Objectives: Progressive neuronal cell death and microglial activation are the key pathological features in most neurodegenerative diseases. Analyzing progressive changes has been obfuscated by the lack of a non-invasive approach that allows long term, serial monitoring of individual neuronal and microglial cells. Because of the clear optical media in the eye, direct visualization of the retinal ganglion cells (RGCs) and microglia is possible with high resolution in vivo imaging technique. In this study, (1) we developed experimental models to visualize and characterize the cellular morphology of RGCs and retinal microglia in vivo in the Thy-1 YFP and the CX3CR1 +/GFP transgenic mice, (2) described the patterns of axonal and dendritic shrinkage of RGCs, (3) discerned the dynamic profile of microglial activation and (4) investigated the relationship between RGC survival and microglial activation after optic nerve crush and retinal ischemic injury induced by acute elevation of intraocular pressure.
Methods: A confocal scanning laser ophthalmoscope (CSLO) was used to image the axonal and dendritic aborizations of RGCs in the Thy-1 YFP mice or microglia in CX3CR1 GFP/+ mice. With quantitative analysis of cell body area, axon diameter, dendritic field, number of terminal branches, total dendritic branch length, branching complexity, symmetry and distance from the optic disc, the morphologies of RGCs and the patterns of axonal and dendritic degeneration were analyzed.
Results: After optic nerve crush, RGC damage was observed prospectively to begin with progressive dendritic shrinkage, followed by loss of the axon and the cell body. Similar pattern of RGC degeneration was observed after 90 minutes of retinal ischemia although no morphological changes were detected when the duration of ischemia was shortened to 30 minutes. The rate of dendritic shrinkage was variable and estimated on average 2.0% per day and 11.7% per day with linear mixed modeling, after optic nerve crush and retinal ischemic injury, respectively. RGCs with a larger dendritic field had a slower rate of dendritic shrinkage. Activation of retinal microglia was characterized with an increase in cell number reaching a peak at a week after optic nerve crush and retinal ischemic injury, which was followed by a gradual decline falling near to the baseline at the 4 the week. The activation of retinal microglia was proportional to the severity of injury. The number of RGCs survival at 4 -12 weeks post-injury was significantly associated with the number of activated retinal microglia.
Conclusions: In summary, we demonstrated that dendritic shrinkage could be evident even before axonal degeneration after optic nerve crush and retinal ischemic injury. We have established a methodology for in vivo and direct visualization of RGCs and retinal microglia, which could provide reliable and early markers for neuronal damage. Measuring the rate of dendritic shrinkage and tracking the longitudinal activation of microglia would provide new paradigms to study the mechanism of neurodegenerative diseases and offer new insights in testing novel therapies for neuroprotection.