Rat Cornea Research Articles

Protective effect of pituitary adenylate cyclase activating polypeptide in diabetic keratopathy

Diabetic keratopathy (DK) is the major complication of the cornea characterizing diabetes-affected patients. This ocular pathology is correlated with the hyperglycemic state leading to delayed corneal wound healing and recurrent corneal ulcers. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with widespread distribution throughout the body, and exerting cytoprotective effects in the neural and non-neuronal parts of the eye, including the cornea. The purpose of the present study was to investigate whether changes in PACAP expression can concur for delayed epithelial wound healing in diabetic cornea and whether the protective effect of the peptide could be mediated through the activation of the EGFR signaling pathway, which has been reported to be impaired in DK. Expression and distribution of PACAP, PAC1R, and EGFR were investigated through immunohistochemistry analysis in the cornea of normal and diabetic rats. The role of the peptide on wound healing during DK was evaluated in an in vitro model represented by rabbit corneal epithelial cells grown in high glucose conditions. Western blotting and immunofluorescence analysis were used to examine the ability of PACAP to induce the activation of the EGFR/ERK1/2 signaling pathway. Our results showed that in diabetic cornea the expression of PACAP, PAC1R, and EGFR is drastically reduced. The treatment with PACAP via PAC1R activation enhanced cell viability and corneal epithelium wound healing in cells grown under high glucose conditions. Furthermore, both EGFR and ERK1/2 signaling was induced upon the peptide treatment. Overall, our results showed the trophic efficiency of PACAP for enhancing the corneal epithelium re-epithelialization suggesting that the peptide could be beneficially valuable as a treatment for DK.

Aurantio-obtusin Alleviates Dry Eye Disease by Targeting NF-κB/NLRP3 Signaling in Rodent Models.

Dry eye disease (DED) is a common inflammatory ocular surface disorder, seriously affecting the quality of life of patients. Aurantio-obtusin (AO) is a bioactive anthraquinone compound isolated from Semen Cassiae which has multiple pharmacological activities. Nonetheless, the specific function of AO in DED is unclarified.In this study, a rodent DED model was established by benzalkonium chloride (BAC) induction, followed by topical administration of AO. The results showed that topical application of AO increased tear production, mitigated ocular surface disruption and maintained the number of goblet cells in BAC-induced DED rats (p˂0.05). ELISA revealed that AO treatment significantly (p˂0.001) reduced the production of proinflammatory cytokines and chemokines in the conjunctiva and cornea of BAC-induced DED rats.Immunohistochemical staining and western blotting showed that AO treatment suppressed the expression levels of NLR family pyrin domain containing 3 (NLRP3)inflammasome-related proteins, and inhibited activation of nuclear factor kappa B (NF-κB)signaling pathway in rat conjunctiva and cornea (p˂0.001). In conclusion,AO treatment alleviates BAC-induced DED in rats by inhibiting NF-κB/NLRP3 inflammasome signaling pathway.

Untargeted metabolomics in the aqueous humor reveals the involvement of TAAR pathway in glaucoma

