Responses of Retinal Ganglion Cells to Optic Nerve Injury

Glaucoma leads to the death of retinal ganglion cells, likely because their processes (axons) are injured at the point where they leave the eye. There appears to be some sort of signal that is sent to retinal ganglion cell bodies when their axons are injured that triggers a sequence of events that lead to cell death. One of the suspected messengers of this signal is glutamate, a neurotransmitter in the retina and brain. Elevated levels of glutamate are known to kill retinal ganglion cells, and glutamate is found in high concentrations in the eyes of people and animals with glaucoma. The series of events that elevated glutamate triggers in retinal ganglion cells is not well understood, but it appears to be dependent on an influx of calcium ions into the cell. I propose to monitor calcium concentration inside retinal ganglion cells before, during, and after optic nerve injury to determine if calcium increases as a result of axonal injury. I hypothesize that axonal injury changes in the way retinal ganglion cells regulate intracellular calcium, either by causing increases in calcium or by altering the ability of retinal ganglion cells to regulate calcium concentration. I have developed a surgical procedure that permits me to monitor intracellular calcium concentration in retinal ganglion cells in vivo in an anesthetized mouse. This approach has an advantage over in vitro and cell culture systems because it does not require that the retinal ganglion cells be removed from the animal. This in vivo approach therefore mimics the clinical situation more closely than other techniques. Secondly, by monitoring calcium in real time with video imaging microscopy, this technique will give us a dynamic picture of the processes going on inside retinal ganglion cells as they occur. This project will involve developing a means to label retinal ganglion cells with a special dye that is sensitive to calcium. When calcium concentration increases, calcium ions bind to this type of dye and changes its fluorescence characteristics in a predictable and measurable way. Therefore, by measuring the changes in fluorescence emitted by the dye, we can measure changes in calcium concentration. I will devise strategies to label retinal ganglion cells with such dyes. Secondly, I will design a device with which to cut the optic nerve while imaging the retinal ganglion cells. Once these technical aspects have been achieved, I will examine changes in calcium concentration in retinal ganglion cells before, during and at various times after optic nerve injury. I will also test the retinal ganglion cells’ ability to recover from increases in calcium that I impose on them with drugs. These experiments will provide some insight into the role that changes in calcium may play in the retinal ganglion cell death that results from axonal injury. This, in turn, will provide a basis for the rational design of therapeutic protocols to prevent vision loss due to glaucoma.