Central Nervous Activity upon Systemic Salicylate Application in Animals with Kanamycin-Induced Hearing Loss--A Manganese-Enhanced MRI (MEMRI) Study.

This study investigated the effect of systemic salicylate on central auditory and non-auditory structures in mice. Since cochlear hair cells are known to be one major target of salicylate, cochlear effects were reduced by using kanamycin to remove or impair hair cells. Neuronal brain activity was measured using the non-invasive manganese-enhanced magnetic resonance imaging technique. For all brain structures investigated, calcium-related neuronal activity was increased following systemic application of a sodium salicylate solution: probably due to neuronal hyperactivity. In addition, it was shown that the central effect of salicylate was not limited to the auditory system. A general alteration of calcium-related activity was indicated by an increase in manganese accumulation in the preoptic area of the anterior hypothalamus, as well as in the amygdala. The present data suggest that salicylate-induced activity changes in the auditory system differ from those shown in studies of noise trauma. Since salicylate action is reversible, central pharmacological effects of salicylate compared to those of (permanent) noise-induced hearing impairment and tinnitus might induce different pathophysiologies. These should therefore, be treated as different causes with the same symptoms.

Reversible neurotoxicity of kanamycin on dorsal cochlear nucleus.

The time course of aminoglycoside neurotoxic effect on cochlear nucleus is still obscure. We examined dynamic pathological changes of dorsal cochlear nucleus (DCN) and investigated whether apoptosis or autophagy was upregulated in the neurotoxic course of kanamycin on DCN after kanamycin treatment. Rats were treated with kanamycin sulfate/kg/day at a dose of 500mg by subcutaneous injection for 10 days. Dynamic pathological changes, neuron density and neuron apoptosis of the DCN were examined at 1, 7, 14, 28, 56, 70 and 140 days after kanamycin treatment. The expressions of JNK1, DAPK2, Bcl-2, p-Bcl-2, Caspase-3, LC3B and Beclin-1 were also detected. Under transmission electron microscopy, the mitochondrial swelling and focal vacuoles as well as endoplasmic reticulum dilation were progressively aggravated from 1 day to 14 days, and gradually recovered from 28 days to 140 days. Meanwhile, both autophagosomes and autolysosomes were increased from 1 day to 56 days. Only few neurons were positive to the TUNEL staining. Moreover, neither the expressions of caspase-3 and DAPK2 nor neurons density of DCN changed significantly. LC3-II was drastically increased at 7 days. Beclin-1 was upgraded at 1 and 7 days. P-Bcl-2 increased at 1, 7, 14 and 28 days. JNK1 increased at 7 days, and Bcl-2 was downgraded at 140 days. LC3-B positive neurons were increased at 1, 7 and 14 days. These data demonstrated that the neurons damage of the DCN caused by kanamycin was reversible and autophagy was upregulated in the neurotoxic course of kanamycin on DCN through JNK1-mediated phosphorylation of Bcl-2 pathway.

Screen of FDA-approved drug library reveals compounds that protect hair cells from aminoglycosides and cisplatin.

Loss of mechanosensory hair cells in the inner ear accounts for many hearing loss and balance disorders. Several beneficial pharmaceutical drugs cause hair cell death as a side effect. These include aminoglycoside antibiotics, such as neomycin, kanamycin and gentamicin, and several cancer chemotherapy drugs, such as cisplatin. Discovering new compounds that protect mammalian hair cells from toxic insults is experimentally difficult because of the inaccessibility of the inner ear. We used the zebrafish lateral line sensory system as an in vivo screening platform to survey a library of FDA-approved pharmaceuticals for compounds that protect hair cells from neomycin, gentamicin, kanamycin and cisplatin. Ten compounds were identified that provide protection from at least two of the four toxins. The resulting compounds fall into several drug classes, including serotonin and dopamine-modulating drugs, adrenergic receptor ligands, and estrogen receptor modulators. The protective compounds show different effects against the different toxins, supporting the idea that each toxin causes hair cell death by distinct, but partially overlapping, mechanisms. Furthermore, some compounds from the same drug classes had different protective properties, suggesting that they might not prevent hair cell death by their known target mechanisms. Some protective compounds blocked gentamicin uptake into hair cells, suggesting that they may block mechanotransduction or other routes of entry. The protective compounds identified in our screen will provide a starting point for studies in mammals as well as further research discovering the cellular signaling pathways that trigger hair cell death.

