Histological and immunohistochemical characterization of the porcine ocular surface

RESEARCH ARTICLE Histological and immunohistochemical characterization of the porcine ocular surface Mario Crespo-MoralID 1☯ , Laura Garcı ´a-Posadas 1☯ , Antonio Lo ´pez-Garcı ´a 1,2 , Yolanda Diebold 1,2 * 1Ocular Surface Group, IOBA – University of Valladolid, Valladolid, Spain, 2Biomedical Research Networking Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Valladolid, Spain ☯These authors contributed equally to this work. *[email protected] Abstract The ocular surface of the white domestic pig (Sus scrofa domestica) is used as a helpful model of the human ocular surface; however, a complete histological description has yet to be published. In this work, we studied porcine eyeballs with intact eyelids to describe and characterize the different structures that form the ocular surface, including the cornea and conjunctiva that covers the bulbar sclera, tarsi, and the nictitating membrane. We determined the distribution of goblet cells of different types over the conjunctiva and analyzed the conjunctival-associated lymphoid tissue (CALT). Porcine eyeballs were obtained from a local slaughterhouse, fixed, processed, and embedded in paraffin blocks. Tissue sections (4 μm) were stained with hematoxylin/eosin, Alcian blue/Periodic Acid Schiff, and Giemsa. Slides were also stained with lectins from Arachis hypogaea (PNA) and Helix pomatia (HPA) agglutinins and immunostained with rabbit anti-CD3. We found that the porcine cornea was composed of 6–8 epithelial cell layers, stroma, Descemet’s membrane, and an endothelial monolayer. The total corneal thickness was 1131.0±87.5 μm (mean±standard error of the mean) in the center and increased to 1496.9±138.2 μm at the limbus. The goblet cell density was 71.25±12.29 cells/mm, ranging from the highest density (113.04±37.21 cells/mm) in the lower palpebral conjunctiva to the lowest density (12.69±4.29 cells/mm) in the bulbar conjunctiva. The CALT was distributed in the form of intraepithelial lymphocytes and subepithelial diffuse lymphoid tissue. Lenticular-shaped lymphoid follicles, about 8 per histological section, were also present within the conjunctival areas. In conclusion, we demonstrated that the analyzed porcine ocular structures are similar to those of humans, confirming the potential usefulness of pig eyes to study ocular surface physiology and pathophysiology. Introduction The ocular surface is the interface between the eye and the environment. Classically, it is comprised of the corneal, limbal, and conjunctival epithelia and the tear film [1]. However, PLOS ONE | https://doi.org/10.1371/journal.pone.0227732 January 13, 2020 1 / 17 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Crespo-Moral M, Garcı ´a-Posadas L, Lo ´pez-Garcı ´a A, Diebold Y (2020) Histological and immunohistochemical characterization of the porcine ocular surface. PLoS ONE 15(1): e0227732. https://doi.org/10.1371/journal. pone.0227732 Editor: Yu-Chi Liu, Singapore Eye Research Institute, SINGAPORE Received: November 26, 2019 Accepted: December 27, 2019 Published: January 13, 2020 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0227732 Copyright: ©2020 Crespo-Moral et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All data files are available from the Figshare database (https:// figshare.com/articles/Histological_and_ immunohistochemical_characterization_of_the_ the concept of the ocular surface has evolved in the last 20 years to a more complex pathophysiological functional unit [2]. In this initial report, we confined our analysis to the components of the traditionally recognized ocular surface and the Meibomian gland. Due to the contact with the external environment, the lacrimal functional unit, and specifically the ocular surface, multiple defensive mechanisms exist. The corneal epithelium forms a tight barrier that impedes the entrance of pathogens. However, because the cornea must be transparent to allow the transmission of light, it has no blood vessels and depends on other tissues to support it. The corneal epithelium is renewed by epithelial stem