顺铂耳毒性大鼠耳聋模型的研究

摘要 目的：探讨顺铂对大鼠造成的听力损伤及耳蜗细胞形态学变化。方法：体内实验,运用顺铂腹腔注射的方法,连续七天注射,通过听性脑干反应检测,观察顺铂对不同日龄的大鼠听力损伤情况;测听后取耳蜗,通过基底膜铺片和冰冻切片的免疫荧光染色,观察听力损伤后对耳蜗毛细胞和螺旋神经元的影响。体外实验,耳蜗器官培养免疫荧光染色,观察顺铂对耳蜗毛细胞和螺旋神经元的影响。结果：顺铂具有耳毒性,会对大鼠听力造成损伤,高频听力损伤更加严重,而且对不同日龄的大鼠造成的听力损失不同,小日龄的大鼠对顺铂耳毒性更加敏感。体内实验,顺铂耳毒性造成听力损失,会引起大鼠耳蜗毛细胞的缺失,但未观察到明显的螺旋神经元缺失,也没有观察到明显的Cleaved caspase-3阳性螺旋神经元细胞。体外实验,可以观察到顺铂同时引起毛细胞和螺旋神经元产生明显的损伤。结论：体、内外实验,都可以建立稳定的顺铂耳毒性大鼠耳聋模型,对研究顺铂损伤耳蜗毛细胞的发生机制和保护奠定了实验基础。     

Therapeutic potential of neurotrophins for treatment of hearing loss

Degeneration of spiral ganglion neurons (SGNs) and hair cells in the cochlea induced by aging, injury, ototoxic drugs, acoustic trauma, and various diseases is the major cause of hearing loss. Discovery of growth factors that can either prevent SGN and hair-cell death or stimulate hair-cell regeneration would be of great interest. Studies over the past several years have provided evidence that specific neurotrophins are potent survival factors for SGNs and protect these neurons from ototoxic drugs in vitro and in vivo. Current research focuses more on understanding the mechanism of hair-cell regeneration/differentiation and identification of growth factors that can stimulate hair-cell regeneration. SGNs are required to relay the signal to the central nervous system even when a cochlear implant is used to replace hair-cell function or in the case that cochlear sensory epithelium can be stimulated to regenerate new hair cells successfully. Therefore, neurotrophins may have their therapeutic value in prevention and treatment of hearing impairment.     

Cochlear Inner Hair Cell Ribbon Synapse is the Primary Target of Ototoxic Aminoglycoside Stimuli

The ribbon synapses of inner hair cells (IHCs) play an important role in sound encoding and neurotransmitter release. However, it remains unclear whether IHC ribbon synapse plasticity can be interrupted by ototoxic aminoglycoside stimuli. Here, we report that quantitative changes in the number of IHC ribbon synapses and hearing loss occur in response to gentamicin treatment in mice. Using 3D reconstruction, we were able to calculate the number of IHC ribbon synapses after ototoxic gentamicin exposure. Mice were injected intraperitoneally with a low dose of gentamicin (100 mg/kg) once a day for 14 days. Double immunostaining was used to identify IHC ribbon synapses; histopathology and scanning electron microscopy were used to observe the morphology of cochlear hair cells and spiral ganglion neurons (SGNs), the hearing threshold shifts were recorded by auditory brainstem response examinations. Our study shows that the maximal number of IHC ribbon synapses appeared at the 7th day after treatment, followed by a significant reduction after the 7th day regardless of ongoing treatment. Correspondingly, the maximal elevation of hearing threshold was observed at the 7th day after treatment. Meanwhile, additional cochlear components included OHCs, IHCs, and SGNs were unaffected, suggesting that IHC ribbon synapses are more susceptible to ototoxic aminoglycoside stimulation. Our study indicated that quantitative changes in the number of IHC ribbon synapses is critical response to lower dose of ototoxic stimulation, and may contribute to moderate hearing loss. Additionally, our data indcated that ribbon synaptic plasticity may require the quantitative changes to play self-protective role adapted to ototoxic aminoglycoside stimuli.     

