Comparative efficacy of 0.1% and 0.15% Sodium Hyaluronate on lipid layer and meibomian glands following cataract surgery: A randomized prospective study.

PURPOSE: To compare the efficacy of a 0.15% HA with that of 0.1% HA eye drops for DES after cataract surgery. METHODS: This study was double blinded, randomized and prospective study, and conducted in 69 participants (70 eyes) from Pusan National University Yangsan Hospital and executed from February 1, 2022 to November 30, 2022. Participants were adult cataract patients with normal lid position, not suffering from any other ocular disease and not meet the exclusion cirteria of clinical trial. Participants were randomly divided into two groups: 35 participants (17 males and 18 females) in the 0.1% HA group and 34 participants (19 males and 15 females) in the 0.15% HA group, receiving treatment six times daily for 6 weeks following cataract surgery. Subjective and objective assessments were performed at preoperative and postoperative visits, including ocular surface disease index score, tear break up time, corneal staining score, Schirmer's I test score, lipid layer thickness), meiboscore, and biochemical analysis of the eye drops. RESULTS: Throughout the study, the postoperative ocular surface disease index score was significantly lower in the group receiving 0.15% hyaluronic acid than in the group receiving 0.1% hyaluronic acid. Additionally, the postoperative ocular surface disease index score showed a significant positive correlation with the postoperative use of 0.15% hyaluronic acid and the preoperative Schirmer's I test score. In multivariate analysis, treatment with 0.15% hyaluronic acid and the preoperative ocular surface disease index score were significant independent parameters affecting the postoperative ocular surface disease index score. CONCLUSION: The use of 0.15% hyaluronic acid is recommended for its potential advantages in alleviating symptoms following cataract surgery, making it a viable alternative to traditional 0.1% hyaluronic acid treatment. TRIAL REGISTRATION: ISRCTN95830348.

The effect of airborne particulate matter 2.5 (PM2.5) on meibomian gland.

Exposure to particulate matters in air pollution of 2.5 µm or less (PM2.5) was associated with loss of meibomian glands. The aim of this study was to verify that PM2.5 could directly impact meibomian gland epithelial cells and damage their function. To investigate the impact of PM2.5 on meibomian gland, immortalized human meibomian gland epithelial cells were treated with various concentrations of PM2.5in vitro. Meibomian gland cell microstructure, cell viability, expression of proliferating cell nuclear antigen and IL-1ß, and intracellular accumulation of acidic vesicles were measured by transmission electron microscopy, cell counting, Western blot and LysoTracker staining, respectively. To further study the effect of PM2.5in vivo, male C57BL/6J mice were treated with 5 mg/ml PM2.5 or vehicle for 3 months. Corneal fluorescein staining and ocular examinations were done before and after the treatment. Eyelids tissues were processed for morphological studies, immunostaining and Oil Red O staining. Our data suggest that exposure to PM2.5 caused significant meibomian gland dropout, clogged gland orifice and increased corneal fluorescein staining that were consistent with the clinical presentations of meibomian gland dysfunction. Prominent changes in the morphology and ultrastructure of meibomian glands was observed with PM2.5 treatment. PM2.5 promoted ductal keratinization, inhibited cell proliferation, induced cell apoptosis and increased Interleukin-1ß production in meibomian gland epithelial cells. This study may explain the association between PM2.5 exposure and meibomian gland dropout observed in clinic. PM2.5 resuspension instillation could be used to induce a meibomian gland dysfunction animal model.

Effects of topical ketorolac tromethamine on tear parameters, meibography, goblet cell density, and conjunctival oxidative stress in healthy dogs.

