Abstract
Purpose :
Glaucoma is a leading cause of irreversible blindness, affecting retinal ganglion cells (RGCs) through sensitivity to intraocular pressure (IOP). A body of evidence supports the hypothesis of compartmentalized degeneration, where varied degenerative programs affect the different RGC components (soma, dendrites, and axon). However, evidence shows deletion of Bax, which is associated with cell apoptosis, partially protects axons. We test whether Bax-/-, with elevated IOP, affects optic nerve axon profiles.
Methods :
We increased IOP unilaterally by microbead occlusion of the anterior chamber in 1- 3-month-old Bax-/- and wild type C57 (WT) mice. An equivalent volume of PBS was injected into the contralateral eye as an internal control. IOP was monitored twice a week using applanation tonometry. Four weeks after IOP elevation, we perfused the animals with 0.1M cacodylate buffer followed by 2.5% glutaraldehyde and isolated 3 mm segments of optic nerve proximal to the globe. We embedded the optic nerves segments in Epon resin and obtained ultra-thin (70nm) cross-sections. Ultra-thin sections were prepared and photographed at 2700X- (ten images per nerve for axon count) and 240X-magnification (one image per nerve to measure cross-sectional nerve area) using a Philips CM-12, 120 keV transmission electron microscope at Vanderbilt Cell Imaging Shared Resource (CISR) Core. A naïve observer manually counted total and degenerated axons using ImageJ software.
Results :
Elevated IOP did not significantly alter the cross-sectional nerve area for either C57 or Bax-/- animals (P=1.0). However, Bax-/- optic nerves were inherently larger than C57 nerves (96309±11609 vs 60420±4974µm2, P=0.017). Increased IOP also did not affect the number of axons within WT or Bax-/- nerves (P≥0.88). Although, Bax-/- nerves possessed greater axon density per nerve (0.86±0.02 vs 0.55±0.05µm2, P>0.001). Finally, Bax-/- and WT nerves showed a similar percentage of degenerating axons (2.1±0.4 vs 2.9±0.9%), and IOP did not significantly change the percentage of degenerating axon profiles in Bax-/- and WT nerves (3.2±0.9 vs 2.1±0.4%, P=0.67).
Conclusions :
Four weeks of IOP elevation did not impact optic nerve cross-sectional area, axon density, or percentage of degenerating axon profiles in WT or Bax-/- animals. To test our hypothesis that IOP-dependent axon degeneration is independent of Bax activation, next we will increase IOP for an extended duration.
This is a 2020 ARVO Annual Meeting abstract.