using-laminating-technique-to-perform-confocal-microscopy-human

Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera


The sclera is a dense connective tissue that covers and protects the eye. It mainly consists of dense collagen bundles (types I, III, IV, V, VI, and ...

University of Cologne

Penetrating keratoplasty has a very poor outcome compared with adults if performed in the first years of life. Rejection in these young patients occurs even in the absence of known immunological risk factors. Recently, a baby rat model was introduced and an essential contribution of natural killer (NK) cells during allograft rejection was suggested. To analyze this, NK cells were depleted in baby rats before keratoplasty.

NK cell depletion delays corneal allograft rejection in baby rats.

Predictive value of serum markers for pulmonary involvement in ocular sarcoidosis.

To investigate whether the normal adult human sclera contains lymphatic vessels and to study their relation to immune cells and blood vessel anatomy.

Enrichment of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1)-positive macrophages around blood vessels in the normal human sclera.

Tumor-derived VEGF-A, apart from expediting sufficient vascularization, subsequent tumor growth, and metastatic spread, can act on malignant cells themselves provided that VEGF receptors 1 or 2 (VEGF-R1, -R2) are co-expressed. The study goal was to investigate whether such autocrine VEGF-A signaling exists in uveal melanoma (UM).

Autocrine impact of VEGF-A on uveal melanoma cells.

To explore the function of natural killer (NK) cells in inflammatory angiogenesis in mice.

A novel pro-angiogenic function for interferon-γ-secreting natural killer cells.

The adult sclera is free of lymphatic vessels, but contains a net of blood vessels. Whether and when this selectively lymphangiogenic privilege is achieved during embryologic development is not known yet. Therefore, we investigated the developing human sclera for blood- and lymphatic vessels in 34 abortions/stillborns (12-38 weeks of gestation). The probes were subdivided into three groups (group 1: 12-18 weeks of gestation, n = 10; group 2: 19-23 weeks of gestation, n = 13; group 3: 24-38 weeks of gestation, n = 11), and prepared for paraffin sections followed by immunohistochemistry against CD31 to detect blood vessels, and against lymphatic vessel endothelial hyaluronan receptor-1 (LYVE1)/podoplanin to detect lymphatic vessels. We could show, that in the human episclera distinct CD31 + blood vessels are present as early as week of gestation 13. Their amount increased during pregnancy, whereas stromal CD31 + blood vessels were elevated in early pregnancy and regressed with ongoing pregnancy. In the lamina fusca CD31 + blood vessels were absent at any time point investigated. Single LYVE1 + cells were identified primarily in the episclera; their amount decreased significantly with increasing gestational ages (group 1 compared to group 3: p < 0.01). However, LYVE1+/podoplanin + lymphatic vessels were not detectable in the sclera at any gestational ages analyzed. In contrast to the conjunctiva where LYVE1+/podoplanin + lymphatic vessels were detectable as early as week 17, the amount of LYVE1 + cells in the sclera was highest in early pregnancy (group 1), with a significant decrease during continuing pregnancy (p < 0.001). These findings are the first evidence for a fetal lymphangiogenic privilege of the sclera and show, that the fetal human sclera contains CD31 + blood vessels, but is primarily alymphatic. Our findings suggest a strong expression of selectively antilymphangiogenic factors, making the developing sclera a potential model to discern antilymphangiogenic mechanisms.

Absence of lymphatic vessels in the developing human sclera.

During human ocular development, expression of proteins varies in different maturation stages. This study aims to characterize structures in human fetal eyes stained by the lymphatic marker podoplanin (D2-40) with emphasis on the stage of maturation and the presence of intraocular lymphatic structures. Formalin-fixed paraffin-embedded eyes from 40 human fetuses between 10 and 38 weeks of gestation (WoG) were investigated. Immunohistochemical stains were performed for D2-40, LYVE-1 as a secondary lymphatic marker, and CD34 as a control for endothelial reactivity. A semiquantitative analysis of antigen expression in different segments of the eye was performed by light microscopy. The intensity of antigen expression was graded with a score ranging from 0 to 3. Podoplanin expression was found with a variable intensity in 97.5% of the eyes, in particular in lymphatic vessels of the conjunctiva (n = 26), conjunctival and corneal epithelium (n = 33), corneal endothelium (n = 4), trabecular meshwork (n = 28), and optic nerve sheaths (n = 23). A slight, equivocal staining reaction was noted in the choroid (n = 14). There was a correlation of antigen reactivity and the gestational age for corneal endothelial reactivity in earlier gestational stages (p = 0.003) and trabecular meshwork in older eyes (p = 0.031). D2-40 positive Müller cells were detected in two eyes ≥32 WoG. Thus, aside from conjunctival lymphatic vessels, podoplanin was expressed in several structures of the human fetal eye and the ocular adnexae at different gestational stages. Podoplanin positive structures were also found in the choroid and the chamber angle. However, lymphatic vessels or its progenitors could not be unequivocally identified in intraocular structures during 10-38 weeks of gestation. There is no evidence from our data that transient intraocular lymphactics develop in the fetal eye between 10 and 38 weeks of gestation.

Expression of the lymphatic marker podoplanin (D2-40) in human fetal eyes.

Lymphangiogenesis is a very early step in lymphatic metastasis. It is regulated and promoted not only by the tumor cells themselves, but also by cells of the tumor microenvironment, including cancer associated fibroblasts, mesenchymal stem cells, dendritic cells, or macrophages. Even the extracellular matrix as well as cytokines and growth factors are involved in the process of lymphangiogenesis and metastasis. The cellular and noncellular components influence each other and can be influenced by the tumor cells. The knowledge about mechanisms behind lymphangiogenesis in the tumor microenvironmental crosstalk is growing and offers starting points for new therapeutic approaches.

