Amyloid precursor protein transport block evaluation as a biomarker for experimental mouse glaucoma

Abstract

Purpose : To develop a quantifiable marker of axonal transport blockade, which occurs early in glaucoma and is associated with eventual retinal ganglion axonal loss.

Methods : Experimental microbead glaucoma (Cone et al. 2012) was induced in one eye for 3 days in CD1 (n=59) mice. A subset of mice were pre-treated with glyceraldehyde (Kimball et al. 2014, n=17), losartan (n=10), or irbesartan (n=9) treatments. Oral treatment with losartan (40-60mg/kg/day) and irbesartan (50mg/kg/day) was began 12 days prior to bead injection. Eyes were cryosectioned and immunostained with DAPI and anti-amyloid precursor protein (APP), then imaged with a LSM710 confocal microscope. Pixel brightness histograms were made in ImageJ (FIJI) and analyzed with MATLAB. The mean intensity of the brightest pixels (brighter than 97.5^th percentile of controls) and fraction of brightest pixels (brightest pixels/total pixels) for the pre-optic nerve head (pre-ONH) and unmyelinated optic nerve (UON) were compared between groups.

Results : With 3 day IOP glaucoma, visible clumps of APP within the pre-ONH and UON (Figure 1) led to significantly greater pixel intensity and fraction in glaucoma than control (non-glaucoma) eyes (p<0.05 for all; Figure 2). There was an increase in intensity and pixel fraction in glaucoma + glyceraldehyde eyes compared to control (non-glaucoma) + glyceraldehyde-treated eyes in the UON region (p<0.05). Losartan treatment reduced transport block in the pre-ONH of glaucoma eyes compared to glaucoma only and glaucoma + irbesartan (pixel fraction, p<0.05). The effects in this new marker are consistent with past findings that glyceraldehyde increased glaucoma axonal loss vs glaucoma only (44.6 ± 32.4% vs, 23.0 ± 30.9%, p<0.01), while losartan lowered axonal loss in glaucoma eyes contrasted with glaucoma only (6.2 ± 2.5% vs 25.4 ± 26.4%, p<0.01).

Conclusions : This APP transport block analysis allows an early look at retinal ganglion cell degeneration process in experimental glaucoma.

This is a 2020 ARVO Annual Meeting abstract.

 

Figure 1. APP (green) and DAPI (blue) expression in mouse optic nerve with the pre-ONH (top) and UON (bottom) outlined in white, with increase of APP over control (A) in glaucoma (B). Scale Bar: 100µm.

 

Figure 2. Fraction and mean intensity of brightest pixels above 97.5^th percentile of controls for (A) glyceraldehyde and (B) oral sartan pretreatment of 3 day bead glaucoma mice. *p<0.05 paired test; #p<0.05 unpaired test