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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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+adverse+effect+profiles+of+platinum+drugs.">Comparative adverse effect profiles of platinum drugs.</a> Drug Saf. 13 (4), 228-244 (1995).</li><li>Boulikas, T., Vougiouka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cisplatin+and+platinum+drugs+at+the+molecular+level.">Cisplatin and platinum drugs at the molecular level.</a> Oncol Rep. 10 (6), 1663-1682 (2003).</li><li>Fouladi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+II+study+of+oxaliplatin+in+children+with+recurrent+or+refractory+medulloblastoma,+supratentorial+primitive+neuroectodermal+tumors,+and+atypical+teratoid+rhabdoid+tumors:+a+pediatric+brain+tumor+consortium+study.">Phase II study of oxaliplatin in children with recurrent or refractory medulloblastoma, supratentorial primitive neuroectodermal tumors, and atypical teratoid rhabdoid tumors: a pediatric brain tumor consortium study.</a> Cancer. 107 (9), 2291-2297 (2006).</li><li>Pasetto, L. 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P., Husain, K., Morris, C., Whitworth, C., Somani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+protective+agents+against+cisplatin+ototoxicity.">Effect of protective agents against cisplatin ototoxicity.</a> Am J Otol. 21 (4), 513-520 (2000).</li><li>Lee, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+reactive+radicals+in+degeneration+of+the+auditory+system+of+mice+following+cisplatin+treatment.">Role of reactive radicals in degeneration of the auditory system of mice following cisplatin treatment.</a> Acta Otolaryngol. 124 (10), 1131-1135 (2004).</li><li>Lee, J. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+auditory+brainstem+response+testing.">Mouse auditory brainstem response testing.</a> Bio Protoc. 6 (6), 1768 (2016).</li><li>Montgomery, S. C., Cox, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+Mount+Dissection+and+Immunofluorescence+of+the+Adult+Mouse+Cochlea.">Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea.</a> J. Vis. Exp. (107), e53561 (2016).</li><li>Whitlon, D. S., Szakaly, R., Greiner, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryoembedding+and+sectioning+of+cochleas+for+immunocytochemistry+and+in+situ+hybridization.">Cryoembedding and sectioning of cochleas for immunocytochemistry and in situ hybridization.</a> Brain Res Brain Res Protoc. 6 (3), 159-166 (2001).</li><li>Londos, C., Cooper, D. M., Wolff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subclasses+of+external+adenosine+receptors.">Subclasses of external adenosine receptors.</a> Proc Natl Acad Sci. 77 (5), 2551-2554 (1980).</li><li>Lohse, M. J., Klotz, K. N., Lindenborn-Fotinos, J., Reddington, M., Schwabe, U., Olsson, R. 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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

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 ...

Watch this Scientific Journal Video about Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity at JoVE.com

Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity

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

9.3K Views.  Southern Illinois University School of Medicine. 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.

