John C.M.J. de Groot

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.

Enhanced Survival of Spiral Ganglion Cells After Cessation of Treatment with Brain-Derived Neurotrophic Factor in Deafened Guinea Pigs

Exogenous delivery of neurotrophic factors into the cochlea of deafened animals rescues spiral ganglion cells (SGCs) from degeneration. To be clinically relevant for human cochlear implant candidates, the protective effect of neurotrophins should persist after cessation of treatment and the treated SGCs should remain functional. In this study, the survival and functionality of SGCs were investigated after temporary treatment with brain-derived neurotrophic factor (BDNF). Guinea pigs in the experimental group were deafened, and 2xa0weeks later, the right cochleae were implanted with an electrode array and drug delivery cannula. BDNF was administered to the implanted cochleae during a 4-week period via a mini-osmotic pump. After completion of the treatment, the osmotic pumps were removed. Two weeks later, the animals were killed and the survival of SGCs was analyzed. To monitor the functionality of the auditory nerve, electrically evoked auditory brainstem responses (eABRs) were recorded in awake animals throughout the experiment. BDNF treatment resulted in enhanced survival of SGCs 2xa0weeks after cessation of the treatment and prevented the decreases in size and circularity that are seen in the untreated contralateral cochleae. The amplitude of the suprathreshold eABR response in BDNF-treated animals was significantly larger than in deafened control animals and comparable to that in normal-hearing control animals. The amplitude in the BDNF-treated group did not decrease significantly after cessation of treatment. The eABR latency in BDNF-treated animals was longer than normal and comparable to that in deafened control animals. These morphological and functional findings demonstrate that neurotrophic intervention had a lasting effect, which is promising for future clinical application of neurotrophic factors in implanted human cochleae.

Time sequence of degeneration pattern in the guinea pig cochlea during cisplatin administration.: A quantitative histological study

We investigated the key tissues that are implicated in cisplatin ototoxicity within the time window during which degeneration starts. Guinea pigs were treated with cisplatin at a dose of 2 mg/kg/day for either 4, 6, or 8 consecutive days. Histological changes in the organ of Corti, the stria vascularis and the spiral ganglion were quantified at the light microscopical level. Outer hair cell (OHC) loss started between 4 and 6 days of cisplatin administration, but is only significantly different from the non-treated group after 8 days of treatment. Midmodiolar OHC counts were comparable to the cytocochleogram data. The cross-sectional area of the stria vascularis did not differ from the non-treated group, nor did an endolymphatic hydrops develop during the course of treatment. Spiral ganglion cell (SGC) densities did not decrease. After 6 days, however, detachment of the myelin sheath of the type-I SGCs was seen in the lower basal turn, whereas after 8 days it was also present in the more apically located turns. Myelin sheath detachment is the result of perikaryal shrinkage and swelling of the myelin sheath. The present study confirms that cisplatin at a daily dose of 2 mg/kg has a detrimental effect on the OHCs as well as on the type-I SGCs. These intracochlear effects occur simultaneously; OHC loss and SGC shrinkage start between the fourth and sixth day of cisplatin administration and appear to develop in parallel. At this dose, no histological effect on the stria vascularis could be observed, although previous electrophysiological experiments demonstrated a clear effect on the endocochlear potential

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.

Immunohistochemical detection of platinated DNA in the cochlea of cisplatin-treated guinea pigs ☆

Cisplatin-induced ototoxicity is correlated with functional and morphological changes in the organ of Corti, the stria vascularis and the spiral ganglion. However, the cochlear sites of cisplatin uptake and accumulation have not been properly identified. Therefore, we have developed an immunohistochemical method to, indirectly, detect cisplatin in semithin cryosections of the guinea pig cochlea (basal turn) using an antiserum containing antibodies against cisplatin-DNA adducts. Platinated DNA was present in the nuclei of most cells in the organ of Corti and the lateral wall after cisplatin administration. Nuclear immunostaining was most pronounced in the outer hair cells, the marginal cells and the spiral ligament fibrocytes. This study is the first to demonstrate the presence of cisplatin in histological sections of the cochlea.

Spiral ganglion cell survival after round window membrane application of brain-derived neurotrophic factor using gelfoam as carrier

Several studies have shown that treatment with various neurotrophins protects spiral ganglion cells (SGCs) from degeneration in hair-cell deprived cochleas. In most of these studies the neurotrophins are delivered by means of intracochlear delivery methods. Recently, other application methods that might be more suited in cochlear implant patients have been developed. We have examined if round window membrane application of gelfoam infiltrated with a neurotrophin resulted in SGC survival in deafened guinea pigs. Two weeks after deafening, gelfoam cubes infiltrated with 6xa0μg of brain-derived neurotrophic factor (BDNF) were deposited onto the round window membrane of the right cochleas. Electric pulses were delivered through an electrode positioned within the round window niche to electrically evoke auditory brainstem responses (eABRs). Two or four weeks after deposition of the gelfoam all cochleas were histologically examined. We found that local BDNF treatment enhances the survival of SGCs in the basal cochlear turn after two and four weeks. The treatment had no effect on SGC size or shape. In animals treated with BDNF, eABR amplitudes were smaller than in normal-hearing control animals and similar to those in deafened controls. We conclude that BDNF delivered by means of local gelfoam application provides a protective effect, which is limited compared to intracochlear delivery methods.

