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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.
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@#AIM: To investigate the effect of L-carnitine(LC)on corneal epithelial repair and its regulatory molecular mechanism in the hypertonic and inflammatory environment caused by alkali burn.<p>METHODS: Ninety-six healthy C57/6J mice were randomly divided into blank control group, phosphate buffered solution(PBS)group and LC group. The blank control group did not receive any treatment, LC group and PBS group were prepared acute alkali burn models. LC group was given 60mmol/L LC eye drops, and PBS group was given PBS eye drops, 6 times/d, for continuous days from one day before alkali burn. The repair of corneal epithelium was observed by fluorescein sodium staining under slit lamp microscope at 0h, 3 and 7d. On the 3d, the expressions of Ki-67 and IL-1β proteins in cornea were detected by immunofluorescence, the total proteins of corneal epithelial were extracted for Western blot to detect the expression of P63, NLRP3, Caspase-1 and phosphorylation level of STAT3.<p>RESULTS: The results of corneal fluorescein sodium staining showed that on the 3 and 7d after alkali burn, the percentage of residual corneal epithelial defect area in PBS group compared with LC group was(29.38±6.83)% <i>vs</i>(17.78±4.11)% and(14.23±4.51)% <i>vs</i>(4.10±2.10)%, respectively(<i>P</i><0.01). The repair of corneal epithelium in LC group was faster than that in PBS group. On the 3d, compared with the blank control group, the expressions of pyroptosis related proteins NLRP3 and Caspase-1 in the corneal epithelium of the alkali burn treated mice were up-regulated, the expression of P63 was decreased, and the p-STAT3/STAT3 level was increased, all the differences were significant except cleaved Caspase-1 of blank control group <i>vs</i> LC group. Compared with PBS group, in LC group, the expression of NLRP3, pro Caspase-1 and cleaved Caspase-1 protein were decreased, P63 was up-regulated, and p-STAT3 /STAT3 was increased, all the differences were significant. Immunofluorescence showed that compared with the blank control group,the expressions of IL-1β and Ki-67 were up-regulated in the alkali burned group. Compared with PBS group, the expression of Ki-67 protein was up-regulated and IL-1β was decreased in LC group.<p>CONCLUSION: LC can promote the proliferation of stem/progenitor cells in the corneal epithelium of mice and further promote the repair of corneal epithelium after alkali burn by inhibiting the pyroptosis signaling pathway and promoting the activation of STAT3 signaling pathway.
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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.
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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.
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@#AIM: To compare the effect of using autologous serum and deproteinised calf serum eye gel in the treatment of corneal alkali burn through establishing corneal alkali burn models. <p>METHODS: Alkali burn model of cornea was established on the right eyes by putting the filter paper with 1.0mol/L NaOH on the center cornea for 1min in 30 white rabbits. The model rabbits were divided randomly into 3 groups after scoring based on Hughes criteria. Normal saline, calf blood deproteinized eye gel and autologous serum eye drops 4 times/day, atropine eye gel 1 times/night, ofloxacin eye gel 1 times/night for 2wk respectively. The morphology of corneal neovascularization was observed on the 7 and 14d, and the area was calculated. On the 14d, the corneas of each group were removed and routine histopathological examinations were performed according to the groups. The concentration of CD45, IL-10, IFN-γ and VEGF in corneal homogenate were determined. <p>RESULTS:Area of corneal neovascularization: on the 7 and 14d, the area of corneal neovascularization of Group DCS(29.48±2.27, 34.19±2.67mm2), AS(34.19±2.67, 33.89±2.74mm2)(<i>P</i>>0.05). Concentration of CD45, IL-10, IFN-γ, VEGF in cornea homogenate(pg/mL): on the 14th day, the concentration of CD45 Group DCS(0.56±0.04ng/mL), AS(0.54±0.05ng/mL)<Group Ctrl(1.27±0.07ng/mL)(<i>P</i><0.05). The concentration of IL-10 Group AS(452.49±11.40pg/mL)>Group DCS>(332.49±13.67pg/mL)>Group Ctrl(111.05±6.95pg/mL)(<i>P</i><0.05). The Concentration of IFN-γ Group DCS(23.20±2.89pg/mL), AS(22.61±2.72pg/mL)<Group Ctrl(41.77±4.26pg/mL). They have a significant difference(<i>P</i><0.05). The concentration of VEGF Group DCS(151.14±18.21pg/mL), AS(149.11±14.75pg/mL)<Group Ctrl(391.35±28.59pg/mL)(<i>P</i><0.05). <p>CONCLUSION:AS has the same effect as DCS in inhibiting the release of inflammatory factors(CD45, IFN-gamma and VEGF)and the formation of corneal neovascularization after alkali burn in rabbits, and AS has the strongest effect in promoting the release of anti-inflammatory factors(IL-10)and inhibiting the infiltration of inflammatory cells, followed by DCS.
