Wound Healing In Cornea Research Articles

Novel nanopolymer RNA therapeutics normalize human diabetic corneal wound healing and epithelial stem cells

Human diabetic corneas develop delayed wound healing, epithelial stem cell dysfunction, recurrent erosions, and keratitis. Adenoviral gene therapy modulating c-Met, cathepsin F and MMP-10 normalized wound healing and epithelial stem cells in organ-cultured diabetic corneas but showed toxicity in stem cell-enriched cultured limbal epithelial cells (LECs). For a safer treatment, we engineered a novel nanobiopolymer (NBC) that carried antisense oligonucleotide (AON) RNA therapeutics suppressing cathepsin F or MMP-10, and miR-409-3p that inhibits c-Met. NBC was internalized by LECs through transferrin receptor (TfR)-mediated endocytosis, inhibited cathepsin F or MMP-10 and upregulated c-Met. Non-toxic NBC modulating c-Met and cathepsin F accelerated wound healing in diabetic LECs and organ-cultured corneas vs. control NBC. NBC treatment normalized levels of stem cell markers (keratins 15 and 17, ABCG2, and ΔNp63), and signaling mediators (p-EGFR, p-Akt and p-p38). Non-toxic nano RNA therapeutics thus present a safe alternative to viral gene therapy for normalizing diabetic corneal cells.

Mammalian Ste20-like kinase 4 inhibits the inflammatory response in Aspergillus fumigatus keratitis

Mammalian Ste20-like kinase 4 (MST4), a new member of the germinal-center kinase STE20 family, was recently demonstrated to be a negative regulator of inflammation. However, whether MST4 participates in the inflammatory response to fungal infection remains unknown. Our study investigated the role and molecular mechanisms of MST4 in mice cornea and corneal epithelial cells exposed to Aspergillus fumigatus (A. fumigatus). Protein level of MST4 was detected in mice corneas and human corneal epithelial cells (HCECs) by Western blot analysis. The MST4 protein level was significantly elevated in mice corneas infected with A. fumigatus and HCECs exposed to A. fumigatus. MST4 expression was also detected in mice corneas by immunofluorescence staining. Furthermore, we found recombinant MST4 inhibited proinflammatory cytokines expressions induced by A. fumigatus at both the mRNA and protein levels in mice corneas and HCECs. To further investigate the mechanism of MST4′s anti-inflammatory effect in A. fumigatus keratitis, we verified recombinant MST4 can inhibit curdlan-mediated proinflammatory cytokines production in HCECs. Surprisingly, recombinant MST4 protein downregulated A. fumigatus-induced Dectin-1 expression in both mRNA and protein levels in mice corneas. Recombinant MST4 can inhibit the mRNA expression level of Dectin-1 which was induced by curdlan in HCECs. MST4 can also inhibit the expression of Dectin-1 in mRNA levels increased by Dectin-1 overexpression plasmid in HCECs. Moreover, A. fumigatus or curdlan significantly induced the phosphorylation of Syk, which was consequently suppressed by recombinant MST4. Finally, recombinant MST4 promotes HCECs proliferation, which contribute to cornea wound healing. Taken together, our results provide evidences that MST4 inhibits inflammatory signaling response in A. fumigatus keratitis by downregulating Dectin-1/p-Syk pathway and simultaneously promotes HCECs proliferation.

S100A4 Silencing Facilitates Corneal Wound Healing After Alkali Burns by Promoting Autophagy via Blocking the PI3K/Akt/mTOR Signaling Pathway.

PurposeThis study investigated the role of S100 calcium binding protein A4 (S100A4) in corneal wound healing and the underlying mechanism of the S100A4-mediated PI3K/Akt/mammalian target of rapamycin (mTOR) pathway.MethodsThe rabbit corneal alkali burn model was established in vivo. S100A4 expression, wound healing, inflammation, and autophagy in rabbit cornea after alkali burn were detected. The NaOH-treated rabbit corneal stromal cells (rCSCs) were transfected with overexpressed S100A4 or silencing S100A4 to examine the effect of S100A4 on corneal wound healing in vitro. The effect of S100A4 on cell viability, proliferation, migration, invasion, fibrosis, and autophagy of rCSCs after alkali burn was analyzed. Then the functional rescue experiments were carried out. The PI3K inhibitor, LY294002, was used to elucidate the PI3K/Akt/mTOR signaling pathway in rCSCs.ResultsS100A4 silencing promoted rabbit corneal wound healing by inhibiting fibrosis and inflammation and promoting autophagy in alkali-burned cornea, corresponding to increased levels of LC3, Beclin 1, and Atg4B but lowered α-smooth muscle actin, TNF-ɑ, and p62 levels. Moreover, silencing S100A4 inhibited proliferation, migration, invasion, and fibrosis of NaOH-treated rCSCs and promoted the differentiation of rCSCs into corneal cells and the autophagy of damaged rCSCs. The inhibitory role of S100A4 in wound healing was achieved via activation of the PI3K/Akt/mTOR pathway.ConclusionsS100A4 silencing confers a promising effect on wound healing of alkali-burned cornea by blocking the PI3K/Akt/mTOR pathway, supporting the advancement of corneal gene therapies for wound healing.

