Lacrimal Gland Tissue Research Articles

Clodronate liposome-mediated macrophage depletion ameliorates iron overload-induced dry eye disease.

Dry eye disease (DED) is a prevalent ophthalmic disease that affects millions of people worldwide. Iron overload and macrophage inflammation have been implicated in the development of murine DED, though the specific role of macrophages under iron overload conditions remains unclear. This study aimed to establish a novel iron overload-induced mouse model of DED and investigate macrophage involvement. The model was induced via intraperitoneal injection of D-glucoside iron. Results showed that macrophage depletion via clodronate liposomes (CL) significantly mitigated iron deposit, decreased ocular surface inflammation, improved tear production and restored the structure of ocular surface tissues. Furthermore, CL specifically targeted pro-inflammatory M1 macrophages and reduced levels of the inflammatory cytokines IL-1β, IL-6, and TNF-α, effectively alleviating symptoms of DED. In conclusion, this study characterized a novel iron overload-induced DED mouse model and demenstrated that macrophage depletion mitigated the pathological changes in ocular surface and lacrimal gland tissues caused by iron overload, suggesting potential therapeutic strategies for further investigation in the treatment of DED.

Characterization and Role of Glucagon-Like Peptide 1 Receptor in the Lacrimal Gland: Novel Insights into Diabetic Dry Eye Pathogenesis.

This study aimed to investigate the expression of glucagon-like peptide 1 receptor (GLP-1R) in the lacrimal gland and explore the effects of topical application of GLP-1R agonist (GLP-1RA) on lacrimal gland function in a murine model of type 1 diabetes. Tear secretion was evaluated using phenol red threads, RNA sequencing (RNA-seq) was used to explore gene expression profiles associated with hyperglycemia-induced lacrimal gland injuries, and histological analysis was conducted to evaluate the degree of damage. The expression of GLP-1R in the lacrimal gland was first identified and a downregulation trend associated with diabetes was observed. RNA-seq data from lacrimal gland tissues revealed that differentially expressed genes (DEGs) were enriched in inflammatory response pathways. Histological analysis demonstrated persistent hyperglycemia-induced infiltration of inflammatory cells and progressive fibrosis in the lacrimal gland, resulting in atrophy and diminished tear secretion. Topical application of liraglutide effectively attenuated inflammation and alleviated fibrosis, thus promoting tear production in diabetic mice. Additionally, local intervention with liraglutide could promote autophagy degradation function in the lacrimal gland. This study represents the first validation of GLP-1R expression in the lacrimal gland and its downregulation induced by diabetes; furthermore, these findings demonstrate that topical administration of liraglutide eye drops, a GLP-1RA, can effectively mitigate hyperglycemia-induced damage in the lacrimal gland while enhancing tear secretion.

The Granulomas of Orbital Sarcoidosis Often Show Foci of Necrosis: A Retrospective Observational Study.

To characterize the frequency and extent of necrosis within granulomas among patients diagnosed with orbital sarcoidosis on biopsy. A retrospective review was conducted of patients diagnosed with orbital sarcoidosis at a single institution from 2018 to 2024. Histopathology slides were re-reviewed for evidence of any necrosis or infection. Demographic, laboratory, histopathologic, and radiographic data were gathered for these patients. All findings were presented using descriptive statistics. There were 19 total cases of orbital sarcoidosis in the study timeframe. The majority were female (15, 78.9%) with a median age of 49 (range: 11-85) presenting with a unilateral, mass-occupying orbital lesion (n = 16, 84.2%). The lacrimal gland and orbital fibroadipose tissue were the most frequently involved and biopsied. Biopsies from 13 patients (68.4%) had granulomas containing varying amounts of necrosis on histopathology on retrospective review. No fungal or acid-fast microorganisms were detected with special stains; a subset of patients had additional molecular genetic-based microbiologic studies which were also unrevealing. Although the histopathology of sarcoidosis is classically described as a "non-necrotizing (noncaseating) granulomas," granulomas with varying extents of necrosis are more frequently seen in orbital sarcoidosis than previously recognized, or than reported in biopsies from patients with sarcoidosis at other anatomic sites. The presence of necrosis within sarcoidal granulomas should not exclude a diagnosis of sarcoidosis at this site, given that infection is adequately excluded.

