Rabbit Cornea Research Articles

Ocular Tissue Distribution of Omidenepag Isopropyl in Rabbits and Cynomolgus Monkeys.

To evaluate the ocular distribution of omidenepag isopropyl (OMDI) and its active form omidenepag (OMD), an EP2 receptor agonist, after topical administration of OMDI into rabbit and monkey eyes, and to determine whether OMDI and OMD interact with target receptors or enzymes of other antiglaucoma agents. Both eyes of six rabbits and of 14 monkeys were topically instilled with 0.03% [14C]-OMDI. Rabbits were sacrificed after one to four hours, and ocular tissues were collected. Monkeys were sacrificed after 0.25 to 24 hours, and blood and ocular tissues were collected. Radioactivity was measured in each sample. The interactions of OMDI and OMD with the receptors and enzymes associated with the mechanisms of action of other antiglaucoma agents were evaluated. Most radioactivity applied to rabbit eyes was recovered as OMD from the cornea, aqueous humor, and iris-ciliary body. Similarly, high concentrations of radioactivity were observed in monkey cornea, bulbar/palpebral conjunctiva, and trabecular meshwork. OMD bound to EP2 receptors, but neither OMD nor OMDI bound to α2A, β1, and β2 adrenergic receptors or inhibited enzymatic activities of CA1 and CA2. OMD and OMDI had little or no effect on ROCK1 and ROCK2. OMDI rapidly permeates rabbit and monkey corneas and is converted to OMD, which distributes into anterior ocular tissues. Neither OMD nor OMDI interacted with the target receptors or enzymes of other antiglaucoma agents, suggesting that OMD interacts highly selectively with EP2 receptors. OMDI is a specific antiglaucoma agent that interacts selectively with ocular EP2 receptors.

αB-crystallin mini-peptides support corneal healing in vitro and in vivo in rabbit model.

To evaluate if topical use of αB-crystallin mini-peptides supports corneal healing following flap surgery. Cultured corneal cells were treated with fluorescent tagged αB-crystallin mini-peptides to assess its internalization. Cultured corneal cells pre-treated with or without the mini-peptides were exposed to H2O2 and cell viability was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Elongation of neurites of cultured trigeminal neurones was examined following treatment either with αB-crystallin mini-peptides or protein. Cultured trigeminal neurones were pre-treated either with αB-crystallin mini-peptides or crystallin protein and exposed to H2O2 and presence of beading in the dendrites and axons was assessed. Corneal flap surgery was conducted on rabbit cornea and treated topically either with αB-crystallin peptide (0.5 mg/mL thrice daily for 14d) or phosphate-buffered saline (PBS). Corneal healing was evaluated under slit-lamp biomicroscope, mRNA expression of inflammatory cytokines were assessed and the corneas were evaluated by histopathology. Internalization of αB-crystallin mini-peptides was ascertained by the detection of fluorescence within the corneal cells. The MTT assay revealed that treatment with αB-crystallin mini-peptide reduced cell death induced by H2O2 treatment. The mini-peptides did not influence the elongation of trigeminal neurites, but significantly (P<0.05) reduced beading in the neurites. In rabbit eye, the treated corneas showed reduced hyper-reflective zones (P<0.05) and suppression in the expression of inflammatory cytokines. Histopathological examination also revealed reduction of inflammatory response in treated corneas. The αB-crystallin mini-peptides restrict the damage to corneal cells and neurons and aids in corneal healing.

The Efficacy of Topical Cefiderocol Treatment of Experimental Extensively Drug-Resistant Pseudomonas aeruginosa Keratitis Is Dependent upon the State of the Corneal Epithelium.

Background: An overlooked factor in the efficacy of topical antibiotics to treat bacterial keratitis is the state of the corneal epithelium. Recently, we evaluated topical cefiderocol for the treatment of extensively drug-resistant (XDR) Pseudomonas aeruginosa (PA) keratitis in eyes with the corneal epithelium abraded. The goal of this study was to use the same model with the corneal epithelium left intact to evaluate the efficacy of cefiderocol and other antibiotics and compare the results to those of the previous study. Methods: NZW rabbit corneas with intact epithelium were inoculated with XDRPA. After 16 h, the rabbits were topically treated with cefiderocol 50 mg/mL, ciprofloxacin 0.3%, tobramycin 14 mg/mL, or saline. Following 8 h of treatment, the corneas were harvested for CFU determinations and cefiderocol concentrations. Results: Only cefiderocol significantly decreased CFU of the XDRPA strain compared with saline. The CFU in the cefiderocol and tobramycin-treated corneas for the XDRPA strain with initially intact epithelium were 1.83-1.4 Log10 greater than those produced in corneas with the abraded epithelium (p < 0.05). Cefiderocol concentrations were 5.02× less in corneas with initially intact epithelium. Conclusions: The efficacy of cefiderocol and tobramycin to treat experimental XDRPA keratitis is dependent on the state of the corneal epithelium.

