Expression patterns of conjunctival mucin 5AC and aquaporin 5 in response to acute dry eye stress.
ABSTRACT: The relationship between aquaporin (AQP) 5 and mucin (MUC) 5AC in the conjunctiva was investigated in response to acute dry eye (DE) stress. A mixed-mechanism rabbit DE model, in which the main lacrimal gland, Harderian gland, and nictitating membrane were resected, was further explored in this study. Conjunctival impression cytology specimens were harvested before excision (BE) and up to 3 months after excision (AE) in 8 (16 eyes) male New Zealand White rabbits, and immunoblotting was employed to assess the expression of AQP5 and MUC5AC. It was observed that AQP5 and MUC5AC showed a positive, synchronous expression pattern with progressive upregulation at protein level up to 2 months AE. At 3 months, the expression of both proteins decreased, but was still higher than that of BE. Such a synchronous relationship was further observed in mouse conjunctiva epithelium primary cells under hyperosmotic condition. Moreover, the co-immunoprecipitation of AQP5 and MUC5AC suggested a possible physical interaction between the two molecules. Our data indicates that conjunctival AQP5 and MUC5AC act synchronously in response to acute DE stress.
Project description:Vitreo-retinal (VR) surgeries induce conjunctival changes. However, there are no study reports regarding prevalence and severity of dry eye after these surgeries. This study evaluated dry eye outcome after VR surgery. Patients undergoing VR surgery classified as scleral buckle and microincision vitrectomy surgery (n = 44, mean age: 56.09±10.2 years) were recruited. Dry eye evaluation was done before and 8 weeks after surgery (2 weeks after omitting topical eye drops). Conjunctival imprint cytology for goblet cell count and tear Mucin 5AC (MUC5AC) protein estimation was done. Gene expressions of MUC5AC, MUC4, MUC16, Aquaporin 4 (AQP4) and AQP5 were analyzed in the conjunctival imprint cells by qPCR. None of the patients exhibited clinical signs of dry eye after VR surgery. But the conjunctival goblet cell density (GCD) was significantly lowered post-VR surgery (63% cases, **p = 0.012) with no alterations in the tear MUC5AC protein. Post-VR surgery, the conjunctival cell gene expression of MUC4, MUC16 and AQP4 were significantly increased (*p = 0.025, *p = 0.05 and *p = 0.02 respectively) and AQP5 was significantly lowered (*p = 0.037), with no change in MUC5AC expression. Tear cytokines were significantly increased post-VR surgery (anti-inflammatory: IL1RA, IL4, IL5, IL9, FGF; PDGFbb and pro-inflammatory: IL2, IL6, IL15, GMCSF and IFNg). Though clinical signs of dry eye were not observed after VR surgery, ocular surface changes in the form of reduced GCD, altered MUC5AC, MUC4, MUC16, AQP4, AQP5 and cytokines are suggestive of dry eye outcome at the molecular level especially inpatients aged above 51 years, especially female gender and those who are diabetic.
Project description:The first aim of this study was to clarify whether cigarette smoking affects tear secretion, goblet cell density, and tear MUC5AC concentration. The second purpose was to evaluate the correlations of conjunctival goblet cell density with tear MUC5AC concentration and other ocular surface evaluation factors. This cross-sectional study included 88 office workers. All subjects were required to fill in dry eye and smoking questionnaires, in addition to ocular surface evaluation. Tear wash fluid was collected from inferior fornix, and conjunctival epithelium was obtained by impression cytology. Tear MUC5AC concentration was quantified using enzyme-linked immunoassay, and conjunctival goblet cell density was counted after Periodic-acid Schiff staining. Tear MUC5AC concentration had significant positive correlation with conjunctival goblet cell density (r = 0.181, P = 0.03). In current smokers, Schirmer I test value, goblet cell density and tear MUC5AC concentration were significantly lower than non-smokers. Pack-years of smoking have significant negative correlation with goblet cell density (r = -0.174, P = 0.036) and tear MUC5AC concentration (r = -0.183, P = 0.028). We concluded that smoking might decrease tear secretion, goblet cell density and tear MUC5AC concentration. In addition, MUC5AC concentration in tears depends on goblet cell density in the conjunctiva among office workers.
