This presentation is a demonstration of the procedures for whole organ cultures of utricle from adult mice. The use of adenovirus to infect supporting cells of cultured utricle will also be demonstrated. This is accomplished by first carefully dissecting the utricle from the inner ear.
The second step is to remove the roof epithelium. Next, the otoconia are removed, leaving the sensory epithelium intact. The final step is to infect the supporting cells of the utricle using adenovirus.
These techniques can be used in a wide variety of experiments designed to examine mature mammalian hair cells and supporting cells. In vitro. The sensory receptor cells in the inner ear are called hair cells, and these cells are responsible for converting sound energy or head movement into neural input to the brain.
Hearing loss is often caused by death of these sensory hair cells. In order to study the mechanisms underlying sensory hair cell death, it is very useful to have an in vitro model system. The method that we will demonstrate utilizes whole organ cultures of utricle from adult mice.
The utricle is a vestibular organ in the inner ear, and the hair cells of the utricle are similar to the hair cells in the hearing organ, which is called the organ of corde. The utricle preparation is the best characterized model system for studies of hair cells and supporting cells from mature mammals. In addition, we will demonstrate a method that our laboratory has recently developed for using adenovirus to infect the supporting cells of the utricle.
The adenovirus technique allows us to examine the roles of supporting cells as mediators of hair, cell survival, death, and regeneration. We use these techniques in our laboratory to address two major questions. First, what are the cellular and molecular signals that determine whether a hair cell under stress ultimately lives or dies?
And secondly, how can we utilize this information to develop therapies, clinical therapies to prevent or reverse hearing loss? Lindsey Maye, a biologist in my laboratory, will demonstrate these Techniques. Visual demonstration of the utricle dissection is important because the tissue is small and fragile, and it's very susceptible to mechanical damage, especially during the steps of removing otoconia and roof epithelium.
After euthanizing and decapitating an adult mouse in accordance with an institutionally approved animal care and use protocol, bisect the skull from coddle to rostral and remove the brain from both halves to reveal the bony labyrinth. Next, trim the skull away from the bony cochlea and transfer the bony labyrinth to a 35 millimeter tissue culture dish containing sterile medium, 1 99. If the auditory bullah is still intact, use two sturdy forceps to break the bullah.
Take a moment to identify the landmarks of the bony preparation, including the apex base, acles, oval, and round windows eighth nerve root and semi-circular canals as seen in these images which are reproduced in the written protocol, orient the preparation in the dish such that the ossicles are on the bottom of the dish, and the eighth nerve root is facing up. Use forceps to hold the preparation in the region of the semicircular canals using a scalpel with a number 11 blade cut off the apex of the cochlea just apical to the nerve root. Use the anterior semicircular canal as a handle to stabilize the preparation and rotate the preparation so that the cut portion faces upward.
Now using number five forceps, remove the cochlea at the modis down to the base. Then use a fine probe made from half a pair of number 55 forceps to lift the last bony shelf of the osseous spiral lamina. To reveal the S beneath the S is the staes footplate.
This image shows the staes footplate labeled sf, which is an important landmark for identifying the position of the utricle labeled U, which is immediately adjacent to the staes footplate. Use the fine probe to chip away enough bone to reveal about one third of the utricle. Then using number 55 forceps, carefully remove the utricle by grasping it either very near the edge or at the nerve root.
If the pigmented roof epithelium is still attached to the utricle, then remove this using fine forceps. If u trickles are to be used for adenovirus infection, then removal of the otoconia is necessary to do this. Fill a syringe with dissecting media and fit it with a 26 or 28 gauge needle.
Hold the utricle at the edge with number 55 forceps and the otoconia using a stream of media. Alternatively, otoconia can be removed using an eyelash tool. Once all U trickles have been dissected, change the dissecting media to sterile culture media and place the plate in an incubator at 37 degrees Celsius with 5%carbon dioxide.
All adenovirus procedures are performed according to biosafety level two precautions. When ready to infect, use a micro curette to transfer one utricle hair cells up into one well of a no mini tray containing 15 microliters of serum free D-M-E-M-F 12. Add one to four times 10 to the seventh plaque forming units of adenovirus to the well containing the utricle culture.
The u trickles and virus containing media at 37 degrees Celsius with 5%carbon dioxide in a humidified chamber for two hours. After two hours transfer the u trickles to a 24 well tissue culture plate containing culture. Media with serum once transferred, the U trickles are again cultured overnight at 37 degrees Celsius with 5%carbon dioxide.
U trickles can be used the next day for live imaging experiments, or they can be fixed for immunochemistry. U trickles are fixed in 4%Para aldehyde on a rocking platform for three hours at room temperature or overnight at four degrees Celsius. Next, wash the utricle three times for 15 minutes each in 0.1 molar sorensens phosphate buffer.
If the otoconia were not removed for adenovirus infection, then remove them at this point by immersing the utricle in calx decalcify for one to two minutes, then wash with 0.1 molar sorens and phosphate buffer immerse the utricle in blocking solution for three hours at room temperature. After the blocking step, dilute the primary antibody in blocking solution. Add to the plate containing the u trickles and incubate overnight at four degrees Celsius.
The next day, wash the U trickles and sorens and phosphate buffer. Then add secondary antibody diluted in blocking solution and incubate for four hours while protected from light at room temperature on a rocking platform. Finally, mount the utricle on glass slides using flora Mount G.These confocal images show the hair cell layer and the supporting cell layer in the same area of one utricle infected with adenovirus driving expression of green fluorescent protein.
Hair cells are labeled with an antibody against myosin seven A, which appears magenta. Supporting cells are labeled with an antibody against socks, two, which appears red Here. It can be seen that adenovirus GFP localizes to supporting cells and not to hair cells.
This graph demonstrates that adenoviral infection with adenovirus GFP at four times 10 to the eighth. Plaque forming units per milliliter does not result in death of hair cells or supporting cells Once the dissection is mastered. Both uracil from one animal can be put into culture in 10 to 15 minutes.
However, it's important while developing this procedure to remember to take your time and be careful when handling the tissue following this procedure. Other methods like live confocal imaging, immunohistochemistry, R-T-P-C-R and Western blotting may be performed to address questions regarding supporting cell and hair cell death, survival and regeneration. After watching this video, you should have a good understanding of how to dissect the utricle and infect supporting cells using adenovirus.
Remember that working with viruses can be hazardous and biosafety. Level two precautions should always be taken when performing these procedures.
