EXPERIMENTAL STUDIES ILSASCHWARTZ, PhD Experimental Study Editor
Effect of irradiation on guinea pig ABR thresholds J. SCOTT GREENE, MD, NEIL A. GIDDINGS, MD, and JOHN T. JACOBSON, PhD, Danville, Pennsylvania, and Houston, Texas
A significant number of patients undergo Irradiation to the temporal bone for mallg· nancles. Conflicting reports exist regarding the effects of Irradiation on hearing thresh· olds. Although radlatlon·lnduced otitis media and osteoradionecrosis of the osslcles with resultant conductive hearing loss are well·documented, there Is disagreement regarding the effect of Irradiation on sensorineural hearing. Previous animal models, relying only on behavioral tests and reflex thresholds, have failed to reveal consistent threshold shifts after Irradiation. However, with the advent of aUditory bralnstem reo sponse (ABR) testing, a reliable objective measurement of hearing In animals Is avail· able. Hearing thresholds were determined bilaterally by ABR testing In 21 albino guinea pigs. Theleft temporal bones of sixteen animals were then Irradiated with a total dose ranging from 5750 to 7000 cGy over 7 weeks. Theright ears of these animals, plus both ears of five nonlrradlated guinea pigs, served as controls. Follow·up threshold ABRs were obtained Immediately post·lrradlatlon (RT), and at 6 and 12 months post·RT. Av· erage thresholds In all groups Increased over time: 60 dB In the control group; 53 dB In the control ears of the Irradiated animals; and 46 dB In the Irradiated ears. There were no statistically significant Increases In ABR thresholds for Irradiated ears vs.control ears. At the 6·month followup, hearing was actually better In the Irradiated ears than the control ears and this difference between ears was significantly greater than the difference at baseline (p < 0.026). Overall, there was no evidence that Irradiation produces changes In ABR thresholds. (OTOLARYNGOL HEAD NECK SURG 1992;107:763.)
A
significant number of patients undergo either therapeutic or prophylactic irradiation involving the temporal bone for either primary or metastatic lesions to the cranium. I·~ Conflicting reports exist regarding the effects of radiation therapy on the temporal bone. Although radiation-induced otitis media" and osteoradionecrosis of the ossicles'" with resultant conductive hearing loss are well-documented, there is disagreement about whether radiation therapy produces sensorineural hearing loss.
From the Department of Otolaryngology (Drs. Greene and Giddings), Geisinger Medical Center, and the Department of Otolaryngology (Dr. Jacobson), University of Texas Health Science Center. Received for publication Jan. 3, 1992; revision received June 16, 1992; accepted July 24, 1992. Reprint requests: J. Scott Greene, MD, Geisinger Medical Center. I Academy Ave., Danville, PA 17822.
23/10/41504
Animal models using behavioral testing'"" and certain auditory reflexes, II which are limited and cumbersome in application, have generally failed to show increased hearing thresholds. Studies in dogs":" and rats" found no evidence of hearing loss after irradiation. A study of cochlear potentials in six guinea pigs did find a reduction of the cochlear potentials, with a maximum reduction corresponding to approximately 8.4 dB. B Another study of the pinna reflex in 20 guinea pigs demonstrated a rise in reflex thresholds after 3 hours and a completely absent reflex after 24 hours." In studies of human beings, a number of authors report evidence of radiation-induced sensorineural hearing loss.2,4.'~.2o The amounts of radiation received by patients in these studies varied from 2500 cGy to more than 23,000 cGy in one case. Hearing loss was not clearly related to amount of radiation. Several other studies reported no evidence of hearing loss after radiation to the head.":" One study suggested that ra-
763
OlolaryngologyHead and Neck Surgery
764 GREENE at al.
30
iii 'a
>!::
20
VI
z
...
w ~
VI ::::I oJ ::::I
==
~
VI
15 15 10 10 5 5
LATENCY 2.00 ms/dlv Fig. 2. Sample guinea pig ABR for determining hearing threshold.
adult values for the ABR are reached by 4 weeks of
Fig. 1. ABR testing of anesthetized guinea pig (Insert probes removed for illustration),
diation led to greater susceptibility to hearing loss after subsequent chemotherapy. 24 In the previous animal studies, behavioral or reflex responses, such as the Preyer reflex, have been used rather than objective measures of hearing thresholds. In addition, the number of animals studied was usually small, and radiation doses were far from the standard doses used in radiation therapy for head and neck cancer patients. The studies of human beings yielded mixed results. They were commonly retrospective and lacked comparison or control groups. With the advent of auditory brainstem response. (ABR) testing, a reliable, objective measure of thresholds suitable for testing animals exists. Using the ABR, it is possible to measure threshold changes reliably on an animal model irradiated with the same radiation dose (both fractionated and total) that patients with malignancies of the head and neck receive. The auditory brainstem response has been used extensively in guinea pigs in a wide variety of experimental studies,":" and
age."
