Protective effects of resolvin D1 in Pseudomonas aeruginosa keratitis.

PURPOSE: Here, we explored the protective effects of resolvin D1 (RvD1) in Pseudomonas aeruginosa (PA) keratitis. METHODS: C57BL/6 (B6) mice were used as an animal model of PA keratitis. Plate counting and clinical scores were used to assess the severity of the infection and the therapeutic effects of RvD1 in the model. Myeloperoxidase assay was used to detect neutrophil infiltration and activity. Quantitative PCR (qPCR) was used to examine the expression of proflammatory and anti-inflammatory mediators. Immunofluorescence staining and qPCR were performed to identify macrophage polarization. RESULTS: RvD1 treatment alleviated PA keratitis severity by decreasing corneal bacterial load and inhibiting neutrophil infiltration in the mouse model. Furthermore, RvD1 treatment decreased mRNA levels of TNF-α, IFN-γ, IL-1ß, CXCL1, and S100A8/9 while increasing those of IL-1RA, IL-10, and TGF-ß1. RvD1 treatment also reduced the aggregation of M1 macrophages and increased that of M2 macrophages. RvD1 provided an auxiliary effect in gatifloxacin-treated mice with PA keratitis. CONCLUSION: Based on these findings, RvD1 may improve the prognosis of PA keratitis by inhibiting neutrophil recruitment and activity, dampening the inflammatory response, and promoting M2 macrophage polarization. Thus, RvD1 may be a potential complementary therapy for PA keratitis.

Therapeutic effect of Atractylenolide I on Aspergillus fumigatus keratitis by affecting MyD88/ NF-κB pathway and IL-1ß, IL-10 expression.

PURPOSE: Atractylenolide I (AT-I) is a natural sesquiterpene with anti-inflammatory effects. The purpose of this study was to research the anti-inflammatory effect of AT-I on Aspergillus fumigatus(A. fumigatus) keratitis in mice. METHODS: Cytotoxicity test and cell scratch test were used to determine the therapeutic concentrations of corneal infections. In vivo and in vitro studies, mouse cornea and human corneal epithelial cells (HCECs) infected with A. fumigatus were treated with AT-I or dimethyl sulfoxide (DMSO). Then, to analyze the effect of AT-I on inflammatory response, namely neutrophil or macrophage recruitment and the expression of cytokines involving MyD88, NF-κB, interleukin 1ß (IL-1ß) and interleukin 10 (IL-10). To study the effects of the drug, the techniques used include slit-lamp photography, immunofluorescence, myeloperoxidase (MPO) detection, quantitative real-time polymerase chain reaction (QRT-PCR), and western blot. At the same time, in order to explore the combined effect of the drug and natamycin, slit-lamp photographs and clinical scores were used to visually display the disease process. RESULTS: No cytotoxicity was observed under the action of AT-I at a concentration of 800 µM. In mouse models, AT-I significantly suppressed inflammatory responses, reduced neutrophil and macrophage recruitment, and decreased myeloperoxidase levels early in infection. Studies have shown that AT-I may reduce the levels of IL-1ß and IL-10 by inhibiting the MyD88/ NF-κB pathway. The drug combined with natamycin can increase corneal transparency in infected mice. CONCLUSION: AT-I may inhibit MyD88 / NF-κB pathway and the secretion of inflammatory factors IL-1 ß and IL-10 to achieve the therapeutic effect of fungal keratitis.

Cationic Polyelectrolyte Nanocapsules of Moxifloxacin for Microbial Keratitis Therapy: Development, Characterization, and Pharmacodynamic Study.

Microbial keratitis (MK) is a vision-threatening disease and the fourth leading cause of blindness worldwide. In this work, we aim to develop moxifloxacin (MXN)-loaded chitosan-based cationic mucoadhesive polyelectrolyte nanocapsules (PENs) for the effective treatment of MK. PENs were formulated by polyelectrolyte complex coacervation method and characterized for their particle size, surface charge, morphology, mucoadhesive property, in-vitro and ex-vivo release, ocular tolerance, and antimicrobial efficacy studies. The pharmacodynamic study was conducted on rabbit eye model of induced keratitis and it is compared with marketed formulation (MF). Developed PENs showed the size range from 230.7 ± 0.64 to 249.0 ± 0.49 nm and positive surface charge, spherical shape along with appropriate physico-chemical parameters. Both in-vitro and ex-vivo examination concludes that PENs having more efficiency in sustained release of MXN compared to MF. Ocular irritation studies demonstrated that no corneal damage or ocular irritation. The in-vivo study proved that the anti-bacterial efficacy of PENs was improved when compared with MF. These results suggested that PENs are a feasible choice for MK therapy because of their ability to enhance ocular retention of loaded MXN through interaction with the corneal surface of the mucous membrane.

