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Many human optic neuropathies lead to crippling conditions resulting in partial or complete loss of vision. While the retina is made up of several different cell types, retinal ganglion cells (RGCs) are the only cell type connecting the eye to the brain. Optic nerve crush injuries, wherein RGC axons are damaged without severing the optic nerve sheath, can serve as a model for traumatic optical neuropathies as well as some progressive neuropathies such as glaucoma. In this chapter, we describe two different surgical methods for establishing an optic nerve crush (ONC) injury in the postmetamorphic frog, Xenopus laevis. Why use the frog as an animal model? Mammals lose the ability to regenerate damaged CNS neurons, but amphibians and fish retain the ability to regenerate new RGC bodies and regrow RGC axons following an injury. In addition to presenting two different surgical ONC injury methods, we highlight their advantages and disadvantages and discuss the distinctive characteristics of Xenopus laevis as an animal model for studying CNS regeneration.
Fig. 1
GFP fluorescence can be used to identify the optic nerve and validate success of the ONC injury. Forceps are used to crush the retinal ganglion cell axons without severing the optic nerve sheath or damaging the blood supply. Representative images from a ventral ONC surgery show an intact optic nerve from a Tg(islet2b:GFP) transgenic frog taken using a standard stereoscopic dissecting microscope with white light (a), under fluorescence (a′), and following a 3 s ONC injury under white light and fluorescence, respectively (b, b′). Transgenic frog lines expressing endogenous GFP in RGC axons are useful for tracking RGC axon regrowth. In the intact frog, axons can be imaged along the nerve (b, b′), at the optic chiasm (c, c′), and in the tectum (d, d′). Representative images using an epifluorescence dissecting microscope of a naïve (c, d) and post-ONC injury day 7 (c′, d′) frog brain show the dorsal side of the optic tectum with the optic chiasm visible (c, c′) and the ventral side of the optic tectum showing entry of the RGC axons into the tectal area (d, d′). By post-ONC injury day 7, GFP is no longer visible in the RGC axons in the right optic nerve chiasm (c′) or tectum (d′). Scale bars = 1 mm (a–b′) and 2 mm (c–d′) (see Note 1)
Fig. 2
Ventral/buccal ONC surgery (right ONC). The frog is placed ventral side up on a vinyl dissecting mat (left and right refer to the frog’s orientation) (a). A simple three-pin placement stabilizes the frog to provide access to the buccal cavity (a). The vasculature (red) running parallel to midline (dashed line) and the creases in the buccal dermal tissue surrounding the buccal cavity (black lines) emanating from the internal nares (black ovals) provide landmarks to inform the position of the initial surgical incision (b, c). A scalpel can be used to make a shallow incision, and blunt forceps (#5) are used to lift the dermal tissue and expand the incision without damaging the underlying tissue (d). A diagram shows the location of the eyes (red), tendons (yellow), and optic nerve sheath (green) containing the RGC axons with the overlying vasculature (e). Note that as the optic nerve crosses through the bones of the skull into the brain, the thick optic nerve dural sheath is absent (e). The RGC axons from each eye cross at the optic chiasm and synapse with tectal cells in the optic tectum which lies on the underside of the brain. Note that the optic nerve is no longer surrounded by the optic sheath upon entry into the skull (e). Panel f, left of midline, shows the fat and muscles located beneath the buccal dermal layer, while to the right of midline a dissection with the fat and muscles removed reveals the optic nerves and tendons (f). Panel g shows a close-up of the optic nerve (ON), tendons (t1, t2), vasculature (vasc.), and eye. Scale bar = 1 mm (see Note 7)
Fig. 3
Dorsal ONC surgery (left ONC). The postmetamorphic frog is placed dorsal side up on a dissecting mat, and pins are placed on either side of the head to limit movement (left and right refer to the frog’s orientation) (a). Forceps and/or a scalpel is used to tease away the conjunctival tissue of the eyelid 180° around the eye (b, c). A pair of iris scissors are used to first cut the skin at a 45° angle to midline (d) to locate the vasculature (vasc.) in the dermal layer beneath the skin (e, f). The dermal layer underlying the skin is then cut while avoiding the vasculature so as to expose the eye cavity behind the eye (e). As surgical skills improve, the dermal layer will no longer need to be cut and forceps can be inserted between the eye and dermal layer and used to widen the gap. Blunt forceps are used to gently move the musculature, while a second pair of forceps is used to grasp the conjunctiva above the eye and roll/tilt the eye forward and to the side in order to expose the optic nerve (ON green) which is attached to the base of the eye (e, f). While holding the eye forward with one hand, with the other hand, use sharp #55 or #5/45° angled forceps to crush the optic nerve (ON) for 3 s approximately 2 mm from the eye orbit (e). Crushing the nerve from an angle beneath the optic nerve helps ensure the eye vasculature remains intact. The eye diagram in panel f illustrates the movement of the eye rotation, the optic nerve inside the ON sheath (green), and the eye blood vasculature (red). Scale bar = 1 mm (see Note 7)
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