Development of dual-functional core-shell electrospun mats with controlled release of anti-inflammatory and anti-bacterial agents for the treatment of corneal alkali burn injuries.

In this study, a novel dual-drug carrier for the co-administration of an anti-inflammatory and antibiotic agent consisting of core-shell nanofibers for the treatment of cornea alkali burns was designed. The core-shell nanofibers were prepared via coaxial electrospinning of curcumin-loaded silk fibroin as the core and vancomycin-loaded chitosan/polyvinyl alcohol (PVA) as the shell. Electron microscopy (SEM and TEM) images confirmed the preparation of smooth, bead-free, and continuous fibers that formed clear core-shell structures. For further studies, nanofiber mats were cross-linked by heat treatment to avoid rapid disintegration in water and improve both mechanical properties and drug release. The release profile of curcumin and vancomycin indicated an initial burst release, continued by the extended release of both drugs within 72 hours. Rabbit corneal cells demonstrated high rates of proliferation when evaluated using a cell metabolism assay. Finally, the therapeutic efficiency of core/shell nanofibers in healing cornea alkali burn was studied by microscopic and macroscopic observation, fluorescence staining, and hematoxylin-eosin assay on rabbit eyes. The anti-inflammatory activity of fabricated fibers was evaluated by enzyme-linked immunosorbent assay and Immunofluorescence analysis. In conclusion, using a robust array of in vitro and in vivo experiments this study demonstrated the ability of the dual-drug carriers to promote corneal re-epithelialization, minimize inflammation, and inhibit corneal neovascularization. Since these parameters are critical to the healing of corneal wounds from alkali burns, we suggest that this discovery represents a promising future therapeutic agent that warrants further study in humans.

Cytoprotective effect of crocin and trans-resveratrol on photodamaged primary human retinal pigment epithelial cells.

PURPOSE: Light-induced damage to retinal pigment epithelium during pars plana vitrectomy remains a hot topic in ophthalmology. Improvements in technology led to a change of light sources, selective filters, and shorter light exposure time. Currently, there is no satisfying solution to the problem. The aim of the study was to investigate the cytoprotective effects of crocin and resveratrol on light-induced damage to primary human retinal pigment epithelial cells in vitro. METHODS: Primary human retinal pigment epithelial cells were exposed to light analogous to the illumination during pars plana vitrectomy. To evaluate the cytoprotective effects and potential toxicity of resveratrol and crocin, human retinal pigment epithelial cells were incubated with varying concentrations of both before 3-[4,5-dimethylthiazol-2-yl] tetrazolium bromide (MTT) viability assay. Furthermore, glutathione levels were measured to investigate synergistic antioxidant potential. Apoptosis of human retinal pigment epithelial cells was determined by a nucleosome detection enzyme-linked immunosorbent assay. RESULTS: Crocin and resveratrol improved cell viability in photodamaged human retinal pigment epithelial cells significantly from 40.65 ± 21.99% in illuminated human retinal pigment epithelial cells and reached a peak viability of 85.64 ± 11.37% in crocin and resveratrol pretreated cells (for all: p < 0.001). In line, the combination of the supplements increased glutathione levels significantly from 39.35 ± 21.96% to 80.74 ± 10.32% (p = 0.017). No toxic effects were detected (p > 0.99). However, no change in apoptosis rates could be observed following pretreatment with crocin and resveratrol (p > 0.99). CONCLUSION: Crocin and trans-resveratrol revealed cytoprotective effects on human retinal pigment epithelial cells supporting both supplement's development as potential perioperative treatments in light-induced retinal pigment epithelial damage.

Cucurbita argyrosperma Seed Extracts Attenuate Angiogenesis in a Corneal Chemical Burn Model.

Severe corneal inflammation produces opacity or even perforation, scarring, and angiogenesis, resulting in blindness. In this study, we used the cornea to examine the effect of new anti-angiogenic chemopreventive agents. We researched the anti-angiogenic effect of two extracts, methanol (Met) and hexane (Hex), from the seed of Cucurbita argyrosperma, on inflamed corneas. The corneas of Wistar rats were alkali-injured and treated intragastrically for seven successive days. We evaluated: opacity score, corneal neovascularization (CNV) area, re-epithelialization percentage, and histological changes. Also, we assessed the inflammatory (cyclooxigenase-2, nuclear factor-kappaB, and interleukin-1ß) and angiogenic (vascular endothelial growth factor A, VEGF-A; -receptor 1, VEGFR1; and -receptor 2, VEGFR2) markers. Levels of Cox-2, Il-1ß, and Vegf-a mRNA were also determined. After treatment, we observed a reduction in corneal edema, with lower opacity scores and cell infiltration compared to untreated rats. Treatment also accelerated wound healing and decreased the CNV area. The staining of inflammatory and angiogenic factors was significantly decreased and related to a down-expression of Cox-2, Il-1ß, and Vegf. These results suggest that intake of C. argyrosperma seed has the potential to attenuate the angiogenesis secondary to inflammation in corneal chemical damage.

