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The cornea is a transparent outer layer of the anterior eye segment, innervated by a high density of neural tissue. In the process of corneal innervation, trigeminal ganglion originated corneal nerves traverse different types of corneal cell in the epithelial and stromal layers. Corneal stromal cells, epithelial cells, immune cells, and other cells interact closely to maintain corneal microenvironmental homeostasis. In addition, corneal nerves is associated with the occurrence and development of many ocular surface diseases. Corneal nerves release various active peptides that regulate corneal sensation, maintain epithelial integrity and proliferation, improve wound healing, and manage local inflammation and immune response. This article reviews the advances in the corneal nerve regulation of the ocular surface microenvironment, providing some new ideas for the further study and treatment of corneal nerve-associated diseases.

AIM: To investigate the effectiveness and safety of subconjunctival injection of conbercept in the treatment of corneal neovascularization (CNV). METHODS: The data on 10 consecutively recruited patients with CNV who received a subconjunctival conbercept (1 mg) once, and measured the area, length, and diameter of neovascularization before and after (1d, 1, 2wk, and 1mo) treatment as well as the occurrence of systemic and ocular complications after treatment were analyzed. RESULTS: There was a statistically significant reduction in the area of CNV one day after treatment (mean±SD: 38.46±11.36 mm2), compared with before treatment (42.46±12.80 mm2, P

AIM: To evaluate the efficacy of transplantation of human umbilical cord mesenchymal stem cells(hUCMSCs)in the treatment of corneal alkali burn in rabbits, and study the infiltration of polymorphonuclear neutrophils(PMNs)and the changes of vascular endothelial growth factor(VEGF)expression.METHODS: Corneal alkali burn models were established in right eyes of 75 healthy Japanese white rabbits, which were divided into three groups(group A, B and C), with 25 rabbits in each group. Group A was treated with amniotic membrane combined with hUCMSCs on the day after corneal alkali burn. Group B was treated with amniotic membrane only. Group C did not give any treatment after corneal alkali burn. At 3, 7, 14, 21 and 28d after corneal alkali burn, the corneal recovery was observed by slit lamp and photographed, the growth of corneal neovascularization(CNV)was scored, and corneal tissue was separated to make pathological sections. PMNs infiltration was observed by hematoxylin-eosin(HE)staining, and the expression of VEGF was determined by immunohistochemical staining.RESULTS: The growth of CNV in group A was much slower than that in group B at 14d after alkali burn. The CNV growth score around lesions of group A was significantly lower than that of group B(P

Conbercept is a novel anti-vascular endothelial growth factor drug independently developed by China. Since it was approved for clinical application by the State Food and Drug Administration of China in 2013, conbercept has shown reliable safety and efficacy in the treatment of ocular neovascular diseases such as wet age-related macular degeneration, choroidal neovascularization and macular edema. For different diseases, the treatment strategies of conbercept are different. This article mainly reviews the application progress of conbercept in ocular neovascularization related diseases including wet age-related macular degeneration, diabetic macular edema, pathologic myopia choroidal neovascularization, neovascular glaucoma, retinopathy of prematurity and corneal neovascularization, and summarizes and explores the indications, administration scheme and therapeutic effect of conbercept. It is expected that the indications of conbercept will be wider and the administration scheme will be more given, and the usage of conbercept will bring new ideas for the treatment of ocular neovascular diseases.

AIM: To observe the curative effect of pterygium resection and autologous limbal stem cell transplantation(LSCT)combined with amniotic membrane transplantation(AMT)in the treatment of pterygium.METHODS: Totally 177 patients(187 eyes)with pterygium treated in ophthalmology clinic of the hospital between January 2017 and January 2020 were selected and randomly divided into group A(59 cases, 64 eyes), group B(59 cases, 60 eyes), and group C(59 cases, 63 eyes). All were treated with pterygium resection. On this basis, patients in the three groups were treated with autologous LSCT, AMT, and autologous LSCT combined with AMT, respectively. All subjects were followed up for 12mo after surgery. Visual acuity, corneal epithelial repair, and neovascularization of the three groups were comparatively analyzed. Postoperative recurrence rate, ocular symptoms, complications, and survival of grafts were statistically analyzed.RESULTS: Visual acuity changes and repair time of corneal epithelial defect showed no statistically significant difference among the three groups(P>0.05). 1mo after surgery, the corneal fluorescein staining(FL)value of group C was significantly lower than that of group A or group B(all P0.05). 1mo after surgery, the tear film breakup time(BUT)of group C was significantly longer than that of group A or group B(all P

Various ocular injuries can induce corneal neovascularization, which promote the development of diseases, causing corneal edema, impaired vision and even blindness. Therefore, with very important clinical significance, inhibiting corneal neovascularization can help to delay the progression of diseases and reduce corneal damage. This article will make the latest systematic discussion on the cells and molecules involved in corneal neovascularization, and analyze the possible inhibitory targets, hoping to provide references for scientific research and clinical practice.

