Assessment of ototoxicity in experimental animals

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Assessment of ototoxicity in experimental animals Charles M. Henley and R. Don Brown Ototoxicity limited the use of the antitubercular drug, streptomycin, and there are many other drugs with similar damaging side-effects. Cochlear toxicity represents one form of ototoxicity, and studies may include analyses of behavioral, anatomical, electrophysiological and biochemical changes. However, each parameter has drawbacks and limitations, and in this review Charles Henley and Don Brown discuss why a combined electrophysiological and anatomical approach represents the most effective method of assessing risk of ototoxicity. The purpose of toxicity testing in animals is to obtain (predictive) information applicable to the human, a formidable task considering the n u m b e r of reports of species, strain and sex differences in response to drugs. Dearborn 1 lists over 40 variables affecting toxicity studies illustrating the difficulty of proper control of comparative toxicity studies and the risk in attributing a difference in toxicity to a single variable. Ototoxicity refers to the vestibular apparatus as well as the cochlea; however this review will be limited to a discussion of cochlear toxicity. Cochlea toxicity Research of the cochlea has received more attention than the vestibular apparatus. Animals' responses to ototoxic agents, especially noise and/or drugs, depend upon m a n y variables; consequently, cochlear toxicity studies include examination of behavioral, anatomical, electrophysiological, and biochemical changes 2. Behavioral studies Behavioral methods are expensive and time consuming, requiring extensive animal and reR. Don Brown is Professor in the Department of Pharmacology and Therapeutics, Louisiana State University School of Medicine, P.O. Box 33932, Shreveport, LA 71130-3932, USA. Charles M. Henley Ill is a Postdoctoral Fellow in the same department. 1985, Elsevier Science P u b l i s h e r s B.V., A m s t e r d a m

searcher training. Behavioral procedures are effectively learned only after first-hand, supervised laboratory instruction. Moreover, sufficient knowledge in behavioral analysis is necessary to confront the numerous and complex problems peculiar to behavioral studies. Behavioral studies are a useful adjunct to anatomical and electrophysiological studies. Moody and Stebbins 3 discuss effectiveness and adaptability of behavioral procedures in assessing several aspects of auditory function. The behavioral response may be the most direct, non-invasive indication of h o w well the animal actually 'hears' during or following exposure to an ototoxic agent. Often auditory-evoked potentials in conjunction with behavioral thresholds provide predictive data; however, the validity of using behavioral thresholds alone has been questioned 4. Anatomical studies Anatomical studies on the effects of ototraumatic agents on the cochlea provide an overall view of pathological changes induced by these agents. Cochlear tissue may be examined by light microscopy, transmission electron microscopy, and scanning electron microscopy (SEM; see Fig. 1). 'Surface preparations' of the organ of Corti are useful in detecting irregularities in hair cells or supportive cells of the reticular lamina. SEM surface preparations may be fractured for

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studying the interior of the organ of Corti. Dissection difficulty varies with the animal model, investigator experience and proper application of techniques. For example, overdecalcified tissue may be too fragile to dissect whereas underdecalcified tissue, too tough. The length of the decalcification period is variable and is often determined only with ' h a n d s - o n ' experience. Once properly prepared, hair cells may be counted and recorded in a cochleogram (a diagrammatic ' m a p ' of cochlear hair cells). Above a certain threshold reduction in hair cell n u m b e r is directly related to ototoxicity. Cochlear lesions may then be compared with electrophysiological findings. Anatomical studies may correlate directly with electrophysiological findings. However, discrepancies may occur making it difficult to determine precisely the nature and extent of the anatomical acoustic lesion through the sole use of cochlear potentials s or, conversely, to assess the functional status on the basis of anatomical findings alone 6. Round w i n d o w recordings of cochlear potentials represent hair cell activity weighted towards cells in the basal region of the cochlea. Output from more distal cells is attenuated through the perilymph; the same technical problems exist with differential, intracochlear electrodes s. Thus, combined electrophysiological and anatomical studies represent the best approach in assessing the effects of ototoxic agents and establishment of damage risk criteria. Electrophysiological studies Electrophysiological studies are used extensively in auditory research and include monitoring of the N1 potential (cochlear comp o u n d action potential), the a.c. cochlear potential (ACCP) or cochlear microphonics, and the positive d.c. endocochlear potential (EP). N1 is a volume conductor recording of the action potentials of the primary, first order auditory afferents, whereas the ACCP is a receptor potential derived from hair cell activity and is an electrical analog of the acoustic stimulus. The EP represents the d.c. polarization of the central compartment of the cochlea (scala media) 7. More