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@#AIM: To investigate the effect of comprehensive treatment of ocular alkali burn in different periods.<p>METHOD: A retrospective analysis was performed on 124 cases(166 eyes)of ocular alkali burns admitted to our hospital from January 2019 to December 12. According to the severity of the disease, a number of comprehensive measures were taken to treat the ocular alkali burn with drugs and surgery respectively. The patients were followed up for 6-12mo to observe the healing of ocular alkali burn and the final outcome of disease.<p>RESULTS: After treatment, the symptoms of all patients were relieved, the corneal conjunctiva healed, and no infection occurred. The average hospitalization time was 13d, totally 118 eyes were cured(71.1%), 43 eyes were improved(25.9%), 5 eyes were ineffective(3.0%). There was no complication in degree I and degree II of ocular alkali burn patients, degree III was better than degree IV, and the complication rate in degree III was lower than that in degree IV.<p>CONCLUSION: According to the corneal conjunctiva and eyelid injury evaluation of ocular alkali burn degree, choose appropriate time to take corresponding treatment measures, and give systemic and local drug treatment. Combined with ocular surface irrigation, anterior chamber puncture, amniotic membrane transplantation, conjunctival flap covering, corneal transplantation, limbal stem cell transplantation and other comprehensive treatment methods can obtain good clinical effect.
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@#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±0.13mm<sup>2</sup> respectively, which were lower than those in PBS group(7.58±0.20 and 9.24±0.08mm<sup>2</sup>; 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.
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@#AIM: To explore a new method to induce the animal model of rabbit partial limbal stem cell deficiency(LSCD).<p>METHODS: LSCD was induced through corneal alkali burn, C57 mice and New Zealand rabbits were used to establish the animal models. Corneal alkali burn manipulation was accomplished in experimental animals under general anesthesia combined with surface anesthesia in the operated eye. Specifically, mice(<i>n</i>=30)were used to induce complete LSCD model. In brief, the filter paper(diameter of 3mm)that immersed in 1mol/L potassium hydroxide solution was placed on the central corneal surface of the left eye for 30s, followed by washing with saline. In addition, rabbits(<i>n</i>=19)were utilized to establish the partial LSCD model. Briefly, the nictitating membrane(third eyelid)was resected, and the filter paper(diameter of 5mm)that immersed in 1mol/L potassium hydroxide solution was placed on the superior temporal peripheral corneal surface of the left for 30s, followed by washing with saline. After surgery, the model eyes were treated with 0.5% Levofloxacin Hydrochloride Eye Drops four times a day. In addition, the slit-lamp microscope was adopted for observation and photo-taking before burn, as well as at 1, 2, 4wk and 2mo after burn; meanwhile, complications such as corneal ulcer and perforation were recorded. 2mo after surgery, the corneal goblet cell distribution was detected with impression cytology, and the severity of LSCD was classified according to slit-lamp microscopic findings and corneal impression cytology. The animals were sacrificed 2mo after surgery, cornea and conjunctiva sections were made to observe angiogenesis and goblet cell distribution in cornea. Animals died accidentally were not counted into the total number, and the successful induction rates of complete LSCD and partial LSCD models were compared.<p>RESULTS: Six out of the 30 mice died accidentally, while 2 developed corneal perforation after burn, and the remaining 22 had developed complete LSCD only, yielding the successful induction rate of 92%. 2mo after burn, extensive angiogenesis distribution in the superficial and deep corneal stromal layers could be observed, and pathological sections revealed corneal angiogenesis. Seven out of the 19 rabbits died accidentally, while the remaining 12 had various degrees of LSCD only(partial LSCD, average involving 1.17±0.39 quadrants). Additionally, no corneal perforation was observed, and the successful induction rate was 100%. The result of Fisher's exact test <i>P</i> value is 0.543, without statistical difference. No goblet cells were observed in the normal corneal region, while goblet cells were observed in the LSCD region, with the average density of 58.60±12.58 cell/HP.<p>CONCLUSION: Central corneal alkali burn can induce complete LSCD; however, some animals will experience failure in model induction due to corneal ulcer and perforation, LSCD is generally serious and may be combined with angiogenesis in deep cornea. Alkali burn in superior temporal cornea can induce partial LSCD, which may be combined with relatively minor corneal lesion, and the corneal angiogenesis is located in the superficial layer.