cells located in the Palisades of Vogt, which are radially-oriented fibrovascular ridges present in the limbus, the area between the cornea and the conjunctiva [3]. The limbal tissue transitions into the conjunctiva, a mucosal tissue that, unlike the cornea, is highly vascularized and displays a strong reaction against antigens and infections without compromising the maintenance and/or recovery of ocular surface homeostasis [4]. Indeed, the conjunctiva is the major supporting tissue of the ocular surface. One of its main functions is attributed to the presence of mucin-secreting goblet cells that play a role in protecting the ocular surface. These mucins, along with the water and electrolytes secreted by the lacrimal gland and the lipids produced by the Meibomian glands, form the tear film [5]. Alterations in the function of goblet cells lead to changes in tear composition that can result in different pathologies [6,7]. The conjunctiva also possesses specific lymphoid components belonging to the mucosaassociated lymphoid tissue (MALT) that can locally initiate and regulate immune responses [8,9]. In the conjunctiva, the MALT is present as the conjunctival-associated lymphoid tissue (CALT) and consists of a diffuse layer of lympho-epithelium and lymphoid follicles composed of B and T lymphocytes, macrophages, and dendritic cells. The CALT functions as the efferent and afferent arms of the conjunctival immune system [10]. The lymphoid follicles are overlain by a specialized follicle-associated epithelium (FAE) that is thinner than the regular conjunctival epithelium, has a fragmented basal lamina, lacks goblet cells, and contains M-cells that transport antigens [8]. Another typical feature of CALT is the presence of high endothelial venules (HEVs) associated with the follicles and that facilitate the migration of lymphocytes to these areas of the conjunctiva. Experiments conducted to study the ocular surface can be performed in different in vitro, ex vivo, or in vivo models. Several cell lines have been widely used to study the corneal [11–14] and conjunctival [13,15] epithelia. However, the use of cell lines is being questioned due to frequent problems of misidentification and cross-contamination [16,17]. For that reason, primary cultures are emerging as the best way to study cell physiology in vitro. In addition, ex vivo models are an excellent tool to deepen the knowledge of physiological features without the disadvantages of in vivo research. Unfortunately, the availability of human tissue to perform in vitro or ex vivo studies is highly limited, a situation that constrains this type of experimentation. Therefore, the use of animal tissue is necessary. Humans and pigs share similar anatomic and physiologic characteristics that make pigs useful as experimental models in biomedical research [18,19]. Several studies have reported the characteristics of pig eyes, including parameters of the whole eyeball [20,21], retina [22,23], cornea [24], limbus [25], and the lacrimal gland [26]. However as far as we know, a thorough description of the conjunctiva of the pig eye has not yet been reported. Thus, the purpose of this study is to provide a detailed description of the pig ocular surface with special emphasis on the conjunctiva. Our goal was to determine the suitability of porcine conjunctiva as a model to advance knowledge of the human ocular surface and associated diseases. Histological and immunohistochemical characterization of the porcine ocular surface PLOS ONE | https://doi.org/10.1371/journal.pone.0227732 January 13, 2020 2 / 17 porcine_ocular_surface_Raw_data_xlsx/ 11417130). Funding: YD, MAT2013-47501-C2-1-R (Spanish Ministry of Economy and Competitiveness and European Regional Development Fund) http:// www.mineco.gob.es/ and https://ec.europa.eu/ regional_policy/en/funding/erdf/ YD and LGP, RTI2018-094071-B-C21 (Spanish Ministry of Science, Innovation and Universities and European Regional Development Fund) http://www.ciencia. gob.es/portal/site/MICINN/ and https://ec.europa. eu/regional_policy/en/funding/erdf/ LGP, Postdoctoral contrats 2017 call (University of