Corneal Epithelial Basement Membrane Research Articles

Biologically inspired bioactive hydrogels for scarless corneal repair.

Corneal injury-induced fibrosis occurs because of corneal epithelial basement membrane (EBM) injury and defective regeneration. Corneal fibrosis inhibition and transparency restoration depend on reestablished EBM, where the collagen network provides structural stability and heparan sulfate binds corneal epithelium-derived cytokines to regulate homeostasis. Inspired by this, bioactive hydrogels (Hep@Gel) composed of collagen-derived gelatins and highly anionic heparin were constructed for scarless corneal repair. Hep@Gel resembled the barrier function of the EBM regarding surface-confined binding, long-time sequestration, and progressive degradation of IL-1, TGF-β, and PDGF-BB, which robustly inhibited the apoptosis and myofibroblast transition of keratocytes. Animal models of rabbits and nonhuman primates confirmed that Hep@Gel effectively limited the influx of inflammatory and fibrotic cytokines from the epithelium into the stroma to down-regulate the wound healing cascade, contributing to better vision quality with 73% reduced fibrosis. Hep@Gel offers a solution for preventing corneal injury-induced scarring and substituting for lamellar keratoplasty to remove scarring.

Pilot Safety Study of a Microfabricated Device for Anterior Stromal Puncture to Treat Corneal Epithelial Disease in the Optical Axis.

Anterior stromal puncture is an inexpensive and widely used technique at the slit lamp to treat recurrent corneal erosions (RCE), common sequelae of corneal abrasions, trauma, and epithelial basement membrane dystrophies (EBMDs). The purpose of this study was to determine safety of a novel anterior stromal micropuncture device (termed a "plexitome") for use in the optical axis of the cornea. We performed an IRB-approved clinical pilot study on 45 patients with diverse corneal pathologies to determine the safety of the plexitome device and examined corneal healing responses clinically for a minimum of 30 days after treatment. Follow on efficacy data was collected for treated patients with RCE.Micropuncture treatment was performed either through loose epithelial tissue or after debridement using a cotton tip at the slit lamp. After "imprinting," patients were treated with topical medications including antibiotics, hypertonic saline/ointment, bandage contact lenses, and/or patching according to the treating physician's routine care for the condition. Micropuncture using the "plexitome" device did not create visible scars in the corneal stroma of the 45 patients treated and followed for at least 30 days. Photographic evidence of imprinting was seen at in 1 patient at 30 days. There were no significant adverse events associated with treatment. Micropuncture of the cornea using the "plexitome" device does not create optically evident anterior stromal scarring after treatment. Micropuncture using the device may be an effective way of treating RCE and other corneal manifestations of EBMD in the optical axis, which is not currently possible using standard anterior stromal puncture methods.

Characterization of Ocular Morphology in Col4a3-/- Mice as a Murine Model for Alport Syndrome.

The purpose of this study was to investigate the ocular morphological characteristics of Col4a3-/- mice as a model of Alport syndrome (AS) and the potential pathogenesis. The expression of collagen IV at 8, 12, and 21 weeks of age was evaluated by immunohistochemistry in wild-type (WT) and Col4a3-/- mice. Hematoxylin and eosin (H&E) staining and thickness measurements were performed to assess the thickness of anterior lens capsule and retina. Ultrastructure analysis of corneal epithelial basement membrane, anterior lens capsule, internal limiting membrane (ILM), and retinal pigment epithelium (RPE) basement membrane was performed using transmission electron microscopy. Finally, Müller cell activation was evaluated by glial fibrillary acidic protein (GFAP) expression. Collagen IV was downregulated in the corneal epithelial basement membrane and ILM of Col4a3-/- mice. The hemidesmosomes of Col4a3-/- mice corneal epithelium became flat and less electron-dense than those of the WT group. Compared with those of the WT mice, the anterior lens capsules of Col4a3-/- mice were thinner. Abnormal structure was detected at the ILM Col4a3-/- mice, and the basal folds of the RPE basement membrane in Col4a3-/- mice were thicker and shorter. The retinas of Col4a3-/- mice were thinner than those of WT mice, especially within 1000µm away from the optic nerve. GFAP expression enhanced in each age group of Col4a3-/- mice. Our results suggested that Col4a3-/- mice exhibit ocular anomalies similar to patients with AS. Additionally, Müller cells may be involved in AS retinal anomalies. This animal model could provide an opportunity to understand the underlying mechanisms of AS ocular disorders and to investigate potential new treatments.