AIM: To evaluate the potential efficacy and mechanisms of nintedanib in corneal neovascularization (NV) in rabbit models. METHODS: Corneal NV was induced using 1 mol/L NaOH. Rabbits (n=21) were randomized to 3 groups: Group 1 were treated with 0.9% NaCl, Group 2 with Avastin (5 mg/mL), and Group 3 with nintedanib (1 mg/mL). All treatments started 1d after alkaline burns and were topically performed 3 times a day for 2wk. Photographs were taken on a slit lamp microscope on day 7 and 14. The NV area, the length of the vascularization and angiogenesis index (AI) were used to evaluate the corneal NV. On day 14, the immunohistochemical (IHC) studies of the cornea were examined. Western blot was performed to test the expression levels of vascular endothelial growth factor (VEGF), Akt, p-Akt, P38, p-P38, MMP-2 and MMP-9. RESULTS: The corneal NV area, vessel length and AI in Group 3 were significantly lower than Group 2, with both being lower than Group 1. IHC staining showed that VEGF was significantly overexpressed in the epithelium and stroma of cornea following alkaline burns. In contrast, the level of VEGF was significantly suppressed in both Group 2 and Group 3. Western blot results further confirmed that, compared with Group 1, Group 3 had significantly reduced expressions of VEGF, Akt, p-Akt, p-P38, MMP-2, and MMP-9 in corneal tissues. Trends of lower levels of MMP-2, AKT, and p-AKT in Group 3 than Group 2 were identified. CONCLUSION: Nintedanib and Avastin can effectively inhibit corneal NV, with P38 MAPK and AKT signaling pathways being possibly involved. Nintedanib seems more effective than Avastin and has the potential to be a novel therapy for preventing corneal NV.

AIM: To evaluate the inhibitory effect of glycyrrhizin(Gly)on acute alkali burn induced corneal neovascularization(CNV)in mice. METHODS: Corneal neovascularization was established in mice by alkali burn. Sixty mice were then randomly distributed into normal group, Gly group and phosphate buffer solution(PBS)group. The mice were treated with subconjunctival injection of 2g/L Gly solution or vehicle alone every other day for 14d. Corneal inflammation and neovascularization were monitored with a slit lamp microscope. At the end of treatment, the corneas were harvested for hematoxylin-eosin(HE)staining as well as immunohistochemical of CD34 and myeloperoxidase(MPO)staining, microvessel density(MVD), neutrophils were then calculated. RESULTS: At the 7 and 14d, the CNV area of Gly group were 4.16±0.00 and 7.33±0.13mm2 respectively, which were lower than those in PBS group(7.58±0.20 and 9.24±0.08mm2; all PPPCONCLUSION: Gly can eliminate corneal inflammation and inhibit corneal neovascularization in mice with acute corneal alkali burn, which provides a new idea for clinical prevention and treatment of corneal neovascularization.

The invasion of the cornea by capillary or lymphatic vessels leads to corneal neovascularization and if not handled in time, it will seriously affect vision, the establishment and application of transgenic mouse models of corneal neovascularization provides a good platform for the study of corneal neovascularization mechanism, the screening of antiangiogenic drugs and the evaluation of treatments. Transgenic mouse model of corneal neovascularization is a very valuable and potential animal model. This paper mainly introduces the application of transgenic mouse models in the research of corneal neovascularization.

Corneal neovascularization(CNV)is a common cause of corneal diseases, but there is still no effective drug or treatment. At present, the common methods cannot inhibit the growth of CNV completely, cannot last for a long the therapeutic effect time, and there are also serious side effects, so the specific treatment of CNV inhibition is still being explored. The pathogenesis and treatment of CNV are the current research hotspots. CNV area is an important indicator to evaluate the efficacy of drugs and treatment regimens. There are multiple methods to develop CNV, including ink perfusion, immunofluorescence staining and so on, in recent years, optical coherence tomography(OCT)technology is also a potential new method. This article reviews the developing methods of CNV, hopes to provide a reference for the study of CNV.

