| BackgroundThe normal cornea is an avascular and transparent structure. Avascularity is actively maintained, and is necessary for corneal transparency and corneal immune privilege. Corneal neovascularization (CNV) involves the sprouting of new vessels from capillaries and venules of the pericorneal(limbal) plexus. Blinding CNV is a sequela of various conditions and lead the cornea to lipid exudation, inflammation, and scarring. Corneal vascularization of the recipient corneal bed is an established risk factor for corneal graft rejection and ultimate failure. Worldwide, CNV is a sight-threatening condition affecting millions of people.The formation of corneal neovascularization is a complex pathological process and the exact pathogenesis remains unclear. Basically has the following several kinds of theory:(1) hypoxia theory (2) inflammation theory (3) damage of cornea mechanical barrier (4) cytokines balance theory. At present most scholars tend to cytokines balance theory and they thinks the unbalance between stimulatory factor and inhibitory factor of angiogenesis is the main mechanism of neovascularization. Research on treatment of corneal neovascularization is a difficult job with a lot of problems unresolved. Based on cytokines balance theory, the current treatment for neovascular diseases research focus on the balance of angiogenesis. On the one hand can enhance angiogenesis inhibitory factor, on the other hand can weaken stimulatory factor. Pigment epithelium derived factor (PEDF) is the most effective endogenous angiogenesis inhibitor discovered. In cornea and lens, the PEDF is supposed to be the main factor of maintaining eye diaphaneity and avascularity. Avastin is a humanized anti-VEGF monoclonal antibody. It can indirectly block the combination of VEGF and its receptors and thus play a role of anti-angiogenesis. There are many experimental studies, also part of the clinical report, showing curative effect. In order to study the inhibitory effect of PEDF on corneal neovascularization, we first set up alkali-induced corneal neovascularization model in rats. Then we use PEDF eye drops to observe its inhibitory effect on new vessels, comparing with Avastin. And dexamethasone group was set at the same time as a comparison standard.Part1Establishment of alkali-induced corneal neovascularization model in ratsObjectiveThe aim of this parts of experiment was to find appropriate rat modles of comeal neovascularization (CNV) induced by alkali injury. Providing a reliable animal model for the next experiment.MethodsAll animals(n=24) were randomly assigned to three groups,each consisting of eight experimental eyes. Alkali injury was induced on day0by application of lmol/L NaOH to right eyes of S-D rats for20s,30s and45s respectively. Biomicmscopic features including comeal neovascularization was observed for7days.ResultThe induced rate of CNV for groups20s,30s,45s were62.5%,100%,100%respectely. CNV distributed sparsely and single in group20s, reaching the margin of burn area. In30s groups, CNV was dense and connected into a net. There were4rats (50%) appearing severe hyphema in45s group, which affects the observation of corneal neovascularization.ConclusionsThe appropriate induced time for S-D rat modles of comeal neovascularization by using filter paper which diameter is3mm and immerged solution of lmol/L sodium hvdroxide was30s. It’s an ideal animal model for CNV induced by chemical injuries.Part2Study of inhibitory effect of PEDF on alkali-induced corneal neovascularization in ratsObjectiveTo evaluate the effect of topically administered pigment epithelium-derived factor (PEDF) on experimentally alkali-induced corneal neovascularization on a rat model, comparing with PEDF and traditional medicines steroids hormones.Methods.1.A11right eyes of40SD rats were chemically cauterized by applying a3mm-diameter filter paper soaked lmol/L NaOH solution at the central cornea for40seconds. The left eyes were as normal control eyes. All animals were randomly assigned to four groups:group A, the10μg/ml PEDF drops was used the day after cauterization; group B, the5mg/ml Avastin drops was used the day after cauterization; group C,0.1%Dexamethasone Sodium Phosphate Eye Drops was used the day after cauterization; group D, normal saline solution drops was used the day after cauterization(control group). All the eye drops were applied10μL every time and4times per day.2.Observation of comeal neovascularization From the first days after burn, CNV were observed by slit-lamp microscopy eyeryday. Burn stimulus were evaluated according to the grading scale at the seventh day. To measure the length of CNV and calculate the area of CNV at the3th,7th,12th day, and digital photographs were taken on the12th