Oxidative lipid and DNA changes in the meibomian glands.

<p>A, Late phase lipid peroxidation marker 4-HNE stainings show dense staining in the 50 week old Cu, Zn-Superoxide Dismutase-1 knockout (<i>Sod1</i>^−/−) mice. Wild type (WT) mice specimens also had some staining but not to the extent observed in the same age <i>Sod1</i>^−/− mice. Bar = 50 micrometer. B, The extent of cellular staining with 4-HNE was significantly higher in the <i>Sod1</i>^−/− than the WT mice at 50 weeks (<i>p</i> = 0.0022). Note the significant timewise elevation in the anti-4-HNE staining from 10 to 50 weeks in the <i>Sod1</i>^−/− mice. C, Staining with 8-OHdG antibodies in <i>Sod1</i>^−/− and WT mice samples at 10 and 50 weeks. Meibomian gland acinar cell nuclei showed scant staining in 10 week <i>Sod1</i>^−/− and WT mice. Bar = 100 micrometer. There was a marked increase in nuclear staining from 10 to 50 weeks, especially in <i>Sod1</i>^−/− mice. Relatively more acinar nuclei were stained with anti-8-OHdG antibodies in the <i>Sod1</i>^−/− mice at 50 weeks compared to meibomian gland specimens from the same age WT mice. D, Quantitative assessment for the cellular staining of anti-8-OHdG antibodies showed a statistically significant timewise increase from 10 to 50 weeks in <i>Sod1</i>^−/− and WT mice (<i>p</i> = 0.0003, <i>p</i> = 0.0011, respectively). A significant timewise elevation in staining was observed in the <i>Sod1</i>^−/− and WT mice at 50 weeks (<i>p</i> = 0.0133). Five tissue sections of each animal's eye were analyzed to produce the figure. Data represent the mean ± standard deviation for 7 mice from the <i>Sod1</i>^−/− group and 6 mice from the wild type group at 10 and 50 weeks.</p

Transmission electron microscopic examination of the mitochondrial alterations.

<p>Marked ultrastructural changes in the mitochondria including swelling, disorientation, shortening, and disorganization of cristae were noted at 50 weeks in the Cu, Zn-Superoxide Dismutase-1 knockout (<i>Sod1</i>^−/−) mice. We observed abnormalities in 40 percent of the 50 week wild type (WT) mice and 80 percent in the age matched <i>Sod1</i>^−/− mice. No such phenotypic alterations were observed in the mitochondria of the 10 to 50 week WT mice. Bar = 1 micrometer. Five tissues sections from each eye of the 6 animals in each mice group were analyzed to produce the representative images.</p

Aqueous tear production, tear stability and ocular surface epithelial cell damage assessment.

<p>A, The weight adjusted tear quantity decreased significantly from 10 to 50 weeks in the Cu, Zn-Superoxide Dismutase-1 knockout (<i>Sod1</i>^−/−) mice (<i>p</i> = 0.0012). The tear quantity was also significantly lower in the <i>Sod1</i>^−/− mice compared to the age matched wild type (WT) mice at 10 and 50 weeks (<i>p</i> = 0.0079 and <i>p</i><0.0001, respectively). B, The mean tear break-up time values decreased significantly from 10 to 50 weeks in the <i>Sod1</i>^−/− mice (<i>p</i> = 0.0330). The tear stability was significantly worse in the <i>Sod1</i>^−/− mice compared to WT mice at 50 weeks (<i>p</i> = 0.0004). C, Fluorescein staining scores were significantly higher in the <i>Sod1</i>^−/− mice compared to the WT mice both at 10 (<i>p</i> = 0.0005) and 50 (<i>p</i> = 0.0006) weeks. Fluorescein staining also increased significantly from 10 to 50 weeks in both the <i>Sod1</i>^−/− and WT mice (<i>p</i> = 0.0113 and <i>p</i> = 0.0032, respectively). Data represent the mean ± standard deviation of 9 mice from the <i>Sod1</i>^−/− groups and 10 mice from the WT groups, at 10 and 50 weeks.</p

Meibomian gland histopathological alterations and tissue fibrosis in the <i>Sod1</i>^−/− and wild type mice.