Valladolid) http://www.uva.es/export/sites/uva/ MCM, Regional JCyL Scholarship/European Social Fund Program ORDEN EDU/128/2015 (Regional JCyL and European Social Fund) https://www.jcyl. es/ and https://ec.europa.eu/esf/home.jsp The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Materials and methods Porcine eyes All experiments were conducted following the Association for Research in Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research (https://www.arvo.org/ About/policies/statement-for-the-use-of-animals-in-ophthalmic-and-vision-research/) and were approved by the Ethics Committee of the University of Valladolid. Eyeballs with eyelids (n = 3) were obtained from white domestic pigs (Sus scrofa domestica) donated by the local slaughterhouse Justino Gutie ´rrez SL (Laguna de Duero, Valladolid, Spain). The animals were between 6 and 8 months of age (pre-adults), and weighed 120–150 kg. The chief veterinarian of the slaughterhouse performed the exenterations immediately after each pig was killed, and the exenterated tissues were immediately placed in 4% buffered paraformaldehyde. Afterwards, tissues were transported to the laboratory where they were maintained in the fixative solution for ten days. Tissue processing All the adjacent muscle, fat, and connective tissue were dissected and removed to finally process the fixed eyeball and eyelids in a tissue processor (Leica Biosystems ASP300, Nussloch, Germany) for 16 hours. Paraffin tissue blocks were prepared and 4-μm–thick sections were obtained using a soft tissue microtome (Microm, Walldorf, Germany). Histological staining and light microscopy analysis Ocular sections were deparaffinized with xylene (Applichem Panreac, Barcelona, Spain) and rehydrated through a decreasing gradient of ethanol (Applichem Panreac) followed by de-ionized water. Then, the sections were stained with hematoxylin/eosin (H/E), Alcian blue/periodic acid Schiff (AB/PAS), or Giemsa. H/E staining was used to identify and describe the different tissues and structures within them. Rehydrated slides were rinsed for 1 min in distilled water, immersed in Mayer’s Hematoxylin (Millipore, Billerica, MA, USA) for 5 min, and rinsed in running tap water for 10 min. Then, they were rinsed in 80% ethanol for 1 min and immersed in eosin (Fluka, Buchs, Switzerland) for 5 min. Finally, the slides were dehydrated, cleared, and mounted with cover slips. AB/PAS-stained slides were used to identify and count goblet cells. Slides were rinsed in distilled water, immersed in 3% acetic acid for 3 min followed by Alcian blue solution (pH 2.5; Sigma Aldrich, St. Louis, MO, USA) for 15 min. After that, the slides were immersed in 0.5% periodic acid for 5 min, rinsed with distilled water, and placed in Schiff’s solution (Millipore) for 15 min. The slides were then rinsed in running tap water for 10 min, counterstained with Mayer´s Hematoxylin (Millipore) for 5 min, and finally rinsed, dehydrated, cleared, and mounted with cover slips. Acidic (blue), neutral (pink), and mixed (purple) goblet cells were counted in each conjunctival area. Goblet cell density (GCD) was calculated by counting the number of goblet cells in a section and dividing that number by the length of the section. At least three different sections of �500 μm were counted for each conjunctival region. Mean values for the number of cells counted independently by two researchers were then calculated. Giemsa-stained sections were used to analyze the presence of inflammatory cells and characterize the CALT in porcine conjunctiva. Briefly, rehydrated slides were placed in 20% Giemsa solution (Merck, Darmstadt, Germany) for 1 h and then rinsed in distilled water. The sections were then differentiated with 0.5% acetic acid, dehydrated rapidly, cleared, and mounted. Histological and immunohistochemical characterization of the porcine ocular surface PLOS ONE | https://doi.org/10.1371/journal.pone.0227732 January 13, 2020 3 / 17 Lectin staining To detect glycoconjugates produced by conjunctival goblet cells, we used lectins from Arachis hypogaea agglutinin (PNA) and from Helix pomatia agglutinin (HPA), which bind