Mapping 3-Dimensional Neovessel Organization Steps Using Micro-Computed Tomography in a Murine Model of Hindlimb Ischemia–Brief Report
Objectives— Studying the mechanisms of neovascularization and evaluating the effects of proangiogenic strategies require accurate analysis of the neovascular network. We sought to evaluate the contribution of the microcomputed tomography (mCT) providing high-resolution 3-dimensional (3D) structural data, to a better comprehension of the well-studied mouse hindlimb postischemic neovascularization.
Methods and Results— We showed a predominant arteriogenesis process in the thigh and a predominant angiogenesis-related process in the tibiofibular region, in response to ischemia during the first 15 days. After 15 days, mCT quantitative analysis reveals a remodeling of arterial neovessels and a regression depending on the restoration of the blood flow. We provided also new mCT data on the rapid and potent angiogenic effects of mesenchymal stem cell therapy on vessel formation and organization. We discussed the contribution of this technique compared with or in addition to data generated by the more conventional approaches.
Conclusion— This study demonstrated that optimized mCT is a robust method for providing new insights into the 3D understanding of postischemic vessel formation.
As mouse models are largely used to study mechanisms of neovascularization, vascular repair, and growth of collateral vessels,1 a throughout analysis of the vascular network has gained of interest. Understanding neovessel formation mechanisms requires a complete view of the vascular architecture over the whole of the tissue analyzed: size, orientation, branching, and organization of collaterality. However, the classical methods of assessment are not always quantitative, restricted to a limited area of view, evaluate capillary density in 2D sections, or report superficial blood flow data.1 Hence, microcomputed tomography (mCT) can, after the injection of a radiopaque contrast agent, image the vascular network in 3D in an entire organ2,3 and give quantitative data. Several studies have investigated the microvascularization of the kidney, heart, and liver in the rat.4–6 Only a few teams have used quantitative tools for studying postischemic angiogenesis in the mouse.2,3
Here, we used a contrast agent and mCT acquisition procedure dedicated to analyze postischemic kinetics of vessel formation and remodeling restricted to arterial network in the mouse hindlimb. We discussed the contribution of these images compared with or in addition to data produced by the conventional laser Doppler and immunolabeling approaches. Our results revealed a predominant arteriogenesis process in the thigh and in contrast a predominant angiogenesis related process in the tibiofibular region. We showed that neovessel remodeling depends on the restoration of the blood flow. We then provided new data on the mesenchymal stem cell therapy on the feature of vessel formation and organization.
Materials and Methods
Materials and methods related to MSC culture, animal care, model of ischemia, micro-CT data processing and analysis, and histological vascularization quantification appear in the supplemental Materials and Methods section (available online at http://atvb.ahajournals.org). Briefly, we studied the formation of neovessels in the mouse hindlimb ischemia model using microCT analysis. We developed a selective arterial contrast agent, a mixture of Neoprene and barium sulfate. This technique allows a lower threshold to be used which is constant from one animal to another, given the maximum contrast provided by the barium sulfate, and therefore allows higher quality images to be obtained. The variability introduced by the micro-CT method was assessed by performing repeated scans, reconstructions, and quantitative analyses of iso-intensity surfaces on the same arterial specimen. All the 3D acquired images were recorded and the different parameters as vessel number, diameter, connectivity, vessel volume, and intervessel space were quantified. To assess the role of MSC in vessel formation, 500 000 MSCs were injected into the thigh and into the anterior tibial muscle 48 hours after surgery and the hindlimb vascular network was analyzed by microCT 8 days after surgery.
Results and Discussion
Use of Neoprene Latex Allows Selective Arterial Filling
We developed and validated a new combination of micronised barium as contrast agent and Neoprene latex as suitable vehicle able to fill only arterial network within a diameter of 20 μm. The viscosity property distinguishes latex vehicule from other contrast agent vectors which more easily fill the whole vascular network (supplemental Results and Discussion section and supplemental Figure I).
3D Quantification of Postischemic Arterial Vascular Growth in the Hindlimb
For the first time, we demonstrated a significant difference in ischemia-induced vascular growth mechanisms between the thigh and the tibiofibular regions as reported on Figure 1.
mCT analysis demonstrated that the vascular response in the thigh is mostly attributable to arteriogenesis mechanism as described.2,3 At D15, the number of vessel and connectivity increased slightly, their diameter decreasing by two times, with no modification of the total arterial vessel volume in ischemic compared to non ischemic muscle. All these parameters return close to the baseline values by D28 when the perfusion defect was compensated (Figure 1A and 1B). In summary, mCT uncovered novel arterial remodeling data in the thigh. Other approaches as microangiography were limited by the low spatial resolution and the absence of quantitative volumetric analysis.7,8 Histological examination is rarely carried out in the thigh, because of the variability in the degree of ischemia from one muscle to another, of the variations in diameter of medium-calibre arteries detectable only on mCT but not on immunohistochemistry images. Finally, measuring blood flow by laser Doppler is not applicable in this region.
In contrast to the thigh, angiogenesis predominated in the tibiofibular region. In the first 21 days, we showed a dramatic increase in arterial network density (9 fold), volume (6 fold), and vessel connectivity or branching (7 fold) with a decrease of the vessel mean diameter. All of these parameters are the hallmark for a dynamic angiogenic related process with a very dense and divided arterial network. For the first time, we evidenced a complex arterial vascular remodeling between D15 and D28 in the tibiofibular region, less pronounced in the thigh. The number, connectivity, and occupied volume of arterial vessels dropped considerably after D21 (Figure 1B), whereas CD31-positive capillary number and the blood flow gradually improved after 21 days (Figure 1C).
Thus, we propose that the arterial network adapts to tissue perfusion; the precocious development of large- and medium-calibre vessels in the thigh would favor the underlying arterial perfusion and angiogenesis on the tibiofibular region. We then reported after D21, in the tibiofibular region, an arterial neovessel regression whereas venous and lymphatic capillaries develop as evidenced by immunohistolabeling.
Application to the Quantification of an Angiogenesis-Focused Cell Therapy
To dissect the mechanisms of mesenchymal stem cell based angiogenic therapy,9 we examined and quantified by mCT the vascular network in MSC injected versus saline injected hindlimb. MSC graft induced a burst of neovessels as soon as D8, predominant in the tibiofibular region with an increase in the number, volume, and the connectivity of the arterial vessels but with no modification of the mean vessel diameter (Figure 2A and 2B). Blood flow increased significantly in the mice treated with MSCs (0.205±0.10 versus 0.280±0.17 for the ischemic/normal limb ratio in control versus MSC-injected animals at D8, respectively, P<0.05; not shown). In summary, these observations uncovered a precocious and potent angiogenic role of MSC as soon as D8, showing that MSC therapy acts essentially on angiogenesis.
See supplemental materials for a discussion of the findings in this report in relationship to relevant articles in the literature.
Sources of Funding
This work was supported by the Fondation de France (grant #2006005678), Fondation pour la Recherche Médicale (DCV20070409258), and Communauté de Travail Pyrénnéenne (CTP). L.L. and M.A.R. are the recipients of grants from the Fondation pour la Recherche Médicale.
Received December 24, 2008; revision accepted August 25, 2009.
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