1. The Effects of Salicylate on Auditory Evoked Potential Amplitudes from the Auditory Cortex and Brainstem Brian Sawka AuD Project

2. Tinnitus Subjective tinnitus is the sensation of sound in the absence of an acoustic stimulus. Subjective tinnitus is the sensation of sound in the absence of an acoustic stimulus. Tinnitus can be induced by Tinnitus can be induced by noise exposure (Axelsson & Hamernik, 1987; Job et al. 2007) noise exposure (Axelsson & Hamernik, 1987; Job et al. 2007) ototoxic drugs (Huang & Schacht, 1989; Day et al. 1989) ototoxic drugs (Huang & Schacht, 1989; Day et al. 1989) Tinnitus is usually associated with cochlear damage Tinnitus is usually associated with cochlear damage (Demeester et al. 2007; Job et al. 2007).

3. Salicylate Causes Hearing Loss and Tinnitus Salicylate causes temporary hearing loss and tinnitus in humans Salicylate causes temporary hearing loss and tinnitus in humans (Hicks & Bacon, 1999; Halla et al. 1991). Tinnitus-like behaviors suggest that animals experience tinnitus with treatment of salicylate Tinnitus-like behaviors suggest that animals experience tinnitus with treatment of salicylate (Bauer et al. 1999; Jastreboff et al. 1988; Lobarinas et al. 2004; (Bauer et al. 1999; Jastreboff et al. 1988; Lobarinas et al. 2004; Yang et al, 2007). Yang et al, 2007). Consequently, tinnitus has been extensively used in research to study tinnitus Consequently, tinnitus has been extensively used in research to study tinnitus

4. Salicylate Enhances Auditory Evoked Potential Amplitude at the Auditory Cortex Yang et. al (2007) showed enhancement of auditory cortex (AC) response amplitude with high intensity 16 and 20 kHz tone burst stimuli post salicylate treatment Yang et. al (2007) showed enhancement of auditory cortex (AC) response amplitude with high intensity 16 and 20 kHz tone burst stimuli post salicylate treatment These frequencies were associated with tinnitus-like behavior in another set of animals (Yang et al. 2007) These frequencies were associated with tinnitus-like behavior in another set of animals (Yang et al. 2007) Time in ms Amplitude in mV Lu et al. (2008)

5. Cochlear/8 th nerve, Cochlear Nucleus, and Inferior Colliculus I/O Functions Pre and Post Noise Exposure Salvi et al. found enhancement at the inferior colliculus with high level 1 kHz tone bursts post noise exposure using local implants in chinchillas (2000) Salvi et al. found enhancement at the inferior colliculus with high level 1 kHz tone bursts post noise exposure using local implants in chinchillas (2000) Salvi et al. 2000

6. Aim of Study Cochlea Cochlear Nucleus Inferior Colliculus 8 th Auditory Cortex  Observe if the enhancement of the AC waveform post salicylate is also present in the ABR post salicylate is also present in the ABR waveform. waveform.  To address this question, four implanted animals will be tested pre and post salicylate treatment for will be tested pre and post salicylate treatment for both: both:  AC response  ABR response  Observe if the enhancement of the AC waveform post salicylate is also present in the ABR post salicylate is also present in the ABR waveform. waveform.  To address this question, four implanted animals will be tested pre and post salicylate treatment for will be tested pre and post salicylate treatment for both: both:  AC response  ABR response

7. Methods: Subjects Subjects: Four male Sprague Dawley rats Subjects: Four male Sprague Dawley rats Implanted at 2-3 months of age Implanted at 2-3 months of age Tested at 6-7 months of age Tested at 6-7 months of age Implants consist of: Implants consist of: Auditory Cortex electrode (right side) Auditory Cortex electrode (right side) Frontal lobe skull electrode Frontal lobe skull electrode Screw for head restraint Screw for head restraint Affixed to skull with dental cement and stainless steel screws Affixed to skull with dental cement and stainless steel screws The implant procedure has been described in detail (Yang et al, 2007; Lobarinas et al, 2006). The implant procedure has been described in detail (Yang et al, 2007; Lobarinas et al, 2006).

8. Methods: Animal Restraint The rats were trained to remain still during awake recordings: The rats were trained to remain still during awake recordings: Small modified plastic cage Small modified plastic cage Head restraint using head screw and a mechanical arm Head restraint using head screw and a mechanical arm Awake recordings: Awake recordings: Required for AC recordings Required for AC recordings Chosen for ABR to avoid influences of anesthesia Chosen for ABR to avoid influences of anesthesia

9. Methods: Auditory Stimuli 4 ms alternating polarity tone bursts of 4, 8, 12, 16 and 20 KHz 4 ms alternating polarity tone bursts of 4, 8, 12, 16 and 20 KHz Created in Tucker Davis Technologies (TDT) SigGen RP 4.4 software Created in Tucker Davis Technologies (TDT) SigGen RP 4.4 software TDT Stimuli Hardware: TDT Stimuli Hardware: Processor Processor Attenuator Attenuator Headphone driver Headphone driver 8 ohm tweeter ~ 2 ¼ inches from ear 8 ohm tweeter ~ 2 ¼ inches from ear Left ear for AC recordings (contralateral to recording electrode) Left ear for AC recordings (contralateral to recording electrode) Right ear for ABR recordings (ispilateral to recording electrode) Right ear for ABR recordings (ispilateral to recording electrode) The auditory stimuli will be binaural as both ears will remain unrestricted The auditory stimuli will be binaural as both ears will remain unrestricted

