Iron Chelator Deferiprone Restores Iron Homeostasis and Inhibits Retinal Neovascularization in Experimental Neovascular Age-Related Macular Degeneration.

Purpose: Retinal neovascularization is a significant feature of advanced age-related macular degeneration (AMD) and a major cause of blindness in patients with AMD. However, the underlying mechanism of this pathological neovascularization remains unknown. Iron metabolism has been implicated in various biological processes. This study was conducted to investigate the effects of iron metabolism on retinal neovascularization in neovascular AMD (nAMD). Methods: C57BL/6J and very low-density lipoprotein receptor (VLDLR) knockout (Vldlr-/-) mice, a murine model of nAMD, were used in this study. Bulk-RNA sequencing was used to identify differentially expressed genes. Western blot analysis was performed to test the expression of proteins. Iron chelator deferiprone (DFP) was administrated to the mice by oral gavage. Fundus fluorescein angiography was used to evaluate retinal vascular leakage. Immunofluorescence staining was used to detect macrophages and iron-related proteins. Results: RNA sequencing (RNA-seq) results showed altered transferrin expression in the retina and RPE of Vldlr-/- mice. Disrupted iron homeostasis was observed in the retina and RPE of Vldlr-/- mice. DFP mitigated iron overload and significantly reduced retinal neovascularization and vascular leakage. In addition, DFP suppressed the inflammation in Vldlr-/- retinas. The reduced signals of macrophages were observed at sites of neovascularization in the retina and RPE of Vldlr-/- mice after DFP treatment. Further, the IL-6/JAK2/STAT3 signaling pathway was activated in the retina and RPE of Vldlr-/- mice and reversed by DFP treatment. Conclusions: Disrupted iron metabolism may contribute to retinal neovascularization in nAMD. Restoring iron homeostasis by DFP could be a potential therapeutic approach for nAMD.

Uveal melanoma distant metastasis prediction system: A retrospective observational study based on machine learning.

Uveal melanoma (UM) patients face a significant risk of distant metastasis, closely tied to a poor prognosis. Despite this, there is a dearth of research utilizing big data to predict UM distant metastasis. This study leveraged machine learning methods on the Surveillance, Epidemiology, and End Results (SEER) database to forecast the risk probability of distant metastasis. Therefore, the information on UM patients from the SEER database (2000-2020) was split into a 7:3 ratio training set and an internal test set based on distant metastasis presence. Univariate and multivariate logistic regression analyses assessed distant metastasis risk factors. Six machine learning methods constructed a predictive model post-feature variable selection. The model evaluation identified the multilayer perceptron (MLP) as optimal. Shapley additive explanations (SHAP) interpreted the chosen model. A web-based calculator personalized risk probabilities for UM patients. The results show that nine feature variables contributed to the machine learning model. The MLP model demonstrated superior predictive accuracy (Precision = 0.788; ROC AUC = 0.876; PR AUC = 0.788). Grade recode, age, primary site, time from diagnosis to treatment initiation, and total number of malignant tumors were identified as distant metastasis risk factors. Diagnostic method, laterality, rural-urban continuum code, and radiation recode emerged as protective factors. The developed web calculator utilizes the MLP model for personalized risk assessments. In conclusion, the MLP machine learning model emerges as the optimal tool for predicting distant metastasis in UM patients. This model facilitates personalized risk assessments, empowering early and tailored treatment strategies.

Unleashing Novel Therapeutic Strategies for Dry Eye: Targeting ROS and the cGAS-STING Signaling Pathway with Tetrahedral Framework Nucleic Acids.

Dry eye affects majority of the global population, causing significant discomfort or even visual impairment, of which inflammation plays a crucial role in the deterioration process. This highlights the need for effective and safe anti-inflammatory treatments to achieve satisfactory therapeutic outcomes. This study focuses on the potential of tetrahedral framework nucleic acids (tFNA), a self-assembled nucleic acid material, as a simple and rapid treatment for oxidative stress and inflammation-induced disorders associated with dry eye. Mechanistically, tFNA is found to effectively alleviate dry eye damage by promoting corneal epithelial healing, restoring goblet cell function, and facilitating tear secretion recovery. Through RNA-seq analysis, it is observed that tFNA treatment normalizes the expression levels of most genes. Further exploration of the mechanism reveals that tFNA reduces excessive production of reactive oxygen species and modulates the inflammatory microenvironment, especially through cGAS-STING pathway thereby levels of inflammatory cytokines, including MMP9 and IL-6, are reduced. Additionally, tFNA demonstrates excellent safety performance without causing damage to the eye. Importantly, this study represents a successful application of nanophase materials with nucleic acid biological features for the effective treatment of dry eye, highlighting the potential clinical use of tFNA in the treatment of dry eye.

