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GABA transporter mRNAs in the mammalian vestibular end organs
P84
OtolaryngologyHead and NeckSurgery August T999
Research Forum--Monday
Poster 20
An Analysis of Vestibular Decruitment JAMES JOHN BOUZOUKISMD (presenter); ARVIND KUMAR MD; Chicago IL
Problem: Vestibular decruitment (VD) is a paradoxical response to stimulation of the ear with the 2 strengths of the Torok monothermal caloric test. The culmination frequency (CF) value of the weaker of the 2 stimuli is equal to or greater than the strong stimulus response. Vestibular decruitment has been shown to be associated with brain stem and cerebellar lesions. However, the underlying neurophysiology of VD is not known. The purpose of this study is to attempt an understanding of VD by analyzing the CF values obtained from a normal group of subjects and comparing the results from a patient cohort who had MRI confirmed brain stem/cerebel!ar lesions. Methods: From our database of patients, we randomly selected 70 patients who had both MRI-confirmed posterior fossa lesions and VD. In 35 patients VD was unilateral, and in the rest it was bilateral. Normative data were obtained prospectively from 27 patients. The CF values from each of these groups were statistically compared and analyzed. Results: Patients with bilateral VD displayed an average CF of 40.9 with the 10-mL stimulus and 40.6 with the 100-mL stimulus. The latter values showed no statistical difference from CF values of normal subjects. However the 10-mL CF values in patients were significantly higher than CF values of normal subjects. Statistical analysis of CF values of unilateral VD showed similar results. Conclustion: Although this study does not explain the underlying neurophysiological mechanisms of VD, statistical analysis of the data shows that in VD it is an increase in the weaker stimulus CF value that results in the paradoxical response rather than a decrease in the strong stimulus responses. This information could form the basis for further experimental studies. Poster 21
Varied Walking Velocities and Dynamic Visual Acuity during Locomotion DIANE M SHIRLEYMD (presenter); JACOB J BLOOMBERG PHD; AJiTKUMAR MULAVARA PHD; LAUREN A MERKLE PHD; HELEN S COHEN EDD; Houston TX
Problem: The maintenance of functional visual acuity during activities of daily living demands that gaze be stabilized during body motion. The ability to read during locomotion requires integration of various vestibular and locomotor reflexes. Change in any system leads to altered dynamic visual acuity (DVA) during locomotion. Testing DVA during locomotion serves as a global indicator of the integration of events responsible for locomotion. The Locomotor Dynamic Visual Acuity Test (LDVA) identifies deficits that may result in functional limitations. This test can indicate potential functional
changes in crew members, evaluate labyrinthine-deficient patients, and document rehabilitation progress. Methods: The LDVA protocol constrained subjects by walking at a single velocity, 6.4 km/hour. Our goal was to improve the test by examining results across a range of walking velocities. Eight normal subjects and 4 patients with bilateral vestibular dysfunction read numerals of different font sizes displayed on a computer while walking at varied velocities: 3.2, 4.0, 4.8, 5.6, 6.4, 7.2, and 8.0 km/hour. Three labyrinthinedeficient patients (LD) were evaluated while walking at 3.2, 4.0, 4.8, and 5.6 km/hour. One patient walked at 2.4, 3.2, and 4.0 km/hour only. Head and trunk kinematic data were collected using a video-based motion analysis system. Results: Normal subject DVA scores were significantly decreased at the faster walking velocities, 5.6 to 8.0 km/hour. Preliminary data analysis showed that the LD scores were worse across all velocities tested. The trend in the kinematic data demonstrated that, in comparison to the normal subjects, the LD subjects had lower magnitudes at the predominant frequency of motion of the head, in the pitch, roll, and yaw planes at the varied walking speeds. The magnitudes at the lower frequencies increased. Conclusion: We conclude that these results indicate an increase in the efficacy of the LDVA test by evaluating scores across a range of walking velocities. The preliminary kinematic results indicate a trend showing that the LD