Understanding the metabolic dysfunctions and underlying complex pathological mechanisms of neurodegeneration in glaucoma could help discover disease pathways, identify novel biomarkers, and rationalize newer therapeutics. Therefore, we aimed to investigate the local metabolomic alterations in the aqueous humor and plasma of primary glaucomatous patients. This study cohort comprised primary open-angle glaucoma (POAG), primary angle-closure glaucoma (PACG), and cataract control groups. Aqueous humor and plasma samples were collected from patients undergoing trabeculectomy or cataract surgery and subjected to high-resolution mass spectrometry (HRMS) analysis. Spectral information was processed, and the acquired data were subjected to uni-variate as well as multi-variate statistical analyses using MetaboAnalyst ver5.0. To further understand the localized metabolic abnormalities in glaucoma, metabolites affected in aqueous humor were distinguished from metabolites altered in plasma in this study. Nine and twelve metabolites were found to be significantly altered (p < 0.05, variable importance of projection >1 and log2 fold change ≥0.58/≤ −0.58) in the aqueous humor of PACG and POAG patients, respectively. The galactose and amino acid metabolic pathways were locally affected in the PACG and POAG groups, respectively. Based on the observation of the previous findings, gene expression profiles of trace amine-associated receptor-1 (TAAR-1) were studied in rat ocular tissues. The pharmacodynamics of TAAR-1 were explored in rabbits using topical administration of its agonist, β-phenyl-ethylamine (β-PEA). TAAR-1 was expressed in the rat's iris-ciliary body, optic nerve, lens, and cornea. β-PEA elicited a mydriatic response in rabbit eyes, without altering intraocular pressure. Targeted analysis of β-PEA levels in the aqueous humor of POAG patients showed an insignificant elevation. This study provides new insights regarding alterations in both localized and systemic metabolites in primary glaucomatous patients. This study also demonstrated the propensity of β-PEA to cause an adrenergic response through the TAAR-1 pathway.

Low Energy Blue Pulsed Light‐Activated Injectable Materials for Restoring Thinning Corneas

AbstractMany alternatives to human donor corneas are being developed to meet the global shortage of donated tissues. However, corneal transplantation remains the gold standard for diseases resulting in thinning corneas. In this study, transparent low energy photoactivated extracellular matrix‐mimicking materials are developed for intrastromal injection to restore stromal thickness. The injectable biomaterials are comprised of short peptides and glycosaminoglycans (chondroitin, hyaluronic acid) that assemble into a hydrogel when pulsed with low‐energy blue light. The dosage of pulsed‐blue light needed for material activation is minimal at 8.5 mW cm−2, thus circumventing any blue light cytotoxicity. Intrastromal injection of these light‐activated biomaterials in rat corneas shows that two iterations of the formulations remain stable in situ without stimulating significant inflammation or neovascularization. The use of low light intensities and the ability of the developed materials to stably rebuild and change the curvature of the cornea tissue make these formulations attractive for clinical translation.

Experimental glaucoma triggers a pro-oxidative and pro-inflammatory state in the rat cornea

BackgroundIncreasing evidence suggests that glaucoma affects the ocular surface. We aimed to investigate the cellular mechanisms underlying the glaucoma-associated corneal alterations in an animal model. MethodsWistar rats underwent the cauterization of two episcleral veins of the left eye to elevate the intraocular pressure (ipsilateral, G-IL). Control animals received a sham procedure (C-IL). Contralateral eyes did not receive any procedure (G-CL or C-CL). Enzymes related to the redox status, oxidative damage to macromolecules, and inflammatory markers were assessed in corneal lysates. ResultsCompared to C-IL, NOX4, NOX2, and iNOS expression was increased in G-IL (68%, p < 0.01; 247%, p < 0.01; and 200%, p < 0.001, respectively). We found an increase in SOD activity in G-IL (60%, p < 0.05). The GSH/GSSG ratio decreased in G-IL (80%, p < 0.05), with a decrease in GR activity (40%, p < 0.05). G-IL displayed oxidative (90%, p < 0.01) and nitrosative (40%, p < 0.05) protein damage, and enhanced lipid peroxidation (100%, p < 0.01). G-IL group showed an increased in CD45, CD68 and F4/80 expression (50%, p < 0.05; 190%, p < 0.001 and 110%, p < 0.05, respectively). G-CL displayed a higher expression of Nrf2 (60%, p < 0.001) and increased activity of SOD, CAT, and GPx (60%, p < 0.05; 90%, p < 0.01; and 50%, p < 0.05, respectively). ConclusionsGlaucoma induces a redox imbalance in the ipsilateral cornea with an adaptive response of the contralateral one. General significanceOur study provides a possible mechanism involving oxidative stress and inflammation that explains the corneal alterations observed in glaucoma. We demonstrate that these changes extend not only to the ipsilateral but also to the contralateral cornea.