The present study uses accepted ABR techniques to prospectively relate use of radiation therapy to sensorineural hearing loss in guinea pigs, using both experimental and control groups. METHODS
This study was reviewed and approved by the Scientific Review Committee and the Animal Care and Use Committee of the Geisinger Medical Center. Twenty-one albino guinea pigs, ranging in weight from 310 to 380 grams each, were placed in the study. Animal care was in compliance with The Principles of Laboratory Animal Care, formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals, prepared by the National Academy of Sciences and published by the National Institutes of Health. The animals were randomly divided into three groups of five animals each (groups I, II, and III) and one group of six animals (group IV). Baseline auditory brains tern response thresholds were obtained on all animals while they were under general anesthesia (Fig. 1). Auditory brainstem responses were measured with the animals under general anesthesia (ketamine HCl
Volume 107 Number 6 Part 1 December 1992
Effect of irradiation of guinea pig ABR thresholds 765
Fig. 3. Restrained shielded guinea pig receives irradiation.
with xylazine IP and Atropine sulfate SQ). The measurement of the ABR required that a small nontraumatic probe be inserted in the ear canal for presentation of the click stimuli and two small electrodes be pasted to each auricle, with a third electrode pasted to the vertex for measurement of brainwave activity. Auditory brainstem responses to 500 alternating monaural click (100 msec) stimuli were averaged by a Bio-Logics Systems Diagnostic Computer (Navigator). Responses were obtained from both the left and right ears of each guinea pig. The click signal was presented monaurally at a rate of 21.1 per second by means of the external ear canal insert probes. Beginning stimulus intensity was set at 70 to 90 dB (human hearing level) and decreased (in 10 dB intervals for high intensities and 5 dB intervals for low intensities) until reproducible waves could not be obtained. The records were differentially amplified by 50,000 and bandpass filtered from 30 to 1500 (3 dB down point, 12 dB/octave). The artifact level was set at IS microvolts and the notch filter was activated to reduce possible line noise interference. Sweep analysis time was 10 milliseconds. Responses were recorded, stored on disk, and printed for off-line analysis (Fig. 2). The absolute wave components and their relative latency values were identified and labeled. ABR threshold was defined as the lowest decibel stimulus that gave
reproducible waves and was determined blindly by one author (J.T.J.) without knowledge of exposure to irradiation. For each radiation treatment, the animals were restrained in a modified restrainer cage (Biodec METRAP Catalog no. 1-282-IOA, Biodec, Inc., Cincinnati, Ohio) without anesthesia. Irradiation exposure was accomplished in the Department of Radiation Medicine on an orthovoltage unit (MA20 KV200 setting for 1.2 minutes) and was limited to the left ear and temporal bone of each test animal by controlling the width and length of the beam and by shielding with lead plates all areas except the left temporal bone (see Fig. 3). Groups II, Ill, and IV all received 5,750 to 7,000 cGy to the left temporal bone, fractionated at 150 to 200 cGy per day, 5 days per week for 7 weeks. Both ears of group I subjects served as control ears and were not irradiated. In addition, the right ear of all irradiated subjects (groups II, 1II, and IV) served as control ears. After completion of radiation therapy, all 21 animals underwent repeat ABR threshold testing under general anesthesia. Group II animals were then killed and their temporal bones harvested. At 6 months after radiation therapy, groups I, 1II, and IV again underwent ABR threshold testing. Group III animals were then killed
OtolaryngologyHead and Neck Surgery
766 GREENE at 01.
Table 1. Average ABR thresholds (dB) by group After radiotherapy Before radiotherapy Group I II III IV
Control (N = 5) Experimental (N Experimental (N Experimental (N
= 5) = 5) = 6)
Left
Right
10
9 7
10
11
9
Immediate
6 months
12 months
Left
Right
Left
Right
Left
Right
10
14
9
14 10 13
16 8 13 13
48
43
67
73
31
52 28
55
62
28
Numbers in boldface italics indicate irradiated ears.