Research progress of photo-crosslink hydrogels in ophthalmology: A comprehensive review focus on the applications.

Hydrogel presents a three-dimensional polymer network with high water content. Over the past decade, hydrogel has developed from static material to intelligent material with controllable response. Various stimuli are involved in the formation of hydrogel network, among which photo-stimulation has attracted wide attention due to the advantages of controllable conditions, which has a good application prospect in the treatment of ophthalmic diseases. This paper reviews the application of photo-crosslink hydrogels in ophthalmology, focusing on the types of photo-crosslink hydrogels and their applications in ophthalmology, including drug delivery, tissue engineering and 3D printing. In addition, the limitations and future prospects of photo-crosslink hydrogels are also provided.

Models for Meibomian gland dysfunction: In vivo and in vitro.

Meibomian gland dysfunction (MGD) is a chronic abnormality of the Meibomian glands (MGs) that is recognized as the leading cause of evaporative dry eye worldwide. Despite its prevalence, however, the pathophysiology of MGD remains elusive, and effective disease management continues to be a challenge. In the past 50 years, different models have been developed to illustrate the pathophysiological nature of MGD and the underlying disease mechanisms. An understanding of these models is crucial if researchers are to select an appropriate model to address specific questions related to MGD and to develop new treatments. Here, we summarize the various models of MGD, discuss their applications and limitations, and provide perspectives for future studies in the field.

Chronic Sleep Deprivation Impairs Visual Functions via Oxidative Damage in Mice.

Sleep deprivation (SD) is a global public health burden, and has a detrimental role in the nervous system. Retina is an important part of the central nervous system; however, whether SD affects retinal structures and functions remains largely unknown. Herein, chronic SD mouse model indicated that loss of sleep for 4 months could result in reductions in the visual functions, but without obvious morphologic changes of the retina. Ultrastructural analysis by transmission electron microscope revealed the deterioration of mitochondria, which was accompanied with the decrease of multiple mitochondrial proteins in the retina. Mechanistically, oxidative stress was provoked by chronic SD, which could be ameliorated after rest, and thus restore retinal homeostasis. Moreover, the supplementation of two antioxidants, α-lipoic acid and N-acetyl-l-cysteine, could reduce retinal reactive oxygen species, repair damaged mitochondria, and, as a result, improve the retinal functions. Overall, this work demonstrated the essential roles of sleep in maintaining the integrity and health of the retina. More importantly, it points towards supplementation of antioxidants as an effective intervention strategy for people experiencing sleep shortages.

Accurate detection and grading of pterygium through smartphone by a fusion training model.

BACKGROUND/AIMS: To improve the accuracy of pterygium screening and detection through smartphones, we established a fusion training model by blending a large number of slit-lamp image data with a small proportion of smartphone data. METHOD: Two datasets were used, a slit-lamp image dataset containing 20 987 images and a smartphone-based image dataset containing 1094 images. The RFRC (Faster RCNN based on ResNet101) model for the detection model. The SRU-Net (U-Net based on SE-ResNeXt50) for the segmentation models. The open-cv algorithm measured the width, length and area of pterygium in the cornea. RESULTS: The detection model (trained by slit-lamp images) obtained the mean accuracy of 95.24%. The fusion segmentation model (trained by smartphone and slit-lamp images) achieved a microaverage F1 score of 0.8981, sensitivity of 0.8709, specificity of 0.9668 and area under the curve (AUC) of 0.9295. Compared with the same group of patients' smartphone and slit-lamp images, the fusion model performance in smartphone-based images (F1 score of 0.9313, sensitivity of 0.9360, specificity of 0.9613, AUC of 0.9426, accuracy of 92.38%) is close to the model (trained by slit-lamp images) in slit-lamp images (F1 score of 0.9448, sensitivity of 0.9165, specificity of 0.9689, AUC of 0.9569 and accuracy of 94.29%). CONCLUSION: Our fusion model method got high pterygium detection and grading accuracy in insufficient smartphone data, and its performance is comparable to experienced ophthalmologists and works well in different smartphone brands.