β-D-galactose-1!3-D-N-acetyl-galactosamine and α-N-acetyl-α-D-galactosamine residues, respectively. Ocular sections were deparaffinized and rehydrated as previously described. The slides were washed three times with phosphate-buffered saline (PBS) and then fluorescein isothiocyanate (FITC)-conjugated PNA (Sigma Aldrich, L7381, Lot 056k4006) and tetramethylrhodamine (TRITC)-conjugated HPA (Sigma Aldrich, L1261, Lot 091k3793) at 1:500 dilution were added and incubated for 40 min at room temperature. After that, the slides were washed 3 times with PBS to remove the excess lectins. Cell nuclei were counterstained with Hoechst 33342 dye (Sigma Aldrich, B2261) at 1:1000 dilution. The preparations were viewed under an epifluorescence microscope (Leica DMI 6000B; Leica Microsystems, Wetzlar, Germany). T lymphocyte immunodetection To determine T lymphocyte locations within the porcine conjunctiva, ocular sections were stained with anti-CD3 antibody. Prior to immunodetection, enzymatic antigen retrieval was performed in the sections with 0.05% trypsin. Then, sections were immunostained with rabbit monoclonal anti-CD3 (Abcam, ab16669, Cambridge, UK) at a 1:200 dilution for 18 h at 4˚ C. After that, the sections were incubated with polyclonal donkey anti-rabbit Alexa Fluor 1 488 (Thermo Fisher Scientific, Waltham, MA, USA) secondary antibody (1:200 dilution) for 1 h at room temperature. Cell nuclei were counterstained with Hoechst 33342 at a 1:1000 dilution. Negative controls included the omission of primary antibody and positive controls included human tonsil. Slides were observed under the Leica DMI 6000B epifluorescence microscope and representative micrographs were taken at different magnifications. Automated image acquisition AB/PAS-stained slides were analyzed using the Automated Cellular Imaging System III (ACIS III; Dako, Glostrup, Denmark). The system automatically acquired digital images of the glass slides using the ACIS scanner at low magnification (x10). Using ACIS III functionalities, we measured the thickness of the cornea in three different regions: at approximately the corneal apex, in the intermediate between the apex and the limbus, and at the periphery near the limbus. Data presentation and statistical analysis Data were presented as means ±standard errors of the mean. Statistical differences in corneal thickness were analyzed with Student’s t-test. Statistical differences in GCD were analyzed by one-way analysis of variance. Then, pairwise comparisons were performed with Tukey’s test. Results were considered significantly different at p �0.05. Statistical analyses were conducted using the Statistical Package for the Social Sciences software (SPSS). Results Macroscopic description of the pig eyeball and eyelids The macroscopic anatomy of the pig eyeball and eyelids is similar to that of humans, although the pig eye has a bigger iris and a thicker cornea. Regarding the eyelids, the main and most obvious difference between pigs and humans was the presence of the nictitating membrane, also known as the third eyelid, in the pig eyes (Fig 1). The nictitating membrane was situated at the medial angle of the eye and contained cartilage that provides structural support as the lid Histological and immunohistochemical characterization of the porcine ocular surface PLOS ONE | https://doi.org/10.1371/journal.pone.0227732 January 13, 2020 4 / 17 moves horizontally across the eyeball. In contrast, the upper and lower eyelids, which were similar in size and shape to one another, move vertically across the eyeball. The porcine cornea Based on histological analysis, the thickness of central cornea, 1,131.0 ±56.3 μm, was slightly thinner than the intermediate area, 1,215.2 ±32.9 μm (p <0.187), that is located between the central cornea and the limbus. The limbal cornea was the thickest, 1,496.9 ±60.8 μm (p <0.001 vs central cornea, p <0.001 vs intermediate cornea). The porcine cornea was composed of four layers: epithelium, stroma, Descemet’s membrane, and endothelium (from outermost to innermost, Fig 2). The epithelium was stratified and composed of 6 to 8 layers, distributed as 2–3 