Interconnections between diabetic corneal neuropathy and diabetic retinopathy:diagnostic and therapeutic implications

Diabetic corneal neuropathy and diabetic retinopathy are ocular complications occurring in the context of diabetes mellitus.Diabetic corneal neuropathy refers to the progressive damage of corneal nerves.Diabetic retinopathy has traditionally been considered as damage to the retinal microvasculature.However,growing evidence suggests that diabetic retinopathy is a complex neurovascular disorder resulting from dysfunction of the neurovascular unit,which includes both the retinal vascular structures and neural tissues.Diabetic retinopathy is one of the leading causes of blindness and is frequently screened for as part of diabetic ocular screening.However,diabetic corneal neuropathy is commonly overlooked and underdiagnosed,leading to severe ocular surface impairment.Several studies have found that these two conditions tend to occur together,and they share similarities in their pathogenesis pathways,being triggered by a status of chronic hyperglycemia.This review aims to discuss the interconnection between diabetic corneal neuropathy and diabetic retinopathy,whether diabetic corneal neuropathy precedes diabetic retinopathy,as well as the relation between the stage of diabetic retinopathy and the severity of corneal neuropathy.We also endeavor to explore the relevance of a corneal screening in diabetic eyes and the possibility of using corneal nerve measurements to monitor the progression of diabetic retinopathy.

Clinical associations of corneal neuromas with ocular surface diseases

Corneal neuromas,also termed microneuromas,refer to microscopic,irregula rly-shaped enlargements of terminal subbasal nerve endings at sites of nerve damage or injury.The formation of corneal neuromas results from damage to corneal nerves,such as following corneal pathology or corneal or intraocular surge ries.Initially,denervated areas of sensory nerve fibers become invaded by sprouts of intact sensory nerve fibers,and later injured axons regenerate and new sprouts called neuromas develop.In recent years,analysis of corneal nerve abnormalities including corneal neuromas which can be identified using in vivo confocal microscopy,a non-invasive imaging technique with microscopic resolution,has been used to evaluate corneal neuropathy and ocular surface dysfunction.Corneal neuromas have been shown to be associated with clinical symptoms of discomfort and dryness of eyes,and are a promising surrogate biomarker for ocular surface diseases,such as neuropathic corneal pain,dry eye disease,diabetic corneal neuropathy,neurotrophic keratopathy,Sjogren's syndrome,bullous keratopathy,post-refra ctive surgery,and others.In this review,we have summarized the current literature on the association between these ocular surface diseases and the presentation of corneal microneuromas,as well as elaborated on their pathogenesis,visualization via in vivo confocal microscopy,and utility in monitoring treatment efficacy.As current quantitative analysis on neuromas mainly relies on manual annotation and quantification,which is user-dependent and labor-intensive,future direction includes the development of artificial intelligence software to identify and quantify these potential imaging biomarkers in a more automated and sensitive manner,allowing it to be applied in clinical settings more efficiently.Combining imaging and molecular biomarkers may also help elucidate the associations between corneal neuromas and ocular surface diseases.

Characteristics of corneal aberration in patients with bilateral keratoconus and unilateral corneal Vogt’s striae

AIM:To assess the corneal high-order aberration(HOA)and its correlation with corneal morphological parameters in patients with bilateral keratoconus(KCN)and unilateral Vogt’s striae.METHODS:A total of 168 eyes of 84 patients with KCN,whose corneas had definite signs of unilateral Vogt’s striae,were enrolled.Corneal HOA and morphological parameters were measured using Pentacam HR.RESULTS:The corneal morphological parameters between KCN eyes with and without Vogt’s striae were evidently different(P<0.001).The 3rd coma 90°,4th spherical aberration,5th coma 90°,root-mean-square(RMS)(total),and RMS(HOA)in the front,back surfaces and total cornea in KCN eyes with Vogt’s striae were significantly higher than those in KCN eyes without Vogt’s striae(P<0.001).In KCN eyes with Vogt’s striae,the 3rd coma 90°and 4th spherical aberration in the front surface and total cornea were negatively correlated with flat keratometry value(K1),steep keratometry value(K2),mean keratometry value(Km),maximum keratometry value(Kmax),anterior corneal elevation(ACE),and posterior corneal elevation(PCE;P<0.05).The 3rd coma 90°,4th spherical aberration in back surface and RMS(total),RMS(HOA)in the front,back surfaces,total cornea were positively correlated with K1,K2,Km,Kmax,ACE,and PCE(P<0.05).CONCLUSION:Corneal HOA especially vertical coma and spherical aberration may increase when Vogt’s striae appeared in KCN eyes.The scale of increase is significantly related with changes in corneal shapes.

