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K. Hockmann, M. Duong, A. K. Ball; Activated Microglia are Neuroprotective Following ET-1 Mediated RGC Death. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3241.
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ET-1 injection into the eye causes gradual, specific retinal ganglion cell (RGC) loss over 4 weeks accompanied by a loss of axons within the optic nerve. We have previously shown that following ET-1 mediated injury microglia (MG) become activated, proliferate, and migrate within retinal layers. We have also previously demonstrated that inhibition of MG activation with minocycline exacerbates ET-1 mediated RGC loss, suggesting MG may be protective. In the present study we demonstrate the neuroprotective role of controlled MG activation following ET-1 mediated injury.
Rat RGCs were retrogradely labelled with Fluorogold (FG) from the superior colliculus (SC) 2 days prior to intraocular injection with ET-1 (5µl; 500µM). Animals were intravitreously injected with saline (0.5 ml) or clodronate liposomes (CL;5µL) immediately post to ET-1 injection to decrease the intraocular MG population. In another experimental group, rats were injected with zymosan (60µg;5µL) 3 days following ET-1 mediated injury. All animals were killed at 7, 14, 21 and 28 days post-ET-1 injection, and their eyes were fixed in phospate buffered 4% formaldehyde and processed for immunohistochemistry. RGCs were identified using antisera directed against FG and examined by confocal microscopy. Micrographs were taken from the midperiphery of each retinal quadrant and the number of RGCs counted in each sample. Retinas were also processed for double label immunohistochemistry using antisera directed against Iba-1 and ED-1.
Normal, untreated retinas expressed only Iba-1 and lacked the microglial activation marker ED-1. ET-1/saline treated animals were immuno-positive for both Iba-1 and ED-1 similar to ET-1/zymosan and ET-1/CL treated animals. ET-1/saline treated animals experienced a 21%, 22%, 33% and 41% loss of RGC density after 7, 14, 21 and 28 days respectively when compared to untreated animals. Inhibitory modulation of MG with minocycline significantly increased RGC loss to 45% and 57% only after 21 and 28 days respectively. CL administration did not significantly alter RGC loss when compared to ET-1/saline treated animals. In contrast, controlled activation of MG with zymosan resulted in a 15%, 18%, 30% and 29% loss of RGCs after 7, 14, 21 and 28 days, respectively.
Inhibition of microglia with anti-inflammatory drugs has deleterious effects on RGC survival at 4 weeks post ET-1 mediated injury whereas controlled activation of microglia induces a neuroprotective effect on RGCs following ET-1 mediated injury. These finding provide evidence for a neuroprotective role for microglia in the ET-1 model of RGC injury.
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