Changes in Meibum Lipid Composition With Ocular Demodex Infestation.

Purpose: The purpose of this study was to understand the impact of Demodex infection in the lipid component of meibum in patients. Methods: The meibum samples were collected from four groups of subjects: (1) Demodex-negative with non-MGD (D-M-; n = 10); (2) Demodex-positive with non-MGD (D+M-; n = 10); (3) Demodex-negative with MGD (D-M+; n = 10); and (4) Demodex-positive with MGD (D+M+; n = 10). A liquid chromatography-mass spectrometry (LC-MS) system consisting of ultra-performance liquid chromatography and a Q Exactive high-resolution mass spectrometer was used for lipids separation and detection. Results: Compared with the D-M- group, the D+M- group had lower levels of phosphatidylcholines (PCs) and lysophosphatidylcholines (LPCs) and higher levels of phosphatidylethanolamines (PEs). Compared with the D-M+ group, the levels of sphingomyelins (SMs) and PCs in the D+M+ group were decreased, whereas the levels of (O-acyl)-ω-hydroxy fatty acids (OAHFAs), ceramides (CERs), LPCs, and diacylglycerols (DGs) were significantly increased. Triacylglycerols (TGs), DGs, CERs, and OAHFAs were decreased in D-M+ group, whereas levels of PEs, phosphatidylinositols, and phosphatidylglycerols were increased in meibum obtained from the D-M+ group compared with those in the D-M- group. TGs, SMs, CERs, and PEs were decreased in the D+M+ group, whereas levels of LPCs, LPEs, PCs, and PEs were increased in meibum from the D-M+ group compared with those in the D+M- group. Conclusions: To the best of our knowledge, this is the first study to assess the changes in meibum from patients with ocular Demodex infestation. The significant increase of OAHFAs in the Demodex-positive group suggest that OAHFAs may be associated with the progress of ocular Demodex infections. Translational Relevance: OAHFAs could be a potential new therapeutic target for ocular Demodex infestation.

Hyperkeratinization and Proinflammatory Cytokine Expression in Meibomian Glands Induced by Staphylococcus aureus.

Purpose: This exploratory study aimed to investigate the morphological and pathological alterations of the meibomian gland (MG) with the Staphylococcus aureus crude extracts (SACEs) treatment. Methods: Mouse MG explants were cultured and differentiated with or without SACEs for 48 hours. Explant's viability and cell death were determined by thiazolyl blue tetrazolium bromide (MTT) assay and TUNEL assay. MG morphology was observed by Hematoxylin and Eosin staining. Lipid droplet production was detected by Nile Red staining and LipidTox immunostaining. The pro-inflammatory cytokines were detected by ELISA. The relative gene and protein expression in MG explants was determined via quantitative RT-PCR, immunostaining, and immunoblotting. The components of the SACEs were analyzed by immunoblotting and silver staining. Results: Our findings demonstrated that the SACEs treatment induced overexpression of keratin 1 (Krt1) in the ducts and acini of MG explants, accompanied by a decrease in viability and an increase in cell death in explants. Furthermore, the SACEs treatment dose-dependently increased the levels of TNF-α, IL-1ß, and IL-6 in MG explants. The SACEs treatment induced activation of the nuclear factor kappa B (NF-κB) and AIM2 (absent in melanoma 2)/ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) inflammasome signaling pathway in explants. Further investigation showed expression of the key adipogenesis-related molecule peroxisome proliferator-activated receptor γ was decreased after SACEs treatment. However, no change was found in the lipid synthesis of MG explants after treatment with the SACEs. Staphylococcal enterotoxins B (SEB) was detected in the SACEs. SEB induced the overexpression of Krt1 and IL-1ß in ducts and acini of MG explants. Conclusions: Our findings confirm that Staphylococcus aureus induced hyperkeratinization and pro-inflammatory cytokines expression in MG explants ducts and acini. These effects might be mediated by SEB. Activation of the NF-κB and AIM2/ASC signaling pathway is involved in this process.

Real dynamic assessment of tear film optical quality for monitoring and early prevention of dry eye.

To evaluate real dynamic assessment of tear film optical quality for monitoring and prevention of dry eye.Right eyes of 62 normal and 39 dry eye subjects were included. Dynamic measurement of objective scatter index (OSI) was performed by using the Optical Quality Analysis System II (OQAS II), correlation coefficient between OSI and time (CCOT) was calculated. According to whether the CCOT was significantly ascending, normal and dry eye groups were further subdivided for comparison. By using Scheimpflug-Placido topographer, non-invasive tear break-up time (NITBUT) was recorded, and a 2-dimensional precorneal tear film map was reconstructed and divided into central, middle, and peripheral corneal zones, distribution of tear break-up spots in the 3 corneal zones were analyzed.The numbers of tear break-up spots were higher in all the 3 corneal zones of the dry eye subjects (P < .01), when compared with the normal subjects. The Dry Eye subjects with ascending CCOT had the shortest NITBUT (P < .001-.034) and the most tear break-up spots over the whole cornea (P < .001-.044). Between the dry eye subjects with non-ascending CCOT and those with ascending CCOT, difference of tear break-up spots was found significant only in the peripheral corneal zone (P < .01).Non-ascending and ascending CCOT of dry eye patients reflect different stability of tear film. Real dynamic assessment of tear film optical quality is potential for monitoring and early prevention of dry eye.

