The overall goal of this trans-tympanic procedure, is to locally deliver high concentrations of antioxidants, otoprotective agents, or biological reagents directly into the cochlea for treatment of hearing loss. The trans-tympanic route of drug delivery allows for the localized delivery of drugs and other agents into the cochlea that may cause significant side effects if administered systemically. The main advantage of this matter is that it permits rapid administration of drugs in significantly higher doses into the cochlea than would be achieved by systemic delivery.
This technique has the potential for the prophylactic delivery of reagents to prevent against cisplatin-induced hearing loss without interfering with the anti-tumor effect. After confirming a lack of response to toe pinch, apply ointment to the eyes of the rat. To asses the auditory brainstem response, place the rat in the prone position on a 37 degree Celsius heating pad inside a controlled acoustical booth and insert a stainless steel ground electrode into the rear flank, a stainless steel positive electrode into the top of the skull between the ears, and one stainless steal negative electrode below the pinna of each ear.
Carefully insert high-frequency transducer tubes into the rat's ears. Then, using high-frequency transducers, apply acoustics stimuli as a five millisecond tone burst at 8, 16, and 32 kilohertz. Place the animal in the left lateral decubitus position.
Insert a 2.5 millimeter disposable ear specula into the ear canal and use a surgical scope to position the specula so that the tympanic membrane is visible. It's important to orient the needles so that the ear drum is still visible, because the ear specula is so small and a successful injection requires visualization of all materials and tissue under the microscope. Using a 29 and a half gauge 0.5 milliliter insulin syringe, aspirate 50 microliters of the drug or reagent of interest into the syringe barrel.
And use the specula to direct the needle into the anterior inferior of the tympanic membrane. Use the needle to poke a hole through the membrane and administer the entire 50 microliter volume of the drug. Allow the rat to rest in this position for 15 minutes.
Then, place the rat in the right lateral decubitus position and administer the drug into the right ear as just demonstrated. For rats receiving cisplatin, place the rat in the supine position on a 37 degree Celsius heating pad and draw cisplatin into a 21 and three-quarter gauge butterfly needle connected to a 12-inch piece of tubing. Then, use a syringe pump to administer one milligram per milliliter of cisplatin via intraperitoneal injection over 30 minutes.
When all of the cisplatin has been delivered, place the rat back into its cage in the prone position, taking care that its breathing is not obstructed, and monitor the animal until it is fully recovered. Following the measurement of the post-treatment ABR, sacrifice the rat and dissect out temporal bone. Place the harvested cochlea in a seven milliliter glass scintillation vial containing four percent paraformaldehyde in PBS and store it overnight at four degrees Celsius.
The next morning, wash the cochlea in room temperature PBS. Followed by complete immersion in 120 millimolar EDTA. Then, place the vial on a rotator to decalcify the cochlea for two to three weeks at room temperature.
Upon completion of the decalcification, place the cochlea in sequential immersions of seven milliliters of sucrose for 24 hours per sucrose solution at four degrees Celsius. After the last submersion, add fresh optimal cutting temperature compound to a 15 by 15 by 5 millimeter disposable embedding mold, and place the cochlea on its side in the mold so that the sample is parallel to the bottom of the mold. Immediately place the mold on dry ice to solidify the OCT and store the mold at negative 80 degrees Celsius overnight.
The next morning, transfer the mold onto dry ice and set a cryostat to negative 30 degrees Celsius. Then, use sharp microtone blade to acquire two ten-micrometer sections of cochlea sample per polylysine coated slide. Insert the slides at four degrees Celsius.
To analyze the sections by immunohistochemistry, place the slides in a glass microscope slide staining dish on a slide rack and wash the section three times with 350 milliliters of PBS for five minutes per wash. After the last watch, use a lab wipe to remove the excess PBS around each sample, and use hydrophobic barrier pen to surround each section. Block the tissues for one hour at room temperature with 150 microliters of blocking solution per sample.
At the end of the incubation, top off the excess blocking solution and incubate the samples with 150 microliters of the primary antibody of interest overnight at four degree Celsius in a humidified chamber. The next morning, return the slides to the staining dish on the side rack and wash the slides three times in PBS as just demonstrated. Incubate the sections with the appropriate secondary antibodies for two to three hours at room temperature in the humidified chamber in the dark.
At the end of the incubation, return the slides to the staining dish and wash the slides three times in fresh PBS. After the last wash, tap the slides to remove the excess PBS and dry the slides with a lab wipe. Place a drop of mounting agent supplemented with DAPI directly onto each tissue section and carefully place a cover slip onto each slide taking care not to create bubbles.
Then, allow the slides to cure overnight at room temperature in the dark before imaging the samples by confocal microscopy. Auditory brainstem responses measured in rats at three days post-cisplatin administration shows significant elevation in thresholds that is reduced in rats treated with transtympanic delivery of the adenosine A1 receptor agonist, R-PIA, prior to cisplatin administration. Conversely, treatment with DPCPX, an A1 receptor-specific antagonist, potentiates the cisplatin-induced auditory brainstem response threshold shifts, demonstrating the specificity of the R-PIA agonist for the A1 receptor.
Cisplatin also increases outer hair cell loss and damage as assessed by scanning electron microscopy, an effect that is significantly reduced by transtympanic R-PIA administration. Further, transtympanic R-PIA reduces basal and cisplatin-induced phospho stat1 immunoreactivity in the outer hair cells of the cochlea, in accordance with the anti inflammatory property of this drug. The transtympanic route can also be used to administer other biologics, such as siRNAs, to reduce stat1 levels and to block the activation of this transcription factor to phospho stat1 by cisplatin, for example.
Addition studies show that transtympanic administration of NOX3 siRNA reduces the ability of drugs, such as capsaicin, to increase the expression of some downstream mediators that are implicated in capsaicin-induced hearing loss. When administering this technique in intratympanic injection, it is imperative that the animal be adequately anesthetized so it does not move during the procedure. Once mastered, this drug delivery system could be used to screen a large number of compounds for efficacy against drug-induced hair loss in experimental animals.