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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.
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Objective To detect the expressions of hypoxia inducible factor-1α (HIF-1α) and aquaporin-1 (AQP1) in the alkali-burn cornea and investigate the roles of HIF-1α and AQP1 in alkali-burn mechanisms.Methods Totally 48 healthy adult female Kunming mice were selected,and the left eyes were treated with saline as the control group,and the model of corneal alkali burn was established with concentration of 1 mol · L-1 NaOH on the right eyes,and then these rats were randomly divided into 1-day,4-day,7-day and 14-day group.The expressions of HIF-1α and AQP1 in the cornea were detected using immunofluorescence and qRT-PCR after corneal alkali burn model was successful.Results In the control group,HIF-1 α was expressed in the corneal epithelial basement membrane,but it was increased in the corneal epithelium layer 1 day after alkali burn,and it was expressed in the corneal epithelium layer and stroma 4 days,and peaked 7 days after alkali burn;AQP1 was weakly expressed only in the endothelial layer in the control group,but it was increased in the corneal endothelial layer 1 day after alkali burn,and it was strongly expressed in the endothelial cell layer and the stroma 4 days and 7 days after alkali burn.qRT-PCR indicated that the relative expression level of HIF-1α mRNA was 269.70 ± 15.68 in 1-day group,350.50 ± 67.26 in 4-day group and 272.10 ±6.88 in 7-day group,respectively,which all were higher than that in the control group (188.70 ± 33.99),with significant difference (P ＜ 0.05).In addition,the relative expression level of AQP1 mRNA was 61.90 ± 5.45 in 1-day group and 48.34 ± 1.33 in 7-day group,which were significantly higher than that in the control group (36.43 ± 3.95),with statistical difference (P ＜ 0.05).Conclusion Alkali burn can cause the pathological changes in the cornea,and HIF-1 α and AQP1 involve in corneal injury after alkali burn.
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Objective:To investigate the effect of vasoactive intestinal peptide on IL-1β expression after corneal alkali burn.Methods:27 healthy adult female Wistar rats were randomly divided into group A(normal blank control group),group B(alkali burn treated with saline group),and group C (alkali burn treated with vasoactive intestinal peptide group).Group A were not processed.Group B,C were made right eyes′moderate corneal alkali burn models.After inducing burn,group B and group C were followed respectively by 0.9% saline and 0.25 g/L vasoactive intestinal peptide eye drops,4 times per day.The corneal epithelial healing rates were calculated by measuring the corneal staining area by methylene blue staining at 3 d,7 d and 14 d after inducing burn.At 3 d,7 d and 21 d,the corneal tissues were removed and divided into two equal parts.The corneal tissues were observed by staining,and the expression of IL-1β were detected by immunohistochemistry.Results:The healing rates of corneal epithelium in group C were greater than those in group B at 7 d and 14 d(P<0.05).The mean optical density of IL-1β was measured by immunohistochem-istry.The mean optical density in group B and group C at 3 d were(1.62 ± 0.96) ×10-2,(0.98 ± 0.45) ×10-2,respectively,which at 7 d were (1.42 ± 0.62) ×10-2,(0.71 ± 0.22) ×10-2,respectively,which at 21 d were(0.37 ± 0.24) ×10-2,(0.13 ± 0.16) × 10-2,respectively.The mean optical density of corneal IL-1β in group C were lower than those in group B(P<0.05).Conclusion:Va-soactive intestinal peptide can decrease the expression of IL-1β after corneal alkali burn,down-regulate the level of immune stress, reduce the inflammatory reaction and promote the recovery of cornea after alkali burn.
