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• W2172434371 abstract "Meibomian gland dysfunction is the most frequent cause of evaporative dry eye, yet its underlying pathophysiology is unknown. To gain insight into this pathophysiology, we characterized the time-dependent tear film and ocular surface changes occurring in X-linked anhidrotic-hypohidrotic ectodermal dysplasia mice (Tabby), which lack the meibomian gland. These mice sequentially developed corneal epithelial defects, central corneal stromal edema, neovascularization, and pannus 8 to 16 weeks after birth. Aqueous tear secretion was normal, whereas tear break-up time and ex vivo tear evaporation times were all shortened. Corneal epithelial microvilli were less numerous, conjunctival goblet cell density was unaffected, and MUC5AC and MUC5B gene expression was increased. Markers of squamous metaplasia (cytokeratin 10 and small proline-rich protein 1B) were noticed in the corneal epithelium of Tabby mice as early as the fourth week. Taken together, the Tabby mouse is a relevant meibomian gland dysfunction-related dry eye model that may lead to a better understanding of how meibomian glands are related to ocular surface health. This model may also help with discovering novel drug options for treating evaporative dry eye. Meibomian gland dysfunction is the most frequent cause of evaporative dry eye, yet its underlying pathophysiology is unknown. To gain insight into this pathophysiology, we characterized the time-dependent tear film and ocular surface changes occurring in X-linked anhidrotic-hypohidrotic ectodermal dysplasia mice (Tabby), which lack the meibomian gland. These mice sequentially developed corneal epithelial defects, central corneal stromal edema, neovascularization, and pannus 8 to 16 weeks after birth. Aqueous tear secretion was normal, whereas tear break-up time and ex vivo tear evaporation times were all shortened. Corneal epithelial microvilli were less numerous, conjunctival goblet cell density was unaffected, and MUC5AC and MUC5B gene expression was increased. Markers of squamous metaplasia (cytokeratin 10 and small proline-rich protein 1B) were noticed in the corneal epithelium of Tabby mice as early as the fourth week. Taken together, the Tabby mouse is a relevant meibomian gland dysfunction-related dry eye model that may lead to a better understanding of how meibomian glands are related to ocular surface health. This model may also help with discovering novel drug options for treating evaporative dry eye. Meibomian glands (MGs) are modified large sebaceous glands embedded in the tarsal plate of the eyelids.1Asbell P.A. Stapleton F.J. Wickstrom K. Akpek E.K. Aragona P. Dana R. Lemp M.A. Nichols K.K. The international workshop on meibomian gland dysfunction: report of the clinical trials subcommittee.Invest Ophthalmol Vis Sci. 2011; 52: 2065-2085Crossref PubMed Scopus (50) Google Scholar Their secretions, that is, meibum, consist of a complex mixture of various polar and nonpolar lipids2Butovich I.A. Uchiyama E. McCulley J.P. Lipids of human meibum: mass-spectrometric analysis and structural elucidation.J Lipid Res. 2007; 48: 2220-2235Crossref PubMed Scopus (126) Google Scholar and >90 different proteins.3Tsai P.S. Evans J.E. Green K.M. Sullivan R.M. Schaumberg D.A. Richards S.M. Dana M.R. Sullivan D.A. Proteomic analysis of human meibomian gland secretions.Br J Ophthalmol. 2006; 90: 372-377Crossref PubMed Scopus (112) Google Scholar The meibum lipids spread onto the tear film and smooth the corneal surface, which stabilizes and reduces tear film evaporation. Moreover, tear lipids also form a barrier to protect the eye from microbial infections.4Holly F.J. Lemp M.A. Tear physiology and dry eyes.Surv Ophthalmol. 