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recently, the b r a i n s t e m a u d i t o r y evoked potential has b e e n s h o w n to b e useful, especially in chronic studies 4. The ACCP is valuable in auditory research for the following reasons: (1) Changes in hair cell activity can often be detected before behavioral changes, suggesting possible physiological mechanisms underlying behavioral deficits; (2) ACCP changes m a y be used to predict changes in N1 and in b r a i n s t e m recorded potentials; (3) the ACCP is a non-fatigable response u n d e r normal acoustic stimulation eliminating variation due to adaptation; and (4) loss and recovery of the ACCP correlate fairly well w i t h behavioral t e m p o r a r y threshold shift's (TTSs) 6. The p h e n o m e n a of the TTS and p e r m a n e n t threshold shift (PTS) seen in cochlear electrophysiology are of p r i m a r y interest to researchers. Damage risk criteria for certain levels of intense s o u n d are difficult to establish; i.e., it is difficult to d e t e r m i n e w h e t h e r the s o u n d will p r o d u c e a TTS or a PTS. In addition, the damage risk criteria established m a y be species-dependent. Furthermore, the d u r a t i o n of the TTS is not easily ascertained. Kisiel a n d Bobbin 6 have discussed long-term recovery of the ACCP following severe acoustic trauma. The a m o u n t of recovery seems indep e n d e n t of the initial change in sensitivity a n d the degree of initial loss does not accurately predict w h e t h e r recovery or cont i n u e d loss of function will occur. ACCP recovery appears to be a complicated process d e p e n d e n t on healing of cells, return to normal functioning of cells receiving m i n i m a l damage, and delayed degeneration. This becomes i m p o r t a n t in noise--drug interaction studies. Recordings before recovery from a TTS i n d u c e d b y noise alone m a y lead one to falsely assume that the observed effects are from the combination of noise plus drug. Cochlear i m p a i r m e n t in noise-drug studies m a y be t e m p o r a r y or p e r m a n e n t and d e p e n d s u p o n variables such as intensity and duration of noise exposure, dose and duration of drug treatment, the animal species and the presence or absence of a concomitant

Fig. 1. Scanning electron micrographs of the upper basal turn of the cochleas of an untreated, control rat (,4) and of a rat exposed to short duration, high intensity noise then treated with chloramphenicol (B). (,4) Normal - note the characteristic 'V' or 'W' pattern formed by the stereocilia of the three rows of outer hair cells and the erect stere~ilia on the single row of inner hair cells. (B) Noise then chloramphenicol pathological changes include loss of outer hair cells ('phalangeal scars') and missing, clumping and abnormally shaped stereocilia on the remaining outer hair cells. Also note the complete absence of inner hair cell stereocilia.

infectious process w i t h i n the m i d d l e or inner ear. In our investigations of noise and chloramphenicol on cochlear function in rats, we have observed both XTSs and PTSs and have concluded that a post exposure stabilization period of 21-30 days is n e e d e d to distinguish b e t w e e n a TTS and a PTS 8. Also, otitis m e d i a m a y be a confounding variable for the production of p e r m a n e n t or temporary cochlear deficits caused b y chlorampheni-

col and noise. The post-treatment stabilization period is also important in anatomical studies. If this period is too long, it m a y be impossible to distinguish the initial (primary) site of action of acoustic trauma a n d / o r drug ototoxicity from seco n d a r y sites. Damage at secondary sites m a y represent nonspecific degenerative changes and thus m a y h i n d e r determination of the mechanism of action of the ototoxic agent(s). On the other

