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. .

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.

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.

Inhibition of EGFR attenuates EGF-induced activation of retinal pigment epithelium cell via EGFR/AKT signaling pathway

AIM:To explore the effect of epidermal growth factor receptor(EGFR)inhibition by erlotinib and EGFR siRNA on epidermal growth factor(EGF)-induced activation of retinal pigment epithelium(RPE)cells.METHODS:Human RPE cell line(ARPE-19 cells)was activated by 100 ng/mL EGF.Erlotinib and EGFR siRNA were used to intervene EGF treatment.Cellular viability,proliferation,and migration were detected by methyl thiazolyl tetrazolium(MTT)assay,bromodeoxyuridine(BrdU)staining assay and wound healing assay,respectively.EGFR/protein kinase B(AKT)pathway proteins and N-cadherin,α-smooth muscle actin(α-SMA),and vimentin were tested by Western blot assay.EGFR was also determined by immunofluorescence staining.RESULTS:EGF treatment for 24h induced a significant increase of ARPE-19 cells’viability,proliferation and migration,phosphorylation of EGFR/AKT proteins,and decreased total EGFR expression.Erlotinib suppressed ARPE-19 cells’viability,proliferation and migration through down regulating total EGFR and AKT protein expressions.Erlotinib also inhibited EGF-induced an increase of proliferative and migrative ability in ARPE-19 cells and clearly suppressed EGF-induced EGFR/AKT proteins phosphorylation and decreased expression of N-cadherin,α-SMA,and vimentin proteins.Similarly,EGFR inhibition by EGFR siRNA significantly affected EGF-induced an increase of cell proliferation,viability,and migration,phosphorylation of EGFR/AKT proteins,and up-regulation of N-cadherin,α-SMA,and vimentin proteins.CONCLUSION:Erlotinib and EGFR-knockdown suppress EGF-induced cell viability,proliferation,and migration via EGFR/AKT pathway in RPE cells.EGFR inhibition may be a possible therapeutic approach for proliferative vitreoretinopathy(PVR).

Amniotic membrane covering promotes healing of cornea epithelium and improves visual acuity after debridement for fungal keratitis

AIM:To investigate the effect of amniotic membrane covering(AMC) on the healing of cornea epithelium and visual acuity for fungal keratitis after debridement.METHODS:Twenty fungal keratitis patients were divided into two groups randomly, the AMC group and the control group, ten patients each group. Both debridement of the infected cornea tissue and standard anti-fungus drugs treatments were given to every patients, monolayer amniotic membrane were sutured to the surface of the entire cornea and bulbar conjunctiva with 10-0 nylon suture for patients in the AMC group.The diameter of the ulcer was determined with slit lamp microscope and the depth of the infiltration was determined with anterior segment optical coherence tomography. Uncorrected visual acuity(UCVA) was tested before surgery and three month after healing of the epithelial layer. The healing time of the cornea epithelium, visual acuity(VA) was compared between the two groups using t- test.RESULTS:There was no statistical difference of the diameter of the ulcer, depth of the infiltration, height of the hypopyon and VA between the two groups beforesurgery(P 】0.05). The average healing time of the AMC group was 6.89 ±2.98 d, which was statistically shorter than that of the control group(10.23±2.78d)(P 【0.05).The average UCVA of the AMC group was 0.138 ±0.083,which was statistically better than that of the control group(0.053±0.068)(P 【0.05).CONCLUSION:AMC surgery could promote healing of cornea epithelium after debridement for fungal keratitis and lead to better VA outcome.

Regulation of interleukin 33/ST2 signaling of human corneal epithelium in allergic diseases

