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DOI: 10.1148/radiol.2403050976
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(Radiology 2006;240:698-707.)
© RSNA, 2006

Experimental Studies

Arteriogenesis: Noninvasive Quantification with Multi–Detector Row CT Angiography and Three-dimensional Volume Rendering in Rodents1

Zhen W. Zhuang, MD, Ling Gao, ME, Masahiro Murakami, MD, PhD, Justin D. Pearlman, MD, ME, PhD, Terry J. Sackett, BS, Michael Simons, MD and Ebo D. de Muinck, MD, PhD

1 From the Angiogenesis Research Center (Z.W.Z., L.G., M.M., J.D.P., M.S., E.D.d.M.) and Departments of Radiology (Z.W.Z., J.D.P., T.J.S.), Medicine (Z.W.Z., L.G., M.M., J.D.P., M.S., E.D.d.M.), and Physiology (E.D.d.M.), Dartmouth Medical School, Borwell Research Building HB 7700, 1 Medical Center Dr, Lebanon, NH 03756. Received June 11, 2005; revision requested August 3; revision received September 2; accepted September 22; final version accepted November 23. Z.W.Z. supported by Society of Interventional Radiology Foundation grant. Address correspondence to Z.W.Z. (e-mail: zzw{at}dartmouth.edu).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
Purpose: To evaluate two-dimensional (2D) multi–detector row computed tomographic (CT) angiography and three-dimensional (3D) volume rendering for depiction of patterns of arterial growth and quantification of blood vessel density and volume.

Materials and Methods: The institutional animal care and use committee approved this study. The right femoral artery and its branches were ligated and excised in 16 inbred Lewis rats; animals were randomly assigned to receive 70 µL Dulbecco's modified Eagle's medium (DMEM) or 1.5 x 107 bone marrow–derived mononuclear cells (BMC) from isogenic donor rats in 70 µL DMEM. At 2 weeks, CT angiography was performed with injection of 0.45 mL barium sulfate suspension at 0.7 mL/min, followed by silver staining. Number of blood vessels, area, mean area, volume, and blood vessel size distribution derived from digitally subtracted 2D CT angiographic sections were quantified; 3D images were reconstructed. Two-way analysis of variance and paired and unpaired Student t tests were performed.

Results: CT angiography showed two patterns of arterial growth: collateral arterial formation and branching arteriogenesis. Two-way analysis of variance indicated that differences within subjects (ischemic vs nonischemic legs) and between subjects (BMC vs DMEM treatment) were significant for total blood vessel area, total blood vessel volume, and mean of blood vessel area (P < .001). In the BMC group, there were significantly more arteries (mean, 241.6 ± 77.0 [standard deviation] vs 196.4 ± 75.2, P = .028), but mean cross-sectional area of these arteries was smaller in ischemic versus nonischemic legs (5.4 mm2 ± 1.2 vs 6.8 mm2 ± 1.3, P = .006). Total arterial area and volume did not differ significantly between ischemic and nonischemic legs.

Conclusion: BMC injection had a substantial effect on arteriogenesis, with normalization of total arterial area and volume in the BMC group; this effect was successfully depicted.

© RSNA, 2006


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
Neovascularization of adult tissues is a complex process involving dissolution of the existing matrix, migration and proliferation of endothelial cells, formation and maturation of new vascular structure, and, finally, deposition of new extracellular matrix. A large number of genes capable of both stimulation and inhibition of many of these steps are involved in the regulation of neovascularization (1,2). During the past 9 years, our group and others have demonstrated the ability of interventions to induce physiologically meaningful neovascularization in a variety of models of ischemia with various angiogenic growth factors, which include fibroblast growth factors 1, 2, 4, and 5; vascular endothelial growth factor 165; platelet-derived growth factor; and multipotent cells of different origins (1–4). The biologic contribution of these factors and cells to an endogenous neovascular response, however, has been harder to define, and the clinical application of proangiogenic therapy has been unsuccessful thus far.

Although results of clinical studies in small numbers of patients have been encouraging, the results in two of the largest clinical growth factor trials to date were not (5–12). Many issues may have contributed to the lack of success of clinical growth factor therapy. Patient selection, choice of growth factor therapy, dose and route of administration, and a lack of understanding of the biologic nature of the process all are likely to have played a role. Two major factors are emerging as essential for progress in this field. One is a better control over concentration and persistence of exogenous therapeutic agents in the target area, and the other is the requirement for reliable noninvasive imaging techniques that allow monitoring of blood vessel development and organ function in response to therapy.

