Lacrimal Gland Repair after Short-term Obstruction of Excretory Duct in Rabbits.
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SUBMITTER: Lin H
PROVIDER: S-EPMC5557958 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundAqueous-deficient dry eye disease (ADDED) resulting from dysfunction of the lacrimal gland (LG) is currently incurable. Although LG stem/progenitor cell-based therapy is considered to be a promising strategy for ADDED patients, the lack of a reliable serum-free culture method to obtain enough lacrimal gland stem cells (LGSCs) and the basic standard of LGSC transplantation are obstacles for further research.MethodsAdult mouse LGSCs were cultured in Matrigel-based 3D culture under serum-free culture condition, which contained EGF, FGF10, Wnt3A, and Y-27632. LGSCs were continuously passaged over 40 times every 7 days, and the morphology and cell numbers were recorded. LGSCs were induced to differentiate to ductal cells by reducing Matrigel rigidity, while fetal bovine serum was used for the induction of acinar cells. RT-PCR or qRT-PCR analysis, RNA-sequence analysis, H&E staining, and immunofluorescence were used for characterization and examining the differentiation of LGSCs. LGSCs were allotransplanted into diseased LGs to examine the ability of repairing the damage. The condition of eye orbits was recorded using a camera, the tear production was measured using phenol red-impregnated cotton threads, and the engraftments of LGSCs were examined by immunohistochemistry.ResultsWe established an efficient 3D serum-free culture for adult mouse LGSCs, in which LGSCs could be continuously passaged for long-term expansion. LGSCs cultured from both the healthy and ADDED mouse LGs expressed stem/progenitor cell markers Krt14, Krt5, P63, and nestin, had the potential to differentiate into acinar or ductal-like cells in vitro and could engraft into diseased LGs and relieve symptoms of ADDED after orthotopic injection of LGSCs.ConclusionWe successfully established an efficient serum-free culture for adult mouse LGSCs aiming at LG repair for the first time. Our approach provides an excellent theoretical and technical reference for future clinical research for ADDED stem cell therapy.
| S-EPMC6951017 | biostudies-literature
Project description:In humans, the lacrimal gland (LG) is the primary contributor to the aqueous layer of the tear film. Production of tears in insufficient quantity or of inadequate quality may lead to aqueous-deficiency dry eye (ADDE). Currently there is no cure for ADDE. The development of strategies to reliably isolate LG stem/progenitor cells from the LG tissue brings great promise for the design of cell replacement therapies for patients with ADDE. We analyzed the therapeutic potential of epithelial progenitor cells (EPCPs) isolated from adult wild-type mouse LGs by transplanting them into the LGs of TSP -1-/- mice, which represent a novel mouse model for ADDE. TSP-1-/- mice are normal at birth but progressively develop a chronic form of ocular surface disease, characterized by deterioration, inflammation, and secretory dysfunction of the lacrimal gland. Our study shows that, among c-kit-positive epithelial cell adhesion molecule (EpCAM+ ) populations sorted from mouse LGs, the c-kit+ dim/EpCAM+ /Sca1 - /CD34 - /CD45 - cells have the hallmarks of an epithelial cell progenitor population. Isolated EPCPs express pluripotency factors and markers of the epithelial cell lineage Runx1 and EpCAM, and they form acini and ducts when grown in reaggregated three-dimensional cultures. Moreover, when transplanted into injured or "diseased" LGs, they engraft into acinar and ductal compartments. EPCP-injected TSP-1-/- LGs showed reduction of cell infiltration, differentiation of the donor EPCPs within secretory acini, and substantial improvement in LG structural integrity and function. This study provides the first evidence for the effective use of adult EPCP cell transplantation to rescue LG dysfunction in a model system. Stem Cells Translational Medicine 2017;6:88-98.
| S-EPMC5442743 | biostudies-literature
Project description:PurposeTo develop a nomenclature for the lacrimal duct system in the rabbit, based on the anatomic and structural characteristics of each duct segment, and to provide RT-PCR and immunofluorescence data to support the notion that the duct system plays important roles in lacrimal function.MethodsParaffin-embedded lacrimal glands (LGs) were stained with hematoxylin and eosin (H&E) and evaluated with a stereomicroscope. Cryosections of LG were stained with cresyl violet, and acinar cells and ductal epithelial cells were isolated from each duct segment by laser capture microdissection (LCM). mRNA levels from these cells were analyzed by real-time RT-PCR. Standard protocol was followed for immunofluorescence detection of ionic transporters.ResultsThe lacrimal duct system was divided into six segments on the basis of morphologic characteristics: the intercalated, intralobular, interlobular, intralobar, interlobar, and main excretory ducts. Although the morphologic features change incrementally along the entire duct system, the gene expression of ionic transporters and aquaporins, including AE3, AQP4, AQP5, CFTR, ClC2gamma, KCC1, NHE1, NKAalpha1, NKAbeta1, NKAbeta2, NKAbeta3, and NKCC1 varied greatly among duct segments. Immunofluorescence results were generally in accordance with the abundance of mRNAs along the acinus-duct axis.ConclusionsMost LG research has focused on the acinar cells, with relatively little attention being paid to the lacrimal ducts. The lack of knowledge regarding the lacrimal ducts was so profound that a precise nomenclature had not been established for the duct system. The present data establish a nomenclature for each segment of the lacrimal duct system and provide evidence that ducts play critical roles in lacrimal secretion.