Role of Somatostatin Receptor-2 in Gentamicin-Induced Auditory Hair Cell Loss in the Mammalian Inner Ear

Hair cells and spiral ganglion neurons of the mammalian auditory system do not regenerate, and their loss leads to irreversible hearing loss. Aminoglycosides induce auditory hair cell death in vitro, and evidence suggests that phosphatidylinositol-3-kinase/Akt signaling opposes gentamicin toxicity via its downstream target, the protein kinase Akt. We previously demonstrated that somatostatin—a peptide with hormone/neurotransmitter properties—can protect hair cells from gentamicin-induced hair cell death in vitro, and that somatostatin receptors are expressed in the mammalian inner ear. However, it remains unknown how this protective effect is mediated. In the present study, we show a highly significant protective effect of octreotide (a drug that mimics and is more potent than somatostatin) on gentamicin-induced hair cell death, and increased Akt phosphorylation in octreotide-treated organ of Corti explants in vitro. Moreover, we demonstrate that somatostatin receptor-1 knockout mice overexpress somatostatin receptor-2 in the organ of Corti, and are less susceptible to gentamicin-induced hair cell loss than wild-type or somatostatin-1/somatostatin-2 double-knockout mice. Finally, we show that octreotide affects auditory hair cells, enhances spiral ganglion neurite number, and decreases spiral ganglion neurite length.     

Glutamate induces apoptosis in cultured spiral ganglion explants

Traumatic sound exposure, aminoglycoside antibiotics, cochlea ischemia or traumatic stress leads to an excessive release of glutamate from inner hair cells into the synaptic cleft. The high glutamate concentration can cause a swelling and destruction of the dendrites of spiral ganglion neurons of type I as well as a reduction in the number of neurons. This may be a cause of hearing loss. The mechanism causing the reduction of neurons is still not known. Apoptosis, also called programmed cell death, could be involved. In this study, cultured spiral ganglion explants were incubated with glutamate in high concentrations. Neurite outgrowth was determined and additionally a new method was established for studying the morphology of single spiral ganglion neurons. For the first time it was shown that glutamate induces apoptosis of spiral ganglion neurons, which could be blocked selectively by a caspase-3 inhibitor. This could offer a new therapeutic strategy for hearing disorders.     

Neurotrophin-4/5, brain-derived neurotrophic factor,and neurotrophin-3 promote survival of cultured vestibular ganglion neurons and protect them against neurotoxicity of ototoxins

The ability of neurotrophin-4/5 (NT-4/5), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and nerve growth factor (NGF) to promote survival of postnatal rat vestibular ganglion neurons (VGNs) was examined in dissociated cell cultures. Of the four neurotrophins, NT-4/5 and BDNF were equally effective but more potent than NT-3 in promoting the survival of VGNs. In contrast, NGF showed no detectable effects. As expected, TrkB-IgG (a fusion protein of extracellular domain of TrkB and Fc domain of human immunoglobulin G) specifically inhibited the survival-promoting effects by NT-4/5 or BDNF and TrkC-IgG fusion protein completely blocked that of NT-3. Immunohistochemistry with TrkB, TrkA, and p75 antisera revealed that VGNs made TrkB and p75 proteins, but not TrkA protein. Ototoxic therapeutic drugs such as cisplatin and gentamicin often induce degeneration of hair cells and ganglion neurons in both auditory and vestibular systems that leads to impairment of hearing and balance. When cisplatin and gentamicin were added to the dissociated VGN culture in which the hair cells were absent, additional cell death of VGNs was induced, suggesting that the two ototoxins may have a direct neurotoxic effect on ganglion neurons in addition to their known toxicity on hair cells. However, if the cultures were co-treated with neurotrophins, NT-4/5, BDNF, and NT-3, but not NGF, prevented or reduced the neurotoxicity of the two ototoxins. Thus, the three neurotrophins are survival factors for VGNs and are implicated in the therapeutic prevention of VGN loss caused by injury and ototoxins. © 1995 John Wiley & Sons, Inc.     

