Apoptosis Of Spiral Ganglion Cells Research Articles

Effects of estrogen on apoptosis of spiral ganglion cells in cochlea of aged C57BL/6J mice

Objective: To observe the effects of estrogen on cochlear spiral ganglia cell apoptosis in aged C57BL/6J mice, and to explore the possible mechanism of estrogen's protective effects on senile deafness. Methods: Forty C57BL/6J mice were divided into the following four groups (10 mice/group): 3 m group (3 months old group), 12 m group (12 months old sham operation group); In the 12 m OVX group (ovariectomized at 12 months), bilateral oophorectomy was performed at the age of 9 months and normal feeding was performed until the age of 12 months.The 12m OVX+E2 group (estrogen intervention group) underwent bilateral oophorectomy at 9 months of age. After the one-month washout period, mice in the other groups were treated with estrogen at the dose of 100 μg/(kg·d) by subcutaneous injection, lasting 2 months to 12 months old. Mice in the other groups were fed normally.Blood samples were collected from the tail vein at the end of the treatment in 12 m OVX+E2 group. Enzyme-linked immunosorbent assays (ELISAs) was used to determine the serum estrogen levels. Auditory brainstem response (ABR) was used to detect the changes of hearing threshold in each group.Mice were anesthetized with 2% pentobarbital sodium. Bilateral cochlea was extracted after neck amputation and paraffin-embedded sections were performed.Hematoxylin eosin (HE) staining was used to observe the morphological changes in the cochlea spiral ganglion neurons (SGN), and TUNEL staining was used to observe the apoptosis of SGN. The expression levels of Caspase-3, Bax and Bcl-2 mRNA of the apoptotic proteins in cochlear spiral ganglion were measured by real-time fluorescence quantitative PCR (QRT-PCR). Results: Compared with the 3 m group, the hearing threshold of the 12 m group was improved, the loss of spiral ganglion cells was aggravated, and the apoptosis of the cells was increased(P＜0.01). After removal of the ovaries, the hearing threshold of the mice in the 12 m OVX group was higher than that in the 12 m control group (P＜0.01), and this increased threshold was accompanied by an increased loss of spiral ganglion cells, and increased apoptosis (P＜0.01). Meanwhile, the mRNA levels of apoptotic protein Caspase-3 and Bax were increased (P＜0.01), while the mRNA level of anti-apoptotic protein Bcl-2 was decreased (P＜0.01). After exogenous estrogen was given to the 12 m OVX+E2 group, the hearing threshold was lower than that in 12 m OVX group(P＜0.01). At the same time, the apoptosis of helical ganglion cells was reduced, the mRNA levels of Caspase-3 and Bax were decreased (P＜0.01), and the Bcl-2 mRNA level was increased (P＜0.01). Conclusion: Estrogen inhibited apoptosis of cochlear spiral ganglion cells in aged C57BL/6J mice ,thus achieving a protective effect on presbycusis.

Effects of Memantine on Aminoglycoside-Induced Apoptosis of Spiral Ganglion Cells in Guinea Pigs.

To explore whether memantine, an N-methyl-D-aspartate receptor antagonist, exerts a neuroprotective effect against apoptosis of spiral ganglion cells (SGCs) induced by gentamicin. An animal experiment. Dong-A University College of Medicine, Busan, Korea. Gentamicin was injected into the left cochleae of guinea pigs to induce apoptosis of SGCs; the contralateral cochleae served as controls. Memantine was intraperitoneally injected 12 hours and 1 hour prior to gentamicin injection. At 1 week after gentamicin and/or memantine injection, the cochleae were removed and stained with hematoxylin and eosin to evaluate morphologic changes and apoptosis. Western blotting was performed to measure FasL expression and the extent of caspase activation in SGCs. SGC numbers remained stable after memantine treatment. Western blotting showed that FasL expression and activation of caspases 3, 8, and 9 were reduced in SGCs after memantine treatment. Memantine attenuated the gentamicin-induced apoptosis of SGCs in guinea pigs. Moreover, memantine may affect Fas-FasL signaling in the receptor-mediated apoptotic pathway and caspase activation involved in the receptor-mediated and mitochondrial apoptotic pathways.

