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Corneal Sensory Experience via Transient Receptor Potential Vanilloid 1 Accelerates the Maturation of Neonatal Tearing

2019; Elsevier BV; Volume: 189; Issue: 9 Linguagem: Inglês

10.1016/j.ajpath.2019.05.015

1525-2191

Kai Jin, Toshihiro Imada, Shigeru Nakamura, Yusuke Izuta, Erina Oonishi, Michiko Shibuya, Hisayo Sakaguchi, Hirotaka Tanabe, Masataka Ito, Kimiaki Katanosaka, Kazuo Tsubota,

Infant Health and Development

Tearing maturates rapidly after birth, and external environmental challenges play a key role in promoting lacrimal functional maturation. However, little is known about the facilitative factors underlying this developmental process or the potential of application of these factors to treat hypofunction of the lacrimal gland. In this study, eye opening and the subsequent ocular surface sensory experience, which is thought to be involved in postnatal maturation of lacrimal function, were investigated. Our results demonstrated that eye opening after birth is essential for the maturation of neonatal tearing. The maturation process of lacrimal function is dependent on the ocular surface sensory experience via transient receptor potential cation channel subfamily member 1 after birth. This study provides, for the first time, important evidence of the sensory experience of the ocular surface in relation to the maturation of functional tear secretion during the postnatal period. Tearing maturates rapidly after birth, and external environmental challenges play a key role in promoting lacrimal functional maturation. However, little is known about the facilitative factors underlying this developmental process or the potential of application of these factors to treat hypofunction of the lacrimal gland. In this study, eye opening and the subsequent ocular surface sensory experience, which is thought to be involved in postnatal maturation of lacrimal function, were investigated. Our results demonstrated that eye opening after birth is essential for the maturation of neonatal tearing. The maturation process of lacrimal function is dependent on the ocular surface sensory experience via transient receptor potential cation channel subfamily member 1 after birth. This study provides, for the first time, important evidence of the sensory experience of the ocular surface in relation to the maturation of functional tear secretion during the postnatal period. When an infant is born, the first challenges that must be faced and completed compose a critical period of physiological adaptations. During this time, the gradual and progressive maturation of effective ventilation, closed circulation, energy absorption, and the nervous system allows the newborn to deal with the sudden drastic changes in the environment and to move from reliance on the maternal resource.1Fitzgerald M. The development of nociceptive circuits.Nat Rev Neurosci. 2005; 6: 507-520Crossref PubMed Scopus (573) Google Scholar, 2Morton S.U. Brodsky D. Fetal physiology and the transition to extrauterine life.Clin Perinatol. 2016; 43: 395-407Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 3Xin M. Olson E.N. Bassel-Duby R. 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Neural circuits underlying crying and cry responding in mammals.Behav Brain Res. 2007; 182: 155-165Crossref PubMed Scopus (142) Google Scholar the physiological aspect of tearing mainly refers to the process where the fluid mixture produced by the lacrimal gland (LG) is secreted and expelled onto the ocular surface to lubricate, nourish, and protect the ocular surface, as a result of the multifunctional characteristics of the tear film, as well as to maintain a uniform and regular refractive surface for optimal optical quality.5Sullivan D.A. Stern M.E. Tsubota K. Dartt D.A. Sullivan R.M. Bromberg B.B. Lacrimal Gland, Tear Film, and Dry Eye Syndromes 3: Basic Science and Clinical Relevance. Springer US, New York, NY2002Crossref Google Scholar, 6Montes-Mico R. Cervino A. Ferrer-Blasco T. Garcia-Lazaro S. Madrid-Costa D. The tear film and the optical quality of the eye.Ocul Surf. 2010; 8: 185-192Crossref PubMed Scopus (75) Google Scholar, 7Stern M.E. Gao J. Siemasko K.F. Beuerman R.W. Pflugfelder S.C. The role of the lacrimal functional unit in the pathophysiology of dry eye.Exp Eye Res. 2004; 78: 409-416Crossref PubMed Scopus (380) Google Scholar Normal infants cry without tearing during the first 2 weeks after birth, and the tear secretion of newborns is lower than that of infants or adults, a phenomenon that can be traced back to Aristotle (Historia Naturalis).8Botelho S.Y. Tears and the lacrimal gland.Sci Am. 1964; 211: 78-86Crossref PubMed Scopus (69) Google Scholar, 9Sjogren H. The lacrimal secretion in newborn premature and fully developed children.Acta Ophthalmol. 