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• W2129476710 abstract "Goblet cell numbers decrease within the conjunctival epithelium in drying and cicatrizing ocular surface diseases. Factors regulating goblet cell differentiation in conjunctival epithelium are unknown. Recent data indicate that the transcription factor SAM-pointed domain epithelial-specific transcription factor (Spdef) is essential for goblet cell differentiation in tracheobronchial and gastrointestinal epithelium of mice. Using Spdef−/− mice, we determined that Spdef is required for conjunctival goblet cell differentiation and that Spdef−/− mice, which lack conjunctival goblet cells, have significantly increased corneal surface fluorescein staining and tear volume, a phenotype consistent with dry eye. Microarray analysis of conjunctival epithelium in Spdef−/− mice revealed down-regulation of goblet cell–specific genes (Muc5ac, Tff1, Gcnt3). Up-regulated genes included epithelial cell differentiation/keratinization genes (Sprr2h, Tgm1) and proinflammatory genes (Il1-α, Il-1β, Tnf-α), all of which are up-regulated in dry eye. Interestingly, four Wnt pathway genes were down-regulated. SPDEF expression was significantly decreased in the conjunctival epithelium of Sjögren syndrome patients with dry eye and decreased goblet cell mucin expression. These data demonstrate that Spdef is required for conjunctival goblet cell differentiation and down-regulation of SPDEF may play a role in human dry eye with goblet cell loss. Spdef−/− mice have an ocular surface phenotype similar to that in moderate dry eye, providing a new, more convenient model for the disease. Goblet cell numbers decrease within the conjunctival epithelium in drying and cicatrizing ocular surface diseases. Factors regulating goblet cell differentiation in conjunctival epithelium are unknown. Recent data indicate that the transcription factor SAM-pointed domain epithelial-specific transcription factor (Spdef) is essential for goblet cell differentiation in tracheobronchial and gastrointestinal epithelium of mice. Using Spdef−/− mice, we determined that Spdef is required for conjunctival goblet cell differentiation and that Spdef−/− mice, which lack conjunctival goblet cells, have significantly increased corneal surface fluorescein staining and tear volume, a phenotype consistent with dry eye. Microarray analysis of conjunctival epithelium in Spdef−/− mice revealed down-regulation of goblet cell–specific genes (Muc5ac, Tff1, Gcnt3). Up-regulated genes included epithelial cell differentiation/keratinization genes (Sprr2h, Tgm1) and proinflammatory genes (Il1-α, Il-1β, Tnf-α), all of which are up-regulated in dry eye. Interestingly, four Wnt pathway genes were down-regulated. SPDEF expression was significantly decreased in the conjunctival epithelium of Sjögren syndrome patients with dry eye and decreased goblet cell mucin expression. These data demonstrate that Spdef is required for conjunctival goblet cell differentiation and down-regulation of SPDEF may play a role in human dry eye with goblet cell loss. Spdef−/− mice have an ocular surface phenotype similar to that in moderate dry eye, providing a new, more convenient model for the disease. Conjunctival goblet cells secrete hydrophilic glycoproteins, termed mucins, which are believed to maintain fluid on the ocular surface and to trap and remove surface debris through movement over the ocular surface by blinking. In humans, the conjunctival goblet cells secrete the mucin MUC5AC; in mice, an additional mucin, Muc5b (by convention, human mucins are designated MUC and mouse mucins, Muc) is also secreted, albeit at lower levels.1Gupta D. Harvey S.A. Kaminski N. Swamynathan S.K. Mouse conjunctival forniceal gene expression during postnatal development and its regulation by Kruppel-like factor 4.Invest Ophthalmol Vis Sci. 2011; 52: 4951-4962Crossref PubMed Scopus (20) Google Scholar It is currently thought that mucin secretion by conjunctival goblet cells is necessary for the maintenance of a healthy ocular surface, because there is a well-documented decrease in goblet cell numbers within the conjunctiva in cicatrizing diseases including Stevens-Johnson syndrome and ocular cicatricial pemphigoid, as well as in dry eye of several etiologies, including Sjögren