Duct system of the rabbit lacrimal gland: structural characteristics and role in lacrimal secretion

Abstract

Purpose: To develop a nomenclature for the lacrimal duct system in the rabbit, based on the anatomic and structural characteristics of each duct segment, and to provide RT-PCR and immunofluorescence data to support the notion that the duct system plays important roles in lacrimal function.

Methods: Paraffin-embedded lacrimal glands (LGs) were stained with hematoxylin and eosin (H&E) and evaluated with a stereomicroscope. Cryosections of LG were stained with cresyl violet, and acinar cells and ductal epithelial cells were isolated from each duct segment by laser capture microdissection (LCM). mRNA levels from these cells were analyzed by real-time RT-PCR. Standard protocol was followed for immunofluorescence detection of ionic transporters.

Results: The lacrimal duct system was divided into six segments on the basis of morphologic characteristics: the intercalated, intralobular, interlobular, intralobar, interlobar, and main excretory ducts. Although the morphologic features change incrementally along the entire duct system, the gene expression of ionic transporters and aquaporins, including AE3, AQP4, AQP5, CFTR, ClC2gamma, KCC1, NHE1, NKAalpha1, NKAbeta1, NKAbeta2, NKAbeta3, and NKCC1 varied greatly among duct segments. Immunofluorescence results were generally in accordance with the abundance of mRNAs along the acinus-duct axis.

Conclusions: Most LG research has focused on the acinar cells, with relatively little attention being paid to the lacrimal ducts. The lack of knowledge regarding the lacrimal ducts was so profound that a precise nomenclature had not been established for the duct system. The present data establish a nomenclature for each segment of the lacrimal duct system and provide evidence that ducts play critical roles in lacrimal secretion.

Figure 1.

Illustration of the lacrimal duct system. The LG is composed of several lobes, which are further subdivided into lobules (shown in the shaded lobe). The lacrimal duct system is a tree-like structure and, on the basis of its anatomic and structural characteristics, it can be divided into, from the acini to the ocular surface: intralobular, interlobular, intralobar, interlobar, and main excretory ducts. Illustration courtesy of Joel Schechter, PhD.

Figure 2.

Segments of the lacrimal duct system. (A) Intercalated ducts (arrow) emerge from acini and merge into larger intralobular duct (arrowhead). Cells of intercalated ducts are simple cuboidal and stain red with H&E. The intracellular contents appear smooth, rather than the pale and foamy appearance of acinar cells. Several intercalated ducts merge into larger intralobular ducts that have much larger lumens. Intercalated and intralobular ducts drain acini and are in immediate continuity with acini and have minimal connective tissues surrounding them. OD ranges from 20 to 30 μm. An intralobar duct can be seen in the upper right corner. Ac, acinus. (B) Interlobular ducts (arrow) are merged from intralobular ducts (∼30 to 50 μm OD). Duct cells range from simple cuboidal to low columnar and stain red with a smooth appearance of intracellular contents. These ducts drain lobules, having connective tissue, and frequently, blood vessels surrounding them. Two intralobular ducts are also visible in this image (arrowheads). (C) Intralobar ducts (arrow) drain individual lobes. Duct cells are low to tall columnar and range from simple to pseudostratified epithelial cells. These ducts are surrounded by increasingly more connective tissues and often with blood vessels (∼50 to 80 μm OD). Note that an intralobular duct (arrowhead) can be seen with a small lumen and in immediate continuity with acini. (D) Interlobar ducts (arrow) are merged from intralobar ducts. Ducts range from simple to stratified columnar epithelia, ∼80 to 200 μm OD, albeit the luminal profiles are highly variable. These duct segments are surrounded by abundant connective tissue. Scale bar, 100 μm.

Figure 3.