Thiel-Behnke Corneal Dystrophy in a Young Man in Denmark—A Case Report

Background: This case report presents a case of bilateral Thiel-Behnke corneal dystrophy in Denmark. Thiel-Behnke is an autosomal dominant inherited epithelial-stromal TGFBI dystrophy causing visual impairment. Methods and Results: This case study presents a 24-year-old Lithuanian man, with no previous ocular history, who had experienced slowly progressive visual impairment since his childhood. He was examined at the Department of Ophthalmology at Vejle Hospital and Aarhus University Hospital, where he was diagnosed with bilateral Thiel-Behnke corneal dystrophy. Histology confirmed the diagnosis. A lamellar corneal transplantation was performed in the right eye;however, due to epithelial growth under the corneal graft, it was later decided to redo the operation. Following the operations, the patient experienced a visual improvement in best corrected visual acuity (BCVA) from 0.1 (20/25 Snellen equivalent) to 0.3 (20/40 Snellen equivalent) in his right eye. Conclusions: This case of Thiel-Behnke corneal dystrophy is to our knowledge the first reported case in Denmark.

Surgical approaches to correct corneal astigmatism at time of cataract surgery: a mini-review

Among refractive errors,astigmatism is the most common optical aberration,where refraction changes in different meridians of the eye.It causes blurred vision at any distance and includes corneal,lenticular,and retinal astigmatism.Cataract surgery used to cause a progressive increase in the pre-exisiting corneal astigmatism because of creating a surgically induced astigmatism,for example,a large size surgery incision.The development of surgical techniques during last decades has made cataract surgery interchange to treat preoperative corneal astigmatism at time of surgery.Nowadays,three surgical approaches can be used.By placing a sutureless clear corneal incision on the steep meridian of the cornea,a preoperative corneal astigmatism less than 1.0 D can be corrected.Single or paired peripheral corneal relaxing incisions(PCRIs)provide 1.0-3.0 D corneal astigmatism correction.PCRIs are typically used for treating 1.0-1.5 D of regular corneal astigmatism,if more than 2.0 D,the risk of overcorrection and irregular astigmatism is increased.When toric intraocular lenses(IOLs)are unavailable in markets,PCRIs are still a reasonable option for patients with up to 3.0 D of pre-existing corneal astigmatism.Toric IOLs implantation can correct 1.0-4.5 D of corneal astigmatism.Several IOLs are approved to correct a high degree of corneal astigmatism with cylinder power up to 12.0 D.These approaches can be used alone or in combination.

Acellular porcine corneal matrix as a carrier scaffold for cultivating human corneal epithelial cells and fibroblasts in vitro

AIM：To investigate the feasibility of corneal anterior lamellar reconstruction with human corneal epithelial cells and fibroblasts,and an acellular porcine cornea matrix（APCM） in vitro.·METHODS：The scaffold was prepared from fresh porcine corneas which were treated with 0.5%sodium dodecyl sulfate（SDS）solution and the complete removal of corneal cells was confirmed by hematoxylin-eosin（HE）staining and 4’,6-diamidino-2-phenylindole（DAPI）staining.Human corneal fibroblasts and epithelial cells were cultured with leaching liquid extracted from APCM,and then cell proliferative ability was evaluated by MTT assay.To construct a human corneal anterior lamellar replacement,corneal fibroblasts were injected into the APCM and cultured for 3d,followed by culturing corneal epithelial cells on the stroma construction surface for another 10d.The corneal replacement was analyzed by HE staining,and immunofluorescence staining.·R ESULTS：Histological examination indicated that there were no cells in the APCM by HE staining,and DAPI staining did not detect any residual DNA.The leaching liquid from APCM had little influence on the proliferation ability of human corneal fibroblasts and epithelial cells.At 10d,a continuous 3 to 5 layers of human corneal epithelial cells covering the surface of the APCM was observed,and the injected corneal fibroblasts distributed within the scaffold.The phenotype of the construction was similar to normal human corneas,with high expression of cytokeratin 12 in the epithelial cell layer and high expression of Vimentin in the stroma.·CONCLUSION：Corneal anterior lamellar replacement can be reconstructed in vitro by cultivating human corneal epithelial cells and fibroblasts with an acellular porcine cornea matrix.This laid the foundation for the further transplantation in vitro.

