Effect of temperature elevation on rabbit cochlear function as measured by distortion-product otoacoustic emissions WILLARDS. NOYES, MD, THOMAS V. McCAFFREY, MD, PhD, DAVID A. FABRY,PhD, MARTIN S. ROBINETTE,PhD, and VERA J. SUMAN, PhD,Rochester, Minnesota Low-intensity laser stapedotomy has been shown to produce temperature elevations of 3 ° to 4 ° C within the cochlea. This study investigates the effect of temperature elevations in this range on cochlear outer hair cell function by use of distortion-product otoacoustic emissions in rabbits. Using esophageal temperature monitoring, we compared 2f 142 distortion-product otoacoustic emissions over a range of frequencies (1806 to 8691 Hz) at rabbit normothermia, normothermia plus 3 ° C, and normothermia after passive cooling. Cochlear temperature was found to exceed changes in esophageal temperature by as much as 1.2° C. We found that a maximum of 3 ° C elevation in esophageal temperature did not permanently impair
outer hair cell function in the rabbit cochlea. Results of this study suggest that moderate changes in cochlear temperature, such as those produced by low-intensity CO2 and holmium-yttrium aluminum garnet lasers, m a y not produce irreversiblethermal d a m a g e to the cochlear outer hair cells. (Otoloryngoi Heod Neck Surg 199(5;]15:548-52.)
O u t e r hair cells (OHCs) are the most abundant and vulnerable sensory cells in the cochlea. ~ Thermal damage to these and other cochlear cell types is a potential complication of otologic laser surgep/. During a laser stapedotomy procedure, laser energy is applied to the stapes footplate. This energy is either absorbed by the footplate or transmitted through the footplate to the cochlear fluid compartment. 2 A technique for assessing thermal damage to the cochlear OHCs is the measurement of otoacoustic emissions (OAE). First described by Kemp, 3 these emissions are generated in the normal cochlea in response to acoustic stimulation and are believed to arise from the motility of the OHCs in the organ of Corti. 4 Studies in laboratory animals have
From the Departments of Otorhinolm'yngology-Headand Neck Surgery (Drs. Noyes, McCaffrey, Fabry, and Robinette) and Health Sciences Research (Dr. Suman), Mayo Clinic. Supported by a research grant from the Mayo Cdnic, Rochester,Minn. (grant no. 4040745100). Presented at the Annual Meeting of the American Academy of Otolaryngology-Headand Neck Surgery, San Diego, Calif., Sept. 18-21, 1994. Received for publication Sept. 19, 1994; revision received Dec. 12, 1995; accepted Dec. 16, 1995. Reprint requests: Willard S. Noyes, MD, J.C. Blair Memorial Hospital, Warm Springs Ave., Huntington, PA i6652. Copyright © 1996 by the American Academy of OtolaryngologyHead and Neck Surgery Foundation, Inc. 0194-5998/96/$5.00+0 23/10/71310 548
demonstrated that OAEs are susceptible to anoxia, 2'5 hydrops, 6 hypoxia, 7 ototoxic agents, 8 and noise exposure. 9 Because of the susceptibility of OHCs to physiologic injury, OAEs may be an indicator of thermalinduced OHC damage. One type of OAE is the distortion-product otoacoustic emission (DPOAE). DPOAEs are elicited in response to two continuous pure tones, f~ and f2, separated in frequency by a set difference. In the mammalian ear, the largest D P O A E occurs at 2fl-fa. ~° The usefulness of DPOAEs is demonstrated by a DPOAE-gram, which reflects the frequency configuration of a standard audiogram. H DPOAEs have been studied extensively in rabbits, mj2't3 These investigations have established normative data on DPOAEs in albino rabbits (Oryctolagus cuniculus), have demonstrated that DPOAEs can accurately be perfoi3ned in anesthetized rabbits (ketamine/xylazine), and have determined that D P O A E data are repeatable. Laser types currently used by otologic surgeons include the CO 2, argon, KTR and holmium-yttrium aluminum garnet (YAG) lasers. These lasers differ according to their spectral properties, tissue absorption, and method of delivery. 14 Several animal studies have measured endocochlear fluid temperature changes after laser stapedotomy. The temperature extremes during a single burst of laser energy to the cochlea vary according to the type of laser used, the power setting, and the burst duration. For the argon laser these extremes are ap-
