Obstruction of the Tear Drainage Altered Lacrimal Gland Structure and Function

Abstract

Purpose: Orbital glands and drainage conduits are two distinct entities that constitute the lacrimal apparatus system, the malfunction of which leads to a range of ocular surface disorders. Despite the close functional relationship, how the two parts interact under pathophysiological conditions has not been directly tested. The study aims to investigate the lacrimal gland (LG) structural and functional changes upon the drainage system obstruction, thus, testing their function link.

Methods: Dacryocystectomy was performed in C57BL/6 mice to create a surgical model for tear duct (TD) obstruction (STDOB). Prickle1 mutant line with congenital nasolacrimal duct dysplasia serves as a genetic model for TD obstruction (GTDOB). Alterations of the LG and the ocular surface in tear duct obstruction mice were examined.

Results: STDOB and GTDOB mice showed similar ocular surface phenotypes, including epiphora, corneal epithelial defects, and conjunctival goblet cell abnormalities. At the molecular and cellular levels, aberrant secretory vesicle fusion of the LG acinar cells was observed with altered expression and localization of Rab3d, Vamp8, and Snap23, which function in membrane fusion. LG secretion was also altered in that lactoferrin, lipocalin2, and lysozyme expression were increased in both LG and tears. Furthermore, STDOB and GTDOB mice exhibited similar LG transcription profiles.

Conclusions: Physical obstruction of tear drainage in STDOB or GTDOB mice leads to LG dysfunction, suggesting a long-distance interaction between the tear drainage conduits and the LG. We propose that various components of the lacrimal apparatus should be considered an integral unit in diagnosing and treating ocular surface diseases.

Figure 1.

Schematic illustration of dacryocystectomy to create a surgical tear drainage obstruction (STDOB) mouse model. (A-C) Schematic diagrams of the lacrimal sac removal procedure. (A) Fast green dye was instilled onto the ocular surface to track the tear path through the drainage duct. (B) The lateral nasal skin was carefully opened to expose the lacrimal sac marked by the fast green dye. (C) The lacrimal sac (L.S.) was removed, and the skin was sutured. (D, E) Representative pictures demonstrating dye-filled lacrimal sac of the sham (left) and experiment (right) eyes 8 weeks post operations. White arrows indicate the position of the lacrimal sac.

Figure 2.

STDOB mice exhibited epiphora, increased corneal fluorescein staining, and increased conjunctival goblet cells. (A) Sham operation: a representative image of the normal ocular surface. (B) Epiphora with white discharge in the inner canthus of the STDOB mice. Arrows point to the inner canthus. (C) Tear flow test using the phenol red thread. (D) Corneal fluorescein staining of the sham-operated mice showing integral surface. (E) Punctate fluorescein staining of the STDOB mouse cornea. (F) Quantifications of the fluorescein-stained corneal area of the STDOB and sham mice. (G) PAS-stained goblet cells in the sham-operated mice had a round shape and uniform size. (H) More conjunctival goblet cells of the STDOB mice stained by the PAS. (I, J) Magnified images of the goblet cells from boxed areas in (G) and (H), respectively. (K) Quantification of the number of goblet cells of the STDOB and sham mice; n = 10 mice for (C) and (F); and n = 5 mice for (K).

Figure 3.

STDOB mice showed similar phenotypes to those observed in Prickle 1 mutant mice. (A, D) Normal ocular surface with intact corneal epithelium showed in the wild-type mice. (B, E) Epiphora with white discharge and increased fluorescein stain on the cornea of the Prickle1 mutant mice. The arrow indicates the white discharge in the inner canthus. (C) Tear flow was significantly increased in the Prickle1 mutant mice using the phenol red thread test. (F) Corneal fluorescein staining score revealed more severe staining of the Prickle1 mutant mice. (G) Periodic acid-Schiff (PAS) staining of the conjunctival epithelium of the wild-type mice showed rounded and uniform staining patches. (H) The Prickle1^a/b group showed an increase in goblet cells with enhanced staining in the apical areas. (I) and (J), Magnified images from boxed areas in (G) and (H), respectively. (K) The number of goblet cells was significantly increased in the Prickle1^a/b group; n = 10 mice for (C) and (F); and n = 5 mice for (K).

Figure 4.

