The association between altered intestinal microbiome, impaired systemic and ocular surface immunity, and impaired wound healing response after corneal alkaline-chemical injury in diabetic mice.

Purpose: We aim to investigate the effect of sustained hyperglycemia on corneal epithelial wound healing, ocular surface and systemic immune response, and microbiome indices in diabetic mice compared to controls after alkaline chemical injury of the eye. Methods: Corneal alkaline injury was induced in the right eye of Ins2Akita (Akita) mice and wild-type mice. The groups were observed at baseline and subsequently days 0, 3, and 7 after injury. Corneal re-epithelialization was observed under slit lamp with fluorescein staining using a cobalt blue light filter. Enucleated cornea specimens were compared at baseline and after injury for changes in cornea thickness under hematoxylin and eosin staining. Tear cytokine and growth factor levels were measured using protein microarray assay and compared between groups and time points. Flow cytometry was conducted on peripheral blood and ocular surface samples to determine CD3+CD4+ cell count. Fecal samples were collected, and gut microbiota composition and diversity pattern were measured using shotgun sequencing. Results: Akita mice had significantly delayed corneal wound healing compared to controls. This was associated with a reduction in tear levels of vascular endothelial growth factor A, angiopoietin 2, and insulin growth factor 1 on days 0, 3, and 7 after injury. Furthermore, there was a distinct lack of upregulation of peripheral blood and ocular surface CD3+CD4+ cell counts in response to injury in Akita mice compared to controls. This was associated with a reduction in intestinal microbiome diversity indices in Akita mice compared to controls after injury. Specifically, there was a lower abundance of Firmicutes bacterium M10-2 in Akita mice compared to controls after injury. Conclusion: In diabetic mice, impaired cornea wound healing was associated with an inability to mount systemic and local immune response to ocular chemical injury. Baseline and post-injury differences in intestinal microbial diversity and abundance patterns between diabetic mice and controls may potentially play a role in this altered response.

The anti-scarring role of Lycium barbarum polysaccharide on cornea epithelial-stromal injury.

PURPOSE: Cornea epithelial-stromal scarring is related to the differentiation of fibroblasts into opaque myofibroblasts. Our study aims to assess the effectiveness of Lycium barbarum polysaccharide (LBP) solution as a pre-treatment in minimizing corneal scarring. METHODS: Human corneal fibroblasts were cultured in a three-dimensional collagen type I-based hydrogel in an eye-on-a-chip model. Fibroblasts were pre-treated with 2 mg/mL LBP for 24 h, followed by another 24-h incubation with 10 ng/mL transforming growth factor-beta 1 (TGF-ß1) to induce relevant physiological events after stromal injury. Intracellular pro-fibrotic proteins, extracellular matrix proteins, and pro-inflammatory cytokines that involved in fibrosis, were assessed using immunocytochemistry and enzyme-linked immunosorbent assays. RESULTS: Compared to the positive control TGF-ß1 group, LBP pre-treated cells had a significantly lower expression of alpha-smooth muscle actin, marker of myofibroblasts, vimentin (p < 0.05), and also extracellular matrix proteins both collagen type II and type III (p < 0.05) that can be found in scar tissues. Moreover, LBP pre-treated cells had a significantly lower secretion of pro-inflammatory cytokines interleukin-6 and interleukin-8 (p < 0.05). The cell-laden hydrogel contraction and stiffness showed no significant difference between LBP pre-treatment and control groups. Fibroblasts pretreated with LBP as well had reduced angiogenic factors expression and suppression of undesired proliferation (p < 0.05). CONCLUSION: Our results showed that LBP reduced both pro-fibrotic proteins and pro-inflammatory cytokines on corneal injury in vitro. We suggest that LBP, as a natural Traditional Chinese Medicine, may potentially be a novel topical pre-treatment option prior to corneal refractive surgeries with an improved prognosis.

Lycium barbarum Polysaccharide Suppresses Expression of Fibrotic Proteins in Primary Human Corneal Fibroblasts.

(1) Objective: To study the anti-fibrotic effects of Lycium barbarum polysaccharides (LBP) on corneal stromal fibroblasts and assess LBP's effect on cell viability. (2) Methods: Primary human corneal keratocytes of passage 3 to 6 were used for all experiments. Cells are pretreated with LBP solution for 24 h and then transforming growth factor beta 1 (TGFß1) for 48 h and collected for experiments. Fibrotic protein analysis was performed using immunofluorescence and Western blot. The effect of LBP on cell viability was assessed using the MTS assay. (3) Results: LBP significantly reduced the expression of fibrotic proteins, including α-SMA and extracellular matrix proteins (collagen type I and III). LBP significantly decreased the viability of myofibroblasts but not the fibroblasts. Conclusions: In this study, LBP was effective in the prevention of fibrosis gene expression. Further studies to assess the underlying mechanism and pharmacological properties will facilitate the formation of a topical LBP solution for in vivo studies.