syndrome, meibomian gland disease, and keratoconjunctivitis sicca of undefined cause.2DEWSResearch in dry eye: report of the Research Subcommittee of the International Dry Eye WorkShop (2007).Ocul Surf. 2007; 5: 179-193Abstract Full Text PDF PubMed Google Scholar Approximately 4.8 million people are affected by dry eye in the United States alone.2DEWSResearch in dry eye: report of the Research Subcommittee of the International Dry Eye WorkShop (2007).Ocul Surf. 2007; 5: 179-193Abstract Full Text PDF PubMed Google Scholar In addition to loss of goblet cells, these dry eye diseases also feature changes in the ocular surface epithelium, including increased corneal surface fluorescein staining, inflammation of the ocular surface tissues, changes in tear volume and composition, alterations in corneal epithelial barrier function, increases in conjunctival epithelial proliferation, and alterations in cell surface and secreted mucins as well as keratinization-related proteins.2DEWSResearch in dry eye: report of the Research Subcommittee of the International Dry Eye WorkShop (2007).Ocul Surf. 2007; 5: 179-193Abstract Full Text PDF PubMed Google Scholar, 3Lemp M.A. Report of the National Eye Institute/Industry workshop on Clinical Trials in Dry Eyes.Clao J. 1995; 21: 221-232PubMed Google Scholar Currently, there are relatively few effective treatments for these diseases and few convenient animal models in which drying and cicatrizing diseases can be studied.4Barabino S. Dana M.R. Animal models of dry eye: a critical assessment of opportunities and limitations.Invest Ophthalmol Vis Sci. 2004; 45: 1641-1646Crossref PubMed Scopus (115) Google Scholar The most commonly used method to create dry eye syndrome in mice involves repeated daily injections of scopolamine to inhibit production of aqueous tears in conjunction with exposure to environmental desiccating stress.5Dursun D. Wang M. Monroy D. Li D.Q. Lokeshwar B.L. Stern M.E. Pflugfelder S.C. A mouse model of keratoconjunctivitis sicca.Invest Ophthalmol Vis Sci. 2002; 43: 632-638PubMed Google Scholar, 6de Paiva C.S. Schwartz C.E. Gjorstrup P. Pflugfelder S.C. Resolvin E1 (RX-10001) reduces corneal epithelial barrier disruption and protects against goblet cell loss in a murine model of dry eye.Cornea. 2012; 31: 1299-1303Crossref PubMed Scopus (71) Google Scholar, 7Okanobo A. Chauhan S.K. Dastjerdi M.H. Kodati S. Dana R. Efficacy of topical blockade of interleukin-1 in experimental dry eye disease.Am J Ophthalmol. 2012; 154: 63-71Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 8Sadrai Z. Stevenson W. Okanobo A. Chen Y. Dohlman T.H. Hua J. Amparo F. Chauhan S.K. Dana R. PDE4 inhibition suppresses IL-17-associated immunity in dry eye disease.Invest Ophthalmol Vis Sci. 2012; 53: 3584-3591Crossref PubMed Scopus (23) Google Scholar Although it is known that goblet cell dropout commonly occurs in drying and cicatrizing diseases, to date, little is known about goblet cell differentiation in the conjunctiva. Early studies have shown that conjunctival epithelial cells and corneal-limbal epithelial cells are from two separate cell lineages that are intrinsically divergent.9Wei Z.G. Wu R.L. Lavker R.M. Sun T.T. In vitro growth and differentiation of rabbit bulbar, fornix, and palpebral conjunctival epithelia. Implications on conjunctival epithelial transdifferentiation and stem cells.Invest Ophthalmol Vis Sci. 1993; 34: 1814-1828PubMed Google Scholar To date, no definitive goblet cell precursors have been identified, although it is known that goblet cells and differentiated conjunctival epithelial cells (keratinocytes) share a common progenitor.10Wei Z.G. Lin T. Sun T.T. Lavker R.M. Clonal analysis of the in vivo differentiation potential of keratinocytes.Invest Ophthalmol Vis Sci. 1997; 38: 753-761PubMed Google Scholar, 11Pellegrini G. Golisano O. Paterna P. Lambiase A. Bonini S. Rama P. De Luca M. Location and clonal analysis of stem cells and their differentiated progeny in the human ocular surface.J Cell Biol. 1999; 145: 769-782Crossref PubMed Scopus (598) Google Scholar Identification of the factors required to induce goblet cell differentiation may be useful in understanding the mechanisms of dry eye pathology and may provide potential therapeutic treatments for replacement of goblet cells lost during dry eye. Recent studies have demonstrated that the transcription factor sterile α motif pointed domain epithelial specific transcription factor (Spdef), is involved in the induction of goblet cell differentiation from precursor cells in the tracheobronchial epithelium. In respiratory epithelia, expression of Spdef in Clara cells (a goblet cell precursor cell) creates goblet cell hyperplasia by inducing their differentiation into goblet cells.12Park K.S. Korfhagen T.R. Bruno M.D. Kitzmiller J.A. Wan H. Wert S.E. Khurana Hershey G.K. Chen G. Whitsett J.A. SPDEF regulates goblet cell hyperplasia in the airway epithelium.J Clin Invest. 