July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Lacking TNFα accelerates development of Argon laser-induced choroidal neovascularization with augmentation of neutrophil population and increased vascular endothelial cell apoptosis in mice.
Author Affiliations & Notes
  • HIroki Iwanishi
    Ophthalmology, Wakayama Medical University, Wakayama, Japan
  • Takayoshi Sumioka
    Ophthalmology, Wakayama Medical University, Wakayama, Japan
  • Shizuya Saika
    Ophthalmology, Wakayama Medical University, Wakayama, Japan
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 2637. doi:
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      HIroki Iwanishi, Takayoshi Sumioka, Shizuya Saika; Lacking TNFα accelerates development of Argon laser-induced choroidal neovascularization with augmentation of neutrophil population and increased vascular endothelial cell apoptosis in mice.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2637.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : To evaluate the effects of lacking TNFα on the development of experimental choroidal neovascularization (CNV) induced by ocular fundus Argon-laser irradiation in mice.

Methods : Male wild-type (WT) and TNFα-null (KO) mice of 6 - 8 week-old were used. (1) Argon laser-photocoagulation (5 shots, 80 μm spot size, 0.05 sec. duration, 200 mW) was performed in the ocular fundus of WT (n = 15) and KO (n = 15) mice. Mice received FITC-labeled dextran angiography and the size of CNV was evaluated in the flat-mounted retino-choroidal tissue at day7, 14 and 21 with computer software assistance (2) Laser photocoagulation (25 shots) was performed in the eye of WT (n = 60) and KO (n = 60) mice. Total RNA was extracted from retino-choroidal tissue at day 0, 1, 3, 5, 7 and 9. Real-time RT-PCR was ran for mRNAs of VEGF, IL-6, F4/80, TGFb1, MPO, MMP-2, MMP-9 and MCP-1. Primary human retinal microvascular endothelial cells (HRMECs) were plated in well of a 96-well plate at a concentration of 2.0 × 104 cells/well. After incubation for 24hrs, cells were treated with 0, 1, 5, 10 ng/ml TNFa (4 wells for each condition). After 6 or 21hrs treatment, cells were stained using Dead Cell Apoptosis Kit (Cell Signaling) or Hoechst. Fluorescence microscopic observation and software-assisted analysis were performed to examine the effects of supplementation of TNFa on cell apoptosis.

Results : The size of laser-induced CNV formation was was significantly larger in KO mice at day 21, but not other timepoints, than in WT mice (P < 0.05). Lacking TNFa augmented MPO-positive neutrophil infiltration at day1, 3, 5. mRNA expression of F4/80 or IL-6 was suppresses by the loss of TNFa at day 1 or day7, respectively. mRNA expression VEGF, MCP-1,MMP-2, MMP-9 and TGFb1 was not altered by TNFa gene ablation at each timepoint. TNFa treatment increased the number of apoptotic HRMECs with positive Annexin and PI and decreased nucleus number at 21hrs post-supplementation in vitro.

Conclusions : Loss of TNFα accelerates the growth of laser-induced CNV in mice. Mechanism of the KO phenotype might include decreased vascular endothelial cell apoptosis and augmentation of neutrophil population in the treated tissue.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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