Sabine Ladrech

Caspase Inhibitors, but not c-Jun NH2-Terminal Kinase Inhibitor Treatment, Prevent Cisplatin-Induced Hearing Loss

Cisplatin (CDDP) is a highly effective chemotherapeutic agent but with significant ototoxic side effects. Apoptosis is an important mechanism of cochlear hair cell loss following exposure to an ototoxic level of CDDP. This study examines intracellular pathways involved in hair cell death induced by CDDP exposure in vivo to develop effective therapeutic strategies to protect the auditory receptor from CDDP-initiated hearing loss. Guinea pigs were treated with systemic administration of CDDP. Cochlear hair cells from CDDP-treated animals exhibited classic apoptotic alterations in their morphology. Several important signaling events that regulate the death of CDDP-injured cochlear hair cells were identified. CDDP treatment induced the activation and redistribution of cytosolic Bax and the release of cytochrome c from injured mitochondria. Activation of caspase-9 and caspase-3, but not caspase-8, was detected after treatment with CDDP, and the cleavage of fodrin by activated caspase-3 was observed within damaged hair cells. Intracochlear perfusions with caspase-3 inhibitor (z-DEVD-fmk) and caspase-9 inhibitor (z-LEHD-fmk) prevent hearing loss and loss of sensory cells, but caspase-8 inhibitor (z-IETD-fmk) and cathepsin B inhibitor (z-FA-fmk) do not. Although the stress-activated protein kinase/c-Jun NH2-terminal kinase (JNK) signaling pathway is activated in response to CDDP toxicity, intracochlear perfusion of d-JNKI-1, a JNK inhibitor, did not protect against CDDP ototoxicity but instead potentiated the ototoxic effects of CDDP. The results of the present study show that blocking a critical step in apoptosis may be a useful strategy to prevent harmful side effects of CDDP ototoxicity in patients having to undergo chemotherapy.

Electrophysiological evidence for the presence of NMDA receptors in the guinea pig cochlea.

An excitatory amino acid, possibly L-glutamate, which probably acts as a neurotransmitter at the inner hair cell-afferent fiber synapses in the cochlea. In the present study, we have used an electrophysiological approach to investigate at this level the presence of a major type of excitatory amino acid receptor, namely the glutamatergic receptor for which N-methyl-D-aspartate is a selective agonist. Our results show that, when N-methyl-D-aspartate and the antagonist 2-amino-5-phosphonovalerate are perfused through the perilymphatic scalae, they induced, by different mechanisms, a significant reduction of the amplitude of the compound action potential and an increase of the N1 latency, both predominant at high intensity tone burst stimulations. No significant difference was found in the presence or absence of Mg2+ in the artificial perilymph used as a vehicle. A further slight N-methyl-D-aspartate-induced decrease of the amplitude of the compound action potential, although non significant, was observed when the Mg2(+)-free perilymph contained 100 or 1000 microM glycine. In all the experimental conditions, no effect was observed on the cochlear microphonic potential. This observation is consistent with an action of N-methyl-D-aspartate and 2-amino-5-phosphonovalerate at receptors located on the auditory nerve dendrites contacting the inner hair cells. In conclusion, our results suggest the presence of N-methyl-D-aspartate receptors in the cochlea.

Contribution of auditory nerve fibers to compound action potential of the auditory nerve

Sound-evoked compound action potential (CAP), which captures the synchronous activation of the auditory nerve fibers (ANFs), is commonly used to probe deafness in experimental and clinical settings. All ANFs are believed to contribute to CAP threshold and amplitude: low sound pressure levels activate the high-spontaneous rate (SR) fibers, and increasing levels gradually recruit medium- and then low-SR fibers. In this study, we quantitatively analyze the contribution of the ANFs to CAP 6 days after 30-min infusion of ouabain into the round window niche. Anatomic examination showed a progressive ablation of ANFs following increasing concentration of ouabain. CAP amplitude and threshold plotted against loss of ANFs revealed three ANF pools: 1) a highly ouabain-sensitive pool, which does not participate in either CAP threshold or amplitude, 2) a less sensitive pool, which only encoded CAP amplitude, and 3) a ouabain-resistant pool, required for CAP threshold and amplitude. Remarkably, distribution of the three pools was similar to the SR-based ANF distribution (low-, medium-, and high-SR fibers), suggesting that the low-SR fiber loss leaves the CAP unaffected. Single-unit recordings from the auditory nerve confirmed this hypothesis and further showed that it is due to the delayed and broad first spike latency distribution of low-SR fibers. In addition to unraveling the neural mechanisms that encode CAP, our computational simulation of an assembly of guinea pig ANFs generalizes and extends our experimental findings to different species of mammals. Altogether, our data demonstrate that substantial ANF loss can coexist with normal hearing threshold and even unchanged CAP amplitude.