Apoptosis of inner hair cells caused by laser ablation of their spiral ganglion neurons in cultures of the mouse organ of Corti

Laser beam ablation of spiral ganglion neurons was performed in seven organotypic cultures of the newborn mouse cochlea between 5 and 8 days in vitro, with a recovery period of from 18 hours to 3 days. Direct somatic injury (laser or mechanical) inflicted on hair cells does not necessarily cause their death; many of them survive, repair damage and re-establish their neurosensory connections. By contrast, laser irradiation and ablation of their afferent spiral ganglion neurons causes a most spectacular degeneration of sensory cells within 18–48 hours after the insult. Ultrastructurally, the degenerated hair cells—characteristically the inner hair cells—display “dark-cell vacuolar degeneration” that combines the signs of apoptotic death (the peripheral condensation of nuclear chromatin and nuclear pyknosis) with signs of cell edema, vacuolization and necrosis. The ultimate condensation of the cytoplasm gives the dead cells a jet black appearance. The irradiated spiral ganglion neurons die displaying similar pathological characteristics. The extent and locus of inner hair cell degeneration correspond to that of ablated spiral ganglion neurons: ultimately the ablation of one neuron causes degeneration of a single inner hair cell within the closest radial segment of the afferent innervation. The elimination of spiral ganglion neurons by mechanical means does not affect hair cell survival. It is inferred that the laser pulse acts as a stimulus depolarizing the neuronal membrane of the spiral ganglion neurons and their radial fibers and causing the excitotoxic death of their synaptic sensory cells through excessive stimulation of the glutamatergic receptors. Reciprocal pre-and postsynaptic synapses between the afferent dendrites and inner hair cells in culture could possibly serve as entryways of the stimulus. The pathogenesis of this apparent transsynaptically-induced apoptotic death of inner hair cells will be further examined in culture.     

Heregulin enhances regenerative proliferation in postnatal rat utricular sensory epithelium after ototoxic damage

Hair cell loss due to acoustic and ototoxic damage often leads to hearing and balance impairments. Although a spontaneous event in chicks and lower vertebrates, hair cell replacement occurs at a much lower frequency in mammals presumably due to a very low rate of supporting cell proliferation following injury. We report here that heregulin, a member of the neuregulin family, dramatically enhances proliferation of supporting cells in postnatal rat utricular epithelial sheet cultures after gentamicin treatment, as revealed by bromo-deoxyuridine (BrdU) immunocytochemistry. A dose-dependent study shows that the maximal effects of heregulin are achieved at 3 nM. The mitogenic effects of heregulin are confirmed in utricular whole mount cultures. Autoradiography of the utricular whole mount cultures shows that heregulin also enhances the number of tritiated thymidine-labeled cells within the hair cell layer. TaqMan quantitative RT-PCR analysis and immunocytochemistry reveal that heregulin and its binding receptors (ErbB-2, ErbB-3 and ErbB-4) are expressed in the inner ear sensory epithelium. Of several ligands activating various ErbB receptors, including heregulin, neuregulin-3, β-cellulin, heparin binding-epidermal growth factor (HB-EGF), transforming growth factor-α (TGF-α) and EGF, heregulin shows the most potent mitogenic effects on supporting cells. Because neuregulin-3 that signals only through ErbB-4 does not show an effect, these data suggest that activation of the ErbB-2-ErbB-3 heterodimeric complexes, rather than ErbB-4, is critical for the proliferative response in the utricular sensory epithelium. In addition, gentamicin treatment induces an upregulation of heregulin mRNA. Considered together, heregulin may play an important role in hair cell regeneration following ototoxic damage.     

