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.

Effect of PMNs and MMP-9 on corneal injury after alkali burn / 国际眼科杂志(Guoji Yanke Zazhi)

@#AMI: To study the relationship between the infiltration of polymorphonuclear neutrophils(PMNs)and the expression of matrix metalloproteinases 9(MMP-9)in corneal stroma injury after alkali burn.<p>METHODS: Cornea alkali-burned model was made in 25 rabbits, then animals were grouped and sacrificed at 3d, 7d, 14d, 21d and 28d, respectively. The condition developing of alkali-burned cornea was observed by slit lamp biommicroscopy. The corneas were enucleated for histopathological examination. The infiltration of PMNs was identified by hematoxylin eosin(HE)staining and the expression of MMP-9 was identified by immunohistochemisty in different periods.<p>RESULTS: The quantity of PMNs and MMP-9 increased on the 3d, reached the lower level on 7d, shown a peak on the 14d, then decreased gradually. The area and depth of corneal stroma after alkali burn were the most severe on the 14d.<p>CONCLUSION: During the wound healing process, alkali-burned cornea has close relation with the infiltration of PMNs and the expression of MMP-9. The infiltration of PMNs and the expression of MMP-9 is positively correlated in corneal stroma injury after alkali burn.

Subconjunctival injection of antagomir-21 alleviates corneal neovascularization in a mouse model of alkali-burned cornea.

Corneal neovascularization may result in loss of corneal transparency and blindness. However, developing successful and inexpensive medical treatments for corneal neovascularization remains an unresolved issue. Recently, several studies have implicated miRNA functions in the regulation of cornea homeostasis. This study aimed to identify the miRNA expression profile in the neovascularized cornea after an alkali burn and to investigate the related underlying mechanisms. Here, alkali-burned corneas and matched normal tissues were pooled to perform miRNA sequencing. MiR-21 in alkali-burned cornea showed the greatest increment of abundance at 4 and 7 d after injury compared to the healthy cornea. The miR-21 expression was positively correlated with both the mRNA and protein level of key angiogenic factors including vascular endothelial growth factor (VEGF)-A and hypoxia-inducible factor-1α (HIF-1α). At 2 and 8 d after alkali burn, the mice received subconjunctival injections of antagomir-21 (1 or 5 nmol per injection). The injection of antagomir-21 (5 nmol) inactivated miR-21 and attenuated neovascularization progression by inhibiting the expression of VEGF-A and HIF-1α. Western blot analysis of the corneas demonstrated that antagomir-21 restored Sprouty 2/4 expression and silenced p-ERK activation. Therefore, these data reveal that antagomir-21 ameliorates the progression of corneal neovascularization likely via Sprouty 2/4-mediated inactivation of p-ERK. Delivery of antagomir-21 might be a potential therapeutic approach to prevent or treat visual loss caused by corneal neovascularization.

Effect of vasoactive intestinal peptide on the wound healing of alkali-burned corneas.

AIM: To study the effect of vasoactive intestinal peptide (VIP) on wound healing in experimental alkali burns of the cornea. METHODS: Twenty-seven albino rabbits, weighing 3.2±0.75 kg were used. Alkali burns were induced on corneas by applying 10 mm Whatman paper No:50 soaked in 1 mol/L NaOH. They have further classified into 5 groups as follows: 1) control group given no treatment (n=5); 2) VIP given subconjunctivally (n=6); 3) VIP injected into anterior chamber (n=6); 4) NaCl 0.9% given subconjunctivally (n=5); 5) NaCl 0.9% given into the anterior chamber (n=5). All treatment protocols except control group were followed by topical eye drops composed of VIP at two hourly intervals for one week from 8 a.m. to 6 p.m. RESULTS: VIP treated groups of rabbits with alkali burns were found to have better wound healing findings histo-pathologically when compared to those of control group who have received no treatment on day 30. No differences were observed between groups in respect to degree of polymorphonuclear leukocytes (PMNL) infiltration and degree of loss of amorphous substrate on day 15. However, PMNL infiltration and degree of loss of amorphous substrate were lower in Groups 2 and 3 when compared to that of control group on day 30 (P<0.05). CONCLUSION: We have shown that VIP has positive effects on alkali induced corneal burns. VIP may inhibit PMNL migration to cornea through an immunomodulatory effect. Inhibition of PMNL migration might reduce the release of collagenases and this might prevent the extracellular amorphous substance loss.

Changes in infiltration of polymorphonuclear neutrophils in alkali-burned cornea of conjunctival flap covering / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To study the infiltration of polymorphonuclear neutrophils ( PMNs ) after conjunctival flap covering in alkali-burned cornea. ●METHODS: Rabbit cornea alkali-burned model was made, then 50 rabbits were randomly divided into the experimental group ( n=25 ) and the control group ( n=25 ) . At the same time the surgery of conjunctival flap covering was given to rabbits of the experimental group. The condition developing of alkali-burned cornea was observed by slit lamp biomicroscopy, and took photos in two groups. The infiltration of PMNs was identified by hematoxylin eosin ( HE) staining in different periods. ●RESULTS:The quantity of PMNs increased on the 3d, reached the lower level on 7d, shown a peak on the 14d, then decreased gradually. PMNs level of the experimental group was significantly lower than that in the control group, and the difference of 3, 14 and 21d was significant (P ●CONCLUSION: During the wound healing process, alkali - burned cornea has close relation with the infiltration of PMNs. The treatment of conjunctival flap covering for the severe alkali-burned cornea was found to have good effect.

Effects of a 5% monosulfated glycosaminoglycan and duasorb topic solution on alkali-burned rabbit corneas

Topical effects of chondroitin sulfate, a monosulfated glycosaminoglycan, on the healing of alkali-burned corneas were studied.Forty albino rabbits were divided into two groups (GI and GII, n =20) submitted to corneal ulceration with 3 M sodium hydroxide(NaOH). GI was treated with a topical solution of 5% chondroitin sulfate 4 and chondroitin sulfate 6 and GII was treated with 0.9%physiological saline at 6 hour intervals in both cases. The rabbits were studied over a period of 60 days by biomicroscopy witha slit lamp in order to evaluate blepharospasm, blepharitis, chemosis, conjunctival congestion, ocular secretion, and cornealedema, neovascularization and macula. The fluorescein test was performed daily to monitor the evolution of the ulcers. Theanimals were submitted to eye enucleation for histopathology on the 1st, 3rd, 10th, 30th and 60th day after ulcer induction. Whencompared to GII, GI showed 4 perforations. In the eyes in which no perforation occurred, no clinical or microscopic differenceswere observed between groups, except for ocular secretion, which persisted for a longer time in GII animals. Except for thosewith perforations, the corneas tended to heal, on average, within 30 days. The results permit us to conclude that the 5%chondroitin sulfate solution, when instilled on alkali-burned rabbit corneas did not alter healing when compared to the animalstreated with 0