实验性角膜新生血管的形态观察

目的观察兔角膜新生血管的形态特点,探讨其发生机制及治疗效果.方法将16只新西兰兔随机分为实验和对照两组,均以75%硝酸银液烧灼兔角膜,实验组于硝酸银烧灼后行β射线照射,观察两组角膜新生血管的生长规律;选期制做新生血管铸型,扫描电镜下比较两组间新生血管形态差异.结果角膜新生血管以典型的芽生方式发生和增殖,经β射线照射后的血管芽形成受到明显的抑制和破坏,新生的血管支干发生萎缩、坏死.结论阻断新生血管的芽生过程可抑制角膜新生血管的形成和发展.     

Effects of salicylate and steroid on neutrophil migration and corneal blood vessel growth

Cauterization of the cornea results in emigration of neutrophils from the limbal blood vessels into the corneal tissue. Blood vessel proliferation follows, the stimulus for which is unknown. In this study, 0.1 M sodium salicylate drops administered topically to cauterized rat corneas over a 48-h period had an inhibitory effect on the migration of neutrophils from the limbal vessels 6 h after injury, but this was not maintained at 48 h. After 6 days of treatment, the salicylate had no effect on vessel growth into the cauterized rat cornea. Application of prednisolone disodium phosphate ointment to cauterized corneas also inhibited neutrophil migration at 6 h, but increased the extravascular neutrophils at 48 h. After 6 days of treatment, corneal blood vessel growth was significantly reduced. It was concluded that there is no consistent relation between the number of extravascular neutrophils at the corneal limbus and the extent of corneal blood vessel growth.     

The effect of selective cyclooxygenase-2 inhibitor on corneal angiogenesis in the rat.

PURPOSE. Eicosanoids that are present in inflamed tissues are thought to play a significant role in angiogenesis. Cyclooxygenase, a key enzyme in eicosanoid synthesis, has recently been shown to exist in two isoforms: the constitutive COX-1 and the inducible COX-2. This study was undertaken to determine the role of COX-2 in the corneal angiogenic response. METHODS. Angiogenesis in the rat cornea was provoked by chemical cautery. Either NS-398, a selective COX-2 inhibitor, or indomethacin, a non-selective COX inhibitor, was applied topically 3 times daily for 4 days. Neovascularization was quantitated by digital image analysis in corneal flat preparations. To test their inhibitory effects on eicosanoid synthesis, normal or cauterized corneas were incubated in the culture medium with the inhibitor. Prostaglandin E2 in the medium was assayed using an enzyme-linked immunosorbent assay. RESULTS. Both NS-398 and indomethacin significantly inhibited corneal neovascularization with the % inhibition of 36.4 +/- 9.6%, and 38.5 +/- 9.0%, respectively, when applied topically at a concentration of 0.1% (p <.001). Neither reduced the angiogenic response at a concentration of 0.01% or below. PGE(2) production in the cauterized cornea was 2.0 times higher than that in the controls. In normal corneas, indomethacin inhibited PGE(2) synthesis by 80%, whereas NS-398 inhibited it by no more than 20%. In contrast, in injured corneas, both indomethacin and NS-398 inhibited PGE(2) synthesis in a similar fashion, with a maximal inhibition rate of 75 to 80%. CONCLUSIONS. Our results suggest that COX-2 induction in cauterized corneas increases the level of eicosanoids, which result in corneal angiogenesis.     

Expression of integrins and MMPs during alkaline-burn-induced corneal angiogenesis

PURPOSE: To determine in a corneal alkaline burn model of angiogenesis whether the expression of integrins and MMPs is consistent with a VEGF-induced angiogenic response. METHODS: Neovascularization in female Sprague-Dawley rats was induced by alkaline cauterization of the central cornea. RT-PCR for integrins alpha(1), alpha(2), beta(3), and beta(5); the endothelial marker CD31; and metalloproteinases MMP-2 and MT1-MMP was performed on naive corneas and on cauterized corneas 72 and 288 hours after cautery. Analyses of protein and MMP expression were conducted on naive corneas and on cauterized corneas 24, 72, 120, and 168 hours after cautery by immunofluorescence microscopy and gelatin zymography. RESULTS: RT-PCR indicated a correlation between the induced angiogenic response and the expression of alpha(1) and beta(3) integrin subunits and MT1-MMP. Immunohistochemical analysis indicated that alpha(1), alpha(2), alpha(5), and beta(5) integrins and MMP-2 and MT1-MMP were expressed on the newly developing vasculature. The beta(3) integrin was preferentially expressed on platelets. CONCLUSIONS: Integrin expression during neovascularization of rat corneas in response to alkaline injury correlates with an angiogenic response that uses the VEGF/alpha(v)beta(5) pathway. MMP-2 and MT1-MMP, but not MMP-9, are expressed in a pattern consistent with their involvement in the angiogenic response.     