Hind limb models of ischemia in rodents are used extensively to develop new approaches to therapeutic neovascularization, and the standard imaging technique in this model is angiography. Angiography offers unsurpassed spatial resolution for vasculature without destruction of the specimen, as occurs in histologic analysis; furthermore, it is fast and, thus, offers a practical method to evaluate large experimental groups. With angiography, however, the viewing angles are predetermined and limited in number, and the method is associated with the problem of blood vessel superimposition, which makes it impossible to distinguish overlapping vessels from a true side branch. Thus, it is often impossible to determine whether a new vessel is the result of maturation of preexisting rudimentary collateral vessels or a new branch that arises from an existing vessel.

Conventional projection angiography does not allow generation of three-dimensional (3D) data, and it is, therefore, impossible to determine the spatial relationship between vessels and between the vasculature and the surrounding tissues. If we are to understand how new vessels grow, this information is essential. Also, quantification of neovascular responses on the basis of conventional x-ray angiography is inherently flawed because of the lack of 3D information. These limitations prompted us to consider multi–detector row computed tomographic (CT) angiography in rats to monitor new blood vessel growth and to quantify neovascular responses. Thus, the purpose of our study was to evaluate two-dimensional (2D) multi–detector row CT angiography and 3D volume rendering for depiction of patterns of arterial growth and quantification of blood vessel density and volume.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
Animals
Twenty-one inbred Lewis rats (Harlan, Indianapolis, Ind), 10–12 weeks of age and weighing 275–325 g, were used for all experiments. Surgical procedures were performed with sterile conditions. The study conformed to the NIH Guide for the Care and Use of Laboratory Animals (publication no. 85–23, revised 1985) published by the U.S. Public Health Service and the National Institutes of Health and was approved by the institutional animal care and use committee of Dartmouth Medical School, Lebanon, NH.

Preparation of Bone Marrow–derived Mononuclear Cells
Bone marrow–derived mononuclear cells (BMC) were used as a stimulus to promote new blood vessel growth because these cells have been shown to promote blood vessel growth in hind limb models of ischemia (13,14). The reason for their positive effect on compensatory blood vessel growth with conditions of impaired organ perfusion is unknown but likely involves a pleiotropic effect that relies on the capacity of these cells to act as a source of multiple chemokines and cytokines that stimulate neovascularization (13). Therefore, we chose BMC from five isogenic Lewis donor rats as a neovascular stimulus.

The donor rats were anesthetized with an intramuscular injection of ketamine hydrochloride (Ketaset; Fort Dodge Animal Health, Fort Dodge, Iowa), 80 mg/kg, and xylazine (Xyla-Ject; Phoenix Pharmaceutical, St Joseph, Mo), 10 mg/kg, and were killed by means of exsanguination. Bone marrow from the femur and tibia was collected and placed in phosphate-buffered saline. The bone marrow was transferred to a sterile tube and mixed with 10 mL Dulbecco's modified Eagle's medium (DMEM) that contained 10% fetal bovine serum and 100 U/mL penicillin G and 100 µg/mL streptomycin sulfate (Penicillin-streptomycin; Invitrogen, Grand Island, NY). The tube was centrifuged at 2000 rpm for 15 minutes, and the cell pellet was resuspended in 10 mL DMEM. To separate BMC from red blood cells, the cells were centrifuged over a Ficoll density gradient (15) at 800 rpm for 30 minutes, washed with phosphate-buffered saline twice to remove the Ficoll, and resuspended in DMEM for in vivo studies.

Rat Hind Limb Model of Ischemia
Sixteen rats were anesthetized with intramuscularly administered ketamine hydrochloride (80 mg/kg) and xylazine (10 mg/kg). A vertical longitudinal (2–3 cm long) incision was made in the right hind limb. The right femoral artery and its branches were dissected and ligated by using 6-0 silk sutures spaced 10 mm apart, and the vessel segment between the ligatures was excised. By using an Internet-based source (http://randomizer.org/form.htm), the rats were randomly assigned to receive a BMC injection (n = 8) or only DMEM (controls, n = 8). With a tuberculin syringe, 1.5 x 107 BMC in 70 µL DMEM or only 70 µL DMEM was injected at five sites in the three major thigh muscles: the adductor (two injections), quadriceps (two injections), and semimembranous (one injection) muscles. To prevent perforation of the muscle from the force of the injection and to prevent back-leakage from the puncture site, each injection was administered during 15 seconds. After the cells were injected, the incision was closed with 4-0 silk sutures in layers. All of these procedures were performed by one individual (Z.W.Z., with 9 years of experience).