AIM:To identify the function of ST2 and explore the role of IL-33/ST2 signaling in regulating the pro-allergic cytokine production in human corneal epithelial cells (HCECs). METHODS:Human corneal tissues and cultured primary HCECs were treated with IL-33 in different concentrations without or with different inhibitors to evaluate the expression, location and signaling pathways of ST2 in regulating production of pro-allergic cytokine and chemokine. The expression of mRNA was determined by reverse transcription and real time PCR, and protein production was measured by enzyme-linked immunosorbent assay (ELISA), immunohistochemical and immunofluorescent staining. ST2 protein was detected in donor corneal epithelium, and ST2 signal was enhanced by exposure to IL-33. ·RESULTS:IL-33 significantly stimulated production of pro-allergic cytokines thymic stromal lymphopoietin (TSLP) and chemokine (CCL2, CCL20, CCL22) in HCECs at both mRNA and protein levels. These stimulated productions of pro-allergic mediators by IL-33 were blocked by ST2 antibody or soluble ST2 protein(P 【0.05). Interestingly, the IκB-α inhibitor BAY11-7082 or NF-κB activation inhibitor quinazoline blocked NF-κB p65 protein nuclear translocation, and also suppressed the productions of these pro-allergic cytokines and chemokine induced by IL-33. CONCLUSION:These findings demonstrate that IL-33/ ST2 signaling plays an important role in regulating IL-33 induced pro-allergic responses. IL-33 and ST2 could become novel molecular targets for the intervention ofallergic diseases in ocular surface.

Expression of S100B during the innate immune of corneal epithelium against fungi invasion

AIM： To explore the expression of SIOOB in corneal epithelial cells under ,Aspergillus stimulation both in vivo and in vitro. METHODS： Immortalized human corneal epithelial cells （HCECs） were exposed to inactive #lsperg///us fumigatus （A. fumigatus） conidia at 0, 4, 8, 12, 16, and 24h respectively. The corneas of Wistar rats were exposed to active A. fumigatus at 0, 12, 24, 48h and the normal rat corneas were used for normal control. The mRNA level of S100B was evaluated by real time quantitative reverse transcription-polymerase chain reaction （qRT-PCR）. Sl00B protein expression in cornea epithelium was detected by immunohistochemical/immunocytochemical staining （IHC/ICC）. RESULTS： Histopathology revealed a significant inflammatory cell infiltration in fungal keratitis human and rat cornea. Corneal epithelial cells didn＇t express or rarely express S100B at baseline. A. fumigatus significantly induced S100B mRNA expression in cultured corneal epithelial cells in a time depended manner in vitro the mRNA began to rise significantly at 8h in vitro （P〈0.05） and continue to rise as time prolonged （P〈0.01）. in vivo S100B mRNA level was low in the normal corneas. However, it was increased in keratitis corneas from 12h after infection （P〈0.05） and reached to a peak at 24h （P〈0.001）. Immunochemistry revealed an obvious staining in fungal keratitis corneas as well as immortalized HCECs compared to the normal ones respectively, indicating an increased expression of SlOOB protein. CONCLUSION： S100B exists in corneal epithelial cells and is over-expressed under A. fumigatus stimulation. Sl00B may play an important role in the innate immune response of the corneal epithelium during A. fumigatus infection.

Netrin-1 promotes epithelium repair in corneal injury

●AIM:To explore netrin-1 functions on corneal epithelium in vitro and in vivo.●METHODS:In vitro the human corneal epithelial(HCE)cells were treated with serum free DMEM-F12 basic media containing 0,50,100,200,300,500,800,and 1000 ng/mL of netrin-1,respectively.The cells viability was detected by cell counting kit-8(CCK-8).The wound-healing assay was applied to assess the migration proficiency of HCE cells.Flow cytometry was used to analyze the cell-cycle distribution and apoptosis.In vivo,normal c57(6 wk)mice were demarcated with a trephine in the middle of the cornea to produce a 3-mm circular wound.Mice corneas were inflicted no epithelium with a 3-mm wound displayed,but remained the limbal epithelium intact.A blunt scalpel blade was used to remove the corneal epithelian cells,followed by topical netrin-1 application(200 ng/mL),and the group treated by PBS as control.The treated group was injected netrin-1 into the normal c57 mice inferior subconjunctival 4 h before trauma.Mouse corneal inflammation and neovascularization were observed under slit lamp microscope.The apoptosis of corneal cells was determined by TUNEL staining.●RESLUTS:A concentration of 200 ng/mL netrin-1 enhanced 25%of the HCE viability.The relative migration rates were 76.3%and 100%in control and netrin-1 treated group after cultured 72 h.Treated with netrin-1(200 ng/mL)decreased the apoptosis of HCE cells,as well as decreased their percentage from 19.3%±0.57%to 12.7%±0.42%of the total.The remaining wound area was 1.22 mm2 in control group but 0.22 mm2 in the netrin-1 treated group.Exogenous Netrin-1 inhibits apoptosis of corneal epithelial cells of c57 mice.TUNEL-positive cells at the epithelial layer of the corneas of the control and netrin-1 treated c57 mice at 24 h after wounding were 43.3%and 16.7%respectively.●CONCLUSION:Netrin-1 can reduce HCE apoptosis as well as promote its proliferation and migration.Topical application of netrin-1 promotes the injuryed cornea epithelial wound repair and inhibits apoptosis of corneal epithelial cells.These findings may offer potential therapies to repair the defects of corneal epithelial based on netrin-1.