OBJECTIVES: The objective of the study was to evaluate whether a twice-daily instillation of 0.45% preservative-free ketorolac tromethamine (FKT) or 0.4% benzalkonium chloride-preserved ketorolac tromethamine (BACKT), every 12 h for 30 days may affect tear film parameters and the meibography in healthy dogs. Additionally, we assessed whether the same treatments irritated the ocular surface, affected goblet cell density (GCD), and the levels of oxidative stress biomarkers (OSB) in the conjunctiva of the same dogs. PROCEDURES: Experimental and masked comparison study. In 11 healthy dogs baseline values of the lipid layer thickness, tear meniscus height, non-invasive tear breakup time (NI-TFBT), and the meibomian gland (MG) loss were assessed by OSAvet®. For each dog, one eye received 40 µL of BACKT, while the other received 40 µL FKT, every 12 h for 30 consecutive days. Tear parameters and meibography were repeated 15, 30, and 60 days post-treatments. Conjunctival hyperemia and blepharospasm were monitored at the same time points. At baseline and Day 30, a conjunctival biopsy was collected for GCD and OSB determination. RESULTS: Conjunctival hyperemia and blepharospasm were not observed. At Day 15, the MG loss increased only in FKT-treated eyes (p < .001). On Day 30, both treatment groups showed increased MG loss, shortened NI-TFBT, and reduced GCD and catalase (p < .05). At Day 30, BACKT-treated eyes showed lower levels of superoxide dismutase (SOD) (p = .006) and higher levels of malondialdehyde (MDA) (p = .02). Differences between treatments were not observed for any parameter at any time point (p > .05). 60 days after treatment, OSAvet® parameters tended to return to values assessed at baseline; however, significant differences remained for MG loss (p < .05). CONCLUSIONS: Twice-daily instillation of KT, containing or not BAC, for 30 consecutive days shortened NI-TFBT, decreased GCD, and increased the MG loss in healthy dogs. KT should be used with caution when prescribed for long periods, particularly in patients with tear film abnormalities. However, future controlled studies using KT, BAC, and other topical NSAIDs are indicated to further support this finding.

Periplocin ameliorates mouse age-related meibomian gland dysfunction through up-regulation of Na/K-ATPase via SRC pathway.

Age-related meibomian gland dysfunction (MGD) is the main cause of evaporative dry eye disease in an aging population. Decreased meibocyte cell renewal and lipid synthesis are associated with age-related MGD. Here, we found an obvious decline of Ki67, ΔNp63, and Na+/K+ ATPase expression in aged meibomian glands. Potential Na+/K+ ATPase agonist periplocin, a naturally occurring compound extracted from the traditional herbal medicine cortex periplocae, could promote the proliferation and stem cell activity of meibocyte cells in vitro. Moreover, we observed that periplocin treatment effectively increased the expression of Na+ /K+ ATPase, accompanied with the enhanced expression of Ki67 and ΔNp63 in aged meibomian glands, indicating that periplocin may accelerate meibocyte cell renewal in aged mice. LipidTox staining showed increased lipid accumulation after periplocin treatment in cultured meibomian gland cells and aged meibomian glands. Furthermore, we demonstrated that the SRC pathway was inhibited in aged meibomian glands; however, it was activated by periplocin. Accordingly, the inhibition of the SRC signaling pathway by saracatinib blocked periplocin-induced proliferation and lipid accumulation in meibomian gland cells. In sum, we suggest periplocin-ameliorated meibocyte cell renewal and lipid synthesis in aged meibomian glands via the SRC pathway, which could be a promising candidate for age-related MGD.

The Ocular Surface Characteristics in Prostate Cancer Patients Treated with Androgen Deprivation Therapy.

BACKGROUND: To investigate the association of long-term androgen deprivation therapy (ADT) with ocular surface characteristics in prostate cancer patients. METHODS: A total of 30 male prostate cancer patients who received ADT were selected. All candidates were scored using the Ocular Surface Disease Index (OSDI) and subsequently divided into two groups containing 9 symptomatic patients (scores >12) and 21 asymptomatic patients (scores ≤ 12). Another 20 healthy age-matched males were selected as the control group. Each candidate was assessed with respect to eyelid margin abnormality, tear film break-up time (NI-BUT), tear meniscus height (TMH), meiboscore, meibum expressibility, and demodex infection. RESULTS: The NI-BUT in the ADT group was significantly shorter than that in the control group. The scores for OSDI, eyelid margin abnormality, meibum expressibility, and meiboscores were significantly higher in the ADT group (P < 0.05). Moreover, the NI-BUT in the symptomatic ADT group was significantly shorter than that in the asymptomatic ADT group (P < 0.05). The meiboscores and meibum expressibility score in the symptomatic ADT group were significantly higher than those in the asymptomatic ADT group (P < 0.05). The presence of demodex in the symptomatic ADT group was also higher than that in the asymptomatic ADT group (P < 0.05).The length of time that patients had been taking ADT was positively correlated with meiboscores and negatively correlated with NI-BUT. CONCLUSION: Androgen levels were associated with significant changes in relative meibomian gland function. Subjective symptoms, such as dryness and foreign body sensation, were more obvious in prostate cancer patients receiving ADT, which may be caused by MGD and demodex infection. It's recommended that more attention be paid to the ocular surface in prostate cancer patients taking ADT by performing examination of NI-BUT and meibomian gland morphology and function with a view to providing more comprehensive prevention and treatment protocols.

Semi-quantification of lipids in human meibomian gland epithelial cells using dual staining microplate assays.

