Experiments were performed in a conventional catheterization laboratory in accordance with the European Community rules of animal care [19]. A pilot study with three adult male New Zealand white rabbits was performed with dummy gold wires (length range, 10-20 mm; diameter range, 0.5-1.0 mm; dwelling time range, 10-30 min) to test the ability of a 2.9-French catheter bearing one straight wire to be pushed into small curved arteries. The protocol using a dummy 0.5 × 15 mm wire and dwelling times of less than 20 min was best tolerated by the rabbits.

We chose ¹⁹⁸Au for our study because it has predominant beta-ray-emission properties and a low-energy gamma-ray-emitting component, theoretically offering good radioprotection. Twenty-one adult New Zealand white rabbits (10 females and 11 males) weighing an average of 3.5 kg (range, 3.2-3.8 kg) who had been fed a regular rabbit chow ad libitum were our study population.

The intraarterial gamma-ray and beta-ray radiation source consisted of a 15-mm-long ¹⁹⁸Au gamma- and beta-ray-emitting wire (0.0578 g; half-life, 2.7 days; maximal beta energy, 0.961 MeV [99%]; average gamma energy, 0.412 MeV [96%]) with an outer diameter of 0.5 mm, obtained by activation under a thermal neutron flux—2.76 × 10¹³ neutrons × cm^-2 × sec^-1 for 5 min—in a nuclear reactor. The delivery system was a 2.9-French catheter (Balt, Montmorency, France; lumen diameter, 0.35 mm; wall thickness, 0.125 mm). The radiation source was pushed towards the occluded end of the catheter by a 0.021-inch (0.5-mm) stiffening guide wire (Terumo, Tokyo, Japan).

Dosimetric simulation was performed by computing the dose distribution from previous data on ¹⁹⁸Au brachytherapy [20]. The source (106 MBq) was modeled according to a beta-point source dose function [21]. The absorbed dose rate (DR) at a distance of x mm was obtained by the following formula: DRx = DRs × e^-μx, with DRs being the absorbed dose rate at the surface of the source and μ being the apparent absorption coefficient in water. The exponential function of (-μx) is expressed as e^-μx. The maximal range of beta particles for 1 MeV of energy is close to 4 mm in water. The radiation dose from gamma photons was considered to be negligible because of their weak average energy.

All procedures were performed by two operators under the control of a physicist. Each operator wore all usual tools of radiation protection. Thermoluminescent dosimeters, film dosimeters, and two ionization chambers were used. Dose rates, exact distances, and exposure times were noted. Because of the rapid dose fall-off of beta-ray emitters over distance, the dose resulting from beta particles is considered to be negligible beyond a distance of 80 cm (through air) [22]. The measurements performed beyond a 1-m distance, therefore, concerned only gamma and X rays.

We used a variant of a previously described endovascularly induced renal artery stenosis model [17]. In this variant, we used a femoral percutaneous approach and an 18-gauge needle to introduce a 0.021-inch (0.5-mm) guidewire and a 4-French sheath (Terumo) into the mid femoral artery. A 5-mm-long nonstenotic reference segment was preserved (Fig. 1A).

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Fig. 1A. —Left-sided renal artery endovascular brachytherapy procedure performed in rabbits is shown. Angiogram reveals bilateral renal artery stenoses (arrowheads).

After 7 weeks, the rabbits were treated with aspirin (5 mg/24 hr) for 48 hr before therapeutic percutaneous transluminal renal angioplasty to reduce the risk of thrombosis or ischemia [23]. After bilaterally induced stenotic lumen irregularities were identified in the renal arteries on angiograms, therapeutic percutaneous transluminal renal angioplasty was performed bilaterally in the renal arteries. A 2.0 × 20 mm coronary angioplasty balloon catheter was inflated to 10.1 × 10⁵ Pa for 30 sec at a site just beyond the prestenotic reference segment. The renal arteries were not deendothelialized at this step of the experiment. The location of the middle of the balloon was marked externally with a 23-gauge needle, which was inserted into the skin and remained in place until brachytherapy was performed (Fig. 1B). A second angiogram was then acquired to assess the immediate postprocedural patency of the renal artery.

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Fig. 1B. —Left-sided renal artery endovascular brachytherapy procedure performed in rabbits is shown. Radiograph obtained immediately after percutaneous transluminal renal angioplasty shows external landmark needle (asterisk) inserted in skin. Tip of needle indicates site of middle of dilatation balloon (arrow).

