(values were determined by one-way ANOVA and Tukeys multiple comparison test. Thinning of the outer nuclear layer, a parameter indicative of the loss of photoreceptor cell viability, was also reduced in DFP-treated albino mice from age 2C6 mo. The null mutant mouse is well known to accumulate bisretinoid fluorophores such as A2E and A2-GPE at elevated rates (27C30). In the DFP-treated albino mice, A2E/iso-A2E were present in amounts that were twofold higher ( 0.05) than in control untreated mice (difference as percentage of control) (Fig. 1). A2E levels in the DFP-treated agouti wild-type mice were 49% higher than in untreated mice ( 0.05). This treatment effect can be explained as a DFP-mediated reduction in the degradative loss of bisretinoid. Since photodegradation of bisretinoid is usually more pronounced in the albino vision (17), high-performance liquid chromatography (HPLC)-quantified bisretinoid was not appreciably different in albino versus agouti mice (Fig. 1 and at and and agouti 129 wild-type mice. A2E (the sum of all-mice, and eight eyes (four mice) were pooled for agouti 129Sv mice. values were determined by one-way ANOVA and Sidaks multiple comparison test. Rt, retention time. Measuring Fundus Autofluorescence in DFP-Treated Mice by qAF. We also measured bisretinoid noninvasively using a previously published in vivo qAF approach (31, 32). Analysis revealed 26% higher levels of fundus autofluorescence in albino mice treated with DFP from age 2C4 mo ( 0.05) and 56% increased qAF in the mice treated from 2 to 6 mo of age ( 0.05) (Fig. 2). The higher qAF in DFP-treated compared with untreated mice is usually indicative of reduced bisretinoid loss due to oxidation. The difference between DFP-treated and control mice when measured by qAF is usually less than when measured by HPLC. We attribute this to the greater baseline short-wavelength fundus autofluorescence (SW-AF) signal recorded in albino mice due to the more pronounced intraocular light. Open in a separate windows Fig. 2. Quantitative fundus autofluorescence (qAF) (488 nm) in albino mice aged 4 and 6 mo. Mice were treated with DFP beginning at 2 mo of age. (value was determined by one-way ANOVA and Sidaks multiple comparison test. (of the image of the DFP-treated mouse is usually darker, indicating higher fundus AF (qAF) levels. Measuring Retinal Fe Levels Through Transferrin Receptor qPCR. When cells need more Fe, transferrin receptor mRNA is usually stabilized, leading to more Fe uptake (33). Transferrin receptor mRNA levels reflect intracellular Fe concentrations (23, 34), since in cells needing more Fe, transferrin receptor mRNA is usually stabilized. In mice receiving DFP in drinking water from age 2 mo, transferrin receptor mRNA levels, quantified in neural retina by qRT-PCR (35) were 1.73-fold greater than controls ( 0.05) at 4 mo of age, while the fold change in RPE/choroid/sclera was 1.66 ( 0.05) (Fig. 3). Open in a separate windows Fig. 3. Effect of DFP on ocular transferrin receptor expression in albino mice. Transferrin receptor mRNA is usually increased in neural retina (age 4 mo) and RPE/choroid/sclera (age 4 and 6 mo) in DFP-treated mice beginning at age 2 mo. Each value is the mean of four eyes analyzed from two mice. values were derived by one-way ANOVA and Sidaks multiple comparison test. Outer Nuclear Layer Thickness. In albino mice, the accelerated formation of bisretinoid leads to reduced photoreceptor cell viability that is detected at age 8 mo by measuring the thickness of the outer nuclear layer (ONL) (31, 36). In the DFP-treated mice, thinning of the ONL was less pronounced (Fig. 4). Interobserver agreement was calculated according to Bland and Altman (37). The mean difference between two observers (bias) was 0.05 m and the 95% limits of agreement was ?1.8C1.7 m. The ONL area, decided using the sum of ONL thicknesses in superior and inferior retina SA-2 (0.2C2 mm), was increased by 25% ( 0.001, unpaired two-tailed test) in superior hemiretina of mice receiving oral DFP and 17% ( 0.001) in inferior hemiretina compared with the control mice. Open in a separate window Fig. 4. Outer nuclear layer (ONL) thickness in 8-mo-old albino mice. (values were determined by one-way ANOVA and Sidaks multiple comparison test. Cp?/?; Heph?