Role of NADPH Oxidase 4 in Corneal Endothelial Cells Is Mediated by Endoplasmic Reticulum Stress and Autophagy.

Human corneal-endothelial cells (hCEnCs) are located on the inner layer of the cornea. Injury to CEnCs leads to permanent corneal edema, requiring corneal transplantation. NADPH oxidase 4 (NOX4) has been reported to be implicated in the pathogenesis of CEnCs diseases. Thus, we investigated the role of NOX4 in CEnCs in this study. In an animal study, siRNA for NOX4 (siNOX4) or plasmid for NOX4 (pNOX4) was introduced into the corneal endothelium of rats by electroporation, using a square-wave electroporator (ECM830, Havard apparatus) to decrease or increase the expression of NOX4, respectively, and the rat corneas were cryoinjured through contact with a metal rod of 3 mm diameter frozen in liquid nitrogen for 10 min. The immunofluorescence staining of NOX4 and 8-OHdG showed that the levels of NOX4 and 8-OHdG were decreased in the siNOX4 group compared to the siControl, and increased in the pNOX4 group compared to the pControl at one week after treatment. Without cryoinjury, corneal opacity was more severe, and the density of CEnCs was lower, in pNOX4-treated rats compared to pControl. After cryoinjury, the corneas were more transparent, and the CEnC density was higher, in siNOX4-treated rats. The hCEnCs were cultured and transfected with siNOX4 and pNOX4. The silencing of NOX4 in hCEnCs resulted in a normal cell shape, higher viability, and higher proliferation rate than those transfected with the siControl, while NOX4 overexpression had the opposite effect. NOX4 overexpression increased the number of senescent cells and intracellular oxidative stress levels. NOX4 overexpression increased ATF4 and ATF6 levels, and nuclear translocation of XBP-1, which is the endoplasmic reticulum (ER) stress marker, while the silencing of NOX4 had the opposite effect. Additionally, the mitochondrial membrane potential was hyperpolarized by the silencing of NOX4, and depolarized by NOX4 overexpression. The LC3II levels, a marker of autophagy, were decreased by the silencing of NOX4, and increased by NOX4 overexpression. In conclusion, NOX4 plays a pivotal role in the wound-healing and senescence of hCEnCs, by modulating oxidative stress, ER stress, and autophagy. The regulation of NOX4 may be a potential therapeutic strategy for regulating the homeostasis of CEnCs, and treating corneal-endothelial diseases.

Distribution of the cysteinyl leukotriene system components in the human, rat and mouse eye

