All the animal procedures were performed in accordance with the guidelines and regulations of the Animal Welfare Committee of Canton Basel City, Switzerland. Postnatal C57BL/6JR mice, Wistar rats, and STAT1-deficient mice (mixed C57BL/6-129/SvEv)7 aged 3-5 days and of either sex were used for the experiments.
1. Coating the multi-well chambers
2. Dissection of the temporal bone
3. Isolation of the cochlea
4. Culture of cochlear explants
5. Test of ototoxic agents
6. Fixation and immunofluorescence
7. Immunofluorescence of live cells from explants
8. Visualization by confocal imaging
The present protocol has been tested on the cochlea of neonatal mice and rats. This paper presents images of explants from different experiments. The explants of the organ of Corti were exposed to gentamicin or cisplatin, and hair cell loss was visible. The explants of the organ of Corti maintained their structure and total length under both normal and stress conditions (Figure 1 and Figure 2). The surviving hair cells along the entire length of the rat explants previously exposed to cisplatin were individually detectable (Figure 1). In addition to the detection of surviving hair cells, hair cells undergoing apoptosis were also detected (Figure 2). This approach facilitates the visualization and counting of surviving cells, which can be performed using a deep learning approach, as described previously12. It was also possible to detect the biological processes in living cochlear cells using appropriate cell-permeable probes (Figure 3).
In the case of cochlear explants containing spiral ganglion neurons, the explants can be cut into two pieces, or the apex region can be cut away to provide better culture conditions. Here we chose to separate the apex region, because it is less affected under stress conditions. Figure 4 shows the base and medial regions of the cochlear explants. Hair cells labeled with the hair cell marker MYO7A were detected. Similarly, healthy and damaged spiral ganglion cell bodies and neurites labeled with the neuronal marker TuJ1 were identified. The analysis of spiral ganglion regions can be performed manually or using open-source software such as FIJI with the NeuronJ plugin for neurite tracing13 or extensions such as TrackMate and Cellpose for morphological segmentation14,15. The closer examination of the mouse explants revealed the high resolution of the cochlear cells and hair cell stereocilia (Figure 5).
Figure 1: Explants of the organ of Corti from rats exposed to gentamicin. Representative images (maximum intensity projection) of (A) control and (B) gentamicin-exposed (200 µM for 24 h) explants. The hair cells are labeled with phalloidin and can be visualized along the entire length of the cochlea. For better illustration, the image is in gray tones. The images were acquired using a spinning disk confocal microscope with a 20x objective (numerical aperture: 0.75). Scale bar = 500 µm. Please click here to view a larger version of this figure.
Figure 2: Explants of the organ of Corti from mice exposed to cisplatin. Representative images (maximum intensity projection) of (A) control and (B) cisplatin-exposed (160 µM for 48 h) explants. The hair cells are labeled with phalloidin (red), and the apoptotic hair cells are labeled with fluorescin. The images were acquired using a point-scanning confocal microscope and a 20x objective (numerical aperture: 0.75). Scale bar = 200 µm. Please click here to view a larger version of this figure.
Figure 3: Explants of the organ of Corti from live imaging experiments. Representative images (maximum intensity projection) of explants from wild-type mice showing (A) control explants and (B) explants with exposure to 125 µM cisplatin for 18 h. The hair cells are labeled with mito-hydroethidine and caspase-3. The images were acquired using a spinning disk confocal microscope and a 20x objective (numerical aperture: 0.75). Scale bar = 50 µm. Please click here to view a larger version of this figure.
Figure 4: Cochlear explants from STAT1 knockout mice exposed to cisplatin. Representative images (maximum intensity projection) of (A) control explants and (D) explants exposed to 40 µM cisplatin for 48 h. The hair cell bodies are labeled with the MYO7A antibody (green), and the spiral ganglion cells with the TuJ1 antibody (red) and DAPI nuclear labeling (blue). The images were acquired using a point-scanning confocal microscope and a 20x objective (numerical aperture: 0.75) with an additional 2.15 zoom (B,C,E,F). Scale bar = (B,C,E,F) 50 µm and (A,D) 200 µm. Please click here to view a larger version of this figure.
