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.     

Corneal lymphangiogenesis: evidence,mechanisms, and implications for corneal transplant immunology

PURPOSE: The normal cornea is devoid of blood and lymphatic vessels but can become vascularized secondary to a variety of corneal diseases and surgical manipulations. Whereas corneal (hem)angiogenesis, i.e., the outgrowth of new blood vessels from preexisting limbal vessels, is obvious both clinically and histologically, proof of associated corneal lymphangiogenesis has long been hampered by invisibility and lack of specific markers. This has changed with the recent discovery of the lymphatic endothelial markers vascular endothelial growth factor receptor 3, LYVE-1 (a lymphatic endothelium-specific hyaluronan receptor), Prox 1, and Podoplanin. METHODS: We herein summarize the current evidence for lymphangiogenesis in the cornea and describe its molecular markers and mediators. Furthermore, the pathophysiologic implications of corneal lymphangiogenesis for corneal transplant immunology are discussed. RESULTS: Whereas corneal angiogenesis in vascularized high-risk beds provides a route of entry for immune effector cells to the graft, lymphangiogenesis enables the exit of antigen-presenting cells and antigenic material from the graft to regional lymph nodes, thus inducing alloimmunization and subsequent graft rejection. CONCLUSIONS: Antilymphangiogenic strategies may improve transplant survival both in the high- and low-risk setting of corneal transplantation.     

Wessely corneal ring phenomenon: An unsolved pathophysiological dilemma

The cornea is a densely innervated avascular tissue showing exceptional inflammatory and immune responses. The cornea is a site of lymphangiogenic and angiogenic privilege devoid of blood and lymphatic vessels that limits the entry of inflammatory cells from the adjacent and highly immunoreactive conjunctiva. Immunological and anatomical differences between the central and peripheral cornea are also necessary to sustain passive immune privilege. The lower density of antigen-presenting cells in the central cornea and the 5:1 peripheral-to-central corneal ratio of C1 are two main features conferring passive immune privilege. C1 activates the complement system by antigen-antibody complexes more effectively in the peripheral cornea and, thus, protects the central corneas’ transparency from immune-driven and inflammatory reactions.Wessely rings, also known as corneal immune rings, are noninfectious ring-shaped stromal infiltrates usually formed in the peripheral cornea. They result from a hypersensitivity reaction to foreign antigens, including those of microorganism origin. Thus, they are thought to be composed of inflammatory cells and antigen-antibody complexes. Corneal immune rings have been associated with various infectious and noninfectious causes, including foreign bodies, contact lens wear, refractive procedures, and drugs. We describe the anatomical and immunologic basis underlying Wessely ring formation, its causes, clinical presentation, and management.     

Lymphatic vessels in vascularized human corneas: immunohistochemical investigation using LYVE-1 and podoplanin

PURPOSE: To determine whether lymphatic vessels exist in vascularized human corneas, by using immunohistochemistry with novel markers for lymphatic endothelium. METHODS: Human corneas exhibiting neovascularization secondary to keratitis, transplant rejection, trauma, and limbal insufficiency (n = 21) were assessed for lymphatic vessel content by conventional transmission electron microscopy and by immunostaining and immunoelectron microscopy with antibodies specific for the lymphatic endothelial markers, lymphatic vessel endothelial hyaluronan receptor (LYVE-1) and the 38-kDa integral membrane glycoprotein podoplanin. In addition, corneas were stained for the lymphangiogenic growth factor VEGF-C, and its receptor VEGFR3 by immunohistochemistry and in situ RNA hybridization, respectively. RESULTS: Thin-walled, erythrocyte-free vessels staining with lymphatic markers (LYVE-1 and podoplanin) were found to constitute 8% of all vessels, to be more common in the early course of neovascularization, to be always associated with blood vessels and stromal inflammatory cells, and to correlate significantly with the degree of corneal hemangiogenesis (r = 0.6; P = 0.005). VEGF-C, VEGFR3, podoplanin, and LYVE-1 colocalized on the endothelial lining of lymphatic vessels. With immunogold labeling, LYVE-1 and podoplanin antigen were found on endothelial cells lining vessels with ultrastructural features of lymph vessels. CONCLUSIONS: Immunohistochemistry with novel lymph-endothelium markers and ultrastructural analyses indicate the existence of lymphatic vessels in vascularized human corneas. Human corneal lymphangiogenesis appears to be correlated with the degree of corneal hemangiogenesis and may at least partially be mediated by VEGF-C and its receptor VEGFR3.     