Therapeutic Strategies for Attenuation of Retinal Ganglion Cell Injury in Optic Neuropathies: Concepts in Translational Research and Therapeutic Implications.

Retinal ganglion cell (RGC) death is the central and irreversible endpoint of optic neuropathies. Current management of optic neuropathies and glaucoma focuses on intraocular pressure-lowering treatment which is insufficient. As such, patients are effectively condemned to irreversible visual impairment. This review summarizes experimental treatments targeting RGCs over the last decade. In particular, we examine the various treatment modalities and determine their viability and limitations in translation to clinical practice. Experimental RGC treatment can be divided into (1) cell replacement therapy, (2) neuroprotection, and (3) gene therapy. For cell replacement therapy, difficulties remain in successfully integrating transplanted RGCs from various sources into the complex neural network of the human retina. However, there is significant potential for achieving full visual restoration with this technique. Neuroprotective strategies, in the form of pharmacological agents, nutritional supplementation, and neurotrophic factors, are viable strategies with encouraging results from preliminary noncomparative interventional case series. It is important to note, however, that most published studies are focused on glaucoma, with few treating optic neuropathies of other etiologies. Gene therapy, through the use of viral vectors, has shown promising results in clinical trials, particularly for diseases with specific genetic mutations like Leber's hereditary optic neuropathy. This treatment technique can be further extended to nonhereditary diseases, through transfer of genes promoting cell survival and neuroprotection. Crucially though, for gene therapy, teratogenicity remains a significant issue in translation to clinical practice.

Experimental modeling of cornea wound healing in diabetes: clinical applications and beyond.

Diabetes mellitus is the most common cause of blindness in working age populations worldwide. While much of the focus for public health has been on secondary prevention in sight-threatening diabetic retinopathy, the cornea, including its epithelium and nerves, represents a major site of damage by chronic hyperglycemia. On injury, the diabetic cornea exhibits a delayed wound-healing response, as well as an altered ocular surface immune response. This suggests a potential association between the dysfunctional wound healing response and altered inflammation on the ocular surface. However, the presence of potential confounders makes this association difficult to investigate in human epidemiological studies. Thus, we turn to animal diabetic models for a better understanding. In this review, 20 original studies, published between 2008 and 2018, describe in vivo and in vitro models of diabetic cornea disease. We compared different models of diabetic cornea wound healing and discussed the relative strengths and drawbacks of each model. A number of molecular and cellular components involved in the corneal wound healing response that are altered in the presence of diabetes have been identified in the reviewed studies. Particularly, altered corneal epithelial protein concentrations of lumician and occludin were detected in diabetic eyes compared with controls. Additionally, the importance of IL-1ß in modulating the inflammatory response after corneal injury in patients with diabetes and controls was further elucidated. Meanwhile, abnormal P2×7 receptor localization and decreased corneal sub-basal nerve density in diabetic eyes were shown to contribute to altered corneal nerve signaling after injury and thus affecting the wound healing response. Finally, the discovery of the therapeutic effects of topically administered aloe vera, Serpine 1, Resolvin D1 (RvD1), pigment epithelium-derived factor (PEDF) and Pro-His-Ser-Arg-Asn in diabetic animal models of cornea epithelial and nerve injury provide encouraging evidence for the future availability of effective treatment for diabetic keratopathy.

A Systematic Review of Potential Therapeutic Use of Lycium Barbarum Polysaccharides in Disease.

OBJECTIVE: To evaluate the effect of Lycium barbarum polysaccharides in the treatment and/or prevention of diseases of different etiologies and systems. METHODS: We performed an Entrez PubMed literature search using keywords "lycium", "barbarum", "polysaccharides", "anti-fibrotic", "anti-apoptotic", "anti-oxidizing", "anti-aging", "neuroprotection", "metabolism", "diabetes", "hyperlipidemia", "neuroprotection", and "immunomodulation" on the 14th of August 2018, resulting in 207 papers, of which 20 were chosen after filtering for 'English language' and 'published within 10 years' as well as curation for relevance by the authors. RESULTS: The 20 selected papers included 2 randomized control trials (1 double-blinded RCT and 1 double-blinded placebo-controlled RCT), 11 in vivo studies, 5 in vitro studies, 1 study with both in vivo and in vitro results, and 1 chemical study. There is good evidence from existing studies on the antifibrotic, antioxidizing, neuroprotective, anticancer, and anti-inflammatory effects of Lycium barbarum polysaccharides. However, there is a need for further studies in the form of large-scale clinical trials to support its use in humans. There is also significant potential for LBP as a safe and effective topical treatment in ocular surface diseases, owing to promising in vitro results and a lack of demonstrated toxic effects to corneal epithelial cells. CONCLUSION: Results from existing studies suggest that LBP is a promising therapeutic agent, particularly in the management of liver disease, hyperlipidemia, and diabetes. One major limitation of current research is a lack of standardization and quality control for the LBP used. The availability of research-grade LBP will inevitably promote future research in this field worldwide.