Figure 5: Mouse organ of Corti explants. (A–D) Representative images (maximum intensity projection) of full-length explants from wild-type mice. (A,B) The explants were labeled with the MYO7A antibody, phalloidin, and DAPI nuclear labeling. (B) In the magnified overview, the cell bodies of the hair cells labeled with MYO7A antibody (green) are clearly visible. (C,D) Phalloidin labels the stereocilia and cuticular plate of the hair cells. The explants were labeled with phalloidin. (D) In the magnified overview, deconvoluted images of individual inner hair cell stereocilia are well identified (lower row), whereas individual outer hair cell stereocilia are more difficult to delineate (upper row). The images in panels A and C were acquired with a spinning disk confocal microscope with a 20x objective (numerical aperture: 0.75). The image in panel B was acquired with the same microscope but with a 40x air objective (numerical aperture: 0.95). The image in panel D was acquired with a point-scanning confocal microscope and a 100x oil objective (numerical aperture: 1.45) with an additional 3.46 zoom. The scans were averaged four times per XY section, and the pixel size was 0.02 µm. Scale bars = (D) 3 µm, (B) 100 µm, and (A,C) 200 µm. Please click here to view a larger version of this figure.
15 mL High-Clarity Polypropylene Conical Tube 17 x 120 mm style | FALCON | 352096 | |
45° Angled Forceps | Fine Science Tools | 11251-35 | |
50 mL Polypropylene Conical Tube 30 x 115 mm style | FALCON | 352070 | |
Antifade Mounting Medium | VECTASHIELD | H-1000 | |
Alexa Fluor 568 phalloidin | Thermofisher | 2151755 | |
Anti-beta III Tubulin antibody [TUJ-1] | Abcam | ab14545 | |
Antifade Mounting Medium With DAPI | VECTASHIELD | H-1200 | |
Anti-myosin VII rabbit polyclonal | Abcam | ab3481 | |
B-27 Supplement (50x), minus antioxidants | Thermofisher | 10889038 | |
CARBON STEEL surgical blades 23 | Swann Moiton | 210 | |
CellEvent™ Caspase-3/7 Green Detection Reagent | Thermofisher | C10723 | |
DMEM/F-12/(1:1)(1x) + GlutaMAX | Thermofisher | 31331028 | |
Double spatulas, one curved end | VWR | RSGA038.150 | |
Ethyl alcohol 70% V/V 1,000 mL | bichsel | 160 0 106 00 | |
Fetal Bovine Serum, certified | Thermofisher | 16000036 | |
Fixative Solution 4% paraformaldehyde prepared in PBS | Thermofisher | 201255309/201255305 | |
High Intensity Cold Halogen Light Source | Intralux® | 5100 | |
Huygens Professional version 21.10 | Scientific Volume Imaging | ||
ibidi µ-Slide 8 well | ibidi | 80826 | |
microscope | LEICA | M80 | |
microscope | LEICA | MS5 | |
MitoSOX™ Red Mitochondrial Superoxide Indicator, for live-cell imaging | Thermofisher | M36008 | |
N2 supplement (100x) | Thermofisher | 17502048 | |
Nikon Eclipse Ti microscope with a Yokogawa CSU-W1 spinning disk confocal unit, and a Photometrics Prime 95B camera. | NIKON | ||
Nikon Eclipse Ti microscope with an A1 point-scanning confocal unit | NIKON | ||
Operating scissors | Fine Science Tools | 14005-16 | |
Operating scissors | Fine Science Tools | 14088-10 | |
Operating tweezers | Fine Science Tools | 11008-15 | |
PBS pH 7.2 (1x), 500mL | Thermofisher | 20012-019 | |
Penicillin | Sigma-Aldrich | P3032 | |
POLY-D-LYSINE HYDROBROMIDE MOL WT GT 30 | Sigma-Aldrich | P7405 | |
Scalpel Handle #4 | Fine Science Tools | 10004-13 | |
Steri 250 Second sterilizer | Simon Keller AG | 031100 | |
Sterilizer, desiccant pellets | Simon Keller AG | 31120 | |
Tissue Culture Dish 60 x 15 mm | FALCON | 353802 | |
Tissue Culture Dish 60 x 15 mm | FALCON | 353004 | |
Trito X-100 | Sigma | T9284 | |
Unconventional myosin-VIIa | Developmental Studies Hybridoma Bank | 138-1s | |
WFI for Cell Culture[-]Antimicrobial, 500 mL | Thermofisher | A12873-01 |
Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals’ quality of life. Sensorineural hearing loss is characterized by the cumulative and irreversible loss of sensory hair cells and auditory nerves in the cochlea. Entire and vital cochlear explants are one of the fundamental tools in hearing research to detect hair cell loss and to characterize the molecular mechanisms of the inner ear cells. Many years ago, a protocol for neonatal cochlear isolation was developed, and although it has been modified over time, it still holds potential for improvement.
