Effects of TPCA-1-loaded PLGA microparticles on macrophage recruitment to the laser lesions.

<p>Retrobulbar injection of 100-μL microparticle suspensions containing either PLGA (10 mg) alone or PLGA loaded with TPCA-1 (10 mg/0.8 mg) was performed on the mice at age of 8-week-old, and followed immediately by laser burn of Bruch’s membrane. After 24, 48, or 72 hours, mice were euthanized, and the eyes were dissected and fixed in 4% PFA. The posterior eye cups with attached RPE layer were washed with 1xPBS, stained with F4/80 antibody, and flat-mounted for fluorescent imaging. (A) The representative flat-mount images show that F4/80-stained macrophages appear to have migrated to CNV lesions at 24, 48, or 72 hrs post-laser. Images 1–8 of the laser lesion area on the whole-mounted eye cups were taken at low magnification, and the images 9–14 were taken at higher-magnification. The white arrows indicate optic disks. The scale bar in image A1 represents the magnification of images A1–8, and the scale bar in A9 represents the magnification of A9–14. Scale bar, 250 μm. (B) The number of anti-F4/80-stained macrophages in the choroid/RPE surface were counted and expressed as mean±SD, n = 4 eyes for each condition. Statistics were performed using a two-tailed Student’s <i>t</i>-test to compare the values between the PLGA and PLGA/TPCA-1-treated eyes. ***<i>p</i> < 0.005. (C) mRNA expression of <i>Vegfa</i> and <i>Ccl2</i> determined via qPCR. RNA samples were collected from the choroid/RPE tissues of control and treated mice. The CT method normalized to <i>Gapdh</i> was used to analyze relative changes in gene expression. The relative expression (arbitrary units) is expressed as a ratio. Data are expressed as mean±SD, n = 3 experiments. Statistics were performed using a two-tailed Student’s <i>t</i>-test to compare the values between the PLGA- and PLGA/TPCA-1- treated eyes. *<i>p</i> < 0.05, ***<i>p</i> < 0.005.