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

Abstract

Glutamate plays an important role in the central nervous system as an excitatory neurotransmitter. However, its abundance can lead to excitotoxicity which necessitates the proper function of active glutamate transporters. The glutamate-aspartate transporter (GLAST) has been shown to exist and function within non-human cochlear specimens regulating the inner ear glutamate concentration. In this study, we examined micro-dissected human cochleas from formalin-fixed celloidin-embedded temporal bone specimens of three different types of patients (Meniere's disease, normal controls, and other otopathologic conditions) and examined the differential expression of GLAST in the spiral ligament of the basal, middle, and apical turns of the cochlea. Immunohistochemical staining was performed with polyclonal antibodies against GLAST and image analysis was carried out with the Image J analysis software. In contrast to other studies with non-human specimens, GLAST was expressed in the spiral ligament fibrocytes but was not detected in the satellite cells of the spiral ganglia or supporting cells of the organ of Corti in the human cochlea. Our data also showed that GLAST expression significantly differs in the basal and apical turns of the cochlea. Lastly, post-hoc analysis showed a difference in the GLAST immunoreactive area of patients with Meniere's disease when compared to that of patients with other otopathologic conditions—such as presbycusis or ototoxicity. These results may potentially lead to further understanding of different disease states that affect hearing.

Immunohistochemical techniques for the human inner ear

“…Using the preparative methods for IHC described previously (O’Malley et al 2009a, b) adopted to our laboratory protocol, we have been able to detect Tom20 (translocase of the outer mitochondrial membrane-20) (Balaker et al 2013), the glutamate transporter (GLAST) (Ahmed et al 2013), the mu-opioid receptor (Nguyen et al 2014) and recently neuroglobin (Vorasubin et al 2016) in celloidin-embedded sections of the human cochlea. The protocol for IF in celloidin-embedded tissue sections described in this review manuscript allows for colocalization of several proteins in a regular fluorescent microscope or confocal microscope (Fig.…”

“…The specimens are serially sectioned at 20 μm, and every tenth section is stained with hematoxylin and eosin. The celloidin-embedding method yields superb morphological and histopathological preservation of the human inner ear that can be used for IHC (Ganbo et al 1997, 1999; Cunningham et al 2001; Keithley et al 1994, 2000; Markaryan et al 2011; Ying and Balaban 2009; Ahmed et al 2013; Balaker et al 2013; Nguyen et al 2014; Vorasubin et al 2016). It is important to have in consideration the length of fixation specially for IHC.…”

“…Methanol–sodium hydroxide method completely removed celloidin and produced good immunostaining with six antibodies: prostaglandin D synthase, Na + K + -ATPase, aquaporin 1, connective tissue growth factor, tubulin and 200kd neurofilaments. We used sodium ethoxide to remove celloidin from the sections and obtain very good immunoreactive signal with almost no background (Calzada et al 2012a, b; Ahmed et al 2013; Balaker et al 2013; Nguyen et al 2014; Vorasubin et al 2016). …”

Neuroglobin immunoreactivity in the human cochlea

“…Celloidin was removed from the temporal bone sections and then subjected to the antigen retrieval technique (Ahmed et al, 2013; Balaker et al, 2013). In brief, celloidin embedded sections containing the cochlea were mounted on Super frost plus slides (Fisher Scientific) and air dried, then slides were immersed in acetone for 30 minutes followed by sodium ethoxide (1:3 in ethanol) for 30 minutes.…”

VEGF-A Inhibition Ameliorates Podocyte Apoptosis via Repression of Activating Protein 1 in Diabetes

“…Snap-frozen left kidney tissues were used for immunofluorescence staining and TUNEL assay. Images were taken with a BX51 light microscope (Olympus, Tokyo) and the staining intensity of VEGF-A, c-jun, c-fos and bcl-2 expression was measured using ImageJ analysis software (ImageJ 1.44, National Institute of Health) [14]. Details are described in the online supplementary Materials and Methods.…”