The effect of platelet-rich plasma and sodium alginate hydrogel on corneal wound healing after corneal alkali burns in rats with computer-assisted anterior segment optical coherence tomography image analysis.

Our objective was to determine the effect of a semi-synthetic sodium alginate hydrogel and its combination with platelet-rich plasma (PRP) on histopathological, biochemical, clinical, and anterior segment optical coherence tomography (AS-OCT) data. Alkali chemical burn of the cornea was induced. Injured rats were randomly divided into five equal groups and topically treated with phosphate-buffered saline (sham), platelet-rich plasma (PRP), 0.5% sodium citrate, a semi-synthetic sodium alginate hydrogel, or a combination of PRP and hydrogel (combined group) three times daily. The degree of corneal opacity (CO), corneal epithelial staining (CES), percentage of corneal epithelial defects (CEDP), degree of ciliary hyperemia (CH), neovascularization size (NVS), and extent of neovascularization (NVE) were evaluated. AS-OCT was performed at nine days, and then rats were sacrificed. Histological examination and enzyme-linked immunosorbent assays were performed to detect the concentrations of IL-1ß and MMP-9 in the cornea. There were no significant differences between the groups regarding CEDP, CO, CES, CH, NVS, or NVE on the first day after corneal alkali burn injury (p > 0,05). At the last examination, CO was significantly lower in the PRP group than in the sham group (p = 0,044), while the CO concentrations were similar in terms of NVS (p > 0,05). Similarly, in terms of tissue MMP-9 levels, there were no significant differences between groups (p > 0,05). However, there was a significant difference in tissue IL-1ß levels between the groups (p < 0,001). In the PRP and combined groups, the level of IL-1ß was significantly lower than that in the sham group (p = 0,043 and p = 0,036, respectively). There was a significant difference in epithelial necrosis between the PRP, and it was the lowest in the combined group (p = 0,003). Epithelial thickness was highest in the combined group (p = 0,002). CEDP was significantly different at the last visit between the groups (p = 0.042). The fastest epithelial closing rate was observed for the combined group (p = 0,026). There was a significant negative correlation between tissue MMP-9 levels and corneal solidity and between tissue MMP-9 levels and the corneal area according to the AS-OCT measurements (p = 0,012 and p = 0,027, respectively). When used alone, topical hydrogel application did not significantly enhance the healing of corneal wounds. However, when combined with PRP, it leads to an increased rate of epithelial closure and neovascularization. This combination did not exacerbate inflammation or corneal opacity compared to PRP alone. The anticoagulant citrate solution in the PRP tube did not prove effective. The synergistic use of PRP and hydrogel could enhance epithelial thickness and reduce epithelial necrosis. The use of new parameters for corneal wound healing assessment was facilitated through AS-OCT image processing.

A Novel Multi-Observation System to Study the Effects of Anterior Ocular Inflammation in Zinn's Zonule Using One Specimen.

Zinn's zonule is a fragile and thin tissue, and little is known about its pathogenesis. The aim of this study was to develop an experimental setup for a comprehensive analysis of Zinn's zonule. Rats were divided into two groups: a control group (n = 4) and an alkali injury group (n = 4). Seven days after injury, the eyes were enucleated, the anterior eye was dissected and embedded in gelatin, and macroscopic observations were made. The gelatin specimens were then embedded in paraffin and observed in detail by low-vacuum scanning electron microscopy, immunofluorescence, and quantitative reverse transcription polymerase chain reaction (RT-qPCR). The results show qualitative changes in Zinn's zonules in both macroscopic and microscopic observations. In addition, macrophage infiltration and increased matrix metalloproteinase 2 (MMP2) expression were observed in the injured group, consistent with the RT-qPCR results. The experimental system in this study allowed us to capture the morphological and molecular biological changes of Zinn's zonule and to gain insight into its pathogenesis. In conclusion, this study presents a new experimental setup for the comprehensive analysis of the rat Zinn's zonule. The results suggest that this system can be used in the future to study and analyze a variety of paraffin-embedded tissues and specimens.

Combination of Peroxisome Proliferator-Activated Receptor (PPAR) Alpha and Gamma Agonists Prevents Corneal Inflammation and Neovascularization in a Rat Alkali Burn Model.

