The ratio of 488 nm AF measurements between
Abca4−/− and WT mice increased steeply within the first 3 months of life to approximately two, followed by a ceiling effect with only a minor further increase until age 9 months. These values are similar to those previously reported in patients with Stargardt disease compared with healthy controls.
24,25 Although the ratio of A2E levels between
Abca4−/− and WT mice changed with a similar time course, its increase was considerably higher. One hypothesis to explain this discrepancy (assuming
Abca4−/− mice had
bis-retinoid and AF levels similar to WT mice at birth) would be that a substantial fraction of the 488-nm fundus AF signal in WT mice derives from fluorophores other than A2E and related
bis-retinoids that do not vary considerably between WT and
Abca4−/− mice (e.g., connective tissue flavoproteins, retinoids that do not depend on functional ABCA4;
Fig. 7). A large increase in A2E content would then be necessary before relevant changes of 488 nm AF may be detected. Thus, increased 488 nm fundus AF levels parallel the accumulation of A2E in the RPE of
Abca4−/− mice, but may not quantitatively represent its accumulation in direct proportion. Such “incongruence” was also reported based on post mortem experiments by Boyer et al. who found a lack of correspondence in the rates of increase between lipofuscin-related AF in eyecups and quantification of A2E.
26 Similar to our data, AF and A2E-levels in eyes from aged
Abca4−/− mice (background strain: 129Sv) compared with WT controls were approximately 2- and 10-fold higher, respectively.
26 The same authors also reported incongruence between lipofuscin-related AF and A2E levels across different WT mouse strains: while eyecup AF intensity in the C57BL/6 strain was higher compared with the 129Sv strain, A2E-levels were lower. Such differences of AF intensity based on genetic background were also observed in our experiments (not reported in detail;
Supplementary Fig. S8) and may be of interest when interpreting quantitative AF measures in humans.
27 Boyer et al.
26 also challenged the current hypothesis on the function of ABCA4 by providing evidence that light exposure and thus all-
trans-retinal formation is not necessary for accumulation of A2E- and lipofuscin in the RPE. Assessment of AF intensity in vivo in dark-reared animals was not performed in our study, but would complement those findings.