Two spectrophotometric microplate assays with dual staining for either fluorescent Nile red (NR) plus 4,6-diamidino-2-phenylindole (DAPI) or non-fluorescent Oil red O (ORO) plus Crystal violet (CV) were applied and optimised to evaluate the lipid producing capacity of immortalised human meibomian gland epithelial cells (iHMGEC). Cells were treated with rosiglitazone (Rosi, 10-50 µM), a known lipid producing inducer for iHMGEC, and were analysed for lipids using the NR-DAPI and ORO-CV microplate assays. The lipid producing capacity of iHMGEC after each treatment was determined by normalising lipid quantity (measured with NR or ORO) to cell number (measured with DAPI or CV). The dye concentrations of NR 1 µg/mL, DAPI 5 µg/mL, ORO 0.3% (v/v) and CV 0.5% (v/v), provided optimal linearity and coverage of signals over a range of cell densities (corresponding to 10-100% cell confluence). Both NR-DAPI and ORO-CV showed a dose-dependent effect of Rosi on lipid production in iHMGEC, consistent with the results reported previously using traditional microscopic imaging methods. The microplate assays offer a rapid, high throughput and objective measurement of the amount of lipids in iHMGEC (and potentially other lipid-producing cells) and can be used for screening the effects of biological agents or incubation changes on lipid production in cells in future studies.

Triacylglycerol lipidome from human meibomian gland epithelial cells: Description, response to culture conditions, and perspective on function.

Preliminary work has shown that select triacylglycerols (TAGs) are upregulated in a preclinical model of MGD, suggesting that TAGs may be an important outcome variable in research involving human meibomian gland epithelial cells (HMGECs). The purpose of this study was to explore the HMGEC TAG lipidome in culture conditions known to influence differentiation. HMGECs were differentiated in DMEM/F12 with 10 ng/ml EGF, FBS (2% or 10%), and rosiglitazone (0, 20, or 50 µM) for two or five days. Following culture, lipids were extracted, processed, and directly infused into a Triple TOF 5600 mass spectrometer (SCIEX, Framingham, MA) with electrospray ionization. MS and MS/MSALL spectra were acquired in the positive ion mode and performed with the SWATH technology. Only the TAGs that were present in all 48 samples were included in the analysis. Multiple regression techniques were utilized to assess the effects of each factor (FBS, rosiglitazone, and culture duration) on each expressed TAG. The HMGEC TAG lipidome consisted of 115 TAGs with 42-62 carbons and zero to 10 double bonds. Fatty acyl chains had 14 to 26 carbons and zero to five double bonds. C18:1 (oleic acid, 25/115, 21.7%) and C16:0 (palmitic acid, 16/115, 13.9%) were the most common fatty acids. FBS, rosiglitazone, and culture duration were significant predictors for 93 TAGs (80.9%) with R2 values ranging from 0.20 to 0.77 (p < 0.05). FBS and rosiglitazone achieved significance (p < 0.05) for 80 (69.6%) and 67 TAGs (58.3%), respectively. Rosiglitazone demonstrated a selective upregulation of TAGs containing 16 or 18 carbons. Culture duration reached significance (p < 0.05) for only 36 TAGs (31.3%). When comparing the 10 most abundant C18:1-containing TAGs in meibum, FBS was a negative predictor for five TAGs (mean standardized coefficient [SC] = -0.58, p < 0.001), rosiglitazone was a positive predictor for six TAGs (mean SC = 0.41, p ≤ 0.03), and culture duration weakly influenced one TAG (SC = 0.27, p = 0.008). FBS and rosiglitazone, unlike culture duration, are powerful modulators of the TAG profile. Rosiglitazone induces changes that could be consistent with fatty acid synthesis, suggesting that quantifying the TAG lipidome could be an indirect measure of lipogenesis. Though both have been described as differentiating agents, FBS and rosiglitazone induce opposing effects on meibum-relevant TAGs. Culturing with rosiglitazone is associated with a TAG profile that is more consistent with the expected outcome of lipogenesis and with the profile observed in normal human meibum.

A prospective multicentre study of intravitreal injections and ocular surface in 219 patients: IVIS study.

