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(Circulation. 1997;95:805-808.)
© 1997 American Heart Association, Inc.

Articles

Use of Synchrotron Radiation Microangiography to Assess Development of Small Collateral Arteries in a Rat Model of Hindlimb Ischemia

Satoshi Takeshita, MD; Takaaki Isshiki, MD; Hidezo Mori, MD; Etsuro Tanaka, MD; Koji Eto, MD; Yoshimichi Miyazawa, DVM; Akira Tanaka, ME; Yoshiro Shinozaki, BE; Kazuyuki Hyodo, PhD; Masami Ando, PhD; Misao Kubota, BE; Kenkichi Tanioka, PhD; Keiji Umetani, ME; Masahiko Ochiai, MD; Tomohide Sato, MD; Hideo Miyashita, MD

the Second Department of Medicine (S.T., T.I., K.E., Y.M., M.O., T.S., H. Miyashita), Teikyo University School of Medicine, Tokyo, Japan; Department of Physiology (H. Mori, E.T., A.T., Y.S.), Tokai University School of Medicine, Isehara, Japan; the Photon Factory (K.H., M.A.), National Laboratory for High Energy Physics, Tsukuba, Japan; Science and Technical Research Laboratories (M.K., K.T.), Japan Broadcasting Corporation (NHK), Tokyo, Japan; and Central Research Laboratory (K.U.), Hitachi Ltd, Tokyo, Japan.

Correspondence to Satoshi Takeshita, MD, The Second Department of Medicine, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi, Tokyo 173, Japan. E-mail stangiol@med.teikyo-u.ac.jp.

	Abstract

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Background Current methods of angiography cannot provide images of arteries measuring <200 µm in diameter. We have recently developed a new angiography system that uses monochromatic synchrotron radiation and a high-definition video system with a spatial resolution of 30 µm. In the present study, we applied this microangiography system to visualize small arteries in normal and ischemic rat limbs and investigated the development of collateral arteries.

Methods and Results Microangiography was performed in the normal and the ischemic limb 4 weeks after the excision of the femoral artery. In the normal limb, up to the fourth branches of the iliac and/or femoral arteries (diameter <100 µm) were readily identified. Some of these branches were found to perfuse the distal thigh area. In the ischemic limb, an extensive structural remodeling of the vascular network was observed. Numerous small arteries had developed from the branches of the iliac artery to constitute a fine arterial network, the so-called "midzone," which was composed of linear, normal-appearing arteries and those with an undulating, unbranched appearance.

Conclusions The small collateral artery network was angiographically visualized with a resolution limit <100 µm. The linear collaterals appeared to result from an opening of preexisting vessels. The undulating, unbranched vessels were not observed in the normal limbs and seemed to be vessels that were newly formed after limb ischemia. Synchrotron radiation microangiography appears to be a powerful means of assessing the development of small collateral arteries, which may help to provide a basis for understanding of the collateral circulation.

Key Words: angiogenesis • angiography • collateral circulation • microcirculation

	Introduction

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Blood flow to the muscles during obstruction of a major limb artery is dependent on the development of collateral vessels. If the capacity of collaterals is insufficient, tissue necrosis may result. To visualize collateral vessels, previous studies have used such methods as corrosion casts^{1 2} and angiography.^{3 4} Corrosion casts allow the visualization of small arteries <100 µm in diameter,¹ although the complete distension of vessels depends on such factors as the elastic properties of the vessel wall, the viscosity of the infused material, and the pressure of infusion. Angiography can be performed in live animals and allows for repeated examinations. However, previous reports indicated that conventional angiography systems have insufficient resolution to fully display the small vessels.^{5 6}

Mori et al^{5 6} recently developed a new angiography system called synchrotron radiation (SR) microangiography. This system uses monochromatic SR as an x-ray source and a high-definition video system as a detector, which has the potential to visualize arteries as small as 30 µm in diameter. In the current study, we used this microangiography system and investigated the morphological features of the small collateral arteries in the ischemic rat limb.

	Methods

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Animal Model
The development of collateral vessels was investigated in a rat model of unilateral hindlimb ischemia described previously,⁷ with minor modifications. All of our experiments in animals conformed to the National Institutes of Health's Guide for the Care and Use of Laboratory Animals. Male Wistar rats (weight, 400 to 450 g; Charles River Japan, Yokohama) were anesthetized with pentobarbital sodium 40 mg/kg IP (Nembutal, Abbott Laboratories). A vertical longitudinal incision was made on one limb, extending from the inguinal ligament to a point just proximal to the knee. The femoral artery and its branches were dissected, ligated, and completely excised through this incision. After the incision was closed, the animals were housed for 4 weeks until angiography was performed.

