The chicken chorioallantoic membrane (CAM) is a simple, highly vascularized extraembryonic membrane, which performs multiple functions during embryonic development, including but not restricted to gas exchange. Over the last two decades, interest in the CAM as a robust experimental platform to study blood vessels has been shared by specialists working in bioengineering, development, morphology, biochemistry, transplant biology, cancer research and drug development. The tissue composition and accessibility of the CAM for experimental manipulation, makes it an attractive preclinical in vivo model for drug screening and/or for studies of vascular growth. In this article we provide a detailed review of the use of the CAM to study vascular biology and response of blood vessels to a variety of agonists. We also present distinct cultivation protocols discussing their advantages and limitations and provide a summarized update on the use of the CAM in vascular imaging, drug delivery, pharmacokinetics and toxicology.

The chicken chorioallantoic membrane model in biology, medicine and bioengineering.

OBJECTIVE—Fractal analysis can quantify the geometric complexity of the retinal vascular branching pattern and may therefore offer a new method to quantify early diabetic microvascular damage. In this study, we examined the relationship between retinal fractal dimension and retinopathy in young individuals with type 1 diabetes.

RESEARCH DESIGN AND METHODS—We conducted a cross-sectional study of 729 patients with type 1 diabetes (aged 12–20 years) who had seven-field stereoscopic retinal photographs taken of both eyes. From these photographs, retinopathy was graded according to the modified Airlie House classification, and fractal dimension was quantified using a computer-based program following a standardized protocol.

RESULTS—In this study, 137 patients (18.8%) had diabetic retinopathy signs; of these, 105 had mild retinopathy. Median (interquartile range) retinal fractal dimension was 1.46214 (1.45023–1.47217). After adjustment for age, sex, diabetes duration, A1C, blood pressure, and total cholesterol, increasing retinal vascular fractal dimension was significantly associated with increasing odds of retinopathy (odds ratio 3.92 [95% CI 2.02–7.61] for fourth versus first quartile of fractal dimension). In multivariate analysis, each 0.01 increase in retinal vascular fractal dimension was associated with a nearly 40% increased odds of retinopathy (1.37 [1.21–1.56]). This association remained after additional adjustment for retinal vascular caliber.

CONCLUSIONS—Greater retinal fractal dimension, representing increased geometric complexity of the retinal vasculature, is independently associated with early diabetic retinopathy signs in type 1 diabetes. Fractal analysis of fundus photographs may allow quantitative measurement of early diabetic microvascular damage.

https://care.diabetesjournals.org/content/diacare/32/1/106.full.pdf

Quantitative Assessment of Early Diabetic Retinopathy Using Fractal Analysis

Ocular fundus imaging plays a key role in monitoring the health status of the human eye. Currently, a large number of imaging modalities allow the assessment and/or quantification of ocular changes from a healthy status. This review focuses on the main digital fundus imaging modality, color fundus photography, with a brief overview of complementary techniques, such as fluorescein angiography. While focusing on two-dimensional color fundus photography, the authors address the evolution from nondigital to digital imaging and its impact on diagnosis. They also compare several studies performed along the transitional path of this technology. Retinal image processing and analysis, automated disease detection and identification of the stage of diabetic retinopathy (DR) are addressed as well. The authors emphasize the problems of image segmentation, focusing on the major landmark structures of the ocular fundus: the vascular network, optic disk and the fovea. Several proposed approaches for the automatic detection of signs of disease onset and progression, such as microaneurysms, are surveyed. A thorough comparison is conducted among different studies with regard to the number of eyes/subjects, imaging modality, fundus camera used, field of view and image resolution to identify the large variation in characteristics from one study to another. Similarly, the main features of the proposed classifications and algorithms for the automatic detection of DR are compared, thereby addressing computer-aided diagnosis and computer-aided detection for use in screening programs.

/pdf/digital-ocular-fundus-imaging-a-review-6uzy4lzfqq.pdf

Digital ocular fundus imaging: a review.

Aim Fractal analysis provides a global assessment of vascular network architecture. We examined the relationship of retinal vascular fractal dimension ( D f) with coronary heart disease (CHD) mortality.