Video: Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity

Thermal Ablation for the Treatment of Abdominal Tumors

Percutaneous thermal ablation is an emerging treatment option for many tumors of the abdomen not amenable to conventional treatments. During a thermal ablation procedure, a thin applicator is guided into the target tumor under imaging guidance. Energy is then applied to the tissue until temperatures rise to cytotoxic levels (50-60 &deg;C). Various energy sources are available to heat biological tissues, including radiofrequency (RF) electrical current, microwaves, laser light and ultrasonic waves. Of these, RF and microwave ablation are most commonly used worldwide.
During RF ablation, alternating electrical current (~500 kHz) produces resistive heating around the interstitial electrode. Skin surface electrodes (ground pads) are used to complete the electrical circuit. RF ablation has been in use for nearly 20 years, with good results for local tumor control, extended survival and low complication rates1,2. Recent studies suggest RF ablation may be a first-line treatment option for small hepatocellular carcinoma and renal-cell carcinoma3-5. However, RF heating is hampered by local blood flow and high electrical impedance tissues (eg, lung, bone, desiccated or charred tissue)6,7. Microwaves may alleviate some of these problems by producing faster, volumetric heating8-10. To create larger or conformal ablations, multiple microwave antennas can be used simultaneously while RF electrodes require sequential operation, which limits their efficiency. Early experiences with microwave systems suggest efficacy and safety similar to, or better than RF devices11-13.
Alternatively, cryoablation freezes the target tissues to lethal levels (-20 to -40 &deg;C). Percutaneous cryoablation has been shown to be effective against RCC and many metastatic tumors, particularly colorectal cancer, in the liver14-16. Cryoablation may also be associated with less post-procedure pain and faster recovery for some indications17. Cryoablation is often contraindicated for primary liver cancer due to underlying coagulopathy and associated bleeding risks frequently seen in cirrhotic patients. In addition, sudden release of tumor cellular contents when the frozen tissue thaws can lead to a potentially serious condition known as cryoshock 16.
Thermal tumor ablation can be performed at open surgery, laparoscopy or using a percutaneous approach. When performed percutaneously, the ablation procedure relies on imaging for diagnosis, planning, applicator guidance, treatment monitoring and follow-up. Ultrasound is the most popular modality for guidance and treatment monitoring worldwide, but computed tomography (CT) and magnetic resonance imaging (MRI) are commonly used as well. Contrast-enhanced CT or MRI are typically employed for diagnosis and follow-up imaging.

A thermal tumor ablation procedure is described. The entire procedure is detailed, including pretreatment planning and imaging studies, anesthesia, adjuvant techniques to facilitate a percutaneous approach, imaging guidance of the ablation device to the tumor, thermal treatment, post-treatment care and follow-up.

Percutaneous thermal ablation is an emerging treatment option for many tumors of the abdomen not amenable to conventional treatments. During a thermal ...

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

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) has been shown to be a useful approach to local drug delivery into the inner ear. The goal of this article is to describe, in detail, the surgical techniques involved in canalostomy in both adult and neonatal mice. As indicated by fast-green dye and adeno-associated virus serotype 8 with the green fluorescent protein gene, the canalostomy facilitated broad distribution of injected reagents in the cochlea and vestibular end-organs with minimal damage to hearing and vestibular function. The surgery was successfully implemented in both adult and neonatal mice; indeed, multiple surgeries could be performed if required. In conclusion, canalostomy is an effective and safe approach to drug delivery into the inner ears of adult and neonatal mice and may be used to treat human inner ear diseases in the future.

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.

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) ...

Genetics

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

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.

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

Neuroscience

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

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and genetic mechanisms that contribute to hearing loss. Introduction of vectors into the inner ear must be done in a way that widely distributes the agent throughout the cochlea while minimizing injury to the existing structures. This manuscript describes a post-auricular surgical approach that can be used for mouse cochlear therapy using molecular, pharmacologic, and viral delivery to mice postnatal day 10 and older via the round window membrane (RWM). This surgical approach enables rapid and direct delivery into the scala tympani while minimizing blood loss and avoiding animal mortality. This technique involves negligible or no damage to essential structures of the inner and middle ear as well as neck muscles while wholly preserving hearing. To demonstrate the efficacy of this surgical technique, the vesicular glutamate transporter 3 knockout (VGLUT3 KO) mice will be used as an example of a mouse model of congenital deafness that recovers hearing after delivery of VGLUT3 to the inner ear using an adeno-associated virus (AAV-1).

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.

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and ...