Fibrocytes, Wound Healing, and Corneal Fibrosis.

PurposeThis review highlights the roles of fibrocytes—their origin, markers, regulation and functions—including contributions to corneal wound healing and fibrosis.MethodsLiterature review.ResultsPeripheral blood fibroblast-like cells, called fibrocytes, are primarily generated as mature collagen-producing cells in the bone marrow. They are likely derived from the myeloid lineage, although the exact precursor remains unknown. Fibrocytes are identified by a combination of expressed markers, such as simultaneous expression of CD34 or CD45 or CD11b and collagen type I or collagen type III. Fibrocytes migrate into the wound from the blood where they participate in pathogen clearance, tissue regeneration, wound closure and angiogenesis. Transforming growth factor beta 1 (TGF-β1) and adiponectin induce expression of α-smooth muscle actin and extracellular matrix proteins through activation of Smad3 and adenosine monophosphate-activated protein kinase pathways, respectively. Fibrocytes are important contributors to the cornea wound healing response and there are several mechanisms through which fibrocytes contribute to fibrosis in the cornea and other organs, such as their differentiation into myofibroblasts, production of matrix metalloproteinase, secretion of tissue inhibitor of metalloproteinase, and release of TGF-β1. In some tissues, fibrocytes may also contribute to the basement membrane regeneration and to the resolution of fibrosis.ConclusionsNew methods that block fibrocyte generation, fibrocyte migration, and their differentiation into myofibroblasts, as well as their production of matrix metalloproteinases, tissue inhibitor of metalloproteinase, and TGF-β1, have therapeutic potential to reduce the accumulation of collagens, maintain tissue integrity and retard or prevent the development of fibrosis.

Procedure for early corneal basement membrane repair and regeneration in corneal penetrating injury in rabbits

Objective To describe the procedure for early corneal epithelial basement membrane(EBM) repair and regeneration in rabbits with corneal penetrating injury. Methods Forty-two New Zealand white rabbits were divided into modeling 1-, 3-, 5-, 7-, 14-, 21-, and 30-day groups using a random number table method, with 6 rabbits in each group; the right eyes were selected as the experimental eyes.Another 6 New Zealand white rabbits without any treatment were taken as the normal control group.A 2.0-mm trephine was used to ablate a full-thickness button of the central corneal tissue of each rabbit.The corneas were observed by slit lamp biomicroscopy at the respective time points after the trephined injury.Corneal epithelial fluorescein staining was used to evaluate re-epithelialization with Image J software and haze grading was evaluated with the Fantes classification.Hematoxylin-eosin staining was used to observe the healing process of the cornea.Transmission electron microscopy was conducted to assess the regeneration of the EBM and the reconstruction of the cornea.The study protocol was approved by the Ethics Committee of Guangxi Medical University (No.201811031). The use and care of the experimental animals complied with the Statement for the Use of Animals in Ophthalmic and Vision Research. Results The corneal epithelial fluorescein areas in modeling 1-, 3-, 5-, 7-, and 14-day group were (4.00±0.10), (3.11±0.10), (2.00±0.06), (0.90±0.04) and (0.67±0.03)mm2, respectively, with a significant difference among them (F=3 398.88, P<0.01). With the increasing of time after modeling, the corneal epithelium fluorescein area was gradually reduced, showing significant differences between any two groups (all at P<0.05), and the staining was disappeared at 21 and 30 days after modeling.The corneal haze grades were 3.44±0.53, 0.67±0.25, 1.33±0.50, 2.11±0.60, 2.44±0.53, 3.22±0.44 and 3.78±0.44 in modeling 1-, 3-, 5-, 7-, 14-, 21-, and 30-day group, respectively. The corneal opacity score gradually decreased during 1-5 days after modeling and gradually increased during 5-30 days after modeling, with a significant difference among them (F=51.182, P<0.01). Hematoxylin-eosin staining revealed that a fibrin clot formed in the wound area, and a single layer of epithelium covered the area at initial 3 days after modeling, and a large number of fibroblasts and some extracellular matrix were found at 5 days after modeling.At 21 and 30 days after modeling, the collagen fibers were tightly arranged in the anterior stroma.Transmission electron microscopy showed that the wound was filled with irregular collagen fibers and myofibroblasts.The stroma was remodeled at 21 days after modeling, and defective regeneration of the EBM was detected at 21 and 30 days after modeling. Conclusions Corneal fibrosis initiates after corneal penetrating injury in rabbits and gradually aggravates, the EBM regenerates defectively. Key words: Epithelial basement membrane; Cornea wound healing; Penetrating injury; Rabbit

