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.
Animals ; Mice ; Hair Cells, Auditory, Inner ; Cochlea/metabolism* ; Hair Cells, Auditory, Outer/metabolism* ; Disease Models, Animal ; Fibroblast Growth Factor 8/metabolism*

BACKGROUND AND OBJECTIVES: Locacorten Vioform (Novartis UK) is frequently prescribed for otomycosis. Its component, Clioquinol, also has anti-bacterial properties. Up to this point, its ototoxic potential has not been evaluated. Our objective aims to evaluate Locacorten Vioform’s potential ototoxicity when applied directly to the middle ear cavity. MATERIALS AND METHODS: We performed an experimental prospective animal study in our animal research center with 20 Hartley guinea pigs divided into 2 groups. The first group (experimental) was treated with Locacorten Vioform in one ear and with a physiologic saline solution in the other. The second group (positive control) was treated with concentrated gentamycin in one ear and physiologic saline in the other. Auditory brainstem response measurements were obtained before and after three sets of injections. Statistics were analyzed using a variance analysis with repeated measures. The histological state of cochlear outer hair cells was compared between the two groups using scanning electron microscopy. RESULTS: Average hearing loss in ears treated with Locacorten Vioform was 32.1 dB, compared with a 2.5 dB average loss in the saline-treated ears. Ears treated with gentamycin lost an average of 33.0 dB. There were clinically and statistically significant differences between the two ears of the guinea pigs in both groups (p < 0.001). Scanning electron microscopy revealed severe pericochlear and cochlear inflammation and ossification in the Locacorten Vioform-treated ears. Gentamycin caused significant destruction of outer hair cell architecture. CONCLUSIONS: Locacorten Vioform induces a hearing loss similar to that caused by gentamycin when applied directly to the middle ear of a guinea pig model. Electron microscopy indicates a pericochlear and cochlear inflammatory reaction with ossification.
Animal Experimentation ; Animals ; Clioquinol ; Ear ; Ear, Middle ; Evoked Potentials, Auditory, Brain Stem ; Gentamicins ; Guinea Pigs ; Guinea ; Hair ; Hair Cells, Auditory, Inner ; Hair Cells, Auditory, Outer ; Hearing Loss ; Inflammation ; Microscopy, Electron ; Microscopy, Electron, Scanning ; Otomycosis ; Prospective Studies ; Sodium Chloride

OBJECTIVE: To detect the expression variation of microRNA-183 family in cochlea of animal model characterized by noise-induced deafness at various time points, and to explore the mechanisms responsible for noise-induced deafness. METHOD: Fifty mice were randomly divided into 5 groups. In the experimental group, 40 mice were exposed to 2-4 kHz narrow band noise at 100 dB SPL 6h per day for 3 consecutive days. The rest 10 mice served as the control group without receiving any noise. Auditory brainsterm response (ABR) were examined at the 1st, 7th, 14th and 28th day compaired with the ABR before the experiment,to confirm noise lead to the permanent threshold shift. The pathological damage processes of hair cell were detected by the basilar membrane stretched techniques. Real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) was apply to quantify the expression of microRNA183 family members. Statistical analysis was performed by the SPSS 17.0 software. RESULT: The hearing of mice in the experimental group was significantly less than that in the control group. In the experimental group, the hearing of mice exposed to noise were markedly less when compared with the one exposure to null-noise. The hearing in the 1st day group was least among experimental groups, and the followed one was mice in the 7th day group. No statistical difference were observed between the 14th and 28th day groups (P > 0.05). The results of surface preparation showed that the outer hair cells were chaotic, deformational, and their number decreased is time-dependent. The missing of the outer hair cells occurred mainly in the first and second rows, while the inner hair cells were not pronouncedly missing. The qRT-PCR showed that the expressions of the three genes (miR-183/96/182)in the 1st day and 7th day group with exposure to noise were less than in the control group (P < 0.01), while no significant difference was found between 1st day and 7th day group (P > 0.05). The expressions rised in the 14th day experimental groups, whereas the 28th day group's expressions of the three genes decreased markedly which were more than that in the 1st day and 7th day group (P < 0.01). CONCLUSION After noise exposure for some time, the expressions of miRNA-183 family members have significant changes in animal model with noise-induced deafness, which indicated that the miRNA183 family members may play important roles in the pathogenesis and development of noise-induced deafness.
Animals ; Disease Models, Animal ; Evoked Potentials, Auditory, Brain Stem ; Female ; Hair Cells, Auditory, Inner ; pathology ; Hair Cells, Auditory, Outer ; pathology ; Hearing Loss, Noise-Induced ; metabolism ; pathology ; Male ; Mice ; MicroRNAs ; metabolism

