The ocular surface phenotype of Muc5ac and Muc5b null mice

Abstract

Purpose: Recent development of mice null for either Muc5ac or Muc5b mucin allows study of their specific roles at the mouse ocular surface. A recent report indicated that Muc5ac null mice show an ocular surface phenotype similar to that seen in dry eye syndrome. The purpose of our study was to determine the effect of lack of Muc5ac or Muc5b on the ocular surface, and to determine if environmental desiccating stress exacerbated a phenotype.

Methods: Muc5ac null and Muc5b null mice, and their wild-type controls were examined for ocular surface defects by fluorescein staining. The number of goblet cells per area of conjunctival epithelium was counted, and levels of mucin gene expression and genes associated with epithelial stress, keratinization, and differentiation, known to be altered in dry eye syndrome, were assayed. To determine if the null mice would respond more to desiccating stress than their wild-type controls, they were challenged in a controlled environment chamber (CEC) and assessed for changes in fluorescein staining, tear volume, and inflammatory cells within the conjunctival and corneal epithelia.

Results: Unlike the previous study, we found no ocular surface phenotype in the Muc5ac null mice, even after exposure to desiccating environmental stress. Similarly, no ocular surface phenotype was present in the Muc5b null mice, either before or after exposure to a dry environment in the CEC.

Conclusions: Our results indicate that deleting either the Muc5ac or Muc5b gene is insufficient to create an observable dry eye phenotype on the ocular surface of these mice.

Keywords: animal models; dry eye; mucins.

Figure 1

Examination of the eye and eyelids of Muc5ac and Muc5b null mice. No obvious changes or defects in the gross appearance of the cornea or eyelids were observed in either Muc5ac (top panels) or Muc5b (bottom panels) null mice compared to wild-type control mice.

Figure 2

Goblet cells do not differ in morphology or number in Muc5ac and Muc5b null mice. (A) Goblet cells are present within the conjunctival epithelium in Muc5ac (top panels) and Muc5b (bottom panels) null mice, and are indistinguishable from those in Muc5ac and Muc5b wild-type mice. Scale bar: 10 μm. (B) No significant differences were observed in the number of goblet cells within the conjunctival epithelium in either Muc5ac or Muc5b null mice compared to their respective wild-type control mice, nor in comparison to each other. Error bars represent ± SEM.

Figure 3

Muc5ac and Muc5b null mice do not display evidence of ocular surface inflammation. The number of CD45-positive cells within the conjunctival epithelium (A), corneal epithelium (B), and corneal stroma (C) of Muc5ac and Muc5b null mice were not statistically different than those of wild-type mice or of each other. Note the decrease in scale for (B, C), as very few CD45-positive cells were found in either the corneal epithelium or stroma. Error bars represent ± SEM.

Figure 4

Localization of Muc5ac and Muc5b in conjunctival epithelial goblet cells of wild-type and Muc5ac and Muc5b null mice. Muc5ac protein was immunolocalized to most goblet cells within the conjunctival epithelium of wild-type (+/+) mice (A), whereas Muc5ac antibody binding was absent in Muc5ac null (−/−) mice (B), showing both knockout of the gene and antibody specificity. Muc5b protein was immunolocalized to only a few goblet cells within the conjunctival epithelium of wild-type mice (C) and Muc5b antibody binding was absent in Muc5b null mice (D) showing Muc5b knockout and antibody specificity. In double labeling experiments (E), Muc5ac (red) and Muc5b (green) localize to different goblet cells in wild-type mice, indicating that conjunctival goblet cells make one mucin exclusively. Comparison of the numbers of Muc5b-positive goblet cells in Muc5ac wild-type (F) to those in Muc5ac null mice (G) showed an increase in Muc5b localization in Muc5ac null mice, corroborating the finding that expression of Muc5b was upregulated significantly in Muc5ac null mice (see Fig. 6). Scale bar: 50 μm.

Figure 5

Expression levels of mucin genes (A), and epithelial stress, differentiation, and keratinization genes (B) in Muc5ac and Muc5b null mice. No significant changes in gene expression levels of Muc1, Muc4, Sprr2h, Tgm1, or K17 were observed in either Muc5ac or Muc5b null mice compared to wild-type controls. Interestingly, while a significant increase in Muc5b gene expression in Muc5ac null mice was observed, Muc5ac gene expression levels in Muc5b null mice were not significantly different from Muc5b wild-type controls. Error bars represent ± SEM. ***P < 0.001.

Figure 6

Exposure of Muc5ac and Muc5b null mice to desiccating environmental stress. (A) Fluorescein staining scores were not significantly different in Muc5ac null mice before entry into the CEC (Day 0) and were not statistically different from wild-type control mice during exposure to desiccating stress in the CEC. (B) Tear volume in Muc5ac null mice was not significantly different from wild-type mice before exposure to desiccating stress and did not change over time in the CEC; however, wild-type mice showed a significant elevation in tear production during CEC exposure at days 12 and 15. Neither fluorescein staining scores (C) nor tear volume (D) were significantly different in Muc5b null mice before entry into the CEC, and both fluorescein staining scores and tear volumes remained similar to wild-type mice throughout the duration of CEC exposure. Error bars represent ± SEM. ***P < 0.001.