Diabetic corneal neuropathy as a surrogate marker for diabetic peripheral neuropathy

Diabetic neuropathy is a prevalent microvascular complication of diabetes mellitus,affecting nerves in all parts of the body including corneal nerves and peripheral nervous system,leading to diabetic corneal neuropathy and diabetic peripheral neuropathy,respectively.Diabetic peripheral neuropathy is diagnosed in clinical practice using electrophysiological nerve conduction studies,clinical scoring,and skin biopsies.However,these diagnostic methods have limited sensitivity in detecting small-fiber disease,hence they do not accurately reflect the status of diabetic neuropathy.More recently,analysis of alterations in the corneal nerves has emerged as a promising surrogate marker for diabetic peripheral neuropathy.In this review,we will discuss the relationship between diabetic corneal neuropathy and diabetic peripheral neuropathy,elaborating on the foundational aspects of each:pathogenesis,clinical presentation,evaluation,and management.We will further discuss the relevance of diabetic corneal neuropathy in detecting the presence of diabetic peripheral neuropathy,particularly early diabetic peripheral neuropathy;the correlation between the severity of diabetic corneal neuropathy and that of diabetic peripheral neuropathy;and the role of diabetic corneal neuropathy in the stratification of complications of diabetic peripheral neuropathy.

Corneal stromal mesenchymal stem cells: reconstructing a bioactive cornea and repairing the corneal limbus and stromal microenvironment

Corneal stroma-derived mesenchymal stem cells(CS-MSCs) are mainly distributed in the anterior part of the corneal stroma near the corneal limbal stem cells(LSCs). CS-MSCs are stem cells with self-renewal and multidirectional differentiation potential. A large amount of data confirmed that CS-MSCs can be induced to differentiate into functional keratocytes in vitro, which is the motive force for maintaining corneal transparency and producing a normal corneal stroma. CS-MSCs are also an important component of the limbal microenvironment. Furthermore, they are of great significance in the reconstruction of ocular surface tissue and tissue engineering for active biocornea construction. In this paper, the localization and biological characteristics of CS-MSCs, the use of CS-MSCs to reconstruct a tissue-engineered active biocornea, and the repair of the limbal and matrix microenvironment by CS-MSCs are reviewed, and their application prospects are discussed.

Anti-inflammatory potential of human corneal stroma-derived stem cells determined by a novel in vitro corneal epithelial injury model

BACKGROUND An in vitro injury model mimicking a corneal surface injury was optimised using human corneal epithelial cells(hCEC).AIM To investigate whether corneal-stroma derived stem cells(CSSC) seeded on an amniotic membrane(AM) construct manifests an anti-inflammatory, healing response.METHODS Treatment of hCEC with ethanol and pro-inflammatory cytokines were compared in terms of viability loss, cytotoxicity, and pro-inflammatory cytokine release, in order to generate the in vitro injury. This resulted in an optimal injury of 20%(v/v) ethanol for 30 s with 1 ng/mL interleukin-1(IL-1) beta. Co-culture experiments were performed with CSSC alone and with CSSC-AM constructs.The effect of injury and co-culture on viability, cytotoxicity, IL-6 and IL-8 production, and IL1 B, TNF, IL6, and CXCL8 mRNA expression were assessed.RESULTS Co-culture with CSSC inhibited loss of hCEC viability caused by injury. Enzyme linked immunosorbent assay and polymerase chain reaction showed a significant reduction in the production of IL-6 and IL-8 pro-inflammatory cytokines, and reduction in pro-inflammatory cytokine mRNA expression during co-culture with CSSC alone and with the AM construct. These results confirmed the therapeutic potential of the CSSC and the possible use of AM as a cell carrier for application to the ocular surface.CONCLUSION CSSC were shown to have a potentially therapeutic anti-inflammatory effectwhen treating injured hCEC, demonstrating an important role in corneal regeneration and wound healing, leading to an improved knowledge of their potential use for research and therapeutic purposes.

