Name: simple surgical induction of conductive hearing loss with verification using otoscope visualization and behavioral clap startle response in rat
Uploaded: 2019-10-26T12:30:00
Description: Watch this Scientific Journal Video about Simple Surgical Induction of Conductive Hearing Loss with Verification Using Otoscope Visualization and Behavioral Clap Startle Response in Rat at JoVE.com

This work was supported in part by the Hong Kong Research Grants Council, Early Career Scheme, Project #21201217 to C. L., for the project Brain mapping guided electrophysiology with applications in hearing and noise pollution research. We thank the Posgrado en Ciencias Biom&#233;dicas, the Instituto de Neurobiolog&#237;a of the Universidad Nacional Aut&#243;noma de M&#233;xico (UNAM), the Consejo Nacional de Ciencia y Tecnolog&#237;a (CONACyT) M&#233;xico for the Graduate Fellowship 578458 to FAM Manno.

The present study and procedures were approved by the animal research ethics committees of the City University of Hong Kong, the University of Hong Kong, and the Department of Health of the Hong Kong Special Administrative Region.
1. Animals
<ol>
	<li>Use Sprague-Dawley (SD) rats of two months (N = 90, 200-250 g). 
	NOTE: Rodents were provided by the accredited Laboratory Animal Unit of the University of Hong Kong.</li>
	<li>Maintain the rats under a constant 25 &#176;C temperature and ...

<ol>
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N., 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=A+sensitive+period+for+the+impact+of+hearing+loss+on+auditory+perception.">A sensitive period for the impact of hearing loss on auditory perception.</a> The Journal of Neuroscience. 34 (6), 2276-2284 (2014).</li><li>Finck, A., Sofouglu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+sensitivity+of+the+Mongolian+gerbil.">Auditory sensitivity of the Mongolian gerbil.</a> The Journal of Auditory Research. 6, 313-319 (1966).</li><li>Finck, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+sensitivity+of+the+Mongolian+Gerbil+(Merionesunguiculatus).">Auditory sensitivity of the Mongolian Gerbil (Merionesunguiculatus).</a> The Journal of the Acoustical Society of America. 41 (60), 1579 (1967).</li><li>Finck, A., Schneck, C. D., Hartman, A. 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We describe a simple surgical induction of CHL with verification using otoscope visualization and behavioral clap startle response in rat. Here we demonstrate the method on rat and previously this method has been applied to gerbils and mice. The method can easily be adopted to other rodents. Induction of CHL allows the study of a subtle form of hearing loss which manifests in auditory cortical alterations and psychophysical behavioral findings4,5,<sup ...

The prevalence of the hearing loss in children and adults is approximately 19.5%1 and 15.2%2 respectively. However, approximately 39.3% of all newborns with an abnormal hearing screening do not receive remedial treatment as reported by the Centers for Disease Control3. Hearing loss is a widely studied condition, and the rodent is a robust model to study normal hearing and hearing related disorders4,5,6,7,8,9,10,11,12,13,14,15. Hearing disorders such as conductive hearing loss (CHL) lead to an increase in the short term synaptic depression in the auditory cortex4, which results in shallower psychometric slopes associated with frequency modulation detection thresholds5. Conductive hearing loss models by surgical removal/displacement of the malleus, tympanic membrane (TM) puncture or earplug are easily employed and allow the rapid induction of the hearing loss model5,14,15,16,17,18. The goal of the present protocol and method is to demonstrate a simple and reproducible CHL model in rodents.
The present protocol is inexpensive (&#8776;USD$300 with all tools), and readily amendable to different research pursuits. The rat has had detailed assessments of middle ear anatomy19,20,21,22,23, surgical approaches24, models in otitis media25,26,27 and TM puncture regeneration16,17,18,28,29,30, making it an ideal model to study hearing loss. Here, a simple CHL induction procedure is described with verification by otoscope and behavioral assessment with clap startle response in rat, which then may be used to explore additional sequelae&#160;of hearing loss. The CHL procedure is induced by surgical puncture of the TM. Verification of the CHL procedure is performed by otoscope visualization to determine absence of the TM. Behavioral assessment is performed by a high decibel (dB) sound pressure level (SPL) hand-clap. This method has been applied previously in a variety of rodents.&#160;It is easy to replicate, produces robust psychometric differences and changes in neural physiological responses4,5,16,17,18.

<table border="0" cellpadding="0" cellspacing="0" style="width:612px;" width="610">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Latex, polyvinyl or nitrile gloves</td>
			<td style="width:95px;">AMMEX</td>
			<td style="width:123px;"></td>
			<td style="width:172px;">Use unpowdered gloves 8-mil</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Micro spring scissors (see Fig. 1b)</td>
			<td style="width:95px;">RWD Life Science</td>
			<td style="width:123px;">S11035-08</td>
			<td>8.0 cm total length, with 3.5mm cutting edge, or similar micro forceps. Standard tweezers with spring action will suffice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Otoscope mini 3000</td>
			<td style="width:95px;">HEINE&#160;</td>
			<td style="width:123px;">D-008.70.120M</td>
			<td>Standard LED otoscope will suffice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Rat or mouse</td>
			<td style="width:95px;">JAX labs</td>
			<td style="width:123px;"></td>
			<td>Any small rodent&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Small rodent cage</td>
			<td style="width:95px;">Tecniplast</td>
			<td style="width:123px;">1284L</td>
			<td>Need two cages to separate CHL rodent from hearing rodent. If rodent are in direct contact with one-another, they will startle each other. Cage dimensions 365 x 207 x 140 mm, floor area: 530 cm2/82.15 in2</td>
		</tr>
	</tbody>
</table>

simple surgical induction of conductive hearing loss with verification using otoscope visualization and behavioral clap startle response in rat

