We thank Shannon M. Lefler for assistance with figures and the Table of Materials. Research reported in this publication was supported by the National Institute of Deafness and Other Communication Disorders within the National Institutes of Health, through the &#34;Development of Clinician/Researchers in Academic ENT&#34; training grant, award number T32DC000022 (C.V.V.) and by R01 DC014997 (J.T.L). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

All procedures listed immediately below in the Protocol section were conducted as described in protocols approved by the Washington University in St. Louis Institutional Animal Care and Use Committee.
1. Anesthetic Induction and Monitoring of Vital Signs
NOTE: This study used pigmented NIH-strain guinea pigs obtained from an in-house breeding colony.
<ol>
	<li>Use guinea pigs of either sex, weighing at least 350 g.</li>
	<li>Place the guinea pig in a neonatal warming isolette and give a ketamine/xylazine mixture intraperitoneally (50 mg/kg ketamine and 10 mg/kg xylazine) for induction anesthesia. Observe the guinea pig until it loses the toe-pinch reflex.</li>
	<li>Once loss of toe-pinch reflexes occurs, shave the posterior neck and head of the guinea pig with a hair trimmer typically advertised for human use.</li>
	<li>Inject a subcutaneous&#160;bolus of 12 mL of lactated Ringer&#39;s solution into the back of the animal.</li>
	<li>Place the guinea pig supine on a warming pad with legs raised and place 27.5 G butterfly needle intraperitoneally. Verify that the butterfly needle is in the correct position in the intraperitoneal space by ensuring only air is aspirated. If blood or fluid is aspirated, there is concern for delivery into the vascular or bowel system.&#160;The butterfly needle is used for repeated administration of anesthesia.</li>
	<li>Flip the guinea pig over to the prone position and secure the head to a stereotactic holder.</li>
	<li>Secure&#160;a pulse oximeter to the foot. If using pigmented guinea pigs, pigmented paws can prevent reading oxygen saturation. Therefore, place the pulse oximeter on any paw that is not pigmented.</li>
	<li>Insert a rectal temperature probe to monitor body temperature. The rectal probe is part of a warming blanket system that maintains body temperature at 38 &#176;C. Do not turn on the warming blanket until the rectal probe is in place to avoid overheating the warming blanket. If having difficulty placing the rectal probe, it can be laid alongside the guinea pig&#39;s body.</li>
	<li>Apply lubricant to both eyes of the guinea pig to prevent corneal abrasions.</li>
	<li>Administer supplemental oxygen as needed via a rubber tubing positioned near the nose to maintain oxygen saturation levels above 90%.</li>
	<li>Give enrofloxacin 0.5 mg/kg subcutaneously as an antibiotic prophylaxis.</li>
	<li>Give 0.25 mg/kg bupivacaine with 1:100,000 epinephrine subcutaneously at the anticipated incision site for local anesthesia and vasoconstrictive effects.</li>
	<li>Provide maintenance anesthesia every 20 min&#160;for 4 cycles and then only as needed based on vital signs. Routinely monitor the depth of anesthesia by body temperature, respiration rate, oxygen saturation, and heart rate.</li>
	<li>Monitor vital signs every 15 minutes (temperature, respiratory rate, heart rate, and oxygen saturation).</li>
</ol>
2. Surgical Preparation
<ol>
	<li>Once the head of the guinea pig is positioned securely in a stereotactic holder, place a piece of masking tape over the back to provide adequate tension along the skin overlying the occiput. Secure the ends of the tape to the stereotactic holder.</li>
	<li>Liberally prep the skin overlying the occiput and posterior neck with iodine solution and 70% ethanol in a sterile fashion three times.</li>
	<li>At this point, use sterile precautions and autoclaved instruments. Place sterile drapes over the guinea pig.</li>
</ol>
3. Surgical Procedure
<ol>
	<li>Using a 15 blade, make a small, midline incision along the posterior occiput extending down into the posterior neck. Once under the skin, use iris scissors to detach the right posterior cervical muscles from the occipital bone. If any bleeding occurs while cutting the muscles, control by applying pressure with a sterile cotton ball.</li>
	<li>Using a combination of a #3 mm, #2 mm, and #1 mm diamond burr with a 5-0 suction and sterile irrigation, perform a craniotomy that is bounded by the external occipital crest, lamboidal ridge, the occipitomastoid suture line, and the dorsal margin of the foramen magnum.
