Inhibition of histone methyltransferase PRMT5 attenuates cisplatin-induced hearing loss through the PI3K/Akt-mediated mitochondrial apoptotic pathway / 药物分析学报

This study aimed to evaluate the therapeutic potential of inhibiting protein arginine methyltransferase 5(PRMT5)in cisplatin-induced hearing loss.The effects of PRMT5 inhibition on cisplatin-induced auditory injury were determined using immunohistochemistry,apoptosis assays,and auditory brainstem response.The mechanism of PRMT5 inhibition on hair cell survival was assessed using RNA-seq and Cleavage Under Targets and Tagment-quantitative polymerase chain reaction(CUT&Tag-qPCR)analyses in the HEI-OC1 cell line.Pharmacological inhibition of PRMT5 significantly alleviated cisplatin-induced damage to hair cells and spiral ganglion neurons in the cochlea and decreased apoptosis by protecting mitochondrial function and preventing the accumulation of reactive oxygen species.CUT&Tag-qPCR analysis demonstrated that inhibition of PRMT5 in HEI-OC1 cells reduced the accumulation of H4R3me2s/H3R8me2s marks at the promoter region of the Pik3ca gene,thus activating the expression of Pik3ca.These findings suggest that PRMT5 inhibitors have strong potential as agents against cisplatin-induced ototoxicity and can lay the foundation for further research on treatment strategies of hearing loss.

Dynamic expression of putative receptor protein related to the angiotensin type 1 receptor in postnatal mouse cochlear hair cells and spiral ganglion neurons / 解剖学报

Objective To study the temporal and spatial expressions of G protein-coupled receptor, putative receptor protein related to angiotensin type 1 receptor (APJ), in mammal cochlea postnatal development. Methods The cochlear tissues of each group 11 C57BL/ 6 mice at postnatal day 7 (P7), P14, P28 and postnatal month 2(P2M) were taken out under a stereo microscope. Real-time PCR, Western blotting and immunofluorescent staining were used to detect the expressions of APJ in hair cells and spiral ganglion neurons. Results The expression pattern of APJ in cochleae showed an upward trend during the period from P7 to P2M. The temporal expressions of APJ in hair cells and spiral ganglion neurons increased obviously at P14 and P2M. The spatial expression patterns of APJ in hair cells and spiral ganglion neurons followed a declined gradient from base turns to apex turns at P14. Conclusion APJ expression exhibits a specific spatial and temporal pattern during mouse cochlea postnatal development, and may play a role in cochleae maturation and hearing formation.

Blocking NR2B Receptor Reversed the Process of Cytotoxicity to Spiral Ganglion Neurons Induced by Sodium Salicylate in Guinea Pig / 听力学及言语疾病杂志

Objective To investigate whether blocking NR2B receptor can reverse the process of cytotoxicity to spiral ganglion neurons induced by sodium salicylate in guinea pig by applying ifenprodil (a NR2B antagonist) at the round window niche .Methods Sixty healthy guinea pigs provided by the experimental animal center of Guangxi medical university were randomly and evenly divided into a control group (Group I ,no treatment) ,an APL group (Group II ,60μl APL directly applied to the round window ) ,a sodium salicylate group (Group III ,60 μl APL di-rectly applied to the round window and then be given intraperitoneal sodium salicylate injection ) ,and an ifenprodil group (Group IV ,60μl of 10μmol/l ifenprodil in APL directly applied to the round window and then be given intra-peritoneal sodium salicylate injection ) .Sodium salicylate was given at 400 mg · kg -1 · d-1 for 7 days .Auditory brainstem responses (ABRs) were recorded before animal sacrifice by decapitation .The left cochlea was removed and prepared for detection of caspase -3 expression in spiral ganglion neurons via immunohistochemistry .From each group ,6 cochleae were used to test apoptosis index in spiral ganglion neurons using the TUNEL technique .Results Before salicylate administration ,the ABR threshold was less than 40 dB SPL in all animals .After salicylate ad-ministration ,the ABR threshold was 33 .33 ± 5 .17 dB SPL in Group II ,64 .17 ± 7 .36 dB SPL in Group III and 49 .17 ± 5 .85 dB SPL in Group IV ,in contrast to 31 .67 ± 5 .16 dB SPL in Group I (controls) .The caspase -3 ex-pression was not changed obviously in Group I and Group II ,but was significantly changed in Group III and Group IV (P<0 .01) .The caspase-3 expression appeared to be decreased in Group IV compared to those in Groups III (P<0 .05) ,but still increased compared to those of in Group I and II (P<0 .05) .The apoptosis index among spiral ganglion neurons in Groups III and IV increased significantly compared to those of in Group I and II (P<0 .001) .It was however ,lower in Group IV than in Group III (P<0 .01) .Conclusion Blocking NR2B receptor with specificity can reverse the process of cytotoxicity to spiral ganglion neurons induced by sodium salicylate in guinea pig .