Conductive hearing loss (CHL) is a prevalent hearing impairment in humans. The goal of the protocol is to describe a simple surgical procedure for inducing CHL in rodents. The protocol demonstrates CHL by tympanic membrane puncture. Verification of CHL surgery was by otoscope examination and behavioral assessment by clap startle response, both replicable and reliable, and are simple methods to demonstrate hearing loss has occurred. The simple CHL procedure is advantageous due to its reproducibility and flexibility to different pursuits in hearing loss research. The limitations of inducing CHL by a surgical approach are associated with the learning curve to perform the surgical procedure and confidence in audiological examination. Inducing a hearing impairment by CHL allows one to readily study the neural manifestations and behavioral outcomes of hearing loss.

The simple CHL procedure was performed on 90 rats and out of this group 2 had significant bleeding and 2 did not have hearing loss the next day as assessed by behavioral clap startle. These four rats were discarded. Rats should be discarded as described by the reasons in the discussion due to complications. Inducing TM puncture and/or malleus displacement/removal (Figure 2B) elicits CHL, which results in behavioral manifestations (i.e. CHL - no response to lo...

Watch this Scientific Journal Video about Simple Surgical Induction of Conductive Hearing Loss with Verification Using Otoscope Visualization and Behavioral Clap Startle Response in Rat at JoVE.com

Simple Surgical Induction of Conductive Hearing Loss with Verification Using Otoscope Visualization and Behavioral Clap Startle Response in Rat

Here, we present a protocol to establish a replicable conductive hearing loss induction via surgical tympanic membrane puncture and verification by otoscope visualization and behavioral assessment by clap startle.

Conductive hearing loss (CHL) is a prevalent hearing impairment in humans. The goal of the protocol is to describe a simple surgical procedure for ...

The overall goal of this study is to establish a reliable hearing loss induction via surgical tympanic membrane puncture and verification by otoscope visualization and behavioral assessment by clap startle. The purpose of our practical is to develop a model for human hearing conductive disorders. We demonstrate in the practical, the conductive hearing loss surgical procedure, which is a simplification in the malleus removal, the method to verify conductive hearing loss by otoscope visualization, and a simple behavioral clap startle, which validate that conductive hearing loss has occurred.The main advantage of the procedure we described are non-invasive procedures, verification of conductive hearing loss by otoscope, and validation by clap startle. The present study and procedures were approved by the animal research ethics committee and the City University of Hong Kong, the University of Hong Kong, and the Department of Health of the Hong Kong Special Administrative Region. For the present protocol, we choose rats, but you may choose any rodent.Weigh animals to ensure proper anesthesia for IP injection. For anesthesia, it is important that animals have the proper dose. Pick up your rat by the scruff.Ensure your correct dose of ketamine and xylazine for IP injection, and inject. Make sure to perform toe pinch to verify no pain sensation. For surgical setup consists of your anesthetized rat, the microscissors, and your otoscope.Please thoroughly clean the surgical area with ethanol. Place the surgical drape on the cleaned bench. Subsequently introduce your scissors, otoscope, and rat.The size of your scissors will depend on the size of your rodent. Under otoscope, visualize the left and right ear of the rat to ensure a healthy tympanic membrane. Grab the helix and extend the external auditory canal to become obscured and blackened.Here you will introduce the microscissors. Introduce the microscissors to the center of the auditory canal, paying close attention not to nick the tissue. Listen for the pop sound, then twist to ensure displacement of the malleus.The confirmation of a successful conductive hearing loss surgery, should be made with an otoscope. Evaluate each rat ear before and after surgery with an otoscope with a small diameter speculum. When visualized with an otoscope, the differences between a normal tympanic membrane and a punctured tympanic membrane should be studied.Here, the normal tympanic membrane on the left has the head of the malleus in place and is drastically different than the punctured tympanic membrane on the right. The punctured tympanic membrane, after the conductive hearing loss surgery, has the malleus displaced and the pars tensa ruptured with the umbo of the malleus and head completely missing. Postoperative care for rodents.Place the rodent in the home cage under a warm lamp and inject with antibacterial agents and glucose. Corroborate connective hearing loss with behavioral clap startle. The CHL rat is placed next to a normal rat in two adjoining cages.These are placed in a silent room. Stand half a meter away and clap in equally spaced durations a specific number of times. Here, we choose five.Here, our representative behavioral results. Note the rat at the bottom does not jump to the loud clap sound. The rat at the top of the screen is startled by these loud clapping sounds.Please study the before and after the clap difference for these two rats. Failure to induce conductive hearing loss. A failure to induce conductive hearing loss could be caused by bleeding in one or both ears.And in this case, the rodent should be euthanized. This would indicate that the scissors did not make contact with the tympanic membrane. A no popping sound during the procedure.A no popping sound would indicate that the conductive hearing loss procedure was poor and not clean. A no popping sound would mean that your microscissors did not make direct contact with the tympanic membrane. Otoscope visualization.If, when visualizing by otoscope, the tympanic membrane appears normal, this would indicate that no CHL has occurred. A response to the loud clap sound test. This would indicate that no CHL has occurred and the rodent does not have conductive hearing loss.This would occur when a rat jumps or is startled by a loud clap sound. The main advantage of the procedure we described, are non-invasive procedures, verification of conductive hearing loss by otoscope, and validation by clap startle. After doing the practical, you can induce conductive hearing loss in a rodent model and explore the behavioral and psychophysical manifestations of this hearing disorder.

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