The expression of VEGF-C and molecular mechanisms of lymphangiogenesis in rat cornea after alkali injury was studied. The rat alkali injured corneal models were made. Under electron microscopy, the lymphatic vessels i...The expression of VEGF-C and molecular mechanisms of lymphangiogenesis in rat cornea after alkali injury was studied. The rat alkali injured corneal models were made. Under electron microscopy, the lymphatic vessels in the rat injured corneas were examined. The expression of VEGF-C proteins was detected by using immunohistochemical assay at day 1, 3, 5, 7, 9 after injury. The expression levels of VEGF-C mRNA were quantified with reverse transcription polymerase chain reaction (RT-PCR). The results showed that the lymphatic vessels were found in the injured rat corneas 14 days after the injury. The VEGF-C protein was detectable 3 days after injury, reached the peak 5 days after injury, and gradually decreased. In the control group, no VEGF-C proteins were detected. The VEGF-C mRNA was minimally detected in the normal rat corneas, but it was highly expressed 5 days after the injury. The difference was statistically significant. It was concluded that VEGF-C might be one of the most important relevant factors in corneal lymphangiogenesis after alkali injury.展开更多
BACKGROUND: The transplantation of limbal epithelial cells cultivated on amniotic membrane is a newly developed treatment for limbal stem cell deficiency. The purpose of our study was to investigate the biological cha...BACKGROUND: The transplantation of limbal epithelial cells cultivated on amniotic membrane is a newly developed treatment for limbal stem cell deficiency. The purpose of our study was to investigate the biological characteristics of limbal epithelial cells and evaluate the effect of transplantation of cultivated human limbal epithelial cells on ocular surface reconstruction in limbal stem cell deficiency rat model. METHODS: Human limbal cells were isolated and cultivated in vitro. Cytokeratins 3, 12, and 19 (K3, K12 and K19) and p63 were detected by immunofluorescent staining or RT-PCR. BrdU labelling test was used to identify the slow cycling cells in the cultures. Limbal stem cell deficiency was established in rat cornea by alkali burn. Two weeks after injury, the rats received transplants of human limbal stem cells cultivated on amniotic membrane carrier. The therapeutic effect was evaluated by slit lamp observation, Hemotoxin and Eosin (HE) staining and immunofluorescent staining. RESULTS: On day 7 in primary culture, p63 and K19 were strongly expressed by most cells but only a few cells expressed K3. On days 14 and 21, p63 and K19 were still expressed by a majority of cells, but the expressive intensity of p63 decreased in a number of cells, while the proportion of K3 positive cells increased slightly and some cells coexpressed p63 and K3. RT-PCR showed that gene expression of both p63 and K12 were positive in cultivated limbal cells, but in mature superficial epithelial cells, only K12 was detected. BrdU labelling test showed that most cells were labelled with BrdU after 7 days' labelling and BrdU label retaining cells were observed after chasing for 21 days with BrdU free medium. For in vivo test, slit lamp observation, HE staining and immunofluorescent staining showed that the rats receiving transplant of human limbal stem cells cultivated on amniotic membrane grew reconstructed corneas with intact epithelium, improved transparency and slight or no neovascularization. A majority of epithelial cells of the reconstructed cornea were positive to antihuman nuclear antibody and cells expressing K3 were found mainly in superfacial epithelium. CONCLUSIONS: Limbal stem cells can be cultivated in vitro: the cells are characterized by high proliferation and slow cycling and identified as p63/K19 positive and K3/K12 negative. During culture, some stem cells can proliferate and differentiate into mature cornea epithelial cells. Amniotic membrane is a suitable carrier for limbal stem cells. Transplantation of human limbal stem cells cultivated on amniotic membrane can functionally reconstruct rat cornea with limbal stem cell deficiency.展开更多
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-Da...AIM： To evaluate the inhibitory effect of subconjunctival bevacizumab as single-and multiple-dose application, and compare their effects on corneal neovascularization in a rat model. METHODS： Thirty adult Sprague-Dawley rats were used in this experimental study. The central cornea of the rats was cauterized chemically. The rats were randomly enrolled into three groups. All groups received subconjunctival injections. In Group 1（control group, n=10）, 0.05 m L 0.9% Na Cl solution was injected on the first day. In Group 2（single-dose group, n=10）, 0.05 m L bevacizumab（1.25 mg） was injected on the first day. In Group 3（multiple-dose group, n=10）, four doses of 0.05 m L bevacizumab（1.25 mg） were injected on the first, third, fifth and seventh day. Slit-lamp examination of all rats was performed at the third and ninth day. Digital images of the corneas were taken and analyzed using image analysis software to calculate corneal neovascularization area. All rats were sacrificed on the tenth day. In corneal sections, the number of blood vessels, state of inflammation and collagen formation was evaluated histopathologically. RESULTS： In Group 3, corneal edema grades were significantly lower than Group 1 and Group 2（P=0.02, and P=0.035, respectively）. The mean percentage of neovascularized corneal area in Group 3 was significantly lower than Group 2（P=0.005）. On histopathological examination, Group 2 and Group 3 showed significantly less number of blood vessels than Group 1（P=0.005, and P=0.001, respectively）. Additionally, Group 3 showed significantly less number of blood vessels compared to Group 2（P=0.019）. Inflammation and edema grades were significantly lower in Group 3 compared to Group 1（P=0.001）. CONCLUSION： Subconjunctival bevacizumab injection is effective in inhibition of newly formed corneal neovascularization. The multiple-dose bevacizumab treatment seems to be more effective compared to single-dose treatment.展开更多
文摘The expression of VEGF-C and molecular mechanisms of lymphangiogenesis in rat cornea after alkali injury was studied. The rat alkali injured corneal models were made. Under electron microscopy, the lymphatic vessels in the rat injured corneas were examined. The expression of VEGF-C proteins was detected by using immunohistochemical assay at day 1, 3, 5, 7, 9 after injury. The expression levels of VEGF-C mRNA were quantified with reverse transcription polymerase chain reaction (RT-PCR). The results showed that the lymphatic vessels were found in the injured rat corneas 14 days after the injury. The VEGF-C protein was detectable 3 days after injury, reached the peak 5 days after injury, and gradually decreased. In the control group, no VEGF-C proteins were detected. The VEGF-C mRNA was minimally detected in the normal rat corneas, but it was highly expressed 5 days after the injury. The difference was statistically significant. It was concluded that VEGF-C might be one of the most important relevant factors in corneal lymphangiogenesis after alkali injury.
