Sjaak F.L. Klis

The cochlear targets of cisplatin: An electrophysiological and morphological time-sequence study

Cisplatin ototoxicity has at least three major targets in the cochlea: the stria vascularis, the organ of Corti, and the spiral ganglion. This study aims to differentiate between these three targets. In particular, we address the question of whether the effects at the level of the organ of Corti and spiral ganglion are mutually dependent or whether they develop in parallel. This question was approached by studying the ototoxic effects while they develop electrophysiologically and comparing these to earlier presented histological data [Van Ruijven et al., 2004. Hear. Res. 197, 44-54]. Guinea pigs were treated with intraperitoneal injections of cisplatin at a dose of 2 mg/kg/day for either 4, 6, or 8 consecutive days. This time sequence has not revealed any evidence of one ototoxic process triggering another. Therefore, we have to stay with the conclusion of Van Ruijven et al. (2004) that both processes run in parallel.

Time course of cochlear electrophysiology and morphology after combined administration of kanamycin and furosemide.

In animal models of deafness, administration of an aminoglycoside in combination with a loop diuretic is often applied to produce a rapid loss of cochlear hair cells. However, the extent to which surviving hair cells remain functional after such a deafening procedure varies. In a longitudinal electrocochleographical study, we investigated the variability of cochlear function between and within guinea pigs after combined administration of kanamycin and furosemide. Concurrently, histological data were obtained at 1, 2, 4 and 8 weeks after deafening treatment. The main measures in our study were compound action potential (CAP) thresholds, percentage of surviving hair cells and packing density of spiral ganglion cells (SGCs). One day after deafening treatment, we found threshold shifts widely varying among animals from 0 to 100dB. The variability decreased after 2 days, and in 18 out of 20 animals threshold shifts greater than 55dB were found 4-7 days after deafening. Remarkably, in the majority of animals, thresholds decreased by up to 25dB after 7 days indicating functional recovery. As expected, final thresholds were negatively correlated to the percentage of surviving hair cells. Notably, the percentage of surviving hair cells might be predicted on the basis of thresholds observed one day after deafening. SGC packing density, which rapidly decreased with the period after deafening treatment and correlated to the percentage of surviving inner hair cells, was not a determining factor for the CAP thresholds.

Morphological changes in spiral ganglion cells after intracochlear application of brain-derived neurotrophic factor in deafened guinea pigs.

When guinea pigs are deafened with ototoxic drugs spiral ganglion cells (SGCs) degenerate progressively. Application of neurotrophins can prevent this process. Morphological changes of rescued SGCs have not been quantitatively determined yet. It might be that SGCs treated with neurotrophins are more vulnerable than SGCs in cochleae of normal-hearing guinea pigs. Therefore, the mitochondria and myelinisation of type-I SGCs were studied and the perikaryal area, cell circularity and electron density were determined. Guinea pigs were deafened with a subcutaneous injection of kanamycin followed by intravenous infusion of furosemide. Brain-derived neurotrophic factor (BDNF) delivery was started two weeks after the deafening procedure and continued for four weeks. Four cohorts of cochleae were studied: (1) cochleae of normal-hearing guinea pigs; (2) of guinea pigs two weeks after deafening; (3) six weeks after deafening; (4) cochleae treated with BDNF after deafening. The deafening procedure resulted in a progressive loss of SGCs. Six weeks after deafening the size of mitochondria, perikaryal area and cell circularity of the remaining untreated SGCs were decreased and the number of layers of the myelin sheath was reduced. In the basal part of the cochlea BDNF treatment rescued SGCs from degeneration. SGCs treated with BDNF were larger than SGCs in normal-hearing guinea pigs, whereas circularity had normal values and electron density was unchanged. The number of layers in the myelin sheath of BDNF-treated SGCs was reduced as compared to the number of layers in the myelin sheath of SGCs in normal-hearing guinea pigs. The morphological changes of SGCs might be related to the rapid loss of SGCs that has been reported to occur after cessation of BDNF treatment.

Neurotrophins and their role in the cochlea

Spiral ganglion cell (SGC) degeneration following hair cell loss can be prevented by administration of exogenous neurotrophic factors. Many of these neurotrophic factors, in particular the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), have been described to be involved in the development of the rodent cochlea. While expression of most of the neurotrophins has decreased to below detectable levels during adulthood (only NT-3 remains highly expressed), their respective receptors remain present in SGCs. Indeed much less is known about the function of neurotrophins in the mature cochlea. Such knowledge is crucial in the search for tools to improve SGC survival following cochlear implantation. In this review, we will critically regard the current experimental findings of neurotrophic treatment of the SGCs in the perspective of fundamental cellular mechanisms underlying neurotrophin signaling. We conclude that, in order to fully apprehend the effects of neurotrophic treatment of degenerating SGCs and in order to consider clinical application of neurotrophins, future research should focus (a) on characterizing the expression pattern of neurotrophins in the cochlea after deafening, (b) on more detailed characterization of functional and morphological changes of SGCs associated with both deafening and neurotrophic treatment and (c) on the possible self-supporting state of SGCs after cessation of short-term neurotrophic treatment.

