Naturally occurring, transgenic and knockout rodent models are instrumental in the study of retinal disease mechanisms and in the development of treatments for human retinal dystrophies. To date, the majority of studies using rodent disease models rely upon retinal histopathology to follow disease progression and the effect of candidate therapies. Histopathology yields high resolution images and morphometric estimates of surviving retinal cells;1
however, the major drawback of this approach is that it does not allow longitudinal studies in the same animals.
imaging of the rodent retina offers the possibility to visualize disease processes and progression in individual animals and to reduce the effects of animal to animal variation, background lighting and genetic background. For example, it has been shown that in various neurodegenerative diseases, substantial modifications in the morphology of axons and dendrites can take place well before cell death.2, 3, 4, 5, 6, 7, 8
These observations highlight the need to develop high resolution in vivo
imaging techniques capable of resolving subcellular structures (such as individual axons and dendrites) in rodent eyes.
The resolution of in vivo imaging is limited by the optical quality of the rodent eye. Compared with the human eye, rodent eyes have smaller axial lengths, higher optical powers, larger average refractive errors and larger numerical apertures (NA) [see ]. Rats typically possess a large hyperopic refractive error. Retinoscopy measurements have shown refractive errors in the range of +5D to +15D in albino rats, with a strong dependence on the strain (Irving EL, et al. IOVS 2005;45:ARVO E-Abstract 4334). A dilated rodent eye also has a larger numerical aperture than a dilated human eye. Thus, in theory, one could resolve smaller retinal features with a perfect correction of the eye's aberrations in a dilated rodent eye than in a dilated human eye.
Ocular parameters for human, rat and mouse eyes.
Many studies have used fluorescence microscopy, fundus photography, two photon microscopy, confocal microscopy, or scanning laser ophthalmoscopy (SLO) to image the living rodent retina, allowing the visualization of structures such as blood vessels, capillaries, nerve fiber bundles, photoreceptors, retinal ganglion cells (RGCs), retinal pigment epithelial (RPE) cells and microglial cells.21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39
Fluorescently labeled RGCs have been imaged in the rodent retina in vivo
over a wide field,21-28,36,38,39
with some studies showing the apparent loss of ganglion cells in diseased retina.23,24,26,27,38,39
One report recently used a confocal laser scanning microscope to image ganglion cells and processes in vivo
in transgenic mice that expressed yellow fluorescent protein (YFP) in a small subset of RGCs.33
Resolution in all of these studies could be improved by correcting the eye's aberrations with adaptive optics (AO) so that many fine features that could previously be resolved only in excised retina could now be imaged in vivo
. Adaptive optics ophthalmoscopes have enabled near diffraction-limited imaging of cellular structures (such as individual photoreceptors, ganglion cells, and RPE cells) in living human and non-human primates,40, 41, 42, 43, 44
as well as the resolution of subcellular features (such as ganglion cell axons and dendrites) in living non-human primates.42,43
Recently, Biss et al.
fluorescently imaged mouse capillaries and microglia cells using an adaptive optics biomicroscope, showing that some of the benefits of adaptive optics found in primates can be realized in rodent eyes.31
We describe here a fluorescence scanning laser ophthalmoscope equipped with adaptive optics (fAOSLO) for the rat eye. We show that cellular and subcellular features in the rat retina, such as fine capillaries and individual fluorescently-labeled ganglion cell dendrites and axons, can be imaged. The in vivo resolution of our instrument was subsequently determined via confocal images of flatmounts of the enucleated retinas.