	<ol>
		<li>Gently place a small piece of saline-moistened cotton ball&#160;under the bone while separating the occipital bone from the dura.</li>
	</ol>
	</li>
	<li>Skeletonize the sigmoid sinus with a #0.5 mm diamond burr and carefully remove the bone overlying it.</li>
	<li>Once the sigmoid sinus is exposed, gently retract the sigmoid sinus medially using a cotton ball and switch to using a 3-0 suction.</li>
	<li>Identify the operculum as a slit like structure that is located within the petrous temporal bone. The subarcuate fossa will be situated superiorly and the sigmoid sinus will be medial to it. The extra-osseous portion of the endolymphatic sac is then visualized as a clear sac entering the operculum and attached to the dura overlying the sigmoid sinus. The operculum is oval shaped, approximately 3 to 4 mm by 1.5 to 2 mm. However, as seen from the surgical view, the operculum appears as an approximate 1 mm slit. The visible portion of the sac from the surgical view is approximately the same size as the visible portion of the operculum, if not smaller.</li>
	<li>Apply gentle retraction to the sigmoid sinus medially in order to clearly visualize the extra-osseous portion of the endolymphatic sac and increase the tension between the extraosseous and intraosseous portions of the endolymphatic sac.
	<ol>
		<li>Use a fine angled pick to gently expunge the intermediate portion of the endolymphatic sac. It is critical that the expungement process leave no visible connection between the dura and the operculum; then place a fine pick inside the operculum to broadly scrape along the inside of the bone to injure it.</li>
		<li>Turn the fine pick in the direction of the endolymphatic duct and blindly disrupt the lining. At this point some bleeding may occur from a vessel within the operculum. It can be controlled with a small piece of cotton.</li>
	</ol>
	</li>
	<li>Dry the empty operculum with a small piece of cotton. Using the 3-0 suction as needed to keep the cotton dry.</li>
	<li>Obtain bone dust by using a small curette to scrape along the squamosal portion of the temporal bone. Generously pack the operculum with bone dust. Use a cotton ball and suction to keep the area dry while packing it with bone dust.</li>
	<li>Apply bone wax to the operculum to seal it. Ensure that there is no excess bone wax dislodged into the skull.</li>
	<li>Use bone wax to cover the skull&#160;defect.</li>
	<li>Approximate the posterior cervical muscles with 4-0 braided, absorbable suture in an interrupted fashion.</li>
	<li>Perform a subcuticular closure using a 4-0 braided, absorbable suture.</li>
</ol>
4. Post-procedure Care
<ol>
	<li>Remove the guinea pig from the custom stereotactic holder and transfer to a warming isolette.</li>
	<li>Give 2 mg/kg Atipamezole and 24 mL of lactated Ringer&#39;s solution (subcutaneously away from the incision). Give lactated Ringer&#39;s solution due to the diuretic effects of xylazine. Administer 0.2 mg/kg meloxicam subcutaneously for post-operative analgesic coverage.</li>
	<li>Obtain vital signs every 15 minutes until the guinea pig fully emerges from anesthesia.</li>
	<li>Give an additional 12 mL fluid bolus of lactated Ringer&#39;s solution about 2 hours from end of surgery during the recovery period.</li>
	<li>Once the guinea pig is alert, ambulating, voiding, and having bowel movements, return the guinea pig to the animal facility. Approximately 2 to 4 hours are needed for the guinea pig to emerge completely from anesthesia.</li>
	<li>Monitor guinea pigs twice daily for the first three post-operative days.&#160;If signs of discomfort are observed, administer 0.2 mg/kg meloxicam subcutaneously every 24 hours as needed.&#160;Alternatively, buprenorphine (0.05 mg/kg) may be administered subcutaneously if symptoms are not sufficiently managed by meloxicam.&#160;</li>
	<li>Give a 12 mL fluid bolus of lactated Ringer&#39;s solution subcutaneously twice a day for up to&#160;three days until the guinea pig reaches the pre-operative weight. If the guinea pig reaches its pre-operative weight prior to the third post-operative day, then stop fluid boluses. If the guinea pig continues to lose weight after the first three days, use a supplement nutrition shake typically advertised for human consumption mixed with crushed guinea pig food pellets.</li>
	<li>Monitor guinea pigs weekly until their end point.</li>
</ol>