| S-EPMC2891459 | biostudies-literature
Project description:PurposeTo determine whether high fructose intake (HFI) influences the daily transcriptional clock rhythms of murine extraorbital lacrimal glands (ELGs).MethodsTimed ELGs were collected from two groups of C57BL/6J mice subjected to a 12-hour light/12-hour dark (LD) cycle for 10 days; the first group received water-only feeding and the second received water with 15% fructose. Total RNA was extracted and subjected to RNA sequencing. A JTK_CYCLE algorithm and computational software were used to determine the periodicity, rhythmicity, and amplitude of the cycling transcripts. The rhythmic transcripts from different conditions were analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.ResultsHFI feeding caused massive remodeling of the preexisting rhythmic genes in the normal control (NC)-fed ELGs. The induced transcripts in HFI-fed mice resulted in a profound reorganization of the coordinated transcriptional oscillations and KEGG pathways. Moreover, HFI feeding significantly altered the distribution of the KEGG pathways over an LD in the ELGs. Finally, we found that the ELGs have a robust core clock machinery and HFI feeding altered amplitude and the peak phase of clock gene transcriptional oscillation in ELGs.ConclusionsShort-term HFI reprograms the daily transcriptomic oscillation of murine ELGs. This information may deepen our understanding of the outcomes of lacrimal glands altered by nutritional challenge.
| S-EPMC6890433 | biostudies-literature
Project description:The role of cystic fibrosis transmembrane conductance regulator (CFTR) in lacrimal gland (LG) function has only recently received some attention, mainly from our group. In the present study, we investigated the potential changes of LG pathology, tear secretion, ocular surface integrity, and fluid secretion in isolated LG ducts from CFTR knockout (KO) mice.Tear production and ocular surface integrity were investigated in anesthetized wild-type (WT) and KO mice using cotton threads and fluorescein staining, respectively. Immunofluorescence was used to localize CFTR protein in the LGs. Ductal fluid secretions evoked by forskolin (10 ?M); cell-permeable cAMP analogue (8-bromo cAMP, 100 ?M); or carbachol (100 ?M) were measured in isolated LG ducts using video-microscopy. Intracellular Ca2+ homeostasis underlying carbachol stimulation was investigated with microfluorometry.Significant decrease in tear secretion and impaired ocular surface integrity were observed in KO mice. Immunofluorescence demonstrated the predominant presence of CFTR protein in the apical membranes of the duct cells from WT mice. Continuous fluid secretion was evoked by forskolin and 8-bromo cAMP in LG ducts from WT mice, while no secretory response was observed in ducts from KO mice. Carbachol caused similar secretory responses in ducts from WT and KO animals without significant differences in cytosolic Ca2+ signaling.Our results suggest the important role of CFTR in LG ductal secretion and in the maintenance of ocular surface integrity, suggesting that CFTR may be a promising target of novel therapeutic approaches in the treatment of dry eye.
| S-EPMC5756044 | biostudies-literature
Project description:Long-term submandibular duct obstruction is thought to cause irreversible atrophy and dysfunction of the submandibular gland. As an atrophic gland may be induced clinically by chronic or recurrent infection, it is generally removed surgically. However, the regenerative capacity of atrophic submandibular glands after long-term obstruction is not completely understood. We studied the regenerative capacity of the mouse submandibular duct using an aneurysm clip ligation model. We documented changes in the size, histologic structure and gene expression of the submandibular gland after 2 months of ligation, and 1 week, 1 month and 2 months after clip removal. Two months of ligation caused atrophy, particularly in the acinar portion. In the 2 months after clip removal, we observed a steady and significant increase in the expression of the acinar cell precursor gene cytokeratin 5 (CK5), and a significant decrease in the expression of the stem cell marker c-kit. These findings suggest that the submandibular gland retains some capacity for regeneration even after long-term obstruction, and that CK5 could serve as a marker of this regenerative process.
