Experimental study of transplantation of human umbilical cord mesenchymal stem cells in the treatment of alkali-burned cornea / 国际眼科杂志(Guoji Yanke Zazhi)

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<0.05). The quantity of PMNs increased on the 3d with the stromal layer of cornea infiltrated, relatively decreased on the 7d, shown a peak on the 14d, and then decreased gradually. Early infiltration after alkali burn was in the corneal stroma of the lesion area, and the extent of infiltration was equal to the ulcer area at later stage. The cell densities of corneal PMNs in group A and group B were significantly lower than those in group C at all time points after alkali burns(P<0.05), and those in group A were significantly lower than group B at 14 and 21d(P<0.05). The expression levels of corneal VEGF in all groups after alkali burn reached peak at 7～14d and decreased significantly at 28d, and the expression levels of VEGF in group A and group B at all time points after alkali burn were significantly lower than those in group C(P<0.05), and group A was significantly lower than that in group B at 7, 14 and 21d(P<0.05).CONCLUSION: The transplantation of hUCMSCs after alkali burn cornea can reduce the formation of CNV and inhibit corneal revascularization after alkali burn. The corneal pathological lesions and vascularization are closely related to PMNs and VEGF.

Inhibitory effect of miR-497 on corneal neovascularization and its regulation mechanism targeting STAT3 / 中华实验眼科杂志

Objective:To investigate the role of microRNA (miR)-497 in the formation of corneal neovascularization (CNV) induced by alkali burn and its mechanism.Methods:Forty-two wild type (WT) C57BL/6 mice aged 6 to 8 weeks, 42 CRISPR/Cas9 mediated miR-497 knockout (KO) and 42 CRISPR/Cas9 mediated overexpression transgenic (TG) C57BL/6 mice were selected and assigned as WT group, KO group and TG group, respectively.The corneal alkali burn model was established.At 3, 7, 14 and 21 days after modeling, corneal epithelium damage and stromal turbidity were scored according to slit lamp microscopy.The area of neovascularization was measured.Corneal structural changes and expression of inflammatory cells were observed by histopathological staining.The expression of CD31 in corneal tissues was detected by immunohistochemistry staining.The targeted binding relationship between miR-497 and signal transducer and activator of transcription 3 (STAT3) was detected by luciferase reporter assay.The relative expressions of miR-497, vascular endothelial growth factor A (VEGFA), tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β and macrophage inflammatory protein (MCP)-1 mRNA were detected by real-time quantitative PCR.At 14 days following modeling, the expression of STAT3 and p-STAT3 proteins in mice corneal tissues was detected by Western blot.The use and care of animals complied with the ARVO statement.The study protocol was approved by the Ethics Committee of Renmin Hospital of Wuhan University (No.2019K-K010).Results:Corneal injury, inflammatory cell infiltration and CNV occurred in mice cornea after alkali burn.Corneal epithelial injury score, corneal stromal turbidity score and CNV area increased first and reached the peak on the 14th day after modeling, and then decreased.There were significant differences in corneal epithelial injury score, corneal stromal turbidity score, CNV area and number of CD31-positive cells among various time points after alkali burn ( Fgroup=49.19, 34.56, 44.56, 77.56; all at P<0.01; Ftime=51.62, 65.62, 71.32, 46.12; all at P<0.01). Corneal epithelial injury score, corneal stromal turbidity score, CNV area and the number of CD31-positive cells were greater in KO group at various time points than in WT and TG groups, and those in WT group were greater than in TG group (all at P<0.05). In WT STAT3 co-transfected cells, the luciferase activity of the miR-497 group was significantly lower than that of the miR-negative control group and normal control group (both at P<0.05). In mutant STAT3-transfected cells, there was no significant difference in luciferase activity among all groups ( F=0.69, P=0.56). On the 14th day after modeling, the relative expression levels of miR-497 in corneal tissue of WT, KO and TG groups were 0.68±0.11, 0.41±0.06 and 1.05±0.14, respectively, which were significantly lower than 1.00±0.04, 0.56±0.07 and 1.34±0.11 before modeling (all at P<0.01). The relative expressions of STAT3 and p-STAT3 were higher in KO group than in WT and TG groups, and were lower in TG group than in WT group, and the differences were statistically significant (all at P<0.05). The expressions of VEGFA, TNF-α, IL-6, IL-1β and MCP-1 mRNA at various time points after modeling in various groups were significantly higher than before modeling, which were higher in KO group than in WT and TG groups and were lower in TG group than in WT group, and the differences were statistically significant (all at P<0.01). Conclusions:MiR-497 inhibits corneal inflammation and CNV formation induced by alkali burn.It might inhibit the activation of the inflammation signal pathway via targeting STAT3.