Clearing the light path: proteoglycans and their important roles in the lens and cornea.

Some of the earliest studies of glycans were performed on mammalian corneas and lenses with many of the key concepts we currently recognize as being fundamental to our understanding of basic cell biology arising from these studies. Proteoglycans and their GAG side chains are essential components of the ECM of the lens capsule. They also are present in the anterior corneal epithelial basement membrane and the posterior (Decemet's) basement membrane, and they organize collagen fiber diameters and spacing in the corneal stroma to maintain stromal clarity. Studies using genetically engineered mice and characterization of spontaneously arising mutations in genes controlling proteoglycan synthesis have generated new insight into the roles played by proteoglycans in signal transduction. We now know that proteoglycans and GAGs can regulate cell signaling and the maintenance of avascularity and immune privilege that are hallmarks of these tissues. In addition, proteoglycan-rich matrices provide the pathways for immune cells to populate the surface of the lens as a response to corneal wounding and in a model of Experimental Autoimmune Uveitis. Here we describe what is known about proteoglycans and GAGs in the cornea and lens. This knowledge has begun to provide promising leads into new proteoglycan-based treatments aimed at restoring and maintaining homeostasis in the cornea. Future studies are needed to determine how these new drugs impact the recruitment of immune cells to the lens for functions in restoring/maintaining homeostasis in the eye.

Epidemiology of and Genetic Factors Associated with Acanthamoeba Keratitis.

Acanthamoeba keratitis (AK) is a severe, rare protozoal infection of the cornea. Acanthamoeba can survive in diverse habitats and at extreme temperatures. AK is mostly seen in contact lens wearers whose lenses have become contaminated or who have a history of water exposure, and in those without contact lens wear who have experienced recent eye trauma involving contaminated soil or water. Infection usually results in severe eye pain, photophobia, inflammation, and corneal epithelial defects. The pathophysiology of this infection is multifactorial, including the production of cytotoxic proteases by Acanthamoeba that degrades the corneal epithelial basement membrane and induces the death of ocular surface cells, resulting in degradation of the collagen-rich corneal stroma. AK can be prevented by avoiding risk factors, which includes avoiding water contact, such as swimming or showering in contact lenses, and wearing protective goggles when working on the land. AK is mostly treated with an antimicrobial therapy of biguanides alone or in combination with diaminidines, although the commercial availability of these medicines is variable. Other than anti-amoeba therapies, targeting host immune pathways in Acanthamoeba disease may lead to the development of vaccines or antibody therapeutics which could transform the management of AK.

Matrix Metalloproteinases and the Pathogenesis of Recurrent Corneal Erosions and Epithelial Basement Membrane Dystrophy.

Matrix metalloproteinases (MMPs) are a group of proteolytic enzymes which are members of the zinc endopeptidase family. They have the ability to degrade extracellular matrix elements, allowing for the release of binding molecules and cell migration. Although metalloproteinases regulate numerous physiological processes within the cornea, overexpression of metalloproteinase genes and an imbalance between the levels of metalloproteinases and their inhibitors can contribute to the inhibition of repair processes, the development of inflammation and excessive cellular proliferation. The involvement of MMPs in the pathogenesis of dystrophic corneal diseases needs clarification. Our analyses focus on the involvement of individual metalloproteinases in the pathogenesis of recurrent corneal erosions and highlight their impact on the development of corneal epithelial basement membrane dystrophy (EBMD). We hypothesize that abnormalities observed in patients with EBMD may result from the accumulation and activation of metalloproteinases in the basal layers of the corneal epithelium, leading to basement membrane degradation. A barrier formed from degradation materials inhibits the normal migration of epithelial cells to the superficial layers, which contributes to the development of the aforementioned lesions. This hypothesis seems to be lent support by the elevated concentrations of metalloproteinases in the corneal epithelium of these patients found in our previous studies on the relationships between MMPs and recurrent corneal erosions.

Increased Matrix Metalloproteinase-2 and Matrix Metalloproteinase-3 Concentrations in Corneal Epithelium of Patients with Recurrent Corneal Erosions.