Transplantation of tissue-engineered human corneal epithelium in limbal stem cell deficiency rabbit models

AIM: To evaluate the biological functions of tissue-engineered human corneal epithelium (TE-HCEP) by corneal transplantation in limbal stem cell deficiency (LSCD) rabbit models. METHODS: TE-HCEPs were reconstructed with DiI-labeled untransfected HCEP cells and denuded amniotic membrane (dAM) in air-liquid interface culture, and their morphology and structure were characterized by hematoxylin-eosin (HE) staining of paraffin-sections, immunohistochemistry and electron microscopy. LSCD models were established by mechanical and alcohol treatment of the left eyes of New Zealand white rabbits, and their eyes were transplanted with TE-HCEPs with dAM surface outside by lamellar keratoplasty (LKP). Corneal transparency, neovascularization, thickness, and epithelial integrality of both traumatic and post transplantation eyes were checked once a week by slit-lamp corneal microscopy, a corneal pachymeter, and periodic acid-Schiff (PAS) staining. At day 120 post surgery, the rabbits in each group were sacrificed and their corneas were examined by DiI label observation, HE staining, immunohistochemistry and electron microscopy. RESULTS: After cultured for 5 days on dAM, HCEP cells, maintaining keratin 3 expression, reconstructed a 6-7 layer TE-HCEP with normal morphology and structure. The traumatic rabbit corneas, entirely opaque, conjunctivalized and with invaded blood vessels, were used as LSCD models for TE-HCEP transplantation. After transplantation, obvious edema was not found in TE-HCEP-transplanted corneas which became more and more transparent, the invaded blood vessels reduced gradually throughout the monitoring period. The corneas decreased to normal thickness on day 25, while those of dAM eyes were over 575 mu m in thickness during the monitoring period. A 45 layer of epithelium consisting of TE-HCEP originated cells attached tightly to the anterior surface of stroma was reconstructed 120 days after TE-HCEP transplantation, which was similar to the normal control eye in morphology and structure. In contrast, intense corneal edema, turbid, invaded blood vessels were found in dAM eyes, and no multilayer epithelium was found but only a few scattered conjunctiva-like cells appeared. CONCLUSION: The TE-HCEP, with similar morphology and structure to those of innate HCEP, could reconstruct a multilayer corneal epithelium with normal functions in restoring corneal transparency and thickness of LSCD rabbits after transplantation. It may be a promising HCEP equivalent for clinical therapy of corneal epithelial disorders.

In vitro reconstruction and characterization of tissue-engineered human corneal epithelium with seeder cells from an untransfected human corneal epithelial cell line

AIM: To demonstrate the morphology and structure of in vitro reconstructed tissue-engineered human corneal epithelium (TE-HCEP) with seeder cells from an untransfected HCEP cell line. METHODS: The TE-HCEPs were reconstructed in vitro with seeder cells from an untransfected HCEP cell line, and scaffold carriers of denuded amniotic membrane (dAM) in air-liquid interface culture for 3, 5, 7 and 9 days, respectively. The specimens were examined with hematoxylin-eosin (HE) staining of paraffin-section, immunocytochemical staining, scanning and transmission electron microscopy. RESULTS: During in vitro reconstruction of TE-HCEP, HCEP cells formed a 3-4, 6-7 and 8-10 layers of an HCEP-like structure on dAMs in air-liquid interface culture for 3, 5 and 7 days, respectively. But the cells deceased to 5-6 layers and the structure of straified epithelium became loose at day 9. And the cells maintained positive expression of marker proteins (keratin 3 and keratin 12), cell-junction proteins (zonula occludens-1, E-cadherin, connexin 43 and integrin beta 1) and membrane transport protein of Na+-K+ ATPase. The HCEP cells in TE-HCEP were rich in microvilli on apical surface and established numerous cell-cell and cell-dAM junctions at day 5. CONCLUSION: The morphology and structure of the reconstructed TE-HCEP were similar to those of HCEP in vivo. The HCEP cells in the reconstructed TE-HCEP maintained the properties of HCEP cells, including abilities of forming intercellular and cell-extracellular matrix junctions and abilities of performing membrane transportation. The untransfected HCEP cells and dAMs could promisingly be used in reconstruction HCEP equivalent for clinical corneal epithelium transplantation.