Topical Pergolide Enhance Corneal Nerve Regrowth Following Induced Corneal Abrasion.

PurposeNeurotrophic keratopathy is a degenerative disease that may be improved by nerve growth factor (NGF). Our aim was to investigate the use of pergolide, a dopamine (D1 and D2) receptor agonist known to increase the synthesis and release of NGF for regeneration of damaged corneal nerve fibers.MethodsPergolide function was evaluated by measuring axon length and NGF levels by enzyme-linked immunosorbent assay in cultured chicken dorsal root ganglion (DRG) cells with serial doses of pergolide (10, 25, 50, 150, and 300 µg/ml) and with different concentrations of a D1 antagonist. Pergolide function was further evaluated by cornea nerve fiber density and wound healing in a cornea scratch mouse model.ResultsPergolide increased DRG axon length significantly at a dose between 50 and 300 µg/ml. Different concentrations of D1 antagonist (12, 24, 48, and 96 µg/ml) inhibited DRG axon length growth with pergolide (300 µg/ml). Pergolide (50 µg/ml) upregulated NGF expression in DRG cells at both 24 hours and 48 hours. Pergolide improved cornea nerve fiber density at both 1 week and 2 weeks. Pergolide also improved cornea wound healing.ConclusionsWe demonstrated that pergolide can act to promote an increase in NGF which promotes corneal nerve regeneration and would therefore improve corneal sensation and visual acuity in eyes with peripheral neurotrophic keratopathy.

Protocols for decellularization of human amniotic membrane.

Human amniotic membrane (HAM) has been used as a very promising biological-based product in health centers, especially for skin and cornea wound healing applications. The excellent properties of this membrane make it a potential candidate in treatment of various skin injuries such as bedsores, burn wounds and diabetic ulcers. Such properties are cytobiocompatibility, a structure very similar to normal skin composed of extracellular matrix (ECM) proteins, various growth factors involved in normal wound healing process and antibacterial agents. HAM contains epithelial cells, fibroblasts and mesenchymal stem cells. Therefore, the successful decellularization of HAM with minimal negative effects on its ECM components is very important to avoid graft rejection and shows improved performance. To date, several approaches have been conducted for decellularization of HAM, which is mainly based on enzyme-, detergent- or mechanical procedures with various ranges of success. Here, we describe a systematic detergent-based decellularization protocol as main protocol. We also explain the enzyme- and mechanical-based methods as the alternative protocols for decellularization of HAM.

Understanding cornea homeostasis and wound healing using a novel model of stem cell deficiency in Xenopus

Limbal Stem Cell Deficiency (LSCD) is a painful and debilitating disease that results from damage or loss of the Corneal Epithelial Stem Cells (CESCs). Therapies have been developed to treat LSCD by utilizing epithelial stem cell transplants. However, effective repair and recovery depends on many factors, such as the source and concentration of donor stem cells, and the proper conditions to support these transplanted cells. We do not yet fully understand how CESCs heal wounds or how transplanted CESCs are able to restore transparency in LSCD patients. A major hurdle has been the lack of vertebrate models to study CESCs. Here we utilized a short treatment with Psoralen AMT (a DNA cross-linker), immediately followed by UV treatment (PUV treatment), to establish a novel frog model that recapitulates the characteristics of cornea stem cell deficiency, such as pigment cell invasion from the periphery, corneal opacity, and neovascularization. These PUV treated whole corneas do not regain transparency. Moreover, PUV treatment leads to appearance of the Tcf7l2 labeled subset of apical skin cells in the cornea region. PUV treatment also results in increased cell death, immediately following treatment, with pyknosis as a primary mechanism. Furthermore, we show that PUV treatment causes depletion of p63 expressing basal epithelial cells, and can stimulate mitosis in the remaining cells in the cornea region. To study the response of CESCs, we created localized PUV damage by focusing the UV radiation on one half of the cornea. These cases initially develop localized stem cell deficiency characteristics on the treated side. The localized PUV treatment is also capable of stimulating some mitosis in the untreated (control) half of those corneas. Unlike the whole treated corneas, the treated half is ultimately able to recover and corneal transparency is restored. Our study provides insight into the response of cornea cells following stem cell depletion, and establishes Xenopus as a suitable model for studying CESCs, stem cell deficiency, and other cornea diseases. This model will also be valuable for understanding the nature of transplanted CESCs, which will lead to progress in the development of therapeutics for LSCD.