AIM: To investigate the ocular biodistribution and clearance of topically administered 7-taurocholic acid conjugated low-molecular weight heparin (LHT7) in a neovascularized mouse cornea using an in vivo optical imaging system. METHODS: A total of 10 eyes of 6 to 8-week-old BALB/c mice were analyzed. Corneal neovascularization (CoNV) was induced in the inferior cornea (IC) of each animal by penetrating the stroma with two interrupted sutures. The development of CoNV was verified after one week and the area of each neovascularized region was measured. A near-infrared fluorescent probe of 20 µmol/L Cy5.5 labeled LHT7 (LHT7-Cy5.5) in 0.02 mL solution was topically instilled onto the cornea in the experimental group (n=5). Free-Cy5.5 of 20 µmol/L in 0.02 mL was instilled in the control group (n=5). In vivo optical images were obtained before instillation and 5min, 2, 4, and 6h after instillation. The intensities were separately measured at the superior cornea (SC) and the IC. RESULTS: The mean CoNV areas were 1.97±0.17 mm2 and 1.92±0.96 mm2 in the experimental and control groups, respectively (P=0.832). The SC remained normal in all 10 subject animals. The IC intensity of the LHT7-Cy5.5 was greater than the SC intensity at 5min (P=0.038), 2h (P=0.041), and 4h (P=0.041) after application. The IC intensity fell to less than half of its initial value (42.9%±8.6%) at 6h in the experimental group. In the control mice, here were no significant differences in the free-Cy5.5 intensity between the IC and SC. CONCLUSION: Topically administered LHT7 shows a high biodistribution in CoNV areas for 4h and should be reapplied accordingly to maintain its effects. In vivo optical imaging can be a useful tool for evaluating the ocular biodistribution of a drug in an animal model.

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 mL 0.9% NaCl solution was injected on the first day. In Group 2 (single-dose group, n=10), 0.05 mL bevacizumab (1.25 mg) was injected on the first day. In Group 3 (multiple-dose group, n=10), four doses of 0.05 mL 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.

AIM: To investigate whether curcumin suppressed corneal neovascularization (CNV) formation via inhibiting activation of Wnt/β-catenin pathway. METHODS: Suture-induced CNV was established on Sprague-Dawley (SD) rats. Curcumin were daily administrated by subconjunctival injection. Phosphorylation of low-density lipoprotein receptor-related protein 6 (LRP6) and nuclear accumulation of β-catenin, two indicators of activated Wnt/β-catenin pathway, were determined by Western-blot analysis in subconfluent/proliferating human microvascular endothelial cells (HMEC) and neovascularized corneas. Wnt3a conditioned medium (WCM) were harvested from Wnt3a expressing cells. WCM-induced cell proliferation and endothelial tubular formation capacity was measured by MTT assay and Matrigel assay, respectively. RESULTS: Phosphorylation of LRP6 and nuclear accumulation of β-catenin was significantly increased in subconfluent/proliferating endothelial cells. Activation of Wnt/β-catenin pathway by WCM markedly promotes HMEC proliferation and tubular formation. Curcumin inhibited LRP6 phosphorylation and nuclear accumulation of β-catenin. In addition, curcumin attenuated WCM-induced HMEC proliferation and disrupted tubular structure of endothelial cells on Matrigel. Meanwhile curcumin suppressed suture-induced CNV and inhibited LRP6 phosphorylation as well as β-catenin accumulation in SD rats. CONCLUSION: Taken together, activation of Wnt/β-catenin pathway could be involved in endothelial proliferation during suture-induced CNV formation and curcumin attenuated CNV formation via inhibition of Wnt/β-catenin pathway activation.

AIM: To study the effect of diallyl trisulfide(DATS)on experimental corneal neovascularization(CNV)in rats induced by corneal suture and detect the expression of vascular endothelial growth factor(VEGF)and p-AKT in rats cornea. METHODS: The rat model of corneal neovascularization(CNV)was induced by corneal suture. Rats were randomly divided into Group A: physiological saline control group containing DMSO(10 rats); Group B: 25μmol/L DATS treatment group(10 rats); Group C: 50μmol/L DATS treatment group(10 rats); Group D: 100μmol/L DATS treatment group(10 rats); Group E: 200μmol/L DATS treatment group(10 rats). The occurrence and development of CNV were observed by slit-lamp microscope at 7d after suture, and the area of CNV were calculated.Two weeks later, HE staining was used to observe the pathological organization form of each cornea, and RT-PCR and Western blot were used to detect the expression of VEGF mRNA and protein expression of VEGF and p-AKT between each groups. RESULTS: The blood vessel area of Group C, D and E was compared with that of Group A, the difference was statistically significant(PPPCONCLUSION: DATS can inhibit corneal neovascularization of the rats induced by suture. Its mechanism may be associated with suppression of VEGF secretion, down-regulation of VEGF and inactivation of p-AKT.