IL-33/ST2 enhances MMP-12 expression by macrophages to mediate inflammatory and immune response in IgG4-Related Ophthalmic Disease

IgG4-Related Ophthalmic Disease (IgG4-ROD) is a chronic autoimmune-mediated fibrotic disease that predominantly affects the lacrimal glands, often leading to loss of function in the involved tissues or organs. Recent studies have demonstrated that MMP-12 is highly expressed in IgG4-ROD and plays a significant role in regulating immune responses. In this study, we reviewed nine patients diagnosed with IgG4-ROD based on clinical manifestations and histological analysis, and we investigated the expression of IL-33/ST2 and MMP-12 in IgG4-ROD lacrimal gland tissues using IHC. We found that IL-33 interacts with its specific receptor ST2, both of which are significantly overexpressed in IgG4-ROD tissues. Additionally, we successfully constructed a mouse model by introducing the LatY136F mutation into C57BL/6 mice to mimic IgG4-ROD lacrimal gland involvement, which helped elucidate the mechanisms involved in the induction of MMP-12. Furthermore, immunofluorescence staining confirmed that most MMP-12+ cells were derived from M2 macrophages, and an ELISA assay demonstrated that IL-33 upregulates MMP-12 in IgG4-ROD. Collectively, these data suggest that the IL-33/ST2/MMP-12 signaling pathway is activated in IgG4-ROD, with IL-33/ST2 potentially promoting M2 macrophage polarization and activation to produce MMP-12, which may serve as a novel therapeutic target for IgG4-ROD.

Measurement of lacrimal gland tissue stiffness for the diagnosis of visual display terminal-associated dry eye disease using shear wave elastography

ObjectiveThis study aimed to evaluate whether lacrimal gland tissue stiffness can aid in diagnosing dry eye disease (DED) using shear wave elastography (SWE). We also aimed to assess the correlation between the subjective symptoms of ocular strain, SWE values, and other ocular examination findings (Schirmer's test and tear film breakup time [TBUT]) contributing to the diagnosis of DED. MethodsThis cross-sectional study recruited 300 participants who were engaged in video display terminal (VDT) work and had been diagnosed with DED by an ophthalmologist for more than one year, and 100 healthy participants without DED symptoms, from August 2020 to December 2021. The modified TFOS DEWS II was used for diagnosing DED. The ocular symptoms, Schirmer's test results, and TBUT of the participants were assessed using standardized methods. Eighty patients with DED and 40 sex- and age-matched healthy participants were selected as the observation and control group, respectively. The lacrimal gland tissue architecture was assessed using ultrasound. ResultThe SWE Emean value reflecting the lacrimal gland tissue stiffness of patients with DED was significantly higher than that of healthy participants (11.06 ± 1.26 for the DED group vs. 7.54 ± 1.27 for the control group, P < 0.001). In patients with DED, the Emean values of the lacrimal glands significantly correlated with the scores for subjective symptoms of ocular dryness (P < 0.001). No significant correlation was observed between the TBUT and Schirmer's test results. The area under the receiver operating characteristic (ROC) curve for SWE was 0.995. According to the ROC analysis results using an Emean cutoff value of 9.32 kPa for the diagnosis of DED, the sensitivity, specificity, positive predictive value（PPV）, and negative predictive （NPV）value were 97.5 %, 95 %, 100 %, and 95 %, respectively. Thus, SWE can be considered for diagnosing VDT-associated DED. ConclusionVDT-associated DED was associated with increased tear gland tissue stiffness. SWE aids in the quantitative detection of lacrimal gland tissue stiffness. Moreover, SWE significantly correlated with the subjective symptoms of DED.