Advances in corneal nerve regulation of ocular surface microenvironment / 国际眼科杂志(Guoji Yanke Zazhi)

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.

Risk of induction of corneal neovascularization with topical erythropoietin: an animal safety study

Purpose: To evaluate the pro-angiogenic effect of topical erythropoietin on cornea in chemical burn-injured rabbit eyes. Methods: The corneal alkali-burn injury was induced in 10 eyes of 10 rabbits using filter paper saturated with 1.0 mol sodium hydroxide. The eyes were categorized into the treatment group (n = 5) that received topical erythropoietin (3000 IU/mL) every 8 hr for one month versus the control group (n = 5) that received normal saline every 8 hr for one month. All eyes were treated with topical ciprofloxacin every 8 hr until corneal re-epithelialization was complete. Corneal epithelial defects, stromal opacity, and neovascularization were evaluated after the injury. At the conclusion of the study, the rabbits were euthanized and their corneas were submitted to histopathological examination. Results: Baseline characteristics including the rabbits' weight and the severity of corneal injury were comparable in two groups. Time to complete corneal re-epithelialization was 37 days in the treatment group and 45 days in the control group (P = 0.83). There was no significant difference between the groups in the rate of epithelial healing or corneal opacification. Clinical and microscopic corneal neovascularization was observed in one eye (20%) in the treatment group and two eyes (40%) in the control group (P = 0.49). Conclusion: Recombinant human erythropoietin administered topically did not induce vessel formation in rabbit corneas after chemical burn.

Comparison of the inhibitory effect of topical cyclosporine A 0.1% and topical anti-VEGF application in an experimental model of corneal neovascularization / Comparação do efeito inibitório da ciclosporina A 0,1% tópica e aplicação de anti-VEGF tópica em um modelo experimental de neovascularização da córnea

ABSTRACT Purpose: The aim of this study was to compare the effects of topical cyclosporine 0.1% and bevacizumab on experimentally induced corneal neovascularization in a rat model. Methods: A total of 30 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 as follows: Group 1 received bevacizumab 1%, Group 2 received cyclosporine 0.1%, and Group 3 received isotonic saline twice a day for 28 days. Slit-lamp examination of all rats was performed at the 3rd and 28th day. The rats were then sacrificed, and the corneas were excised. The number of blood vessels, state of inflammation, and collagen formation were evaluated histopathologically in the corneal sections. Results: Corneal opacity and edema grades were significantly lower in Group 2 than in Group 3 (p=0.04 and 0.00, respectively). In the histopathological examination, Group 2 demonstrated significantly lesser number of blood vessels than Group 3 (p=0.001). Regarding collagen formation, Group 2 exhibited more regular collagen formation than Groups 1 and 3 (p=0.03). Inflammation grades were significantly lower in Groups 1 and 2 than in Group 3 (p=0.014 and 0.001, respectively). Conclusion: Topical bevacizumab is effective in inhibiting newly formed corneal neovascularization. The topical cyclosporine 0.1% treatment appears to be more effective than the topical bevacizumab treatment.

RESUMO Objetivo: Comparar os efeitos da ciclosporina tópica 0,1% e do bevacizumabe na neovascularização da córnea produzida experimentalmente em um modelo com ratos. Métodos: Trinta ratos Sprague-Dawley adultos foram usados neste estudo experimental. A córnea central dos ratos foi cauterizada quimicamente. Os ratos foram distribuídos aleatoriamente em três grupos. O grupo 1 recebeu bevacizumabe a 1%, o grupo 2 recebeu ciclosporina tópica a 0,1% e o grupo 3 recebeu solução salina isotônica duas vezes ao dia durante 28 dias. O exame de lâmpada de fenda de todos os ratos foi realizado no terceiro e no vigésimo oitavo dias. Os ratos foram então sacrificados e as córneas excisadas. Nos cortes da córnea, o número de vasos sanguíneos, o estado de inflamação e a formação de colágeno foram avaliados em uma análise anatomopatológica. Resultados: No Grupo 2, os graus de opacidade e de edema da córnea foram significativamente menores que no Grupo 3 (p=0,04 e 0,00, respectivamente). No exame histopatológico, o Grupo 2 apresentou um número significativamente menor de vasos sanguíneos do que o Grupo 3 (p=0,001). Em relação à avaliação da formação de colágeno, esta mostrou-se mais regular no Grupo 2 que no Grupo 1 e no Grupo 3 (p=0,03). Os graus de inflamação foram significativamente menores no Grupo 1 e no Grupo 2 em comparação com o Grupo 3 (p=0,014 e 0,001, respectivamente). Conclusão: O bevacizumabe tópico é eficaz na inibição da neovascularização da córnea recém-formada. O tratamento tópico com ciclosporina a 0,1% parece ser mais eficaz em comparação ao tratamento tópico com bevacizumabe.

