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Elizabeth Fassbender, Yubin Qiu, Siyuan Shen, Amber Woolfenden, Andrea Delpero, Yong Kim, Bruce D Jaffee, Stephen H Poor; Intravitreal (IVT) injection exacerbates murine choroidal neovascularization (CNV) area by a MYD88 dependent mechanism. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1194. doi: https://doi.org/.
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To compare the effect of route of administration of vehicle and the role of innate immunity in the mouse laser-induced CNV model.
We performed an analysis of CNV area in mice dosed vehicle by different routes of administration. CNV area in age and sex matched C57/BL6 mice were investigated after systemic or IVT administration of a toll receptor 3 activator, Polyinosinic:polycytidylic acid (poly I:C) or vehicle. A single 50µg dose or vehicle was administered i.v. at day -1 or day 2. A single IVT injection of 1.5 or 3.8µg poly I:C or vehicle was injected immediately after laser. CNV area was assessed in littermate age and sex matched MYD88 KO and toll receptor (TLR) 2 KO mice. Laser pulses were applied with an Iridex Oculight® GLx 532nm laser, 3 lesions/eye, 6 lesions/mouse, 9-15 mice/group yielding 54-90 data points per treatment group. On day 7 after laser, blood vessels were labeled with an i.v. injection of FITC-ConA and mice were euthanized. Eyes were enucleated and fixed in paraformaldehyde. Posterior ocular segments were dissected and mounted onto slides. CNV lesions were captured by fluorescent microscopy and CNV area was analyzed with Axiovision software. Analysis and exclusions were completed on masked data. Statistics were analyzed by an unpaired two tailed t test in Graph pad prism Vs6.
CNV area was increased 42% in mice injected IVT with vehicle compared to mice receiving vehicle systemically (n=21 IVT vehicle studies, mean CNV area=17.0 ± 5.5 mm2 x 10-3; n=20 systemic vehicle studies, mean CNV area=12.8 ± 3.4 mm2 x 10-3, p=0.0066). CNV area in mice administered i.v. (n=2 studies) or IVT (n=3 studies) poly I:C was similar in size to CNV area in mice injected with saline (p>0.05). CNV area was similar in male MYD 88 KO mice and littermate controls (n=3 studies, p>0.05). Both male and female MYD88 KO mice injected with IVT saline had smaller CNV than control mice (male n=2 studies, mean % CNV inhibition in KO compared to WT=47%, p≤0.0038; females n=2 studies, mean % CNV inhibition in KO compared to WT=48%, p≤0.0042). CNV area was similar in size between TLR2 KO mice and controls injected IVT with saline (n=2 studies, p>0.05).
IVT injection exacerbates CNV area compared to non-injected mice. IVT injection increases CNV area through a MYD88-dependent mechanism, not through TLR2. Activation of TLR3 does not alter CNV area.
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