2.1.

Embryo Culture

Fertilized Japanese quail (Coturnix coturnix japonica) eggs (Boyd’s Bird Co., Pullman, Washington) were disinfected and incubated at $37°\mathrm{C}$ under ambient atmosphere until E2.5. They were then opened under sterile conditions in a laminar flow hood, and each embryo was place in one well of a six-well cell culture cluster (Costar Inc., Corning, New York) and cultured further under the same conditions.

2.2.

Embryo Treatment

Some of the embryos were kept without perturbation to evaluate the normal angiogenesis and these served as baseline controls. Other embryos were treated with phosphate-buffered saline (PBS), mouse leptin 4.0 and $0.4\mu \mathrm{g}∕\mathrm{ml}$ [AFP352C, National Hormone & Peptide Program, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (NIDDK, NIH)], rabbit polyclonal antibodies to mouse leptin at different dilutions (1:10, 1:100, 1:1000) (lot AFP3011199, National Hormone & Peptide Program, NIDDK, NIH), chicken leptin at different concentrations (0.1, 0.05, 0.025, 0.012, 0.006, and $0.003\mu \mathrm{g}∕\mathrm{ml}$ ) (lot AFP8942E, National Hormone & Peptide Program, NIDDK, NIH), and rabbit polyclonal antibodies to chicken leptin at different dilutions (1:500, 1:1000, 1:10000) (lot AFP1082300, National Hormone & Peptide Program, NIDDK, NIH). ³ Human recombinant VEGF-165 ( ${\mathrm{hrVEGF}}_{165}$ ) $500\mathrm{ng}∕\mathrm{ml}$ and $5\mu \mathrm{g}∕\mathrm{ml}$ (Genentech, Inc., San Francisco, California) and monoclonal antibodies to ${\mathrm{hrVEGF}}_{165}$ $5\mu \mathrm{g}∕\mathrm{ml}$ (lot 74839, G180CL, Genentech, Inc., San Francisco, California) were used as a positive and negative controls, respectively, in all the experiments. All of the substances used were prepared in a sterile laminar flow hood to the desired concentration and prewarmed at $37°\mathrm{C}$ . Each treatment was prepared in a total volume of $500\mu \mathrm{l}$ and was added gently onto each CAM at E7.

2.3.

Fixation and Processing of the CAM

After 24 $48\mathrm{h}$ of treatment, the embryos were fixed in situ with Karnovsky fixative (16% paraformaldehyde, 25% glutaraldehyde, and $0.2\mathrm{M}$ sodium phosphate) (Electron Microscopy Sciences, Hatfield, Pennsylvania), and allowed to fix for at least $24\mathrm{h}$ . The whole embryos were then taken out of their wells and placed in PBS for further dissection to separate the CAM from the rest of the embryo. Each CAM was carefully rinsed with PBS to flush out the blood within the veins. Arterial blood remained inside the vessels after the fixation. Then the edges of the CAM were dissected without affecting the midterminal zone of the arteries. Finally, each CAM was mounted onto a glass slide and covered with a glass coverslip or with Crystal Mount (Biomeda, Foster City, California).

2.4.

Image Analysis

Images from the midterminal region of the CAM were acquired at $1600\times 1200\text{pixels}$ , a magnification of $10\times$ , and a resolution of $1041\text{pixels}∕\mathrm{cm}$ with a stereoscopic microscope (MZ 12.5, Leica, Bannockburn, Illinois) attached to a CCD camera (IN1120, Diagnostic Instruments, Inc., Sterling Heights, Michigan). We used two methods to analyze the images. In the first method, we evaluated the vascular complexity and density using the tools of ImageJ 1.30v software (Wayne Rasband National Institutes of Health; United States). Each image was converted to $8\text{-}\text{bit}$ binary (black and white) and then the image was skeletonized. In the images taken with our system, each pixel corresponds to $9.6\mu \mathrm{m}$ . The skeletonized images were analyzed by fractal dimension $\left(D_f\right)$ with the box-counting method. ⁶ This method overlays the image with a series of square boxes of decreasing size. The number of boxes that contain at least one black pixel is counted. The value of the slope of a squared regression yields $D_f$ value. The $D_f$ value was used as an indicator of vascular complexity that combines branching and tortuousity but does not discriminate among these two parameters. The method of grid intersection was used to evaluate vessel density with the same software²⁸ (ImageJ, 1.30v). This method takes into account the number of intersections between a skeletonized vessel and a superimposed rectangular vertical and horizontal grid of $64\text{-}\text{pixel}$ spacing.