The impact of hearing experience on signal integration in the auditory brainstem: a c-Fos study of the rat.

In this study we investigated the pattern of c-Fos expression in anteroventral (AVCN) and dorsal cochlear nucleus (DCN) and central inferior colliculus (CIC) following electrical intracochlear stimulation (EIS) of anesthetized adult rats that were neonatally deafened. The animals never experienced acoustic sensations as their hair cells were destroyed by daily kanamycin injections between postnatal days 10 to 20, resulting in a rise of hearing threshold by about 90 dB. Unilateral EIS was applied through a cochlear implant inserted into the medial turn of the left cochlea and lasted for 45 or 73 min, 2, 3:15, or 5h. Following EIS at 50Hz, a high number of c-Fos positive nuclei were observed showing only marginal tonotopic order in ipsilateral AVCN, in DCN bilaterally, and in contralateral CIC. Quantifying the number of c-Fos positive nuclei in ipsilateral AVCN, we found a steady increase with stimulation time. By contrast, the population of neurons expressing c-Fos in DCN and CIC revealed a transient maximum at 73 min. A direct comparison with our previous study (Rosskothen-Kuhl, N., Illing, R.-B., 2010. Nonlinear development of the populations of neurons expressing c-Fos under sustained electrical intracochlear stimulation in the rat auditory brainstem. Brain Res. 1347, 33-41) reveals that absence of hearing experience has far-reaching consequences for the interneuronal communication within networks of the auditory brainstem. When hearing fails, EIS entails expression of c-Fos in populations of neurons that are much larger than normally, essentially disregard tonotopic order, and lack much of spatio-temporal variations seen in hearing-experienced rats.

Effects of restricted basilar papillar lesions and hair cell regeneration on auditory forebrain frequency organization in adult European starlings.

The frequency organization of neurons in the forebrain Field L complex (FLC) of adult starlings was investigated to determine the effects of hair cell (HC) destruction in the basal portion of the basilar papilla (BP) and of subsequent HC regeneration. Conventional microelectrode mapping techniques were used in normal starlings and in lesioned starlings either 2 d or 6-10 weeks after aminoglycoside treatment. Histological examination of the BP and recordings of auditory brainstem evoked responses confirmed massive loss of HCs in the basal portion of the BP and hearing losses at frequencies >2 kHz in starlings tested 2 d after aminoglycoside treatment. In these birds, all neurons in the region of the FLC in which characteristic frequencies (CFs) normally increase from 2 to 6 kHz had CF in the range of 2-4 kHz. The significantly elevated thresholds of responses in this region of altered tonotopic organization indicated that they were the residue of prelesion responses and did not reflect CNS plasticity. In the long-term recovery birds, there was histological evidence of substantial HC regeneration. The tonotopic organization of the high-frequency region of the FLC did not differ from that in normal starlings, but the mean threshold at CF in this frequency range was intermediate between the values in the normal and lesioned short-recovery groups. The recovery of normal tonotopicity indicates considerable stability of the topography of neuronal connections in the avian auditory system, but the residual loss of sensitivity suggests deficiencies in high-frequency HC function.

Fenestration of the calyx of Held occurs sequentially along the tonotopic axis, is influenced by afferent activity, and facilitates glutamate clearance.

