碱烧伤后角膜新生淋巴管与炎症反应指数间的关联

目的 探讨角膜碱烧伤后的角膜新生淋巴管与炎症反应指数间的关联.方法 实验研究.制备大鼠角膜碱烧伤模型.采用5'核苷酸酶-碱性磷酸酶(5'-NA-ALP)双重酶组织化学染色及全角膜免疫荧光法分别检测碱烧伤后1、3 d,1、2、3、4、5、6、7及8周的角膜新生淋巴管和血管的动态变化,并进行淋巴管计数(LVC)和血管计数(BVC).同时,于裂隙灯显微镜下观察角膜炎症反应的变化,记录炎症反应指数(IF),并比较LVC和IF之间的关联.11例人角膜取自碱烧伤后行角膜移植的11例患者.淋巴管内皮细胞受体(LYVE-1)免疫组织化学染色法标记人角膜中的新生淋巴管,LVC和IF之间的关联运用Pearson's相关分析,采用配对t检验比较角膜中存在淋巴管和不存在淋巴管的患者之间IF、炎性细胞计数、碱烧伤病史、年龄的差异.结果 碱烧伤后,角膜基质层存在着新生淋巴管.碱烧伤后3 d时出现角膜新生淋巴管,2周末达到高峰,5周末消退.新生淋巴管的出现滞后于炎症反应,但先于炎症反应和新生血管而消退.LVC与IF之间呈正相关(r=0.572,P＜0.01).11例患者中3例存在着角膜新生淋巴管.与另8例角膜中无新生淋巴管的患者相比,前者IF显著性升高(t=3.28,P＜0.05)、炎性细胞计数显著性增加(t=2.42,P＜0.05),年龄显著性下降(t=2.62,P＜0.05),而碱烧伤病史无显著性差异(t=1.28,P＞0.05).结论 角膜碱烧伤后有淋巴管生成,角膜新生淋巴管和炎症反应指数之间存在着密切的关联.     

Crucial role of corneal lymphangiogenesis for allograft rejection in alkali‐burned cornea bed

Backgroud: To examine the time course of hemangiogenesis, lymphangiogenesis, inflammation after corneal alkaline burns and compare with the importance of corneal hemangiogenesis, lymphangiogenesis and inflammation in allograft rejection on alkali‐burned cornea bed, respectively. Methods: Rat corneal hemangiogenesis and lymphangiogenesis were examined by whole mount immunofluorescence and double enzyme‐histochemistry, and the state of corneal inflammation was evaluated by inflammation index scoring and histopathology. Then, corneal transplantations were divided into six groups and performed before the burn (group A) and on day 3 (group B), 2 weeks (group C), 5 weeks (group D), 6 weeks (group E) and 8 weeks (group F) after alkaline burns, respectively. The immune rejection of grafts was evaluated by interferon‐γ, interleukin‐2 enzyme‐linked immunosorbent assay and slit‐lamp examination. Results: Both corneal lymphatic and blood vessels reached the top 2 weeks after the burn. Corneal lymphangiogenesis disappeared 5 weeks after the burn, and corneal hemangiogenesis regressed completely 3 weeks later. Corneal inflammation was strong on day 3, but resolved 6 weeks after the burn. Compared with other groups, the mean survival time of groups B (4.67 ± 1.03 days) and C (5.00 ± 0.63 days) was significantly shorter (P < 0.05). The difference of mean survival time of grafts between group D (9.50 ± 1.05 days) and group E (9.83 ± 0.75 days), between group D and group F (10.00 ± 0.89 days) was not significant (P > 0.05). Conclusions: Corneal lymphangiogenesis presents for a shorter duration than corneal hemangiogenesis or corneal inflammation but plays a crucial role in allograft rejection on alkali‐burned cornea bed.     

