| Objective: Corneal alkali burn is one of the serious and common chemical injury of eye in clinic. Severe alkali burn may deprive visual acuity since serious pathological change of corneal dilapsus, corneal ulcer, corneal penetration and corneal neovascularization. Especially the corneal neovascularization strongly degrade the achievement ratio of corneal transplant after alkali burn. Therefore, it has being studied generaly by dometic and overseas scholars that the cure of alkali burn and the prevention of the emerge of neovascularization after it. Recent years, Chitosan have drawed close attention because of its specific character of biology. It has been confirmd that Chitosan as a high molecular polymer of glucidamin possess many kinds of biological activity such as anti-inflammatory, anti-microbial, promoting tissue repair, inhibiting accrementition of the fibers, preventing oxidative damage and so on. However the investigations concerning it's effection of inhibiting corneal neovascularization are seldom. In order to evaluate the effect of Chitosan on inhibiting corneal neovascularization after alkia burn and investigate its mechanism during this process, PMN, VEGF and MMP-9 in cautery cornea was semi-quantitative analysed. All this were expected to provide experimental and theoretical accordance of chitsoan's clinical application in the future.Methods: Corneal alkali-burn model of 48 New Zealand white rabbits was made by burning right eyes with NaOH. The rabbits were divided into Cautery group(cautery) and Chitosan group (cautery and put drops in the eyes with 2% chitsan) in random. The rabbits were observed by slit lamp microscope after cautery and taken photoshops with digital camera. 6 rabbits were killed at 1,4,7 and 14 day randomly in each groups and the right cornea was extirpated. Half of every cornea were fixed in 4% paraformaldehyde, imbedded in paraffin to make tissue sections and stained with HE for light microscope study. Poly-morphonuclear leukocytes(PMNs) whose nuclear are poly-morphon were counted one by one visual field when the multiplying factor of objective was 40. The computer image analysis system was used to analyse the expression of VEGF, MMP-2 in cornea after immunohistochemistry stain. The rest of cornea were fixed in 4% glutaraldehyde-phosphate buffer quickly at the size of 1mm3 to make samples for transmission electron microscope for observing the ultrastructure changes of the corneal epithelium.SPSS12.0 was used to analyse data of this study.Results: 1 Through lit lamp microscope: In Cautery group, the secretion increased, the vascular nets on limbus corneae engorged, the corneal epithelium amoticed on the 1st day after cautery; there were neovascularizations grew in limbus corneae fastly, the corneal epithelium repaired on the 3rd day; the neovascularizations encroached on corneal center on the 7th day; the neovascularizations became subsided though concentrate, and there were leukoma on the cautery region expressly on the 14th. In Chitosan group, the secretion were less than Cautery group, the vascular nets on limbus corneae did not engorged, the corneal epithelium amoticed punctiformly on the 1st day; the neovascularizations grew in limbus corneae slowly, the corneal epithelium repaired on the 3rd day; the neovascularizations did not encroach on corneal center on the 7th day; the neovas- cularizations were rarefaction, and there no leukoma were detected on the 14th.2 Statistic results of area of CNV: On the 1st day after cautery, there were not new vessels in Chitosan group and Cautery group. On the 4th, 7th and 14th day after cautery, the area (mm2) of CNV in Cautery group and Chitosan group were respectively 21.30±6.24, 38.86±6.38, 40.37±5.71; 13.06±4.45, 27.69±4.87, 30.81±3.07.The area of CNV in Chitosan group were decreased significantly compared with Cautery group in every time after cautery( P<0.05 on the 4th day, P<0.01 on the 7th,14th day).3 Statistic results of content of PMNs: On the 1st, 4th, 7th and 14th day after cautery, the content(μmol/gprot) of PMNs in Cautery group and Chitosan group were respectively 14.23±1.72,19.20±2.40,28.70±1.63,15.10±2.09;11.23±1.33,15.30±2.24,20.00±1.60,12.22±1.09.The content of PMNs in Chito- san group were decreased significantly compared with Cautery group in every time after cautery(P<0.01).4 Immunohistochemistry analysis of VEGF, MMP-2Immunolocalization of VEGF, MMP-2 in cornea were intracytoplasm of corneal epithelium cells, matrix fiber cells, infiltrative inflammatory cells and neovascular endothelium cells.4.1 Area density value of VEGF: On the 1st,4th,7th and 14th day after cautery, the area density value of VEGF in Cautery group and Chitosan group were respectively 0.0844±0.0348,0.1029±0.0379,0.7256±0.0100,0.0316±0.0074; 0.0346±0.0080,0.0699±0.0150,0.5334±0.1602,0.0388±0.0455. On the 1st,4th,7th day after cautery, the area density value of VEGF in Chitosan group were decreased significantly compared with Cautery group(P<0.01 on the 1st day, P<0.05 on the 4th,14th day), but there were not significant difference between them (P>0.05) on the 14th day after cautery.4.2 Area density value of MMP-2: On the 1st,4th,7th and 14th day after cautery, the area density value of MMP-2 in Cautery group and Chitosan group were respectively 0.0537±0.0090,0.0616±0.0126,0.1315±0.0605,0.0713±0.0112; 0.0275±0.0112,0.0365±0.0083,0.0698±0.0107,0.0616±0.0121. On the 1st,4th ,7th and 14th day after cautery, the area density value of MMP-2 in Chitosan group were decreased significantly compared with Cautery group(both 1st and 4th P<0.01,both 7th and 14th P<0.05).5 Histopathology change 5.1 Light microscope:Cautery group: The corneal epithelium healing was slowly; cell shape was irregularity; inflammatory cell infiltration was critical; corneal neovascularization was early and development was quick; substantia propria layel collagen fibers had segmentation and recovery was slowly. Chitosan group: The corneal epithelium healing was quick; cell shape was integrity; inflammatory cell infiltration was slight; corneal neovascularization was late, slow-moving and rarefaction; substantia propria layel collagen fibers arrange irregularity but its recovery was fast.5.2 Electron microscope: Cautery group: microvilli of the corneal epithelium cells had amoticed and reduced in quantity , cell junction, hemidesmosomea, depressed; the perinuclear cisterna widened, there were lots of vacuolus and hydroncus in the cytoplasm; rough endoplasmic reticulum distended and had a diversely degranulation in cytoplasm of the basal cells; mitochondria hydroncus partly, crista reduced in quantity arrangements were disorderly, crista confluenced or disappeared partly or mostly. Chitosan group: the quantity of the microvilli decreased were not obviously, and lined up in order, the quantity of the hemidesmosomea were normal basicly; the perinuclear cisterna widened slightly; there were less vacuolus and no hydroncus in the cytoplasm; rough endoplasmic reticulum distended and had a slightly degranulation in cytoplasm of the basal cells; mitochondria had a little hydroncus, crista of the mitochondria confluenced or disappeared partly. Conclusion: 1 All of PMN, VEGF, MMP-2 participate the process of corneal neovascularization through different pathway.2 Chitosan can depress the contents of VEGF, MMP-2 in the corneal epithelium cells, matrix fiber cells and inflammatory cells through inhibited inflammatory reactionfunction, accord- ingly inhibited the formation of corneal neovascularization.3 Chitsoan play a role in protecting the corneal epithelium cells, relieving damage and promoting the recovery of cornea after cautery.
|
PDF Full Text Request