Distinctive small molecules blend: Promotes lacrimal gland epithelial cell proliferation in vitro and accelerates lacrimal gland injury repair in vivo.

PURPOSE: This study aims to develop a novel serum-free culture strategy containing only two small molecules, Y27632 and SB431542 (2C), for in vitro expansion of mouse lacrimal gland epithelial cells (LGECs) and investigate an innovative therapeutic approach for lacrimal gland (LG) injury. METHODS: LGECs proliferative capacity was assessed by cell counting, crystal violet staining, qRT-PCR and immunofluorescence. Cell differentiation was achieved by manipulating culture conditions and assessed by qRT-PCR and AQP5 immunofluorescence. LGECs were seeded in Matrigel for three-dimensional culture and assessed by qRT-PCR and immunofluorescence. Secretory function of the cultures was assayed by ELISA. In vivo, 2C injection verified its reparative capacity in a mouse LG injury model. Corneal fluorescein staining, phenol red cotton thread, H&E, immunofluorescence and Western blot were used to assess LG injury repair. RESULTS: LGECs cultured with 2C exhibited high expression of stemness/proliferation markers and maintained morphology and proliferative capacity even after the tenth passage. Removal of 2C was efficacious in achieving LGECs differentiation, characterized by the increased AQP5 expression and LTF secretion. 3D spheroids cultured with 2C demonstrated differentiation potential, forming microglandular structures containing multiple LG cell types with secretory functions after 2C removal. In vivo, 2C improved the structural integrity and function of the injured LG. CONCLUSIONS: We present a small molecule combination, 2C, that promotes LGECs expansion and differentiation in vitro and accelerates LG injury repair in vivo. This approach has potential applications for providing a stable source of seed cells for tissue engineering applications, providing new sights for LG-related diseases treatment.

Organotypic culture model of mouse meibomian gland as a screening platform for risk factors related to meibomian gland dysfunction.

PURPOSE: Meibomian glands (MGs) are crucial for maintaining tear film stability and ocular surface health. Here, we aim to establish a novel organotypic culture model of MGs and explore the risk factors of MG dysfunction (MGD). METHODS: We developed a novel organotypic culture model for MGs at the air-liquid interface. The viability and cell proliferation of MGs were assessed using CCK-8, immunofluorescence, and qPCR. Lipid accumulation was evaluated by Nile red staining and microscopic examination. Protein expression levels were evaluated by immunofluorescence and Western blot assay. EdU assay was employed to track the proliferation of acinar cells. The validity of the model was confirmed through culturing MGs from mice of different ages and incorporating certain drugs (Dex) into the culture system. RESULTS: Utilizing the novel culture model, the MG tissue exhibited sustained viability, cellular division, and continuous production of lipids for a duration of 7 days. Lipid droplets formed were directly visualized using light field microscopy. Through the cultivation of aged mice's MGs, it was discovered that aging resulted in diminished proliferation and lipid synthesis, along with an aberrant increase in Krt10 expression. Further application of this model showed that Dex treatment diminished MG's proliferation and lipid synthesis. Finally, an in vivo study was conducted to provide additional confirmation of the phenomenon of Dex-induced abnormalities. CONCLUSIONS: In this study, a stable organotypic culture model of the MGs was established. The organotypic culture model offers a valuable tool to investigate the pathophysiological mechanisms and facilitate drug screening for MG-related diseases.

A cocktail of small molecules maintains the stemness and differentiation potential of conjunctival epithelial cells.

PURPOSE: The conjunctival epithelial cells cultured with bovine serum or feeder cells were not suitable for clinical application. Therefore, we developed a novel serum-free and feeder cell-free culture system containing only a cocktail of three chemicals (3C) to expand the conjunctival epithelial cells. METHODS: The cell proliferative ability was evaluated by counting, crystal violet staining and Ki67 immunostaining. Co-staining of K7 and MUC5AC was performed to identify goblet cells. PAS staining was used to assess the ability of cells to synthesis and secrete glycoproteins. In vivo, eye drops containing 3C was administered to verify the role of 3C in the mouse conjunctival injury model. PAS, HE and immunofluorescence staining were performed to show conjunctival epithelial repair. RESULTS: Compared with other small molecule groups and the serum group, the cells in 3C group showed superior morphology and proliferative ability. Meanwhile, 3C maintained the well-proliferative capacity of cells even after fifth passage. The 3C group also exhibited more K7 and MUC5AC double positive cells, and the PAS staining positive areas were present in both the cytoplasm and extracellular matrix. The cell sheets treated with 3C in air-lifted culture were obviously stratified. In vivo, more goblet cells in the conjunctival epithelium were observed in the 3C group. CONCLUSION: Overall, our culture system can expand the conjunctival epithelial cells and retain their potential to differentiate into mature goblet cells, which provided a promising source of seed cells for conjunctival reconstruction. Furthermore, this system provides new insights for the clinical treatment of ocular surface diseases.