鼠角膜碱烧伤后新生淋巴管上血管内皮生长因子受体-3的表达和意义

目的探讨鼠角膜碱烧伤后血管内皮生长因子受体-3(VEGFR-3)在新生淋巴管上的表达和意义,进一步证实角膜新生淋巴管的存在。方法在大鼠角膜上制作碱烧伤模型,应用免疫组化法检测第3、5、7天角膜中VEGFR-3蛋白的表达。电镜观测伤后第5、7、10、14天角膜新生淋巴管的情况。结果碱烧伤后,大鼠角膜组织中VEGFR-3表达从第3天开始明显上升,并于第5天达到最高峰,伤后第14天出现新生淋巴管。结论角膜碱烧伤后有新生淋巴管长入,VEGFR-3可能成为抑制角膜新生淋巴管的一个新靶点。     

高危角膜移植大鼠排斥反应及VEGF-C/D表达的研究

目的：探讨鼠角膜碱烧伤后VEGF-C/D的表达和意义,以及新生淋巴管在高危角膜移植后排斥反应中的作用。
　　方法：制作角膜碱烧伤模型,取不同时间段角膜进行电镜观察,观察角膜血管化情况;采用免疫组织化学方法检测l、3、5、7、14、28 d角膜组织VEGF-C/D及VEGFR-3的表达;并在角膜中仅有血管(A组),同时存在新生血管及新生淋巴管(B组),新生淋巴管消退期(C组),角膜新生血管消退期(D组)以及正常组(N 组)进行穿透性角膜移植,比较不同角膜植片的排斥反应指数( rejection index, RI)值及存活时间。
　　结果：电镜观察发现,在碱烧伤后第7d时鼠角膜出现新生血管,未出现新生淋巴管,在碱烧伤2 wk时出现新生血管的同时出现淋巴管,5wk时无明显的新生淋巴管,8wk时新生血管逐渐消退;大鼠角膜组织中 VEGF-C/D 及VEGFR-3的表达从第3 d开始明显上升,并于第5 d达到最高峰。角膜移植后N、A、B、C、D组的植片平均存活时间分别为14.25±0.62、9.35±1.02、5.06±1.13、8.71±0.83、9.44±1.05d。组间比较发现,B组植片平均存活时间显著性缩短(P<0.05),A、C、D的存活时间均显著性延长(P<0.05)。
　　结论：角膜碱烧伤后存在VEGF-C/D及VEGFR-3的高表达,而且新生淋巴管能加速高危角膜移植后的免疫排斥反应。     

角膜移植后角膜新生淋巴管与新生血管间的关联

目的:研究角膜移植后角膜新生淋巴管与新生血管间关联。方法:人角膜取自行二次角膜移植的19名患者。5核苷酸酶-碱性磷酸酶(5-nase-Alkaline phosphatase,5-NA-ALP)双重酶组织化学染色及淋巴内皮细胞受体(lymphatic vessel endothelial receptor,LYVE-1)、内皮细胞黏附因子-1(platelet endothelial cell adhesion modecule-1,PECAM-1)双重免疫组化法标记角膜中的新生血管和淋巴管,并进行淋巴管计数(lymphatic vessels counting,LVC)和血管计数(blood vessels counting,BVC),比较BVC与LVC之间的关联。结果:角膜中存在角膜新生血管12例(63%),存在角膜新生淋巴管5例(26%)。角膜新生淋巴管仅出现在血管化角膜中。角膜移植后BVC与LVC间呈显著性正相关(r=0.725;P<0.01)。结论:人角膜移植后角膜新生淋巴管与新生血管之间存在密切关联。     

Corneal lymphangiogenesis correlates closely with hemangiogenesis after keratoplasty

AIM: To examine the relationship between corneal lymphangiogenesis and hemangiogenesis after keratoplasty. · METHODS: Nineteen human corneas were obtained from 19 patients undergoing a second corneal transplantation in Zhongshan Ophthalmic Center in 2005. Blood and lymphatic vessels in human transplanted corneas were identified by lymphatic vessel endothelial receptor (LYVE-1) and platelet endothelial cell adhesion modecule-1 (PECAM-1) immunohi- stochemistry, and double enzyme-histochemistry; then the association of corneal blood vessel counting (BVC) with lymphatic vessel counting (LVC) was examined. · RESULTS: Corneal hemangiogenesis was present in 12 cases (63%), and lymphangiogenesis occurred in 5 cases (26%) human transplanted corneas. In addition, corneal lymphangiogenesis was only present in vascularized corneas. LVC was strongly and positively correlated with BVC(r=0.725, P <0.01). · CONCLUSION: Corneal lymphangiogenesis develops after keratoplasty and strongly associates with hemangiogenesis.     

