The granulation (t)issue: A narrative and scoping review of basic and clinical research of the equine distal limb exuberant wound healing disorder.

Exuberant granulation tissue (EGT) is often observed during second intention wound healing in horses. Despite its impact on wound care, the basic mechanisms leading to EGT are still unclear and effective strategies to prevent and/or treat EGT are lacking. The development of EGT is a poorly understood, multifactorial process involving hyperproliferating fibroblasts and malfunctional differentiation of keratinocytes, suboptimal wound contraction, dysfunctional vascularisation, and chronic inflammation. To consolidate and describe basic and clinical research literature on EGT and to identify knowledge gaps and opportunities for future research, a search was systematically conducted using predefined search terms. Subsequently, a scoping review was conducted using specific criteria to select the peer-reviewed literature that described methods to treat and/or prevent EGT. Proposed mechanisms of effects as well as results and main conclusions were extracted and tabulated. The systematic search resulted in 1062 publications in PubMed and 767 in Web of Science. Twenty additional studies were later included. Of these, 327 studies were reviewed for the narrative review on basic research and 35 controlled clinical trials were eligible for the scoping review. All 35 studies were conducted in university hospitals, and all but one involved surgically induced non-infected wounds. The study population was predominantly horses (n = 230) with a small number of ponies (n = 18) and donkeys (n = 14). In conclusion, there remains a strong need for evidence-based recommendations on EGT treatment, preferably using multi-centre studies that represent the general population of horses, include higher numbers of animals, and are performed in naturally occurring wounds. This narrative and scoping review also emphasises the importance of incorporating basic research knowledge in the study design of clinical trials.

High Numbers of CD163-Positive Macrophages in the Fibrotic Region of Exuberant Granulation Tissue in Horses.

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 (µCT) 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 µm 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.

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.

The complex TIE between macrophages and angiogenesis.

Macrophages are primarily known as phagocytic immune cells, but they also play a role in diverse processes, such as morphogenesis, homeostasis and regeneration. In this review, we discuss the influence of macrophages on angiogenesis, the process of new blood vessel formation from the pre-existing vasculature. Macrophages play crucial roles at each step of the angiogenic cascade, starting from new blood vessel sprouting to the remodelling of the vascular plexus and vessel maturation. Macrophages form promising targets for both pro- and anti-angiogenic treatments. However, to target macrophages, we will first need to understand the mechanisms that control the functional plasticity of macrophages during each of the steps of the angiogenic cascade. Here, we review recent insights in this topic. Special attention will be given to the TIE2-expressing macrophage (TEM), which is a subtype of highly angiogenic macrophages that is able to influence angiogenesis via the angiopoietin-TIE pathway.