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

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research involving the cochlea and its associated neural pathways entails exposure of model cochleae to a variety of ototoxic agents. This allows investigators to study the effects of targeted damage to cochlear structures, and in some cases, the self-repair or regeneration of those structures. Various techniques exist for delivery of ototoxic agents to the cochlea. When selecting a particular technique, investigators must consider a number of factors, including the induction of inadvertent systemic toxicity, the amount of cochlear damage produced by the surgical procedure itself, the type of lesion desired, animal survivability, and reproducibility/reliability of results. Currently established techniques include parenteral injection, intra-peritoneal injection, trans-tympanic injection, endolymphatic sac injection, and cochleostomy with perilymphatic perfusion. Each of these methods has been successfully utilized and is well described in the literature; yet, each has various shortcomings. Here, we present a technique for topical application of ototoxic agents directly to the round window niche. This technique is non-invasive to inner ear structures, produces rapid onset of reliably targeted lesions, avoids systemic toxicity, and allows for an intra-animal control (the contra-lateral ear). Results stemming from this approach have helped deeper understanding of auditory pathophysiology, cochlear cell degeneration, and regenerative capacity in response to an acute injury. Future investigations may use this method to conduct interventional studies involving gene therapy and stem cell transplantation to combat hearing loss.

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.

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research ...

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

Hearing research has long been facilitated by rodent models, although in some diseases, human symptoms cannot be recapitulated. The common marmoset (Callithrix jacchus) is a small, easy-to-handle New World monkey which has a similar anatomy of the temporal bone, including the middle ear ossicular chains and inner ear to humans, than in comparison with that of rodents. Here, we report a reproducible, safe, and rational surgical approach to the cochlear round window niche for the drug delivery to the inner ear of the common marmoset. We adopted posterior tympanotomy, a procedure used clinically in human surgery, to avoid manipulation of the tympanic membrane that may cause conductive hearing loss. This surgical procedure did not lead to any significant hearing loss. This approach was possible due to the large bulla structure of the common marmoset, although the lateral semicircular canal and vertical portion of the facial nerve should be carefully considered. This surgical method allows us to perform the safe and accurate administration of drugs without hearing loss, which is of great importance in obtaining pre-clinical proof of concept for translational research.

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

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.

Hearing research has long been facilitated by rodent models, although in some diseases, human symptoms cannot be recapitulated. The common marmoset ...

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

Endoscopic middle ear surgery is a widely employed minimally invasive surgical technique to address middle ear and mastoid pathology. Bone drilling is the main technical challenge of endoscopic middle ear surgery. The accompanying video describes the detailed protocol of a constant-suction bone-drilling technique and the procedure of endaural exclusive endoscopic atticoantrotomy (retrograde mastoidectomy) using this technique. The main components of this bone-drilling technique include a soft and flexible suction tube, which is placed into the tympanic cavity to provide constant suction, and a soft sleeve, which is wrapped around the drill shaft to prevent the high-speed rotating shaft from damaging the lens of the endoscope. With these simple modifications, the traditional otological electrodrill can be used for drilling a tiny endaural incision in endoscopic middle ear surgery. Based on this bone-drilling technique, endaural endoscopic atticoantrotomy (retrograde mastoidectomy) can be successfully established for the removal of various amounts of bone, depending on the extent of the lesion. The short-term postoperative outcome seems promising.

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

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

Endoscopic middle ear surgery is a widely employed minimally invasive surgical technique to address middle ear and mastoid pathology. Bone drilling is the ...

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

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. The first procedure consists of perforation of the tympanic bulla, termed bullostomy; the second one is a transtympanic injection. Both emulate human clinical intratympanic procedures.
Chitosan-glycerophosphate (CGP) and Ringer&#180;s Lactate buffer (RL) were used as biocompatible vehicles for local drug delivery. CGP is a nontoxic biodegradable polymer widely used in pharmaceutical applications. It is a viscous liquid at RT&#160;but it congeals to a semi solid phase at body temperature. RL is an isotonic solution used for intravenous administrations in humans. A small volume of this vehicle is precisely placed on the Round Window (RW) niche by means of a bullostomy. A transtympanic injection fills the middle ear and allows less control but broader access to the inner ear.
The safety profiles of both techniques were studied and compared by using functional and morphological tests. Hearing was evaluated by registering the Auditory Brainstem Response (ABR) before and several times after microsurgery. The cytoarchitecture and preservation level of cochlear structures were studied by conventional histological techniques in paraformaldehyde-fixed and decalcified cochlear samples. In parallel, unfixed cochlear samples were taken and immediately frozen to analyze gene expression profiles of inflammatory markers by quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR).
Both procedures are suitable as drug delivery methods into the mouse middle ear, although transtympanic injection proved to be less invasive compared to bullostomy.

