Rho GTPases are essential regulators of the actin cytoskeleton.They are involved in various physiological and biochemical processes such as the regulation of cytoskeleton dynamics,development,proliferation,survival,an...Rho GTPases are essential regulators of the actin cytoskeleton.They are involved in various physiological and biochemical processes such as the regulation of cytoskeleton dynamics,development,proliferation,survival,and regeneration.During the development of cochlear hair cells,Rho GTPases are activated by various extracellular signals through membrane receptors to further stimulate multiple downstream effectors.Specifically,RhoA,Cdc42,and Rac1,members of the classical subfamily of the Rho GTPase family,regulate the development and maintenance of cilia by inducing the polymerization of actin monomers and stabilizing actin filaments.In addition,they also regulate the normal morphology orientation of ciliary bundles in auditory hair cells,which is an important element of cell polarity regulation.Moreover,the actin-related pathways mediated by RhoA,Cdc42,and Rac1 also play a role in the motility of outer hair cells,indicating that the function of Rho GTPases is crucial in the highly polar auditory sensory system.In this review,we focus on the expression of RhoA,Cdc42,and Rac1 in cochlear hair cells and how these small molecules participate in ciliary bundle morphogenesis and cochlear hair cell movement.We also discuss the progress of current research investigating the use of these small molecules as drug targets for deafness treatment.展开更多
The purpose of the present study was to determine protectivie effects of basic fibroblast growth factor (bFGF) on cochlear neurons and hair cells in vitro and in vivo. In experiment I, cultured spiral ganglion neurons...The purpose of the present study was to determine protectivie effects of basic fibroblast growth factor (bFGF) on cochlear neurons and hair cells in vitro and in vivo. In experiment I, cultured spiral ganglion neurons (SGNs) prepared from P3 mice were exposed to 20mM glutamate for 2 hours before the culture medium was replaced with fresh medium containing 0, 25, 50, and 100 ng/ml bFGF, respectively. Fourteen days later, all cultures were fixed with 4% paraformaldehyde, and stained with 1% toluidine blue. The number of surviving SGNs were counted and the length of SGNs neurites were measured. Exposure to 20 mM glutamate for 24 hours resulted in an inhibition on neurite outgrowth of SGNs and elevated cell death. Treatment of the cultures with bFGF led to promotion of neurite outgrowth and elevated number of surviving SGNs. Effects of bFGF were dose dependent with the highest potency at 100 ng/ml. In experiment Ⅱ, in vivo studies were carried out with guinea pigs in which bFGF or artificial perilymph was perfused into the cochlea to assess possible protective effects of bFGF on cochlear hair cells and compound action potentials(CAP). The CAPs were measured before, immediatly and 48 hours after exposure to noise. Significant differences in CAP were observed (p＜0. 05 ) among the bFGF perfused group, control group(t =3. 896 ) and artificial perilymph perfused group (t =2. 520) at 48 hours after noise exposure, Cochleae were removed and hair cell Loss was analyzed in surface preparations prepared from all experimental animals. Acoustic trauma caused loss of 651 and 687 inner hair cells in the control and artificial perilymph perfused group, respectively. In sharp contrast, only 31 inner hair cells were lost in the bFGF perfused ears. Similarly, more outer hair cells died in the control and perilymph perfuesed group (41830 and 41968, respectively) than in the group treated with bFGF (34258). Our results demonstrate that bFGF protected SGNs against glutmate neurotoxicity in vitro. In addition, treatment with bFGF also protected hair cells from acoustic trauma.展开更多
Atoh1 overexpression in cochlear epithelium induces new hair cell formation. Use of adenovirus-mediated Atoh1 overexpression has mainly focused on the rat lesser epithelial ridge and induces ectopic hair cell regenera...Atoh1 overexpression in cochlear epithelium induces new hair cell formation. Use of adenovirus-mediated Atoh1 overexpression has mainly focused on the rat lesser epithelial ridge and induces ectopic hair cell regeneration. The sensory region of rat cochlea is difficult to transfect, thus new hair cells are rarely produced in situ in rat cochlear explants. After culturing rat cochleae in medium containing 10% fetal bovine serum, adenovirus successfully infected the sensory region as the width of the supporting cell area was significantly increased. Adenovirus encoding Atoh1 infected the sensory region and induced hair cell formation in situ. Combined application of the Notch inhibitor DAPT and Atoh1 increased the Atoh1 expression level and decreased hes1 and hes5 levels, further promoting hair cell generation. Our results demonstrate that DAPT enhances Atoh1 activity to promote hair cell regeneration in rat cochlear sensory epithelium in vitro.展开更多
Objective To study the recovery of the outer hair cells in the bat cochlea after gentamicin exposure. Methods Bats were injected with a daily dose of gentamicin for 15 consecutive days and bromodeoxyuridine (BrdU) was...Objective To study the recovery of the outer hair cells in the bat cochlea after gentamicin exposure. Methods Bats were injected with a daily dose of gentamicin for 15 consecutive days and bromodeoxyuridine (BrdU) was given from day 16 to day 40 of this recovery phase. Hearing was assessed by overt acoustic behavior and auditory brainstem responses analysis, which was performed one day prior to the first injection and a day after the last injection (day 16). On day 40 animals were sacrificed for detection of cells that could take up BrdU. Results After 15 days of gentamicin treatment, all of the animals were proved to be deafened with significant increases of ABR thresholds, compared with control group. The findings in immunocytochemical stained samples and scanning electron microscopy revealed that BrdU labeled nuclei were observed in the cochlea in all of the deafened animals most commonly in the regions of the first-row and second-row Deiter's cells (DCs) and occasionally in the regions of the third-row DCs. Conclusion We suggest that, under sufficient drug and enough time, the bat cochlear supporting cells can directly transdifferentiate into the outer hair cells after aminoglycoside exposure. This transdifferentation process is essential for repair of outer hair cells and recovery of normal function after gentamicin exposure.展开更多
Objective This study is to explore the relationship between acetylcholine(ACh)-induced calcium release from intracellular Ca2+ stores and function of outer hair cell(OHC) motors, in an attempt to elucidate the mechani...Objective This study is to explore the relationship between acetylcholine(ACh)-induced calcium release from intracellular Ca2+ stores and function of outer hair cell(OHC) motors, in an attempt to elucidate the mechanism of OHC electromotility at resting state. Methods OHCs were isolated from adult guinea pig (200-300 g) cochlea and loaded with Fluo-3/AM. The cells were treated with ACh/dHBSS, ACh/HBSS, dHBSS only or HBSS only. Intracellular [Ca2+]i variations in cells under the four treatments were observed using an Ar-Kr laser scan confocal microscope. Results [Ca2+]i oscillations were recorded in five OHCs treated with ACh/dHBSS but not in other cells. This is the first time that Ach-excited [Ca2+]i oscillations are reported in guinea pig OHCs independent of extracellular calcium. Conclusions ACh-excited [Ca2+]i oscillations in OHCs originates from intracellular calcium release and may play a crucial role in maintaining active mechanical motility of the OHC at resting and modulating OHC electromotility.展开更多
Hair cells in the mammalian inner ear are very fragile and are often injured as a result of acoustic trauma or exposure to ototoxic drugs (cisplatin, aminoglycosides, etc) [1].
