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Comments of Dr. Ishiyama

One important aspect of the temporal bone research is the need for the development of new techniques and the need for the use of modern scientifically sound techniques. I feel that it is critical that temporal bone researchers integrate with basic scientists and clinicians from other disciplines to develop new fields of study in human temporal bone research. I have been active in developing and utilizing the microdissection technique. I work closely with Dr. Yong Tang, an experienced research scientist in the field of anatomy and unbiased stereology. Together we have developed the application of unbiased stereology in temporal bone research. While we are able to obtain unbiased estimates of Scarpa's ganglia and spiral ganglia neuronal number using archival temporal bones, we are not able to use unbiased techniques in the neuroepithelium nor the vestibular and auditory nerves using archival specimens. Therefore, we altered the temporal bone harvest protocol by conducting immediate microdissection of the temporal bone specimens at the time of autopsy. The microdissection technique allows for the proper orientation of each endorgan and the nerve bundle such that unbiased stereology can be applied. Furthermore, because specimens can be sectioned ultra-thin (1-2 microns), and because of the elimination of the decalcification and dehydration steps, morphological preservation is excellent. Using microdissection and the physical fractionator-stereology technique, we obtained total type I and type II vestibular hair cell counts in the human utricular macula. Unbiased stereology has undoubtedly become the gold standard for morphometric measures, and therefore it is important that we use unbiased stereology for morphometric measures.

Other advantages of the microdissection technique include the ability to use pre-embedded immunohistochemistry. Whole mount endorgans can be stained and properly oriented and photographed. The tissue can then be removed from the slide, re-embedded in plastic, properly oriented, and thin cross-sections made. Antigen retrieval techniques can be used to restore antigenicity of specimens which had been in formalin storage. Other potential studies include the combination of the microdissection technique with molecular biological techniques such as in situ hybridization, or polymerase chain reaction.

We need to standardize the clinical history and objective audiovestibular testing from prospective temporal bone donors. It would be ideal to use standardized questionnaires by all of the active temporal bone research laboratories for comparative purposes. In cases of inheritable audiovestibular disorders, outcome of genetic testing should be included. It is not uncommon to encounter temporal bone specimens with limited clinical information. In order to avoid this problem, continued efforts need to be made to contact the temporal bone donors periodically to update the clinical information. If funds can be made available by NIDCD for each active temporal bone research laboratory, an administrative assistant can organize and facilitate these efforts. Also, laboratories which are inactive or active and harbor valuable archival temporal bones should allow for the sharing of the temporal bone specimens if not currently being used.

With regard to the logistics of training of future temporal bone researchers, I think we should establish workshops sponsored by NIDCD. These workshop should be constructed by identifying active temporal bone research laboratories with particular areas of strength. For example, to train future temporal bone histopathologists, a workshop organized by an institution such as the Mass Eye and Ear would be ideal. For morphometric study of the temporal bone, UCLA can organize a workshop on unbiased stereology.

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