Methods and results We examined the relationship of D f with 14-year CHD mortality in a prospective, population-based cohort of 3303 participants aged 49 years or older. D f was measured from digitized fundus photographs using computer-automated methods; CHD mortality was documented from Australian National Death Index records. Mean D f in this population was 1.441 (standard deviation, 0.024). Over 14 years, there were 468 (14.2%) CHD deaths. Participants with suboptimal D f (lowest and highest quartiles) had 50% higher 14-year CHD mortality than those with optimal D f (middle quartiles), after adjusting for age, blood pressure, and other risk factors. Among participants aged ≤70 years, suboptimal D f was associated with a nearly two-fold higher risk of CHD mortality [adjusted hazard ratio (HR) 1.89, 95% confidence interval (CI), 1.25, 2.84 for the lowest quartile and HR 1.87, CI 1.30, 2.69 for the highest quartile, compared with middle quartiles].

Conclusions D f is a novel means of quantifying microvascular branching that independently predicted 14-year CHD mortality. These findings suggest that suboptimal microvascular branching may play a role in development of clinical cardiovascular disease.

Fractal analysis of retinal microvasculature and coronary heart disease mortality

Objective Classical measures of vessel morphology, including diameter and density, are employed to study microvasculature in endothelial membrane labeled mice. These measurements prove sufficient for some studies; however, they are less well suited for quantifying changes in microcirculatory networks lacking hierarchical structure. We demonstrate that automated multifractal analysis and lacunarity may be used with classical methods to quantify microvascular morphology. Methods Using multifractal analysis and lacunarity, we present an automated extraction tool with a processing pipeline to characterize 2D representations of 3D microvasculature. We apply our analysis on four tissues and the hyaloid vasculature during remodeling. Results We found that the vessel networks analyzed have multifractal geometries and that kidney microvasculature has the largest fractal dimension and the lowest lacunarity compared to microvasculature networks in the cortex, skin, and thigh muscle. Also, we found that, during hyaloid remodeling, there were differences in multifractal spectra reflecting the functional transition from a space filling vasculature which nurtures the lens to a less dense vasculature as it regresses, permitting unobstructed vision. Conclusion Multifractal analysis and lacunarity are valuable additions to classical measures of vascular morphology and will have utility in future studies of normal, developing, and pathological tissues.

Multifractal and lacunarity analysis of microvascular morphology and remodeling.

Quantification of microvascular remodeling as a meaningful discovery tool requires mapping and measurement of site-specific changes within vascular trees and networks. Vessel density and other critical vascular parameters are often modulated by molecular regulators as determined by local vascular architecture. For example, enlargement of vessel diameter by vascular endothelial growth factor (VEGF) is restricted to specific generations of vessel branching (Parsons-Wingerter et al., Microvascular Research72: 91, 2006). The averaging of vessel diameter over many successively smaller generations is therefore not particularly useful. The newly automated, user-interactive software VESsel GENeration Analysis (VESGEN) quantifies major vessel parameters within two-dimensional (2D) vascular trees, networks, and tree-network composites. This report reviews application of VESGEN 2D to angiogenic and lymphangiogenic tissues that includes the human and murine retina, embryonic coronary vessels, and avian chorioallantoic membrane. Software output includes colorized image maps with quantification of local vessel diameter, fractal dimension, tortuosity, and avascular spacing. The density of parameters such as vessel area, length, number, and branch point are quantified according to site-specific generational branching within vascular trees. The sole user input requirement is a binary (black/white) vascular image. Future applications of VESGEN will include analysis of 3D vascular architecture and bioinformatic dimensions such as blood flow and receptor localization. Branching analysis by VESGEN has demonstrated that numerous regulators including VEGF(165), basic fibroblast growth factor, transforming growth factor beta-1, angiostatin and the clinical steroid triamcinolone acetonide induce 'fingerprint' or 'signature' changes in vascular patterning that provide unique readouts of dominant molecular signaling.