<p>A, Representative images for meibomian glands from the 10 and 50 week Cu, Zn-Superoxide Dismutase-1 knockout (<i>Sod1</i>^−/−) and wild type (WT) mice show normal meibomian gland acinar units morphology. Note the extensive periglandular inflammatory infiltration, and meibomian gland changes in the <i>Sod1</i>^−/− mice at 50 weeks. Bar = 50 micrometer. B, Mallory stainings show increased fibrosis (dark blue stained areas) from 10 to 50 weeks in the <i>Sod1</i>^−/− mice. Similar changes were observed in the WT mice but not to the extent observed in the <i>Sod1</i>^−/− mice. Bar = 50 micrometer. C, Meibomian gland acinar unit density significantly decreased from 10 to 50 weeks in both <i>Sod1</i>^−/− mice and the WT mice (<i>p</i> = 0.0007 and <i>p</i> = 0.0175, respectively). Meibomian gland acinar unit density was also significantly lower in the <i>Sod1</i>^−/− mice compared to the WT mice at 50 weeks (<i>p</i> = 0.0003). Five tissue sections of each mouse eye were analyzed to produce the meibomian gland acinar unit density values. Data represent the mean ± standard deviation for at least 7 mice from the <i>Sod1</i>^−/− groups and 10 mice from the WT groups, at 10 and 50 weeks. Five tissues sections from each eye of 7 animals (5 images per animal's eye) for each group were analyzed to produce the representative Mallory staining images.</p

Inflammatory changes in the meibomian glands, serum and tears of the <i>Sod1</i>^−/− and wild type mice.

<p>A, Specimens stained with CD45 from 10 and 50 weeks Cu, Zn-Superoxide Dismutase-1 knockout (<i>Sod1</i>^−/−) and wild type (WT) mice. Note the timewise increase in inflammatory cell staining from 10 to 50 weeks in the <i>Sod1</i>^−/− mice. Wild type mice specimens showed scant inflammatory cell staining, which increased significantly in the 50 week WT mice but not to the extent observed in the <i>Sod1</i>^−/− mice at 50 weeks. Bar = 50 micrometer. B, A significant timewise increase in the mean inflammatory cell densities from 10 to 50 weeks was observed in both the <i>Sod1</i>^−/− and WT mice (<i>p</i> = 0.0143 and <i>p</i> = 0.0286, respectively). Note the significantly higher inflammatory cell density in the <i>Sod1</i>^−/− mice at 50 weeks compared to the age matched WT mice (<i>p</i> = 0.0317). Five tissue sections of each eye of 6 animals (5 per mouse eye) were analyzed to produce the figure. Data represent the mean ± standard deviation for 6 mice from the <i>Sod1</i>^−/− groups and 6 mice from the WT groups at 10 and 50 weeks. C, The mean serum IL-6 concentration increased significantly from 10 to 50 weeks in both the <i>Sod1</i>^−/− (<i>p</i> = 0.0195) and WT mice (<i>p</i> = 0.0001). Serum IL-6 concentration was significantly higher in the 50 week <i>Sod1</i>^−/− compared to 50 week WT mice (<i>p</i> = 0.0100). Serum TNF-α levels were also significantly higher (<i>p</i> = 0.0011) in the 50 week <i>Sod1</i>^−/− mice compared to the age matched WT mice. The mean serum TNF-α concentration significantly increased from 10 to 50 weeks in the <i>Sod1</i>^−/− mice (<i>p</i> = 0.0457). Data represent the mean ± standard deviation for 14 and 8 <i>Sod1</i>^−/− mice at 10 and 50 weeks as well as 11 and 9 wild type mice at 10 and 50 weeks, respectively. D, There was also a significant (<i>p</i> = 0.0148) timewise increase in the mean tear IL-6 concentration in the <i>Sod1</i>^−/− mice from 10 to 50 weeks. Note the significantly higher IL-6 concentration in the <i>Sod1</i>^−/− mice at 10 (<i>p</i> = 0.0414) and 50 weeks (<i>p</i> = 0.0022) compared to the age matched WT mice. Tear TNF-α concentrations increased significantly from 10 to 50 weeks in the <i>Sod1</i>^−/− (<i>p</i> = 0.0115). Also note the significantly higher TNF-α concentration in the <i>Sod1</i>^−/− mice compared to the WT mice at 50 weeks (<i>p</i> = 0.0087). Data represent the mean ± standard deviation for 7 mice from the <i>Sod1</i>^−/− groups and 9 mice from the WT groups at 10 and 50 weeks.</p