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Comparison of Inner Ear and Esophageal Temperature
proximately 8 ° to 10 ° C (0.2- to 0.5-second pulse duration, 1.9 W), and if the argon laser burst is applied directly to the perilymph, temperature elevations as high as 100 ° C have been recorded. 15 Using the holmiumYAG laser (250-gsec pulse duration, 2 Hz, 40 to 60 mJ), Reder ~6 demonstrated an endocochlear temperature elevation of 3.6 ° C in chinchillas. Temperature extremes for the CO 2 laser tend to be lower and range from 1.1 ° to 1.7 ° C (0.2-second pulse duration, 0.51 to 3.05 W). ~7 Laser studies on cadaver temporal bones ~4'18substantiate these findings. Simultaneous measurement of endocochlear fluid temperature and DPOAEs during laser manipulation of the cochlea or stapes footplate is difficult because (1) surgical exposure of the stapes requires disruption of the middle ear transformer mechanism; (2) cochlear trauma resulting from intracochlear thermocouple insertion could affect the OHC response; and (3) a temperature reading obtained from the bony otic capsule might not accurately reflect endocochlear temperature. Difficulties might also arise in distinguishing thermoacoustic laser damage from OHC injury induced by cochlear fluid hyperthermia alone) 9 One way tO overcome these limitations is to estimate endocochlear temperature by monitoring esophageal temperature during core hyperthermia. Esophageal temperature is believed to most closely reflect the temperature of the blood because of its close proximity with the ascending thoracic aorta. 2° With application of this method, no mechanical disruption of the hearing mechanism would occur, and accurate OAE data could be gathered. This study was designed to examine the effect of temperature on DPOAEs.
The first study involved four separate experiments. Each rabbit was anesthetized and endoscopically intubated with a truncated 00 pediatric endotracheal tube. The periauricular region was shaved, and an esophageal temperature probe (model 402; Yellow Springs Instrument Co., Inc., Yellow Springs, Ohio) was inserted approximately 11 cm from the incisor teeth into the esophagus. With an otologic microscope, the left round window membrane was surgically exposed through a postauricular incision. A polystyrene-insulated, 24gauge, hypodermic temperature probe (model 524; Yellow Springs Instrument Co.) was inserted through the round window membrane and secured. The incision was closed, and the animal was placed in a closed, singlewalled sound booth (Industrial Acoustics Co., Inc., Bronx, N.Y.) equipped with a heating blanket and a 250-W heat lamp. Temperature probes were connected to a calibrated telethermometer (model 42SC; Yellow Springs Instrument Co.). Temperature conversions were made according to manufacturer-supplied conversion tables. Two rabbits were heated with a warming blanket. The animal was positioned on its side with the operated ear away from the heat source. Simultaneous measurement of esophageal and cochlear temperatures were made at esophageal normothermia (NT1) and normothermia plus 3 ° C (NT~ + 3 ° C). The remaining two rabbits were warmed by a heat lamp positioned 60 cm above the operated ear. Thermoprobe cables were shielded from direct light contact with tin foil, and temperature comparisons were made as before.
METHODS
Temperature Effects on DPOAEs
Experimental animals were rabbits (Oryctolagus cuniculus) from albino New Zealand strains. Animal protocols were approved by our institutional animal care and use committee. Experiments were carried out in compliance with all federal, state, and local regulations concerning the humane use of animals in research. All rabbits were male and weighed between 2.8 and 3.5 kg. Two studies were performed. The first study was designed to compare response to core hyperthermia. The second study evaluated the DPOAE response to core hyperthermia. Esophageal temperature was used as the reference (core) temperature for all experiments. All experiments were performed on anesthetized rabbits by use of intramuscular ketamine (50 mg/kg) and xylazine (10 mg/kg) injections. After completion of each experiment, rabbits were killed with intracardiac sodium pentothal injection.