1977; 22: 69-87Abstract Full Text PDF PubMed Scopus (464) Google Scholar MG dysfunction (MGD) is a chronic and diffuse abnormality of the MGs, commonly characterized by terminal duct obstruction and/or possible qualitative/quantitative changes in the glandular secretion.5Nelson J.D. Shimazaki J. Benitez-del-Castillo J.M. Craig J.P. McCulley J.P. Den S. Foulks G.N. The international workshop on meibomian gland dysfunction: report of the definition and classification subcommittee.Invest Ophthalmol Vis Sci. 2011; 52: 1930-1937Crossref PubMed Scopus (556) Google Scholar MGD may cause tear film instability and increase tear evaporation, resulting in symptoms such as eye irritation and clinically apparent inflammation.5Nelson J.D. Shimazaki J. Benitez-del-Castillo J.M. Craig J.P. McCulley J.P. Den S. Foulks G.N. The international workshop on meibomian gland dysfunction: report of the definition and classification subcommittee.Invest Ophthalmol Vis Sci. 2011; 52: 1930-1937Crossref PubMed Scopus (556) Google Scholar It is now well accepted that MGD is the most common cause of evaporative dry eye.6Nichols K.K. Foulks G.N. Bron A.J. Glasgow B.J. Dogru M. Tsubota K. Lemp M.A. Sullivan D.A. The international workshop on meibomian gland dysfunction: executive summary.Invest Ophthalmol Vis Sci. 2011; 52: 1922-1929Crossref PubMed Scopus (627) Google Scholar, 7Bron A.J. Tiffany J.M. Gouveia S.M. Yokoi N. Voon L.W. Functional aspects of the tear film lipid layer.Exp Eye Res. 2004; 78: 347-360Crossref PubMed Scopus (582) Google Scholar, 8Bron A.J. Tiffany J.M. The contribution of meibomian disease to dry eye.Ocul Surf. 2004; 2: 149-165Abstract Full Text PDF PubMed Scopus (269) Google Scholar, 9Foulks G.N. Bron A.J. Meibomian gland dysfunction: a clinical scheme for description, diagnosis, classification, and grading.Ocul Surf. 2003; 1: 107-126Abstract Full Text PDF PubMed Scopus (365) Google Scholar Hence, more attention is being paid to the effect of MGD on changes in ocular surface integrity. Many human generated and naturally occurring MGD animal models exist such as those generated by cauterization of the MG orifices,10Gilbard J.P. Rossi S.R. Heyda K.G. Tear film and ocular surface changes after closure of the meibomian gland orifices in the rabbit.Ophthalmology. 1989; 96: 1180-1186Abstract Full Text PDF PubMed Scopus (137) Google Scholar topical application of epinephrine,11Jester J.V. Nicolaides N. Kiss-Palvolgyi I. Smith R.E. Meibomian gland dysfunction. II. The role of keratinization in a rabbit model of MGD.Invest Ophthalmol Vis Sci. 1989; 30: 936-945PubMed Google Scholar, 12Jester J.V. Rife L. Nii D. Luttrull J.K. Wilson L. Smith R.E. In vivo biomicroscopy and photography of meibomian glands in a rabbit model of meibomian gland dysfunction.Invest Ophthalmol Vis Sci. 1982; 22: 660-667PubMed Google Scholar, 13Lambert R. Smith R.E. Hyperkeratinization in a rabbit model of meibomian gland dysfunction.Am J Ophthalmol. 1988; 105: 703-705Abstract Full Text PDF PubMed Scopus (9) Google Scholar systemic administration of isotretinoin,14Lambert R.W. Smith R.E. Pathogenesis of blepharoconjunctivitis complicating 13-cis-retinoic acid (isotretinoin) therapy in a laboratory model.Invest Ophthalmol Vis Sci. 1988; 29: 1559-1564PubMed Google Scholar and polychlorinated biphenyl poisoning.15Ohnishi Y. Kohno T. Polychlorinated biphenyls poisoning in monkey eye.Invest Ophthalmol Vis Sci. 1979; 18: 981-984PubMed Google Scholar The genetic mutant mice that showed abnormal MGs include ACAT1 gene,16Yagyu H. Kitamine T. Osuga J. Tozawa R. Chen Z. Kaji Y. Oka T. Perrey S. Tamura Y. Ohashi K. Okazaki H. Yahagi N. Shionoiri F. Iizuka Y. Harada K. Shimano H. Yamashita H. Gotoda T. Yamada N. Ishibashi S. Absence of ACAT-1 