Research progress on animal models of corneal epithelial-stromal injury

A corneal epithelial-stromal defect is recognized as a major contributor to corneal scarring.Given the rising prevalence of blindness caused by corneal scarring,increasing attention has been focused on corneal epithelialstromal defects.Currently,the etiology and pathogenesis of these defects remain inadequately understood,necessitating further investigation through experimental research.Various modeling methods exist both domestically and internationally,each with distinct adaptive conditions,advantages,and disadvantages.This review primarily aims to summarize the techniques used to establish optimal animal models of corneal epithelial-stromal injury,including mechanical modeling,chemical alkali burns,post-refractive surgery infections,and genetic engineering.The intention is to provide valuable insights for studying the mechanisms underlying corneal epithelial-stromal injury and the development of corresponding therapeutic interventions.

The Limbal Niche and Its Role in Maintaining Corneal Regeneration

In recent years, stem cells have been a focal point in research designed to evaluate the efficacy of ophthalmologic therapies, specifically those for corneal conditions. The corneal epithelium is one of the few regions of the body that maintains itself using a residual stem cell population within the adjacent limbus. Stem cell movement has additionally captivated the minds of researchers due to its potential application in different body regions. The cornea is a viable model for varying methods to track stem cell migratory patterns, such as lineage tracing and live imaging from the limbus. These developments have the potential to pave the way for future therapies designed to ensure the continuous regeneration of the corneal epithelium following injury via the limbal stem cell niche. This literature review aims to analyze the various methods of imaging used to understand the limbal stem cell niche and possible future directions that might be useful to consider for the better treatment and prevention of disorders of the cornea and corneal epithelium. .

Preliminary studies of constructing a tissue-engineered lamellar corneal graft by culturing mesenchymal stem cells onto decellularized corneal matrix

AIM:To construct a competent corneal lamellar substitute in order to alleviate the shortage of human corneal donor.METHODS:Rabbit mesenchymal stem cells(MSCs)were isolated from bone marrow and identified by flow cytometric,osteogenic and adipogenic induction.Xenogenic decellularized corneal matrix(XDCM)was generated from dog corneas.MSCs were seeded and cultured on XDCM to construct the tissueengineered cornea.Post-transplantation biocompatibility of engineered corneal graft were tested by animal experiment.Rabbits were divided into two groups then underwent lamellar keratoplasty(LK)with different corneal grafts:1)XDCM group(n=5):XDCM;2)XDCM-MSCs groups(n=4):tissue-engineered cornea made up with XDCM and MSCs.The ocular surface recovery procedure was observed while corneal transparency,neovascularization and epithelium defection were measured and compared.In vivo on focal exam was performed 3 mo postoperatively.RESULTS:Rabbit MSCs were isolated and identified.Flow cytometry demonstrated isolated cells were CD90 positive and CD34,CD45 negative.Osteogenic and adipogenic induction verified their multipotent abilities.MSC-XDCM grafts were constructed and observed.In vivo transplantation showed the neovascularization in XDCMMSC group was much less than that in XDCM group postoperatively.Post-transplant 3-month confocal test showed less nerve regeneration and bigger cell-absent area in XDCM-MSC group.CONCLUSION:This study present a novel corneal tissue-engineered graft that could reduce post-operatively neovascularization and remain transparency,meanwhile shows that co-transplantation of MSCs may help increase corneal transplantation successful rate and enlarge the source range of corneal substitute to overcome cornea donor shortage.

Corneal neurotization: a narrative review of techniques, outcomes, and surgical considerations

