Name: Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity (Video) | JoVE Journal
Uploaded: 2018-03-16T12:30:00.000+00:00
Duration: 9 min 52 s
Description: Learn trans-tympanic drug delivery for treating ototoxicity using rat models. This method enables localized cochlear administration of protective agents to prevent cisplatin-induced hearing loss while preserving anti-cancer efficacy.

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5 minutes

The work described in this article was supported by a NCI RO1 CA166907, NIDCD RO1-DC 002396 and RO3 DC011621.

Male Wistar rats were handled in accordance with the National Institutes of Health animal use guidelines and a protocol approved by the Southern Illinois University School of Medicine Laboratory Animal Care and Use Committee. Auditory brainstem response (ABR) was performed on rats while under anesthesia prior to drug administration and 72 h after to verify the effect of the trans-tympanic drug delivery.
1. Auditory Brainstem Response (ABR)
Note:&#160;ABR measurements were collected using the auditory testing equipment and software. ABR represents evoked potentials or high-frequency waves generated by the eighth cranial nerve (wave I and II) and other higher auditory brain stem structures including anteroventral cochlear nucleus (wave III), lateral lemniscus (wave IV), and inferior colliculus (wave V). These waves are differentiated depending on the latency. ABR measurements were performed as described previously12.
<ol>
	<li>Anesthetize rats with a mixture of 90 mg/kg ketamine and 17 mg/kg xylazine&#160;via&#160;intraperitoneal injection. Confirm the depth of anesthesia&#160;via&#160;toe-pinch reflex. Apply ophthalmic lubrication (or mineral oil drops) to both eyes to prevent the eyes from drying and avoid ulceration during anesthesia.</li>
	<li>Place the rat in the prone position on a heating pad (37 &#176;C) inside of a controlled acoustical booth. Audiology testing equipment is located outside and next to the booth.</li>
	<li>Insert stainless steel electrodes accordingly: the ground electrode in the rear flank, the positive electrode between the two ears directly atop the skull, and the negative electrodes below the pinna of each ear.</li>
	<li>Apply acoustic stimuli using High-Frequency Transducers as a 5 ms tone burst at 8, 16, and 32 kHz. Determine stimulus intensities as decibel sound pressure level (dB SPL). This begins at 10 dB SPL and reaches 90-dB SPL with a 10-dB step size. Calibrate the sound intensities to a maximum output of 90-dB SPL using an impulse precision sound level meter. 
	Note:&#160;The test result gives an indication of the integrity of the peripheral hearing organ, the cochlea, and the aforementioned auditory structures. The waveforms are generated within 15 ms of stimulus input and the latency of the waves depends on conduction time, which in turn is regulated by three critical factors: volume of the brain, the intensity, and the frequency of the sound. Auditory evoked potentials were recorded using software provided by the manufacturer.</li>
	<li>Consider the minimum intensity which can evoke two different waveforms (II and III) of 0.5 &#181;V amplitude as threshold prominently. 
	Note:&#160;Threshold shift represents the difference in the threshold measured after treatment compared with the threshold obtained prior to treatment.</li>
</ol>
2. Trans-Tympanic Injections
<ol>
	<li>To administer the drug trans-tympanically, place the rat in the left lateral decubitus position.
	<ol>
		<li>Insert 2.5 mm disposable ear specula into the ear canal.</li>
		<li>With the use of a surgical scope, position the specula so that the tympanic membrane is visible.</li>
		<li>Using a 29 G X &#189;, 0.5 mL insulin syringe, draw up 50 &#181;L of the [R]-N-phenyl isopropyl adenosine (R-PIA) solution (1 &#181;M), 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX) solution (3 &#181;M), or siRNA solution (0.9 &#181;g) to inject (5 units).</li>
		<li>Use the specula to direct the needle to the anterior inferior region of the tympanic membrane.</li>
		<li>Poke a hole through the membrane with the needle and administer the drugs mentioned in 1.2.3. Allow the rat to rest in this position for 15 min. 
		Note: 50 &#181;L should be the adequate volume to fit behind the tympanic membrane. No fluid should be in the ear canal after administration.</li>
		<li>Place the rat in the right lateral decubitus position and repeat steps 1.2.1 through 1.2.5 for administration in the other ear.</li>
	</ol>
	</li>
	<li>For rats receiving cisplatin intraperitoneally, place the rat in the supine position on a heating pad at 37 &#176;C.
	<ol>
		<li>Using a 21 G X 3/4 butterfly needle (12&#34; length tubing), draw up cisplatin (11 mg/kg, 1 mg/mL solution in sterile phosphate buffer saline [PBS]).</li>