Impact of the prolymphangiogenic crosstalk in the tumor microenvironment on lymphatic cancer metastasis.

There is currently considerable controversy about existence and classification of "lymphatic vessels" in the eye. Some of the confusion is certainly caused by inappropriate use (or nonuse) of the correct immunohistochemical markers. Many experts in the field expressed the need for a consensus statement, and, in this perspective, authors offer arguments and solutions to reliably continue with immunohistochemical ocular lymphatic research.

Consensus statement on the immunohistochemical detection of ocular lymphatic vessels.

The lymphatic vasculature is - amongst other tasks - essentially involved in inflammation, (auto)immunity, graft rejection and cancer metastasis. The eye is mainly devoid of lymphatic vessels except for its adnexa, the conjunctiva and the limbus. However, several pathologic conditions can result in the secondary ingrowth of lymphatic vessels into physiologically alymphatic parts of the eye such as the cornea or the inner eye. Therefore, the cornea has served as an excellent in vivo model system to study lymphangiogenesis, and findings from such studies have substantially contributed to the understanding of central principles of lymphangiogenesis also with relevance outside the eye. Grafting experiments at the cornea have been extensively used to analyze the role of lymphangiogenesis in transplant immunology. In this regard, we recently demonstrated the crucial role of lymphatic vessels in mediating corneal allograft rejection and could show that antilymphangiogenic therapy increases graft survival. In the field of cancer research, we recently detected tumor-associated lymphangiogenesis in the most common malignant tumors of the eye, such as conjunctival carcinoma and melanoma, and ciliochoroidal melanoma with extraocular extension. These neolymphatics correlate with an increased risk of local recurrence, metastasis and tumor related death, and may offer potential therapeutic targets for the treatment of these tumors. This review will focus on corneal and tumor-associated ocular lymphangiogenesis. First, we will describe common experimentally used corneal lymphangiogenesis models and will recapitulate recent findings regarding the involvement of lymphatic vessels in corneal diseases and transplant immunology. The second part of this article will summarize findings about the participation of tumor-associated lymphangiogenesis in ocular malignancies and their implications for the development of future therapeutic strategies.

Antilymphangiogenic therapy to promote transplant survival and to reduce cancer metastasis: what can we learn from the eye?

Reports of lymphatics in the anterior human uvea are contradictory. This might be caused due to a certain topography, which has not been considered yet. Therefore, here we systematically analyze iris and adjacent ciliary body with immunohistochemistry by combining various lymphatic markers.

Topography of Lymphatic Markers in Human Iris and Ciliary Body.

Malignant melanomas of the ocular surface (conjunctival melanoma [CM]) and within the eye (uveal melanoma [UM]) show different types of metastatic behavior. While CM has a propensity to spread first to regional lymph nodes, UM metastasizes almost exclusively via the hematogenic route to the liver. We investigated whether these different metastatic patterns might be attributable to differential hem- and lymphangiogenic characteristics of CM and UM cells.

Comparing the Hem- and Lymphangiogenic Profile of Conjunctival and Uveal Melanoma Cell Lines.

Conjunctival melanoma (CM) is an ocular surface tumor that can lead to fatal metastases. Patients developing, tumor-associated lymphangiogenesis have a significantly increased risk of metastatic disease, because tumor spread primarily occurs via lymphatic vessels to the draining lymph node. Here, we describe a novel immune-competent mouse model of CM that displays tumor-associated lymphangiogenesis with development of metastatic tumors.

Article	Year
NK cell depletion delays corneal allograft rejection in baby rats. Molecular vision\| PubMed ID: 21031017	2010
Predictive value of serum markers for pulmonary involvement in ocular sarcoidosis. Acta ophthalmologica\| PubMed ID: 23910667	2014
Enrichment of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1)-positive macrophages around blood vessels in the normal human sclera. Investigative ophthalmology & visual science\| PubMed ID: 24436193	2014
Autocrine impact of VEGF-A on uveal melanoma cells. Investigative ophthalmology & visual science\| PubMed ID: 24677103	2014
A novel pro-angiogenic function for interferon-γ-secreting natural killer cells. Investigative ophthalmology & visual science\| PubMed ID: 24713481	2014
Absence of lymphatic vessels in the developing human sclera. Experimental eye research\| PubMed ID: 24975208	2014
Expression of the lymphatic marker podoplanin (D2-40) in human fetal eyes. Experimental eye research\| PubMed ID: 25135789	2014
Impact of the prolymphangiogenic crosstalk in the tumor microenvironment on lymphatic cancer metastasis. BioMed research international\| PubMed ID: 25254213	2014
Consensus statement on the immunohistochemical detection of ocular lymphatic vessels. Investigative ophthalmology & visual science\| PubMed ID: 25315233	2014
Antilymphangiogenic therapy to promote transplant survival and to reduce cancer metastasis: what can we learn from the eye? Seminars in cell & developmental biology\| PubMed ID: 25460541	2015
Topography of Lymphatic Markers in Human Iris and Ciliary Body. Investigative ophthalmology & visual science\| PubMed ID: 26225635	2015
Comparing the Hem- and Lymphangiogenic Profile of Conjunctival and Uveal Melanoma Cell Lines. Investigative ophthalmology & visual science\| PubMed ID: 26313304	2015
A Novel Model of Metastatic Conjunctival Melanoma in Immune-Competent Mice. Investigative ophthalmology & visual science\| PubMed ID: 26377082	2015
Sufficient Evidence for Lymphatics in the Developing and Adult Human Choroid? Investigative ophthalmology & visual science\| PubMed ID: 26588256	2015

Simona L. Schlereth

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