The cysteinyl leukotrienes (CysLTs) have important functions in the regulation of inflammation and cellular stress. Blocking the CysLT receptors (CysLTRs) with specific antagonists is beneficial against progression of retinopathies (e.g. diabetic retinopathy, wet AMD). However, the exact cellular localization of the CysLTRs and their endogenous ligands in the eye have not been elucidated in detail yet. It is also not known whether the expression patterns differ between humans and animal models. Therefore, the present study aimed to describe and compare the distribution of two important enzymes in CysLT biosynthesis, 5-lipoxygenase (5-LOX) and 5-lipoxygenase-activating protein (FLAP), and of CysLTR1 and CysLTR2 in healthy human, rat and mouse eyes.Human donor eyes (n = 10) and eyes from adult Sprague Dawley rats (n = 5) and CD1 mice (n = 8) of both sexes were collected. The eyes were fixed in 4% paraformaldehyde and cross-sections were investigated by immunofluorescence with specific antibodies against 5-LOX, FLAP (human tissue only), CysLTR1 and CysLTR2. Flat-mounts of the human choroid were prepared and processed similarly. Expression patterns were assessed and semiquantitatively evaluated using a confocal fluorescence microscope (LSM710, Zeiss).We observed so far unreported expression sites for CysLT system components in various ocular tissues. Overall, we detected expression of 5-LOX, CysLTR1 and CysLTR2 in the human, rat and mouse cornea, conjunctiva, iris, lens, ciliary body, retina and choroid. Importantly, expression profiles of CysLTR1 and CysLTR2 were highly similar between human and rodent eyes. FLAP was expressed in all human ocular tissues except the lens. Largely weak immunoreactivity of FLAP and 5-LOX was observed in a few, yet unidentified, cells of diverse ocular tissues, indicating low levels of CysLT biosynthesis in healthy eyes. CysLTR1 was predominantly detected in ocular epithelial cells, supporting the involvement of CysLTR1 in stress and immune responses. CysLTR2 was predominantly expressed in neuronal structures, suggesting neuromodulatory roles of CysLTR2 in the eye and revealing disparate functions of CysLTRs in ocular tissues.Taken together, we provide a comprehensive protein expression atlas of CysLT system components in the human and rodent eye. While the current study is purely descriptive and therefore does not allow significant functional conclusions yet, it represents an important basis for future studies in diseased ocular tissues in which distribution patterns or expression levels of the CysLT system might be altered. Furthermore, this is the first comprehensive study to elucidate expression patterns of CysLT system components in human and animal models that will help to identify and understand functions of the system as well as mechanisms of action of potential CysLTR ligands in the eye.

Mesenchymal Stem Cell Exosomes as Immunomodulatory Therapy for Corneal Scarring.

Corneal scarring is a leading cause of worldwide blindness. Human mesenchymal stem cells (MSC) have been reported to promote corneal wound healing through secreted exosomes. This study investigated the wound healing and immunomodulatory effects of MSC-derived exosomes (MSC-exo) in corneal injury through an established rat model of corneal scarring. After induction of corneal scarring by irregular phototherapeutic keratectomy (irrPTK), MSC exosome preparations (MSC-exo) or PBS vehicle as controls were applied to the injured rat corneas for five days. The animals were assessed for corneal clarity using a validated slit-lamp haze grading score. Stromal haze intensity was quantified using in-vivo confocal microscopy imaging. Corneal vascularization, fibrosis, variations in macrophage phenotypes, and inflammatory cytokines were evaluated using immunohistochemistry techniques and enzyme-linked immunosorbent assays (ELISA) of the excised corneas. Compared to the PBS control group, MSC-exo treatment group had faster epithelial wound closure (0.041), lower corneal haze score (p = 0.002), and reduced haze intensity (p = 0.004) throughout the follow-up period. Attenuation of corneal vascularisation based on CD31 and LYVE-1 staining and reduced fibrosis as measured by fibronectin and collagen 3A1 staining was also observed in the MSC-exo group. MSC-exo treated corneas also displayed a regenerative immune phenotype characterized by a higher infiltration of CD163+, CD206+ M2 macrophages over CD80+, CD86+ M1 macrophages (p = 0.023), reduced levels of pro-inflammatory IL-1β, IL-8, and TNF-α, and increased levels of anti-inflammatory IL-10. In conclusion, topical MSC-exo could alleviate corneal insults by promoting wound closure and reducing scar development, possibly through anti-angiogenesis and immunomodulation towards a regenerative and anti-inflammatory phenotype.

Effects of noopept on ocular, pancreatic and renal histopathology in streptozotocin induced prepubertal diabetic rats