促红细胞生成素(Epo)及其受体(EpoR)在正常及碱烧伤诱导的鼠新生血管化角膜上的表达(英文)

目的:促红细胞生成素(Epo)是促红细胞生成的因子,由胎儿肝脏和成人肾脏产生,是贫血及缺氧时的一种应答反应。视网膜异常血管形成,如早产儿视网膜病变(ROP),增殖性糖尿病性视网膜病变(PDR)时Epo水平增高,提示Epo在病理性眼部血管生成中的作用。Epo参与视网膜血管生成,但其与角膜新生血管是否有关尚未见报道。本研究旨在探讨Epo/EpoR是否在正常和新生血管化角膜表达及角膜内注射Epo是否可诱发角膜新生血管的产生,从而了解其与角膜新生血管的联系。方法:(1)制备碱烧伤诱导鼠角膜新生血管模型,免疫组织化学的方法检测Epo及EpoR是否在正常角膜及新生血管化角膜表达;(2) Epo的克隆、表达及纯化;(3)角膜基质内分别注射Epo(6μL,1μg)及Epo对照(载体对照)及盐水,第14d观察角膜是否有新生血管产生。结果:Epo及EpoR在正常角膜及碱诱发的新生血管化角膜的角膜上皮细胞,角膜内皮细胞,基质细胞均有表达,并在新生血管化角膜表达加强,同时也在基质内炎症细胞及新生血管均有表达。角膜基质内注射Epo后第14d,6眼中5眼产生新生血管,对照组6眼均未见新生血管。结论:本文首次报道了Epo及其受体表达于正常角膜和新生血管化角膜。角膜内注射注射Epo可诱发角膜新生血管。Epo及其受体系统与角膜新生血管化的形成有关。     

Time course of angiogenesis and lymphangiogenesis after brief corneal inflammation

PURPOSE: To study the time course of angiogenesis and lymphangiogenesis in the cornea after a short inflammatory insult. This might be helpful for the timing of corneal transplantation in high-risk eyes. METHODS: The mouse model of suture-induced inflammatory corneal neovascularization was used. After placement of 3 interrupted 11-0 sutures into the corneal stroma of BALB/c mice (left in place for 14 days), corneas were excised 2, 3, 5, 7, 14, and 21 days as well as 1, 2, 3, 6, and 8 months after surgery. Hem- and lymphangiogenesis were evaluated using double immunohistochemistry of corneas with CD31/PECAM1 as panendothelial and LYVE-1 as lymphatic endothelial marker. RESULTS: Both blood and lymphatic vessels grew into the cornea as early as day 2 after suture placement. The outgrowth was initially parallel. Hem- and lymphangiogenesis peaked around day 14. Thereafter, both vessel types started to regress. Regression of lymphatic vessels started earlier and was more pronounced than that of blood vessels. Whereas at 6 and 8 months (partly) perfused CD31+++/LYVE-1(-) blood vessels and (nonperfused) ghost vessels could still be observed, there were no CD31+/LYVE-1+++ lymphatic vessels detectable beyond 6 months after this short inflammation. CONCLUSIONS: After a temporary inflammatory insult to the cornea, there is initially parallel outgrowth of both blood and lymphatic vessels. But thereafter, lymphatic vessels regress earlier than blood vessels and are completely regressed by 6 months. Earlier regression of pathologic corneal lymph versus blood vessels suggests that corneal graft survival in high-risk eyes might best be delayed for a prolonged interval following an inflammatory insult.     