Background and Objective:Corneal neurotization is a novel surgical technique used to restore corneal sensation in patients with neurotrophic keratopathy.Neurotrophic keratopathy is a disorder characterized by dysfunction of the ophthalmic division of the trigeminal nerve,which provides sensory innervation to the cornea.Without sensation,the cornea is at risk of infection,ulceration,perforation,and ultimately,vision loss.Corneal neurotization has emerged as an innovative technique to reinnervate anesthetized corneas by transferring a healthy donor nerve to the affected eye around the corneoscleral limbus.As the field of corneal neurotization rapidly grows,there is a need to synthesize the existing body of literature on corneal neurotization and identify important areas for further research.In this review,we will discuss neurotrophic keratopathy and its current management strategies,followed by an overview of corneal neurotization techniques,outcomes,surgical considerations,and future directions.Methods:PubMed and Google Scholar searches were conducted to retrieve and analyze relevant original papers and reviews on neurotrophic keratopathy and corneal neurotization up until April 2022.Key Content and Findings:Currently,numerous techniques for corneal neurotization exist,including direct nerve transfers,as well as indirect neurotization via interposition nerve grafts.So far,corneal neurotization has been shown to be highly successful in restoring corneal sensation,improving visual acuity,and improving corneal epithelial health.To date,there have been no significant differences in outcomes between direct versus indirect neurotization techniques,different donor nerves,or autologous versus allogeneic interposition grafts.However,there is some evidence that corneal neurotization procedures may be more successful in pediatric patients.Conclusions:Corneal neurotization shows great promise in treating neurotrophic corneas and represents the first management option to date that addresses the underlying pathophysiological mechanism of neurotrophic keratopathy by restoring corneal sensation.As the use of corneal neurotization continues to broaden,additional studies will become important to compare techniques in a systematic manner,with larger sample sizes,as well as standardized outcome measures and follow-up time.

AB027.Varying pattern of proteases secretion in Fuchs corneal endothelial dystrophy

Background:The goal of this project was to analyze the relationship between cell morphology and proteases/proteases inhibitors(PIs)secretion profile in fuchs endothelial corneal dystrophy(FECD)corneal endothelial cells(CECs).Methods:Cell morphology was determined using a circularity index(4π×area/perimeter2)for each CECs population extracted from surgical FECD specimens(N=2)and healthy Eye bank corneas(N=3).CECs were cultured 28 days post-confluency.Supernatant was collected and analysed using Proteome Profiler Array detecting 35 proteases and 32 PIs(R&D Systems).Proteome signal was analyzed using Image Studio Lite and correlated with the population’s circularity index.Results:Calculation of circularity index reported different morphologies among FECD populations(0.59±0.18 and 0.64±0.17)and healthy populations(0.44±0.18,0.66±0.13 and 0.71±0.11).Proteome arrays revealed the presence of 10 proteases(ADAMTS1,Cathepsin A,B,D,and X/Z/P,DPPIV/CD26,MMP-2,3 and 12,uPA/Urokinase)and 10 PIs(Protease Nexin II,Cystatin B and C,EMMPRIN/CD147,Latexin,Lipocalin-1,Serpin E1,TFPI,TFPI-2,TIMP-1,2 and 4).Healthy and FECD specimens showed similar variation patterns according to morphology for secretion of ADAMTS1,MMP-3 and 12.However,opposing patterns between healthy and FECD populations were observed for Cathepsin B and D.Moreover,some proteins did not show variation according to phenotype in healthy CECs,but did in FECD CECs:Cathepsin A,Cystatin C,TFPI-2 and total TIMPs.For the other proteins,secretion did not vary according to morphology or no specific pattern was distinguishable.Conclusions:To conclude,our results suggest that cell phenotype is linked to the secretion of certain proteases/PIs in both groups.However,there seems to be differences in secretion of particular proteases and PIs between FECD and healthy specimens as morphology did not have a similar influence.These differences might initiate an imbalance between proteases and PIs explaining the irregular thickening of the Descemet membrane seen in FECD.

Evaluation of corneal cell growth on tissue engineering materials as artificial cornea scaffolds

The keratoprosthesis(KPro;artificial cornea)is a special refractive device to replace human cornea by using heterogeneous forming materials for the implantation into the damaged eyes in order to obtain a certain vision.The main problems of artificial cornea are the biocompatibility and stability of the tissue particularly in penetrating keratoplasty.The current studies of tissue-engineered scaffold materials through comprising composites of natural and synthetic biopolymers together have developed a new way to artificial cornea.Although a wide agreement that the long-term stability of these devices would be greatly improved by the presence of cornea cells,modification of keratoprosthesis to support cornea cells remains elusive.Most of the studies on corneal substrate materials and surface modification of composites have tried to improve the growth and biocompatibility of cornea cells which can not only reduce the stimulus of heterogeneous materials,but also more importantly continuous and stable cornea cells can prevent the destruction of collagenase.The necrosis of stroma and spontaneous extrusion of the device,allow for maintenance of a precorneal tear layer,and play the role of ensuring a good optical surface and resisting bacterial infection.As a result,improvement in corneal cells has been the main aim of several recent investigations;some effort has focused on biomaterial for its well biological properties such as promoting the growth of cornea cells.The purpose of this review is to summary the growth status of the corneal cells after the implantation of several artificial corneas.

