Losing axons in glaucoma may be stopped by gene

December 1, 2007

Mouse model studies explore role of matrix metalloproteinase 9 (MMP9) in glaucoma-related axon loss. Initial findings revealed a significant difference in relative axon loss between the MMP9 knockout mice and wild-type control mice.

Key Points

Durham, NC-The method of inducing glaucoma by injecting hypertonic saline into the episcleral veins in an experimental model can be adapted to study the pathophysiology of matrix metalloproteinase 9 (MMP9) involvement in axon loss in glaucoma.

In a preliminary experiment using MMP9 knockout mice and wild-type control mice, researchers were able to increase IOP in the treated eyes of all animals, showing that MMP9 is upregulated in the controls in association with elevated IOP, and demonstrating that the knockout mice lost fewer axons than the controls, said Frankie-Lynn Silver, MD, clinical associate, ophthalmology, Duke University, Durham, NC.

Dr. Silver began this line of research in the lab of Stuart McKinnon, MD, PhD, after reading a paper published in 2002 that suggested MMP9 deficiency might be protective against ganglion cell loss in an acute nerve injury model. She theorized that MMPs might offer another way of exploring the pathology of glaucoma and the pathways that lead to ganglion cell loss, ultimately leading to a pharmacologic intervention to prevent vision loss.

"Having a secondary way to intervene in a different area of the pathology of glaucoma would be very helpful," Dr. Silver said.

To begin pursuing her hypothesis, Dr. Silver obtained 10 MMP9 knockout mice and 10 matched controls. Glaucoma was induced in the right eye of each animal, while the left served as a control. IOP was measured weekly in conscious mice using a TonoLab rebound tonometer. Control and treated eyes were collected when the group average change in IOP was greater than 600 mmdays. Axon counts were obtained from optic nerve cross-sections stained with toluidine blue.

"We wanted to see if we saw any difference with and without MMP9," Dr. Silver said. "Our initial results are promising, and there appears to be some difference. We are continuing our research so we can get a larger number of data points to see if this is consistent."

If additional data support the initial findings of a significant difference in relative axon loss between the two groups, the research will be pursued further, Dr. Silver added.

She and her colleagues found that IOP increased with duration after induction and that both the knockout mice and controls developed typical IOP elevations in the eye with glaucoma. The team also confirmed that MMP9 is upregulated in the control mouse retina in the experimental eye but absent in the knockout retina.

At baseline, axons appeared to be fairly evenly distributed and of similar size in both the knockout and control eyes. After the injection of hypertonic saline to induce elevated IOP, a greater degree of change was found in the control animals than in the knockout mice. Changes included less even distribution of axons, greater variability of axon size, and a relative increase in the proportion of non-staining glial tissue and darkly staining areas of myelin from degenerating axons in the experimental right eye than the control left eye.

Evaluation of the axon counts generated by an imaging program found a moderately wide standard deviation and a statistically significant difference in axon counts between the treated and untreated eyes in both groups.