Irradiation was performed immediately after percutaneous transluminal renal angioplasty. During irradiation, the rabbits were placed in the supine position on the angiography table under a 1-cm-thick acrylic tunnel (beta-ray protection) and surrounded by 2-cm-thick lead screens (gamma-ray protection). The rabbits then were randomly divided into three groups—A, B, and C—immediately after therapeutic percutaneous transluminal renal angioplasty. The right renal artery was irradiated in group A and the left renal artery was irradiated in group B. Group C received a dummy wire, randomly placed in either the right or left side. In rabbits of groups A and B, the renal artery contralateral to wire placement served as a control artery. In group C, both renal arteries were controls. A 3.6-French curved carrier sheath (Cook, Bloomington, IN) was advanced into the renal artery selected for irradiation. The location of the delivery system was tracked with fluoroscopy so that the middle of the source wire was in exactly the same position as the middle of the balloon (which had been marked with the 23-gauge needle) (Fig. 1C). Therefore, only 15 mm of the 20-mm-long dilated portion of the artery was irradiated—that is, a 2.5-mm-long segment at each extremity of the dilated portion was less irradiated. The 0.7-mm-diameter endovascular brachytherapy delivery system did not become wedged into the dilated (2-mm diameter) renal arteries. We planned for all vessels to receive 25 Gy at a radial 2.0-mm depth. After irradiation, the endovascular access equipment was withdrawn, and the groin was manually compressed for 10 min. The rabbits were thereafter kept in individual housing on an aspirin-free diet.

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Fig. 1C. —Left-sided renal artery endovascular brachytherapy procedure performed in rabbits is shown. Radiograph obtained after withdrawal of carrier-sheath from renal artery during irradiation shows middle of gold-198 wire (arrowheads) is projected over needle tip. After irradiation, carrier-sheath is advanced beyond wire end. Complete delivery device may be withdrawn from rabbit via femoral 4-French introducer sheath.

The goal of the study was to evaluate the impact of irradiation on patent stenotic renal arteries treated by percutaneous transluminal renal angioplasty. After a 3-week period (representing the time interval to reach the maximal inflammatory response of the arterial wall after percutaneous transluminal renal angioplasty), the rabbits were deeply anesthetized by IV injection of 50 mg/kg of body weight of pentobarbital. The renal arteries were exposed by blunt dissection via a midline abdominal incision. The infrarenal aorta and vena cava were cannulated with a 14-gauge angiocatheter (Introcan; B. Braun, Melsungen, Germany). The supra- and infrarenal aorta and vena cava were isolated from the general circulation by ligation, and the renal veins were sectioned.

The sections were examined by two experienced observers unaware of which artery had been irradiated. The renal arteries and aortic local segments were perfusion-fixed via a flexible aortic angiocatheter at a driving pressure of 100 mm Hg with neutral buffered 10% solution of formaldehyde (Sigma Chemical, St. Louis, MO) for 60 min to fix expanded arteries in situ. The right kidney was marked by a sagittal cut. Perirenal aorta, renal arteries, and kidneys were then removed and immersion-fixed in the same formaldehyde fixative. The rabbits were killed by exsanguination at the suprarenal aorta.

Tissue harvesting and preparation.—In harvesting the tissue, we used an embedding technique. Each renal artery was cut into seven (right artery) or eight (left artery) 3- to 4-mm-long segments. An aorta-sided ink label indicated the proximal pole of the sample. Another similar baseline landmark represented the distal extremity of each segment. Each identified lesion of the renal artery was therefore localized by its distance in millimeters from the ostium. Four-micrometer-thick serial sections of renal artery were sampled transversely (one series of eight slices per 250-μm-long portion of each artery) and dehydrated in graded ethanol. Among the slices, 40 per renal artery represented the segment close to the middle of the balloon (i.e., the wire), and two represented the reference segment. Twenty slices and one reference segment were stained with H and E and safran for histologic study, and another 20 slices and one reference segment were stained with orcein for histomorphometric analysis. Each specimen was evaluated for the presence of neointimal formation, medial dissection, alteration of the internal and external elastic laminae, and morphologic appearance of the adventitial, medial, and neointimal layers. Sections were also evaluated for the presence of intraluminal thrombus, intraluminal hemorrhage, and inflammatory cells.

Histomorphometry.—Reference and stenotic segments of the irradiated and nonirradiated renal arteries were examined. Neointima was defined as any tissue layer circumscribed by the internal elastic lamina, excluding intramural deposits of fibrin as well as larger, organized mural thrombotic material. Other specific changes were defined as the reshaping of the medial layer, tearing of one of the elastic laminae, and the presence of adventitial fibrosis.