Transplantation of Neural Differentiated Human Mesenchymal Stem Cells into the Cochlea of an Auditory-neuropathy Guinea Pig Model

The aim of this study was to determine the effects of transplanted neural differentiated human mesenchymal stem cells (hMSCs) in a guinea pig model of auditory neuropathy. In this study, hMSCs were pretreated with a neural-induction protocol and transplanted into the scala tympani of the guinea pig cochlea 7 days after ouabain injury. A control model was made by injection of Hanks balanced salt solution alone into the scala tympani of the guinea pig cochlea 7 days after ouabain injury. We established the auditory neuropathy guinea pig model using 1 mM ouabain application to the round window niche. After application of ouabain to the round window niche, degeneration of most spiral ganglion neurons (SGNs) without the loss of hair cells within the organ of Corti and increasing the auditory brain responses (ABR) threshold were found. After transplantation of neural differentiated hMSCs, the number of SGNs was increased, and some of the SGNs expressed immunoreactivity with human nuclear antibody under confocal laser scanning microscopy. ABR results showed mild hearing recovery after transplantation. Based on an auditory neuropathy animal model, these findings suggest that it may be possible to replace degenerated SGNs by grafting stem cells into the scala tympani.

N-Acetylcysteine and N-Nitroarginine Methyl Ester Attenuate Carboplatin-Induced Ototoxicity in Dissociated Spiral Ganglion Neuron Cultures

OBJECTIVES: Carboplatin, a platinum-containing anti-cancer drug used to treat a variety of cancers, induces ototoxicity. Since, reactive oxygen species (ROS) and nitric oxide (NO) seem to be responsible for this toxicity, the antioxidant, N-acetyl-L-cysteine (L-NAC), and NO synthetase inhibitor, N-nitro-L-arginine methyl ester (L-NAME) were predicted to have protective effects against carboplatin ototoxicity. The aim of this study was to test for the protective effects of L-NAC and L-NAME on cochlear hair cells and spiral ganglion neurons (SGNs). METHODS: Cochlear organotypic cultures and dissociated spiral ganglion neuron cultures, from mice postnatal day 5 cultures were used in this study. The cultures were treated with carboplatin alone or in combination with L-NAC or L-NAME, and carboplatin-induced damage was monitored. RESULTS: Treatment with carboplatin induced a significant loss of outer hair cells, while inner hair cells were preserved in the cochlear organotypic cultures. Addition of L-NAC or L-NAME reduced the amount of carboplatin-induced hair cell damage; the differences did not reach statistical significance. However, carboplatin significantly decreased the number of surviving SGNs in dissociated cultures. The toxic effects were significantly reduced by addition of L-NAC or L-NAME. In addition, carboplatin induced the loss of neurites from the SGN somata, and this was not blocked with L-NAC or L-NAME. CONCLUSION: The results of this study suggest that ROS and NO are involved in carboplatin-induced damage to hair cells and SGNs, and administration of L-NAC/L-NAME can be used to attenuate the toxicity.

