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 cochlea's 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.

Partial deletion of both the spermine synthase gene and the Pex gene in the X-linked hypophosphatemic, gyro (Gy) mouse.

Gy, along with Hyp, is a dominant mutation of the normal gene Pex causing X-linked hypophosphatemia in the mouse. Hemizygous Gy male mice, however, have greater defects in survival, bodily growth, skeletal mineralization, and neurological function than those found in heterozygous Gy females or in Hyp mice. Since the gene for spermine synthase is immediately upstream of the homologous human gene PEX, we compared the effects of the Gy and Hyp mutations on both the spermine synthase gene and the Pex gene. Barely detectable levels of spermine (< 5% of normal) with elevated levels of its precursor, spermidine, were found in organs of Gy male mice compared to normal male littermates. Neither Gy females nor Hyp male mice were significantly affected. Four missing introns of the spermine synthase gene were identified in Gy male mice, suggesting extensive gene disruption. A pseudogene for spermine synthase was also identified in the mouse genome. Pex mRNA was found in several but not all tissues studied in adult normal mice. Pex mRNA was altered in both Gy and Hyp mice. All male Hyp mice were lacking the 3' end of the Pex message, whereas all male Gy mice were deficient at the 5' end. In summary, the Gy mutation is associated with a recessively expressed mutation of the spermine synthase gene, leading to spermine deficiency, and a dominantly expressed mutation of the Pex gene, leading to hypophosphatemia. Alterations in two contiguous genes in Gy may explain the additional phenotypic abnormalities present in the Gy male mouse.

Quantification of furosemide from serum and tissues using high-performance liquid chromatography.

Since pharmacokinetics may play a significant role in furosemide (FSM) developmental ototoxicity, we developed an assay for the extraction and quantification of FSM in tissue and fluid from neonatal and adult rats. Rats from post-natal day (PND) 10, 30 and 50, were given an intravenous dose of FSM (35 mg/kg). Blood and tissues were analyzed by HPLC. FSM in serum, perilymph and liver was elevated in PND ten rats as was the body burden of FSM. Renal concentrations were higher in older rats. Altered clearance of FSM in developing rats may result in higher concentrations in the cochlea and ototoxicity.

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

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 posttraumatic 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.

Iron deficiency anemia and hearing.

Iron deficiency anemia is a frequently occurring clinical disorder. Despite the suggested association with hearing loss in the literature, cochlear sequelae of iron deficiency have yielded conflicting results in experimental studies. Auditory function was tested in iron-deficient and normal male Wistar albino rats using distortion product otoacoustic emissions and auditory brainstem response audiometry for the clarification of the opposing results in the literature. Hemoglobin, hematocrit, serum iron and albumin levels were monitored to verify iron deficiency. Although dramatic differences in weight gain and blood test parameters were noted, no significant change in auditory function due to iron deficiency was detected.

Polyamines increase in the brain stem and cerebellum following labyrinthectomy.

The goal of this investigation was to test the hypothesis that unilateral damage to the vestibular end-organ (labyrinthectomy) stimulates polyamine synthesis in central vestibular neural structures that mediate the process of behavioral recovery (vestibular compensation). Pharmacological studies have shown that compensation can be altered by alpha-difluoromethylornithine (DFMO), a specific inhibitor of polyamine synthesis. Because polyamines are important in regeneration, development and modulation of N-methyl-D-aspartate (NMDA) excitatory amino acid receptors, which mediate vestibular synaptic plasticity, we investigated changes in polyamines in specific central vestibular structures after unilateral labyrinthectomy. The supernatant fraction of brain tissue homogenates was reacted with dansyl chloride. Dansylated polyamine derivatives were quantified in the vestibular nuclei, cerebellum, and inferior olive in both the control and the unilaterally labyrinthectomized guinea pig by high-performance liquid chromatography-fluorometric detection. No left-right differences in putrescine, spermidine, or spermine were detected in any brain parenchyma of controls. Polyamine imbalance, characterized by increased spermidine in the ipsilateral medial and lateral vestibular nuclei, was noted 12 and 24 h after unilateral labyrinthectomy (UL). In contrast, spermidine, spermine, and putrescine were elevated bilaterally in the cerebellum and inferior olive after UL. These biochemical changes may represent neuronal modifications to establish a balance between the vestibular nuclei after unilateral labyrinthectomy. Elucidation of the role of polyamines in central vestibular function and in vestibular compensation offers promise for the development of novel therapeutic strategies for treatment of vestibular disorders.

Effects of anesthesia on polyamine metabolism and water content in the rat brain.