Evaluation of artificial tears on cornea epithelium healing

AIM： To observe the efficacy of different artificial eye drops on corneal epithelium healing in rabbit. METHODS： Thirty-five rabbits with 6 mm diameter central corneal epithelium removed were randomly assigned to six groups： 0.9% normal saline（NS） group, 0.1% hyaluronate（HA） group, 0.3% HA group, Tears Naturale Free？（TNF） group, 0.4% polyethylene glycol（PEG） group, 0.5% carboxymethyl cellulose（CMC） group and blank control group. Treatments were administered topically four times daily. Corneal epithelium healing was evaluated by the percentage reduction in wound area at 24, 36, 48, 60, and 72 h after removal of the corneal epithelium. Cornea re-epithelialization was also assessed by histological analysis and electron microscopy. RESULTS： All corneal wounds completely re-epithelialized in less than 72 h. The average re-epithelialization time was 47.61±4.25 h in the 0.3% HA group and 49.72±1.05 h in the 0.9% NS group, followed by 0.1% HA, TNF, 0.4% PEG, 0.5% CMC, and lastly by the control group. Compared to the control group, there were significant differences among 0.3% HA, 0.9% NS, PEG, and TNF（P〈0.05） groups. At the first 24 h, re-epithelialization at the 0.3% HA, TNF, and 0.9% NS treatment groups were significantly faster than the other groups. At 48 h post-wounding, corneal epithelium is nearly completing re-epithelialization at 0.3% HA and 0.9% NS treatment groups. Electron microscopy revealed that there were a large number of vacuoles in the cells of the 0.9% NS group at 72 h. CONCLUSION： Artificial tears promote corneal reepithelium varied in the efficacy. Obviously, all artificial eye drops better than blank group. In the process of corneal healing, corneal epithelium cells suffered from hypoxia caused by NS.

TREM-1 expression in rat corneal epithelium with Aspergillus fumigatus infection

AIM: To investigate the expression of triggering receptor expressed on myeloid cells-1(TREM-1) in the aberrant inflammation within the corneal epithelium at early period of fungal infection.METHODS: A total of 65 Wistar rats were randomly divided into control group, sham group and fungal keratitis(FK) group, in which the cornea was infected by Aspergillus fumigatus(A. fumigatus). After executed randomly at 8, 16, 24, 48 and 72 h after experimental model being established, the severity of keratomycosis in rats was scored visually with the aid of a dissecting microscope and slit lamp. Then corneas in three groups were collected to assess the expression of TREM-1through quantitative reverse transcription-polymerase chain reaction(RT-PCR), immunofluorescence technique and Western blot analysis. The correlation between FK inflammation and expression of TREM-1 was also analyzed.RESULTS: Corneal inflammation scores increased with time after fungal infection(F =49.74, P =0.000). The inflammation scores in FK group were obviously higher than those in sham group on the whole(F =137.78, P =0.000). Levels of TREM-1 in the infected rat corneal epithelium had elevated at 8h and peaked at 48h(P 【0.001,compared with control group). Western blot analysis also showed an obviously elevated TREM-1 level in rat corneal epithelium at 24 h and 48 h after fungal infection.Immunofluorescence technique showed that TREM-1mainly existed in corneal epithelium and infected corneal stoma of rat. TREM-1 protein expression was enhanced after fungal infection. Moreover, severity of FK inflammation was significantly related to TREM-1expression in FK(r =0.942, P =0.000).CONCLUSION: TREM-1 may contribute to amplify theinflammation in the cornea infected with A. fumigatus and play critical roles in the battle against A. fumigatus in the innate immune responses.