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Objective To dynamically observe the expressions of Toll like-receptor 2 (TLR-2),TLR-4 and inflammatory cytokine IL-1 beta and TNF alpha,and analyze the correlation between TLR-2,TLR-4 and inflammatory response on rat cornea alkali burn.Methods Forty SPF healthy SD rats were excluded the anterior segment disease,the right eye was set as burn experiment and the left eye as normal control,Ⅲ level corneal alkali burn model was established with concentration of 1 mol · L-1 NaOH(At the establishment and after the establishment of the model,the inconsistent degree of corneal bums,the corneal perforation and hyphema in rats,etc.,were removed,and finally randomly selected 32 eligible rats),then were randomly grouped into 3 days,7 days,14 days and 21 days group,8 eyes in each group.The rats were observed and photographed anterior segment of each group,evaluated the corneal inflammation index,then removed the eyeball of the rats in the corresponding time points.The eyeballs were made into pathological tissue section and stained with HE method.The number of inflammatory cells in the cornea was calculated,and the expression of TLR-2,TLR-4,IL-1 beta and TNF alpha was detected in the rat cornea with method of Western blot protein detection at the same time.The differences of each group were analyzed,and the correlation was assessed between TLRs and inflammatory factor.Results There were the expressions of TLR-2,TLR-4,IL-1 beta and TNF alpha in the normal rat cornea,and its expressions were gradually increased after alkali burn,reached the peak at the 7 days,then decreased gradually,the difference of each group (the 3 day,the 7 day,the 14 day,the 21 day) was statistically significant (all P ＜ 0.05).The expressions of TLR-2 and IL-1 beta (r =0.986,P ＜ 0.05),TNF alpha (r =0.986,P ＜ 0.05) were positive correlated.The expressions of TLR-4 and IL-1 beta(r =0.975,P ＜ 0.05),TNF alpha (r =0.990,P ＜0.05) were positive correlated.Conclusion TLRs participates and may start immune inflammatory response after corneal alkali burn,mediates the production of inflammatory cytokine.
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AIM: To analyze the effect of human amniotic homogenate extract on corneal neovascularization after corneal alkali burn in the process of pigment epithelium derived factor (PEDF) and vascular endothelial growth factor (VEGF) expression and the effect of corneal neovascularization.METHODS: Totally 32 patients with corneal alkali burn were selected from June 2015 to June 2016 in Foshan,and were randomly divided into Group A and Group B,with a total of 37 eyes.Group A of 17 cases,with a total of 19 eyes,were treated with 40mg/L human amniotic homogenate extract;Group B (n=15),and 18 eyes,treated with 3g/L prednisolone eye drops.In the treatment of 1,4,7,14,21 and 28d at different time points,we observed the growth of corneal neovascularization,and detected the expression of PEDF and VEGF during angiogenesis.RESULTS: Group A of patients in the use of human amniotic homogenate extract after the treatment,the expression level of PEDF was significantly higher than that in Group B(P=0.001),after 28d treatment,the expression level of PEDF reached 0.721±0.314.While patients in Group B the expression level of PEDF was only 0.538±0.253.Two groups had significant difference between the expression level of PEDF (P<0.05).The expression level of VEGF in Group A was lower than in Group B at different time points in the test.After the treatment of 28d patients in the Group A,the expression level of VEGF was 0.152±0.020,in Group B the expression level of VEGF was0.302±0.031.Two groups of patients with VEGF expression level between the differences were statistically significant (P<0.05).The patients number in Group A with corneal neovascularization was significantly lower than that in Group B,the difference was statistically significant (P<0.05).CONCLUSION: Human amniotic homogenate extract can increase the expression of PEDF in corneal neovascularization after corneal alkali burn,inhibit the expression of VEGF and the proliferation of corneal neovascularization.