Ethyl benzene-induced ototoxicity in rats: a dose-dependent mid-frequency hearing loss.

Rats were exposed to ethyl benzene at 0, 300, 400 and 550 ppm for 8 hours/day for 5 consecutive days. Three to six weeks after the exposure, auditory function was tested by measuring compound action potentials (CAP) in the frequency range of 1-24 kHz and 2f1-f2 distortion product otoacoustic emissions (DPOAEs) in the frequency range of 4-22.6 kHz. In addition, outer hair cell (OHC) loss was quantified by histological examination. The lowest concentration ethyl benzene had no effect on any of the above measures. At 400 ppm, auditory thresholds were increased by 15 and 16 dB at 12 and 16 kHz, respectively, and at 550 ppm by 24, 31, and 22 dB at 8, 12, and 16 kHz, respectively. DPOAE amplitude growth with stimulus level was affected only after 550 ppm at 5.6, 8, and 11.3 kHz. OHC loss was found in two of the five examined locations in the cochlea. At 400 ppm, 25% OHC loss was found at the 11- and 21-kHz region. The highest concentration evoked 40% and 75% OHC loss at the 11- and 21-kHz location, respectively. Thus, the mid-frequency region of rats is affected after exposure to relatively low concentrations of ethyl benzene (400-550 ppm). These results indicate that ethyl benzene is one of the most potent ototoxic organic solvents known today.

The ototoxic effects of ethyl benzene in rats

Exposure to organic solvents has been shown to be ototoxic in animals and there is evidence that these solvents can induce hearing loss in humans. In this study, the effects of inhalation of the possibly ototoxic solvent ethyl benzene on the cochlear function and morphology were evaluated using three complementary techniques: (1) reflex modification audiometry (RMA), (2) electrocochleography and (3) histological examination of the cochleas. Rats were exposed to either ethyl benzene (800 ppm, 8 h/day for 5 days) or to control conditions. The RMA threshold increased significantly by about 25 dB, 1 and 4 weeks after the exposure, irrespective of the stimulus frequency tested (4-24 kHz). Electrocochleography was performed between 8 and 11 weeks after exposure to the organic solvent. The threshold for the compound action potential increased significantly by 10-30 dB at all frequencies tested (1-24 kHz). Histological examination of the cochlea showed outer hair cell (OHC) loss, especially in the upper basal and lower middle turns (corresponding to the mid-frequency region) to an extent of 65%. We conclude that exposure to 800 ppm ethyl benzene for 8 h/day during 5 days induces hearing loss in rats due to OHC loss.

Simultaneous exposure to ethyl benzene and noise : Synergistic effects on outer hair cells

The effects on hearing of simultaneous exposure to the ototoxic organic solvent ethyl benzene and broad-band noise were evaluated in rats. The effects of three ethyl benzene concentrations (0, 300 or 400 ppm) and three noise levels (95 or 105 dB(lin) SPL or background noise at 65 dB(lin) SPL) and all their combinations were investigated for a 5 day exposure at 8 h/day. Distortion product otoacoustic emissions and compound action potentials were affected after 105 dB noise alone, and after 105 dB noise in combination with ethyl benzene (300 and 400 ppm). However, the amount of loss for these combinations did not exceed the loss for 105 dB noise alone. Outer hair cell (OHC) loss after exposure to 300 ppm ethyl benzene was located in the third row of OHCs. At 400 ppm, the loss spread out to the second and first row of OHCs. Noise alone hardly affected the OHC counts except for a minor loss in the first row of OHCs after 105 dB SPL. Noise at 105 dB in combination with ethyl benzene at 300 and 400 ppm, however, showed OHC loss greater than the sum of the losses induced by noise and ethyl benzene alone.

