September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Changes in immunostaining of five elastic fiber components in Bruch’s membrane and laser-induced choroidal neovascularization in mice
Author Affiliations & Notes
  • Hidetsugu Mori
    Opthalmology, Kansai Medical University, Hirakata, Osaka, Japan
  • Haruhiko Yamada
    Opthalmology, Kansai Medical University, Hirakata, Osaka, Japan
  • Kanji Takahashi
    Opthalmology, Kansai Medical University, Hirakata, Osaka, Japan
  • Tomoya Akama
    Pharmacology, Kansai Medical University, Hirakata, Osaka, Japan
  • Tomoyuki Nakamura
    Pharmacology, Kansai Medical University, Hirakata, Osaka, Japan
  • Footnotes
    Commercial Relationships   Hidetsugu Mori, None; Haruhiko Yamada, None; Kanji Takahashi, None; Tomoya Akama, None; Tomoyuki Nakamura, None
  • Footnotes
    Support  none
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 5794. doi:
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      Hidetsugu Mori, Haruhiko Yamada, Kanji Takahashi, Tomoya Akama, Tomoyuki Nakamura; Changes in immunostaining of five elastic fiber components in Bruch’s membrane and laser-induced choroidal neovascularization in mice. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5794.

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

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Abstract

Purpose : Fibrillin-1, Developmental Arteries and Neural Crest Epidermal growth factor-like (DANCE), tropoelastin and Latent Transforming growth factor beta-Binding Protein-2 and -4 (LTBP-2 and -4) are essential proteins for elastic fiber formation. In this study, we analyzed the distribution of these five elastic fiber components in the elastic lamina of the Bruch’s membrane and laser-induced choroidal neovascularization (LiCNV) using immunofluorescence staining.

Methods : Twenty eyes of ten C57BL/6 female mice (8-10 weeks) were used. LiCNVs were induced in ten eyes by intense laser photocoagulation (532nm, 50μm, 350mW; GYC-2000®, NIDEK, Aichi, Japan). One week after photocoagulation, the eyes were enucleated and immediately frozen (-80°C). Eight micrometers thin-sliced sections were cut using a cryostat. Frozen sections were fixed with methanol at -20°C for 5 minutes, washed with phosphate-buffered saline (PBS) for 5 minutes and blocked with 4% blocking agent for 20 minutes. They were incubated with primary antibodies (Fibrillin-1, DANCE, tropoelastin, LTBP-2 or -4) for 1 hour and were visualized with Alexa-488/546 and DyLight650 secondary antibodies for 1 hour at room temperature. After washing 3 times with PBS, all slides were mounted with ProLongGold® (Thermo Fisher Scientific, Waltham, USA) and observed under a fluorescence microscope (BZ-X700®; KEYENCE, Osaka, Japan).

Results : These five proteins (Fibrillin-1, DANCE, tropoelastin, LTBP-2 and -4) were expressed both in the elastic lamina of the Bruch’s membrane and inside LiCNVs in all eyes. The localization of these five proteins was similar. However, the signal intensity of DANCE was very faint compared to a positive control. Moreover, these five proteins were stained more intensely inside LiCNVs compared to the elastic lamina of the Bruch’s membrane.

Conclusions : In this study, we elucidated that Fibrillin-1, DANCE, tropoelastin and LTBP-2 and -4 were expressed in the elastic lamina of the Bruch’s membrane and LiCNVs. The weak signal of DANCE implies that DANCE may have less involvement with elastic fiber formation in eyes. The roles of five elastic proteins in the formation of CNV will be investigated in future studies using knockout mice for each individual gene.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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