0-8, group SI) and salicylate + noise (days 8-16, group SN2). Group 3, 6 rats: quinine only (days 0-8, group C l), quinine + noise (days 8-16, group CN2). Group 4, 6 rats: gentamycin only (days 0-8, group G l), gentamycin + noise (days 8-16, group GN2). Group 5, 6 rats: salicylate + noise (days 0-8, group SN1), salicylate only (days 8-16, group S2). Group 6, 6 rats: quinine + noise (days 0-8, group CN1), quinine only (days 8-16, group C2). Group 7, 6 rats: gentamycin + noise (days 0-8, group GN1), and gentamycin only (days 8-16, group G2). The experiments were initiated after an acclimatization period of 6 days. During these 6 days the mean weight increased from 214 ± 10 to 257 ± 12 g. The experimental period lasted 16 days. After 8 days, the drugonly groups were exchanged for the drug and noise groups in order to exclude any undue influence of possibly biasing factors such as slightly different room conditions. At the same time, the exchange could test the reversibility of the drug/noise effects. The daily doses were 300 mg/kg body weight for sodium salicy­ late (intraperitoneally administered), 50 mg/kg for quinine (subcuta­ neously administered) and 80 mg/kg for gentamycin (subcuta­ neously administered). The noise-only animals were injected physi­ ological saline subcutaneously. Food and water were available ad libitum, and the rats were maintained on a 12 h light/dark cycle throughout the experiment. Daily weight measurements were per­ formed at the beginning of the daylight period before the noise exposure and before the drug injections. Noise exposure was delivered by a double car horn driven by compressed air. The daily exposure time was 1 h divided into 4 exposure periods irregularly distributed over 12 h. The horn was placed at 1 m in front of the cage rack. The noise intensity at this

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Fig. 1. Frequency spectrum of the noise (double car horn). Fig. 2. Mean weight change (noise and salicylate groups). N = Noise only, S = salicylate only, SN = salicylate and noise.

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distance was 120-125 dBA, the frequency spectrum is shown in figure 1. The drug-only animals were kept in an adjacent room with an ambient noise of less than 50 dBA. After the experiment, one sample animal of each group was taken for autopsy.

Results The pilot study had shown that an initial weight loss occurred especially in the noise/drug animals. Later on, most animals seemed to recover at least partially in all experimental groups. After 8 days, prolongation of the experiment thus was ineffective in sharpening the inter­ group differences. The pilot study also showed that intra­ peritoneal quinine administration resulted in deleterious side effects (paralytic ileus) which unduly influenced the body weight. In the present study, too, a more or less pronounced initial weight loss was followed by a new weight increase after some days in the groups S, SN, C, CN (fig. 2, 3).

The intergroup differences tended to decrease toward the end of the experimental period in the S/SN and C/CN groups. The mean daily aberration from the initial body weight was found to discriminate the groups better than the mean final weight difference. For all drugs tested, the drug/noise groups showed greater weight loss or growth inhibition than the noise-only and drug-only groups (tables 1, 2). For salicylate, there was statistical significance (table 2) for S ^ SN and N 4 SN, whereas there was no statistical significance for S 4 N. Even higher statistical evidence was found for C 4 CN and N 4 CN. Again, there was no statistical significance for C 4 N. For the gentamycin groups, no such statistical evi­ dence was obtained, although during the first 8 days, gentamycin animals showed almost normal weight in­ crease, whereas GN animals soon started to loose weight (fig. 4). During the second experimental period, all gen­ tamycin animals showed an enormous weight decrease, which was almost equal in the gentamycin-only and the gentamycin/noise groups (fig. 5).

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Fig. 3. Mean weight change (noise and quinine groups). N = Noise only, C = quinine only, CN - quinine and noise. Fig. 4. Mean weight change (noise and gcntamycin groups). N 1 + N2 (Noise (16 days), G1 = gentamycin (first 8 days), GN1 = gentamycin and noise (first 8 days). Fig. 5. Mean weight change (noise and gcntamycin groups). N1 + N2 = Noise only (16 days), G 1 + G2 = gentamycin (both experimen­ tal periods), GN1 + GN2 = gentamycin and noise (both experimen­ tal periods).

