Merkel cells, first described by Fredrich S. Merkel, are mostly located in the basal layer of the epidermis in distinct areas of hairy mammalian skin ¹ . Merkel cells have also been shown in vertebrates and cyclostomes ² and can be widely found in hairy skin, the eccrine glandular ridges of the glabrous skin (palms and soles), ectoderm-derived mucosal tissues (palatine mucosa), and whisker pads or taste buds ^{3, 4} . Merkel cells are generally classified into two distinct subpopulations based on whether or not they are associated with nerves. Those that are associated with nerves are referred to as the Merkel cell-nerve complexes and have mechanoreceptive and chemo-sensitive functions. Those that do not associate with nerves possess endocrine functions ^{4– 6} . Recent studies have shown that Merkel cells are important in light-touch responses and can work as neurotransmitters, it is suggested that Merkel cells are essential for the perception of space experienced by individuals ⁷ .

However, Merkel cells have, thus far, not been described on the ocular surface. In this study, we investigated the distribution and ultrastructure of Merkel cells on the ocular surface and revealed a morphological basis for further study of the function of Merkel cells on the ocular surface.

As previously reported, Merkel cells express CK 8 ⁴ . In this study, we first identified the location of CK8-positive cells on the ocular surface. The ocular surface whole-mounts show that CK8-positive cells were localized in the fornix and the palpebral conjunctiva, and CK8-positive immunofluorescence was observed in the membrane, nucleus and cytoplasm of specific cells. We also found that the CK8-positive cells exhibited circular nuclei and visible nucleoli, and the cytoplasmic fluorescence revealed reticular and granular shapes ( Figure 1). However, we did not find any CK8-immunofluorescence-positive cells in the bulbar conjunctiva.

Double immunofluorescence stain showed that there were two kinds of CK8-positive cells on the ocular surface. The types that also stained with Math1 ( Figure 2) were located in the stromal layer of the fornix and the palpebral conjunctiva, the other in the epithelial basal layer (we also identified that the CK8-positive cells in the epithelial basal layer were goblet cells). So we speculated that the Merkel cells were located in the stromal layer and could express Ngn3 ( Figure 3) and beta-tubulin III ( Figure 4) in the cytoplasm.

The classical method to identify Merkel cells has been electron microscopy, so we used TEM to analyze the ultrastructure of the conjunctival cells. As shown in Figure 5, there is a class of cells contain electron-dense membrane-surrounded granules, mitochondria and intermediate filaments ( Figure 5A). In addition, these cells exhibited spine-like protrusions of variable lengths from the cell surface ( Figure 5B) and located in the conjunctival stroma ( Figure 5C). These characteristics are similar to the ultrastructural features of Merkel cells ⁴ . The granules were not only observed within these cells ( Figure 5A) but were also found in the extracellular regions ( Figure 5B). We also observed some cells that were associated with nerve terminals, forming Merkel cell-neurite complexes with vesicles at the nerve endings ( Figure 5D). Shrinkage of the membrane-surrounded granules and Merkel cells in close proximity to the conjunctival epithelium and nuclear lobulation were investigated after corneal wounding ( Figure 5E). CK8-specific Merkel cells were observed in the conjunctival stroma by immuno-electron microscopy ( Figure 5F). A small number of CK8-specific particles were observed in the conjunctival epithelium cells under high magnification ( Dataset-1).

It has been reported that the FM1-43 dye is a useful marker for live Merkel cells ¹³ . Therefore, we used FM1-43 dye to identify conjunctival Merkel cells. Twelve hours after intraperitoneal injection of FM1-43, green fluorescent granules were observed in the cytoplasm of Merkel cells, and nuclear labeling was found in the conjunctiva of the stroma using the deconvolution microscopy ( Figure 1B).

The conjunctiva is an important part of the ocular surface. It plays a very important role in the process of mucus secretion and immunological reactions. The conjunctiva can be divided into two parts, the conjunctival epithelium and the stroma. The epithelial cells can be further classified into superficial cells, intermediate cells and basal cells ¹⁴ . The conjunctiva, similar to the skin and some mucosal tissues, is developmentally derived from the ectoderm. Previous studies have shown that Merkel cells are located in the epithelia of the skin and in the oral mucosa, where they participate in endocrine and mechanoreceptor functions ^{3, 4} . However, their presence on the ocular surface had not yet been confirmed.