		<li>Using a syringe pump, administer cisplatin (1 mg/mL) via intraperitoneal injection over 30 min. 
		Note: For a 250-g rat, the volume would be 2.75 mL at a rate of approximately 0.1 mL per min.</li>
	</ol>
	</li>
	<li>Continue to monitor the depth of anesthesia throughout these procedures. Once cisplatin administration is complete, place the rat back in the cage in the prone position, making sure there is nothing to obstruct its breathing.</li>
	<li>Monitor the rat until fully recovered.</li>
</ol>
3. Cochlea Dissection and Decalcification
<ol>
	<li>Following the final ABR (after 72 h), anesthetize the rat with a mixture of 90 mg/kg ketamine and 17 mg/kg xylazine via intraperitoneal injection. Confirm anesthesia with a toe-pinch reflex. Euthanize the rat via decapitation.</li>
	<li>Dissect out the temporal bone as described previously13.</li>
	<li>Place cochlea in 4% paraformaldehyde in 1x PBS solution in a 7-mL glass scintillation vial (completely covering the cochlea). Refrigerate overnight at 4 &#176;C. Remove paraformaldehyde and wash with 1x PBS at room temperature.</li>
	<li>Remove PBS and fill the tube completely with a 120-mM solution of EDTA (pH 7.3). Place on a rotator at room temperature and allow decalcification for 2 to 3 weeks, changing the EDTA solution daily.</li>
</ol>
4. Cryosectioning
<ol>
	<li>Upon the completion of decalcification, completely immerse the cochlea in the following solutions (7 mL each) for 24 h at 4 &#176;C: 10% sucrose, 20% sucrose, 1:1 mixture of 20% sucrose, and optimal cutting temperature (OCT) embedding compound.</li>
	<li>Place fresh OCT compound to fill and completely cover the cochlea in a 15 mm x 15 mm x 5 mm disposable embedding mold. Orient the cochlea on its side so that it is parallel to the bottom of the embedding mold, as described by Whitlon et al.14</li>
	<li>Immediately place the mold on dry ice to solidify the OCT and store at -80 &#176;C overnight.</li>
	<li>Immerse microscope slides in 0.01% Poly-L Lysine at room temperature for 30 min. Remove the slides, do not rinse, and allow to dry overnight. Use these slides for cryosections.</li>
	<li>Remove the cochlea in OCT from the -80 &#176;C freezer and place it on dry ice. Using a sharp microtome blade (L x W: 80 mm x 8 mm, thickness 0.25 mm, cutting angle of 34 &#176;), section the OCT blocks at 10 &#181;m using a cryostat at -30 &#176;C. Place two sections per slide.</li>
	<li>Refrigerate slides at 4 &#176;C when finished.</li>
</ol>
5. Immunohistochemistry
<ol>
	<li>Place the slides in a glass microscope slide staining dish on a slide rack. Fill the dish with 350 mL of 1x PBS and wash the slides for 5 min three times at room temperature.</li>
	<li>Remove the slides from the dish and dry off the area surrounding the tissue using a dry wipe, being sure not to dry off the tissue.
	<ol>
		<li>Using a liquid blocking pen, draw a circle around the tissue section.</li>
	</ol>
	</li>
	<li>Block the tissue for 1 h at room temperature by adding 150 &#181;L of blocking solution containing 10% normal (donkey) serum, 1% nonionic detergent, and 1% BSA in 1x PBS.</li>
	<li>Tap off excess blocking solution and incubate the tissue overnight at 4 &#176;C in a humidified chamber with 150 &#181;L of 10% normal (donkey) serum, 0.1% nonionic detergent, and primary antibody (see the Note below) in 1x PBS. 
	Note: For the following antibodies, these dilutions were used: For Figure 2 and 3, p-STAT1 Ser727 1:300. For Figure 4, p-STAT1 Ser727, 1:100 and TRPV1 1:100.</li>
	<li>Place the slides in the glass microscope slide staining dish on a slide rack. Fill the dish with 350 mL of 1x PBS and wash slides for 5 min three times at room temperature.</li>
	<li>Incubate with the secondary antibodies diluted in a solution containing 0.01% nonionic detergent and 10% normal (donkey) serum in 1x PBS for 2 to 3 h at room temperature in a humidified chamber in the dark. 
	Note: For the following secondary antibodies, these dilutions were used: For Figure 2 and 3, Donkey Anti-Rabbit IgG 1:600. For Figure 4, Rhodamine (TRITC) Donkey Anti-Rabbit IgG 1:500 and Donkey Anti-Goat IgG 1:500.</li>
	<li>Place the slides in the glass microscope slide staining dish on a slide rack. Fill the dish with 350 mL of 1x PBS and wash slides for 5 min three times at room temperature.</li>
	<li>Tap excess liquid off the slide, and dry the slide with a dry wipe around the tissue. Mount slides by adding one drop of the mounting agent with DAPI directly onto the tissue. Slowly place the coverslip on top, ensuring that no bubbles are formed underneath, and allow the slides to cure overnight at room temperature in the dark. Store slides at 4 &#176;C.</li>
	<li>Image slides using confocal microscopy. Use lasers for imaging as follows: UV laser for DAPI, 488 nm laser for Donkey Anti-Rabbit IgG, and 543 nm laser for rhodamine TRITC.</li>
</ol>