AIM: To observe the expression of erythropoietin(EPO)on the corneal of rabbit and evaluate the inhibition effect of EPO siRNA on corneal neovascularization(CNV). METHODS: Totally 22 healthy rabbits were randomly divided into 2 groups, which were experimental group and normal control group. Both eyes of rabbits in experimental group were chosen to establish corneal neovascularization model by alkali burn. The morphologic change of corneal was observed with slit lamp microscope and the area of CNV was calculated every day. After alkali burn, the right eye of the experimental group was accepted EPO siRNA injection under the conjunctiva, and the left eye was assigned to be experimental control group. The corneal with CNV was collected for immunohistochemistry at 3d, 7d, 14d, 21d after alkali burn, and the expression of EPO was measured. RESULTS: CNV began growing at the 3d after alkali burn in experimental group, and it was vigorous growing at 7d-14d period. The result of immunohistochemistry shows that the expression of EPO increased after the operation. Compared with experimental group, the rabbits who were treated by EPO siRNA was found with less neovascularization on their corneal, and the expression of EPO decreased. There were statistical significance between the two group at different time(PCONCLUSION: EPO is likely to play an important role on CNV growth, and EPO siRNA can inhibit the growth of CNV by restraining the expression of EPO.

Corneal neovascularization(CNV)is caused by the unbalance of corneal avascularity maintaining factors, and then the limbus capillary invades the transparent corneal tissue. CNV is one of the common causes of blindness. In recent years, with the developing of immunology, molecular biology and pharmacology,we have made a lot of breakthroughs about the pathogenesis and treatment of CNV. However, there is no effective medicine and therapy for CNV. In this article we reviewed the pathogenesis, molecular mechanism of CNV, and focus on the progress of therapies.

AIM: To choose appropriate concentration of sodium hydroxide (NaOH) solution to establish a stable and consistent corneal alkali burn mouse model in grade II. METHODS: The mice (n=60) were randomly divided into four groups and 15 mice each group. Corneal alkali burns were induced by placing circle filter paper soaked with NaOH solutions on the right central cornea for 30s. The concentrations of NaOH solutions of groups A, B, C, and D were 0.1 mol/L, 0.15 mol/L , 0.2 mol/L, and 1.0 mol/L respectively. Then these corneas were irrigated with 20 mL physiological saline (0.9% NaCl). On day 7 postburn, slit lamp microscope was used to observe corneal opacity, corneal epithelial sodium fluorescein staining positive rate, incidence of corneal ulcer and corneal neovascularization, meanwhile pictures of the anterior eyes were taken. Cirrus spectral domain optical coherence tomography was used to scan cornea to observe corneal epithelial defect and corneal ulcer. RESULTS: Corneal opacity scores () were not significantly different between the group A and group B (P=0.097). Incidence of corneal ulcer in group B was significantly higher than that in group A (P=0.035). Incidence of corneal ulcer and perforation rate in group B was lower than that in group C. Group C and D had corneal neovascularization, and incidence of corneal neovascularization in group D was significantly higher than that in group C (P=0.000). CONCLUSION: Using 0.15 mol/L NaOH can establish grade II mouse model of corneal alkali burns.

Corneal neovascularization (CNV) is a global important cause of visual impairment. The immune mechanisms leading to corneal heme- and lymphangiogenesis have been extensively studied over the past years as more attempts were made to develop better prophylactic and therapeutic measures. This article aims to discuss immune cells of particular relevance to CNV, with a focus on macrophages, Th17 cells, dendritic cells and the underlying immunology of common pathologies involving neovascularization of the cornea. Hopefully, a thorough understanding of these topics would propel the efforts to halt the detrimental effects of CNV.

AIM: To explore the inhibiting effect of FTY720 on corneal neovascularization(CNV)of rat.METHODS: MTT assay and cells scratch were adopted to observe hyperplasia of human umbilical vein endothelial cells(HUVECs)and cell migration induced by sphingosine-1-phosphate(S1P)after using FTY720 of different concentration. The effect of FTY720 on CNV induced by S1P in a rat corneal micropocket model was detected. 30SD rats were randomly divided into group A, group B and group C with 10 rats per group. S1P and 0μg, 5μg, and 20μg FTY720 controlled-released particles were implanted into the corneal stroma. The growth of CNV and having pathological examination on 12d after the operation was observed. Findings was analyzed by one-way ANOVA.RESULTS: 10, 102, 103, and 104 nmol/L FTY720 and HUVECs co-incubate 72h could inhibit cell proliferation(P2(F=145.155, PPCONCLUSION: FTY720 can inhibit S1P-induced corneal neovascularization.