PURPOSE: To assess the impact of intravitreal injections (IVTI) on ocular surface of patients treated with multiple injections. METHODS: Prospective, tricentric study conducted in patients treated with unilateral IVTI. An asepsis protocol with povidone-iodine was used for all patients during IVTI. The primary endpoint was the difference between the pre-IVTI Ocular Surface Disease Index (OSDI 1) score and that measured on day one (D1) post-IVTI (OSDI 2). Secondary endpoints were the evaluation of predictive factors for OSDI scores, pain assessment on D1, and the Lacrydiag® analysis of tears from the injected eye versus contralateral eye before IVTI. RESULTS: Two hundred and nineteen patients with a mean age of 75.9 ± 10 years were included. The mean OSDI2-OSDI1 difference was 19.2 ± 20.6 (p < 0.001). The mean noninvasive tear break-up time was 6.41 ± 4.59 seconds in the injected eye versus 7.36 ± 4.36 seconds in the contralateral eye (p < 0.001). In the multivariate analysis, the factors significantly associated with the OSDI 2 score were the OSDI 1 score (p < 0.001), the pain score on D1 (p < 0.001) the number of instilled glaucoma eye drop (p = 0.01) and a centre effect (centres 2 and 3 versus centre 1, p < 0.001). CONCLUSION: Our results confirm the impairment of the ocular surface and quality of life immediately after an IVTI. These results suggest 3 levels of action to improve the immediate tolerance: improving the basal status of the ocular surface, reducing the contact time with povidone-iodine that might be toxic to the surface, and improving immediate post-IVTI treatment.

Efficacy of 0.05% cyclosporine A on the lipid layer and meibomian glands after cataract surgery: A randomized, double-masked study.

PURPOSE: To quantitatively evaluate the effects of 0.05% cyclosporine A (CsA) on lipid layer thickness (LLT) and meibomian glands after cataract surgery using the LipiView® ocular surface interferometer. METHODS: This study was a prospective randomized double-masked clinical trial conducted by Pusan National University Yangsan Hospital between April 04, 2019, and November 31, 2019. Sixty-two participants were recruited, and 12 of them were not enrolled because they had undergone previous treatments for ocular surface diseases. The participants were adult patients with cataract, exhibiting normal lid position; they did not present any other ocular disease and did not meet the exclusion criteria of the clinical trial. Fifty subjects were enrolled in the study. The randomized subjects received treatment with 0.05% CsA (group A) or 0.5% carboxymethyl cellulose (CMC) (group B) over the 3 months following the cataract surgery. Subjective and objective assessments were performed at preoperative and postoperative visits. Ocular Surface Disease Index (OSDI), tear breakup time (TBUT), and Schirmer's I test were performed by the same surgeon, and LLT and meiboscore were determined using the LipiView® interferometer. RESULTS: Fifty subjects subjects enrolled consisted of men (50%) and women (50%), with a mean (SD) age of 65.94 (10.35) years. Four subjects in group A and five in group B were excluded from the analysis as they were lost to follow-up within 1 month after cataract surgery. Thus, the study comprised 41 eyes of 41 subjects; 21 subjects were treated with CsA and 20 subjects with CMC. Comparing the clinical measurements between groups A and B taken at the last visit, while controlling the effects of the preoperative values, TBUT and LLT showed significant differences (p = 0.035 and p = 0.047, respectively, by ANCOVA). The TBUT between the subjects using CsA and those using CMC after cataract surgery showed a significant difference during follow up (p = 0.003 by repeated measures ANOVA). In the multivariate analysis, preoperative LLT and the use of CsA were found to be independent parameters for postoperative LLT (R2 = 0.303; p = 0.008 and p = 0.045, respectively), whereas the follow-up duration exhibited a positive correlation with the difference between the preoperative and postoperative values of LLT in the group treated with CsA (R2 = 0.738 and p < 0.001). CONCLUSION: Treatment with 0.05% CsA following cataract surgery is effective in improving TBUT and LLT in comparison with 0.5% CMC. A higher preoperative value of LLT and the postoperative use of CsA could be significant determinants of a higher postoperative LLT value. TRIAL REGISTRATION: ISRCTN registry with ISRCTN 10173448.

Effects of Terpinen-4-ol on Meibomian Gland Epithelial Cells In Vitro.

PURPOSE: Infestation with demodex mites has been linked to the development of chalazion, meibomian gland dysfunction, and blepharitis. An effective treatment is the eyelid application of terpinen-4-ol (T4O), a tea tree oil component. However, T4O is also known to be toxic to nonocular epithelial cells. We hypothesize that T4O toxicity also extends to human meibomian gland epithelial cells (HMGECs). METHODS: Immortalized (I) HMGECs were cultured with varying concentrations (1.0%-0.001%) of T4O under proliferating or differentiating conditions up to 5 days. Experimental procedures included analyses of cell appearance, survival, P-Akt signaling, lysosome accumulation, and neutral lipid content. RESULTS: Our findings show that T4O causes a dose- and time-dependent decrease in the cell survival of IHMGECs. After 15 minutes of exposure to 1% T4O, IHMGECs exhibited rounding, atrophy, and poor adherence. Within 90 minutes of such treatment, almost all cells died. Reducing the T4O concentration to 0.1% also led to a marked decrease in P-Akt signaling and cell survival of IHMGECs. Decreasing the T4O amount to 0.01% caused a slight, but significant, reduction in the IHMGEC number after 5 days of culture and did not influence the ability of these cells to differentiate. CONCLUSIONS: T4O, even at levels 10-fold to 100-fold lower than demodicidal concentrations, is toxic to HMGECs in vitro.

