Assessment of rat and mouse RGC apoptosis imaging in vivo with different scanning laser ophthalmoscopes

Abstract

Purpose: We have recently described a novel way of imaging apoptosing retinal ganglion cells in vivo in the rat. This study investigated if this technique could be used in the mouse, and whether the Heidelberg Retina Angiograph II (HRAII) was appropriate.

Methods: Retinal ganglion cell (RGC) death was induced by intravitreal injections in rat and mouse eyes using staurosporine. Fluorescent-labeled apoptosing cells were detected by imaging with both the HRAII and a prototype Zeiss confocal scanning laser ophthalmoscope (cSLO). Averaged in vivo images were analyzed and results compared with histologic analysis.

Results: Fluorescent points (FPs) used as a measure of RGC apoptosis in vivo were detected in the mouse eye but only with the HRAII and not the Zeiss cSLO. The HRAII was able to detect 62% more FPs in rat than the Zeiss cSLO. Both cSLOs showed peak FP counts at the 5- to 10-microm range in rat and mouse. Maximal FP counts were detected in the superior and superior temporal regions in the rat, with no obvious pattern of distribution in the mouse. The HRAII was found to have more FP correspondence with histologically identified apoptosing RGCs.

Conclusions: To our knowledge, this is the first demonstration of visualized apoptosing RGC in vivo in a mouse. The improved image quality achieved with the HRAII compared with the Zeiss cSLO was validated by histology. This together with its enhanced maneuverability and the fact that it is already commercially available make the HRAII a potential tool for the early detection and diagnosis of glaucomatous disease in patients.

FIGURE 1

Retinal reflective images obtained in vivo in the rat with the (a) HRAII and the (b) Zeiss cSLO and in the mouse with the (c) HRAII and the (d) Zeiss cSLO.

FIGURE 2

Retinal fluorescence images obtained in vivo with the (a, c) HRAII and the (b, d) Zeiss cSLO of the same retinae in (a, b) rat and (c, d) mouse. The numerous fluorescent points (FPs) are evident in the enlarged area outlined in (c) The increased resolution of the HRAII is clearly visible.

FIGURE 3

Comparison of the FP size distribution between rat and mouse obtained by the HRAII. (a) Most fluorescent counts were obtained in the 5- to 10-μm size range in both rats and mice (a). (b) A comparison of the average number of FPs between rat and mouse according to its respective retinal image location is shown. (c) There is no pattern of distribution of FPs in relation to retinal image location, although the fluorescence is more evenly distributed in the mouse retina.

FIGURE 4

Comparison of apoptotic FP density (HRA vs. Zeiss). (a) The average number of apoptotic FPs per mm² in rats was 25 for the HRAII and 13 for the Zeiss cSLO. This suggest that the HRAII picks up 1.9 times more fluorescence signal compared with the Zeiss cSLO. (b) The difference between HRAII and Zeiss FP counts plotted against the average combined HRAII and Zeiss count shows a positive trend suggesting the HRAII is more sensitive at detecting smaller point signals. The marked data points highlight typical points, detailed in the text for comparison.

FIGURE 5

FP distribution in relation to (a) their diameter size and (b, c) the retinal image location in the rat. On average, the Zeiss detects 60% less fluorescent signal compared with the HRAII. The majority of FPs were within the 5- to 10-μm size range and the count number gradually decreased with increasing point diameter (a). The average number of FPs per single frame according to its respective retinal image location is shown in (b) with the corresponding retinal image map (c).

FIGURE 6

Histologic confirmation that apoptotic cells (a, labeled with Annexin V) were localized to RGC (b, retrogradely labeled with DiI) as shown in the combined micrograph (c) in the SSP model in a mouse.

FIGURE 7

Comparisons between the average apoptotic FP density detected by in vivo imaging and average apoptotic RGC density detected by confocal histology. (a) In the rat, confocal histology detects 42 cells/mm² compared with HRAII (25 cells/mm²; 60% of histology) and Zeiss cSLO (13 cells/mm²; 32% of histology). (b) In the mouse, the HRAII shows 54% (25 apoptotic cells/mm²) of the confocal histology (48 apoptotic cells/mm²).