attenuates atherosclerosis but causes dry eye and cutaneous xanthomatosis in mice with congenital hyperlipidemia.J Biol Chem. 2000; 275: 21324-21330Crossref PubMed Scopus (158) Google Scholar tumor necrosis factor receptor-associated factor 6 deficiency,17Naito A. Yoshida H. Nishioka E. Satoh M. Azuma S. Yamamoto T. Nishikawa S. Inoue J. TRAF6-deficient mice display hypohidrotic ectodermal dysplasia.Proc Natl Acad Sci U S A. 2002; 99: 8766-8771Crossref PubMed Scopus (149) Google Scholar CCAAT/enhancer binding protein α and β,18House J.S. Zhu S. Ranjan R. Linder K. Smart R.C. C/EBPalpha and C/EBPbeta are required for Sebocyte differentiation and stratified squamous differentiation in adult mouse skin.PLoS One. 2010; 5: e9837Crossref PubMed Scopus (34) Google Scholar ectodysplasin-A and its receptor,19Cui C.Y. Smith J.A. Schlessinger D. Chan C.C. X-linked anhidrotic ectodermal dysplasia disruption yields a mouse model for ocular surface disease and resultant blindness.Am J Pathol. 2005; 167: 89-95Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar bone morphogenetic protein signaling,20Huang J. Dattilo L.K. Rajagopal R. Liu Y. Kaartinen V. Mishina Y. Deng C.X. Umans L. Zwijsen A. Roberts A.B. Beebe D.C. FGF-regulated BMP signaling is required for eyelid closure and to specify conjunctival epithelial cell fate.Development. 2009; 136: 1741-1750Crossref PubMed Scopus (72) Google Scholar, 21Plikus M. Wang W.P. Liu J. Wang X. Jiang T.X. Chuong C.M. Morpho-regulation of ectodermal organs: integument pathology and phenotypic variations in K14-Noggin engineered mice through modulation of bone morphogenic protein pathway.Am J Pathol. 2004; 164: 1099-1114Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar homeodomain transcription factor BARX2,22Tsau C. Ito M. Gromova A. Hoffman M.P. Meech R. Makarenkova H.P. Barx2 and Fgf10 regulate ocular glands branching morphogenesis by controlling extracellular matrix remodeling.Development. 2011; 138: 3307-3317Crossref PubMed Scopus (57) Google Scholar kruppel-like factor 5,23Kenchegowda D. Swamynathan S. Gupta D. Wan H. Whitsett J. Swamynathan S.K. Conditional disruption of mouse Klf5 results in defective eyelids with malformed meibomian glands, abnormal cornea and loss of conjunctival goblet cells.Dev Biol. 2011; 356: 5-18Crossref PubMed Scopus (48) Google Scholar and fatty acid transport protein 4 mutation.24Lin M.H. Hsu F.F. Miner J.H. Requirement of fatty acid transport protein 4 for development, maturation, and function of sebaceous glands in a mouse model of ichthyosis prematurity syndrome.J Biol Chem. 2013; 288: 3964-3976Crossref PubMed Scopus (27) Google Scholar The transgene models include mice overexpressing apolipoprotein C1 gene25Jong M.C. Gijbels M.J. Dahlmans V.E. Gorp P.J. Koopman S.J. Ponec M. Hofker M.H. Havekes L.M. Hyperlipidemia and cutaneous abnormalities in transgenic mice overexpressing human apolipoprotein C1.J Clin Invest. 1998; 101: 145-152Crossref PubMed Scopus (131) Google Scholar and with deletion of stearoyl-CoA desaturase 1 gene.26Miyazaki M. Man W.C. Ntambi J.M. Targeted disruption of stearoyl-CoA desaturase1 gene in mice causes atrophy of sebaceous and meibomian glands and depletion of wax esters in the eyelid.J Nutr. 2001; 131: 2260-2268Crossref PubMed Scopus (224) Google Scholar, 27Zheng Y. Eilertsen K.J. Ge L. Zhang L. Sundberg J.P. Prouty S.M. Stenn K.S. Parimoo S. Scd1 is expressed in sebaceous glands and is disrupted in the asebia mouse.Nat Genet. 