<ol>
	<li>Kimura, R. S., Schuknecht, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membranous+Hydrops+in+the+Inner+Ear+of+the+Guinea+Pig+after+Obliteration+of+the+Endolymphatic+Sac.">Membranous Hydrops in the Inner Ear of the Guinea Pig after Obliteration of the Endolymphatic Sac.</a> Pract oto-rhino-laryng. 27, 343-354 (1965).</li><li>Salt, A. N., DeMott, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+of+endolymph+volume+increase+in+experimental+hydrops+measured+in+vivo+with+an+ionic+volume+marker.">Time course of endolymph volume increase in experimental hydrops measured in vivo with an ionic volume marker.</a> Hearing Research. 74 (1-2), 165-172 (1994).</li><li>Walsted, A., Garbarsch, C., Michaels, L. <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+craniotomy+and+cerebrospinal+fluid+loss+on+the+inner+ear.+An+experimental+study.">Effect of craniotomy and cerebrospinal fluid loss on the inner ear. An experimental study.</a> Acta Oto-Laryngologica. 114 (6), 626-631 (1994).</li><li>Andrews, J. C., Bohmer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+surgical+approach+to+the+endolymphatic+sac+and+the+cochlear+aqueduct+in+the+guinea+pig.">The surgical approach to the endolymphatic sac and the cochlear aqueduct in the guinea pig.</a> American Journal of Otolaryngology. 10 (1), 61-66 (1989).</li><li>Lee, J. R., Wright, C. G., Meyerhoff, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+occipital+approach+to+the+endolymphatic+sac+and+cochlear+aqueduct+of+the+guinea+pig.">Modified occipital approach to the endolymphatic sac and cochlear aqueduct of the guinea pig.</a> American Journal of Otolaryngology. 14 (2), 165-169 (1993).</li><li>Megerian, C. A., 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=Surgical+induction+of+endolymphatic+hydrops+by+obliteration+of+the+endolymphatic+duct.">Surgical induction of endolymphatic hydrops by obliteration of the endolymphatic duct.</a> Journal of Visualized Experiments. (35), (2010).</li><li>Lichtenhan, J. T., Cooper, N. P., Guinan, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+auditory+threshold+estimation+technique+for+low+frequencies:+proof+of+concept.">A new auditory threshold estimation technique for low frequencies: proof of concept.</a> Ear and Hearing. 34 (1), 42-51 (2013).</li><li>Lichtenhan, J. T., Hartsock, J., Dornhoffer, J. R., Donovan, K. M., Salt, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+delivery+into+the+cochlear+apex:+Improved+control+to+sequentially+affect+finely+spaced+regions+along+the+entire+length+of+the+cochlear+spiral.">Drug delivery into the cochlear apex: Improved control to sequentially affect finely spaced regions along the entire length of the cochlear spiral.</a> Journal of Neuroscience Methods. 273, 201-209 (2016).</li><li>Lichtenhan, J. T., Hartsock, J. J., Gill, R. M., Guinan, J. J., Salt, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+auditory+nerve+overlapped+waveform+(ANOW)+originates+in+the+cochlear+apex.">The auditory nerve overlapped waveform (ANOW) originates in the cochlear apex.</a> Journal of the Association for Research in Otolaryngology. 15 (3), 395-411 (2014).</li><li>Lichtenhan, J. T., Hirose, K., Buchman, C. A., Duncan, R. K., Salt, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+administration+of+2-Hydroxypropyl-Beta-Cyclodextrin+into+guinea+pig+cochleae:+Effects+on+physiological+and+histological+measurements.">Direct administration of 2-Hydroxypropyl-Beta-Cyclodextrin into guinea pig cochleae: Effects on physiological and histological measurements.</a> PloS One. 12 (4), e0175236 (2017).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9 (7), 671-675 (2012).</li></ol>