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AIM:To investigate the effects of amnion epithelial cell ( AEC) suspension liquid on the biological behavior of the rabbit's corneal epithelium, combined with the in vitro and in vivo experiments. · METHODS: The rabbit's corneal epithelium were cultured in the lower chamber of transwell, and AEC suspension liquid was dropwised in the upper chamber. There was only culture medium in the upper chamber of the control group. The proliferation of rabbit's corneal epithelium was observed with CCK-8 automated colorimetry and the expression of PCNA was detected by immunocytochemistry. We used the scratch wound assay to detect the migration of corneal epithelial cell ( CEC ) . The in vivo models were established by placing a 10mm diameter corneal trephine in the center of the cornea, within 1mol/L NaOH for 1min. We divided those into three groups: treatment group of AEC suspension liquid eye drop, AEC suspension liquid subconjunctival injection and the control group without any treatment. Using the slit- lamp biomicroscope and fluorescence staining to observe the cornea per week. After 28d we took the eyeballs with the HE staining. The expression of VEGF was detected by immunohistochemistry. ·RESULTS: The activity of CEC with AEC treatment was much higher than the control group ( P< 0. 05 ). The expression of PCNA increased in AEC group (P<0. 05). And the migration of CEC in the AEC group was faster than the control one. In vivo, the inflammation of the corneal and the CNV of the AEC group were all significantly reduced compared with the control group (P<0. 05 ). There were less invasive cells and more ordered organization arrangement in ACE group observed by the HE staining. The expression of VEGF and mcp-1 in these two AEC treatment groups all significantly decreased compared with the control group (P<0. 05). ·CONCLUSION: AEC suspension liquid can promote the proliferation and migration of the rabbit's corneal epithelium. The potential of AEC suspension liquid as a therapy for acute corneal alkali burn.
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Objective To investigate the effect of the novel curcumin-loaded chitosan liposomes on rabbit alkaline burns. Methods Curcumin-loaded chitosan liposomes were prepared with a film method. The particle sizes,zeta potentials and encapsula?tion efficiencies of liposomes were determined. Rabbits were randomly divided into four groups:the physiological saline group,the blank chitosan coated liposomes group,the dexamethasone group,and the curcumin-loaded chitosan liposomes group. The rabbit cor?neal alkaline burn models were built and respectively processed with the above medicines. The corneal neovascularization(CNV)and proportion of corneal epithelium healing were analyzed with the slit-lamp microscope and digital photographs. The expression of vascu?lar endothelial growth factor(VEGF)was examined with an immunohistochemical method. Results The curcumin-loaded chitosan li?posomes had the particle size of 96.6 ± 14.7 nm,with the average zeta potential of 58.8 ± 2.3 mV,and the encapsulation efficiency of 51.41±1.1%. The CNV was effectively inhibited and the expression of VEGF decreased due to the curcumin-loaded chitosan liposomes and dexamethasone. Furthermore,the curcumin-loaded chitosan liposomes improved epithelial healing of corneal more effectively than dexamethasone. Conclusion The encapsulation efficiency of the curcumin-loaded chitosan liposomes was high. The effects of the cur?cumin-loaded chitosan liposomes on inhibition of CNV and improvement of healing of cornea epithelium were obvious. Curcumin-load?ed chitosan liposomes are novel ophthalmic delivery formulations for the treatment of corneal alkaline burns.
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Objective Corneal alkali burn is a major cause of corneal neovascularization ( CNV) .Confocal microscopy was used to observe the therapeutic effects of KH902 eye drops for the treatment of corneal alkali burns. Methods 24 adult rabbits were randomly divided into two groups( n=12) , alkali burn model being established:experimental group by KH902 eye drops and control group by saline solution 3 times a day.Confocal microscopy was given at 3, 7, 14, 28 d post-operatively (po). Results Corneal epithelium deletion or vacuolar necrosis was found at 3 d po, along with stromal edema.There was no obvious inflammatory cell or no immunocyte infiltration.No difference was found in the corneal structures between two groups.CNV appeared in the peripheral stroma in both groups at 7d po.Inflammatory cell infiltration was more severe in the limbus and peripheral sections in control group than in control group.At 14 d po, inflammatory cells declined gradually and CNV took shape.At 28 d po, scarring and decreased inflammatory cells were found in both groups, while the experimental group had fe-wer and smaller blood vessels than the control group.As to the area of CNV, it was respectively (35.42 ±6.40) mm2, (60.23 ±5.35) mm2, (60.23 ±5.35)mm2 at 7, 14, 28d po in control group, statis-tical difference being found among different time points(P0.05).The CNV area in experiment group is significantly less than that in the control group at 14 d and 30 d po(P<0.05).The inflammatory cell density in control group was respectively (74.21 ±9.33)mm2, (1883.39 ±43.11)mm2, (2532.10 ±98.00)mm2, (723.05 ± 23.34)mm2at 3,7, 14, 28 d po, and (58.0 ±10.22)mm2, (656.90 ±33.01)mm2, (432.32 ±60.11)mm2, (122.11 ±30.37) mm2respectively at 3,7, 14, 28d po in experiment group, significant difference was found at different time points in both groups(P<0.05), among which significant decreased inflammatory cells was found in experiment group compared with control group (P<0.05). Conclusion KH902 eye drops can be used for the inhibition of CNV and inflammatory cell infiltration after alkali burn.