1955; 33: 557-560Crossref PubMed Scopus (26) Google Scholar The current understanding of neonatal tearing during the postnatal period based on observational and clinical evaluation approaches showed that the basal tear volume of newborns without any stimulation is limited but increases rapidly in early infancy.10Esmaeelpour M. Watts P.O. Boulton M.E. Cai J. Murphy P.J. Tear film volume and protein analysis in full-term newborn infants.Cornea. 2011; 30: 400-404Crossref PubMed Scopus (13) Google Scholar Preterm infants have reduced reflex and basal tear secretion.11Isenberg S.J. Apt L. McCarty J. Cooper L.L. Lim L. Del Signore M. Development of tearing in preterm and term neonates.Arch Ophthalmol. 1998; 116: 773-776Crossref PubMed Scopus (43) Google Scholar Accumulating evidence indicates that the vital organs are still immature when infants are born. However, the mechanisms that underlie the maturation of LG and neonatal tearing, in response to environmental changes during a brief period after birth, remain largely unknown. Sensory experience is information perceived from sensing the environment and changes surrounding an organism and is performed by sensory organs, including the eyes, ears, nose, tongue, and skin.12Dorrn A.L. Yuan K. Barker A.J. Schreiner C.E. Froemke R.C. Developmental sensory experience balances cortical excitation and inhibition.Nature. 2010; 465: 932-936Crossref PubMed Scopus (214) Google Scholar, 13Katz L.C. Shatz C.J. Synaptic activity and the construction of cortical circuits.Science. 1996; 274: 1133-1138Crossref PubMed Scopus (2368) Google Scholar This information is collected from sensory receptors that are located throughout the body, processed, and stored by networks of neurons.12Dorrn A.L. Yuan K. Barker A.J. Schreiner C.E. Froemke R.C. Developmental sensory experience balances cortical excitation and inhibition.Nature. 2010; 465: 932-936Crossref PubMed Scopus (214) Google Scholar, 13Katz L.C. Shatz C.J. Synaptic activity and the construction of cortical circuits.Science. 1996; 274: 1133-1138Crossref PubMed Scopus (2368) Google Scholar, 14Hensch T.K. Critical period plasticity in local cortical circuits.Nat Rev Neurosci. 2005; 6: 877-888Crossref PubMed Scopus (1556) Google Scholar The structure of the sensory circuits is formed by individual genetic conditions, as well as the presence and enhancement of neural activity induced by sensory experience, and helps to refine the functional output of the target organs.12Dorrn A.L. Yuan K. Barker A.J. Schreiner C.E. Froemke R.C. Developmental sensory experience balances cortical excitation and inhibition.Nature. 2010; 465: 932-936Crossref PubMed Scopus (214) Google Scholar, 13Katz L.C. Shatz C.J. Synaptic activity and the construction of cortical circuits.Science. 1996; 274: 1133-1138Crossref PubMed Scopus (2368) Google Scholar, 14Hensch T.K. Critical period plasticity in local cortical circuits.Nat Rev Neurosci. 2005; 6: 877-888Crossref PubMed Scopus (1556) Google Scholar The cornea is the most densely innervated tissue in the body.15Acosta M.C. Belmonte C. Gallar J. Sensory experiences in humans and single-unit activity in cats evoked by polymodal stimulation of the cornea.J Physiol. 