Smart coating by thermo-sensitive Pluronic F-127 for enhanced corneal healing via delivery of biological macromolecule progranulin.

As a leading cause of vision impairment and blindness, corneal alkali burns lead to long-term visual deterioration or even permanent visual impairment while effective treatment strategies remain a challenge. Herein, a thermo-sensitive hydrogel with the combination of multi-functional protein progranulin (PGRN), a biological macromolecule consisting of several hundred amino acids and possessing a high molecular weight, is efficiently prepared through a convenient stirring and mixing at the low temperature. The hydrogel can be easily administrated to the ocular surface contacting with the cornea, which can be immediately transformed into gel-like state due to the thermo-responsive behavior, realizing a site-specific coating to isolate further external stimulation. The smart coating not only exhibits excellent transparency and biocompatibility, but also presents a constant delivery of PGRN, creating a nutritious and supportive micro-environment for the ocular surface. The results show that the prepared functional hydrogel can efficiently suppress inflammation, accelerate re-epithelization, and intriguingly enhance axonal regeneration via modulation of multiple signaling pathways, indicating the novel designed HydrogelPGRN is a promising therapy option for serious corneal injury.

A Fully Automatic Estimation of Tear Meniscus Height Using Artificial Intelligence.

Purpose: Accurate quantification measurement of tear meniscus is vital for the precise diagnosis of dry eye. In current clinical practice, the measurement of tear meniscus height (TMH) relies on doctors' manual operation. This study aims to propose a novel automatic artificial intelligence (AI) system to evaluate TMH. Methods: A total of 510 photographs obtained by the oculus camera were labeled. Three thousand and five hundred images were finally attained by data enhancement to train the neural network model parameters, and 60 were used to evaluate the model performance in segmenting the cornea and tear meniscus region. One hundred images were used to test generalization ability of the model. We modified a segmentation model of the cornea and the tear meniscus based on the UNet-like network. The output of the segmentation model is followed by a calculation module that calculates and reports the TMH. Results: Compared with ground truth (GT) manually labeled by clinicians, our modified model achieved a Dice Similarity Coefficient (DSC) and Intersection over union (Iou) of 0.99/0.98 in the corneal segmentation task and 0.92/0.86 for the detection of tear meniscus on the validation set, respectively. On the test set, the TMH automatically measured by our AI system strongly correlates with the results manually calculated by the ophthalmologists. Conclusions: We developed a fully automated and reliable AI system to obtain TMH. After large-scale clinical testing, our method could be used for dry eye screening in clinical practice.

Berberine ameliorate inflammation and apoptosis via modulating PI3K/AKT/NFκB and MAPK pathway on dry eye.

BACKGROUND: Dry eye disease (DED) is a multifactorial disease in ocular surface, and inflammation plays an etiological role. Berberine (BBR) has shown efficacy in treating inflammatory diseases. Yet, there was no adequate information related to the therapeutic effects of BBR for DED. PURPOSE: To detect the effects and explore the potential mechanisms of BBR on DED. STUDY DESIGN: In vitro, in vivo study and network pharmacology analysis were involved. METHOD: The human corneal epithelium cells viability was evaluated with different concentrations of BBR. Dry eye murine model was established by exposing to the desiccating stress, and Ciclosporin (CSA), BBR eye drops or vehicle were topical administration for 7 days. The phenol red cotton tests, Oregon-green-dextran staining and Periodic acid-Schiff staining were performed and evaluated the dry eye after treatment. Inflammation and apoptosis levels of ocular surface were quantified. The potential targets related to berberine and dry eye were collected from databases. The Protein-Protein interaction network analysis and GO & KEGG enrichment analysis were realized by STRING database, Metascape platform and Cytoscape software to find core targets and signaling pathways. The SchrÖdinger software was used to molecular docking and PyMOL software to visualization. Finally, the levels of PI3K/AKT/NFκB and MAPK pathways were detected. RESULT: The data revealed BBR could rescue impaired HCE under hyperosmotic conditions. In addition, BBR eye drops could ameliorate dry eye. And BBR eye drops suppressed the inflammatory factors and CD4+T cells infiltration in conjunctiva. Besides, BBR eye drops protected ocular surface by avoiding the severe apoptosis and decreasing the level of MMP-3 and MMP-9. 148 common targets intersection between BBR and dry eye were found via network pharmacology analysis. Core proteins and core pathways were identified through PPI and GO&KEGG enrichment analysis. Molecular docking displayed excellent binding between BBR and those core targets. Finally, in vivo study verified that BBR eye drops had a therapeutic effect in dry eye by inhibiting PI3K/AKT/NFκB and MAPK pathways. CONCLUSION: The research provided convincing evidence that BBR could be a candidate drug for dry eye.