/pdf/vesgen-2d-automated-user-interactive-software-for-avckuc1dvf.pdf

VESGEN 2D: Automated, User‐Interactive Software for Quantification and Mapping of Angiogenic and Lymphangiogenic Trees and Networks

PURPOSE To quantify the effects of the steroid triamcinolone acetonide (TA) on branching morphology within the angiogenic microvascular tree of the chorioallantoic membrane (CAM) of quail embryos METHODS Increasing concentrations of TA (0-16 ng/mL) were applied topically on embryonic day (E) 7 to the chorioallantoic membrane (CAM) of quail embryos cultured in petri dishes and incubated for an additional 24 or 48 hours until fixation Binary (black/white) microscopic images of arterial end points were quantified by generational analysis of vessel branching (VESGEN) software to obtain major vascular parameters that include vessel diameter (D(v)), fractal dimension (D(f)), tortuosity (T(v)), and densities of vessel area, length, number, and branch point (A(v), L(v), N(v), and Br(v)) For assessment of specific changes in vascular morphology induced by TA, the VESGEN software automatically segmented the vascular tree into branching generations (G(1) G(10)) according to changes in vessel diameter and branching RESULTS Vessel density decreased significantly up to 34% as the function of increasing concentration of TA according to A(v), L(v), Br(v), N(v), and D(f) TA selectively inhibited the growth of new, small vessels because L(v) decreased from 1314 +/- 061 cm/cm(2) for controls to 8012 +/- 082 cm/cm(2) at 16 ng TA/mL in smaller branching generations (G(7)-G(10)) and for N(v) from 47383 +/- 2985 cm(-2) to 30232 +/- 3309 cm(-2) In contrast, vessel diameter (D(v)) decreased throughout the vascular tree (G(1)-G(10)) CONCLUSIONS By VESGEN analysis, TA selectively inhibited the angiogenesis of smaller blood vessels, but decreased the vessel diameter of all vessels within the vascular tree

Selective inhibition of angiogenesis in small blood vessels and decrease in vessel diameter throughout the vascular tree by triamcinolone acetonide.

The steroid triamcinolone acetonide (TA) is a potent anti-angiogenesis drug used to treat retinal vascular diseases that include diabetic retinopathy, vascular occlusions and choroidal neovascularization. To quantify the effects of TA on branching morphology within the angiogenic microvascular tree of the chorioallantoic membrane (CAM) of quail embryos. Increasing concentrations of TA (0-16 ng/ml) were applied topically on embryonic day 7 (E7) to the chorioallantoic membrane (CAM) of quail embryos cultured in Petri dishes, and incubated for an additional 24 or 48 hours until fixation. Binary (black/white) microscopic images of arterial end points were quantified by VESGEN software (for Generational Analysis of Vessel Branching) to obtain major vascular parameters that include vessel diameter (Dv), fractal dimension (Df), tortuosity (Tv) and densities of vessel area, length, number and branch point (Av, Lv, Nv and Brv). For assessment of specific changes in vascular morphology induced by TA, the VESGEN software automatically segmented the vascular tree into branching generations (G1...G10) according to changes in vessel diameter and branching. Vessel density decreased significantly up to 34% as the function of increasing concentration of TA according to Av, Lv, Brv, Nv and Df. TA selectively inhibited the growth of new, small vessels, because Lv decreased from 13.14plus or minus 0.61 cm/cm2 for controls to 8.012 plus or minus 0.82 cm/cm2 at 16 ng TA/ml in smaller branching generations (G7-G10), and for Nv from 473.83 plus or minus 29.85 cm(-)2 to 302.32 plus or minus 33.09 cm-()2. In contrast, vessel diameter (Dv) decreased throughout the vascular tree (G1-G10).

/pdf/triamcinolone-acetonide-selectively-inhibits-angiogenesis-in-2o4i22vobk.pdf

Daniela Ribita

Papers

VESGEN 2D: Automated, User‐Interactive Software for Quantification and Mapping of Angiogenic and Lymphangiogenic Trees and Networks

Selective inhibition of angiogenesis in small blood vessels and decrease in vessel diameter throughout the vascular tree by triamcinolone acetonide.

Triamcinolone Acetonide Selectively Inhibits Angiogenesis in Small Blood Vessels and Decreases Vessel Diameter within the Vascular Tree