Graphic illustration of femtosecond laser creating corneal flap during LASIK surgery.
Lasers have become as essential to the practicing ophthalmologist as the scalpel, the magnifier and sterile gloves. A beam of light can reshape a cornea to improve its focus, create a channel within the eye to relieve the intraocular pressure of glaucoma or cauterize tiny hemorrhages.
During the past decade, lasers have been branching out from ophthalmologic treatments into the diagnostic realm. Some of the hottest areas in vision research involve boosting laser-based microscopic techniques with adaptive optics to enable physicians to detect glaucoma and other devastating eye diseases in their earliest stages. Since the retina is the only part of the central nervous system that physicians can see without having to perform surgery, eye imaging with laser light could even give neurologists a non-invasive view into Alzheimer’s disease in its early stages.
Retinal fluorescence and disease diagnosis
Thanks to a combination of scanning laser ophthalmoscopy and computer processing, scientists can study naturally occurring retinal fluorescence, which could serve as an early-warning marker of glaucoma, macular degeneration and other eyesight-stealing diseases.
Before those technologies existed, researchers could not detect this autofluorescence, which comes from a pigment called lipofuscin, in the living eye. Lipofuscin is made up of yellow-brown granules and accumulates in various parts of the human body—including the retina—as part of the “wear and tear” of aging at the cellular level. An abnormal amount of this buildup in the retinal ganglion cells and retinal pigment epithelium has been implicated in age-related macular degeneration, a disorder of the macula of the eye that makes it difficult for people to see fine details. The macula is the portion of the retina that is responsible for central vision.
At first, scientists knew about the existence of the fluorescence from lipofuscin only from post-mortem studies because the signal was too low in living subjects, said Frederick W. Fitzke, professor of visual optics and psychophysics at University College London (UCL)’s Institute of Ophthalmology (U.K.). The fluorescence occurs under 488-nm light from an argon laser, and the confocal imaging technology excludes the fluorescence from the eye’s crystalline lens, which would otherwise mask the target signal from the retina.
The confocal scanning laser ophthalmoscope (SLO) creates an image of the retina by raster-scanning a narrow beam of light across the retina and picking up the reflected light. The confocal technique greatly improves resolution and allows the user to probe tissue at different depths of interest. Longer wavelengths activate different fluorophores and probe deeper layers of the tissue. Compensation for the patient’s eye movements is typically done in post-processing, when the researcher co-registers the images and averages the values for the aligned image stack.
Anatomy of the eye.
One of the things that scientists seek to detect with the high resolution of confocal SLO is the apoptosis of retinal cells. Apoptosis is one of the two ways in which cells die. Necrosis refers to the premature death of a cell by acute infection, toxin or trauma. Apoptosis, on the other hand, is controlled by a series of biochemical events. It is a necessary function for multicellular organisms—for example, cell development and selective apoptosis together shape an embryo into a fully formed fetus. “It’s a way the cell commits suicide,” said M. Francesca Cordeiro, professor of retinal neurodegeneration and glaucoma studies at UCL.
However, the process of apoptosis can also be triggered abnormally in glaucoma and other neurodegenerative conditions. Glaucoma affects the optic nerve of the eye, which carries visual information to the brain. It is typically caused by increased pressure on the eyeball that can be related to aging, an eye injury, hereditary factors or other things. Glaucoma can lead to decreased vision, facial pain, eye swelling, sensitivity to light and other problems.
In the past, ophthalmologists have not been able to detect retinal ganglion cells that have died through apoptosis in human eyes, but if they could do so, they could identify glaucoma sufferers before these patients experience vision loss. The earlier glaucoma is caught, the more treatable it is.
Fitzke and Cordeiro have been working on a project called DARC, for Detection of Apoptosing Retinal Cells, for the past six years. The scientists use a fluorescent marker called annexin 5 that identifies cells dying through the process of apoptosis. “You can actually see these cells die in front of you,” she said.
So far, the team has worked with animal models only. In one set of experiments, the UCL researchers induced glaucoma in rats by boosting the intraocular pressure in one eye with a shot of saline solution. They imaged the living eyes with a confocal SLO before and after the injection of the fluorophore, and they checked the amount of apoptosis in retinal ganglion cells several times over a 16-week period. The DARC technique identified apoptosis in 15 percent of cells at peak in a glaucoma model.
Cordeiro and colleagues have hypothesized from their animal glaucoma models that the greatest rate of apoptosis of retinal ganglion cells occurs in the first 10 years of the course of the disease—possibly 10 years before current techniques identify a problem, and an even longer time before the patient starts to notice his or her reduced vision.
Early-phase clinical trials in human glaucoma patients may start as early as this year, Cordeiro said. In the future, this work could also allow physicians to monitor how well they are treating their patients and to assess—with quick turnaround—whether these treatments are working.
Lasers could also play a role in helping researchers to provide an early warning of the onset of Alzheimer’s disease, again by enabling the detection of apoptosis. Amyloid-beta—a peptide that makes up the sticky “plaques” that form inside the brains of people suffering from Alzheimer’s—is also deposited on the retina, where its toxicity causes apoptosis. After all, retinal ganglion cells are nerve tissue too—and thanks to the structure of the eye, these ganglia are much easier to image than the deep regions of the brain. Cordeiro is now studying the connections between glaucoma and Alzheimer’s in the hope of developing new screening tools.