Synaptic Repair Mechanisms Responsible for Functional Recovery in Various Cochlear Pathologies

In some cochlear pathologies, temporary hearing loss can be followed by complete or partial functional recovery. Our previous findings suggest the involvement of an excitotoxic (glutamate-related) disruption of inner hair cell (IHC)-auditory nerve synapses, followed by synaptic regeneration. It is essential to understand the molecular mechanisms responsible for this synaptic repair if new therapeutic strategies are to be developed. In guinea pig cochleas, acute synaptic excitotoxic damage (mimicking what occurs with acoustic trauma or local ischemia) is achieved by locally applying AMPA, a glutamate agonist. This results in a total disruption of all IHC-auditory dendrite synapses, together with a disappearance of cochlear potentials. Within the next 5 days, however, a recovery of both the normal pattern of IHC innervation and the physiological responses is observed. The fact that the blockage of the NMDA receptors during functional recovery delayed the regrowth of neurites and the restoration of hearing suggests that glutamate plays a neurotrophic role via activation of NMDA receptors. Experiments are in progress to investigate, among other factors, the role of other glutamate receptor subunits. A reversible in vivo antisense strategy is being developed to overcome the lack of specificity of some antagonists. First results bode well for future pharmacological therapies in cochlear pathologies where glutamatergic synapses are likely to be involved; i.e., noise trauma, ischemia-related sudden deafness, and neural presbycusis.

Correlation Between the Length of Outer Hair Cells and the Frequency Coding of the Cochlea

When comparing outer hair cell morphology, both within and across species, a striking feature emerges: while their cylindrical shapes and diameters are almost invariable, the length of the cell body varies by a factor of 4 (within a species) or 8 (across species). A good correlation seems to exist between the outer hair cell length and the cochlear tonotopy. This paper presents and analyses results taken from serial sections of guinea-pig cochleae. These data, as well as other available values from different mammals, are discussed along the idea that the length of outer hair cells could play a role in an intrinsic tuning mechanism.

Synaptic connections and putative functions of the dopaminergic innervation of the guinea pig cochlea

We report our findings in the guinea pig involving dopamine in postsynaptic regulation of the activity of glutamatergic inner hair cells (IHCs) and in protection of primary auditory neurons during transient ischemia. Seven days after intracochlear perfusion of 6-hydroxydopamine, no immunoreactivity to tyrosine hydroxylase (TH) was demonstrable within the organ of Corti. TH and aromatic amino acid decarboxylase were immunolocalized at an ultrastructural level within lateral olivocochlear varicosities synapsing with radial auditory dendrites postsynaptic to the IHCs. The D2 agonist piribedil induced a dose-dependent decrease in the amplitude of the compound action potential of the auditory nerve. Piribedil also prevented appearance of ischemia-induced swelling of the radial dendrites.

Protein kinase C may be involved in synaptic repair of auditory neuron dendrites after AMPA injury in the cochlea

A suitable model of sudden deafness occurring after acoustic trauma or ischemia, is obtained in guinea pigs by an acute intracochlear perfusion of 200 microM alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), a glutamate analog. By overloading the AMPA/kainate receptors, located post-synaptically to inner hair cells (IHCs), it induces a massive swelling of primary auditory neuron dendrites, which disconnects the IHCs. This synaptic uncoupling and the resulting hearing loss are followed by a progressive regrowth of dendrites, which make new synapses with IHCs, leading to a functional recovery of auditory responses that is completed after 5 days. Knowing the role of protein kinase C in neuroplastic events, we studied the expression of its isoforms alpha,beta(I,II) and gamma, respectively pre- and post-synaptic, in auditory neurons at various times after AMPA administration. In untreated cochleas, we observed an expression of PKC alpha,beta(I,II) and gamma in cell bodies of primary auditory neurons. After the intracochlear administration of AMPA, both isozymes were transiently overexpressed, with a peak at 3-6 h, followed by a decrease after about 24 h. At this point in time immuno-electron microscopy revealed some regrowing dendrites immunoreactive for PKCgamma. Five days after AMPA, when the auditory responses were restored, PKCgamma levels were still elevated in ganglion cell bodies.