All transverse sections were digitized via a CCD video camera (Cohu, San Diego, CA) and then morphometrically analyzed. In each artery, measurements were performed in the proximal healthy reference slice and in 20 stenosed slices (Fig. 2). The extent of restenosis was determined as the percentage of area of restenosis that is eaual to 100% × (1 - stenosis lumen area/reference segment lumen area).

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Fig. 2. —Morphometric measurement of vessel area was obtained by tracing vessel perimeter, delineated by perimeter of external elastic lamina. A represents adventitial area. Perimeter of lumen was traced to define lumen area (L). Perimeter of internal elastic lamina allows definition of area of internal elastic lamina, total area of neointima (N), and L. Medial area (M) was obtained by difference derived from the following formula; vessel area - area of internal elastic lamina. N was obtained by difference derived from subtracting L from area of internal elastic lamina.

Renal artery restenosis was estimated by morphometric data, expressed as mean ± standard error of the mean. The one-way analysis of variance test was used to test for an overall treatment effect. We applied Wilcoxon's signed rank test assuming abnormal distribution between the comparative data after assessment of overall treatment effect. A p value less than 0.05 was required to reject the null hypothesis at the 95% confidence level. Statistical analysis was performed with StatView software (4.5 version; Abacus Concepts, Berkeley, CA).

Among the five rabbits in group C, two received a left-sided and three, a right-sided dummy wire, representing 10 control renal arteries. One rabbit was irradiated bilaterally. The total number of nonirradiated renal arteries for all three groups was 25; these arteries composed the overall control artery group. The overall rate of thrombosis after percutaneous transluminal renal angioplasty was not significantly different (p > 0.05) between the irradiated (n = 2, 11.7%) and control (n = 3, 12%) arteries. Fifteen irradiated and 22 nonirradiated patent arteries were available for morphometric analysis. Data are displayed in Table 1.

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TABLE 1 Comparison Between Renal Arteries of Rabbits Irradiated After Angioplasty and Nonirradiated Control Arteries

The right renal artery had a length of 30 ± 4 mm (mean ± SD), and the left renal artery had a length of 35 ± 4 mm. Forty-two renal arteries were cut into a series of 4-μm-thick slices. A total of 882 slices (20 slices + 1 reference segment × 42 renal arteries) of the portions close to the middle of the balloon (i.e., the wire) were analyzed. A variable degree of rupture of the internal elastic lamina and media was observed in injured segments of both control and beta-particle irradiated arteries, resulting in vessel wall irregularities. However, rupture of the internal elastic lamina was observed in each studied segment as a marker of overdilatation efficacy.

The presence or absence of the dummy wire induced no significant difference in histologic findings in control group arteries. Most sections of the dilated arteries (639/840, 76%) showed evidence of two or more fractures of the internal elastic lamina, with a variable mass of neointima associated with the various medial gaps. In irradiated arteries, the neointima was generally found to be a smooth thin tunic delineated by a thin fibrin layer with apparently moderately smooth muscle proliferation. In our model (one injury and one treatment by percutaneous transluminal renal angioplasty), the difference between nonirradiated and irradiated arteries relative to medial disorganization and adventitial fibrosis was not significant. When present, the cells of the neointima morphologically resembled cells in the control arteries.

Mural fibrinous deposits were observed in a small number of samples. Complete coverage of the luminal surface by a monolayer of endotheliallike cells was seen in all samples. Because the incidence of thrombosis did not differ between irradiated arteries and control arteries (p > 0.05), thrombosis was assumed to be unrelated to irradiation. Perivascular nerve fibers and adipose tissue appeared to be normal. The brachytherapy delivery system did not become wedged in the dilated renal arteries, so no ischemic renal damage was reported.

We evaluated 315 slices of irradiated arteries and 462 slices of nonirradiated arteries. The neointimal area of beta-irradiated arteries was much smaller than that of control arteries, with a few sections showing a total absence of neointimal formation (Table 2), whether the internal elastic lamina on the analyzed slices was ruptured or not. Because the measurements of the medial areas in one group did not differ significantly from the other group, irradiation seemed to have no apparent effect on preexisting remodeling.

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TABLE 2 Computer-Assisted Histomorphometric Analysis of ¹⁹⁸Au-Irradiated Rabbit Renal Arteries Versus Control Arteries (Orcein-Stained)

The duration of fluoroscopic exposure was 244 ± 82 sec. The duration of wire exposure was 63 ± 20 sec outside the rabbit and 30 ± 18 sec inside the rabbit during the source placement.