Peroxisome proliferator-activated receptor alpha (PPARα) and gamma (PPARγ) agonists have anti-inflammatory and anti-neovascularization effects, but few reports have tested the combination of PPARα and PPARγ agonists. In this study, we investigated the therapeutic effects of ophthalmic solutions of agonists of PPARα, PPARγ, and the combination in a rat corneal alkali burn model. After alkali injury, an ophthalmic solution of 0.05% fenofibrate (PPARα group), 0.1% pioglitazone (PPARγ group), 0.05% fenofibrate + 0.1% pioglitazone (PPARα+γ group), or vehicle (vehicle group) was topically instilled onto the rat's cornea twice a day. After instillation, upregulation was seen of PPAR mRNA corresponding to each agonist group. Administration of agonists for PPARα, PPARγ, and PPARα+γ suppressed inflammatory cells, neovascularization, and fibrotic changes. In addition, the PPARγ agonist upregulated M2 macrophages, which contributed to wound healing, whereas the PPARα agonist suppressed immature blood vessels in the early phase. Administration of PPARα+γ agonists showed therapeutic effects in corneal wound healing, combining the characteristics of both PPARα and PPARγ agonists. The results indicate that the combination of PPARα and γ agonists may be a new therapeutic strategy.

Trimebutine prevents corneal inflammation in a rat alkali burn model.

Alkaline burns to the cornea lead to loss of corneal transparency, which is essential for normal vision. We used a rat corneal alkaline burn model to investigate the effect of ophthalmic trimebutine solution on healing wounds caused by alkaline burns. Trimebutine, an inhibitor of the high-mobility group box 1-receptor for advanced glycation end products, when topically applied to the burned cornea, suppressed macrophage infiltration in the early phase and neutrophil infiltration in the late phase at the wound site. It also inhibited neovascularization and myofibroblast development in the late phase. Furthermore, trimebutine effectively inhibited interleukin-1ß expression in the injured cornea. It reduced scar formation by decreasing the expression of type III collagen. These findings suggest that trimebutine may represent a novel therapeutic strategy for corneal wounds, not only through its anti-inflammatory effects but also by preventing neovascularization.

Melatonin exerts anti-angiogenic and anti-inflammatory effects in alkali-burned corneas.

Background: Corneal neovascularization (CNV) caused by alkali burn injury is tightly associated with an inflammatory reaction and can lead to vision loss. Melatonin is involved in anti-inflammation and anti-angiogenesis, but its role in CNV has not yet been investigated. Methods: We induced CNV using sodium hydroxide (NaOH) and compared the reactions of vehicle control and melatonin-treated male C57BL/6 mice at 7 and 14 days following the corneal burn. The infiltration of inflammatory cells and the expression of proangiogenic factors, chemokines, and inflammation-related molecules were quantified via immunohistochemical (IHC) analysis and quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR), respectively. Murine peritoneal macrophages were used in vitro to further verify the effect of melatonin in inflammatory CNV. Results: Compared with the vehicle control mice, the melatonin-treated mice showed significant inhibition of angiogenesis and reduction of corneal epithelial defects in alkali-burned corneas. Concomitantly, the infiltration of inflammatory cells and F4/80+ cells were dramatically reduced after melatonin treatment. The messenger RNA (mRNA) expression of proangiogenic factors [vascular endothelial growth factors (VEGF) and matrix metalloproteinase-9 (MMP-9)], monocyte chemotactic protein-1 (MCP-1), nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3), and pro-inflammatory cytokines [tumor necrosis factor-alpha (TNF-α) and interleukin-1ß (IL-1ß) and IL-6] were down-regulated in the melatonin-treated mice. Moreover, melatonin inhibited the expression of these factors in murine peritoneal macrophages. Conclusions: Melatonin inhibits the neovascular and inflammatory responses in corneal alkali burn injury, suggesting that it may be a potential therapy for CNV.

Daphnetin inhibits corneal inflammation and neovascularization on a mouse model of corneal alkali burn.