Absence of blood and lymphatic vessels in the developing human cornea

PURPOSE: The normal human cornea is devoid of both blood and lymphatic vessels and actively maintains this avascularity (corneal angiogenic privilege). Whether and when corneal angiogenic privilege is achieved during development is unknown. METHODS: This study analyzed whether the cornea is primarily devoid of both blood and lymphatic vessels during intrauterine development or whether secondary regression of pre-existing vessels occurs before delivery. Indirect double immunohistochemistry was performed on 4-microm serial pupil-optic disc sections of paraffin-embedded human eyes stillborn at gestational ages of 17 to 41 weeks with antibodies against von Willebrand factor (vWF; factor VIII-associated antigen) as a panendothelial marker and with antibodies against lymphatic vessel endothelial hyaluronate receptor 1 (LYVE1) as a marker specific for lymphatic vascular endothelium. RESULTS: Human corneas were devoid of both vWF+++/LYVE-1(-) blood vessels and vWF+/LYVE-1+++ lymphatic vessels at all time-points analyzed. In contrast, there were numerous blood and lymphatic vessels detectable in the adjacent conjunctiva. CONCLUSION: The normal human cornea is primarily avascular and devoid of both blood and lymphatic vessels. Corneal angiogenic privilege is already achieved very early during fetal intrauterine development. This suggests early and strong expression of both antiangiogenic and antilymphangiogenic factors in the human cornea during development.     

Comparison of subconjunctivally injected bevacizumab, ranibizumab, and pegaptanib for inhibition of corneal neovascularization in a rat model

AIM:To compare the efficacies of subconjunctival bevacizumab, ranibizumab, and pegaptanib sodium injections for the inhibition of corneal neovascularization in an experimental rat model. METHODS:Sixteen corneas of 16 rats were chemically cauterized and randomized into four groups:bevacizumab group that treated with 0.05mL/1.25mg bevacizumab, ranibizumab group that treated with 0.05mL/0.5mg ranibizumab, pegaptanib group that treated with 0.05mL/0.15mg pegaptanib sodium, and control group that treated with 0.05mL saline solution. Digital photographs of the corneas were taken and analyzed using an image analysis software program. All corneas were excised and examined histologically on the 15^th day. RESULTS: Each treatment group had significantly less neovascularized corneal areas and fewer blood vessels than the control group (all P<0.05). In addition, bevacizumab group had significantly less neovascularized corneal areas and fewer blood vessels than ranibizumab and pegaptanib groups (both P<0.05). However, there was no significant difference between the ranibizumab and pegaptanib groups regarding percentage of neovascularized corneal areas and number of blood vessels (both P>0.05). CONCLUSION:Subconjunctival bevacizumab, ranibizumab, and pegaptanib sodium were effective with no corneal epitheliopathy for inhibiting corneal neovascularization after corneal burn in rats. Bevacizumab was more effective than ranibizumab and pegaptanib sodium.     