Perfusion Fixation, Contrast Agent Injection, and Staining of Endothelial Cells
At 2 weeks after injection of BMC, rats were anesthetized with an intramuscular injection of ketamine and xylazine, the abdomen was opened, and a blunt catheter (PE50; Becton Dickinson, Sparks, Md) was inserted into the abdominal aorta just above the bifurcation and ligated with 4-0 silk. The inferior vena cava was dissected to allow outflow of blood and perfusate. To achieve maximum vasodilatation, fixation, staining of endothelial cells, and optimal infusion of radiographic contrast material, serial perfusion of the extremities was performed at 110 mm Hg pressure by using different perfusates. Silver nitrate was used to stain endothelial cells. First, the vasculature was perfused with saline containing heparin (3000 IU/L) and adenosine (1 g/L) for 5 minutes. Then, the arteries were perfusion fixed for 3 minutes with 1% paraformaldehyde and 0.5% glutaraldehyde in 75 mmol/L cacodylate (fixative) at pH 7.4. Then, they were perfused with saline for 1 minute, 10 mL of 5% glucose for 10 seconds, 10 mL of 0.1% silver nitrate for 10 seconds, 10 mL of 5% glucose for 10 seconds, and fixative for 60 seconds. Two individuals (Z.W.Z., with 9 years of experience, and M.M., with 7 years of experience) performed these perfusions together.

CT Angiography
A mixture of barium sulfate suspension (Maxibar; E-Z-Em, Westbury, NY) and laboratory-grade latex (Ward's, Rochester, NY) was used as a vascular contrast agent. Latex stabilizes the barium sulfate suspension and allows subsequent dissection of the specimen. Prior to mixing, both components were filtered through a cell strainer (pore size, 100 µm) to eliminate large particles that might obstruct the injection apparatus. Subsequently, barium sulfate suspension and latex were added to a sterile bottle at a 40:60 volume ratio, mixed for 3 minutes, and drawn into syringes; 0.45 mL was injected into the hind limbs through the catheter that had been positioned proximal to the aortic bifurcation at a rate of 0.7 mL/min by using a syringe pump (Harvard Apparatus, Holliston, Mass). More details about the particular barium sulfate mixture and injection technique we used can be obtained by contacting one of the authors (Z.W.Z.).

The rats underwent 2D CT scanning before and after contrast agent injection by using a four–detector row CT scanner (LightSpeed QX/i; GE Medical Systems, Milwaukee, Wis), with scanning parameters of 120 kV, 300–440 mAs, and 1-second rotation time. The scans extended from several millimeters proximal to the aortic bifurcation down to the ankles, with a 96-mm field of view. The acquisition time was approximately 23 seconds (range, 20–30 seconds). The acquisition protocol was as follows: detector collimation, 1.25 mm; section thickness, 1.25 mm; data reconstruction interval, 0.8 mm; table increment, 3.75 mm per rotation; and pitch, 3:1. After CT scanning, all rats were frozen in liquid nitrogen to prevent dissipation of the contrast medium into the venous system. CT angiography and CT scanning were performed by two individuals (Z.W.Z., with 12 years of experience, and T.J.S., with 15 years of experience).

Data Transfer, Display and Volume Rendering, and Quantification
The multi–detector row CT angiographic data were transferred over the Ethernet to a workstation (Precision; Dell, Round Rock, Tex) with 400-MB double data rate and two central processing units with real-time 3D volume-rendering software (Vitrea 2, version 3.1; Vital Images, Plymouth, Minn). Traditional transverse viewing methods were applied in combination with alternative viewing techniques, such as multiplanar reformatting and volume rendering. The workstation was capable of real-time interactive volume rendering at rates of 10–20 frames per second. The volume-rendering algorithm has been described in detail for CT angiography (16). A transfer function is used to map the CT number to color and opacity. By interactively changing the transfer function, the user can easily detect the origin of blood vessels and 3D spatial relationships of the vascular tree (main arteries and side branches, including small arterioles).

After a CT data set was transferred to the workstation, we chose the bone protocol to display only the high-attenuation voxels to identify and subtract osseous structures and to create angiographic volume-rendered images because the contrast agent had x-ray attenuation similar to that of the bone. Thus, we were able to generate 2D and 3D arteriographic images. All volume rendering was performed by one radiologist (Z.W.Z., with 3 years of experience in 3D reconstruction) who was familiar with the volume-rendering algorithm and hardware. Multiplanar reformatting techniques allowed us to view the data set in transverse, sagittal, coronal, and hybrid planes. Rotation of 3D multi–detector row CT angiograms also is important for the exclusion of blood vessel foreshortening and especially for the discrimination between arterialization of preexisting collateral vessels and branching arteriogenesis.