BACKGROUND AND OBJECTIVES: The antioxidant ebselen will be able to limit or prevent the ototoxicity arising from 2-hydroxypropyl-β-cyclodextrin (HPβCD). Niemann-Pick Type C (NPC) disease is a disorder of lysosomal storage manifested in sphingolipidosis. Recently, it was noted that experimental use of HPβCD could partially resolve the symptoms in both animals and human patients. Despite its desirable effect, HPβCD can induce hearing loss, which is the only major side effect noted to date. Understanding of the pathophysiology of hearing impairment after administration of HPβCD and further development of preventive methods are essential to reduce the ototoxic side effect. The mechanisms of HPβCD-induced ototoxicity remain unknown, but the resulting pathology bears some resemblance to other ototoxic agents, which involves oxidative stress pathways. To indirectly determine the involvement of oxidative stress in HPβCD-induced ototoxicity, we tested the efficacy of an antioxidant reagent, ebselen, on the extent of inner ear side effects caused by HPβCD. MATERIALS AND METHODS: Ebselen was applied prior to administration of HPβCD in mice. Auditory brainstem response thresholds and otopathology were assessed one week later. Bilateral effects of the drug treatments also were examined. RESULTS: HPβCD-alone resulted in bilateral, severe, and selective loss of outer hair cells from base to apex with an abrupt transition between lesions and intact areas. Ebselen co-treatment did not ameliorate HPβCD-induced hearing loss or alter the resulting histopathology. CONCLUSIONS: The results indirectly suggest that cochlear damage by HPβCD is unrelated to reactive oxygen species formation. However, further research into the mechanism(s) of HPβCD otopathology is necessary.
Animals ; Ear, Inner ; Evoked Potentials, Auditory, Brain Stem ; Hair Cells, Auditory, Outer ; Hearing Loss ; Hearing ; Humans ; Mice ; Oxidative Stress ; Pathology ; Reactive Oxygen Species ; Sphingolipidoses ; Tight Junctions

OBJECTIVETo study the effects of Erlong Zuoci Pill (, ELZCP) and its disassembled: prescriptions on gentamicin (GM)-induced ototoxicity model in vitro.

METHODSAfter the spiral organ of cochleae: of newborn mice (postnatal days: 2-3) cultured for 24 h, GM alone or combined with water extracting-alcohol precipitating solution of ELZCP or with its disassembled prescriptions was added. Hair cells were observed under a fluorescence microscope after TRITC-phalloidin staining, and the cochlear hair cell loss rate was calculated by counting the whole cochlear hair cells and analyzed by whole cochlear hair cells analyzing software.

RESULTSGM induced cochlear outer hair cells (OHCs) and inner hair cells (IHCs) injuries in a dose-dependent manner, and they were significantly different as compared with those in the normal control group (P<0.05, P<0.01). ELZCP at the concentration of 0.003-3 mg/mL could decrease the hair cells loss induced by the 0.3 mmol/L GM (P<0.05, P<0.01), the effects was in a dose-dependent manner, and the concentration of 0.3 mg/mL showed the optimal protective effect. For the ELZCP disassembled prescriptions, Liuwei-Dihuang could decrease OHC loss rate than that in the 0.3 mmol/L GM model group (P<0.05), but the OHC loss rate was still higher than that in the ELZCP group (P<0.01), which indicated that the protective effect of hair cells by Liuwei-Dihuang was not better than that of ELZCP. Poria decreased OHC loss rate from 72.1 % +/-3.7 % to 58.8 %+/- 8.2 % (P<0.05).

