Angiogenesis in the Avian Embryo Chorioallantoic Membrane: A Perspective on Research Trends and a Case Study on Toxicant Vascular Effects

Abstract

The chorioallantoic membrane (CAM) of the avian embryo is an intrinsically interesting gas exchange and osmoregulation organ. Beyond study by comparative biologists, however, the CAM vascular bed has been the focus of translational studies by cardiovascular life scientists interested in the CAM as a model for probing angiogenesis, heart development, and physiological functions. In this perspective article, we consider areas of cardiovascular research that have benefited from studies of the CAM, including the themes of investigation of the CAM's hemodynamic influence on heart and central vessel development, use of the CAM as a model vascular bed for studying angiogenesis, and the CAM as an assay tool. A case study on CAM vascularization effects of very low doses of crude oil as a toxicant is also presented that embraces some of these themes, showing the induction of subtle changes in the pattern of the CAM vasculature growth that are not readily observed by standard vascular assessment methodologies. We conclude by raising several questions in the area of CAM research, including the following: (1) Do changes in patterns of CAM growth, as opposed to absolute CAM growth, have biological significance?; (2) How does the relative amount of CAM vascularization compared to the embryo per se change during development?; and (3) Is the CAM actually representative of the mammalian systemic vascular beds that it is presumed to model?

Keywords: angiogenesis; chicken embryo; chorioallantoic membrane; crude oil.

Figure 1

Some examples of major themes in chorioallantoic membrane (CAM) research involving respiratory gas and ion and water exchange (A), cardiac development (B), angiogenesis (C), toxicology and oncology (D), and general physiology (E). See text for additional discussion.

Figure 2

Vasculature and potential pathways of blood flow in the chicken embryo at mid-incubation. In this highly schematic depiction, cardiovascular structures, vascular confluences, and branch points are not to scale, and not all vessels are depicted or labelled. Blue and red arrows indicate flow of deoxygenated and oxygenated blood, respectively, with purple arrows depicting mixed oxygenated and deoxygenated blood resulting from intra- or extra-cardiac shunts. AF, atrial foramina; LV, left ventricle; RV, right ventricle; LA, left atrium; RA, right atrium. Modified from [13,14,15].

Figure 3

Arterial blood pressure traces and calculated heart rate in a Day 21 chicken embryo before and after injection of the hypertensive agent, angiotensin II. Blood pressure was measured in a chronically indwelling cannula that had been implanted in a CAM artery by keyhole surgery through a small hole in the eggshell. CAM arterial blood pressure, assumed to be a proxy for systemic embryonic pressure, increased dramatically within less than a minute after injection, and stayed elevated for at least 15 min. Heart rate was largely unchanged. These data indicate that receptors for ang II are present at hatching, typically occurring on Day 21 post-fertilization. Modified from [83].

Figure 4

Vascular index of the CAM. (A) A commonly employed methodology for determining a vascular index for the chorioallantoic membrane of the chicken embryo grown ex ovo involves digital analysis of images of the living CAM. Variations on this methodology have been long established for both chicken and alligator embryos [40,92,93]. Embryos are first grown ex ovo on yolk in glass dishes, which provides for normal development of the circulation superficially on the yolk [4,94], allowing for easy observation of the vasculature. This photo of the CAM of a 3-day-old embryo was digitally superimposed upon its concentric rings centered on the base of the umbilical artery, and extending out every 2 mm past the edge of the growing CAM. The vascular index is calculated from the number of visible blood vessels (not discriminating between arterial or venous vessels) that intersect any point on each concentric ring. No distinction is made between arteries and veins in this analysis, as the two vessels are usually visually indistinguishable without surgical intervention at this early stage of development. (B) Developmental changes in in vivo vascularization in the chicken embryos from Day 2 to Day 4 is revealed by a plot of vascular index as a function of distance from the embryo. Mean values ± SE are shown. n= 11 for each point for each day. Arrows indicate superior and inferior vessels subjected to additional quantification in Figure 4B.

Figure 5

Effects of crude oil on survival and CAM vascular growth in chicken embryos grown in ovo. Doses of crude oil of 0, 0.5, 1, or 2 µL were topically applied to the base of the umbilical artery early in Day 2 post-fertilization. (A) Survival curves across all embryonic development. N values of surviving embryos at the start and end of the incubation are indicated (and n at Day 15 for 2.0 µL crude oil exposure). (B,C) Changes in length of specific posterior and inferior CAM vessels, respectively, as a function of crude oil dose on Days 2, 3, and 4 post-fertilization. Capital letters (A,B) indicate significant differences between crude oil doses. NS, not significant. Note: Because the arteries and veins of the CAM vasculature typically run very closely together and in parallel to each other, it was not practical to specify a specific type of artery—hence, the general term “vessels” is used. Mean ± 1 SE are presented.

Figure 6

Computer-aided digital analysis of the length and branches of the vessels of the CAM of the 3-day-old chicken embryo. (A) A sample image of the CAM of a 3-day-old embryo grown ex ovo. (B) CAM vasculature evident in a digitally enhanced image. (C) Pathway of the CAM vessels (red lines) and branch points (blue dots). Images in (B,C) were analyzed using the AngioTool ImageJ plugin. (D) Vascular tree topology reconstructed from microscopic images [60]. See text for further details.

Figure 7

Subtle, reproducible pattern changes in CAM vascular growth induced by low levels of crude oil exposure in the Day 3 post-fertilization embryo. (A) In the control embryos, the major vessels at the edge of the CAM branch (enhanced in the image by dashed lines) at nearly right angles (arrow) are shown. In these controls, the major vessels of the CAM emerge from a few vitelline vessels (blue circle). In the sham embryos, in which 2.0 µL of chick Ringer was pipetted onto the embryo’s surface at the base of the umbilical vessels, no change in pattern of CAM vascularization is observed compared to the controls. (B) To assess the effect of crude oil exposure on CAM vascularization, embryos received 0.5, 1.0, and 2.0 µL doses of crude oil (Source Oil A from the April 10, 2010 Deepwater Horizon oil spill) topically applied to the base of the umbilical arteries. At any dose of crude oil exposure, the major vessels develop a characteristic deep fork immediately after the feeder vessel has split, further from the CAM edge (black arrow). Additionally, multiple major vessels emerged from the embryo (dashed lines, blue circle). Complicating the analysis of overall CAM vascular density, these additional vessels adjacent to the embryo wall do not continue to branch at the same rate as the controls, and as a consequence the peripherally measured vascular index of the CAM generally is not increased in the oil-exposed embryos, even though the vascular pattern itself changes. dpf, days post-fertilization.