N-Acetylcysteine and N-Nitroarginine Methyl Ester Attenuate Carboplatin-Induced Ototoxicity in Dissociated Spiral Ganglion Neuron Cultures

OBJECTIVES: Carboplatin, a platinum-containing anti-cancer drug used to treat a variety of cancers, induces ototoxicity. Since, reactive oxygen species (ROS) and nitric oxide (NO) seem to be responsible for this toxicity, the antioxidant, N-acetyl-L-cysteine (L-NAC), and NO synthetase inhibitor, N-nitro-L-arginine methyl ester (L-NAME) were predicted to have protective effects against carboplatin ototoxicity. The aim of this study was to test for the protective effects of L-NAC and L-NAME on cochlear hair cells and spiral ganglion neurons (SGNs). METHODS: Cochlear organotypic cultures and dissociated spiral ganglion neuron cultures, from mice postnatal day 5 cultures were used in this study. The cultures were treated with carboplatin alone or in combination with L-NAC or L-NAME, and carboplatin-induced damage was monitored. RESULTS: Treatment with carboplatin induced a significant loss of outer hair cells, while inner hair cells were preserved in the cochlear organotypic cultures. Addition of L-NAC or L-NAME reduced the amount of carboplatin-induced hair cell damage; the differences did not reach statistical significance. However, carboplatin significantly decreased the number of surviving SGNs in dissociated cultures. The toxic effects were significantly reduced by addition of L-NAC or L-NAME. In addition, carboplatin induced the loss of neurites from the SGN somata, and this was not blocked with L-NAC or L-NAME. CONCLUSION: The results of this study suggest that ROS and NO are involved in carboplatin-induced damage to hair cells and SGNs, and administration of L-NAC/L-NAME can be used to attenuate the toxicity.

An Experimental Study on the Protective Effects of Ginkgo Biloba Extract on the Spiral Ganglion Neuron of the Rat Cochlea / 听力学及言语疾病杂志

Objective To investigate the protective effects of the extract of ginkgo biloba(EGb)on the spiralganglion neuron(SGNs)in cochlea tissues on the hearing loss induced by noise in rats.Methods Thirty-six healthy animals were randomly divided into three groups:the normal control group(n=12).the noise exposured group(n =12)and the EGb treamment group(n=12).The control group received no noise and no medications.The other two groups were exposed to the noise of 110 dB SPL for consecutively 10 days,6 hours per day.The treatment group rats were injected with 10 ml/d EGb while the other two groups with 0.9%saline of the same amount.The experiment lasted for ten days.The rats were measured by auditory brainsterm response(ABR)before and after niose exposure.The ultrastructural changes of SGNs were detected by tranismision electron microscpoe(TEM) and the contents of malondiadehyde(MDA) and activities of superoxide dismutase(SOD)were also measured.Results Hearing were signifcantlly decreased in the experimental group.Nevertheless,EGb relatively reduced the contents of MDA while increased the activities of SOD.Conclusion EGb seems to be able to moderately pretect SGNs and to play a preventive and remedial role in noise-induced hearing loss.

Expression of Neural Cell Adhesion Molecule and Polysialic Acid in Cultured Spiral Ganglion Neurons / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Neural cell adhesion molecule (NCAM) and polysialic acid (PSA) function basically in cell adhesion and migration. In neural development, they are closely associated with axon pathfinding, synaptogenesis, neural cell migration, differentiation and myelination. The purpose of this study is to assess expression of NCAM and PSA expression in spiral ganglion neurons and Schwann cells and to postulate their functions. MATERIALS AND METHOD: Guinea pig spiral ganglion cells were harvested and cultured in vitro. The cells were grown and differentiated in culture medium together with brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3) and glial cell derived neurotrophic factor (GDNF). After 1 week of culturing, the cells were fixed and immunocytochemical staining with beta-III tubulin, S-100, polysialic acid (PSA) and neural cell adhesion molecule (NCAM) were performed. We then checked axon growth rate with Axon Analyzer System(R). RESULTS: In the spiral ganglion culture, cultured neurons showed positive staining for beta-III tubulin, NCAM, and different expressions of PSA. S-100 positive glial cells (Schwann cells) showed different expressions of NCAM and no expression of PSA. Some NCAM positive neurons and Schwann cells were in contact each other. The growth rate of neuron was about 10-30 micrometer/h using Axon Analyzer System(R). CONCLUSION: We postulated that NCAM may play an important role in neural cell adhesion, myelination, fasciculation and ganglion formation. But PSA did not express the adhesive function of NCAM ; its absence may have been due to developmental reason. The differential expression of NCAM in the Schwann cells may indicate its different immunocytochemical characteristics and functions as shown in the CNS glial cells, astrocytes and oligodendrocytes.