Guidelines for the application of artificial intelligence in the diagnosis of anterior segment diseases (2023).

The landscape of ophthalmology has observed monumental shifts with the advent of artificial intelligence (AI) technologies. This article is devoted to elaborating on the nuanced application of AI in the diagnostic realm of anterior segment eye diseases, an area ripe with potential yet complex in its imaging characteristics. Historically, AI's entrenchment in ophthalmology was predominantly rooted in the posterior segment. However, the evolution of machine learning paradigms, particularly with the advent of deep learning methodologies, has reframed the focus. When combined with the exponential surge in available electronic image data pertaining to the anterior segment, AI's role in diagnosing corneal, conjunctival, lens, and eyelid pathologies has been solidified and has emerged from the realm of theoretical to practical. In light of this transformative potential, collaborations between the Ophthalmic Imaging and Intelligent Medicine Subcommittee of the China Medical Education Association and the Ophthalmology Committee of the International Translational Medicine Association have been instrumental. These eminent bodies mobilized a consortium of experts to dissect and assimilate advancements from both national and international quarters. Their mandate was not limited to AI's application in anterior segment pathologies like the cornea, conjunctiva, lens, and eyelids, but also ventured into deciphering the existing impediments and envisioning future trajectories. After iterative deliberations, the consensus synthesized herein serves as a touchstone, assisting ophthalmologists in optimally integrating AI into their diagnostic decisions and bolstering clinical research. Through this guideline, we aspire to offer a comprehensive framework, ensuring that clinical decisions are not merely informed but transformed by AI. By building upon existing literature yet maintaining the highest standards of originality, this document stands as a testament to both innovation and academic integrity, in line with the ethos of renowned journals such as Ophthalmology.

The cGAS-STING pathway-dependent sensing of mitochondrial DNA mediates ocular surface inflammation.

The innate immune response is the main pathophysiological process of ocular surface diseases exposed to multiple environmental stresses. The epithelium is central to the innate immune response, but whether and how innate immunity is initiated by ocular epithelial cells in response to various environmental stresses in ocular surface diseases, such as dry eye, is still unclear. By utilizing two classic experimental dry eye models-a mouse ocular surface treated with benzalkonium chloride (BAC) and a mouse model with surgically removed extraorbital lachrymal glands, as well as dry eye patient samples-along with human corneal epithelial cells (HCE) exposed to hyperosmolarity, we have discovered a novel innate immune pathway in ocular surface epithelial cells. Under stress, mitochondrial DNA (mtDNA) was released into the cytoplasm through the mitochondrial permeability transition pore (mPTP) and further activated the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, aggravating downstream inflammatory responses and ocular surface damage. Genetic deletion or pharmacological suppression of STING and inhibition of mtDNA release reduced inflammatory responses, whereas mtDNA transfection supported cytoplasmic mtDNA-induced inflammatory responses by activating the cGAS-STING pathway. Our study clarified the cGAS-STING pathway-dependent sensing of mitochondrial DNA-mediated ocular surface inflammation, which elucidated a new mechanism of ocular surface diseases in response to multiple environmental stresses.

Limbal Stem Cell Dysfunction Induced by Severe Dry Eye via Activation of the p38 MAPK Signaling Pathway.