大鼠角膜化学伤后PEDF和VEGF的不平衡表达

目的比较硝酸银化学伤后大鼠角膜和正常角膜色素上皮衍生因子（PEDF）和血管内皮生长因子（VEGF）表达水平，揭示两者与角膜新生血管的相关性。方法10只大鼠左眼角膜硝酸银化学伤后为实验组，右眼为正常对照组，伤后15d行免疫组织化学法定位及Western blot定量检测样本角膜PEDF、VEGF等的表达。结果免疫组织化学检查：实验组角膜VEGF、碱性成纤维细胞生长因子（bFGF）强表达，PEDF未见表达或弱表达。正常组角膜PEDF高表达，VEGF弱表达，bFGF几乎不表达。Western Blot分析：实验组角膜PEDF表达明显下降（t=8．0049，P〈0．01），VEGF表达显著升高（t=48．3637，P〈0．01）。结论角膜严重化学伤后新生血管抑制因子PEDF破坏，刺激因子VEGF产生增加，PEDF／VEGF比值降低，角膜血管新生。     

Toward electron-beam sterilization of a pre-assembled Boston keratoprosthesis

PurposeTo evaluate the effects of electron-beam (E-beam) irradiation on the human cornea and the potential for E-beam sterilization of Boston keratoprosthesis (BK) devices when pre-assembled with a donor cornea prior to sterilization.MethodsHuman donor corneas and corneas pre-assembled in BK devices were immersed in recombinant human serum albumin (rHSA) media and E-beam irradiated at 25 kGy. Mechanical (tensile strength and modulus, and compression modulus), chemical, optical, structural, and degradation properties of the corneal tissue after irradiation and after 6 months of preservation were evaluated.ResultsThe mechanical evaluation showed that E-beam irradiation enhanced the tensile and compression moduli of human donor corneas, with no impact on their tensile strength. By chemical and mechanical analysis, E-beam irradiation caused a minor degree of crosslinking between collagen fibrils. No ultrastructural changes due to E-beam irradiation were observed. E-beam irradiation slightly increased the stability of the cornea against collagenase-induced degradation and had no impact on glucose diffusion. The optical evaluation showed transparency of the cornea was maintained. E-beam irradiated corneal tissues and BK-cornea pre-assembled devices were stable for 6 months after room-temperature preservation.ConclusionsE-beam irradiation generated no detrimental effects on the corneal tissues or BK-cornea pre-assembled devices and improved native properties of the corneal tissue, enabling prolonged preservation at room temperature. The pre-assembly of BK in a donor cornea, followed by E-beam irradiation, offers the potential for an off-the-shelf, ready to implant keratoprosthesis device.     

Endothelin-1 and ETA/ETB receptor protein and mRNA: expression in normal and vascularized human corneas

PURPOSE: Neovascularization of the cornea causes blindness and increases the risk of immune rejections after keratoplasty. The purpose of this study was to investigate involvement of the potent angiogenic growth factor endothelin (ET)-1 and its receptors, ETA and ETB, in corneal neovascularization. METHODS: ET-1, ETA, and ETB receptor protein expression was evaluated in nonvascularized and vascularized human corneas by immunohistochemistry. Epithelial ET-1 protein expression of both groups was compared using a semiquantitative scoring system. Double immunofluorescence was used to colocalize ETA and ETB receptor with CD31. In situ hybridization and immunoelectron microscopy analyzed ET-1 and its receptors in normal and vascularized corneas. RESULTS: Nonvascularized corneas displayed ET-1 and ETA/ETB receptor protein and mRNA in epithelial and some corneal endothelial cells. ETA more than ETB receptors were expressed on some keratocytes. In vascularized corneas, ET-1 and ETA/ETB receptor expression was found in the endothelial lining of new blood vessels (as shown by CD31-colocalization). ET-1 protein expression was significantly increased in the epithelium of vascularized corneas (P < 0.001). Immunogold localized ET-1 and its receptors to the nuclear/perinuclear space and to the luminal side of endothelial cells of new blood vessels. CONCLUSIONS: In corneal neovascularization, ET-1 protein and mRNA expression is upregulated in epithelial cells. Together with ET-1, ETA, and ETB receptor expression on endothelial cells of ingrown new blood vessels, this points to an involvement of ET-1 and its receptors in corneal angiogenesis. As potent ETA and ETB receptors are available, the endothelin system may represent an additional target for corneal antiangiogenic therapy.     