Lacrimal gland Alterations and the Effect of artesunate on experimental induced diabetes rat models and related mechanisms

Diabetic patients are at high risk of developing lacrimal gland dysfunction, and the antimalarial drug artesunate (ART) was recently used to induce experimental-induced diabetes mellitus. This study’s objective is to investigate the lacrimal gland alteration and the effect of ART on experimentally induced diabetes rat models and its related mechanisms. Forty rats were divided into five groups (8 rats/group): healthy control group (HC), diabetic group (DM), 50 mg/kg ART intervention diabetic group [DM + ART (50 mg/kg)], 100 mg/kg ART intervention diabetic group [DM + ART (100 mg/kg)] and 6 U/kg Insulin intervention diabetic group (DM + INS). The morphology of the eyeball and lacrimal gland tissues was determined using hematoxylin and eosin staining. In addition, external lacrimal glands were harvested for electronic microscopic examination, NFκB1, and TNF-α protein expression evaluation by immunohistochemistry and mRNA expression analysis by RT-PCR. Histopathological and ultrastructural changes suggest ART intervention has an improved structural effect. Protein expression of NFκB1 in the DM + ART (100 mg/kg) group was decreased. TNF-α significantly decreased in the DM + ART (50 mg/kg) and insulin groups. We concluded that ART improves structural changes in a lacrimal gland in diabetic rats. The present study provides further evidence of the therapeutic effect of ART on the lacrimal gland of diabetic rats by decreasing the expression of NFκB1 and TNF-α.

Modulation of Decellularized Lacrimal Gland Hydrogel Biodegradation by Genipin Crosslinking.

Hydrogels derived from decellularized tissues are promising biomaterials in tissue engineering, but their rapid biodegradation can hinder in vitro cultivation. This study aimed to retard biodegradation of a hydrogel derived from porcine decellularized lacrimal glands (dLG-HG) by crosslinking with genipin to increase the mechanical stability without affecting the function and viability of lacrimal gland (LG)-associated cells. The effect of different genipin concentrations on dLG-HG stiffness was measured rheologically. Cell-dependent biodegradation was quantified over 10days, and the impact on matrix metalloproteinase (MMP) activity was quantified by gelatin and collagen zymography. The viability of LG epithelial cells (EpCs), mesenchymal stem cells (MSCs), and endothelial cells (ECs) cultured on genipin-crosslinked dLG-HG was assessed after 10days, and EpC secretory activity was analyzed by β-hexosaminidase assay. The 0.5-mM genipin increased the stiffness of dLG-HG by about 46%, and concentrations > 0.25mM caused delayed cell-dependent biodegradation and reduced MMP activity. The viability of EpCs, MSCs, and ECs was not affected by genipin concentrations of up to 0.5mM after 10days. Moreover, up to 0.5-mM genipin did not negatively affect EpC secretory activity compared to control groups. A concentration of 0.5-mM genipin increased dLG-HG stiffness, and 0.25-mM genipin was sufficient to prevent MMP-dependent degradation. Importantly, concentrations of up to 0.5-mM genipin did not compromise the viability of LG-associated cells or the secretory activity of EpCs. Thus, crosslinking with genipin improves the properties of dLG-HG for use as a substrate in LG tissue engineering.

Sonographic findings of COVID-19-related acute dacryoadenitis

Objectives: To evaluate the ability of grayscale sonography and color Doppler sonography in determining the involvement of the lacrimal glands in coronavirus disease (COVID-19). Methods: A retrospective analysis was performed on a total of 25 COVID-19 patients with symptoms of acute dacryoadenitis and 25 healthy participants. The study’s inclusion criteria encompassed pain, swelling, and discomfort in the superior temporal aspect of the upper eyelid and orbit, consistent with acute dacryoadenitis occurring within 30 days of a positive test for SARS-CoV-2. PCR testing yielded positive results for all patients. Inclusion criteria for healthy participants included asymptomatic orbit and upper lid, no prior trauma or surgery involving the orbit, no evidence of upper respiratory tract infection consistent with SARS-CoV-2 in the past 6 months, and no history of systemic inflammatory disorders. The evaluation of the lacrimal gland and periorbital adipose tissue involved gray-scale and color Doppler ultrasonography to assess echogenicity, homogeneity, vascularity and enlargement of the lacrimal gland. The patients involved in the study underwent orbital examination and US evaluations repeated at 3 weeks and 3 months. Results: The mean age of the patients were 41.5±12.2 years (range, 18 to 63 years), while for the healthy participants, it was 34.4±5.2 years (range, 18 to 47 years). Significant differences were observed in the echogenicity (p=0.025), homogeneity (p=0.018), and vascularity (p&lt;0.001), size (p&lt;0.001) of the lacrimal gland between healthy participants and COVID-19 patients exhibiting symptoms of acute dacryoadenitis. However, no difference was noted in the perilacrimal fat tissue changes between COVID-19 patients with symptoms of acute dacryoadenitis and the control group (p=0.054). Conclusion: Gray-scale and color Doppler ultrasonography demonstrates as a valuable radiologic technique for assessing the acute onset involvement of the lacrimal glands in COVID-19.

