Charles M. Henley

Ototoxicity in developing mammals

Developing mammals are more sensitive to noise, chemical and drug-induced ototoxicity than adults, with maximum sensitivity occurring during periods of anatomical and functional maturation of the cochlea. Normal physiological development of resting potentials (the endocochlear potential) and sound-evoked potentials including cochlear microphonics, summating potentials, compound action potentials, auditory brainstem responses and more recently distortion-product otoacoustic emissions have been characterized in several species including rats, mice, kittens, gerbils and guinea pigs. All of these responses are significantly impaired following acoustic trauma and/or exposure to a variety of ototoxic agents including aminoglycoside antibiotics, loop diuretics, antithyroid and antitumor drugs (alpha-difluoromethylornithine) and excitatory amino acids. Coupled with physiological and anatomical development is the maturation of specific biochemical pathways, which may be vulnerable targets of environmental noise and chemicals, excitatory amino acids and therapeutic drugs with ototoxic potentials.

Distortion product otoacoustic emissions and outer hair cell defects in the hyt/hyt mutant mouse.

Thyroid hormone plays an important role in hearing development. Hereditary hypothyroidism is frequently associated with sensorineural hearing loss as identified in both animal models and human patients. Building upon our original demonstration of congenital deafness and hair cell abnormality in a hyt/hyt mouse model which carries an autosomal recessive mutation causing hereditary hypothyroidism, we investigated the functional capacity of the outer hair cell (OHC) system in these animals using distortion product otoacoustic emissions (DPOAEs). In particular, the amplitude and detection features of DPOAEs were correlated with measures of the auditory brainstem response (ABR) as well as the cellular structure and ultrastructure of the organ of Corti. Input-output (I/O) functions for the 2f(2)-f(1) DPOAEs were obtained for frequencies from 2 to 18 kHz. The thresholds were significantly higher and amplitudes were significantly lower in the homozygous mice (hyt/hyt) than in both heterozygous mice (hyt/+) and wild-type controls at DPOAE frequencies recorded above 6 kHz. Hearing thresholds were significantly elevated in the mutant compared to control mice. In addition, morphological studies revealed consistent inner ear defects in hyt/hyt animals including distortion of the tectorial membrane, dysplasia of the tunnel of Corti and distinct OHC abnormalities. The most striking histopathological finding was a contiguous membrane along the apices of all of the OHC stereocilia. Such ultrastructural changes in the stereocilia of the OHC may limit the deflection of the stereocilia and therefore affect an active cochlear function that produces otoacoustic emissions as well as cause a failure to evoke the normal action potentials in the auditory nerve. From both functional and morphologic evaluations, it was concluded that the OHC system is the most susceptible to the developmental effects of congenital hypothyroidism in the hyt/hyt mouse. The normal OHCs with well-developed ciliary bundles are crucial to maintain the activity of biological mechanisms within the cochlea.

Increased susceptibility of male rats to kanamycin-induced cochleotoxicity

Although clinical observations suggest that males are more susceptible than females to ototoxic drugs, controlled experimental studies investigating gender susceptibility have not been performed. Aminoglycosides initially attack the cochleas outer hair cells (OHCs). We investigated the effects of the aminoglycoside, kanamycin, on electrophysiological function of OHCs in male and female rats. Animals were grouped by gender and treated with kanamycin (400 mg/kg/day kanamycin base, intramuscular injection) or equivolume normal saline. Administration was continued until distortion product otoacoustic emissions (DPOAEs) suggested a loss in OHC function in kanamycin-treated rats. Males treated with kanamycin showed changes in DPOAE thresholds and amplitudes as early as treatment day 10 which spread to all test frequencies by treatment day 13. In contrast, females treated with kanamycin did not show significant changes in thresholds or amplitudes until treatment day 22. The mechanism of increased male susceptibility to kanamycin cochleotoxicity has not been determined.

Sensitive developmental periods for kanamycin ototoxic effects on distortion-product otoacoustic emissions

The developing rat is hypersensitive to aminoglycoside toxicity, which is expressed early on as a destruction of outer hair cells (OHC). In the current study, distortion-product otoacoustic emissions (DPOAE), which specifically measure the micromechanical activity of OHCs, were used to assess functional effects of administering a regimen of kanamycin to three groups of neonatal rats representing discrete postnatal developmental periods. In this manner, pigmented rats were treated at postnatal days 1-10, 11-20, and 21-30. A series of input-output (I/O) functions obtained for the 2f1-f2 DPOAE during the post-treatment period indicated that detection thresholds were significantly elevated for the animals treated on postnatal days 1-10 and 11-20, with the greatest elevations observed at the higher test frequencies.

DFMO reduces cortical infarct volume after middle cerebral artery occlusion in the rat

Ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, is induced in ischemic tissue and may mediate vasogenic edema and delayed neuronal death. We determined the effects of α-difluoromethylornithine (DFMO), a specific inhibitor of ODC, on infarct size and ODC activity in a rat model of transient focal ischemia. DFMO blocked the ischemia-induced increase in ODC and significantly reduced infarct volumes by 57–45%, depending upon the treatment regimen. These studies suggest that polyamine metabolism plays a role in the development of cerebral infarction after focal ischemia and that DFMO may be useful in limiting injury after a stroke.