AIM: To investigate anti-angiogenic effects of polysulfated heparin endostatin (PSH-ES) on alkali burn induced corneal neovascularization (NV) in rabbits. METHODS: An alkali burn was made on rabbit corneas to induce corneal NV in the right eye of 24 rabbits. One day after burn creation, a 0.2 mL subconjunctival injection of 50 μg/mL PSH-ES, 50 μg/mL recombinant endostatin (ES), or normal saline was administered every other day for a total of 14d (7 injections). Histology and immunohistochemisty were used to examine corneas. Corneal NV growth was evaluated as microvessel quantity and corneal vascular endothelial growth factor (VEGF) expression was measured by immunohistochemical assay. RESULTS: Subconjunctival injection of ES and PSH-ES resulted in significant corneal NV suppression, but PSH-ES had a more powerful anti-angiogenic effect than ES. Mean VEGF concentration in PSH-ES treated corneas was significantly lower than in ES treated and saline treated corneas. Histological examination showed that corneas treated with either PSH-ES or ES had significantly fewer microvessels than eyes treated with saline. Additionally corneas treated with PSH-ES had significantly fewer microvessels than corneas treated with ES. CONCLUSION: Both PSH-ES and recombinant ES effectively inhibit corneal NV induced by alkali burn. However, PSH-ES is a more powerful anti-angiogenic agent than ES. This research has the potential to provide a new treatment option for preventing and treating corneal NV.

AIM: To assess cultured limbal epithelial stem cell transplantation in patients with limbal stem cell deficiency by analyzing and quantifying corneal neovascularization.METHODS: This retrospective, interventional case series included eight eyes with total limbal stem cell deficiency. Ex vivo limbal epithelial stem cells were cultured on human amniotic membrane using an animal-free culture method. The clinical parameters of limbal stem cell deficiency, impression cytology, and quantification of corneal neovascularization were evaluated before and after cultured limbal stem cell transplantation. The area of corneal neovascularization, vessel caliber (VC), and invasive area (IA) were analyzed before and after stem cell transplantation by image analysis software. Best-corrected visual acuity (BCVA), epithelial transparency, and impression cytology were also measured.RESULTS: One year after surgery, successful cases showed a reduction (improvement) of all three parameters of corneal neovascularization [neovascular area (NA), VC, IA], while failed cases did not. NA decreased a mean of 32.31% (P=0.035), invasion area 29.37% (P=0.018) and VC 14.29% (P=0.072). BCVA improved in all eyes (mean follow-up, 76±21mo). Epithelial transparency improved significantly from 2.00±0.93 to 0.88±1.25 (P=0.014). Impression cytology showed that three cases failed after limbal epithelial stem cell therapy before 1y of follow-up.CONCLUSION: This method of analyzing and monitoring surface vessels is useful for evaluating the epithelial status during follow-up, as successful cases showed a bigger reduction in corneal neovascularization parameters than failed cases. Using this method, successful cases could be differentiated from failed cases.

AIM: To describe the expression of hypoxia-inducible factor-1α(HIF-1α)and erythropoietin(EPO)in rats' corneal and evaluate its potential effect on corneal neovascularization(CNV)growth. METHODS: The young SD rats(3mo)was chosen and randomly divided into 2 groups, which were experimental group and normal control group. CNV model was established by alkali burn, and the length and area of CNV was observed everyday after operation by slit lamp. After that, the expression of HIF-1α and EPO was measured by SABC and RT-PCR methods at 1, 3, 5, 7, and 14d after alkali burn. The data was analyzed by SPSS 20.0. RESULTS: The area of CNV was increasing at 1, 3, 5, 7, and 14d after alkali burn, and the peak point appear at 7d. The growth speed was decreased after 14d. SABC method told us that no HIF-1α and very tiny amount EPO was detected at normal rats' corneal. The expression of the two factors increased at 1d after alkali burn in corneal epithelium and endoderm. The results of RT-PCR showed that a few amounts of HIF-1α and EPO mRNA were detected at normal group. The expression of the two factors was increased at 3d after alkali burn, and the peak value was found at 7d, however, it was decreased at 14d. Statistical difference was found at different time(PCONCLUSION: HIF-1α and EPO is closely related to CNV.

AIM: To discuss the influence of dexamethasion on IL-1β and TNF-α expression in suture-induced rabbit corneal neovascularization(CNV)and analyze the potential mechanism. METHODS: For 43 healthy rabbits, 40 were randomly selected for establishing CNV model in corneal stroma. The right eyes(group A)were received no medicine and the left eyes(group B)were injected dexamethasone after successfully establishing the model. The no modeling 3 rabbits were normal control group. The morphologic change of corneal was observed with slit lamp microscope and the areas of CNV was calculated every day, then 8 rabbits were randomly chosen for sacrificing at 1, 4, 7, 14, 21d respectively. The pathological characteristics of CNV were observed after HE staining, and IL-1β and TNF-α expression was detected by immunohistochemistry. RESULTS: CNV was grown at the 4d after suture, and the 7-14d was vigorous growth period. inflammatory cell infiltration appeared after HE staining, and CNV was located at the superficial stroma of cornea. Immunohistochemistry results showed that IL-1β and TNF-α expression was gradually increased with prolonged suture time. Compared with corneal stitch group, the rabbits cured by dexamethasone were found with less inflammatory cells infiltrating and neovescularization, moreover, the expression of IL-1β and TNF-α decreased. There were statistical significance between the two groups(PCONCLUSION: Dexamethasone can inhibit the CNV growth by controlling the inflammation of corneal and restraining IL-1β and TNF-α expression.