Project description:The conjunctiva is a clear tissue covering the white part of the eye and lines the back of the eyelids. Conjunctival diseases, such as symblepharon, cause inflammation, discharges, and photophobia. The treatment often requires excision of large parts of conjunctiva. Tissue engineering of conjunctival cells using human amniotic membrane (HAM) denuded of its epithelium as a basement membrane scaffold has been shown to be effective for covering conjunctival defects. However, most epithelial denudation protocols are time-consuming and expensive or compromise HAM's basement membrane structure and matrix components. We have previously described a method to de-epithelialize HAM using ice-cold urea (uHAM). In this report, we used this method to provide tissue-engineered constructs with cultivated conjunctival epithelial cells on uHAM in two patients, one with a giant conjunctival nevus and the other with a large symblepharon. Autologous conjunctival epithelial cells harvested from incisional biopsies of these two patients were cultured on the uHAM scaffold. The transplantation of tissue-engineered constructs to patients' ocular surface immediately after the removal of lesions showed successful reconstruction of the ocular surface. Postoperatively, there were neither recurrence of lesions nor epithelial defects throughout the follow-up (up to 7 and 19 months, respectively). This report highlights the translational potential of an efficient and inexpensive method to prepare de-epithelialized HAM as a basement membrane scaffold for cell-based tissue-engineered treatments of ocular surface disorders. Stem Cells Translational Medicine 2019;8:620&626.
Project description:The ocular surface of the white domestic pig (Sus scrofa domestica) is used as a helpful model of the human ocular surface; however, a complete histological description has yet to be published. In this work, we studied porcine eyeballs with intact eyelids to describe and characterize the different structures that form the ocular surface, including the cornea and conjunctiva that covers the bulbar sclera, tarsi, and the nictitating membrane. We determined the distribution of goblet cells of different types over the conjunctiva and analyzed the conjunctival-associated lymphoid tissue (CALT). Porcine eyeballs were obtained from a local slaughterhouse, fixed, processed, and embedded in paraffin blocks. Tissue sections (4 ?m) were stained with hematoxylin/eosin, Alcian blue/Periodic Acid Schiff, and Giemsa. Slides were also stained with lectins from Arachis hypogaea (PNA) and Helix pomatia (HPA) agglutinins and immunostained with rabbit anti-CD3. We found that the porcine cornea was composed of 6-8 epithelial cell layers, stroma, Descemet's membrane, and an endothelial monolayer. The total corneal thickness was 1131.0±87.5 ?m (mean±standard error of the mean) in the center and increased to 1496.9±138.2 ?m at the limbus. The goblet cell density was 71.25±12.29 cells/mm, ranging from the highest density (113.04±37.21 cells/mm) in the lower palpebral conjunctiva to the lowest density (12.69±4.29 cells/mm) in the bulbar conjunctiva. The CALT was distributed in the form of intraepithelial lymphocytes and subepithelial diffuse lymphoid tissue. Lenticular-shaped lymphoid follicles, about 8 per histological section, were also present within the conjunctival areas. In conclusion, we demonstrated that the analyzed porcine ocular structures are similar to those of humans, confirming the potential usefulness of pig eyes to study ocular surface physiology and pathophysiology.
