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Z. Li, B. Liu, M. Pillai, S. Yeh, R. N. Fariss, M. Campos, R. B. Nussenblatt; Inflammatory Response and Involvement of Immune Components in a Laser Induced CNV (Choroidal Neovascularization) Mouse Model. Invest. Ophthalmol. Vis. Sci. 2008;49(13):201.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the nature and kinetics of the involvement of the immune response using a laser induced CNV mouse model.
C57/B6 and SCID or Rag-1 transgenic mice were used for analysis. A previously established laser induced CNV mouse model was used to generate CNV lesions. Three additive and complimentary cellular and immunological techniques were applied to quantitatively and qualitatively analyze the involvement of immune responses, including 1) immunohistochemistry staining for confocal microscopic analysis for CNV lesions on either flat mount or vibrotome tissue preparations. 2) flow cytometry analysis for immune cell infiltration and 3) antibody-based depleting (for CD25+ Treg) or blocking strategies (for LFA-1) for a functional analysis.
Flat-mount tissue preparation enables quantitatively and qualitatively measuring CNV lesions while vibrotome tissue preparation is best for visualizing the infiltration of inflammatory cells. Flow cytometry is suitable for qualitatively and quantitatively assessing globally the infiltrating immune cells during CNV development. There is an early influx of neutrophils, macrophages, NK cells and microglia cells within 72 hours. Early CNV lesions (within 72hrs) are marked by edema and a random infiltration of immune cells, while lesions at a later stage (7 days) are characterized by a much reduced edema but very organized infiltrating membrane along the region of laser disruption. Microglial cells are also among the earliest to arrive to the laser lesion and persist along with the development of CNV lesions. While no significant differences for CNV development were observed in comparing wild type mice to SCID, or Rag-1 or CD25+ Treg cells depleted mice, there was a significant increase of CNV lesion volume when an LFA-1 blocking antibody was administered.
We have successfully applied several strategies to qualitatively and quantitatively analyze the kinetic involvement of immune cells during a typical laser induced CNV. Our data revealed a complicated but a very well orchestrated infiltration of immune components, mostly innate immune cells, during CNV. It appears that LFA-1 may play a beneficial role for the reversal of CNV. Our data suggest a pivotal role of immune response during CNV development.
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