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Objective To investigate the effect of the novel curcumin-loaded chitosan liposomes on rabbit alkaline burns. Methods Curcumin-loaded chitosan liposomes were prepared with a film method. The particle sizes, zeta potentials and encapsulation efficiencies of liposomes were determined. Rabbits were randomly divided into four groups: the physiological saline group, the blank chitosan coated liposomes group, the dexamethasone group, and the curcumin-loaded chitosan liposomes group. The rabbit corneal alkaline burn models were built and respectively processed with the above medicines. The corneal neovascularization (CNV) and proportion of corneal epithelium healing were analyzed with the slit-lamp microscope and digital photographs. The expression of vascular endothelial growth factor (VEGF) was examined with an immunohistochemical method. Results The curcumin-loaded chitosan liposomes had the particle size of 96.6±14.7 nm, with the average zeta potential of 58.8±2.3 mV, and the encapsulation efficiency of 51.41±1.1%. The CNV was effectively inhibited and the expression of VEGF decreased due to the curcumin-loaded chitosan liposomes and dexamethasone. Furthermore, the curcumin-loaded chitosan liposomes improved epithelial healing of corneal more effectively than dexamethasone. Conclusion The encapsulation efficiency of the curcumin-loaded chitosan liposomes was high. The effects of the curcumin-loaded chitosan liposomes on inhibition of CNV and improvement of healing of cornea epithelium were obvious. Curcumin-loaded chitosan liposomes are novel ophthalmic delivery formulations for the treatment of corneal alkaline burns.
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AIM:To investigate the expression and the significance of VEGF-C/D in rat cornea after alkali burning as well as the role of lymphangiogenesis in the high-risk corneal transplantation rejection. ●METHODS:The model of alkali burn corneal was made. Different times corneas were taken to electron microscope for vascularization, and examined the expression of VEGF-C/D and VEGFR-3 in l, 3, 5, 7, 14, 28d. The other rat cornea after alkali burn were divided into four parts to penetrate keratoplasty, containing only blood vessels in the cornea ( group A ) , angiogenesis and lymphangiogenesis ( group B ) , lymphangiogenesis degenerating period ( group C ) , angiogenesis degenerating period ( group D ) . ln addition, there are also normal groups ( group N ) to compare the Rl values and survival time of corneal graft. ●RESULTS: Electron microscopy showed that, when the first 7d rat cornea appeared neovascularization after alkali burn, but not lymphangiogenesis. The occurrence of new blood vessels and lymphatic in 2wk. There were no obvious lymphangiogenesis in 5wk and the angiogenesis gradually subside in 8 wk. The expression of VEGF-C/D and VEGFR-3 in the corneas of rats were up-regulated in the third days after the injury, and reached its peaks at 5d. The average survival time of group N, A, B, C, D were (14.25±0.62)d, (9.35±1.02)d, (5.06±1.13)d, (8.71±0.83) d, (9. 44±1. 05)d after transplant cornea. Compared to the rest of the group, group B plant average survival time significantly shortened (P ● CONCLUSION: VEGF - C/D and VEGFR - 3 are expressed significantly after corneal alkali burn. New lymphatic vessels can accelerate high - risk corneal transplantation immune rejection.