Quantitative assessment of corneal biomechanical changes in vivo after photorefractive intrastromal corneal cross-linking using optical coherence elastography.

Photorefractive intrastromal corneal cross-linking (PiXL) treatment corrects myopia by enhancing localized central corneal biomechanics. However, the dose-effect relationship between the changes in corneal biomechanics and alterations in corneal curvature resulting from this treatment remain unclear. We therefore developed an acoustic radiation force optical coherence elastography (ARF-OCE) technique to investigate the dose-effect relationship in PiXL. ARF-OCE measurements and corneal topography were performed 3 days before and 1 week after PiXL treatment. Depth-resolved Young's modulus images of the in vivo corneas were obtained based on the phase velocity of the Lamb wave. PiXL treatments with five ultraviolet-A (UVA) energy doses (5.4, 15, 25, 35, and 45 J/cm2) were administered to rabbit corneas in vivo (n=15). The percentage change in Young's modulus (ΔE%) of the cornea increased from 0.26 to 1.71 as the UVA energy dose increased from group I (5.4 J/cm2) to group V (45 J/cm2). Meanwhile, the change in the mean keratometry (ΔKm ) of the cornea increased from 0.40 to 2.10 diopters (D) as the UVA energy dose increased from group I to group IV (35 J/cm2). Furthermore, a statistically significant positive correlation was observed between ΔE% and ΔKm in groups I to IV. With increasing UVA energy dose, the corneal Young's modulus significantly increased. Given the observed correlation, ΔE% holds promise as a new quantitative biomechanical parameter for determining the dose-effect relationship in PiXL treatment. It should be emphasized that there may be an inflection point of ΔE%, at which corneal keratometry ceases to flatten and begins to increase. The ARF-OCE system has demonstrated its efficacy in quantitatively assessing changes in corneal biomechanics in vivo following PiXL treatment. This technique has great potential in facilitating the quantitative determination of the dose-effect relationship in PiXL treatment.

Comparison of the structural integrity and quality of corneal endothelium stored in organ culture storage medium versus Eusol-C.

In this experimental study, we compared the structural integrity and cell quality of corneal endothelium stored in organ culture medium (OCS) and Eusol-C. The experiment included rabbit and human cornea experiments in vitro. Thirty rabbit corneas and thirty-two human corneas were collected and divided into two groups. All right corneas were allocated in experiment group and left corneas were placed in control group. The corneas in experimental group were stored in OCS at 34°C, and the corneas in control group were stored in Eusol-C at 4°C for 7, 14, 21, 28, and 35days, respectively. Endothelial cell morphology, cell count, and trypan blue staining for viability were assessed before storage (Day 0) and at days 7, 14, 21, 28 and 35. The structural integrity of human corneal endothelial cell was analyzed using immunohistochemistry. The samples of storage solution for microbial culture were collected on the third day and at the end of storage. The results show that no bacterial and fungal infections were found in both groups. After 14days of storage, the morphology of endothelial cell was better in the experimental group than in the control group. The endothelial cell stored in OCS were better than those stored in Eusol-C at the end of storage times, except human cornea 14days storage group. The ZO-1 protein staining showed the typical polygonal morphology of endothelial cell stored in the OCS. Corneal endothelial cells stored in the OCS had better quality up to 28days. It can be applied to Chinese eye banks as a method of corneal preservation.

Evaluation of plant-derived biomaterials for the development of tissue-engineered corneal substitutes.

