Rodent anterior ischemic optic neuropathy (rAION) induces regional retinal ganglion cell apoptosis with a unique temporal pattern

Abstract

Purpose: Nonarteritic anterior ischemic optic neuropathy (NAION) results in optic nerve damage with retinal ganglion cell (RGC) loss. An NAION model, rodent anterior ischemic optic neuropathy (rAION), was used to determine AION-associated mechanisms of RGC death and associated regional retinal changes.

Methods: rAION was induced in male Wistar rats, and the retinas analyzed at various times after induction. RGCs were positively identified by both retrograde fluorogold labeling and brain-expressed X-linked protein-1/2 (Bex1/2) immunoreactivity. RGC death was analyzed by fluorescein-tagged annexin-V labeling (FITC-annexin-V), as well as by terminal nucleotide nick-end labeling (TUNEL). Retinal flatmount preparations enabled regional retinal analysis of labeled dying cells. Apoptosis pathway activation was confirmed by Western analysis, with an antibody that recognizes cleaved caspase-3.

Results: Post-rAION, RGCs die by apoptosis over a longer period than previously recognized. Cleaved caspase-3 immunoreactivity was greatest between 11 and 15 days. rAION-induced RGC death occurs regionally, with sparing of large contiguous regions of RGCs.

Conclusions: rAION results in later RGC death than in traumatic optic nerve damage models. Apoptosis, measured by FITC-annexin, occurs maximally in the second to third week after infarct. Cleaved caspase-3 activation confirms that after rAION, RGCs undergo apoptosis by the caspase activation pathway. The regional pattern in dying RGCs after rAION implies that a measure of retinotopic organization occurs in the rodent optic nerve. The prolonged period from insult to death suggests that the window for successful treatment after ON infarct may be longer than previously recognized.

Figure 1

Annexin-V-positive labeling of cells in the RGC layer. (A) Control. Few annexin-V-positive cells were apparent. (B) Three days after induction. More annexin-V-positive cells were present within the RGC layer (arrows). (C) Three days after induction, high-power view. Annexin-V-positive cells (arrow) were present in the NFL plane. (D) Ten days after induction. Numerous annexin-V-positive cells were scattered throughout the retinal region. (E) Thirty-one days after induction. Annexin-V-positive cells were still visible, although much diminished in number. (F) Thirty-one days after induction, high-power view. Annexin-V-positive cells (arrow) were still present at the level of RGC axons. Double-tailed arrowheads: RGC axons in the NFL; arrowheads: Bex1/2-positive cells; VS, retinal vessels. Magnification, ×200. Scale bar (D for A, B, E) 200 μm; (F for C) 100 μm.

Figure 2

RGC colocalization of fluorogold, Bex1/2, and annexin V, 28 days after rAION induction. (A) RGCs were retrograde fluorogold labeled. (B) Bex1/2 staining showed a consistent overlap with fluorogold retrograde labeling. (C) Several annexin-positive cells were visible in this field and appeared outlined by annexin V binding. (D) Merged image. Bex1/2 and fluorogold colocalized. Most annexin-V-positive cells were also fluorogold and Bex1/2 positive. Arrows: RGCs that were fluorogold, Bex1/2, and annexin positive. Scale bar: 50 μm.

Figure 3

rAION results in RGC layer apoptosis. Ten-μm-thick sections of rat retina were analyzed by TUNEL assay. (A) In negative control (uninduced) retinas, no TUNEL-positive cells were apparent. (B) Nine days after rAION induction, scattered TUNEL-positive cells were present in the RGC layer (arrows). There was artifactual light staining surrounding the cells in the inner and outer nuclear layers. (C) Positive control. TUNEL-control retina (DNase-1 treated). All nuclear layers were TUNEL positive. RGC, RGC layer; INL, inner nuclear layer; ONL, outer nuclear layer; Prc, photoreceptors. Scale bar, 100 μm.

Figure 4

Evaluation of caspase 3 (35 kDa) and cleaved caspase-3 (17 kDa) fragment in retinas and PC-12 cells after rAION induction. Controls included total protein homogenates from uninduced PC-12 cells (C) and PC-12 cells treated with 100 ng/mL staurosporine (C+S). Protein-transferred membranes were reacted with anti cleaved caspase 3. Signal was developed with ECL reagents. Protein loading was confirmed by reprobing the stripped membrane with antibody to β-actin. β-Actin-normalized values of cleaved caspase-3 expression are shown.

Figure 5

rAION resulted in regional RGC apoptosis. Different retinal regions of the same 10-day post-rAION flatmounted retina. Red: immunolabeling with Bex-1/2 (red). Green: FITC-annexin-V positivity. (A) Positive region. Images obtained from the peripheral region of one retinal quadrant. Numerous annexin-V-positive cells were seen in the RGC layer. (B) Negative region. Photograph obtained from the contralateral side of the same retina. No annexin-V-positive cells were seen. Scale bar, 100 μm.

Figure 6

Time course of RGC death after rAION induction. (A) Total retina. The y-axis shows the mean number of annexin-V-positive cells/retina (n = 12 fields retina; 3 fields/region). Each time point is the mean average of four retinas. The x-axis is the number of days after infarct. Two peaks are apparent: one at 10 days, and a second at 21 days. (B) Regional analysis. The y-axis shows the mean number of annexin-V-positive cells in three contiguous fields. The x-axis is the number of days after infarct. RGC apoptosis was greatest at 10 days after induction, but the SE was greatly reduced at nearly all time points, suggesting that rAION-induced RGC apoptosis occurs regionally. All data are the mean ± SEM.