Regulation of Axon Guidance by Slit2 and Netrin-1 Signaling in the Lacrimal Gland of Aqp5 Knockout Mice.

Dry eye disease (DED) is multifactorial and associated with nerve abnormalities. We explored an Aquaporin 5 (AQP5)-deficiency-induced JunB activation mechanism, which causes abnormal lacrimal gland (LG) nerve distribution through Slit2 upregulation and Netrin-1 repression. Aqp5 knockout (Aqp5-/-) and wild-type (Aqp5+/+) mice were studied. LGs were permeabilized and stained with neuronal class III β-tubulin, tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP), and calcitonin gene-related peptide (CGRP). Whole-mount images were acquired through tissue clearing and 3D fluorescence imaging. Mouse primary trigeminal ganglion (TG) neurons were treated with LG extracts and Netrin-1/Slit2 neutralizing antibody. Transcription factor (TF) prediction and chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) experiments verified the JunB binding and regulatory effect on Netrin-1 and Slit2. Three-dimensional tissue and section immunofluorescence showed reduced LG nerves in Aqp5-/- mice, with sympathetic and sensory nerves significantly decreased. Netrin-1 was reduced and Slit2 increased in Aqp5-/- mice LGs. Aqp5+/+ mice LG tissue extracts (TEs) promoted Aqp5-/- TG neurons axon growth, but Netrin-1 neutralizing antibody (NAb) could inhibit that promotion. Aqp5-/- mice LG TEs inhibited Aqp5+/+ TG axon growth, but Slit2 NAb alleviated that inhibition. Furthermore, JunB, a Netrin-1 and Slit2 TF, could bind them and regulate their expression. SR11302, meanwhile, reversed the Netrin-1 and Slit2 shifts caused by AQP5 deficiency. AQP5 deficiency causes LG nerve abnormalities. Persistent JunB activation, the common denominator for Netrin-1 suppression and Slit2 induction, was found in Aqp5-/- mice LG epithelial cells. This affected sensory and sympathetic nerve fibers' distribution in LGs. Our findings provide insights into preventing, reversing, and treating DED.

Marginal Zone B (MZB) Cells: Comparison of the Initial Identification of Immune Activity Leading to Dacryoadenitis and Sialadenitis in Experimental Sjögren's Syndrome.

Although multiple mouse strains have been advanced as models for Sjögren's syndrome (SS), which is a human systemic autoimmune disease characterized primarily as the loss of lacrimal and salivary gland functions, the C57BL/6.NOD-Aec1Aec2 recombinant inbred (RI) mouse derived from the NOD/ShiLtJ line is considered one of the more appropriate models exhibiting virtually all the characteristics of the human disease. This mouse model, as well as other mouse models of SS, have shown that B lymphocytes are essential for the onset and development of observed clinical manifestations. Recently, studies carried out in the C57BL/6.IL14α transgenic mouse have provided clear evidence that the marginal zone B (MZB) cell population is directly involved in the early pathological events initiating the development of the clinical SS disease, as well as late-stage lymphomagenesis resulting in B-cell lymphomas. Since MZB cells are difficult to study in vivo and in vitro, we carried out a series of ex vivo investigations that utilize temporal global RNA transcriptomic analyses to profile differentially expressed genes exhibiting temporal upregulation during the initial onset and subsequent development of pathophysiological events within the lacrimal and salivary gland tissues per se or associated with the leukocyte cell migrations into these glands. The initial transcriptomic analyses revealed that while the upregulated gene expression profiles obtained from lacrimal and salivary glands overlap, multiple genetic differences exist between the defined activated pathways. In the current study, we present a concept suggesting that the initial pathological events differ between the two glands, yet the subsequent upregulated TLR4/TLR3 signal transduction pathway that activates the type-1 interferon signature appears to be identical in the two glands and indicates an autoimmune response against dsRNA, possibly a virus. Here, we attempt to put these findings into perspective and determine how they can impact the design of future therapeutic protocols.