Real-time RT-PCR of AQPs. The level of AQP4 mRNA was the lowest in the acini, but increased significantly in the intralobular, intralobar, and interlobar ducts. The expression of AQP4 mRNA was also significantly higher in intralobular ducts than in interlobular ones. AQP5 mRNA levels showed a completely different pattern. It was nine times higher in the acini than in the intralobular ducts and three times higher in the intralobular than in the interlobar ducts. Moreover, although AQP5 mRNA was 40 times more abundant than AQP4 mRNA in the acini, the two transcripts were at similar levels in the intralobular ducts. D4, intralobular duct; D3, interlobular duct; D2, intralobar duct; D1, interlobar duct. Data are presented as the mean ± SEM.

Figure 4.

Real-time RT-PCR of Na⁺/K⁺-ATPase (NKA) subunits. α1 mRNA was the least abundant in acini, whereas its level in the intralobular ducts was significantly more higher. β1 mRNA was similarly present in intralobular ducts and acini, but less so in interlobular ducts. Significant levels of NKAβ2 mRNA were detected in the interlobular and interlobar ducts, but not in the acini and intralobular ducts. NKAβ3 mRNA was significantly more abundant in the intralobular ducts than in the acini and the other duct segments. We were unable to detect a meaningful presence of α2. Labeling and data are as in Figure 3.

Figure 5.

Real-time RT-PCR of transporters involved in Na⁺-Cl⁻-coupled entry mechanisms. The level of AE3 mRNA was the lowest in acini and significantly increased in intralobular, intralobar, and interlobar ducts. NHE1 and NKCC1 showed a similar pattern of mRNA expression in acini and duct segments (i.e., the highest was found in acini, whereas the duct segments had less mRNA, although the difference between expression in acini and duct segments was much more dramatic for NKCC1). Labeling and data are as in Figure 3.

Figure 6.

Real-time RT-PCR of K-2Cl⁻, which is involved in a coupled efflux mechanism. KCC1 is responsible for the efflux of Cl⁻ and K⁺ across the basolateral membranes in LG epithelial cells. The level of mRNA was the lowest in acini, and its abundance increased significantly in intralobular, intralobar, and interlobar ducts. Labeling and data are as in Figure 3.

Figure 7.

CFTR and ClCγ mRNA levels in acini and duct segments. Both transporters are membrane channels that transport chloride across plasma membranes. Minimal CFTR mRNA was found in acini, whereas its level was significantly higher in every duct segment, with the highest observed in the interlobar duct. The expression of ClC2γ mRNA was the lowest in the intralobular duct, whereas the acini and interlobular duct had significantly higher levels. Labeling and data are as in Figure 3.

Figure 8.

Immunofluorescence of AQPs and transporters. (A) AQP4-IR was observed on the basolateral sides of acinar and duct cells, with duct cells (arrows) showing a stronger IR than acinar cells (arrowheads). (B) AQP5-IR was distributed among acini in a mosaic pattern, with some acini and/or acinar cells demonstrating much stronger IR (arrows) than the rest of the acini/acinar cells. However, virtually no IR was detected in duct cells (C, arrowheads, image of the same section used for double labeling of AQP5 and NKAβ₁). (C) NKAβ₁-IR was present in all acinar cells, but the levels differed in a mosaic pattern: higher in some acinar cells and/or acini (arrows) than in others. NKAβ₁-IR in the ductal cells was uniformly high (arrowheads). (D) NHE1-IR was found at the basolateral membranes and within the cytoplasm of all acinar (arrowhead) and ductal cells, whereas the level in ductal cells was considerably higher (arrows). (E) NKCC1-IR was also present at the basolateral membranes and within the cytoplasm of all acinar (arrowheads) and ductal cells (arrow), but the levels were higher in the acinar cells. (F) CFTR-IR (green) was present in punctate aggregates within the apical cytoplasm of all acinar (arrows) and ductal cells (arrowheads), but the level in ductal cells was considerably higher. Rhodamine-conjugated phalloidin, which stains F-actin, was used to outline the morphologic profile (red, also in G). (G) ClC2γ. Like CFTR, ClC2γ-IR (green) was also found in the apical cytoplasm as punctate aggregates (arrowheads). However, no ClC2γ-IR was seen in ductal cells (arrow). Ac, acinus. Scale bar, 50 μm.