Table 1.
Primers.
Figure 1.
Lack of Muc5ac in the ocular tissues of Muc5ac knockout mice.
Cornea (C), Conjunctiva (Cj), and Lacrimal gland (L) were dissected and total RNA was extracted for RT-PCR assay. M: molecular weight marker. Actin was used as a loading control. #1, 2, 3, 4, 5, 6 were different mice. WT: wild-type mice. KO: Muc5ac knockout mice.
Figure 2.
Mucin gene expressions in the conjunctiva tissues of wild-type and Muc5ac knockout mice.
Conjunctiva was dissected and total RNA was extracted for realtime PCR assay. n = 5, $, *: p<0.05. NS: not significant. WT: wild-type mice. KO: Muc5ac knockout mice.
Figure 3.
Significantly increased mucous cell staining positive areas.
Mouse conjunctiva tissues were harvested, fixed and the tissue sections were stained with AB/PAS or anti-Muc5ac and anti-Muc5b antibodies. A) Representative images (60X, 10 um scale bar was shown) from AB/PAS staining on mouse conjunctiva tissues. B) Statistical analysis of AB/PAS staining positive area per basement membrane length. n = 5, *: p<0.05. C) Representative images (60X, 10 um scale bar was shown) from dual staining of Muc5ac and Muc5b on mouse conjunctiva tissues. D) Statistical analysis of Muc5b staining positive area per basement membrane length. n = 5, *: p<0.05. WT: wild-type mice. KO: Muc5ac knockout mice.
Figure 4.
Representative photographs of corneal opacification are shown.
Corneal opacification of various degrees was noted in both eyes of 4 out of 35 Muc5ac-deficient mice (KO), but not in any of the wild-type mice (WT).
Figure 5.
Tear stability is decreased in Muc5ac-deficient mice (KO) compared to wild-type mice (WT).
This is determined by measuring tear film breakup time (TBUT). Bar graph shows the mean TBUT in KO and WT. TBUT values were obtained in a masked fashion under a cobalt blue filter after the application of fluorescein dye. Three measurements were averaged to generate the final value, which represent the data presented. Total 31 KO and 15 WT were compared. These two groups were gender and age matched. (*: p = 0.001, independent t-test, error bars indicate 95%CI).
Figure 6.
Corneal epithelium is disrupted in Muc5ac-deficient mice (KO) compared to wild-type mice (WT).
This is determined by corneal fluorescein staining. Liquid fluorescein (1 ul, 0.25%) was applied for 1 minute and the excess fluorescein was rinsed with 0.2 mL of HBSS and blotted with q-tips. The fluorescein staining was analyzed under a cobalt blue filter and scored as described in the method section. Eyes were closed between each evaluation to prevent excessive exposure and irritation of ocular surface. A) Representative photographs of fluorescein staining demonstrate the corneal staining scores (scores 1, 3, and 4 are shown). B) Bar graph shows the mean score for corneal staining in KO and WT. The mean fluorescein scores from total 31 KO and 15 WT were compared. These two groups were gender and age matched. (*: p<0.001, independent t-test, error bars indicate 95%CI).
Figure 7.
Tear volume is similar in Muc5ac-deficient mice (KO) compared to wild-type mice (WT).
This is determined by red phenol thread test. Bar graph shows the mean thread measurement in Muc5ac-deficient mice and wild-type. Three measurements (mm) were obtained with the thread placed in the lower lid for 15 seconds with the eyes open. These 3 values were averaged to generate the final measurement, representing the data presented. The mean values from total 31 KO and 15 WT were compared. These two groups were gender and age matched. (NS: Not statistically significant. p = 0.061, independent t-test, error bars indicate 95%CI).