2007; 117: 978-988Crossref PubMed Scopus (200) Google Scholar, 13Chen G. Korfhagen T.R. Xu Y. Kitzmiller J. Wert S.E. Maeda Y. Gregorieff A. Clevers H. Whitsett J.A. SPDEF is required for mouse pulmonary goblet cell differentiation and regulates a network of genes associated with mucus production.J Clin Invest. 2009; 119: 2914-2924Crossref PubMed Scopus (51) Google Scholar Furthermore, studies from intestinal epithelia have shown that Spdef also plays an important role in regulating intestinal epithelial cell homeostasis and differentiation. Loss of Spdef severely impairs maturation of goblet and Paneth cells in the intestine14Gregorieff A. Stange D.E. Kujala P. Begthel H. van den Born M. Korving J. Peters P.J. Clevers H. The ets-domain transcription factor Spdef promotes maturation of goblet and paneth cells in the intestinal epithelium.Gastroenterology. 2009; 137 (e1331–1333): 1333-1345Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar and expression of Spdef promotes goblet cell differentiation in the intestinal epithelium at the expense of absorptive, Paneth, and enteroendocrine cell types.15Noah T.K. Kazanjian A. Whitsett J. Shroyer N.F. SAM pointed domain ETS factor (SPDEF) regulates terminal differentiation and maturation of intestinal goblet cells.Exp Cell Res. 2010; 316: 452-465Crossref PubMed Scopus (123) Google Scholar The purpose of this study was to determine whether, as in the tracheobronchial and gastrointestinal epithelium, the transcription factor Spdef regulates goblet cell differentiation in the conjunctiva, and if so, to determine the effect of loss of goblet cells on ocular surface function and phenotype. To address this, we characterized the ocular surface phenotype of mice null for the Spdef gene, and conducted microarray and real-time quantitative RT-PCR (real-time RT-qPCR) analyses to identify changes in expression patterns in inflammatory mediators and genes associated with epithelial cell stress and differentiation that have been shown to be altered in dry eye syndrome. Spdef null mice were also challenged with desiccating environmental stress. To determine the potential role of SPDEF in human dry eye disease, we assayed the levels of SPDEF in conjunctival epithelia derived from patients with Sjögren syndrome dry eye known to have diminution of expression of the goblet cell mucin MUC5AC. Our results indicate that Spdef is critical for goblet cell differentiation in the conjunctiva, that SPDEF is down-regulated in the conjunctival epithelium of patients with Sjögren dry eye, and that the Spdef null mouse serves as an animal model to study the effects of dry eye disease. Mice null for the transcription factor (Spdef−/−) were developed by Alex Gregorieff in the laboratory of Hans Clevers (Netherlands Institute of Developmental Biology)14Gregorieff A. Stange D.E. Kujala P. Begthel H. van den Born M. Korving J. Peters P.J. Clevers H. The ets-domain transcription factor Spdef promotes maturation of goblet and paneth cells in the intestinal epithelium.Gastroenterology. 