ABSTRACT Diabetes mellitus (DM) is a chronic disease at all ages including childhood and puberty. Failure to treat DM can cause retinopathy, nephropathy and neuropathy. Endocrine and metabolic changes during the pubertal period complicate management of DM. Noopept is a cognitive enhancer that exhibits antidiabetic properties. We investigated the effect of noopept on the histopathology of the cornea, retina, kidney and pancreas in pubertal diabetic rats. We allocated 60 prepubertal male rats randomly into six groups of 10: untreated control (C), DM control (DC), noopept control (NC), DM + noopept (D + N), DM + insulin (D + I) and DM + insulin + noopept (D + I + N). DM was induced by streptozotocin in the DC, D + N, D + I and D + I + N groups. Noopept was administered to the NC, D + N and D + I + N groups; insulin was administered to the D + I and D + I + N groups for 14 days. On day 18 of the experiment, animals were sacrificed and eyes, kidneys and pancreata were excised for histological investigation. Renal tubule diameter and corneal and retinal thickness were increased significantly in DC groups compared to the control group. The D + I, D + N and D + I + N groups exhibited fewer DM induced pathological changes than the DC group. The D + I + N group exhibited no significant differences in renal tubule diameter and corneal and retinal thickness compared to the DC group. Our findings suggest that noopept is protective against DM end organ complications in streptozotocin induced diabetic pubertal rats.

APX‑115A, a pan‑NADPH oxidase inhibitor, reduces the degree and incidence rate of dry eye in the STZ‑induced diabetic rat model.

Dye eye disease (DED) is a common ocular disorder in patients with diabetes. It has been reported that APX-115A, a pan-nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase inhibitor, has an apoptosis-inducing effect on Epstein-Barr virus-infected retinal epithelial cells, but its effects in DED are poorly understood. Therefore, a rat model of diabetes was used in the present study to investigate whether APX-115A has an impact on DED in diabetic rats. A diabetic model was established in male Sprague Dawley rats via the intraperitoneal injection of streptozotocin. The eyeballs of the rats were treated with a solution containing APX-115A or a saline control. Tear secretion was measured with the phenol red thread tear test, and the morphology of the eyeball and lacrimal gland tissues was determined using hematoxylin and eosin staining. In addition, localization of NAPDH oxidase 2 (NOX2) in the eyeball and lacrimal gland tissues was detected by immunohistochemistry. The APX-115A treatment had no effect on body weight, blood glucose level or the size of the lacrimal glands. However, morphological changes, namely intracellular vacuoles and acinar atrophy, were observed in the lacrimal glands of the diabetic rats, and APX-115A treatment attenuated these changes. Immunohistochemistry revealed that NOX2 expression was decreased in the lacrimal glands of the diabetic rats, and APX-115A treatment did not attenuate the reduction in NOX2. The corneas of the diabetic rats treated with APX-115A exhibited no change in thickness but had lower NOX2 expression levels compared with those of the control diabetic rats. APX-115A also increased tear secretion and ameliorated the histological changes associated with diabetes. Furthermore, the NOX2 expression levels in the corneas of the diabetic rats treated with APX-115A were restored to the levels observed in normal rats. These findings suggest that APX-115A has potential as a therapeutic agent for DED.

Investigation of the effects of intracameral carbachol on oxidative stress and apoptosis in rat corneas

Investigation of the effects of intracameral carbachol on oxidative stress and apoptosis in rat corneas

Effect of Nintedanib Nanothermoreversible Hydrogel on Neovascularization in an Ocular Alkali Burn Rat Model

Purpose To compare the therapeutic effects of different forms of nintedanib ophthalmic preparations on neovascularization corneal alkali burns in rats. Methods Forty rat models of left eye corneal alkali burns were constructed, and the five groups (N = 8) were treated with normal saline, dexamethasone ointment (dexamethasone), 0.2% nintedanib aqueous solution and nintedanib nano thermoreversible hydrogel (NNTH). A slit lamp microscope was used to observe the area of neovascularization. The levels of the inflammatory factors were detected by ELISA. HE staining was performed on the rat corneas. Vascular endothelial growth factor (VEGFA) was detected by immunohistochemistry, and the expression of corneal VEGFA and CD31 was detected by western blotting. An MTT assay was performed to detect the cytotoxicity of nintedanib on human corneal epithelial cells (HCECs) and human umbilical vein vascular endothelial cells (HUVECs). Cell migration was detected by a cell scratch assay, and the proportion of apoptotic cells was detected by Annexin/PI double staining. Immunofluorescence and western blotting were performed to detect the protein expression of VEGFA and CD31. Results NNTH had a stronger inhibitory effect on corneal neovascularization (CNV) in alkali-burned rats while reducing the level of inflammatory factors. NNTH had a longer drug duration of release than nanoformulations in vitro. Nintedanib at low concentrations (<8 μM) had no significant cytotoxicity to HCECs but significantly induced apoptosis and inhibited the expression of VEGFA and CD31 and the migration of HUVECs. Conclusions Nanomorphic thermoreversible hydrogel is superior among the nintedanib ophthalmic preparations, showing better inhibition of CNV in alkali-burned eyeballs and it blocked the migration and proangiogenic ability of HUVECs.

