Automatic quantification of angiogenesis in 2D sections: a precise and timesaving approach

Abstract

The quantitative evaluation method, using microscopic images of stained histological sections, 'positive- and negative-experts'-based vessel segmentation, and nearest neighbour classification, provides a user-independent and precise but also time- and cost-effective tool for the analysis of vascularized constructs. Our algorithm, which is freely available to the public, outperforms previous approaches especially in terms of unambiguous vessel classification and statistical analyses.

Haemodynamically stimulated and in vivo generated axially vascularized soft‐tissue free flaps for closure of complex defects: Evaluation in a small animal model

“…Stained slices were viewed by means of conventional microscopy and recorded using image processing software (Axio Vision 4, Carl Zeiss Microscopy, Jena, Germany). For the quantification of vessel cross‐sections around the AV loop constructs, algorithms especially developed for this analysis were employed (Weis et al, ). The evaluation of cell count and proliferation was carried out manually using imageJ (http://www.imagej.net).…”

“…This indicates that cell migration into the matrix continues even after 2 weeks of in vivo flap maturation and that an alignment of cell distribution within the matrix occurs with time. A previous study of our group, which examined AV loop‐based neovascularization within a fibrin matrix, revealed comparable total vessel counts between 10 and 15 days after loop creation (Weis et al, ) but an increase in total vessel area. These findings indicate that a collagen‐ and elastin‐based matrix promotes neovascularization more efficiently and, hence, is more suitable for tissue engineering of vascularized soft‐tissue flaps.…”

“…Standardized digital photographs were taken on postoperative days 2, 5, 7, 9, 12 and 14 after defect coverage until explantation. (Weis et al, 2015). The evaluation of cell count and proliferation was carried out manually using imageJ (www.imagej.net).…”

Flow Induced Microvascular Network Formation of Therapeutic Relevant Arteriovenous (AV) Loop-Based Constructs in Response to Ionizing Radiation

“…Hematoxylin and eosin (H&E) staining was performed using an autostainer (ST5010, Leica Microsystems) according to standard protocol. Isolectin B4 (Sigma L 2140, Sigma-Aldrich, Hamburg, Germany) staining was used to detect luminal endothelial cells by means of immunohistochemistry as described elsewhere [ 17 ]. Histological cross sections were visualized using an inversed microscopy (Olympus IX81, 10× magnification, Olympus Corporation, Hamburg, Germany) and analyzed using an automatic two-dimensional quantification algorithm established previously by our group for superior morphometric quantification [ 17 ].…”

“…We wanted to demonstrate that the graft possessed vital intrinsic factors that were at least partially responsible for triggering flow-mediated angiogenesis in the AV loop model, and we generally wanted to investigate the effect of radiation in the developing AV loop microvasculature. Beside three-dimensional micro-CT, a previously described observer-independent automatic software algorithm was used to assess vessel number, density, and area by analyzing two-dimensional histological cross sections in order to characterize the differences of angiogenesis in detail [ 17 ].…”