The primary report is really stress importance of accurately locating the specific frequency region of the basilar membrane in most models, where synapse numbers altered and function can be analyzed. The cochlear localization of the specific frequency regions is reliably performed using, place frequency maps in conjunction with cochleogram data. The microscope provides insight into several research studies.
It supporting the notion that the cochlear neuropathy is the primary initial events specifically preceding hearing loss, tinnitus, and hyperacusis. After confirming a lack of response to toe pinch in an anesthetized eight week old adult C57 black 6J male mouse. Place the mouse on a 37 degree celsius heat pad in an electrically and acoustically shielded room.
Next, place a subdermal 20 millimeter 28 gauge needle recording electrode, with the depth of three millimeters under the skin at the vertex of the skull. A reference electrode in the ipsilateral parotid region, below the pinna of measured ear and a ground electrode in the contralateral parotid region. Place a closed field speaker equipped with a two centimeter plastic tube with a cone shaped tip next to the mouse and fit the tip into the external ear canal.
For an auditory brainstem response, or ABR recording, generate tone pips at decreasing sound pressure levels from 90 to 10 decibels, in five to 10 decibels sound pressure level steps. During this step, the responses are amplified, filtered, and averaged. At each frequency, determine the ABR threshold, which is the minimal sound pressure level that results in a reliable ABR recording with one or more distinguishable waves that can be clearly identified by visual inspection.
After the ABR recording, expose the bulla from the ventral side and open the bulla with sharp scissors to gain access to the cochlea. Using fine forceps, remove the temporal bones and sever the stapes artery. Then remove the stapes from the oval window and rupture the round window membrane.
Gently rotate the tip of a 13 millimeter 27 gauge needle to make a small hole at the apex of the cochlea, before using a fine tipped pipette to gently flush 4%paraformaldehyde through the window into the perilymphatic spaces. Next, rinse the bones three times for five minutes per wash with cold fresh 0.1 molar PBS per wash. Followed by decalcification of the bones with 10%EDTA for four hours at room temperature and 20 rotations per minute on a horizontal shaker.
At the end of the incubation, transfer a decalcified temporal bone into fresh 0.1 molar PBS under a dissecting microscope. Use 3 and 5 Dumont forceps and a 27 gauge needle to dissect the apical, middle, and basal cochlea regions in turn. Use a razor blade to make a small series of cuts along the spiral ligament and remove tectorial membrane and Reissner's membrane.
Further dissect the remaining auditory epithelium, including the spiral limbus, into individual cochlear turns for whole mount preparations. Then use the 40X oil objective of a light microscope to measure the basilar membrane length with 250 micrometer scale placed in the eye piece. It can be adjusted along the stereocilia of the inner hair cells.
After dissection, place each cochlear turn into individual 2.5 milliliter centrifuge tubes. Block any nonspecific binding with 10%goat serum in PBS in 0.1%Triton X-100 for one hour at room temperature on a rotator. At the end of incubation, use 200 microliter pipette tip to remove the solution under a dissection microscope.
Incubate the specimens with the primary antibodies of interest diluted in 5%goat serum and PBS in 0.1%Triton X-100 overnight at 4 degrees Celsius on a rotator. The next morning, rinse the tissue samples three times for five minutes with cold 0.1 molar PBS per wash to remove any residual primary antibody. Then label the specimens with the appropriate secondary antibodies diluted in 5%goat serum in PBS in 0.1%Triton X-100 for two to three hours at room temperature on the rotator, protected from light.
At the end of incubation, wash the samples three times with 0.1 molar PBS as demonstrated and transfer the specimens into individual 35 millimeter plates containing 0.1 molar PBS. Place a drop of DAPI supplemented mounting medium onto the slide and transfer the specimens from PBS to the mounting medium. Place one edge of a cover slip onto each slide and release to let the cover slips fall gently.
Then dry the slides in a slide box at four degrees Celsius overnight. To image the specimens, use a confocal microscope with the appropriate lasers and 63X high resolution oil immersion lens to acquire eight micrometer confocal Z stacks from each cochlear turn. For synaptic puncta counts, set the Z stacks with the 0.3 micrometer step size to span the entire length of inner hair cells, insuring all of the synaptic puncta can be imaged and merge the puncta containing images in a Z stack to obtain the Z access projection.
Import the merged images into an appropriate image processing software program. Divide the synaptic total counts in each Z stack at specific frequency regions by the number of DAPI positive inner hair cells to calculate the number of synaptic puncta for each cell. At each specific frequency region, average all of the synaptic puncta in three images of different microscopic fields, containing nine to 11 inner hair cells.
To better visualize the cytoskeletal architecture and synaptic localization, use the brush tool to visually assess the synaptic structure and distribution of the individual inner hair cells. To inspect the juxtaposition of presynaptic ribbons and postsynaptic receptor patches, use the rectangular marquee tool extract the voxel space around the ribbons and use image cutting to isolate the individual ribbons. Then click image and image size to acquire a thumbnail array of these miniature projections that can be used to identify paired synapses versus orphan ribbons.
All ten of the mice in this representative experiment exhibited ABR thresholds in response to tone bursts ranging between 25 and 70 decibels per sound pressure level depending on the frequency of the stimulus. The hearing threshold for this experiment was lowest at 16 kilohertz corresponding to a approximately 43%of the distance from the cochlear apex, suggesting that the acoustic sensitivity is significantly reduced in other cochlear regions. Whole mounts of the auditory epithelium is dissected into three pieces and the lengths are measured and converted into their percent distance from the cochlear apex.
The frequency location on the basilar membrane of each cochlear turn is calculated using logarithmic function. In normal ears of adult mice, immunostaining reveals juxtaposed pairs of synaptic ribbons and glutamate receptor patches. Studying the surface of the basolateral membrane of the inner hair cells, with eight to 20 pairs per cell.
Although the vast majority of the puncta appear as juxtaposed pairs in normal ears, orphan ribbons are infrequently observed at high magnifications. The number of inner hair cell ribbon synapses is highest at the 16 kilohertz region and significantly decreases as the distance from this location increases. The most important thing to remember during this procedure is that one must guarantee the integrity of cochlear basal membrane.
We need to stress accuracy until technical proficiency is reached. Following this procedure, the gene therapy can be performed to answer whether the cochlear neuropathy can be repaired by this measure. This measure is wider technique for exploring cochlear neuropathy, which if the primary initial events associate with having hearing loss, tinnitus, and hyperacusis.