Inhibitory effect of vorinostat encapsulated hydroxypropyl-β-cyclodextrin eye drops on corneal neovascularization induced by alkali burns in mouse / 中华实验眼科杂志

Objective:To prepare vorinostat encapsulated hydroxypropyl-β-cyclodextrin (SAHA-CD) eye drops and investigate its inhibitory effect on corneal neovascularization (CNV) induced by alkali burns in mouse.Methods:The SAHA-CD eye drops at concentrations of 0.1%, 0.2%and 0.4%were prepared by inclusion technology with hydroxypropyl-β-cyclodextrin, and the content was assayed by high performance liquid chromatography.Seventy-five SPF mice with alkali burn-induced CNV were randomized into 0.1%SAHA-CD group, 0.2%SAHA-CD group, 0.4%SAHA-CD group, dexamethasone group and normal control group according to a random number table, 15 for each group, among which the SAHA-CD groups and dexamethasone group were treated with corresponding drugs, and model control group was treated with normal saline immediately after modeling, four times a day and five microliters each time, lasting for six days.The healing of corneal epithelium was examined with a slit lamp microscope after fluorescein sodium staining, and the areas of cornea epithelial defects were calculated using Eyestudio software.The corneal flat mount was prepared, and the length and areas of CNV were calculated with ImageJ software.The histology of mouse corneas was observed through hematoxylin and eosin staining.The expression level of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and matrix metalloproteinase-9 (MMP-9) in cornea were measured with enzyme linked immunosorbent assay (ELISA) kits.The use and care of animals complied with the ARVO statement and this study protocol was approved by the Experimental Animal Ethics Committee of Henan Eye Institute (No.HNEECA-2020-01).Results:The actual drug contents of the 0.1%, 0.2% and 0.4%SAHA-CD eye drops were 97.62%, 98.33%and 98.14%of the labeled amount.The cornea showed edema and opacification after modeling.On the sixth day after treatment, significant differences were found in the length and areas of CNV among various groups ( F=7.655, 8.802; both at P<0.01).The areas of CNV in 0.2%SAHA-CD, 0.4%SAHA-CD and dexamethasone groups were significantly smaller than model control group, and the length of CNV in 0.1%SAHA-CD, 0.2%SAHA-CD and dexamethasone groups were significantly smaller than model control group (all at P<0.05).On the third and sixth day following modeling, significant differences in the expression levels of VEGF, bFGF and MMP-9 were found among the five groups (third day: F=6.345, 7.149, 18.650; all at P<0.01; sixth day: F=6.749, 5.105, 5.023; all at P<0.01), and the expression levels of VEGF, bFGF and MMP-9 in 0.2%SAHA-CD group were significantly lower than those in 0.1%SAHA-CD group, 0.4%SAHA-CD group and model control group (all at P<0.05). Conclusions:SAHA-CD eye drops can inhibit alkali burn-induced CNV in mouse.

Progress of the application of Conbercept in ocular neovascular diseases / 国际眼科杂志(Guoji Yanke Zazhi)

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.

Research progress of β-adrenergic receptor antagonists in the treatment of ocular neovascular diseases / 中华眼底病杂志

Ocular neovascularization is a pathological change in various ocular diseases such as diabetic retinopathy, retinopathy of prematurity, central retinal vein occlusion and age-related macular degeneration, which seriously affects patient's vision. β receptors are expressed in conjunctiva, corneal epithelial cells, corneal endothelial cells, extraocular muscles, trabecular meshwork, ciliary muscle, lens and retina. β adrenergic receptor antagonists bind to β receptors to exert anti-angiogenic effects by inhibiting the expression of vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1, interleukin-6 and other angiogenic cytokines; reducing macrophage-related inflammatory response; increasing the expression of anti-angiogenic factors. In the treatment of corneal neovascularization, choroidal neovascularization, and retinopathy of prematurity, it can significantly reduce the area of neovascularization and delay disease progression. Co-administration of anti-VEGF drugs can reduce the frequency of administration of anti-VEGF drugs. At effective therapeutic concentrations, β-adrenergic receptor antagonists are well tolerated; they have broader targets than anti-VEGF drugs, which offers new treatment strategies for ocular neovascularization such as corneal, choroidal and retinal neovascularization.

Research progress of cells and molecules in corneal neovascularization / 国际眼科杂志(Guoji Yanke Zazhi)

@#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.