Co-administration of the neurotrophic ACTH(4-9) analogue, ORG 2766, may reduce the cochleotoxic effects of cisplatin

In this study the effect of the neurotrophic ACTH(4-9) analogue, ORG 2766, on cisplatin cochleotoxicity was investigated with both light- and transmission electron microscopy. Guinea pigs were treated with either cisplatin+ORG 2766 (n = 11) or cisplatin + physiological saline (n = 9). All animals treated with cisplatin + physiological saline showed complete loss of outer hair cells (OHC) and degeneration of the organ of Corti in the basal cochlear turns, while partial OHC loss was found in the middle and apical turns. The inner hair cells (IHC) and other cochlear tissues were not affected. Eight animals from the group treated with cisplatin + ORG 2766 demonstrated similar pathological changes, but to a lesser degree, especially in the middle turns. The three remaining animals demonstrated no cochlear alterations at all, light-microscopically, and only minor subcellular changes in the OHCs at the ultrastructural level. Electrophysiologically, these three animals showed normals compound action potential (CAP) amplitudes at stimulus frequencies from 0.5 to 16 kHz and normal cochlear microphonics (CM) in the frequency range from 0.5 to 8 kHz. The other animals treated with cisplatin + ORG 2766 showed a severe loss in their CAPs and CM, except for one showing intermediate loss. All animals from the group treated with cisplatin alone showed a severe loss in their CAPs and CM. Endolymphatic hydrops was present in all animals from the cisplatin- and the cisplatin + ORG 2766-treated groups. These data indicate that daily, concomitant administration of ORG 2766 may reduce OHC loss and subsequent degeneration of the organ of Corti in cisplatin-treated guinea pig cochleas.

Alterations in the stria vascularis in relation to cisplatin ototoxicity and recovery

We have investigated whether or not cisplatin-induced depression of the endocochlear potential (EP), and its subsequent recovery, possesses a morphological correlate in the stria vascularis. Guinea pigs implanted with round window electrodes were treated daily with cisplatin (1.5 mg/kg/day) until the compound action potential showed a profound hearing loss (> or =40 dB at 8 kHz after 5-18 days). Animals were either sacrificed immediately after the shift in hearing threshold (SHORT group) or allowed to recover for > or =4 weeks and subsequently sacrificed (LONG group). Control animals (CONTROL group) were not treated with cisplatin. Using stereological methods we measured the total strial cross-sectional area together with the areas occupied by the different strial components: the marginal, intermediate and basal cells. The total strial cross-sectional area in the basal turn of the LONG group was found to be significantly smaller than that of the SHORT and the CONTROL groups, whereas the EP was normal in the LONG group (in comparison to the CONTROL group) and markedly decreased in the SHORT group. The smaller area in the LONG group was mainly due to a decrease in the area occupied by the intermediate cells and to a lesser extent to a decrease in the marginal cell area. The area occupied by the basal cells did not change. Thus, the marked decrease in EP after 5-18 days of cisplatin administration was not related to shrinkage of the stria vascularis. Moreover, 4 weeks later the EP showed full recovery, whereas the stria vascularis had shrunk markedly.

Chronic electrical stimulation does not prevent spiral ganglion cell degeneration in deafened guinea pigs

Several studies have demonstrated that treatment with intracochlear chronic electrical stimulation (CES) protects spiral ganglion cells (SGCs) from degeneration in deafened animals. Other studies could not confirm this effect of CES. The present study examined whether CES in a mode as presented in cochlear implant users (amplitude modulated, high pulse rate) affects survival, morphology and functionality of SGCs in deafened guinea pigs. Eleven guinea pigs were implanted in the right cochlea with an electrode array to monitor the electrically evoked auditory brainstem responses (eABRs). The guinea pigs were deafened four weeks later. Two days after deafening, monopolar CES was started in five animals through three electrodes in the basal cochlear turn. CES lasted 4 hours per day, five days per week, for six weeks. SGC packing densities, perikaryal area, cell circularity, amplitudes of suprathreshold eABRs and eABR thresholds were not affected by CES. SGCs of all implanted cochleae were larger and more circular than SGCs in unimplanted cochleae, but this did not depend on CES treatment. Interestingly, an increase in eABR latencies observed after deafening, occurred faster in CES-treated than in untreated animals. In conclusion, amplitude-modulated chronic electrical stimulation with a high pulse rate does not affect survival, morphology and functionality of spiral ganglion cells with the exception of eABR latencies.