Novel expression and characterization of lymphatic vessel endothelial hyaluronate receptor 1 (LYVE-1) by conjunctival cells

PURPOSE: Lymphatic vessel endothelial hyaluronic acid receptor (LYVE-1) is a newly discovered lymphatic-specific marker. To date, there is no report of its expression on conjunctival cells. The purpose of this study was to investigate the expression of LYVE-1 in normal conjunctiva, to phenotype LYVE-1+ cells, and to study changes in their expression levels during corneal inflammation. METHODS: Flat-mounted conjunctivae or cross sections of eyeballs were harvested from BALB/c mice (6-8 weeks of age) for immunofluorescent confocal microscopic studies. RESULTS: The data demonstrate, for the first time, that in addition to its expression on lymphatic vessels, LYVE-1 was expressed on CD45+, CD11b+, and CD31- conjunctival cells, indicating a bone-marrow-derived monocytic lineage. Surprisingly, the number of cells that expressed LYVE-1 decreased during corneal inflammation, in conjunction with ingrowth of lymphatics into the cornea. CONCLUSIONS: A new population of monocytic cells has been found to express LYVE-1 in normal conjunctiva. These cells that normally express LYVE-1 may act as a reservoir for lymphangiogenesis and cell recruitment when the immune system is challenged.     

Corneal lymphangiogenesis as a potential target in dry eye disease - a systematic review

Dry eye disease (DED) is a common ocular surface condition causing symptoms of significant discomfort, visual disturbance, and pain. With recent advancements, DED has become recognized as a chronic self-perpetuating inflammatory condition triggered by various internal and environmental factors. DED has been shown to arise from the activation of both the innate and adaptive immune systems, leading to corneal epithelium and lacrimal gland dysfunction. While the cornea is normally avascular and thus imbued with angiogenic and lymphangiogenic privilege, various DED models have revealed activated corneal antigen-presenting cells in regional lymph nodes, suggesting the formation of new corneal lymphatic vessels in DED. The recent availability of reliable lymphatic cell surface markers such as LYVE-1 has made it possible to study lymphangiogenesis. Accordingly, numerous studies have been published within the last decade discussing the role of lymphangiogenesis in DED pathology. We systematically review the literature to identify and evaluate studies presenting data on corneal lymphangiogenesis in DED. There is considerable evidence supporting corneal lymphangiogenesis as a central mediator of DED pathogenesis. These findings suggest that anti-lymphangiogenic therapeutic strategies may be a viable option for the treatment of DED, a conclusion supported by the limited number of reported clinical trials examining anti-lymphangiogenic modalities in DED.     

Angiogenesis and lymphangiogenesis in the cornea. Pathogenesis,clinical implications and treatment options

BACKGROUND: Whereas the normal cornea is devoid of blood and lymphatic vessels,both can invade the cornea secondary to a variety of corneal diseases and after surgery.This not only reduces visual acuity, but also renders such a cornea high-risk, if subsequent corneal transplantation is performed. METHODS: A PUBMED-based literature search was carried out. RESULTS: Current knowledge on pathogenesis, clinical implications and treatment modalities for corneal neovascularization is discussed. CONCLUSIONS: Novel anti-angiogenic and antilymphangiogenic therapeutic strategies should reduce blindness associated with corneal neovascularization and subsequent graft rejection.     

Bevacizumab as a potent inhibitor of inflammatory corneal angiogenesis and lymphangiogenesis

