Bilirubin and the Auditory System

Original Article &&&&&&&&&&&&&& Bilirubin and the Auditory System

Steven M. Shapiro, MD kernicterus have normal intelligence with severe movement disorders; Hajime Nakamura, MD they are trapped in writhing, contorted bodies that make speaking, keyboarding, and communicating difficult. Visual motor integration problems and deafness or auditory processing problems make the situation all more difficult for these children and adults. The auditory system is highly sensitive to bilirubin toxicity. Damage to the The central auditory system is highly sensitive to bilirubin toxicity. auditory nervous system includes auditory neuropathy or auditory There is now evidence that isolated auditory processing problems may dyssynchrony and auditory processing problems which may occur with or occur in children without other signs of classical kernicterus. without deafness, hearing loss. Auditory dysfunction may occur in children Damage to the auditory nervous system may occur at levels of total with or without other signs of classical kernicterus. Bilirubin selectively serum bilirubin previously considered to be safe. The auditory system damages the brainstem auditory nuclei, and may also damage the auditory is not only important in the diagnosis and treatment of kernicterus, nerve and spiral ganglion containing cell bodies of primary auditory but noninvasive auditory neurophysiological tests play an important neurons. The inner ear, thalamic and cortical auditory pathways appear to role in the early detection of central nervous dysfunction due to be spared. Noninvasive auditory neurophysiological tests such as the bilirubin in the neonate. auditory brainstem response (ABR) or brainstem auditory response (BAER) This article describes the areas of damage to the auditory nervous play an important role in the early detection of bilirubin-induced auditory system, which we have seen in the laboratory. The discussion begins and central nervous system dysfunction in the neonate. at the cellular level and moves to neuroanatomic areas of damage Journal of Perinatology 2001; 21:S52 – S55. and their relation to changes in the auditory brainstem response (ABR), also known as brainstem auditory-evoked potential (BAEP) or brainstem auditory-evoked response (BAER). It then relates these findings to kernicterus and the auditory system in humans, to the use OVERVIEW of the ABR to detect early auditory system dysfunction, and to newly Classic kernicterus, permanent brain damage due to excessive reported auditory sequelae of newborn jaundice (i.e., auditory hyperbilirubinemia, is a tragic and usually preventable disorder of neuropathy, also known as auditory dyssynchrony). the brain that occurs in the neonatal period. Kernicterus or bilirubin A classic study of kernicterus and the auditory system by Dublin1 encephalopathy primarily involves the auditory system as well as in 1951 was entitled ‘‘Neurological lesions in erythroblastosis fetalis central nervous systems that control movement. Classic kernicterus is in relation to nuclear deafness,’’ in which nuclear referred to the a tetrad, which includes deafness or dysfunction of the auditory abnormalities found in the brainstem auditory nuclei, specifically the system movement disorders such as athetosis, dystonia, and cochlear nuclei. Dublin found abnormalities of the brainstem hypotonia oculomotor disturbances, especially paresis of upgaze, and auditory structures without finding abnormalities of the inner ear. A dental enamel hypoplasia of primary teeth. more recent study in premature infant by Ahdab-Barmada and Formerly, kernicterus accounted for approximately 10% of cases of Moossy in 19842 clearly showed damage to the brainstem cochlear static encephalopathy or cerebral palsy. Typically, these children had nuclei in premature human infants with kernicterus, and their normal intelligence without significant cerebral cortical involvement findings were similar to those observed with classic kernicterus in and infrequent seizures. The athetotic and dystonic movement term infants and in Gunn rats. Kernicterus does not affect the inner disorders of kernicterus correspond to lesions in the basal ganglia, ear, either in humans3,4 or in Gunn rats.5,6 especially the globus pallidus and subthalamic nucleus. The Purkinje Audiometric findings in humans generally show bilateral high- cells of the cerebellum are also particularly vulnerable and, as a frequency symmetric sensory neural hearing loss with recruitment.7 result, hypotonia is a common finding. Many patients with severe Recruitment or abnormal loudness growth functions are also found. Although this problem is often due to hair cell loss in the inner ear, it can also be due to damage of the auditory nerve or the brainstem Department of Neurology ( S.M.S. ), Medical College of Virginia, Richmond, VA; and Department of Pediatrics ( S.M.S., H.N. ), Kobe University School of Medicine, Kobe, Japan. cochlear nuclei, as emphasized by Dublin.1 Also, Matkin and Carhart7