Conditioned medium from neonatal rat olfactory ensheathing cells promotes the survival and proliferation of spiral ganglion cells

Conclusion: Neonatal olfactory ensheathing cells conditioned medium (OEC-CM) can promote growth and proliferation of neonatal spiral ganglion cells (SGCs), as well as protecting them from apoptosis. Objective: To investigate the effects of neonatal OEC-CM on the survival, apoptosis, and proliferation of spiral ganglion. Methods: SGCs were cultured with OEC-CM, the conditioned medium from olfactory nerve fibroblasts (ONF-CM) or control medium containing 10% serum (DF12-10S). After different periods of culture, the cell types present were identified by immunofluorescent confocal imaging analysis, cell proliferation was determined by BrdU incorporation, and apoptosis was detected with the double-labeled NeuroTACS in situ Apoptosis Detection Kit. Results: SGCs cultured with regular control medium died off via apoptosis. Culturing cells with ONF-CM had no significant effects on the natural process of survival and apoptosis of SGCs. However, culture with OEC-CM significantly increased survival and decreased the apoptotic percentage of SGCs. In addition, OEC-CM temporarily stimulated the proliferative capacity of SGCs on the third day of culture.

Gentamicin-induced spiral ganglion cell death: Apoptosis mediated by ROS and the JNK signaling pathway

Conclusion: Reactive oxygen species (ROS) and the c-Jun N-terminal kinase (JNK) signaling pathway may be involved in secondary apoptosis of spiral ganglion cells (SGCs) induced by intracochlear gentamicin injection. Objectives: The purpose of this study was to ascertain the role of ROS and the JNK signaling pathway in secondary apoptosis of SGCs induced by intracochlear gentamicin treatment. Methods: Gentamicin (40 mg/ml) was injected into the cochlea of guinea pigs (n = 18) to destroy the hair cells and induce secondary apoptosis of SGCs. At 1 (n = 6), 2 (n = 6), and 3 (n = 6) weeks after gentamicin treatment, the cochleas were removed and stained with hematoxylin and eosin, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling to observe the morphologic changes and apoptosis of SGCs. A dihydroethidium (DHE) assay was performed to detect ROS generation, and RT-PCR and Western blot analysis were used to assess the expression of Fas ligand (FasL), JNK, and c-Jun. Results: After gentamicin was injected into the cochlea, apoptosis and progressive loss of SGCs were observed. RT-PCR and Western blot analysis showed increased expression of FasL after gentamicin treatment. ROS generation detected by DHE fluorescence increased progressively, and the expression of JNK, phospho-JNK, c-Jun, and phospho-c-Jun also increased.

Conditioned medium from neonatal rat olfactory ensheathing cells promotes the survival and proliferation of spiral ganglion cells

Neonatal olfactory ensheathing cells conditioned medium (OEC-CM) can promote growth and proliferation of neonatal spiral ganglion cells (SGCs), as well as protecting them from apoptosis. To investigate the effects of neonatal OEC-CM on the survival, apoptosis, and proliferation of spiral ganglion. SGCs were cultured with OEC-CM, the conditioned medium from olfactory nerve fibroblasts (ONF-CM) or control medium containing 10% serum (DF12-10S). After different periods of culture, the cell types present were identified by immunofluorescent confocal imaging analysis, cell proliferation was determined by BrdU incorporation, and apoptosis was detected with the double-labeled NeuroTACS in situ Apoptosis Detection Kit. SGCs cultured with regular control medium died off via apoptosis. Culturing cells with ONF-CM had no significant effects on the natural process of survival and apoptosis of SGCs. However, culture with OEC-CM significantly increased survival and decreased the apoptotic percentage of SGCs. In addition, OEC-CM temporarily stimulated the proliferative capacity of SGCs on the third day of culture.

Secondary Apoptosis of Spiral Ganglion Cells Induced by Aminoglycoside: Fas–Fas Ligand Signaling Pathway

Hair cell loss results in the secondary loss of spiral ganglion neurons (SGNs), over a period of several weeks. The death of the SGNs themselves results from apoptosis. Previous studies have shown that several molecules are involved in the apoptosis of SGNs that occurred secondary to hair cell loss. However, the precise mechanism of apoptosis of the SGNs remains unclear. The aim of this study was to ascertain the secondary apoptosis of spiral ganglion cells induced by aminoglycoside and to investigate the role of the Fas-FasL signaling pathway using guinea pigs as an experimental animal model. Laboratory study using experimental animals. Guinea pigs weighing 250 to 300 g (n = 21) from 3 to 4 weeks of age were used. Gentamicin (60 microL) was injected through a cochleostomy site on their left side. At 1 (n = 7), 2 (n = 7), and 3 (n = 7) weeks after gentamicin treatment, their cochleas were obtained from their temporal bone. Hematoxylin and eosin and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling staining were performed to observe apoptosis. To investigate the involvement of the Fas-FasL signaling pathway in the secondary apoptosis of SGNs, we performed reverse transcription-polymerase chain reaction (RT-PCR), western blotting, and immunohistochemistry. A progressive loss of spiral ganglion cells with increasing time after gentamicin treatment was observed on light microscopic examination. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling staining demonstrated induction of apoptotic cell death in SGNs after gentamicin treatment. Expression of FasL increased over time after gentamicin treatment as determined by RT-PCR and western blotting. On immunohistochemical staining, we observed the localization of FasL in the SGNs. The proapoptotic molecules Bax and Bad were increased, but levels of the antiapoptotic molecule Bcl-2 were decreased at increasing survival times after gentamicin treatment on RT-PCR. The gentamicin-treated group displayed initial activation of caspase-8 and increased the cleavage of caspase-3, caspase-8, and PARP protein in a time-dependent manner. The secondary apoptosis of SGNs could be a result of the apoptotic Fas-FasL signaling pathway. Blocking the Fas-FasL signaling pathway could be considered as a method for preventing secondary degeneration of SGNs, and further studies are needed to confirm this.

