Treatment of alkali-injured cornea by cyclosporine A-loaded electrospun nanofibers - An alternative mode of therapy.

In this study we tried to develop a new approach to suppress inflammation and neovascularization in the alkali-injured rabbit cornea. For this reason Cyclosporine A (CsA)-loaded electrospun nanofibers were transferred onto the ocular surface injured with alkali (0.25 N NaOH). Damaged corneas were divided into the following groups: untreated, treated with CsA eye drops, treated with nanofibers drug-free and treated with CsA-loaded nanofibers. Healthy rabbit corneas served as controls. Drug-free nanofibers and CsA-loaded nanofibers were transferred onto the damaged corneal surface immediately after the injury and sutured to conjunctiva. On day five after the injury the nanofibers were removed. The animals from all groups were sacrificed on day twelve after the injury. The extent of the inflammatory reaction and corneal healing were examined macroscopically, immunohistochemically and biochemically. The central corneal thickness was measured using an ultrasonic pachymeter. When compared with untreated injured corneas, injured corneas treated with drug-free nanofibers or injured corneas treated with CsA eye drops, the number of CD3-positive cells (T lymphocytes) and the production of pro-inflammatory cytokines were strongly reduced in corneas treated with CsA-loaded nanofibers, which was associated with the significantly decreased expression of matrix metalloproteinase 9, inducible nitric oxide synthase, vascular endothelial growth factor and active caspase-3. CsA-loaded nanofibers effectively suppressed corneal inflammation and corneal neovascularization. Central corneal thickness restored to levels before injury only in corneas treated with CsA-loaded nanofibers. Corneal transparency was highly restored in these corneas. It is suggested that the beneficial effect of CsA-loaded nanofibers was associated with the continuous release of CsA from nanofibers and continuous affection of damaged cornea by CsA. The suture of nanofibers to conjunctiva and the closed eyes contributed to beneficial corneal healing. This is in contrast to CsA eye drops, which are quickly washed from the ocular surface and the contact of CsA with the damaged cornea was limited. In conclusion, the approach with CsA-loaded nanofibers could represent an effective alternative mode of therapy for corneal chemical burns.

Cejkova J ，Cejka C ，Trosan P ，Zajicova A ，Sykova E ，Holan V ... -  《-》

Transfer of mesenchymal stem cells and cyclosporine A on alkali-injured rabbit cornea using nanofiber scaffolds strongly reduces corneal neovascularization and scar formation.

Cejka C ，Cejkova J ，Trosan P ，Zajicova A ，Sykova E ，Holan V ... -  《-》

Suppression of alkali-induced oxidative injury in the cornea by mesenchymal stem cells growing on nanofiber scaffolds and transferred onto the damaged corneal surface.