The second study involved 13 rabbits (1 ear/rabbit). Two weeks before emissions data were collected on a rabbit, its ears were cleaned of cerumen and inspected for the presence of middle ear disease or tympanic perforation. No animal was excluded on this basis. The rabbit was then anesthetized so that custom silicone ear molds could be cast for each ear (Per Form H/H; Hal-Hen products, Long Island City, N.Y.). The ear molds were commercially drilled to accommodate an ER-10B probe. The stimulus generation and DPOAE measurement system used consisted of a 386 25-mHz PC-compatible computer equipped with a 80 x 87 math coprocessor and an Ariel DSP-16 high-performance data-acquisition processor (Ariel Corp., Highland Park, N.J.). An Etymotic transducer package consisting of two ER-2 tube phones, an ER-10B preamplifier, and an ER-10B low-
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Table 1. Comparison of inner ear temperature and esophageal temperature in four rabbits with two different heating methods Radiant heat lamp
Warming blanket Rabbit 1
Rabbit 2
Rabbit 3
Rabbit 4
Parameters
Ear
Eso
Ear
Eso
Ear
Eso
Ear
Eso
Normothermia Normothermia + 3 ° C Temperature increase T ~ - T~so
39.40 42.55 3.15
40.0 43.0 3.0
39.3 42.4 3.1
39.7 42.7 3.0
40.25 44.50 4.25
39.6 42.6 3.0
38.2 42.35 4.15
38.3 41.3 3.0
+0.15
+0.1
+1.25
4-1.15
ALl values in degrees Celsius. eso, Esophagus; Tear, ear temperature; T~o, esophagus temperature.
noise microphone (Etymotic Research, Inc., Elk Grove Village, Ill.) was connected to the Ariel port. A CUBeDIS software package (version 2.40; AT&T Bell Labs, Murray Hill, N.J.) was used to generate and record DPOAE data. 2~ The primary tones (fl and f2) were scaled so that ear canal pressure was maintained constant as the frequencies were varied. Emissions were detected in the ear canal by an ER-t0B microphone. Output was then amplified 40 dB and averaged in real time. The distortion product monitored was the cubic difference tone, at a frequency 2fl-fa, where f2/f1 = 1.2. Each animal was placed on its side in a sound booth equipped with a 250-W radimat heat lamp positioned 60 cm above the rabbit's head and thorax. An esophageal temperature probe was inserted 11 cm beyond the incisor teeth and secured. Warming was achieved through ipsilateral radiant heat. Cooling was passive. The ER-10B probe was sealed in the custom ear mold and inserted in the rabbit's uppermost ear. Proper ear canal seals were documented by software calibration of ear canal pressure levels. Two primary tones, f~ and f2, were presented at five points per octave from 1500 Hz through 12,000 Hz (f2/fl = 1.2, L 1= L 2= 55 dB SPL). These parameters resulted in recordaNe distortion products at 1806, 2050, 2392, 2783, 3222, 3710, 4248, 4931, 5712, 6542, 7568, and 8691 Hz. Responses were averaged over a 4-second period to minimize the noise floor (NF). NF was calculated by averaging six Fourier transtorm bins together. These six bins were contiguous to the distortion-product frequency, three above and three below. At each temperature and frequency, several DPOAE recordings were obtained for each rabbit--normothermia (NT~, four sets), normothermia + 3 ° C (NT~ + 3 ° C, three sets) and normothermia after heating (NT 2, three sets). Two rabbits did not survive the cooling phase and were excluded from the study. For each temperature group at each frequency, the mean of each rabbit's DPOAE and NF values was calculated and used in the