patients relied a greater amount on voluntary mechanisms for head stabilization in comparison with the normal subjects, who relied predominantly on the reflex mechanisms of head stabilization in the plane of progression during walking. This may contribute to the decrease in gaze stabilization in the LD patients resulting in the worsening of their DVA scores. Clinical Significance: DVA is a global indicator of sensorimotor integration. The LDVA test is an easily administered, sensitive test of crew member neurologic function on return from spaceflight. It takes approximately 30 minutes to administer and serves as a guide for flight surgeons in determining the readiness of crew members to return to their activities of daily living such as flying, driving, and operating machinery. It also has an extension into clinical medicine and is currently being evaluawxtas a test to follow the progression of rehabilitation in labyrinthine-deficientpatients. In the above-stated situations, it offers a viable alternative to the more traditional, time-consuming vestibular testing. (Supported by Baylor College of Medicine [D.M.S.], National Research Council [L.A.M.], and the Clayton Foundation for Balance Disorders [H.S.C.].) Poster 22
GABA Transporter mRNAs in the Mammalian Vestibular End Organs MANU GUJRATI (presenter); ALAN ROBINSON PHD; DIMITRI PITOVSKI MD; Chicago IL
Problem Addressed: In the vertebrate vestibular periphery
OtolaryngologyHead and Neck Surgery Research F o r u m - - M o n d a y
Volume 121 Number 2
y-aminobutyric acid (GABA) has long been presumed to be a neurotransmitter candidate. Recent molecnlar biological studies from our laboratory suggest that GABA receptor subunits (al-6, b-l-8, gl-3) are expressed in the vestibular end organs. In a continuation of the investigation of GABA action in the vestibular peripheral system, in this investigation, we set out to determine which GABA transporters are expressed in the vestibular end organs. Methods~Measures: Specific primers, 21 to 25 nucleotides in length, were designed based on published cDNA sequences for GABA transporters types GAT 1, GAT2, GAT3, and GAT4. Total RNA was extracted with guanidine thiocyanate containing 1% Sarkosyl and 50 mM EGTA from individual vestibular end organs of adult male Sprague-Dawley rats. Results: After reverse transcription, resulting cDNA was amplified by PCR, with primer pairs specific for nucleotide sequences representing the GABA transporters. PCR amplification of gene fragments of the GABA transporters was first demonstrated by amplification of cDNA derived from rat brain mRNA, which yielded bands of predicted molecular mass products for each primer pair. In controls designed to check for spurious amplification products due to contaminating genomic DNA, mRNA was treated with DNase, and PCR was carried out without reverse transcription. Conclusions: The results of this study provide direct evidence for the presence of GABA transporters of the detected types in the mammalian vestibular end organs. Clinical Significance: This finding lends new support to previous studies implicating GABA as a vestibular neurotransmitter. The therapeutic effects of various agents, including benzodiazepines, by facilitating GABA-mediated inhibitor neurotransmission may now be fuither elucidated and better understood. Further distribution studies by in situ localization of transporter mRNAs will help define the roles of these transporters in vestibular GABAergic neurotransmission. Poster 23
Auditory Cortical Responses in Patients with Unilateral Bell's Palsy TIMO LAHIN MD (presenter); JUHA-PEKKA VASAMA MD; JYRKI M~,KEI_A MD; Helsinki Finland
Purpose: To examine the effect of unilateral Bell's palsy on auditory-evoked cortical responses, and to seek evidence of possible auditory pathway involvement. Methods: We measured auditory-evoked magnetic responses with a whole-scalp 122-channel SQUID neuromagnetometer from 14 adult patients with unilateral Bell's palsy (9 left sided and 5 right sided) and from 10 normal-hearing control subjects. The method allows simultaneous and noninvasive monitoring of neural activity over both hemispheres with good temporal and spatial resolution. The stimuli were 50-ms 1kHz tone bursts, delivered first to the left and then to the right ear at interstimulus intervals (ISis) of 1, 2, and 4 seconds.