Corneal blindness affects over 10 million patients worldwide. Due to the limited supply of donor corneas and frequent graft failure, bioengineered alternatives are crucial. To overcome drawbacks associated with corneal substitutes from synthetic biomaterials, fabrication from plant-derived biomaterials is a potential alternative. Herein, soy protein and glutenin in combination with different crosslinkers were evaluated for fabrication of corneal substitutes. Optical, mechanical, and biochemical properties of fabricated constructs and control rabbit corneas were evaluated in vitro. Soy protein crosslinked with peroxidase/H202 possessed transparency and mechanical properties comparable to controls, although their water content and biocompatibility were inferior. In contrast, soy protein crosslinked with tannic acid showed similar water content, tensile strength, and biocompatibility as rabbit corneas; however, these constructs displayed significantly lower transparency and higher strain to failure. Finally, glutenin cross-linked using formaldehyde showed excellent transparency, strain to failure, and biocompatibility, however; they exhibited significantly lower water content and tensile strength than controls. This study is the first to establish CIELAB color values for the rabbit cornea, allowing quantitative optical evaluation of tissue-engineered substitutes. Thus, a crosslinking strategy utilizing plant-derived proteins for fabrication of constructs with properties comparable to rabbit corneas is a promising direction for development of tissue-engineered corneal substitutes.

An injectable hydrogel based on sodium alginate and gelatin treats bacterial keratitis through multimodal antibacterial strategy

Bacterial keratitis is among the most prevalent causes of blindness. Currently, the abuse of antibiotics in clinical settings not only lacks bactericidal effects but also readily induces bacterial resistance, making the clinical treatment of bacterial keratitis a significant challenge. In this study, we present an injectable hydrogel (GS-PNH-FF@CuS/MnS) containing self-assembled diphenylalanine dipeptide (FF) and CuS/MnS nanocomposites (CuS/MnS NCs) that destroy bacterial cell walls through a synergistic combination of mild photothermal therapy (PTT), chemodynamic therapy (CDT), ion release chemotherapy, and self-assembled dipeptide contact, thereby eliminating Pseudomonas aeruginosa. Under 808 nm laser irradiation, the bactericidal efficiency of GS-PNH-FF@CuS/MnS hydrogel against P. aeruginosa in vitro reach up to 96.97 %. Furthermore, GS-PNH-FF@CuS/MnS hydrogel is applied topically to kill bacteria, reduce inflammation, and promote wound healing. Hematoxylin-eosin (H&E) staining, Masson staining, immunohistochemistry and immunofluorescence staining are used to evaluate the therapeutic effect on infected rabbit cornea models in vivo. The GS-PNH-FF@CuS/MnS demonstrate good biocompatibility with human corneal epithelial cells and exhibit no obvious eyes side effects. In conclusion, the GS-PNH-FF@CuS/MnS hydrogel in this study provides an effective and safe treatment strategy for bacterial keratitis through a multimodal approach.

Limbal stem cells carried by a four-dimensional -printed chitosan-based scaffold for corneal epithelium injury in diabetic rabbits.

Methods: Herein, we obtained and characterized deltaN p63- and adenosine triphosphate-binding cassette subfamily G member 2-expressing limbal stem cells (LSCs). Chitosan and carboxymethyl chitosan (CTH) were cross-linked to be an in situ thermosensitive hydrogel (ACH), which was printed through four-dimensional (4D) printing to obtain a porous carrier with uniform pore diameter (4D-CTH). Rabbits were injected with alloxan to induce diabetes mellitus (DM). Following this, the LSC-carrying hydrogel was spread on the surface of the cornea of the diabetic rabbits to cure corneal epithelium injury. Results: Compared with the control group (LSCs only), rapid wound healing was observed in rabbits treated with LSC-carrying 4D-CTH. Furthermore, the test group also showed better corneal nerve repair ability. The results indicated the potential of LSC-carrying 4D-CTH in curing corneal epithelium injury. Conclusion: 4D-CTH holds potential as a useful tool for studying regenerative processes occurring during the treatment of various diabetic corneal epithelium pathologies with the use of stem cell-based technologies.