Apical membrane recycling has been proposed to be important for normal hair cell function. The current study reports an in vitro work that demonstrates the presence of phosphatidylserine (PS) and PS-positive vesicles ...Apical membrane recycling has been proposed to be important for normal hair cell function. The current study reports an in vitro work that demonstrates the presence of phosphatidylserine (PS) and PS-positive vesicles labeled by Annexin V in the apical portion of hair cells. The following characteristics of the PS-positive vesicles were noticed using scanning confocal fluorescence microscopy:(1) variable sizes around 200 nm; (2) variable distribution patterns (either uniformly along individual stereocilia in the hair bundle or irregular) in the stereocilia from cell to cell; (3) variable sizes and numbers at locations along the border of the cuticular plate (CP), with a large number of them located at the vestigal kinocilial location; (4) motility with some of the vesicles during the observation period; (5) increase in PS labeling and the number of PS-positive vesicles after loud sound stimulation; and (6) decreased PS labeling and PS-positive vesicle numbers following treatment with LY-294002, a PI3 -kinase inhibitor. These results suggest that the presence of PS-positive vesicles at the apical area of hair cells may be indicative of vesicle shedding or transportation of a protein or rafts.展开更多
Auditory hair cells(HCs)are the mechanosensory receptors of the cochlea,and HC loss or malfunction can result from genetic defects.Dock4,a member of the Dock180-related protein superfamily,is a guanine nucleotide exch...Auditory hair cells(HCs)are the mechanosensory receptors of the cochlea,and HC loss or malfunction can result from genetic defects.Dock4,a member of the Dock180-related protein superfamily,is a guanine nucleotide exchange factor for Rac1,and previous reports have shown that Dock4 mutations are associated with autism spectrum disorder,myelodysplastic syndromes,and tumorigenesis.Here,we found that Dock4 is highly expressed in the cochlear HCs of mice.However,the role of Dock4 in the inner ear has not yet been investigated.Taking advantage of the piggyBac transposon system,Dock4 knockdown(KD)mice were established to explore the role of Dock4 in the cochlea.Compared to wild-type controls,Dock4 KD mice showed significant hearing impairment from postnatal day 60.Dock4 KD mice showed hair bundle deficits and increased oxidative stress,which eventually led to HC apoptosis,late-onset HC loss,and progressive hearing loss.Furthermore,molecular mechanism studies showed that Rac1/β-catenin signaling was significantly downregulated in Dock4 KD cochleae and that this was the cause for the disorganized stereocilia and increased oxidative stress in HCs.Overall,our work demonstrates that the Dock4/Rac1/β-catenin signaling pathway plays a critical role in the maintenance of auditory HCs and hearing function.展开更多
The cochlear auditory epithelium contains two types of sound receptors,inner hair cells(IHCs)and outer hair cells(OHCs).Mouse models for labelling juvenile and adult IHCs or OHCs exist;however,labelling for embryonic ...The cochlear auditory epithelium contains two types of sound receptors,inner hair cells(IHCs)and outer hair cells(OHCs).Mouse models for labelling juvenile and adult IHCs or OHCs exist;however,labelling for embryonic and perinatal IHCs or OHCs are lacking.Here,we generated a new knock-in Fgf8^(P2A-3×GFP/+)(Fgf8^(GFP)/+)strain,in which the expression of a series of three GFP fragments is controlled by endogenous Fgf8 cis-regulatory elements.After confirming that GFP expression accurately reflects the expression of Fgf8,we successfully obtained both embryonic and neonatal IHCs with high purity,highlighting the power of Fgf8^(GFP)/+.Furthermore,our fate-mapping analysis revealed,unexpectedly,that IHCs are also derived from inner ear progenitors expressing Insm1,which is currently regarded as an OHC marker.Thus,besides serving as a highly favorable tool for sorting early IHCs,Fgf8^(GFP)/+will facilitate the isolation of pure early OHCs by excluding IHCs from the entire hair cell pool.展开更多
Hair follicle stem cells(HFSCs)in the bulge are a multipotent adult stem cell population.They can periodically give rise to new HFs and even regenerate the epidermis and sebaceous glands during wound healing.An increa...Hair follicle stem cells(HFSCs)in the bulge are a multipotent adult stem cell population.They can periodically give rise to new HFs and even regenerate the epidermis and sebaceous glands during wound healing.An increasing number of biomarkers have been used to isolate,label,and trace HFSCs in recent years.Considering more detailed data from single-cell transcriptomics technology,we mainly focus on the important HFSC molecular markers and their regulatory roles in this review.展开更多
Inner ear hair cells are important for maintaining hearing.Irreversible damage to hair cells is an important cause of sensorineural deafness.Electromagnetic radiation,especially high-power microwave,is an important th...Inner ear hair cells are important for maintaining hearing.Irreversible damage to hair cells is an important cause of sensorineural deafness.Electromagnetic radiation,especially high-power microwave,is an important threat to human health in modern society and war.However,it is not clear whether high-power microwave has an effect on cochlea hair cells.This study aimed to assess the effects of high-power microwave on cochlear hair cells in guinea pigs,and investigate the potential protection of these cells against high-power microwave-induced damage by recombinant adenovirus A20.Based on experimental results,a 65 W/cm^(2) irradiation density applied to guinea pigs in this study to establish a high-power microwave inner ear injury model.In addition,pAdEeay-1/A20 was injected via a round window into experimental guinea pig cochlea,whereas artificial perilymph was injected into the control group.Auditory function was assessed by testing the auditory brainstem response threshold,and damage to cochlear hair cells was investigated by cell counting and scanning electron microscopy observations of the basilar membrane.Inner ear injury was observed 6 hours after 65 W/cm^( 2 ) of irradiation and the auditory brainstem response threshold was significantly higher in the irradiation group(P<0.05)compared with other groups.Propidium iodide staining and scanning electron microscopy results indicated that significant morphological changes occurred after radiation,especially to inner hair cells,which exhibited remarkable damage and the presence of several unknown spherical substances.Auditory brainstem response threshold was decreased in the pAdEeay-1/A20 group compared with the artificial perilymph group;moreover,damage to hair cells was milder in the pAdEeay-1/A20 group compared with the control group(P<0.01).Thus,high-power microwave can cause damage to cochlear hair cells,as well as hearing loss with prolonged exposure and/or high dosage.In this regard,65 W/cm^( 2 ) of irradiation for 6 hours is a reliable target dose for observation of damage.The zinc finger protein A20 can protect cochlear hair cells from high-power microwave-induced damage and prevent further hearing loss.This study was approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University,China on April 18,2017.展开更多
