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S. Zandi, S. Nakao, A. Schering, M. I. Melhorn, S. Miyahara, K. Noda, T. Hisatomi, T. Nakazawa, K. L. Thomas, A. Hafezi-Moghadam; A Novel Method for Analysis of Leukocyte Infiltration Rate During Inflammatory Corneal Angiogenesis. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1490.
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Leukocyte infiltration plays a critical role in pathologic angiogenesis. However, the mechanistic details remain to be investigated. Here we develop a new physiologic method for monitoring leukocyte recruitment in the mouse cornea during inflammatory angiogenesis.
Angiogenesis was induced using the Corneal Micropocket Assay. Briefly, male BALB/c mice were anesthetized, and hydron pellets (0.3 µl), containing mouse IL-1β (30 ng), were implanted into the corneas 1 mm from the the corneal limbus. At 2, 4 and 6 days after pellet implantation, 500 µl of Acridine Orange hemi (zinc chloride) salt (AO, 200 µg/ml) was injected intravenously. The animals were perfused with rhodamine-labeled concanavalin A lectin (ConA, 10 µl/mL in PBS) at 0.5, 2 and 6 h after AO injection to label vascular endothelial cells. Subsequently, animals were perfused with 10 ml PBS to wash out intravascular content and the non-adhering leukocytes. Immediately after perfusion, the corneas were carefully removed, and flatmounts were prepared using a fluorescence anti-fading medium. Photographs were taken using Openlab software and leukocyte numbers were quantified using Image J software’s particle analysis function. Corneal topography was divided into three zones (1-3), the area covering 0 to 500 µm, 500 to 1000 µm, and 1000 to 1500 µm from the limbal vessels.
Two days after pellet implantation 52% and 73% of the leukocytes were located in zone 1, 30 min (410 ± 238) and 2 h (464 ± 137) after AO injection, whereas 6 h (1025 ± 224) after AO injection 40% were found in zone 2.Four days after pellet implantation 92% and 63% of the leukocytes accumulated in zone 1, 30 min (25) and 2 h (480 ± 136) after AO injection, while 6 h (117 ± 41) after AO injection 76% of leukocytes were found in zone 3.Six days after pellet implantation 58% of the leukocytes were situated in zone 1, 30 min (139 ± 27) after AO injection, whereas 93% of leukocytes were located in zone 2, 2 h (301) after AO injection, and 64% in zone 3, 6 h (206 ± 174) after AO injection.
This novel technique allows the measurement of migration rate, the distance traveled from the corneal limbus, and the velocity of infiltrating leukocytes in the inflamed cornea. These parameters may shed light on the mechanisms underlying the various stages of inflammatory angiogenesis and help to develop more effective antiangiogenic ocular treatments.
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