Purpose To assess the role of selected matrix metalloproteinases in defective corneal re-epithelization in patients with recurrent corneal erosions. Subjects The study group (group 1) included patients with recurrent corneal erosions qualified for phototherapeutic keratectomy. The group 1 was divided into two subgroups regarding the etiology of recurrent corneal erosions: group 1A, Cogan's basement membrane dystrophy, and group 1B, trauma. The control group (group 2) included patients with healthy eyes qualified for Epi-Bowman Keratectomy. Methods The analyzed material was the corneal epithelium collected during phototherapeutic keratectomy or Epi-Bowman Keratectomy in the study or control group, respectively. Matrix metalloproteinases concentration was determined by an immunohistochemical method using Human Magnetic Luminex® Assay. Results The study revealed a statistically significantly higher concentration of matrix metalloproteinase-2 in group 1 compared to the control and a statistically significantly higher concentration of matrix metalloproteinase-3 in group 1 compared to the control. Conclusions The results obtained in the study can prove that matrix metalloproteinase-2 and matrix metalloproteinase-3 having the ability to dissolve anchoring fibers and the corneal epithelial basement membrane could be responsible for epithelial instability and their accumulation in the corneal epithelium may induce recurrence of erosion.

An Epithelial Abrasion Model for Studying Corneal Wound Healing.

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its transparency. However, due to its external position, the cornea is highly susceptible to a wide variety of injuries that can lead to the loss of corneal transparency and eventual blindness. Efficient corneal wound healing in response to these injuries is pivotal for maintaining corneal homeostasis and preservation of corneal transparency and refractive capabilities. In events of compromised corneal wound healing, the cornea becomes vulnerable to infections, ulcerations, and scarring. Given the fundamental importance of corneal wound healing to the preservation of corneal transparency and vision, a better understanding of the normal corneal wound healing process is a prerequisite to understanding impaired corneal wound healing associated with infection and disease. Toward this goal, murine models of corneal wounding have proven useful in furthering our understanding of the corneal wound healing mechanisms operating under normal physiological conditions. Here, a protocol for creating a central corneal epithelial abrasion in mouse using a trephine and a blunt golf club spud is described. In this model, a 2 mm diameter circular trephine, centered over the cornea, is used to demarcate the wound area. The golf club spud is used with care to debride the epithelium and create a circular wound without damaging the corneal epithelial basement membrane. The resulting inflammatory response proceeds as a well-characterized cascade of cellular and molecular events that are critical for efficient wound healing. This simple corneal wound healing model is highly reproducible and well-published and is now being used to evaluate compromised corneal wound healing in the context of disease.

Bowman's layer in the cornea– structure and function and regeneration

Bowman's layer lies immediately posterior to the epithelial basement membrane (EBM) and anterior to the stroma proper in humans, chickens, quail, zebra fish, deer, giraffe, antelope, California sea lions, guinea pig and several other species. It is not found in dog, wolf, cat, tiger, lions, rabbit, pigs, cows, goats, or horses. Developmental anomalies of Bowman's layer are rare, but acquired damage to Bowman's layer, or even complete destruction, is frequently seen in advanced bullous keratopathy or Fuchs' endothelial dystrophy. No detrimental effects of removal of Bowman's layer over the central 6–7 mm of central cornea have been noted in millions of patients who've had photorefractive keratectomy (PRK). Recent studies have suggested the randomly-oriented collagen fibrils that make up Bowman's layer do not have a significant barrier function in modulating the passage of moderate- to large-sized proteins. It is hypothesized that Bowman's layer develops in the corneas of those species that have one because of cytokine-mediated interactions occurring between corneal epithelial cells and underlying keratocytes, including negative chemotactic and apoptotic effects on the keratocytes by low levels of cytokines such as interleukin-1α that are gradually released as epithelial cells die and slough during their normal development. A “Bowman's like layer” can generate around stromal epithelial plugs after radial keratotomy, and possibly beneath the central corneal epithelial basement membrane many years after PRK.

