Table 1.
Experimental study groups and the number of animals within each.
Table 2.
Primary antibodies used for histological studies and Immunoblot.
Fig 1.
Calibration of Tonolab tonometer in cannulated eyes from drug-treated and water control eyes after treatment for 1 and 6 weeks. The regression lines for each group closely track with ideal calibration of manometrically-set IOP (error bars are standard error).
Table 3.
Average IOP after bead injection glaucoma.
Table 4.
Blood pressure data.
Table 5.
Axon loss by treatment group.
Table 6.
Multivariable model for percent axon loss.
Fig 2.
Axonal transport effects, APP labeling with losartan-treated vs water control.
Immunolabeling for APP in the optic nerve head of mice treated with oral losartan (A and C) and water alone (B and D) shows axonal transport obstruction 3 days after IOP elevation. The examples show 4 of the 5 grade levels for APP accumulation (level 1, not illustrated, had no label, indicating that there was no accumulation and no background or non-specific labeling). The grades of losartan-treated eyes ranged from 2 to 4 (A and C; mean = 2.6 (sd 0.8), while water-treated eyes ranged from 3 to 5 (B and D; mean = 4.0 (sd 0.9); difference from losartan, p = 0.007, multivariable regression adjusted for IOP exposure). Bar equals 50 um.
Fig 3.
Losartan is not neuroprotective in a crush model of axon injury.
Images of RGCs stained with Sncg from the sham losartan group (A), crush water group (B) and crush losartan group (C), showing no difference in RGC survival from losartan treatment from axon crush. The graph shows all four groups, with the sample sizes for Sham Water, Crush Water, Sham Losartan and Crush Losartan groups n = 9, 9, 10 and 13 respectively.
Table 7.
Axial length and width.
Table 8.
Unfixed Peripapillary Thickness.
Table 9.
Fixed Peripapillary Thickness.
Fig 4.
The effect of losartan on creep rate in ramp—hold inflation studies.
The creep rate during a 30 minute period at 30 mm Hg for losartan- and water-treated glaucoma eyes was compared to control eyes (represented by the horizontal line drawn at the ratio of one), along with losartan without glaucoma compared to control eyes as a ratio. Losartan treatment alone and losartan-glaucoma eyes had no difference in creep rate from controls, but water-treated glaucoma eyes had a greater creep rate than controls.
Fig 5.
Pressure—strain estimates in load—unload protocol for losartan and control groups.
Pressure—strain relationships in load—unload inflation tests show steeper (stiffer) curves for glaucoma eyes of losartan (triangle) and water-treated (diamond) compared to their respective fellow, control eyes (x and square, respectively). Y axis = inflation pressure in mm Hg.
Fig 6.
RTPCR of angiotensin receptor expression.
Graph plots the relative expression of five tissues tested for the presence of AT1a, AT1b, and AT2 receptors- kidney, adrenal gland, retina, sclera and optic nerve from a CD1 mouse. To help normalize the results, a value was calculated by dividing the GAPDH primer treated tissue value by the mean quantification cycle (Cq) value for AT1A, AT1B, or AT2 primer tissue (relative expression). A lower value than 1 indicates a lower value for that receptor in tissue compared to GAPDH.
Fig 7.
Western blots comparing losartan and water groups.
Immunoblots for pERK1/2, ERK1/2, pSmad2, pSmad3, Smad2/3 and GAPDH for 12 scleral specimen groups. (1) water control; (2) losartan control; (3) fellow eyes of water-treated glaucoma; (4) fellow eyes of losartan-treated glaucoma; (5) water-treated glaucoma; and (6) losartan-treated glaucoma. Graphs show quantification by densitometry of blots, indicating the ratio of the activated molecules to the total amount of that molecular species: pERK/total ERK, pSmad2/total Smad2/3, and pSmad3/total Smad2/3. The comparisons are among all types of glaucoma eyes compared to naïve control eyes at 3 days (left group) and 1 week (right group) after glaucoma. Water-glaucoma group in blue shows 3-fold elevation in pERK/ERK ratio, while no difference from control is seen in losartan-glaucoma eyes. At one week, both water and losartan glaucoma eyes have substantial reduction in pERK/ERK ratio compared to controls. Very mild increases are seen in pSmad ratios in either water or losartan groups. See S1 Fig for original western blots.
Fig 8.
Immunohistochemical labeling for thrombospondin 1.
Immunostaining for thrombospondin-1 shows mild scleral label (arrowheads) in water-treated fellow eye (A) and dramatically increased label in both sclera and retina of water-glaucoma eye (B). Thrombospondin 1 label was less in sclera and retina of both the losartan fellow eye (C) and losartan-glaucoma eye than the corresponding water controls (D, scale bar = 50μm; DAPI counterstain).