A Ligation of the Lacrimal Excretory Duct in Mouse Induces Lacrimal Gland Inflammation with Proliferative Cells

Abstract

The lacrimal gland secretes tear fluids to ocular surface, which plays an indispensable role in maintaining the health of the ocular epithelia and protecting the ocular surface from the external environment. The dysfunction of the lacrimal glands causes dry eye disease due to a reduction in tear volume. The dry eye disease is becoming a popular public disease, for the number of patients is increasing, who have subjective symptom and loss of vision, which affect the quality of life. Inflammatory change in the damaged lacrimal gland has been reported; however, a major challenge is to establish a simple animal model to observe the changes. Here, we demonstrated an injury model by ligating the main excretory duct of the lacrimal gland, which is a simple and stable way to clearly understand the mechanism of lacrimal gland inflammation. We observed the process of injury and proliferation of the lacrimal gland and detected a population of lacrimal gland epithelial cells with proliferation potential which were also nestin-positive cells following duct ligation. This study successfully established an injury model to observe the tissue injury process of the lacrimal gland, and this model will be useful for analysis of the inflammation and proliferation mechanism in the future.

Figure 1

The lacrimal duct ligation in mouse. (a) The photos of the lacrimal duct ligation procedure in mice. The white circle showed the excretory duct of the LG and the black circle showed the main LG. The procedure before ligation (left) and after ligation (right) was shown. (b) The macroscopic changes in the LG before and after ligation. The scale bar is 2 mm. (c) The change in the average of tear secretion volume before and after ligation. Each group has more than 20 samples. ^∗p < 0.05, ^∗∗p < 0.01. (d) The change in the gland weight before and after surgery. Each group has more than 20 samples.

Figure 2

Histological changes in the LG after the ligation. (a) The representative images of the H&E staining of the LG before and after surgery. ★ showed the enlarged and generated duct cavities. The scale bar is 50 μm. (b) The change in the average number of acini after surgery. ^∗p < 0.05, ^∗∗p < 0.01. (c) The change in the average number of duct after surgery. ^∗p < 0.05.

Figure 3

Expression changes in representative markers in the LG after surgery. (a) The histoimmunochemical staining of KRT14 and α-SMA in the normal LG (red showed the KRT14, green showed the α-SMA, and arrow showed the cells with both expression). The scale bar is 50 μm. (b) The difference of relative mRNA expression level of the representative markers before and after surgery. ^∗p < 0.05 and ^∗∗p < 0.01.

Figure 4

The inflammation and proliferation of the LG after surgery. (a) The time-course observation of cells with CD45 expression before and after surgery (red showed the CD45). The scale bar is 50 μm. (b) The time-course observation of cell apoptosis by TUNEL staining before and after surgery (red arrows showed the stained cells). The scale bar is 50 μm. (c) The time-course observation of cells with PCNA expression before and after surgery (red showed the PCNA). The scale bar is 50 μm. (d) The enlarged image of PCNA-positive cells at days 3 and 7 after surgery (red showed the PCNA). The scale bar is 20 μm.

Figure 5

The increase in nestin-positive cells in the LG after surgery. (a) The time-course observation of cells with nestin expression before and after surgery (red and arrows showed the nestin). The scale bar is 50 μm. (b) The change in the average number of cells per visual field with nestin expression before and after surgery. More than 10 samples in each group were included. ^∗p < 0.05.