/? ML604440 Mice. Cp and its homolog Heph are copper-containing ferroxidase proteins that convert Fe2+ to Fe3+ so as to enable cellular Fe export. Accordingly, deficiency in Heph and Cp leads to elevated intracellular Fe in RPE (38). We found that mice also exhibited reduced bisretinoid measured as A2E/iso-A2E (Fig. 5). We interpret this finding as indicative of increased A2E oxidation and degradation due to elevated Fe. Open in a separate window Fig. 5. UPLC quantitation of A2E and iso-A2E in mice deficient in ceruloplasmin (Cp) and.Rt, retention time. Measuring Fundus Autofluorescence in DFP-Treated Mice by qAF. effects of bisretinoids in juvenile and age-related macular degeneration. and agouti wild-type mice exhibited elevated bisretinoid levels as measured by high-performance liquid chromatography or noninvasively by quantitative fundus autofluorescence. Thinning of the outer nuclear layer, a parameter indicative of the loss of photoreceptor cell viability, was also reduced in DFP-treated albino mice from age 2C6 mo. The null mutant mouse is well known to accumulate bisretinoid fluorophores such as A2E and A2-GPE at elevated rates (27C30). In the DFP-treated albino mice, A2E/iso-A2E were present in amounts that were twofold higher ( 0.05) than in control untreated mice (difference as percentage of control) (Fig. 1). A2E levels in the DFP-treated agouti wild-type mice were 49% higher than in untreated mice ( 0.05). This treatment effect can be explained as a DFP-mediated reduction in the degradative loss of bisretinoid. Since photodegradation of bisretinoid is more pronounced in the albino eye (17), high-performance liquid chromatography (HPLC)-quantified bisretinoid was not appreciably different in albino versus agouti mice (Fig. 1 and at and and agouti 129 wild-type mice. A2E (the sum of all-mice, and eight eyes (four mice) were pooled for agouti 129Sv mice. values were determined by one-way ANOVA and Sidaks multiple comparison test. Rt, retention time. Measuring Fundus Autofluorescence in DFP-Treated Mice by qAF. We also measured bisretinoid noninvasively using a previously published in vivo qAF approach (31, 32). Analysis revealed 26% higher levels of fundus autofluorescence in albino mice treated with DFP from age 2C4 mo ( 0.05) and 56% increased qAF in the mice treated from 2 to 6 mo of age ( 0.05) (Fig. 2). The higher qAF in ML604440 DFP-treated compared with untreated mice is indicative of reduced bisretinoid loss due to oxidation. The ML604440 difference between DFP-treated and control mice when measured by qAF is less than when measured by HPLC. We attribute this to the greater baseline short-wavelength fundus autofluorescence (SW-AF) signal recorded in albino mice due to the more pronounced intraocular light. Open in a separate window Fig. 2. Quantitative fundus autofluorescence (qAF) (488 nm) in albino mice aged 4 and 6 mo. Mice were treated with DFP beginning at 2 mo of age. (value was determined by one-way ANOVA and Sidaks multiple comparison test. (of the image of the DFP-treated mouse is darker, indicating higher fundus AF (qAF) levels. Measuring Retinal Fe Levels Through Transferrin Receptor qPCR. When cells need more Fe, transferrin receptor mRNA is stabilized, leading to more Fe uptake (33). Transferrin receptor mRNA levels reflect intracellular Fe concentrations (23, 34), since in cells needing more Fe, transferrin receptor mRNA is stabilized. In mice receiving DFP in drinking water from age 2 mo, transferrin receptor mRNA levels, quantified in neural retina by qRT-PCR (35) were 1.73-fold greater than controls ( 0.05) at 4 mo of age, while the fold change in RPE/choroid/sclera was 1.66 ( 0.05) (Fig. 3). Open in a separate window Fig. 3. Effect of DFP on ocular transferrin receptor expression in albino mice. Transferrin receptor mRNA is increased in neural retina (age 4 mo) and RPE/choroid/sclera (age 4 and 6 mo) in DFP-treated mice beginning at age 2 mo. Each value is the mean of four eyes analyzed from two mice. values were derived by one-way ANOVA and Sidaks multiple comparison test. Outer Nuclear Layer Thickness. In albino mice, the accelerated formation of bisretinoid leads to reduced photoreceptor cell viability that is detected at age 8 mo by measuring the thickness of the outer nuclear layer (ONL) (31, 36). In the DFP-treated mice, thinning of the ONL was less pronounced (Fig. 4). Interobserver agreement was calculated according to Bland and Altman (37). The mean difference between two observers (bias) was 0.05 m and the 95% limits of agreement was ?1.8C1.7 m. The ONL area, determined using the sum of ONL thicknesses in superior and inferior retina (0.2C2 mm), was increased by 25% ( 0.001, unpaired two-tailed test) in superior hemiretina of mice receiving oral DFP and 17% ( 0.001) in inferior hemiretina compared with the control mice. Open in a separate window Fig. 4. Outer nuclear layer (ONL) thickness in 8-mo-old albino mice. (values were determined by one-way ANOVA and Sidaks multiple comparison test. Cp?/?; Heph?/? Mice. Cp and its homolog Heph are copper-containing ferroxidase.