Exposure to intense noise can damage cochlear hair cells,leading to hearing loss in mammals.To avoid this constraint,most mammals have evolved in relatively quiet environments.Echolocating bats,however,are naturally e...Exposure to intense noise can damage cochlear hair cells,leading to hearing loss in mammals.To avoid this constraint,most mammals have evolved in relatively quiet environments.Echolocating bats,however,are naturally exposed to continuous intense sounds from their own and neighboring sonar emissions for maintaining sonar directionality and range.Here,we propose the presence of intense noise resistance in cochlear hair cells of echolocating bats against noise-induced hearing loss(NIHL).To test this hypothesis,we performed noise exposure experiments for laboratory mice,one nonecholocating bat species,and five echolocating bat species.Contrary to nonecholocating fruit bats and mice,the hearing and the cochlear hair cells of echolocating bats remained unimpaired after continuous intense noise exposure.The comparative analyses of cochleae transcriptomic data showed that several genes protecting cochlear hair cells from intense sounds were overexpressed in echolocating bats.Particularly,the experimental examinations revealed that ISL1 overexpression significantly improved the survival of cochlear hair cells.Our findings support the existence of protective effects in cochlear hair cells of echolocating bats against intense noises,which provides new insight into understanding the relationship between cochlear hair cells and intense noises,and preventing or ameliorating NIHL in mammals.展开更多
It has been revealed in recent years that contralateral acoustic stimulation can affect cochlear active mechanisms through activating medial olivocochlear system (MOC) of the cochlear efferent nerve fibers. The MOC is...It has been revealed in recent years that contralateral acoustic stimulation can affect cochlear active mechanisms through activating medial olivocochlear system (MOC) of the cochlear efferent nerve fibers. The MOC is therefore postulated to exert protective effects on outer hair cells (OHCs) under intense sound condition. In this study the effects of 4 kHz intense tone exposure on distortion product otoacoustic emissions (DPOAEs) in guinea pigs with and without contralateral white noise stimulation were observed so that to investigate the protective effects of MOC on OHCs. The results showed that DPOAEs obviously deceased after the intense tone exposure in all animals, while both the amplitude reduction and the affected frequency range of DPOAEs were smaller in animals with simultaneously delivered contralateral white noise during the tone exposure than that in animals without colltralateral acoustic stimulation. The above results may suggest some protective nature of the contralateral sound stimulating effects which might be mediated through the activity of MOC. These perhaps can serve as the evidence that the protective mechanism against intense sound operates in the outer hair cells which are strongly innervated by展开更多
Oxidative stress is the key determinant in the pathogenesis of noise-induced hearing loss(NIHL). Given that cellular defense against oxidative stress is an energyconsuming process, the aim of the present study was to ...Oxidative stress is the key determinant in the pathogenesis of noise-induced hearing loss(NIHL). Given that cellular defense against oxidative stress is an energyconsuming process, the aim of the present study was to investigate whether increasing energy availability by glucose supplementation protects cochlear hair cells against oxidative stress and attenuates NIHL. Our results revealed that glucose supplementation reduced the noiseinduced formation of reactive oxygen species(ROS) and consequently attenuated noise-induced loss of outer hair cells, inner hair cell synaptic ribbons, and NIHL in CBA/J mice. In cochlear explants, glucose supplementation increased the levels of ATP and NADPH, as well as attenuating H_2O_2-induced ROS production and cytotoxicity. Moreover, pharmacological inhibition of glucose transporter type 1 activity abolished the protective effects of glucose against oxidative stress in HEI-OC1 cells. These findings suggest that energy availability is crucial for oxidative stress resistance and glucose supplementation offers a simple and effective approach for the protection of cochlear hair cells against oxidative stress and NIHL.展开更多
Recent studies have revealed great functional and structural heterogeneity in the ribbon-type synapses at the basolateral pole of the isopotential inner hair cell(IHC).This feature is believed to be critical for audit...Recent studies have revealed great functional and structural heterogeneity in the ribbon-type synapses at the basolateral pole of the isopotential inner hair cell(IHC).This feature is believed to be critical for audition over a wide dynamic range,but whether the spatial gradient of ribbon morphology is fine-tuned in each IHC and how the mitochondrial network is organized to meet local energy demands of synaptic transmission remain unclear.By means of three-dimensional electron microscopy and artificial intelligence-based algorithms,we demonstrated the cell-wide structural quantification of ribbons and mitochondria in mature mid-cochlear IHCs of mice.We found that adjacent IHCs in staggered pairs differ substantially in cell body shape and ribbon morphology gradient as well as mitochondrial organization.Moreover,our analysis argues for a location-specific arrangement of correlated ribbon and mitochondrial function at the basolateral IHC pole.展开更多