Induction of conjunctival transdifferentiation on vascularized corneas by photothrombotic occlusion of corneal neovascularization

Previous studies have established that conjunctival transdifferentiation (transformation into cornea-like morphology) is inhibited by corneal vascularization. Conversely, occlusion of corneal vessels may induce conjunctival transdifferentiation on vascularized corneas. To test this hypothesis, the corneal epithelia of New Zealand albino rabbits were debrided 3 mm beyond the limbus with n-heptanol. Sixteen corneas healed by conjunctival epithelium, with vascularization persisting for 20 months, were used in this study. Photochemically induced occlusion of the corneal vessels was achieved by intravenous administration of rose bengal-saline solution (40 mg/kg body weight) with subsequent argon laser irradiation of the vessels (514.5 nm, 130 mW, 63 micron and 0.2 sec). The treated vessels remained occluded in an 18-week study, as confirmed by corneal fluorescein angiography. Corneal clarity and epithelial integrity were improved after treatment. Goblet cell loss and morphologic transformation into a cornea-like epithelium were verified by flat-mount preparations, histology, impression cytology, and immunofluorescence studies using a mucin-specific monoclonal antibody. These results indicate that conjunctival transdifferentiation can be induced on vascularized corneas after occlusion of corneal vessels by photothrombosis.     

The effects of hypertonic salicylate on neutrophil migration and vascularization in the rat cornea

Thermal cauterization of the center of the rat cornea results in emigration of neutrophils into the extravascular limbal tissue and blood vessel growth into the cornea. In this study, 1.0 M sodium salicylate, 1.0 M sodium chloride, and ointment vehicle were administered to normal and cauterized rat corneas for periods of 6, 48, and 144 h. When applied to the normal cornea, salicylate resulted in a marked increase in neutrophils in the limbal tissue at 6 h, but an inhibition at 48 h. Similarly, for the cauterized corneas, administration of salicylate increased the extent of neutrophil emigration at 6 h, but this effect was not sustained at 48 h. Neither vehicle nor sodium chloride had any effect on the extravascular neutrophil population. After 6 days, administration of the vehicle resulted in a slight increase in vascular growth into the cornea, whereas sodium salicylate caused a decrease. These findings indicate that hypertonic (1 M) sodium salicylate does not inhibit the emigration of neutrophils from limbal vessels of cauterized rat corneas, but does appear to have a cytotoxic effect on the tissues and on blood vessel endothelial cells.     

Experimental autoimmune keratitis induced in rats by anti-cornea T-cell lines.

PURPOSE: Idiopathic inflammation of the cornea, keratitis, has been proposed to result from an autoimmune process, but thus far no convenient animal model of keratitis exists. An attempt was made to establish an animal model for keratitis, to investigate possible autoimmune mechanisms. METHODS: T-cell lines were established from lymph node cells removed from rats immunized with bovine corneal epithelium (BCE) extract. After restimulation in vitro with BCE or a specific corneal antigen, the cells were transferred by intraperitoneal injection into naive rats, rats subjected to total body irradiation, or rats in which only one eye was irradiated. RESULTS: Neither direct immunization with corneal antigens nor transfer of activated anti-corneal T-cells into naive rats gave any signs of keratitis. Irradiation alone did not induce corneal inflammation. Transfer of corneal-specific activated T cells into irradiated rats produced keratitis starting around day 4 and culminating around day 8. The disease was self-limiting and the severity dependent on the dose and site of radiation. Keratitis was characterized by corneal haze, conjunctival and episcleral hyperemia, episcleral hemorrhages, chemosis, corneal infiltrates, and vascularization. Immunohistochemistry showed T-cell and macrophage infiltration of epithelium and stroma in the affected corneas. CONCLUSIONS: Thus, keratitis may be produced by T cells reactive to corneal antigens, provided that the target tissue has been made susceptible by irradiation. The effectiveness of T-cell vaccination in preventing adoptive keratitis suggests that systemic as well as local tissue factors may regulate the disease process.     