Profiling drug-induced cell death pathways in the zebrafish lateral line

Programmed cell death (PCD) is an important process in development and disease, as it allows the body to rid itself of unwanted or damaged cells. However, PCD pathways can also be activated in otherwise healthy cells. One such case occurs in sensory hair cells of the inner ear following exposure to ototoxic drugs, resulting in hearing loss and/or balance disorders. The intracellular pathways that determine if hair cells die or survive following this or other ototoxic challenges are incompletely understood. We use the larval zebrafish lateral line, an external hair cell-bearing sensory system, as a platform for profiling cell death pathways activated in response to ototoxic stimuli. In this report the importance of each pathway was assessed by screening a custom cell death inhibitor library for instances when pathway inhibition protected hair cells from the aminoglycosides neomycin or gentamicin, or the chemotherapy agent cisplatin. This screen revealed that each ototoxin likely activated a distinct subset of possible cell death pathways. For example, the proteasome inhibitor Z-LLF-CHO protected hair cells from either aminoglycoside or from cisplatin, while d-methionine, an antioxidant, protected hair cells from gentamicin or cisplatin but not from neomycin toxicity. The calpain inhibitor leupeptin primarily protected hair cells from neomycin, as did a Bax channel blocker. Neither caspase inhibition nor protein synthesis inhibition altered the progression of hair cell death. Taken together, these results suggest that ototoxin-treated hair cells die via multiple processes that form an interactive network of cell death signaling cascades.     

Reinnervation of hair cells by auditory neurons after selective removal of spiral ganglion neurons

Hearing loss can be caused by primary degeneration of spiral ganglion neurons or by secondary degeneration of these neurons after hair cell loss. The replacement of auditory neurons would be an important step in any attempt to restore auditory function in patients with damaged inner ear neurons or hair cells. Application of beta-bungarotoxin, a toxin derived from snake venom, to an explant of the cochlea eradicates spiral ganglion neurons while sparing the other cochlear cell types. The toxin was found to bind to the neurons and to cause apoptotic cell death without affecting hair cells or other inner ear cell types as indicated by TUNEL staining, and, thus, the toxin provides a highly specific means of deafferentation of hair cells. We therefore used the denervated organ of Corti for the study of neuronal regeneration and synaptogenesis with hair cells and found that spiral ganglion neurons obtained from the cochlea of an untreated newborn mouse reinnervated hair cells in the toxin-treated organ of Corti and expressed synaptic vesicle markers at points of contact with hair cells. These findings suggest that it may be possible to replace degenerated neurons by grafting new cells into the organ of Corti.     

Deafness due to degeneration of cochlear neurons in caspase-3-deficient mice

Mice that lack caspase-3, which functions in apoptosis, were generated by gene targeting and shown to undergo hearing loss. The ABR threshold of the caspase-3(-/-) mice was significantly elevated compared to that of caspase-3(+/+) mice at 15 days of age and was progressively elevated further by 30 days. Distortion product otoacoustic emissions were not detectable in caspase-3(-/-) mice at 15 days of age. Caspase-3(-/-) mice exhibited marked degeneration of spiral ganglion neurons and a loss of inner and outer hair cells in the cochlea at 30 days of age, although no such changes were apparent at 15 days. The degenerating neurons manifested features, including cytoplasmic vacuolization, distinct from those characteristic of apoptosis. Spiral ganglion neurons and cochlear hair cells thus appear to require caspase-3 for survival but not for initial development. The mapping of both the human caspase-3 gene and the locus responsible for an autosomal dominant, nonsyndromic form of hearing loss (DFNA24) to chromosome 4q35 suggests that the caspase-3(-/-) mice may represent a model of this human condition.     

