Immunohistochemical distribution of basement membrane proteins in the human inner ear from older subjects

Abstract

The immunolocalization of several basement membrane (BM) proteins was investigated in vestibular endorgans microdissected from temporal bones obtained from subjects with a documented normal auditory and vestibular function (n = 5, average age = 88 years old). Fluorescent immunostaining using antibodies directed at collagen IVα2, nidogen-1, laminin-β2, α-dystroglycan, and tenascin-C was applied to cryosections from human cochlea, cristae ampullares, utricular and saccular maculae. Collagen IVα2, nidogen-1, and laminin-β2 localized to all subepithelial cochlear BMs, Reissner's membrane, strial and spiral ligamental perineural and perivascular BMs, and the spiral limbus. Tenascin-C localized to the basilar membrane and the osseous spiral lamina. α-dystroglycan localized to most cochlear BMs except those in the spiral ligament, basilar membrane and spiral limbus. Collagen IV, nidogen-1, and laminin-β2 localized to the subepithelial BMs of the maculae and cristae ampullares, and the perineural and perivascular BMs within the underlying stroma. The BM underlying the transitional and dark cell region of the cristae ampullares also expressed collagen IV, nidogen-1, and laminin β2. Tenascin-C localized to the subepithelial BMs of the utricular maculae and cristae ampullares, and to calyx-like profiles throughout the vestibular epithelium, but not to the perineural and perivascular BMs. α-dystroglycan colocalized with aquaporin-4 in the basal vestibular supporting cell, and was also expressed in the subepithelial BMs, as well as perivascular and perineural BMs. This study provides the first comprehensive immunolocalization of these ECM proteins in the human inner ear. The validity of the rodent models for inner ear disorders secondary to BM pathologies was confirmed as there is a high degree of conservation of expression of these proteins in the human inner ear. This information is critical to begin to unravel the role that BMs may play in human inner ear physiology and audiovestibular pathologies.

Immunohistochemical techniques for the human inner ear

“…Earlier studies utilized cytologic descriptions and graphic reconstructions of the cochlea (Guild 1921). Temporal bone science has advanced such that we are now entering a phase of methodological integration, whereby the same temporal bone can be used for light microscopy, transmission electron microscopy (TEM), immunohistochemistry (IHC), non-radioactive in situ hybridization, DNA and proteomics analysis (Aarnisalo et al 2010; Kong et al 1998; Merchant et al 2008; Ishiyama et al 2009, 2010; Lopez et al 2005a, b, 2007; Markaryan et al 2008a, b, c, 2009a, b, c, 2010a, b; Nguyen et al 2014; Richard et al 2015; Schrott-Fischer et al 1994, 2002a, b, 2007; Wackym et al 1990). The study of the human inner ear has lagged behind other areas of pathology, in large part due to the inaccessibility of the membranous labyrinth.…”

Morphometric analysis of human sciatic nerve perineurial collagen type IV content

“…Distribution of collagen type IV isoforms in human Bruch's membrane was evaluated by Das et al (1990) and Chen et al (2003). Immunohistochemical distribution of BM proteins in human inner ear was studied by Ishiyama et al (2009). They performed their research on temporal bone samples of older cases, but their study was basically morphological, without quantification and consequent comparison with younger healthy control group.…”

“…There are literature data about aging changes of collagen type IV in BM of the human brain blood vessels (Uspenskaia et al, 2004), then blood vessels of the eye (Chen et al, 2003), ear (Ishiyama et al, 2009), and glomeruli in kidney (Anderson and Brenner, 1986;Bloom et al, 1959). First analysis of peripheral nerves extracellular matrix components were performed on rats (Lorimier et al, 1992).…”

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

“…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.…”