Name: a revised surgical approach to induce endolymphatic hydrops in the guinea pig
Uploaded: 2020-06-04T13:30:00
Description: Watch this Scientific Journal Video about A Revised Surgical Approach to Induce Endolymphatic Hydrops in the Guinea Pig at JoVE.com

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 i...

<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 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>
			<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) ...

Research

JoVE Journal

Neuroscience

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

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 ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster

A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster

Aggressive behavior in Drosophila melanogaster is composed of the sequential expression of stereotypical behavioral patterns (for analysis see 1). This ...

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 ...

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

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, ...

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 ...

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 ...

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 ...

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 ...