Expression of extracellular matrix components in the meibomian gland.

Purpose: Extracellular matrix (ECM) is a key component of the stem cell local microenvironment. Our study aims to explore the periglandular distribution of major components of ECM in the Meibomian gland (MG). Methods: Human eyelids and mouse eyelids were collected and processed for immunofluorescence staining. Results: Human MG tissues stained positive for collagen IV α1, collagen IV α2, collagen IV α5, and collagen IV α6 around the acini and duct, but negative for collagen IV α3 and collagen IV α4. The mouse MG were stained positive for the same collagen IV subunits as early as postnatal day 15. Laminin α2, laminin ß1 and perlecan stained the regions surrounding the acini and the acinar/ductal junction in the human MG, but not the region around the duct. Tenascin-C was found specifically located at the junctions between the acini and the central ducts. Neither agrin nor endostatin was found in the human MG tissues. Conclusion: The ECM expresses specific components in different regions around the MG, which may play a role in MG stem cell regulation, renewal, and regeneration.

Eccrine poroma of the eyelid.

Clinical and histopathologic case of an eyelid eccrine poroma, a benign adnexal neoplasm rarely found on the periorbital skin.

The role of routine nasolacrimal sac biopsy during endoscopic dacryocystorhinostomy.

OBJECTIVES/HYPOTHESIS: Most patients who undergo endoscopic dacryocystorhinostomy (DCR) have a diagnosis of idiopathic nasolacrimal duct obstruction. The purpose of this study was to examine the impact of routine biopsy of the lacrimal sac performed at time of DCR on subsequent patient diagnosis and treatment. STUDY DESIGN: Retrospective review. METHODS: The histopathology of nasolacrimal specimens (n = 769), obtained from 654 consecutive patients undergoing endoscopic DCR by a single surgeon over a 30-year period, were reviewed. Specific focus included the identification of unanticipated pathologic findings as they related to pertinent patient demographics, clinical presentation, radiologic findings, and intraoperative observations. RESULTS: The study population was 69.6% female, with an average age of 56.1 ± 18.2 years. Pathological findings of tissue from the nasolacrimal sac, which was routinely sampled in all cases, showed inflammation (n = 566 [73.6%]), normal histology (n = 147 [19.1%]), granulomas (n = 8 [1.0%]), and neoplastic process (n = 7 [0.9%]). Patient history, preoperative CT scan, and/or intraoperative findings alerted the surgeon to the possibility of an unusual diagnosis in 12 of the 15 patients. An unsuspected neoplastic or granulomatous cause of lacrimal obstruction was identified on intraoperative biopsy in three patients (0.46%). CONCLUSIONS: Although neoplastic and granulomatous diseases are relatively rare causes of lacrimal obstruction necessitating DCR surgery, they may be identified by through patient evaluation in most cases and by routine intraoperative biopsy of the lacrimal sac in all cases. LEVEL OF EVIDENCE: 4 Laryngoscope, 130:584-589, 2020.

Ophthalmic Plastic Surgery in Patients 100 Years and Older.

PURPOSE: The centenarian population is growing and ophthalmic plastic surgeons are providing care to an increasing number of elderly patients. Outcomes of centenarians have not been previously studied in the ophthalmic plastic surgery literature. The goal of the current review was to examine the baseline characteristics, surgical problems, and outcomes of this select group of patients. METHODS: A retrospective chart review was performed. Patients who underwent ophthalmic plastic surgery at age 100 or older between January 2000 and June 2016 by a member of the New England Oculoplastics Society were included in the study. RESULTS: Fifteen patients met inclusion criteria. The majority (66%) were female. More than half (60%) presented with a surgical problem of an urgent nature. Most disorders involved the lacrimal system or eyelids, and many were the result of trauma or infection. There were no cases of orbital tumor or thyroid eye disease. There were no surgical or anesthesia-related complications. Most patients (80%) had no documented history of dementia, and only 1 was diabetic. Notably, 33% of patients presented with no light perception vision in at least 1 eye. CONCLUSIONS: Ophthalmic plastic surgery can be performed safely in select patients 100 years of age and older. Formal prospective studies are needed to improve surgical care in this group.

Hypoxia: A breath of fresh air for the meibomian gland.