Arteriogenesis occurs primarily around the occluded blood vessel segment. Because the common femoral artery was ligated in our model, the entire thigh was defined as a region of interest. Scanning before and after contrast agent injection enabled us to apply a subtraction technique and bone editing to the images by using modified software (ImageJ; National Institutes of Health, Bethesda, Md). These postprocessing steps were applied to 35 matched images obtained from areas throughout the thigh in both hind limbs. Minimal edge effect was furthermore attenuated by our modified ImageJ software with an in-house plug-in function. After postprocessing, the regions of interest (222–543 mm2) in both the treated and the nontreated hind limbs were selected manually in two groups. Information from a reconstructed stack of these 35 sections was recorded. Number of detected arteries, size distribution, cross-sectional area, and mean of blood vessel area from the subtracted and region-of-interest–selected transverse images were measured semiautomatically by using modified ImageJ software. Thresholds were set between 60 and 255 to highlight all vessels. The total volume of the depicted arteries was calculated by multiplying the total arterial area in all transverse images by the section thickness (0.45-mm data reconstruction overlap subtracted from 1.25-mm section thickness). All quantitative measurements were performed by our biostatistician (L.G., with 20 years of experience), who was blinded to the treatment that the animals had received. Postprocessing was time consuming because the bone had to be subtracted by using a semiautomatic technique, as mentioned earlier.

Tissue Harvest and Histologic Analysis
After injection of contrast agent and CT angiography, adductor muscles in two hind limbs from both BMC-injected and DMEM-injected rats were harvested, and after paraffin embedment, 4-µm-thick sections were cut from each sample with muscle fibers oriented in the transverse direction. These sections were exposed to light for 15 minutes to develop the silver, were dehydrated in an alcohol gradient, and were mounted whole in glycol methacrylate embedding medium (Permount; Fischer Scientific, Fair Lawn, NJ). Arteries, lumen filled with the contrast agent, and endothelium stained with silver were assessed by using a microscope (Olympus BH-2; GMI, Ramsey, Minn) equipped with a camera (SSC C374; Sony, Cambridge, Mass) and attached to a computer (G3; Macintosh, Cupertino, Calif) with a public domain image processing and analysis program (NIH Image; National Institutes of Health, available at http://rsb.info.nih.gov/nih-image/).

Statistical Analysis
Continuous variables were described as mean ± standard deviation and were derived from 2D-subtracted CT angiography. A two-way analysis of variance model was used to evaluate the difference between treatments (BMC vs DMEM) and the ischemic versus nonischemic legs for the variables of total blood vessel area, total blood vessel volume, mean of blood vessel area, and total number of arteries. With this approach, the ischemic versus the nonischemic leg was used as the within-subject factor, and treatment (BMC or DMEM) was used as the between-subject factor. Furthermore, paired Student t tests were used to compare the difference between ischemic and nonischemic legs of each variable for BMC and DMEM treatments separately. The unpaired Student t test was used to test the difference between treatments (BMC vs DMEM) and in the ischemic leg–nonischemic leg ratio for each variable between treatment groups. The statistical analysis was performed by using software (SPSS 12.0; SPSS, Chicago, Ill), and differences with P < .05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
Types of Arteriogenesis
Reconstructed 3D multi–detector row CT angiograms showed two types of arteriogenesis in the ischemic leg. One pattern showed interconnecting arteries (type 1) that originated from the arteria circumflexa femoris lateralis and connected to the arteria genus descendens and that originated from the arteria profunda femoris and connected to the arteria saphena and arteria caudalis femoris distalis. These arteries presumably arise from preexisting collateral vessels. The other growth pattern was branching arteriogenesis (type 2), which arose from the arteria circumflexa femoris lateralis and the arteria profunda femoris. Finally, 3D multi–detector row CT angiograms demonstrated enlargement and lengthening of small branches that do not perfuse the ischemic leg in comparison with the nonischemic leg. In the BMC-treated group, both types of arteriogenesis were clearly seen in all eight rats (Figs 1a, 2). In contrast with the BMC group, type 1 arteriogenesis was seen in only four of eight rats, and type 2 arteriogenesis was seen in two of eight rats, with a substantially smaller amount in the DMEM group (Fig 1b). In the nonischemic leg, there was no collateral vessel development.