Severe dry eye (SDE) can cause grievous damage to the ocular surface and result in vision impairment and even blindness. To investigate the fate of limbal stem cells in SDE and the underlying mechanism, the current study established an SDE rat model by removing the extraorbital and infraorbital lacrimal glands and maintaining them in a low-humidity environment. One month after the surgery, aqueous tear secretion was reduced dramatically, blood vessels invaded into the central cornea, and inflammatory cells infiltrated into the limbal stroma. The expressions of keratin 12 and paired box gene 6 were down-regulated dramatically, while those of keratin 10, small proline-rich protein 1b, and mucin 5AC were up-regulated in the corneal epithelium of the SDE rats. Cell proliferation in the limbal epithelium was up-regulated, while the stem/progenitor marker adenosine 5'-triphosphate-binding cassette member 2 and the limbal epithelial colony-forming efficiency were decreased in the SDE condition. Furthermore, the p38 mitogen-activated protein kinase signaling pathway was activated in the limbal corneal epithelium of SDE rats. The abnormal differentiation and stemness loss in the corneal epithelium could be reversed upon treatment with a p38 inhibitor in a SDE in vivo model and in vitro hyperosmolar corneal epithelial culture conditions. These data suggest that SDE can lead to limbal stem cell dysfunction, and p38 mitogen-activated protein kinase signaling pathway activation plays an essential role in this process.

Decellularized squid mantle scaffolds as tissue-engineered corneal stroma for promoting corneal regeneration.

Corneal blindness is a worldwide major cause of vision loss, and corneal transplantation remains to be the most effective way to restore the vision. However, often there is a shortage of the donor corneas for transplantation. Therefore, it is urgent to develop a novel tissue-engineered corneal substitute. The present study envisaged the development of a novel and efficient method to prepare the corneal stromal equivalent from the marine biomaterials-squid. A chemical method was employed to decellularize the squid mantle scaffold to create a cell-free tissue substitute using 0.5% sodium dodecyl sulfate (SDS) solution. Subsequently, a novel clearing method, namely clear, unobstructed brain imaging cocktails (CUBIC) method was used to transparent it. Decellularized squid mantle scaffold (DSMS) has high decellularization efficiency, is rich in essential amino acids, and maintains the regular fiber alignment. In vitro experiments showed that the soaking solution of DSMS was non-toxic to human corneal epithelium cells. DSMS exhibited a good biocompatibility in the rat muscle by undergoing a complete degradation, and promoted the growth of the muscle. In addition, the DSMS showed a good compatibility with the corneal stroma in the rabbit inter-corneal implantation model, and promoted the regeneration of the corneal stroma without any evident rejection. Our results indicate that the squid mantle can be a potential new type of tissue-engineered corneal stroma material with a promising clinical application.

MAP4Ks inhibition promotes retinal neuron regeneration from Müller glia in adult mice.

Mammalian Müller glia (MG) possess limited regenerative capacities. However, the intrinsic capacity of mammalian MG to transdifferentiate to generate mature neurons without transgenic manipulations remains speculative. Here we show that MAP4K4, MAP4K6 and MAP4K7, which are conserved Misshapen subfamily of ste20 kinases homologs, repress YAP activity in mammalian MG and therefore restrict their ability to be reprogrammed. However, by treating with a small molecule inhibitor of MAP4K4/6/7, mouse MG regain their ability to proliferate and enter into a retinal progenitor cell (RPC)-like state after NMDA-induced retinal damage; such plasticity was lost in YAP knockout MG. Moreover, spontaneous trans-differentiation of MG into retinal neurons expressing both amacrine and retinal ganglion cell (RGC) markers occurs after inhibitor withdrawal. Taken together, these findings suggest that MAP4Ks block the reprogramming capacity of MG in a YAP-dependent manner in adult mammals, which provides a novel avenue for the pharmaceutical induction of retinal regeneration in vivo.

Transitory alkali exposure on meibomian gland orifices induces meibomian gland dysfunction.