2009; 137 (e1331–1333): 1333-1345Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar and were generously provided by Jeffrey Whitsett (Children’s Hospital, Cincinnati, OH). Spdef−/− mice, as obtained from Dr. Whitsett, were on a mixed background of C57BL/6 and 129. Spdef+/+ mice, also on a C57BL/6-129 mixed background were backcrossed with C57BL/6 mice to expand the colony. Young adult 8-week-old Spdef−/− mice (n = 14) and their wild-type control Spdef+/+ mice (n = 13), as well as aged adult animals >8-month-old Spdef−/− mice (n = 8) and Spdef+/+ mice (n = 10) were used. All animal protocols were approved by the Schepens Eye Research Institute Institutional Animal Care and Use Committee (IACUC). Animals were euthanized by CO2, and eyes with intact lids were excised and fixed in 10% formalin, embedded in methacrylate, sectioned, and stained with PAS stain or H&E to determine presence of goblet and inflammatory cells, or they were embedded in optimal cutting temperature compound, frozen on dry ice, and stored at −80°C until use for laser capture microdissection (LCM) and immunofluorescence microscopy. Inflammatory cells within the conjunctival epithelium were counted either in H&E-stained sections from Spdef+/+ and Spdef−/− mice. Linear measurements of conjunctival epithelial basal lamina were made using Spot RT software version 3.1 (Spot Diagnostic Instruments, Sterling Heights, MI). Results are expressed as the number of inflammatory cells per 1-mm linear length of basal lamina. Inflammatory cell counts were done in a blind manner, with the genotype (+/+ or −/−) unknown to two independent observers (I.K.G. and Sandra Spurr-Michaud). Counts from the two blind observers were averaged for data analysis. CD45-positive cells within the conjunctival epithelium of frozen sections from Spdef+/+ and Spdef−/− mice were identified and quantified using immunofluorescence microscopy. Sections were incubated with either Alexa Fluor 488 anti-mouse CD45 antibody (dilution 1:250; BioLegend, San Diego, CA) or the isotype control antibody Alexa Fluor 488 rat IgG2b (dilution 1:100; BioLegend) for 1.5 hours at room temperature and coverslipped in Vectashield mounting medium with propidium iodide (Vector Laboratories, Burlingame, CA).16Zhang X. Shen L. Jin Y. Saban D.R. Chauhan S.K. Dana R. Depletion of passenger leukocytes from corneal grafts: an effective means of promoting transplant survival?.Invest Ophthalmol Vis Sci. 2009; 50: 3137-3144Crossref PubMed Scopus (18) Google Scholar CD45-positive cells were counted in a blind manner (genotype unknown) by two independent observers (I.K.G. and Ann Tisdale) on a Zeiss Photoscope III fluorescent microscope at ×25. Linear measurements of conjunctival epithelium basal lamina were made from ×10 images using ImageJ version 1.42Q (NIH, Bethesda, MD). Results are expressed as the number of CD45-positive cells per 1-mm linear length of basal lamina. Counts from the two blind observers were averaged together for data analysis. Spdef−/− mice and their wild-type controls were examined for gross ocular surface and/or eyelid phenotype, and then assayed for corneal fluorescein staining and tear volume. Corneal fluorescein staining was imaged with a Topcon SL-07 slit lamp biomicroscope (Topcon Corporation, Tokyo, Japan) using a cobalt blue filter 3 minutes after application of 1 μL of 2.5% sodium fluorescein (Sigma-Aldrich, St. Louis, MO) in sterile saline.17Barabino S. Shen L. Chen L. Rashid S. Rolando M. Dana M.R. The controlled-environment chamber: a new mouse model of dry eye.Invest Ophthalmol Vis Sci. 2005; 46: 2766-2771Crossref PubMed Scopus (139) Google Scholar Fluorescein staining was assayed daily for 5 days, and images were scored using a standardized National Eye Institute grading system. The cornea is divided into five areas: superior, inferior, temporal, nasal, and central; punctate fluorescein staining in each area was graded on a scale of 0 to 3, and the scores for all five areas were summed for a total score (0 to 15).3Lemp M.A. Report of the National Eye Institute/Industry workshop on Clinical Trials in Dry Eyes.Clao J. 1995; 21: 221-232PubMed Google Scholar, 17Barabino S. Shen L. Chen L. Rashid S. Rolando M. Dana M.R. The controlled-environment chamber: a new mouse model of dry eye.Invest Ophthalmol Vis Sci. 2005; 46: 2766-2771Crossref PubMed Scopus (139) Google Scholar, 18Barabino S. Rolando M. Chen L. Dana M.R. Exposure to a dry environment induces strain-specific responses in mice.Exp Eye Res. 2007; 84: 973-977Crossref PubMed Scopus (28) Google Scholar Fluorescein scoring was performed in a blind manner, with the age (8 weeks or >8 months) and genotype (+/+ or −/−) unknown to the two independent scorers (C.K.M. and Sandra Spurr-Michaud). Scores were averaged for a final score used in all subsequent data analysis. Aqueous tear volume was measured using the phenol red thread test (Zone-Quick; Lacrimedics, Eastsound, WA).5Dursun D. Wang M. Monroy