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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>4x4 gauze sponges</td>
			<td>Dukal (Ordered form McKesson)</td>
			<td>374454</td>
			<td></td>
		</tr>
		<tr>
			<td>60mL 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><a href="https://www.fishersci.com/shop/products/fisher-scientific-isotemp-general-purpose-heating-drying-ovens/151030508">15-103-0508</a></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><a href="https://www.findacode.com/code.php?set=NDC&amp;c=63739-0456">63739-0456</a></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><a href="https://www.fishersci.com/shop/products/bd-general-use-precisionglide-hypodermic-needles-20/1482648">14-826-48 14-826-5D</a></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 O2 administration</td>
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			<td>Cardinal Health</td>
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			<td>suction tube by Baron</td>
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			<td>#3 and #5 Suction</td>
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			<td>325112FX</td>
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			<td>Olympus Grace Medical</td>
			<td>BL200011 100313FX</td>
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			<td>xylazine</td>
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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) ...

a-revised-surgical-approach-to-induce-endolymphatic-hydrops-guinea

Research

JoVE Journal

Neuroscience

6.0K 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

Using a Comparative Species Approach to Investigate the Neurobiology of Paternal Responses

A goal of behavioral neuroscience is to identify underlying neurobiological factors that regulate specific behaviors. Using animal models to accomplish this goal, many methodological strategies require invasive techniques to manipulate the intensity of the behavior of interest (e.g., lesion methods, pharmacological manipulations, microdialysis techniques, genetically-engineered animal models). The utilization of a comparative species approach allows researchers to take advantage of naturally occurring differences in response strategies existing in closely related species. In our lab, we use two species of the Peromyscus genus that differ in paternal responses. The male California deer mouse (Peromyscus californicus) exhibits the same parental responses as the female whereas its cousin, the common deer mouse (Peromyscus maniculatus) exhibits virtually no nurturing/parental responses in the presence of pups. Of specific interest in this article is an exploration of the neurobiological factors associated with the affiliative social responses exhibited by the paternal California deer mouse. Because the behavioral neuroscience approach is multifaceted, the following key components of the study will be briefly addressed: the identification of appropriate species for this type of research; data collection for behavioral analysis; preparation and sectioning of the brains; basic steps involved in immunocytochemistry for the quantification of vasopressin-immunoreactivity; the use of neuroimaging software to quantify the brain tissue; the use of a microsequencing video analysis to score behavior and, finally, the appropriate statistical analyses to provide the most informed interpretations of the research findings.

The comparative species approach allows behavioral neuroscientists to explore various neurobiological factors associated with specific behaviors viewed as characteristic of a specific animal model. Taking advantage of naturally occurring differences in behavior between closely related species, this technique doesn&#x2019;t require invasive techniques to manipulate the expression of the behavior.

A goal of behavioral neuroscience is to identify underlying neurobiological factors that regulate specific behaviors.  Using animal models to accomplish ...

A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by immunizing mice with neurogenic peptides to direct an inflammatory attack toward components of the peripheral nervous system (PNS). Recent advances have enabled the induction of EAN in the relatively resistant C57BL/6 mouse line using myelin protein zero (P0)106-125 or P0180-199 peptides delivered in adjuvant combined with the injection of pertussis toxin. The ability to induce EAN in the C57BL/6 strain allows for the use of the numerous genetic tools that exist on this mouse background, and thus allows the sophisticated study of disease pathogenesis and interrogation of the mechanistic action of novel therapeutics in combination with transgenic approaches. In this study, we demonstrate a simple approach to successfully induce EAN using the P0180-199 peptide in C57BL/6 mice. We also outline a protocol for the assessment of functional deficits that occur in this model, accompanied by an array of neuropathological features. Thus, this model is a powerful experimental model to study the pathogenesis of human peripheral demyelinating neuropathies, and to determine the efficacy of potential therapies that aim to promote myelin repair and protect against nerve damage in autoimmune neuritis.

This report outlines a simple approach to successfully induce experimental autoimmune neuritis (EAN) using the myelin protein zero (P0)180-199 peptide in combination with Freund&#39;s complete adjuvant and pertussis toxin. We present a sophisticated paradigm capable of accurately assessing the extent of functional deficits and neuropathology that occur in this EAN.

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

A Benchtop Approach to the Location Specific Blood Brain Barrier Opening using Focused Ultrasound in a Rat Model

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic delivery including precise localization to a target brain region and reduction of off target side effects. However, stereotaxic surgery is highly invasive which limits its translational efficacy, requires long recovery times, and provides challenges when targeting multiple brain regions. Focused ultrasound (FUS) can be used in combination with circulating microbubbles to transiently open the blood brain barrier (BBB) in millimeter sized regions. This allows intracranial localization of systemically delivered agents that cannot normally cross the BBB. This technique provides a noninvasive alternative to stereotaxic surgery. However, to date this technique has yet to be widely adopted in neuroscience laboratories due to the limited access to equipment and standardized methods. The overall goal of this protocol is to provide a benchtop approach to FUS BBB opening (BBBO) that is affordable and reproducible and can therefore be easily adopted by any laboratory.