2001; 534: 511-525Crossref PubMed Scopus (124) Google Scholar, 16Acosta M.C. Peral A. Luna C. Pintor J. Belmonte C. Gallar J. Tear secretion induced by selective stimulation of corneal and conjunctival sensory nerve fibers.Invest Ophthalmol Vis Sci. 2004; 45: 2333-2336Crossref PubMed Scopus (80) Google Scholar, 17Belmonte C. Nichols J.J. Cox S.M. Brock J.A. Begley C.G. Bereiter D.A. Dartt D.A. Galor A. Hamrah P. Ivanusic J.J. Jacobs D.S. McNamara N.A. Rosenblatt M.I. Stapleton F. Wolffsohn J.S. TFOS DEWS II pain and sensation report.Ocul Surf. 2017; 15: 404-437Crossref PubMed Scopus (320) Google Scholar Ocular surface sensory experiences are evoked by distinct functional types of corneal sensory neurons that are broadly classified as polymodal nociceptor neurons, cold thermoreceptor neurons, or selective mechanonociceptor neurons.15Acosta M.C. Belmonte C. Gallar J. Sensory experiences in humans and single-unit activity in cats evoked by polymodal stimulation of the cornea.J Physiol. 2001; 534: 511-525Crossref PubMed Scopus (124) Google Scholar, 16Acosta M.C. Peral A. Luna C. Pintor J. Belmonte C. Gallar J. Tear secretion induced by selective stimulation of corneal and conjunctival sensory nerve fibers.Invest Ophthalmol Vis Sci. 2004; 45: 2333-2336Crossref PubMed Scopus (80) Google Scholar, 17Belmonte C. Nichols J.J. Cox S.M. Brock J.A. Begley C.G. Bereiter D.A. Dartt D.A. Galor A. Hamrah P. Ivanusic J.J. Jacobs D.S. McNamara N.A. Rosenblatt M.I. Stapleton F. Wolffsohn J.S. TFOS DEWS II pain and sensation report.Ocul Surf. 2017; 15: 404-437Crossref PubMed Scopus (320) Google Scholar Cornea sensitivity is necessary to detect and timely respond to the environmental changes or pathologic stresses, interact with the central nerve system, protect against potential damage, and maintain the healthy integrity of the ocular surface. Herein, we investigated the effect of eye opening after birth on the maturation of neonatal tearing. We determined whether the ocular surface sensory experience after eye opening plays a significant role in the maturation of LGs by modulating the intensity of ocular surface sensory activity. C57BL/6J wild-type (WT), homozygous, TRPV1 knockout (KO) mice (stock number 3770; The Jackson Laboratory, Bar Harbor, ME)18Caterina M.J. Leffler A. Malmberg A.B. Martin W.J. Trafton J. Petersen-Zeitz K.R. Koltzenburg M. Basbaum A.I. Julius D. Impaired nociception and pain sensation in mice lacking the capsaicin receptor.Science. 2000; 288: 306-313Crossref PubMed Scopus (2898) Google Scholar and homozygous TRPM8 KO mice (stock number 8198; The Jackson Laboratory), aged from 4 to 28 days, were used in this study. All procedures were performed in accordance with the Association of Research and Vision in Ophthalmology statement for the Use of Animals in Ophthalmology and Vision Research and were approved by the Animal Experimentation Ethics Committee of Keio University School of Medicine (Tokyo, Japan; number 11008) and by the Animal Experiment Committee of Chubu University (Kasugai, Japan; number 2910071). All animals were housed under the general conditions [room temperature was 23°C ± 2°C, humidity was 60% ± 10%, there was an alternating 12-hour light-dark cycle (8 am to 8 pm), and water and food were available ad libitum]. Eyelid suture was performed binocularly in 10-day–old WT mice under inhalation anesthesia with isoflurane (Pfizer, Tokyo, Japan) by the same operator (K.J.). This procedure involved suturing the unopened eyelids with three interrupted 8-0 stitches (Vicryl; Ethicon Inc., Somerville, MA). The eyelids of the mice were kept closed by sutures until postnatal day (P) 21, after which the eyelid sutures were removed. Tear secretion was measured using a modified Schirmer test19Sakamoto R. Bennett E.S. Henry V.A. Paragina S. Narumi T. Izumi Y. Kamei Y. Nagatomi E. Miyanaga Y. Hamano H. Mitsunaga S. The phenol red thread tear test: a cross-cultural study.Invest Ophthalmol Vis Sci. 1993; 34: 3510-3514PubMed Google Scholar, 20Imada T. Nakamura S. Hisamura R. Izuta Y. Jin K. Ito M. Kitamura N. Tanaka K.F. Mimura M. Shibuya I. Tsubota K. Serotonin hormonally regulates lacrimal gland secretory function via the serotonin type 3a receptor.Sci Rep. 2017; 7: 6965Crossref PubMed Scopus (17) Google Scholar with a phenol red thread (Zone-Quick; Showa Yakuhin Kako, Tokyo, Japan). This was placed on the temporal side of the conjunctiva between the limbus and the outer canthus for 15 seconds. The length of the moistened area from the edge was measured to within 0.5 mm. To determine the change in tear secretion during the eyelid-suture period, tear secretion was measured