Особенности ультраструктурных изменений роговичного коллагена экспериментальных животных после уф кросслинкинга роговицы

Purpose. Electron microscopy analysis of collagen fibrils in the corneal stroma of rats after UV cross-linking. Material and methods. Experiments were carried out on rats in 2 groups: 1st – control (intact), 2nd – experiment (UV corneal cross-linking with riboflavin). The observation period is 3, 90 days. The UV irradiation power of the cornea is 3 mW / cm², the duration is 10 minutes. For electron microscopic examination, corneal pieces were fixed in a solution of 2 % glutaraldehyde. Dehydration and pouring were carried out according to the generally accepted method. Ultrathin sections were obtained using the ultramicrotome "Leica EM UC7" (Leica Microsystems) and viewed in the electron microscope JEM-1011 (Jeol). To determine the density of fibrillar packing of corneal collagen fibers, microphotographs were analyzed at magnification ×15,000. The number of collagen fibrils on an area of 250,000 nm² was calculated. The same microphotographs were used to measure the diameter of collagen fibrils. All numerical data were processed by the method of variational statistics. Histograms of the normal diameter distribution of collagen fibrils were analyzed. Results. The ultrastructure of corneal collagen on the 3rd day after UV corneal crosslinking was characterized by an increase in the interfibrillary space. This indicator was partially restored to its original values on the 90th day. At the same time, the fibrils retained the correct shape and sequence. The results of the analysis of the normal distribution of the diameter of collagen fibrils indicated an increase of an average of 10 %. Conclusion. An increase in the interfibrillary space after UV corneal crosslinking is probably associated with the development of postoperative stroma edema, which can be observed up to several months at the ultrastructural level. The revealed increase in the diameter of collagen fibrils is one of the factors increasing the strength and mechanical properties of the cornea. Keywords: UV cross-linking of rat cornea, electron microscopy, diameter of collagen fibrils, interfibrillary space.

Prophylactic Instillation of Hydrogen-Rich Water Decreases Corneal Inflammation and Promotes Wound Healing by Activating Antioxidant Activity in a Rat Alkali Burn Model.

Many studies have demonstrated the therapeutic effects of hydrogen in pathological conditions such as inflammation; however, little is known about its prophylactic effects. The purpose of this study is to investigate the prophylactic effects of hydrogen-rich water instillation in a rat corneal alkali burn model. Hydrogen-rich water (hydrogen group) or physiological saline (vehicle group) was instilled continuously to the normal rat cornea for 5 min. At 6 h after instillation, the cornea was exposed to alkali. The area of corneal epithelial defect (CED) was measured every 6 h until 24 h after alkali exposure. In addition, at 6 and 24 h after injury, histological and immunohistochemical observations were made and real-time reverse transcription polymerase chain reaction (RT-PCR) was performed to investigate superoxide dismutase enzyme (SOD)1, SOD2, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) mRNA expression. CED at 12 h and the number of inflammatory infiltrating cells at 6 h after injury were significantly smaller in the hydrogen group than the vehicle group. Furthermore, SOD1 expression was significantly higher in the hydrogen group than the vehicle group at both 6 and 24 h, and the number of PGC-1α-positive cells was significantly larger in the hydrogen group than the vehicle group at 6 h after injury. In this model, prophylactic instillation of hydrogen-rich water suppressed alkali burn-induced inflammation, likely by upregulating expression of antioxidants such as SOD1 and PGC-1α. Hydrogen has not only therapeutic potential but also prophylactic effects that may suppress corneal scarring following injury and promote wound healing.