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing.

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

Wound-Healing Studies in Cornea and Skin: Parallels, Differences and Opportunities.

The cornea and the skin are both organs that provide the outer barrier of the body. Both tissues have developed intrinsic mechanisms that protect the organism from a wide range of external threats, but at the same time also enable rapid restoration of tissue integrity and organ-specific function. The easy accessibility makes the skin an attractive model system to study tissue damage and repair. Findings from skin research have contributed to unravelling novel fundamental principles in regenerative biology and the repair of other epithelial-mesenchymal tissues, such as the cornea. Following barrier disruption, the influx of inflammatory cells, myofibroblast differentiation, extracellular matrix synthesis and scar formation present parallel repair mechanisms in cornea and skin wound healing. Yet, capillary sprouting, while pivotal in proper skin wound healing, is a process that is rather associated with pathological repair of the cornea. Understanding the parallels and differences of the cellular and molecular networks that coordinate the wound healing response in skin and cornea are likely of mutual importance for both organs with regard to the development of regenerative therapies and understanding of the disease pathologies that affect epithelial-mesenchymal interactions. Here, we review the principal events in corneal wound healing and the mechanisms to restore corneal transparency and barrier function. We also refer to skin repair mechanisms and their potential implications for regenerative processes in the cornea.

Topical Ophthalmic Formulation of Trichostatin A and SurR9-C84A for Quick Recovery Post-alkali Burn of Corneal Haze.

Alkali burn injury is a true ocular emergency of the conjunctiva and cornea that requires immediate precision. Lack of an immediate therapy can lead to a substantial damage in the ocular surface and anterior segment further causing visual impairment and disfigurement. We explored the regenerative capability of dominant negative survivin protein (SurR9-C84A) and histone deacetylase inhibitor trichostatin-A (TSA) in vivo, in a rat alkali burn model. A topical insult in rat eyes with NaOH led to degradation of the conjunctival and corneal epithelium. The integrity of the conjunctival and corneal tissue was increased by TSA and SurR9-C84A by improving the clathrin and claudin expressions. Wound healing was initiated by an increase in TGF-beta-1 and, increased endogenous survivin which inhibited apoptosis post-TSA and SurR9-C84A treatments. Protein expressions of fibronectin and alpha-integrin 5 were found to increase promoting corneal integrity. The cytokine analysis confirmed increased expressions of IL-1beta, IL-6, IL-12, IL-13, IFN-gamma, TNF-alpha, GMCSF, Rantes, and MMP-2 in injured cornea, which were found to be significantly downregulated by the combined treatment of SurR9-C84A and TSA. The ocular and systemic pharmacokinetic (PK) parameters were measured post-topical ocular administration of TSA and SurR9-C84A. The SurR9-C84A and TSA sustained relatively longer in the cornea, conjunctiva, and aqueous humor than in the tear fluid and plasma. Our results confirmed that a combination of TSA with SurR9-C8A worked in synergy and showed a promising healing and anti-inflammatory effect in a very short time against alkali burn. Therefore, a combination of TSA and SurR9-C84A can fulfill the need for an immediate response to wound healing in alkali burnt cornea. We also synthesized ultra-small chitosan nanoparticles (USC-NPs) targeted with alpha-SMA antibodies that can be used for delivery of TSA and SurR9-C84A specifically to the ocular burn site.

