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 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 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 (≈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 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 responses4,5,16,17,18.
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.
- 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.
- Maintain the rats under a constant 25 °C temperature and 60-70% humidity at the Laboratory Animal Research Unit.
- House rodents in 12/12-h light/dark cycles with access to food and drinking water, ad libitum.
- Acclimate rodents to the housing environment for at least one day prior to CHL surgery.
- 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.
- 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
- Sterilize all equipment in an autoclave or hot glass bead sterilizer prior to beginning surgery. Clean the surgical area with 70% ethanol.
- Use latex gloves and lab coat before starting the procedure.
- Place a sterile surgical drape on the clean bench (Figure 1A).
- Sterilize micro-scissors and an otoscope prior to the surgery to minimize ear infection (Figure 1B).
- Place the micro-scissors and otoscope in the sterile area.
- Place the rodent in the surgical field and in alignment with the surgeon.
- Proceed with the surgical induction of CHL.
3. Surgical induction of conductive hearing loss
- Place the tail and the head of the rodent aligned in a prone position, head closest to the surgeon.
- 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.
- 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.
- 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.
- 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).
- 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.
- Induce bilateral CHL by proceeding as above in the opposite ear.
4. Otoscope visualization
- Confirm a successful CHL surgery with an otoscope using a small diameter speculum to visualize the rodent middle ear.
- 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).
- Post-operative care for the rodent
- Place the rodent in home cage under a warm lamp.
- Observe the rodent post-CHL induction until the rodent rights itself.
- 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.
- Inject the rodent via intramuscular with anti-bacterial Enrofloxacin 0.05 mg/kg twice during the 24 h recovery period.
- Observe the rodent regularly for pain behavior or symptoms after surgery.
5. Behavioral assessment (validation of CHL induction) - clap startle response
- Corroborate CHL (after otoscope confirmation) 24 h post-surgery with the behavioral assessment consisting of the clap-startle-test.
- Place the CHL induced rat next to a normal rat in two separate adjoining cages.
- Place the rodents in a silent room.
- 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.
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 loud dB SPL startle). Rats should be checked by otoscope before surgery to ensure normal tympanic membrane and after surgery to ensure tympanic membrane puncture (Figure 2; Supplementary Figure 1). Note, no bleeding or significant inflammation should occur (Supplementary Figure 2). The CHL induced-rodent will not respond to a loud dB SPL clap sound whereas a control hearing cage mate will jump to the loud clap. The CHL rat is not deaf to sound, but has a CHL and therefore subtle behavioral manifestations occur5. A control hearing rodent reacts vigorously with leaps to a loud dB SPL sound, such as the clap startle. The jump/startle response is clearly visible in the video from the hearing cage mate, while the CHL induced rat does not respond. The behavior for a CHL rat may be subtler. We demonstrate the general clap startle reflex for a representative CHL rat and control hearing rat. Over time post-CHL surgery some rats may regain clap startle response.
Using the CHL procedure, earlier studies have investigated the neural manifestations of hearing loss. For example, the degree of hearing loss following the procedure has been quantified by measuring auditory evoked potentials15,16,17, round window recordings4,18, and behavioral hearing thresholds5. Neural activity following CHL has been studied with the 2-deoxyglucose method and whole-cell recordings4,16,17. Auditory nerve activity has also been examined with round window recordings18. Frequency-modulation detection thresholds have been studied behaviorally5. This CHL procedure is clearly compatible with a wide-range of experimental methods for detailed studies of hearing loss.
Figure 1: Surgical setup. A) Place rodent in a well-lighted area with a surgical lamp. Anesthetize the rodent. Make ready an assortment of micro-scissors, if the smallest does not work, a larger pair may ensure TM puncture. Ensure the otoscope is ready. B) Spring loaded micro-scissors. Tweezers will suffice if experimenter can control the opening and closing. Please click here to view a larger version of this figure.
Figure 2: Representative tympanic membrane. A) Tympanic membrane (TM) prior to puncture. B) TM after puncture. When performing CHL and TM puncture confirmation with the otoscope, it is imperative to first note the head of the malleus and after CHL induction, a successful removal and puncture of TM. Please click here to view a larger version of this figure.
Supplementary Information Figure 1: Before and after CHL surgery as visualized by an otoscope. Otoscope visualization before and after CHL surgery (immediate) in identical rat. Left column of figures are representative rat auditory canals visualized before CHL procedure by otoscope with normal TM and middle ear ossicles. Right column of figures are representative rat auditory canals visualized after CHL procedure by otoscope with punctured TM and displaced middle ear ossicles. Significant bleeding consists of blood accumulation in ears. Study middle ear anatomy of rodents before performing the procedure19,20,21,22,23,24,25,26,27,28,29. Otoscope visualization was conducted 24 hours after surgery. Please click here to download this figure.
