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Yan Guo, Zara Mehrabyan, Carol Charniga, Sally Temple, Steven Bernstein; Primary Culture and Characterization of Mouse Laminar Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5162.
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The optic nerve lamina (ONL) is the ON region where retinal ganglion cell axons become myelinated, and plays an important role in many optic nerve diseases. Many studies have looked at this unique region, but the conditions needed for mouse lamina primary cell culture are unknown. We wanted to establish a method to isolate and characterize the mouse ONL to further aid the study of this unique region.
The first 2mm of ONL were obtained from p10-p16 CD-1 mice. Laminae were isolated by removing the ON sheath and sclera followed by enzymatic dissociation. The dissociated cells were seeded in a poly-ornithine coated plate. Cells were grown in a serum free culture media for up to 14 days and evaluated on phase-contrast microscope. We performed immunohistochemistry (IHC), using Nestin, Ng2, GFAP and CNPase antibodies, and examined staining by confocal microscopy. We also evaluated the gene expression from lamina and optic nerve by qPCR from P15 mice. qPCR was performed using SYBR Green Supermix on an iCycler. Only reactions yielding a single melting curve were used in the quantitative analysis.
Phase contrast microscopy showed cells with different morphologies. Both round and polygonal cell bodies were seen, with thin and long processes. These were consistent with astrocytes and oligodendrocytes. Immunofluorescent staining showed Nestin (+) differentiating cells with and without Ng2 immunopositivity, and Ng2(+) with or without GFAP or CNPase. Gene expression analysis revealed that there was differential gene expression between lamina and optic nerve. Ng2 expression is higher in the lamina, while GFAP, MBP, oligo-2 and S100β were higher in the optic nerve.
Primary cultures of mouse ONL can be generated and used to study gene expression with current technology. Immunohistochemistry and gene expression analysis reveals that the ONL possesses cells capable of differentiating, and also that there are different cells possessing unique gene signatures in the laminar region, consistent with its unique functions. This approach enables the use of isolated mouse ONL in primary cultures and will certainly aid the study of this unique region.
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