S-adenosylmethionine decarboxylase Activity is decreased in the rat cortex after traumatic brain injury

Abstract: S‐Adenosyl‐l‐methionine decarboxylase (SAMdc) and l‐ornithine decarboxylase (ODC) are major enzymes regulating polyamine synthesis. Following ischemia, putrescine content increases as a result of post‐traumatic activation of ODC and inhibition of SAMdc. These alterations are thought to mediate edema and cell death. The purpose of this study was to quantify SAMdc activity and edema in the brain following controlled cortical impact injury. Anesthetized adult male rats underwent a right parietal craniectomy and were subjected to cortical impact injury. Tissues were obtained from three bilateral regions: parietal cortex, motor area (CPm); parietal cortex, somatosensory area (CPs); and the pyriform cortex (CPF). SAMdc activity was determined in the postmitochondrial fraction from homogenates of fresh, unfrozen tissues by measuring the decarboxylation of S‐adenosyl‐l‐[carboxyl‐14C]methionine. Basal SAMdc activity was determined in unoperated rats, and regional differences were noted: Activity was lower in the CPF than in the CPm and CPs. SAMdc activity decreased to the greatest extent in the ipsilateral CPm (impact site) from 1 to 72 h following traumatic brain injury. Significant edema was found in the ipsilateral CPm 1, 8, 16, 24, and 48 h after injury. Decreased SAMdc activity impairs the conversion of putrescine to polyamines and may contribute to delayed pathological changes in the brain after traumatic injury.

X. Auditory aspects of seizure in the genetically epilepsy prone rat

The organ of Corti of Genetically Epilepsy Prone Rats was examined anatomically and electrophysiologically using scanning electron microscopy (SEM) and electrophysiological recording of alternating current cochlear potentials (ACCP) and N1, a volume conductor recording of the primary auditory afferent action potentials. ACCPs for GEPRs with low intensity seizures (Acoustic Response Score (ARS) = 2 or 3) and high intensity seizures (ARS = 9) showed similar impairment in cochlear function. Approximately a 25-35 dB shift in input-output functions was present in GEPRs as compared to controls. SEM revealed several types of possible genetic abnormalities which explain the deficits in cochlear function and could serve as the basis for seizure predisposition in these animals.

Postnatal developmental changes in inner ear ornithine decarboxylase (ODC).

Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis, is important in cellular growth, differentiation and development. Although ODC has been quantitated in cochlear tissues of the adult rat, it has not been assessed quantitatively in developing inner-ear tissues. The purpose of the present study was to quantitate ODC in cochlear tissues of the rat during the period of development of hearing. Cochlear ODC was significantly elevated throughout the period of cochlear maturation in that it increased rapidly during the first 10 days, peaked on day 10 and then declined thereafter. ODC in the lateral wall/organ of Corti tissues was significantly higher than in the cochlear nerve in developing, but not in adult rats. Further examination of separate cochlear tissues from 10-day old rats revealed that ODC activity was higher in the organ of Corti than in the lateral wall or cochlear nerve. Postnatal changes in ODC paralleled functional maturation of hearing and the hypersensitive period for aminoglycoside ototoxicity in the rat. Since aminoglycosides have been shown to inhibit ODC in vitro, aminoglycoside inhibition of polyamine synthesis may mediate the hypersensitivity of developing animals to the effects of these drugs.

Pharmacokinetics of kanamycin in the developing rat.

The developing rat is hypersensitive to aminoglycoside ototoxicity during the period of anatomical and functional development of the cochlea. Toxicity is expressed only after a few days of treatment when kanamycin is given during the most sensitive period for production of ototoxicity (postnatal days 11-20). In contrast, when the drug is administered after the 20th postnatal day, the same dose and duration of treatment do not produce an ototoxic effect. Only after prolonged treatment (e.g., > or = 20 days) is there an observed effect. We characterized the pharmacokinetics of kanamycin in the serum of 12- and 25-day-old rats and observed a greater than 2.5-fold increase in elimination half-life in the 12- versus 25-day-old rat. The longer duration half-life of kanamycin in younger rats may explain the hypersensitivity of immature mammals to aminoglycoside ototoxicity.

Amino Acid Assay of Vestibular Nuclei 10 Months after Unilateral Labyrinthectomy in Squirrel Monkeys

Amino acids were assayed by HPLC in bilateral vestibular nuclei from normal squirrel monkeys (n = 3) and those 10-month post-unilateral labyrinthectomy (n = 4). Findings of vestibulo-spinal and vestibulo-oculomotor functions were identical for both groups. No left-right asymmetry of amino acids was found within either group, nor between groups with the exception of GABA: GABA was significantly reduced in the bilateral vestibular nuclei of the 10-month post-lesion animals. This may be indicative of a reduction of cerebello-vestibular inhibitory control which could be secondary to the reduction of excitatory inputs to the system.