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Jenifer Van, Jing Cui, Eleanor To, Patrick McGeer, Joanne Matsubara; Systemic administration of aurin tricarboxylic acid (ATA) reduces C5b-9 immunoreactivity and TUNEL+ cells in the rodent eye: A potential treatment for suppressing complement activation and apoptosis associated with AMD. Invest. Ophthalmol. Vis. Sci. 2013;54(15):158.
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Several key components of the complement cascade are associated with the pathogenesis of age related macular degeneration (AMD), including the Y402H variation in the gene encoding complement factor H (CFH), which confers a several-fold increased risk for developing AMD. Earlier studies, including ours, demonstrated the presence of complement proteins, including C5b-9, in the outer retina of postmortem eyes with drusen, the hallmark deposit of early stage AMD. It is hypothesized that early treatment to inhibit the complement cascade may prevent the progression to late stage AMD. This study investigates the effect of ATA, on C5b-9 accumulation and apoptosis in the rodent retina.
Twelve C57B/6 mice, aged 3.5 month, were divided into a control group (Group 1) and two experimental groups receiving a subcutaneous injection of 200mg/kg (Group 2) or 500mg/kg (Group 3) of ATA dissolved in PBS. After 24 hrs, animals were sacrificed, eyes enucleated and fixed in 4% paraformaldehyde. Tissue was embedded in paraffin and 4 µm thick sections subjected to TUNEL assay following manufacturer’s protocol (Millipore) or immunohistochemistry using a 1:500 dilution of rabbit polyclonal antibody against C5b-9 (BIoss). Under 40X, TUNEL+ cells were counted, C5b-9 immunoreactivity was scored semiquantitatively and retinal thicknesses measured.
ATA treatment reduced retinal C5b-9 immunoreactivity from 3.0 + 0.00 (no treatment), to 1.4+ 0.83 (200mg/kg) and 1.1+ 0.68 (500mg/kg), reaching significance at p<0.05 between Group 1 vs 2 and 3. ATA treatment reduced numbers of TUNEL+ cells in the ONL from 93.0+ 30.47 (no treatment) to 90.7 + 65.69 (200mg/kg, p>0.05) and to 10.5+ 3.69 (500mg/kg, p<0.05) per 8379 µm2 area of ONL, indicating significant anti-apopotic effects at the higher dosage of 500 mg/kg ATA. Significant differences in retinal thickness measurements were not observed.
Systemic administration of ATA (MW=422) has been shown to block the addition of C9 to C5b-8 in several tissues (Lee et al 2012). This study demonstrates significant lowering of C5b-9 immunoreactivity and apoptosis in rodent retina. Future studies are needed to assess whether inhibitors of C5b-9 formation, such as ATA, may be useful in combination treatment for multifactorial diseases such as AMD.
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