Erratum in: Topical Administration of 0.3% Tofacitinib Suppresses M1 Macrophage Polarization and Allograft Corneal Rejection by Blocking STAT1 Activation in the Rat Cornea.

Erratum in: Topical Administration of 0.3% Tofacitinib Suppresses M1 Macrophage Polarization and Allograft Corneal Rejection by Blocking STAT1 Activation in the Rat Cornea.

Using Convolutional Neural Network as a Statistical Algorithm to Explore the Therapeutic Effect of Insulin Liposomes on Corneal Inflammation.

Aiming at the disadvantages of easy recurrence of keratitis, difficult eradication by surgery, and easy bacterial resistance, insulin-loaded liposomes were prepared, and convolutional neural network was used as a statistical algorithm to build SD rat corneal inflammation model and study insulin-loaded liposomes, alleviating effect on corneal inflammatory structure in SD rats. The INS/PFOB@LIP was developed by means of thin-film dispersive phacoemulsification, its structure was monitored using a transmission electron microscope, particle size and appearance potential were monitored using a Malvern particle sizer, and ultraviolet consumption spectrum was monitored using a UV spectrophotometer. The encapsulation rate, drug loading, and distribution of insulin liposomes in rat corneal inflammatory model were measured and calculated. The cytotoxicity of liposome materials was evaluated by CCK-8 assay, and the toxic effects of insulin and insulin liposomes on cells were detected. The cornea of SD rats was burned with NaOH solution (1 mol/L), and the SD rat corneal inflammation model was created. The insulin liposome was applied to the corneal inflammation model, and the therapeutic effect of insulin liposome on corneal inflammation was evaluated by slit lamp, corneal immunohistochemistry, corneal HE staining, and corneal Sirius red staining. Insulin-loaded liposomes were successfully constructed with an average particle size of (130.69 ± 3.87) nm and a surface potential of (−38.24 ± 2.57) mV. The encapsulation rate of insulin liposomes was (48.89 ± 1.24)%, and the drug loading rate was (24.45 ± 1.24)%. The SD rat corneal inflammation model was successfully established. After insulin liposome treatment, the staining area of corneal fluorescein sodium was significantly reduced, the corneal epithelium was significantly thickened, the content of corneal collagen was increased, the expression of inflammatory factors was significantly reduced, and new blood vessels (corneal neovascularization, CNV) growth was inhibited.

Structural Alterations in the Rat Cornea and Retina Induced by Topiramate, A Histological and Immunohistochemical Study.

Structural Alterations in the Rat Cornea and Retina Induced by Topiramate, A Histological and Immunohistochemical Study.

Fullerenol protects cornea from ultraviolet B exposure

The eyes are highly susceptible to the oxidative stress induced by ultraviolet B (UVB, wavelength between 280 ∼ 320 nm), which could cause severe damage to the cornea. Fullerenols are effective antioxidants to alleviate UVB-induced injury, while their application for the eyes is still rare. In present study, we investigated the protective performance and mechanism of fullerenols on cornea under UVB radiation in vivo and in vitro. The synthesized fullerenols exhibited broad-spectrum free radical scavenging properties (applicable to both reactive oxygen species (ROS) and reactive nitrogen species (RNS)) and photo-stability. When compared with another widely used antioxidant glutathione (GSH), the administration of fullerenols markedly decreased the injured area, corneal edema, cell death, and increased the cell proliferation in UVB-induced rat cornea. The effects of fullerenols were confirmed in UVB-exposed human corneal epithelial cells (hCECs), where elevated cell viability and proliferation, decreased oxidative free radical production, repaired mitochondrial dysfunction and DNA lesions were observed. RNA sequencing (RNA-Seq) analysis demonstrated that fullerenol alleviated UVB-induced corneal injury through down-regulation of oxidative stress-related genes and up-regulation of proliferation-associated genes. Our results demonstrate the suitability of fullerenols as a potential exogenous treatment in ameliorating UVB-induced cornea damage.