Evaluating alternative stem cell hypotheses for adult corneal epithelial maintenance

In this review we evaluate evidence for three different hypotheses that explain how the corneal epithelium is maintained. The limbal epithelial stem cell(LESC)hypothesis is most widely accepted. This proposes that stem cells in the basal layer of the limbal epithelium, at the periphery of the cornea, maintain themselves and also produce transient(or transit) amplifying cells(TACs). TACs then move centripetally to the centre of the cornea in the basal layer of the corneal epithelium and also replenish cells in the overlying suprabasal layers. The LESCs maintain the corneal epithelium during normal homeostasis and become more active to repair significant wounds. Second, the corneal epithelial stem cell(CESC) hypothesis postulates that, during normal homeostasis, stem cells distributed throughout the basal corneal epithelium, maintain the tissue. According to this hypothesis, LESCs are present in the limbus but are only active during wound healing. We also consider a third possibility, that the corneal epithelium is maintained during normal homeostasis by proliferation of basal corneal epithelial cells without any input from stem cells. After reviewing the published evidence, we conclude that the LESC and CESC hypotheses are consistent with more of the evidence than the third hypothesis, so we do not consider this further. The LESC and CESC hypotheses each have difficulty accounting for one main type of evidence so we evaluate the two key lines of evidence that discriminate between them. Finally, we discuss how lineage-tracing experiments have begun to resolve the debate in favour of the LESC hypothesis. Nevertheless, it also seems likely that some basal corneal epithelial cells can act as long-term progenitors if limbal stem cell function is compromised. Thus, this aspect of the CESC hypothesis may have a lasting impact on our understanding of corneal epithelial maintenance, even if it is eventually shown that stem cells are restricted to the limbus as proposed by the LESC hypothesis.

Acellular ostrich corneal stroma used as scaffold for construction of tissue-engineered cornea

AIM： To assess acellular ostrich corneal matrix used as a scaffold to reconstruct a damaged cornea. METHODS： A hypertonic saline solution combined with a digestion method was used to decellularize the ostrich cornea. The microstructure of the acellular corneal matrix was observed by transmission electron microscopy （TEM） and hematoxylin and eosin （H＆E） staining. The mechanical properties were detected by a rheometer and a tension machine. The acellular corneal matrix was also transplanted into a rabbit cornea and cytokeratin 3 was used to check the immune phenotype, RESULTS： The microstructure and mechanical properties of the ostrich cornea were well preserved after the decellularization process, in vitro, the methyl thiazolyl tetrazoUum results revealed that extracts of the acellular ostrich corneas （AOCs） had no inhibitory effects on the proliferation of the corneal epithelial or endothelial cells or on the keratocytes, The rabbit lamellar keratoplasty showed that the transplanted AOCs were transparent and completely incorporated into the host cornea while corneal turbidity and graft dissolution occurred in the acellular porcine cornea （APC） transplantation, The phenotype of the reconstructed cornea was similar to a normal rabbit cornea with a high expression of cytokeratin 3 in the superficial epithelial cell layer, CONCLUSION： We first used AOCs as scaffolds to reconstruct damaged corneas. Compared with porcine corneas, the anatomical structures of ostrich corneas are closer to those of human corneas. In accordance with the principle that structure determines function, a xenograft lamellar keratoplasty also confirmed that the AOC transplantation generated a superior outcome compared to that of the APC graft.