Stem cells as therapy for hearing loss

One of the greatest challenges in the treatment of inner-ear disorders is to find a cure for the hearing loss that is caused by the loss of cochlear hair cells or spiral ganglion neurons. The recent discovery of stem cells in the adult inner ear that are capable of differentiating into hair cells, as well as the finding that embryonic stem cells can be converted into hair cells, raise hope for the future development of stem-cell-based treatment regimens. Here, we propose different approaches for using stem cells to regenerate the damaged inner ear and we describe the potential obstacles that translational approaches must overcome for the development of stem-cell-based cell-replacement therapies for the damaged inner ear.     

Caspase-Mediated Apoptosis in the Cochleae Contributes to the Early Onset of Hearing Loss in A/J Mice

A/J and C57BL/6 J (B6) mice share a mutation in Cdh23 (ahl allele) and are characterized by age-related hearing loss. However, hearing loss occurs much earlier in A/J mice at about four weeks of age. Recent study has revealed that a mutation in citrate synthase (Cs) is one of the main contributors, but the mechanism is largely unknown. In the present study, we showed that A/J mice displayed more severe degeneration of hair cells, spiral ganglion neurons, and stria vascularis in the cochleae compared with B6 mice. Moreover, messenger RNA accumulation levels of caspase-3 and caspase-9 in the inner ears of A/J mice were significantly higher than those in B6 mice at 2 and 8 weeks of age. Immunohistochemistry localized caspase-3 expression mainly to the hair cells, spiral ganglion neurons, and stria vascularis in cochleae. In vitro transfection with Cs short hairpin RNA (shRNA) alone or cotransfection with Cs shRNA and Cdh23 shRNA significantly increased the levels of caspase-3 in an inner ear cell line (HEI-OC1). Finally, a pan-caspase inhibitor Z-VAD-FMK could preserve the hearing of A/J mice by lowering about 15 decibels of the sound pressure level for the auditory-evoked brainstem response thresholds. In conclusion, our results suggest that caspase-mediated apoptosis in the cochleae, which may be related to a Cs mutation, contributes to the early onset of hearing loss in A/J mice.     

The Cochlear CRF Signaling Systems and their Mechanisms of Action in Modulating Cochlear Sensitivity and Protection Against Trauma

A key requirement for encoding the auditory environment is the ability to dynamically alter cochlear sensitivity. However, merely attaining a steady state of maximal sensitivity is not a viable solution since the sensory cells and ganglion cells of the cochlea are prone to damage following exposure to loud sound. Most often, such damage is via initial metabolic insult that can lead to cellular death. Thus, establishing the highest sensitivity must be balanced with protection against cellular metabolic damage that can lead to loss of hair cells and ganglion cells, resulting in loss of frequency representation. While feedback mechanisms are known to exist in the cochlea that alter sensitivity, they respond only after stimulus encoding, allowing potentially damaging sounds to impact the inner ear at times coincident with increased sensitivity. Thus, questions remain concerning the endogenous signaling systems involved in dynamic modulation of cochlear sensitivity and protection against metabolic stress. Understanding endogenous signaling systems involved in cochlear protection may lead to new strategies and therapies for prevention of cochlear damage and consequent hearing loss. We have recently discovered a novel cochlear signaling system that is molecularly equivalent to the classic hypothalamic–pituitary–adrenal (HPA) axis. This cochlear HPA-equivalent system functions to balance auditory sensitivity and susceptibility to noise-induced hearing loss, and also protects against cellular metabolic insults resulting from exposures to ototoxic drugs. We review the anatomy, physiology, and cellular signaling of this system, and compare it to similar signaling in other organs/tissues of the body.     