Alterations of tear component and lacrimal gland-secreted proteins in STDOB and Prickle1 mutant mice. (A, B) Representative images of Western blotting of lactoferrin, lipocalin, and lysozyme in the tear fluid (A) and LG (B). The same amount of proteins (see Methods and materials) were loaded for polyacrylamide gel electrophoresis, with the Gapdh serving as a control for LG protein loading (B). (C), Quantification of Western blotting analysis showed increased lactoferrin, lipocalin, and lysozyme from the LG of the STDOB and Prickle1 mutant mice. Protein levels were normalized to Gapdh, and relative expression was compared between the sham and the treated groups (n = 3 LGs/mice).

Figure 5.

Morphologic alterations of lacrimal glands in the STDOB and Prickle1 mutant mice. (A-C) Stereomicroscopy of the LGs from the sham (A), STDOB (B), and Prickle1 mutant mice (C). (D) Quantification of relative LG weights. The wet LG weights were normalized to the body weights presented as ratio indices (LG/body, mg/g); N =10 LGs. (E-J) H&E staining of the LGs from the sham controls (E, H), STDOB (F, I), and Prickle1 mice (G, J). (H), (I), and (J) are magnified images from the boxed areas of (E), (F), and (G), respectively. (K) Average individual acinus areas. Ten acini from each imaging field are roughly randomly chosen for measuring individual acinus areas. The results are presented as average area/acinus. The n = 5 mice/group. (L), The total average acinar area per imaging field was presented as percentages (n = 3 mice/group).

Figure 6.

LG ultrastructure revealed by transmission electron microscopy (TEM). (A, B) LG showed uniform electron density of the acinus secretory vesicles in the sham-operated mice. (C-F), Altered electron density of the LG acinar vesicles in the STDOB and Prickle1 mutant groups. (B), (D), (F) Higher magnification of the boxed areas from (A), (C), and (E), respectively. The green arrow in (B) indicates the clear membrane-bound secretory vesicles in the sham mice. The red arrow in (D) (STDOB) and the purple arrow in (F) (Prickle1 mutant) point to the enlarged fused vesicles with lighter electron density and heterogenous membrane curvatures.

Figure 7.

Altered expression and localization of proteins relevant to lacrimal gland fusion and secretion. (A) Rab3d immunofluorescence was abundant in the apical lumen side of the acinar cells in the sham LGs (left panels), but uniformly distributed in the STDOB (middle panels) and more basally localized in the Prickle1 mutant acini. The bottom panels are magnified images from the top panels. Apical and basal positions of the acinus lumen are illustrated in the right corner of the first panel. (B) Western blotting demonstrated reduced Rab3d expression in the STDOB and Prickle1 mutant LGs compared with the sham. (C) Vamp8 immunofluorescence showed a similar altered pattern as the Rab3d in the STDOB and Prickle1 mice. (D) Western blotting showed increased LG expression of Vamp8 in the STDOB and Prickle1 mice. (E) Expression of Snap23 in LG acini was also basally shifted and showed increased levels in STDOB and Prickle1 mice compared with the sham (F). The white dotted lines indicate acini boundaries. For Western blotting and quantification, n = 4 LGs/mice.

Figure 8.

RNA sequencing and KEGG pathway analysis. (A) Differentially expressed genes (DEGs) of LGs from respective STDOB and Prickle1 mutant mice compared with the sham. Three hundred seventy-five common genes were shared by STDOB and Prickle1 mutant groups. (B) A Venn diagram of intersections between up- and downregulated DEGs. (C) Top 6 KEGG pathways enriched for the total 682 STDOB DEGs. (D) Top 6 KEGG pathways enriched for the total 1351 Prickle 1^a/b DEGs. (E) The top 6 KEGG pathways enriched for the shared 375 DEGs between STDOB and Prickle1 mutant mice.

Figure 9.

Prickle1 is not expressed in the lacrimal gland. (A) The mRNA expression amplitudes of the Prickle1 gene in RNA-seq data from the three experimental groups. FPKM, fragments per kilobase of transcript per million mapped reads. (B) RT-qPCR to determine Prickle1 mRNA levels in the lacrimal gland; CT, cycle threshold. (C) GFP reporter protein was detected by immunohistochemistry in hair follicles (serving as a positive control) but not in the LG acini of the Prickle1^b/+ mice.