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KM Engelsberg, J Wassélius, B Ehinger, K Johansson; Microglia/Macrophage response in Cultured Rat Retina . Invest. Ophthalmol. Vis. Sci. 2002;43(13):758.
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Purpose: Optic nerve transection results in degeneration of ganglion cells, which is accompanied by microglial reactions. To study proliferation, activation and migration of microglia cells and macrophages after axotomy, we investigated the immunohistochemical expression of PCNA, ED-1 and actin on cultured rat retinas. Methods: Explanted neonatal rat retinas were kept for 1, 2 and 6 days in vitro in DMEM/F12 under standard culture conditions, followed by paraformaldehyde fixation and cryostat sectioning for immunohistochemistry. Microglial cells were identified using isolectin B4 labeling. Combined lectin staining and immunolabeling was performed with antibodies against PCNA, ED-1and actin. Double immunolabeling protocols for ED-1/PCNA and ED-1/actin were used to visualize dividing and migrating cells. Lectin staining combined with ED-1/caspase-3 double immunolabeling was used to disclose phagocytosing cells in the vicinity of apoptotic ganglion cells. Results: After one day in culture, numerous cell somata displayed caspase-3 immunoreactivity in the ganglion cell layer (GCL), and were approached by a low number of lectin stained and ED-1 positive cells. Only a part of the lectin positive microglia expressed ED-1 labeling. PCNA labeling revealed the presence of dividing microglia. This pattern was also evident after 2 days in culture, but the fraction of lectin/ED-1 positive cells had increased. At this stage, activated and migrating ED-1/actin positive microglia was observed at different levels in the neuroblast layer (NBL). Also, there was a considerable reduction of caspase-3 positive cells in the GCL. After 6 days in culture, neither caspase-3 positive cells nor PCNA positive cells were observed in the GCL. Almost all of the microglia cells were also positive for ED-1. Conclusion: Axotomy followed by apoptosis among ganglion cells appears to trigger the proliferation and activation of microglial cells already after 1 day in culture or less. Proliferation declines during continued culture whereas the number of activated microglial cells increases with time. Migration of microglia also appears to be an early response following explantation.
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