Project description:The aim of the study was to investigate the effects of the Allergan Intranasal Tear Neurostimulator (ITN) on conjunctival goblet cell (GC) degranulation.A randomized, double-masked, placebo-controlled crossover trial.A total of 15 subjects (5 normal and 10 dry eye) were enrolled in a 3-visit study consisting of 1 screening and 2 separate randomized-masked ITN treatments (sham extranasal or intranasal). Tear meniscus height (TMH) was measured by anterior segment optical coherence tomography before and after applications. Impression cytology (IC) was taken from the bulbar conjunctiva of the right eye for periodic acid-Schiff staining and from the left eye for MUC5AC mucin immunostaining at baseline and after each treatment. The ratio of degranulated to nondegranulated GCs was measured as a marker of secretion.In all participants, both inferior bulbar (IB) and temporal bulbar (TB) cytology specimens stained for MUC5AC revealed a significantly higher ratio of degranulated to nondegranulated GCs after the ITN (IB: 2.28 ± 1.27 and TB: 1.81 ± 1.01) compared to baseline (IB: 0.56 ± 0.55, P = .015) (TB: 0.56 ± 0.32, P = .003) and extranasal sham application (IB: 0.37 ± 0.29, P = .001) (TB: 0.39 ± 0.33, P = .001). When the same analysis was repeated in the dry eye or control groups, the ratio was significantly higher after ITN than the baseline ratio and ratio after extranasal application in both groups (P < .05). Moreover, although control subjects had a higher ratio of degranulated to nondegranulated GCs at baseline (0.75 ± 0.52) compared with the dry eye group (0.41 ± 0.27), the ratio became slightly higher in dry eye (2.04 ± 1.12 vs 1.99 ± 1.21 in control) after the ITN application. There was no significant difference between the IB or TB conjunctiva locations in terms of the effectiveness of the ITN application on conjunctival goblet cell secretory response.These preliminary results document that the Allergan ITN can stimulate degranulation of goblet cells in the conjunctiva, which is a promising new approach for the management of dry eye.
Project description:To investigate the effects of IL-13 on goblet cell proliferation, differentiation, and expression of mucin and immunomodulatory genes.Explants were excised from the conjunctiva of young C57BL/6 mice. Cultures received 200 ?L per week of either Keratinocyte media (KSFM) or KSFM supplemented with 10 ng/mL IL-13 and were incubated for 3 (D3), 7 (D7), or 14 (D14) days. Subsequently, cell proliferation was assessed or cultures were immunostained, collected for dot blot, or for reverse transcription (RT) and quantitative real-time PCR (qPCR) or for RT-PCR gene array.The cultured conjunctival epithelium expressed goblet cell associated keratin 7 and mucins MUC5AC and MUC2 and when stimulated with IL-13 showed increased proliferation at D3 and D7 (P < 0.05) compared with control. MUC5AC expression was increased in the IL-13-treated group at D3 and D14 (P < 0.05). IL-13-treated cultures showed increased chemokine ligand 26 (CCL26), chloride channel calcium activated channel 3 (CLCA3), fas ligand (FasL), and Relm-? at D7. All conjunctival cultures expressed MUC2, and its expression was decreased at D3 (P < 0.05) and increased at D14 (P < 0.05) with IL-13 treatment.This study demonstrated that conjunctival goblet cells are IL-13 responsive cells that produce factors known to maintain epithelial barrier, stimulate mucin production, and modulate immune response in nonocular mucosa when treated with IL-13. The functional significance of IL-13-stimulated factors remains to be determined.
Project description:Leiomyosarcomas of soft tissue are malignant tumours that are not infrequently encountered in clinical medicine and histopathology. Such sarcomas of the eye are, however, a rare occurrence.Herein, the histopathological features of a case of conjunctival leiomyosarcoma are described. A 38-year-old HIV-positive male, who was otherwise fit and healthy, presented with redness of his right eye and a mass of the conjunctiva. Clinically, he did not have any soft-tissue masses elsewhere in the body.He underwent monoblock excision of the conjunctival mass which was confirmed histologically to be a leiomyosarcoma. Unfortunately, the patient had not returned for follow-up examination.The differential diagnosis of a conjunctival spindle cell neoplasm is broad. While a spindle cell carcinoma is the most likely tumour, other tumours must be borne in mind so as not to misdiagnose primary sarcomas in this unusual location.