Human Lacrimal Gland Derived Mesenchymal Stem Cells - Isolation, Propagation, and Characterization.

The existing treatment options for dry eye disease (DED) due to lacrimal gland (LG) dysfunction are mainly palliative. Mesenchymal stem cells (MSCs) based therapies and 3D-LG organoids have been explored as a curative option for LG regeneration in animal models. Human LG epithelial cultures are previously established and, here, we aim to isolate and characterize the spindle-shaped cells obtained from primary human LG cultures in order to unveil its MSC property. Normal human lacrimal glands were obtained from individuals undergoing LG debulking surgery. The conditions for human LG-MSC culture were standardized to obtain pure population of LG-MSCs at passage 3. Population doubling time (PDT), expression of phenotypic markers, tri-lineage differentiation, colony forming potential, and gene expression analysis were carried out to assess the phenotypic and genotypic characteristics compared to bone marrow-MSCs (BM-MSCs). Our data show that these spindle-shaped cells exhibit similar phenotypic expression, colony-forming ability, and trilineage differentiation like BM-MSCs. Moreover, the gene expression also did not show any significant difference, except for increased IL1-β in LG-MSCs. The LG-MSCs do not express any lacrimal epithelial markers unlike LG tissue. This study reveals the first-time evidence for the presence of MSC population within the human LGs, and these cells might play a role in maintaining healthy microenvironment within normal LG and repair in diseased LGs.

Dietary Alcohol Consumption Elicits Corneal Toxicity Through the Generation of Cellular Oxidative Stress.

Purpose: Clinical data suggest that alcohol use is associated with the development of signs and symptoms of dry eye disease. However, preclinical data investigating ocular toxicity after dietary alcohol consumption are lacking. In this study, we investigated the effects of alcohol on the ocular surface, in human corneal epithelial cells (HCE-T) in vitro and in C57BL/6JRj mice in vivo. Methods: HCE-T were exposed to clinically relevant doses of ethanol. To determine the effects of dietary alcohol consumption in vivo, wild-type mice were administered the Lieber-DeCarli liquid diet (5% vol/vol ethanol or isocaloric control) for 10 days ad libitum. Corneal fluorescein staining was performed to assess ocular surface damage. Histopathological and gene expression studies were performed on cornea and lacrimal gland tissue. Results: Sublethal doses of ethanol (0.01%-0.5%) resulted in a dose-dependent increase of cellular oxidative stress in corneal epithelial cells and a significant increase in NFE2L2 and downstream antioxidant gene expression, as well as an increase in NFκB signaling; short-term exposure (0.5%, 4 h) triggered significant corneal epithelial cell barrier breakdown. Exposure to the alcohol-containing diet caused a 3-fold increase in corneal fluorescein staining, with no effect on tear volumes. Corneal thickness was significantly reduced in the alcohol diet group, and corneal tissue revealed dysregulated antioxidant and NFκB signaling. Our data provide the first published evidence that alcohol exposure causes ocular toxicity in mice. Conclusions: Our results are consistent with clinical studies linking past alcohol consumption to signs of ocular surface disease.

Effective encoder-decoder neural network for segmentation of orbital tissue in computed tomography images of Graves' orbitopathy patients.

