We next performed scotopic and photopic ERGs on wt and
lgals3−/− mice at 2.5 months of age. Analysis of a- and b-wave amplitudes of both photopic and scotopic recordings revealed no difference between wt and
lgals3−/− responses (
Figs. 4A,
4B). These results suggest normal rod and cone photoreceptor dependent light responses in the absence of gal-3. Moreover, we found no difference in photoreceptor opsins or other photoreceptor or RPE marker proteins comparing 3-month-old
lgals3−/− and wt eyes (
Fig. 5). In this experiment, we included analysis of 3-month-old
mertk−/− eyes as positive control for retinal degeneration. Retinal degeneration in
mertk−/− mice progresses rapidly from approximately PN25, such that all outer segments and indeed the majority of photoreceptor cells are lost by 3 months of age leading to diminished levels of opsin and synapse proteins.
13 To quantify opsins, we tested whole eye tissues without cornea and lens to minimize variability due to manual neural retina dissection, which involves some damage to the outer segments. As expected, the
mertk−/− samples showed dramatically reduced content of rhodopsin and cone opsins as well as of the neuronal synapse marker PSD95 (see
Fig. 5A). Comparing the ratios of opsin levels relative to wt between
lgals3−/− and
mertk−/− tissues quantified these observations (see
Fig. 5B). The side-by-side tissue comparisons showed no difference in RPE marker RPE65 among the three different mouse strains (see
Fig. 5A). Next, we compared dissected neural retina and posterior eyecups enriched in RPE and choroid of the three mouse strains (see
Fig. 5C). We found that gal-3 content was higher in eyecups than in neural retina (by about 7-fold), but it should be noted that each tissue fraction represents a mix of several cell types (e.g. Müller cells are not an abundant cell type in the neural retina). Müller cell gal-3 (easily observed by microscopy) is thus diluted in the neural retina fraction to which Müller cell proteins make only a minor contribution. Notably,
mertk−/− eyecups show higher gal-3 levels than wt eyecups suggesting that gal-3 in the posterior eye is upregulated during retinal degeneration (see
Figs. 5C,
5D). In contrast,
lgals3−/− eye tissue fractions have normal levels of MerTK (see
Figs. 5C,
5D). Altogether, our experiments do not support any abnormalities in retinal or RPE markers or degeneration in
lgals3−/− mouse retina.