366 hand, if the post-treatment p e r i o d is too short, d a m a g e as a result of p r i m a r y metabolic effects m a y not have occurred or m a y not be readily apparent, especially if surface preparations alone are examined. Therefore, the nature of the s t u d y should dictate the length of the stabilization period. N~ has not b e e n u s e d as extensively as the ACCP in s t u d y i n g the effects of acoustic exposure. A t first N1 was b e l i e v e d to reflect neural activity from only the basal turnT; however, nerve fibers from the h i g h e r turns m a y contribute to N1 (Ref. 9). W i t h this in m i n d , i n p u t / o u t p u t functions of N1 (outp u t voltages v. s o u n d pressure levels) m a y b e the most sensitive index of hair cell loss. A l t h o u g h it is useful to m o n i t o r N1 in conjunction w i t h the ACCP, N1 is a p p a r ently more susceptible to acoustic trauma than is the ACCP, limiting its utility. Davis 7 felt that N1 is too labile for m o n i t o r i n g the effects of acoustic trauma. In recent noise studies, we f o u n d significant differences in ACCPs b e t w e e n different exposure groups TM. H o w ever, N1 responses were low or non-existent in all noise exposed groups, m a k i n g intergroup comp a r i s o n s of N1 difficult or i m p o s s ible. It is possible that noise exposure d i s r u p t e d the integrity of the organ of Corti such that asynchronous discharges of the nerve fibers occurred, p r e v e n t i n g the 'volleying' w h i c h p r o d u c e s N1. Thus, N1 m a y be too labile for some noise studies although it is clearly a very sensitive index of cochlear i m p a i r m e n t caused b y noise.

Surgical and recording procedures Studies of i n n e r ear function are complicated, difficult to interpret, a n d often h a n d i c a p p e d b y surgical procedures necessary to record cochlear potentials, n In a d d i t i o n to altering the normal conditions of the a u d i t o r y a p p a r a t u s and changing the acoustic p r o p e r t i e s of the m i d d l e ear a n d the transfer of mechanical energy, n u m e r o u s pitfalls a c c o m p a n y the surgery and electrode placement. W h e n placing an electrode on the r o u n d w i n d o w m e m b r a n e (RWM), rupture and leakage of p e r i l y m p h m a y occur. Surgically-traumatized tissue m a y leak serous fluid onto

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the RWM, significantly depressing recorded potentials. The rat is susceptible to fluid collection since the RWM is located in a niche formed b y the posteriomedial b o r d e r of the t y m p a n i c bulla a n d the patent, dilated, stapedial artery. (Extreme care to avoid r u p t u r e of the stapedial artery u p o n RWM exposure obviates the resultant excessive b l e e d i n g w h i c h makes recordings impossible.) RWM exposure and electrode placement is much easier in the g u i n e a - p i g a n d cat; the RWM is more accessible and the stapedial artery is absent. Fluid accumulation, checked at intervals d u r i n g the recording period, can be r e m o v e d w i t h dental p a p e r tips; often, the recording procedure can continue. In the rat, removal of fluid is more difficult, time c o n s u m i n g and can interrupt or prevent further recording. The presence of otitis m e d i a also h a m p e r s RWM recordings. Serous otitis m e d i a m a y lead to fluid over-accumulation which cannot be effectively removed. Purulent or m u c o i d otitis m e d i a m a y interfere w i t h ossicular chain function a n d / o r completely occlude the RWM m a k i n g electrode placement impossible. In chronic otitis media, the t y m p a n i c bulla becomes thick, brittle, a n d highly vascularized m a k i n g dissection difficult. Excessive fluid effusion often results. The guinea-pig, rabbit, chinchilla, and particularly the rat are susceptible to m i d d l e ear and respiratory infections (probably linked). In an earlier d r u g - n o i s e s t u d y w i t h rats, high incidence of otitis m e d i a (92%) was observed, which resulted in loss of valuable research time (Henley, C. M. and Brown, R. D., u n p u b l i s h e d observation). In a s u b s e q u e n t study, the incidence of otitis m e d i a (57%) dictated that the experiment be repeated 8. Through special care procedures, w e have reduced the incidence of infections. I m m e d i a t e removal of infected animals, a d e q u a t e temperature and h u m i d i t y control, use of sterilized water bottles, a n d placement of a b s o r b e n t material on waste p a n s b e n e a t h wire cages are precautionary measures taken. In a d d i t i o n to h a m p e r i n g recordings, acute otitis m e d i a m a y make the animal more susceptible

to ototoxic agents s. Experimentally i n d u c e d bacterial otitis m e d i a appears to significantly affect cochlear function in the rat 12. Thus, w h e n investigating the effects of ototoxic agents, animals w i t h observable otitis m e d i a should be excluded. W h e n the incidence is high, the study should be repeated or evaluated w i t h caution since resolution of earlier infections m a y have occurred in some animals.