角膜移植后患者角膜新生淋巴管与新生血管和炎症的关联

目的:研究角膜移植后角膜新生淋巴管与新生血管和炎症的关联。方法:人角膜取自行二次角膜移植的患者19例。淋巴内皮细胞受体(lymphatic vessel endothelial hyaluronan receptor,LYVE-1)和内皮细胞黏附因子-1(platelet endothelial celladhesion modecule-1,PECAM-1)双重免疫组化法标记角膜中的新生血管和淋巴管,进行淋巴管计数(lymphatic ves-sels counting,LVC)和血管计数(blood vessels counting,BVC),比较BVC、炎症指数(inflammation index,IF)、移植历史(transplantation history,TH)与LVC之间的关联。结果:角膜移植后BVC,IF与LVC间均呈显著性正相关,而TH与LVC间呈显著性负相关。角膜移植后新生淋巴管、血管、眼表炎症间大致成平行发展,新生淋巴管最先退化,其次是眼表炎症,新生血管最后消退。结论:人角膜移植后角膜新生淋巴管与新生血管、眼表炎症之间存在着极为密切的关联。     

角膜新生淋巴管与血管内皮生长因子C

角膜新生淋巴管（CL）存在于血管化角膜上，常引起并加重角膜混浊和角膜移植排斥反应，是重要的致盲原因之一。血管内皮生长因子C（VEGF—C）是血管内皮生长因子（VEGF）家族成员之一，是VEGFR-2和VEGFR-3受体的配体。VEGF-C通过与VEGFR-3结合促进淋巴管增生，是一个特异性的淋巴管生长因子，在CL形成中起着重要的作用。就近年来CL研究进展及与VEGF—C的关系进行综述。     

角膜共焦显微镜早期诊断兔高危角膜移植排斥反应的价值

目的:角膜共焦显微镜检查对兔碱烧伤角膜移植排斥反应进行研究,找寻排斥反应早期诊断的客观指标。方法:制作兔角膜碱烧伤模型,36d后行穿透性角膜移植,于角膜移植术后4,9,14,21~28d诊断排斥反应时,角膜共焦显微镜检查角膜。结果:排斥反应时角膜共焦显微镜检查见角膜植片炎性细胞浸润,角膜细胞丢失,新生血管生长。结论:角膜共焦检查有助于早期诊断排斥反应。     

Allograft survival enhancement using doxycycline in alkali‐burned mouse corneas

Purpose: To explore the inhibitory effects of doxycycline on allograft rejection in alkali‐burned cornea beds. Methods: The corneas of BALB/c mice were injured using a 1 mol/l NaOH solution. Following the injury, the corneas from C57BL/6 mice were transplanted into the eyes of BALB/c mice after being randomized into three groups: allogeneic corneal transplantation (group A), topical use of doxycycline after allogeneic corneal transplantation (group B) and syngeneic corneal transplantation (group C). Corneal angiogenesis was examined using whole‐mount immunofluorescence, and corneal inflammation was evaluated using inflammation index scoring. The immune rejection of the grafts was examined using a slit lamp. In addition, the expression of vascular endothelial growth factor A and interleukin‐1β in the transplanted corneas was examined using a real‐time polymerase chain reaction, immunohistochemistry and an enzyme‐linked immunosorbent assay. Results: The outgrowth of the corneal blood vessels in the group A mice was faster than that in the group B and group C mice. The inflammation index levels were highest in the group A mice, intermediate in the group B mice and lowest in the group C mice. Vascular endothelial growth factor and the interleukin‐1β protein and mRNA levels decreased dramatically in the group B mice compared with the group A mice (all p‐values < 0.01). In addition, the mean survival time in the group B mice (27.00 ± 2.00 days) was significantly longer than that in the group A mice (11.67 ± 1.51 days; p < 0.05). Conclusions: Doxycycline may have had a significant role in preventing corneal angiogenesis and inflammation in alkali‐burned corneal beds, which resulted in higher allograft survival rates.     