layers of superficial stratified squamous cells, 2–3 layers of intermediate wing cells, and 2 layers of basal cells. The basal cells rested on a continuous basement membrane. Bowman’s layer was not evident in the porcine cornea. The stroma was the thickest layer of the cornea. It was composed of a structured collagen fiber matrix in which elongated keratocytes were embedded. Attached to the innermost part of the stroma was Descemet’s membrane, and underneath it there is a single layer of endothelial cells. The porcine limbus The limbus is the transition zone between the cornea and the conjunctiva. The limbal epithelium had a special anatomical conformation with the palisades of Vogt (Fig 3), where limbal stem cells are found [27]. In the pig eye, the limbal epithelium was composed of 12 layers of Fig 1. Macroscopic photograph of porcine eye. Transverse section of a fixed pig eyeball with the eyelids. The white line marks the limits of the conjunctiva. https://doi.org/10.1371/journal.pone.0227732.g001 Histological and immunohistochemical characterization of the porcine ocular surface PLOS ONE | https://doi.org/10.1371/journal.pone.0227732 January 13, 2020 5 / 17 epithelial cells: 3 layers of flattened superficial squamous cells, 6 layers of intermediate wing cells, and 3 layers of basal cells. The porcine conjunctiva The porcine conjunctiva was composed of a stratified epithelium and the substantia propria or stroma. The conjunctiva had three main anatomical zones: palpebral conjunctiva, bulbar conjunctiva, and fornix (cul-de-sac) (Fig 4). The palpebral conjunctiva lined the posterior surface of the eyelids. It consisted of the marginal conjunctiva at the edges of the lids and the tarsal conjunctiva. The bulbar conjunctiva was attached to the sclera. Finally, the fornix connected the palpebral conjunctiva with the bulbar conjunctiva. The appearance of the porcine conjunctiva varied in the different regions, presenting diverse characteristics and a variable number of epithelial cell layers. At the marginal conjunctiva between the tarsal and palpebral regions, several infoldings of the epithelium (crypts) were present (Fig 5A). In this area, the conjunctiva consisted of 6 epithelial cell layers, including 2 superficial layers of squamous cells and 4 deeper layers of cuboidal cells (Fig 5A). In the tarsal conjunctiva, the crypts disappeared, and the epithelium consisted of 8 layers (Fig 5B). In the fornix, the histological staining revealed 6 epithelial cell layers (Fig 5C), whereas in the bulbar conjunctiva there were only 4 layers, but the cells were larger so that the total epithelial Fig 2. Histological analysis of the porcine cornea. Tissue section stained with hematoxylin-eosin showing the four layers of the cornea. https://doi.org/10.1371/journal.pone.0227732.g002 Histological and immunohistochemical characterization of the porcine ocular surface PLOS ONE | https://doi.org/10.1371/journal.pone.0227732 January 13, 2020 6 / 17 thickness was maintained (Fig 5D). As described in a following section, the densities of goblet cells varied in these regions. The organization of the conjunctival stroma was dissimilar to that of the cornea. It was composed of loose connective tissue that included a superficial lymphoid layer and a deeper Fig 3. Histological analysis of the porcine limbus. Tissue section of porcine limbus stained with Alcian blue/periodic acid Schiff showing the characteristic palisades of Vogt (arrows) where limbal epithelial stem cells reside. https://doi.org/10.1371/journal.pone.0227732.g003 Fig 4. Histological analysis of the porcine ocular surface. Low magnification tissue section of porcine anterior ocular surface stained with Alcian blue/periodic acid Schiff. Bar = 1 mm. https://doi.org/10.1371/journal.pone.0227732.g004 Histological and immunohistochemical characterization of the porcine ocular surface PLOS ONE | https://doi.org/10.1371/journal.pone.0227732 January 13, 2020 7 / 17 fibrous layer with a large number of fibroblasts. In addition, there was a significant presence of blood vessels and immune cells (see Location and characteristics of lymphoid tissue