P8w
Results: In patients, the mean pure-tone average was 12 dB on the affected side and 11 dB on the healthy side. The N100m, the 100-ms response originating in the auditory cortices, peaked, on average, 8 ms earlier, and the response strengths were, on average, 7 nAm stronger over the contralateral than the ipsilateral hemisphere. The response latencies and strengths did not differ significantly from those of control subjects. In patients, there were no significant differences in response parameters between the stimulation of the affected and healthy sides. On average, the response latencies and strengths increased as a function of ISI over both hemispheres in both patients and controls. Conclusion: These findings suggest that Bell's palsy does not affect the function of the auditory pathways. Poster 24
Analysis of Chromosome 20QI 3 by FISH in Head and Neck Squamous Cell Carcinoma K KAY DURAIRAJ MD (presenter); LEAH E MINTZ MD; RELEF KRONING PFID; ERI S SRIVATSAN PHD; MARILENE B WANG MD; Los Angeles CA
Problem: Head and neck squamous cell carcinomas remain one of the most aggressive of human cancers. While its etiology is unclear, chromosomal amplifications and deletions within tumor cells are suspected to be a possible avenue of tumorigenesis and immortalization. Chromosome 20 amplification has been detected and previously reported in several human carcinomas, including head and neck squamous carcinomas. Previous studies utilizing comparative genomic hybridization have shown chromosome 20Q amplification in head and neck cancers, perhaps contributing to the mechanism of tumor progression by upregulating cellular oncogenes. We sought to verify the previous reports of chromosome 20Q amplification in head and neck squamous cancers using fluorescence in situ hybridization (FISH). FISH is a technique that permits analysis of chromosomal aberrations on paraffin sections, using fluorescence-labeled DNA probes. Methods: Archived, paraffin-embedded tissue slides of 14 patients with primary squamous carcinoma of the head and neck were analyzed using a standard FISH technique. Chromosome 11 centromeric DNA probes were labeled with fluorophore spectrum green as control probes. Chromosome 20Q DNA was prepared from the BAC plasmid 3B23, containing the chromosome 20q13, and labeled with the fluorophore spectrum orange. After hybridization of the DNA probes to the tissue slides, microscopic analysis of the slides was performed using dual band-pass filter fluorescence microscopy. Signals were then counted. Results: Our preliminary results indicate that, contrary to previous reports of chromosome 20q amplifications and gains in head and neck cancers by comparative genomic hybridization, the technique of FISH demonstrates no significant amplification or deletion at this chromosomal locus.
OtolaryngologyHead and NeckSurgery August T999
Research Forum--Monday
Poster 20
An Analysis of Vestibular Decruitment JAMES JOHN BOUZOUKISMD (presenter); ARVIND KUMAR MD; Chicago IL
Problem: Vestibular decruitment (VD) is a paradoxical response to stimulation of the ear with the 2 strengths of the Torok monothermal caloric test. The culmination frequency (CF) value of the weaker of the 2 stimuli is equal to or greater than the strong stimulus response. Vestibular decruitment has been shown to be associated with brain stem and cerebellar lesions. However, the underlying neurophysiology of VD is not known. The purpose of this study is to attempt an understanding of VD by analyzing the CF values obtained from a normal group of subjects and comparing the results from a patient cohort who had MRI confirmed brain stem/cerebel!ar lesions. Methods: From our database of patients, we randomly selected 70 patients who had both MRI-confirmed posterior fossa lesions and VD. In 35 patients VD was unilateral, and in the rest it was bilateral. Normative data were obtained prospectively from 27 patients. The CF values from each of these groups were statistically compared and analyzed. Results: Patients with bilateral VD displayed an average CF of 40.9 with the 10-mL stimulus and 40.6 with the 100-mL stimulus. The latter values showed no statistical difference from CF values of normal subjects. However the 10-mL CF values in patients were significantly higher than CF values of normal subjects. Statistical analysis of CF values of unilateral VD showed similar results. Conclustion: Although this study does not explain the underlying neurophysiological mechanisms of VD, statistical analysis of the data shows that in VD it is an increase in the weaker stimulus CF value that results in the paradoxical response rather than a decrease in the strong stimulus responses. This information could form the basis for further experimental studies. Poster 21
