RESEARCH ARTICLEThe distribution of conjunctival goblet cells in mice
Introduction
A stable tear film is essential to maintain the health of the ocular surface by protecting it from light and pathogens. In 1946 Wolff, (1946) established the three-layer model of the tear film which consists of an outer lipid layer, a middle aqueous component and an inner mucous component. Each layer/component is essential to either lower hydrophobicity, reduce evaporation or release nutrients and antimicrobial proteins. In order to meet the demand for highly dynamic environmental feedback and physical conditions, the lacrimal apparatus must adapt very quickly to protect the ocular surface from irreparable damage.
The tear film itself is a complex fluid produced by the so-called functional unit including cornea, lacrimal and accessory lacrimal glands in human (intra- and extraortbital lacrimal glands in mice), the Meibomian glands and the conjunctival epithelium including conjunctival goblet cells. Goblet cells are small, intraepithelial glands located in high numbers in the airways and the gastrointestinal tract and occur in the lacrimal system within the conjunctiva, in the epithelium of the lacrimal sac and nasolacrimal duct. Depending on the species, these glandular cells may be located as single cells or in form of clusters building intraepithelial mucous glands between stratified squamous epithelial cells. Their main products are mucins with the mucin 5AC (MUC5AC, Muc5ac in mice) as the most prominent glycoprotein (Inatomi et al., 1995; Jumblatt et al., 1999). The mucous products contribute to the health of the ocular surface by maintaining surface moistening, lubrication, prevention of infections and are also involved in clearing processes (Gipson, 2016a; Marko et al., 2013).
It has been shown that changes in conjunctival goblet cell numbers are associated with various diseases. Patients with Stevens Johnson’s syndrome, who frequently suffer from inflammation and necrosis of the conjunctiva, show a reduction of conjunctival goblet cells up to 95% (Lehman, 1999; Nelson and Wright, 1986). It is known that many forms of dry eye disease (DED) are associated with a reduced number of goblet cells (Kunert et al., 2002). Although it is still difficult to determine the prevalence, some reports estimate the incidence of DED to be as high as 75% (Nelson et al., 2017). As age is one of the major risk factors (Moss et al., 2000) and the average age of the population is increasing, further research is needed.
However, in contrast to their respiratory relatives, the characteristics, differentiation and function of conjunctival goblet cells have not been sufficiently investigated so far. Mouse models have proven to be helpful in closing the knowledge gap. Several knockout models such as Spdef−/−, Muc5ac−/−, Muc5b−/−, conditional Tgb2−/−, conditional Notch−/−, and conditional Krüppel-like factor−/− mice, have been established and used successfully since then (Gipson, 2016a). For the investigation of pathological conditions, it is essential to first document the healthy status quo. While the conjunctival goblet cell patterns of humans, cats and other species are available for the public, to our knowledge there are no data from mice without any background of eye diseases (Kessing, 1968a; Moore et al., 1987). Since it is known that the total numbers and distribution of conjunctival goblet cells varies greatly from species to species, it is more important to define the baseline value in advance (Gipson, 2016b).
In this study, we systematically investigated the distribution of mouse conjunctival goblet cells in healthy C57BL/6 J animals without disease background using serial sections. This baseline will serve as a guideline for future studies and help to determine comparative analyses.
Section snippets
Animals
Mice were handled in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research (TS 12/14). 9−12-week-old male C57BL/6 J mice were purchased from Janvier labs and housed in clear cages, kept in rooms at 21 °C with a 12 -h light-dark cycle. All mice had access to food and water ad libitum. Following arrival from the commercial supplier, mice were allowed to acclimatize for at least one week before surgery. General anesthesia was performed by 5-minute exposure to
Total number of conjunctival goblet cells
The quantification of PAS+ conjunctival goblet cells mice of upper lids (UL) and lower lids (LL) revealed interindividual differences. The total number of goblet cells per eye varied between a minimum of 14,042 and a maximum of 37,889 cells with a standard deviation of 7506 cells (Fig. 2A). These interindividual differences were seen in the UL as well as in the LL (Fig.2B). Altogether, the comparison of the number of goblet cells in the UL and LL did not reveal a significant difference (Fig.2C).
Discussion
In 1968, researcher led by Kessing postulated that the distribution of goblet cells in the human conjunctiva is not uniform but irregular (Kessing, 1968a). They showed that the density of goblet cells in the nasal area was significantly higher compared to the temporal area. It is known that the number and distribution of these cells is species-dependent (Gipson, 2016b). As shown in Table 2, several studies analysed the normal distribution and density of conjunctival goblet cells in numerous
Conclusion
On the basis of our data, we conclude that a precise definition of sampling is essential to obtain comparable data. We further suggest that analysis of the middle region of the UL or LL instead of serial sections might be sufficient for goblet cell studies in mice.
Ethical statement
The study was conducted in compliance with institutional review board regulations, informed consent regulations, and the provisions of the Declaration of Helsinki.
The tissue samples of 11 eyes of 9 male mice (C57BL/6) originated of the animal stable of the Institute of Anatomy of the Friedrich-Alexander-University Erlangen-Nürnberg, which were used within the scope of approved animal test projects, which, however, had nothing to do with the present project. The animals were kept under
Financial disclosure
The authors have no proprietary or commercial interest in any materials discussed in this manuscript. FP receives royalties from Elsevier for the 24th Ed. of the anatomy atlas “Sobotta” and the “Sobotta Textbook of Anatomy”. The work was supported by Sybille Kalkhof-Rose Foundation (JW, FP), by Sicca Forschungsförderung of the Association of German Ophthalmologists (JW) and in part by Deutsche Forschungsgemeinschaft (DFG) grant PA738/15−1 (FP). The Funding organizations had no role in the
Disclosure
The author declares that there are no conflicts of interest.
Acknowledgement
We thank Hong Nuygen for her excellent technical assistance and especially Prof. Dr. Elke Lütjen-Drecoll for her great advice and support in finalising this paper.
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