Influence of Gentamycin Electrophysiological Properties on Murine Cochlea Spiral Ganglion Neurons / 听力学及言语疾病杂志

Objective To explore the influence of gentamycin on murine cochlea spiral ganglion neurons' electrophysiological properties and its significance.Methods Using whole-cell voltage clamp technique, we studied gentamycin's influence on the peak currents of the potassium and sodium ion channels on cell membranes of acutely dissociated murine spiral ganglion neurons,the relationship to gentamycin's concentration in extracellular fluid, and the currents' recovery after gentamycin being washed out.Results Gentamycin could inhibit voltage-dependent potassium channels, but it couldn't inhibit voltage-dependent sodium channels. Gentamycin's inhibitation on potassium currents had dose-dependence with gentamycin's concerntration in extracellular fluid and the currents recoverd incompletely after gentamycin being washed out.Conclusion This research explained the ototoxic mechanism of gentamycin through its action on keeping from spiral ganglion neurons' potassium ion channels from the electrophysiological aspect, and set a foundation for further research.

Effect of Neurotrophins and Depolarization on Survival of Spiral Ganglion Neurons in Dissociation Cell Culture / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Several neurotrophic factors have been shown to play an essential trophic role in the development, maintenance and regulation of neuronal function. Specific neurotrophins are currently used in clinical trials for the treatment of some neurodegenerative diseases. The purposes of this experiment were twofold. Firstly, we aimed to determine the trophic effects of BDNF, NT-3, and 25 mM K+ on auditory neurons in dissociated cultures of early postnatal spiral ganglia. Secondly, we tried to collect pure neural cells after dissociating the spiral ganglions using the immunomagnetic sorting method with one of neuronal surface antigens. MATERIALS AND METHODS: Dissociated spiral ganglion cell cultures were pre-pared from cochleae of Sprague Dawley rats of 5-6 days old, and maintained in a neurobasal medium with modified N2 supplements. BDNF (50 ng/ml), NT-3 (50 ng/ml), and 25 mM K+ were added to the cultures, respectively. These cells were grown during the time course (24hr, 48hr, 72hr, 98hr) and stained with NF-200 to identify survival of spiral ganglion neurons. Immunomagnetic cell sorting for separation of spiral ganglion neurons in dissociated cells was carried out using the MiniMACS Separating System. Magnetically separated cells were analysed by flow cytometry. RESULTS: Survival of the auditory neurons in the dissociated cells was significantly increased by addition of BDNF, NT-3, and 25K. The effect of 25 mM K+ on neuronal survival showed the highest in the experimental conditions. BDNF dramatically increased the neurite length compared with those under other conditions. After immunomagnetic sorting in dissociated cultures, spiral ganglion neurons were shown to contain 50% of the fluorescently labeled positive cells. CONCLUSIONS: Neurotrophins (BDNF, NT-3) and depolarization by 25 mM K+ were essential trophic factors for postnatal auditory neurons and BDNF stimulated neuritogenesis in cultured spiral ganglion neurons. The immunomagnetic cell sorting method is not appropriate for collecting pure neural cells from the dissociated cells of spiral ganglia (50% purity).

The Influence of Furosemide on the Currents of the Delayed Rectification Potassium Channels and Sodium Channels of Mice's Spiral Ganglion Neurons / 听力学及言语疾病杂志

Objective To study the influence of furosemide on the currents of the delayed rectification potassium channel and sodium channel of mice's spiral ganglion neurons.Methods Postnatal mice(P1～P6) spiral ganglion neurons were obtained by mechanical dissociation and enzymolysis.Delayed rectification potassium channels' currents and sodium channels' currents were recorded with whole-cell patch clamp techniques.Observed was the influence of furosemide on potassium channels and sodium channels.Results When furosemide was added around the spiral ganglion neurons,the delayed rectification potassium currents were inhibited to around +200～+300 pA,and sodium currents were inhibited to about 30% of peak current.Furosemide was washed away after working steadily for 1 minute,5 and 10 minutes.The delayed rectification potassium currents could recover to 98%,64%,and 25% of the peak currents before and sodium currents could recover to 96%,76% and 54% of the peak currents accordingly.Conclusion The currents of delayed rectification potassium channels and sodium channels could be inhibited by furosemide to different degrees.The longer furosemide was used,the greater damage could occur in the ion channels.