PURPOSE: Optimal meibomian gland (MG) function is critically important for the health and wellbeing of the ocular surface. We hypothesize that low oxygen (O2) conditions promote the function of human MG epithelial cells (HMGECs) and that human MGs exist in a relatively hypoxic environment. The purpose of this study was to test our hypotheses. METHODS: We used human and mouse eyelid segments, and immortalized human MG epithelial cells (IHMGECs) in our studies. To evaluate oxygen (O2) levels in the mouse MG and vicinity, we injected pimonidazole (pimo), a hypoxia marker, before sacrifice. Human eyelid samples were stained with the hypoxia marker glucose transporter 1 (Glut-1). To determine the effect of low O2 levels on IHMGECs, we cultured cells under proliferating and differentiating conditions in both normoxic (21% O2) and hypoxic (3% O2) conditions for 5-15 days. IHMGECs were evaluated for cell number, neutral lipid content, lysosome accumulation, expression of biomarker proteins and DNase II activity. RESULTS: Our results demonstrate that human and mouse MGs, but not the surrounding connective tissue, exist in a relatively hypoxic environment in vivo. In addition, our findings show that hypoxia does not influence IHMGEC numbers in basal or proliferating culture conditions, but does stimulate the expression of SREBP-1 in differentiating IHMGECs. Hypoxia also significantly increased DNase II activity, and apparently IHMGEC terminal differentiation. CONCLUSIONS: Our Results support our hypotheses, and indicate that relative hypoxia promotes MG function.

Biomarkers for Progenitor and Differentiated Epithelial Cells in the Human Meibomian Gland.

The meibomian gland (MG) is a sebaceous gland that secretes through a holocrine process. Because such secretion requires the destruction of MG acinar epithelial cells, they need constant renewal and differentiation. The processes that promote these regenerative events in the human MG are unknown, nor is it known how to distinguish MG progenitor and differentiated cells. We discovered that Lrig1 and DNase2 serve as biomarkers for human MG progenitor and differentiated cells, respectively. Lrig1 is expressed in MG basal epithelial cells in the acinar periphery, a location where progenitor cells originate in sebaceous glands. DNase2 is expressed in the differentiated epithelial cells of the MG central acinus. Furthermore, proliferation stimulates, and differentiation suppresses, Lrig1 expression in human MG epithelial cells. The opposite is true for DNase2 expression. Our biomarker identification may have significant value in clinical efforts to restore MG function and to regenerate MGs after disease-induced dropout. Stem Cells Translational Medicine 2018;7:887-892.

A Common Procedure With an Uncommon Pathology: Triamcinolone Acetonide Eyelid Injection.

Local corticosteroid injections are frequently employed by ophthalmologists to treat a variety of ocular, periocular, and orbital inflammatory conditions. Triamcinolone acetonide is a slowly dissolving crystalline corticosteroid that is often used for this purpose because of its prolonged anti-inflammatory effect. On occasion, previously injected corticosteroid material persists in tissues longer than anticipated, creating nodules that may masquerade as other disease conditions, or appearing incidentally in excised lesions on histopathologic examination. The histopathologic features of corticosteroid residues are unfamiliar to most ophthalmic pathologists and general pathologists. These features are described herein. Triamcinolone acetonide deposits in the skin appear as pale eosinophilic lakes of acellular frothy material on hematoxylin-eosin staining and are occasionally surrounded by a mild inflammatory reaction.

Causes of dacryocystorhinostomy failure: External versus endoscopic approach.

OBJECTIVES: To compare the causes of failure between external and endoscopic dacryocystorhinostomy (DCR) techniques for the treatment of lacrimal obstruction. STUDY DESIGN: A retrospective cohort study. METHODS: The study population consisted of 53 consecutive patients who underwent revision endoscopic DCR from 2002 to 2013 for lacrimal duct obstruction. Identified causes of previous DCR failure were compared between patients whose initial surgery was performed through an external versus an endoscopic approach. RESULTS: Reasons for surgical failure after external (n = 21) versus endoscopic (n = 32) DCR included cicatricial closure of the internal lacrimal ostium (52.4 versus 53.1%; p = 0.96), inadequate removal of bone overlying the lacrimal sac (23.8 versus 9.4%; p = 0.15), sump syndrome (9.5 versus 9.4%; p = 0.99), and intranasal adhesions (65 versus 37.5%; p = 0.05). Adhesions that involved the middle turbinate were a particularly impactful cause of failure when the DCR was performed through an external versus the endoscopic approach (57.1 versus 28.1%; p = 0.04). Septoplasty was more likely to be needed at the time of revision surgery if the initial procedure was performed externally (71.1 versus 15.6%; p = 0.02). Surgical success rates for revision DCR were comparable between the groups (75.0% external versus 73.3% endoscopic; p = 0.90), with a mean follow-up of 12.7 months. CONCLUSION: DCR failure associated with intranasal adhesions was more likely to occur when the surgery was performed through an external rather than an endoscopic approach. Endoscopic instrumentation allowed for identification and correction of intranasal pathology at the time of DCR, including an enlarged middle turbinate or a deviated septum, which may improve surgical outcome.