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

Recurrent corneal erosion: modern view of the problem

Recurrent corneal erosion syndrome (RCES) is a common chronic disease associated with repeated episodes of epithelial disruption and is characterized by sudden onset of sharp pain in the eye, usually at night or upon waking, as well as redness, photophobia and lacrimation. The cause of the disease is considered to be impaired adhesion of the epithelial layer to the stroma, which is a consequence of abnormal regeneration of the basement membrane of corneal epithelium. A number of biological factors, including inflammatory mediators and proteinases in high concentrations, contribute to the destruction of the formed adhesion complexes. The basis of RCES diagnosis is characteristic medical history and biomicroscopy. An area of epithelium absence can be detected under direct illumination - more often in paracentral view - mainly in the lower third of the cornea, where an area of loose and irregular epithelium with microcysts or grayish inclusions can be seen. There may also be a card-like or «fingerprint» pattern. First of all, it is necessary to differentiate RCES from infectious lesions, which require a fundamentally different treatment regimen. It is believed that the first stage of treatment should include lubricants, drugs that promote epithelialization, inhibitors of matrix metalloproteinases and lipases. The «second line» of conservative treatment can include blood derivatives. If conservative therapy is ineffective, it is advised to use phototherapeutic keratectomy (PTK) or perform diamond burr polishing of Bowman's layer. The choice of a method for treating recurrent erosion should be based on the effectiveness of drug therapy, frequency of recurrence, duration of the disease, severity of clinical symptoms, availability of specific ophthalmic equipment and experience with it.

Tropical Keratopathy (Florida Spots) in Cats.

The authors used microscopy and synchrotron-based small-angle X-ray scattering analysis (SAXS) to describe lesions macroscopically typical of tropical keratopathy ("Florida spots") from 6 cats on St Kitts. Microscopically, there were varying degrees of epithelial hyperplasia and thinning of the cornea (by 4% to 18%) due to loss of corneal stroma associated with dense accumulations of collagen in the superficial stroma. The collagen fibrils in lesions were wider and had more variable diameters (39.5 ± 5.0 nm, mean ± SD) than in normal corneas (25.9 ± 3.6 nm; P < .01). There were occasional vacuoles (<1 μm) in the corneal epithelial basement membrane but no evidence of inflammation, edema, stromal neovascularization, fibrosis, acid-fast organisms, or structures suggestive of a fungal organism. SAXS analysis showed collagen fibril diameters and variation in size were greater in stroma containing the lesions compared to normal corneas (48.8 ± 4.5 nm vs 35.5 ± 2.6; P < .05). The d-spacing of collagen in the stroma of lesions and normal corneas was the same, but the average orientation index of collagen in lesions was greater (0.428 ± 0.08 vs 0.285 ± 0.03; P < .05). A survey revealed Florida spots lesions were static over time and became less obvious in only 1 of 6 affected cats adopted on St Kitts and taken to areas in the US where lesions are not reported. An anterior stromal collagen disorder with various degrees of epithelial hyperplasia is the pathologic hallmark of lesions clinically identical to Florida spots in cats from St Kitts.

Research progress of corneal epithelial basal cells and basement membrane

The cylinder cells at the bottom of corneal epithelial cells are basal cells. Their cytoplasm contains keratin intermediate filament which is important in secretion of basement membrane. Corneal epithelial dysfunction due to diabetes or ocular surgery is intimately related with basal cell abnormality. Corneal epithelial basement membrane is a highly specific extracellular matrix which is made up of lamina lucida and lamina densa. It plays an extremely important role in renewal and restoration. Many ocular abnormalities and diseases have been described to relate to the corneal epithelial basement membrane, such as traumatic recurrent corneal erosion, corneal dystrophy and keratoconus. (Chin J Ophthalmol, 2016, 52: 703-707).

Topical Mitomycin-C enhances subbasal nerve regeneration and reduces erosion frequency in the debridement wounded mouse cornea

Corneal epithelial basement membrane dystrophies and superficial injuries caused by scratches can lead to recurrent corneal erosion syndrome (RCES). Patients and animals with reduced corneal sensory nerve innervation can also develop recurrent erosions. Multiple wild-type mouse strains will spontaneously develop recurrent corneal erosions after single 1.5 mm debridement wounds. Here we show that this wound is accompanied by an increase in corneal epithelial cell proliferation after wound closure but without a commensurate increase in corneal epithelial thickness. We investigated whether excess corneal epithelial cell proliferation contributes to erosion formation. We found that topical application of Mitomycin C (MMC), a drug used clinically to improve healing after glaucoma and refractive surgery, reduces erosion frequency, enhances subbasal axon density to levels seen in unwounded corneas, and prevents excess epithelial cell proliferation after debridement wounding. These results suggest that topically applied MMC, which successfully reduces corneal haze and scarring after PRK, may also function to enhance subbasal nerve regeneration and epithelial adhesion when used to treat RCES.