AIM:To explore the suitable conditions in rapid model of corneal neovascularization(CNV)after thermal burn under different constant temperature in rabbit. METHODS: Total 45 New Zealand white rabbits were divided randomly into five groups(A, B, C, D, E). A groups: 100℃(n=10), B groups: 200℃(n=10), C groups: 300℃(n=10), D groups: 400℃(n=10), and E groups: control group(n=5). All left eyes of rabbits in A,B,C,D groups were induced corneal neovascularization by constant temperature burning device. The growth of CNV was observed by slit lamp microscope and the area of CNV were recorded on 4 th, 7 th, 14th, 30th days postoperatively. SPSS 19.0 statistical package was used for data analysis, and the data was recorded by mean±standard deviation. Comparison by analysis of variance was made by repeated measures in the area of neovascularization at each time point in groups. Statistical tests were considered significantly when P values were less than 0.05. RESULTS: On postoperative 4th, 7th, 14th, 30th days: no neovascularization was found after corneal thermal burn in A group, but only a few nebula left(n=2); the area of CNV were(9.16±1.45)mm2,(37.73±5.49)mm2,(62.44±7.54)mm2,(40.28±7.39)mm2 in B group respectively; and(11.45±1.04)mm2,(44.51±4.64)mm2,(66.13±4.13)mm2,(43.04±2.33)mm2 in C group respectively; and(13.23±0.86)mm2,(47.26±4.59)mm2,(67.57±4.56)mm2,(45.59±4.44)mm2 in D group respectively, and part corneal carbide(n=4)was observed as well as corneal perforation(n=6)were found on 3d in D group. No neovascularization was found in normal control group. Comparison of the areas of CNV at each time point between groups was statistically different, PPCOCLUSION: In 4 to 7d, the higher the temperature is, the more the neovascularization area of CNV are. It has no significant difference in 14 to 30d. But corneal carbide and corneal perforation are often found in 400℃ group, so its modeling failure rate is high. It is between 200℃ and 300℃ that repeatability and uniformity of the corneal neovascularization model of rabbit are superior.

AIM:To evaluate the inhibitory effects of regorafenib (BAY 73-4506), a multikinase inhibitor, on corneal neovascularization (NV).METHODS:Thirty adult male Sprague-Dawley rats weighing 250-300 g, were used. Corneal NV was induced by NaOH in the left eyes of each rat. Following the establishment of alkali burn, the animals were randomized into five groups according to topical treatment. Group 1 (n = 6) received 0.9% NaCl, Group 2 (n = 6) received dimethyl sulfoxide, Group 3 (n = 6) received regorafenib 1 mg/mL, Group 4 (n =6) received bevacizumab 5 mg/mL and Group 5 (n = 6) received 0.1% dexamethasone phosphate. On the 7d, the corneal surface covered with neovascular vessels was measured on photographs as the percentage of the cornea’s total area using computer-imaging analysis. The corneas obtained from rats were semiquantitatively evaluated for caspase-3 and vascular endothelial growth factor by immunostaining.RESULTS:A statistically significant difference in the percent area of corneal NV was found among the groups (P P >0.005). There was a statistically significant difference among the groups in apoptotic cell density (P = 0.002). The staining intensity of vascular endothelial growth factor in the epithelial and endothelial layers of cornea was significantly different among the groups (P CONCLUSION: Topical administration of regorafenib 1 mg/mL is partly effective for preventing alkali-induced corneal NV in rats.