Alkali burn is a significant contributor to corneal injury. Alkali burn-induced corneal inflammation often causes vision loss due to corneal neovascularization. Daphnetin (DAP) has been studied for its anti-inflammatory and antiangiogenic properties with encouraging results. Driven by those encouraging results, we sought to explore the effects of DAP in treating alkali burn-induced corneal inflammation and neovascularization and its mechanism of action. We found that the angiogenesis processes of human umbilical vein endothelial cells (HUVECs) induced by vascular endothelial growth factor A (VEGF-A) were primarily attenuated by treatment with DAP, including proliferation, migration, and tube formation. Treatment of DAP significantly suppressed the VEGF-A-induced protein expression of VEGF receptor2 (VEGFR2), as well as the activation of downstream signal transducer and activator of transcription 3 (STAT3), AKT, and extracellular signal-regulated kinase (ERK) signaling. In the mouse corneal alkali burn model, the inflammatory cell infiltrations and neovascularization in the cornea caused by alkali burn were inhibited by 10 µM DAP eye drops. Alkali burn-induced corneal protein expression of VEGF-A, VEGFR2, phosphorylated (p-)STAT3, p-AKT, and p-ERK in corneal tissue were reduced mainly by DAP. Moreover, the upregulation of inflammatory caused by alkali burn in the pathological process was significantly neutralized by DAP. Mechanistically, the inflammatory response could be alleviated by DAP in the way of inhibiting the expression levels of TLR4, p-NF-κB, NLRP3, ASC, Cleaved-caspase-1 (p20), mature-IL-1ß (p17), and N-GSDM. In conclusion, our findings confirmed that the corneal inflammation and neovascularization caused by alkali burn could be inhibited by DAP in vitro and in vivo, elucidating the underlying mechanisms of its protective effects. DAP may have tremendous therapeutic potential for the treatment of corneal alkali burn.

Controlled delivery of pirfenidone through vitamin E-loaded contact lens ameliorates corneal inflammation.

Chemical injury by alkali burn is a major cause of corneal blindness in the clinical setting. Current management advocates multiple therapies aimed to prevent inflammation, initiate quick re-epithelialization, arrest the fibrosis, and avoid dry eye and pain by using bandage contact lenses. We hypothesized sustained delivery of the anti-inflammatory, antifibrotic drug pirfenidone through vitamin E-loaded contact lenses as a logical single approach to counter the pathology involved. Vitamin E particles were created in situ in commercial silicon hydrogel contact lenses by soaking the lenses in a vitamin E-ethanol solution. The vitamin E-laden lenses were then placed into pirfenidone-saline solution to load the drug into the lens. The contact lenses were evaluated by both in vitro and in vivo means. For in vitro, lenses were placed into 3 mL of saline solution. The concentration of pirfenidone released was measured by UV-vis spectrophotometry. The contact lenses were implanted in rabbit eyes following the alkali burn; the drug availability in the aqueous humor was evaluated by HPLC at various time points 10 min, 30 min, 2 h, and 3 h; and gene expression of inflammatory cytokines IL-1ß, TNF-α, and TGF-ß1 was evaluated in the cornea at the end of the study period. In another group of rabbits inflicted with alkali injury, the corneas were graded after 7 days of contact lens implantation with and without pirfenidone. A mathematical model was developed for delivery of the drug to the cornea and aqueous humor after a contact lens is inserted in the eye. The model was validated with experimental data and used to determine the bioavailability both for contact lenses and eye drops. In vitro release of unmodified commercial contact lenses saw a release time of approximately 20 min, with a partition coefficient of 2.68 ± 0.06. The release of pirfenidone from 20% vitamin E-loaded lenses saw a release time of approximately 80 min, with a partition coefficient of 4.20 ± 0.04. In vivo, the drug was available in the aqueous humor for up to 3 h. Gene expression of inflammatory cytokine IL-ß1 and profibrotic growth factor TGF-ß1 was significantly suppressed in corneas treated with pirfenidone contact lenses. A week after the alkali burn, the eyes with pirfenidone contact lenses showed significant improvement in corneal haze in comparison to the control eyes. About 50% of the drug loaded in the lens reached the aqueous humor compared to 1.3% with eye drops. Vitamin E-loaded contact lenses serve as a suitable platform for delivery of pirfenidone following alkali burn in rabbit eyes; positive pre-clinical outcome identifies it as promising therapy for addressing corneal inflammation and fibrosis. The bioavailability is about 40-fold higher for contact lenses compared to that for eye drops.