Depletion of embryonic macrophages leads to a reduction in Angiogenesis in the Ex OVO chick Chorioallantoic membrane assay

Macrophages play an important but poorly understood role in angiogenesis. To investigate their role in vessel formation, relevant in vivo models are crucial. Although the chick chorioallantoic membrane (CAM) model has been frequently used as an angiogenesis assay, limited data are available on the involvement of chicken macrophages in this process. Here, we describe a method to deplete macrophages in the ex ovo chick CAM assay by injection of clodronate liposomes and show that this depletion directly affects vascularisation of collagen onplants. Chicken embryos were injected intravenously with either clodronate or phosphate-buffered saline (PBS) liposomes, followed by placement of collagen type I plugs on the CAM to quantify angiogenic ingrowth. Clodronate liposome injection led to a significant 3.4-fold reduction of macrophages compared with control embryos as measured by immunohistochemistry and flow cytometry. Furthermore, analysis of vessel ingrowth into the collagen plugs revealed a significantly lower angiogenic response in macrophage-depleted embryos compared with control embryos, indicating that chicken embryonic macrophages play an essential function in the development of blood vessels. These results demonstrate that the chick CAM assay provides a promising model to investigate the role of macrophages in angiogenesis

High numbers of CD163-positive macrophages in the fibrotic region of exuberant granulation tissue in horses

Simple Summary Horses are prone to develop a wound healing disorder on their limbs called exuberant granulation tissue (EGT). The exact mechanism for the formation of this tissue remains unknown but the inflammatory response is supposed to be an important contributing factor. In this article, we investigated this inflammatory response in both EGT wounds as well as in control horse wounds. In biopsies, we detected two types of immune cells: (1) immune cells involved in early inflammation (MAC387+ cells) and (2) immune cells important in the later phases of inflammation (CD163+ cells). We detected a higher number of immune cells in EGT wounds compared with the control wounds of 19 days old. This suggests that EGT wounds may not be able to proceed through further phases in the wound healing process or that the inflammation phase is prolonged. Exuberant granulation tissue (EGT) is a frequently encountered complication during second intention healing in equine distal limb wounds. Although it is still unknown what exactly triggers the formation of this tissue, previous research has revealed a persistent inflammatory response in these wounds. In this preliminary study we examined this inflammatory response in EGT-developing wounds as well as in experimental induced wounds. Immunohistological stainings were performed to detect primary inflammatory immune cells (MAC387 staining) as well as pro-resolution immune cells (CD163 staining). Our results show a significantly higher amount of MAC387+ and CD163+ cells in the fibrotic regions of EGT compared with the 19-day-old experimental wounds. This persistent high amount of fibrosis-promoting CD163+ cells in EGT suggests that the wound healing processes in EGT-developing wounds are arrested at the level of the proliferation phase

Techniques used to assess intussusceptive angiogenesis: a systematic review

Intussusceptive angiogenesis (IA) is an important physiological form of angiogenesis in which an existing vessel splits in two by the formation of an intraluminal tissue pillar. The presence of these intraluminal pillars form the hallmark of ongoing IA in growing vascular beds. However, their visualization is technically challenging. The goal of this systematic review was to investigate which techniques are being used to identify intraluminal pillars and to formulate important points to keep in mind when studying IA. A systematic literature search resulted in 154 evaluated articles of which the majority (65%) provided sufficient data to unambiguously demonstrate the presence of intraluminal pillars. Scanning electron microscopy imaging of vascular corrosion casts and serial sectioning of ultrathin sections are the most used techniques. New methods such as serial block face scanning electron microscopy and micro computed tomography (mu CT) are gaining importance. Moreover, our results indicate that IA was studied in a variety of animals and tissues. IA is a biologically very relevant form of angiogenesis. Techniques to visualize intraluminal pillars need to have a minimal resolution of 1 mu m and should provide information on the 3D-nature of the pillars. Optimally, several techniques are combined to demonstrate ongoing IA

Techniques used to assess intussusceptive angiogenesis : a systematic review

Intussusceptive angiogenesis (IA) is an important physiological form of angiogenesis in which an existing vessel splits in two by the formation of an intraluminal tissue pillar. The presence of these intraluminal pillars form the hallmark of ongoing IA in growing vascular beds. However, their visualization is technically challenging. The goal of this systematic review was to investigate which techniques are being used to identify intraluminal pillars and to formulate important points to keep in mind when studying IA. A systematic literature search resulted in 154 evaluated articles of which the majority (65%) provided sufficient data to unambiguously demonstrate the presence of intraluminal pillars. Scanning electron microscopy imaging of vascular corrosion casts and serial sectioning of ultrathin sections are the most used techniques. New methods such as serial block face scanning electron microscopy and micro computed tomography (mu CT) are gaining importance. Moreover, our results indicate that IA was studied in a variety of animals and tissues. IA is a biologically very relevant form of angiogenesis. Techniques to visualize intraluminal pillars need to have a minimal resolution of 1 mu m and should provide information on the 3D-nature of the pillars. Optimally, several techniques are combined to demonstrate ongoing IA

Development of a 3’:5’ digital PCR assay to determine horse mRNA integrity

Accurate tools to measure RNA integrity are essential to obtain reliable gene expression data. The reverse transcription quantitative PCR (RT-qPCR) based 3’:5′ assay permits a direct determination of messenger RNA (mRNA) integrity. However, the use of standard curves and the possible effect of PCR inhibitors make this method cumbersome and prone to variation, especially in small samples. Here we developed a triplex digital PCR (dPCR) 3’:5’ assay for assessing RNA integrity in equine samples as rapid and simple alternative to RT-qPCR. This dPCR assay not only provides a straight forward analysis of the mRNA integrity, but also of its quantity