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Discussion Noise overstimulation leads to hearing loss and tinni­ tus. Both sequelae can be temporary or permanent. Sodium salicylate produces reversible tinnitus and hear­ ing loss, which subside within 2-3 days. Quinine can lead to permanent inner ear damage in exceptional cases, normally it causes reversible tinnitus and hearing loss only. Gentamycin is well known to produce permanent hair cell losses in the organ of Corti as well as in the labyrinthine end organs. In man, tinnitus is an early sign of inner ear damage due to aminoglycoside treatment, preceding the development of permanent hearing loss. Rats are known to be very sensitive to stress, but in no experimental animal, experimentally induced deafness was ever followed by weight losses or mortality. The del­ eterious effect of the combined exposure to noise and tinnitus producing agents thus cannot be due to the hear­ ing loss. Stress by tinnitus only can explain these obser­ vations, although further experiments are needed in or­ der to find the physiological mechanisms, how the tinni­ tus-induced stress influences the rat metabolism. Our results clearly favor this hypothesis: For salicylate and quinine, the noise-only and drug-only groups thrived significantly better than the animals exposed to noise and drugs. The lack of analogous statistical proof in the gen­ tamycin groups could be explained by presumable general drug toxicity. During the first half of the experimental period, gentamycin/noise animals had a significantly lower weight increase than gentamycin-only animals. In the second half of the experimental period, however, gen­ eral toxicity seemed to be more effective than tinnitusinduced stress. In experimental animals, gentamycin is known to produce inner ear damage after 10 or more days only, whereas salicylate and quinine in humans lead to tinnitus almost immediately. Therefore gentamycin toxic­ ity probably preceded the onset of tinnitus in our experi­ mental set-up with elevated gentamycin doses. The results of the second experimental period after exchange of drug-only and the drug/noise groups showed perfect reversibility of the drug/noise effects in the sali­ cylate and quinine animals. This reversibility further con­ firms the specific effect of the noise/drug combination.

In contrast to Carson et al. [4] and Brennan and Jastreboff [5], no sudden deaths were observed in our series, neither occurred such mortality in our pilot study with an observation time of more than 3 weeks. The difference between the noise-only or drug-only groups and the combined groups tends to diminish with increasing observation time after some days. This fact, however, does not contradict our hypothesis: long-last­ ing stress-inducing conditions are well known to lead to habituation mechanisms tending to diminish the stress reactions with time. Thus quinine injections combined with noise seem to be the most reliable animal model for tinnitus-induced stress effects in rats. Salicylate com­ bined with noise is slightly less reliable, whereas genta­ mycin proved to be useless for our purpose. Further studies should concentrate on the determina­ tion of the presumed hormonal stress factors that lead to growth inhibition or weight loss under the combined influence of noise overstimulation and tinnitus-produc­ ing drugs. The question arises, too, if salicylate and qui­ nine ingestion in humans exposed to professional noise shoud be avoided. The drug and noise doses used in the present study were higher than the usual human dosage. In order to minimize any risks, however, high doses of salicylate or quinine should not be recommended in per­ sons exposed to elevated professional noise levels.

Acknowledgment The study was supported by grants from the Zogg Kellerhals Foundation for Inner Ear Research

References 1 Evered D, Lawrenson G (eds): Tinnitus. Ciba Foundation Sym­ posium 85. Bath, Pitman Press, 1981. 2 Evans EF, Wilson JP, Borerwe TA: Animal models of tinnitus; in Evered D, Lawrenson G (eds); Tinnitus. Ciba Foundation Symposium 85. Bath, Pitman Press, 1981, pp 108-129. 3 JastrebofT PJ, Sasaki CT: Salicylate-induced changes in sponta­ neous activity of single units in the inferior colliculus of the guinea pig. J Acoust Soc Am 1986;80:1384-1391. 4 Carson SS, Prazma J, Pulver SH, Anderson T; Combined effects of aspirin and noise in causing permanent hearing loss. Arch Otolaryngol 1989;115:1070-1075. 5 Brennan JF, JastrebofT PJ: Interaction of salicylate and noise. Results in mortality of rats. Experientia 1989;45:731-734. Received: January 17, 1991 Accepted: January 30, 1991 Prof. B. Kellerhals, Universitats-HNO-Klinik Inselspital, CH-3010 Bern (Switzerland)

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The sample autopsies did not reveal any gross pathol­ ogy in all experimental groups. Preyer’s reflex was regu­ larly lost immediately after the noise exposure in all ani­ mals, but in most animals it recovered during the silent intervals throughout the whole experimental period.

Tinnitus-induced weight loss in rats. An animal model for tinnitus research.

Growing rats were exposed to noise and/or tinnitus-producing drugs (sodium salicylate, quinine, gentamycin). Growth inhibition or weight loss was abse...
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