Figure 1
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  		Figure 1a: Representative reconstructed anteroposterior 3D CT angiograms in the right thigh of a rat 2 weeks after ligation of the right common femoral artery (arteria femoralis) and intramuscular injection of (a) 1.5 x 107 BMC and (b) 70 µL DMEM. (a) An extensive network of bridging collateral vessels (solid arrows) and branching arteriogenesis is shown. In the right limb, a 1-cm segment of the arteria femoralis was excised. In the left, there is an overlapping branch (open arrow), a problem that can be resolved by rotating the image at arbitrary angles. (b) In contrast, after DMEM treatment the arteriogenic response is limited to one collateral vessel. L = left leg, R = right leg, 1 = arteria circumflexa femoris caudalis, 2 = arteria iliaca interna, 3 = arteria epigastrica caudalis, 4 = circumflexa femoris lateralis, 5 = arteria femoralis, 6 = arteria genus descendens, 7 = arteria saphena, 8 = arteria poplitea, 9 = arteria caudalis femoris distalis; 10 = arteria profunda femoris; 11 = arteria ductus deferentis, 12 = arteria caudalis dorsolateralis.

 

Figure 1
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  		Figure 1b: Representative reconstructed anteroposterior 3D CT angiograms in the right thigh of a rat 2 weeks after ligation of the right common femoral artery (arteria femoralis) and intramuscular injection of (a) 1.5 x 107 BMC and (b) 70 µL DMEM. (a) An extensive network of bridging collateral vessels (solid arrows) and branching arteriogenesis is shown. In the right limb, a 1-cm segment of the arteria femoralis was excised. In the left, there is an overlapping branch (open arrow), a problem that can be resolved by rotating the image at arbitrary angles. (b) In contrast, after DMEM treatment the arteriogenic response is limited to one collateral vessel. L = left leg, R = right leg, 1 = arteria circumflexa femoris caudalis, 2 = arteria iliaca interna, 3 = arteria epigastrica caudalis, 4 = circumflexa femoris lateralis, 5 = arteria femoralis, 6 = arteria genus descendens, 7 = arteria saphena, 8 = arteria poplitea, 9 = arteria caudalis femoris distalis; 10 = arteria profunda femoris; 11 = arteria ductus deferentis, 12 = arteria caudalis dorsolateralis.

 

Figure 2
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  		Figure 2: Three-dimensional multi–detector row CT angiograms of the hind limbs injected with a contrast agent consisting of 40% barium and 60% latex 2 weeks after induction of ischemia of unilateral hind limb. With 3D volume rendering, a panoramic view (anteroposterior view and every 30° rotation until 150° left anterior oblique position is reached) is generated of the entire arterial vascular tree and the arteriogenic response in the ischemic leg. Two patterns of arteriogenesis were observed: bridging collateral vessel formation and branching arteriogenesis. This CT angiogram demonstrates the quality of imaging possible with the current clinical helical CT system and illustrates the potential of these types of images for use in quantification of blood vessel density changes and blood vessel network analysis.

 
Although we could not quantitatively distinguish between arteriogenesis of types 1 and 2, we could quantify the total arteriogenic response. We quantified several morphologic parameters: the number of arteries, cross-sectional area of each artery (arterial size), total arterial area, total arterial volume, and the distribution of arterial size for both the nonischemic and ischemic hind limbs on the basis of findings at subtracted cross-sectional CT angiography (Fig 3). These data have been summarized in the Table and Figure 4. Results of analysis of variance indicated that the differences within subjects (ischemic vs nonischemic legs) and between subjects (BMC vs DMEM treatment) were significant for the variables of total blood vessel area, total blood vessel volume, and mean of blood vessel area (P < .001) (Table). BMC-treated rats had a significantly higher ischemic leg–nonischemic leg ratio for all morphologic parameters than did DMEM-treated rats, as follows: total blood vessel area, 0.98 ± 0.24 [mean ± standard deviation] versus 0.33 ± 0.17 (P < .001); total blood vessel volume, 0.98 ± 0.24 versus 0.33 ± 0.17 (P < .001; mean of blood vessel area, 0.80 ± 0.13 versus 0.62 ± 0.18 (P = .038); and total number of arteries, 1.28 ± 0.26 versus 0.50 ± 0.18 (P < .001). These values were determined with the unpaired Student t test.


