Our laboratories are dedicated to studying the anatomy, pathology and cellular and molecular biology of the human inner ear. We use a multidisciplinary approach that includes the use of classical anatomical techniques as well as modern cellular and molecular biological methods: immunocytochemistry, unbiased quantitative stereology, transmission electron microscopy, real-time RT-PCR (for gene expression), and recently, proteomics.
Cross-sections from the crista ampullaris (A and B) and utricular macula (C) from specimens of group 2.
(A) Specific neurofilament immunoreactivity is seen within nerve fibers throughout the crista stroma (St) (arrows). (B) A higher magnification view from the insert in (A). Immunoreactive calyceal terminals surrounding type I hair cells (I) within the sensory epithelia can be seen. (C) In the utricular macula, immunoreactive nerve fibers and calyceal terminals surrounding type I (I) hair cells can be identified. Arrows point to transversal section of immunoreactive axoplasm located in the tissue stroma. Lu: lumen. Magnification bar is (A) is 150μm, in (B) is 40μm and in (C) is 25μm.
The Microdissection Technique
To perform state-of-the-art cellular and molecular studies in the human inner ear, our laboratory has implemented the microdissection technique, which allows for rapid fixation due to the immediate removal of the encasing temporal bone, with resultant morphological preservation. Also it is possible to properly orient the different endorgans on the inner ear.
- Principal Investigator: Akira Ishiyama, MD
- Collaborators: Gail Ishiyama, MD, Neurology, UCLA; Robert Baloh, MD, Neurology, UCLA; Ivan A. Lopez PhD, Head and Neck Surgery, UCLA
1. Quantitative stereological studies in normal aging and pathology
During the past 10 years, our laboratory has been dedicated to implementing the most modern methodology for quantification and identification of pathological changes in the human inner ear. For this purpose, our laboratory is equipped with state-of-the-art microscopes coupled to digital cameras and sophisticated hardware and software systems (Cast Grid System) to apply unbiased stereological methods to obtain counts of cells in 3-dimensional structure by sampling in 2 dimensions, free of sampling and systematic bias. Using this methodology we have detected changes in the number of hair cells and supporting cells in the human vestibular periphery, and changes in the number of neurons, volume of the stria vascularis and spiral ligament with age as well as disease (i.e. Meniere's disease).
a) Immunocytochemical studies
Using the microdissected endorgans, we have been able to detect changes in structural and functional proteins in the human inner ear using immunocytochemistry. With this technique we are able to localize in situ the expression of proteins found in the basement membranes, proteins involved in water transport and proteins affected in disease (i.e. cochlin, the most abundant protein in the inner ear). We use vestibular endorgans surgically obtained, normal tissue microdissected from temporal bones (obtained at autopsy) and more recently from archival temporal bone celloidin-embedded (type of plastic) tissue sections.
b) In situ hybridization studies
Using a similar approach, we are implementing techniques to detect changes in the acid nucleic of the inner ear (mRNA and DNA). Using cryostat sections, we have been able to detect opiod receptors present in the inner ear.
2. Molecular biological studies
Using vestibular endorgans obtained during surgery, we are studying the expression of genes involved in ionic and water homeostasis, and the expression of extracellular-matrix-related genes. Our laboratory is equipped with a real-time polymerase chain reaction machine that allow us to detect changes in 84 genes per sample.
3. Pathological studies using the UCLA temporal bone bank
The UCLA Temporal Bone laboratory houses more than 1000 specimens, many of which have been processed (approximately 400). Information regarding age, gender, race, clinical diagnosis, and histopathology is entered into a database compatible with the National Registry database. The UCLA temporal bone laboratory contains specimens across a wide range of disease. With this archive, we have been able to apply unbiased stereology and study changes with age and disease (Meniere's disease, Sjorgren's syndrome), and effects after ototoxic treatment. We are currently using archival celloidin sections to detect quantitative changes in basement membrane proteins. This temporal bone bank collection is used for many research studies in United States and Europe, as well as by fellows interested in pursuing careers in otology, and by medical students. We constantly interchange temporal bone stained sections with the temporal bone at Harvard and the House Ear Institute at Los Angeles, interchange possible by the Human Temporal bone Consortium for Research Resource Enhancement, from which UCLA is part and supported by NIH.