1999; 23: 268-270Crossref PubMed Scopus (196) Google Scholar Both types of models show abnormal development, atrophy, or dystrophy of MGs. Most of these animal model studies focused on pathology or pathophysiology of the MG per se; however, the contribution of MG atrophy or dystrophy to the development of ocular surface abnormalities in these mice remains largely unknown. The X-linked anhidrotic-hypohidrotic ectodermal dysplasia mouse (Tabby) is a naturally developed ectodysplasin A (EDA) mutant strain.28Srivastava A.K. Pispa J. Hartung A.J. Du Y. Ezer S. Jenks T. Shimada T. Pekkanen M. Mikkola M.L. Ko M.S. Thesleff I. Kere J. Schlessinger D. The Tabby phenotype is caused by mutation in a mouse homologue of the EDA gene that reveals novel mouse and human exons and encodes a protein (ectodysplasin-A) with collagenous domains.Proc Natl Acad Sci U S A. 1997; 94: 13069-13074Crossref PubMed Scopus (257) Google Scholar Besides having abnormalities in skin appendages and teeth development, this strain also lacks MGs.29Gruneberg H. The glandular aspects of the tabby syndrome in the mouse.J Embryol Exp Morphol. 1971; 25: 1-19PubMed Google Scholar This strain represents an extreme MGD condition in which no secretions spread onto the ocular surface. Therefore, through evaluating the ocular surface pathophysiologic changes in this model, it is possible to obtain additional insight into the contribution made by MG secretory activity to ocular surface health. The changes in ocular surface phenotype of Tabby mice include corneal epithelial defects, keratitis, corneal ulceration, neovascularization, keratinization, blepharitis, and conjunctivitis.19Cui C.Y. Smith J.A. Schlessinger D. Chan C.C. X-linked anhidrotic ectodermal dysplasia disruption yields a mouse model for ocular surface disease and resultant blindness.Am J Pathol. 2005; 167: 89-95Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar However, most observations focused on the late stage from 12 to 36 weeks, when corneal pathology became prominent. There are no reports at any time during development about any tear film changes and their association with ocular surface pathology. We describe here the ocular surface changes occurring between 4 and 16 weeks after birth in Tabby mice. The results indicate that at the early interval from 6 to 8 weeks changes are already occurring similar to those seen in evaporative dry eye disease. The appearance of changes at this early age suggests that they may also be a valuable model to gain insight into the contribution of MGD to the pathophysiologic changes underlying evaporative dry eye disease. EDA mutant Tabby mice (Eda<Ta-6J>/Y) and wild-type C57BL/6J mice, originally purchased from The Jackson Laboratory (Bar Harbor, ME), were used in this study. The mice were kept in standard pathogen-free environment at 25°C ± 1°C, relative humidity 60% ± 10%, and alternating 12 hours light-dark cycles (8:00 AM to 8:00 PM). All procedures were performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.30Association for Research in Vision and Ophthalmology: Statement for the Use of Animals in Ophthalmic and Visual Research. Copyright, 2015, Association for Research in Vision and Ophthalmology, Rockville, MD. Available at http://www.arvo.org/about_arvo/policies/statement_for_the_use_of_animals_in_ophthalmic_and_visual_researchGoogle Scholar This study received the approval of the Animal Ethical Committee of Xiamen University. In total, 30 male Tabby mice and 30 male wild-type mice were used in this study. Among them, six Tabby mice and six wild-type mice were used for ocular surface observation and assessment. Others were sacrificed at ages of 4, 5, 6, 7, 8, 10, 12, and 16 weeks, with three animals at each time point. The eyelid, eyeball, and lacrimal gland tissues from both sides were harvested for histology, immunostaining, or RNA extraction. Aqueous tear volume was measured with phenol red thread (ZONE-QUICK; Yokota, Tokyo, Japan) tear secretion test at 3:00 PM in the standard environment