10. Methods: Recording Hardware and Parameters TDT Recording hardware: TDT Recording hardware: Four channel head stage Four channel head stage Medusa Preamp Medusa Preamp Medusa Base Station Medusa Base Station Recording Software: TDT BioSig RP 4.41 Recording Software: TDT BioSig RP 4.41 BioSig and electrode settings for AC recording: BioSig and electrode settings for AC recording: Recording electrode placed at the auditory cortex and reference at the frontal lobe of the skull (contralateral recording) Recording electrode placed at the auditory cortex and reference at the frontal lobe of the skull (contralateral recording) Band pass filter 3 Hz - 1000 Hz and 60 Hz notch Band pass filter 3 Hz - 1000 Hz and 60 Hz notch Stimulation rate 2/second and averaged 100 sweeps for each waveform. Stimulation rate 2/second and averaged 100 sweeps for each waveform. BioSig and electrode settings for ABR recording: BioSig and electrode settings for ABR recording: Recording electrode placed at the skull and reference at the auditory cortex (ipsilateral recording) Recording electrode placed at the skull and reference at the auditory cortex (ipsilateral recording) Band pass 100 Hz - 3000 Hz and 60 Hz notch Band pass 100 Hz - 3000 Hz and 60 Hz notch Stimulation rate 19/second and averaged 512 sweeps for each waveform. Stimulation rate 19/second and averaged 512 sweeps for each waveform.

11. Methods: Amplitude Measure for AC and ABR AC recordings AC recordings Large positive peak ~ 11-15 ms Large positive peak ~ 11-15 ms Amplitude recorded peak to following trough in micro volts Amplitude recorded peak to following trough in micro volts ABR recordings ABR recordings Positive peak ~ 4.5 - 5 ms (estimated latency of inferior colliculus) Positive peak ~ 4.5 - 5 ms (estimated latency of inferior colliculus) Amplitude recorded peak to following trough in micro volts Amplitude recorded peak to following trough in micro volts

12. Results: Auditory cortex response enhancement was observed for most conditions Results: Auditory cortex response enhancement was observed for most conditions

13. Results: Auditory Cortex Statistical Analysis Paired t test comparisons of pre and post salicylate AC amplitudes for all four animals: Paired t test comparisons of pre and post salicylate AC amplitudes for all four animals: 4k: p =.047 4k: p =.047 8k: p =.0045 8k: p =.0045 12k: p =.032 12k: p =.032 16k: p =.068 16k: p =.068 20k: p =.032 20k: p =.032 Alpha =.05 Alpha =.05 Micro Volts

14. Results: ABR results for high intensity stimuli were typically smaller amplitude for 2 hours post salicylate compared to baseline

15. Results: ABR Statistical Analysis Paired t test comparisons of pre and post salicylate ABR amplitudes for all four animals: Paired t test comparisons of pre and post salicylate ABR amplitudes for all four animals: 4k: Missing data 4k: Missing data 8k: Missing data 8k: Missing data 12k: p =.001 12k: p =.001 16k: p =.061 16k: p =.061 20k: Missing data 20k: Missing data Alpha =.05 Alpha =.05 Micro Volts

16. Conclusions The AC results confirm high intensity tone burst enhancement post salicylate treatment The AC results confirm high intensity tone burst enhancement post salicylate treatment The ABR data suggests that the amplitudes are overall decreased post salicylate at the inferior colliculus The ABR data suggests that the amplitudes are overall decreased post salicylate at the inferior colliculus

17. Discussion: Comparison of Results Cochlea Cochlear Nucleus Inferior Colliculus 8 th Cochlea Cochlear Nucleus Inferior Colliculus Auditory Cortex Auditory Cortex 8 th Salvi et al. studies using noise exposure Current observations for salicylate treatment

18. Discussion This study is useful as a small pilot study This study is useful as a small pilot study The results suggest that hearing damage and tinnitus from salicylate may have a different physiological mechanism than noise exposure. The results suggest that hearing damage and tinnitus from salicylate may have a different physiological mechanism than noise exposure. A follow up study is recommended that would use local inferior collicului implants to verify these results. A follow up study is recommended that would use local inferior collicului implants to verify these results.

19. Recognition I would like to thank my research committee for their advisement and assistance for this project: I would like to thank my research committee for their advisement and assistance for this project: Wei Sun, Ph.D Wei Sun, Ph.D Richard Salvi, Ph.D Richard Salvi, Ph.D Joan Sussman, Ph.D Joan Sussman, Ph.D Special thanks to: Special thanks to: Lu Jianzhong, Ph.D Lu Jianzhong, Ph.D

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