just after removing the eyelid suture at P14 and P21 in a separate group. C57BL/6J WT mice at P12 and P21 and eyelid-sutured mice at P21 were euthanized with an overdose of pentobarbital sodium; then, the LGs were dissected. The LGs were fixed in 4% paraformaldehyde at 4°C overnight. After fixation, LGs were blocked with 1% bovine serum album in phosphate-buffered saline (PBS) containing 0.25% Triton X-100 for 1 hour at room temperature and then incubated overnight at 4°C with a rabbit polyclonal antibody against calponin, a specific myoepithelial cell marker21Satoh Y. Oomori Y. Ishikawa K. Ono K. Configuration of myoepithelial cells in various exocrine glands of guinea pigs.Anat Embryol. 1994; 189: 227-236Crossref PubMed Scopus (31) Google Scholar, 22Satoh Y. Sano K. Habara Y. Kanno T. Effects of carbachol and catecholamines on ultrastructure and intracellular calcium-ion dynamics of acinar and myoepithelial cells of lacrimal glands.Cell Tissue Res. 1997; 289: 473-485Crossref PubMed Scopus (38) Google Scholar (1:300 dilution; Abcam, Cambridge, MA). After being washed with PBS, LGs were incubated with Alexa Fluor 488–conjugated donkey–anti-rabbit secondary antibody (1:300 dilution; Molecular Probes, Eugene, OR) for 3 hours at room temperature (25°C ± 5°C) and washed with PBS. For visualization of the cell surface, LGs were stained with phalloidin-labeled wheat germ agglutinin (1:500 dilution; Vector Laboratories, Burlingame, CA), which binds to lectins and acts as a marker for the cell surface,23Borges L.F. Sidman R.L. Axonal transport of lectins in the peripheral nervous system.J Neurosci. 1982; 2: 647-653Crossref PubMed Google Scholar at room temperature for 30 minutes. Wheat germ agglutinin–stained LGs were washed three times with PBS for 3 minutes. The LG was observed with a two-photon microscope (FV1200MPE; Olympus, Tokyo, Japan) equipped with a water-immersion objective lens (XLPlaN25×1.05 WMP; Olympus). The excitation wavelength for wheat germ agglutinin and Alexa-488 was 830 nm, and the emission was simultaneously detected through a bandpass filter for wheat germ agglutinin (575 to 630 nm) and for Alexa-488 (510 to 550 nm). The fluorescence images at a depth of approximately 200 μm from the surface of the LG were reconstructed from images (200 μm thick) acquired at z-step sizes of 1 μm using Imaris 8.4.0 software (Bitplane AG, Zurich, Switzerland). For hematoxylin and eosin staining, mice of different postnatal ages (P12 and P21) and eyelid-sutured mice at P21 were euthanized with an overdose of pentobarbital sodium; then, their LGs were dissected. The whole LG was fixed in a 10% formalin solution and embedded in paraffin. Sections (5 μm thick) were obtained from the middle of the horizontal direction in the whole LG. The sections were stained with filtered hematoxylin and eosin. Images were captured using optical microscope BIOREVO BZ-9000 (Keyence, Osaka, Japan). For quantification of the sizes of the acinar cells, the sizes of 20 acinar cells were measured in three randomly selected areas (10,000 μm2) from each section using BZ-Analyzer 2.1 software (Keyence). For the determination of the acinar cell density, the area of the whole specimens was quantified using BZ-Analyzer software in Hybrid Cell Count mode (Keyence), and the occupied area ratio of the individual acinar cells/whole specimens was calculated in each section. For transmission electron microscopy, mice at P12 and P21 and eyelid-sutured mice at P21 were perfused with Karnovsky's fixative (2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 mol/L sodium cacodylate; pH 7.4) under anesthesia, and the excised LG was immersed in the fixative. Sections (1 μm thick) were stained with methylene blue, and ultrathin sections were made using a diamond knife. The ultrathin sections were collected on mesh grids, stained with uranyl acetate and lead citrate, and examined using an electron microscope (JEM-1400Plus; JEOL Ltd, Tokyo, Japan). Total RNA was isolated from the LG using an RNA extraction reagent (ISOGEN; Nippon Gene, Tokyo, Japan), according to the manufacturer's instructions. The RNA was used for reverse transcription; and then, cDNA synthesis was performed using the ReverTra Ace qPCR