analysis. For each frequency level, the paired Student's t test was used to assess whether the DPOAE or NF differed significantly between either NT~ and NT 1 + 3 ° C levels or the NT 1 and NT 2 levels. For each comparison, both the corrected p value for multiple comparisons with Bonferroni's method and the uncorrected p value is examined. A corrected p value < 0.05 was considered significant. Each family of tests compares the DPOAE response at one temperature level with the DPOAE response at another temperature level for all 12 frequency levels. RESULTS Comparison of Inner Ear and Esophageal Temperature Ear and esophageal temperatures at NT 1 and NT~ + 3 ° C are compared in Table 1. With esophageal temperature as the reference, contralateral warming (heating blanket) elevated cochlear temperature an average of 0.125 ° C higher than esophageal temperature (n = 2). Ipsilateral warming (heat lamp) elevated cochlear temperature an average of 1.2 ° C higher than esophageal temperature (n = 2). Temperature Effects on DPOAEs The average normothermic temperature among the 11 rabbits was 39.9 ° C (range, 39.2 ° to 40.2 ° C). Figure 1 displays the mean 2f~-f2 DPOAE response obtained at DPOAE levels ranging from 1806 to 8691 Hz taken at 0.2 octave steps for each of the temperature groups. Standard deviations among the responses typically ranged from 3 to 5 dB. DPOAE amplitude was smallest and most variable at 1806 Hz for each temperature group. Note that DPOAE amplitudes increased with frequency. The DPOAE response pattern was remarkably consistent between temperature groups. NF measurements were not as consistent. NF was found to be higher at NT1 + 3 ° C than NT~, especially at the lower frequencies (difference ranged from 1.4 to 8.0 dB).
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25-
o.. 0'~ 3
Em ~" ILl < O n I~1
,#
W~
10. ~l ~;
50-
~
--
~ o
t
h
e
r
m
Normothermia
Normotherrnia + 3oC i a post heating
i,.Jc I X ~~ ,--~
0.
,..:~ -1
!
.;2. -3--
2fl-f2 DPOAE frequency (Hz)
2f~-f2 DPOAEfrequency(kHz)
Fig. I. M e a n 2fl-f 2 DPOAE responses recorded at 0.2 octave steps from ] 806 to 869 ] Hz. Values obtained at animal normothermia, normothermia plus 3 ° C, and normothermia after passive cooling are plotted together for comparison. 2fl-f2 DPOAE responses were elicited by two primary tones, f~ and f2, with f2/f~ = 1.2 and SPL= 55 dB
(LI : I_2).
No differences in DPOAE amplitudes were found between normothermic groups (NT~-NT2) at or below 8691 Hz. The mean difference in DPOAE amplitude from 1806 to 8691 Hz after 3 ° C temperature elevation (NT~ - [NT~ + 3 ° C]) is plotted in Fig. 2. There is no evidence to suggest that DPOAE levels at normothermia differ from DPOAE at 3 ° C temperature elevation for any of the frequencies below 7568 Hz. There is a suggestion of a decrease in DPOAE at 3 ° C temperature elevation at both 7568 Hz and 8691 Hz. Differences at 7568 Hz and 8691 Hz ranged from -4.80 dB to 3.46 dB (median, -1.68) and -5.13 to 0.14 dB (median, -1.11), respectively. After correcting for multiple comparisons, these differences were not significant (p = 0.444 and p = 0.094, respectively). Thus further studies will be needed to clarify whether there is a clinically important difference at these levels. There is a suggestion of an increase in NF amplitudes after 3 ° C temperature elevation at 1806 to 2783 Hz and 4931 to 7568 Hz (uncorrected p values < 0.05); however, after correcting for multiple comparisons, we found only the increase in NF amplitude at 1806 Hz to be significant. DISCUSSION
This study demonstrates that in rabbits cochlear fluid temperature can be elevated higher than core body temperature by either ipsilateral radiant heat or a contralateral warming blanket. The greater influence of ipsilateral radiant heat on inner ear fluid temperature (1.2 ° C vs. 0.15 ° C) probably results from arteriovenous countercurrent heat exchange in the ipsilateral ear. This mechanism has been shown to occur in human beings. 22-24Direct heating of local tissues surrounding the
r-
1806 2050 239;2 2783 3922 3710 4;248 4931 5712 654;2 7568 8691
Fig. 2. M e a n (+1 SD) difference in 2fl-f 2 DPOAE amplitude from ]806 to 860] Hz offer 3 ° C e s o p h a g e a l temperature elevation (NT1 - (NT 1 + 3 ° C)). Positive values reflect a decrease in amplitude (in decibels SPL) at normothermia
+3°C.