Acoustic stimulation procedures W i t h regard to acoustic stimulation in particular, the use of an o p e n v. closed system, and the importance of e m p l o y i n g reliable, reproducible m e t h o d s of sound presentation cannot be overemphasized. W i t h an open system pure tones or clicks are emitted such that the resultant sound source may not be placed in the same position relative to each animal's ear drum. Though not as critical at low frequencies, at higher frequencies this can result in differences in the intensities of s o u n d presented due to cancellation or summation of reflected s o u n d waves. At high frequencies, m o v e m e n t of the source of only a quarter of the wavelength (which can be less then a centimeter; for example at 10 kHz, the quarter-wave-length = 0.89 cm) can cause significant sound pressure variations reaching the ear d r u m 13. The open system is sensitive enough to make intergroup comparisons with sufficient numbers of animals. In our experience, the standard deviation of response w i t h i n a group m a y be somewhat high, p r o b a b l y as a result of differences in the individual animal's susceptibilities to noise a n d / o r drugs c o m b i n e d with variability induced b y the method of acoustic stimulation. This variability can be reduced b y employing a closed system of acoustic stimulation. W i t h a closed system, sound is presented in a reproducible manner via 'cannulation' of the external auditory canal with a sealed tube and its attached s o u n d transducer. A p r o b e tube can be included to calibrate the sound. The closed system, though more desirable, is more expensive to e q u i p for.

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TIPS - September 1985 Animal models, biochemical considerations M a n y ototoxic d r u g s result in p e r m a n e n t i m p a i r m e n t in cochlear f u n c t i o n . In a d d i t i o n , o p p o r t u n i s t i c d a t a f r o m p a t i e n t s receivi n g a c o m b i n a t i o n of d r u g s or suffering from severe pathophysiology, s u c h as renal failure, c o m p l i c a t e s i n t e r p r e t a t i o n of d r u g i n d u c e d ototoxicity. T h e r e f o r e , a n i m a l m o d e l s are e s s e n t i a l to cochlear toxicity studies, a n d also in s t u d i e s of t h e m e c h a n i s m of ototoxic action of t h e s e drugs. Predictions from animal studies agree w e l l w i t h the clinical experie n c e in h u m a n s in relation to r e l a t i v e ototoxic p o t e n c i e s of d r u g s s u c h as the a m i n o g l y c o sides 14. A n a n i m a l m o d e l w h i c h yields data likely to b e r e l e v a n t to the human m u s t be chosen. For e x a m p l e , if c h l o r a m p h e n i c o l w a s b e i n g i n v e s t i g a t e d for its ototoxic potential, v a r i o u s factors w o u l d i n f l u e n c e the choice of a p p r o priate model. Chloramphenicol primarily undergoes glucuronid a t i o n in its m e t a b o l i c d i s p o s i t i o n in the h u m a n 15. T h e cat, w i t h a glucuronyl transferase deficie n c y 16, e l i m i n a t e s c h l o r a m p h e n i col at a s l o w e r rate t h a n the h u m a n ; thus, is an i n a p p r o p r i a t e c h o i c e for c h l o r a m p h e n i c o l c o m p a r a t i v e toxicity studies. O n the o t h e r h a n d , the cat m a y b e a u s e f u l m o d e l for the h u m a n n e o nate, w h i c h has an u n d e v e l o p e d microsomal enzyme system and an i m p a i r e d ability to c o n j u g a t e chloramphenicol with glucuronic acid TM. In the rat, 80% of the chlora m p h e n i c o l g l u c u r o n i d e is excreted in the bile ( c o m p a r e d w i t h