Curative effect of pterygium resection and autologous limbal stem cell transplantation combined with amniotic membrane transplantation in the treatment of pterygium / 国际眼科杂志(Guoji Yanke Zazhi)

@#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.<p>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.<p>RESULTS: Visual acuity changes and repair time of corneal epithelial defect showed no statistically significant difference among the three groups(<i>P</i>>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 <i>P</i><0.05). No angiogenesis or recurrent true pterygium was observed. 6mo and 12mo after surgery, the grades of conjunctival fibroplasia in group A and group C were significantly different from that in group B(<i>P</i><0.05). There was no statistically significant difference in the wet length of the filter paper in Schirmer I test in terms of time, inter-group and interaction effects(<i>P</i>>0.05). 1mo after surgery, the tear film breakup time(BUT)of group C was significantly longer than that of group A or group B(all <i>P</i><0.05). There were different degrees of conjunctival edema in the three groups after surgery, which disappeared within 2wk after suture removal. Grafts all survived, vascularization of amniotic membrane grafts ended.<p>CONCLUSION:Autologous LSCT, AMT and autologous LSCT combined with AMT all are effective in the treatment of pterygium. However, autologous LSCT combined with AMT can achieve better short-term effect, with milder conjunctival fibroplasia and dry eye symptoms.

Inhibition of Glycyrrhizin on acute alkali burn induced corneal neovascularization in mice / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To evaluate the inhibitory effect of glycyrrhizin(Gly)on acute alkali burn induced corneal neovascularization(CNV)in mice. <p>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. <p>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 <i>P</i><0.05). The HE pathological staining showed that there were no changes in morphology as well as no neovascularization or inflammatory cell infiltration in the cornea of control group. In the Gly group, blood vessels and inflammatory cell infiltration nearly diminished with collagen in normal shape. While in the PBS group, extensive infiltration of inflammatory cells and neovascularization was examed in the corneal stroma. The immunohistochemical CD34 staining performed that the MVD in the Gly group was 11.13±1.46 bars per square millimeter, which was lower than that in PBS group(34.08±2.46)bars per square millimeter(<i>P</i><0.001). Additionally, the immunohistochemical MPO staining showed that the number of neutrophils in Gly group was 17.50±1.98 cells per 200-fold field of view, lower than that in PBS group(59.56±4.79, <i>P</i><0.001). <p>CONCLUSION: 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.

In vivo investigation of the effectiveness of reactive oxygen species-responsive nanomedicine in suppressing corneal neovascularization / 中华实验眼科杂志

Objective To evaluate the effectiveness of reactive oxygen species (ROS)-responsive nanomedicine in suppressing corneal neovascularization (CNV) in vivo.Methods ROS-responsive nanomedicine (ROS-TK-5/siVEGF),which consists of vascular endothelial growth factor (VEGF) small interfering RNA (siRNA) and thioketal linkage was synthesized by the Michael addition.The cumulative release of siVEGF from nanomedicine under oxidant conditions was assessed by agarose gel electrophoresis.Thirty-nine VEGFR2-1uc-KI transgenic mice were used in this study,of which 30 mice were randomly divided into a normal control group,a PBS control group,an ROS-TK-5/NC group,an ROS-TK-5/siVEGF group,and a ranibizumab group,with 6 mice in each group.The ROS levels in the corneal tissue after alkali burning were tested by dihydroethidium (DHE) staining in the other 9 mice.In each group,alkali-burned mice were subconjunctivally injected with 10 μl of a different formula every two days.The effectiveness of nanomedicine in attenuating CNV was evaluated by slit-lamp microscopy and an in vivo imaging system (IVIS) at 7,14,and 21 days after alkali burning.The use and care of the animals complied with the Statement of the Association for Research in Vision and Ophthalmology (ARVO) and the Guidelines of the Animal Experimental Committee of Liberation Army General Hospital.The study protocol was approved by the Ethics Committee of Liberation Army General Hospital (No.2018-X14-82).Results After treathrent with an aqueous solution without ROS,only 5％-10％ of the siVEGF was released from the nanoparticles within 10 hours.In contrast,about 70％ of the siVEGF was released from the nanoparticles after treatment with 10 mmol/L H2O2 within 10 hours.The relative fluorescent intensities in the corneal stromal layer at 7 days and 14 days after alkali burning were 5.403±0.306 and 2.930±0.255,respectively,which was significantly greater than those in the normal control group (1.003±0.015) (both at P＜0.05).The CNV areas were statistically different among the four groups at various time points (Fgroup =49.855,P＜0.01;Ftime =65.556,P＜0.01).The CNV area was significantly reduced in the ROS-TK-5/siVEGF and ranibizumab groups compared with the PBS control and ROS-TK-5/NC groups at 7 days and 14 days after modeling,and the CNV area was more effectively reduced in the ROS-TK-5/siVEGF group than the ranibizumab group at 7 days and 14 days after modeling (all at P＜0.05).At day 21 after modeling,the CNV area was significantly reduced in the ROS-TK-5/siVEGF and ranibizumab groups compared to the PBS control and ROS-TK-5/NC groups (all at P＜ 0.05).IVIS showed that the corneal fluorescent intensity was statistically different among the four groups at various times (Fgroup =27.193,P =0.003;Ftime =51.062,P ＜ 0.01).The corneal fluorescent intensities were significantly reduced in the ROS-TK-5/siVEGF and ranibizumab groups compared to the PBS control and ROS-TK-5/NC groups at 7 days and 14 days after modeling;in addition,the corneal fluorescent intensity was more effectively reduced in the ROS-TK-5/siVEGF group in comparison with the ranibizumab group at 7 days and 14 days after modeling (all at P＜ 0.05).At 21 days after modeling,the corneal fluorescent intensity was significantly reduced in the ROS-TK-5/ siVEGF and ranibizumab groups compared to the PBS control and ROS-TK-5/NC groups (all at P ＜ 0.05).Conclusions ROS-TK-5/siVEGF nanomedicine effectively attenuates alkali burn-induced CNV formation and appears to have a better effect in comparison with ranibizumab at an early stage.