Histopathologic Clues in Diagnosing Oral Mucosal Grafts to the Conjunctiva.

Excised redundant, forniceal "conjunctival" tissue from a 67-year-old man who experienced a chemical injury to his OS 25 years earlier was evaluated histopathologically with the hematoxylin-eosin, periodic acid Schiff (PAS) with and without diastase, mucicarmine, and Alcian blue methods. Additional immunoperoxidase testing for gross cystic disease fluid protein-15 (GCDFP-15) was undertaken. Non-keratinizing squamous epithelium composed of 8 to 10 layers of swollen keratinocytes without goblet cells surmounted a variably dense and well-vascularized collagenized lamina propria deep to which, in submucosal fibroadipose tissue, was embedded an accessory gland. The acini of the gland were composed of both GCDFP-15-positive serous cells and mucicarmine-positive goblet cells, indicating they were seromucinous rather than entirely serous, as is characteristic of normal lacrimal glandular tissue. Different features of the surface epithelium, the lamina propria, and the submucosa can separate the conjunctival and oral mucous membranes. A close analysis of the cytologic composition of associated accessory glands can reinforce the correct diagnosis of an oral mucous membrane graft when the past surgical history is unclear, because only serous cells but not mucocytes comprise the lacrimal glandular units.

Serum-induced differentiation of human meibomian gland epithelial cells.

PURPOSE: We hypothesize that culturing immortalized human meibomian gland epithelial cells in serum-containing medium will induce their differentiation. The purpose of this investigation was to begin to test our hypothesis, and explore the impact of serum on gene expression and lipid accumulation in human meibomian gland epithelial cells. METHODS: Immortalized and primary human meibomian gland epithelial cells were cultured in the presence or absence of serum. Cells were evaluated for lysosome and lipid accumulation, polar and neutral lipid profiles, and gene expression. RESULTS: Our results support our hypothesis that serum stimulates the differentiation of human meibomian gland epithelial cells. This serum-induced effect is associated with a significant increase in the expression of genes linked to cell differentiation, epithelium development, the endoplasmic reticulum, Golgi apparatus, vesicles, and lysosomes, and a significant decrease in gene activity related to the cell cycle, mitochondria, ribosomes, and translation. These cellular responses are accompanied by an accumulation of lipids within lysosomes, as well as alterations in the fatty acid content of polar and nonpolar lipids. Of particular importance, our results show that the molecular and biochemical changes of immortalized human meibomian gland epithelial cells during differentiation are analogous to those of primary cells. CONCLUSIONS: Overall, our findings indicate that immortalized human meibomian gland epithelial cells may serve as an ideal preclinical model to identify factors that control cellular differentiation in the meibomian gland.

Ectopic (choristomatous) orbital respiratory cyst: histopathology and immunohistochemistry.

A 24-year-old woman underwent excision of a slowly growing mass located in the right superomedial orbit that had histopathologic and immunohistochemical findings consistent with a choristomatous respiratory cyst. This rare condition may either arise primarily from embryologic respiratory epithelium rests in the orbit or develop secondarily as the result of trauma or chronic sinus disease complicated by mucocele formation.

Effect of growth factors on the proliferation and gene expression of human meibomian gland epithelial cells.

PURPOSE: We hypothesize that growth factors, including epidermal growth factor (EGF) and bovine pituitary extract (BPE), induce proliferation, but not differentiation (e.g., lipid accumulation), of human meibomian gland epithelial cells. We also hypothesize that these actions involve a significant upregulation of genes linked to cell cycle processes, and a significant downregulation of genes associated with differentiation. Our objective was to test these hypotheses. METHODS: Immortalized human meibomian gland and conjunctival epithelial cells were cultured for varying time periods in the presence or absence of EGF, BPE, EGF + BPE, or serum, followed by cell counting, neutral lipid staining, or RNA isolation for molecular biological procedures. RESULTS: Our studies show that growth factors stimulate a significant, time-dependent proliferation of human meibomian gland epithelial cells. These effects are associated with a significant upregulation of genes linked to cell cycle, DNA replication, ribosomes, and translation, and a significant decrease in those related to cell differentiation, tissue development, lipid metabolic processes, and peroxisome proliferator-activated receptor signaling. Serum-induced differentiation, but not growth factor-related proliferation, elicits a pronounced lipid accumulation in human meibomian gland epithelial cells. This lipogenic response is unique, and is not duplicated by human conjunctival epithelial cells. CONCLUSIONS: Our results demonstrate that EGF and BPE stimulate human meibomian gland epithelial cells to proliferate. Further, our findings show that action is associated with an upregulation of cell cycle and translation ontologies, and a downregulation of genetic pathways linked to differentiation and lipid biosynthesis.