Figure 3
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  		Figure 3: A–F, CT image postprocessing (subtraction technique) for clinical CT angiography (transverse view) by using modified ImageJ software in a rat after surgical ligation of the right common femoral artery and intramuscular injection of 1.5 x 107 BMC. Image C was obtained by subtracting image A (before contrast agent injection) from image B (after contrast agent injection). Images D–F demonstrate how to select a region of interest that surrounds the ligated site in the thigh, how to set up a certain threshold for highlighting all vessels, and how to quantify vessels by using the modified ImageJ software. The native common femoral artery is shown in the nonischemic limb (arrow on D), and the number of small vessels (n = 13, collateral vessels and branches) in the ischemic limb was greater than in the nonischemic limb (n = 5, native common femoral artery and its branches). On this CT section, the total arterial area is comparable in both limbs (39.35 mm2 in the ischemic limb vs 38.12 mm2 in the nonischemic limb).

 

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  		Morphologic Parameters Assessed with Multi–Detector Row CT Angiography in Rats 2 Weeks after Induction of Ischemia of Hind Limb and Intramuscular Injection of DMEM or BMC

 

Figure 4
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  		Figure 4a: Graphs show results of quantitative assessment for mean arterial size distribution in ischemic limbs (right legs) and contralateral nonischemic limbs (left legs) in (a) BMC-treated and (b) control rats at 2 weeks follow-up. Nonischemic limbs represent the normal anatomic distribution of arterial diameters in the thigh. In the BMC group, the number of small arteries is significantly greater in the ischemic leg versus the nonischemic leg, whereas in the control group, the number of arteries is significantly smaller in the ischemic leg across the entire diametral spectrum. ** = P < .01 versus control group, * = P < .05 versus control group. (c) When expressed as right leg–left leg ratio, the ischemic limbs in the BMC-treated rats show a significantly greater number of both small- and mid-sized arteries. Data are presented as mean ± standard error of the mean. ** = P < .01 versus control group, * = P < .05 versus control group.

 

Figure 4
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  		Figure 4b: Graphs show results of quantitative assessment for mean arterial size distribution in ischemic limbs (right legs) and contralateral nonischemic limbs (left legs) in (a) BMC-treated and (b) control rats at 2 weeks follow-up. Nonischemic limbs represent the normal anatomic distribution of arterial diameters in the thigh. In the BMC group, the number of small arteries is significantly greater in the ischemic leg versus the nonischemic leg, whereas in the control group, the number of arteries is significantly smaller in the ischemic leg across the entire diametral spectrum. ** = P < .01 versus control group, * = P < .05 versus control group. (c) When expressed as right leg–left leg ratio, the ischemic limbs in the BMC-treated rats show a significantly greater number of both small- and mid-sized arteries. Data are presented as mean ± standard error of the mean. ** = P < .01 versus control group, * = P < .05 versus control group.

 

Figure 4
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  		Figure 4c: Graphs show results of quantitative assessment for mean arterial size distribution in ischemic limbs (right legs) and contralateral nonischemic limbs (left legs) in (a) BMC-treated and (b) control rats at 2 weeks follow-up. Nonischemic limbs represent the normal anatomic distribution of arterial diameters in the thigh. In the BMC group, the number of small arteries is significantly greater in the ischemic leg versus the nonischemic leg, whereas in the control group, the number of arteries is significantly smaller in the ischemic leg across the entire diametral spectrum. ** = P < .01 versus control group, * = P < .05 versus control group. (c) When expressed as right leg–left leg ratio, the ischemic limbs in the BMC-treated rats show a significantly greater number of both small- and mid-sized arteries. Data are presented as mean ± standard error of the mean. ** = P < .01 versus control group, * = P < .05 versus control group.

 
In the two-way analysis of variance model, the variable of total number of arteries was significant for neither the difference between ischemic and nonischemic legs (P = .246) nor the difference between treatments (P = .125) when both legs were included in the model; however, the total number of blood vessels in the BMC group was significantly higher than that in the DMEM group for the ischemic leg (P = .002). If we look at only the nonischemic leg between treatment groups by using the unpaired Student t test, the differences in total blood vessel area, total blood vessel volume, and total blood vessel number are not significant (P > .05).

On the basis of image resolution (188 µm), we binned the vessel diameters into 21 intervals (an even multiple of the resolution) to analyze the distribution of the arteries. Naturally, the distribution of arteries of different sizes is clearly skewed to the right (more numbers in small blood vessels). Figure 4c shows that the differences between BMC and DMEM treatment in regard to the number of arteries for the ischemic leg–nonischemic leg ratio arise in the small and medium blood vessel intervals after BMC injection.