Table 2. Mean Interaural threshold differences (dB)
Baseline Post-irradiation 6 months 12 months
Control animals
Experimental animals
0.0 -2.0 5.0 -6.7
-0.3 0.6 -13.6t
-6.lt
Mann-Whitney U test" p < P< P< P
Effect of irradiation on guinea pig ABR thresholds J. SCOTT GREENE, MD, NEIL A. GIDDINGS, MD, and JOHN T. JACOBSON, PhD, Danville, Pennsylvania, and Houston, Texas
A significant number of patients undergo Irradiation to the temporal bone for mallg· nancles. Conflicting reports exist regarding the effects of Irradiation on hearing thresh· olds. Although radlatlon·lnduced otitis media and osteoradionecrosis of the osslcles with resultant conductive hearing loss are well·documented, there Is disagreement regarding the effect of Irradiation on sensorineural hearing. Previous animal models, relying only on behavioral tests and reflex thresholds, have failed to reveal consistent threshold shifts after Irradiation. However, with the advent of aUditory bralnstem reo sponse (ABR) testing, a reliable objective measurement of hearing In animals Is avail· able. Hearing thresholds were determined bilaterally by ABR testing In 21 albino guinea pigs. Theleft temporal bones of sixteen animals were then Irradiated with a total dose ranging from 5750 to 7000 cGy over 7 weeks. Theright ears of these animals, plus both ears of five nonlrradlated guinea pigs, served as controls. Follow·up threshold ABRs were obtained Immediately post·lrradlatlon (RT), and at 6 and 12 months post·RT. Av· erage thresholds In all groups Increased over time: 60 dB In the control group; 53 dB In the control ears of the Irradiated animals; and 46 dB In the Irradiated ears. There were no statistically significant Increases In ABR thresholds for Irradiated ears vs.control ears. At the 6·month followup, hearing was actually better In the Irradiated ears than the control ears and this difference between ears was significantly greater than the difference at baseline (p < 0.026). Overall, there was no evidence that Irradiation produces changes In ABR thresholds. (OTOLARYNGOL HEAD NECK SURG 1992;107:763.)
A
significant number of patients undergo either therapeutic or prophylactic irradiation involving the temporal bone for either primary or metastatic lesions to the cranium. I·~ Conflicting reports exist regarding the effects of radiation therapy on the temporal bone. Although radiation-induced otitis media" and osteoradionecrosis of the ossicles'" with resultant conductive hearing loss are well-documented, there is disagreement about whether radiation therapy produces sensorineural hearing loss.
From the Department of Otolaryngology (Drs. Greene and Giddings), Geisinger Medical Center, and the Department of Otolaryngology (Dr. Jacobson), University of Texas Health Science Center. Received for publication Jan. 3, 1992; revision received June 16, 1992; accepted July 24, 1992. Reprint requests: J. Scott Greene, MD, Geisinger Medical Center. I Academy Ave., Danville, PA 17822.
23/10/41504
Animal models using behavioral testing'"" and certain auditory reflexes, II which are limited and cumbersome in application, have generally failed to show increased hearing thresholds. Studies in dogs":" and rats" found no evidence of hearing loss after irradiation. A study of cochlear potentials in six guinea pigs did find a reduction of the cochlear potentials, with a maximum reduction corresponding to approximately 8.4 dB. B Another study of the pinna reflex in 20 guinea pigs demonstrated a rise in reflex thresholds after 3 hours and a completely absent reflex after 24 hours." In studies of human beings, a number of authors report evidence of radiation-induced sensorineural hearing loss.2,4.'~.2o The amounts of radiation received by patients in these studies varied from 2500 cGy to more than 23,000 cGy in one case. Hearing loss was not clearly related to amount of radiation. Several other studies reported no evidence of hearing loss after radiation to the head.":" One study suggested that ra-
763
OlolaryngologyHead and Neck Surgery
764 GREENE at al.
30
iii 'a
>!::
20
VI
z
...
w ~
VI ::::I oJ ::::I
==
~
VI
15 15 10 10 5 5
LATENCY 2.00 ms/dlv Fig. 2. Sample guinea pig ABR for determining hearing threshold.
adult values for the ABR are reached by 4 weeks of
Fig. 1. ABR testing of anesthetized guinea pig (Insert probes removed for illustration),
diation led to greater susceptibility to hearing loss after subsequent chemotherapy. 24 In the previous animal studies, behavioral or reflex responses, such as the Preyer reflex, have been used rather than objective measures of hearing thresholds. In addition, the number of animals studied was usually small, and radiation doses were far from the standard doses used in radiation therapy for head and neck cancer patients. The studies of human beings yielded mixed results. They were commonly retrospective and lacked comparison or control groups. With the advent of auditory brainstem response. (ABR) testing, a reliable, objective measure of thresholds suitable for testing animals exists. Using the ABR, it is possible to measure threshold changes reliably on an animal model irradiated with the same radiation dose (both fractionated and total) that patients with malignancies of the head and neck receive. The auditory brainstem response has been used extensively in guinea pigs in a wide variety of experimental studies,":" and
age."