The calyx of Held is a type of giant glutamatergic presynaptic terminal in the mammalian auditory brainstem that transmits afferent information from the cochlear nucleus to the medial nucleus of the trapezoid body (MNTB). It participates in sound localization, a process that requires very high temporal precision. Consistent with its functional role, the calyx shows a number of specializations for temporal fidelity, one of them being the giant terminal itself with its many release sites. During the first 3 weeks of postnatal development, the calyx transforms from a spoon-shaped, closed morphology to a highly fenestrated open structure. Calyces in Mongolian gerbils (Meriones unguiculatus) were labeled via injection of fluorescent tracers and their morphology was reconstructed at various timepoints during early postnatal development. We show that the fenestration process does not occur simultaneously in all calyces. Calyces transmitting high-frequency sound information fenestrate significantly earlier than those transmitting low-frequency information, such that a temporary developmental gradient along the tonotopic axis is established around the time of hearing onset. Animals that were deprived of afferent activity before hearing onset, either via cochlear removal or administration of ototoxic drugs, do not show this developmental gradient. Glial processes containing glutamate transporters occupy the newly created windows in the calyx and thus could augment the fast clearance of neurotransmitter. The physiological consequences of this faster clearance include a faster decay time course of synaptic currents as well as a lower amount of residual current accumulating during the processing of repeated activity such as stimulus trains.

Sox2 and JAGGED1 expression in normal and drug-damaged adult mouse inner ear.

Inner ear hair cells detect environmental signals associated with hearing, balance, and body orientation. In humans and other mammals, significant hair cell loss leads to irreversible hearing and balance deficits, whereas hair cell loss in nonmammalian vertebrates is repaired by the spontaneous generation of replacement hair cells. Research in mammalian hair cell regeneration is hampered by the lack of in vivo damage models for the adult mouse inner ear and the paucity of cell-type-specific markers for non-sensory cells within the sensory receptor epithelia. The present study delineates a protocol to drug damage the adult mouse auditory epithelium (organ of Corti) in situ and uses this protocol to investigate Sox2 and Jagged1 expression in damaged inner ear sensory epithelia. In other tissues, the transcription factor Sox2 and a ligand member of the Notch signaling pathway, Jagged1, are involved in regenerative processes. Both are involved in early inner ear development and are expressed in developing support cells, but little is known about their expressions in the adult. We describe a nonsurgical technique for inducing hair cell damage in adult mouse organ of Corti by a single high-dose injection of the aminoglycoside kanamycin followed by a single injection of the loop diuretic furosemide. This drug combination causes the rapid death of outer hair cells throughout the cochlea. Using immunocytochemical techniques, Sox2 is shown to be expressed specifically in support cells in normal adult mouse inner ear and is not affected by drug damage. Sox2 is absent from auditory hair cells, but is expressed in a subset of vestibular hair cells. Double-labeling experiments with Sox2 and calbindin suggest Sox2-positive hair cells are Type II. Jagged1 is also expressed in support cells in the adult ear and is not affected by drug damage. Sox2 and Jagged1 may be involved in the maintenance of support cells in adult mouse inner ear.

[The effect of polikatan on the ototoxic action of kanamycin]. / Vliianie polikatana na ototoksicheskoe deistvie kanamitsina.

Experiments on guinea pigs demonstrated that preliminary injection of polycatan (standardized magnesium solution containing the mineral bischofite) into the parotid region by means of electrophoresis reduces the ototoxic effect of the aminoglycoside antibiotic kanamycin. Polycatan prevents kanamycin-induced degenerative changes of the hair cells found in the labyrinth of the internal ear and improves the local blood flow.

The ototoxicity of 3,3'-iminodipropionitrile: functional and morphological evidence of cochlear damage.

