Relocalization of Apoptosis-Inducing Factor in Photoreceptor Apoptosis Induced by Retinal Detachment in Vivo

Abstract

Apoptosis-inducing factor (AIF) is a novel mediator in apoptosis. AIF is a flavoprotein that is normally confined to the mitochondrial intermembrane space, yet translocates to the nucleus in several in vitro models of apoptosis. To investigate the role of AIF in the apoptotic process in vivo, we induced retinal detachment (RD) by subretinal injection of sodium hyaluronate, either in Brown Norway rats or in C3H mice. Apoptotic DNA fragmentation, as determined by terminal nick-end labeling, was most prominent 3 days after RD. The subcellular localization of AIF was examined by immunohistochemistry and immunoelectron microscopy. In normal photoreceptor cells, AIF was present in the mitochondrion-rich inner segment. However, AIF was found in the nucleus after RD. Photoreceptor apoptosis developed similarly in C3H control mice, and in mice bearing the gld or lpr mutations, indicating that cell death occurs independently from the CD95/CD95 ligand system. Both the mitochondrio-nuclear transition of AIF localization and the nuclear DNA fragmentation were inhibited by subretinal application of brain-derived neurotrophic factor. To our knowledge, this is the first description of AIF relocalization occurring in a clinically relevant, in vivo model of apoptosis.

Intraocular expression and release of high-mobility group box 1 protein in retinal detachment

“…Previous studies indicated that HMGB1 could not be released from apoptotic cells 6 and the apoptotic photoreceptors were prominent in this RD model at day 3 after RD. 22 We also confirmed remarkable numbers of apoptotic photoreceptors in the detached retina at day 3 (Figure 3b), and found that the early faint TUNEL-positive nuclei had relatively low levels of HMGB1 and fragmented nuclei, which were brightly stained by TUNEL, had almost no apparent HMGB1 immunoreactivity (Figure 3c), suggesting that apoptotic dying cells might lose the expression of HMGB1 to maintain the proper gene transcription. It might be indispensable for the surviving photoreceptors to maintain and/or boost the nuclear HMGB1 in RD.…”

“…22 Briefly, the rats were anesthetized with an intramuscular injection of ketamine and xylazine, and their pupils were dilated with topical 1% tropicamide and 2.5% phenylephrine hydrochloride. The retinas were detached using a subretinal injection of 1% sodium hyaluronate (Opegan; Santen, Osaka, Japan) with an anterior chamber puncture to reduce intraocular pressure.…”

“…After overnight incubation, sections were washed and probed with Alexa-Fluor 594-conjugated goat anti-rabbit IgG F(ab 0 )2 fragment (Molecular Probes, Carlsbad, CA) for 1 h. In some experiments, TUNEL co-staining was also performed according to the manufacturer's protocol (ApopTag Fluorescein In situ Apoptosis Detection kit; Chemicon, Temecula, CA) as previously described. 22 Slides were counterstained with DAPI, mounted with Shandon PermaFlour (Thermo Scientific, Waltham, MA), and viewed with a Zeiss fluorescence microscope. Images were captured using the same exposure time for each comparative section.…”

Expression of antiapoptotic and proapoptotic molecules in diabetic retinas

“…42,43 In addition, the distribution of AIF expression in nondiabetic retinas observed in the present study is consistent with a previous animal study. 44 Diabetic retinas showed upregulation of cytochrome c and AIF immunoreactivity. Our data for cytochrome c in diabetic retinas are in agreement with several studies reporting increased cytochrome c immunoreactivity and activity in the retina postaxotomy and following optic nerve crush 43,45 suggesting that one of the early responses in the retina after optic nerve injury is to scale up the energy production.…”

Toxic effects of extracellular histones and their neutralization by vitreous in retinal detachment

“…The sections were incubated with the secondary antibody for 1 h. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) costaining was performed using an ApopTag Fluorescein In Situ Apoptosis Detection Kit according to the manufacturer's protocol (Chemicon, Temecula, CA, USA). 17 The slides were counterstained with 4,6-diamidino-2-phenylindole (DAPI), mounted with Shandon PermaFluor (Thermo Scientific, Waltham, MA, USA), and viewed with an Olympus fluorescence microscope (Olympus, Tokyo, Japan). Images were obtained using the same exposure time for each comparative section.…”