Baicalein Protects Against Aspergillus fumigatus Keratitis by Reducing Fungal Load and Inhibiting TSLP-Induced Inflammatory Response.

Purpose: To investigate the antifungal and anti-inflammatory effects of baicalein on Aspergillus fumigatus (A. fumigatus) keratitis and the underlying mechanisms. Methods: The noncytotoxic antifungal concentration of baicalein was determined using CCK8, cell scratch assay, minimum inhibitory concentration, biofilm formation, scanning electron microscopy, propidium iodide uptake test and adherence assay in vitro and Draize test in vivo. In fungal keratitis (FK) mouse models, clinical score and plate count were used to evaluate FK severity, and myeloperoxidase assay and immunofluorescence staining were performed to examine neutrophil infiltration and activity. Real-time PCR, ELISA, and Western blot were performed to explore the anti-inflammatory activity of baicalein and the underlying mechanisms in vivo and in vitro. Results: Baicalein at 0.25 mM (noncytotoxic) significantly inhibited A. fumigatus growth, biofilm formation, and adhesion in vitro. In A. fumigatus keratitis mice, baicalein mitigated FK severity, reduced fungal load, and inhibited neutrophil infiltration and activity. Baicalein not only suppressed mRNA and protein levels of proinflammatory factors IL-1ß, IL-6, and TNF-α, but also inhibited the expression of thymic stromal lymphopoietin (TSLP) and TSLP receptor (TSLPR) in vivo and in vitro. In HCECs, mRNA and protein levels of IL-1ß, IL-6, and TNF-α were significantly lower in the TSLP siRNA-treated group, while higher in the rTSLP-treated group than in the corresponding control. Baicalein treatment significantly inhibited rTSLP induced the expression of IL-1ß, IL-6, and TNF-α. Conclusions: Baicalein plays a protective role in mouse A. fumigatus keratitis by inhibiting fungal growth, biofilm formation, and adhesion, and suppressing inflammatory response via downregulation of the TSLP/TSLPR pathway.

Isorhamnetin Ameliorates Aspergillus fumigatus Keratitis by Reducing Fungal Load, Inhibiting Pattern-Recognition Receptors and Inflammatory Cytokines.

Purpose: Isorhamnetin is a natural flavonoid with both antimicrobial and anti-inflammatory properties, but its effect on fungal keratitis (FK) remains unknown. The current study aims to investigate the antifungal and anti-inflammatory effects of isorhamnetin against mouse Aspergillus fumigatus keratitis. Methods: In vitro, the lowest effective concentration of isorhamnetin was assessed by minimum inhibitory concentration and cytotoxicity tests in human corneal epithelial cells (HCECs) and RAW264.7 cells. The antifungal property was investigated by scanning electron microscopy and propidium iodide uptake test. The anti-inflammatory effect of isorhamnetin in HCECs and RAW264.7 cells was observed by quantitative real-time polymerase chain reaction (qRT-PCR). In the eyes of mice with A. fumigatus keratitis, FK severity was evaluated using clinical score, plate counting, histological staining and periodic acid Schiff staining. In vivo, the anti-inflammatory effect of isorhamnetin was examined by immunofluorescence staining, myeloperoxidase assay, Western blot, enzyme-linked immunosorbent assay, and qRT-PCR. Results: In HCECs and RAW264.7 cells, isorhamnetin significantly inhibited A. fumigatus conidia growth and hyphae viability at 80 µg/mL without affecting cell viability. In vitro, isorhamnetin altered A. fumigatus hyphal morphology and membrane integrity. In A. fumigatus keratitis mouse model, isorhamnetin treatment alleviated the severity of FK by reducing corneal fungal load and inhibiting neutrophil recruitment. In addition, the mRNA and protein expression levels of TLR-2, TLR-4, Dectin-1, IL-1ß, and tumor necrosis factor-α were significantly decreased in isorhamnetin-treated groups in vivo and in vitro. Conclusions: Isorhamnetin improves the prognosis of A. fumigatus keratitis in mice by inhibiting the growth of A. fumigatus, reducing the recruitment of neutrophils and downregulating inflammatory factors.

Drugs - Do we need them? Applications of non-pharmaceutical therapy in anterior eye disease: A review.