PURPOSE: To analyze whether bevacizumab can inhibit inflammatory angiogenesis and lymphangiogenesis in the cornea. Bevacizumab (Avastin; Roche, Welwyn Garden City, UK) is a recombinant, humanized, monoclonal antibody against VEGF-A that has been approved by the U.S. Food and Drug Administration for the treatment of colon carcinomas. METHODS: The mouse model of suture-induced corneal neovascularization was used to assess the antihemangiogenic and antilymphangiogenic effect of bevacizumab by systemic and topical application. Corneal flatmounts were stained with LYVE-1 as a specific lymphatic vascular endothelial marker and CD31 as a pan-endothelial marker, and blood and lymph vascularized areas were analyzed morphometrically. The inhibitory effect of bevacizumab on lymphatic endothelial cells (LECs) was analyzed with a colorimetric (BrdU) proliferation ELISA. The binding ability of bevacizumab to murine VEGF-A was analyzed by Western blot, ELISA, and surface plasmon resonance. RESULTS: The systemic and topical applications of bevacizumab significantly inhibited the outgrowth of blood (P < 0.006 and P < 0.0001, respectively) and lymphatic (P < 0.002 and P < 0.0001, respectively) vessels. Inhibition of the proliferation of LECs was also significant (P < 0.0001). Western blot analysis, ELISA, and the surface plasmon resonance assay showed that bevacizumab binds murine VEGF-A. CONCLUSIONS: Topical or systemic application of bevacizumab inhibits both inflammation-induced angiogenesis and lymphangiogenesis in the cornea. This finding suggests an important role of VEGF-A in corneal lymphangiogenesis. Bevacizumab may be useful in preventing immune rejections after penetrating keratoplasty or tumor metastasis via lymphatic vessels.     

Relationship between angiogenesis and lymphangiogenesis in recurrent pterygium

AIM: To examine the relationship between angiogenesis and lymphangigenesis in recurrent pterygia. METHODS: Tissues from 34 excised recurrent pterygia (including 12 Grade 1, 10 Grade 2, and 12 Grade 3) were involved in the study and tissues from 7 nasal epibulbar conjunctivae segments were used as controls. Sections from each pterygium were immunostained with CD₃₁ and LYVE-1 monoclonal antibodies to evaluate lymphatic microvessel density (LMVD) and blood microvessel density (BMVD), and the relationship between LMVD and BMVD in the pterygium was examined. RESULTS: There was a large number of CD₃₁⁽⁺⁾LYVE-1^(-) blood vessels but only a few CD₃₁⁽⁺⁾LYVE-1⁽⁺⁾ lymphatic vessels in grades 1 and 2 pterygium. However, lymphatic vessels were dramatically increased in grade 3 pterygium. LMVD correlated closely with BMVD in all pterygia, including grades 1, 2 and 3 peterygium patients (all P values <0.01). Although both the density of blood and lymphatic vessels increased in recurrent pterygia, lymphatic vessels developed much faster than blood vessels, especially in grade 3 pterygia. CONCLUSION: There is a significant but not parallel relationship between angiogenesis and lymphangiogenesis in recurrent pterygium. The outgrowth of blood and lymphatic vessels provide evidence that immunological mechanism may play a role in the development and recurrence of pterygium.     

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。     

Horizons in therapy for corneal angiogenesis

Corneal neovascularization can lead to a devastating disease process that involves the breakdown of the limbal barrier and the formation of blood vessels in the cornea, leading to severe visual impairment. This review discusses the delicate balance between antiangiogenic and angiogenic factors that govern the antiangiogenic privilege of the cornea. Current treatment methods, clinical trials, and future prospects in the management of corneal neovascularization also are discussed.     

Short- and long-term corneal vascular effects of tafluprost eye drops

Background

Prostaglandin analogs are first line therapy in the treatment of glaucoma, but also display side effects during ocular inflammation. In this context, the potential side effects of prostaglandin analogs on the normally avascular cornea, the main application route for eye drops, are so far not fully defined. Therefore, the aim of this study was to evaluate the vascular effects of the prostaglandin analog tafluprost on the healthy and inflamed cornea.

Methods

For in vitro studies, blood and lymphatic endothelial cells were treated with tafluprost; cell proliferation was assessed after 48 h. For long-term in vivo studies under healthy conditions, naïve corneas of BALB/c mice were treated with tafluprost eye drops for 4 weeks. For short-term in vivo studies under inflammatory conditions, corneal inflammation was induced by suture placement; mice then received tafluprost eye drops for 1 week. Afterwards, corneas were stained with CD31 as panendothelial and LYVE-1 as lymphendothelial (and macrophage) marker.

Results

In vitro, tafluprost did not alter blood or lymphatic endothelial cell proliferation. In vivo, there was no change in limbal blood or lymphatic vessel anatomy after long-term treatment with tafluprost. Short-term treatment with tafluprost under inflammatory conditions did not influence the recruitment of LYVE-1 positive macrophages into the cornea. Moreover, treatment of inflamed corneas with tafluprost did not significantly influence corneal hem- and lymphangiogenesis.

Conclusions

Tafluprost does not affect blood and lymphatic vessel growth, neither under resting nor under inflammatory conditions. These findings suggest a safe vascular profile of tafluprost eye drops at the inflammatory neovascularized cornea.     