The authors have nothing to disclose.

The presented extradural method had a success rate of 86% in achieving histologically confirmed endolymphatic hydrops and low-frequency hearing loss. The method reliably achieved histological evidence of endolymphatic hydrops by post-operative day 30, consistent with prior studies that used an intradural approach2. The significance of the method with respect to existing methods is that a CSF leak is not required, thus removing a potential confounding variable that has been suggested to result in a...

Endolymphatic hydrops is an enlargement of scala media. The presence of endolymphatic hydrops can be measured using the cross-sectional area of scala media. It is thought that clinical endolymphatic hydrops can be associated with low-frequency sensorineural hearing loss, such as that seen in Meniere&#39;s disease.&#160;But the origin(s) of the hearing loss remain unclear. To adequately study the origins of low-frequency hearing loss associated with endolymphatic hydrops, a reliable model is needed.
In 1965, Kimura and Schuknecht described how to induce endolymphatic hydrops in the guinea pig using an intradural approach1. Their technique involved using a posterior cranial fossa approach to access the operculum and subarcuate fossa. The steps involved incising dura, retracting the cerebellum with a Ringer&#39;s solution soaked cotton pad, and drilling across the endolymphatic duct and the intermediate portion of the endolymphatic sac. Bone wax was then placed into the operculum to separate the endolymphatic duct from the distal endolymphatic sac. The craniotomy defect was closed by placing absorbable gelatin powder (e.g., Gelfoam) and reapproximating the overlying muscles. Histologic evidence of endolymphatic hydrops was consistently found at post-operative days 1, 3, 7, 14, 21, and 30, demonstrating that the intradural approach was a reliable method&#160;to induce histologically-confirmed endolymphatic hydrops. Using the same intradural approach as Kimura and Schuknecht, but with different time points, Salt and DeMott confirmed that scala media in the second turn of the cochlea was significantly enlarged at day 4 and beyond2. While the actual morbidity of inducing a cerebrospinal fluid (CSF) leak using Kimura and Schuknecht&#39;s intradural approach was not reported in the original study, the presence of a CSF leak could increase the risk of meningitis. It has been suggested that loss of CSF could lead to an outflow of perilymph, resulting in a simultaneous temporary expansion of the endolymphatic volume in the guinea pig3. An extradural approach to inducing endolymphatic&#160;hydrops would be a safer option.
In 1989, Andrews and Bohmer described two extradural surgical approaches to reach the endolymphatic sac and duct, via either a middle cranial fossa approach or posterior cranial fossa approach, to obliterate the endolymphatic sac4. They described removing the operculum with a diamond drill, and then either drilling off the intermediate portion of the endolymphatic sac or using a fine pick to disrupt the endolymphatic sac and duct. In 1993, Lee, Wright, and Meyerhoff described a similar approach, which included drilling through the endolymphatic sac and duct, but differed in that they also simultaneously obstructed the cochlear aqueduct5. They demonstrated the presence of endolymphatic hydrops, as assessed via histology, at four weeks after obliterating the endolymphatic sac and obstructing the cochlear aqueduct. Megerian et al. was the first to publish a video article demonstrating an extradural obliteration of the endolymphatic sac and duct that involved drilling directly on the medial portion of the operculum to enter into the endolymphatic sac and duct6. They demonstrated histologic evidence of endolymphatic hydrops in a guinea pig sacrificed at 28 weeks after surgery, as well as hearing loss in the 16 kHz region6. Whether it was possible to induce histologically confirmed endolymphatic hydrops and low-frequency hearing loss at an early time point using extradural approaches was unknown.
The overall goal of this report is to demonstrate an extradural approach to induce experimental endolymphatic hydrops at 30 days post-operatively by obliterating the endolymphatic sac and injuring the endolymphatic duct with a fine pick. The rationale behind the use of this technique is the advantage of avoiding the need to drill on the petrous temporal bone, thereby removing the risk of accidentally injuring the dura and causing a CSF leak, mitigating the possibility of injuring the posterior semicircular canal, and reducing the risk of injury to the sigmoid sinus.