Supplementary Information Figure 2: Control and after CHL surgery rat auditory canal as assessed by histology. Histological view of the middle ear in control and after CHL surgery (Bar = 500 µm). Auditory canals were processed by hematoxylin and eosin (H&E) and Masson's Trichrome (MT) staining in control and 24 hours post-CHL surgery. Left column of figures are representative histological sections of the auditory canal in control rats. Right column of figures are representative histological sections of the auditory canal after CHL procedure. Note that no significant inflammation occurs following CHL surgery. No difference was noticed up to one week. Please click here to download this figure.
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 findings4,5,16,17,18.
The failure to induce CHL could be due to several reasons as listed below. In each case, we recommend discarding the rat. Euthanize the rat if one of the ears is bleeding. Bleeding indicates that the scissors made contact with the auditory canal and the surgical approach was not clean. Otherwise, it can signify that the puncture was deeper than preferred, causing internal damage, possibly brain damage. Internal damage could mean a deep insertion or nicking the auditory canal. The procedure is a minimally invasive tympanic membrane and/or malleus removal/displacement; therefore, no significant bleeding should occur (Supplemental Figure 1). If significant bleeding occurs, euthanize the rat. If no "pop" sound is heard during the tympanic membrane puncture, this could indicate a poor CHL, which means the scissors did not make direct contact with the tympanic membrane. Discard the rodent from the group and euthanize. Using the otoscope, if visualization confirms the middle ear is similar to control ears, this indicates the tympanic membrane has not been punctured. No CHL was induced and therefore the rodent should be discarded. If the rodent responds to the clap-sound-test, it should be discarded. If a CHL induced rodent leaps or jumps after clap sounds, similar to its cage mate, this indicates the surgical procedure was not successful. A response to a clap sound indicates no CHL has occurred and the rat should be discarded or euthanized. The limitations of inducing CHL by a surgical approach are associated with the learning curve to perform the surgical procedure and confidence in audiological examination19,20,22,23,27,28,29. Furthermore, this protocol did not use ABR or DPOAE for assessing hearing loss15 in order to establish a simple protocol for scientists unfamiliar with audiological procedures. The audiological procedures ABR and DPOAE can be used for scientists who want further assessment of CHL15.
A modification of the technique is using TM puncture with malleus displacement. For a novice this may be difficult to establish, but the malleus is clearly visualized with an otoscope in nearly all rodent ears. The TM is a fibrous membrane with a pellucid appearance that transmits light. Practice otoscope verification to establish consistent visualization based on previous literature19,20,22,23,27,28,29. After one is confident in malleus visualization, its displacement during surgery is easily confirmed by a loud 'pop' as can be heard at ≈ 2 min 52 s. This pop is a loud dB SPL change in the audio file of approximately 20 dB SPL prior to the twisting of the spring-loaded micro-scissors. The loud pop confirms TM puncture and success without damaging the auditory canal. Two aspects of the surgical procedure must be performed with care: 1) ensure the micro-scissors are placed in the center of the auditory canal, and 2) ensure the angle of the micro-scissors is parallel with the auditory canal during the downward puncture. Ensure the puncture motion is quick and approximately 5 mm downward thrust from the opening of the auditory canal. Rotate and open the micro-scissors for malleus displacement if desirable.
The key steps of the CHL protocol are ensuring TM puncture and verifying behavioral clap startle. Ensuring verification of malleus removal/puncture of the TM confirms CHL has been performed. Verification is done through otoscopic examination. The critical steps for the behavioral assessment are to ensure a loud enough clap that the rodent is startled. Differences have been noted when a CHL rat is placed in a similar cage as a hearing rat; here, one rat is startled by the other, even if the rat cannot hear the sound. A startled rat/rodent will often jump or hop, but this is not a criterion. As can be observed, the CHL rat does not move to the clap. Since the TM puncture is a CHL, different levels of db SPL sounds may be heard by the rodent5.
The primary significance of this simple CHL method is that it can be performed by a wide range of researchers, even with just modest resources. Verification of hearing loss is performed by 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 findings4,5,16,17,18 The present technique can be combined with electrophysiology and behavioral techniques to determine the cortical consequences of hearing loss, which is of great interest to hearing loss research31.
The authors declare no financial or non-financial conflicts of interests.
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 Biomédicas, the Instituto de Neurobiología of the Universidad Nacional Autónoma de México (UNAM), the Consejo Nacional de Ciencia y Tecnología (CONACyT) México for the Graduate Fellowship 578458 to FAM Manno.
|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|
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