Functional variants in NOS1 and NOS2A are not associated with progressive hearing loss in Ménière's disease in a European Caucasian population

Hearing loss in Ménière's disease (MD) is associated with loss of spiral ganglion neurons and hair cells. In a guinea pig model of endolymphatic hydrops, nitric oxide synthases (NOS) and oxidative stress mediate loss of spiral ganglion neurons. To test the hypothesis that functional variants of NOS1 and NOS2A are associated with MD, we genotyped three functional variants of NOS1 (rs41279104, rs2682826, and a cytosine-adenosine microsatellite repeat in exon 1f) and the CCTTT repeat in the promoter of NOS2A gene (rs3833912) in two independent MD sets (273 patients in total) and 550 controls. A third cohort of American patients was genotyped as replication cohort for the CCTTT repeat. Neither allele nor genotype frequencies of rs41279104 and rs2682826 were associated with MD, although longer alleles of the cytosine-adenosine microsatellite repeat were marginally significant (corrected p?=?0.05) in the Mediterranean cohort but not in a second Galicia cohort. Shorter numbers of the CCTTT repeat in NOS2A were significantly more frequent in Galicia controls (OR?=?0.37 [CI, 0.18-0.76], corrected p?=?0.04), but this finding could not be replicated in Mediterranean or American case-control populations. Meta-analysis did not support an association between CCTTT repeats and risk for MD. Severe hearing loss (>75?dB) was also not associated with any functional variants studied. Functional variants of NOS1 and NOS2A do not confer susceptibility for MD.     

5,7-Dihydroxy-4-methylcoumarin modulates the JNK/FoxO1 signaling pathway to attenuate cisplatin-induced ototoxicity by suppressing oxidative stress and apoptosis in vitro

5,7-Dihydroxy-4-methylcoumarin (D4M) is attributed to free radical scavenging effects, with wide application for anti-oxidation. This work aimed to assess D4M's impact on cisplatin-induced ototoxicity. The cell viability was estimated with CCK-8 assay. Apoptosis was detected by the Annexin V-FITC and PI assay. The reactive oxygen species (ROS) level was determined by MitoSOX-Red and CellROX-Green probes. Mitochondrial membrane potential was analyzed with TMRM staining. Immunofluorescence was utilized for hair cells and spiral ganglion neuron detection. Apoptosis-associated proteins were assessed by cleaved caspase-3 and TUNEL staining. These results showed that D4M pretreatment protected hair cells from cisplatin-induced damage, increased cell viability, and decreased apoptosis in House Ear Institute-Organ of Corti1 (HEI-OC1) cells and neonatal mouse cochlear explants. D4M significantly inhibited cisplatin-induced mitochondrial apoptosis and reduced ROS accumulation. In addition, the protective effect of D4M on cisplatin-induced ototoxicity was also confirmed in cochlear hair cells and spiral ganglion neurons in neonatal mice. Mechanistic studies showed that D4M markedly downregulated p-JNK and elevated the expression ratio of p-FoxO1/FoxO1, thereby reducing cisplatin-induced caspase-dependent apoptosis. Meanwhile, D4M-related protection of HEI-OC1 cells was significantly blunted by JNK signaling induction with anisomycin. This study supports the possibility that D4M may be used as a new compound to prevent cisplatin-related hearing loss.     

The role of PKCζ in amikacin-induced apoptosis in the cochlea: Prevention by aspirin

Aminoglycoside antibiotics are ototoxic, inducing irreversible sensorineural hearing loss mediated by oxidative and excitotoxic stresses. The NF-κB pathway is involved in the response to aminoglycoside damage in the cochlea. However, the molecular mechanisms of this ototoxicity remain unclear. We investigated the expression of PKCζ, a key regulator of NF-κB activation, in response to aminoglycoside treatment. Amikacin induced PKCζ cleavage and nuclear translocation. These events were concomitant with chromatin condensation and paralleled the decrease in NF-κB (p65) levels in the nucleus. Amikacin also induced the nuclear translocation of apoptotic inducing factor (AIF). Prior treatment with aspirin prevented PKCζ cleavage and nuclear translocation. Thus, aspirin counteracts the early effects of amikacin, thereby protecting hair cells and spiral ganglion neurons. These results demonstrate that PKCζ acts as sentinel connecting specific survival pathways to mediate cellular responses to amikacin ototoxicity. E. Lecain and B. Omri both authors contributed equally.