with modification.31Lin Z. Liu X. Zhou T. Wang Y. Bai L. He H. Liu Z. A mouse dry eye model induced by topical administration of benzalkonium chloride.Mol Vis. 2011; 17: 257-264PubMed Google Scholar In brief, the lower eyelid was pulled down slightly, and 10 mm of the thread was placed on the palpebral conjunctiva one-third of the distance away from the lateral canthus of the lower eyelid for 15 seconds. The values indicated by the red color position of the aqueous front on the thread were recorded in millimeters. Anterior and lateral views of the mice eyeballs were observed under a slit-lamp microscope (Kanghua Science & Technology Co., Ltd., Chongqing, China). After that, 1 μL of 0.1% liquid sodium fluorescein (Jingmingxin Co., Ltd., Tianjin, China) was dropped into the conjunctival sac. After three compulsory blinks, tear break-up time (TBUT) was recorded in seconds as previously reported.31Lin Z. Liu X. Zhou T. Wang Y. Bai L. He H. Liu Z. A mouse dry eye model induced by topical administration of benzalkonium chloride.Mol Vis. 2011; 17: 257-264PubMed Google Scholar Ninety seconds later, corneal epithelial fluorescein staining was recorded under the slit-lamp microscope with a cobalt blue filter. For grading of the fluorescein staining, the cornea was divided into four quadrants, staining was scored separately, and the scores of four quadrants were summed and analyzed.31Lin Z. Liu X. Zhou T. Wang Y. Bai L. He H. Liu Z. A mouse dry eye model induced by topical administration of benzalkonium chloride.Mol Vis. 2011; 17: 257-264PubMed Google Scholar One microliter of phosphate buffer [0.1 mol/L phosphate-buffered saline (PBS), pH7.4] was dripped onto the ocular surface of 8-week-old mice. After 1 minute, 0.5 μL of diluted tears mix was collected with a micropipette and dripped onto a glass slide. The same PBS volume was used as a control. The tear smear was then observed under a microscope, and the time required for crystal formation was recorded as ex vivo tear evaporation time. The images of tear smears were taken before and after crystallization. This assay was conducted at 22.4°C and relative humidity of 48.6% in a stable environment. Scanning electron microscopy was performed to observe the surface ultra-microstructure of the corneal epithelium. In brief, diced central cornea blocks of 1 mm2 were carefully dissected, then immediately fixed by 2.5% glutaraldehyde for 2 hours. After three washes with PBS for 10 minutes each, tissues were further fixed in osmic acid for another 2 hours. After three additional washes with PBS for 10 minutes each, tissue was dehydrated with ethanol and acetone for 2 hours, and then tertiary butanol for 30 minutes at room temperature. Afterward, they were soaked in tertiary butanol at 4°C for 16 hours, followed by drying them in a vacuum evaporator. Finally, the surface of the samples was sprayed with a metallic film and observed under a JSM6390 scanning electron microscope (JEOL, Tokyo, Japan). Periodic acid-Schiff staining was performed to evaluate the distribution of goblet cells in conjunctival tissue. Each paraffin section was deparaffinized twice in xylene for 15 minutes and rehydrated four times in ethanol for 5 minutes. After rinsing with deionized water for 10 minutes, sections were stained in periodic acid alcohol for 10 minutes. After a 10-minute rinse with deionized water, sections were stained in Schiff reagent (Leica Biosystems, Nussloch, Baden, Germany) for 10 minutes. After three rinses first with sulfurous acid for 2 minutes and then tap water for 15 minutes, each section was dehydrated four times in ethanol for 2 minutes and twice in xylene for 2 minutes. Finally, sections were mounted with the use of mounting medium. Oil Red O staining was conducted