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</ol>

No conflict of interest declared.

The trans-tympanic administration route allows for localized delivery of drugs and other agents to the cochlea which could otherwise produce significant systemic side effects if administered systemically. This method of drug administration allows rapid access of drugs to the site of action at significantly higher doses than would be achieved through the systemic route. Results presented here and published previously showed that trans-tympanic administration of [R]-N-phenyl isopropyl adenosine (R-PIA) pr...

The peripheral auditory system is exquisitely sensitive to drugs such as cisplatin and aminoglycoside antibiotics. Cisplatin is a widely used chemotherapeutic agent for the treatment of a variety of solid tumors, such as ovarian, testicular, and head and neck cancers. Ototoxicity experienced with the use of this drug is dose-limiting and quite common, affecting 75-100% of patients treated1. Other drugs, such as carboplatin and oxaliplatin, have emerged as alternatives to cisplatin2,3,4,5, but their usefulness is limited to a few cancers.
Early studies have shown the critical role of reactive oxygen species (ROS) in mediating the ototoxicity produced by cisplatin and aminoglycosides. Subsequent studies showed that the NOX3 isoform of NADPH oxidase is the primary source of ROS in the cochlea, and is activated by cisplatin6,7. The generation of ROS compromises the antioxidant buffering capacity of cells, leading to increased lipid peroxidation of cellular membranes8. Furthermore, cisplatin increases the production of hydroxyl radicals which generate highly toxic aldehyde 4-hydroxynonenal (4-HNE), an initiator of cell death9,10. Based on these findings, several antioxidants have been examined for the treatment of cisplatin ototoxicity. These include N-acetyl cysteine (NAC), sodium thiosulfate (STS), amifostine, and D-methionine. However, a major concern of antioxidant therapy is that these antioxidants could reduce cisplatin chemotherapeutic efficacy when administered systemically11 through the interaction of cisplatin with thiol groups in the antioxidant molecules.
In view of these problems with antioxidant therapy, the goal of this study was to examine the trans-tympanic route of delivering antioxidants and other drugs to the cochlea to reduce hearing loss. The trans-tympanic route of drug and short interfering (si) RNA, described below, appears particularly promising.

