Immunohistochemical localization and mRNA expression of aquaporins in the macula utriculi of patients with Meniere’s disease and acoustic neuroma

Abstract

Meniere’s disease is nearly invariably associated with endolymphatic hydrops (the net accumulation of water in the inner ear endolymphatic space). Vestibular maculae utriculi were acquired from patients undergoing surgery for Meniere’s disease and acoustic neuroma and from autopsy (subjects with normal hearing and balance). Quantitative immunostaining was conducted with antibodies against aquaporins (AQPs) 1, 4, and 6, Na+K+ATPase, Na+K+2Cl co-transporter (NKCC1), and α-syntrophin. mRNA was extracted from the surgically acquired utricles from subjects with Meniere’s disease and acoustic neuroma to conduct quantitative real-time reverse transcription with polymerase chain reaction for AQP1, AQP4, and AQP6. AQP1 immunoreactivity (−IR) was located in blood vessels and fibrocytes in the underlying stroma, without any apparent alteration in Meniere’s specimens when compared with acoustic neuroma and autopsy specimens. AQP4-IR localized to the epithelial basolateral supporting cells in Meniere’s disease, acoustic neuroma, and autopsy. In specimens from subjects with Meniere’s disease, AQP4-IR was significantly decreased compared with autopsy and acoustic neuroma specimens. AQP6-IR occurred in the sub-apical vestibular supporting cells in acoustic neuroma and autopsy samples. However, in Meniere’s disease specimens, AQP6-IR was significantly increased and diffusely redistributed throughout the supporting cell cytoplasm. Na+K+ATPase, NKCC1, and α-syntrophin were expressed within sensory epithelia and were unaltered in Meniere’s disease specimens. Expression of AQP1, AQP4, or AQP6 mRNA did not differ in vestibular endorgans from patients with Meniere’s disease. Changes in AQP4 (decreased) and AQP6 (increased) expression in Meniere’s disease specimens suggest that the supporting cell might be a cellular target.

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.…”

The expression of glutamate aspartate transporter (GLAST) within the human cochlea and its distribution in various patient populations

“…Immunoperoxidase staining was performed using Immpact DAB solution (Vector Labs). The reaction was stopped with PBS washes (5 min × 3) after 10–15 s. Slides were then mounted with Vectamount AQ aqueous mounting media and glass cover slips (Lopez et al, 2005; Lopez et al, 2007; Ishiyama et al, 2010). …”

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.…”

“…Quantitative immunocytochemistry for Tom20 in the cochlea was performed as described by Ishiyama et al (2010). To minimize bias in the analysis, the observer was ''blinded'' to the identity of the tissue samples to be analyzed.…”

“…The area immunostained was quantified using the computer image analysis software ImageJ (http://rsb. info.nih.gov/ij/index.html, version v1.47a) with the protocol described by Ishiyama et al (2010) and Calzada et al (2012). Tom20 immunoreactive area was measured at the apical, middle, and basal region of each cochlea section to determine whether there were regional variations in its expression.…”