Toxicity of the cosmetic preservatives parabens, phenoxyethanol and chlorphenesin on human meibomian gland epithelial cells.

Recently, we discovered that the cosmetic preservatives, benzalkonium chloride and formaldehyde, are especially toxic to human meibomian gland epithelial cells (HMGECs). Exposure to these agents, at concentrations approved for human use, leads within hours to cellular atrophy and death. We hypothesize that these effects are not unique, and that other cosmetic preservatives also exert adverse effects on HMGECs. Such compounds include parabens, phenoxyethanol and chlorphenesin, which have been reported to be toxic to corneal and conjunctival epithelial cells, the liver and kidney, as well as to irritate the eye. To test our hypothesis, we examined the influence of parabens, phenoxyethanol and chlorphenesin on the morphology, signaling, survival, proliferation and lipid expression of immortalized (I) HMGECs. These cells were cultured under proliferating or differentiating conditions with varying concentrations of methylparaben, ethylparaben, phenoxyethanol and chlorphenesin for up to 5 days. We monitored the signaling ability, appearance, number and neutral lipid content of the IHMGECs, as well as their lysosome accumulation. Our findings show that a 30-min exposure of IHMGECs to these preservatives results in a significant reduction in the activity of the Akt pathway. This effect is dose-dependent and occurs at concentrations equal to (chlorphenesin) and less than (all others) those dosages approved for human use. Further, a 24-h treatment of the IHMGECs with concentrations of methylparaben, ethylparaben, phenoxyethanol and chlorphenesin close to, or at, the approved human dose induces cellular atrophy and death. At all concentrations tested, no preservative stimulated IHMGEC proliferation. Of particular interest, it was not possible to evaluate the influence of these preservatives, at close to human approved dosages, on IHMGEC differentiation, because the cells did not survive the treatment. In summary, our results support our hypothesis and show that methylparaben, ethylparaben, phenoxyethanol and chlorphenesin are toxic to IHMGECs.

Ocular surface changes in the treatment of rosacea: comparison between low-dose oral isotretinoin and doxycycline / Alterações da superfície ocular no tratamento da Rosácea: comparação entre isotretinoína oral em baixa dosagem e doxiciclina

ABSTRACT Purpose: To compare the impact of ocular changes between systemic treatment with doxycycline and low-dose oral isotretinoin in patients with moderate-to-severe papulopustular rosacea. Methods: Patients were randomized to receive either isotretinoin 0.3-0.4 mg/kg (group A) or doxycycline 100 mg/day (group B) for 16 weeks. Ocular symptoms were searched and evaluated, including best-corrected visual acuity (BCVA), Schirmer test, breakup time, rose bengal staining score, and meibomian gland dysfunction grading. The patients were retested at the end of treatment. Results: The present study included 39 patients (30 females and 9 males). Best-corrected visual acuity was > 20/30 in >90% of patients in both groups and did not change after treatment. After treatment, improvement in ocular symptoms and meibomian gland dysfunction was more pronounced in group B (p<0.05); the other parameters did not reach statistical significance. Conclusion: Doxycycline improved meibomian gland dysfunction, ocular symptoms, and ocular surface in patients with rosacea. Even though some patients experienced worsening meibomian gland dysfunction and symptoms, no subject experienced any serious complications after administration of low-dose isotretinoin.

