Abstract
Purpose:
Microglia are the principal immune cells of the central nervous system, and have several important functions in immune surveillance and neuronal homeostasis. However, there is increasing evidence that microglia can produce and release angiogenic factors and may be involved in neovascularisation. We examined the role of the proangiogenic factor Angiotensin II (AngII) in activation of microglia in the retina.
Methods:
Transgenic heterozygote (Cx3cr1+/GFP) and control (C57bl6) mice were studied in these experiments. Cx3cr1+/GFP animals are normal with the exception that microglia are labeled with Enhanced Green Fluorescent Protein (eGFP). Three to 6-month old Cx3Cr1+/GFP mice received 1µl intravitreal injections of 10mM AngII, with the fellow eye receiving 1µl saline. Retinae were collected after 24hrs and processed for immunocytochemistry, neuronal apoptosis, and microglia/neuronal contacts. Labelling and microglia morphology were assessed in both vertical sections and wholemount retinae. Metamorph Offline® software was used for morphological analyses of microglia.
Results:
All retinal microglia in both Cx3cr1+/GFP and control mice expressed the Angiotensin type 1 receptor (AT1-R). The injection of AngII resulted in altered microglial morphology consistent with activation, characterised by increased soma size (n=5, p<0.001) and decreased process length (n=5, p<0.05) compared to vehicle injected retinae. There was no significant differences in the number of microglia in Ang II injected retina compared to vehicle injected control eyes (n=5, p>0.05). Analysis of neuronal apoptosis and Müller cell gliosis revealed no significant differences between vehicle and AngII injected retinae.
Conclusions:
We show for the first time that retinal microglia express the AT1-R, and that increases in AngII cause microglial activation within 24 hours. These changes are independent of cell death and Müller cell gliosis. The results suggest that AngII may directly activate AT1-Rs on microglia to induce an activation response and a morphological phenotype suggestive of retinal inflammation. This may have implications for diseases like diabetic retinopathy in which increases in retinal AngII and neuroinflammation have been found to have an important role.
Keywords: 595 microglia •
540 glia •
700 retinal neovascularization