Investigation the effect of Hypericum perforatum on corneal alkali burns.

Purpose: To investigate the effect of Hypericum perforatum on corneal alkali burn. Methods: We studied 45 250 g weighing, 4 months old Wistar albino rats. Alkaline burns were performed in the corneas of all experimental animals with 2 mol/L NaOH after general anaesthesia. Rats were divided into five groups according to the subsequent process applied to them: group 1 was the topical Hypericum perforatum group, group 2 was the topical pure olive oil group, group 3 was the oral Hypericum perforatum group, group 4 was the oral pure olive oil group, and group 5 was the control untreated group. Rats were sacrificed under general anaesthesia on the 14 day. The rate of corneal inflammation, neovascularization, fibroblastic activity, and cluster of differentiation 31 (CD31) staining was investigated. Result: There were 45 rats at the beginning of the study. One rat in groups 1, 2, and 3 died during the study; therefore, 42 rats could be evaluated. There were 8 rats in group 1, 8 rats in group 2, 8 rats in group 3, and 9 rats in group 4. We found less corneal neovascularization (CNV), inflammation, and fibroblastic activity in group 1 and group 2 than in the other groups (p ˂ 0.001 for all parameters). CNV, inflammation, fibroblastic activity, and CD31 staining rates were lower in group 1 than in group 2 (p ˂ 0.001 for all parameters). There was no difference between groups 3, 4, and 5 (respectively, p = 0.436, 0.634, and 0.750). Conclusions: We found that both topical Hypericum perforatum oily extract and olive oil have anti-inflammatory, anti-angiogenic, and anti-fibroblastic effects when applied after corneal alkali burns in rat corneas. Further studies should be conducted in this field.

Danshensu Decreases UVB-Induced Corneal Inflammation in an Experimental Mouse Model via Oral Administration.

BACKGROUND: Danshensu is a bioactive constituent of Salvia miltiorrhiza, a plant commonly used in traditional Chinese medicine. In this study, we investigated the pharmacological efficacy of sodium danshensu, or named salvianic acid A sodium (SAS) on ultraviolet B (UVB)-mediated corneal inflammatory injury in mice. METHODS: Albino mice were divided into one blank control group, and three UVB radiation groups, i.e. SAS-untreated group, and prophylactic treatment groups with SAS at 1 and 10 mg/kg via oral administration. The structure integrity and inflammatory changes of cornea were assessed by surface evaluation of smoothness, topographic distortion, opacity, lissamine green staining, and histologic tissue staining. The inflammatory cytokines was measured by bead-based ELISA assays. RESULTS: Prophylactic treatment of SAS significantly inhibited pathologic changes, improved tissue structural integrity, and reduced inflammatory injury in the cornea after UVB exposure. Dosing with SAS treatment attenuated the incidence rate of leukocyte influx by inhibit increase of interleukin (IL)-1ß, IL-6, monocyte chemoattractant protein-1, and tumor necrosis factor-α. Treatment with 10 mg/kg SAS was more effective in preventing the onset of corneal damage than that with 1 mg/kg SAS. CONCLUSIONS: These results indicate that SAS exhibit the pharmacological efficacy on corneal protection through its inhibition of UVB induced photodamage and subsequently inflammatory injury in vivo.

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.

Mechanisms of Retinal Damage after Ocular Alkali Burns.

Alkali burns to the eye constitute a leading cause of worldwide blindness. In recent case series, corneal transplantation revealed unexpected damage to the retina and optic nerve in chemically burned eyes. We investigated the physical, biochemical, and immunological components of retinal injury after alkali burn and explored a novel neuroprotective regimen suitable for prompt administration in emergency departments. Thus, in vivo pH, oxygen, and oxidation reduction measurements were performed in the anterior and posterior segment of mouse and rabbit eyes using implantable microsensors. Tissue inflammation was assessed by immunohistochemistry and flow cytometry. The experiments confirmed that the retinal damage is not mediated by direct effect of the alkali, which is effectively buffered by the anterior segment. Rather, pH, oxygen, and oxidation reduction changes were restricted to the cornea and the anterior chamber, where they caused profound uveal inflammation and release of proinflammatory cytokines. The latter rapidly diffuse to the posterior segment, triggering retinal damage. Tumor necrosis factor-α was identified as a key proinflammatory mediator of retinal ganglion cell death. Blockade, by either monoclonal antibody or tumor necrosis factor receptor gene knockout, reduced inflammation and retinal ganglion cell loss. Intraocular pressure elevation was not observed in experimental alkali burns. These findings illuminate the mechanism by which alkali burns cause retinal damage and may have importance in designing therapies for retinal protection.

