Investigators working with a new method of analysis based on topical coenzyme Q10, a molecule shown to have neuroprotective activity, have successfully profiled different phases of cell death in vivo.
Fort Lauderdale, FL-Investigators working with a new method of analysis based on topical coenzyme Q10 (CoQ10), a molecule shown to have neuroprotective activity, have successfully profiled different phases of cell death in vivo. They hypothesize that CoQ10 may be modifying the profile and could possibly target early apoptosis and even reverse it.
These findings suggest that the retina may be ideal for quantitative monitoring of early dynamic and early disease processes, said M. Francesca Cordeiro, MBBS, MRCP, FRCOphth, PhD, in a presentation at the annual meeting of the Association for Research in Vision and Ophthalmology.
Dr. Cordeiro explained that investigation of what happens before a cell dies has relied on tissue culture studies of staining characteristics to determine whether a cell has gone into early or late apoptosis or necrosis. It was thought until recently that once a cell started down the pathway toward apoptosis, it was committed to die.
In earlier work, Dr. Cordeiro and colleagues demonstrated that annexin V and propidium iodide (PI) could be used in vivo to identify different phases of cell death. Three weeks after inducing intraocular hypertension in a rat model, animals exhibited different cell death profiles from those treated with MK801. PI-positive cells were necrotic, those stained with annexin V were in early apoptosis, and those stained with both markers were in late apoptosis.
Following up on that study, Dr. Cordeiro and colleagues set out to determine whether the phase of retinal ganglion cell (RGC) death varied in glaucoma, whether treatment with topical CoQ10 could alter that profile, and whether apoptosis could be reversed.
CoQ10 was used because it is a familiar topical agent and affects mitochondria.
"The idea is that in vivo mitochondrial dysfunction occurs before apoptosis," said Dr. Cordeiro, also head of the Glaucoma and Neurodegenerative Disease Research Group and consultant ophthalmologist at Western Eye Hospital, London.
Dark agouti rats were randomly assigned to treatment with control or CoQ10 eyedrops (Visufarma srl, Italy) 30 minutes before and 1 hour and 1 week after IOP elevation following an established protocol. The animals were imaged at baseline and 1, 3, and 8 weeks after IOP elevation. At each time point, imaging was performed 2 hours after administration of annexin V and PI.
"Three weeks and 8 weeks later, there was a significant reduction in the level of apoptosis in annexin-positive cells," Dr. Cordeiro said.
As early as 1 week, levels of both early and late apoptosis were significantly different between treated rats and untreated controls. Since peak annexin staining and apoptosis in this model occur at 3 weeks after surgical induction of elevated IOP, this finding indicated that CoQ10 treatment had decreased the level of both early and late apoptosis at an early stage of disease, Dr. Cordeiro said.
At 3 weeks, the majority of cells were in late apoptosis in the untreated controls, but interestingly there was a reduction in the level of late, but not early, apoptosis and in the level of necrosis with treatment.
Differences in phases of RGC death
To investigate the difference between phases of RGC death in the early and late stages of the experimental model of glaucoma, Dr. Cordeiro and colleagues shifted from a chronic model to an acute model of disease; this enabled them to perform the imaging portion of the evaluation more efficiently.
"One hypothesis (for this difference) is that in a state of disease you tend to see a predominance of late apoptosis in early disease, and as the disease progresses there may be less apoptosis and more necrosis," Dr. Cordeiro said.
In the experiments with the acute model, animals were followed for 3 hours. Annexin V staining was suppressed in the treated rats, although there was little effect on necrosis and late apoptosis. These findings suggest that CoQ10 had an effect on the very early phases of apoptosis.
"The presence of CoQ10 significantly reduced the level of the first peak of early apoptosis, suggesting that it could indeed make a difference in the level of apoptosis," Dr. Cordeiro concluded. "A wealth of literature now suggests that apoptosis in the membrane can be reversible in the early stages."
M. Francesca Cordeiro, MBBS, MRCP, FRCOphth, PhDPhone: 020-7608-6938
Visufarma provided the topical CoQ10 but no other funding. Dr. Cordeiro reported commercial relationships with Alcon Laboratories, Allergan, Heidelberg Engineering, and Merck and is a named inventor on a patent application for technology discussed in this study.