In vivo investigation of the effectiveness of reactive oxygen species-responsive nanomedicine in suppressing corneal neovascularization / 中华实验眼科杂志

Objective@#To evaluate the effectiveness of reactive oxygen species (ROS)-responsive nanomedicine in suppressing corneal neovascularization (CNV) in vivo.@*Methods@#ROS-responsive nanomedicine (ROS-TK-5/siVEGF), which consists of vascular endothelial growth factor (VEGF) small interfering RNA (siRNA) and thioketal linkage was synthesized by the Michael addition.The cumulative release of siVEGF from nanomedicine under oxidant conditions was assessed by agarose gel electrophoresis.Thirty-nine VEGFR2-luc-KI transgenic mice were used in this study, of which 30 mice were randomly divided into a normal control group, a PBS control group, an ROS-TK-5/NC group, an ROS-TK-5/siVEGF group, and a ranibizumab group, with 6 mice in each group.The ROS levels in the corneal tissue after alkali burning were tested by dihydroethidium (DHE) staining in the other 9 mice.In each group, alkali-burned mice were subconjunctivally injected with 10 μl of a different formula every two days.The effectiveness of nanomedicine in attenuating CNV was evaluated by slit-lamp microscopy and an in vivo imaging system (IVIS) at 7, 14, and 21 days after alkali burning. The use and care of the animals complied with the Statement of the Association for Research in Vision and Ophthalmology (ARVO) and the Guidelines of the Animal Experimental Committee of Liberation Army General Hospital.The study protocol was approved by the Ethics Committee of Liberation Army General Hospital (No.2018-X14-82).@*Results@#After treathrent with an aqueous solution without ROS, only 5%-10% of the siVEGF was released from the nanoparticles within 10 hours.In contrast, about 70% of the siVEGF was released from the nanoparticles after treatment with 10 mmol/L H2O2 within 10 hours.The relative fluorescent intensities in the corneal stromal layer at 7 days and 14 days after alkali burning were 5.403±0.306 and 2.930±0.255, respectively, which was significantly greater than those in the normal control group (1.003±0.015) (both at P<0.05). The CNV areas were statistically different among the four groups at various time points (Fgroup=49.855, P<0.01; Ftime=65.556, P<0.01). The CNV area was significantly reduced in the ROS-TK-5/siVEGF and ranibizumab groups compared with the PBS control and ROS-TK-5/NC groups at 7 days and 14 days after modeling, and the CNV area was more effectively reduced in the ROS-TK-5/siVEGF group than the ranibizumab group at 7 days and 14 days after modeling (all at P<0.05). At day 21 after modeling, the CNV area was significantly reduced in the ROS-TK-5/siVEGF and ranibizumab groups compared to the PBS control and ROS-TK-5/NC groups (all at P<0.05). IVIS showed that the corneal fluorescent intensity was statistically different among the four groups at various times (Fgroup=27.193, P=0.003; Ftime=51.062, P<0.01). The corneal fluorescent intensities were significantly reduced in the ROS-TK-5/siVEGF and ranibizumab groups compared to the PBS control and ROS-TK-5/NC groups at 7 days and 14 days after modeling; in addition, the corneal fluorescent intensity was more effectively reduced in the ROS-TK-5/siVEGF group in comparison with the ranibizumab group at 7 days and 14 days after modeling (all at P<0.05). At 21 days after modeling, the corneal fluorescent intensity was significantly reduced in the ROS-TK-5/siVEGF and ranibizumab groups compared to the PBS control and ROS-TK-5/NC groups (all at P<0.05).@*Conclusions@#ROS-TK-5/siVEGF nanomedicine effectively attenuates alkali burn-induced CNV formation and appears to have a better effect in comparison with ranibizumab at an early stage.