Safety,penetration and efficacy of topically applied bevacizumab: evaluation of eyedrops in corneal neovascularization after chemical burn

Purpose: That vascular endothelial growth factor (VEGF) plays a major role in inflammatory angiogenesis has been well established. This pilot study was designed to evaluate experimental treatment with bevacizumab eyedrops in corneal neovascularization induced by alkali burn. The feasibility of topical administration, corneal cell viability and corneal penetration were investigated in an animal model. Methods: Eighteen chinchilla bastard rabbit corneas injured with 1 m NaOH were divided into three groups: untreated, early and late treatment groups. Eyedrops of bevacizumab solution (25 mg/ml) were administered five times daily. Clinical examination under stereoscopic microscope was performed to evaluate corneal opacity, neovascularization, vessel size and oedema. Histopathology was analysed for vessel density and apoptotic reaction. Additionally, intracameral bevacizumab concentration was measured with enzyme‐linked immunosorbent assay (ELISA) after repeated topical applications. Results: A fast increase in aqueous bevacizumab concentration was achieved when the solution was instilled every minute onto a healthy eye surface. As well as clear anti‐angiogenic effects, anti‐fibrotic effects were also seen after corneal burn, maintaining corneal transparency. Early treatment of actively growing vessels showed a significantly better outcome, although apoptosis of pre‐existing vessels could also be induced by the late treatment. No specific toxicity was seen regarding epithelium, keratocytes or endothelium. Conclusions: The data from this pilot study suggest that bevacizumab eyedrops can sufficiently penetrate the corneal stroma and anterior chamber. When administered soon after alkali burn, bevacizumab seems to significantly reduce corneal damage. Combinations of established treatment regimens with topical bevacizumab might be considered in severe injuries with otherwise devastating prognoses.     

碱烧伤后角膜干细胞活性的实验研究

目的 了解大鼠角膜烧伤后角膜干细胞的活性。方法 对大鼠角膜中央区，上半周及全周３个不同区域分别进行碱性烧灼，于伤后２４小时取材做离体角膜＾３Ｈ－Ｔｄｒ掺５入，计算角膜基底细胞标记率。结果 中央区及上半周烧伤组角膜基底细胞标记率分别为６．７８４和６．００２，与对照组１．２９相比，Ｈ＝３０．５５，Ｐ〈０．００１，有显著性差异，而全周烧伤组未见标记细胞。     

The release of a neutrophil chemotactic factor from UV-B irradiated rabbit corneas in vitro

Rabbit corneas were isolated, mounted on plastic rings to form a cup and the endothelium was covered with RPMI tissue culture medium. The preparation was then irradiated with 1 J. cm-2 of 300 nm light over 1 hour and then incubated for a further two hours in the dark. The supernatant fluid was assayed for chemotactic activity toward rabbit neutrophils in an in vitro Boyden chamber assay. The results indicated that medium from irradiated corneas had a chemotactic activity that was 42% of that produced by the standard chemoattractant f-met-leu-phe, (10(-9) M) while medium from unexposed corneas and exposed medium alone had less than 3% activity. An in vivo assay using sub-epidermal injection into the back of a rabbit gave qualitatively similar results, only f-met-leu-phe and the medium from irradiated corneas causing neutrophil infiltration of the tissue. A checkerboard analysis confirmed that the activity was chemotactic rather than chemokinetic. Release of a chemotactic factor following UV-B irradiation provides a mechanism for the recruitment of neutrophils, at specific localized areas of the endothelium, that is seen after discrete in vivo irradiation. The results also confirm the importance of corneal inflammatory mediators in the development of tissue damage subsequent to exposure to toxic agents.     

Pericyte recruitment in human corneal angiogenesis: an ultrastructural study with clinicopathological correlation

BACKGROUND/AIM: During angiogenesis-that is, the outgrowth of new from pre-existing blood vessels, new capillaries undergo a period of "fine tuning" when vascular endothelial cells become apoptotic if sufficient supply of angiogenic factors is lacking. Morphologically, this period correlates with the absence of pericyte coverage of new vessels. Mature, pericyte covered vessels, in contrast, do not depend on elevated levels of angiogenic factors for survival. This study analyses whether, and if so when, pathological vessels in human corneal neovascularisation (CN) acquire pericyte coverage. This can be of importance for future angioregressive therapeutic strategies. METHODS: Vascularised human corneas obtained by keratoplasty were evaluated by electron microscopy for pericyte coverage of new vessels. These data were correlated with the duration of CN (mean 73 (SD 95) (range 0.5-360) months; n = 15). CN was secondary to keratitis, transplant rejection, aniridia, or trauma. RESULTS: Overall, 196 blood vessels were analysed ultrastructurally (72 (37%) capillaries, 122 (62%) venules, and two (1%) arterioles). Electron microscopically, 170 (87%) vessels were covered by pericytes and two (1%) in addition by smooth muscle cells. Pericyte recruitment increased with time, evolving between clinically noted onset of CN and keratoplasty. Already 2 weeks after onset of CN, more than 80% of new vessels were covered by pericytes. CONCLUSION: Pathological new vessels in human corneal angiogenesis are rapidly covered by pericytes. Therapeutic strategies aimed at regression of immature, not yet pericyte covered vessels by antagonising angiogenic factors should thus be most effective if applied very early in the course of corneal neovascularisation.     