RT kit (TOYOBO, Osaka, Japan). SYBR Green–based quantitative real-time PCR was performed using the Step One Plus system (Applied Biosystems, Framingham, MA). Each PCR amplification was performed using a specific primer set. The primer sequences were as follows: GAPDH, 5′-ACCCAGAAGACTGTGGATGG-3′ (sense) and 5′-GGATGCAGGGATGATGTTCT-3′ (antisense); and lactoperoxidase (Lpo), 5′-AAGGCACACTGCAGTGATGAAA-3′ (sense) and 5′-TTGGCATGCTTGAGATAATCTGAC-3′ (antisense). Data were normalized to GAPDH. The tear component proteins extracted from a phenol red thread were diluted in PBS to 0.4 mg/mL protein concentrations. The same volume of 2× Laemmli sample buffer was added to the tear samples, as well as 5% β-mercaptoethanol. The tear samples were then boiled at 100°C and separated by PAGE. After electrophoresis, the gels were stained in 50% methanol/5% acetic acid/0.25% Coomassie brilliant blue R-250 and destained in 5% methanol/7% acetic acid. Band intensities were quantified using ImageJ software version 1.52a (NIH, Bethesda, MD; http://imagej.nih.gov/ij). After P14 (when eye opening occurs), mouse pups were treated with eye drops of capsaicin (1 μmol/L; Wako Pure Chemical, Osaka, Japan), menthol (50 μmol/L; Tokyo Chemical, Tokyo, Japan), or vehicle saline solution three times per day for 7 consecutive days. A 1 μL drop of capsaicin (1 μmol/L), menthol (50 μmol/L), or vehicle saline solution was softly applied to the center of the cornea using a micropipette. On P5 and P6, the mouse pups were given a single s.c. injection of capsaicin per day, at a dose of 50 mg/kg, dissolved in saline containing 10% Tween-80 and 10% ethanol. Vehicle-treated mouse pups received an injection of saline containing 10% Tween-80 and 10% ethanol at the same dose. The mouse pups injected with capsaicin or saline solution were then housed with their mothers and fed under normal conditions. A sustained decrease of corneal sensitivity in capsaicin-treated neonatal mice was confirmed, as reported in previous studies.24Aita M. Maeda T. Seo K. The effect of neonatal capsaicin treatment on the CGRP-immunoreaction in the trigeminal subnucleus caudalis of mice.Biomed Res. 2008; 29: 33-42Crossref PubMed Scopus (8) Google Scholar, 25Kagawa Y. Itoh S. Shinohara H. Investigation of capsaicin-induced superficial punctate keratopathy model due to reduced tear secretion in rats.Curr Eye Res. 2013; 38: 729-735Crossref PubMed Scopus (4) Google Scholar, 26Nakao A. Takahashi Y. Nagase M. Ikeda R. Kato F. Role of capsaicin-sensitive C-fiber afferents in neuropathic pain-induced synaptic potentiation in the nociceptive amygdala.Mol Pain. 2012; 8: 51Crossref PubMed Scopus (53) Google Scholar Capsaicin (1 μmol/L), menthol (50 μmol/L), or vehicle (saline) was pipetted onto the corneal surface of the mice at the volume of 1 μL. Immediately after the ocular application of capsaicin or vehicle, the mice were placed into a plexiglass chamber; and the number of ipsilateral eye wipes with the forelimb was counted for 3 minutes. Facial grooming with both forepaws and hind paw scratches were also included. After the assessment of wipe behavior, the mice were removed from the chamber and returned to the initial cage. Corneal sensitivity was measured without topical anesthesia using a modified Cochet-Bonnet filament aesthesiometer. The testing was initiated using a nylon filament of 4.0 cm. This was the end of the nylon filament used to touch the central part of the cornea. If the mouse blinked, the length of the filament was recorded. If the mouse did not blink, the nylon filament was shortened by 0.5 cm and the test was repeated until the blinking reflex was observed and recorded. This process was repeated for each eye three times. To evaluate the changes in corneal sensitivity after repetitive corneal stimulation with eye drops of capsaicin or menthol, measurements were performed before first instillation (P14) and 24 hours after final instillation of capsaicin or menthol (P21). Statistical analyses were performed using JMP12 software version 12.2 (SAS Institute, Cary, NC). Comparisons between the two groups were performed using an F-test, followed by the t-test for parametric variables and the U-test for nonparametric variables. Multiple comparisons were performed using a one-way analysis of variance, followed by the Tukey-Kramer or Dunnett test. The differences between the measurement variables were considered significant if P ≤ 0.05. The time of natural eye opening in the mice was from P12 to P14. Herein, >95% of the mouse pups opened their eyes naturally at P14 (Figure 1A), as reported previously.27Ko H. Cossell L. Baragli C. Antolik J. Clopath C. Hofer S.B. Mrsic-Flogel T.D. The emergence of functional microcircuits in visual cortex.Nature. 