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

Abstract

Abstract:In this study, we introduce a technique for repeated, microscopic observation of single regressing capillaries in vivo in inflamed murine corneas. Natural capillary regression was initiated by removal of inflammatory stimulus during an active pro-angiogenic phase, while the additional impact of anti-angiogenic treatment with triamcinolone or bevazicumab was investigated. Capillaries regressed naturally within one week and treatments did not further enhance the natural regression. Morphologically, early-phase regression was characterized by significant lumen narrowing and a significant reduction in CD11b+ myeloid cell infiltration of the extracellular matrix. By one week, vascular remodeling occurred concomitant with CD11b+CD68+KiM2R+ mature macrophage localization on capillary walls. Empty conduits without blood flow, positive for collagen IV and devoid of vascular endothelium and pericytes, were apparent in vivo and by three weeks were more numerous than perfused capillaries. By three weeks, macrophages aggregated around remaining perfused capillaries and were observed in apposition with degrading capillary segments. Abrupt termination of capillary sprouting in our regression model further revealed vascular endothelial abandonment of sprout tips and perfused capillary loop formation within a single angiogenic sprout, possibly as an intussusceptive response to cessation of the stimulus. Finally, we observed lumen constriction and macrophage localization on capillary walls in vivo in a clinical case of corneal capillary regression that paralleled findings in our murine model.

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.…”

Surgical outcomes of phototherapeutic keratectomy on Epithelial basement membrane dystrophy, and the characterisation of Bowman's Layer

“…IVCM has also been used for clinical diagnosis of pathology of the cornea and as a screening/monitoring tool for patients undergoing treatment [112][113][114][115]. Other applications are distinguishing different inflammatory cell subtypes in corneal inflammation, and feature recognition for diagnosis, such as acanthamoeba and fungal keratitis.…”

Anin VivoMethod for Visualizing Flow Dynamics of Cells within Corneal Lymphatics

“…At day 7 after suture placement, the cornea was fully vascularized from the limbus to the suture, as previously depicted. 16 Both prior to, and at 1, 4, 7, 14, and 21 days after the initial surgery, rats were anesthetized and laser scanning in vivo confocal microscopy (IVCM; HRT3-RCM, Heidelberg Engineering, Heidelberg, Germany) of the corneas was performed.…”