To propose a neural network (NN) that can effectively segment orbital tissue in computed tomography (CT) images of Graves' orbitopathy (GO) patients. We analyzed orbital CT scans from 701 GO patients diagnosed between 2010 and 2019 and devised an effective NN specializing in semantic orbital tissue segmentation in GO patients' CT images. After four conventional (Attention U-Net, DeepLab V3+, SegNet, and HarDNet-MSEG) and the proposed NN train the various manual orbital tissue segmentations, we calculated the Dice coefficient and Intersection over Union for comparison. CT images of the eyeball, four rectus muscles, the optic nerve, and the lacrimal gland tissues from all 701 patients were analyzed in this study. In the axial image with the largest eyeball area, the proposed NN achieved the best performance, with Dice coefficients of 98.2% for the eyeball, 94.1% for the optic nerve, 93.0% for the medial rectus muscle, and 91.1% for the lateral rectus muscle. The proposed NN also gave the best performance for the coronal image. Our qualitative analysis demonstrated that the proposed NN outputs provided more sophisticated orbital tissue segmentations for GO patients than the conventional NNs. We concluded that our proposed NN exhibited an improved CT image segmentation for GO patients over conventional NNs designed for semantic segmentation tasks.

Purple Sweet Potato Powder Containing Anthocyanin Mitigates High-Fat-Diet-Induced Dry Eye Disease.

Purple sweet potato (PSP) powder with anthocyanins possesses the ability to reduce oxidative stress and inflammation. Studies have presumed a positive correlation between body fat and dry eye disease (DED) in adults. The regulation of oxidative stress and inflammation has been proposed as the mechanism underlying DED. This study developed an animal model of high fat diet (HFD)-induced DED. We added 5% PSP powder to the HFD to evaluate the effects and underlying mechanisms in mitigating HFD-induced DED. A statin drug, atorvastatin, was also added to the diet separately to assess its effect. The HFD altered the structure of lacrimal gland (LG) tissue, reduced LG secretory function, and eliminated the expression of proteins related to DED development, including α-smooth muscle actin and aquaporin-5. Although PSP treatment could not significantly reduce body weight or body fat, it ameliorated the effects of DED by preserving LG secretory function, preventing ocular surface erosion, and preserving LG structure. PSP treatment increased superoxide dismutase levels but reduced hypoxia-inducible factor 1-α levels, indicating that PSP treatment reduced oxidative stress. PSP treatment increased ATP-binding cassette transporter 1 and acetyl-CoA carboxylase 1 levels in LG tissue, signifying that PSP treatment regulated lipid homeostasis maintenance to reduce the effects of DED. In conclusion, PSP treatment ameliorated the effects of HFD-induced DED through the regulation of oxidative stress and lipid homeostasis in the LG.

Metagenomic Deep Sequencing for Orbital Inflammatory Disease

ABSTRACT Purpose Orbital inflammatory disease (OID) is a heterogeneous group of immunologic disorders whose etiology is often non-specific despite routine investigation. In this proof-of-concept study, metagenomic deep sequencing (MDS) is applied to examine host gene expression in two subtypes of OID. Methods Prospectively collected lacrimal gland tissue from patients with OID was processed for MDS. Differential gene expression analysis was performed to evaluate for host transcriptome signatures. Proof-of-concept comparison was made between histologically confirmed samples of idiopathic dacryoadenitis and IgG4-related disease (IgG4-RD). Results Twelve genes were identified to be differentially expressed between idiopathic dacryoadenitis and IgG4-RD. Differences in innate humoral immunity gene expression were observed. Several additional genes of interests were also found to be upregulated in idiopathic dacryoadenitis. Conclusions A unique transcriptome signature was found when comparing idiopathic dacryoadenitis to IgG4-RD. This suggests that MDS can identify differentially expressed genes in OID. Such insight could potentially provide a better understanding of host gene expression and the inflammatory pathways involved in OID.

Identification and validation of ferroptosis-related genes and immune cell infiltration in thyroid associated ophthalmopathy.