颈淋巴结切除术抑制碱烧伤后角膜移植免疫排斥反应的实验研究

目的：探讨颈淋巴结切除术对碱烧伤后角膜移植免疫排斥反应的抑制作用． 方法：建立大鼠同种异体角膜移植模型，SD鼠为受体，Wistar鼠为供体，受体鼠再随机分为A，B，C，D四组，A组为对照组，B组为颈淋巴结切除组，C组为碱烧伤后角膜移植组，D组为碱烧伤后角膜移植合并颈浅淋巴结切除组。每组均为6只，其中1只于术后14d行植片的巨噬细胞免疫组化染色。其余5只通过裂隙灯观察角膜免疫排斥的状况，检测并比较各组植片平均存活时间（mean survival time，MST）。 结果：各组MST分别是10．40±1．14d；46．30±9．46d；7．00±1．58d和15．00±3．39d。B组MST较A组明显延长（P〈0．05），而D组MST较C组明显延长，差异具有显著性意义（P〈0．05）。角膜移植后14d，B组植片中无CD68阳性细胞出现，A组和D组植片中有不同程度的CD68阳性的巨噬细胞浸润，而C组植片中CD68呈强阳性表达。 结论：颈淋巴结切除术能有效抑制正常及碱烧伤后角膜移植术后的免疫排斥反应。     

Experimental study on cervical lymph nodes removal enhance allograft survival in alkali-burned cornea

AIM: To explore the inhibitive effects of cervical lympha- denectomy on keratoplasy after alkaline burns. METHODS: The Wistar rats' corneas were transplanted into Sprague-Dawley (SD) rats' eyes which were randomly divided into 4 groups: group A (control group); group B, the cervical lymphadenectomy group; group C, corneal transplantation after the alkali burn injury; group D, cervical lymphaden- ectomy following group C. Out of 6 rats in each group, the cornea of one rat was used for macrophage immuno- histochemistry at day 14 after the transplantation, and the remaining 5 rats were used for studying corneal immune rejection with a slit lamp. The time when allograft rejection occurred was recorded and mean survival times (MST) were compared among the groups. RESULTS: Compared with the MST of group A (10.40±1.14 days),the MST of group B(46.30±9.46 days) was significantly longer (P <0.05). MST of grafts between group C (7.00±1.58 days) and group D (15.00±3.39 days) was also significant (P < 0.05). At 14^th day after the transplantation, there was no CD₆₈immunoreactivity in the graft of group B, and CD₆₈ proteins were expressed to some extent in the grafts of group A and D. However, in the graft of group C, the expression of proteins was dramatically up-regulated. CONCLUSION: Cervical lymphadenectomy therapy has a significant effect in preventing corneal allograft rejection in normal and alkali burned corneal beds.     

In vivo observations on experimental corneal neovascularization with a newly developed macroscope

The process of corneal neovascularization induced by alkali burns was periodically observed with a newly developed macroscope. The central corneas were burned using filter discs measuring 6 mm in diameter that had been immersed in 1 N NaOH. At 0, 1, 3 and 7 days and at 2, 3 and 4 weeks after injury, the corneas were observed with the macroscope and then examined histologically. At 1 day post-burn, the limbal vascular plexus was engorged but no new vessel formation was detected. By 3 days, many vascular sprouts had arisen from the limbal vascular arcade. At 7 days, the vascular sprouts grew and became fine new vessels. At 2 weeks, the new vessels lengthened further to the central cornea. At 3 weeks, trunk vessels extended and branched like a vascular tree. Blood in the trunk vessels appeared to flow slowly to and fro. The ends of the vessels swelled in a fusiform shape on the application of slight pressure of the macroscope probe. Histological examination revealed that the ends of the vessels consisted of single vascular endothelial cells and the trunk vessels were covered by pericytes. By 4 weeks, the branch vessels around the burned lesion had degenerated and collapsed. Thus, our in vivo study using the new macroscope not only clarified the process of corneal neovascularization from the early to the regressive phases but also provided some valuable new information.     