cochlea may have also played a role. From the results of this experiment, it is reasonable to assume that, when ipsilateral radiant heat was used to elevate esophageal temperature 3 ° C, the temperature within the endocochlear fluid compartment increased to approximately 4.2 ° C. Because cochlear temperature was not actually measured in those rabbits undergoing DPOAE testing, this value must remain an estimate. Because high-frequency eliciting tones can be partially reflected from the tympanic membrane, a pressure null, at the location of the ER-10B microphone, can develop. 21To maintain a constant ear canal pressure, the CUBeDIS software will turn the primary eliciting tones to maximal levels once this pressure null is detected. This happening may in part explain why we were unable to consistently detect DPOAE amplitudes at frequencies greater than 6542 to 8691 Hz. Despite multiple pretest manipulations of the ER-10B probe, we were unable to overcome this software limitation. Interanimal variability likely reflected normal biologic differences or may have resulted from variable placement of the ER-10B probe within each rabbit's ear canal. The observed elevation in NF during hyperthermia at 1806 Hz was undoubtedly related to an increase in cardiorespiratory noise. CUBeDIS software parameters used in this study were in accordance with those outlined by Whitehead et al.10 Using low-level f l and f2 tones at L 1 and L 2 less than 60 dB SPL, Whitehead et al. demonstrated that the rabbit ear canal 2fl-f2 distortion product is strong from 2 kHz to above 10 kHz. This is in accordance with findings of other researchers who determined that acoustic sensitivity in the rabbit is strongest at frequencies between 1 and 16 Hz. 25'26 A decreased DPOAE amplitude recorded below 2 kHz was attributed to either a reduction of the efficiency of DPOAE generation by the cochlea or a reduction of transmission efficiency of
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t h e m i d d l e e a r at l o w e r f r e q u e n c i e s . 27 F o r t h e s e r e a s o n s , we analyzed only those DPOAEs measurements equal to or g r e a t e r t h a n 1806 Hz. V o l l r a t h a n d S c h r e i n e r 19 b e l i e v e d t h a t c o c h l e a r t e m perature elevation after argon laser stapedotomy does not significantly impact basilar membrane function. P r o l o n g e d c o m p o u n d a c t i o n p o t e n t i a l r e c o v e r y w a s att r i b u t e d to a d e s y n c h r o n i z a t i o n o f n e u r a l activity resulting f r o m a t e m p e r a t u r e - r e l a t e d c h e m o e l e c t r i c a l u n c o u P l i n g b e t w e e n t h e h a i r cells a n d t h e e i g h t h n e r v e . O u r r e s u l t s d o n o t c o n f i r m or o p p o s e this theory. W h e t h e r t h e i m p a c t o f h y p e r t h e r m i a w o u l d h a v e b e e n m o r e pron o u n c e d at f r e q u e n c i e s h i g h e r t h a n 8691 H z r e m a i n s u n k n o w n . L i k e w i s e , w e w e r e u n a b l e to c o n f i r m w h e t h e r a f u r t h e r rise in t e m p e r a t u r e , to a l e v e l p r e v i o u s l y d o c u m e n t e d f o r a r g o n l a s e r s t a p e d o t o m y (8 ° to 10 ° C), 15 would have substantially altered OHC function. This study confirms that cochlear temperature elevat i o n in t h e r a n g e o f 3 ° to 4.2 ° C d o e s n o t i m p a i r r a b b i t O H C f u n c t i o n m e a s u r e d t h r o u g h D P O A E s at 1806 to 8691 Hz. E n d o c o c h l e a r t e m p e r a t u r e e l e v a t i o n o f this m a g n i t u d e e x c e e d s t h a t r e c o r d e d i n c t h e r studies a f t e r low-intensity
CO 2 and
holmium-YAG
laser
stape-
dotomy.
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