Varied Walking Velocities and Dynamic Visual Acuity during Locomotion DIANE M SHIRLEYMD (presenter); JACOB J BLOOMBERG PHD; AJiTKUMAR MULAVARA PHD; LAUREN A MERKLE PHD; HELEN S COHEN EDD; Houston TX
Problem: The maintenance of functional visual acuity during activities of daily living demands that gaze be stabilized during body motion. The ability to read during locomotion requires integration of various vestibular and locomotor reflexes. Change in any system leads to altered dynamic visual acuity (DVA) during locomotion. Testing DVA during locomotion serves as a global indicator of the integration of events responsible for locomotion. The Locomotor Dynamic Visual Acuity Test (LDVA) identifies deficits that may result in functional limitations. This test can indicate potential functional
changes in crew members, evaluate labyrinthine-deficient patients, and document rehabilitation progress. Methods: The LDVA protocol constrained subjects by walking at a single velocity, 6.4 km/hour. Our goal was to improve the test by examining results across a range of walking velocities. Eight normal subjects and 4 patients with bilateral vestibular dysfunction read numerals of different font sizes displayed on a computer while walking at varied velocities: 3.2, 4.0, 4.8, 5.6, 6.4, 7.2, and 8.0 km/hour. Three labyrinthinedeficient patients (LD) were evaluated while walking at 3.2, 4.0, 4.8, and 5.6 km/hour. One patient walked at 2.4, 3.2, and 4.0 km/hour only. Head and trunk kinematic data were collected using a video-based motion analysis system. Results: Normal subject DVA scores were significantly decreased at the faster walking velocities, 5.6 to 8.0 km/hour. Preliminary data analysis showed that the LD scores were worse across all velocities tested. The trend in the kinematic data demonstrated that, in comparison to the normal subjects, the LD subjects had lower magnitudes at the predominant frequency of motion of the head, in the pitch, roll, and yaw planes at the varied walking speeds. The magnitudes at the lower frequencies increased. Conclusion: We conclude that these results indicate an increase in the efficacy of the LDVA test by evaluating scores across a range of walking velocities. The preliminary kinematic results indicate a trend showing that the LD patients relied a greater amount on voluntary mechanisms for head stabilization in comparison with the normal subjects, who relied predominantly on the reflex mechanisms of head stabilization in the plane of progression during walking. This may contribute to the decrease in gaze stabilization in the LD patients resulting in the worsening of their DVA scores. Clinical Significance: DVA is a global indicator of sensorimotor integration. The LDVA test is an easily administered, sensitive test of crew member neurologic function on return from spaceflight. It takes approximately 30 minutes to administer and serves as a guide for flight surgeons in determining the readiness of crew members to return to their activities of daily living such as flying, driving, and operating machinery. It also has an extension into clinical medicine and is currently being evaluawxtas a test to follow the progression of rehabilitation in labyrinthine-deficientpatients. In the above-stated situations, it offers a viable alternative to the more traditional, time-consuming vestibular testing. (Supported by Baylor College of Medicine [D.M.S.], National Research Council [L.A.M.], and the Clayton Foundation for Balance Disorders [H.S.C.].) Poster 22
GABA Transporter mRNAs in the Mammalian Vestibular End Organs MANU GUJRATI (presenter); ALAN ROBINSON PHD; DIMITRI PITOVSKI MD; Chicago IL
Problem Addressed: In the vertebrate vestibular periphery
OtolaryngologyHead and Neck Surgery Research F o r u m - - M o n d a y
Volume 121 Number 2
y-aminobutyric acid (GABA) has long been presumed to be a neurotransmitter candidate. Recent molecnlar biological studies from our laboratory suggest that GABA receptor subunits (al-6, b-l-8, gl-3) are expressed in the vestibular end organs. In a continuation of the investigation of GABA action in the vestibular peripheral system, in this investigation, we set out to determine which GABA transporters are expressed in the vestibular end organs. Methods~Measures: Specific primers, 21 to 25 nucleotides in length, were designed based on published cDNA sequences for GABA transporters types GAT 1, GAT2, GAT3, and GAT4. Total RNA was extracted with guanidine thiocyanate containing 1% Sarkosyl and 50 mM EGTA from individual vestibular end organs of adult male Sprague-Dawley rats. Results: After reverse transcription, resulting cDNA was amplified by PCR, with primer pairs specific for nucleotide sequences