Trauma-induced hearing loss due to apoptotic auditory neuronal death in cerebellopontine angle manipulations: an experimental study

This study reports for the first time the appearance, extent and duration of auditory neuron apoptosis following injury to the central processes. Apoptosis was studied in a rat model that consisted of compression of the auditory nerve in the cerebellopontine angle cistern with intraoperative recordings of auditory nerve compound action potentials to ensure highly reproducible results. Rats were killed between days 0 and 14 after compression, and apoptosis of spiral ganglion cells (SGCs) was evaluated quantitatively and qualitatively. The average number of TUNEL-positive apoptotic SGCs in each cochlear turn increased from days 1 to 5, and then decreased gradually to an undetectable level on day 14 after compression. The average proportion of apoptotic SGCs identified in any cochlear turn on any day was always lower than 10%. These results of our present study should be useful in determining the therapeutic time window for rescuing auditory neurons undergoing apoptosis due to injury during surgery in the cerebellopontine angle.

Intrathecal injection of HVJ-E containing HGF gene to cerebrospinal fluid can prevent and ameliorate hearing impairment in rats.

Hearing impairment, which is the most prevalent sensory deficit of human beings, needs a breakthrough in therapeutic technologies. One technology is the usage of a vector system to reach the inner ear, and another is by a therapeutic molecule. Here we developed a novel gene therapy strategy by combining hepatocyte growth factor (HGF) with hemagglutinating virus of Japan envelope (HVJ-E) vector. When HVJ-E containing human HGF gene was injected intrathecally into the cerebrospinal fluid via cisterna magna of rats, the vector reached the inner ear region, and human HGF gene expression was detected in the spiral ganglion cells (SGCs) of the inner ear. Expression of endogenous rat HGF and its receptor, c-Met, was also induced in SGCs by human HGF. Kanamycin treatment results in hearing impairment by inducing degeneration of hair cells (HCs) and apoptosis of SGCs in rats. By HGF gene transfer before kanamycin treatment, both loss of HCs and apoptosis of SGCs were prevented. Furthermore, hearing function, evaluated by auditory brainstem response, was maintained at a normal level. When HGF gene transfer was performed 2 wk after kanamycin treatment, hearing impairment was significantly recovered. These results indicate a novel and effective therapeutic strategy against sensorineural hearing impairment.

Regeneration of auditory nerve following complete sectioning and intrathecal application of the IN-1 antibody.

The cochlear nerve of adult Lewis rats was following microsurgical exposure in the cerebellopontine angle (CPA). The lesions completely interrupted the auditory nerve axons at the lesion site producing ipsilateral deafness in all animals. The rats were then treated with a recombinant Fab fragment of the antibody IN-1 against nerve growth inhibitory proteins for one to two weeks. An age-matched control group of rats was treated with unspecific mouse IgG antibody. Because the cochlear nerve lesions resulted in significant neuronal apoptosis of spiral ganglion cells, neurotrophin-3 (NT-3) was applied to the lesion site immediately post-injury in some rats. Electrophysiological studies were carried out by recording the brainstem auditory evoked potentials (BAEP) before and immediately after the lesion, and at regular intervals up to 2 months after injury. Cochlear nerve fibres were anterogradely traced by horseradish peroxidase (HRP) or biotinylated dextran amine (BDA) injected into the spiral ganglion. The results achieved in this study were consistent with the following conclusions: 1) transection of the adult rat cochlear nerve at the CPA results in functional deafness, disappearance of BAEP, apoptosis of parent axotomized neurons of the spiral ganglion, and interruption of labelled axons close to the lesion site; 2) NT-3 is able to partially rescue axotomized neurons of the spiral ganglion; 3) injured cochlear nerve fibres show a limited spontaneous sprouting and regrowth response which does not lead to BAEP recovery; 4) intrathecal treatment with IN-1 directed against myelin-associated neurite growth inhibitory proteins promotes significant elongation of the injured fibres; and 5) the regenerating fibres seem to navigate to correct targets, and be able to establish synaptic connections for functional recovery as depicted by BAEP examinations.