Organotypic Culture of Mouse Meibomian Gland: A Novel Model to Study Meibomian Gland Dysfunction In Vitro.

Purpose: Meibomian glands are essential in maintaining the integrity and health of the ocular surface. Meibomian gland dysfunction (MGD), mainly induced by ductal occlusion, is considered as the major cause of dry eye disease. In this study, a novel in vitro model was established for investigating the role of inflammation in the process of MGD. Methods: Mouse tarsal plates were removed from eyelids after dissection and explants were cultured during various time ranging from 24 to 120 hours. Meibomian gland epithelial cells were further enzymatically digested and dissociated from tarsal plates before culturing. Both explants and cells were incubated in different media with or without serum or azithromycin (AZM). Furthermore, explants were treated with IL-1ß or vehicle for 48 hours. Analyses for tissue viability, histology, biomarker expression, and lipid accumulation were performed with hematoxylin and eosin (H&E) staining, immunofluorescence staining, and Western blot. Results: Higher viability was preserved when explants were cultured on Matrigel with immediate addition of culture medium. The viability, morphology, biomarker expression, and function of meibomian glands were preserved in explants cultured for up to 72 hours. Lipid accumulation and peroxisome proliferator-activated receptor γ (PPARγ) expression increased in both explants and cells cultured in media containing serum or AZM. Treatment with IL-1ß induced overexpression of Keratin (Krt) 1 in meibomian gland ducts. Conclusions: Intervention with pro-inflammatory cytokine IL-1ß induces hyperkeratinization in meibomian gland ducts in vitro. This novel organotypic culture model can be used for investigating the mechanism of MGD.

Intense Pulsed Light Therapy for Patients with Meibomian Gland Dysfunction and Ocular Demodex Infestation.

To observe the clinical changes of meibomian gland dysfunctipn (MGD) and ocular Demodex infestation after intense pulsed light (IPL) treatment to further examine the mechanism of IPL treating patients with MGD and ocular Demodex infestation. The medical records of 25 patients (49 eyes) with MGD treated with IPL, were retrospectively examined to determine outcomes. Associated ocular-surface parameters (ocular surface disease index, OSDI; lipid layer thickness, LLT; noninvasive first breakup time, NIF-BUT; noninvasive average breakup time, NIAvg-BUT; tear film breakup area, TBUA; Schirmer I Test, SIT; corneal fluorescein staining, CFS), eyelid margin abnormalities, meibum quality and expressibility, MG morphological parameters (macrostructure and microstructure), and the number of Demodex infestation were examined before and after treatment. The MG microstructure and the Demodex infestation were examined via in vivo confocal microscopy (IVCM). The results showed that there were statistically significant differences in associated ocular-surface parameters (all P<0.05) before and after IPL treatment, except SIT (P=0.065). Eyelid margin abnormalities, meibum quality and expressibility obviously improved in upper and lower eyelid after IPL treatment (all P<0.0001). MG macrostructure (MG dropouts) decreased in upper (P=0.002) and lower eyelid (P=0.001) after IPL treatment. The nine parameters of MG microstructure in upper and lower eyelid all distinctly improved after IPL treatment (all P<0.0001). The mean number of Demodex mites on the upper lid margin (6.59±7.16 to 3.12±3.81/9 eyelashes) and lower lid margin (2.55±2.11 to 1.29±1.53/9 eyelashes) significantly reduced after IPL treatment (all P<0.0001). The Demodex eradication rate was 20% (8/40) in upper lid margin and 34.15% (14/41) in lower lid margin. These findings indicate that IPL shows great therapeutic potential for patients of MGD and ocular Demodex infestation.

[Observation on treatment of lacrimal passage obstruction under lacrimal endoscope].