Subconjunctival bevacizumab and argon laser photocoagulation for preexisting neovascularization following deep lamellar anterior keratoplasty

We report a rare case of deep anterior lamellar keratoplasty (DALK) neovascularization managed with combination of subconjunctival bevacizumab and argon laser photocoagulation. A 24 year old male underwent Deep anterior lamellar keratoplasty for corneal stromal opacity following presumed viral keratitis. Deep corneal neovascularization was observed postoperatively which was successfully managed using a combination of subconjunctival bevacizumab and argon laser photocoagulation within one week of DALK. The neovascularization resolved by 3 months and at 2 years follow up, patient maintained good visual acuity of 6/12 Snellen's without recurrence of vascularization. A combination of bevacizumab and argon laser may be an effective approach to manage neovascularisation in the immediate postoperative phase (Post DALK) and improve graft survival.

Inhibitory effect of bromfenac sodium hydrate ophthalmic solution on alkali burn induced-corneal neovascularization in rats / 中华实验眼科杂志

Objective To investigate the inhibitory effect of bromfenac sodium hydrate ophthalmic solution on corneal neovascularization (CNV) induced by alkali burn.Methods A total of 192 specific pathogen free (SPF) degree adult male Sprague-Dawley (SD) rats were used in this study.One hundred and seventy-two rats were chosen to establish CNV model with alkali burn in the right eyes.Following alkali burn,rats were randomly divided into CNV group,model control group,bromfenac sodium group and fluorometholone group,with 43 rats (43 eyes) in each group.Another 20 rats (40 eyes) served as normal control group.One day after modeling,the model control group,bromfenac sodium group and fluorometholone group received phosphate buffer saline (PBS),bromfenac sodium hydrate ophthalmic solution and 0.1％ fluorometholone eye drops,respectively.The state of cornea and anterior chamber and the growth of CNV of rats in each group were observed by slit-lamp microscope every day after modeling.At 1,3,7,14,21 and 28 days after modeling,the anterior segment photos of the experimental eyes were captured,and the percent of cornea areas covered by CNV was calculated.At 7,14 and 28 days after modeling,the eye tissue sections were stained with hematoxylin and eosin staining and immunohistochemistry staining to evaluate the expressions of CD45 and VEGF-A.Real-time quantitative PCR and enzyme-linked immunosorbent assay (ELISA)were used to detect the expression of COX-2 and VEGF mRNA and protein level.The use and care of the animals complied with the Statement of the Association for Research in Vision and Ophthalmology(ARVO).Results Each model group showed corneal edema and opacification 1 day after modeling.The corneal edema was aggravated 7 days after modeling.On the 14th day after modeling,the degree of corneal opacity and edema decreased gradually.On the 28th day after modeling,leucoma was observed in CNV group and model control group,and nebula was observed in bromfenac sodium group and fluorometholone group.At 7,14,21 and 28 days after modeling,the percentages of CNV areas in bromfenac sodium group and fluorometholone group were significantly lower than those in CNV group and model control group (all at P＜0.05).No significant difference was found in the percentage of CNV areas between bromfenac sodium group and fluorometholone group at various time points (all at P＞0.05).On the 7th day after modeling,the thinning of corneal epithelial layer,edema and arrangement disorder of stroma layer were observed,and the expression of VEGF-A was positive in all model groups;a small amount of CD45 positive inflammatory cell infiltrations were observed in CNV group and model control group.On the 14th and 28th day after modeling,CNV was seen in the center of cornea in CNV group and model control group;the epithelial keratosis and reduction of corneal edema were seen in each group,and no inflammatory cell infiltration was observed in each group.On the 7th day after modeling,the expressions of COX-2 and VEGF mRNA in CNV group and model control group were significantly higher than those in normal control group,bromfenac sodium group and fluorometholone group (all at P ＜ 0.05),the expressions of COX-2 and VEGF protein in bromfenac sodium group were significantly lower than those in CNV group (all at P＜0.05).The corneal peroration rate in model control group and bromfenac sodium group was 10％ (1 case in 10 rats).The corneal perforation rate in fluorometholone group was 30％ (3 cases in 10 rats).In each model group,10％ to 30％ rats had hyphema.Conclusions Bromfenac sodium hydrate ophthalmic solution can inhibit the formation and growth of CNV after alkali burn in rats.This effect may be mediated by regulating COX-2 expression,reducing inflammation and inhibiting VEGF production.

Application progress of transgenic mouse models of corneal neovascularization / 国际眼科杂志(Guoji Yanke Zazhi)

@#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.