Sodium salicylate is an anti-inflammatory medication with a side-effect of tinnitus.Here,we used mouse cochlear cultures to explore the effects of salicylate treatment on cochlear inner hair cells(IHCs).We found that ...Sodium salicylate is an anti-inflammatory medication with a side-effect of tinnitus.Here,we used mouse cochlear cultures to explore the effects of salicylate treatment on cochlear inner hair cells(IHCs).We found that IHCs showed significant damage after exposure to a high concentration of salicylate.Whole-cell patch clamp recordings showed that 1–5 mmol/L salicylate did not affect the exocytosis of IHCs,indicating that IHCs are not involved in tinnitus generation by enhancing their neuronal input.Instead,salicylate induced a larger peak amplitude,a more negative half-activation voltage,and a steeper slope factor of Ca^(2+)current.Using noise analysis of Ca^(2+)tail currents and qRT-PCR,we further found that salicylate increased the number of Ca^(2+)channels along with CaV1.3 expression.All these changes could act synergistically to enhance the Ca^(2+)influx into IHCs.Inhibition of intracellular Ca^(2+)overload significantly attenuated IHC death after 10 mmol/L salicylate treatment.These results implicate a cellular mechanism for tinnitus generation in the peripheral auditory system.展开更多
Voltage-gated sodium channels(VGSCs)are transiently expressed in cochlear hair cells before hearing onset and play an indispensable role in shaping spontaneous activity.In this study,we showed that Na^+currents shaped...Voltage-gated sodium channels(VGSCs)are transiently expressed in cochlear hair cells before hearing onset and play an indispensable role in shaping spontaneous activity.In this study,we showed that Na^+currents shaped the spontaneous action potentials in developing mouse inner hair cells(IHCs)by decreasing the time required for the membrane potential to reach the action-potential threshold.In immature IHCs,we identified 9 known VGSC subtypes(Navl.la-l.9ot),among which Navl.7a was the most highly expressed subtype and the main contributor to Na+currents in developing hair cells.Electrophysiological recordings of two cochlea-specific Navi.7 variants(CbmNavl.7a and CbmNavl.7b)revealed a novel loss-of-function mutation(C934R)at the extracellular linker between segments 5 and 6 of domain II.In addition,post-transcriptional modification events,such as alternative splicing and RNA editing,amended the gating properties and kinetic features of CbmNavl.7a(C934).These results provide molecular and functional characteristics of VGSCs in mammalian IHCs and their contributions to spontaneous physiological activity during cochlear maturation.展开更多
Objective Adipose-derived stem cells(ADSCs) are suggested to possess a highly plastic ability to differentiate into several specific cell types in addition to adipocyte lineages, including germ layer tissue-specific c...Objective Adipose-derived stem cells(ADSCs) are suggested to possess a highly plastic ability to differentiate into several specific cell types in addition to adipocyte lineages, including germ layer tissue-specific cell lineages such as chondrocyte, myocyte, neuronal, and osteoblast lineages. The aim of this study is to establish an in vitro culture technique for ADSCs in an adult guinea pig model that facilitate their differentiation into hair cell-like cells. Materials and Methods Cells from inguinal fat pads in adult guinea pigs were cultured with β-mercaptoethanol, RA, Forskorin, Heregulin, bFGF, BDNF and EGF. Cellular differentiation was examined using immunocytochemistry techniques. Results The ADSCs demonstrated hair cell immunophenotypes with expression of epitopes of the hair cell marker protein myosin Ⅶa. Conclusion ADSCs from adult guinea pig adipose tissue can differentiate into hair cell-like cells when cultured in vitro. ADSCs may serve as seed cells for tissue engineering.展开更多
Background:Cisplatin is a chemotherapeutic agent commonly used clinically for the treatment of various human cancers.Patients often reduce the use of cisplatin due to its side effects,which in turn affects its treatme...Background:Cisplatin is a chemotherapeutic agent commonly used clinically for the treatment of various human cancers.Patients often reduce the use of cisplatin due to its side effects,which in turn affects its treatment.This study explored the mechanism of action of safflower extract as an adjuvant traditional Chinese medicine for chemotherapy.Methods:Primary human follicle dermal papilla cells(HFDPCs)were used as target cells for cisplatininduced damage to hair cells.Western blotting was used to investigate the molecular targets of cisplatin and safflower extract in causing HFDPCs damage.Cell survival and cell cycle were analyzed by mitochondrial staining reagent WST-1 and propidium iodide.Results:Cisplatin could reduce the viability of HFDPCs without causing cell death.Cisplatin increased the level of phospho-Rad17 in HFDPCs and activated the Chk1/Cdc25C signaling to reduce the expression of Cdc2 protein,thereby arresting the cells in the G2/M phase.The combination of safflower extract and the flavonoids could effectively inhibit the signal transduction of Rad17/Chk1/Cdc25 in cisplatin-treated cells and reduce the cell population in the G2/M phase.Finally,we also confirmed that safflower extract could effectively inhibit the damage to HFDPCs caused by cisplatin,mainly at the level of reducing the DNA damage caused by cisplatin.Conclusions:Safflower extract can be used as an adjuvant Chinese medicine for chemotherapy to reduce the damage caused by chemotherapy to normal hair follicle cells.展开更多
基金supported by the Natural Science Foundation of Jiangsu Province,No.BK20221377(to JG)the Natural Science Foundation of the Jiangsu Higher Education Institutions of China,No.22KJB180023(to JG)。
文摘Rho GTPases are essential regulators of the actin cytoskeleton.They are involved in various physiological and biochemical processes such as the regulation of cytoskeleton dynamics,development,proliferation,survival,and regeneration.During the development of cochlear hair cells,Rho GTPases are activated by various extracellular signals through membrane receptors to further stimulate multiple downstream effectors.Specifically,RhoA,Cdc42,and Rac1,members of the classical subfamily of the Rho GTPase family,regulate the development and maintenance of cilia by inducing the polymerization of actin monomers and stabilizing actin filaments.In addition,they also regulate the normal morphology orientation of ciliary bundles in auditory hair cells,which is an important element of cell polarity regulation.Moreover,the actin-related pathways mediated by RhoA,Cdc42,and Rac1 also play a role in the motility of outer hair cells,indicating that the function of Rho GTPases is crucial in the highly polar auditory sensory system.In this review,we focus on the expression of RhoA,Cdc42,and Rac1 in cochlear hair cells and how these small molecules participate in ciliary bundle morphogenesis and cochlear hair cell movement.We also discuss the progress of current research investigating the use of these small molecules as drug targets for deafness treatment.