<table><tbody><tr><td>Ketathesia (100 mg/ml) 10 ml</td><td>Henry Schein</td><td>56344</td><td>Controlled substance&amp;nbsp;</td></tr><tr><td>AnaSed Injection/Xylazine (20 mg/ml) 20 ml</td><td>Henry Schein</td><td>33197</td><td></td></tr><tr><td>2.5 mm disposable ear specula</td><td>Welch Allyn</td><td>52432</td><td></td></tr><tr><td>Surgical Scope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>29 G X 1/2 insulin syringe</td><td>Fisher Scientific</td><td>14-841-32&amp;nbsp;</td><td>Can be purchased through other vendors</td></tr><tr><td>cis-Diammineplatinum(II) dichloride</td><td>Sigma Aldrich</td><td>P4394</td><td>TOXIC - wear proper PPE</td></tr><tr><td>Harvard 50-7103 Homeothermic Blanket Control Unit</td><td>Harvard Apparatus</td><td>Series 863</td><td></td></tr><tr><td>Excel International 21 G X 3/4 butterfly needle</td><td>Fisher</td><td>14-840-34&amp;nbsp;</td><td>Can be purchased through other vendors</td></tr><tr><td>BSP Single Speed Syringe Pump</td><td>Brain Tree Sci, Inc</td><td>BSP-99</td><td></td></tr><tr><td>Pulse Sound Measurement System</td><td>Bruel &amp;amp; Kjaer</td><td>Pulse 13 software</td><td></td></tr><tr><td>High-Frequency Module</td><td>Bruel &amp;amp; Kjaer</td><td>3560C</td><td></td></tr><tr><td>1/8&amp;Prime; Pressure-field Microphone &amp;mdash;-Type 4138</td><td>Bruel &amp;amp; Kjaer</td><td>bp2030</td><td></td></tr><tr><td>High Frequency Transducer</td><td>Intelligent Hearing System</td><td>M014600</td><td></td></tr><tr><td>Opti-Amp Power Transmitter</td><td>Intelligent Hearing System</td><td>M013010P</td><td></td></tr><tr><td>SmartEP ABR System</td><td>Intelligent Hearing System</td><td>M011110</td><td></td></tr><tr><td>Disposable Subdermal EEG Electrodes</td><td>CareFusion</td><td>019-409700</td><td></td></tr><tr><td>16% Formaldehyde, Methanol-free</td><td>Fisher Scientific</td><td>28908</td><td>TOXIC - wear proper PPE&amp;nbsp;</td></tr><tr><td>7 mL Borosilicate Glass Scintillation Vial</td><td>Fisher Scientific</td><td>03-337-26</td><td>Can be purchased through other vendors</td></tr><tr><td>EDTA</td><td>Fisher Scientific</td><td>BP118-500</td><td>Can be purchased through other vendors</td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>S5-500</td><td>Can be purchased through other vendors</td></tr><tr><td>Tissue Plus OCT Compound</td><td>Fisher Scientific</td><td>4585</td><td></td></tr><tr><td>CryoMolds (15 mm x 15 mm x 5mm)</td><td>Fisher Scientific</td><td>22-363-553</td><td>Can be purchased through other vendors</td></tr><tr><td>Microscope Slides (25mm x 75mm)</td><td>MidSci</td><td>1354W</td><td>Can be purchased through other vendors</td></tr><tr><td>Coverslips (22 x 22 x 1)</td><td>Fisher Scientific</td><td>12-542-B</td><td>Can be purchased through other vendors</td></tr><tr><td>Poly-L-Lysine Solution (0.01%)</td><td>EMD Millipore</td><td>A-005-C</td><td>Can be purchased through other vendors</td></tr><tr><td>HM525 NX Cryostat</td><td>Thermo Fischer Scientific</td><td>956640</td><td></td></tr><tr><td>MX35 Premier Disposable Low-Profile Microtome Blades</td><td>Thermo Fischer Scientific</td><td>3052835</td><td></td></tr><tr><td>Wheaton&amp;trade;&amp;nbsp;Glass 20-Slide Staining Dish with Removable Rack</td><td>Fisher Scientific</td><td>08-812</td><td></td></tr><tr><td>Super Pap Pen Liquid Blocker</td><td>Ted Pella, Inc.</td><td>22309</td><td></td></tr><tr><td>Normal Donkey Serum</td><td>Jackson Immuno Research</td><td>017-000-121</td><td>Can be purchased through other vendors</td></tr><tr><td>TritonX-100</td><td>Acros</td><td>21568</td><td>Can be purchased through other vendors</td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>A7906</td><td>Can be purchased through other vendors</td></tr><tr><td>Phospho-Stat1 (Ser727) antibody</td><td>Cell Signaling</td><td>9177</td><td></td></tr><tr><td>VR1 Antibody (C-15)</td><td>Santa Cruz</td><td>sc-12503</td><td></td></tr><tr><td>DyLight 488 Donkey anti Rabbit</td><td>Jackson Immuno Research</td><td>711-485-152</td><td>Discontinued</td></tr><tr><td>DyLight 488 Donkey anti Goat</td><td>Jackson Immuno Research</td><td>705-485-003</td><td>Discontinued</td></tr><tr><td>Rhodamine (TRTIC) Donkey anti Rabbit</td><td>Jackson Immuno Research</td><td>711-025-152</td><td>Discontinued</td></tr><tr><td>ProLong&amp;reg; Diamond Antifade Mountant w/ DAPI</td><td>Thermo Fisher</td><td>P36971</td><td></td></tr><tr><td>(&amp;minus;)-N6-(2-Phenylisopropyl)adenosine</td><td>Sigma Aldrich</td><td>P4532</td><td></td></tr><tr><td>8-Cyclopentyl-1,3-dipropylxanthine</td><td>Sigma Aldrich</td><td>C101</td><td></td></tr><tr><td>siRNA pSTAT1</td><td>Qiagen</td><td>Custome Made</td><td>Kaur et al. 2011&lt;sup&gt;20&lt;/sup&gt;</td></tr><tr><td>siRNA NOX3</td><td>Qiagen</td><td>Custome Made</td><td>Kaur et al. 2011&lt;sup&gt;20&lt;/sup&gt;</td></tr><tr><td>Scrambled Negative Control siRNA</td><td>Qiagen</td><td>1022076</td><td>Kaur et al. 2011&lt;sup&gt;20&lt;/sup&gt;</td></tr></tbody></table>