Development and evaluation of hot-melt–extruded diquafosol tetrasodium formulations for ophthalmic inserts: A design of experiments approach

This study aimed to develop, optimize, and evaluate hot-melt–extruded ophthalmic inserts capable of sustained release of diquafosol tetrasodium (DQS) via a design of experiments approach. DQS, a tear stimulant for dry eye management, faces challenges of frequent administration and low bioavailability. The developed insert uses biodegradable polymers in varied proportions to achieve sustained release. Optimized through mixture design, the insert completely dissolved within 24 h and maintained a stable drug content, thickness, and surface pH over three months at room temperature. In vitro corneal permeation studies on excised rabbit corneas demonstrated increased bioavailability, suggesting a reduced dosing frequency compared with conventional eye drops. Therefore, this insert has potential to enhance treatment outcomes by improving patient compliance and providing sustained drug effects.

Moxifloxacin-loaded nanoparticles of thiolated xyloglucan for ocular drug delivery: Permeation, mucoadhesion and pharmacokinetic evaluation

The current study goal was to improve mucoadhesive potential and ocular pharmacokinetics of nanoparticles of thiolated xyloglucan (TXGN) containing moxifloxacin (MXF). Thiolation of xyloglucan (XGN) was achieved with esterification with 3-mercaptopropionic acid. TXGN was characterized by NMR and FTIR analysis. The nanoparticles of TXGN were prepared using ionic-gelation method and evaluate the antibacterial properties. TXGN and nanoparticles were determined to possess 0.06 and 0.08 mmol of thiol groups/mg of polymer by Ellman's method. The ex-vivo bioadhesion time of TXGN and nanoparticles was higher than XGN in a comparative assessment of their mucoadhesive properties. The creation of a disulfide link between mucus and TXGN is responsible for the enhanced mucoadhesive properties of TXGN (1-fold) and nanoparticles (2-fold) over XGN. Improved MXF penetration in nanoparticulate formulation (80 %) based on TXGN was demonstrated in an ex-vivo permeation research utilizing rabbit cornea. Dissolution study showed 95 % release of MXF from nanoparticles. SEM images of nanoparticles showed spherical shape and cell viability assay showed nontoxic behavior when tested on RPE cell line. Antibacterial analysis revealed a zone of inhibition of 31.5 ± 0.5 mm for MXF, while NXM3 exhibited an expanded zone of 35.5 ± 0.4 mm (p < 0.001). In conclusion, thiolation of XGN improves its bioadhesion, permeation, ocular-retention and pharmacokinetics of MXF.

Solanum nigrum Toxicity and Its Neuroprotective Effect Against Retinal Ganglion Cell Death Through Modulation of Extracellular Matrix in a Glaucoma Rat Model.

Purpose: Glaucoma is a complex degenerative optic neuropathy characterized by loss of retinal ganglion cells (RGCs) leading to irreversible vision loss and blindness. Solanum nigrum has been used for decades in traditional medicine system. However, no extensive studies were reported on its antiglaucoma properties. Therefore, this study was designed to investigate the neuroprotective effects of S. nigrum extract on RGC against glaucoma rat model. Methods: High performance liquid chromatography and liquid chromatography tandem mass spectrometry was used to analyze the phytochemical profile of aqueous extract of S. nigrum (AESN). In vitro, {3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide} (MTT) and H2DCFDA assays were used to determine cell viability and reactive oxygen species (ROS) production in Statens Seruminstitut Rabbit Cornea cells. In vivo, AESN was orally administered to carbomer-induced rats for 4 weeks. Intraocular pressure, antioxidant levels, and electrolytes were determined. Histopathological and immunohistochemical analysis was carried out to evaluate the neurodegeneration of RGC. Results: MTT assay showed AESN exhibited greater cell viability and minimal ROS production at 10 μg/mL. Slit lamp and funduscopy confirmed glaucomatous changes in carbomer-induced rats. Administration of AESN showed minimal peripheral corneal vascularization and restored histopathological alterations such as minimal loss of corneal epithelium and moderate narrowing of the iridocorneal angle. Immunohistochemistry analysis showed increased expression of positive BRN3A cells and decreased matrix metalloproteinase (MMP)-9 activation in retina and cornea, whereas western blot analysis revealed downregulation of extracellular matrix proteins (COL-1 and MMP-9) in AESN-treated rats compared with the diseased group rats. Conclusions: AESN protects RGC loss through remodeling of MMPs and, therefore, can be used for the development of novel neurotherapeutics for the treatment of glaucoma.