Previous reports have suggested that IDPN may be ototoxic (Wolff et al., 1977; Crofton and Knight, 1991). The purpose of this research was to investigate the ototoxicity of IDPN using behavioral, physiological and morphological approaches. Three groups of adult rats were exposed to IDPN (0-400 mg/kg/day) for three consecutive days. In the first group, at 9-10 weeks post-exposure, thresholds for hearing of 5.3- and 38-kHz filtered clicks were measured electrophysiologically and brainstem auditory evoked responses (BAERs) were also recorded to a suprathreshold broadband click stimulus. A second set of animals was tested at 9 weeks for behavioral hearing thresholds (0.5- to 40-kHz tones) and at 11-12 weeks post-exposure for BAER thresholds (5- to 80-kHz filtered clicks). A third group of animals was exposed (as above), and killed at 12-14 weeks post-exposure for histological assessment. Kanamycin sulfate was used as a positive control for high-frequency selective hearing loss. Surface preparations of the organ of Corti were prepared in order to assess hair cells, and mid-modiolar sections of the cochlea were used to examine Rosenthal's canal and the stria vascularis. Functional data demonstrate a broad-spectrum hearing loss ranging from 0.5 kHz (30 dB deficit) to 80 kHz (40 dB deficit), as compared to a hearing deficit in kanamycin-exposed animals that was only apparent at frequencies greater than 5 kHz. Surface preparations revealed IDPN-induced hair cell loss in all turns of the organ of Corti, with a basal-to-apical gradient (more damage in the basal turns) at the lower dosages. At higher dosages there was complete destruction of the organ of Corti. There was also a dosage-related loss of spiral ganglion cells in all turns of the cochlea, again with a basal-to-apical gradient at the lower dosages. These data demonstrate that IDPN exposure in the rat results in extensive hearing loss and loss of neural structures in the cochlea.

Hair cell regeneration in the European starling (Sturnus vulgaris): recovery of pure-tone detection thresholds.

Behavioral detection thresholds were obtained from four starlings before, during, and after 11 days of subcutaneous injections of kanamycin. Birds were operantly conditioned to respond to pure-tones ranging in frequency from 0.25 kHz to 7 kHz using the method of constant stimuli and were tested daily for 141 days after the first injection of aminoglycoside. All four birds sustained hearing losses greater than 60 dB at frequencies from 4 kHz to 7 kHz by the end of the 11 day injection schedule. Two birds had a slight shift in threshold at 3 kHz. No change in threshold occurred for any of the birds at lower frequencies. Recovery of detection thresholds began soon after the injections ceased and continued for approximately 50 days. In all four birds there was some degree of permanent hearing loss: 5 dB to 15 dB at frequencies between 4 kHz and 6 kHz, and approximately 25 dB at 7 kHz. Scanning electron microscopy (SEM) was performed at 0 and 5 days post-injection in a separate group of starlings given the same injection schedule. Hair cell loss and damage was observed across the basal 34% to 36% of the basilar papilla. SEM in two behaviorally tested birds sacrificed 142 days after the first injection showed that there was regeneration of hair cells to populate the previously damaged region, but that disorientation of stereocilia bundles in the basal third of the basilar papilla was common. The other two behaviorally tested birds were treated with kanamycin again for 16 days beginning at 142 days after the first injection. Thresholds shifted again, but less than during the first dosing period. SEM of these birds' basilar papillae showed less hair cell loss than observed in the birds given only a single, 11 day dosing of kanamycin. This result suggests that birds may be less susceptible to the ototoxic effects of kanamycin in repeated treatments. In all four birds, the degree and position of damage observed with SEM corresponded with the extent and frequency of hearing loss.

Recovery of CAP threshold and amplitude in chickens following kanamycin ototoxicity.