Project description:BACKGROUND:Until recently, synthetic fiber conjunctival granuloma (SFCG) is rarely reported and has been poorly understood. Our study was to explore the clinical features, histopathologic characteristics, surgical outcomes, and prognosis of SFCG after surgical excision. METHODS:Retrospective review of clinical findings, histopathological and immunohistochemical studies identified 18 cases of SFCG. Specimens were routinely processed and stained with H&E. Immunohistochemical stains for GMS, PAS, CD68 and CK-pan were also performed. RESULTS:Eighteen patients with an average age of 9.3?±?6.6?years had a tender white to red mass on the conjunctiva. All the lesions were completely removed, and none of the patients relapsed. Histologically, all of the specimens revealed inflammatory granulation tissues with a large number of inflammatory cells infiltration and the presence of synthetic fibers. Immunohistochemical stains were positive for CD68, CK, GMS and PAS. CONCLUSIONS:Synthetic fiber conjunctival granuloma is an uncommon lesion with foreign body sensation caused by inoculation of synthetic exogenous materials. These lesions are mostly unilateral and occur in the inferior conjunctival fornix. SFCGs are characterized by a large number of inflammatory cells infiltration and the presence of synthetic fibers. Surgical excision followed by topical corticosteroids has been clinically proven to be effective.
Project description:<h4>Purpose</h4>To evaluate the expression patterns of cytokeratin (K) 12, 13, and 19 in normal epithelium of the human ocular surface to determine whether K13 could be used as a marker for conjunctival epithelium.<h4>Methods</h4>Total RNA was isolated from the human conjunctiva and central cornea. Those transcripts that had threefolds or higher expression levels in the conjunctiva than the cornea were identified using microarray technique. Expression levels of three known signature genes and of two conjunctival genes, K13 and K19 were confirmed by using quantitative real-time PCR (qRT-PCR). Protein expression of K12, K13, and K19 was confirmed by immunostaining with specific antibodies on histologic sections of human sclerocornea that contained the conjunctiva, limbus, and cornea and on impression cytology (IC) specimens of the cornea and conjunctiva from normal donors. Double staining of K13/K12 and K19/K12 on histologic sections and IC specimens was performed.<h4>Results</h4>There were 337 transcripts that were preferentially expressed in the conjunctiva. K13 and K19 were among the top twenty transcripts in the conjunctiva and this preferential expression pattern of K13 and K19 was confirmed by qRT-PCR. Immunohistochemical studies showed that K13 was expressed at the posterior limbal epithelium and conjunctival epithelium but was totally absent in the cornea. K12 was expressed in the corneal and anterior limbal epithelia except for the basal layer and was absent from the conjunctiva. In contrast, K19 was detected in the corneal, limbal and conjunctival epithelia. Immunostaining of the IC specimens showed K12(+) epithelial cells in the corneal region, K13(+) cells in the conjunctival area, and K19(+) cells in the corneal and conjunctival specimens. Expression of K13 and K12 on the ocular surface was mutually exclusive on both the histologic and IC samples using double immunostaining.<h4>Conclusions</h4>K13 is more specific to the conjunctival epithelial cells than K19 and potentially could be used as a marker to identify conjunctival epithelial cells in limbal stem cell deficiency.
Project description:Conjunctivochalasis is a common cause of tear dysfunction due to the conjunctiva becoming loose and wrinkly with age. The current solutions to this disease include either surgical excision in the operating room, or thermoreduction of the loose tissue with hot wire in the clinic. We developed a near-infrared laser thermal conjunctivoplasty system. The system utilizes a 1460-nm programmable laser diode system as the light source. At this wavelength, a water absorption peak exists and the blood absorption is minimal, so the heating of redundant conjunctiva is even and there is no bleeding. A miniaturized handheld probe delivers the laser light and reshapes the laser into a 10 × 1 mm2 line on the working plane. A foot pedal is used to deliver a preset number of calibrated laser pulses. A fold of loose conjunctiva is grasped by a pair of forceps. The NIR laser light is delivered through an optical fiber and a laser line is aimed exactly on the conjunctival fold by a cylindrical lens. Ex vivo experiments using porcine eye was performed to investigate the induced shrinkage of conjunctiva and decide the optimal laser parameters. It was found that up to 45% of conjunctiva shrinkage could be achieved.