文摘The purpose of the present study was to determine protectivie effects of basic fibroblast growth factor (bFGF) on cochlear neurons and hair cells in vitro and in vivo. In experiment I, cultured spiral ganglion neurons (SGNs) prepared from P3 mice were exposed to 20mM glutamate for 2 hours before the culture medium was replaced with fresh medium containing 0, 25, 50, and 100 ng/ml bFGF, respectively. Fourteen days later, all cultures were fixed with 4% paraformaldehyde, and stained with 1% toluidine blue. The number of surviving SGNs were counted and the length of SGNs neurites were measured. Exposure to 20 mM glutamate for 24 hours resulted in an inhibition on neurite outgrowth of SGNs and elevated cell death. Treatment of the cultures with bFGF led to promotion of neurite outgrowth and elevated number of surviving SGNs. Effects of bFGF were dose dependent with the highest potency at 100 ng/ml. In experiment Ⅱ, in vivo studies were carried out with guinea pigs in which bFGF or artificial perilymph was perfused into the cochlea to assess possible protective effects of bFGF on cochlear hair cells and compound action potentials(CAP). The CAPs were measured before, immediatly and 48 hours after exposure to noise. Significant differences in CAP were observed (p＜0. 05 ) among the bFGF perfused group, control group(t =3. 896 ) and artificial perilymph perfused group (t =2. 520) at 48 hours after noise exposure, Cochleae were removed and hair cell Loss was analyzed in surface preparations prepared from all experimental animals. Acoustic trauma caused loss of 651 and 687 inner hair cells in the control and artificial perilymph perfused group, respectively. In sharp contrast, only 31 inner hair cells were lost in the bFGF perfused ears. Similarly, more outer hair cells died in the control and perilymph perfuesed group (41830 and 41968, respectively) than in the group treated with bFGF (34258). Our results demonstrate that bFGF protected SGNs against glutmate neurotoxicity in vitro. In addition, treatment with bFGF also protected hair cells from acoustic trauma.
基金supported by the National Natural Science Foundation of China,No.81420108010,81271084,81200740,81371093
文摘Atoh1 overexpression in cochlear epithelium induces new hair cell formation. Use of adenovirus-mediated Atoh1 overexpression has mainly focused on the rat lesser epithelial ridge and induces ectopic hair cell regeneration. The sensory region of rat cochlea is difficult to transfect, thus new hair cells are rarely produced in situ in rat cochlear explants. After culturing rat cochleae in medium containing 10% fetal bovine serum, adenovirus successfully infected the sensory region as the width of the supporting cell area was significantly increased. Adenovirus encoding Atoh1 infected the sensory region and induced hair cell formation in situ. Combined application of the Notch inhibitor DAPT and Atoh1 increased the Atoh1 expression level and decreased hes1 and hes5 levels, further promoting hair cell generation. Our results demonstrate that DAPT enhances Atoh1 activity to promote hair cell regeneration in rat cochlear sensory epithelium in vitro.
基金supported by the National Natural Science Foundation of China (No.39970785) International Collaborate Research Foundation of National Natural Science of China (No.322200462).
文摘Objective To study the recovery of the outer hair cells in the bat cochlea after gentamicin exposure. Methods Bats were injected with a daily dose of gentamicin for 15 consecutive days and bromodeoxyuridine (BrdU) was given from day 16 to day 40 of this recovery phase. Hearing was assessed by overt acoustic behavior and auditory brainstem responses analysis, which was performed one day prior to the first injection and a day after the last injection (day 16). On day 40 animals were sacrificed for detection of cells that could take up BrdU. Results After 15 days of gentamicin treatment, all of the animals were proved to be deafened with significant increases of ABR thresholds, compared with control group. The findings in immunocytochemical stained samples and scanning electron microscopy revealed that BrdU labeled nuclei were observed in the cochlea in all of the deafened animals most commonly in the regions of the first-row and second-row Deiter's cells (DCs) and occasionally in the regions of the third-row DCs. Conclusion We suggest that, under sufficient drug and enough time, the bat cochlear supporting cells can directly transdifferentiate into the outer hair cells after aminoglycoside exposure. This transdifferentation process is essential for repair of outer hair cells and recovery of normal function after gentamicin exposure.
文摘Objective This study is to explore the relationship between acetylcholine(ACh)-induced calcium release from intracellular Ca2+ stores and function of outer hair cell(OHC) motors, in an attempt to elucidate the mechanism of OHC electromotility at resting state. Methods OHCs were isolated from adult guinea pig (200-300 g) cochlea and loaded with Fluo-3/AM. The cells were treated with ACh/dHBSS, ACh/HBSS, dHBSS only or HBSS only. Intracellular [Ca2+]i variations in cells under the four treatments were observed using an Ar-Kr laser scan confocal microscope. Results [Ca2+]i oscillations were recorded in five OHCs treated with ACh/dHBSS but not in other cells. This is the first time that Ach-excited [Ca2+]i oscillations are reported in guinea pig OHCs independent of extracellular calcium. Conclusions ACh-excited [Ca2+]i oscillations in OHCs originates from intracellular calcium release and may play a crucial role in maintaining active mechanical motility of the OHC at resting and modulating OHC electromotility.
