Effect of EDE on <i>P.</i><i>aeruginosa</i> corneal colonization in SP-D knockout mice.

<p>Corneal colonization by <i>P. aeruginosa</i> in normal Black Swiss mice (A) or SP-D deficient age/sex-matched Black Swiss mice (B) under normal (NC) and experimental dry eye (EDE) conditions. After 5 days EDE induction, otherwise uninjured corneas were challenged with 10⁹ cfu of <i>P. aeruginosa</i> strain PAO1 (T = 0). EDE did not affect bacterial colonization in wild-type mice after 6 h. However, EDE in SP-D knockout mice (<i>sp-d</i> −/−) resulted in a ∼5-fold increase in corneal colonization after 6 h. Data shown is representative of two independent experiments with SP-D-deficient Black Swiss mice (n≥5 animals per group). P values were obtained using the Mann-Whitney Test. Data for each sample are shown as the median (black square) with upper and lower quartiles (boxed area), and range of the data (error bars).</p

Induction of experimental dry eye.

<p>Tear volumes (A) and fluorescein staining (B) in the eyes of C57BL/6 mice under experimental dry eye (EDE) conditions versus normal controls (NC). (A) EDE resulted in significant decreases in tear volume after 2 days. Tears were collected from the lateral canthus using cotton thread and reported as millimeters of wetted thread. Data are expressed as the mean +/− standard deviation per group from three independent experiments (≥3 mice per group for each experiment). * Denotes significance differences between treatment groups (determined with a parametric repeated measures ANOVA and Bonferroni post-test), p<0.001 in each instance). (B) Corneal integrity was assessed by fluorescein staining in EDE mice or normal controls after 5 or 10 days. Eyes were examined under blue light illumination at 20-x magnification. Photographs are representative of three independent experiments (≥3 mice per group for each experiment). Control mouse eyes are shown in the upper panels (a, b), EDE mouse eyes are shown in the lower panels (c, d). Arrows denote regions of fluorescein staining on the ocular surface.</p

SP-D expression in EDE before and after <i>P.</i><i>aeruginosa</i> challenge.

<p>Western immunoblot blot analysis of SP-D expression in pooled ocular surface washes from EDE and control mice (10 mice per group) after 5 days EDE induction, and before and 6 h after inoculation with <i>P. aeruginosa</i> strain PAO1 (10⁹ cfu). To normalize for differences in tear volume, equivalent amounts of protein (2 µg) were used in the analysis (BCA protein assay). Purified recombinant SP-D (rSP-D, ∼43 kDa monomer), and a relevant number of bacteria suspended in PBS (5×10³ cfu, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065797#pone-0065797-g002" target="_blank">Fig. 2B</a>), were included as positive and negative controls, respectively. SP-D expression in ocular surface washes was increased under EDE conditions before bacterial inoculation. The experiment was repeated once.</p

Ocular clearance of <i>P.</i><i>aeruginosa</i> in EDE.

<p>Levels of viable <i>P. aeruginosa</i> (cfu) in corneal homogenates (A) or ocular surface washes (B) of C57BL/6 EDE mice compared to normal controls (NC) at 6 h post-inoculation with 10⁹ cfu of <i>P. aeruginosa</i> strain PAO1 (T = 0). EDE was induced for 5 days prior to bacterial inoculation. Bacteria were rapidly cleared from the murine ocular surface of both groups of mice after 6 h. Similar bacterial levels were found in corneal homogenates (A), but fewer bacteria were recovered from the ocular surface washes of EDE mice compared to controls (p = 0.049, Mann-Whitney test) (B). Data are representative of three independent experiments (≥5 animals per group in each experiment). Data for each sample are shown as the median (black square) with upper and lower quartiles (boxed area), and range of the data (error bars).</p

Incidence and severity of <i>P. aeruginosa</i> infections in EDE mice.

<p>C57BL/6 mice were exposed to EDE or control conditions for 10 d prior to topical challenge with 10⁹ cfu of <i>P. aeruginosa</i> strain PAO1. Mice were monitored for corneal infiltrates, opacities, and changes in epithelial surface regularity. Pathology was graded at 96 h post-inoculation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065797#s2" target="_blank">Materials and Methods</a>). Incidences of pathology in EDE and control groups were not significantly different (Chi-square analysis). Data is representative of two independent experiments.</p