Progressive damage along the optic nerve following induction of crush injury or rodent anterior ischemic optic neuropathy in transgenic mice

Abstract

Purpose: To characterize the histological changes that occur in response to induction of ischemic or mechanical optic nerve damage in transgenic mice.

Methods: Either optic nerve crush injury or rodent anterior ischemic optic neuropathy (rAION) were induced in the right eye of mice transgenic for the Thy1 gene promoter expressing cyan fluorescent protein (CFP; n=40) and mice transgenic for the cyclic nucleotide phosphodiesterase (CNPase) gene promoter expressing green fluorescent protein (GFP; n=40). The left eye served as a control. The mice were euthanized at different times after injury. Eyes were enucleated, and the brain together with the optic nerves was completely dissected. Cryopreserved sections of both optic nerves were analyzed by fluorescence microscopy. In addition, flat-mounted retinas from the Thy1-CFP mice were analyzed for retinal ganglion cell (RGC) loss.

Results: Axonal loss was detected in the right eye of the Thy1-CFP mice, and demyelination was detected in the CNPase-GFP mice. Both processes occurred simultaneously in the two models of injury. The damage proceeded retrogradely and, in the crush-injury group, crossed the chiasm within 4 days. At 21 days after injury, RGC loss measured 70% in the crush-injury group and 25% in the rAION group.

Conclusions: Axonal injury and demyelination along the optic nerves occur simultaneously in transgenic mice exposed to ischemic or crush injury. The degree of RGC loss reflects the severity of the injury. Loss of oligodendrocytes and myelin apparently leads to axonal loss. Transgenic mice offer a promising model for exploring the damage caused by optic nerve injury. Use of fluorescence labeling makes it possible to better understand the underlying pathophysiology, which can help researchers formulate neuroprotective agents.

Figure 1

Axonal loss in optic nerves of Thy1-CFP mice at various intervals after crush injury. A: Control optic nerve; no damage. Note the absence of damage to the optic nerve. B: Four days after crush injury; 30% axonal loss can be detected. C: Fourteen days after crush injury; maximal (75%) axonal loss is seen. Note the central loss of Thy1-CFP-labeled axons.

Figure 2

RGC loss in flat-mounted retinas of Thy1-CFP mice at various intervals after crush injury. A: Control retina; no damage is detected. B: Four days after crush injury; approximately 20%–30% RGC cell loss was detected. C: Seven days after crush injury; 50% RGC loss was detected. D: Fourteen days after crush injury; maximal 75% RGC loss can be detected. Note the diffuse loss of the labeled cells.

Figure 3

Oligodendrocyte cell loss in chiasm and optic nerves of CNPase-GFP mice at various intervals after crush injury. A: Fourteen days after crush injury; approximately 50% oligodendrocyte loss in right optic nerve (white arrow) as compared to the left optic nerve. Note the thin right optic nerve and the reduced fluorescence signal in the right axonal fibers crossing the chiasm toward the contralateral lateral geniculate body. B Ipsilateral (right) and C contralateral (left) lateral geniculate body (LGB) with 25% oligodendrocyte cell loss in the latter. D and E: Hoechst staining of both LGB can be seen, with 20% cell loss contralateral to the injured nerve (same magnification, 10X).

Figure 4

Retinal and optic nerve findings at various intervals after rAION induction. A: Control optic nerve from Thy1-CFP transgenic mouse; no axonal loss is detected. B: Histologicial section of the optic nerve at 21 days after rAION induction; arrows point to moderate axonal loss (maximal, 25%). C: Flat-mounted retina from control (untreated) eye of same mouse showing normal density of RGC nuclei. D. Flat-mounted retina from study eye showing RGC loss, as compared to control (C). E: Histological section of control (untreated) optic nerve from CNPase-GFP transgenic mouse. Note the complete myelinization and number of oligodendrocyte nuclei. F: Twenty-one days after rAION induction, maximal (20%–30%) oligodendrocyte loss is demonstrated.

Figure 5

Apoptosis assay: retina. A: Control (untreated) eye; no staining for apoptosis was detected. B: Three days after rAION induction, TUNEL staining yielded positive cells along the retina C: Three days after crush injury, more positive cells were detected. D: Retinal section served for positive control of the apoptosis assay.

Figure 6

Apoptosis assay: optic nerve, after crush and rAION. A: Normal optic nerve; no apoptotic cells are detected. B: Positive control. C: TUNEL-positive (red) cells along the optic nerve 3 days following crush injury of wild type mice. D: TUNEL staining of optic nerve of GFP-CNPase mice 3 days following crush showing positive staining. Oligodendrocytes apoptotic cells are yellow (green for GFP and red represent positive staining for apoptosis), suggesting that oligodendrocytes in the optic nerve proximal to the globe undergo apoptosis 3 days following induction of crush injury. E: The whole optic nerve of same GFP-CNPase mice (D) is shown. F: Optic nerve head, 3 days following crush injury; showing hemorrhage (asterisk), immediately posterior to the globe. G: Same damaged area at higher magnification, demonstrating loss of oligodendrocytes and focal hemorrhagic area. H: Optic nerve head 3 days following rAION induction, showing preserved architecture of retina and intraocular optic nerve, without apoptotic cells. I: Same nerve as H, 3 days after rAION, demonstrating the anterior segment of the optic nerve behind the globe. Note few TUNEL-positive cells (red staining, arrows) at the center of the anterior optic nerve.