RiTe conjugate mediated corneal collagen crosslinking, a novel therapeutic intervention for keratoconus – in vitro and in vivo study

Corneal collagen crosslinking (CXL) is an effective method to halt the disease progression of keratoconus, a progressive corneal dystrophy leading to cone shaped cornea. Despite the efficacy of standard protocol, the concerning step of this procedure is epithelial debridement performed to facilitate the entry of riboflavin drug. Riboflavin, a key molecule in CXL protocol, is a sparsely permeable hydrophilic drug in corneal tissues. The present study has employed cell penetrating peptide (CPP), Tat2, to enhance the penetration of riboflavin molecule, and thereby improve currently followed CXL protocol. This study demonstrates approximately two-fold enhanced uptake of CPP riboflavin conjugate, Tat2riboflavin-5′Phosphate (RiTe conjugate), both in vitro and in vivo. Two different CXL protocols (Epi ON and Epi OFF) have been introduced and implemented in rabbit corneas using RiTe conjugate in the present study. The standard and RiTe conjugate mediated CXL procedures exhibited an equivalent extent of crosslinking in both the methods. Reduced keratocyte loss and no endothelial damage in RiTe conjugate mediated CXL further ascertains the safety of the proposed CXL protocols. Therefore, RiTe conjugate mediated CXL protocols present as potential alternatives to the standard keratoconus treatment in providing equally effective, less invasive and patient compliant treatment modality.

Novel artemisinin derivative P31 inhibits VEGF-induced corneal neovascularization through AKT and ERK1/2 pathways

Corneal neovascularization (CoNV)is a major cause of blindness in many ocular diseases. Substantial evidence indicates that vascular endothelial growth factor (VEGF) plays an important role in the pathogenesis of corneal neovascularization. Previous evidence showed that artemisinin may inhibit angiogenesis through down regulation of the VEGF receptors. We designed and synthesized artemisinin derivatives, and validated their inhibitory effect on neovascularization in cell and animal models, and explored the mechanisms by which they exert an inhibitory effect on CoNV. Among these derivatives, P31 demonstrated significant anti-angiogenic effects in vivo and in vitro. Besides, P31 inhibited VEGF-induced HUVECs angiogenesis and neovascularization in rabbit model via AKT and ERK pathways. Moreover, P31 alleviated angiogenic and inflammatory responses in suture rabbit cornea. In conclusion, as a novel artemisinin derivative, P31 attenuates corneal neovascularization and has a promising application in ocular diseases.

Application of direct electric current to the corneal and conjunctival epithelia regulates the tight junctional assembly for ocular iontophoretic drug delivery

Objectives: In this study, we determined how iontophoresis (IP) affects tight junctions (TJs) in isolated rabbit corneas and conjunctiva.&#x0D; Methods: Direct electric current in the range of 0.5–2.0 and 0.5–10 mA/cm2 were applied to the cornea and conjunctiva, respectively, for 30 min. The localization and expression levels of TJ-associated proteins were assessed before and after the application of the electric currents using immunostaining and western blotting.&#x0D; Results: In both corneal and conjunctival epithelia, the localization of proteins, such as claudin-1, claudin-4, occludin, and ZO-1, was temporarily altered by anodal and cathodal IP; however, the protein relocalization was slower at higher currents. Additionally, in both anodal and cathodal IP, the expression levels of claudin-1 and occludin in the cornea and conjunctiva remained unchanged after the application of the electric currents compared with those before.&#x0D; Conclusion: Our results indicated that the application of a direct electric current temporarily regulated TJ assemblies without altering the levels of TJ-associated proteins in both the cornea and conjunctiva. This temporary weakening of the paracellular barrier by the current may be responsible for the enhanced drug transport across the cornea and conjunctiva induced by ocular IP.&#x0D; Keywords: ocular drug delivery, iontophoresis, electric current, cornea, conjunctiva, tight junction

Deoxynivalenol Damages Corneal Epithelial Cells and Exacerbates Inflammatory Response in Fungal Keratitis.