Chickens were given a dose of kanamycin (400 mg/kg/d x 10 d) which destroyed hair cells over the basal 37-58% of the basilar papilla. Afterwards, the threshold and amplitude of the compound action potential were measured at recovery times ranging from 2 days to 10-20 weeks post-kanamycin treatment. At 2 days post-treatment, the thresholds at 1000, 2000 and 4000 Hz were elevated 40-60 dB while the thresholds at 250 and 500 Hz were elevated only 25 dB. By 10-20 weeks post-treatment, the threshold at 250 and 500 Hz had completely recovered whereas a residual threshold shift of 5 dB to 25 dB was present between 1000 to 4000 Hz. The maximum amplitude of the compound action potential was also reduced by more than 60% at all frequencies at 2 days post-treatment; however by 10-20 weeks post-treatment, the amplitude of the compound action potential had completely recovered at 500, 1000 and 2000 Hz. By contrast, the amplitude of the compound action potential at 4000 Hz was still reduced by more than 50% of its normal value 10-20 weeks post-treatment. The results of the present study indicate that the time course of recovery of the compound action potential is extremely slow and may lag behind the regeneration of hair cells by many weeks. The permanent deficits observed at the high frequencies could conceivably be due to functional deficits in regenerated hair cells, their afferent synapses or the loss of cochlear ganglion cells.

Behavioural evidence for recovery of auditory function in guinea pigs following kanamycin administration.

Deterioration followed by recovery of behavioural absolute threshold and frequency selectivity has been observed in guinea pigs following kanamycin administration of 200 mg/kg body weight daily for 16 days. Deterioration in function consistently follows a high-to-low frequency pattern and recovery generally occurs at the lowest of the high (8-32 kHz) frequencies affected. The degree of recovery is related to the magnitude of the threshold elevation; where large (40-45 dB) elevations occur initially, the process appears to be partial since threshold recovers only to within 5-12 dB of pre-administration levels. In instances where smaller threshold elevations (5-20 dB) take place initially, recovery can sometimes be complete. However, when threshold elevations of over 50 dB occur, no recovery is apparent. Recovery is relatively slow, taking place over periods of up to 100 days post-kanamycin administration. Hair cell counts have established that the threshold elevation which remains in instances of partial recovery is not related to a reduction in hair cell numbers at the light microscope level.

Reflex modification as a test for sensory function.

Reflex modification is a versatile procedure for the assessment of sensory function because it can provide information about the responses of several sensory systems to both weak and intense stimuli. The procedure has two elements: The elicitation of some reflex, such as the acoustic startle reflex, and the modification of that reflex by preliminary stimuli. In these experiments we used reflex modification and reflex elicitation procedures to examine the normal development of auditory function in rats and to evaluate alterations in auditory function produced by physical and toxic insult. Adult rats exposed to octave bands of noise demonstrated frequency-specific deficits on a test of reflex modification, but not reflex elicitation. In the studies of developing rats, reflex elicitation appeared by postnatal day 12 and modification around day 14. Frequency-specific increases in both measures suggested that the phenomena were sensitive to auditory development and, not simply, motor development. Exposure to kanamycin on postnatal days 8 to 16 produced dose-related deficits in the ability to detect stimuli at 32 and 16 kHz, but not 4 and 0.8 kHz. These effects were observed in the absence of changes in reflex elicitation. The results demonstrate that reflex modification procedures provide more sensitive and specific information than that provided by the use of reflex elicitation alone.

[Toxicity and antimicrobial properties of the new aminoglycoside antibiotic 535]. / Izuchenie toksichnosti i antimikrobnykh svoistv novogo aminoglikozidnogo antibiotika 535.

LD50 of antibiotic 535 (3'-desoxykanamycin C) on its intravenous, subcutaneous and oral administration to albino mice was 225, 1150 and at least 5000 mg/kg respectively. After a single subcutaneous administration to rabbits in a dose of 10 mg/kg antibiotic 535 was rapidly absorbed and detected in the blood and organs of the animals for 24 hours. The antibiotic was mainly excreted with the urine. Comparative investigation of the pharmacokinetics of antibiotic 535, tobramycin and kanamycin in rabbits revealed no significant differences. Antibiotic 535 showed a broad antibacterial spectrum and inhibited both grampositive and gramnegative bacteria. It was highly active against infections caused by S. aureus, E. coli and Pr. vulgaris and somewhat less active against infections caused by Ps. aeruginosa. In treatment of experimental tuberculosis of albino mice antibiotic 535 and tobramycin were inferior by their efficacy to kanamycin.

