establishment-of-a-mouse-model-of-aqueous-deficiency-dry-eye

Establishment of A Mouse Model of Aqueous Deficiency Dry Eye

Dry eye disease is a prevalent condition affecting 5%-50% of the global population. Animal model investigations play a crucial role in understanding its ...

establishing-severe-corneal-inflammation-model-rats-based-on-corneal

Establishing a Severe Corneal Inflammation Model in Rats Based on Corneal Epithelium Curettage Combined with Corneal Sutures

Corneal inflammation, especially severe corneal inflammation, plays a significant role in the development of corneal limbal stem cell dysfunction. ...

a-protocol-for-harvesting-single-cell-suspension-from-mouse-corneas

A Protocol for Harvesting Single-cell Suspension from Mouse Corneas

As the outermost layer of the eyeball, the cornea plays a critical role in maintaining ocular surface homeostasis and vision. Recent advances in ...

the Affiliated Hospital of Guizhou Medical University

Guizhou Medical University

Trichilemmal carcinoma (TLC) is a rare malignant adnexal tumor most commonly found in the elderly, usually affecting the scalp, eyelids, neck and face. Here, we first reported a rare case of corneal perforation caused by eyelid margin TLC.

Corneal Perforation Caused by Eyelid Margin Trichilemmal Carcinoma: A Case Report and Review of Literature.

The single-cell RNA-sequencing (scRNA-seq) technique is used to explore the biological characteristics of tissues under pathological and physiological conditions that include certain chronic eye diseases. Harvesting of single-cell suspensions is one challenge inherent to scRNA-seq procedures. This study aimed to use an optimized method to digest a whole mouse cornea to harvest single-cell suspensions. We utilized five different mouse cornea digestion methods to obtain single-cell suspensions: (1) 5 dissected mouse corneas were cut into pieces (∼0.5 mm) and digested in trypsin for 10 min, and this digestion was repeated for 10 cycles; (2) 5 dissected mouse corneas were cut into pieces and incubated with 5 mg/ml collagenase A at 37 °C for 1h and then further digested in trypsin at 37 °C for 10 min; (3) used the same approach as that used in method 2, but the second digestion step was performed in TrypLE for 20 min; (4) used the same approach as that used in method 2, but the concentration of collagenase A was 2 mg/ml and the incubation time was 2h; (5) used the same approach as that used in method 3, but the corneas were incubated in 2 mg/ml collagenase A at 37 °C for 2h. Trypan blue staining was used to calculate the cell viability and agglomeration rate. The cell types and percentages were determined using immunofluorescence staining. RNA integrity number (RIN) was measured by Agilent 2100. Method 1 showed the lowest cell yield (0.375 × 10), epithelial cell percentage, and less than 70% cell viability, thus not a proper protocol. Method 2 showed the highest cell viability (over 90%), percentage of single-cell (89.53%), and high cell quantity (1.05 × 10). Method 3 had a significantly lower cell viability (55.30%). Cell agglomeration rates of method 4 and 5 reached up to 20% and 13%, and with lower cell viability (72.51%, 59.87%, respectively) and decreased epithelial cell rate compared to method 2 and 3. The results suggest that method 2 (5 mg/ml collagenase A and trypsin) is a preferred protocol for digesting mouse cornea to obtain single-cell suspension which achieves the criterion of single-cell RNA sequencing.

Optimization of method for achieving a single-cell suspension from mouse corneas.

In humans, the lacrimal gland is located in the socket of the frontal bone above the outer orbital area. As an essential part of the eye surface, the gland is fixed to the orbital periosteum by connective tissue. The lacrimal gland passes through the outer tendon membrane, which divides the gland into larger orbital and minor eyelid glands. The lacrimal glands are the main contributors to tear film. They secrete electrolytes, proteins, and water to help nourish and protect the eye's surface. Furthermore, clinically, lacrimal glands are associated with a variety of inflammatory reactions and immune factors and are also vulnerable sites for tumors. Changes in tear gland morphology or secretory function affect tear film stability and tear secretion quality. Various technological devices have been developed and applied to lacrimal glands. This article systematically reviewed the clinical examination of the lacrimal gland to help inform personalized strategies for the diagnosis of lacrimal gland-related diseases.

Advances in clinical examination of lacrimal gland.