小鼠角膜碱烧伤后前炎性因子、趋化因子及其受体的表达

目的检测前炎性因子、趋化因子及其受体在小鼠角膜碱烧伤后表达的动态变化。方法建立小鼠碱烧伤模型，用RT—PCR法检测角膜碱烧伤后不同时间点前炎性因子、趋化因子及其受体mRNA的表达。结果RT-PCR检测显示，角膜碱烧伤后角膜组织中前炎性因子IL-1d、IL-1B及IL-6表达增强；调控单核细胞迁移的CCR1-CCL2、CCL3、CCIA、CCR2-CCL2以及调控中性粒细胞迁移的CXCR2-CXCL2表达增强。结论角膜碱烧伤后前炎性因子以及参与调控白细胞迁移的趋化因子及其受体明显上调，提示其参与角膜碱烧伤后白细胞向局部的迁移。     

碱烧伤人角膜组织新生淋巴管的检测

目的 探讨人角膜碱烧伤后新生淋巴管的生长情况及其影响因素.方法 回顾性系列病例研究.分析2005年1至12月期间在中山大学中山眼科中心因角膜碱烧伤住院手术治疗的22例(22只眼)患者.记录患眼的烧伤时间(IT)和损伤等级(ID),并测量炎性反应指数(II)和角膜新生血管的相对面积(BVA);应用双重酶组织化学、免疫组织化学染色及透射电镜检测角膜标本的新生淋巴管(LVC)和血管(BVC);采用苏木素-伊红染色检测多形核白细胞(PMN)浸润情况.结果 采用配对Student t检验、Pearson相关检验及Stepwise回归进行统计学分析.结果 22例患者观测结果 IT为(57.62±31.72)个月、ID为(12.00±2.76)分、II为(2.32±2.63)分、BVA为29.79%±18.61%、BVC为(14.45±9.29)个、LVC为(2.73±4.57)个、PMN为(13.45±13.09)个,其中7例(占32%,IT<64个月)同时存在角膜新生淋巴管(8.6±3.8)个和血管(22.3±11.1)个,LVC总数为60个,占发生新生淋巴管的角膜中总管腔数(BVC和LVC,378个)的16%.LVC与IT、ID、BVA、PMN、II的相关系数分别为-0.673、0.604、0.755、0.806、0.873,均P<0.05;进一步回顾分析得出LVC近似等于II和BVA分别和特定的常数相乘后取和所得的淋巴管指数(LI).透射电镜从微观上证实了碱烧伤人角膜组织中存在具有典型结构特征的新生淋巴管和炎性细胞浸润.结论 碱烧伤后部分角膜组织存在新生淋巴管,通过II和BVA可间接估计其发生情况.LI是一项评估碱烧伤角膜新生淋巴管的有用临床参数.(中华眼科杂志,2009,45:115-121)     

Plasminogen kringle 5 inhibits alkali-burn-induced corneal neovascularization

PURPOSE: Plasminogen kringle 5 (K5) is a potent angiogenic inhibitor. The purpose of the present study was to evaluate the therapeutic effect of K5 on alkali-burn-induced corneal neovascularization (NV) and to investigate its mechanism of action. METHODS: Corneal NV was induced in rabbits by NaOH. The rabbits received eye drops containing K5 or vehicle alone, four times per day. Corneal NV and inflammation were monitored every other day with a slit lamp microscope, and the length of the vessels in the cornea and the area of NV were measured. Vascular endothelial growth factor (VEGF) was determined by immunohistochemical and Western blot analyses. The TUNEL assay was used to assess the apoptosis of endothelial cells. The effects of K5 on primary bovine aortic endothelial cells (BAECs) were determined by MTT assay, flow cytometry, transmission electron microscopy, and DNA fragmentation assay. RESULTS: Alkali-burn-induced progressive corneal NV and inflammation in the cornea. K5 delayed the onset of corneal NV (P < 0.05) and decreased NV areas (P < 0.05) in a dose-dependent manner. K5 treatment, after the formation of corneal NV, induced regression of newly formatted vessels in the cornea. K5 decreased the inflammatory index in the corneas at different time points after the alkali burn. Corneal VEGF levels were reduced by K5 treatment. K5 inhibits proliferation and induces apoptosis in BAECs. CONCLUSIONS: Topical application of K5 may have therapeutic potential for the chemical burn-induced corneal NV and inflammation. The inhibitory effect of K5 on corneal NV may be by downregulation of VEGF expression.