RESUMO Objetivos: Comparar o impacto das alterações oculares entre o tratamento sistêmico de doxiciclina e isotretinoína em baixa dosagem em pacientes com rosácea papulopustulosa moderada a grave. Métodos: Os pacientes form randomizados para receber isotretinoína 0,3 a 0,4 mg/kg (grupo A) ou doxiciclina 100mg/dia (grupo B) por 16 semanas. Os sintomas oculares foram pesquisados e avaliados, incluindo melhor acuidade visual corrigida, teste de Schirmer, tempo de ruptura do filme lacrimal, coloração de rosa bengala e graduação da disfunção de glândula de Meibomius. Os pacientes foram novamente testados no final do tratamento. Resultados: O presente estudo incluiu 39 pacientes (30 mulheres e 9 homens). A melhor acuidade visual corrigida foi >20/30 em >90% dos pacientes em ambos os grupos e não se alterou após o tratamento. A melhora dos sintomas oculares e da disfunção de glândula de Meibomius foi mais pronunciada no grupo B (p<0,05) após o tratamento; as demais variáveis não atingiram significância estatística. Conclusão: A doxiciclina melhorou a disfunção de glândula de Meibomius, os sintomas oculares e a superfície ocular de pa cientes com rosácea. Mesmo que alguns pacientes tenham piorado a disfunção e os sintomas da glândula de Meibomius, nenhum indivíduo apresentou complicações graves após a admi nistração de baixas doses de isotretinoína.

The Role of Hypoxia-Inducible Factor 1α in the Regulation of Human Meibomian Gland Epithelial Cells.

Purpose: We recently discovered that a hypoxic environment is beneficial for meibomian gland (MG) function. The mechanisms underlying this effect are unknown, but we hypothesize that it is due to an increase in the levels of hypoxia-inducible factor 1α (HIF1α). In other tissues, HIF1α is the primary regulator of cellular responses to hypoxia, and HIF1α expression can be induced by multiple stimuli, including hypoxia and hypoxia-mimetic agents. The objective of this study was to test our hypothesis. Methods: Human eyelid tissues were stained for HIF1α. Immortalized human MG epithelial cells (IHMGECs) were cultured for varying time periods under normoxic (21% O2) or hypoxic (1% O2) conditions, in the presence or absence of the hypoxia-mimetic agent roxadustat (Roxa). IHMGECs were then processed for the analysis of cell number, HIF1α expression, lipid-containing vesicles, neutral and polar lipid content, DNase II activity, and intracellular pH. Results: Our results show that HIF1α protein is present in human MG acinar epithelial cells in vivo. Our findings also demonstrate that exposure to 1% O2 or to Roxa increases the expression of HIF1α, the number of lipid-containing vesicles, the content of neutral lipids, and the activity of DNase II and decreases the pH in IHMGECs in vitro. Conclusions: Our data support our hypothesis that the beneficial effect of hypoxia on the MG is mediated through an increased expression of HIF1α.

Effects of Different Doses of Systemic Isotretinoin on Eyes: A Histopathological and Immunohistochemical Study in Rats.

PURPOSE: To evaluate ocular side effects associated with systemic isotretinoin histopathologically. METHODS: In this multicenter study, a total of 15 male and 15 female rats were randomly divided into 3 equal groups according to the oral dose of isotretinoin they were administered: 0 mg/kg/d (group A), 7.5 mg/kg/d (group B), and 15 mg/kg/d (group C). Biopsy specimens were taken from the globe conjunctiva, cornea, and eyelid conjunctiva. Expression levels of human beta-defensin-1, human beta-defensin-2, toll-like receptor (TLR)-2, and TLR-4 were evaluated by immunohistochemical methods. RESULTS: The number of goblet cells in eyelid conjunctiva was significantly lower in group B than that in group A and group C (P = 0.002). The sizes of meibomian gland acini were significantly smaller in group B and group C than those in group A (P < 0.001). Fibrosis of eyelid conjunctiva was significantly higher in group C and group B than that in group A (P = 0.002). The levels of staining of TLR-4 in the cornea with group B were significantly lower compared with group C (P = 0.035). CONCLUSIONS: Our study suggests that isotretinoin in the early period affects eyelid conjunctiva and meibomian glands without affecting the globe conjunctiva and cornea. Occurrence of the initial symptoms of isotretinoin on the eyelids, especially on the meibomian glands, suggests that the symptoms of patients occur because of evaporative dry eye.

Ocular surface changes in the treatment of rosacea: comparison between low-dose oral isotretinoin and doxycycline.

PURPOSE: To compare the impact of ocular changes between systemic treatment with doxycycline and low-dose oral isotretinoin in patients with moderate-to-severe papulopustular rosacea. METHODS: Patients were randomized to receive either isotretinoin 0.3-0.4 mg/kg (group A) or doxycycline 100 mg/day (group B) for 16 weeks. Ocular symptoms were searched and evaluated, including best-corrected visual acuity (BCVA), Schirmer test, breakup time, rose bengal staining score, and meibomian gland dysfunction grading. The patients were retested at the end of treatment. RESULTS: The present study included 39 patients (30 females and 9 males). Best-corrected visual acuity was > 20/30 in >90% of patients in both groups and did not change after treatment. After treatment, improvement in ocular symptoms and meibomian gland dysfunction was more pronounced in group B (p<0.05); the other parameters did not reach statistical significance. CONCLUSION: Doxycycline improved meibomian gland dysfunction, ocular symptoms, and ocular surface in patients with rosacea. Even though some patients experienced worsening meibomian gland dysfunction and symptoms, no subject experienced any serious complications after administration of low-dose isotretinoin.