D. Li D.Q. Lokeshwar B.L. Stern M.E. Pflugfelder S.C. A mouse model of keratoconjunctivitis sicca.Invest Ophthalmol Vis Sci. 2002; 43: 632-638PubMed Google Scholar Fine forceps were used to place the thread into the lateral canthus of the conjunctival fornix, and the thread was held in place for 30 seconds. Wetting of the thread was measured in millimeters using the scale on the thread box under a light microscope. Tear volume measurements were assayed twice daily (AM and PM) for 3 consecutive days. Conjunctival tissue from Spdef+/+ and Spdef−/− mice was cryostat-sectioned at −20°C. Sections (6 μm thick) were collected on Arcturus PEN Membrane glass slides (Applied Biosystems, Carlsbad, CA) and stained immediately or stored overnight at −80°C. Before laser microdissection, sections were fixed in 70% ethanol, washed in Nuclease-Free water (Ambion, Austin, TX), stained with Mayer’s Hematoxylin Solution (Sigma-Aldrich), and alcoholic Eosin Y solution (Sigma-Aldrich), dehydrated in 95% and 100% ethanol and xylene, and air dried. For microarray analysis and real-time RT-qPCR, 40% of the conjunctival epithelium, as measured from the deepest point of the fornix cul-de-sac, was captured using a laser microdissection microscope (Model AS LMD; Leica, Wetzlar, Germany). In studies investigating the location of Frzb and Spdef mRNA, multiple clusters of goblet cells and regions of stratified epithelial cells (where no goblet cells were present) were collected into two separate samples by LCM. Total RNA was isolated from laser-captured sections of conjunctival epithelium using an Arcturus PicoPure Isolation Kit (Applied Biosystems). RNA integrity (RIN number) and concentration was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). The RNA samples were analyzed on Affymetrix murine genome MOE430 chips (Affymetrix, Santa Clara, CA), which have approximately 45,000 genes for array. Additional assessment of RNA quality and quantity, as well as probe preparation, labeling, hybridization, and image scans were performed by the Dana Farber Cancer Institute Microarray Core Facilities (Boston, MA). Three replicate arrays were performed for Spdef+/+ and Spdef−/− mice. One eye from one male and one female animal were pooled into a single sample. Microarray data analysis was performed using dChip software (Cheng Li Lab, Dana Farber Cancer Institute and Harvard School of Public Health, Boston, MA; http://www.hsph.harvard.edu/cli/complab/dchip, last accessed January 20, 2011), and differences in gene expression between Spdef+/+ and Spdef−/− mice were considered significant if fold changes were greater than 3.0 and P < 0.01. Gene ontology (GO) analysis was performed using DAVID Bioinformatics Resources 6.7 (http://david.abcc.ncifcrf.gov, last accessed January 26, 2011),19Huang da W. Sherman B.T. Lempicki R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.Nat Protoc. 2009; 4: 44-57Crossref PubMed Scopus (24210) Google Scholar, 20Huang da W. Sherman B.T. Lempicki R.A. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists.Nucleic Acids Res. 2009; 37: 1-13Crossref PubMed Scopus (9790) Google Scholar and genes were sorted by functional annotation clustering with the KEGG: Kyoto Encyclopedia of Genes and Genomes (http://www.genome.ad.jp/kegg, last accessed; also accessible from within the DAVID website). Microarray data are available at the National Center for Biotechnology Information Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/projects/geo; accession number GSE44101). Real-time RT-qPCR was performed to validate expression levels of five genes selected from the microarray data: Sprr2h, Tgm1, K17, Frzb, and Wnt5b. Additionally, real-time RT-qPCR was performed to assess changes in Il-1α, Il-1β, and Tnf-α gene expression levels in the conjunctival epithelium of Spdef−/− mice compared to wild-type controls. Frzb and Spdef expression in conjunctival goblet cells, as compared to stratified conjunctival epithelium from Spdef+/+ mice, and SPDEF expression in human subjects with Sjögren syndrome dry eye, as compared to normal control subjects, were also analyzed. RNA was isolated using a Qiagen RNeasy Micro Isolation Kit (Qiagen, Valencia, CA). RNA integrity and concentration was determined using the NanoDrop 2000 Spectrophotometer (Thermo Scientific, Waltham, MA) before