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 alpha-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 alpha-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.

Fig. 1

Immunohistochemical controls. a AQP1 immunoreactivity (red) in the human kidney. a’ AQP1 immunoreactivity (red) in human red blood cells. AQP1 localizes on the membrane (arrowheads). b AQP4 immunoreactivity (red) in a cross section of the rat organ of Corti. Inner sulcus and outer sulcus cells are AQP4-immunoreactive (arrowheads). Phalloidin Oregon green staining was used to identify actin in the apical portion of supporting cells (green). b’ AQP4 immunoreactivity in the rat kidney (amber) counterstained with hematoxylin (blue nuclei). c AQP6 immunoreactivity in the rat kidney (amber, arrows) counterstained with hematoxylin (purple nuclei). d Cross section of a human macula utriculi from an autopsy; AQP1 was absorbed with the corresponding peptide. No specific immunoreaction was detected. e Na⁺K⁺ATPase (red) in the rat stria vascularis (SV) and spiral ligament (sl). f NKCC1 in the rat SV, sl, and spiral limbus (right; dark amber). g α-Syntrophin in blood vessels of the mice cerebellum (green). Bars 50 µm (a, b, e, f), 10 µm (a’), 120 µm (b’), 40 µm (c), 45 µm (d), 25 µm (g)

Fig. 2

AQP 1 (aqp1), AQP4 (aqp4), and AQP6 (aqp6) immunoreactivity in the utricular macula of Meniere’s, acoustic, and autopsy patients. AQP1 immunoreactivity (red) in Meniere’s disease (a), acoustic neuroma (a’), and autopsy specimens (a’’) is localized to the fibrocytes beneath the overlying sensory epithelium (se, arrow) and to the fibrocytes in the underlying stroma (st, double arrows) in the utricular maculae (lu luminal region). AQP1 immunoreactivity was not present in the sensory epithelium itself. A quantitative comparison of AQP1 immunoreactivity between the three types of specimens (a’’’) showed no statistically significant differences. AQP4 immunoreactivity (red) in Meniere’s disease (b), acoustic neuroma (b’), and autopsy specimens (b’’) was localized to the basal pole of supporting cells. The AQP4 immunoreactivity in the utricular macula from subjects with Meniere’s disease (b) was significantly diminished in the sensory epithelium (arrows) when compared with that in acoustic neuroma (b’, arrows) and in autopsy material (b’’, arrows). Quantitative immunoreactivity analysis (b’’’) shows a statistically significant decrease in AQP4 immunoreactivity in Meniere’s specimens when compared with acoustic neuroma and autopsy specimens. AQP6 immunoreactivity in Meniere’s disease (c), acoustic neuroma (c’), and autopsy specimens (c’’). In the utricular macula from subjects with Meniere’s disease (c), AQP6 immunoreactivity (red) was diffusely distributed throughout the supporting cell in the sensory epithelium (arrows). In utricular maculae from patients with acoustic neuroma (c’) and normative subjects (c’’), AQP6 immunoreactivity was polarized to the sub-apical portion of the supporting cells. An increased intensity and more diffuse expression of AQP6 (red) was noted in Meniere’s disease (c) compared with acoustic neuroma (c’) and normative (c’’) material. Quantitative immunoreactivity analysis (c’’’) shows a statistically significant increase in AQP6 immunoreactivity in Meniere’s specimens when compared with acoustic neuroma and autopsy specimens. Phalloidin Oregon green staining (green) was used to identify actin at the apical portion of the sensory epithelium. No co-localization was observed between AQP6 (red) and phalloidin (green) corroborating a sub-apical distribution in the supporting cell of utricular maculae derived from acoustic neuroma (b) and normative (c) samples. Bars 50 µm (a–a’’, b–b’’, c–c’’)

Fig. 3

Immunohistochemical localization of protein related to ion homeostasis in Meniere’s disease patients. a Immunoreactivity for Na⁺K⁺ATPase (green) and NKCC1 (red) is similarly located (double arrows; lu lumen, st stroma, se sensory epithelium). b Na⁺K⁺ATPase in nerve fibers terminals and calyces (arrows). Co-localization with myosin VIIa (red) helps to visualize the cytoplasm of vestibular hair cells (hc). c α-Syntrophin shows a ubiquitous distribution in the sensory epithelium. Hair cell (hc) cytoplasm (arrows) and the basal portion of supporting cells (sc, double arrowheads) are syntrophin immunoreactive. d Quantitative comparison of Na⁺K⁺ATPase, NKCC1, and syntrophin immunoreactivity between autopsy and Meniere’s specimens reveals no statistically significant changes. Bars 26 µm (a, b), 28 µm (c)

Fig. 4

Quantification by real-time reverse transcription with the polymerase chain reaction (PCR) of AQP mRNAs extracted from Meniere’s and acoustic neuroma utricles. a Individual mRNA expression in utricles from subjects with Meniere’s disease (md) and acoustic neuroma (an) patients. b Electrophoresis of real-time PCR products of AQP1 (1), AQP4 (4), and AQP6 (6) amplified from Meniere’s utricle. Molecular size markers (m) are indicated (ladder amplicon sizes of 100 bp, 50 bp, and 25 bp)