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Background Corneal chemical burn is one of blinding eye diseases.Previous therapies for corneal chemical burn is limited to certain extent.However,transplantation of adipose-derived mesenchymal stem cells (ADSCs) for corneal diseases is drawing more and more attention.Objective This study was to observe the effect of rabbit ADSCs transplantation for ocular alkali burns and explore its mechanism.Methods Rabbit corneal stromal cells (CSCs) were isolated and cultured by suspended matrix method,and rabbit ADSCs were obtained and digested from inguinal fat tissue with enzyme digestion method (0.25％ trypsin) and identified by flow cytometry.CSCs cocultured with ADSCs,and CSCs-induced ADSCs were identified by double-label of with immunofluorescence and reverse transcription PCR (RT-PCR).Then induced or uninduced ADSCs were inoculated on amniotic membrane to prepared ADSCs-amnion patch.Corneal alkali burn models were established in the right eyes of 60 New Zealand rabbits by placing a filter paper with 1％ NaOH solution at the central cornea for 50 seconds.The models were randomized into the induced ADSCs+ amnion implanted group,the uninduced ADSCs + amnion implanting group,amnion implanted group and model group.Corneal opacification and neovascular area were examined and corneal inflammation was graded by slit lamp microscope 1 week,2 weeks and 1 month after surgery.The contents of CD45,interferon-γ (IFN-γ) and interleukin-10 (IL-10) in corneal homogenate as well as vascular endothelial growth factor (VEGF) in aqueous humor were detected by ELISA assay.The use and care of experiment animals followed the Statement of ARVO.Results ADSCs showed the positive responses for CD105,CD29,CD44 with the positive rate 90.23 ％,88.56％ and 98.88％,respectively.CSCs was positively reactive for vimentin.The double-label staining was positive after coculture of CSCs with ADSCs.Hematoxylin-eosin stain exhibited that ADSCs grew well on the amnion.Corneal porcelain opacity and a lot of new blood vessels were seen in the model group,and corneal was clear in the induced ADSCs+ amnion implanted group 1 month after surgery.The inflammatory scores were 1.65 ±0.18,2.05 ± 0.17,2.68±0.25,2.90 ±0.18,and the areas of neovasculization were (10.59 ± 1.78),(22.58 ± 1.63),(37.98 ± 1.90),(45.37±1.65)mm2 respectively in the induced ADSCs+ amnion implanted group,uninduced ADSCs+ amnion implanted group,amnion implanted group and the model group.The inflammatory scores of 1 week,2 weeks,1 month after operation among the four groups had statistically significant differences (F =280.826,330.172,465.707,all at P =0.000),and the areas of neovasculization of 1 week,2 weeks,1 month after operation among the four groups had statistically significant differences (F=60.020,670.811,1 510.231,all at P =0.000),the inflammatory scores in the induced ADSCs + amnion implanted group were remarkably lower than those of the other groups,the areas of neovasculization in the induced ADSCs+ amnion inplanted group were smaller than those of the other groups (all at P＜0.01).In 1 month after surgery,the contents of CD45,IL-10,IFN-γ in cornea and VEGF in aqueous humor were statistically different among the groups(F =916.545,1 739.358,462.134,129.126,all at P =0.000).Compared with the uninduced ADSCs+ amnion implanted group,amnion implanted group and the model group,CD45 and IFN-γ contents were declined,and IL-10 content was elavated in the induced ADSCs+ amnion implanted group (all at P＜ 0.01).In addition,VEGF contents in aqueous humor were significantly lower than those in the other groups (all at P＜0.01).Conclusions Rabbit CSCs-induced ADSCs amnion patch transplantation is effective for the reconstruction of ocular surface after alkali damage probably by differentiation of ADSCs into epithelial-like cell after CSCs induced.Moreover,amnion can alleviate immuno-inflammatory response and suppress neovascularization.
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AlM:To find better ways of treating ocular alkali burn, and to reduce the suffering of patients and social burden.METHODS:Totally 100 patients were graded according to the degree of chemical burns to four major groups, each half were randomly divided into the control group and the treatment group. Control group underwent conventional treatment. ln addition to conventional therapy, patients in each treatment group were also added a Breviscapine intravenous injection of 40mg daily. Corneal recovery time, changes in vision, degree of corneal opacity, number of corneal neovascularization and other complications were observed. Curative effects were analyzed statistically. RESULTS:There was no significant difference in levelⅠgroup between control group and treatment group ( P>0. 05); There were significantly different in level Ⅱ, Ⅲand Ⅳ group ( P CONCLUSlON: Breviscapine in the treatment of ocular alkali burns can shorten the course of treatment, reduce corneal scarring, and improve vision.