基金supported by the follow grants:1. The StateKey Program of National Natural Science of China (Grant No. 30730040)2. The National High Technology Research and Development Program of China (Grant No.2007AA02Z150)+2 种基金3. The National Natural Science Foundation of China (30871398)Key Projects in the National Science & Technology Supporting Program during the Eleventh Five-Year Plan Period (2008BAI50B08 2007 BAI18B12, 2007BAI18B14)
文摘Hair cells in the mammalian inner ear are very fragile and are often injured as a result of acoustic trauma or exposure to ototoxic drugs (cisplatin, aminoglycosides, etc) [1].
文摘Apical membrane recycling has been proposed to be important for normal hair cell function. The current study reports an in vitro work that demonstrates the presence of phosphatidylserine (PS) and PS-positive vesicles labeled by Annexin V in the apical portion of hair cells. The following characteristics of the PS-positive vesicles were noticed using scanning confocal fluorescence microscopy:(1) variable sizes around 200 nm; (2) variable distribution patterns (either uniformly along individual stereocilia in the hair bundle or irregular) in the stereocilia from cell to cell; (3) variable sizes and numbers at locations along the border of the cuticular plate (CP), with a large number of them located at the vestigal kinocilial location; (4) motility with some of the vesicles during the observation period; (5) increase in PS labeling and the number of PS-positive vesicles after loud sound stimulation; and (6) decreased PS labeling and PS-positive vesicle numbers following treatment with LY-294002, a PI3 -kinase inhibitor. These results suggest that the presence of PS-positive vesicles at the apical area of hair cells may be indicative of vesicle shedding or transportation of a protein or rafts.
基金This work was supported by grants from National Key R&D Program of China(2021YFA1101300,2020YFA0112503,2020YFA0113600)the Strategic Priority Research Program of the Chinese Academy of Science(XDA16010303)+5 种基金the National Natural Science Foundation of China(82030029,81970882,82071013,81870721,92149304,82000984)the Natural Science Foundation of Jiangsu Province(BE2019711)the Science and Technology Department of Sichuan Province(2021YFS0371)the Shenzhen Fundamental Research Program(JCYJ20190814093401920,JCYJ20210324125608022)the China National Postdoctoral Program for Innovative Talents(BX20200082)the Open Research Fund of State Key Laboratory of Genetic Engineering,Fudan University(SKLGE-2109).
文摘Auditory hair cells(HCs)are the mechanosensory receptors of the cochlea,and HC loss or malfunction can result from genetic defects.Dock4,a member of the Dock180-related protein superfamily,is a guanine nucleotide exchange factor for Rac1,and previous reports have shown that Dock4 mutations are associated with autism spectrum disorder,myelodysplastic syndromes,and tumorigenesis.Here,we found that Dock4 is highly expressed in the cochlear HCs of mice.However,the role of Dock4 in the inner ear has not yet been investigated.Taking advantage of the piggyBac transposon system,Dock4 knockdown(KD)mice were established to explore the role of Dock4 in the cochlea.Compared to wild-type controls,Dock4 KD mice showed significant hearing impairment from postnatal day 60.Dock4 KD mice showed hair bundle deficits and increased oxidative stress,which eventually led to HC apoptosis,late-onset HC loss,and progressive hearing loss.Furthermore,molecular mechanism studies showed that Rac1/β-catenin signaling was significantly downregulated in Dock4 KD cochleae and that this was the cause for the disorganized stereocilia and increased oxidative stress in HCs.Overall,our work demonstrates that the Dock4/Rac1/β-catenin signaling pathway plays a critical role in the maintenance of auditory HCs and hearing function.
基金funded by the National Key R&D Program of China(2021YFA1101804)the Strategic Priority Research Program of the Chinese Academy of Science(XDB32060100)+3 种基金the National Natural Science Foundation of China(91949119,82101212,and 82101209)a Shanghai Municipal Science and Technology Major Project(2018SHZDZX05)the Science and Technology Commission of Shanghai Municipality(21ZR1440200)the Shanghai Sailing Program(20YF1426400).
文摘The cochlear auditory epithelium contains two types of sound receptors,inner hair cells(IHCs)and outer hair cells(OHCs).Mouse models for labelling juvenile and adult IHCs or OHCs exist;however,labelling for embryonic and perinatal IHCs or OHCs are lacking.Here,we generated a new knock-in Fgf8^(P2A-3×GFP/+)(Fgf8^(GFP)/+)strain,in which the expression of a series of three GFP fragments is controlled by endogenous Fgf8 cis-regulatory elements.After confirming that GFP expression accurately reflects the expression of Fgf8,we successfully obtained both embryonic and neonatal IHCs with high purity,highlighting the power of Fgf8^(GFP)/+.Furthermore,our fate-mapping analysis revealed,unexpectedly,that IHCs are also derived from inner ear progenitors expressing Insm1,which is currently regarded as an OHC marker.Thus,besides serving as a highly favorable tool for sorting early IHCs,Fgf8^(GFP)/+will facilitate the isolation of pure early OHCs by excluding IHCs from the entire hair cell pool.
基金National Natural Science Foundation of China,No.82173446the Youth Training Program of the Army Medical University,No.2018XQN01.
文摘Hair follicle stem cells(HFSCs)in the bulge are a multipotent adult stem cell population.They can periodically give rise to new HFs and even regenerate the epidermis and sebaceous glands during wound healing.An increasing number of biomarkers have been used to isolate,label,and trace HFSCs in recent years.Considering more detailed data from single-cell transcriptomics technology,we mainly focus on the important HFSC molecular markers and their regulatory roles in this review.
基金supported by grants from the National Natural Science Foundation of China(No.81873702,81470694,to WY)Chongqing Natural Science Foundation of China(No.cstc2017jcyjAX0407,to WY)Southwest Hospital Foundation of China(No.SWH2016JCZD-02,to WY).