RGTA-based matrix therapy in severe experimental corneal lesions: safety and efficacy studies.

Corneal alteration potentially leading to ulceration remains a major health concern in ocular surface diseases. A treatment that would improve both the quality and speed of healing and control the inflammation would be of great interest. Regenerating agents (RGTAs) have been shown to stimulate wound healing and modulate undesired fibrosis in various in vivo systems. We investigated the effects of RGTA-OTR4120(®) in a rabbit corneal model in order to assess its potential use in ocular surface diseases. First, we assessed its safety for 7 and 28 days using the Draize test criteria in healthy rabbit eyes; then, we investigated the effect of a single dose (50µl, 5µg) in an alkali-burned cornea model. Daily follow-up of clinical signs of healing was scored, and histology was performed at D7. RGTA was well tolerated; no signs of ocular irritation were observed. In the corneal alkali-burn model, non-RGTA-treated eyes showed inflammatory clinical signs, and histology confirmed a loss of superficial corneal layers with epithelial disorganization, neovascularization and infiltration of inflammatory cells. When compared to NaCl control, RGTA treatment appeared effective in reducing clinical signs of inflammation, enhancing re-epithelialization, and improving histological patterns: edema, fibrosis, neovascularization and inflammation. Three to four layers of epithelial cells were already organized, stroma was virtually unvascularized and keratocytes well implanted in parallel collagen fibers with an overall reorganization similar to normal cornea. RGTA appears to be a promising agent for controlling ocular surface inflammation and promoting corneal healing and was well tolerated. This study offers preclinical information and supports the findings of other (compassionate or pilot) studies conducted in patients with various ocular surface diseases.

Wound-healing effect of micronized sacchachitin (mSC) nanogel on corneal epithelium.

The extraction residue of the Ganoderma fruiting body, named sacchachitin, has been demonstrated to have the potential to enhance cutaneous wound healing by inducing cell proliferation. In this study, a nanogel formed from micronized sacchachitin (mSC) was investigated for the potential treatment of superficial chemical corneal burns. Reportedly, mSC has been produced successfully and its chemical properties confirmed, and physical and rheological properties characterized. An in vitro cell proliferation study has revealed that at the concentrations of 200, 300, and 400 microg/mL, mSC nanogel significantly increased Statens Seruminstitut rabbit corneal (SIRC) cell proliferation after 24 hours of incubation. In cell migration assay, migration of SIRC cell to wound closure was observed after 24 hours of incubation with the addition of 200 microg/mL mSC of nanogel. In an animal study, acceleration of corneal wound healing was probably due to the inhibition of proteolysis. In conclusion, the findings of this study substantiate the potential application of sacchachitin in the form of mSC nanogel for the treatment of superficial corneal injuries.

Anti-inflammatory and antioxidant effects of Tualang honey in alkali injury on the eyes of rabbits: experimental animal study.

BACKGROUND: Alkali injury is one of the most devastating injuries to the eye. It results in permanent unilateral or bilateral visual impairment. Chemical eye injury is accompanied by an increase in the oxidative stress. Anti-inflammatory and antioxidant agents play a major role in the treatment of chemical eye injuries. The purpose of this study is to evaluate the anti-inflammatory (clinical and histopathological) and antioxidant effects of Tualang honey versus conventional treatment in alkali injury on the eyes of rabbits. METHODS: A preliminary study was carried out prior to the actual study to establish the alkali chemical injury on rabbit's cornea and we found that alkali chemical injury with 2 N NaOH showed severe clinical inflammatory features. In actual study, alkali injury with 2 N NaOH was induced in the right eye of 10 New Zealand White rabbits' cornea. The rabbits were divided into two groups, Group A was given conventional treatment and Group B was treated with both topical and oral Tualang honey. Clinical inflammatory features of the right eye were recorded at 12 hours, 24 hours, 72 hours, 5th day and 7th day post induction of alkali burn on the cornea. The histopathological inflammatory features of the right corneas of all rabbits were also evaluated on day-7. The level of total antioxidant status and lipid peroxidation products in the aqueous humour, vitreous humour and serum at day-7 were estimated biochemically. Fisher's Exact, Chi-Square and Mann-Whitney test were used to analyse the data. RESULTS: There was no statistically significant difference in clinical inflammatory features (p > 0.05) between honey treated and the conventional treated group at different times of examination. Histopathological examination of the cornea showed the number of polymorphonuclear leucocytes was below 50 for both groups (mild grade). There was also no significant difference in the level of total antioxidant status as well as lipid peroxidation products in aqueous humour (p = 0.117, p = 0.382 respectively), vitreous humour (p = 0.917, p = 0.248 respectively) and serum (p = 0.917, p = 0.332 respectively) between honey treated and the conventional treated group. CONCLUSION: Tualang honey has almost the equal effects when compared with the conventional treatment in treating alkali injury on rabbit's eye. Future research with more number of rabbits and control group is warranted to explore the anti-inflammatory and antioxidant effects of Tualang honey.