5 % in the h u m a n ) , a n d there is c o n s i d e r a b l e e n t e r o h e p a t i c circulation 1s,17. Thus, a c c u m u l a t i o n of c h l o r a m p h e n i c o l is p r o b a b l y different in the rat t h a n in m a n . In the g u i n e a - p i g a n d t h e rat, a larger p e r c e n t a g e of m e t a b o l i t e s are excreted in the b i l e a n d feces t h a n in m a n a n d t h e r e is also a s i g n i f i c a n t a m o u n t of n i t r o - r e d u c t i o n p r o d u c t s f o r m e d 17, w h i c h m a y also b e r e a b s o r b e d . To c o m p l i c a t e m a t t e r s further, sex differences h a v e b e e n r e p o r t e d in the rat for the nitroreduction metabolic pathway and a n u m b e r of m e t a b o l i c o x i d a t i o n reactions TM. Thus, s i g n i f i c a n t species differences exist in the m e t a b o l i c d i s p o s i t i o n of drugs. Also, p r o b l e m s m a y d e v e l o p as a result of species a n d i n d i v i d u a l differences in d r u g a b s o r p t i o n , d i s t r i b u t i o n , excretion, p r o t e i n b i n d i n g a n d m e m b r a n e t r a n s p o r t 19. W h e n cond u c t i n g a n i m a l e x p e r i m e n t s for the p u r p o s e of a s s e s s i n g t h e ototoxic p o t e n t i a l of a drug, it is of u t m o s t i m p o r t a n c e to k n o w , for t h e particular d r u g in q u e s t i o n , h o w close the test a n i m a l is to man. Acknowledgements The authors wish to acknowledge the help of Vicki Davis, Judy Horner, D a w n Britt and Bob Jankowski for their editorial assistance. References 1 Dearborn, E.H. (1967) Fed. Proc. 26, 1075-1077 2 Brown, R.D. and Daigneault, E.A. (eds) (1981) Pharmacology of Hearing: Experimental and Clinical Bases, John Wiley, New York 3 Moody, D.B. and Stebbins, W.C. (1982) in Nervous System Toxicology (Mitchell, C. L., ed.) pp. 109-131. Raven

Press, New York 4 Henderson, D., Hamernik, R. P., Salvi, R.J. and Ahroon, W. A. (1983) Audiology 22, 172-180 5 Durrant, J. D. (1976) in Effects of Noise on Hearing (Henderson, D., Hamernick, R.P., Dosanjh, D.S. and Mills, J.H., eds), 179-197, Raven Press, New York 6 Kisiel, D. L. and Bobbin, R. P. (1981) in Pharmacology of Hearing: Experimental and Clinical Bases (Brown, R.D. and Daigneault, E.A.), pp. 231-270. John Wiley, New York 7 Davis, H., Benson, R. W., Covell, W. P., Fernandez, C., Goldstein, R., Katsuki, Y., Legouix, J.-P., McAuliffe, D. R. and Tasaki, I. (1953) J. Acoust. Soc. Am. 25, 1180-1189 8 Henley, C.M., Brown, R.D., Penny, J. E., Kupetz, S. A., Hodges, K. B. and Jobe, P. C. (1984) Neuropharmacology 23, 197-202 9 Davis, H. (1961) in Sensory Communication (Rosenblith, W. A., ed.), pp. 119, Wiley Press, New York 10 Henley, C.M., Brown, R.D., Penny, J. E. and Jobe, P. C. (1984) Fed. Proc. 43, 220 11 Lawrence, M. (1976) in Handbook of Auditory and Vestibular Research Methods (Vernon, J. and Smith, C., eds), pp. 181-207. Charles C. Thomas, Springfield 12 Henley, C.M., Purdy, K.A., Brown, R.D., Penny, J.E. and Wallace, M.S. (1985) Association for Research in Otolaryngology. Eighth Midwinter Research Meeh'n~, Abs. 172, 131-132 13 Vernon, J., Katz, B. and Meikle, M. (1976) in Handbook of Auditory and Vestibular Research Methods (Vernon, J. A. and Smith, C. A., eds) pp. 306-356. Charles C. Thomas, Springfield 14 Brummett, R. and Fox, K. (1982) in The Aminoglycosides (Whelton, A. and Neu, H. C., eds), pp. 419-451, Marcel Dekker, New York, Basel 15 Smith, A.L. and Weber, A. (1983) Pediatric Clinics of N. America 30, 209236 16 Dutton, G.J. and Greig, C.G. (1957) Biochem. J. 66, 52 17 Glazko, A.J., Dill, W.A. and Wolf, L. M. (1952) J. Pharmacol. Exp. Ther. 104, 452-458 18 Kato, R. and Gillette, J.R. (1965) J. Pharmacol. Exp. Ther. 150, 279-284 19 Williams, R.T. (1967) Fed. Proc. 26, 1029-1039