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1National Eye Institute, NIH, 2Ophthalmology Department, The Second Hospital of Harbin Medical University
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This protocol shows how to retrogradely label retinal ganglion cells, and how to subsequently make an optic nerve crush injury in order to analyze retinal ganglion cell survival and apoptosis. It is an experimental disease model for different types of optic neuropathy, including glaucoma.
Tang, Z., Zhang, S., Lee, C., Kumar, A., Arjunan, P., Li, Y., et al. An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival. J. Vis. Exp. (50), e2685, doi:10.3791/2685 (2011).
Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. Examples of such diseases in human include traumatic optic neuropathy and optic nerve degeneration in glaucoma. It is characterized by typical changes in the optic nerve head, progressive optic nerve degeneration, and loss of retinal ganglion cells, if uncontrolled, leading to vision loss and blindness.
The optic nerve crush (ONC) injury mouse model is an important experimental disease model for traumatic optic neuropathy, glaucoma, etc. In this model, the crush injury to the optic nerve leads to gradual retinal ganglion cells apoptosis. This disease model can be used to study the general processes and mechanisms of neuronal death and survival, which is essential for the development of therapeutic measures. In addition, pharmacological and molecular approaches can be used in this model to identify and test potential therapeutic reagents to treat different types of optic neuropathy.
Here, we provide a step by step demonstration of (I) Baseline retrograde labeling of retinal ganglion cells (RGCs) at day 1, (II) Optic nerve crush injury at day 4, (III) Harvest the retinae and analyze RGC survival at day 11, and (IV) Representative result.
All equipments and reagents used are sterile. All animal experiments were approved by the Animal Care and Use Committee (ACUC) at the NEI/NIH (animal study protocol NEI-570), and were performed according to the NIH guidelines and regulations.
1. Baseline Retrograde Labeling of Retinal Ganglion Cells (RGCs) At Day 1
The purpose of this procedure is to label the retinal ganglion cells retrogradely by injecting a neural tracer dye into the superior colliculus of the mice three days before the optic nerve crush injury. The dye will be retrogradely taken up by the retinal ganglion cells and provides a marker for the living RGCs (Figure 1). This approach yields reproducible labeling of viable RGCs with little variation 1-5.
2. Optic Nerve Crush Injury At Day 4
In this procedure, we will apply a crush injury to the optic nerve to cause a primary damage to the axons (Figure 2), which will lead to a gradual death of the retinal ganglion cells.
3. Harvest the Retinae and Analyze RGC Survival At Day 11
The purpose of this procedure is to harvest the retinaeto analyze retinal ganglion cell survival.
4. Representative Results:
To analyze retinal ganglion cell survival, seven days after optic nerve crush injury, the retinae were harvested, fixed, flattened and mounted. Retinal images were taken using a fluorescent microscope. Compared with the uninjured normal retina (left, Normal), the number of viable RGCs (green, retrogradely labeled by the FluoroGold neural tracer dye injected into the superior colliculus) is significantly lower in the retinae with an optic nerve crush injury (right, ONC) (Figure 3).
Figure 1. Baseline retrograde labeling of retinal ganglion cells
In order to count the viable retinal ganglion cells at different time points after optic nerve crush injury, the retinal ganglion cells are labeled retrogradely by injecting a neural tracer dye (green color) into the superior colliculus in the brain three days before the optic nerve (red) crush injury. Since the axons of the retinal ganglion cells reside in the superior colliculus, the tracer dye will be taken up by the RGCs retrogradely and provides a marker for the living RGCs.
Figure 2. Optic nerve crush injury
In order to analyze the status of retinal ganglion cell survival, a crush injury is applied to the optic nerve (red) to cause a primary damage to the axons. This will lead to a gradual death of the retinal ganglion cells. Three days after the tracer dye (green) injection, the mouse is anesthetized. The conjunctiva of one eye is incised. Deflect the orbital muscles and put them aside. Expose the optic nerve at its exit from the eye globe. Apply a crush injury to the optic nerve at about 2 mm from the eyeball for about 3 seconds using a pair of cross-action forceps.
Figure 3. Fluorescence-labeled retinal ganglion cells with or without optic nerve crush injury
To analyze retinal ganglion cell survival, seven days after optic nerve crush injury, the retinae were harvested, fixed, flattened and mounted. Retinal images were taken using a fluorescent microscope. Compared with the uninjured normal retina (left, Normal), the number of viable RGCs (green, retrogradely labeled by the FluoroGold neural tracer dye injected into the superior colliculus) was significantly lower in the retinae with an optic nerve crush injury (right, ONC) (Figure 3). Scale bar: 50 μm
The optic nerve crush injury murine model is useful to study the process of RGC death and survival. This model is also often used to investigate the effects of different reagents and genes on RGC apoptosis and survival. One advantage of this model is that it has a high degree of reproducibility with minimal variations.
However, special care is required in several steps in this model. First, when making the optic nerve crush injury, it is essential not to use too much force and not to crush for too long, because they may lead to damage to the ophthalmic artery and therefore subsequent retinal ischemia. In addition, when trying to expose the optic nerve, one should take great care not to damage other blood vessels surrounding the mouse eye.
No conflicts of interest declared.
Our research is supported by the Intramural Research Program of the NIH, National Eye Institute.
|Stereotaxic apparatus||ASI Instruments||SAS-4100|
|Dissecting microscope||World Precision Instruments, Inc.||PZMIV-BS|
|Scalpel handle||Fine Science Tools||10003-12|
|Scalpel Blades #15||Fine Science Tools||10015-00|
|Microdrill||Fine Science Tools||18000-17|
|Suture needle||Fine Science Tools||12050-03|
|Spring scissors||Fine Science Tools||16144-13|
|Dumont #5 forceps||Fine Science Tools||11252-50|
|Hamilton syringe||Hamilton Co||88400|
|Gauze sponges||Office Depot||674889|
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