to evaluate the lipid secretion of the MG. Briefly, frozen eyelid sections were immediately fixed in 4% paraformaldehyde for 5 minutes, washed in PBS for 5 minutes, and stained for 7 minutes in filtered Oil Red O solution that was freshly prepared by mixing the stock solution (0.8% Oil Red O in 98% isopropyl alcohol) and distilled water at a ratio of 1.5 to 1, followed by washing in PBS for 5 minutes. Sections were then counterstained with hematoxylin and mounted in 90% glycerol. Real-time PCR was performed to detect gene expression in corneal epithelium and conjunctival tissues. Total RNA was extracted from mouse corneal epithelia and conjunctival tissues with the use of TRIzol (Invitrogen, Carlsbad, CA) and was reverse-transcribed to cDNA with the use of the ExScript RT Reagent kit (Takara, Dalian, China). The primers used to amplify specific gene products are listed in Table 1. Real-time PCR was performed with a StepOne Real-Time PCR detection system (Applied Biosystems, Darmstadt, Germany) with the use of a SYBR Premix Ex Taq Kit (Takara) according to the manufacturer's instructions. The amplification program included an initial denaturation step at 95°C for 10 minutes, followed by denaturation at 95°C for 10 seconds and annealing and extension at 60°C for 30 seconds for 40 cycles. The results of relative quantitative real-time PCR were analyzed by the comparative threshold cycle method and normalized to β-actin expression as an internal control.Table 1Mouse Primer Sequences Used for Quantitative Real-Time PCRGeneSense primerAntisense primerSPRR1B5′-GCGACCACACTACCTGTCCT-3′5′-CTGGCAAGGCTGTTTCACTT-3′K105′-TCGAGGACCTTAAGGGGCAG-3′5′-GTCAGCTCATCCAGTACCCTG-3′K125′-TGACCCTGACTAGAGCCGAC-3′5′-ACATTGACCTCACCTGGACC-3′MUC5AC5′-ACACATGTTCTGGAGGGCA-3′5′-ACACTTTCGCAGCTCAACCA-3′MUC5B5′-GGAATGGGGGCTGTATTGCT-3′5′-CAGGGCTTGTTGGTGCATTC-3′MUC15′-CCCAGGACACCTACCATCCT-3′5′-ACTGCCATTACCTGCCGAAA-3′MUC45′-GTCCACTTCTTCCCCATCTCG-3′5′-CCATTGTGACAGTAGCCCTCA-3′MUC135′-TTTGGCTACAGCGGGATGAA-3′5′-TCTTTGACCTCGCAGAGACG-3′MUC155′-CCTGGGTGCTTCACTGCTTA-3′5′-TGGTGCATTGTCTAATCGCAG-3′ACTB5′-GCTATTTGGCGCTGGACTT-3′5′-GCGGCTCGTAGCTCTTCTC-3′ Open table in a new tab Immunofluorescence staining on K12, K10, small proline-rich protein 1B (SPRR1B), and mucin (MUC) 5AC was performed to evaluate the phenotypic change of corneal and conjunctival epithelia in Tabby mice. Cryostat sections (4 μm) of mice eyeballs and eyelid tissues of different ages from 4 to 8 weeks were fixed in acetone for 10 minutes at −20°C. Sections were rehydrated in PBS and then incubated in 0.2% Triton X-100 for 10 minutes. After rinsing sections three times with PBS for 5 minutes each and preincubating with 2% bovine serum albumin to block nonspecific staining for 60 minutes, sections were incubated with anti-K12 (sc-17101; Santa Cruz Biotechnology, Inc., Dallas, TX), anti-K10 (ab24638; Abcam, Cambridge, MA), anti-SPRR1B (LS-C146187; Lifespan Bioscience, Seattle, WA), and anti-MUC5AC (sc-16902; Santa Cruz Biotechnology, Inc.) antibodies (all dilution 1:50) at 4°C overnight. After three washes with PBS for 5 minutes, they were then incubated with Alexa Fluor 594-conjugated IgG (dilution 1:300; A11058) or Alexa Fluor 488-conjugated IgG (dilution 1:300; A11055, A21206; Life Technologies, Carlsbad, CA) for 60 minutes. After three additional PBS washes for 5 minutes, they were counterstained with DAPI, mounted, and photographed with the use of a confocal laser scanning microscope (Fluoview 1000; Olympus, Tokyo, Japan). Statistical analysis was performed with SPSS 16.0.0 (SPSS Inc., Chicago, IL). Summary data are reported as means ± SD. Independent Sample t-test and one-way analysis of variance were applied to evaluate significance between groups. P < 0.05 was considered