2013; 496: 96-100Crossref PubMed Scopus (257) Google Scholar, 28Sullivan D.A. Yee L. Conner A.S. Hann L.E. Olivier M. Allansmith M.R. Influence of ocular surface antigen on the postnatal accumulation of immunoglobulin-containing cells in the rat lacrimal gland.Immunology. 1990; 71: 573-580PubMed Google Scholar, 29Farmer D.T. Nathan S. Finley J.K. Shengyang Yu K. Emmerson E. Byrnes L.E. Sneddon J.B. McManus M.T. Tward A.D. Knox S.M. Defining epithelial cell dynamics and lineage relationships in the developing lacrimal gland.Development. 2017; 144: 2517-2528Crossref PubMed Scopus (24) Google Scholar To determine whether the rapid maturation/growth of neonatal tearing that follows eye opening could be attributed solely to endogenous developmental mechanisms, binocular eyelid suture was performed on the mouse pups before the time of eye opening (P10) through P21, at which point the eyelid suture was removed (Figure 1B). Linear elevation of the body weight was observed in the normal and eyelid-sutured group from P12 to P21 at the same level (Figure 1C). In the normal group, tear secretion was gradually increased after eye opening (Figure 1D). A significant increase in tear secretion was observed at P18, P21, and P28 compared with that at P14. In the eyelid-sutured group, tear secretion in the pups that delayed eye opening by 1 week was unchanged at P21 compared with that at P12. At P21, tear secretion was significantly lower in the eyelid-sutured group than in the normal group. Tear secretion in the eyelid-sutured group was increased from P21 to P28, and significance was observed between P21 and P28. No difference in tear secretion was observed at P28 between the normal and eyelid-sutured groups. The LG of mouse pups at P7 was small, with a size of approximately 3.0 mm (vertical) and 2.5 mm (horizontal). No obvious changes in the LG size were observed before the eye-opening period (P7 to P14) in both the normal and eyelid-sutured groups. During the postnatal age before eye opening, no significant changes in the LG weight were observed (P7 to P14). In the normal mouse pup, the LG weight increased significantly after eye opening and the weight at P21 was approximately fourfold of the weight at P14 (Figure 1E). In the eyelid-sutured mouse pups, the LG weight significantly increased with postnatal age; however, the weight at P21 was significantly lower than that in the age-matched normal mouse pups. To further assess the relevance of eye opening to LG maturation, the changing ratio of the weight of LG, other organs, and the whole body was calculated at P21 versus those at P12. In the normal group, a significant difference was observed in the LG, salivary gland (SG), eyeball, and kidney weight at P21 compared with P12 (Figure 1F). At P21, the changing ratio of the LG weight was significantly higher than that of the SG, eyeball, and kidney weight in the normal group. In the eyelid-sutured group, the changing ratio of the LG weight was significantly lower than in the age-matched normal controls. There was no significant difference in the changing ratio of the weight of the other organs and the whole body in the eyelid-sutured group compared with the age-matched normal group. The gross appearance of the LG from the eyelid-sutured pups at P21 was evidently smaller compared with the age-matched normal group (Figure 1F). The size of the SG and heart in the eyelid-sutured pups was the same as that of the age-matched controls (P21). These findings indicate that the occurrence of eye opening is important for the rapid growth of the LG during the postnatal period. The LG is a tubule-acinar exocrine gland that initiates by branching morphogenesis during the embryonic period and continues to develop during the postnatal period, during which it approaches maturity.29Farmer D.T. Nathan S. Finley J.K. Shengyang Yu K. Emmerson E. Byrnes L.E. Sneddon J.B. McManus M.T. Tward A.D. Knox S.M. Defining epithelial cell dynamics and lineage relationships in the developing lacrimal gland.Development. 