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Cellular-Level Characterization of Lymph Vessels in Live, Unlabeled Corneas by In Vivo Confocal Microscopy
Linköping University, Department of Clinical and Experimental Medicine, Ophthalmology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of Ophthalmology UHL/MH.
Linköping University, Department of Clinical and Experimental Medicine, Ophthalmology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of Ophthalmology UHL/MH.
Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pathology and Clinical Genetics. (Landstinget i Östergötland)
Östergötlands Läns Landsting, Reconstruction Centre, Department of Ophthalmology UHL/MH. Linköping University, Department of Clinical and Experimental Medicine, Ophthalmology. Linköping University, Faculty of Health Sciences.
2010 (English)In: Investigative Ophthalmology and Visual Science, ISSN 0146-0404, Vol. 51, no 2, 830-835 p.Article in journal (Refereed) Published
Abstract [en]

PURPOSE. To determine whether in vivo confocal microscopy (IVCM) of the cornea can be used for the label-free detection and monitoring of lymph vessels in live corneas.

METHODS. Parallel corneal hemangiogenesis and lymphangiogenesis was induced by the placement of a single suture in one cornea of male Wistar rats. Fourteen days after suture placement and under general anesthesia, laser-scanning IVCM was performed in the vascularized region. Corneas were subsequently excised for flat-mount double immunofluorescence with a pan-endothelial marker (PECAM-1/CD31) and a lymphatic endothelial specific marker (LYVE-1). Using the suture area and prominent blood vessels as points of reference, the identical microscopic region was located in both fluorescent and archived in vivo images. Additionally, vessel diameter, lumen contrast, and cell diameter and velocity within vessels were quantified from in vivo images.

RESULTS. Comparison of identical corneal regions in fluorescence and in vivo revealed prominent CD31(+)/LYVE-1(3+) lymph vessels that were visible in vivo. In vivo, corneal lymph vessels were located in the vascularized area in the same focal plane as blood vessels but had a darker lumen (P andlt; 0.001) sparsely populated by highly reflective cells with diameters similar to those of leukocytes in blood vessels (P = 0.61). Cell velocity in lymph vessels was significantly reduced compared with blood particle velocity (P andlt; 0.001). Morphologic characteristics enabled subsequent identification of corneal lymphatics in live, vascularized rat corneas before immunofluorescence labeling.

CONCLUSIONS. IVCM enabled the nondestructive, label-free, in vivo detection of corneal lymphatics. IVCM provides the possibility of observing lymphatic activity in the same live corneas longitudinally and, as a clinical instrument, of monitoring corneal lymphatics in live human subjects.

Place, publisher, year, edition, pages
Rockville, MD, United States, 2010. Vol. 51, no 2, 830-835 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:liu:diva-53820DOI: 10.1167/iovs.09-4407ISI: 000273704700030OAI: diva2:292220
Available from: 2010-02-05 Created: 2010-02-05 Last updated: 2014-09-10Bibliographically approved
In thesis
1. Angiogenesis from a new perspective
Open this publication in new window or tab >>Angiogenesis from a new perspective
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Angiogenesis is the emergence of new blood and lymph vessels from existing ones. In the pathologic form it contributes to the onset and progression of numerous different human disorders such as cancer, inflammation, atherosclerosis and blinding eye diseases. There exist a number of models to study angiogenesis, both in vitro and in vivo, but there is no single perfect model so far. Consequently there is a need to develop new angiogenesis assays for evaluating blood and lymph vessel behaviour in different physiologic settings.

The aim of this thesis was to gain insight into in vivo angiogenesis introducing a new technique in an inflammatory corneal model. The method involved in vivo examination of the cornea and subsequent comparison of in vivo findings with ex vivo immunohistochemical analysis of the same tissue samples. An existing suture model for inflammatory angiogenesis in the cornea was modified for in vivo observations with a clinically-approved corneal confocal microscope.

In this thesis, corneal lymph vessels were characterized for the first time in vivo and findings from the experimental bench could be applied in a clinical setting, where presumed lymphatics were observed in a corneal transplant patient with rejection. Furthermore, the technique was extended to investigate time-lapse processes in sprouting and regressing capillaries, and led to a number of new observations. CD11b+ myeloid cells constitute the first bulk of infiltrating inflammatory cells and contribute to inflammatory sprouting and regression in numerous ways including pre-patterning of the corneal stroma and guiding of capillary sprouts. Newly formed hemangiogenic sprouts are perfused with a slow-moving fluid and have a lumen. In blood vessel regression, capillary remodeling occurred by abandonment of sprout tips in close association with macrophages and vascular loops formed by presumed intussusceptive angiogenesis. In addition, a network of pericyte- and endothelium-free basement membrane tubes was formed after desertion or degradation of vascular endothelium in former corneal capillaries.

In conclusion, we introduce a new in vivo technique for investigating angiogenesis in a corneal model were in vivo findings can be interpreted with ex vivo definitions of specific cell types by immunohistochemistry. Findings from pre-clinical experiments have been possible to apply in a clinical setting when examining patients with corneal pathology.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2012. 91 p.
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1284
National Category
Medical and Health Sciences
urn:nbn:se:liu:diva-73137 (URN)978-91-7519-999-3 (ISBN)
Public defence
2012-01-20, Nils-Holger salen, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 13:00 (Swedish)
Available from: 2011-12-19 Created: 2011-12-19 Last updated: 2014-06-05Bibliographically approved

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Bourghardt Peebo, BeatriceFagerholm, PerTraneus-Rockert, CatharinaLagali, Neil
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OphthalmologyFaculty of Health SciencesDepartment of Ophthalmology UHL/MHDepartment of Clinical Pathology and Clinical Genetics
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