Imaging methods of corneal neovascularization / 国际眼科杂志(Guoji Yanke Zazhi)

@#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.

The dynamic expression of CD146 in corneal neovascularization and its significance / 中华实验眼科杂志

Objective To investigate the expression and localization of CD146 in corneal neovascularization.Methods Fifty SPF male SD rats with the age of 8 weeks were randomly divided into 1 day group,3 days group,7 days group,14 days group and normal control group by using random number table method,10 rats for each group.Corneal neovascularization models were established by sticking the filter papers with NaOH on the central corneas.The area of corneal neovascularization was observed at different time points by slit lamp microscopy.The expression of CD146 mRNA and protein were detected by reverse transcription PCR and Western blot,respectively.The location of CD146 expression was observed by using immunohistochemical staining method.The animal feeding and use was in accordance with the standards set by the ARVO,and the experiment was approved by the Ethic Committee for Experimental Animal of Affiliated Hospital of Xuzhou Medical University (2016012).Results Corneal neovascularization occurred at 3 days and peaked at 7 days after alkali burn,then gradually subsided.The area of cornealneovascularization was (1.9±0.7),(10.3±1.1),(29.6±2.4) and (11.8±1.0)mm2 in 1 day,3 days,7 days and 14 days after alkali burn,respectively,the overall difference was statistically significant (F =650.976,P =0.000),and the area of corneal neovascularization in 1 day group,3 days group,14 days group was significantly shrinked compared with that in the 7 days group after alkali burn,with significant differences between them (t =-33.293,-20.475,20.744,all at P =0.000),while there was no significant differences between the other groups (all at P＞0.05).The relative expression levels of CD146 mRNA in the 1 day,3 days,7 days and 14 days group after alkali burn was 0.3±0.1,1.1±0.2,3.5±0.4 and 1.3±0.3,and the relative expression levels of CD146 protein was 0.2±0.1,1.4 ± 0.2,4.1 ± 0.5 and 1.3 ± 0.2,respectively,the overall differences between the four groups were statistically significant (F =1 176.920,P =0.000;F =233.127,P =0.000),and the relative expression levels (o)(f) CD146 mRNA and protein in the 1 day,3 days,7 days and 14 days group were lower than those in the 7 days group after alkali burn,with significant differences between them (mRNA:t =-58.109,-33.725,31.006;all at P =0.000.protein:t =-59.873,-38.762,39.153,all at P =0.000).Immunohistochemical results showed that CD146 was highly expressed in corneal neovascular endothelial cells at 7 days after alkali burn,forming lumen structure,while only weak expression of CD146 could be detected in mature corneal neovascularization at 14 days after modeling.Conclusions CD146 is closely related to corneal neovascularization formation,and it is promising as a new target for treatment.

Inhibitory effect of Tum5 on angiogenic activity of human umbilical vein endothelial cells and corneal neovascularization induce by alkali burn in rats / 眼科新进展