Fungal keratitis (FK) is a kind of infectious keratopathy with a high rate of blindness worldwide. Deoxynivalenol (DON) has been proven to have multiple toxic effects on humans and animals. The aim of this study was to explore a possible pathogenic role of DON in FK. We first made an animal model of FK in New Zealand white rabbits, and then attempted to detect DON in a culture medium in which Fusarium solani had been grown and also in the corneal tissue of the animal model of Fusarium solani keratitis. Next, a model of DON damage in human corneal epithelial cells (HCECs) was constructed to evaluate effects of DON on the activity, migration ability, cell cycle, and apoptosis in the HCECs. Then, putative the toxic damaging effects of DON on rabbit corneal epithelial cells and the impact of the repair cycle were studied. The expression levels of inflammatory factors in the corneas of the animal model and in the model of DON-damaged HCECs were measured. The Fusarium solani strain used in this study appeared to have the potential to produce DON, since DON was detected in the corneal tissue of rabbits which had been inoculated with this Fusarium solani strain. DON was found to alter the morphology of HCECs, to reduce the activity and to inhibit the proliferation and migration of HCECs. DON also induced the apoptosis and S-phase arrest of HCECs. In addition, DON was found to damage rabbit corneal epithelial cells, to prolong the corneal epithelial regeneration cycle, and to be associated with the upregulated expression of inflammatory factors in HCECs and rabbit corneas. DON appears to have a toxic damaging effect on HCECs in FK, and to induce the expression of inflammatory factors, leading to the exacerbation of keratitis and the formation of new blood vessels. Future studies will explore the possibility of developing a test to detect DON in ophthalmic settings to aid the rapid diagnosis of FK, and to develop DON neutralizers and adsorbents which have the potential to improve keratocyte status, inhibit apoptosis, and alleviate inflammation, therein providing new thinking for therapy of clinical FK.

Inflammation-suppressing cornea-in-a-syringe with anti-viral GF19 peptide promotes regeneration in HSV-1 infected rabbit corneas

Pathophysiologic inflammation, e.g., from HSV-1 viral infection, can cause tissue destruction resulting in ulceration, perforation, and ultimately blindness. We developed an injectable Cornea-in-a-Syringe (CIS) sealant-filler to treat damaged corneas. CIS comprises linear carboxylated polymers of inflammation-suppressing 2-methacryloyloxyethyl phosphorylcholine, regeneration-promoting collagen-like peptide, and adhesive collagen-citrate glue. We also incorporated GF19, a modified anti-viral host defense peptide that blocked HSV-1 activity in vitro when released from silica nanoparticles (SiNP-GF19). CIS alone suppressed inflammation when tested in a surgically perforated and HSV-1-infected rabbit corneal model, allowing tissue and nerve regeneration. However, at six months post-operation, only regenerated neocorneas previously treated with CIS with SiNP-GF19 had structural and functional features approaching those of normal healthy corneas and were HSV-1 virus-free. We showed that composite injectable biomaterials can be designed to allow regeneration by modulating inflammation and blocking viral activity in an infected tissue. Future iterations could be optimized for clinical application.

A novel intraocular pressure predicting method based on hyperelastic mechanical model of cornea

Measuring intraocular pressure (IOP) is crucial and remains challenging in diagnosing glaucoma, as it is associated with cornea deformation during inflation. In this study, a three-dimensional analytical model based on hyperelastic constitutive relationship to predict correlation between cornea vertex displacement and the IOP is proposed. The analytical model is validated by rigorous experiments. Rabbit corneas were selected for this study and their mechanical properties were obtained using uniaxial tensile tests. To mimic the environment in which the cornea exists, an artificial anterior chamber equipped with water-injection pipelines was constructed to study the relationship between the corneal vertex displacement with IOP value in practical situation. The experimental results of rabbits corneas prove that the IOP can be deduced based on the measured corneal vertex displacement by the analytical model. Furthermore, subtle difference occurs when comparing the calculated human IOPs with those measured by medical equipment, demonstrating that the proposed method is suitable for monitoring the IOP of human. This novel IOP predicting method provides new inspiration for the design of eyepieces, as well as the preoperative preparation for laser surgery and evaluation of corneal damage.