Because variances have been noted in brain putrescine levels of anesthetized rats (control, SHAM-operated), we investigated the effects of several anesthetics on polyamine metabolism and water content in the adult rat brain. Short duration (5 min) anesthesia was studied in three groups: ketamine:xylazine [KX; 40 and 8 mg/kg, respectively, intraperitoneal injection (IP)], urethane (UR; 1.5 g/kg, IP), and isoflurane (IF, initially 3.5% in 100% O2, followed by a maintenance dose of 2.5% IF in 100% O2). Effects of IF at longer duration (30 min) were also studied because this paradigm is often used in our laboratory. Control rats received no anesthesia (NA). Following decapitation, tissue samples were obtained from 3 bilateral brain regions: parietal cortex, motor area (CPm); parietal cortex, somatosensory area (CPs); and the pyriform cortex (CPF). The polyamines, spermidine and spermine, and their precursor, putrescine, were quantified by HPLC-fluorometric detection and brain water content was determined by wet-to-dry weight measures. KX decreased putrescine (54%) and spermidine (20%) in the CPs, increased spermine (24%) in the CPF, and increased water content in all brain regions. UR decreased putrescine (51%) and slightly increased water content (0.7%) in the CPF. Short duration IF decreased putrescine and spermidine in all brain regions; decreased spermine in the CPm, and increased water content in the CPF (0.8%). In contrast, longer duration IF increased putrescine (181%) and spermidine (23%) in the CPm, with no change in water content. Anesthetics produce region-specific changes in putrescine, polyamines, and water content in the rat brain which could contribute to the experimental variability.

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.

Activation of ornithine decarboxylase and accumulation of putrescine after traumatic brain injury.

Activation of ornithine decarboxylase (ODC), the initial enzyme in polyamine synthesis, and accumulation of putrescine are thought to mediate pathological processes in the ischemic and traumatized brain. Past studies have separately investigated either ODC or polyamines after head injury. The purpose of the present study was to quantify both ODC activity and polyamines in the rat parietal cortex before and after controlled cortical impact injury. Adult, male rats underwent a right craniectomy and were subjected to a 5 m/sec, 2-mm deformation impact injury. Rats were sacrificed 1, 4, 8, and 24 h postimpact and tissues from the injured (right) and contralateral (left) hemisphere were analyzed for ODC and polyamines. ODC activity was determined by measuring the decarboxylation of [14C]ornithine to putrescine. Putrescine, spermidine, and spermine were determined by high performance liquid chromatography. Cortical impact induced a 10- to 20-fold increase in ODC activity and a 4- to 5-fold increase in putrescine in the ipsilateral cortex. Spermidine and spermine did not significantly increase in the ipsilateral (right) cortex compared to controls (right cortex). In contrast, there was a slight increase in spermidine content in the contralateral (left) cortex after injury. The delayed increase in ODC activity and accumulation of putrescine may mediate pathophysiological changes observed after head injury.

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.

Localization of ornithine decarboxylase (ODC) in the cochlea of the immature rat.

Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis, is important in cochlear development. Whereas tissue specific differences in cochlear ODC activity have been demonstrated, cellular localization of ODC protein in the inner ear of the immature rat has not. ODC was localized in inner ear structures using an ODC polyclonal antibody and the effects of cycloheximide on ODC immunoreactivity and enzymatic activity were determined. Tissues demonstrating elevated enzymatic activity contained cells with the strong immunoreactivity. ODC activity was highest in the organ of Corti and lateral wall followed by the cochlear nerve. Immunoreactivity was demonstrated throughout the cochlea with intense staining of the hair cells, pillar cells, Deiter's cells, inner sulcus cells, basilar membrane, stria vascularis, spiral ganglion cell bodies and cochlear nerve fibers. Cycloheximide rapidly diminished cochlear ODC activity and expression of ODC protein. The half-life of cochlear ODC was 30 min. Localization of cellular sites of ODC is important in understanding the role of the ODC-polyamine pathway in cochlear development and will be a valuable marker for tissue damage from ototoxic agents.

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.

Spermine and spermidine as gating molecules for inward rectifier K+ channels.

Inward rectifier K+ channels pass prominent inward currents, while outward currents are largely blocked. The inward rectification is due to block by intracellular Mg2+ and a Mg(2+)-independent process described as intrinsic gating. The rapid loss of gating upon patch excision suggests that cytoplasmic factors participate in gating. "Intrinsic" gating can be restored in excised patches by nanomolar concentrations of two naturally occurring polyamines, spermine and spermidine. Spermine and spermidine may function as physiological blockers of inward rectifier K+ channels and "intrinsic" gating may largely reflect voltage-dependent block by these cations.

Polyamine changes in the vestibular nuclei of guinea pigs following labyrinthectomy.

Vestibular compensation is a process of behavioral recovery from ocular, motor and postural disorders following unilateral damage to the vestibular end-organ. Although restoration of the normal resting discharge rate in the ipsilateral vestibular nuclei is important in compensation, the biochemical and molecular mechanisms mediating recovery are largely unknown. The ornithine decarboxylase polyamine pathway is activated in the nervous system following axotomy or denervation. The authors postulate that changes in polyamines mediate vestibular compensation. Within 150-micron brain stem coronal section micropunches analyzed by high performance liquid chromatography techniques, the polyamine spermidine was significantly increased in the ipsilateral lateral vestibular nucleus 8 hours following labyrinthectomy in the guinea pig model. Because naturally occurring polyamines modulate excitatory amino acid receptors (N-methyl-D-aspartate [NMDA]) which in turn mediate neurotransmission between primary afferents and second order vestibular neurons, stimulation of polyamine pathways following neural injury may play a critical role in compensation.

alpha-Difluoromethylornithine delays behavioral recovery and induces decompensation after unilateral labyrinthectomy.