In this report, a standard nomenclature defined by Longland⁸ will be used to facilitate communication. "Midzone collateral arteries" are the plexus of branching arteries that interconnect the stem and reentry arteries. The "stem" arteries are the "feeders" from which collateral arteries are derived. "Reentrant" arteries are those that rejoin the midzone plexus with the distal circulation.

Study Design
Four rats did not undergo limb surgery and were used for the microangiographic assessment of the vasculature of the normal limb. Ten rats underwent surgical resection of the femoral artery as described above. Four weeks after the limb surgery, the animals were reanesthetized with pentobarbital sodium. An incision was made on the thigh of the contralateral, nonischemic limb to expose the femoral artery, and a 24-gauge catheter (Surflo, Terumo) was cannulated into the artery via a small cutdown. The tip of the catheter was positioned just above the aortic bifurcation in the lower abdominal aorta. Angiography was performed by injecting a total of 2 mL nonionic contrast media (Iomeprol, Eisai) by use of an automated angiographic injector (Medrad). After the angiography had been completed, the animals were killed with an overdose of pentobarbital.

Imaging System
Angiographic procedures were performed at the National Laboratory for High Energy Physics in Tsukuba, Japan. Details of the imaging system used in the present study were described previously.^{5 6} In brief, SR was derived from an accumulation ring of electrons with an accelerated energy of 6.5 GeV and an average beam current of 25 mA. This SR beam with a wide energy band was monochromatized and magnified by an asymmetrically cut silicon crystal that was placed in front of the animal. The final photon density of the SR beam after monochromatization and magnification was estimated to be lxl0⁹ photons·mm^-2·s^-1, which was almost equal to that obtained from a conventional x-ray source (8 to 10x10⁸ photons·mm^-2·s^-1) with a 15- to 80-keV energy band. This high photon energy of SR resulted in an adequate signal-to-background ratio (300:1 to 1000:1) at the detector even after the x-ray beam had passed through the tissue.^{5 9}

Obtained images of limb arteries were formed on a fluorescent screen (HR3 or HR-mammo Fine, Fuji Medical Systems), scanned by a high-definition video camera (Harpicon, Hitachi; Super Harp, Japan Broadcasting Corp), and stored as an analog video signal (SR-W310, Victor; Uni-High, Sony) or as a digital image on computer (1024x1024, 12-bit).¹⁰ The "avalanche" type of high-definition video system used here was 30 to 80 times more sensitive than a conventional charge-coupled device (CCD) in terms of quantum efficiency (ie, the conversion ratio of photons to electrons). The combination of SR and this high-definition video system resulted in a spatial resolution of 30 µm.⁶

	Results

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Microangiographic Findings in Normal Limbs
The global (low-resolution) view of the normal limb arterial network is presented in Fig 1A. SR microangiography was capable of visualizing small arteries with an internal diameter <100 µm, including the third to fourth branches of the iliac and/or femoral arteries. These arteries observed in the normal limbs were generally linear in their morphology. By branching "daughter" arteries that were also linear in appearance, these arteries progressively narrowed to a diameter below the limit of resolution and finally became undetectable. Importantly, some of the small branches of the iliac artery were found to extend and perfuse the distal area of the thigh. SR microangiography documented such small branches of the iliac artery perfusing the distal thigh in all normal limbs examined (Figs 1A and 2A, arrows).

View larger version (145K): [in this window] [in a new window]   Figure 1. Low-resolution microangiography of normal and ischemic limbs. A, Representative microangiogram showing the appearance of the arterial network of a normal limb. Some of the small branches of the iliac artery extend to perfuse the distal thigh area (arrows). B, Representative microangiogram showing the appearance of the arterial network of an ischemic limb. The midzone arteries consisted of relatively linear arteries (small arrows) and undulating or tortuous unbranched arteries (open arrows). The large part of the branched midzone arteries rejoined before they anastomosed with reentrant arteries (large arrow). The diameter of the reference copper wire (arrowheads) was 130 µm.