Advantages of Cubosomal Formulation for Gatifloxacin Delivery in the Treatment of Bacterial Keratitis: In Vitro and In Vivo Approach Using Clinical Isolate of Methicillin-Resistant Staphylococcus aureus.

The objective of this study was to enhance the corneal permeation of gatifloxacin (GTX) using cubosomal nanoparticle as a delivery system. Cubosomal nanoparticle loaded with GTX was prepared and subjected for in vitro and in vivo investigations. The prepared GTX-loaded cubosomal particles exhibited nanoparticle size of 197.46 ± 9.40 nm and entrapment efficiency of 52.8% ± 2.93. The results of ex vivo corneal permeation of GTX-loaded cubosomal dispersion show approximately 1.3-fold increase compared to GTX aqueous dispersion. The incorporation of GTX into cubosomal particles resulted in a fourfold reduction in the minimum inhibitory concentration (MIC) value for the GTX cubosomal particles relative to GTX aqueous dispersion. Furthermore, the enhanced corneal penetration of GTX-loaded cubosomal dispersion compared was evident by a significant decrease in the area % of corneal opacity in MRSA infected rats. Moreover, these results were confirmed by photomicrographs of histological structures of corneal tissues from rats treated with GTX-cubosomal dispersion which did not present any change compared to that of the normal rat corneas. In conclusion, treatment of ocular bacterial infections and reduction in the probability of development of new resistant strains of MRSA could be accomplished with GTX-loaded cubosomal nanoparticles.

Effect of Ultraviolet-A and Riboflavin treatment on the architecture of the center and periphery of normal rat cornea: 7 days post treatment

Corneal collagen cross-linking (CXL) is a treatment that is widely applied to halt the progression of ectatic diseases such as keratoconus by creating biomechanical strength in the cornea. Most of the studies assessed the effect of the CXL on the cornea without any differentiation of its effect between periphery and the center of the untreated control cornea especially after the 7 days of CXL application. We investigate the ultrastructural changes in the architecture of the center and periphery of rat corneas, 7 days after standard CXL application. Five Wistar rats (10 corneas) were used in the present study. The left eye corneas (5 mm area) were de-epithelialized and irradiated with standard CXL application using riboflavin and Ultraviolet-A (UVA) (3 mW/cm2 for 30 min). The right eye corneas were used as a control. The sclera-cornea button was removed and processed for electron microscopy. Digital images were captured with a bottom mounted Quemesa camera and analyzed using the iTEM software. The ultrastructure of epithelium, hemi-desmosomes, Bowman's layer and stroma were organized in both untreated control and CXL rat cornea in both untreated control and CXL rat cornea. Within the same CXL cornea, both the collagen fibril (CF) diameter and interfibrillar spacing at the center were significantly smaller compared to the peripheral diameter and spacing of the cornea. When comparing the untreated control and CXL cornea, the central interfibrillar spacing of the CXL cornea was significantly smaller than the central spacing the untreated control cornea. In the CXL cornea the peripheral spacing was significantly higher compared to the peripheral interfibrillar spacing of the untreated control cornea. Within the CXL cornea, the proteoglycans (PGs) area and density of the periphery was significantly higher compared to the area and density of the center of the cornea. It suggests that CXL was more effective at the periphery of the cornea. This could be due to the higher amount of leucine rich PG lumican and higher diffusion of oxygen and riboflavin at the periphery cornea.