文摘Inner ear hair cells are important for maintaining hearing.Irreversible damage to hair cells is an important cause of sensorineural deafness.Electromagnetic radiation,especially high-power microwave,is an important threat to human health in modern society and war.However,it is not clear whether high-power microwave has an effect on cochlea hair cells.This study aimed to assess the effects of high-power microwave on cochlear hair cells in guinea pigs,and investigate the potential protection of these cells against high-power microwave-induced damage by recombinant adenovirus A20.Based on experimental results,a 65 W/cm^(2) irradiation density applied to guinea pigs in this study to establish a high-power microwave inner ear injury model.In addition,pAdEeay-1/A20 was injected via a round window into experimental guinea pig cochlea,whereas artificial perilymph was injected into the control group.Auditory function was assessed by testing the auditory brainstem response threshold,and damage to cochlear hair cells was investigated by cell counting and scanning electron microscopy observations of the basilar membrane.Inner ear injury was observed 6 hours after 65 W/cm^( 2 ) of irradiation and the auditory brainstem response threshold was significantly higher in the irradiation group(P<0.05)compared with other groups.Propidium iodide staining and scanning electron microscopy results indicated that significant morphological changes occurred after radiation,especially to inner hair cells,which exhibited remarkable damage and the presence of several unknown spherical substances.Auditory brainstem response threshold was decreased in the pAdEeay-1/A20 group compared with the artificial perilymph group;moreover,damage to hair cells was milder in the pAdEeay-1/A20 group compared with the control group(P<0.01).Thus,high-power microwave can cause damage to cochlear hair cells,as well as hearing loss with prolonged exposure and/or high dosage.In this regard,65 W/cm^( 2 ) of irradiation for 6 hours is a reliable target dose for observation of damage.The zinc finger protein A20 can protect cochlear hair cells from high-power microwave-induced damage and prevent further hearing loss.This study was approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University,China on April 18,2017.
基金the National Natural Science Foundation of China(31930011,31922010,31871270),Chinathe China National Center for Biotechnology Development(2020YFC0847000)+3 种基金the Key Research Program of the Chinese Academy of Sciences(KJZD-SWL11)the Yunnan Fundamental Research Project(No.2019FI008),China.J.T.was supported by the Program for Changjiang Scholars and Innovative Research Team in University(IRT_16R37).L.B.Z.was supported by the GDAS Special Project of Science and Technology Development(2018GDASCX-0107).
文摘Exposure to intense noise can damage cochlear hair cells,leading to hearing loss in mammals.To avoid this constraint,most mammals have evolved in relatively quiet environments.Echolocating bats,however,are naturally exposed to continuous intense sounds from their own and neighboring sonar emissions for maintaining sonar directionality and range.Here,we propose the presence of intense noise resistance in cochlear hair cells of echolocating bats against noise-induced hearing loss(NIHL).To test this hypothesis,we performed noise exposure experiments for laboratory mice,one nonecholocating bat species,and five echolocating bat species.Contrary to nonecholocating fruit bats and mice,the hearing and the cochlear hair cells of echolocating bats remained unimpaired after continuous intense noise exposure.The comparative analyses of cochleae transcriptomic data showed that several genes protecting cochlear hair cells from intense sounds were overexpressed in echolocating bats.Particularly,the experimental examinations revealed that ISL1 overexpression significantly improved the survival of cochlear hair cells.Our findings support the existence of protective effects in cochlear hair cells of echolocating bats against intense noises,which provides new insight into understanding the relationship between cochlear hair cells and intense noises,and preventing or ameliorating NIHL in mammals.
文摘It has been revealed in recent years that contralateral acoustic stimulation can affect cochlear active mechanisms through activating medial olivocochlear system (MOC) of the cochlear efferent nerve fibers. The MOC is therefore postulated to exert protective effects on outer hair cells (OHCs) under intense sound condition. In this study the effects of 4 kHz intense tone exposure on distortion product otoacoustic emissions (DPOAEs) in guinea pigs with and without contralateral white noise stimulation were observed so that to investigate the protective effects of MOC on OHCs. The results showed that DPOAEs obviously deceased after the intense tone exposure in all animals, while both the amplitude reduction and the affected frequency range of DPOAEs were smaller in animals with simultaneously delivered contralateral white noise during the tone exposure than that in animals without colltralateral acoustic stimulation. The above results may suggest some protective nature of the contralateral sound stimulating effects which might be mediated through the activity of MOC. These perhaps can serve as the evidence that the protective mechanism against intense sound operates in the outer hair cells which are strongly innervated by
基金supported by the National Natural Science Foundation of China (81570916, 81771018, and 81873699)。
文摘Oxidative stress is the key determinant in the pathogenesis of noise-induced hearing loss(NIHL). Given that cellular defense against oxidative stress is an energyconsuming process, the aim of the present study was to investigate whether increasing energy availability by glucose supplementation protects cochlear hair cells against oxidative stress and attenuates NIHL. Our results revealed that glucose supplementation reduced the noiseinduced formation of reactive oxygen species(ROS) and consequently attenuated noise-induced loss of outer hair cells, inner hair cell synaptic ribbons, and NIHL in CBA/J mice. In cochlear explants, glucose supplementation increased the levels of ATP and NADPH, as well as attenuating H_2O_2-induced ROS production and cytotoxicity. Moreover, pharmacological inhibition of glucose transporter type 1 activity abolished the protective effects of glucose against oxidative stress in HEI-OC1 cells. These findings suggest that energy availability is crucial for oxidative stress resistance and glucose supplementation offers a simple and effective approach for the protection of cochlear hair cells against oxidative stress and NIHL.
基金the National Natural Science Foundation of China(81800901)the Program for Professor of Special Appointment(Eastern Scholar)at Shanghai Institutions of Higher Learning(QD2018015)+2 种基金the Shanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases(14DZ2260300)the Strategic Priority Research Program of the Chinese Academy of Sciences(XDB32030200)the Bureau of International Cooperation,Chinese Academy of Sciences(153D31KYSB20170059).