Focused ultrasound with microbubble agents can open the blood brain barrier focally and transiently. This technique has been used to deliver a wide range of agents across the blood brain barrier. This article provides a detailed protocol for the localized delivery to the rodent brain with or without MRI guidance.

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic ...

Combining Behavior and EEG to Study the Effects of Mindfulness Meditation on Episodic Memory

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has examined the behavioral and neural effects of mindfulness meditation on episodic memory. Here we present a protocol that combines mindfulness meditation training, an episodic memory task, and EEG to examine how mindfulness meditation changes behavioral performance and the neural correlates of episodic memory. Subjects in a mindfulness meditation experimental group were compared to a waitlist control group. Subjects in the mindfulness meditation experimental group spent four weeks training and practicing mindfulness meditation. Mindfulness was measured before and after training using the Five Facet Mindfulness Questionnaire (FFMQ). Episodic memory was measured before and after training using a source recognition task. During the retrieval phase of the source recognition task, EEG was recorded. The results showed that mindfulness, source recognition behavioral performance, and EEG theta power in right frontal and left parietal channels increased following mindfulness meditation training. In addition, increases in mindfulness correlated with increases in theta power in right frontal channels. Therefore, results obtained from combining mindfulness meditation training, an episodic memory task, and EEG reveal the behavioral and neural effects of mindfulness meditation on episodic memory.

Here we present a protocol for combining mindfulness meditation training, an episodic memory task, and EEG to understand the behavioral and neural effects of mindfulness meditation on episodic memory.

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has ...

Extraction and Dissection of the Domesticated Pig Brain

Use of the pig as a preclinical and translatable animal model has been well-documented and accepted by research fields investigating cardiovascular systems, gastrointestinal systems, and nutrition, and the pig is increasingly being used as a large animal model in neuroscience. Furthermore, the pig is an accepted model to study neurodevelopment as it displays brain growth and development patterns similar to what occurs in humans. As a less common animal model in neuroscience, surgical and dissection procedures on pigs may not be as familiar or well-practiced among researchers. Therefore, a standardized visual protocol detailing consistent extraction and dissection methods may prove valuable for researchers working with the pig. The following video showcases a technique to remove the pig brain while keeping the cortex and brainstem intact and reviews methods to dissect several commonly investigated brain regions including the brainstem, cerebellum, midbrain, hippocampus, striatum, thalamus, and medial prefrontal cortex. The purpose of this video is to provide researchers with the tools and knowledge necessary to consistently perform a brain extraction and dissection on the four-week-old pig.

This protocol details the technique for removal of the pig brain in its entirety and dissection of several brain regions commonly studied in neuroscience.

Use of the pig as a preclinical and translatable animal model has been well-documented and accepted by research fields investigating cardiovascular ...

Pre-Chiasmatic, Single Injection of Autologous Blood to Induce Experimental Subarachnoid Hemorrhage in a Rat Model

Despite advances in treatment over the last decades, subarachnoid hemorrhage (SAH) continues to carry a high burden of morbidity and mortality, largely afflicting a fairly young population. Several animal models of SAH have been developed to investigate the pathophysiological mechanisms behind SAH and to test pharmacological interventions. The pre-chiasmatic, single injection model in the rat presented in this article is an experimental model of SAH with a predetermined blood volume. Briefly, the animal is anesthetized, intubated, and kept under mechanical ventilation. Temperature is regulated with a heating pad. A catheter is placed in the tail artery, enabling continuous blood pressure measurement as well as blood sampling. The atlantooccipital membrane is incised and a catheter for pressure recording is placed in the cisterna magna to enable intracerebral pressure measurement. This catheter can also be used for intrathecal therapeutic interventions. The rat is placed in a stereotaxic frame, a burr hole is drilled anteriorly to the bregma, and a catheter is inserted through the burr hole and placed just anterior to the optic chiasm. Autologous blood (0.3 mL) is withdrawn from the tail catheter and manually injected. This results in a rise of intracerebral pressure and a decrease of cerebral blood flow. The animal is kept sedated for 30 min and given subcutaneous saline and analgesics. The animal is extubated and returned to its cage. The pre-chiasmatic model has a high reproducibility rate and limited variation between animals due to the pre-determined blood volume. It mimics SAH in humans making it a relevant model for SAH research.

Subarachnoid hemorrhage continues to carry a high burden of mortality and morbidity in man. To facilitate further research into the condition and its pathophysiology, a pre-chiasmatic, single injection model is presented.

Despite advances in treatment over the last decades, subarachnoid hemorrhage (SAH) continues to carry a high burden of morbidity and mortality, largely ...

Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice

Stereotaxic surgery to target brain sites in mice is commonly guided by skull landmarks. Access is then obtained via burr holes drilled through the skull. This standard approach can be challenging for targets in the caudal brainstem and upper cervical cord due to specific anatomical challenges as these sites are remote from skull landmarks, leading to imprecision. Here we outline an alternative stereotaxic approach via the cisterna magna that has been used to target discrete regions of interest in the caudal brainstem and upper cervical cord. The cisterna magna extends from the occipital bone to the atlas (i.e., the second vertebral bone), is filled with cerebrospinal fluid, and is covered by dura mater. This approach provides a reproducible route of access to select central nervous system (CNS) structures that are otherwise hard to reach due to anatomical barriers. Furthermore, it allows for direct visualization of brainstem landmarks in close proximity to the target sites, increasing accuracy when delivering small injection volumes to restricted regions of interest in the caudal brainstem and upper cervical cord. Finally, this approach provides an opportunity to avoid the cerebellum, which can be important for motor and sensorimotor studies.

Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice

Stereotaxic surgery to target brain sites in mice commonly involves access through the skull bones and is guided by skull landmarks. Here we outline an alternative stereotaxic approach to target the caudal brainstem and upper cervical spinal cord via the cisterna magna that relies on direct visualization of brainstem landmarks.

Stereotaxic surgery to target brain sites in mice is commonly guided by skull landmarks. Access is then obtained via burr holes drilled through the skull. ...

Pupillometry to Assess Auditory Sensation in Guinea Pigs

Noise exposure is a leading cause of sensorineural hearing loss. Animal models of noise-induced hearing loss have generated mechanistic insight into the underlying anatomical and physiological pathologies of hearing loss. However, relating behavioral deficits observed in humans with hearing loss to behavioral deficits in animal models remains challenging. Here, pupillometry is proposed as a method that will enable the direct comparison of animal and human behavioral data. The method is based on a modified oddball paradigm - habituating the subject to the repeated presentation of a stimulus and intermittently presenting a deviant stimulus that varies in some parametric fashion from the repeated stimulus. The fundamental premise is that if the change between the repeated and deviant stimulus is detected by the subject, it will trigger a pupil dilation response that is larger than that elicited by the repeated stimulus. This approach is demonstrated using a vocalization categorization task in guinea pigs, an animal model widely used in auditory research, including in hearing loss studies. By presenting vocalizations from one vocalization category as standard stimuli and a second category as oddball stimuli embedded in noise at various signal-to-noise ratios, it is demonstrated that the magnitude of pupil dilation in response to the oddball category varies monotonically with the signal-to-noise ratio. Growth curve analyses can then be used to characterize the time course and statistical significance of these pupil dilation responses. In this protocol, detailed procedures for acclimating guinea pigs to the setup, conducting pupillometry, and evaluating/analyzing data are described. Although this technique is demonstrated in normal-hearing guinea pigs in this protocol, the method may be used to assess the sensory effects of various forms of hearing loss within each subject. These effects may then be correlated with concurrent electrophysiological measures and post-hoc anatomical observations.

Pupillometry, a simple and non-invasive technique, is proposed as a method to determine hearing-in-noise thresholds in normal hearing animals and animal models of various auditory pathologies.

Noise exposure is a leading cause of sensorineural hearing loss. Animal models of noise-induced hearing loss have generated mechanistic insight into the ...

Magnetic Resonance-Guided Stereotaxy for Infusions to the Pig Brain

The overall goal of this procedure is to perform stereotaxy in the pig brain with real-time magnetic resonance (MR) visualization guidance to provide precise infusions. The subject was positioned prone in the MR bore for optimal access to the top of the skull with the torso raised, the neck flexed, and the head inclined downward. Two anchor pins anchored on the bilateral zygoma held the head steady using the head holder. A magnetic resonance imaging (MRI) flex-coil was placed rostrally across the head holder so that the skull was accessible for the intervention procedure. A planning grid placed on the scalp was used to determine the appropriate entry point of the cannula. The stereotactic frame was secured and aligned iteratively through software projection until the projected radial error was less than 0.5 mm. A hand drill was used to create a burr hole for insertion of the cannula. A gadolinium-enhanced co-infusion was used to visualize the infusion of a cell suspension. Repeated T1-weighted MRI scans were registered in real time during the agent delivery process to visualize the volume of gadolinium distribution. MRI-guided stereotaxy allows for precise and controlled infusion into the pig brain, with concurrent monitoring of cannula insertion accuracy and determination of the agent volume of distribution.

The protocol presented here demonstrates stereotaxy to the pig brain using convection-enhanced infusions, with real-time magnetic resonance imaging (MRI) visualization guidance and real-time infusion distribution visualization.

The overall goal of this procedure is to perform stereotaxy in the pig brain with real-time magnetic resonance (MR) visualization guidance to provide ...