<table><tbody><tr><td>12 mL syringe</td><td>Henke-Sass Wolf</td><td>5100-X00V0</td><td></td></tr><tr><td>1 mL and 3 mL syringe</td><td>BD Precision(Ordered from Fischer Sci)</td><td>14-826-87 15859152</td><td></td></tr><tr><td>27.5 butterfly gauge needle</td><td>Terumo Surflo Winged Infusion Set, Terumo Corporation, Japan) (Ordered from McKesson)</td><td>448407</td><td></td></tr><tr><td>4-0 suture</td><td>McKesson</td><td>1034507</td><td></td></tr><tr><td>4 x 4 gauze sponges</td><td>Dukal (Ordered form McKesson)</td><td>374454</td><td></td></tr><tr><td>60 mL syringe</td><td>Fisher Sci</td><td>22-031-375</td><td></td></tr><tr><td>Anspach otologic drill</td><td>Anspach</td><td>SC2100</td><td></td></tr><tr><td>atipamezole</td><td>Zoetis</td><td>107204-6</td><td></td></tr><tr><td>autoclave</td><td>Fisher sci</td><td>&lt;a href=&quot;https://www.fishersci.com/shop/products/fisher-scientific-isotemp-general-purpose-heating-drying-ovens/151030508&quot;&gt;15-103-0508&lt;/a&gt;</td><td></td></tr><tr><td>autoclave bags</td><td>McKesson</td><td>524881</td><td></td></tr><tr><td>bayonet separator</td><td>Olympus</td><td>AL 130564</td><td></td></tr><tr><td>bupivicaine</td><td>auro Medics Pharma</td><td>555150-169-10</td><td></td></tr><tr><td>clear sterile drape</td><td>3M</td><td>1020</td><td></td></tr><tr><td>cotton balls</td><td>Fisherbrand (ordered from Fisher Sci)</td><td>22-456-885</td><td></td></tr><tr><td>cotton swabs</td><td>McKesson</td><td>508716</td><td></td></tr><tr><td>diamond burrs #3, #2, #1, and #0.5 mm</td><td>Anspach</td><td>QD8-3SD; QD8-2SD; QD8-1SD; QD8-05SD</td><td></td></tr><tr><td>diaper pad</td><td>McKesson</td><td>945330</td><td></td></tr><tr><td>disposable 15 blade</td><td>Swann-Morton</td><td>0305</td><td></td></tr><tr><td>enrofloxacin</td><td>Hospira</td><td>0409-4888-01</td><td></td></tr><tr><td>epinephrine</td><td>McKesson</td><td>&lt;a href=&quot;https://www.findacode.com/code.php?set=NDC&amp;amp;c=63739-0456&quot;&gt;63739-0456&lt;/a&gt;</td><td></td></tr><tr><td>eye ointment</td><td>Dechra Vet Products</td><td>17033-211-38</td><td></td></tr><tr><td>Freer elevator</td><td>Grace Medical</td><td>215100FX</td><td></td></tr><tr><td>gelfoam</td><td>Pfizer (Ordered from McKesson)</td><td>82830</td><td></td></tr><tr><td>hair trimmers</td><td>Oster Power Pro Cordless (ordered from Amazon)</td><td>078400-020-000</td><td></td></tr><tr><td>iodine scrub</td><td>Purdue Pharma (ordered from mcKesson)</td><td>521243</td><td></td></tr><tr><td>iris scissors</td><td>Olympus</td><td>CL-542114</td><td></td></tr><tr><td>ketamine</td><td>Henry Schein Animal Health</td><td>55853</td><td></td></tr><tr><td>lactated ringers</td><td>B. Braun Medical (ordered from McKesson)</td><td>186662</td><td></td></tr><tr><td>lancet knife by Rosen</td><td>Grace Medical</td><td>151100FX</td><td>referred to as curette in the text</td></tr><tr><td>lubricant</td><td>Milex (ordered from Cooper Surgical)</td><td>MX5030</td><td></td></tr><tr><td>masking tape</td><td>3M (ordered from fisher sci</td><td>19047259</td><td></td></tr><tr><td>metal rectangle basin</td><td>Amazon</td><td>B07NQDBC6T</td><td></td></tr><tr><td>needle holder</td><td>Olympus</td><td>CR 213015-ENT</td><td></td></tr><tr><td>needles: 27 gage, 18 gauge</td><td>BD Precision(Ordered from Fischer Sci)</td><td>&lt;a href=&quot;https://www.fishersci.com/shop/products/bd-general-use-precisionglide-hypodermic-needles-20/1482648&quot;&gt;14-826-48 14-826-5D&lt;/a&gt;</td><td></td></tr><tr><td>neonatal warming isollete</td><td>Air Borne Life Support Systems</td><td>731-1800</td><td></td></tr><tr><td>operating microscope</td><td>Carl Zeiss</td><td>OPMI pico</td><td></td></tr><tr><td>oxygen tank</td><td>AirGas</td><td>OX USP200</td><td></td></tr><tr><td>pulse ox</td><td>CapnoTrue (Ordered from Medacx)</td><td>M-3090112001</td><td></td></tr><tr><td>rectal probe with heating blanket</td><td>Harvard Apparatus</td><td>probe: PY2 50-7217 Heating Blanket: PY2 50-7214</td><td></td></tr><tr><td>red body holder</td><td>Lichtenhan Lab</td><td>N/A</td><td>In-house product</td></tr><tr><td>right angle</td><td>Olympus</td><td>BV-230337</td><td></td></tr><tr><td>rosen needle</td><td>Olympus</td><td>AM-130566</td><td>customized, it is the instrument I use to tear the sac</td></tr><tr><td>rubber tubing for O&lt;sub&gt;2&lt;/sub&gt; administration</td><td>Fisher Sci</td><td>14-171-104</td><td></td></tr><tr><td>saran wrap</td><td>Fisher Sci</td><td>NC9617977</td><td></td></tr><tr><td>stereotactic head holder</td><td>WUSTL Instrument Machine Shop</td><td>N/A</td><td>In-house product</td></tr><tr><td>sterile drapes</td><td>Cardinal Health</td><td>7553</td><td></td></tr><tr><td>suction tube by Baron</td><td>Grace Medical</td><td>034903FX 034905FX</td><td>#3 and #5 Suction</td></tr><tr><td>tissue forceps adson brown</td><td>Grace Medical</td><td>325112FX</td><td></td></tr><tr><td>Weitlander retractor</td><td>Olympus Grace Medical</td><td>BL200011 100313FX</td><td></td></tr><tr><td>xylazine</td><td>Akorn</td><td>59399-110-20</td><td></td></tr></tbody></table>

