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

Summary

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.

Abstract

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.

Introduction

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 disorders^{4,5,6,7,8,9,10,11,12,13,14,15}. 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 model^{5,14,15,16,17,18}. The goal of the present protocol and method is to demonstrate a simple and reproducible CHL model in rodents.

The present protocol is inexpensive (≈USD$300 with all tools), and readily amendable to different research pursuits. The rat has had detailed assessments of middle ear anatomy^{19,20,21,22,23}, surgical approaches²⁴, models in otitis media^25,26,27 and TM puncture regeneration^{16,17,18,28,29,30}, 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 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. It is easy to replicate, produces robust psychometric differences and changes in neural physiological responses^4,5,16,17,18.

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Protocol

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

1. 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.
2. Maintain the rats under a constant 25 °C temperature and 60-70% humidity at the Laboratory Animal Research Unit.
3. House rodents in 12/12-h light/dark cycles with access to food and drinking water, ad libitum.
4. Acclimate rodents to the housing environment for at least one day prior to CHL surgery.
5. Pick up the rodent by the scruff and anesthetize the rodent with a cocktail of ketamine and xylazine (80-100 mg/kg: 5-10 mg/kg, respectively) via intraperitoneal injection (combine 1.0 mL of ketamine with 0.5 mL of xylazine as a final concentration).
   NOTE: Use a 1 mL of syringe with a 23-25G needle. Inject approximately 0.2 mL per 100 g body weight of the rat for 30 min of anesthesia.
6. Perform the toe pinch to verify pain sensation and to corroborate proper anesthesia. A reaction to deep toe pinch (by withdrawal of hind limb) indicates insufficient anesthesia.

2. Surgical setup

1. Sterilize all equipment in an autoclave or hot glass bead sterilizer prior to beginning surgery. Clean the surgical area with 70% ethanol.
2. Use latex gloves and lab coat before starting the procedure.
3. Place a sterile surgical drape on the clean bench (Figure 1A).
4. Sterilize micro-scissors and an otoscope prior to the surgery to minimize ear infection (Figure 1B).
5. Place the micro-scissors and otoscope in the sterile area.
6. Place the rodent in the surgical field and in alignment with the surgeon.
7. Proceed with the surgical induction of CHL.

3. Surgical induction of conductive hearing loss

1. Place the tail and the head of the rodent aligned in a prone position, head closest to the surgeon.
2. Visualize the left and right ear of the rodent under otoscope to ensure healthy tympanic membrane (Figure 2a). Evaluate both the right and left ear of every rodent to verify the healthy condition prior to the CHL induction.
3. Grab the ear helix and extend the external auditory canal (i.e., ear canal, external auditory meatus, herein auditory canal) to cause the inside to become obscured and blackened by depth. Here, the auditory canal is made perpendicular to the surface of the TM.
   NOTE: Ensure the auditory cannel is directly parallel now and the TM forms a right angle with the axis of insertion of the micro-scissors. It will be visualized as a black tube without light. Sometimes the auditory canal needs to be tilted at a slight angle, approximately 15° to the planar surface of the skull. This ensures the surgeon hand is perpendicular to the surface of the TM.
4. Introduce the micro-scissors in the center of the auditory canal paying attention not to skim or nick the tissue of the auditory canal and proceed slightly, approximately ≈5 mm from the center of obscurity, thrusting forward gently through the center of the TM.
   NOTE: TM puncture can be confirmed by a pop sound when the micro-scissor tips puncture the TM. The popping sound can be heard at approximately ≈ 2 min 52 s in the video. This is not a scissor sound; this is a TM puncture sound. As measured post-analysis, the "pop" is approximately ≈ 20 dB SPL greater than background sound as recorded by a high frequency microphone. There is no need to verify the "pop" sound to this extent, otoscope visualization is sufficient. The investigator may need to practice ensuring a 'pop' is heard during each CHL procedure.
5. Immediately open the spring-loaded micro-scissors and rotate three times after puncturing the TM to ensure displacement of the head of the malleus away from the TM (only if malleus displacement is desired).
6. Remove the micro-scissors and place the rodent under the otoscope for visualization.
   NOTE: It is important to note that no significant bleeding should occur after the surgical procedure. Euthanize the rodent and do not proceed to behavioral assessment if bleeding occurs.
7. Induce bilateral CHL by proceeding as above in the opposite ear.

4. Otoscope visualization

1. Confirm a successful CHL surgery with an otoscope using a small diameter speculum to visualize the rodent middle ear.
2. Evaluate every rodent prior to and after the CHL surgical procedure underneath the otoscope. Ensure confirmation of normal TM (Figure 2a) and damaged TM after CHL induction (Figure 2b).
3. Post-operative care for the rodent
   1. Place the rodent in home cage under a warm lamp.
   2. Observe the rodent post-CHL induction until the rodent rights itself.
   3. Inject the rodent with glucose (dextrose/saline) serum to recover consciousness and place rodent in homecage for recovery.
      NOTE: Use a 23G needle with a 10 mL syringe to inject 5 mL of saline after CHL surgery.
   4. Inject the rodent via intramuscular with anti-bacterial Enrofloxacin 0.05 mg/kg twice during the 24 h recovery period.
   5. Observe the rodent regularly for pain behavior or symptoms after surgery.

5. Behavioral assessment (validation of CHL induction) - clap startle response

1. Corroborate CHL (after otoscope confirmation) 24 h post-surgery with the behavioral assessment consisting of the clap-startle-test.
2. Place the CHL induced rat next to a normal rat in two separate adjoining cages.
3. Place the rodents in a silent room.
4. Stand approximately 0.5 m away from the rodents and proceed to clap in equally spaced durations a number of times (5 claps were chosen and spaced over 1 second).
   NOTE: The clap startle produced by the hand clap measured ≈ 40 dB SPL greater than background sound as recorded by a high frequency microphone.

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Results

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...

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Discussion

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 findings^4,5,

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Materials

Name	Company	Catalog Number	Comments
Latex, polyvinyl or nitrile gloves	AMMEX		Use unpowdered gloves 8-mil
Micro spring scissors (see Fig. 1b)	RWD Life Science	S11035-08	8.0 cm total length, with 3.5mm cutting edge, or similar micro forceps. Standard tweezers with spring action will suffice
Otoscope mini 3000	HEINE	D-008.70.120M	Standard LED otoscope will suffice
Rat or mouse	JAX labs		Any small rodent
Small rodent cage	Tecniplast	1284L	Need two cages to separate CHL rodent from hearing rodent. If rodents 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