Optic Nerve Degeneration after Retinal Ischemia/Reperfusion in a Rodent Model

Abstract

Retinal ischemia is a common pathomechanism in many ocular disorders such as age-related macular degeneration (AMD), diabetic retinopathy, glaucoma or retinal vascular occlusion. Several studies demonstrated that ischemia/reperfusion (I/R) leads to morphological and functional changes of different retinal cell types. However, little is known about the ischemic effects on the optic nerve. The goal of this study was to evaluate these effects. Ischemia was induced by raising the intraocular pressure (IOP) in one eye of rats to 140 mmHg for 1 h followed by natural reperfusion. After 21 days, histological as well as quantitative real-time PCR (qRT-PCR) analyses of optic nerves were performed. Ischemic optic nerves showed an infiltration of cells and also degeneration with signs of demyelination. Furthermore, a migration and an activation of microglia could be observed histologically as well as on mRNA level. In regard to macroglia, a trend toward gliosis could be noted after ischemia induction by vimentin staining. Additionally, an up-regulation of glial fibrillary acidic protein (GFAP) mRNA was found in ischemic optic nerves. Counting of oligodendrocyte transcription factor 2 positive (Olig2⁺) cells revealed a decrease of oligodendrocytes in the ischemic group. Also, myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) mRNA expression was down-regulated after induction of I/R. On immunohistological level, a decrease of MOG was detectable in ischemic optic nerves as well. In addition, SMI-32 stained neurofilaments of longitudinal optic nerve sections showed a strong structural damage of the ischemic optic nerves in comparison to controls. Consequently, retinal ischemia impacts optic nerve degeneration. These findings could help to better understand the course of destruction in the optic nerve after an ischemic insult. Especially for therapeutic studies, the optic nerve is important because of its susceptibility to be damaged as a result to retinal ischemic injury and also its connecting function between the eye and the brain. So, future drug screenings should target not only the retina, but also the functionality and structure of the optic nerve. In the future, these results could lead to the development of new therapeutic strategies for treatment of ischemic injury.

Keywords: ischemia/reperfusion; macroglia; microglia; neurofilament; oligodendrocytes; optic nerve; retinal ischemia.

Figure 1

(A) Longitudinal optic nerve sections were stained with hematoxylin & eosin (H&E). Cell cluster and a resolution of the tissue could be noted after ischemia/reperfusion (I/R). In the control group, the cells were arranged in series. (B) The cell infiltration was determined using a scoring system ranging from 0 (no infiltration) to 4 (massive infiltration). Significantly more cells infiltrated the optic nerves of ischemic animals. Arrows = cluster of cells. Scale bar: 20 μm. ***p < 0.001.

Figure 2

(A) Representative pictures of the toluidine blue staining. While in the control group a uniform coloring was present, various light areas were visible in the ischemia group. (B) Staining of longitudinal optic nerve sections with luxol fast blue (LFB). Comparable to toluidine blue staining, large bright areas were seen in the ischemic optic nerves. In contrast to this, the control optic nerves were uniformly colored. (C) The loss of myelin was valued based on a scoring system. Many areas of degeneration with subsequent loss of myelin and a dissolution of the tissue could be observed 21 days after ischemia induction. Arrows = demyelination area, asterisks = structure resolution. Scale bar: toluidine blue 50 μm, LFB 20 μm. ***p < 0.001.

Figure 3

(A) Optic nerve sections labeled with SMI-32 for neurofilament (green) and 4′,6-Diamidin-2-phenylindol (DAPI) for cell nuclei (blue). In comparison to the control group, many retraction bulbs, short axons and holes could be noted in ischemic optic nerve tissue. (B) Scoring of the SMI-32 stained sections revealed a significant structural distortion of the optic nerves of the ischemic group. Arrows = retraction bulbs, asterisks = holes. Scale bar: 20 μm. ***p < 0.001.

Figure 4

(A) Myelin basic protein (MBP, green), and DAPI (cell nuclei; blue) staining of optic nerve sections. No differences in immunoreactivity of MBP could be seen, but a destruction of the ischemic tissue could be observed. (B) Statistical analysis showed no differences in MBP⁺ area between both groups. (C) Otherwise, a significant decrease of MBP mRNA was noted in optic nerves of ischemic eyes in relation to controls. (D) Oligodendrocyte transcription factor 2 (Olig2) was used to stain oligodendrocytes (maturing oligodendrocyte precursor cells (OPCs), red), DAPI was used for cell nuclei (blue). Regarding the Olig2⁺ cells, fewer cells were observed in the ischemic group. (E) Counting of Olig2⁺ cells revealed a significantly decreased cell number in the ischemic group compared to controls. (F) On mRNA level, no differences could be observed between both groups. (G) With myelin oligodendrocyte glycoprotein (MOG) the myelin glycoprotein of oligodendrocytes was marked (green). DAPI was used for cell nuclei (blue). A lower MOG signal and a resolution of the tissue could be shown in ischemic optic nerves. (H) Statistical analyses revealed a significant reduction in the MOG⁺ area after ischemia induction. (I) Also via quantitative real-time PCR (qRT-PCR), a significant decrease of MOG mRNA expression was observed in ischemic optic nerves in comparison to controls. Scale bar: 20 μm. *p < 0.05, ***p < 0.001.

Figure 5

(A) Macroglial staining of optic nerve sections with glial fibrillary acidic protein (GFAP; red) and DAPI for cell nuclei (blue). An unstructured GFAP immunoreactivity could be observed after I/R. (B) Evaluation of GFAP stained sections revealed no differences in GFAP⁺ area between the optic nerves of both groups. (C) On mRNA level, a significant up-regulation in GFAP expression could be shown in the ischemia group in relation to controls. (D) Additional staining of macroglia with vimentin (red). DAPI was used to label cell nuclei (blue). (E) No differences could be noted in vimentin⁺ area between both groups, but an increasing trend could be observed in the ischemic group. (F) Compared to controls, also on mRNA level, no differences in vimentin expression were measured in ischemic optic nerves. Scale bar: 20 μm. *p < 0.05.

Figure 6

(A) Optic nerve sections were stained with Iba1 (microglia; green), ED1 (active microglia; red) and DAPI (cell nuclei; blue). Much more microglia as well as activated ones were present in the tissue of ischemic optic nerves. (B) Significantly more Iba1⁺ microglia were detected in the optic nerves of the ischemic group. (C) Also, via qRT-PCR, a significant increase of Iba1 mRNA expression was observed in ischemic optic nerves in comparison to controls. (D) A significant activation of microglia could be revealed in the ischemic group. Significantly more ED1⁺ and Iba1⁺ microglia were counted. (E) Equally, CD68 mRNA (activated microglia) expression was significantly up-regulated in ischemic optic nerves in relation to controls. Arrows = co-localization of ED1⁺ and Iba1⁺ cells. Scale bar: 20 μm. *p < 0.05, **p < 0.01, ***p < 0.001.