RESEARCH ARTICLE
The distribution of conjunctival goblet cells in mice

https://doi.org/10.1016/j.aanat.2020.151664Get rights and content

Abstract

Purpose

To evaluate the density and distribution of conjunctival goblet cells in mice without clinical evidence of ocular surface diseases.

Methods

Immediately after euthanasia of C57BL/6 wild-type mice, the eyes including eyelids were removed and fixed in paraformaldehyde. Entire eyeballs and eyelids were cut in series along the sagittal axis from nasal to temporal on a microtome and then stained with Periodic Acid-Schiff acid to visualize the goblet cells. At each section stained in this way, the conjunctival goblet cells of the entire upper and lower lid conjunctiva were counted by light microscopy. Additional (transmission electron microscopy) (TEM)-Analysis on ultrathin sections was performed to evaluate morphological differences.

Results

The total number of conjunctival goblet cells differs markedly between individual animals. Categorisation into upper eyelid (UL) and lower eyelid (LL) and into regions (nasal, middle, temporal) revealed a significant increase of goblet cells from nasal to temporal in the UL and a significant decrease in the LL.

Conclusion

The distribution of conjunctival goblet cells in mice differs considerably from humans and between individual animals. Therefore, precise selection of sampling and methods are needed to obtain comparable data. We recommend to use the middle region of the conjunctiva of UL/LL for goblet cell studies in mice. These findings are of particular interest for dry eye mouse models as well as pharmacological studies on mice with influence on their goblet cells.

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.

References (43)

  • C.S. De Paiva et al.

    Dry eye–induced conjunctival epithelial squamous metaplasia is modulated by interferon-γ

    Invest. Ophthalmol. Vis. Sci.

    (2007)
  • M.J. Doughty

    Assessment of goblet cell orifice distribution across the rabbit bulbar conjunctiva based on numerical density and nearest neighbors analysis

    Curr. Eye Res.

    (2013)
  • R. Eördögh et al.

    Density and distribution of feline conjunctival goblet cells

    J. Feline Med. Surg.

    (2017)
  • K. Gasser et al.

    Investigations on the conjunctival goblet cells and on the characteristics of glands associated with the eye in the guinea pig

    Vet. Ophthalmol.

    (2011)
  • B.H. Grahn et al.

    Qualitative tear film and conjunctival goblet cell assessment of cats with corneal sequestra

    Vet. Ophthalmol.

    (2005)
  • M. Hiraishi et al.

    Histopathological changes in tear-secreting tissues and cornea in a mouse model of autoimmune disease

    Exp. Biol. Med. (Maywood)

    (2020)
  • A. Huang et al.

    Morphogenesis of rat conjunctival goblet cells

    Invest. Ophthalmol. Vis. Sci.

    (1988)
  • T. Inatomi et al.

    Human corneal and conjunctival epithelia express MUC1 mucin

    Invest. Ophthalmol. Vis. Sci.

    (1995)
  • M.M. Jumblatt et al.

    MUC5AC mucin is a component of the human precorneal tear film

    Invest. Ophthalmol. Vis. Sci.

    (1999)
  • S. Kessing

    Mucus gland system of the conjunctiva. A quantitative normal anatomical study

    Acta Ophthalmol. (Copenh)

    (1968)
  • S. Kessing

    Topographical quantitative studies of the conjunctival goblet cells

    Acta Ophthalmol. (Copenh)

    (1968)
  • Cited by (6)

    • The mystery behind the nostrils – technical clues for successful nasal epithelial cell cultivation

      2021, Annals of Anatomy
      Citation Excerpt :

      Considering this important method, the NAEPCs cultures can be also observed through SEM as presented in and the readers are kindly referred to (Aydin et al., 2020; Schildgen et al., 2018; Aydin et al., 2021). To determine the cell integrity and the morphology of cell organelles and nucleus, the NAEPCs cultures can be also analyzed through TEM and a brief technical description was previously described (Hampel et al., 2015; Aydin et al., 2021, 2020; Culemann et al., 2019; Seselja et al., 2019; Welss et al., 2021). After sufficient fixation, the preparations are drained and dehydrated in an increasingly concentrated alcohol series (30%, 50%, 70%, 90%, 100% ethanol, and 100% propylene oxide).

    View full text