Expression of vitamin D receptor and cathelicidin in human corneal epithelium cells during fusarium solani infection

AIMTo observe the expression of vitamin D receptor (VDR) in human specimen and immortalized human corneal epithelium cells (HCEC) when challenged with fusarium solani. Moreover, we decided to discover the pathway of VDR expression. Also, we would like to detect the expression of cathelicidin antimicrobial peptide (CAMP) in the downstream pathway of VDR.METHODSImmunohistochemistry was used to examine the VDR expression in HCEC from healthy and fungal keratitis patients. Real time quantitative polymerase chain reaction (qPCR) was performed to observe the messenger ribonucleic acid (mRNA) change of VDR when immortalized HCEC were challenged with fusarium solani for different hours. CAMP was detected at both mRNA and protein levels.RESULTSWe found out that the VDR expression in fusarium solani keratitis patients' specimen was much more than that in healthy people. The mRNA and protein expression of VDR increased when we stimulated HCEC with fusarium solani antigen (P<0.01) and it could be inhibited by toll like receptor 2 (TLR2) monoclonal antibody. The CAMP expression was decreased because of fusarium solani antigen stimulation (P<0.01).CONCLUSIONThe VDR expression can be increased via TLR2/1-VDR pathway while the CAMP expression is decreased by the stimulation of fusarium solani antigen.

Anti-inflammatory and anti-apoptotic effects of N-acetylcysteine in diabetic rat corneal epithelium

AIM:To characterize the anti-inflammatory and antiapoptotic effects of N-acetylcysteine(NAC)in streptozotocin(STZ)-induced diabetic rat corneal epithelium and human corneal epithelial cells(HCECs)exposed to a high-glucose environment.METHODS:HCECs were incubated in 0,5,50 mmol/L glucose medium,or 50 mmol/L glucose medium with NAC for 24h.Diabetes was induced in rats by intraperitoneal injection of 65 mg/kg STZ and some of these rats were topically administered NAC to corneas with 3 mice per group.We characterized receptor for advanced glycation end-products(RAGE)expression using immunofluorescence,and interleukin(IL)-1βand cleaved caspase-3(CCAP-3)expression using immunohistochemistry.Circulating tumor necrosis factor(TNF)-αconcentration was measured by ELISA and cleaved poly-ADP ribose polymerase(PARP)concentration was quantified by Western blotting.Apoptotic cells were detected using TUNEL assay and annexin V and propidium iodide staining.RESULTS:Diabetic rats had higher expression of RAGE(2.46±0.13 fold),IL-1β,and CCAP-3 in apoptotic cells of their corneas than control rats.The expression of RAGE(1.83±0.11 fold),IL-1β,and CCAP-3,and the number of apoptotic cells,were reduced by topical NAC treatment.HCECs incubated in 50 mmol/L glucose medium showed high concentrations of TNF-α(310±2.00 pg/mL)and cleaved PARP(7.43±0.56 fold),and more extensive apoptosis than cells in 50 mmol/L glucose medium.However,the addition of NAC reduced the concentrations of TNF-α(153.67±2.31 pg/mL)and cleaved PARP(5.55±0.31 fold)and the number of apoptotic cells.CONCLUSION:NAC inhibits inflammation and apoptosis in the corneas of diabetic rats and HCECs maintained in a high-glucose environment.

Central corneal epithelium self-healing after ring-shaped glycerin-cryopreserved lamellar keratoplasty in Terrien marginal degeneration

Dear Sir, I am Dr Yan-Long Bi, from the Department of Ophthalmology, Tongji University Affiliated to Tongji University School of Medicine, Shanghai, China. I write to present a case report of total limbal stem cells deficiency after treatment with ring-shaped lamellar keratoplasty secondary to Terrien marginal degeneration. During 3 years

STUDY IN CYTOTOXICITY OF GENTAMYCIN TO CORNEAL EPITHELIUM AND ENDOTHELIUM IN TISSUE CULTURE

A study in cytotoxicity of gentamyein to tissue-cultured bovine corneal endothelial cells and rabbit corneal epithelial cells is reported. When the cultured cells reached confluence, they were exposed to tissue culture media containing gentamycin for 6 hours. We founl that 0.5% gentamycin caused significant damage to corneal epithelial cells---diffuse plasmolysis, with scattered cell necrosis and 5% loss.While corneal endothelial cells were exposed to 1.6 mg/ml gentamycin, extensive cell loss (approximate- ly 15%) was observed. The damaged cells recovered their normal morphology after 24 hours. When the concentration of gentamycin increased twice, serious damage to cells occured. The area of cell loss reached 40%, and the recovery of cellular morphology Was much slower. This study demonstrates that gentamycin potential cytotoxicity to corneal epithelium and endothelium, suggesting that gentamycin should be rationally used in the treatment of ocular diseases.