2017; 144: 2517-2528Crossref PubMed Scopus (24) Google Scholar, 30Tsau 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 The mature LG is composed of terminally differentiated acinar cells, myoepithelial cells, and ductal cells.29Farmer D.T. Nathan S. Finley J.K. Shengyang Yu K. Emmerson E. Byrnes L.E. Sneddon J.B. McManus M.T. Tward A.D. Knox S.M. Defining epithelial cell dynamics and lineage relationships in the developing lacrimal gland.Development. 2017; 144: 2517-2528Crossref PubMed Scopus (24) Google Scholar, 31Makarenkova H.P. Dartt D.A. Myoepithelial cells: their origin and function in lacrimal gland morphogenesis, homeostasis, and repair.Curr Mol Biol Rep. 2015; 1: 115-123Crossref PubMed Google Scholar These structures are organized as secretory lobes that cooperate to synthesize, modify, and secrete tear fluid. The morphologic changes in the acinar cells and myoepithelial cells of the LG, accompanying the occurrence of eye opening, were evaluated with two-photon, light, and electron microscopy. Using two-photon microscopy, numerous secretory lobes composed of acinar cells and myoepithelial cells in the LG were observed in the normal group at P12 and P21 and the eyelid-sutured group at P21 (Figure 2A). In the normal mouse pups, individual secretory lobes were found enlarged at P21 compared with P12. The secretory lobes in the LG of the eyelid-sutured mouse pups at P21 were larger and smaller than those of the normal mouse pups at P12 and P21, respectively. Using two-photon microscopy at high magnification, enlarged acinar cells of the secretory lobes were observed in the LG of the normal group at P21 compared with P12 (Figure 2A). The myoepithelial cells surrounding the surface of the acinar cells grew with more elongated processes at P21 compared with P12 in the normal mouse pups. In the LG of the eyelid-sutured mouse pups at P21, a small size of acinar cells and a high density of myoepithelial cells on the LG surface were observed compared with the normal LG of P21. Most myoepithelial cell processes in the LG of eyelid-sutured pups at P21 were elongated compared with the normal LG at P12; however, some appeared shorter and thinner than the age-matched normal LG. Using light microscopy to observe the hematoxylin and eosin–stained LG specimens, a gradual increase in the size of acinar cells with neonatal age was clearly observed in normal mouse pups, in particular in the cytoplasmic area of each individual acinar cell (Figure 2B). The acinar cell size was smaller in the LG of eyelid-sutured pups at P21 compared with the normal LG at P21, and the size was found to decrease between the normal P14 and P21 groups. Quantitative analysis of acinar cell size was performed at different postnatal ages in normal mouse and at P21 in eyelid-sutured mouse. Acinar cell size was found to significantly increase with postnatal age in the normal and eyelid-sutured groups compared with P12 (Figure 2B). In the eyelid-sutured mouse at P21, acinar cell size was approximately 75% of that in the age-matched normal mice, and significant differences were observed between the normal and eyelid-sutured mice at P21 (Figure 2B). Acinar cell density did not change throughout the postnatal age after eye opening in normal mice, and that of the eyelid-sutured mice was the same as the age-matched normal mice (Figure 2B). These findings suggest that an enlargement of acinar cell size and not an increase in the number of acinar cells occurred in the LG during neonatal age after eye opening. On transmission electron microscopy observation of the LG acinar cells of the normal mouse pups, a dilation of the stacked cisternae, composing the rough endoplasmic reticulum and the Golgi apparatus, as well as an increase in the number of secretory vesicles (SVs), was observed to be dependent on the age after eye opening (Figure 2C). In the LG of eyelid-sutured pup