Address correspondence to Steven M. Shapiro, MD, Department of Neurology, Medical College of reported central auditory nervous system abnormalities including Virginia, Randolph Minor Hall, 307 College Street, 7th Floor, Richmond, VA 23219. patients with decreased binaural fusion, auditory aphasia and

imperception, and word deafness. They described patients labeled as neurons from the brainstem cochlear nuclei also produces ‘‘deaf’’ when the audiogram showed normal thresholds. Systematic hyperexcitability, with depolarization of neurons that normally studies of the central auditory processing problems were not done, but would be quiescent. clearly these reports agree with the pathology of the central auditory nervous system. More recent retrospective studies by Bergman et al.8 and de Vries et al.10 have shown a relationship between LOCALIZATION OF ABNORMALITIES IN THE AUDITORY hyperbilirubinemia and deafness. De Vries et al. showed that not only SYSTEM the amount of bilirubin but also the duration of exposure to high Neuroanatomic Studies levels of bilirubin was related to deafness. Prospective studies We investigated the expression of two calcium binding proteins, describing no significant risk of hearing loss with hyperbilirubinemia calbindin and parvalbumin, in the auditory system of jaundiced must be examined closely, since risk is not well assessed by using only Gunn rats given sulfonamide to exacerbate bilirubin toxicity. Rats a single total serum bilirubin in the neonate, and since dependent were given sulfa on day 15; on day 17 ABRs were performed and the variables that use audiograms or survey reports of hearing loss may animals were sacrificed. Immunohistochemistry, performed by my miss children with auditory neuropathy or central auditory colleague (Dr. Robert F. Spencer) revealed that the reduced processing disorders. expression of these calcium-binding proteins is a very sensitive index of brain damage due to bilirubin. The reduced expression is selective. Brainstem nuclei sensitive to bilirubin neurotoxicity show loss of LABORATORY STUDIES activity whereas neighboring nuclei are unaffected. We have studied bilirubin toxicity in cultured neurons, in In the auditory nerve terminals in the brainstem ventral cochlear hippocampal tissue slices, and in the jaundiced Gunn rat. The nucleus, the density and immunoreactivity of calcium-binding Gunn rat, derived from the Wistar strain,11 is the classical proteins are reduced in jaundiced Gunn rats compared to experimental model of bilirubin toxicity. These rats have an nonjaundiced controls. Striking abnormalities are also seen in other autosomal recessively inherited deficiency of hepatic UDP auditory brainstem nuclei including the lateral superior olivary and glucuronyl transferase.12 The heterozygous Gunn rat (Nj) has superior paraolivary nuclei. Similarly, we have seen abnormalities in approximately 50% enzyme activity and is phenotypically normal. the vestibular nuclei, which control truncal tone and posture, as well The homozygous jaundiced (jj) rat has virtually no enzyme activity as the oculomotor nuclei including Dieter’s nucleus, which controls and is jaundiced, ataxic, and may or may not develop other overt vertical gaze. signs of kernicterus. We have studied ABRs, a binaural interaction The cell bodies of the auditory nerve in the spiral ganglia of the wave ABR, and cochlear microphonics and have correlated these cochlea are also abnormal. Cell size and density in kernicteric Gunn neurophysiological tests with biochemical measures such as total rats are reduced compared to nonjaundiced littermates. Preliminary serum bilirubin, unbound bilirubin. transcutaneous bilirubin, findings indicate that there is a selective loss of large myelinated neuroanatomy, and immunohistochemistry. fibers