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R. Kumar-Singh, S. Cashman; Activation of C3 Causes Significant Functional and Anatomical Changes in Mouse Retina. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1934.
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© ARVO (1962-2015); The Authors (2016-present)
Complement activation has been implicated as one of the major causes of age-related macular degeneration (AMD). While the complement cascade can be initiated in one of three distinct pathways, all three become activated by the breakdown of complement component 3 (C3) into the anaphylatoxin C3a and opsonin C3b. With the ever-increasing data implicating complement in AMD, we considered it imperative to determine the effects of complement activation in the retina.
Due to the ability of C3 to be spontaneously converted to the active components, C3a and C3b, we elected to increase expression of C3 in mouse retina by sub-retinal injection of a C3-expressing adenovirus. Scotopic electroretinography (ERG) and fluorescein angiography were performed 7 days post-injection. Eyes were harvested for detailed histological, immunohistochemical and quantitative RT-PCR analysis.
ERGs recorded at two different light intensities showed b-waves to be significantly reduced in C3-injected eyes when compared with both GFP-injected and uninjected (p<0.001) eyes. B-wave amplitudes of C3-injected mice were reduced by 28.5% (0dB) and 26.8% (-10dB) when compared with GFP-injected mice. A-wave amplitudes were observed to be reduced in C3-injected eyes at the higher light intensity (0dB; p<0.047 ) but were not reduced at the lower intensity. Compared with GFP-injected eyes, a-waves of C3-injected mice at 0dB were observed to be reduced by 15.7%. As expected, the ratio of b-wave to a-wave was reduced significantly in C3-injected mice when compared with GFP-injected eyes. This reduction in ratio ranged from 15.6% at 0dB (p=0.0054) to 31.4% at -10dB (p<0.0001). Among the histological changes incurred by C3-injected retinas, we observed loss of pigment and morphological changes in RPE cells, loss of outer segments, as well as retinal detachment. Staining of C3-injected eyes with the endothelial cell marker, GSL I, showed endothelial cells throughout all layers of the retina and along the RPE-retinal interface in the region of detachment. Flatmounts of C3-injected eyecups showed that 43% had GSL I-positive cells covering 14-23% of the RPE-retinal surface. Fluorescein angiography showed greatly increased permeability of retinal vessels, while staining of retinas for glial fibrillary acidic protein indicated significant muller cell activation within the region of increased GSL I reactivity. qRT-PCR confirmed an increase in C3 mRNA levels (~60-fold above uninjected).
C3 causes significant functional and anatomical changes within 1 week of increased expression in mouse retina. This model will be very useful for the development of anti-complement therapies for retinal diseases, such as AMD.
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