Thyroid associated ophthalmopathy (TAO) is an orbital autoimmune inflammatory disease that is commonly associated with thyroid dysfunction. Although the etiology of TAO is unclear, ROS accumulation and oxidative stress have been closely linked to the pathogenesis of TAO. Ferroptosis is an iron-dependent programmed cell death characterized by intracellular labile iron levels, excessive accumulation of reactive oxygen species (ROS) and lipid peroxidation. Currently, there are few reports regarding the role of ferroptosis in TAO. This article aimed to identify ferroptosis-related genes (FRGs) with diagnostic and therapeutic potential in TAO and explore their relationship with immune cells and lncRNAs. GSE58331 was downloaded from Gene Expression Omnibus (GEO) database. A total of 162 DEGs were identified between 27 TAO samples and 22 health samples from GSE58331, among which six FRGs (CYBB, CTSB, SLC38A1, TLR4, PEX3, and ABCC1) were obtained. The AUC of SLC38A1, TLR4, PEX3 in lacrimal gland tissues was greater than 80 which suggested high diagnostic value in TAO. The result of immune cell infiltrate analysis indicated increased infiltration of monocytes (p < 0.001), macrophages M0(p = 0.039), mast cells activated (p = 0.008), and neutrophils (p = 0.045) in orbital tissues from TAO patients. Meanwhile, mast cells resting (p = 0.043) and macrophages M2 (p = 0.02) showed reduced infiltration in TAO samples. There were no gender differences in immune cell infiltration in the TAO patients. Two differentially expressed lncRNAs, LINC01140 and ZFHX4-AS1, in TAO groups were identified as ferroptosis-related lncRNAs. CYBB-LINC01140-TLR4, CYBB- LINC01140- SLC38A1, TLR4- LINC01140- SLC38A1, and CTSB- ZFHX4-AS1- CYBB may be potential RNA regulatory pathways in TAO. Targeted drugs and transcription factors for differential expressed FRGs were also screened out in our study. In vitro, experiments revealed that CTSB, PEX3, ABCC1 and ZFHX4-AS1(lncRNA) were differentially expressed in orbital fibroblasts (OFs) between TAO groups and healthy controls at the transcriptional level.

APX‑115A, a pan‑NADPH oxidase inhibitor, reduces the degree and incidence rate of dry eye in the STZ‑induced diabetic rat model.

Dye eye disease (DED) is a common ocular disorder in patients with diabetes. It has been reported that APX-115A, a pan-nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase inhibitor, has an apoptosis-inducing effect on Epstein-Barr virus-infected retinal epithelial cells, but its effects in DED are poorly understood. Therefore, a rat model of diabetes was used in the present study to investigate whether APX-115A has an impact on DED in diabetic rats. A diabetic model was established in male Sprague Dawley rats via the intraperitoneal injection of streptozotocin. The eyeballs of the rats were treated with a solution containing APX-115A or a saline control. Tear secretion was measured with the phenol red thread tear test, and the morphology of the eyeball and lacrimal gland tissues was determined using hematoxylin and eosin staining. In addition, localization of NAPDH oxidase 2 (NOX2) in the eyeball and lacrimal gland tissues was detected by immunohistochemistry. The APX-115A treatment had no effect on body weight, blood glucose level or the size of the lacrimal glands. However, morphological changes, namely intracellular vacuoles and acinar atrophy, were observed in the lacrimal glands of the diabetic rats, and APX-115A treatment attenuated these changes. Immunohistochemistry revealed that NOX2 expression was decreased in the lacrimal glands of the diabetic rats, and APX-115A treatment did not attenuate the reduction in NOX2. The corneas of the diabetic rats treated with APX-115A exhibited no change in thickness but had lower NOX2 expression levels compared with those of the control diabetic rats. APX-115A also increased tear secretion and ameliorated the histological changes associated with diabetes. Furthermore, the NOX2 expression levels in the corneas of the diabetic rats treated with APX-115A were restored to the levels observed in normal rats. These findings suggest that APX-115A has potential as a therapeutic agent for DED.

Expression of Androgen and Estrogen Receptors in the Human Lacrimal Gland

Lacrimal gland dysfunction causes dry eye disease (DED) due to decreased tear production. Aqueous-deficient DED is more prevalent in women, suggesting that sexual dimorphism of the human lacrimal gland could be a potential cause. Sex steroid hormones are a key factor in the development of sexual dimorphism. This study aimed to quantify estrogen receptor (ER) and androgen receptor (AR) expression in the human lacrimal gland and compare it between sexes. RNA was isolated from 35 human lacrimal gland tissue samples collected from 19 cornea donors. AR, ERα, and ERβ mRNA was identified in all samples, and their expression was quantified using qPCR. Immunohistochemical staining was performed on selected samples to evaluate protein expression of the receptors. ERα mRNA expression was significantly higher than the expression of AR and ERβ. No difference in sex steroid hormone (SSH) receptor mRNA expression was observed between sexes, and no correlation was observed with age. If ERα protein expression is found to be concordant with mRNA expression, it should be investigated further as a potential target for hormone therapy of DED. Further research is needed to elucidate the role of sex steroid hormone receptors in sex-related differences of lacrimal gland structure and disease.