4. Studies on aging
The effect of age on the human inner ear and brain is also investigated in our laboratory. Specifically, we study the changes in the number of vestibular sensory hair cells and nerve fibers during age and or disease. Histopathological, immunohistochemical and quantitative analysis is performed at the light and TEM level in postmortem specimens —inner ear and brain— as well as inner ear tissue obtained from surgery.
5. Additional projects
Additional projects performed in our laboratory include the study of expression of ion channels, transporters, receptors and neurotransmitters in the human and animal inner ear using immunohistochemistry (Dr. Gail Ishiyama and Dr. Ivan Lopez).
Human temporal bone celloidin embedded sections (from the UCLA temporal bone bank) stained with hematoxylin and eosin. (A) Normal view of the human cochlea (organ of Corti). Arrows point to the normal Reissner's membrane (B) and (C) shows endolymphatic hydrops from two patients diagnosed with Meniere's disease. Arrows point to the distension of the Reissner's Membrane. Magnification bar is 250 μm.
- Ishiyama A, López I, Wackym P. Subcellular innervation patterns of the calcitonin gene-related peptidergic efferent terminals in the chinchilla vestibular periphery, Otolaryngology-Head and Neck Surgery 111, 385-395, 1994.
- Ishiyama A, López I, Wackym P. Choline acetyltransferase immunoreactivity in the human vestibular end-organs, Cell Biol. Int. 18, 979-984, 1994.
- Ishiyama A, López I, Wackym P. Distribution of efferent cholinergic terminals and alfa-bungarotoxin binding to putative nicotinic acetylcholine receptors in the human vestibular periphery, Laryngoscope. 105, 1167-1172, 1995.
- Baloh RW, López I, Honrubia V, Ishiyama A, Wackym P. Vestibular Neuritis: Clinical-pathological correlation. Head and Neck-Otol. 114:586-592, 1996.
- Ishiyama A, Ishiyama G, López I, Eversole L, Honrubia V, Baloh RW. Histopathology of idiopathic chronic recurrent vertigo. Laryngoscope 106:1340-1346, 1996.
- Ishiyama A, López I, Wackym P. Molecular characterization of muscarinic receptors in the human vestibular periphery. Am J Otology, 18, 648-654, 1997.
- Baloh RW, López I, Beykirch K, Ishiyama A, Honrubia V. Clinical-pathological correlation in a patient with selective loss of hair cells in the vestibular endorgans. Neurology, 49, 1377-1382, 1997.
- Kim JS, López I, DiPatre PL, Liu F, Ishiyama, A, Baloh, RW. Internal auditory artery infarction: clinico pathologic correlation. Neurology, 52, 40-44. 1999.
- Ishiyama G, López I, Ishiyama A. Subcellular immunolocalization of NMDA receptor subunit NR-1 in the chinchilla vestibular periphery. Brain Research. 851,270-276, 1999.
- Park JJ, Tang Y, López I, Ishiyama A. Unbiased stereological quantification of neurons in the human vestibular ganglion. Neuroreport. 11,853-857, 2000.
- Lee H, López I, Ishiyama A, Baloh RW. Can migraine damage the inner ear? Arch Neurology 57, 1631-1634, 2000.
- Park J, Tang Y, López I, Ishiyama A. Age-related changes in the number of neurons in the human vestibular ganglion. J Comp Neurol 431,437-443, 2001.
- Ishiyama A, Agena J, López, I and Tang Y. Unbiased stereological quantification of neurons in the human spiral ganglion. Neuroscience Letters, 304, 93-96, 2001.
- Park J, Tang Y, López I, Ishiyama A. Unbiased estimation of human vestibular ganglion neurons. Ann NY Acad Sci, 942, 475-478, 2001.