View larger version (92K): [in this window] [in a new window]   Figure 2. High-resolution microangiography of normal and ischemic limbs. A, Representative microangiogram showing the arterial network of a normal limb. Some of the small branches of the iliac artery extend to perfuse the distal thigh area (arrows). B, Representative microangiogram showing the arterial network of an ischemic limb. Small arrows indicate linear collaterals with side branches. Open arrows indicate undulating unbranched collaterals. The diameter of the reference copper wire (arrowheads) was 130 µm. The imaging field of each angiogram was 20x20 mm.

Microangiographic Findings in Ischemic Limbs
Four weeks after surgery, SR microangiography demonstrated that the excision of the femoral artery led to an increase in the number of small arteries in the thigh (Fig 1B). Most of them originated from the hypogastric trunk of the ipsilateral iliac artery and traversed the entire thigh with fine arterial anastomoses (ie, midzone collaterals). The large part of these branched arteries came together again before they anastomosed with the reentrant arteries (Fig 1B, large arrow). This regathering feature of midzone arteries was not observed in the branching pattern of normal limb arteries.

Midzone collaterals consisted of arteries with two distinct morphologies. One group of collateral vessels appeared to be relatively linear (Figs 1B, small arrows; 2B, arrows), as observed in the normal limbs (Figs 1A and 2A, arrows). The other group of collaterals exhibited an undulating or tortuous appearance and typically lacked side branches (Figs 1B and 2B, open arrows). Such undulating, unbranched vessels were observed in all of the ischemic limbs examined but were not observed in the normal limbs. Some of these undulating vessels exceeded 200 µm in diameter, while others were <100 µm, depending on the rat and the section of the collateral. Occasionally, both types of collateral vessels (linear and undulating) originated from the same linear parent vessel.

	Discussion

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SR microangiography proved capable of displaying arteries having a diameter <100 µm, which is well beyond the limits of conventional angiography.^{5 6} In the normal limb, the third to fourth branches of the iliac and/or femoral arteries could be identified. In the ischemic limb, an extensive structural remodeling of the small vascular network was observed. A number of arteries <100 µm in diameter developed from branches of the iliac artery and constituted the fine arterial anastomoses, ie, midzone collaterals.

The most intriguing finding of the current study was the presence of two types of collateral arteries with distinct morphologies (linear and undulating). Linear collaterals had a branching pattern similar to that observed in normal arteries. Furthermore, such linear vessels that traversed the entire thigh and perfused the distal limb also existed in the normal rat limbs. This suggests that linear collaterals may result from the functional opening or the dilation of preexisting vessels. Conversely, no undulating arteries were observed in the normal limbs. It is possible that such undulating collaterals may result not from the simple opening of preexisting vessels, but rather from a remodeling of preexisting vessels or from newly formed vessels. The fact that undulating collaterals exhibited branching patterns that differed from those of normal limb arteries supports this hypothesis. Regarding the apparent absence of side branches in undulating collaterals, Longland more than three decades ago proposed an interesting hypothesis by stating, "...once a collateral pathway has become established, it simply serves to conduct blood from the arteries proximal to those distal to the block, very little blood is being lost en route into side branches. Certainly such branches are not to be seen in arteriograms...."⁸ Additional study is required to differentiate between the apparent lack of side branches and the branches that are functionally closed.

In conclusion, the present study angiographically demonstrated for the first time the morphological features of small arterial networks in normal and ischemic limbs. The SR microangiography system appears to be a powerful new method for assessing the development of small collateral arteries, which may help to provide a basis for understanding of the collateral circulation.

	Acknowledgments

 
This work was supported in part by grants-in-aid for scientific research (No. 07670809 to Dr Takeshita; Nos. 07557060 and 07807073 to Dr Mori; and No. 08877118 to Dr Tanaka) from the Ministry of Education, Science, and Culture, Tokyo, Japan; a grant from the Ryoichi Naito Foundation for Medical Research, Osaka, Japan (to Dr Takeshita); a grant from the Mochida Memorial Foundation for Medical and Pharmaceutical Research, Tokyo, Japan (to Dr Takeshita); and a grant from Eisai Co Ltd, Tokyo, Japan (to Dr Mori). This project was approved as a Joint Research Program of the National Laboratory for High Energy Physics, Tsukuba, Japan (93G241, 95G113, and 95G287).

Received October 29, 1996; revision received December 12, 1996; accepted December 17, 1996.

	References

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