In BMC-treated rats, there were significantly more arteries in the ischemic legs compared with those in the nonischemic legs by using the paired t test (P = .028) (Table). The mean size of these arteries, however, was significantly smaller (P = .006) in the ischemic limb. The increased number of small arteries resulted in a comparable total arterial area and volume, which did not differ significantly between the ischemic legs and the nonischemic legs (Table). Figure 4a showed that BMC in the ischemic legs enhanced small blood vessel growth in comparison with the nonischemic legs, which revealed a larger number of small blood vessels after BMC injection.

In contrast to BMC-treated rats, in DMEM-treated rats all four measurements were significantly smaller in the ischemic hind limb than in the nonischemic hind limb (Table, Fig 1b). The analysis of distribution of blood vessel size showed that the inability to respond to the loss of a large conductance artery with effective arteriogenesis involved blood vessels across the entire diametral spectrum, from small to large (Fig 4b). Thus, BMC enhancement of arteriogenesis in this model is characterized by normalization of arterial area and volume, which compensates for the loss of a large conductance artery (the femoral artery) through the generation of a multitude of small and medium arteries.

Histologic Analysis
Microvasculature was analyzed in the midportion of the adductor muscles by means of silver staining. Silver staining clearly showed that the contrast agent filled both small arteries (>188 µm) and surrounding arterioles (10–40 µm) (Fig 5). The contrast agent clearly penetrated vessels of 10 µm or larger in diameter.


Figure 5
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  		Figure 5: Tissue sample harvested from the right adductor muscle of the rat shown in Figure 2. Both small arteries (≥188 µm in diameter) and arterioles (10–40 µm in diameter) are filled with contrast agent (black dots). Scale bar is 10-µm wide and 50-µm long. (Silver stain; original magnification, x40.)

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ADVANCES IN KNOWLEDGE
 References
 
To our knowledge, we showed for the first time that 2D multi–detector row CT angiography and 3D reconstruction of CT sections obtained with a multi–detector row clinical CT scanner can be used to image the compensatory arteriogenic response to the loss of a large conductance artery in a rat hind limb model of ischemia. First, we saw that there were two patterns of arterial growth, with one pattern that resulted in bridging collateral vessels that most likely originated from preexisting rudimentary vessels (17) and another pattern that showed branching arteriogenesis. Second, we were able to quantify total arterial area and volume for the entire thigh, thus generating a comprehensive picture of arterial density that is not affected by the selection bias of blood vessel counts on a limited number of histologic sections and the impossibility to distinguish between branching and overlapping blood vessels on 2D angiograms. Third, we identified a response to occlusion of a conductance artery (ie, the enlargement of other branches from the feeding trunk). Finally, this imaging method taught us that, in this model, the compensatory response to the loss of a large conductance artery is characterized by the generation of a multitude of small arteries, and this response can be enhanced with BMC treatment to a degree to which total arterial area and volume are the same in the ischemic leg and in the nonischemic leg.

The fact that results of large clinical trials involving angiogenesis have not corroborated the success of earlier smaller studies has led to the realization that the therapeutic goal of neovascularization should be refocused on arteriogenesis. The importance of arteriogenesis in maintaining cardiac function and patient survival after myocardial infarction is illustrated by the observation that patients with myocardial infarction are less likely to develop ventricular aneurysms and they show improved survival if they have collateral arteries (18). In mouse strains that respond to ischemia of the hind limb with a blunted arteriogenic response, ischemia of the calf is significantly more pronounced than it is in strains that have a normal arteriogenic response (19). Unfortunately there is no reference to document arteriogenesis in clinical and experimental studies (20).

Angiography is widely used, but the inherent problems are such that some investigators have used angiography and microangiography only for qualitative assessment of blood vessel growth (5,21,22). In attempts to approach the problem more quantitatively, researchers in some studies have reported blood vessel density as the percentage of pixels per digitized image occupied by blood vessels in both mice (23) and rats (24). Other investigators have quantified neovascular responses by counting the crossing points of arterial branches at perpendicular lines (25) or the intersection of the contrast material–opacified arteries that cross over an overlapping composite of 2-mm2 grids in rats (26) or of 5-mm2 grids in rabbits (14,27,28). For the grid method, an angiographic score was calculated for each angiogram as the ratio of grid intersections crossed by the contrast material–opacified arteries divided by the total number of grid intersections in the medial thigh. Conventional angiography is neither sufficiently sensitive nor accurately quantitative for assessment of this process because of superimposition of vessels and other structures, and this superimposition makes it impossible to differentiate bridging collateral vessels, new arterial branches, and overlapping preexisting blood vessels. In our study, we demonstrated that different patterns of arterial growth can be seen after femoral artery occlusion and that 3D CT angiography can be applied successfully to make these distinctions.