in the auditory nerve, whereas the smaller fibers appear normal or less affected. However, the inner ears of these animals, even those with severely abnormal or absent ABRs, are normal. These findings PATHOGENESIS are in agreement with previous histologic studies on Gunn rats.5,6 We have considered several hypotheses of how bilirubin damages neurons, and currently favor the theory that bilirubin interferes with ABR Studies in the Gunn Rat intracellular calcium homeostasis. Several different lines of evidence The pathologic findings correspond to abnormalities of the ABR in support this hypothesis. The activity of the enzyme calcium/ jaundiced Gunn rats that we have previously reported. There are calmodulin kinase II (CaM-Kinase II) is inhibited in vitro by differences in ABR wave latencies and amplitudes in jaundiced Gunn physiologic doses of calcium.13 Using cultured hippocampal rats during development and in adulthood.14,15 When infant jaundiced pyramidal neurons, we found increased intracellular calcium occurs Gunn rats are given a sulfonamide to displace bilirubin from blood immediately after micropipeting a solution containing 32 mol/l (albumin) into brain tissue,16 there were acute increases in interwave bilirubin plus 32 mol/l human serum albumin. The increase in intervals and decreases in amplitudes of ABR waves II and III, which intracellular calcium peaks within a minute and then decreases to a correspond to waves III and V of the human, respectively.17 These steady state slightly above baseline approximately 45 minutes after changes occur within an hour or two after toxicity, sometimes within exposure. This increased intracellular calcium may sensitize the cell minutes, and are reversible.18 In more severely affected rats, the to other agents, which may be involved in hypoxic and/or ischemic response from the auditory nerve, wave I, is affected. In the most brain injury, such as glutamate. Physiological experiments using severe kernicteric rats, all waves are lost, similar to that observed with patch-clamping show that application of bilirubin to these severe bilirubin neurotoxicity in humans. hippocampal neurons causes hyperexcitability and seizure-like We have also studied at binaural interaction evoked potentials in activity. Application of bilirubin solution to cultured auditory the Gunn rats. We stimulated the left and then the right ear

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monaurally and then stimulated binaurally. We then digitally added or demyelination, with desynchronization of the ascending auditory the left and right ear together to get a summed wave that appears impulses rendering them undetectable or nearly undetectable by the quite similar to the binaural wave, but with a difference that occurs ABR technique. at approximately 4 to 5 msec. When the summed monaural wave is Auditory neuropathy has been described in infants.21,24 One third to subtracted from the binaural wave, a binaural difference or binaural half of reported cases are associated with significant hyper- interaction wave results. This wave is thought to arise from neurons bilirubinemia. We recently surveyed parents of children diagnosed in the superior olivary nucleus, which detect whether sounds arrive at with classic kernicterus. Ten of 12 had ABRs done and all were both ears simultaneously by comparing the timing, intensities, and abnormal and met clinical criteria of auditory neuropathy. phases of responses from the two ears. The abnormalities of these Auditory neuropathy in adults can be found in the presence or waves correlate neuroanatomically with the abnormal expression of absence of hearing loss. In all cases, neural synchrony is abnormal. calcium-binding proteins in the superior olivary complex. These Difficulties occur with sound localization or discrimination of speech waves disappear with sulfonamide-induced bilirubin neurotoxicity.19 and noise or discrimination of speech in situations where there are Preliminary studies suggest that this binaural interaction ABR is even no visual cues, such as on the telephone. The audiogram may or may more sensitive than the conventional ABR in detecting bilirubin not be normal. In these cases hearing aids may be harmful, as they neurotoxicity. may overstimulate the normal inner ear. Early indications are that In another set of experiments, Ahlfors CE, Shapiro SM, we auditory neuropathy or dyssynchrony