We use a second method to quantify more precisely other vascular parameters such as vessel length, diameter, tortuousity, and branching. This image-analysis method consisted of a program that we developed based on the image processing toolbox of MATLAB® (The MathWorks, Natick, Massachusetts). An automatic-histogram-derived threshold selection, as proposed by Otsu,²⁹ was used to obtain the CAM vascular binarization images. A sequential thinning method was then used to skeletonize images from the binarized vascular images (Fig. 1 ). The distance $D_{sb}$ between each skeleton pixel in the skeletonized image and its nearest background pixel in the corresponding binarized image can be calculated based on Euclidean distances. In this method, the vascular radius of each skeleton/center pixel is equal to the corresponding $D_{sb}$ , thus generating a diameter map. With this map, several measurements were evaluated, includins length, area, and average diameter of the vessels. For example, the length of a branch was defined as the total number of branch pixels after the preceding processing.

Fig. 1

Image processing of the CAM. The images were taken in a stereoscopic microscope at a $10\times$ magnification. Then, they were transformed to (a) $8\text{-}\text{bit}$ gray scale, (b) binarized, and (c) skeletonized images before applying morphometry quantification. $\text{Bar}=1\mathrm{mm}$ .

We obtained a node-structural map from every skeletonized vascular image. This node-structural map provided a functional tool for reconstructing the total vessel network and we could completely characterize the vascular organization by specifying the connectivity properties of each pixel. We also could evaluate the total number of branches (end nodes) and the degree of branching (furcation/junction nodes) in these regions as well as vessel tortuousity. The vessel tortuousity has been quantified in the vessels of the retina and is defined as the arc to chord ratio^{30, 31} (see Fig. 4 in Sec. 3.2). The arc length $L_{\mathrm{arc}}$ is defined as the length of the vessel section, and the chord length is the Euclidean distance between start point $\left(S\right)$ and endpoint $\left(E\right)$ of this vessel section. According the definition, the greater the ratio value, the greater the tortuousity.^{30, 31}

Fig. 4

Evaluation of vessel tortuousity based on the node-structure map. The vessel tortuousity is defined as the arc-to-chord ratio. The arc length $\left(L_{\mathrm{arc}}\right)$ is defined as the length of blood vessel section. The chord length is the ED between start point $\left(S\right)$ and endpoint $\left(E\right)$ of the blood vessel. According to the definition, the greater the ratio value, the greater the tortuousity.

2.5.

Statistics

Data are expressed as $\text{mean}\pm \text{standard}$ error from 50 samples for each treatment group of five independent experiments. The statistical significance was assessed by Student’s $t$ test.

3.1.

Effect of Mouse Leptin, Chicken Leptin, and VEGF on CAM

Similarities between the chicken and mammalian leptin receptor genes have been reported.³² We treated the CAM with mouse leptin and chicken leptin (as proangiogenic factors) and the corresponding antimouse leptin and antichicken leptin antibodies (as antiangiogenic factors) to evaluate the angiogenic responses. The ${\mathrm{hrVEGF}}_{165}$ and anti-hVEGF effects were used as controls. The CAM treated with mouse leptin for $24\mathrm{h}$ showed a clear increase in vascular density, complexity, and tortuousity [Fig. 2b ] compared to the CAM controls [Fig. 2a]. An increase in vessel tortuousity and density was also observed in CAM treated with ${\mathrm{hrVEGF}}_{165}$ [Fig. 2c]. Antimouse leptin antibodies clearly diminished the vascular density and complexity [Fig. 2d] Quantification of these vascular parameters using morphometric measurements such as grid intersection and fractal dimension are shown in Fig. 3 . A significant increase in vessel density and complexity is evident with mouse leptin and ${\mathrm{hrVEGF}}_{165}$ in E8-E9 embryos compared to controls treated with PBS. These effects were totally repressed by antimouse leptin and anti-hVEGF antibodies. Surprisingly, when these antibodies were used directly, and alone, on the CAM, a decrease in these parameters was observed, particularly at higher concentrations.

Fig. 2

Effect of mouse leptin on CAM from E8-E9 embryos. Examples of some binarized CAM images captured at $10\times$ of magnification; (a) CAM without any treatment, (b) CAM treated with mouse leptin $(0.4\mu \mathrm{g}∕\mathrm{ml})$ , (c) CAM treated with ${\mathrm{hrVEGF}}_{165}$ $(500\mathrm{ng}∕\mathrm{ml})$ , and (d) CAM treated with antimouse leptin $(5\mu \mathrm{g}∕\mathrm{ml})$ . $\text{Bar}=1\mathrm{mm}$ .