Cisplatin-induced ototoxicity; electrophysiological evidence of spontaneous recovery in the albino guinea pig

For 8 days albino guinea pigs (n = 48) were treated with cisplatin (cis-diamminedichloroplatinum(II), 1.5 mg/kg body weight/day). Compound action potentials (CAP), cochlear microphonics (CM) and summating potentials (SP) were recorded from the apical surface of the cochlea in response to tone bursts ranging in frequency from 0.5 to 16 kHz. The recordings were collected in different groups of animals, 1 day, 1 week, 2, 4, 8 and 16 weeks after cisplatin treatment, respectively. One day after the 8-day treatment we found frequency-dependent loss in the amplitudes of the three cochlear potentials, with the larger losses occurring at the higher frequencies. In terms of threshold shift the losses were larger for the CAP than for the hair cell-related potentials SP and CM. A salient improvement in both CAP and CM amplitude occurred over the next 8 weeks. Also, the SP showed improvement. These results indicate that guinea pig cochlear transduction recovers spontaneously after cisplatin injury. Recovery of the hair cell-related potentials suggests that recovery occurs already at the hair cell level. The question whether this recovery originates with the formation of new hair cells or with repair of damaged hair cells should be answered on the basis of subsequent morphological investigations.

Cisplatin Ototoxicity Involves Organ of Corti, Stria Vascularis and Spiral Ganglion: Modulation by αMSH and ORG 2766

It has been shown that αMSH and the nonmelanotropic ACTH/MSH(4–9) analog ORG 2766 can ameliorate cisplatin-induced neurotoxicity and ototoxicity. Here, we investigated whether these peptides delay the occurrence of the cisplatin-induced shift in auditory threshold, and whether they affect the subsequent recovery of cochlear potentials. Chronically implanted round window electrodes were used to obtain daily recordings of auditory nerve compound action potentials (CAP) and cochlear microphonics at frequencies ranging from 2 to 16 kHz. Cisplatin (1.5 mg/kg i.p.) plus αMSH, ORG 2766 (75 µg/kg s.c.), or saline were injected daily until the 40-dB CAP threshold shift at 8 kHz was reached. Endocochlear potential (EP) was measured either 1–2 days or 28 days later, followed by morphometric analysis of the cochlea. Peptide cotreatment did not consistently delay the threshold shift; however, the CAP threshold recovered faster and to a greater extent, with the potency order being αMSH > ORG 2766 > saline. Significant recovery at the 2 highest frequencies was seen in the αMSH-treated animals only. CAP amplitude at high sound pressures, which depends more on nerve function than on outer hair cell (OHC) function, decreased severely in all groups but recovered significantly in the αMSH- and completely in the ORG-2766-cotreated group. EP was significantly lower in the first days after the threshold shift but had completely recovered at 28 days. Morphometric analysis of the spiral ganglion also indicated involvement of ganglion cells. OHC loss was most severe in the basal turn of saline-cotreated animals. These data suggest that the cisplatin-induced acute threshold shift might be due to reversible strial failure, whereas subsequent OHC survival determines the final degree of functional recovery. Both OHC loss and neuronal function were ameliorated by peptide cotreatment.

Auditory-Nerve Responses to Varied Inter-Phase Gap and Phase Duration of the Electric Pulse Stimulus as Predictors for Neuronal Degeneration

After severe hair cell loss, secondary degeneration of spiral ganglion cells (SGCs) is observed—a gradual process that spans years in humans but only takes weeks in guinea pigs. Being the target for cochlear implants (CIs), the physiological state of the SGCs is important for the effectiveness of a CI. For assessment of the nerve’s state, focus has generally been on its response threshold. Our goal was to add a more detailed characterization of SGC functionality. To this end, the electrically evoked compound action potential (eCAP) was recorded in normal-hearing guinea pigs and guinea pigs that were deafened 2 or 6 weeks prior to the experiments. We evaluated changes in eCAP characteristics when the phase duration (PD) and inter-phase gap (IPG) of a biphasic current pulse were varied. We correlated the magnitude of these changes to quantified histological measures of neurodegeneration (SGC packing density and SGC size). The maximum eCAP amplitude, derived from the input–output function, decreased after deafening, and increased with both PD and IPG. The eCAP threshold did not change after deafening, and decreased with increasing PD and IPG. The dynamic range was wider for the 6-weeks-deaf animals than for the other two groups. Excitability increased with IPG (steeper slope of the input–output function and lower stimulation level at the half-maximum eCAP amplitude), but to a lesser extent for the deafened animals than for normal-hearing controls. The latency was shorter for the 6-weeks-deaf animals than for the other two groups. For several of these eCAP characteristics, the effect size of IPG correlated well with histological measures of degeneration, whereas effect size of PD did not. These correlations depend on the use of high current levels, which could limit clinical application. Nevertheless, their potential of these correlations towards assessment of the condition of the auditory nerve may be of great benefit to clinical diagnostics and prognosis in cochlear implant recipients.