resulting from neonatal recorded ABRs in jaundiced Gunn rats and then drew blood. Samples hyperbilirubinemia is more likely to be reversible than other types of were spun in the dark; plasma was separated and frozen at À708C auditory neuropathy. There are anecdotal reports of auditory and express-mailed on dry ice to Dr. Charles Ahlfors, who assayed processing problems in these children. The thought of unsuspected bilirubin, albumin, and unbound bilirubin by peroxidase-diazo learning disabilities and auditory processing problems resulting from method. There were no correlations found between total bilirubin or bilirubin neurotoxicity unsuspected by measuring only total serum the bilirubin/albumin molar ratio and ABR intervals; however, a bilirubin in term or preterm infants is sobering. If a subset of correlation was found between the unbound bilirubin and ABR I–III learning disorders in children could be prevented by more vigorous (corresponding to the I–V interwave interval in humans) with a and/or more focused and directed treatment of infants with correlation coefficient of 0.63 and a p value of 0.004. This finding hyperbilirubinemia, then the effort to better detect or define what is suggests that unbound bilirubin may be a better indicator of central excessive bilirubin in neonates has potentially important benefits to nervous system dysfunction than total bilirubin or the bilirubin/ society. albumin molar ratio in our model. Currently, we can only detect cases of classic kernicterus. We do Neurophysiologic studies show abnormalities of ABRs in not know the incidence of subtle bilirubin encephalopathy or isolated humans. The initial electrophysiologic study of children with central nervous system dysfunction that may occur. Nor do we know deafness and hearing loss resulting from hyperbilirubinemia were how many cases are transient and reversible, how many are subtle done in the late 70s by Chisin et al.20 who showed normal but irreversible, or how many are not so subtle and irreversible but cochlear microphonic responses and abnormal or absence of not recognizable as classic kernicterus. My colleagues and I have auditory brainstem responses. The present cochlear microphonics been collecting ABR data from an automated ABR machine, which is are the reflection of normal outer hair cell function in the currently approved for use as a hearing screener. In the future, we cochlea. In fact, this early study by Chisin et al. would fit the may be able to obtain ABRs or perhaps automated ABRs from all recently coined definition of auditory neuropathy by Starr et al.21 infants at risk from excessive hyperbilirubinemia to estimate how (see below). Improvements during exchange transfusions were often hyperbilirubinemia affects CNS function in newborns, how reported almost 20 years ago by several authors, and generally the mild or severe this dysfunction is, how quickly or slowly it is waves affected are the brainstem waves III and V, although other reversible, and how it relates to outcomes, including learning authors have also shown abnormalities of wave I.22,23 ABR disorders involving the auditory system. abnormalities may partially resolve. Auditory neuropathy, also known as auditory dyssynchrony, has SUMMARY been recently described21,24 and functionally defined as abnormal or absent ABRs with normal inner ear function as tested by cochlear Where does bilirubin damage the auditory system? We would say the microphonic responses and/or otoacoustic emissions. In this following: Inner ear, no. Auditory nerve and spiral ganglion condition, the inner ear or cochlea is believed to be normal, but the containing the cell bodies of the auditory nerve, yes. It is our belief ascending auditory pathway in the nerve or the brainstem is that the nerve is affected only at higher degrees of bilirubin toxicity in abnormal, although involvement of the inner hair cells, the receptors the Gunn rat, but this remains to be determined in humans. in the inner ear, could also cause auditory neuropathy. ABRs are Brainstem auditory nuclei are clearly involved, including the either absent or severely abnormal. Audiograms may be either cochlear nuclei, the superior olivary complex, the lateral lemnisci, normal or abnormal. This may reflect the selective loss of nerve fibers the trapezoid bodies, and the inferior colliculi of the midbrain. The