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

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.

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. ...

Biology

Hearing Preservation with Long Electrodes in Electric-Acoustic Stimulation

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

Electric-acoustic stimulation (EAS) is a promising treatment to improve hearing ability in patients with high-frequency hearing loss (HL). In EAS surgeries, shorter electrodes have been preferred to avoid the presence of an electrode covering the residual hearing region. However, our earlier studies showed that EAS with longer electrodes (28 mm) could preserve acoustic hearing. Additionally, we reported that the hearing preservation (HP) scores were independent of the length of the inserted electrodes, consistent with the systematic review. As most EAS patients gradually lose residual hearing over time due to the natural course of HL, in these cases, providing broader cochlear coverage using longer electrodes was beneficial toward better place-pitch matching. In addition to preparing for the deterioration in&#160;hearing in&#160;the future, EAS with longer electrodes could offer various types of map strategies. Herein, we show the pre-, intra-, and post-procedures for EAS surgery. Appropriate preoperative evaluation, less invasive surgery, flexible lateral-wall electrodes, and steroid administration resulted in good HP following EAS with longer electrodes.

Author Spotlight: Optimizing EAS with Long Electrodes for Enhanced Cochlear Coverage and Hearing Preservation

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.

Electric-acoustic stimulation (EAS) is a promising treatment to improve hearing ability in patients with high-frequency hearing loss (HL). In EAS ...

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

Begin by placing the anesthetized mouse on a pre-heated electric pad. Set the temperature of the electric pad to approximately 37 degrees Celsius. Cover the animal&#39;s eyes with eye ointment, then shave the left postauricular region with an electric animal clipper, and disinfect the skin three times with 75% ethanol.
Next place, the animal in the right lateral position to facilitate surgery on the left ear. Make a one to 1.5-centimeter postauricular incision, three millimeters from the left retroauricular groove. Note, when the root of the pinna is defined as the origin, and the plane parallel to the calvarium as 3 to 9 o&#39;clock, the posterior semicircular canal and the lateral semicircular canal are typically located about three millimeters from the root of the pinna between 2 and 3 o&#39;clock.
Bluntly dissect the muscle covering the temporal bone with micro forceps to expose the posterior semicircular canal and lateral semicircular canal, whose margins are clearly visible as dark stripes in the temporal bone. Then, collect a small piece of muscle with micro-forceps, and let it dry.
Next, make a small hole in the middle portion of the posterior semicircular canal using a 26-gauge needle, such that fluid leakage through the hole indicates successful penetration of the bony wall of the posterior semicircular canal. Enlarge the hole to slightly larger than the diameter of the polyamide tubing.
Then, clean the effusion surrounding the whole of the posterior semicircular canal using a cotton pellet. Insert the tip of the polyamide tubing gently into the posterior semicircular canal towards the crus commune to a depth of one to two millimeters.
Start the injection by pressing the &#39;Run&#39; button on the pump. After the injection, wait two minutes to allow the reagent to spread. Then, remove the injection cannula, and immediately place the muscle into the hole in the posterior semicircular canal.
Return the separated muscles and subcutaneous tissues together. Suture the incision using a 5-0 suture. Disinfect the incision region with povidone-iodine. Finally, after the surgery is complete, position the animal in the right lateral position on an electric pad preheated to 37 degrees Celsius for recovery.

Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity

Podsumowanie

Przeglądaj więcej filmów

Thermal Ablation for the Treatment of Abdominal Tumors

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

Optogenetic Stimulation of the Auditory Nerve

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

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

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

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

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

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

Kanatotomia do miejscowego dostarczania leku do ucha wewnętrznego dorosłej myszy