Fig. 3

Quantification of the effects of mouse leptin on CAM using the image tool box of ImageJ, 1.30v. Conventional vascular parameters were applied to CAM images. (a) Vascular density was measured applying grid intersection analysis $\left({\rho }_v\right)$ and (b) vascular complexity was evaluated using fractal dimension analysis $\left(D_f\right)$ . Note: Cont, CAM controls without treatment; Lep, CAM treated with mouse leptin, $\alpha \mathrm{Lep}$ , CAM treated with antibodies antimouse leptin; VEGF, CAM treated with VEGF; $\alpha \mathrm{VEGF}$ , CAM treated with antibodies antiVEGF; ${}^{*}$ , $P<0.05$ ; and ${}^{**}$ , $P<0.0001$ .

Table 1 summarizes these results and data from those CAM treated with chicken leptin and antibodies antichicken leptin. A significant increase was observed in vascular complexity and density at lower concentrations $(0.025\mu \mathrm{g}∕\mathrm{ml})$ of chicken leptin compared to mouse leptin $(0.4\mu \mathrm{g}∕\mathrm{ml})$ . Those CAM treated with antichicken leptin antibodies alone also showed a significant decrease in vascular density and complexity compared to controls.

Table 1

Quantification of the effects of chicken leptin, antichicken leptin, hrVEGF165 , and anti-hVEGF (controls) on the quail CAM using conventional vascular parameters.

3.2.

Other Specific Vascular Parameters Measured on the CAM

As mentioned, mouse leptin and ${\mathrm{hrVEGF}}_{165}$ increased the vascular complexity and density in CAM. These vascular parameters have been measured previously using morphometric techniques.^{6, 10, 11, 12, 13, 14} To measure other vascular parameters for specific angiogenic changes such as vessel length, vessel diameter, branching, and tortuousity, we designed a new computer-assisted method, similar to the one used by Parsons-Wingerter ¹³ We used transformed images used in the binarized and skelenotized mode for these analyses. Based on the node-structure map that we developed with the software, we could easily evaluate the vessel length, diameter, branching, and tortuousity.

As mentioned in Sec. 2, the vessel tortuousity is defined as the arc to chord ratio.^{30, 31} Figure 4 shows an example of two branches with different tortuousity. The arc length $\left(L_{\mathrm{arc}}\right)$ is defined as the branch length of the vessel and the chord length is the Euclidean distance (ED) between start point $\left(S\right)$ and endpoint $\left(E\right)$ of that branch. By this definition, the radio $(L_{\mathrm{arc}}∕\mathrm{ED})$ will be greater in those vessels that exhibit more tortuousity.

Figure 5 shows the results of the image quantification of the vascular parameters already mentioned in those CAM treated with mouse leptin, antimouse leptin, ${\mathrm{hrVEGF}}_{165}$ and anti-hVEGF. Table 2 summarizes the results obtained using chicken leptin, antichicken leptin, ${\mathrm{hrVEGF}}_{165}$ , and anti-hVEGF on CAM. Mouse leptin increased the total vascular length [Fig. 5a] along with a decrease in vessel diameter [Fig. 5b]. Similar results were observed in those CAM treated with ${\mathrm{hrVEGF}}_{165}$ . Antimouse leptin and anti-hVEGF antibodies significantly decreased the vascular length without any modification in the vascular average diameter [Figs. 5a and 5b]. Chicken leptin also increases the total vascular length and decreases the average diameter of CAM blood vessels (Table 2). Antichicken leptin affects only the total vascular length.

Fig. 5

Quantification of specific vascular parameters on CAM treated with different angiogenic and antiangiogenic factors using the image processing toolbox of MATLAB®: (a) total vessel length, (b) vascular diameter, (c) tortuousity, and (d) vascular branching (end and furcation branch number) were measured with this method ( ${}^{*}$ , $P<0.05$ ; ${}^{**}$ , $P<0.001$ ).

Table 2

Quantification of the effects of chicken leptin, antichicken leptin, hrVEGF165 and anti-hVEGF (controls) on quail CAM using other specific vascular parameters.

A tortuousity pattern was evident in most of the first-, second-, and third-generation blood vessels treated with mouse leptin and ${\mathrm{hrVEGF}}_{165}$ [see Figs. 2b and 2c]. A increase in tortuousity was observed in CAM treated with mouse leptin, chicken leptin, and ${\mathrm{hrVEGF}}_{165}$ [Fig. 5c and Table 2]. The vessel tortuousity was more dramatic in those CAM treated with mouse and chicken leptin [Fig. 2b, Table 2]. Antimouse leptin, anti-hVEGF, and antichicken leptin antibodies had no effect on vessel tortuousity [Fig. 5c, Table 2].

A drastic effect in the formation of end nodes, which represent terminal branches, and furcation nodes, which represent emerging branches, was observed after treatment with mouse leptin, chicken leptin, and ${\mathrm{hrVEGF}}_{165}$ [Fig. 5d, Table 2]. Antimouse leptin, antichicken leptin, and anti-hVEGF antibodies produced a significant decrease, however, in the number of furcation nodes. No changes were observed in the number of end nodes.