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.     

角膜血管新生机制与调控新进展

正常角膜在多种因素的共同作用下处于稳定的"血管赦免"状态,这也是角膜发挥正常生理功能的基础。但在某些特定情况,如缺氧、炎症和角膜干细胞缺乏时,角膜血管生成因子和抑制因子的平衡被打破,角膜血管化开关开启,角膜缘毛细血管网通过血管新生和血管生成产生新生血管,使角膜无血管状态被破坏。我们从正常角膜的无血管状态、角膜血管新生启动机制、血管的生长过程和几种主要调控因子等方面对角膜血管新生机制与调控机制进行综述。     

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。     

LYVE-1-positive macrophages are present in normal murine eyes

PURPOSE: A functioning lymphatic system is necessary not only to permit the organism to mount a rapid and effective immune response but, even more so, to maintain tissue fluid homeostasis. However, no clear evidence of lymphatic vessels draining intraocular and orbital tissues--retina, choroid, sclera, and extraocular muscles--exists. METHODS: Ocular tissue flatmounts from normal or enhanced green fluorescence protein (EGFP) chimeric mice were immunostained for lymphatic endothelium hyaluronan receptor (LYVE-1, a routinely used lymphatic endothelial marker), podoplanin, Flt4/VEGFR3, Sca-1, CD11b, or F4/80 and were observed by confocal microscopy. Single-cell suspensions from ocular tissues were also prepared and were analyzed by flow cytometry. RESULTS: Lymphatic vessels were detected in the posterior regions of the extraocular muscles and the connective tissues of the extraocular muscle cones in the normal mouse. No typical lymphatic vessels were found within the eye. A large population of LYVE-1(+) nonendothelial cells, distributed as single cells, was detected in all ocular tissues except the central cornea. These cells also express another lymphatic endothelial cell marker, Flt4/VEGFR3, but not podoplanin, and they have hyaluronan-binding ability. Bone marrow chimerism studies indicated that the LYVE-1(+) cell populations are bone marrow derived and have a slow turnover in ocular tissues (3-6 months). Phenotype analysis revealed that nonendothelial LYVE-1(+)cells in the sclera, choroid, and iris included CD11b(+)F4/80(+) macrophages, CD11b(+)F4/80(-) macrophages, and CD11b(-)F4/80(-) bone marrow-derived cells. All LYVE-1(+) cells in the retina were CD11b(+)F4/80(+) macrophages. Cells in the limbus and the iris root also express Sca-1, suggesting that they are hematopoietic lymphatic vessel progenitor cells. CONCLUSIONS: These observations suggest that a lymphatic system exists for the transport of immune cells and fluids from the posterior segment of the eye, that ocular tissues are rich in bone marrow-derived LYVE-1(+) macrophages under normal physiological conditions, and that a subpopulation of these cells may represent resident precursor cells necessary for the de novo formation of ocular/orbital lymphatic vessels in pathologic conditions.     

Lymphangiogenesis concurrent with haemangiogenesis in the human cornea

BACKGROUND: Corneal transplant rejection can occur with and without neovascularization; therefore, it is necessary to elucidate what other factors allow for rejection. It has been suggested that the lymphatic system may play a role in graft failure, but it has also been held that the cornea is devoid of lymphatics. Use of a new monoclonal antibody against a lymphatic endothelial marker, D2-40, has been used to detect lymphatics in other tissues. The purpose of this study was to use this new tool to determine if the human cornea can undergo lymphangiogenesis. METHODS: Twelve corneal buttons submitted for routine pathology were subjected to immunohistochemical staining with a monoclonal antibody against D2-40 to detect the presence/absence of lymphatics by light microscopy. RESULTS: By the criteria defined, lymphatic vessels were identified in seven out of 12 corneal buttons. In these cases, there was also evidence of neovascularization. Lymphatic positive buttons included four cases where there were histological markers of inflammation. There were no identifiable lymphatics in the remaining five cases and no sign of vascularization. CONCLUSIONS: Corneal lymphatics were identified in association with corneal neovascularization, via the use of a monoclonal antibody against D2-40. In non-vascularized corneas, lymphatics were absent.     