a revised surgical approach to induce endolymphatic hydrops in the guinea pig

Endolymphatic hydrops is an enlargement of scala media that is most often associated with Meniere&#39;s disease, though the pathophysiologic mechanism(s) remain unclear. In order to adequately study the attributes of endolymphatic hydrops, such as the origins of low-frequency hearing loss, a reliable model is needed. The guinea pig is a&#160;good model because it hears in the low-frequency regions that are putatively affected by endolymphatic hydrops. Previous research has demonstrated that endolymphatic hydrops can be induced surgically via intradural or extradural approaches that involve drilling on the endolymphatic duct and sac. However, whether it was possible to create an endolymphatic hydrops model using an extradural approach that avoided dangerous drilling on the endolymphatic duct and sac was unknown. The objective of this study was to demonstrate a revised extradural approach to induce experimental endolymphatic hydrops at 30 days post-operatively by obliterating the endolymphatic sac and injuring the endolymphatic duct with a fine pick. The sample size consisted of seven guinea pigs. Functional measurements of hearing were made and temporal bones were subsequently harvested for histologic analysis. The approach had a success rate of 86% in achieving endolymphatic hydrops. The risk of cerebrospinal fluid leak was minimal. No perioperative deaths or injuries to the posterior semicircular canal occurred in the sample. The presented method demonstrates a safe and reliable way to induce endolymphatic hydrops at a relatively quick time point of 30 days. The clinical implications are that the presented method provides a reliable model to further explore the origins of low-frequency hearing loss that can be associated endolymphatic hydrops.

The presented method used an extradural approach to obliterate the endolymphatic sac and injure the endolymphatic duct with a fine pick in seven guinea pigs consisting of two males and five females. The average duration of surgery was 2 hours from incision to closure. The total drill time ranged from 5-10 minutes. Up to&#160;4 hours was needed for the guinea pig to fully emerge from anesthesia. There were no intra-operative or post-operative deaths in the sample. There were no injuries to...

Watch this Scientific Journal Video about A Revised Surgical Approach to Induce Endolymphatic Hydrops in the Guinea Pig at JoVE.com

A Revised Surgical Approach to Induce Endolymphatic Hydrops in the Guinea Pig

This article demonstrates an extradural approach to obliterate the guinea pig endolymphatic sac and injure the endolymphatic duct with a fine pick in order to induce experimental endolymphatic hydrops.

Endolymphatic hydrops is an enlargement of scala media that is most often associated with Meniere&#39;s disease, though the pathophysiologic mechanism(s) ...

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6.1K Views.  Washington University School of Medicine in St. Louis. This article demonstrates an extradural approach to obliterate the guinea pig endolymphatic sac and injure the endolymphatic duct with a fine pick in order to induce experimental endolymphatic hydrops.

Video: A Revised Surgical Approach to Induce Endolymphatic Hydrops in the Guinea Pig

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

Surgical Induction of Endolymphatic Hydrops by Obliteration of the Endolymphatic Duct

Surgical induction of endolymphatic hydrops (ELH) in the guinea pig by obliteration and obstruction of the endolymphatic duct is a well-accepted animal model of the condition and an important correlate for human Meniere's disease. In 1965, Robert Kimura and Harold Schuknecht first described an intradural approach for obstruction of the endolymphatic duct (Kimura 1965). Although effective, this technique, which requires penetration of the brain's protective covering, incurred an undesirable level of morbidity and mortality in the animal subjects. Consequently, Andrews and Bohmer developed an extradural approach, which predictably produces fewer of the complications associated with central nervous system (CNS) penetration.(Andrews and Bohmer 1989) 
The extradural approach described here first requires a midline incision in the region of the occiput to expose the underlying muscular layer. We operate only on the right side. After appropriate retraction of the overlying tissue, a horizontal incision is made into the musculature of the right occiput to expose the right temporo-occipital suture line. The bone immediately inferio-lateral the suture line (Fig 1) is then drilled with an otologic drill until the sigmoid sinus becomes visible. Medial retraction of the sigmoid sinus reveals the operculum of the endolymphatic duct, which houses the endolymphatic sac. Drilling medial to the operculum into the area of the endolymphatic sac reveals the endolymphatic duct, which is then packed with bone wax to produce obstruction and ultimately ELH. 
In the following weeks, the animal will demonstrate the progressive, fluctuating hearing loss and histologic evidence of ELH.

This video shows how to surgically obstruct the guinea pig's endolymphatic duct to produce endolymphatic hydrops.

Surgical induction of endolymphatic hydrops (ELH) in the guinea pig by obliteration and obstruction of the endolymphatic duct is a well-accepted animal ...

Biology

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 ...

Medicine

Cochlear Implantation in the Guinea Pig

Cochlear implants are highly efficient devices that can restore hearing in subjects with profound hearing loss. Due to improved speech perception outcomes, candidacy criteria have been expanded over the last few decades. This includes patients with substantial residual hearing that benefit from electrical and acoustical stimulation of the same ear, which makes hearing preservation during cochlear implantation an important issue. Electrode impedances and the related issue of energy consumption is another major research field, as progress in this area could pave the way for fully implantable auditory prostheses. To address these issues in a systematic way, adequate animal models are essential. Therefore, the goal of this protocol is to provide an animal model of cochlear implantation, which can be used to address various research questions. Due to its large tympanic bulla, which allows easy surgical access to the inner ear, as well as its hearing range which is relatively similar to the hearing range of humans, the guinea pig is a commonly used species in auditory research. Cochlear implantation in the guinea pig is performed via a retroauricular approach. Through the bullostomy a cochleostomy is drilled and the cochlear implant electrode is inserted into the scala tympani. This electrode can then be used for electrical stimulation, determination of electrode impedances and the measurement of compound action potentials of the auditory nerve. In addition to these applications, cochlear implant electrodes can also be used as drug delivery devices, if a topical delivery of pharmaceutical agents to the cells or fluids of the inner ear is intended.