Novel model of innate immunity in corneal infection.

The cornea functions as the major refractive interface for vision and protects the internal eye from insult. Current understanding of innate immune responses to corneal infection derives from a synthesis of in vitro and in vivo analyses. However, monolayer cell cultures and mouse models do not accurately duplicate all aspects of innate immunity in human patients. Here, we describe a three-dimensional culture system that incorporates human cells and extracellular matrix to more completely simulate the human cornea for studies of infection. Human corneal stromal fibroblasts were mixed with type I collagen in 3-μm pore size transwell inserts, and overlayed with Matrigel to simulate a human corneal stroma and epithelial basement membrane. These were then infected with a cornea-tropic adenovirus, and exposed on their inferior side to leukocytes derived from human peripheral blood. Subsequent analyses were performed with histology, confocal microscopy, ELISA, and fluorescence-activated cell sorting (FACS). CXCL8, a neutrophil chemokine shown previously as the first cytokine induced in infection of human corneal cells, increased upon adenovirus infection of facsimiles in a dose-responsive fashion. Myeloperoxidase-positive cells infiltrated infected corneal facsimiles in a sub-Matrigel location, possibly due to CXCL8 colocalization with heparan sulfate, a Matrigel constituent. Cellular infiltration was significantly inhibited by treatment with chemical inhibitors of p38 MAPK and Src kinase, both constituents of a signaling cascade previously suggested to regulate inflammation after adenovirus infection. FACS analysis determined that both virus and corneal fibroblasts were necessary for the induction of leukocyte migration into the facsimiles. The corneal facsimile, literally a cornea in a test tube, permits mechanistic studies on human tissue in a highly tractable system.

Epithelial basement membrane proteins perlecan and nidogen-2 are up-regulated in stromal cells after epithelial injury in human corneas

The epithelial basement membrane (BM) is a specialized extracellular matrix that has been shown to have a critical role in corneal development, wound healing, and disease. Although the epithelial BM contributes to corneal homeostasis, relatively little is know about non-epithelial production of its components that may be important in defective regeneration of the epithelial basement membrane associated with opacity after photorefractive keratectomy. The purpose of the current study was to investigate stromal production of corneal epithelial BM proteins in wounded human corneas using immunohistochemistry. A total of five unwounded control eyes and five 30-min epithelial-wounded corneas were obtained from fresh corneoscleral buttons removed from human eyes enucleated due to choroidal melanoma with normal anterior segments. In the wounded corneas, an eight mm patch of central corneal epithelium and epithelial BM was removed with a Beaver blade when the patient was under general anesthesia. Immunohistochemical analyses were performed to detect perlecan and nidogen-2 proteins–important components of the epithelial BM lamina lucida and lamina densa zones. Perlecan and nidogen-2 proteins were detected in the BM itself and at low levels in keratocytes in all unwounded corneas. After epithelial injury, both perlecan and nidogen-2 were expressed at high levels in stromal keratocytes, including superficial keratocytes in the early phases of apoptosis. Thus, after epithelial and epithelial BM injury, stromal keratocytes contribute important perlecan and nidogen-2 components to the regenerating epithelial BM.

Fourier-Domain Optical Coherence Tomography Imaging in Corneal Epithelial Basement Membrane Dystrophy: A Structural Analysis