Objective To investigate the inhibitory effects of Tum5 on the angiogenesis of human umbilical vein endothelial cells (HUVECs) and alkali-induced corneal neovascularization.Methods HUVECs in logarithmic growth phase were divided into 4 groups,cells with untreated as normal control group,cells with the infection of rAdGFP virus as rAd-GFP group,cells with the infection of rAd-Tum5 virus as rAd-Tum5 group,and cells with the infection of rAd-Tum5 virus followed by VEGF treatment as rAd-Tum5 + VEGF group.Then cell proliferation,migration,and tube formation of HUVECs were examined by CCK-8,Transwell and Matrigel assays,respectively.Sixty-four healthy male SD rats were randomly divided into 4 groups (n =16) by using random number table,and they were normal control group,alkali-burn group,alkali-burn + rAd-GFP group,and alkali-burn + rAd-Tum5 group.The alkali-burn rat model was then established except normal control group,and the normal control group received no treatment,whereas the alkali-burn,alkali-burn + rAd-GFP,and alkali-burn + rAd-Tum5 groups received subconjunctival injection of equal volumes of sterilized saline,rAd-GFP virus,and rAd-Tum5 virus,respectively following the alkaline burn.The relative area of corneal neovascularization and the number of infiltrating inflammatory cells were recorded on day 1,7,and 14 after injection.Results The CCK-8 assay showed that the proliferative rate of rAd-Tum5 group was lower than that of the normal control group and rAd-GFP group (both P ＜0.01),while rAd-Tum5 + VEGF group exhibited a significantly greater cell proliferative capability than rAd-Tum5 group (P =0.004).There were no statistical differences between rAd-Tum5 + VEGF group,normal control group and rAdGFP group (all P ＞ 0.05).Transwell assay showed the significantly lower number of migrating cells in the rAd-Tum5 group than those in the normal control group and rAdGFP group (both P ＜ 0.01).The number of migrating cells in rAd-Tum5 + VEGF group was higher than those in rAd-Tum5 group (P =0.000);however,the migration capacity had not been restored to normal level,and rAd-Tum5 + VEGF group had significant difference with normal control group and rAd-Tum5 group (both P ＜0.05).Matrigel assay showed that the number of meshes in rAd-Tum5 group was lower than that in the normal control group and rAd-GFP group (both P ＜0.01);while the number of meshes in rAd-Tum5 + VEGF group was significantly increased compared with rAd-Tum5 group(P =0.001).The density and number of corneal neovascularization increased gradually from day 1 to day 14 after alkali burn,while the relative neovascularization area in the alkali-burn + rAd-Tum5 group was significantly reduced as compared to those in the alkali-burn group and alkali-burn + rAd-GFP group on day 7 and day 14,suggesting that Tum5 could reduce the growth rate and density of corneal neovascularization,so as to inhibit corneal neovascularization induced by alkali burn.On day 7 and 14 after alkali burn,in the normal control group,the corneas were intact,no infiltrating inflammatory cells and cells were arranged in an orderly manner.On day 7 after alkali burn,there were disordered epithelial structure,corneal edema and infiltrating inflammatory cells in alkali-burn group and alkali-burn + rAd-GFP group.The number of infiltrating inflammatory cells in alkaliburn + rAd-Tum5 group was significantly lower than that in alkali-burn group and alkali-burn + rAd-GFP group (both P ＜0.01).On day 14 after alkali burn,the number of infiltrating inflammatory cells in alkali-bum,alkali-burn + rAd-GFP and alkali-burn + rAd-Tum5 group were significantly lower than those on day 7,while the corneal epitheliums were intact and dropsy was alleviated,while the number of inflammatory cells was significantly lower than that in alkali burn group and alkali-burn + rAd-GFP group,with significant difference (both P ＜ 0.01).Conelusion Tum5 can inhibit the angiogenic capability of HUVECs by VEGF pathway,as well as suppress the alkali-burn-induced corneal neovascularization and inflammatory cell infiltration in rats.

Inhibitory effects of hydrogen on corneal neovascularization in rats with alkali burn / 眼科新进展

ObJective To observe the inhibitory effects of hydrogen on corneal neovascularization (CNV) induced by alkali burn in rats and its underlying mechanisms Methods A total of 48 SPF-grade SD rats (48 eyes) were selected and randomly divided into blank group,control group and treatment group.Then the model of CNV was established successfully induced by alkali burn in these animals.The treatment group was given 1.6 × 10-6 hydrogen eye drops,the control group received tobramycin dexamethasone eye drops,and blank group left untreated.And CNV length was measured on day 1,3,7 and 14 after model establishment,followed by calculation of the CNV area.The corneal and aqueous humor of the rats was removed on day 14 after alkali burn.Then PEDF protein was detected by immunohistochemical staining,and RT-PCR was used to detect the expression of PEDF mRNA in the cornea.Additionally,TNF-α in the aqueous humor was detected by enzyme e-linked immunosorbent assay (ELISA).Resuits The area of corneal neovascularization on day 3,7 and 14 after alkali burn was (3.26 ± 1.82) mm2,(8.59 ± 3.98)mm2 and (3.24 ± 2.34) mm2 in the treatment group,respectively,(4.27 ± 2.68) mm2,(6.44 ± 4.67) mm2 and (15.98 ± 4.75) mm2 in the control group,respectively,(9.49 ±2.79)mm2,(17.71 ±4.06)mm2 and (25.35 ± 1.40)mm2 in blank group,respectively.The CNV areas of the treatment group and the control group were less than that of the blank group on day 3,7 and 14 after alkali bum,and the differences were statistically significant (all P ＜ 0.01),but there was no significant difference between the treatment group and the control group on day 3,7 after alkali bum (P ＞ 0.05),while the area of the treatment group was significantly less than that of the control group on 14th day,and the difference was statistically significant (P ＜0.01).On day 14 after alkali bum,the results of immunohistochemistry showed that the expression of PEDF protein in corneal epithelium was significantly increased in the treatment group than that in the control group and the blank group,and there were few new blood vessels in the stromal layer.RT-PCR results showed that the expression of PEDF mRNA in the treatment group was significantly higher than that in the control group and the blank group,and the difference was statistically significant (all P ＜ 0.01).ELISA results showed that the content of TNF-α protein in the treatment group was significantly lower than that in the control group and the blank group,and the difference was statistically significant (all P ＜0.01).Conclusion 1.6 × 10-6 hydrogen eye drops can effectively inhibit the corneal neovascularization which induced by alkali burn in rats.