and after the pooling of one eye of one male and one female animal into a single sample. Real-time RT-qPCR was performed as previously described21Kunert K.S. Keane-Myers A.M. Spurr-Michaud S. Tisdale A.S. Gipson I.K. Alteration in goblet cell numbers and mucin gene expression in a mouse model of allergic conjunctivitis.Invest Ophthalmol Vis Sci. 2001; 42: 2483-2489PubMed Google Scholar, 22Gipson I.K. Spurr-Michaud S. Argueso P. Tisdale A. Ng T.F. Russo C.L. Mucin gene expression in immortalized human corneal-limbal and conjunctival epithelial cell lines.Invest Ophthalmol Vis Sci. 2003; 44: 2496-2506Crossref PubMed Scopus (284) Google Scholar using the Roche LightCycler 480 (Roche Applied Science, Indianapolis, IN), with either TaqMan chemistry (SPDEF human data) or RT2 SYBR Green qPCR Mastermix chemistry (mouse microarray confirmation; Frzb and Spdef expression). Prevalidated primer sets (SABiosciences, Frederick, MD) were used. In mouse samples, 18S RNA was used as the endogenous control gene; GAPDH mRNA was used for human samples. Relative levels of mRNA were calculated using the ΔΔCt method described in the Qiagen RT2 qPCR Primer Assay Handbook with the mean of the Spdef+/+ (mouse), mean of the goblet cell (Frzb and Spdef localization), or normal control (human) samples as the calibrator. Frzb and SPDEF proteins were localized in mouse conjunctival goblet cells and human conjunctival epithelium, respectively, using immunofluorescence microscopy of frozen sections of mouse and human conjunctiva. Sections were incubated with either anti–sFRP-3 (Frzb) goat polyclonal primary antibody (dilution 1:50; R&D Systems, Minneapolis, MN) overnight at 4°C or anti-Pdef (SPDEF) mouse monoclonal primary antibody (dilution 1:50; Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hour at room temperature, followed by incubation with either fluorescein isothiocyanate donkey anti-goat IgG or fluorescein isothiocyanate donkey anti-mouse IgG secondary antibody (dilution 1:50; Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 hour at room temperature. Sections were coverslipped in Vectashield mounting medium with propidium iodide (Vector Laboratories) and then viewed under a fluorescence microscope. For controlled-environment chamber (CEC)-exposure experiments, 4- to 5-month-old Spdef+/+ (n = 10) and Spdef−/− (n = 8) mice were exposed to desiccating environmental stress in a CEC (XDry Corporation, Las Vegas, NV) with an average temperature of 20.4 ± 0.5°C and an average relative humidity of 13.4 ± 2.9% for 15 days. Corneal fluorescein staining and tear volume measurements were collected and analyzed as described above before entering the CEC and subsequently every 3 days. Animals were euthanized after 15 days in the CEC (experimental day 16), and eyes with intact eyelids were excised. Conjunctival tissue used for immunolocalization of SPDEF and for measurement of SPDEF mRNA levels was archived material from a previously reported study.23Argueso P. Balaram M. Spurr-Michaud S. Keutmann H.T. Dana M.R. Gipson I.K. Decreased levels of the goblet cell mucin MUC5AC in tears of patients with Sjogren syndrome.Invest Ophthalmol Vis Sci. 2002; 43: 1004-1011PubMed Google Scholar Subject selection, as well as tear fluid and conjunctival epithelium sample collection, was performed as described.23Argueso P. Balaram M. Spurr-Michaud S. Keutmann H.T. Dana M.R. Gipson I.K. Decreased levels of the goblet cell mucin MUC5AC in tears of patients with Sjogren syndrome.Invest Ophthalmol Vis Sci. 2002; 43: 1004-1011PubMed Google Scholar Tear fluid and conjunctival epithelium from normal subjects and patients with Sjögren syndrome dry eye were collected to determine MUC5AC protein levels and mRNA expression, respectively. Remnant cDNA samples from the study were used in the current study to determine SPDEF mRNA expression levels in normal subjects (n = 6) and in patients with Sjögren syndrome dry eye (n = 5). The original 2002 study, from which the archived material was used, was conducted in compliance with good clinical practice, institutional review board regulations, informed-consent regulations, and the tenets of the Declaration of Helsinki. Statistics were performed using GraphPad InStat version 3.1a (GraphPad Software, La Jolla, CA). U-tests were used to evaluate differences