Isolation and characterization of Tn5 insertion mutants of Erwinia chrysanthemi that are deficient in polygalacturonate catabolic enzymes oligogalacturonate lyase and 3-deoxy-D-glycero-2,5-hexodiulosonate dehydrogenase.

Mutants of Erwinia chrysanthemi EC16 deficient in the polygalacturonate catabolic enzymes oligogalacturonate lyase (Ogl-) and 3-deoxy-D-glycero-2,5-hexodiulosonate (ketodeoxyuronate) dehydrogenase (KduD-) were obtained by Tn5 mutagenesis using the R plasmid pJB4JI. Ogl- Exu+ (Exu+, D-galacturonate utilization) and KduD- Exu- strains macerated potato tuber tissue and utilized glucose, glycerol, and gluconate, but they did not utilize polygalacturonate, unsaturated digalacturonate, or saturated digalacturonate. Genetic and physical evidence indicated that the Ogl- mutants and a KduD- recombinant contained a single copy of Tn5 and that Tn5 (Kmr) was linked to the mutant phenotypes. In the Ogl+ parents, basal levels of oligogalacturonate lyase were present in glycerol-grown cells and induced levels were present with saturated or unsaturated digalacturonate, while oligogalacturonate lyase was undetectable under similar conditions in Ogl- strains. Pectate lyase, polygalacturonase, and ketodeoxyuronate dehydrogenase were induced in an Ogl- strain by 3-deoxy-D-glycero-2,5-hexodiulosonate and by the enzymatic products of unsaturated digalacturonate but not by the digalacturonates. The KduD- strains lacked the dehydrogenase activity but in the presence of the digalacturonates produced higher levels of pectate lyase, polygalacturonase, and oligogalacturonate lyase than the KduD+ parents did. In the KduD- strains, pectate lyase and oligogalacturonate lyase were induced by unsaturated digalacturonate in a "gratuitous" manner, suggesting an intracellular accumulation of the inducer(s). We conclude that an intermediate(s) of the ketodeoxyuronate pathway induces pectate lyase, polygalacturonase, oligogalacturonate lyase, and ketodeoxyuronate dehydrogenase in E. chrysanthemi.

Scanning electron microscopy of the nerve endings of Corti's organ.

Scanning electron microscopy (SEM) study of the innervation of the organ of Corti has been carried out for many years. Various methods have been used in attempts to observe more details of the endings of nerve fibers on the sensory hair cells. In this study, a new method of microdissection of Corti's organ was done using a small piece of double-sided adhesive tape. This method is simple and facilitates observation of Corti's organ from the basal to the apical turns. Using it we found variations of the nerve endings and the Deiters' cells in each half turn beginning from the lower basal turn to the upper fourth turn. Nerve endings at the lower poles of outer hair cells are arranged in clusters, and are reduced in size and number as the upper turn is approached. On the contrary, the Deiters' cells are increased in size toward the upper turn, therefore nerve endings in the upper turn are often covered by Deiters' cells. Small nerve endings high on the side of the outer hair cell were also observed. Although there was clear evidence of ultrastructural change in the hair cells' cilia after administration of ototoxic drugs or after hyperstimulation by sound, there was no evidence of concomitant ultrastructural change in the nerve endings. Nerve endings appeared to be more resistant than the hair cells.

The influence of pigmentation of rats and guinea-pigs on the ototoxicity of kanamycin and neomycin.

Following the finding that melanin pigment played a role in the accumulation of ototoxic drugs in the inner ear, an investigation was made of the possible influence of the pigmentation of animals on their susceptibility to the ototoxic effects of drugs. Hearing acuity was assessed by measurement of acoustic startle reaction. Preliminary experiments suggested that pigmented animals might be more likely to suffer hearing impairment following ototoxic drug administration. However, in a controlled study using rats treated with kanamycin, it was not possible to confirm this and albino animals appeared no less vulnerable than pigmented animals to kanamycin-induced deafness.