PURPOSE: To determine pathological changes of meibomian glands (MGs) after transient exposure of the rat eyelid margin to alkali solution. METHODS: Filter paper infiltrated with 1 N sodium hydroxide solution was applied to the eyelid margin of Sprague-Dawley rats for 30 s under general anesthesia, without touching the conjunctiva, after which the ocular surface and eyelid margin were examined by slit-lamp microscopy. In vivo confocal microscopy and stereomicroscopy were subsequently applied to observe MG morphology on day 5, day 10 and day 30 post alkali injury. Eyelid cross-sections were processed for H&E staining, Oil red O staining and immunofluorescent staining. RESULTS: After alkali injury, there was marked plugging of MG orifices, telangiectasia and hypertrophy of the eyelid margin, while corneal epithelium was intact at post-injury days 5 and 10. However, 30 days after alkali injury, mild corneal epithelial damage was observed. Degeneration of MG acini was observed at days 5 and became aggravated at days 10 and 30, along with MG duct dilation and acini loss. Oil red O staining showed lipid accumulation in the dilated duct. Inflammatory cell infiltration and the presence of apoptotic cells was seen in the MG loci 5 days post injury, but diminished at days 10 and 30. Cytokeratin 10 expression was increased in dilated duct, while cytokeratin 14, PPAR-γ, Ki67 and LRIG1 expression were decreased in the acini of injured loci. CONCLUSIONS: Transitory alkali exposure of the rat eyelid margin obstructs the MG orifice and induces pathological changes of MG dysfunction.

eIF2α incites photoreceptor cell and retina damage by all-trans-retinal.

Dry age-related macular degeneration (AMD) and recessive Stargardt's disease (STGD1) lead to irreversible blindness in humans. The accumulation of all-trans-retinal (atRAL) induced by chaos in visual cycle is closely associated with retinal atrophy in dry AMD and STGD1 but its critical downstream signaling molecules remain ambiguous. Here, we reported that activation of eukaryotic translation initiation factor 2α (eIF2α) by atRAL promoted retinal degeneration and photoreceptor loss through activating c-Jun N-terminal kinase (JNK) signaling-dependent apoptosis and gasdermin E (GSDME)-mediated pyroptosis. We determined that eIF2α activation by atRAL in photoreceptor cells resulted from endoplasmic reticulum homeostasis disruption caused at least in part by reactive oxygen species production, and it activated JNK signaling independent of and dependent on activating transcription factor 4 and the activating transcription factor 4/transcription factor C/EBP homologous protein (CHOP) axis. CHOP overexpression induced apoptosis of atRAL-loaded photoreceptor cells through activating JNK signaling rather than inhibiting the expression of antiapoptotic gene Bcl2. JNK activation by eIF2α facilitated photoreceptor cell apoptosis caused by atRAL via caspase-3 activation and DNA damage. Additionally, we demonstrated that eIF2α was activated in neural retina of light-exposed Abca4-/-Rdh8-/- mice, a model that shows severe defects in atRAL clearance and displays primary features of human dry AMD and STGD1. Of note, inhibition of eIF2α activation by salubrinal effectively ameliorated retinal degeneration and photoreceptor apoptosis in Abca4-/-Rdh8-/- mice upon light exposure. The results of this study suggest that eIF2α is an important target to develop drug therapies for the treatment of dry AMD and STGD1.

A Rational Design of Metal-Organic Framework Nanozyme with High-Performance Copper Active Centers for Alleviating Chemical Corneal Burns.

Metal-organic frameworks (MOFs) have attracted significant research interest in biomimetic catalysis. However, the modulation of the activity of MOFs by precisely tuning the coordination of metal nodes is still a significant challenge. Inspired by metalloenzymes with well-defined coordination structures, a series of MOFs containing halogen-coordinated copper nodes (Cu-X MOFs, X = Cl, Br, I) are employed to elucidate their structure-activity relationship. Intriguingly, experimental and theoretical results strongly support that precisely tuning the coordination of halogen atoms directly regulates the enzyme-like activities of Cu-X MOFs by influencing the spatial configuration and electronic structure of the Cu active center. The optimal Cu-Cl MOF exhibits excellent superoxide dismutase-like activity with a specific activity one order of magnitude higher than the reported Cu-based nanozymes. More importantly, by performing enzyme-mimicking catalysis, the Cu-Cl MOF nanozyme can significantly scavenge reactive oxygen species and alleviate oxidative stress, thus effectively relieving ocular chemical burns. Mechanistically, the antioxidant and antiapoptotic properties of Cu-Cl MOF are achieved by regulating the NRF2 and JNK or P38 MAPK pathways. Our work provides a novel way to refine MOF nanozymes by directly engineering the coordination microenvironment and, more significantly, demonstrating their potential therapeutic effect in ophthalmic disease.