Currently, in vitro cultured corneal epithelial transplantation is effective in treating limbal stem cell dysfunction (LSCD). Selecting carriers is crucial for constructing the corneal epithelium through tissue engineering. In this study, the traditional amniotic membrane (AM) was modified, and mesenchymal stem cells (MSCs) were inoculated into the ultra-thin amniotic membrane (UAM) stroma to construct a novel UAM-MSC tissue-engineered corneal epithelial carrier, that could effectively simulate the limbal stem cells (LSCs) microenvironment. The structure of different carriers cultured tissue-engineered corneal epithelium and the managed rabbit LSCD model corneas were observed through hematoxylin-eosin staining. Cell phenotypes were evaluated through fluorescence staining, Western blotting, and RT-qPCR. Additionally, cell junction genes and expression markers related to anti-neovascularization were evaluated using RT-qPCR. Corneal epithelium cell junctions were observed via an electron microscope. The tissue-engineered corneal epithelium culture medium was analyzed through mass spectrometry. Tissue-engineered corneal epithelial cells expanded by LSCs on UAM-MSCs had good transparency. Simultaneously, progenitor cell (K14, PNCA, p63) and corneal epithelial (PAX6) gene expression in tissue-engineered corneal epithelium constructed using UAM-MSCs was higher than that in corneal epithelial cells amplified by UAM and de-epithelialized amniotic membrane. Electron microscopy revealed that corneal epithelial cells grafted with UAM-MSCs were closely connected. In conclusion, the UAM-MSCs vector we constructed could better simulate the limbal microenvironment; the cultured tissue-engineered corneal epithelium had better transparency, anti-neovascularization properties, closer intercellular connections, and closer resemblance to the natural corneal epithelial tissue phenotype.

A novel tissue-engineered corneal epithelium based on ultra-thin amniotic membrane and mesenchymal stem cells.

Fuchs endothelial corneal dystrophy (FECD) stands as the most prevalent primary corneal endothelial dystrophy worldwide, posing a significant risk to corneal homeostasis and clarity. Corneal endothelial cells exhibit susceptibility to oxidative stress, suggesting a nuanced relationship between oxidant-antioxidant imbalance and FECD pathogenesis, irrespective of FECD genotype. Given the constrained availability of corneal transplants, exploration into non-surgical interventions becomes crucial. This encompasses traditional antioxidants, small molecule compounds, biologics, and diverse non-drug therapies, such as gene-related therapy, hydrogen therapy and near infrared light therapy. This review concentrates on elucidating the mechanisms behind oxidant-antioxidant imbalance and the evolution of strategies to restore oxidant-antioxidant balance in FECD. It provides a comprehensive overview of both conventional and emerging therapeutic approaches, offering valuable insights for the advancement of non-surgical treatment modalities. The findings herein might establish a robust foundation for future research and the therapeutic strategy of FECD.

Evolution of therapeutic strategy based on oxidant-antioxidant balance for fuchs endothelial corneal dystrophy.

Dry eye can damage the ocular surface and result in mild corneal epithelial defect to blinding corneal pannus formation and squamous metaplasia. Significant progress in the treatment of dry eye has been made in the last two decades; progressing from lubricating and hydrating the ocular surface with artificial tear to stimulating tear secretion; anti-inflammation and immune regulation. With the increase in knowledge regarding the pathophysiology of dry eye, we propose in this review the concept of ocular surface microenvironment. Various components of the microenvironment contribute to the homeostasis of ocular surface. Compromise in one or more components can result in homeostasis disruption of ocular surface leading to dry eye disease. Complete evaluation of the microenvironment component changes in dry eye patients will not only lead to appropriate diagnosis, but also guide in timely and effective clinical management. Successful treatment of dry eye should be aimed to restore the homeostasis of the ocular surface microenvironment.

Dry Eye Management: Targeting the Ocular Surface Microenvironment.

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.

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

The purpose of this study was to investigate the clinical characteristics of patients with corneal Mooren's ulcers (MU) and explore potential risk factors for recurrence after keratoplasty.

ArticleTotal : 3	Year
Publication title Cited by 2	2024
Publication title Cited by 2	2024
Publication title	2026

Article	Year
Corneal Perforation Caused by Eyelid Margin Trichilemmal Carcinoma: A Case Report and Review of Literature. Frontiers in medicine\| PubMed ID: 35646982	2022
Optimization of method for achieving a single-cell suspension from mouse corneas. Experimental eye research\| PubMed ID: 37336469	2023
Advances in clinical examination of lacrimal gland. Frontiers in medicine\| PubMed ID: 37720501	2023
A novel tissue-engineered corneal epithelium based on ultra-thin amniotic membrane and mesenchymal stem cells. Scientific reports\| PubMed ID: 39075142	2024
Evolution of therapeutic strategy based on oxidant-antioxidant balance for fuchs endothelial corneal dystrophy. The ocular surface\| PubMed ID: 39111696	2024
Dry Eye Management: Targeting the Ocular Surface Microenvironment. International journal of molecular sciences\| PubMed ID: 28661456	2017
Limbal Stem Cell Dysfunction Induced by Severe Dry Eye via Activation of the p38 MAPK Signaling Pathway. The American journal of pathology\| PubMed ID: 37634709	2023
Risk factors for recurrence of Mooren's ulcer after keratoplasty. Heliyon\| PubMed ID: 39553563	2024

Shangkun Ou

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