Effects of vitamin e, pentoxifylline and aprotinin on light-induced retinal injury.

PURPOSE: A considerable amount of clinical and experimental evidence exists suggesting the involvement of reactive oxygen species (ROS) in the etiology of light-induced retinal injury. The aim of this study was to investigate the protective role of vitamin E, pentoxifylline (PTX) and aprotinin against light-induced retinal injury in guinea pigs. METHODS: Thirty adult male guinea pigs were divided into 5 groups of 6 animals each. The first group was used as control. The guinea pigs were kept in cyclic light for 2 weeks before the experiments. The animals were maintained in 12-hour light-dark cycles, before and after exposure to intense white fluorescent light, for as long as 12 h and then returned to cyclic light. Groups 3-5 received intraperitoneal injections of vitamin E, PTX and aprotinin, respectively. One eye of each animal was selected for histopathological evaluation and the other for biochemical assay. Retinal malondialdehyde (MDA) levels and the thickness of the outer nuclear layers were measured. RESULTS: The compounds had the following relationships: vitamin E more than PTX more than aprotinin in preventing light-induced retinal damage. All 3 gave significant protection against the formation of MDA. Retinas of all 3 treatment groups had been protected from light-induced injury. CONCLUSION: The intraperitoneal vitamin E, PTX and aprotinin supplementations may strengthen the antioxidant defense system because of decreased ROS, and these agents may play a role in treating light-induced retinal injury.

[Establishing human, conjunctival fibroblast cultures as a test system for evaluating ophthalmic drugs]. / Etablierung humaner, konjunktivaler Fibroblastenkulturen als Testsystem zur Beurteilung von Ophthalmika.

BACKGROUND: Chronic inflammation of the anterior eye segment caused by severe burns has to be treated by several drugs. Often it is very difficult to recognize the relationship between drug and side effects. Although the results were usually satisfying, proliferation of the conjunctival tissue was observed. Therefore the question was raised whether these proliferations might have been a side effect of the drugs. To answer this question, we established cultures of human conjunctival fibroblasts for ocular toxicity testing of drugs used in the therapy of severe eye burns. MATERIALS AND METHODS: Conjunctival fibroblasts from young donors were cultured under standard conditions (37 degrees C, 5% C02, 95% RH) in Medium 199 supplemented with 20% FCS without antibiotics. At the time of inoculation the following drugs were added: aprotinin, prednisolone, chloramphenicol and methylhydroxypropylcellulose. Cell growth was observed and growth kinetics were estimated by hemocytometer over a period of 7 days. RESULTS: The investigations of prednisolone showed the well-known dose-dependent anti-proliferative effect. The application of methylhydroxypropylcellulose resulted in decreased cell growth and in total cell detachment. Experiments with aprotinin and chloramphenicol showed no effects on the growth behaviour. The application of a drug mixture lead to similar results as in experiments with prednisolone. DISCUSSION: The presented cell-culture system is able to reproduce specific effects, especially the toxicity of ophthalmic drugs but a complex interaction of an inflammatory reaction, e.g. after severe eye burns, cannot be simulated. The network of reactions and the interaction of many substances playing an important role during this process are too complex.

[A comparative pharmacological evaluation of sea buckthorn, rose and plantain oils in experimental eye burns]. / Sravnitel'naia farmakologicheskaia otsenka masel oblepikhi, shipovnika i podorozhnika pri éksperimental'nom ozhoge glaz.

Hippophae, Rosa and plantain oils have been shown to produce and essential therapeutic action on chemical burns of rabbit eye. The effect was attained in the phases of trophic disturbances and epithelialization.

The combined effect of citrate/ascorbate treatment in alkali-injured rabbit eyes.