T-Cell Repertoire Analysis in the Conjunctiva of Murine Dry Eye Model.

Purpose: Dry eye is closely related to the activation and proliferation of immune cells, especially T cells. However, the determination of the preferential T-cell clonotypes is technically challenging. This study aimed to investigate the characterization of T-cell receptor (TCR) repertoire in the conjunctiva during dry eye. Methods: A desiccating stress animal model was established using C57/BL6 mice (8-10 weeks, female). After 7 days of stress stimulation, the slit-lamp image and Oregon-green-dextran staining were used to evaluate the ocular surface injury. Periodic acid-Schiff staining was used to measure the number of goblet cells. Flow cytometry was used to detect the activation and proliferation of T cells in the conjunctiva and cervical lymph nodes. Next-generation sequencing was used to detect the αß TCR repertoire of the conjunctiva. Results: The αß TCR diversity increased significantly in the dry eye group, including the higher CDR3 amino acid length, marked gene usage on TCR V and J gene segments, extensive V(D)J recombination, and distinct CDR3 aa motifs. More important, several T-cell clonotypes were uniquely identified in dry eye. Furthermore, these perturbed rearrangements were reversed after glucocorticoid administration. Conclusions: A comprehensive analysis of the αß TCR repertoire in the conjunctiva of the dry eye mouse model was performed. Data in this study contributed significantly to the research on dry eye pathogenesis by demonstrating the TCR gene distribution and disease-specific TCR signatures. This study further provided some potential predictive T-cell biomarkers for future studies.

Effect of palmitoylethanolamide on degeneration of a human-derived retinal pigment epithelial cell induced by all-trans retinal.

AIM: To study the effect of palmitoylethanolamide (PEA) on apoptosis of retinal pigment epithelial (RPE) cells induced by all-trans retinal (atRAL) and to explore the possible molecular mechanism. METHODS: CellTiter 96® Aqueous One Solution Cell Proliferation Assay (MTS) was used to detect the effect of PEA on human-derived retinal epithelial cells (ARPE-19) viability induced by atRAL. A Leica DMi8 inverted microscope was used to observe cell morphology. Reactive oxygen species (ROS) production was evaluated with 2',7'-dichlorodihydrof-luorescein diacetate (H2DCFDA) staining and fluorescence microscopy. Expression of c-Jun N-terminal kinase (JNK), phosphorylated JNK (p-JNK), c-Jun, phosphorylated c-Jun (p-c-Jun), Bak, cleaved caspase-3, C/EBP homologous protein (CHOP), and binding (Bip) protein levels were tested by Western blot. Abca4 -/- Rdh8 -/- mice, mouse models of atRAL clearance defects which displays some symbolic characteristics of dry age-related macular degeneration (AMD) and Stargardt disease (STGD1). In the animal models, PEA was injected intraperitoneally. The full-field electroretinogram was used to detect visual function under scotopic conditions traced from mice. Optical coherence tomography showed reconstitution or thickening of the retinal pigment epithelium layer. Effect of PEA on fundus injury induced by light in Abca4-/-Rdh8-/- mice was observed by fundus photography. RESULTS: PEA ameliorated ARPE-19 cells apoptosis and inhibited ROS (including mitochondrial ROS) production induced by atRAL. PEA improved the retinal functional, prohibited both RPE and photoreceptor from death, ameliorates light-induced fundus impairment in Abca4 -/- Rdh8 -/- mice. In vitro and in vivo, PEA inhibited JNK, p-JNK, c-Jun, p-c-Jun, Bak, cleaved caspase-3, CHOP, and Bip protein levels induced by all-trans retinal in ARPE-19 cells. CONCLUSION: PEA has effect on treating RPE cells apoptosis in retinopathy caused by atRAL accumulation. PEA is a potential treatment strategy for dry AMD and STGD1. The molecular mechanism is affecting the ROS-JNK-CHOP signaling pathway partly.