Natural products have been in use long before the introduction of modern drug therapies and are still used in various communities worldwide for the treatment of anterior eye disease. The aim of this review is to look at the current non-pharmaceutical modalities that have been tried and assess the body of existing evidence behind them. This includes alternative medicine, existing non-pharmaceutical therapy and more recent low and high tech solutions. A detailed search of all available databases including MEDLINE, Pubmed and Google was made to look for English-language studies for complementary and alternative treatment modalities (CAM), natural therapies and new modalities for anterior eye disease such as blepharitis, dry eye and microbial keratitis. We have included a broad discussion ranging from traditional treatments like honey and aloe vera which have been used for centuries, to the more recent technological advances like Intense Pulsed Light (IPL), LipiFlow and photoactivated chromophore for corneal cross linking in infectious keratitis (PACK-CXL). Alternative management strategies may have a role in anterior eye diseases and have a potential in changing the way we currently approach them. Some of the available CAM could play a role if incorporated in to current management practices of not only chronic diseases like blepharitis and dry eye, but also acute conditions with significant morbidity like microbial keratitis. Further large-scale randomized control trials stratified by disease severity are required to improve our understanding and to evaluate the use of non-pharmaceutical therapy against current practice.

Involvement of pigment epithelium-derived factor, docosahexaenoic acid and neuroprotectin D1 in corneal inflammation and nerve integrity after refractive surgery.

Alterations in corneal innervations result in impaired corneal sensation, severe dry eye and damage to the epithelium that may in turn lead to corneal ulcers, melting and perforation. These alterations can occur after refractive surgery. We have discovered that pigment epithelium-derived factor (PEDF) plus docosahexaenoic acid (DHA or the docosanoid bioactive neuroprotectin D1 (NPD1)) induces nerve regeneration after corneal surgery that damages the stromal nerves. We found that PEDF is released from corneal epithelial cells after injury, and when DHA is provided to the cells it stimulates the biosynthesis of NPD1 by an autocrine mechanism. The combination of PEDF plus DHA also decreased the production of leukotriene B4 (LTB4), a neutrophil chemotactic factor, thereby decreasing the inflammation induced after corneal damage. These studies suggest that PEDF plus DHA and its derivative NPD1 hold promise as a future treatment to restore a healthy cornea after nerve damage.

Bioadhesive sulfacetamide sodium microspheres: evaluation of their effectiveness in the treatment of bacterial keratitis caused by Staphylococcus aureus and Pseudomonas aeruginosa in a rabbit model.

The aim of this study was to prepare bioadhesive sulfacetamide sodium (SA) microspheres to increase their residence time on the ocular surface and to enhance their treatment efficacy on ocular keratitis. Microspheres were fabricated by spray drying method using mixture of polymers such as pectin, polycarbophil and hydroxypropylmethyl cellulose (HPMC) at different ratios. The particle size and distribution, morphological characteristics, thermal behavior, encapsulation efficiency, mucoadhesion and in vitro drug release studies on formulations have been investigated. After optimisation studies, SA-loaded polycarbophil microsphere formulation with polymer:drug ratio of 2:1 was found to be the most suitable for ocular application and used in in vivo studies. In vivo studies were carried out on New Zealand male rabbit eyes with keratitis caused by Pseudomonas aeruginosa and Staphylococcus aureus. Sterile microsphere suspension in light mineral oil was applied to infected eyes twice a day. Plain SA suspension was used as a positive control. On 3rd and 6th days of the antimicrobial therapy, the eyes were examined in respect to clinical signs of infection (blepharitis, conjunctivitis, iritis, corneal oedema and corneal infiltrates) which are the main symptoms of bacterial keratitis and then cornea samples were counted microbiologically. The rabbit eyes treated with microspheres demonstrated significantly lower clinical scores than those treated with SA alone. A significant decrease in the number of viable bacteria in eyes treated with microspheres was observed in both infection models when compared to those treated with SA alone. In conclusion, in vitro and in vivo studies showed that SA-loaded microspheres were proven to be highly effective in the treatment of ocular keratitis.

Evaluation and prediction of fluoroquinolone pharmacodynamics in bacterial keratitis.

Observational studies suggest that a pharmacodynamic index helps to predict the therapeutic outcome of respiratory and other infections. This study explored the prognostic importance of a ratio of the achievable corneal level for a fluoroquinolone to the fluoroquinolone's minimal inhibitory concentration (MIC) for corneal isolates of 391 patients with bacterial keratitis. The peak concentration and the area under the concentration curve (AUC) in the cornea were estimated from reported values achieved with topical ciprofloxacin. The inhibitory quotient (IQ) was calculated as the ratio of the estimated peak achievable corneal ciprofloxacin concentration to the ciprofloxacin MIC of keratitis isolates, and the area under the inhibitory curve (AUIC) was defined as the expected 24-hour AUC divided by the MIC. The probability of clinical improvement of ciprofloxacin-treated bacterial keratitis was 90% or more if ciprofloxacin's IQ was above 8 or the AUIC was greater than 151. A pharmacodynamic index relating corneal pharmacokinetic and susceptibility concentrations may correlate with the clinical response of bacterial keratitis to fluoroquinolone therapy.