The goal of this protocol is to provide an animal model of cochlear implantation, which can be used to address a multitude of research questions. Potential applications include the evaluation of pharmaceutical interventions or electrical stimulation for beneficial effects on hearing thresholds or electrode impedances.

Cochlear implants are highly efficient devices that can restore hearing in subjects with profound hearing loss. Due to improved speech perception ...

Subretinal Implantation of RPE on a Carrier in Minipigs: Guidelines for Preoperative Preparations, Surgical Techniques, and Postoperative Care

Degenerative disorders of the retina (including age-related macular degeneration), which originate primarily at or within the retinal pigmented epithelial (RPE) layer, lead to a progressive disorganization of the retinal anatomy and the deterioration of visual function. The substitution of damaged RPE cells (RPEs) with in vitro cultured RPE cells using a subretinal cell carrier has shown potential for re-establishing the anatomical structure of the outer retinal layers and is, therefore, being further studied. Here, we present the principles of a surgical technique that allows for the effective subretinal transplantation of a cell carrier with cultivated RPEs into minipigs. The surgeries were performed under general anesthesia and included a standard lens-sparing three-port pars plana vitrectomy (PPV), subretinal application of a balanced salt solution (BSS), a 2.7 mm retinotomy, implantation of a nanofibrous cell carrier into the subretinal space through an additional 3.0 mm sclerotomy, fluid-air exchange (FAX), silicone oil tamponade, and closure of all the sclerotomies. This surgical approach was used in 29 surgeries (18 animals) over the past 8 years with a success rate of 93.1%. Anatomic verification of the surgical placement was carried out using in vivo fundus imaging (fundus photography and optical coherence tomography). The recommended surgical steps for the subretinal implantation of RPEs on a carrier in minipig eyes can be used in future preclinical studies using large-eye animal models.

The subretinal implantation of retinal pigmented epithelium (RPE) is one of the most promising approaches for the treatment of degenerative retinal diseases. However, the performance of preclinical studies on large-eye animal models remains challenging. This report presents guidelines for the subretinal transplantation of RPE on a cell carrier into minipigs.

Degenerative disorders of the retina (including age-related macular degeneration), which originate primarily at or within the retinal pigmented epithelial ...

Bioengineering

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. ...

Endolymphatic Duct Blockage as a Surgical Treatment Option for M&#233;ni&#232;re's Disease

Endolymphatic duct blockage is a relatively new treatment option for M&#233;ni&#232;re's disease, aiming to reduce vertigo attacks while sparing hearing and equilibrium. After a regular mastoidectomy, the posterior semicircular canal is identified, and Donaldson's line is determined. This is a line through the horizontal semicircular canal, crossing the posterior semicircular canal. The endolymphatic sac is usually found at this site under the posterior semicircular canal. The bone of the endolymphatic sac and the dura are thinned until the sac is skeletonized, after which the endolymphatic duct is identified. The duct is then blocked with a titanium clip. Using a computerized tomography (CT) scan, the position is confirmed. Follow-up visits take place 1 week, 6 weeks and 1 year after surgery. To this day, only one prospective trial assessing this method has been conducted, comparing this new method to endolymphatic sac decompression. Results of the duct blockage are promising, with 96.5% of the patients free of vertigo after 2 years. However, further research is required.

Endolymphatic duct blockage is a relatively new surgical method for patients suffering from M&#233;ni&#232;re's disease. Following a regular mastoidectomy, the endolymphatic duct is identified and ligated using a regular titanium hemoclip. The effectiveness of this procedure is currently being assessed in a randomized trial.

Endolymphatic duct blockage is a relatively new treatment option for M&#233;ni&#232;re's disease, aiming to reduce vertigo attacks while sparing hearing ...

Surgical Treatment of an Endolymphatic Sac Tumor

Endolymphatic sac tumors (ELST) are low-grade papillary adenocarcinoma originating from the endolymphatic sac. Usually slow-growing, with local aggressiveness and a low risk of distant metastases, ELST can be sporadic but also frequently associated with von Hippel Lindau disease. The current treatment of ELST is primarily surgical resection. A 55-year-old woman accessed our otologic tertiary level referral center for a sudden worsening of hearing loss in her left ear and vertigo. A magnetic resonance (MRI) and computer tomography scan study subsequently showed a mass in the petrous bone; hence, the presence of an ELST was hypothesized. After embolization of the mass, the patient underwent surgical removal of the lesion. The resection of the mass was done through a translabirinthine approach, with an uneventful procedure. No residual disease remained after surgery. After 24 months of radiologic follow up with MRI, there are no signs of recurrence disease. This paper reports the management of this sporadic ELST, as well as the follow up results, providing clinicians this protocol for the handling of such a challenging otologic skull base surgery and rare disease.