in porcine conjunctiva section below). The characteristics of the conjunctival epithelium in the nictitating membrane varied depending on the area (Fig 6A). In the zones over the cartilage, the epithelium consisted of 4 to 6 layers of stratified squamous cells (Fig 6B). At the edges, the nictitating membrane was composed of 12 epithelial cell layers (Fig 6C), whereas in the center it had only 8 layers (Fig 6D). Goblet cell characteristics and distribution in porcine conjunctiva. Numerous goblet cells were present in the porcine conjunctival epithelium. The large, rounded cells were filled with glycoconjugate (mucin) granules. The glycoconjugate components of these cells differed from cell to cell and were differentiated by AB/PAS staining (Fig 7A). The acidic glycoconjugates were stained blue by AB, and the neutral glycoconjugates were stained pink by PAS. Some goblet cells contained a mixture of blueand pink-stained glycoconjugates, appearing as purple granules. In the porcine conjunctival epithelium, 7.59 ±0.94% of the total goblet cells were neutral, 5.38 ±1.65% were acidic, and 87.02 ±1.36% were mixed. The distribution of goblet cells varied along the conjunctival epithelium topography, resulting in variations of GCDs in the different eyelids. GCD was 84.07 ±9.59 cells/mm in the upper eyelid and upper fornix, 87.51 ±9.16 cells/mm in the lower eyelid and lower fornix, and 71.44 ±4.86 cells/mm in the anterior and posterior surfaces of the nictitating membrane. In addition to the conjunctival epithelium, goblet cells were also present in stromal structures similar to human pseudogland of Henle These crypts were sparsely distributed in both the superior and inferior conjunctival fornices, but they were absent in other areas (Fig 7B). Fig 5. Histological analysis of porcine conjunctiva. Tissue sections of pig conjunctiva stained with AB/PAS. (A) The marginal conjunctiva between the tarsal and palpebral surfaces was covered by a stratified squamous epithelium and deeper cuboidal epithelial cells. Epithelial downgrowths into the stroma appeared as crypts (arrowheads). (B) The tarsal conjunctiva had a large number of goblet cells containing acidic glycoconjugates. (C) Conjunctiva in the fornix. (D) The bulbar conjunctiva had 4 epithelial cell layers and very few goblet cells. https://doi.org/10.1371/journal.pone.0227732.g005 Histological and immunohistochemical characterization of the porcine ocular surface PLOS ONE | https://doi.org/10.1371/journal.pone.0227732 January 13, 2020 8 / 17 For further analysis of goblet cell distribution, we divided the conjunctiva into 7 different areas: (1) upper palpebra, (2) upper fornix, (3) bulbar, (4) lower palpebra, (5) lower fornix, (6) anterior nictitating membrane, and 7) posterior nictitating membrane. The bulbar conjunctiva had the lowest GCD, 12.69 ±4.29 cells/mm (Fig 8). The highest GCDs were in the upper and lower palpebral conjunctivas, 103.20 ±15.19 cells/mm and 113.04 ±5.76 cells/mm respectively, and both were greater than each of the other areas (p <0.05 for all comparisons). We also analyzed goblet cell contents based on lectin staining. Porcine conjunctival goblet cells stained with HPA and PNA lectins, indicating the presence of α-N-acetyl-α-D-galactosamine and β-D-galactose-1!3-D-N-acetyl-galactosamine, respectively (Fig 9). Fig 6. Porcine nictitating membrane. (A) Low magnification micrograph of the nictitating membrane union with upper eyelid. Bar = 500 μm. (B) Goblet cells in the conjunctival epithelium over the nictitating membrane cartilage. (C) Conjunctival edge between nictitating membrane and lower eyelid. (D) Conjunctiva over the center of the nictitating membrane anterior surface showing great abundance of goblet cells containing acidic, neutral, or both types of glycoconjugates. https://doi.org/10.1371/journal.pone.0227732.g006 Fig 7. Porcine conjunctival goblet cells. (A) The different types of goblet cells can be distinguished with AB/PAS staining. Acidic glycoconjugates were stained blue (arrow) by AB, and neutral glycoconjugates were pink (arrowhead) by PAS. 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