Estimation of the number of nerve fibers in the human vestibular endorgans using unbiased stereology and immunohistochemistry

López, Iván; Ishiyama, Gail; Tang, Yong; Frank, Michael; Baloh, Robert W.; Ishiyama, Akira

doi:10.1016/j.jneumeth.2004.11.024

Cited by 38 publications

(54 citation statements)

References 43 publications

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“…The microdissection technique that we used was as previously described (Hawkins and Johnsson 1976;LarsGoran and Hawkins 1972;Lee et al 1990;Lopez et al 2005b;Wright and Meyerhoff 1989). In brief, under the operating microscope, the middle ear was opened, and the ossicles were carefully removed.…”

Section: Microdissection Of Auditory and Vestibular Endorgansmentioning

confidence: 99%

Immunohistochemical localization of aquaporins in the human inner ear

et al. 2007

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We report the immunolocalization of aquaporins (AQPs) 1, 4, and 6 in the human auditory and vestibular endorgans. A rapid protocol was applied to audiovestibular endorgans microdissected from postmortem human temporal bones from six subjects (ages ranging from 75 to 97 years) with no history of audiovestibular disease. Temporal bones were fixed in formalin, and the endorgans were immediately microdissected. Cryostat sections were obtained from audiovestibular endorgans and were subjected to double-immunohistochemical staining with antibodies against AQPs and several cellular markers. In the human cochlea, AQP1 immunoreactivity was localized to the fibrocytes of the spiral ligament and the sub-basilar tympanic cells; AQP4 immunoreactivity was localized to the outer sulcus cells, Hensen's cells, and Claudius' cells; AQP6 immunoreactivity was localized to the apical portion of interdental cells in the spiral limbus. In the vestibular endorgans (macula utriculi and cristae), AQP1 was localized to fibrocytes and blood vessels of the underlying stroma and trabecular perilymphatic tissue; AQP4 immunoreactivity was localized to the basal pole of vestibular supporting cells; AQP6 was localized to the apical portion of vestibular supporting cells. Cochlear and vestibular hair cells and nerve fibers were not immunoreactive for any AQP. Supporting cells were identified with antibodies against glial fibrilar acidic protein. Nerve fibers and terminals were identified with antibodies against neurofilaments and Na(+)K(+)ATPase. The high degree of conservation of AQP expression in the human inner ear suggests that AQPs play a critical role in inner ear water homeostasis.

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Section: Microdissection Of Auditory and Vestibular Endorgansmentioning

confidence: 99%

Immunohistochemical localization of aquaporins in the human inner ear

et al. 2007

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“…Temporal bone removal and tissue processing: methods for human tissue processing have been described in detail (Lopez et al, 2005Ishiyama et al, 2009Ishiyama et al, , 2010. In brief, at autopsy the whole brain, including the brainstem and blood vessels, was removed from the cranial cavity.…”

Section: Methodsmentioning

confidence: 99%

“…Thereafter, the fixative was removed by washing with PBS (10 min Â 3). The temporal bones were placed under a dissecting microscope (Nikon SMZ1500), and using forceps, the muscle, connective tissue, and bone surrounding the membranous labyrinth were carefully removed as previously described (Lopez et al, 2005). Individual endorgans (i.e., crista, macula, and cochlea) were then removed and immersed in 30% sucrose in PBS for 7 days.…”

Section: Methodsmentioning

confidence: 99%

Immunocytochemical Localization of the Translocase of the Outer Mitochondrial Membrane (Tom20) in the Human Cochlea

Balaker

Ishiyama

López

et al. 2012

The Anatomical Record

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Mitochondrial degeneration in the inner ear is likely a contributing factor in age-related hearing loss and other otopathologies such as Meniere's disease. Most mitochondrial proteins are synthesized in the cytosol and imported through the mitochondrial membranes by translocators. The translocase of the outer membrane (Tom) is the universal entry gate for all proteins that are imported into mitochondria. Altered function of the translocator could alter protein transport into the mitochondria, and disrupt function. In this study, we determined the immunolocalization of Tom20, a major mitochondrial protein import receptor, in microdissected human cochlea frozen sections obtained from postmortem autopsy and celloidin-embedded archival specimens. We used affinity purified rabbit polyclonal antibodies against Tom20. We also determined the Tom20 immunolocalization in the mouse inner ear. In the human and mouse cochlea, Tom20 was ubiquitously distributed in the organ of Corti, allowing well-delineated visualization of inner and outer hair cells. Tom20 immunoreactivity localized in the cytoplasm of spiral ganglia neurons. In the inner ear of aged subjects with Meniere's disease, there was decreased expression of Tom20. These results suggest that Tom20 can be used in the inner ear as a marker for mitochondrial protein import. Anat Rec,