Effects of Preservative-free 3% Diquafosol in Patients with Pre-existing Dry Eye Disease after Cataract Surgery: A Randomized Clinical Trial.

Dry eye disease (DED) after cataract surgery has become a critical concern, and various therapeutic options have been developed. Recently, preservative-free diquafosol ophthalmic solution has been introduced; however, its therapeutic effect on DED after cataract surgery has not been reported. We investigated the efficacy of preservative-free diquafosol in patients with pre-existing DED after cataract surgery. We divided subjects who were diagnosed with DED and scheduled to undergo cataract surgery, into 3 groups (preservative-free diquafosol, group 1; preservative-containing diquafosol, group 2; preservative-free hyaluronate, group 3), and each eye drops was administered 6 times daily after surgery. Tear break up time (TBUT), Ocular Surface Disease Index (OSDI), corneal staining score, lid margin abnormality, and meibum quality improved over time in group 1. Groups 1 and 2 had significantly superior TBUT, meibomian gland dysfunction grade, and meibomian gland expressibility throughout the study period than group 3. Meibum quality of group 1 was significantly better than group 2 at 1 and 3 months after surgery. Preservative-free diquafosol showed better efficacy in treating DED after cataract surgery than preservative-containing diquafosol or preservative-free hyaluronate. Preservative-free diquafosol may serve as a reliable option for the management of patients with pre-existing DED after phacoemulsification.

Further Investigations on the Crosslinking of Tarsal Collagen as a Treatment for Eyelid Laxity: Optimizing the Procedure in Animal Tissue.

PURPOSE: A follow-up experimental study on the exposure of animal tarsal plate to ultraviolet-A radiation aimed at establishing an optimum range for safe irradiation conditions. METHODS: Sheep tarsus specimens were excised postmortem and then subjected to irradiation with ultraviolet-A rays (wavelength 365 nm) at higher irradiances than those reported in an initial study, using a laboratory radiation source. The mechanical properties (tensile strength and Young's modulus) of irradiated and nonirradiated samples were evaluated in a mechanical tester. The test and control specimens were examined histologically with an aim to assess the effects of radiation upon the meibomian glands and tarsal collagen networks, and to establish a safe range for the exposure irradiance level. RESULTS: As expected, irradiation induced both stiffening and strengthening of the tarsal plate specimens. At an irradiance of 50 mW/cm for 3-minute exposure, these effects were at their maximum level, after which a decline in mechanical characteristics were observed. No destruction of the tarsal connective tissue or the meibomian glands were noticed up to an irradiance of 125 mW/cm for 3-minute exposure, corresponding to a fluence of 22.5 J/cm. Histology revealed that the collagen network surrounding the glands were packed more compactly following irradiation. At a fluence of 45 J/cm, massive destruction of periglandular collagen-rich network and meibocytes were demonstrated histologically. CONCLUSIONS: The study indicates that irradiation of tarsal collagen leading to tissue stiffening shall be carried out at levels of fluence between 10 and 15 J/cm, a region that is deemed safe. The exposure time can be adjusted according to the surgeon's decision.Safe irradiation conditions are established for the exposure of ex vivo ovine tarsus to ultraviolet-A radiation as a potentially effective treatment for eyelid laxity in human patients.

In vitro effects of benzalkonium chloride and prostaglandins on human meibomian gland epithelial cells.

