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Rumi Kawashima, Kenji Matsushita, Shinichi Usui, Kohji Nishida; Functional Analysis of Cultured Astrocytes from the Murine Optic Nerve Head. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5595.
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Astrocytes are the major glial cells in the non-myelinated optic nerve head (ONH). Chronic astrocyte activation might affect the optic nerve axons in glaucoma. To study the physiologic functions of ONH astrocytes in vitro, we cultured murine ONH astrocytes, observed the expression of gap junction and aquaporins that allow the ONH astrocytes to form a syncytium, and examined the response to ATP.
We dissected ONH cells from postnatal 1 day C57BL/6 mice ONH and plated the cells onto a dish or the coated coverslips. Pre-confluent and confluent cells were immunostained with antibodies against GFAP, Cx43, AQP4, and AQP9. For flow cytometry, confluent cells were treated and divided into three tubes: 1, an antibody against GFAP, followed by IgG Alexa dye; 2, only IgG Alexa dye; 3, no antibodies. The cells were incubated and analyzed by FACS. Confluent astrocytes were loaded with Fluo4-AM (Dojindo). ATP (100 and 200 μM) was applied and calcium responses were monitored by microscopy (AQUACOSMOS, Hamamatsu Photonics).
A few days after plating, rounded compact cells accumulated; some cells extended branching processes and others extended sheet-like processes. One week after plating the astrocyte count was 555±94 cells/mm2 and 2 weeks after plating the count was 2,315±360 cells/mm2 (n=4). At confluence, all primary cultured cells were stained positively by GFAP. FACS showed a 95.1% GFAP-positive cell population. The astrocytic gap junction protein Cx43-immunoreactive plaques appeared as strings of dots localized to the perinuclear areas and the plasma membrane, especially at areas of cell-to-cell apposition. AQP4 expression in the cultured ONH astrocytes was weak, but AQP9 expression was strong in the membrane domains. ATP generated increased intracellular calcium in the ONH astrocytes for 2 to 4 seconds, which reached the highest levels in a concentration-dependent manner.
We cultured highly purified murine ONH astrocytes. The cultured astrocytes expressed the specific markers for ONH astrocytes as GFAP, Cx43 and AQP9. And they had an ATP-mediated response of increased intracellular calcium. The results indicated that cultured murine ONH astrocytes might regulate the neuronal function by forming a syncytium.
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