The visual cycle in 5 to 8 mo-old <i>Fatp1</i>^−/− mice.

<p>A. Expression levels of the main visual cycle genes, Rpe65, Lrat and Rdh5. Bars represent the mean of triplicates ± SEM. * <i>p</i><0.05. The genes were slightly deregulated with a 9.6% increase observed for <i>Rpe65</i>, a 12.5% increase for <i>Lrat</i> and a 19.5% increase for <i>Rdh5</i>. B. Recovery of the b-wave amplitude after bleaching in 5 to 8 mo-old mice (<i>n</i> = 29 <i>Fatp1</i>^+/+ and <i>n</i> = 21 <i>Fatp1^−/−</i>). Error bars represent SEM. * <i>p</i><0.05. C. Quantification of the 11-<i>cis</i> retinal and all-<i>trans</i> retinal levels in the whole eye. Error bars represent SEM. The recovery of the electroretinogram is delayed in <i>Fatp1^−/−</i> mice but this difference is not linked to a visual cycle kinetic defect.</p

Aging of the <i>Fatp1</i><i>^−/−</i> retina.

<p>A. Electroretinography of <i>Fatp1</i>^+/+ (<i>n</i> = 5) and <i>Fatp1^−/−</i> (<i>n</i> = 11) mice, * <i>p</i> = 0.021 for the whole curve. B. Quantification of autofluorescence in young (<i>n</i> = 3 <i>Fatp1</i>^+/+ and <i>n</i> = 4 <i>Fatp1^−/−</i>) and old (<i>n</i> = 6 <i>Fatp1</i>^+/+ and <i>n</i> = 5 <i>Fatp1^−/−</i>) mice. The values are expressed in arbitrary fluorescent units from the machine. For each mouse, the result is obtained by summing up all values between 520 and 640 nm taken every 10 nm. Error bars represent SEM. <i>Fatp1^−/−</i> PRs remains less responsive to light with age. <i>Fatp1^−/−</i> mice do not accumulate more autofluorescence, thus presumably more lipofuscin, than <i>Fatp1</i>^+/+ with age. C. Upper panel: neutral lipid labeling of <i>Fatp1</i>^+/+ (WT) and <i>Fatp1^−/−</i> (KO) retinas. Ch: choroid. RPE: retinal pigment epithelium. POS: PR outer segments. PIS: PR inner segments. ONL: outer nuclear layer. OPL: outer plexiform layer. INL: inner nuclear layer. IPL: inner plexiform layer. GCL: ganglion cell layer. Arrows indicate the BM. Lower panel: positive control at the same magnification (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050231#s2" target="_blank">Material and methods</a>). Arrows indicate the BM as in the upper panel. The asterisk indicates positive ORO labeling. <i>Fatp1^−/−</i> or <i>Fatp1</i>^+/+ mice showed no Oil red O labeling in the BM.</p

Abnormalities observed in aged mice by TEM.

<p>Abnormalities observed in aged mice by TEM.</p

Visual phenotype of the <i>Fatp1</i>^−/− mouse.

<p>A. Histology of the retina. Ch: choroid. RPE: retinal pigment epithelium. POS: PR outer segments. PIS: PR inner segments. ONL: outer nuclear layer. OPL: outer plexiform layer. INL: inner nuclear layer. IPL: inner plexiform layer. GCL: ganglion cell layer. WT: <i>Fatp1</i>^+/+. KO: <i>Fatp1^−/−</i>. B. TEM of WT and KO retinas. BM: Bruch membrane. C. Electroretinography of <i>Fatp1</i>^+/+ (<i>n</i> = 29) and <i>Fatp1^−/−</i> (<i>n</i> = 23) mice. * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001. The statistical analyses were performed initially for the whole curve (line above the graph) and subsequently for each point. D. b-wave on a-wave ratio of the ERG experiments. Error bars represent SEM. E. Quantification of the retinal coupled-rhodopsin (<i>n</i> = 4 <i>Fatp1</i>^+/+ and <i>n</i> = 4 <i>Fatp1^−/−</i>) in mice aged 5 to 8 months. Error bars represent SEM. The retinal histology is unchanged in the <i>Fatp1^−/−</i> mice. <i>Fatp1^−/−</i> PRs present a lower response to light flashes and this decrease is not due to a decrease in the level of retinal coupled-rhodopsin in these animals.</p

Ultrastructural aging of the <i>Fatp1</i><i>^−/−</i> retina.

<p>A: TEM at low magnification of a <i>Fatp1</i>^+/+ retina. PR outer segments in <i>Fatp1^−/−</i> at low magnification (B), in <i>Fatp1</i>^+/+ at high magnification (C), in <i>Fatp1^−/−</i> mice at high magnification (D–F). Certain <i>Fatp1^−/−</i> mice had PR outer segment disorganization. G: <i>Fatp1^−/−</i> retina at low magnification showing a thick BM. H: <i>Fatp1</i>^+/+ BM at high magnification. I-K: <i>Fatp1^−/−</i> retina at high magnification showing a thicker BM than age-matched control. L: Box plot graph showing the thickness of the BM between <i>Fatp1</i>^+/+ and <i>Fatp1^−/−</i> retinas (solid line indicates the median; <i>p</i><0.05). M-O: <i>Fatp1^−/−</i> retinas at low magnification showing numerous vessels along the BM (M), no vessels (N) and large vessels (O). Arrows indicate the thickness of the BM. Asterisks indicate vessels. The majority of <i>Fatp1^−/−</i> mice exhibited an irregular or laminar thicker BM. Most of the <i>Fatp1^−/−</i> mice showed strikingly modified choroidal vessels, ranging from presence of only large vessels to absence of vessels.</p

Fatty acid composition in the NR and RPE in 8 mo-old <i>Fatp1</i>^+/+ and <i>Fatp1^−/−</i> mice.

<p><i>Fatp1</i>^+/+ (n = 5) and <i>Fatp1^−/−</i> (n = 7). 16:0: palmitate. 22:6n-3: DHA. The functional visual difference between <i>Fatp1^−/−</i> and <i>Fatp1</i>^+/+ mice is not due to a different fatty acid composition.</p

Expression of Fatp1 and Fatp4 in the retinas of <i>Fatp1</i>^+/+ and <i>Fatp1</i><i>⁻</i>^/<i>−</i> mice.

<p>Expression of <i>Fatp1</i> (A) and <i>Fatp4</i> (B) in the NR and RPE of <i>Fatp1</i>^+/+ and <i>Fatp1^−/−</i> mice as determined by qPCR. Results are expressed as a percentage of <i>Actin</i> mRNA expression for NR and as a percentage of <i>Mertk</i> mRNA expression for RPE. mRNA was extracted from tissue pools of 12 <i>Fatp1</i>^+/+ and 10 <i>Fatp1⁻</i>^/<i>−</i> mice aged 5 months old. Bars represent the mean of triplicates ± SEM. * <i>p</i><0.05. <i>Fatp4</i> expression is decreased in the <i>Fatp1^−/−</i> NR as compared to wt and expression levels are unchanged in the RPE. There is no compensatory up-regulation of <i>Fatp4</i> in the <i>Fatp1^−/−</i> retinas.</p