文摘BACKGROUND: The transplantation of limbal epithelial cells cultivated on amniotic membrane is a newly developed treatment for limbal stem cell deficiency. The purpose of our study was to investigate the biological characteristics of limbal epithelial cells and evaluate the effect of transplantation of cultivated human limbal epithelial cells on ocular surface reconstruction in limbal stem cell deficiency rat model. METHODS: Human limbal cells were isolated and cultivated in vitro. Cytokeratins 3, 12, and 19 (K3, K12 and K19) and p63 were detected by immunofluorescent staining or RT-PCR. BrdU labelling test was used to identify the slow cycling cells in the cultures. Limbal stem cell deficiency was established in rat cornea by alkali burn. Two weeks after injury, the rats received transplants of human limbal stem cells cultivated on amniotic membrane carrier. The therapeutic effect was evaluated by slit lamp observation, Hemotoxin and Eosin (HE) staining and immunofluorescent staining. RESULTS: On day 7 in primary culture, p63 and K19 were strongly expressed by most cells but only a few cells expressed K3. On days 14 and 21, p63 and K19 were still expressed by a majority of cells, but the expressive intensity of p63 decreased in a number of cells, while the proportion of K3 positive cells increased slightly and some cells coexpressed p63 and K3. RT-PCR showed that gene expression of both p63 and K12 were positive in cultivated limbal cells, but in mature superficial epithelial cells, only K12 was detected. BrdU labelling test showed that most cells were labelled with BrdU after 7 days' labelling and BrdU label retaining cells were observed after chasing for 21 days with BrdU free medium. For in vivo test, slit lamp observation, HE staining and immunofluorescent staining showed that the rats receiving transplant of human limbal stem cells cultivated on amniotic membrane grew reconstructed corneas with intact epithelium, improved transparency and slight or no neovascularization. A majority of epithelial cells of the reconstructed cornea were positive to antihuman nuclear antibody and cells expressing K3 were found mainly in superfacial epithelium. CONCLUSIONS: Limbal stem cells can be cultivated in vitro: the cells are characterized by high proliferation and slow cycling and identified as p63/K19 positive and K3/K12 negative. During culture, some stem cells can proliferate and differentiate into mature cornea epithelial cells. Amniotic membrane is a suitable carrier for limbal stem cells. Transplantation of human limbal stem cells cultivated on amniotic membrane can functionally reconstruct rat cornea with limbal stem cell deficiency.
文摘AIM： To evaluate the inhibitory effect of subconjunctival bevacizumab as single-and multiple-dose application, and compare their effects on corneal neovascularization in a rat model. METHODS： Thirty adult Sprague-Dawley rats were used in this experimental study. The central cornea of the rats was cauterized chemically. The rats were randomly enrolled into three groups. All groups received subconjunctival injections. In Group 1（control group, n=10）, 0.05 m L 0.9% Na Cl solution was injected on the first day. In Group 2（single-dose group, n=10）, 0.05 m L bevacizumab（1.25 mg） was injected on the first day. In Group 3（multiple-dose group, n=10）, four doses of 0.05 m L bevacizumab（1.25 mg） were injected on the first, third, fifth and seventh day. Slit-lamp examination of all rats was performed at the third and ninth day. Digital images of the corneas were taken and analyzed using image analysis software to calculate corneal neovascularization area. All rats were sacrificed on the tenth day. In corneal sections, the number of blood vessels, state of inflammation and collagen formation was evaluated histopathologically. RESULTS： In Group 3, corneal edema grades were significantly lower than Group 1 and Group 2（P=0.02, and P=0.035, respectively）. The mean percentage of neovascularized corneal area in Group 3 was significantly lower than Group 2（P=0.005）. On histopathological examination, Group 2 and Group 3 showed significantly less number of blood vessels than Group 1（P=0.005, and P=0.001, respectively）. Additionally, Group 3 showed significantly less number of blood vessels compared to Group 2（P=0.019）. Inflammation and edema grades were significantly lower in Group 3 compared to Group 1（P=0.001）. CONCLUSION： Subconjunctival bevacizumab injection is effective in inhibition of newly formed corneal neovascularization. The multiple-dose bevacizumab treatment seems to be more effective compared to single-dose treatment.