To investigate the features of corneal epithelial basement membrane dystrophy using spectral-domain optical coherence tomography (SD OCT) and to examine the reliability of SD OCT in distinguishing epithelial basement membrane dystrophy from the normal cornea. Diagnostic test study. Forty-five individuals with epithelial basement membrane dystrophy and 45 age- and sex-matched controls with normal corneas were examined, and SD OCT scans of their corneas were performed. Invivo confocal microscopy was performed to confirm or rule out the diagnosis of epithelial basement membrane dystrophy. The structural corneal changes occurring in eyes with epithelial basement membrane dystrophy based on SD OCT findings were described. Epithelial abnormalities were observed in 86 of 87 eyes with epithelial basement membrane dystrophy (45 patients) on SD OCT scans. The 2 main features were the presence of an irregular and thickened epithelial basement membrane duplicating or insinuating into the corneal epithelium layer, or both, and the presence of hyperreflective dots. In some cases, we detected hyporeflective spaces between the corneal epithelial layer and the Bowman layer similar to a corneal epithelial detachment. This corneal epithelial detachment sometimes was associated with a cleavage with a stair-step appearance within the corneal epithelial layer. We found a perfect correlation between invivo confocal microscopy and SD OCT findings in the diagnosis of epithelial basement membrane dystrophy (κ= 0.98). SD OCT provides an accurate assessment of the structural changes occurring in eyes with epithelial basement membrane dystrophy. These changes, visible on SD OCT scans, are easily detectable and permit an accurate diagnosis, especially in patients with no biomicroscopically visible corneal changes.

The corneal epithelial basement membrane: Master regulator of the stromal myofibroblast response to injury

AbstractThe corneal epithelial basement membrane (EBM) is positioned between basal epithelial cells and the stroma. This highly specialized extracellular matrix functions not only to anchor epithelial cells to the stroma but also during migration, differentiation, and maintenance of the differentiated epithelial phenotype. Basement membranes are composed of a diverse assemblage of extracellular molecules, some of which are likely specific to the cornea. In general, they are composed of four primary components—collagens (types IV and VII), laminins, heparan sulfate proteoglycans, and nidogens. Our recent studies have demonstrated defective regeneration of the EBM in corneas that develop haze after high correction photorefractive keratectomy (PRK) in rabbits or irregular PTK in mice. Likely this is attributable to myofibroblast generation from bone marrow‐derived and keratocyte‐derived precursor cells that persist in the subepithelial stroma and block keratocyte/corneal fibroblast contribution of EBM components. In turn, persistent EBM defects facilitate the development and persistence of myofibroblasts via augmented TGF beta and PDGF penetration into the anterior stroma. Similar mechanisms underlie corneal scaring after most injuries.

Cytokine Deposition Alters Leukocyte Morphology and Initial Recruitment of Monocytes and γδT Cells After Corneal Injury

An in vivo mouse model reproducibly induces recurrent epithelial erosions in wild-type mice spontaneously 2 weeks after a single 1.5-mm corneal debridement wound made with a dulled blade. When 1.5-mm wounds are made by a rotating burr so that the corneal epithelial basement membrane is removed, corneas heal without developing erosions. Here, we characterize differences in cytokine deposition and changes in leukocytes between 0 and 6 hours after dulled-blade and rotating-burr wounding. BALB/c mice were used to study 1.5-mm corneal wounds made using a dulled blade or a rotating burr. Mice were studied immediately after wounding (0 hour) and at 6 hours in vivo and in vitro in organ culture. Corneas, corneal extracts, and collagenase digests from naïve and wounded mice were used for three-dimensional (3D) confocal imaging, cytokine arrays, and flow cytometry. Confocal imaging showed CD45, a protein derived from leukocytes, accumulates at the wound edge by 3 and 6 hours after wounding in vivo but not in vitro with more CD45 accumulating after dulled-blade compared with rotating-burr wounds. Morphologic changes occurred in CD45+ leukocytes and higher levels for several cytokines were detected in the stromal wound bed within minutes following dulled-blade wounds. Flow cytometry showed significantly more monocytes (CD45+/CD11b+/Ly6C+) and γδT cells (CD45+/GL3+) recruited into the corneas of mice with dulled-blade wounds by 6 hours. Differences in cytokine-driven leukocyte responses are seen after dulled-blade debridement compared with rotating-burr injury.

Diabetic keratopathy: new progresses and challenges

The incidence of diabetes mellitus and its complications, including diabetic keratopathy, was significantly elevated with the improved living standards in China recently. Diabetic keratopathy mainly exhibited several clinical features, including the delay of corneal epithelial regeneration, the decreasing of corneal sensitivity, neurotrophic corneal ulcers. The features were mainly caused by the changes of corneal epithelial basement membrane compositions, glycation products deposition, corneal nerve ending damage, reduced tear secretion and oxidative stress in the hyperglycemic conditions. This review focuses on the progress of molecular and cellular mechanism related with the clinical features, and discusses the main problems in the basic research of diabetic keratopathy.