- Tang Y, López I, Ishiyama A. Application of unbiased stereology in archival human temporal bone. Laryngoscope 112, 526-533, 2002.
- Ishiyama G, López I, Williamson R, Acuna D, Ishiyama A. Subcellular distribution of NMDA subunit NR1,2A ,2B in the rat vestibular periphery. Brain Research, 935, 16-23, 2002.
- Ishiyama G, López I, Baloh RW, Ishiyama A. Cannavan's leukodystrophy is associated with defects in cochlear neurodevelopment and deafness. Neurology, 60:1702-1704, 2003.
- Gopen Q, López I, Ishiyama G, Baloh RW and Ishiyama A. Unbiased stereological quantification of type I and type II hair cells in the human utricular macula. Laryngoscope 113:1132-1138, 2003.
- Ishiyama A. López I, Ishiyama G, Tang Y. Unbiased quantification of the microdissected human Scarpa's ganglion neurons. Laryngoscope, 114:196-1499, 2004.
- Ishiyama A, Ishiyama G, López I , Jen J, Kim G, Robert W. Baloh RW. Temporal bone histopathology in dominantly inherited audiovestibular syndrome. Neurology, 63:1859-1862, 2004.
- López I , Ishiyama G, Tang Y, Frank M, Baloh RW, Ishiyama A. Estimation of the number of nerve fibers in the human vestibular endorgans using unbiased stereology and immunohistochemistry. Journal of Neuroscience Methods, 145: 37-46, 2005.
- López I, Ishiyama G, Tang Y, Tokita J, Baloh RW, Ishiyama A. Regional estimates of hair cells and supporting cells in the human crista ampullaris. Journal of Neuroscience Research, 82:421-431, 2005.
- Ishiyama G, Finn M, López I, Tang Y, Ishiyama G. Unbiased quantification of Scarpa's ganglion neurons in aminoglycoside ototoxicity. J Vestibular Research, 15,197-202, 2005.
- Kho S, López IA, Evans C, Ishiyama A, Ishiyama G. Immunolocalization of Orphanin/FQ in Rat Cochlea. Brain Research, 113:146-152, 2006.
- Ishiyama G, López IA, Ishiyama A. Aquaporins and Meniere's disease. Curr Opin Otolaryngol Head and Neck Surg, 14:332-336, 2006.
- Ishiyama G, López IA, Ishiyama A. Histopathology of the vestibular end organs after intratympanic gentamicin failure for Meniere's disease. Acta Oto-Laryngologica, 127:34-40, 2007.
- Ishiyama G, Tokita J , López IA, , Tang Y, Ishiyama A. Unbiased stereological analysis of the human spiral ligament and stria vascularis: a temporal bone study. JARO, 8:8-17, 2007.
- López IA, Ishiyama G, Lee M, Baloh RW, Ishiyama A. Immunohistochemical localization of aquaporins in the human inner ear. Cell & Tissue Research, 328:453-460, 2007.
- Merchant SN, McKenna MJ, Adams JC, Nadol JB Jr, Fayad J, Gellibolian R, Linthicum FH Jr, Ishiyama A, López IA, Ishiyama G, Baloh R, Platt C. Human temporal bone consortium for research resource enhancement. J Assoc Res Otolaryngol, 9:1-4, 2008.
- Ishiyama A, Mowry SE, López IA Ishiyama G. Immunohistochemical distribution of basement membrane proteins in the human inner ear. Hearing Research, 2009, 254:1-14.
- McCall A, Ishiyama G, López IA, Sunita B, Ishiyama A. Histopathological and ultrastructural analysis of vestibular endorgans obtained from patients with Meniere's disease. BMC Ear Nose Throat Disord. 2009;9:4.
- Miller M, Ishiyama G, López IA, Ishiyama A. Endolymphatic Hydrops in Otologic Syphilis: A temporal bone study. Otology Neurotology, 2010, In Press.
Director: Akira Ishiyama, MD
Email: [email protected]
Phone: (310) 206-2041
Fax: (310) 794- 5089