Cellular-Level Characterization of Lymph Vessels in Live, Unlabeled Corneas by In Vivo Confocal Microscopy

Abstract

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.

Early effects of dexamethasone and anti-VEGF therapy in an inflammatory corneal neovascularization model

“…The microscope was equipped with a 63x/0.95 NA water-immersion objective (Zeiss, Oberkochen, Germany), that provides an en face view of a 400 µm x 400 µm corneal area at a selectable corneal depth. The IVCM imaging technique has been described in detail elsewhere (Bourghardt Peebo et al, 2011;Peebo et al, 2011Peebo et al, ,2010. The focal depth of the HRT3 was initially adjusted to image the corneal epithelial surface, and the lateral and transverse microscope alignments were adjusted to locate the suture in the real-time image display window.…”

“…Digital images were recorded at 5 frames/s while lateral, transverse, and axial controls were adjusted during image capture to locate and follow the path of cells or vessels from the limbus to the suture area. IVCM has been shown to be a valuable tool for imaging of various cells in transparent tissue, noninvasively, in a time-lapse manner, without the need for cell labeling (Bourghardt Peebo et al, 2011;Peebo et al, 2011Peebo et al, ,2010.…”

“…Current methods to assess the efficacy of anti-angiogenic treatments have overwhelmingly focused on examination with slit lamp and photography (Chen et al, 2009;Cheng et al, 2012;Chung and Lee, 2012;Dastjerdi et al, 2009;Yoeruek et al, 2008), partly due to the lack of specific methods to detect early inflammation and tissue damage in corneal neovascularization. We have previously showed that it is possible to detect early signs of inflammation by in vivo confocal microscopy (IVCM) (Peebo et al, 2010). A more complete assessment of antiangiogenic therapy would therefore include quantification of its suppressive effect on the dynamics of tissue inflammation at both the tissue and pathway level.…”

Cellular level characterization of capillary regression in inflammatory angiogenesis using an in vivo corneal model

“…All animals were treated following the Association for Research in Vision and Ophthalmology (ARVO) guidelines for the Use of Animals in Ophthalmic and Vision Research. With approval from the Linköping regional animal ethics review board, eighteen rats were anesthetized using intraperitoneal injection of dexmeditomedine (Orion Pharma AB, Sollentuna, Sweden) and xylazine (Pfizer AB, Sollentuna, Sweden) and after topical administration of 1% tetracaine (Chauvin Pharmaceuticals Ltd, London, England) a corneal stromal suture (10-0 nylon) was placed 1.5 mm from the temporal limbus as previously described [34]. A second stromal suture was placed at the 3 o'clock position, 1.5mm from the nasal limbus, to provide additional tissue samples for ex vivo analysis.…”

“…Digital images were recorded at 5 frames/s, and the probed region was adjusted in lateral, transverse, and axial directions during image capture to locate and follow the path of vessels from the suture area to the limbus. IVCM has been shown to be a valuable tool for imaging of various cells in transparent tissue, noninvasively, in a time-lapse manner, without the need for cell labeling [33,34]. With IVCM it is possible to identify and follow the same structures and thereby observe dynamic processes in live tissue.…”