| S-EPMC5519490 | biostudies-literature
Project description:PurposeThe aim of this study was to describe the transcriptional changes of individual cellular components in the lacrimal sac in patients with primary acquired nasolacrimal duct obstruction (PANDO) and attempt to construct the first lacrimal sac cellular atlas to elucidate the potential mechanisms that may drive the disease pathogenesis.MethodsLacrimal sac samples were obtained intra-operatively during the endoscopic dacryocystorhinostomy (EnDCR) procedure from five patients. Single-cell RNA sequencing was performed to analyze each individual cell population including epithelial and immune cells during the early inflammatory and late inflammatory phases of the disease.ResultsEleven cell types were identified among 25,791 cells. T cells and B cells were the cell populations with the greatest variation in cell numbers between the two phases and were involved in immune response and epithelium migration-related pathways. The present study showed that epithelial cells highly expressed the genes of senescence-associated secretory phenotype (SASP) and were involved in influencing the inflammation, neutrophil chemotaxis, and migration during the late inflammatory stage. Enhanced activity of CXCLs-CXCRs between the epithelial cells and neutrophils was noted by the cell-cell communication analysis and is suspected to play a role in inflammation by recruiting more neutrophils.ConclusionsThe study presents a comprehensive single-cell landscape of the lacrimal sac cells in different phases of PANDO. The contribution of T cells, B cells, and epithelial cells to the inflammatory response, and construction of the intercellular signaling networks between the cells within the lacrimal sac has further enhanced the present understanding of the PANDO pathogenesis.
| S-EPMC10981439 | biostudies-literature
Project description:Lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes the aqueous layer of the tear film. LG epithelium is composed of ductal, acinar, and myoepithelial cells (MECs) bordering the basal lamina and separating the epithelial layer from the extracellular matrix. Mature MECs have contractile ability and morphologically resemble smooth muscle cells; however, they exhibit features typical for epithelial cells, such as the presence of specific cytokeratin filaments. Increasing evidence supports the assertion that myoepithelial cells (MECs) play key roles in the lacrimal gland development, homeostasis, and stabilizing the normal structure and polarity of LG secretory acini. MECs take part in the formation of extracellular matrix gland and participate in signal exchange between epithelium and stroma. MECs have a high level of plasticity and are able to differentiate into several cell lineages. Here, we provide a review on some of the MEC characteristics and their role in LG morphogenesis, maintenance, and repair.
| S-EPMC4683023 | biostudies-literature
Project description:BackgroundThe study of human lacrimal gland biology and development is limited. Lacrimal gland tissue is damaged or poorly functional in a number of disease states including dry eye disease. Development of cell based therapies for lacrimal gland diseases requires a better understanding of the gene expression and signaling pathways in lacrimal gland. Differential gene expression analysis between lacrimal gland and other embryologically similar tissues may be helpful in furthering our understanding of lacrimal gland development.MethodsWe performed global gene expression analysis of human lacrimal gland tissue using Affymetrix ® gene expression arrays. Primary data from our laboratory was compared with datasets available in the NLM GEO database for other surface ectodermal tissues including salivary gland, skin, conjunctiva and corneal epithelium.ResultsThe analysis revealed statistically significant difference in the gene expression of lacrimal gland tissue compared to other ectodermal tissues. The lacrimal gland specific, cell surface secretory protein encoding genes and critical signaling pathways which distinguish lacrimal gland from other ectodermal tissues are described.ConclusionsDifferential gene expression in human lacrimal gland compared with other ectodermal tissue types revealed interesting patterns which may serve as the basis for future studies in directed differentiation among other areas.
| S-EPMC5231359 | biostudies-literature
Project description:Ongoing studies demonstrate that the murine lacrimal gland is capable of repair after experimentally induced injury. It was recently reported that repair of the lacrimal gland involved the mobilization of mesenchymal stem cells (MSCs). These cells expressed the type VI intermediate filament protein nestin whose expression was upregulated during the repair phase. The aim of the present study was to investigate the roles of vimentin, a type III intermediate filament protein and a marker of epithelial-mesenchymal transition (EMT) in repair of the lacrimal gland.Injury was induced by direct injection of interleukin (IL)-1 into the exorbital lacrimal gland. MSCs were prepared from injured glands using tissue explants. Expression of vimentin and the transcription factor Snai1, a master regulator of EMT, was determined by RT-PCR, Western blotting analysis, and immunofluorescence.These data show that vimentin expression, at both the mRNA and the protein levels, was upregulated during the repair phase (2-3 days postinjury) and returned to the control level when repair ended. Temporal expression of Snai1 mirrored that of vimentin and was localized in cell nuclei. Cultured MSCs isolated from injured lacrimal glands expressed Snai1 and vimentin alongside nestin and alpha smooth muscle actin (another biomarker of EMT). There was a strong positive correlation between Snai1 expression and vimentin expression.It was found that EMT is induced during repair of the lacrimal gland to generate MSCs to initiate repair, and that mesenchymal-epithelial transition is then activated to form acinar and ductal epithelial cells.
| S-EPMC3292353 | biostudies-literature