The present study uses accepted ABR techniques to prospectively relate use of radiation therapy to sensorineural hearing loss in guinea pigs, using both experimental and control groups. METHODS
This study was reviewed and approved by the Scientific Review Committee and the Animal Care and Use Committee of the Geisinger Medical Center. Twenty-one albino guinea pigs, ranging in weight from 310 to 380 grams each, were placed in the study. Animal care was in compliance with The Principles of Laboratory Animal Care, formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals, prepared by the National Academy of Sciences and published by the National Institutes of Health. The animals were randomly divided into three groups of five animals each (groups I, II, and III) and one group of six animals (group IV). Baseline auditory brains tern response thresholds were obtained on all animals while they were under general anesthesia (Fig. 1). Auditory brainstem responses were measured with the animals under general anesthesia (ketamine HCl
Volume 107 Number 6 Part 1 December 1992
Effect of irradiation of guinea pig ABR thresholds 765
Fig. 3. Restrained shielded guinea pig receives irradiation.
with xylazine IP and Atropine sulfate SQ). The measurement of the ABR required that a small nontraumatic probe be inserted in the ear canal for presentation of the click stimuli and two small electrodes be pasted to each auricle, with a third electrode pasted to the vertex for measurement of brainwave activity. Auditory brainstem responses to 500 alternating monaural click (100 msec) stimuli were averaged by a Bio-Logics Systems Diagnostic Computer (Navigator). Responses were obtained from both the left and right ears of each guinea pig. The click signal was presented monaurally at a rate of 21.1 per second by means of the external ear canal insert probes. Beginning stimulus intensity was set at 70 to 90 dB (human hearing level) and decreased (in 10 dB intervals for high intensities and 5 dB intervals for low intensities) until reproducible waves could not be obtained. The records were differentially amplified by 50,000 and bandpass filtered from 30 to 1500 (3 dB down point, 12 dB/octave). The artifact level was set at IS microvolts and the notch filter was activated to reduce possible line noise interference. Sweep analysis time was 10 milliseconds. Responses were recorded, stored on disk, and printed for off-line analysis (Fig. 2). The absolute wave components and their relative latency values were identified and labeled. ABR threshold was defined as the lowest decibel stimulus that gave
reproducible waves and was determined blindly by one author (J.T.J.) without knowledge of exposure to irradiation. For each radiation treatment, the animals were restrained in a modified restrainer cage (Biodec METRAP Catalog no. 1-282-IOA, Biodec, Inc., Cincinnati, Ohio) without anesthesia. Irradiation exposure was accomplished in the Department of Radiation Medicine on an orthovoltage unit (MA20 KV200 setting for 1.2 minutes) and was limited to the left ear and temporal bone of each test animal by controlling the width and length of the beam and by shielding with lead plates all areas except the left temporal bone (see Fig. 3). Groups II, Ill, and IV all received 5,750 to 7,000 cGy to the left temporal bone, fractionated at 150 to 200 cGy per day, 5 days per week for 7 weeks. Both ears of group I subjects served as control ears and were not irradiated. In addition, the right ear of all irradiated subjects (groups II, 1II, and IV) served as control ears. After completion of radiation therapy, all 21 animals underwent repeat ABR threshold testing under general anesthesia. Group II animals were then killed and their temporal bones harvested. At 6 months after radiation therapy, groups I, 1II, and IV again underwent ABR threshold testing. Group III animals were then killed
OtolaryngologyHead and Neck Surgery
766 GREENE at 01.
Table 1. Average ABR thresholds (dB) by group After radiotherapy Before radiotherapy Group I II III IV
Control (N = 5) Experimental (N Experimental (N Experimental (N
= 5) = 5) = 6)
Left
Right
10
9 7
10
11
9
Immediate
6 months
12 months
Left
Right
Left
Right
Left
Right
10
14
9
14 10 13
16 8 13 13
48
43
67
73
31
52 28
55
62
28
Numbers in boldface italics indicate irradiated ears.
Table 2. Mean Interaural threshold differences (dB)
Baseline Post-irradiation 6 months 12 months
Control animals
Experimental animals
0.0 -2.0 5.0 -6.7
-0.3 0.6 -13.6t
-6.lt
Mann-Whitney U test" p < P< P< P