AIM:To solidify the involvement of Saa-related pathway in corneal neovascularization (CorNV). The pathogenesis of inflammatory CorNV is not fully understood yet, and our previous study implicated that serum amyloid A (Saa) 1 (Saa1) and Saa3 were among the genes up-regulated upon CorNV induction in mice.METHODS:Microarray data obtained during our profiling project on CorNV were analyzed for the genes encoding the four SAA family members (Saa1-4), six reported SAA receptors (formyl peptide receptor 2, Tlr2, Tlr4, Cd36, Scarb1, P2rx7) and seven matrix metallopeptidases (Mmp) 1a, 1b, 2, 3, 9, 10, 13 reportedly to be expressed upon SAA pathway activation. The baseline expression or changes of interested genes were further confirmed in animals with CorNV using molecular or histological methods. CorNV was induced in Balb/c and C57BL/6 mice by placing either three interrupted 10-0 sutures or a 2 mm filter paper soaked with sodium hydroxide in the central area of the cornea. At desired time points, the corneas were harvested for histology examination or for extraction of mRNA and protein. The mRNA levels of Saa1, Saa3, Fpr2, Mmp2 and Mmp3 in corneas were detected using quantitative reverse transcription-PCR, and SAA3 protein in tissues detected using immunohistochemistry or western blotting.RESULTS:Microarray data analysis revealed that Saa1, Saa3, Fpr2, Mmp2, Mmp3 messengers were readily detected in normal corneas and significantly up-regulated upon CorNV induction. The changes of these five genes were confirmed with real-time PCR assay. On the contrary, other SAA members (Saa2, Saa4), other SAA receptors (Tlr2, Tlr4, Cd36, P2rx7, etc), or other Mmps (Mmp1a, Mmp1b, Mmp9, Mmp10, Mmp13) did not show consistent changes. Immunohistochemistry study and western blotting further confirmed the expression of SAA3 products in normal corneas as well as their up-regulation in corneas with CorNV.CONCLUSION:SAA-FPR2 pathway composing genes were expressed in normal murine corneas and, upon inflammatory stimuli challenge to the corneas, their expressions were up-regulated, suggesting their roles in pathogenesis of CorNV. The potential usefulness of SAA-FPR2 targets in future management of CorNV-related diseases deserves investigation.

AIM: To compare the effect of topically administered and subconjunctivally injected bevacizumab on experimental corneal neovascularization in rats for two weeks after treatment.METHODS: Twenty-eight Sprague-Dawley rats were divided into four groups of 7 animals. Each corneal center of right eye was cauterized with silver/potassium nitrate for 8s. After corneal burning, bevacizumab (12.5mg/mL) was topically administered three times per day (TB group) for two weeks or subconjunctivally injected on days 2 and 4 after cauterization (0.02mL; SB group). As negative controls, rats received 0.9% saline topically three times per day (TS group) or subconjunctivally on days 2 and 4 (0.02mL; SS group). Digital photographs of the cornea were taken 1 and 2 weeks after treatment and analyzed to determine the area of cornea covered by neovascularization as the percentage of corneal neovascularization.RESULTS: One week after treatment, the percentage of corneal neovascularization was significantly lower in the TB and SB groups than in the TS and SS groups (all PPPCONCLUSION: Topically administered bevacizumab has longer standing anti-angiogenic effect than subconjunctivally injected bevacizumab in corneal neovascularization following chemical injury in rats.

AIM: To study the effect of amniotic membrane(AM)suspension on corneal neovascularization(CNV)and ultrastructure in corneal alkali burn, and to explore the best concentration of AM for the clinical treatment.METHODS: Forty eyes from 40 Sprague-Dawley male rats were used. After alkali injuries at central corneal using 1mol/L NaOH,the control group(Group A)received topical PBS four times a day. The rest groups(Group B, C, D)received topical AM suspension with different concentration(240, 400, 560μg/mL). Using slit lamp biomicroscopy, CNV were evaluated and scored. Tissue ultrastructure changes of cornea were analyzed by corneal confocal microscope.RESULTS:The area of CNV were statistically different at each time point between group C and group A, B(all PCONCLUSION: AM suspension leads to significant reduce in CNV with increasing AM concentrations. However, when the concentration reached 400μg/mL, the effects on restraining CNV cannot be enhanced by concentration. This effect may be elicited in part through the inhibiting of inflammatory cells.

AIM:To compare the efficacies of subconjunctival bevacizumab, ranibizumab, and pegaptanib sodium injections for the inhibition of corneal neovascularization in an experimental rat model.METHODS:Sixteen corneas of 16 rats were chemically cauterized and randomized into four groups:bevacizumab group that treated with 0.05mL/1.25mg bevacizumab, ranibizumab group that treated with 0.05mL/0.5mg ranibizumab, pegaptanib group that treated with 0.05mL/0.15mg pegaptanib sodium, and control group that treated with 0.05mL saline solution. Digital photographs of the corneas were taken and analyzed using an image analysis software program. All corneas were excised and examined histologically on the 15th day.RESULTS: Each treatment group had significantly less neovascularized corneal areas and fewer blood vessels than the control group (all P0.05). CONCLUSION:Subconjunctival bevacizumab, ranibizumab, and pegaptanib sodium were effective with no corneal epitheliopathy for inhibiting corneal neovascularization after corneal burn in rats. Bevacizumab was more effective than ranibizumab and pegaptanib sodium

BIGH3 gene plays an important role in ocular diseases. On the one hand, it is closely related to the occurrence of corneal diseases. BIGH3 gene can inhibit corneal neovascularization, lead to corneal dystrophy, participate in keratoconus formation. On the other hand, it can lead to the formation of neovascularization in diabetic retinopathy. The latest experiments show that TGF beta secreted by macrophages can promote the expression of BIGH3 mRNA and BIGH3 protein, and promote apoptosis of retinal endothelial cells and pericytes, which leads to the formation of neovascularization in diabetic retinopathy. This article will describe the new progress of BIGH3 gene in ocular diseases from several aspects as mentioned above.