The classical method used to identify Merkel cells is to check for their ultrastructure using TEM. Recently, various studies have confirmed that Merkel cells express specific cytokeratins (CK), such as CK8, CK18, CK19, and CK20 ^{15– 17} , and neuropeptides, such as vasoactive intestinal peptide (VIP) ¹⁸ , substance P ¹⁹ and calcitonin gene-related peptide (CGRP) ²⁰ , and other proteins, such as Math1, neurofilament-H, and neurotrophin-3 ³ . These bio-markers provide a basis for the identification of Merkel cells using light microscopy. Currently, there are two hypotheses for the origin of Merkel cells: one suggests that Merkel cells differentiate from epidermal keratinocyte-like cells and the other states that Merkel cells arise from stem cells of neural crest origin that migrated during embryogenesis ^{3, 4} .

In this study, we observed Merkel cells located at the bottom of the basal adjacent stroma in the fornix and the palpebral conjunctiva and the cells could be stained with CK8, Math1, Ngn3 and beta-tubulin III. This suggests that the Merkel cells in the conjunctival might originate from the neural crest.

The ultrastructure of Merkel cells in the conjunctival stroma exhibits characteristics similar to those of other tissues, such as an electron-dense core with membrane-surrounded granules and intermediate filaments ( Figure 5C). They have spine-like protrusions of variable lengths ( Figure 5B) ^{1, 4, 21} , and some Merkel cells make contact with nerve terminals to form Merkel cell-neurite complexes ( Figure 5D). The conjunctival Merkel cells, however, also have unique features, as they are distributed in the conjunctival stroma and were not associated with keratinocytes ( Figure 5A, B). In addition, their granules can be visualized not only within the cells ( Figure 5A) but also in extracellular regions ( Figure 5B).

The technique of marking live Merkel cells with quinacrine has been employed to differentiate these cells from other epidermal cells in vitro and in vivo. However, the quinacrine dye incorporated by Merkel cells is rapidly lost, thus making the fluorescence very difficult to identify ^{22– 24} . Another important reason that chloroquine is no longer used is due to the agent’s severe toxicity to the optic nerve. Fukuda et al. ¹³ examined the absorption of FM dyes in Merkel cell cultures and found that FM dyes produce a specific fluorescence that remains sufficiently long to allow for clear identification of Merkel cells. The FM1-43 dye exhibited the best effects as compared with other FM dyes. Our results showed that an intraperitoneal injection of FM1-43 could effectively label the conjunctival stromal Merkel cells in vivo, which may be a useful technique for future studies of Merkel cells.

There is still some controversy regarding the function of Merkel cells. In vitro, neuropeptides secreted from Merkel cells (e.g., VIP and substance P) can promote cell growth and differentiation of keratinocytes and fibroblasts. These neurosecretory substances are released from the Merkel cells and act as neurotransmitters in the Merkel cell-axon complexes ⁴ . However, the exact function of Merkel cells in the conjunctiva stroma is still unclear. Thus, we established a corneal abrasion model to observe the response of these cells to stress. In this study, we demonstrated that wounding the corneal epithelium can induce nuclear lobulation, and shrinkage of cell-volume and an increase in the membrane-surrounded granules of Merkel cells ( Figure 5E). These results indicate that Merkel cells in the conjunctival stroma might play an important physiological and pathological role in the stress response of corneal epithelium wounding. Further research work about the functions of the cells is ongoing in our laboratory.

In conclusion, our data showed that Merkel cells are mainly present in the conjunctival stroma, with the following findings: (1) The cells are distributed in the forniceal conjunctival stroma. (2) The Merkel cells express CK8, Math1, Ngn3 and β-tubulin III. (3) The ultrastructure of the Merkel cells contains an electron-dense core with membrane-surrounded granules, intermediate filaments and mitochondria. (4) Some Merkel cells make contact with nerve terminals to form Merkel cell-neurite complexes. (5) The cells exhibit endocytosis of FM1-43. (6) After corneal epithelial wounding, nuclear lobulation, shrinkage of cell-volume, and an increase in the membrane-surrounded granules of the Merkel cells was also observed. However, further studies are needed to verify the exactly physiological function and role of these cells in specific ocular surface diseases.