Ophthalmic Combination of SurR9-C84A and Trichostatin-A Targeting Molecular Pathogenesis of Alkali Burn

Background: Alkali burn is a frequently occurring ocular injury that resembles ocular inflammation caused by eye allergies, infection, and refractive surgeries.Methods: We investigated the synergistic regenerative potential of dominant negative survivin mutant (SurR9-C84A) and histone deacetylase (HDAC) inhibitor trichostatin-A (TSA) against alkali burn and corneal haze using human keratocytes and rabbit alkali burn model (Female New Zealand white rabbits).Results: Combination of SurR9-C84A and TSA suppressed levels of transforming growth factor (TGF)-β, alpha smooth-muscle actin (α-SMA), fibronectin and HDAC1, leading to apoptosis in myofibroblast cells and, showed the potential to clear the corneal haze. An insult with 0.5 N NaOH for 1 min led to neutrophils infiltration and formation of large vacuoles in the stroma. Treatments with TSA and SurR9-C84A for 40 min led to improvement in the conjunctival and corneal tissue integrity, marked by an increase in clathrin, and claudin expressions. An increase in TGF-β and endogenous survivin confirmed wound healing and cell proliferation in rabbit cornea. The blood analysis revealed a substantial decrease in the RBC, WBC, platelets, or the hemoglobin content post alkali burn. The cytokine array analysis revealed that NaOH induced expressions of IL-1α and MMP-9, which were found to be significantly downregulated (1.8 and 11.5 fold respectively) by the combinatorial treatment of SurR9-C84A and TSA.Conclusion: Our results confirmed that combination of SurR9-C84A with TSA worked in synergy to heal ocular injury and inflammations due to alkali burn and led to the regeneration of ocular tissue by increasing clathrin, claudin, survivin, and TGF-β and reversal of alkali burn by suppressing IL-1α and MMP-9 without inducing haze.

MK2 inhibitor reduces alkali burn-induced inflammation in rat cornea.

MK2 activation by p38 MAPK selectively induces inflammation in various diseases. We determined if a MK2 inhibitor (MK2i), improves cornea wound healing by inhibiting inflammation caused by burning rat corneas with alkali. Our study, for the first time, demonstrated that MK2i inhibited alkali burn-induced MK2 activation as well as rises in inflammation based on: a) blunting rises in inflammatory index, inflammatory cell infiltration, ED1+ macrophage and PMN+ neutrophil infiltration; b) suppressing IL-6 and IL-1β gene expression along with those of macrophage inflammatory protein-1α (MIP-1α), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1); c) reducing angiogenic gene expression levels and neovascularization (NV) whereas anti-angiogenic PEDF levels increased. In addition, this study found that MK2i did not affect human corneal epithelial cell (HCEC) proliferation and migration and had no detectable side effects on ocular surface integrity. Taken together, MK2i selectively inhibited alkali burn-induced corneal inflammation by blocking MK2 activation, these effects have clinical relevance in the treatment of inflammation related ocular surface diseases.

NorLeu3A(1-7) Accelerates Clear Corneal Full Thickness Wound Healing.

We evaluated the effect of the renin angiotensin system (RAS) peptide NorLeu3-Angiotensin (1-7) (NLE) formulated in a viscoelastic gel (USB004) on the healing of full-thickness corneal injuries. Dutch pigmented rabbits received conjunctival administration of 0.3% USB004, 0.03% USB004, or vehicle-control to healthy and full-thickness injured eyes administered once daily for 28 consecutive days. Safety was evaluated using IOP measurement, slit-lamp examination, and confocal microscopy. Evaluations for both efficacy studies included an oblique light examination, modified Seidel test (Seidel test with gentle ocular pressure) as well as during elevated IOP test, confocal microscopy imaging, and histologic analysis. Application of 0.3% USB004, 0.03% USB004, and vehicle-control was safe in healthy and incised eyes. Further, application of 0.3% and 0.03% USB004 following full-thickness corneal incision resulted in a 2-fold acceleration of resolution of edema and inflammation, reduction in duration of wound leakage on a modified Seidel test (Seidel test with gentle ocular pressure) as well as during elevated IOP test, and healing with near normal architecture without evidence of fibrosis and angiogenesis when compared to vehicle-control animals. Topical ocular application of 0.3% and 0.03% USB004 promotes full-thickness cornea wound healing without the evidence of fibrosis and angiogenesis. Further studies are warranted to determine the cornea-specific mechanism of action(s) that promotes regeneration leading to clear corneal healing.