AIM: To observe the clinical effect subconjunctival bevacizumab and triamcinolone acetonide(TA)on corneal neovascularization(CNV)induced by penetrating keratoplasty.METHODS: Nineteen patients(20 eyes)with CNV induced by penetrating keratoplasty after 3mo were selected and were divided into 4 groups randomly: Group A was control group, group B was treated by subconjunctival of bevacizumab(2.5mg, 0.1mL), group C was treated by subconjunctival of bevacizumab(2.5mg, 0.1mL)and TA(4mg, 0.1mL), group D was treated by subconjunctival of TA(4mg, 0.1mL), one time each month, twice.RESULTS: The average follow-up period was 3a. Visual acuity of groups B and C kept stable or increase slightly. CNV and immunologic rejection were lighten(PCONCLUSION: Treatment of subconjunctival bevacizumab and TA can effectively inhibite CNV and immunologic rejection, also has the therapeutical effect to maintain implant transparence after penetrating keratoplasty.

AIM: To explore the roles of neuronal axon-guidance molecules Slit3 and Robo4 receptor in corneal neovascularization(CNV)by study their expression in neovascularized cornea of rats. METHODS: CNV models were established by implantation pellets containing basic fibroblast growth factor(bFGF)into corneal stroma. CNV models were measured by biomicroscopy photography. Immunohistochemical staining and imaging analysis system were used to detect the expression of Slit3 and Robo4 in the models after 1, 4, 7, 10 and 14d.RESULTS: The area of CNV and the expression of Slit3, Robo4 were increased in CNV models compared to that in normal cornea and reached highest level on 7d. And the expression level of Slit3 and Robo4 were significantly correlated with the size of CNV on every time point except 1d(r=0.84-0.91, all PCONCLUSION: The expression of Slit3 and Robo4 may be related to the CNV development. They are potential therapeutical target for CNV.

AIM: To evaluate the effect of inhibition of 99Tc-MDP on experimental corneal neovascularization in rats.METHODS: Chemical cauterization of the cornea was performed by touching alkali. Twenty-four healthy SD rats were divided randomly into two equal groups: experimental group underwent i.p. injection of 99Tc-MDP everyday until 14th day after alkali burn. Control group underwent i.p. injection of normal saline as control. Rats were examined for detection of the first signs of neovascularization. 21 days later, lengths of neovascular and total corneal neovascularization area were assessed.RESULTS: The time of neovascularization in experimental group was 6.25±1.93 days, the control group was 5.83±1.86 days, and there was no statistically significant difference(P=0.30). The lengths of neovascular were 1.74±0.33mm and 2.82±0.25mm in two groups and the difference was statistically significant. The total neovascularization area was 17.3±3.26mm2, 24.8±5.10mm2 respectively and the difference between the two groups was statistically significant(PCONCLUSION: 99Tc-MDP has a significant effect on inhibition of alkali burn-induced corneal neovascularization.

AIM: To compare the inhibitory effect of curcunmin and Avastin on the rat corneal neovascularization(CNV), and approach the mechanism of the curcunmin's inhibition. METHODS: CNV was established in thirty SD rats by alkaline burning. Rats were divided equally to group A and group B at random. In group A, right eyes were experimental group A1, treated by 40μmol/L curcunmin solution, and left eyes were control group A2, treated by 0.09% sodium chloride. In group B, right eyes were experimental group group B1, treated by 5g/L avastin, and left eyes were control group B2, treated by 0.09% sodium chloride. Cornea and aqueous humor were collected by time spot. The capillary vessels were study, and the expressions of VEGF were detected by Enzyme-Linked immunosorbnent Assay(ELISA). RESULTS: No toxic effects of the drugs were found. The capillary vessels in experimental group were less than those of control group(P

AIM:To investigate the appropriate methods to establish corneal alkali chemical injury on rats. METHODS:The rats(n=87)were randomly divided into three groups. Corneal alkali injury was induced by placing 1mol/L NaOH soaked filter paper on the limbus of right cornea for 20 seconds(group A, n=34)or 40 seconds(group B, n=23), and on the central axis of the right cornea for 40 seconds(group C, n=30)respectively. Corneal transparency, corneal ulceration, and corneal neovascularization were observed and recorded under slit- lamp biomicroscope on day 7 post-operation. RESULTS: Incidence of corneal ulceration, corneal perforation and positive rate of corneal fluorescein staining in limbal corneal injury groups(group A and B)were significantly higher than that of central corneal injury group(group C)(P