W&L Team Uses Latest Technology to Study the Optic Nerve Regeneration

Optic nerve and crushed optic nerve

The optic nerve of adult frogs, left, expressing green fluorescent protein can be easily visualized through the perineural sheath that surrounds the retinal ganglion cell axons and connects the eye and brain. The post-crush optic nerve, right, shows how the retinal ganglion cell axons within the optic nerve are severed as a result of crushing the optic nerve using fine needle forceps.

As baby boomers age over the next 10 years, the number of cases of blindness in the United States is expected to jump dramatically. According to the National Eye Institute, glaucoma alone will account for 3.6 million cases in 2020, up from about 2.2 million today.

Blindness occurs in glaucoma patients because the optic nerve has been damaged, typically from built-up pressure within the eye. In humans and other mammals, retinal ganglion cells (RGCs) in the optic nerve cannot regenerate and heal after an injury. In frogs, fish and cold-blooded invertebrates, they can.

“The question is, why can they do it but we can’t? What have we lost or gained over the course of evolution so that we can no longer do this?” asked Fiona Watson, assistant professor of biology and neuroscience at Washington and Lee University.

Watson and two Washington and Lee undergraduate research scholars from the Class of 2015,  Andrew Watson of Great Falls, Va. and Bayan Misaghi of Charleston, W.Va., have tackled these questions this summer.

“Our ultimate goal is to generate a profile, a list of genes that are turned on or off specifically in response to this injury,” said Watson. Once a gene profile is complete, scientists can compare it with the profile of a mouse to determine which genes are reacting differently after damage to the optic nerve. The goal is then to determine the functional role of these genes in the regenerative process. This knowledge could help to develop treatments for a range of eye diseases.

Watson’s team is studying retinal ganglion cells (RGCs), one of seven major eye cell types.  Visual perception begins when millions of visual stimuli hit rod and cone photoreceptor cells.  These visual signals are ultimately transmitted to the RGCs before being transmitted to the area of the brain where these signals are integrated and vision occurs.  The RGCs extend their axons from the retina in the eye to the optic tectum, the area in the frog brain where visual input is integrated. These RGC axons are contained in one large bundle that connect the eye to the brain. “You’ve got one set of cells that are carrying all the information from the eye to the brain. You knock out this set of cells, [or if there’s] any kind of injury to these cells, you lose your vision,” said Watson. “It’s a very fragile system.”

To prepare, Andrew Watson, a Levy Neuroscience Fellow, and Misaghi, a Howard Hughes Medical Institute Fellow, reviewed literature about optic nerve regeneration in amphibians as well as in mice. Watson and Misaghi also took a spring term course ‘Research Preparation in the Biosciences’ (Bio 200) to prepare them for the rigors of scientific research.

The optic nerves of live tadpoles expressing green fluorescent protein (GFP) in the retinal ganglion cells and their axons can be visualized using a fluorescent stereomicroscope.

The optic nerves of live tadpoles expressing green fluorescent protein (GFP) in the retinal ganglion cells and their axons can be visualized using a fluorescent stereomicroscope.

In June, the students began developing eye surgery techniques on tadpoles. Tadpoles were their initial test subjects because their axons regenerate more quickly than a frog’s. Tadpoles are also transparent, making it easier to conduct surgeries; they can also be used in surgeries only 14 days after breeding.

“We would penetrate the skin of the tadpole near the eye with fine-tipped capillary needles using micro-manipulators in order to move in tiny increments, just iota movements until we were able to cut the optic nerve sandwiched between two glass capillary needles” said Misaghi.

“It was quite challenging if you can imagine using 0.5 mm thick chop sticks,” said Misaghi. “Our vitality rate was pretty low. The surgeries took at least 20 minutes each, only about 30% of the tadpoles survived and since we needed several dozen tadpoles to collect sufficient tissue for a single time point, this strategy was going to be too labor intensive.” The students also experimented with electrolytic lesions, a method that  ‘zaps’ the tadpoles’ optic nerve instead of cutting it. Zapping the optic nerve was faster but the method still needed to be optimized.

The team ultimately decided that it would be more efficient to operate on post-metamorphic frogs as the retinas were larger and easier to manipulate. Using frogs that express the green fluorescent protein (GFP) so the RGCs and their axons appear green under fluorescent light microscopy, students were able to visualize the optic nerve crush, the subsequent degeneration of the RGC axons and finally, its recovery.

To generate a gene profile, Watson and her research assistants plan to use a method of cellular analysis called Translating Ribosome Affinity Purification, or TRAP. The students will implement the TRAP method by crushing the frogs’ optic nerves, removing the eyes and grinding up these tissues for analysis.  For the study, Watson created a line of transgenic tadpoles and frogs whose retinal ganglion cells express the GFP gene fused to a ribosomal gene (rpl10a), a protein that is part of the cell machinery used to convert genes into proteins.   An antibody against GFP can then be used to isolate messenger RNAs (mRNAs) that are actively being translated into proteins.

The next step? The team will use GFP to isolate ribosomal proteins and by association the mRNAs that actively transcribes signals from the eye to the brain. These mRNA proteins will then be sequenced to generate a list of genes referred to as a transcriptome, that are active during during the process of nerve regeneration.  As a control, the team will compare mRNAs from eyes removed from frogs whose optic nerves have not been damaged.

“I’ve really liked doing the surgeries, actually getting experience doing dissections, taking out eyes. It’s interesting,” said Andrew Watson. “I’m thinking about med school and going into medicine. It’s good to get a sense of what that might be like.”

The project has also prepared the students for graduate level study. “I’ve gained a huge amount of respect for people who are pursuing a PhD,” said Misaghi. “We’ve only been here 10 weeks, but I think I have a flavor of the triumphs and setbacks that PhD students must face.”

— by Amy Balfour '87, '91L

News Contact:
Jeffery G. Hanna
Executive Director of Communications and Public Affairs
jhanna@wlu.edu
(540) 458-8459

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