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thalamus and cortical auditory pathways do not appear to be 10. de Vries LS, Lary S, Whitelaw AG, Dubowitz LM. Relationship of serum involved. Humans with classic kernicterus or isolated bilirubin- bilirubin levels and hearing impairment in newborn infants. Early Hum Dev induced neurologic damage may have abnormalities of brainstem 1987;15:269–77. auditory nuclei and central auditory pathways, the auditory nerve cell 11. Gunn CK. Hereditary acholuric jaundice in a new mutant strain of rats. bodies or processes, or some combination of abnormalities. J Hered 1938;29:137–9. In closing, the auditory system provides a sensitive and objective 12. Strebel L, Odell GB. Bilirubin uridine disphosphoglucuronyltransferase in rat liver microsomes: genetic variation and maturation. Pediatr Res window into central nervous system functioning in the jaundiced 1971;5:548–59. neonate. It is an important component of classic kernicterus. It is 13. Churn SB, DeLorenzo RJ, Shapiro SM. Bilirubin induces a calcium- involved in auditory neuropathy or dyssynchrony of which dependent inhibition of multifunctional Ca2+/calmodulin-dependent kinase hyperbilirubinemia has reported to be a significant cause. We predict II activity in vitro. Pediatr Res 1995;38:949–54. the auditory system will have an important role in determining 14. Shapiro SM, Hecox KE. Developmental studies of brainstem auditory evoked whether we are able to prevent neurologic sequelae and learning potentials in jaundiced Gunn rats. Dev Brain Res 1988;41:147–57. disabilities with more focused and aggressive treatment of neonatal 15. Shapiro SM, Hecox KE. Brain stem auditory evoked potentials in jaundiced hyperbilirubinemia. Gunn rats. Ann Otol Rhinol Laryngol 1989;98:308–17. 16. Diamond I, Schmid R. Experimental bilirubin encephalopathy: The mode of entry of bilirubin- 14C into the CNS. J Clin Invest 1966;45:678–89. 17. Shapiro SM. Acute brainstem auditory evoked potential abnormalities in References jaundiced Gunn rats given sulfonamide. Pediatr Res 1988;23:306–10. 1. Dublin W. Neurological lesions in erythroblastosis fetalis in relation to 18. Shapiro SM. Reversible brainstem auditory evoked potential abnormalities in nuclear deafness. Am J Clin Pathol 1951;21:935–9. jaundiced Gunn rats given sulfonamide. Pediatr Res 1993;34:629–33. 2. Ahdab-Barmada M, Moossy J. The neuropathology of kernicterus in the 19. Shapiro SM. Binaural effects in brainstem auditory evoked potentials of premature neonate: diagnostic problems. J Neuropathol Exp Neurol jaundiced rats. Hearing Res 1991;53:41–8. 1984;43:45–56. 20. Chisin R, Perlman M, Sohmer H. Cochlear and brain stem responses in 3. Gerrard J. Nuclear jaundice and deafness. J Laryngol Otol 1952;66:39–46. hearing loss following neonatal hyperbilirubinemia. Ann Otol 1979;88: 4. Kelemen G. Erythroblastosis fetalis. Pathologic report on the hearing organs 352–7. of a newborn infant. AMA Arch Otolaryngol 1956;63:392–8. 21. Starr A, Picton TW, Sininger Y, Hood LJ, Berlin CI. Auditory neuropathy. 5. Belal A. Effect of hyperbilirubinemia on the inner ear in Gunn rats. Brain 1996;119:741–53. J Laryngol Otol 1975;89:259–65. 22. Funato M, Tamai H, Shimada S, Nakamura H. Vigitiphobia, unbound 6. Uziel AM, Marot M, Pujol R. The Gunn rat: An experimental model for bilirubin, and auditory brainstem responses. Pediatrics 1994;93:50–3. central deafness. Acta Otolaryngol 1983;95:651–6. 23. Nakamura H, Takada S, Shimabuku R, Matsuo M, Matsuo T, Negishi H. 7. Matkin ND, Carhart R. Auditory profiles associated with Rh incompatibility. Auditory nerve and brainstem responses in newborn infants with Arch Otolaryngol 1966;84:502–13. hyperbilirubinemia. Pediatrics 1985;75:703–8. 8. Bergman I, Hirsch RP, Fria TJ, Shapiro SM, Holzman I, Painter MJ. Cause of 24. Deltenre P, Mansbach AL, Bozet C, Clerex A, Hecox KE. Auditory neuropathy: hearing loss in the high-risk premature infant. J Pediatr 1985;106:95–101. A report on three cases with early onsets and major neonatal illnesses. 9. de Vries LS, Lary S, Dubowitz LMS. Relationship of serum bilirubin levels to Electroencephalogr Clin Neurophysiol 1997;104:17–22. ototoxicity and deafness in high-risk, low birth-weight infants. Pediatrics 25. Ahlfors CE, Shapiro SM. Auditory brainstem response and unbound bilirubin 1985;76:351–4. in jaundiced (jj) Gunn rat pups. Biol Neonate 2001;80(2):158–62 (Aug).

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