CONCLUSIONSELZCP could play a role in antagonizing the injury of cochlear hair cells induced by GM ototoxicity,: and its disassembled prescriptions, Liuwei-Dihuang was the main component to protect the cochlear hair cells from GM-induced ototoxicity, and Magnetitum combined with Radix Bupleurui could strengthen the action of the whole prescription; Poria could reduce GM-induced OHC loss.

Animals ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; pharmacology ; Gentamicins ; toxicity ; Hair Cells, Auditory, Inner ; drug effects ; pathology ; Hair Cells, Auditory, Outer ; drug effects ; pathology ; Mice ; Organ of Corti ; drug effects ; pathology ; Prescriptions ; Tablets

In order to study the functional and structural alterations of the retina in SD rat model after methanol intoxication, 35 rats were divided randomly into five groups administrated with saline, 3-day high dose, 7-day high dose, 3-day low dose and 7-day low dose methanol separately. The retinal function of each group was assessed by flash electroretinogram (F-ERG) 3 and 7 days after methanol poisoning. The microstructure and ultrastructure of the retina were observed at the same time. The high-dose methanol intoxication induced irreversible retinal functional and structural damages 3 days after poisoning, which included prolonged latency and reduced amplitude of the Max-reaction of F-ERG. These injuries were aggravated 7 days after poisoning. Meanwhile, the latency and amplitude of the Cone-reaction of F-ERG were also affected 3 days after poisoning, but there were no further worsening tendency 7 days after poisoning. The retinal histological analysis showed cellular edema, heteromorphy and disarrangement, tissular loosen of the inner nuclear layer and photoreceptors layer. The mitochondrial damage began at the photoreceptors layer and developed further into the inner nuclear layer. The low-dose methanol intoxication only caused transient damage of the retina. Our results showed that the function and structure of the photoreceptor and inner nuclear layer were the primary target of methanol intoxication and that the rod cells were more sensitive to methanol intoxication than the cone cells. The mitochondrial damage developed from outer layer to inner layer of the retina.
Animals ; Edema/pathology* ; Electroretinography ; Forensic Medicine ; Male ; Methanol/poisoning* ; Mitochondria/pathology* ; Photoreceptor Cells/pathology* ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Retina/physiopathology* ; Retinal Cone Photoreceptor Cells/pathology* ; Retinal Diseases/pathology* ; Retinal Rod Photoreceptor Cells/pathology* ; Time Factors

OBJECTIVETo study the effect of Vaccinium uliginosum L., (VU) on the electroretinogram (ERG) and retinal pathological changes in rabbits after light-induced damage.

METHODSTwenty-eight Chinchilla rabbits were randomly divided into four groups: administration beforehand (A), administration after injury (B), light injury without administration (C), and blank (D) groups. After a 4-week administration of VU homogenate at 4.8 g/(kg·d) once a day in group A, ERG in groups A, B and C were recorded according to the standards set by the International Society for Clinical Electrophysiology of Vision (ISCEV). Except for group D, the groups were then exposed to strong light. Just after that, group A stopped receiving VU treatment and group B started to receive it. Then ERGs in all groups were recorded after 1 day, 1 week, and 2 weeks. Throughout the whole process groups which were not fed with VU were fed with normal saline. Finally, the tissues and structures of all the groups were observed and the thickness of the outer nuclear layers (ONL) was measured.

RESULTS(1) After 4-week feeding with VU, the latency time of ERG in group A became shorter than those in the other groups and the amplitude increased. After being exposed to strong light, the latency time lengthened and amplitude decreased in all the injury groups, but comparing at each time point, the measured values in group A were better than those in group C. With the accumulation of VU, the ERG in group B improved, and finally, all of the detected values became better than those in group C. (2) Retinae in group D were normal in histology and the layers were in order but those in group C became disarranged. The injuries in groups A and B were minor compared with those in group C. The thickness of the ONL in group C was significantly thinner than in the other groups (P=0.000), and that in groups A and B was thicker than that in group C, although thinner than in group D. That in group A was thicker than in group B.

