Changes in RGC axon survival measured in microbead rat model

Key Points

Fort Lauderdale, FL- A new latex bead rat model is a viable means of increasing IOP to induce loss of retinal ganglion cell (RGC) axons, according to Rebecca M. Sappington, PhD, postdoctoral fellow, and her laboratory director, David J. Calkins, PhD, associate professor, both of the Vanderbilt Eye Institute at Vanderbilt University Medical Center in Nashville, TN. Dr. Sappington and postdoctoral fellow Sam Crish, PhD, have used the laboratory's adapted model to measure IOP and compare the rate of axon loss throughout the eye and optic nerve.

Their model is an adaptation of two existing models: one by Weber and Zelenak and the other by Urcola et al. In the new model, IOP is elevated in Brown Norway rats by injection of 15 μm polystyrene microbeads into the anterior chamber. Differences from the existing models include the size of the microbeads, which are 5 μm larger, and the significantly lower injection volume, which is either 2.5 or 5 μl. The beads are injected with a 25-μl Hamilton syringe equipped with a 31-gauge needle. Each animal serves as its own control; beads are injected into one eye and saline into the other. Both eyes are dilated prior to injection to avoid nicking the iris. Dr. Sappington described the first results with the model used in the Calkins laboratory during a presentation at the annual meeting of the Association for Research in Vision and Ophthalmology.

The group reported that at 2 weeks post-injection, beads are still visible in the outflow canals of the anterior chamber. Large clusters can typically be found near the anterior and posterior rims of the iris. In addition, individual beads are dispersed throughout the anterior chamber between the anterior and posterior poles of the iris.

Used with cannulation

To determine how well tonometer measurements correlated with actual IOP, the Calkins team followed the tonometer measurements with cannulation.

"Consistently, the cannulation showed that the true IOP was about 40% to 45% less than what the [tonometer] read," Dr. Sappington said. Data indicate, however, that when IOP in the rat eyes is elevated with an injection of 10 μl saline, the cannulation shows a higher increase in IOP than is seen with the tonometer, and therefore the two readings are more closely associated.

"It appears in this case that the [tonometer] is able to more accurately assess IOP as it increases," Dr. Sappington said.

Although the team would like to use cannulation in the bead-injected eyes as well as the saline-injected controls, this is not currently possible since the microbeads clog the cannulation needle, she added.

As they have experimented with their model, the Calkins laboratory has made several discoveries about the relationship between the beads and elevation in IOP. With only two injections of the smaller amount of beads (2.5 μl), IOP remains elevated for at least 5 weeks. Pressure begins to rise after the first injection and increases more significantly after the second, which is given a week later.

"We believe this is due to the fact that after 2 weeks, we can see that beads still remain in the intraocular chamber, so a second injection would add beads on top of those already present," Dr. Sappington said.

Investigators also discovered that the volume of bead injection correlates with the rate and amplitude of IOP elevations.

"With the 2.5-μl injection, IOP rises but rises at a slower rate than that of the 5.0-μl injection, which is rather intuitive, " Dr. Sappington said. "It also doesn't rise to as high a level prior to the second injection but does so after the second injection. With the 5-μl injection, IOP rises quickly and stays elevated through the second injection and to the end of the experiment."

In addition to measuring IOP elevation, the Calkins group has used the microbead rat model to assess RGC axon loss under the two paradigms. Axon loss was measured in two locations: proximal, which was immediately behind the globe, and distal, just prior to the optic chiasm.