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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.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.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)% vs(17.78±4.11)% and(14.23±4.51)% vs(4.10±2.10)%, respectively(P

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

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. 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. 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)(P>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)PGroup DCS>(332.49±13.67pg/mL)>Group Ctrl(111.05±6.95pg/mL)(PPPCONCLUSION: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.

AIM: To explore a new method to induce the animal model of rabbit partial limbal stem cell deficiency(LSCD).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(n=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(n=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.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 P 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.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.

AIM: To investigate the effect of recombinant canstatin proteins with different concentration on the expressions of matrix metalloproteinase-2(MMP-2)and tissue inhibitor of metalloproteinase-2(TIMP-2)in mice with corneal alkali burn.METHODS: Sixty BALB/c mice were divided into three groups(experimental group A, experimental group B and control group C), 20 mice in every group and their corneas in the right eyes were burned with alkali(1mol/L NaOH). The experimental group A received recombinant canstatin proteins drops with 3μg/mL. The experimental group B received recombinant canstatin proteins drops with 5μg/mL and the control group C was treated with physiologic saline. At different time points(1, 3, 7 and 14 d)after alkali burns, the mice were killed and the growth of epithelial defect and corneal neovascularization(CNV)were observed with an operation microscope. The expressions of MMP-2 and TIMP-2 in cornea were measured by the Western blot technique, and the results were analyzed by enhanced chemiluminescent(ECL).RESULTS: The areas of epithelial defect and corneal neovasularization significantly reduced in mice treated with recombinant canstatin proteins compared to mice treated with physiologic saline at 3, 7 and 14d after alkali-induced injury( all P); the neovasularization was suppressed and the area of CNV was less than that in control group C(all P). Western blot analysis showed that the expression levels of MMP-2 in experimental group A and B were significantly lower than that in control group C(PPP), while the level of TIMP-2 in experimental group B were significantly higher than that in experimental group A on day 7 and day 14(P). CONCLUSION: Recombinant canstatin proteins may suppress the expression of MMP-2, upregulate the expression of TIMP-2 in cornea cells and the infiltrated inflammatory cells, lower the rapid resolution of cornea and ulceration, and play a vital role in the remodeling of alkali treated cornea in mice.

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

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

AIM: To 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. In 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 level Ⅰ group between control group and treatment group(P>0.05); There were significantly different in level Ⅱ, Ⅲ and Ⅳ group(PPCONCLUSION: Breviscapine in the treatment of ocular alkali burns can shorten the course of treatment, reduce corneal scarring, and improve vision.

AIM: To investigate the inhibited effect of Doxycycline on the infiltration of inflammatory cells in rat alkali burn corneas. METHODS: Corneas of 32 SD rats were injured with 1mol/L NaOH, then divided into two groups: control and doxycycline-treated. All agents were administered topically 4 times daily. Slit-lamp microscope was performed and inflammatory index was calculated at 3, 7, 14 and 21 days after injury. Then 4 rats were randomly sacrificed and each cornea was divided into two parts, one for histopathology, the other for ICAM-1 ELISA assay. RESULTS:In control group, the inflammatory index and the number of inflammatory cell was higher than the doxycycline-treated dramatically at all time points(P