trans-tympanic drug delivery for the treatment of ototoxicity

Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could interfere with the chemotherapeutic efficacy of the primary drugs. This is especially true for the drug cisplatin, whose anticancer actions are attenuated by antioxidants which provide adequate protection against hearing loss. Other current or potential otoprotective agents could pose a similar problem, if administered systemically. The application of various biologicals or protective agents directly to the cochlea would allow for high levels of these agents locally with limited systemic side effects. In this report, we demonstrate a trans-tympanic method of delivery of various drugs or biological reagents to the cochlea, which should enhance basic science research on the cochlea and provide a simple way of directing the use of otoprotective agents in the clinics. This report details a method of trans-tympanic drug delivery and provides examples of how this technique has been used successfully in experimental animals to treat cisplatin ototoxicity.

ABR responses measured in rats at three days following cisplatin administration showed a significant elevation in thresholds. Elevation of these thresholds was significantly reduced in rats administered with trans-tympanic [R]-N-phenylisopropyladenosine (R-PIA), adenosine A1 receptor agonist15, prior to cisplatin. The specificity of the action of R-PIA at the adenosine A1 receptor was demonstrated by the observation ...

We present a technique for localized administration of drugs through the trans-tympanic route into the cochlea. Drug delivery through this route would not interfere with the anti-cancer efficacy of the chemotherapeutic drugs such as cisplatin.

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could ...

trans-tympanic-drug-delivery-for-the-treatment-of-ototoxicity

Research

JoVE Journal

Medicine

This video showcases canalostomy, a procedure that allows local drug delivery into the inner ear of a mouse through the semicircular canal with minimal damage to hearing and vestibular function. This method enables the inoculation of viral vectors, pharmaceuticals, and small molecules into the mouse's inner ear.

Canalostomy for Local Drug Delivery into the Inner Ear of an Adult Mouse

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

Optogenetic Stimulation of the Auditory Nerve

The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane.

Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

Various methods exist for introducing ototoxic agents to the cochleae of animal models. Presented is a surgical protocol for delivery of ototoxic agents to the round window niche. The procedure is reliable, creates targeted intra-cochlear lesions, and avoids mechanical damage to the microarchitecture. Examination of cochlear self-repair/regeneration is possible.

The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration

Two microsurgery approaches for local drug delivery to the inner ear are described here and compared in terms of impact on hearing parameters, cochlear cytoarchitecture and expression of inflammatory markers.

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

We report a surgical method to administrate drugs to the inner ear of a non-human primate, the common marmoset (Callithrix jacchus), via the round window membrane.

A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset (Callithrix jacchus)

Here we describe canalostomy procedure which allows local drug delivery into the inner ears of adult and neonatal mice through the semicircular canal with minimum damage to hearing and vestibular function. This method can be used to inoculate viral vectors, pharmaceuticals, and small molecules into the mouse inner ear.

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Animal models of cochlear implants can advance knowledge of the technological bases of treating permanent sensorineural hearing loss with electrical stimulation. This study presents a surgical protocol for acute deafening and cochlear implantation of an electrode array in mice as well as the functional assessment with auditory brainstem response.

Cochlear Implant Surgery and Electrically-evoked Auditory Brainstem Response Recordings in C57BL/6 Mice

In this paper, we present the protocol and short-term outcomes of endaural exclusive endoscopic atticoantrotomy using a constant suction bone-drilling technique.

Endaural Endoscopic Atticoantrotomy (Retrograde Mastoidectomy) using a Constant Suction Bone-drilling Technique

Electric acoustic stimulation (EAS) with longer electrodes can offer broader cochlear coverage and various types of maps in cases of high-frequency hearing loss. Combining less invasive surgery, flexible lateral-wall electrodes, and steroid administration permits deeper insertion with little or no surgical trauma, resulting in good preservation of hearing.

Enhanced Cochlear Coverage and Hearing Preservation in High-Frequency Hearing Loss via Electric Acoustic Stimulation with Longer Electrode

Learn trans-tympanic drug delivery for treating ototoxicity using rat models. This method enables localized cochlear administration of protective agents to prevent cisplatin-induced hearing loss while preserving anti-cancer efficacy.

Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity

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