The purpose of this study was to investigate whether rabbit bone marrow-derived mesenchymal stem cells (MSCs) effectively decrease alkali-induced oxidative stress in the rabbit cornea. The alkali (0.15 N NaOH) was applied on the corneas of the right eyes and then rinsed with tap water. In the first group of rabbits the injured corneas remained untreated. In the second group MSCs were applied on the injured corneal surface immediately after the injury and eyelids sutured for two days. Then the sutures were removed. In the third group nanofiber scaffolds seeded with MSCs (and in the fourth group nanofibers alone) were transferred onto the corneas immediately after the injury and the eyelids sutured. Two days later the eyelid sutures were removed together with the nanofiber scaffolds. The rabbits were sacrificed on days four, ten or fifteen after the injury, and the corneas were examined immunohistochemically, morphologically, for the central corneal thickness (taken as an index of corneal hydration) using an ultrasonic pachymeter and by real-time PCR. Results show that in untreated injured corneas the expression of malondialdehyde (MDA) and nitrotyrosine (NT) (important markers of lipid peroxidation and oxidative stress) appeared in the epithelium. The antioxidant aldehyde dehydrogenase 3A1 (ALDH3A1) decreased in the corneal epithelium, particularly in superficial parts, where apoptotic cell death (detected by active caspase-3) was high. (In control corneal epithelium MDA and NT are absent and ALDH3A1 highly present in all layers of the epithelium. Cell apoptosis are sporadic). In injured untreated cornea further corneal disturbances developed: The expressions of matrix metalloproteinase 9 (MMP9) and proinflammatory cytokines, were high. At the end of experiment (on day 15) the injured untreated corneas were vascularized and numerous inflammatory cells were present in the corneal stroma. Vascular endothelial growth factor (VEGF) expression and number of macrophages were high. The results obtained in injured corneas covered with nanofiber scaffolds alone (without MSCs) or in injured corneas treated with MSCs only (transferred without scaffolds) did not significantly differ from the results found in untreated injured corneas. In contrast, in the injured corneas treated with MSCs on nanofiber scaffolds, ALDH3A1 expression remained high in the epithelium (as in the control cornea) and positive expression of the other immunohistochemical markers employed was very low (MMP9) or absent (NT, MDA, proinflammatory cytokines), also similarly as in the control cornea. Corneal neovascularization and the infiltration of the corneal stroma with inflammatory cells were significantly suppressed in the injured corneas treated with MSCs compared to the untreated injured ones. The increased central corneal thickness together with corneal opalescency appearing after alkali injury returned to normal levels over the course of ten days only in the injured corneas treated with MSCs on nanofiber scaffolds. The expression of genes for the proinflammatory cytokines corresponded with their immunohistochemical expression. In conclusion, MSCs on nanofiber scaffolds protected the formation of toxic peroxynitrite (detected by NT residues), lowered apoptotic cell death and decreased matrix metalloproteinase and pro-inflammatory cytokine production. This resulted in reduced corneal inflammation as well as neovascularization and significantly accelerated corneal healing.

Cejkova J ，Trosan P ，Cejka C ，Lencova A ，Zajicova A ，Javorkova E ，Kubinova S ，Sykova E ，Holan V ... -  《-》

Therapeutic effects of zerumbone in an alkali-burned corneal wound healing model.

Cornea is an avascular transparent tissue. Ocular trauma caused by a corneal alkali burn induces corneal neovascularization (CNV), inflammation, and fibrosis, leading to vision loss. The purpose of this study was to examine the effects of Zerumbone (ZER) on corneal wound healing caused by alkali burns in mice. CNV was induced by alkali-burn injury in BALB/C female mice. Topical ZER (three times per day, 3μl each time, at concentrations of 5, 15, and 30μM) was applied to treat alkali-burned mouse corneas for 14 consecutive days. Histopathologically, ZER treatment suppressed alkali burn-induced CNV and decreased corneal epithelial defects induced by alkali burns. Corneal tissue treated with ZER showed reduced mRNA levels of pro-angiogenic genes, including vascular endothelial growth factor, matrix metalloproteinase-2 and 9, and pro-fibrotic factors such as alpha smooth muscle actin and transforming growth factor-1 and 2. Immunohistochemical analysis demonstrated that the infiltration of F4/80 and/or CCR2 positive cells was significantly decreased in ZER-treated corneas. ZER markedly inhibited the mRNA and protein levels of monocyte chemoattractant protein-1 (MCP-1) in human corneal fibroblasts and murine peritoneal macrophages. Immunoblot analysis revealed that ZER decreased the activation of signal transducer and activator of transcription 3 (STAT3), with consequent reduction of MCP-1 production by these cells. In conclusion, topical administration of ZER accelerated corneal wound healing by inhibition of STAT3 and MCP-1 production.

Kim JW ，Jeong H ，Yang MS ，Lim CW ，Kim B ... -  《-》

The healing of alkali-injured cornea is stimulated by a novel matrix regenerating agent (RGTA, CACICOL20): a biopolymer mimicking heparan sulfates reducing proteolytic, oxidative and nitrosative damage.

Cejkova J ，Olmiere C ，Cejka C ，Trosan P ，Holan V ... -  《-》