Foldable capsular vitreous body implantation for treatment of traumatic retinal detachment: two case reports.

Retinal detachment caused by severe ocular trauma is a type of refractory vitreoretinal disease. Current treatment methods include vitrectomy combined with silicone oil tamponade. However, long-term use of silicone oil tamponade has various complications, including a risk of silicone oil dependence that eventually leads to eyeball atrophy and enucleation. Foldable capsular vitreous bodies (FCVBs) offer a good solution for these problems. However, FCVBs have not been used in large-scale clinical applications and few cases have been reported in the published literature. The main use of FCVBs, based on current evidence, is in the treatment of the relatively few (but important) patients whose eyes have no visual potential; the aim of treatment in these patients is globe preservation, rather than restoration of vision. Here, we describe two patients who underwent FCVB implantation. The findings in these patients indicated that FCVBs can effectively support the vitreous cavity and detached retina. FCVB implantation may thus offer a safe and effective method for treatment of severe retinal detachment, avoiding the inconvenience caused by silicone oil dependence and enucleation. To confirm its long-term usefulness in clinical applications, many additional case reports are needed.

Melatonin ameliorates ANIT­induced cholestasis by activating Nrf2 through a PI3K/Akt­dependent pathway in rats.

Cholestasis is a devastating liver condition which is increasing in prevalence worldwide; however, its underlying pathogenic mechanisms remain to be fully elucidated. It was hypothesised that melatonin may alleviate the hepatic injury associated with cholestasis due to its established antioxidant effects. Therefore, the effect and potential anticholestatic properties of melatonin were investigated in rats with α­naphthylisothiocyanate (ANIT)­induced liver injury, a common animal model that mimics the cholestasis­associated liver injury in humans. The rats received intraperitoneal injection of ANIT with or without subsequent treatment with melatonin, and were sacrificed 24 h later. The serum biochemistry parameters of the liver were measured using conventional laboratory assays, and the liver tissue was subjected to conventional histological examination, reverse transcription­quantitative polymerase chain reaction analysis and western blotting. The levels of alanine transaminase, aspartate transaminase, total bilirubin, direct bilirubin, total bile acids, alkaline phosphatase, γ­glutamyl transferase and glutathione were restored in rats treated with melatonin. Histological examination provided further evidence supporting the protective effect of melatonin against ANIT­induced cholestasis. In addition, the mRNA and protein expression levels of glutamate cysteine ligase, phosphorylated Akt and nuclear factor­erythroid 2­related factor­2 were restored in rats treated with melatonin. These findings indicate that melatonin is a natural agent that appears to be promising for the treatment of cholestasis, and that the anticholestatic effects of melatonin involve the alleviation of oxidative stress.

Identification of potential biomarkers in cholestasis and the therapeutic effect of melatonin by metabolomics, multivariate data and pathway analyses.

The present study investigated the anti­cholestatic effect of melatonin (MT) against α­naphthyl isothiocyanate (ANIT)­induced liver injury in rats and screened for potential biomarkers of cholestasis. Rats were administered ANIT by intraperitoneal injection and then sacrificed 36 h later. Serum biochemical parameters were measured and liver tissue samples were subjected to histological analysis. Active components in the serum were identified by gas chromatography­mass spectrometry, while biomarkers and biochemical pathways were identified by multivariate data analysis. The results revealed that the serum levels of alanine aminotransferase, aspartate aminotransferase, total bilirubin, direct bilirubin, γ­glutamyl transpeptidase, and alkaline phosphatase were reduced in rats with ANIT­induced cholestasis that were treated with MT. The histological observations indicated that MT had a protective effect against ANIT­induced hepatic tissue damage. Metabolomics analysis revealed that this effect was likely to be associated with the regulation of compounds related to MT synthesis and catabolism, and amino acid metabolism, including 5­aminopentanoate, 5­methoxytryptamine, L­tryptophan, threonine, glutathione, L­methionine, and indolelactate. In addition, principal component analysis demonstrated that the levels of these metabolites differed significantly between the MT and control groups, providing further evidence that they may be responsible for the effects induced by MT. These results provide an insight into the mechanisms underlying cholestasis development and highlight potential biomarkers for disease diagnosis.