Multi–detector row CT angiography is evolving as a practical tool in the diagnosis and treatment of peripheral arterial disease (29,30). It is a rapidly developing and promising technique, which may replace digital subtraction angiography as the method of choice to establish the extent of peripheral arterial disease for clinical assessment. Its advantages are low cost, availability, detailed depiction of small blood vessels in terms of the blood vessel calibers encountered in patients, and rapid image acquisition. Multi–detector row CT angiography, however, has not been used to quantify neovascular responses to cell therapy or single–growth factor therapy in experimental settings. Clearly, our imaging system is limited in that it can only depict arteries greater than a certain size; the field of view of the current CT scanner is 96 x 96 mm, with a pixel density of 512 x 512, resulting in a resolution of 188 µm. Hence, we will have missed any arteries that are smaller than 188 µm in diameter, although our contrast agent opacified blood vessels as small as 10 µm in diameter. It has been shown, however, that arteriogenic substrates typically are 30–50-µm-diameter rudimentary blood vessels that evolve into arteries that are 25-fold larger in diameter (0.75–1.25 mm) (31). Therefore, it is likely that our imaging technique captured any major arteriogenic event in the rat hind limb model of ischemia.

Thus far, two types of arteriogenesis have been described. Collateral arteries may originate from preexisting rudimentary vessels, which are the underdeveloped arteries that nevertheless have an intima and a tunica media with vascular smooth muscle cells (19), and they can arise through budding of new vessels from postcapillary venules on the adventitial surface of the occluded artery that gradually expand and form new branches or connect to the distal arterial segment (4). Our 3D multi–detector row CT angiographic images clearly indicate that both collateral arterial formation and branching arteriogenesis occur in the rat hind limb model of ischemia. Finally, 3D multi–detector row CT angiography enabled detection of enlargement of other branches that arise from the feeding trunk of the occluded femoral artery. For this finding, it is likely that increased flow and shear stress in these branches contribute to this outward vascular remodeling response (32). Detailed assessment of hemodynamics, shear and wall stress in these branches, by using tip microsensors needs to be performed to unravel the mechanisms of this response in the future.

An important limitation of our study was that the animals did not survive the contrast agent injection. A contrast agent that is compatible with survival of rodents will open the possibility of sequential imaging and will further increase the utility of multi–detector row CT angiography because 3D anatomic information will be supplemented with functional information (ie, measurement of blood flow). Another limitation of this study was the nonisotropic nature of the images generated with the four–detector row scanner, and this nature resulted in a partial volume effect in the z-axis on the 1.25-mm thickness of sections. This effect may result in an overestimation of vascular area and volume and an underestimation of the number of arteries because of bifurcation or trifurcation. The nonisotropic nature, however, was the same for all images; thus, it did not influence the comparison between the BMC group and the control group.

In conclusion, to our knowledge we demonstrated for the first time the utility of 2D and 3D multi–detector row CT angiography in the assessment of arteriogenic responses to the occlusion of a large conductance artery. The method allowed discrimination between side branches and overlapping vessels, and two patterns of arterial growth were observed. We observed an increase in the length and diameter in branches that do not feed the ischemic leg, and this increase is a positive remodeling response that merits further investigation.

Practical application: The technique described in this article appears to be promising for use in the quantitative measurement of the progress of the development of arteriogenesis. Further study of the diagnostic and prognostic accuracy of this technique is required to assess its value in clinical practice. The emerging field of therapeutic angiogenesis and arteriogenesis may benefit from this technique, which allows serial noninvasive assessment of collateral vessel development. This assessment of neovascularization with multi–detector row CT angiography also may prove useful in the monitoring of arteriogenesis-suppressive therapy for treatment of cancer.


   ADVANCES IN KNOWLEDGE
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
References
 


   ACKNOWLEDGMENTS
 
We gratefully acknowledge Peter K. Spiegel, MD, for his critical review of this manuscript.


   FOOTNOTES
 

Abbreviations: BMC = bone marrow–derived mononuclear cells • DMEM = Dulbecco's modified Eagle's medium • 3D = three-dimensional • 2D = two-dimensional

Authors stated no financial relationship to disclose.

Author contributions: Guarantor of integrity of entire study, Z.W.Z.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; manuscript final version approval, all authors; literature research, Z.W.Z., M.M., M.S., E.D.d.M.; experimental studies, Z.W.Z., L.G., M.M., T.J.S.; statistical analysis, Z.W.Z., L.G.; and manuscript editing, Z.W.Z., J.D.P., M.S., E.D.d.M.


   References
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 

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