Supporting cells regulate the remodelling of aminoglycoside‐injured organ of Corti, through the release of high mobility group box 1

To examine whether an inflammatory process occurs in the amikacin‐poisoned cochlea, we investigated the presence of the cytokines tumour necrosis factor‐α (TNF‐α), interleukin (IL)‐1β, and IL‐10. No TNF‐α, IL‐1β or IL‐10 was detected in the cochlear perilymph after the loss of most auditory hair cells, indicating the absence of severe inflammation. In contrast, we observed a significant and temporary increase in the level of extracellular high mobility group box 1 (HMGB1), a late mediator of inflammation that also functions as a signal of tissue damage. This increase coincided with epithelial remodelling of the injured organ of Corti, and occurred concomitantly with robust and transient cytoplasmic expression of acetylated HMGB1 within the non‐sensory supporting cells, Deiters cells. Here, HMGB1 was found to be enclosed within vesicles, a number of which carried the secretory vesicle‐associated membrane‐bound protein Rab 27A. In addition, transient upregulation of receptor for advanced glycation end‐products (RAGE), an HMGB1 membrane receptor, was found in most epithelial cells of the scarring organ of Corti when extracellular levels of HMGB1 were at their highest. Altogether, these results strongly suggest that, in stressful conditions, Deiters cells liberate HMGB1 to regulate the epithelial reorganization of the injured organ of Corti through engagement of RAGE in neighbouring epithelial cells.

Changes in MAP2 and tyrosinated α-tubulin expression in cochlear inner hair cells after amikacin treatment in the rat

The expression of MAP2 (microtubule‐associated protein 2) and of tyrosinated α‐tubulin was investigated immunocytochemically in the cochleas of normal and amikacin‐treated rats. For MAP2, two different antibodies were used: anti‐MAP2ab, against the high molecular weight forms, and anti‐MAP2abc, additionally against the embryonic form c. In the cochlea of the normal rat, the outer (OHCs) and inner (IHCs) hair cells were labeled for MAP2abc. The labeling was weaker in IHCs than in OHCs. The hair cells were rarely labeled for MAPab. Both OHCs and IHCs were labeled for tyrosinated α‐tubulin. In the cochlea of the amikacin‐treated rat, aggregates of anti‐MAP2abc and anti‐tyrosinated α‐tubulin antibodies were seen in the apical region of the IHCs as early as the end of the antibiotic treatment. In rats investigated during the following week, the cell body of most of the surviving IHCs were not labeled for MAP2abc and tyrosinated α‐tubulin. Then, labeling for these two antibodies reappeared in the surviving IHCs, including their giant stereocilia. Fewer surviving IHCs were labeled for tyrosinated α‐tubulin than for MAP2abc. The amikacin‐poisoned IHCs were rarely labeled for MAP2ab. These results suggest that cochlear hair cells essentially express form c of MAP2. In the amikacin‐damaged cochlea, the apical aggregation of MAP2c and tyrosinated α‐tubulin within the poisoned IHCs could be implicated in a cell degenerative process. By contrast, the extinction and recovery of MAP2c and tyrosinated α‐tubulin labeling in the remaining IHCs suggest the occurrence of a limited repair process. A possible role of MAP2 and tubulin in hair cell survival is discussed. J. Comp. Neurol. 451:70–78, 2002.

High mobility group box 1 (HMGB1): dual functions in the cochlear auditory neurons in response to stress?

High mobility group box 1 (HMGB1) is a DNA-binding protein that facilitates gene transcription and may act extracellularly as a late mediator of inflammation. The roles of HMGB1 in the pathogenesis of the spiral ganglion neurons (SGNs) of the cochlea are currently unknown. In the present study, we tested the hypothesis that early phenotypical changes in the SGNs of the amikacin-poisoned rat cochlea are mediated by HMGB1. Our results showed that a marked downregulation of HMGB1 had occurred by completion of amikacin treatment, coinciding with acute damage at the dendrite extremities of the SGNs. A few days later, during the recovery of the SGN dendrites, the protein was re-expressed and transiently accumulated within the nuclei of the SGNs. The phosphorylated form of the transcription factor c-Jun (p-c-Jun) was concomitantly detected in the nuclei of the SGNs where it often co-localized with HMGB1, while the anti-apoptotic protein BCL2 was over-expressed in the cytoplasm. In animals co-treated with amikacin and the histone deacetylase inhibitor trichostatin A, both HMGB1 and p-c-Jun were exclusively found within the cytoplasm. The initial disappearance of HMGB1 from the affected SGNs may be due to its release into the external medium, where it may have a cytokine-like function. Once re-expressed and translocated into the nucleus, HMGB1 may facilitate the transcriptional activity of p-c-Jun, which in turn may promote repair mechanisms. Our study therefore suggests that HMGB1 can positively influence the survival of SGNs following ototoxic exposure via both its extracellular and intranuclear functions.