in tear volume, fluorescein staining, and the number of inflammatory cells and CD45-positive cells between two groups of mice based on age, genotype, or time in the CEC, with P < 0.05 considered statistically significant. Student t-tests were used to evaluate differences in relative expression of the genes of interest in all real-time RT-qPCR experiments. P < 0.05 was considered statistically significant. Assessment of the gross appearance of the eye and eyelids of Spdef−/− mice showed no significant changes, as they were indistinguishable from eyes of wild-type mice (Figure 1A). In addition, there were no changes in gross appearance with aging, as the eyes and eyelids of 8-week-old Spdef+/+ (wild-type) and Spdef−/− (null) mice (Figure 1A) were indistinguishable from those of Spdef+/+ and Spdef−/− mice that were >8 months of age (Figure 1A). Despite a normal exterior appearance, Spdef−/− mice lack goblet cells in the conjunctival epithelium (Figure 1B). Other than the lack of goblet cells, Spdef−/− mice do not appear to have any additional major detectable histological defects. Comparison of the number of epithelial cell layers and thickness of the conjunctiva and corneal epithelium in Spdef−/− mice compared to wild-type controls were not statistically significant, although a trend toward increased cell layers and thickness in the Spdef−/− mice was found (data not shown). However, inflammatory cells, identified both by morphology (Figure 1C) and by the inflammatory cell marker CD45 (Figure 1E), were observed within both the conjunctival epithelium and subjacent connective tissue in Spdef−/− mice. Counts of inflammatory cells and CD45-positive cells per 1 mm of conjunctival epithelium basal lamina were both significantly higher in the conjunctival epithelium of Spdef−/− mice compared to that of wild-type mice (Figure 1, D and F). A solution of sodium fluorescein was applied to the ocular surface in Spdef+/+ and Spdef−/− mice to assess damage to the ocular surface (corneal) epithelium. Increased fluorescein dye uptake is a hallmark of human dry eye disease, and is often used in the clinic to diagnose dry eye syndrome2DEWSResearch in dry eye: report of the Research Subcommittee of the International Dry Eye WorkShop (2007).Ocul Surf. 2007; 5: 179-193Abstract Full Text PDF PubMed Google Scholar, 3Lemp M.A. Report of the National Eye Institute/Industry workshop on Clinical Trials in Dry Eyes.Clao J. 1995; 21: 221-232PubMed Google Scholar (Figure 2A). Minimal to no corneal fluorescein staining was observed in 8-week-old wild-type mice, whereas scattered punctate fluorescein staining was observed in 8-week-old Spdef−/− mice (Figure 2A). In >8-month-old Spdef−/− mice, patches of punctate and diffuse corneal fluorescein staining were observed (Figure 2A). Scoring of the amount of fluorescein staining showed a significant increase in Spdef−/− mice at 8 weeks of age and >8 months of age, as compared to age-matched Spdef+/+ animals (Figure 2B). A significant increase in fluorescein staining was seen in aged wild-type mice (>8 months old) as compared to 8-week-old wild-type mice, consistent with data previously reported with aging in humans.24Carlson K.H. Bourne W.M. McLaren J.W. Brubaker R.F. Variations in human corneal endothelial cell morphology and permeability to fluorescein with age.Exp Eye Res. 1988; 47: 27-41Crossref PubMed Scopus (130) Google Scholar, 25Chang S.W. Hu F.R. Changes in corneal autofluorescence and corneal epithelial barrier function with aging.Cornea. 1993; 12: 493-499Crossref PubMed Scopus (33) Google Scholar The increase in fluorescein staining with age in wild-type mice was not observed in Spdef−/− mice, as there was no statistical difference in the fluorescein staining scores of 8-week-old Spdef−/− mice and >8-month-old Spdef−/− mice. As alterations in tear volume have been commonly reported to occur in dry eye disease, aqueous tear volume was compared in Spdef−/− and wild" @default.
• W2129476710 created "2016-06-24" @default.
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• W2129476710 date "2013-07-01" @default.
• W2129476710 modified "2023-10-16" @default.
• W2129476710 title "Spdef Null Mice Lack Conjunctival Goblet Cells and Provide a Model of Dry Eye" @default.
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