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RC Hunt, R Ganti, H Lu, K Dutt, A Davis, M Hunt; Identification of the Metallothionein Isoforms Expressed by Human Retinal Cells Using Laser Capture Microdissection . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4560.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:Metallothioneins (MTs) are small proteins that may act as anti-oxidants. They are expressed in many tissues including retina in which anti-oxidant proteins may be important since the retina is the most highly oxygenated tissue in the body. Diseases such as retinopathy of prematurity may result from insufficient anti-oxidant protection. Humans express several MTs: MT-1 and MT-2 are found in many tissues while MT-3 is found in the brain and MT-4 in stratified squamous epithelial cells. There are several types of MT-1 and MT-2. We investigated the MTs that are expressed in various cells of the retina. Methods:MTs were detected in retinal sections by immunocytochemistry. To identify the MTs expressed by various cells in the human retina, donor eyes were fixed, frozen and sectioned. Cells of the pigment epithelium, inner and outer nuclear layers and the ganglion cell layer were isolated using laser capture micro-dissection with an Arcturus PixCell system. RNA was extracted, copied to cDNA and RT-PCR, with specific primers, was used to identify MT species present in each cell type. Results:Using immunocytochemistry, it was found that MTs are expressed in all layers of the retina including inner and outer nuclear layer, pigment epithelium and ganglion cells, with the most intense staining in the latter cell type. Neither immuno-cytochemistry nor in situ hybridization can readily distinguish the expression of the various classes of MT because of the similarity of the proteins and their mRNAs. It was found that retinal pigment epithelial cells express MT-1a and MT-2a but MT-3 could not be detected. The three neural cell types expressed MT-3 together with MT-2a but not MT-1a. Conclusion: Laser capture micro-dissection allows identification of the MT species made by as few as 100 captured cells. MT expression in the retina is cell type-specific with brain-associated MT-3 being made only by cells of the neural retina and not the pigment epithelium while MT-1 was made only by retinal pigment epithelial cells. All cell types made MT-2.
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