Biochemical and pharmacologic studies suggest a role for the ornithine decarboxylase-polyamine system as a modulator of behavioral changes during vestibular compensation. alpha-Difluoromethylornithine specifically blocks the rate-limiting step of polyamine biosynthesis. To assess the effects of alpha-difluoromethylornithine on the acute phase of postural compensation, guinea pigs were divided into groups subjected to either unilateral labyrinthectomy only (n = 7), alpha-difluoromethylornithine (500 mg/kg/day) for 4 days before labyrinthectomy (n = 10), equivalent volumes of saline for 4 days before labyrinthectomy (n = 8), and sham operations (n = 5). Yaw head tilt and roll head tilt, trunk curvature, and air-righting reflex were measured at baseline and at regular intervals up to 4 weeks. alpha-Difluoromethylornithine significantly delayed recovery of normal air-righting but had no effect on yaw head tilt, roll head tilt, and trunk curvature. We also evaluated effects of alpha-difluoromethylornithine in compensated guinea pigs. Fully compensated animals from phase 1 were randomly assigned to receive alpha-difluoromethylornithine (500 mg/kg/day) or saline once daily for 4 days. Only 33% of alpha-difluoromethylornithine animals maintained air-righting, compared with 100% of saline-treated animals (p = 0.003). Maximum trunk curvature was greater in the alpha-difluoromethylornithine group (p = 0.02). Thus alpha-difluoromethylornithine not only delayed the time course for postural recovery after unilateral labyrinthectomy, it also transiently disrupted the maintenance of the compensated state.

Cochlear polyamines: markers of otitis media-induced cochlear damage.

High-performance liquid chromatography (HPLC) set to the femtomole [corrected] sensitivity level was used to identify and quantify the polyamines spermidine and spermine as well as the diamine putrescine in the different tissues of the inner ears of guinea pigs with experimentally induced otitis media. The tissues examined were the lateral wall (stria vascularis and the spiral ligament), the organ of Corti, and the cochlear nerve. The difference in polyamine profile in the different tissues of the control noninfected guinea pigs suggests a relation to the particular function of each of these tissues [see erratum notice re: preceding sentence]. The difference in polyamine profile in infected different inner ear tissues compared to controls encourages the assumption that the polyamines may be involved in a repair process of the inner ear after injury and that they may be considered as biochemical markers for inner ear damage secondary to acute otitis media.

Postnatal changes in cochlear polyamine metabolism in the rat.

Ornithine decarboxylase (ODC), the initial enzyme in the polyamine biosynthetic pathway, is increased in the developing rat cochlea, suggesting that polyamine biosynthesis is important in cochlear development. Although cochlear polyamines have been detected in adult rats, they have not been identified in developing rats. We quantified polyamines in the developing and mature rat cochlea and further characterized ODC in the early postnatal period. Putrescine and spermidine in combined tissues of the organ of Corti and lateral wall of the cochlea were highest during the first 10 postnatal days, then declined to adult levels shortly thereafter. Spermine demonstrated a similar developmental trend. A high spermidine to spermine ratio was noted during this period as was rapidly increasing ODC activity. A high spermidine/spermine ratio was also noted in the cochlear nerve of developing and mature rats, suggesting that spermidine may be necessary for function and maintenance of the nerve. This is the first report of polyamines in the developing rat cochlea. The period of increased polyamine synthesis coincides with the critical period for ototoxicity induced by alpha-difluoromethylornithine, a specific ODC inhibitor, and the period of rapid cochlear development.

Kanamycin depletes cochlear polyamines in the developing rat.

Developing mammals are more sensitive to aminoglycoside antibiotics and other ototoxic agents than adults, with maximum sensitivity occurring during the period of anatomic and functional maturation of the cochlea. For the aminoglycoside antibiotics, the hypersensitive period in rats occurs during the second and third postnatal weeks. Toxicity is initially expressed as outer hair cell (OHC) damage in the high-frequency, basal region of the cochlea. Distortion-product otoacoustic emissions (DPOAEs), physiologic measures of OHC function, are particularly sensitive to aminoglycoside exposure during the period of rapid cochlear physiologic development. Toxicity is characterized by increased DPOAE thresholds and decreased amplitudes. The mechanism of developmental sensitivity to aminoglycosides is unknown. A potential biochemical target of aminoglycosides is the ornithine decarboxylase (ODC)-polyamine pathway. ODC activity is elevated in the developing rat cochlea, aminoglycosides inhibit cochlear ODC in developing rats, and alpha-difluoromethylornithine (a specific ODC inhibitor) impairs development of cochlear function. In the present study we demonstrate an incomplete polyamine response to aminoglycoside damage, characterized by inhibition of the polyamines spermidine and spermine and accumulation of putrescine in the organ of Corti. Aminoglycoside inhibition of polyamine synthesis may mediate developmental ototoxic hypersensitivity by interfering with developmental and repair processes.

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 alpha-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.