NF-κB activation in retinal microglia is involved in the inflammatory and neovascularization signaling in laser-induced choroidal neovascularization in mice

Exp Cell Res. 2021 Jun 1;403(1):112581. doi: 10.1016/j.yexcr.2021.112581. Epub 2021 Mar 31.

Abstract

Purpose: To evaluate Nuclear Factor NF-κB (NF-κB) signaling on microglia activation, migration, and angiogenesis in laser-induced choroidal neovascularization (CNV).

Methods: Nine-week-old C57BL/6 male mice were randomly assigned to IMD-0354 treated or untreated groups (5 mice, 10 eyes per group). CNV was induced with a 532-nm laser. Laser spots (power 250 mW, spot size 100 μm, time of exposure 50 ms) were created in each eye using a slit-lamp delivery system. Selective inhibitor of nuclear factor kappa-B kinase subunit beta (IKK2) inhibitor IMD-0354 (10 μg) was delivered subconjunctivally; vehicle-treated mice were the control. The treatment effect on CNV development was assessed at five days post-CNV induction in vivo in C57BL/6 and Cx3cr1gfp/wt mice by fluorescent angiography, fundus imaging, and ex vivo by retinal flatmounts immunostaining and Western blot analysis of RPE/Choroidal/Scleral complexes (RCSC) lysates. In vitro evaluations of IMD-0354 effects were performed in the BV-2 microglial cell line using lipopolysaccharide (LPS) stimulation.

Results: IMD-0354 caused a significant reduction in the fluorescein leakage and size of the laser spot, as well as a reduction in microglial cell migration and suppression of phospho-IκBα, Vascular endothelial growth factor (VEGF-A), and Prostaglandin-endoperoxide synthase 2 (COX-2). In vivo and ex vivo observations demonstrated reduced lesion size in mice, CD68, and Allograft inflammatory factor 1 (IBA-1) positive microglia cells migration to the laser injury site in IMD-0354 treated eyes. The data further corroborate with GFP-positive cells infiltration of the CNV site in Cx3cr1wt/gfp mice. In vitro IMD-0354 (10-25 ng/ml) treatment reduced NF-κB activation, expression of COX-2, caused decreased Actin-F presence and organization, resulting in reduced BV-2 cells migration capacity.

Conclusion: The present data indicate that NF-κB activation in microglia and it's migration capacity is involved in the development of laser CNV in mice. Its suppression by NF-κB inhibition might be a promising therapeutic strategy for wet AMD.

Keywords: IKK-β; IKK2; IMD-0354; Laser CNV; Microglia; NF-κB; Retina.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Choroidal Neovascularization / drug therapy
  • Choroidal Neovascularization / pathology
  • Disease Models, Animal
  • Gene Expression Regulation / physiology*
  • I-kappa B Proteins / metabolism
  • Inflammation / metabolism
  • Lasers
  • Mice
  • Mice, Inbred C57BL
  • Microglia / metabolism*
  • NF-kappa B / metabolism*
  • Retina / metabolism*

Substances

  • I-kappa B Proteins
  • NF-kappa B