statistically significant. Wild-type corneas remained transparent with a normal ocular surface throughout the observation period from 4 to 16 weeks. However, scabrous corneal surface reflection was seen in 6-week-old Tabby mice. Their central or pericentral corneal stromal regions became mildly edematous at 8 weeks of age. Neovascularization of the edematous regions became evident at 12 weeks of age, followed by pannus formation in the central cornea at 16 weeks. In a lateral eyeball view, congestion and ingrowth of the limbal blood vessels was seen at 10 weeks that were mainly distributed in the corneal edematous area at 12 weeks and accompanied by pannus tissue formation in 16-week-old mice (Figure 1). Fluorescein staining of the Tabby mice cornea was scarce at 4 and 6 weeks; however, it progressively increased from weeks 8 to 12. By approximately 16 weeks, a staining mass could be easily found under the slit-lamp microscope (Figure 2A). However, the fluorescein staining was restricted at most to just one quadrant in the wild-type mice from 4 to 16 weeks (data not shown). The fluorescein score was dramatically different between the Tabby mice and their age-matched wild-type counterparts at all of the time points from 8 to 16 weeks (Figure 2B). Phenol red thread tear secretion test results in Tabby mice whose ages ranged from 4 to 16 weeks indicate that aqueous tear secretion remained relatively stable with only a transient increase at 10 weeks (Figure 3A). Lacrimal gland hematoxylin and eosin staining of 8-week-old mice showed no obvious structural difference between wild-type mice (Figure 3C) and Tabby mice (Figure 3D). These negative effects indicate that the declines in ocular surface integrity were not attributable to disruption of either lacrimal gland secretory activity or structure. Tear film stability was invariant in wild-type mice throughout the first 16 weeks of their life. From 4 to 16 weeks, TBUT in Tabby mice gradually decreased, and it was significantly shorter than that in age-matched wild-type mice at 4, 8, 10, 12, and 16 weeks (Figure 3B). Eyelid blinking was complete in 8-week-old Tabby mice, and the blinking frequency was 2.1 blinks per minute in average, whereas it was 0.2 blink per minute in age-matched wild-type mice, as recorded by digital camera. Ex vivo tear evaporation assay in 8-week-old mice showed different crystal patterns between age-matched wild-type mice and Tabby mice (Figure 4A), suggesting a tear component difference between the two groups. Tear smear ex vivo evaporation time was significantly shorter in Tabby mice than that in wild-type mice (Figure 4B). These results further supported the notion that tear evaporation is increased in Tabby mice. Together with TBUT, fluorescein staining, and aqueous tear secretion, the results obtained with the Tabby mice are in accord with Dry Eye WorkShop criteria established for identifying evaporative dry eye in humans.32The definition and classification of dry eye disease: report of the Definition and Classification Subcommittee of the International Dry Eye WorkShop (2007).Ocul Surf. 2007; 5: 75-92Abstract Full Text PDF PubMed Google Scholar Oil Red O and hematoxylin double staining showed acini gland formation in the tarsal plate of the eyelid and lipid secretion inside the acini in 4- and 8-week-old wild-type mice. In contrast, neither acinar development nor lipid formation was found in the MG loci in 4- and 8-week-old Tabby mice (Figure 5A), indicating absence of the MG in Tabby mice.19Cui C.Y. Smith J.A. Schlessinger D. Chan C.C. X-linked anhidrotic ectodermal dysplasia disruption yields a mouse model for ocular surface disease and resultant blindness.Am J Pathol. 