文摘Recent studies have revealed great functional and structural heterogeneity in the ribbon-type synapses at the basolateral pole of the isopotential inner hair cell(IHC).This feature is believed to be critical for audition over a wide dynamic range,but whether the spatial gradient of ribbon morphology is fine-tuned in each IHC and how the mitochondrial network is organized to meet local energy demands of synaptic transmission remain unclear.By means of three-dimensional electron microscopy and artificial intelligence-based algorithms,we demonstrated the cell-wide structural quantification of ribbons and mitochondria in mature mid-cochlear IHCs of mice.We found that adjacent IHCs in staggered pairs differ substantially in cell body shape and ribbon morphology gradient as well as mitochondrial organization.Moreover,our analysis argues for a location-specific arrangement of correlated ribbon and mitochondrial function at the basolateral IHC pole.
基金This work was supported by the National Natural Science Foundation of China(81770999 and 81670281)the Shanghai Municipal Commission of Science and Technology Research Project(18140900304,and 19140900902)the Big Data and Artificial Intelligence Project(2020DSJ07).
文摘Sodium salicylate is an anti-inflammatory medication with a side-effect of tinnitus.Here,we used mouse cochlear cultures to explore the effects of salicylate treatment on cochlear inner hair cells(IHCs).We found that IHCs showed significant damage after exposure to a high concentration of salicylate.Whole-cell patch clamp recordings showed that 1–5 mmol/L salicylate did not affect the exocytosis of IHCs,indicating that IHCs are not involved in tinnitus generation by enhancing their neuronal input.Instead,salicylate induced a larger peak amplitude,a more negative half-activation voltage,and a steeper slope factor of Ca^(2+)current.Using noise analysis of Ca^(2+)tail currents and qRT-PCR,we further found that salicylate increased the number of Ca^(2+)channels along with CaV1.3 expression.All these changes could act synergistically to enhance the Ca^(2+)influx into IHCs.Inhibition of intracellular Ca^(2+)overload significantly attenuated IHC death after 10 mmol/L salicylate treatment.These results implicate a cellular mechanism for tinnitus generation in the peripheral auditory system.
基金We thank Prof.Lin Chen(University of Science and Technology of China)and Dr.Juanmei Yang(Eye and ENT Hospital of Fudan University)for valuable help with cochlear microscopic anatomy.This work was supported by the National Natural Science Foundation of China(31571032,31771191.81730028)the National Basic Research Development Program of China(SQ2017YFSF080012)the Postdoctoral Science Foundation of China(2018M640407).
文摘Voltage-gated sodium channels(VGSCs)are transiently expressed in cochlear hair cells before hearing onset and play an indispensable role in shaping spontaneous activity.In this study,we showed that Na^+currents shaped the spontaneous action potentials in developing mouse inner hair cells(IHCs)by decreasing the time required for the membrane potential to reach the action-potential threshold.In immature IHCs,we identified 9 known VGSC subtypes(Navl.la-l.9ot),among which Navl.7a was the most highly expressed subtype and the main contributor to Na+currents in developing hair cells.Electrophysiological recordings of two cochlea-specific Navi.7 variants(CbmNavl.7a and CbmNavl.7b)revealed a novel loss-of-function mutation(C934R)at the extracellular linker between segments 5 and 6 of domain II.In addition,post-transcriptional modification events,such as alternative splicing and RNA editing,amended the gating properties and kinetic features of CbmNavl.7a(C934).These results provide molecular and functional characteristics of VGSCs in mammalian IHCs and their contributions to spontaneous physiological activity during cochlear maturation.
文摘Objective Adipose-derived stem cells(ADSCs) are suggested to possess a highly plastic ability to differentiate into several specific cell types in addition to adipocyte lineages, including germ layer tissue-specific cell lineages such as chondrocyte, myocyte, neuronal, and osteoblast lineages. The aim of this study is to establish an in vitro culture technique for ADSCs in an adult guinea pig model that facilitate their differentiation into hair cell-like cells. Materials and Methods Cells from inguinal fat pads in adult guinea pigs were cultured with β-mercaptoethanol, RA, Forskorin, Heregulin, bFGF, BDNF and EGF. Cellular differentiation was examined using immunocytochemistry techniques. Results The ADSCs demonstrated hair cell immunophenotypes with expression of epitopes of the hair cell marker protein myosin Ⅶa. Conclusion ADSCs from adult guinea pig adipose tissue can differentiate into hair cell-like cells when cultured in vitro. ADSCs may serve as seed cells for tissue engineering.
基金supported by the Taipei Tzu Chi Hospital through grants from the Buddhist Tzu Chi Medical Foundation under the Numbers TCRD-TPE-110-13 and TCRD-TPE-111-23,Taipei,Taiwan.
文摘Background:Cisplatin is a chemotherapeutic agent commonly used clinically for the treatment of various human cancers.Patients often reduce the use of cisplatin due to its side effects,which in turn affects its treatment.This study explored the mechanism of action of safflower extract as an adjuvant traditional Chinese medicine for chemotherapy.Methods:Primary human follicle dermal papilla cells(HFDPCs)were used as target cells for cisplatininduced damage to hair cells.Western blotting was used to investigate the molecular targets of cisplatin and safflower extract in causing HFDPCs damage.Cell survival and cell cycle were analyzed by mitochondrial staining reagent WST-1 and propidium iodide.Results:Cisplatin could reduce the viability of HFDPCs without causing cell death.Cisplatin increased the level of phospho-Rad17 in HFDPCs and activated the Chk1/Cdc25C signaling to reduce the expression of Cdc2 protein,thereby arresting the cells in the G2/M phase.The combination of safflower extract and the flavonoids could effectively inhibit the signal transduction of Rad17/Chk1/Cdc25 in cisplatin-treated cells and reduce the cell population in the G2/M phase.Finally,we also confirmed that safflower extract could effectively inhibit the damage to HFDPCs caused by cisplatin,mainly at the level of reducing the DNA damage caused by cisplatin.Conclusions:Safflower extract can be used as an adjuvant Chinese medicine for chemotherapy to reduce the damage caused by chemotherapy to normal hair follicle cells.