Gene expression in human accessory lacrimal glands of Wolfring.

PURPOSE: The accessory lacrimal glands are assumed to contribute to the production of tear fluid, but little is known about their function. The goal of this study was to conduct an analysis of gene expression by glands of Wolfring that would provide a more complete picture of the function of these glands. METHODS: Glands of Wolfring were isolated from frozen sections of human eyelids by laser microdissection. RNA was extracted from the cells and hybridized to gene expression arrays. The expression of several of the major genes was confirmed by immunohistochemistry. RESULTS: Of the 24 most highly expressed genes, 9 were of direct relevance to lacrimal function. These included lysozyme, lactoferrin, tear lipocalin, and lacritin. The glands of Wolfring are enriched in genes related to protein synthesis, targeting, and secretion, and a large number of genes for proteins with antimicrobial activity were detected. Ion channels and transporters, carbonic anhydrase, and aquaporins were abundantly expressed. Genes for control of lacrimal function, including cholinergic, adrenergic, vasoactive intestinal polypeptide, purinergic, androgen, and prolactin receptors were also expressed in gland of Wolfring. CONCLUSIONS: The data suggest that the function of glands of Wolfring is similar to that of main lacrimal glands and are consistent with secretion electrolytes, fluid, and protein under nervous and hormonal control. Since these glands secrete directly onto the ocular surface, their location may allow rapid response to exogenous stimuli and makes them readily accessible to topical drugs.

IgG4-related systemic disease as a cause of "idiopathic" orbital inflammation, including orbital myositis, and trigeminal nerve involvement.

IgG4-related systemic disease (IgG4-RD) is an inflammatory condition of unknown etiology that has been identified as the cause of tumefactive lesions in a number of tissues and organs. The role of the IgG4 remains to be clarified fully, but the histopathologic diagnosis hinges upon the finding of IgG4-bearing plasma cells in addition to characteristic morphologic features, with or without elevated seum IgG4. We present a 56-year-old man with orbital pseudotumor in whom, after 30 years of intractable disease, biopsy showed IgG4-RD involving the lacrimal gland, extraocular muscles, intraconal fat, and trigeminal nerve. Six months after initiating treatment with rituximab, his disease remained dormant, with improvement in his proptosis and normalization of serum IgG4 levels. We review the differential of idiopathic orbital inflammatory disease, including IgG4-RD, and emphasize the need for biopsy for accurate diagnosis and to guide appropriate treatment.

Eyelid basal cell carcinoma developing in an epidermoid cyst: a previously unreported event.

A 72-year-old woman presented with a 3 × 2 × 1-mm right lower eyelid micronodular, whitish solid lesion at the nasal eyelid margin that had caused madarosis. Local excision led to the microscopic discovery of 2 epidermoid cysts, one of which harbored a basal cell carcinoma arising from its orthokeratinizing squamous cell lining. Poral openings indicated that the cysts represented follicular infundibular ectasias. BER-EP4-positive immunostaining confirmed the basal cell nature of the neoplasm. This is the first example in the eyelid of an epidermoid cyst displaying malignant transformation. No matter how innocent they may appear, all small cystic or noncystic eyelid lesions should be submitted for pathologic evaluation.

Culture, immortalization, and characterization of human meibomian gland epithelial cells.

PURPOSE: Meibomian gland epithelial cells are essential in maintaining the health and integrity of the ocular surface. However, very little is known about their physiological regulation. In this study, the cellular control mechanisms were explored, first to establish a defined culture system for the maintenance of primary epithelial cells from human meibomian glands and, second, to immortalize these cells, thereby developing a preclinical model that could be used to identify factors that regulate cell activity. METHODS: Human meibomian glands were removed from lid segments after surgery, enzymatically digested, and dissociated. Isolated epithelial cells were cultured in media with or without serum and/or 3T3 feeder layers. To attempt immortalization, the cells were exposed to retroviral human telomerase reverse transcriptase (hTERT) and/or SV40 large T antigen cDNA vectors, and antibiotic-resistant cells were selected, expanded, and subcultured. Analyses for possible biomarkers, cell proliferation and differentiation, lipid-related enzyme gene expression, and the cellular response to androgen were performed with biochemical, histologic, and molecular biological techniques. RESULTS: It was possible to isolate viable human meibomian gland epithelial cells and to culture them in serum-free medium. These cells proliferated, survived through at least the fifth passage, and contained neutral lipids. Infection with hTERT immortalized these cells, which accumulated neutral lipids during differentiation, expressed multiple genes for lipogenic enzymes, responded to androgen, and continued to proliferate. CONCLUSIONS: The results show that human meibomian gland epithelial cells may be isolated, cultured, and immortalized.