Clinical and experimental research of corneal lymphangiogenesis after keratoplasty

The objective of this study is to provide further evidence that corneal lymphangiogenesis occurs after keratoplasty, and to explore the association of corneal hemangiogenesis, corneal inflammation and transplantation history with corneal lymphangiogenesis. Rat corneal lymphangiogenesis was examined by electron microscopy, lymphatic vessel endothelial receptor (LYVE-1) immunohistochemistry, and whole-mount immunofluorescence at 1, 3, 7, 10 and 14 days after corneal transplantation. Blood and lymphatic vessels in human transplanted corneas were identified by LYVE-1 and CD(31) immunohistochemistry, then the association between corneal blood vessel counting, inflammatory index and transplantation history with the lymphatic vessel counting was examined. The results showed that corneal lymphangiogenesis was present in all rat corneas and 26% of human transplanted corneas. Lymphatic vessel counting was significantly associated with blood vessel counting, inflammatory index and transplantation history (all p values <0.0001). We conclude that corneal lymphangiogenesis develops after keratoplasty, and is strongly associated with hemangiogenesis, inflammation and the history of transplantation.     

Inhibition of hemangiogenesis and lymphangiogenesis after normal-risk corneal transplantation by neutralizing VEGF promotes graft survival

PURPOSE: To evaluate the occurrence and time course of hem- and lymphangiogenesis after normal-risk corneal transplantation in the mouse model and to test whether pharmacologic strategies inhibiting both processes improve long-term graft survival. METHODS: Normal-risk allogeneic (C57BL/6 to BALB/c) and syngeneic (BALB/c to BALB/c) corneal transplantations were performed and occurrence and time course of hem- and lymphangiogenesis after keratoplasty was observed, by using double immunofluorescence of corneal flatmounts (with CD31 as a panendothelial and LYVE-1 as a lymphatic vascular endothelium-specific marker). A molecular trap designed to eliminate VEGF-A (VEGF Trap(R1R2); 12.5 mg/kg) was tested for its ability to inhibit both processes after keratoplasty and to promote long-term graft survival (intraperitoneal injections on the day of surgery and 3, 7, and 14 days later). RESULTS: No blood or lymph vessels were detectable immediately after normal-risk transplantation in either donor or host cornea, but hem- and lymphangiogenesis were clearly visible at day 3 after transplantation. Both vessel types reached donor tissue at 1 week after allografting and similarly after syngeneic grafting. Early postoperative trapping of VEGF-A significantly reduced both hem- and lymphangiogenesis and significantly improved long-term graft survival (78% vs. 40%; P < 0.05). CONCLUSIONS: There is concurrent, VEGF-A-dependent hem- and lymphangiogenesis after normal-risk keratoplasty within the preoperatively avascular recipient bed. Inhibition of hem- and lymphangiogenesis (afferent and efferent arm of an immune response) after normal-risk corneal transplantation improves long-term graft survival, establishing early postoperative hem- and lymphangiogenesis as novel risk factors for graft rejection even in low-risk eyes.     

Improved semiautomatic method for morphometry of angiogenesis and lymphangiogenesis in corneal flatmounts

Purpose of the study was to describe a novel semiautomatic, quantitative image analysis method based on threshold analysis for morphometry of corneal (lymph)angiogenesis and to test its validity, reliability and objectivity. Murine corneas were vascularized by using a suture-induced neovascularization assay. For immunohistochemistry, flatmounts of the vascularized corneas were stained with LYVE-1 as a specific lymphatic vascular endothelial marker and with CD31 as panendothelial marker. Morphometry of corneal hem and lymphangiogenesis was performed semi-automatically on digital images using image analysis software. Data were analyzed by a paired t-test, intraclass-correlation and systemic difference analysis compared to a manual method. The semiautomatic method based on threshold analysis was more valid in measuring the area covered by blood or lymphatic vessels. Both methods had a good reproducibility with respect to both vessel types (blood vessels: manual: 0.969, semiautomatic: 0.982; lymphatic vessels: manual: 0.951, semiautomatic: 0.966), whereas the systemic difference was significant for both groups measuring lymphatic vessels (manual: p < 0.003; semiautomatic: p < 0.035) and for the manual method measuring blood vessels (manual: p < 0.0001; semiautomatic: p < 0.419). The new semiautomatic morphometry method based on threshold analysis provides higher accuracy, is more valid than and at least as reproducible and objective as the manual outlining method. Therefore the semiautomatic method can be used to detect even small effects on hem and lymphangiogenesis in murine corneal flatmounts with greater precision.