Pseudomonas aeruginosa Keratitis in Rats: Study of the Effect of Topical 5% Hesperidin Practice on Healing.

The aim of this study is to investigate the effect of topical 5% hesperidin application on healing. After 48 rats were randomized and divided into 7 groups, on the first day, an epithelial defect was created in the center of the cornea with the help of microkeratome under intraperitoneal ketamine+xylazine and topical 5% proparacaine anesthesia for the groups to be infected with keratitis according to the groups mentioned below. An amount of 0.05 mL of the solution containing 108 colony-forming units/ mL Pseudomonas aeruginosa (PA-ATC27853) will be taken and inoculated per rat. At the end of the 3 days incubation period, rats with keratitis will be added to the groups, and active substances and antibiotics will be given topically together with other groups for 10 days. At the end of the study, the ocular tissues of the rats will be removed and examined histopathologically. A clinically significant reduction in inflammation was detected in the groups using hesperidin. No transforming growth factor-β1 staining was detected in the group in which keratitis+hesperidin was treated topically. In the group in which hesperidin toxicity was examined, mild inflammation and thickening of the corneal stroma layer were observed, and it was evaluated as a negative transforming growth factor-β1 expression in the lacrimal gland tissue. Corneal epithelial damage was minimal in the keratitis group, and the toxicity group was treated with only hesperidin when compared to the other groups. Topical hesperidin drops may be an important therapeutic factor in tissue healing and in the fight against inflammation in the treatment of keratitis.

Effects of Fibulin-5 Gene Silencing on Proliferation and Apoptosis of IgG4-ROD Lacrimal Gland Fibroblasts.

This study is aimed at discussing the value of RNA interference technology on inhibiting lacrimal gland fibrosis in IgG4-related ocular disease (IgG4-ROD). Six patients with IgG4-ROD who came to the hospital for surgical treatment from October 2018 to August 2019 were selected, and their diseased lacrimal glands were taken for primary cell culture and fibroblast identification. High efficiency and specificity small interference RNA (siRNA) plasmid vector was constructed, its inhibitory effect on fibroblast proliferation was determined by CCK-8 assay, and the appropriate concentration was selected as the siRNA concentration for subsequent experiments. RT-PCR and Western blot detected the relative expression levels of Fibulin-5 mRNA and protein in the cells 48 hours after transfection. The apoptosis rate of each group of cells at 24 hours, 48 hours, and 72 hours after transfection was detected by flow cytometry, and the proliferation and apoptosis of cells after silencing Fibulin-5 were analyzed and compared. 24 hours after transfection, there was no significant difference in the proliferation rate among the four groups (P > 0.05); 48 hours and 72 hours after Fibulin-5 siRNA transfection, the proliferation activity of the transfected cells was significantly decreased compared with the 0 nM group, and the inhibitory effect of 75 nM siRNA was the strongest. The expression of Fibulin-5 mRNA and protein in the siRNA-transfected cells was significantly decreased compared with the blank and empty vector negative siRNA groups, and the difference was statistically significant (P < 0.05). The apoptosis rate of cells in the Fibulin-5 siRNA transfection group was significantly higher than that of cells in the blank and empty vector negative siRNA groups, and the difference was statistically significant (P < 0.05). Fibulin-5 siRNA recombinant plasmid can significantly downregulate the mRNA and protein expressions of target gene Fibulin-5 and promote apoptosis after transfection into IgG4-ROD lacrimal gland fibroblasts. It is speculated that Fibulin-5 can be used as a target to effectively inhibit the fibrosis of lacrimal gland tissues by RNAi technique.