OBJECTIVE: To evaluate the therapeutic efficacy of lacrimal endoscope treatment for lacrimal passage obstruction, and to compare the effectiveness of endoscopically controlled laser surgery and micro-drill surgery for lacrimal passage obstruction. METHODS: It was a prospective random controlled trial. Eighty nine patients (104 eyes) with lacrimal passage obstruction, including presacral canalicular obstruction (PSCO) and nasolacrimal duct obstruction (NLDO), were collected from September 2006 to December 2006 in Department of Ophthalmology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology. Patients were examined by endoscopy of the lacrimal drainage system under local anesthesia to detect the obstruction and changes of lacrimal mucous membrane. The obstructions were treated with laser or microdrill. Irrigation was performed to prove the recanalization of the lacrimal passage followed by injected ointment with 0.3% tobramycin and 0.1% dexamethasone into the lacrimal passage. All patients were followed up after the operation for 9-12 months. The difference between the laser and the microdrill treatment was observed. Chi-square test was used to evaluate the curative effect and complications differences between these two groups. RESULTS: The obstruction scene in the lacrimal passage of 89 patients could be observed effectively. All obstructions (104/104 eyes) were eliminated after the operation. Through the follow-up, the cure rate reached 78.85% (82/104 eyes). The cure rate of PSCO group and NLDO group, reached 77.78% (42/54 eyes) and 80.00% (40/50 eyes), respectively (chi2 = 0.077, P = 0.782). The cure rate of laser group and micro-drill group, was 80.43% (37/46 eyes) and 77.59% (45/58 eyes), respectively (chi2 = 0.125, P = 0.724). The cure rate of laser treatment was 89.66% (26/29 eyes) in the PSCO group and 64.71% (11/17 eyes) in the NLDO group (P = 0.040). The cure rate of micro-drill treatment was 64.00% (16/25 eyes) in the PSCO group and 87.88% (29/33 eyes) in the NLDO group (chi2 = 4.664, P = 0.031). Hemorrhage and palpebral edema occurred in 10.87% (5/46 eyes) and 4.35% (2/46 eyes) after laser treatment, respectively. Percentage of hemorrhage and palpebral edema after the micro-drill treatment was 55.17% (32/58 eyes) (compared to the laser group, chi2 = 21.969, P = 0.000) and 6.90% (4/58 eyes) (compared to the laser group, chi2 = 0.017, P = 0.896). CONCLUSIONS: Lacrimal passage obstruction can be observed and treated directly through the endoscopy of lacrimal drainage system. Choosing an appropriate surgical procedure according to the locations of the obstruction can be helpful for improving the effectiveness of the operation.

[Effect on the proliferation of bovine corneal endothelial cells by small hairpin RNA interference targeting p27Kip1].

OBJECTIVE: To clarify the proliferation of bovine corneal endothelial cells (bCEC) by interference with the recombinant plasmid of short hairpin RNA (shRNA) against p27Kip1, a kind of cyclin-dependent kinase inhibitor (CKI). METHODS: It was an experimental study. Three p27Kip1-shRNA template DNA sequences containing small hairpin structure were designed and synthesized as experimental groups. Plasmid expressing irrelevant shRNA with a random combination was used as negative shRNA. The products were inserted into the Pgensil-1 plasmid and the recombinant plasmid of Pgenesil-P1, Pgenesil-P2, Pgenesil-P3 and Pgenesil-HK were constructed. The recombinant plasmids were transfected into bCEC cells with liposome and a blank group. The expression of mRNA and protein of p27Kip1 was detected by RT-PCR and Western blot after stable transfection, and the plasmid with the best inhibitory effect was selected. The growth of the experimental group, Pgenesil-HK group and blank group were assessed by MTT. The influence of shRNA-p27Kip1 on bCEC cell cycle was deteceted by flow cytometry (FCM). All statistical analyses were performed using one-way ANOVA. RESULTS: Restrictive enzyme digestion and sequence analysis showed that four recombinant plamids were constructed successfully and the aim sequence was obtained. The expression of p27Kip1 mRNA and p27Kip1 protein of Pgenesil-P1 group, Pgenesil-P2 group and Pgenesil-P3 group were all lower than that in the control group, including blank group and negative siRNA group. The inhibitive rate of mRNA reached 32.71%, 67.76% and 80.28% (F = 453.102, P = 0.000 in each group) and the inhibitive rate of protein reached 29.27%, 64.73% and 76.13% (F = 75.385, P = 0.000 in each group) compared with the blank group. As the lowest expression among the three positive shRNA group, Pgenesil-P3 was selected for the next steps. There was no significant difference between blank group and negative Pgenesil-HK of the expression of p27Kip1 protein (P = 0.356) and the express of p27Kip1 mRNA (P = 0.246). Compared with the control group and the blank group, the growth of the bCEC transfected by Pgenesil-P3 was significantly promoted with increased cell percent of S-phrase (F = 334.957, P = 0.000) and decreased cell percent of G1-phrase (F = 134.224, P = 0.000). CONCLUSIONS: shRNA-p27Kip1 can down-regulate the expression of bCEC effectively and increase the growth of bCEC. shRNA-p27Kip1 RNA interference may be an effective method to promote the proliferation of CEC.