2005; 167: 89-95Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar Corneal tissue hematoxylin and eosin staining showed smooth epithelial surface was maintained in wild-type mice of both 4 and 16 weeks of age. Tabby mice also showed smooth corneal epithelial surface at 4 weeks; however; it became irregular from 6 to 10 weeks. At 12 weeks, corneal epithelial thickness increased along with prominent cell infiltration into the anterior stroma of the central cornea in Tabby mice. At 16 weeks, the pannus tissue loci in Tabby mice showed a dramatically thickened stroma with cell infiltration and neovascularization and significant stratification and keratinization of the corneal epithelium (Figure 5B). Scanning electron microscopy of the corneal tissue identified numerous microvilli on the apical surface of the superficial epithelial cells in the wild-type mice at 4 and 8 weeks of age. In contrast, in Tabby mice at 4 weeks the microvilli were scarce and shortened. This trend became more pronounced at 8 weeks because the superficial epithelial cells became more jagged (Figure 6). Goblet cell density declines in aqueous tear-deficient dry eye.33Nelson J.D. Wright J.C. Conjunctival goblet cell densities in ocular surface disease.Arch Ophthalmol. 1984; 102: 1049-1051Crossref PubMed Scopus (268) Google Scholar, 34Zhang X. De Paiva C.S. Su Z. Volpe E.A. Li D.Q. Pflugfelder S.C. Topical interferon-gamma neutralization prevents conjunctival goblet cell loss in experimental murine dry eye.Exp Eye Res. 2014; 118: 117-124Crossref PubMed Scopus (61) Google Scholar However, it is unknown whether such an effect also occurs in MGD. Periodic acid-Schiff staining in the Tabby and wild-type mice revealed that goblet cells were mainly distributed in the fornix conjunctiva. No obvious difference was found in goblet cell density between 4- and 8-week-old mice in the two groups (Figure 7A). MUC5AC staining of the conjunctival tissues showed mild up-regulation in 8-week-old Tabby mice (Figure 7B). Real-time PCR of MUC5AC (Figure 7C) and MUC5B (Figure 7D) gene expression revealed that they were both significantly up-regulated in these Tabby mice, indicating that their goblet cell density did not decrease. Instead, goblet cell mucin secretion even underwent up-regulation. Squamous metaplasia is the hallmark of dry eye whereby ocular surface epithelium undergoes keratinization.35Tseng S.C. Staging of conjunctival squamous metaplasia by impression cytology.Ophthalmology. 1985; 92: 728-733Abstract Full Text PDF PubMed Scopus (446) Google Scholar This process is accompanied by loss of cornea-specific cytokeratin K3 and K12 expression and emergence of epidermis-specific cytokeratin K1 and K10.36Tseng S.C. Hatchell D. Tierney N. Huang A.J. Sun T.T. Expression of specific keratin markers by rabbit corneal, conjunctival, and esophageal epithelia during vitamin A deficiency.J Cell Biol. 1984; 99: 2279-2286Crossref PubMed Scopus (93) Google Scholar, 37Nakamura T. Nishida K. Dota A. Matsuki M. Yamanishi K. Kinoshita S. Elevated expression of transglutaminase 1 and keratinization-related proteins in conjunctiva in severe ocular surface disease.Invest Ophthalmol Vis Sci. 2001; 42: 549-556PubMed Google Scholar SPRR1B is a validated squamous metaplasia biomarker in dry eye disease.38Li S. Nikulina K. DeVoss J. Wu A.J. Strauss E.C. Anderson M.S. McNamara N.A. Small proline-rich protein 1B (SPRR1B) is a biomarker for squamous metaplasia in dry eye disease.Invest Ophthalmol Vis Sci. 2008; 49: 34-41Crossref PubMed Scopus (60) Google Scholar To thoroughly assess the de" @default.
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• W2172434371 title "Meibomian Gland Absence Related Dry Eye in Ectodysplasin A Mutant Mice" @default.
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