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A study in a rat model found retinal ganglion cell loss and retinal thinning following excitotoxic damage. This finding has led researchers to speculate that axonal damage and excitotoxicity are related, and that the connection may be through the Wallerian degeneration gene. Future research into this pathway could lead to new forms of treatment for glaucoma and other neurologic diseases.
Adelaide, Australia-Researchers studying the pathology of excitotoxic injury have made discoveries about dying back-like axonopathy and Wallerian degeneration that may be applicable to glaucoma as well as other neurodegenerative diseases.
Research on the basic pathology of excitotoxicity has led to clues about the genetic mechanisms involved, a step to be followed by testing in transgenic rats, with the ultimate goal of new drug treatments or gene therapy for a host of disorders, said Robert J. Casson, MBBS, DPhil, FRANZCO, director, Ophthalmic Research Laboratory, South Australia Institute of Ophthalmology, and associate professor, University of Adelaide, Australia.
Although Dr. Casson's research is centered on glaucoma, he collaborated with the neuropathology department at the University of Adelaide on this work, because it applies to other neurologic diseases as well. The initial objective was to investigate pathologic changes in the optic nerve after an injection of excitotoxin, a substance that activates nerve cells and damages them through paroxysmal overactivity-in essence it excites them to death.
"The eye provides an excellent model to do that because the tails or axons are isolated from the heads because the heads are inside the eye and the tails are outside the eye. You get an opportunity to study the secondary degeneration of the axon after a primary excitotoxic insult to the cell body," Dr. Casson said.
For their study, the researchers used 40 Sprague Dawley rats that were killed 0 hours, 24 hours, 72 hours, and 7 days after the injection of 20 nmoles of N-methyl-D-aspartate (NMDA) into the vitreous of the left eye. The right eye, with saline injected, served as a control.
Tissues from half of the animals in each group were embedded in paraffin for light microscopy and immunohistochemistry for neurofilament-light (NF-L) and amyloid precursor protein (APP). APP is a marker of dysfunctional fast axonal transport.
The other half of the tissues were resin-embedded for electron microscopy. Proximal (intraorbital) and distal (intracranial) optic nerves were compared using axon counts, axonal swellings, and myelin changes.
Retinal ganglion cell (RGC) loss and reduction in the inner retinal thickness were observed 72 hours after NMDA injection (p < 0.05) and increased after 7 days (p < 0.001). In addition, RGC somata counts strongly correlated with the axonal counts (r = 0.929, p < 0.001), and axon loss, axon swelling, and myelin damage were seen throughout the optic nerve after 72 hours (p < 0.05), with the changes increasing at 7 days (p < 0.001). Early degradation of NF-L was observed with immunohistochemistry and electron microscopy, but APP was not evident. Pathologic changes were more prominent in the distal segments than the proximal segments at all time points (p < 0.05).
Dying back-like axonopathy
These results showed that the type of degeneration occurring in the axons appeared to be a dying back-like axonopathy in which the most pronounced damage was further toward the brain.
"That was unexpected. I thought we would see more damage closer to the eye and that it would spread along the nerve toward the brain," Dr. Casson said.
A second discovery was that the dying back-like axonopathy resembled Wallerian degeneration. The withering away that takes place during the degeneration is an active, genetically determined process. This was learned through experiments in mice and rats with a specific genetic defect. When their axons were cut, the stump withered away slowly.
Exactly what the gene defect does and how it translates that particular effect to the axon are not clear, Dr. Casson said.
"Our hypothesis is that the Wallerian degeneration genes may also be involved in excitotoxicity-induced axonal degeneration. That would be a much bigger finding. It would have important implications for all sorts of neurologic diseases where excitotoxicity is involved, like Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis," he added.
Axonal degeneration is an early feature in all of those diseases, which also are characterized by excitotoxicity. This finding suggests a link between the axonal degeneration and the excitotoxicity involving the Wallerian degeneration genes. The pathway can be manipulated with drugs, a discovery that could open a new pharmacologic strategy for treatment of neurologic diseases with a new class of drugs, Dr. Casson said.
He and his colleagues are just beginning to study this theory, collaborating with ophthalmologist Keith Martin, MD, at the University of Cambridge Centre for Brain Repair, who has a population of rats with the slow Wallerian degeneration phenotype. The same type of experiment will be conducted in the Wld(S) rats that Dr. Casson and colleagues performed in their animal model, consisting of injection of NMDA into the vitreous cavity of the transgenic animals for comparison with controls at various timepoints.