representing the GABA transporters. PCR amplification of gene fragments of the GABA transporters was first demonstrated by amplification of cDNA derived from rat brain mRNA, which yielded bands of predicted molecular mass products for each primer pair. In controls designed to check for spurious amplification products due to contaminating genomic DNA, mRNA was treated with DNase, and PCR was carried out without reverse transcription. Conclusions: The results of this study provide direct evidence for the presence of GABA transporters of the detected types in the mammalian vestibular end organs. Clinical Significance: This finding lends new support to previous studies implicating GABA as a vestibular neurotransmitter. The therapeutic effects of various agents, including benzodiazepines, by facilitating GABA-mediated inhibitor neurotransmission may now be fuither elucidated and better understood. Further distribution studies by in situ localization of transporter mRNAs will help define the roles of these transporters in vestibular GABAergic neurotransmission. Poster 23
Auditory Cortical Responses in Patients with Unilateral Bell's Palsy TIMO LAHIN MD (presenter); JUHA-PEKKA VASAMA MD; JYRKI M~,KEI_A MD; Helsinki Finland
Purpose: To examine the effect of unilateral Bell's palsy on auditory-evoked cortical responses, and to seek evidence of possible auditory pathway involvement. Methods: We measured auditory-evoked magnetic responses with a whole-scalp 122-channel SQUID neuromagnetometer from 14 adult patients with unilateral Bell's palsy (9 left sided and 5 right sided) and from 10 normal-hearing control subjects. The method allows simultaneous and noninvasive monitoring of neural activity over both hemispheres with good temporal and spatial resolution. The stimuli were 50-ms 1kHz tone bursts, delivered first to the left and then to the right ear at interstimulus intervals (ISis) of 1, 2, and 4 seconds.
P8w
Results: In patients, the mean pure-tone average was 12 dB on the affected side and 11 dB on the healthy side. The N100m, the 100-ms response originating in the auditory cortices, peaked, on average, 8 ms earlier, and the response strengths were, on average, 7 nAm stronger over the contralateral than the ipsilateral hemisphere. The response latencies and strengths did not differ significantly from those of control subjects. In patients, there were no significant differences in response parameters between the stimulation of the affected and healthy sides. On average, the response latencies and strengths increased as a function of ISI over both hemispheres in both patients and controls. Conclusion: These findings suggest that Bell's palsy does not affect the function of the auditory pathways. Poster 24
Analysis of Chromosome 20QI 3 by FISH in Head and Neck Squamous Cell Carcinoma K KAY DURAIRAJ MD (presenter); LEAH E MINTZ MD; RELEF KRONING PFID; ERI S SRIVATSAN PHD; MARILENE B WANG MD; Los Angeles CA
Problem: Head and neck squamous cell carcinomas remain one of the most aggressive of human cancers. While its etiology is unclear, chromosomal amplifications and deletions within tumor cells are suspected to be a possible avenue of tumorigenesis and immortalization. Chromosome 20 amplification has been detected and previously reported in several human carcinomas, including head and neck squamous carcinomas. Previous studies utilizing comparative genomic hybridization have shown chromosome 20Q amplification in head and neck cancers, perhaps contributing to the mechanism of tumor progression by upregulating cellular oncogenes. We sought to verify the previous reports of chromosome 20Q amplification in head and neck squamous cancers using fluorescence in situ hybridization (FISH). FISH is a technique that permits analysis of chromosomal aberrations on paraffin sections, using fluorescence-labeled DNA probes. Methods: Archived, paraffin-embedded tissue slides of 14 patients with primary squamous carcinoma of the head and neck were analyzed using a standard FISH technique. Chromosome 11 centromeric DNA probes were labeled with fluorophore spectrum green as control probes. Chromosome 20Q DNA was prepared from the BAC plasmid 3B23, containing the chromosome 20q13, and labeled with the fluorophore spectrum orange. After hybridization of the DNA probes to the tissue slides, microscopic analysis of the slides was performed using dual band-pass filter fluorescence microscopy. Signals were then counted. Results: Our preliminary results indicate that, contrary to previous reports of chromosome 20q amplifications and gains in head and neck cancers by comparative genomic hybridization, the technique of FISH demonstrates no significant amplification or deletion at this chromosomal locus.