CONCLUSIONSVU can relieve the injury to rabbit retinae exposed to normal day and night rhythm, alleviate the harm caused by light when used beforehand, and repair the light damage to the retina.

Animals ; Electroretinography ; Light ; Plant Extracts ; pharmacology ; Rabbits ; Retina ; drug effects ; pathology ; physiopathology ; radiation effects ; Retinal Cone Photoreceptor Cells ; drug effects ; pathology ; radiation effects ; Retinal Rod Photoreceptor Cells ; drug effects ; pathology ; radiation effects ; Time Factors ; Vaccinium ; chemistry

OBJECTIVES: To investigate the otoprotective effects of mouse nerve growth factor (mNGF) in A/J mice. METHODS: The mice at postnatal day 7 (P7) were randomly separated into a mNGF treated group (mNGF group) and a distilled water (for injection) treated group (control group). The mNGF dissolved in distilled water or distilled water alone was given to the mice once every other day from P7 by intramuscular injection in the hips. The otoprotective effects of mNGF in A/J mice were observed in a time course manner. The thresholds of auditory-evoked brainstem response (ABR) were tested from the age of the 3rd to the 8th week. Sections of the inner ears were stained by hematoxylin and eosin, and spiral ganglion neurons (SGNs) were observed at the age of the 3rd, the 6th,and the 8th week. Counts of whole mount outer hair cells (OHCs) in the cochleae were made at the age of 8 weeks. Expression of apoptosis related genes was determined by quantitative real-time polymerase chain reaction and Western blotting. RESULTS: ABR thresholds of the mNGF group were significantly lower than those of the control group at the age of the 6th and the 8th week. Moreover, the mNGF preserved OHC and SGN in the mouse cochleae in this period. Further experiments showed that the expression of caspase genes (including caspase-3) was inhibited in the mouse inner ears in the mNGF group. CONCLUSION: The mNGF improves hearing in A/J mice by preserving SGN and OHC in the cochleae.
Animals ; Apoptosis ; Blotting, Western ; Brain Stem ; Cochlea ; Ear, Inner ; Eosine Yellowish-(YS) ; Hair Cells, Auditory, Outer ; Hearing* ; Hematoxylin ; Hip ; Injections, Intramuscular ; Mice* ; Nerve Growth Factor* ; Neurons ; Real-Time Polymerase Chain Reaction ; Spiral Ganglion ; Water

Hair ; Hair Cells, Auditory, Inner ; Humans ; Regeneration ; Wound Healing

Hearing loss has become increasingly prevalent and causes considerable disability, thus gravely burdening the global economy. Irreversible loss of hair cells is a main cause of sensorineural hearing loss, and currently, the only relatively effective clinical treatments are limited to digital hearing equipment like cochlear implants and hearing aids, but these are of limited benefit in patients. It is therefore urgent to understand the mechanisms of damage repair in order to develop new neuroprotective strategies. At present, how to promote the regeneration of functional hair cells is a key scientific question in the field of hearing research. Multiple signaling pathways and transcriptional factors trigger the activation of hair cell progenitors and ensure the maturation of newborn hair cells, and in this article, we first review the principal mechanisms underlying hair cell reproduction. We then further discuss therapeutic strategies involving the co-regulation of multiple signaling pathways in order to induce effective functional hair cell regeneration after degeneration, and we summarize current achievements in hair cell regeneration. Lastly, we discuss potential future approaches, such as small molecule drugs and gene therapy, which might be applied for regenerating functional hair cells in the clinic.
Infant, Newborn ; Humans ; Hair Cells, Auditory, Inner/physiology* ; Ear, Inner/physiology* ; Hair Cells, Auditory/physiology* ; Regeneration/genetics* ; Stem Cells