PURPOSE: Benzalkonium chloride is the most widely used preservative in ophthalmic topical solutions. The aim of this study was to investigate the influence of BAC as a single substance or as a component of several commercially available ophthalmic solutions on meibomian gland epithelial cells in vitro. MATERIALS AND METHODS: An immortalized human meibomian gland epithelial cell line (HMGEC) was used and cells were cultured in the absence or presence of fetal bovine serum to assess cell morphology, cell proliferation, cell viability (MTS assay) and impedance sensing (ECIS) after stimulation with BAC. Further, the viability of HMGECs stimulated with BAC-containing and BAC-free bimatoprost, travoprost and latanoprost was evaluated using the MTS assay. Real-time PCR analysis for hyperkeratinization associated genes (cornulin, involucrin) was performed. RESULTS: In the absence of serum, the proliferation rate of HMGECs decreased starting with 0.1µg/ml BAC. At concentrations of 50µg/ml BAC and higher, cell viability was reduced after 10min exposure with a corresponding change in cell morphology. Toxicity of BAC-containing ophthalmic solutions was greater than that of BAC alone, whereas BAC-free alternative products did not significantly influence cell viability. Confluence, cell-cell contacts and serum-containing medium appeared to facilitate HMGECs survival. Expression rate of involucrin and cornulin declined after exposure to preserved bimatoprost and BAC. CONCLUSIONS: BAC showed cytotoxic effects on HMGECs starting with a concentration of 0.1µg/ml. The combination of BAC and prostaglandin-analogs might have a synergistic effect which results in higher toxicity than BAC alone. Unpreserved eye drops and eye drops preserved with Polyquaternium-1 are less damaging to HMGECs.

Efficacy of intra-meibomian gland injection of the anti-VEGF agent bevacizumab for the treatment of meibomian gland dysfunction with lid-margin vascularity.

PURPOSE: To investigate the efficacy of a novel treatment - intra-meibomian gland (MG) injection of the anti-VEGF agent bevacizumab - for MG dysfunction (MGD) with eyelid-margin vascularity. METHODS: A total of 26 eyes from 13 patients diagnosed with MGD and eyelid-margin vascularity were included in our study. Patients received intra-meibomian gland injections of bevacizumab (150 µL, 2.5 mg/0.1 mL) at multiple sites with a 29 G needle where telangiectasia was severe. The Ocular Surface Disease Index (OSDI), tear film, tear-breakup time (TBUT), eyelid-margin features, MG features, conjunctiva, and corneal staining were assessed at 1 day before injection and 1 week, 1 month, and 3 months after injection. Blood pressure, best-corrected visual acuity, intraocular pressure, and slit lamp examinations were performed to assure the safety of patients at 1 day before and 1 day, 1 week, 1 month, and 3 months after injection. RESULTS: Lid-margin vascularity, conjunctival injection, expressed secretion quality, expressivity of the MG, TBUT, corneal staining, and OSDI were significantly improved 1 week, 1 month, and 3 months after injection compared to baseline values. Lid-margin vascularity, conjunctival injection, meibomian gland expressivity, TBUT, and OSDI continued to improve; the greatest improvements were observed at 1 month and sustained for 3 months. Spearman's correlation analysis indicated that age and sex significantly influenced TBUT improvement. Females and older patients tended to have shorter baseline TBUT that followed a different trend from that of males and younger patients during postinjection visits, revealed by subgroup analysis. No local or systemic side effects were observed at follow-up visits. CONCLUSION: This study is the first to explore a novel therapy for MGD - intra-MG injection of the anti-VEGF agent bevacizumab - and it demonstrates that the treatment is effective and safe in eliminating eyelid-margin vascularity, improving MG function and relieving clinical signs and symptoms of MGD.

Influence of lipopolysaccharide on proinflammatory gene expression in human corneal, conjunctival and meibomian gland epithelial cells.

PURPOSE: Lipopolysaccharide (LPS), a bacterial endotoxin, is known to stimulate leuokotriene B4 (LTB4) secretion by human corneal (HCECs), conjunctival (HConjECs) and meibomian gland (HMGECs) epithelial cells. We hypothesize that this LTB4 effect represents an overall induction of proinflammatory gene expression in these cells. Our objective was to test this hypothesis. METHODS: Immortalized HCECs, HConjECs and HMGECs were cultured in the presence or absence of LPS (15 µg/ml) and ligand binding protein (LBP; 150 ng/ml). Cells were then processed for RNA isolation and the analysis of gene expression by using Illumina BeadChips, background subtraction, cubic spline normalization and GeneSifter software. RESULTS: Our findings show that LPS induces a striking increase in proinflammatory gene expression in HCECs and HConjECs. These cellular reactions are associated with a significant up-regulation of genes associated with inflammatory and immune responses (e.g. IL-1ß, IL-8, and tumor necrosis factor), including those related to chemokine and Toll-like receptor signaling pathways, cytokine-cytokine receptor interactions, and chemotaxis. In contrast, with the exception of Toll-like signaling and associated innate immunity pathways, almost no proinflammatory ontologies were upregulated by LPS in HMGECs. CONCLUSIONS: Our results support our hypothesis that LPS stimulates proinflammatory gene expression in HCECs and HConjECs. However, our findings also show that LPS does not elicit such proinflammatory responses in HMGECs.