Reconstruction of Rabbit Corneal Layer Composed of Corneal Fibroblasts and Corneal Epithelium on the Lyophilized Amniotic Membrane

Many researchers have employed the cryopreserved amniotic membrane（CAM） and corneal epithelial cells in the treatment of a severely damaged burned cornea, with corneal epithelial cells cultured on an amniotic membrane （AM）. The lyophilized amniotic membrane（LAM） has a higher graft take and a longer shelf life; it is easier to store and safer because of gamma irradiation. Two Teflon rings（ Ahn＇s supporter） were made for culturing the cells on the LAM, and were then used to support the LAM. To reconstruct a corneal layer composed of corneal fibroblasts and epithelium, the corneal fibroblasts were first cultivated on the stromal side of LAM for five days, foUowed by epithelial cells culture on the epithelial side, by using the air-liquid interface culture. The reconstructed corneal layer composed of corneal fibroblasts and corneal epithelial cells has a much healthier basal layer of corneal epithelium than the reconstructed corneal epithelium, which was got by using only corneal epithelial cells, and resembles the epithelium of normal corneas, without the horny layer. Thus, the reconstruction of the corneal layer by using a LAM is considered to be a good in vitro model, not only for its application in toxicological test kits, but also for transplantation in patients with a severely damaged cornea.

Oxidative stress in retinal pigment epithelium degeneration:from pathogenesis to therapeutic targets in dry age-related macular degeneration

Age-related macular degeneration is a primary cause of blindness in the older adult population. Past decades of research in the pathophysiology of the disease have resulted in breakthroughs in the form of anti-vascular endothelial growth factor therapies against neovascular age-related macular degeneration;however, effective treatment is not yet available for geographical atrophy in dry agerelated macular degeneration or for preventing the progression from early or mid to the late stage of age-related macular degeneration. Both clinical and experimental investigations involving human agerelated macular degeneration retinas and animal models point towards the atrophic alterations in retinal pigment epithelium as a key feature in age-related macular degeneration progression. Retinal pigment epithelium cells are primarily responsible for cellular-structural maintenance and nutrition supply to keep photoreceptors healthy and functional. The retinal pigment epithelium constantly endures a highly oxidative environment that is balanced with a cascade of antioxidant enzyme systems regulated by nuclear factor erythroid-2-related factor 2 as a main redox sensing transcription factor. Aging and accumulated oxidative stress triggers retinal pigment epithelium dysfunction and eventually death. Exposure to both environmental and genetic factors aggravates oxidative stress damage in aging retinal pigment epithelium and accelerates retinal pigment epithelium degeneration in age-related macular degeneration pathophysiology. The present review summarizes the role of oxidative stress in retinal pigment epithelium degeneration, with potential impacts from both genetic and environmental factors in age-related macular degeneration development and progression. Potential strategies to counter retinal pigment epithelium damage and protect the retinal pigment epithelium through enhancing its antioxidant capacity are also discussed, focusing on existing antioxidant nutritional supplementation, and exploring nuclear factor erythroid-2-related factor 2 and its regulators including REV-ERBα as therapeutic targets to protect against age-related macular degeneration development and progression.

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.

Single-cell RNA sequencing in cornea research:Insights into limbal stem cells and their niche regulation

The corneal epithelium is composed of stratified squamous epithelial cells on the outer surface of the eye,which acts as a protective barrier and is critical for clear and stable vision.Its continuous renewal or wound healing depends on the proliferation and differentiation of limbal stem cells(LSCs),a cell population that resides at the limbus in a highly regulated niche.Dysfunction of LSCs or their niche can cause limbal stem cell deficiency,a disease that is manifested by failed epithelial wound healing or even blindness.Nevertheless,compared to stem cells in other tissues,little is known about the LSCs and their niche.With the advent of single-cell RNA sequencing,our understanding of LSC characteristics and their microenvironment has grown considerably.In this review,we summarized the current findings from single-cell studies in the field of cornea research and focused on important advancements driven by this technology,including the heterogeneity of the LSC population,novel LSC markers and regulation of the LSC niche,which will provide a reference for clinical issues such as corneal epithelial wound healing,ocular surface reconstruction and interventions for related diseases.