Targeting Imbalance between IL-1β and IL-1 Receptor Antagonist Ameliorates Delayed Epithelium Wound Healing in Diabetic Mouse Corneas

Patients with diabetes mellitus often develop corneal complications and delayed wound healing. How diabetes might alter acute inflammatory responses to tissue injury, leading to delayed wound healing, remains mostly elusive. Using a streptozotocin-induced type I diabetes mellitus mice and corneal epithelium-debridement wound model, we discovered that although wounding induced marked expression of IL-1β and the secreted form of IL-1 receptor antagonist (sIL-1Ra), diabetes suppressed the expressions of sIL-1Ra but not IL-1β in healing epithelia and both in whole cornea. In normoglycemic mice, IL-1β or sIL-1Ra blockade delayed wound healing and influenced each other's expression. In diabetic mice, in addition to delayed reepithelization, diabetes weakened phosphatidylinositol 3-kinase-Akt signaling, caused cell apoptosis, diminished cell proliferation, suppressed neutrophil and natural killer cell infiltrations, and impaired sensory nerve reinnervation in healing mouse corneas. Local administration of recombinant IL-1Ra partially, but significantly, reversed these pathological changes in the diabetic corneas. CXCL10 was a downstream chemokine of IL-1β-IL-1Ra, and exogenous CXCL10 alleviated delayed wound healing in the diabetic, but attenuated it in the normal corneas. In conclusion, the suppressed early innate/inflammatory responses instigated by the imbalance between IL-1β and IL-1Ra is an underlying cause for delayed wound healing in the diabetic corneas. Local application of IL-1Ra accelerates reepithelialization and may be used to treat chronic corneal and potential skin wounds of diabetic patients.

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells.

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

Desiccating Stress-Induced MMP Production and Activity Worsens Wound Healing in Alkali-Burned Corneas.

To evaluate the effects of dry eye on ocular surface protease activity and sight threatening corneal complications following ocular surface chemical injury. C57BL/6 mice were subjected to unilateral alkali burn (AB) with or without concomitant dry eye for 2 or 5 days. Mice were observed daily for appearance of corneal perforation. Whole corneas were harvested and lysed for RNA extraction. Quantitative real-time PCR was performed to measure expression of inflammation cytokines, matrix metalloproteinases (MMP). Matrix metalloproteinase-9 activity, gelatinase activity, and myeloperoxidase (MPO) activity were evaluated in corneal lysates. Presence of infiltrating neutrophils was evaluated by immunohistochemistry and flow cytometry. Eyes subjected to the combined model of AB and dry eye (CM) had 20% sterile corneal perforation rate as soon as 1 day after the initial injury, which increased to 35% by 5 days, delayed wound closure and increased corneal opacity. Increased levels of IL-1β, -6, and MMPs-1, -3, -8, -9, and -13, and chemokine (C-X-C motif) ligand 1 (CSCL1) transcripts were found after 2 days in CM compared with AB corneas. Increased MMP-1, -3, -9, and -13 immunoreactivity and gelatinolytic activity were seen in CM corneas compared with AB. Increased neutrophil infiltration and MPO activity was noted in the CM group compared with AB 2 days post injury. Desiccating stress worsens outcome of ocular AB, creating a cytokine and protease storm with greater neutrophil infiltration, increasing the risk of corneal perforation.

Characteristics of Reconstituted Tight Junctions After Corneal Epithelial Wounds and Ultrastructure Alterations of Corneas in Type 2 Diabetic Rats

Purpose: To investigate the characteristics of reconstituted tight junctions after corneal epithelial wounds and ultrastructure alterations of corneas in a high-fat diet combined with streptozocin (STZ)-induced rat model of type 2 diabetes mellitus (DM).Materials and Methods: One-hundred Sprague-Dawley male rats were randomly divided into diabetic and normal control groups (n = 50 each). A high-fat diet combined with intraperitoneal STZ injection was used to induce type 2 DM. Wound healing was determined using an in vivo corneal epithelial debridement model. Scanning electron microscopy (SEM) was used to study differences in corneal epithelium between diabetic and normal rats. The rate of corneal wound healing was observed by fluorescein and hematoxylin and eosin staining. Diabetic and healthy rats were sacrificed before debridement and 16, 48, 72, and 120 hours afterwards for Western blot and immunofluorescence.Results: Corneal epithelial cell connection abnormalities, dropsy, and microvillus reduction were evident by SEM. Corneal epithelial wound closure rates were decreased in diabetic rats compared with normal rats. Immunofluorescence and Western blot analysis demonstrated that occludin expression in the corneal epithelium of the diabetic group was weaker compared to the normal group at the 16 and 48 hour time-points after debridement.Conclusions: Abnormal occludin expression may contribute to delayed epithelial wound healing in diabetic corneas.