September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
BAX Oligomerization Kinetics in Retinal Ganglion Cells after Optic Nerve Injury are comparable to in vitro Oligomerization Kinetics
Author Affiliations & Notes
  • Margaret Maes
    Opthalmology, University of Wisconsin- Madison, Madison, Wisconsin, United States
  • Footnotes
    Commercial Relationships   Margaret Maes, None
  • Footnotes
    Support  NIH T32 GM081061
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 605. doi:
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      Margaret Maes; BAX Oligomerization Kinetics in Retinal Ganglion Cells after Optic Nerve Injury are comparable to in vitro Oligomerization Kinetics. Invest. Ophthalmol. Vis. Sci. 2016;57(12):605.

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

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Abstract

Purpose : Glaucoma is an optic neuropathy characterized by BAX-dependent apoptosis of retinal ganglion cells (RGCs). BAX forms oligomers at the mitochondrial outer membrane, which marks the ‘point of no return’ for an apoptotic cell. Once initiated, BAX oligomerization proceeds rapidly in tissue culture systems, however the process in living RGCs is undocumented. Understanding the pattern of progression and the kinetic profile of BAX oligomerization in RGCs after injury is imperative to developing effective therapeutics to prevent RGC death.

Methods : BAX oligomerization was monitored after optic nerve crush (ONC) in CB6F1 and BALB/cByJ mice, and in a chronic injury model using DBA2/J mice. Intravitreal injections of AAV2-GFP-BAX were used to introduce GFP-BAX into mouse RGCs. Static images of whole mounted retina were collected at 1, 3, 5, 7 and 14 days after ONC or at 6, 8, 10 and 12 months in DBA2/J mice. Longitudinal imaging of GFP-BAX was performed using a blue light confocal scanning laser ophthalmoscopy with a Heidelberg spectral domain OCT with image capture at 1, 3, 5, 8 and 14 days after ONC. Live imaging of retinal explants was performed four days after ONC. Two photon intravital imaging of BAX oligomerization kinetics in BALB/cByJ mice is underway. IMARIS 7.7 was used for kinetic analysis of time-lapse videos.

Results : The percentage of transduced RGCs exhibiting BAX oligomers after ONC in CB6F1 mice increased at days one (18%, p < 0.05) and three (39%, p < 0.05), then peaked at five days (47%, p < 0.05). The same pattern of progression to peak oligomerization was represented in BALB/cByJ mice. Studies in DBA2/J mice are ongoing. Longitudinal in vivo imaging using the Heidelberg Spectralis showed that BAX expressing RGCs begin to be cleared by 8 days after injury. Live cell imaging of retinal explants revealed that once activated, the BAX oligomerization process in RGCs occurred rapidly and was complete within 25 mins, comparable to tissue culture cells.

Conclusions : The rapid completion of the oligomerization process, along with the consistent pattern of progression to peak BAX oligomer formation among mouse strains and optic nerve injury models, demonstrates that effective therapies preventing RGC death need to be administered prior to peak BAX oligomerization.

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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