day.3.Histopathological examination12th day after cautery, five rats of each group were killed. Corneas were removed carefully, fixed in4%paraformaldehyde, and embedded in paraffin. The corneas were then sectioned at thickness of4mm. Slecting one section to stain using hematoxylin and eosin. Images were then evaluated under a microscope and captured with a digital camera.4.1mmunohistochemical Analysis for VEGF and CD31The sections were deparaffinized in xylene and incubated in3%H2O2solution for10minutes and washed with PBS, followed by incubation with0.05%trypsin containing goat serum albumin for30min. The sections were then incubated overnight at4℃with primary antibodies. After thorough washes in PBS, the sections were incubated with the secondary antibody for30minutes. After3washes, the sections were then visualized using the DAB staining kit until satisfactory color was developed. The sections were washed with water, and the nuclei were counterstained with hematoxylin. Images were examined under a light microscope and captured with a digital camera.5.Western blot detect the expression of VEGF Other five rats of each group were killed at each time points described above. The corneas were removed and frozen in liquid nitrogen immediately. The cornea samples were homogenized in the lysis buffer. Equal amounts of total proteins were resolved by SDS-PAGE and blotted with primary antibody. membranes were stripped and reblotted with a mouse anti-b-actin antibody. Pictures was analysed using image J software.Results 1.Observation of CNV24hours after alkali burn, corneal limbal vascular network of each groups are filled, corneal is turbid without neovascularization. On the3th day, neovascular buds of group D grow into corneal limbal. Neovascular is dense and mushroom in the cornea. Neovascular of group A, B, C is relatively sparse, and the growth is more moderate. On the12th day, neovascular of group A is not up to the cautery area. Neovascular is tiny, pale, sparse. Neovascular of group B and C is short and just reached the cauterization area. The vascular is crossed and thin. Neovascular of group D is close to central cornea, and vascular is intensive and thick.2. Area of CNV at different time points The area of CNV in group A and B and C was smaller than that of group D at all time point(P<0.01). On the3th day, there is no significant difference in terms of CNV areas between group A and B and D (P>0.05). On the7th day,the area of CNV in group A was smaller than that of group B and C (P<0.01). There is no significant difference in terms of CNV areas between group B and C (P>0.05). On the12th day, the area of CNV in group A was smaller than that of group B and C (P<0.01) and there is no significant difference in terms of CNV areas between group B and C (P>0.05).3.Histopathological observation On the12th day, neovascular of group A israre, the vascular lumen is small and relatively sparse. There is little inflammatory cells infiltration, and corneal tissue structures tend to be trim Neovascular of group B and C increase, without a large number of inflammatory cells infiltration, and corneal tissue structure is relatively trim. There is a large number ofneovascular in group D, surrounding a large number of inflammatory cells infiltration. The vascular lumen is large and dense, filling with many mature red blood cells corneal. The corneal tissue structure is disorganized. Normal control eyes have no neovascular, and the corneal tissue structure is tidy. 4.1mmunohistochemical Analysis for VEGF and CD31The cornea of normal control eyes had a weak VEGF staining in the epithelium basal cells and endothelium. VEGF expressions was limited in the corneal epithelium and stroma in group A. There was increased VEGF expression in group B and C. VEGF expressions was substantially higher in the corneal epithelium and stroma in group D, mainly distributing in the epithelium layer and neovascular. There was no detectable expression on of CD31, a vascular endothelial marker, in the normal corneal tissue, demonstrating the lack of vascular endothelial cells in the normal cornea. Expression of CD31in other group is similar with that of VEGF.5. Western Blot Analysis of VEGF Similar to the results of immunohistochemistry, group D expresses high levels of VEGF, which expression level is low in group A. In group B and C, VEGF expression levels is increased comparing with group A.Conclusions1. Topical application of PEDF can inhibit alkali-induced corneal NV of rats.2.The inhibitory effect of PEDF is stronger than that of Avastion and dexamethasone.3. PEDF may restrain corneal neovascularization through lowering VEGF and controlling inflammation.4. PEDF had the effect of anti-inflammatory. |
PDF Full Text Request