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“…Equipment setup was identical to that described in a previous study by Lopez et al (2005a). The volume was estimated with unbiased stereological techniques using Stereo Investigator version 5.0 (MicroBrightField Inc., Colchester, VT, USA) on a Windows-based personal computer.…”

Section: Equipmentmentioning

confidence: 99%

“…The present study uses the unbiased stereological technique, Cavalieri principle, to estimate the volume of the SV and the SL in archival human temporal bone specimens. Our laboratory has successfully applied unbiased stereological techniques by using postmortem human temporal bone specimens to obtain type I and type II hair cell counts in the human vestibular endorgans (Lopez et al 2005b;Gopen et al 2003), vestibular ganglion neuronal number in aminoglycoside ototoxicity (Ishiyama et al 2005) and in aging (Ishiyama et al 2004;Park et al 2001), normative spiral ganglion number , and vestibular nerve fiber counts and diameter distribution (Lopez et al 2005a). Previous studies have applied unbiased stereology to obtain cell volume density estimates of SV in the chinchilla by using a pointcounting technique (Santi and Lakhani 1983;Santi and Muchow 1979).…”

Section: Introductionmentioning

confidence: 99%

Unbiased Stereological Estimation of the Spiral Ligament and Stria Vascularis Volumes in Aging and Ménière’s Disease Using Archival Human Temporal Bones

Ishiyama

Tokita

López

et al. 2006

JARO

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The present study applies the unbiased stereological technique-Cavalieri principle to measure the volumes of the stria vascularis (SV) and the spiral ligament (SL) using postmortem archival human temporal bones from normal young and older subjects and subjects with Ménière_s disease. Normative data was obtained from subjects without ages ranging from 15 to 84 years old who had no history of audiovestibular disease (N = 25). For comparison purposes, the normative specimens were divided into three groups: group 1 (n = 8) had ages ranging from 15 to 38 years old, average age = 23.9; group 2 (n = 8) had ages ranging from 51 to 59 years old, average age = 55.1; group 3 (n = 9) had ages ranging from 64 to 84 years old, average age = 74.3. The average SV volume of group 3 (0.479 mm 3 ) was significantly lower than that of group 1 (0.705 mm 3 ) (p G 0.0005) and was significantly lower than that of group 2 (0.603 mm 3 ) (p = 0.01). The average SL volume of group 3 (8.42 mm 3 ) was significantly lower than that of group 1 (9.54 mm 3 ) (p G 0.05), but was not significantly lower than that of group 2 (8.58 mm 3 ). Five subjects with Ménière_s disease, confirmed by histopathological examination (ages ranging from 63 to 91 years old, average age = 73.4), were studied. The average SV volume in Ménière_s subjects (0.378 mm 3 ) was significantly lower than age-matched controls (p G 0.05). The average SL volume in Ménière_s subjects (7.01 mm 3 ) was also significantly lower than age-matched controls (p G 0.05). The SV and SL volumes were unaffected by gender. The present study demonstrates for the first time the use of the unbiased stereological technique-Cavalieri principle-as a reliable and efficient method to obtain volumetric estimates of the SV and the SL by using archival human temporal bone specimens.

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Estimation of the number of nerve fibers in the human vestibular endorgans using unbiased stereology and immunohistochemistry

Cited by 38 publications

References 43 publications

Immunohistochemical localization of aquaporins in the human inner ear

Immunohistochemical localization of aquaporins in the human inner ear

Immunocytochemical Localization of the Translocase of the Outer Mitochondrial Membrane (Tom20) in the Human Cochlea

Unbiased Stereological Estimation of the Spiral Ligament and Stria Vascularis Volumes in Aging and Ménière’s Disease Using Archival Human Temporal Bones

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