Abstract
Purpose :
Fluorescence lifetime imaging microscopy (FLIM) was previously used to study the metabolic state of wild-type (WT) mice retina. The rd10 mice model has a mutation in the Pde6b gene and can be used to understand the retinal metabolic state in inherited retinal degeneration. We propose to use FLIM to determine differences in the retinal structure and metabolic states for 8 age groups in (WT) and rd10 mice.
Methods :
WT (C57BL6J) and rd10 (Pde6b rd1-J ) mice were used. A total of 121 eyes postnatal day (P)0, 5, 10, 15, 20, 30, 56, and 84 were analyzed. After euthanasia and enucleation, the cornea, lens, and iris were removed, and soaked in 4%PFA for 3 hours. The eyecups were embedded in a polyacrylamide solution and 100 μm sections were created using the Leica Microtome VT1200. These sections were imaged using Leica SP9 DIVE FALCON with a 25x/0.95 NA water immersion objective. An excitation wavelength of 740nm and emission collected between 425-475 nm
were chosen to capture NAD(P)H fluorescence. Statistical analysis was performed using SPSS and Excel.
Results :
The structure of WT and rd10 mice retinae were compared using autofluorescence images. After P15, there was degeneration of the inner and outer photoreceptor segments and outer nuclear layer in the rd10 retinae. The retinal metabolic states were studied using FLIM. The attached table compares the mean+/-SD bound NAD(P)H percentage (bound NAD(P)H / total NAD(P)H) between WT and rd10; a higher percentage of bound NAD(P)H denotes more oxidative phosphorylation (OXPHOS) compared to glycolysis. In most age groups, there was a statistically significantly higher OXPHOS in rd10 compared to WT retinae. Furthermore, a statistically significant more OXPHOS was seen in the inner retina compared to the outer retina in all age groups of rd10 mice and most age groups of WT retinae, as reflected in our previous study with WT mice, which showed a statistically significant more OXPHOS in inner compared to the outer retina.
Conclusions :
Using FLIM, our study verified that OXPHOS has a downward trend in the early time points of retinal development (until P10 for rd10 retina and P30 for WT retina), following which OXPHOS increases and plateaus.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.