Characterization of rabbit corneal biomechanical properties after corneal refractive surgery based on rapid loading-unloading uniaxial tensile test

In order to understand how the biomechanical properties of rabbit cornea change over time after corneal ablation, 21 healthy adult rabbits were used in this study, with the left eye as experimental side and the right eye as the control side. Firstly, a lamellar knife was used to remove a portion of the anterior corneal surface tissue (30%~50% of the original corneal thickness) from the left eye of each rabbit, as an animal model simulating corneal refractive surgery. Secondly, postoperative experimental rabbits were kept for one, three, or six months until being euthanized. Strip specimens were produced using their corneas in vitro to perform a uniaxial tensile test with an average loading-unloading rate of approximately 0.16 mm/s. Finally, the visco-hyperelastic material constitutive model was used to fit the data. The results showed that there was a significant difference in the viscoelastic parameters of the corneas between the experimental and the control eyes at the first and third postoperative months. There was a difference in tangential modulus between the experimental and the control eyes at strain levels of 0.02 and 0.05 at the third postoperative month. There was no significant difference in biomechanical parameters between the experimental and the control eyes at the sixth postoperative month. These results indicate that compared with the control eyes, the biomechanical properties of the experimental eyes vary over postoperative time. At the third postoperative month, the ratio of corneal tangential modulus between the experimental and the control eyes significantly increased, and then decreased. This work lays a preliminary foundation for understanding the biomechanical properties of the cornea after corneal refractive surgery based on rapid testing data obtained clinically.

Multiple effects of angiostatins in injured cornea

Prolonged inflammation and excessive neovascularization of the cornea due to severe injury can impair optical clarity and lead to vision impairment. Plasminogen kringle (K) fragments, known as angiostatins (AS), play a well-established role as inhibitors of neovascularization by suppressing pro-angiogenic signaling­. However, AS effects in the cornea, beyond inhibiting the angiogenesis, are still unexplored. In this study, we estimate the protective effect of two AS variants (K1-3 and K5) against alkali burn injury induced in rabbit and rat corneas. AS K1-3 in the single doses of 0.075 or 0.75 μg (0.1 or 1.0 μM, respectively) or 0.3 μg of AS K5 (1.0 μM) were applied locally as eye drops daily for 14 days after the injury. A significant regression of corneal vessels in-growth in injured eyes treated with AS was revealed. Western blot analysis of corneal tissue lysates revealed that injury-induced overexpression of protein markers­ of hypoxia (HIF-1α), angiogenesis (VEGF), tissue remodeling and fibrosis (MMP-9), autophagy (beclin-1) and endoplasmic reticulum stress (GRP-78) was significantly reduced under AS treatment. Besides, the level of tight junctions protein ZO-1 was shown to be up-regulated after the treatment of the damaged cornea with AS K1-3. Summarizing, our study uncovered novel biological functions of the kringle-containing plasminogen fragments indicating its beneficial effects during corneal healing in the experimental model of alkali burn. The data obtained can be helpful for the development of novel efficient formulations to manage complications of ocular surface injuries.

In Vivo Evaluation of Corneal Biomechanics Following Cross-Linking Surgeries Using Optical Coherence Elastography in a Rabbit Model of Keratoconus.

Validation of the feasibility of novel acoustic radiation force optical coherence elastography (ARF-OCE) for the evaluation of biomechanical enhancement of the in vivo model of keratoconus by clinical cross-linking (CXL) surgery. Twelve in vivo rabbit corneas were randomly divided into two groups. Both groups were treated with collagenase type II, and a keratoconus model was obtained. Then, the two groups were treated with CXL procedures with different irradiation energy of 15 J and 30 J (CXL-15 J and CXL-30 J, respectively). An ARF-OCE probe with an ultrasmall ultrasound transducer was used to detect the biomechanical properties of cornea. An antisymmetric Lamb wave model was combined with the frequency dispersion relationship to achieve depth-resolved elastography. Compared with the phase velocity of the Lamb wave in healthy corneas (approximately 3.96 ± 0.27 m/s), the phase velocity of the Lamb wave was lower in the keratoconus region (P < 0.05), with an average value of 3.12 ± 0.12 m/s. Moreover, the corneal stiffness increased after CXL treatment (P < 0.05), and the average phase velocity of the Lamb wave was 4.3 ± 0.19 m/s and 4.54 ± 0.13 m/s after CXL-15 J and CXL-30 J treatment. The Young's moduli of the keratoconus regions were significantly lower than the healthy corneas. Moreover, the Young's modulus of the keratoconus regions was significantly higher after CXL-30 J treatment than after CXL-15 J treatment. We demonstrated that the ARF-OCE technique has great potential in screening keratoconus and guiding clinical CXL treatment. This work accelerates the clinical translation of OCE systems using ultrasmall ultrasound transducers and is used to guide CXL procedures.