Comparison of the inhibitory effect of different doses of subconjunctival bevacizumab application in an experimental model of corneal neovascularization

AIM： To evaluate the inhibitory effect of subconjunctival bevacizumab as single-and multiple-dose application, and compare their effects on corneal neovascularization in a rat model. METHODS： Thirty adult Sprague-Dawley rats were used in this experimental study. The central cornea of the rats was cauterized chemically. The rats were randomly enrolled into three groups. All groups received subconjunctival injections. In Group 1（control group, n=10）, 0.05 m L 0.9% Na Cl solution was injected on the first day. In Group 2（single-dose group, n=10）, 0.05 m L bevacizumab（1.25 mg） was injected on the first day. In Group 3（multiple-dose group, n=10）, four doses of 0.05 m L bevacizumab（1.25 mg） were injected on the first, third, fifth and seventh day. Slit-lamp examination of all rats was performed at the third and ninth day. Digital images of the corneas were taken and analyzed using image analysis software to calculate corneal neovascularization area. All rats were sacrificed on the tenth day. In corneal sections, the number of blood vessels, state of inflammation and collagen formation was evaluated histopathologically. RESULTS： In Group 3, corneal edema grades were significantly lower than Group 1 and Group 2（P=0.02, and P=0.035, respectively）. The mean percentage of neovascularized corneal area in Group 3 was significantly lower than Group 2（P=0.005）. On histopathological examination, Group 2 and Group 3 showed significantly less number of blood vessels than Group 1（P=0.005, and P=0.001, respectively）. Additionally, Group 3 showed significantly less number of blood vessels compared to Group 2（P=0.019）. Inflammation and edema grades were significantly lower in Group 3 compared to Group 1（P=0.001）. CONCLUSION： Subconjunctival bevacizumab injection is effective in inhibition of newly formed corneal neovascularization. The multiple-dose bevacizumab treatment seems to be more effective compared to single-dose treatment.

Corneal stem cells and tissue engineering: Current advances and future perspectives

Major advances are currently being made in regenerative medicine for cornea. Stem cell-based therapies represent a novel strategy that may substitute conventional corneal transplantation, albeit there aremany challenges ahead given the singularities of each cellular layer of the cornea. This review recapitulates the current data on corneal epithelial stem cells, corneal stromal stem cells and corneal endothelial cell progenitors. Corneal limbal autografts containing epithelial stem cells have been transplanted in humans for more than 20 years with great successful rates, and researchers now focus on ex vivo cultures and other cell lineages to transplant to the ocular surface. A small population of cells in the corneal endothelium was recently reported to have self-renewal capacity, although they do not proliferate in vivo. Two main obstacles have hindered endothelial cell transplantation to date: culture protocols and cell delivery methods to the posterior cornea in vivo. Human corneal stromal stem cells have been identified shortly after the recognition of precursors of endothelial cells. Stromal stem cells may have the potential to provide a direct cell-based therapeutic approach when injected to corneal scars. Furthermore, they exhibit the ability to deposit organized connective tissue in vitro and may be useful in corneal stroma engineering in the future. Recent advances and future perspectives in the field are discussed.

A short-term study of corneal collagen cross-linking with hypo-osmolar riboflavin solution in keratoconic corneas

AIM: To report the 3mo outcomes of collagen crosslinking(CXL) with a hypo-osmolar riboflavin in thin corneas with the thinnest thickness less than 400 μm without epithelium.METHODS: Eight eyes in 6 patients with age 26.2±4.8y were included in the study. All patients underwent CXL using a hypo-osmolar riboflavin solution after its de-epithelization. Best corrected visual acuity, manifest refraction, the thinnest corneal thickness, and endothelial cell density were evaluated before and 3mo after the procedure.RESULTS: The mean thinnest thickness of the cornea was 408.5 ±29.0 μm before treatment and reduced to369.8 ±24.8 μm after the removal of epithelium. With the application of the hypo-osmolar riboflavin solution, the thickness increased to 445.0 ±26.5 μm before CXL and recover to 412.5 ±22.7 μm at 3mo after treatment, P =0.659). Before surgery, the mean K-value of the apex of the keratoconus corneas was 57.6 ±4.0 diopters, and slightly decreased(54.7±4.9 diopters) after surgery(P =0.085). Mean best-corrected visual acuity was 0.55 ±0.23 logarithm of the minimal angle of resolution, and increased to 0.53±0.26 logarithm after surgery(P =0.879).The endothelial cell density was 2706.4 ±201.6 cells/mm2 before treatment, and slightly decreased( 2641. 2 ±218.2 cells/mm2) at last fellow up(P =0.002).CONCLUSION: Corneal collagen cross-linking with a hypo-osmolar riboflavin in thin corneas seems to be a promising treatment. Further study should be done to evaluate the safety and efficiency of CXL in thin corneas for the long-term.