In vitro effect of corneal collagen cross-linking on corneal hydration properties and stiffness

Background

The purpose of this study is to evaluate in-vitro the immediate effect of corneal collagen cross-linking (CXL) on corneal hydration and stiffness.

Methods

Forty-two corneal buttons from freshly enucleated porcine eyes were immersed in riboflavin 0.1% in dextran 20% dilution for 3 h in order for their hydration to reach equilibrium. Corneal buttons where divided into two groups; the first group was stored in dark conditions while the other group was irradiated with UV radiation (370 nm) for 30 min to simulate CXL according to the clinically applied protocol. After irradiation, all corneas were immersed in dextran 20% solution for 3 additional hours. Subsequently, each button underwent weighing, thickness measurement, and was mounted in a special device for the measurement of force versus deformation by compression. Finally, all corneal buttons were dehydrated for 48 h in a desiccating oven set at 62 °C and weighed again to obtain their dry mass. Hydration (%) of each button was calculated.

Results

Mean corneal hydration in the irradiated and the non-irradiated group of corneas was 69.8 and 72.2%, respectively (p?<?0.001). Differences in thickness and compressibility were not statistically significant. Thickness and hydration were positively correlated (Pearson’s r?=?0.714, p?<?0.001).

Conclusions

CXL causes corneal dehydration that can be detected immediately after the procedure. This phenomenon may contribute to increased mechanical stiffness of the cornea. A change in stiffness by means of compressibility could not be detected in porcine corneas.     

Limbal vascular response prior to corneal vascularization

In spite of claims of a significant latent period of up to 2 days between corneal injury and the beginning of corneal vascularization, there is evidence of a limbal reaction occurring only hours after injury. In this study, the central area of rat corneas was cauterized to give an injury which was remote from the limbal vessels. The resulting changes in vascular permeability were investigated. Significant emigration of neutrophil polymorphonuclear leucocytes began at 36 min and leakage of Trypan blue was discernible at about 1 hr after injury. Eosinophilic leucocytes do not appear to be involved in the response and mast cells, while not showing signs of increased degranulation, may be decreased in number over several days. The significance of these results in relation to the stimulus factors involved in these changes, and the importance of separating inflammatory and proliferative changes, are diseussed.     

Suppression of experimental corneal angiogenesis by focal X-ray irradiation.

PURPOSE: To investigate the effect of focal X-ray irradiation on experimental corneal angiogenesis in the rabbit. METHODS: A gelatin hydrogel sheet impregnated with basic fibroblast growth factor was implanted into the corneal stroma of rabbits; this induced corneal angiogenesis. After the first sign of corneal angiogenesis was noted, the corneal region was irradiated with a dose of 10 Gy or 20 Gy. The control rabbits received no irradiation. The eyes were examined by slitlamp biomicroscopy and photographed, over a period of 28 days. The maximum length and total surface area of corneal angiogenesis were quantified by computerized image analysis. RESULTS: Corneal angiogenesis was noted on day 3 following implantation of the hydrogel sheet. In the rabbits irradiated with 10 Gy, the maximum length and total surface area of corneal angiogenesis were both significantly lower on day 4 and 7 following irradiation, compared to the respective measurement in the controls. In the rabbits irradiated with 20 Gy, the maximum length and total surface area of corneal angiogenesis were significantly lower between days 4 and 21, and between days 4 and 14, respectively, compared to the respective measurement in the controls. CONCLUSIONS: Focal X-ray irradiation to the corneal region suppressed corneal angiogenesis in a dose-dependent manner. Focal X-ray irradiation may be beneficial in treating ocular angiogenesis.