This paper presents an optimized protocol for isolating and culturing whole neonatal cochlear explants in multi-well culture chambers that enables the study of hair cells and spiral ganglion neuron cells along the entire length of the cochlea. The protocol was tested using cochlear explants from mice and rats. Healthy cochlear explants were obtained to study the interaction between hair cells, spiral ganglion neuron cells, and the surrounding supporting cells.
One of the main advantages of this method is that it simplifies the organ culture steps without compromising the quality of the explants. All three turns of the organ of Corti are attached to the bottom of the chamber, which facilitates in vitro experiments and the comprehensive analysis of the explants. We provide some examples of cochlear images from different experiments with live and fixed explants, demonstrating that the explants retain their structure despite exposure to ototoxic drugs. This optimized protocol can be widely used for the integrative analysis of the mammalian cochlea.
Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals’ quality of life. Sensorineural hearing loss is characterized by the cumulative and irreversible loss of sensory hair cells and auditory nerves in the cochlea. Entire and vital cochlear explants are one of the fundamental tools in hearing research to detect hair cell loss and to characterize the molecular mechanisms of the inner ear cells. Many years ago, a protocol for neonatal cochlear isolation was developed, and although it has been modified over time, it still holds potential for improvement.
This paper presents an optimized protocol for isolating and culturing whole neonatal cochlear explants in multi-well culture chambers that enables the study of hair cells and spiral ganglion neuron cells along the entire length of the cochlea. The protocol was tested using cochlear explants from mice and rats. Healthy cochlear explants were obtained to study the interaction between hair cells, spiral ganglion neuron cells, and the surrounding supporting cells.
One of the main advantages of this method is that it simplifies the organ culture steps without compromising the quality of the explants. All three turns of the organ of Corti are attached to the bottom of the chamber, which facilitates in vitro experiments and the comprehensive analysis of the explants. We provide some examples of cochlear images from different experiments with live and fixed explants, demonstrating that the explants retain their structure despite exposure to ototoxic drugs. This optimized protocol can be widely used for the integrative analysis of the mammalian cochlea.
Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals’ quality of life. Sensorineural hearing loss is characterized by the cumulative and irreversible loss of sensory hair cells and auditory nerves in the cochlea. Entire and vital cochlear explants are one of the fundamental tools in hearing research to detect hair cell loss and to characterize the molecular mechanisms of the inner ear cells. Many years ago, a protocol for neonatal cochlear isolation was developed, and although it has been modified over time, it still holds potential for improvement.
This paper presents an optimized protocol for isolating and culturing whole neonatal cochlear explants in multi-well culture chambers that enables the study of hair cells and spiral ganglion neuron cells along the entire length of the cochlea. The protocol was tested using cochlear explants from mice and rats. Healthy cochlear explants were obtained to study the interaction between hair cells, spiral ganglion neuron cells, and the surrounding supporting cells.
One of the main advantages of this method is that it simplifies the organ culture steps without compromising the quality of the explants. All three turns of the organ of Corti are attached to the bottom of the chamber, which facilitates in vitro experiments and the comprehensive analysis of the explants. We provide some examples of cochlear images from different experiments with live and fixed explants, demonstrating that the explants retain their structure despite exposure to ototoxic drugs. This optimized protocol can be widely used for the integrative analysis of the mammalian cochlea.