Here, we discuss clinical and radiological features of endolymphatic sac tumors and report the methodology and results of a surgical removal in a case in care at our otologic referral center.

Endolymphatic sac tumors (ELST) are low-grade papillary adenocarcinoma originating from the endolymphatic sac. Usually slow-growing, with local ...

Endoscopic Balloon Dilatation of the Eustachian Tube via the Soft Palate Approach in Miniature Pigs

The eustachian tube (ET) is one of the most complex organs in the human body, and its dysfunction may lead to a variety of diseases. In recent years, an increasing number of scholars have opted to conduct ET-related studies using large experimental animals such as miniature pigs or sheep, yielding promising results. Typically, conventional endoscopic procedures are performed through the nasal approach for large experimental animals. However, due to the elongated and narrow nasal cavity in these animals, transnasal surgeries are challenging. 
To address this issue, we explored an ET surgery approach via the soft palate. The animal was placed in a supine position. After endotracheal intubation under general anesthesia, a mouth opener was used to fully expose the upper palate. Local infiltration with diluted adrenal fluid was performed for anesthesia of the area. A sickle knife was then used to make a longitudinal soft palate incision at the junction of the soft and hard palates. After hemostasis, an endoscope was inserted into the nasopharynx cavity, allowing the visualization of the pharyngeal opening of the ET on the posterior lateral wall of the nasal cavity. 
Subsequently, a specialized pusher was used to insert a balloon into ET. The balloon was inflated, maintained at 10 bar for 2 min, and then removed. The incision in the soft palate was then sutured to ensure proper alignment. The soft palate healed well after the operation. This surgical approach is suitable for ET-related procedures in large experimental animals (e.g., miniature pigs, sheep, and dogs). The surgical procedure is simple, with a short surgical time, and wound healing is rapid. Under endoscopy, the pharyngeal opening of the ET is visible, and it is thus a good choice for procedures such as balloon dilation of the ET.

In this study, we present a protocol to explore an eustachian tube surgery approach via the soft palate in miniature pigs. The surgical procedure is simple, with short surgical time, and wound healing is rapid; it is thus a good choice for procedures such as balloon dilation of the eustachian tube.

The eustachian tube (ET) is one of the most complex organs in the human body, and its dysfunction may lead to a variety of diseases. In recent years, an ...

Dissection of the Endolymphatic Sac from Mice

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the human phenotypes. Although important progress has been done in the understanding of the development and function of the neurosensory epithelia of the cochlea and vestibula, limited knowledge is available regarding the development, cellular composition, molecular pathways and functional characteristics of the endolymphatic sac. This is, in large part, due to the difficulty of visualizing and microdissecting this tissue, which is an epithelium comprised of only one cell layer. The study presented here describes an approach to access and microdissect the endolymphatic sac from the wild-type mouse inner ear at different ages. The result of a similar dissection is shown in a pendrin-deficient mouse model of enlargement of the vestibular aqueduct. A transgenic mouse with a fluorescent endolymphatic sac is presented. This reporter mouse can be used to readily visualize the endolymphatic sac with limited dissection and determine its size. It can also be used as an educational tool to teach how to dissect the endolymphatic sac. These dissection procedures should facilitate further characterization of this understudied part of the inner ear.

This study describes how to dissect the endolymphatic sac from the inner ear of the mouse at different ages. The result of a similar dissection is shown in an Slc26a4-null mouse model of enlargement of the endolymphatic sac. A transgenic mouse with a fluorescent reporter expressed in the endolymphatic sac is presented as a model to readily visualize the endolymphatic sac, and educational tool.

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the ...

A Revised Surgical Approach to Induce Endolymphatic Hydrops in the Guinea Pig

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Chapters in this video

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

Surgical Induction of Endolymphatic Hydrops by Obliteration of the Endolymphatic Duct

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

Cochlear Implantation in the Guinea Pig

Subretinal Implantation of RPE on a Carrier in Minipigs: Guidelines for Preoperative Preparations, Surgical Techniques, and Postoperative Care

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

Endolymphatic Duct Blockage as a Surgical Treatment Option for Ménière's Disease

Surgical Treatment of an Endolymphatic Sac Tumor

Endoscopic Balloon Dilatation of the Eustachian Tube via the Soft Palate Approach in Miniature Pigs

Dissection of the Endolymphatic Sac from Mice