The incidence of ulceration and perforation in the cornea of alkali-injured eyes is significantly reduced by treatment with trisodium citrate or sodium ascorbate. Topical citrate reduces the inflammatory response in the cornea by inhibiting polymorphonuclear leukocytes. Topical ascorbate elevates the depressed level of this vitamin in the alkali-injured cornea, reversing a scorbutic condition. The purpose of the current study was to determine whether combined treatment with topical citrate and ascorbate has an additional therapeutic value when compared with citrate alone. Adsorbotear without EDTA was used as the vehicle for both medications. Rabbit eyes were injured with 1N NaOH for 35 s using a 12-mm well and were rinsed. Group I (47 eyes) received two drops of 10% citrate every hour on the hour and Adsorbotear on the half-hour for 14 h/day. Group II (48 eyes) received two drops of 10% citrate every hour on the hour and 10% ascorbate every hour on the half-hour for 14 h/day. The citrate/ascorbate group had significantly fewer ulcerations during the experiment than did the group receiving citrate alone (2 of 48 versus 10 of 47, 0.01 less than p less than 0.02). Both anterior ulcers in the citrate/ascorbate group and five ulcers in the citrate group healed to no ulcer by the end of the experiment (0 of 48 versus 5 of 47, 0.02 less than p less than 0.05). The average depth of ulceration was significantly less for the citrate/ascorbate group (p less than 0.001). No descemetoceles or perforations were observed in either group.(ABSTRACT TRUNCATED AT 250 WORDS)

[Experimental validation of the use of RNA hydrolysate (ENKAD) in chemical burns of the cornea]. / Eksperimental'noe obosnovanie primeneniia gidrolizata RNK (ENKAD) pri khimicheskikh ozhogakh rogovitsy.

The influence of ENKAD on the course of a severe alkaline burn of the cornea was studied in 158 rabbits (316 eyes). Cytochemically, regularity in correlations of separate amino acids: cystin, cysteine, arginine and glutaminic acid in cellular proteins of the intact cornea was established and their disorder in dynamics of the burn process is shown. Comparative assessment of therapeutic effectiveness of ultrasound, applications, subconjunctival injections and phonophoresis of ENKAD has shown that a combined usage of ultrasound and ENKAD has the most expressed positive action on the course of the burn process of the corneal burn, such as acceleration of corneal epithelialization that accomplished, on the average, by the 9.3 +/- 0.3 day (in the control--by the 20.8 +/- 1.8 day), rapid disappearance of its perifocal edema--11.05 +/- 0.7 (in the control - 29.7 +/- 1.1) day, better outcomes after the burn (in 37.5% of cases superficial limited opacifications were formed, in the control--in 7.1%). One of the factors of therapeutic action of ENKAD phonophoresis is its normalizing influence on disturbed correlation of amino acids in proteins. The results obtained allow to recommend to include ENKAD phonophoresis into a complex treatment of patients with eye burns.

[A new pyrimidine-series preparation in the treatment of experimental chemical eye burns]. / Novyi preparat pirimidinovogo riada v lechenii khimicheskikh ozhogov glaz v éksperimente.

The effectiveness of a substance of a group of pyrimidine derivatives LUS-3 has been studied in 87 rabbits (174 eyes) with chemical burns of the cornea of degree III. In parallel series of experiments, its action was compared with one of traditional methods of treatment of eye burns using sodium sulfacetamide in combination with subconjunctival injections of autologous blood with penicillin as well as with a pyrimidine derivative--methyluracil. The assessment of the effectiveness of the preparations was made by the picture of the clinical course as well as by data of histologic investigations. The results obtained have shown a pronounced effectiveness of the preparation LUS-3: it accelerated regenerative processes, prevented development of secondary infection, and, as a result, conditioned formation of low-intensive opacifications of the cornea.

[Tear enzymes in the treatment of an experimental alkaline corneal burn with gordox]. / Fermenty slezy pri lechenii éksperimental'nogo shchelochnogo ozhoga rogovitsy gordoksom.

The study of the influence of hordox, in treatment of experimental alkaline burn of the cornea, on the activity of trypsin-like proteases, elastases, callicreine, beta-N-acetylglucosaminidase and beta-glucuronidase in a tear fluid has shown that activity of these enzymes in a tear after burn remarkably increases, especially within first 24 hours and at the end of the second week after burn. In treatment by hordox, the activity of all enzymes in the tear, except elastase, reduces as compared with untreated animals, that speaks about antiinflammatory action of the preparation. On the basis of the data obtained it is suggested that investigation of hydrolytic enzymes in a tear can serve as a criterion for aimed correction of proteolysis in inflammatory processes in the cornea.