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Kristopher G. Sheets, Bokkyoo Jun, Yongdong Zhou, Jeremy Winkler, Min Zhu, Nicos Petasis, William C. Gordon, Nicolas G. Bazan; Topical Neuroprotectin D1 Attenuates Experimental CNV And Induces Activated Microglia Redistribution. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5470.
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Neuroprotectin D1 (NPD1) attenuates laser-induced CNV when administered by intraperitoneal injection (PMID 21035444). Topical application, however, is less invasive and targets tissue locally rather than systemically. We tested retinal bioavailability after delivery of deuterium labeled NPD1 to the eye surface. We then assessed the effectiveness of topical NPD1 against laser-induced CNV.
For bioavailability, 10 ng of NPD1-d2 was applied topically to right eyes of C57/BL6 mice, retinas collected after 1h, and analyzed by LC/HESI-tandem mass spectrometry. For CNV, 3 laser-induced lesions were made per eye. Right eyes received 16 ng NPD1 once daily for seven days; left eyes were vehicle. Leakage was analyzed by high-resolution angiography at 7 and 14 days post-laser. Choroid flatmounts were prepared at 15 days post-laser, labeled with isolectin B4, CD11b, and Hoechst, then visualized by confocal microscopy. Lesion area was measured using isolectin B4 and microglia distribution was determined using CD11b. Images were quantified with ImageJ and statistics were performed with SAS.
NPD1-d2 was detected in ipsilateral retinal tissue only. Leakage in vehicle-treated eyes improved between 7 and 14 days post-laser. NPD1 treatment, however, reduced 7 day leakage 32%, to a level below the natural resolution of the model. Neovascular area in NPD1-treated eyes was 12,043 µm2, 56% smaller than the 27,266 µm2 of controls. Amoeboid microglia covered the sub-retinal surface of lesions while peripheral microglia at the RPE surface had hyper-ramified morphology. The number microglia was similar between vehicle- and NPD1-treated eyes. Distribution of peripheral hyper-ramified microglia, however, was significantly different. In vehicle-treated eyes, the average distance between microglia and the neovascular perimeter was 403 µm. NPD1 treatment reduced this 51% to a mean distance of 199 µm.
These results demonstrate that NPD1 applied topically reaches the retina in effective concentrations, reducing leakage and neovascularization associated with laser-induced CNV. Interestingly, NPD1 alters the distribution of microglia associated with CNV. NPD1 binds leukocytes and RPE cells with high-affinity (PMID 19520558). Leukocytes and microglia are of myeloid origin, thus NPD1 may also bind microglia. NPD1 triggered signaling attenuates injury induced inflammation and pathoangiogenesis.
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