The whole-body dose was negligible. The first operator is closer to the source than the second operator, which may explain the difference in thermoluminescent dosimetric measurements for the two operators. The doses measured at wrist and finger were also low (Table 3). The presence of low-energy gamma rays remained undetectable by thoracic dosimeters.

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TABLE 3 Radioprotection Measurements

Performance of intraarterial brachytherapy to prevent postangioplastic restenosis by inhibiting the neointimal formation response to arterial injury has been described in coronary and iliofemoral arteries [25, 26]. Because ¹⁹⁸Au is a relatively low-energy gamma-ray emitter and high-energy beta-ray emitter, its use may allow brachytherapy to be safely performed in a regular interventional radiology facility.

Most published studies have evaluated the action of irradiation on the neointima. A1-though the favorable effect of radiation treatment on remodeling was previously reported in a swine coronary model [27], our results confirm preventive efficacy of treatment targeted to neointimal growth. These data are presumably related to the difficulty of separating recent posttreatment remodeling of the media from the initial postinduction remodeling. In the only study performed in the carotid arteries of rabbits [28], the internal elastic lamina remained intact, and no medial remodeling effect was investigated. Several ¹⁹²Ir or ⁹⁰Y irradiation experiments have been recently reported, using after-loader techniques or hand-delivery ribbons [9, 29]. Like ⁹⁰Y, the reference pure beta-ray emitter, ¹⁹⁸Au has a half-life of just 2.7 days and seems to be attractive in terms of safety. Because gold has no known metabolic toxicity, it does not need to be covered, in contrast with yttrium or strontium that must be isolated from the organism by titanium [9]. Our pilot study data suggested that the mechanical properties of gold, such as flexibility, facilitate advancement of the substance into curved arteries. The nonferromagnetic properties of gold could also make this element suitable for MR-guided percutaneous transluminal renal angioplasty [30] and related endovascular brachytherapeutic procedures in the near future.

For rabbits in the weight range of those in our study, the average diameter of a safe renal artery is 1.5 ± 0.2 mm [17]. A 2.0 × 20 mm balloon, therefore, induces an overdilatation of 33% (range, 20-50%). Under these conditions, the internal elastic lamina is ruptured in each artery by the induction procedure, but the fracture length is possibly shorter than the luminal area recovered by neointimal growth [13].

The irradiation protocol has to be followed by precise tissue harvesting. Highly accurate placement of the source into a 5-mm segment is achieved by carefully positioning the middle of the balloon and wire in the X-ray beam center of a small field of view. Irradiation efficacy is thus comparatively evaluated in the area of the middle of the wire, corresponding to the middle of the angioplasty balloon. In our study, the dilated portion of each renal artery was longer than the irradiated segment containing the delivery system to eliminate any risk of vessel obstruction. Although we knew that this technique was likely to spoil chances of further histomorphometric evaluation because of the edge effects it would produce, only the postirradiation evaluation of artery segments close to the middle region of the balloon (i.e., wire) were considered for morphometric analysis in this experimental study. In human clinical conditions, the likelihood is that having an irradiation device that is longer than the dilated portion could be helpful in eliminating this “candy wrapper” effect.

Because our model was developed to investigate a pure renal restenosis problem, our experimental results are relevant not to initial treatment but to restenosis after angioplasty or possibly after stenting. In fact, stenting may unfortunately be complicated by increased neointimal hyperplasia (an incidence rate of 20%). Although immunosuppressor-coated stents appear to be effective against short-term in-stent restenosis, endovascular brachytherapy seems to present favorable long-term outcome. Long-term studies are still required to validate stents coated with immunosuppressive substances [7].

Our model differs from a repeated-injury model previously developed using swine [31], the aim of which was to develop additional medial damage and marked intimal hyperplasia. In our study, the second percutaneous transluminal renal angioplasty performed was designed to be therapeutic rather than to create a second injury. However, our approach mimics clinical conditions: the response of a renal artery with preexisting deeper lesions of the arterial wall to recent therapeutic percutaneous transluminal renal angioplasty. The small diameter and thickness of the rabbit renal artery present fewer difficulties in terms of source centering than would be encountered in larger animals [9, 28].

Thrombosis was assessed at histology but was not angiographically detected either after the overdilatation—deendothelialization step of stenosis induction or after the therapeutic percutaneous transluminal renal angioplasty, although the presence of deendothelialized areas may constitute a risk factor for thrombosis. The goal of this study was to evaluate the impact of ¹⁹⁸Au irradiation after restenosis of patent renal arteries rather than to determine the incidence of thrombosis in our irradiated restenosis model, so the thrombosed arteries were not included in our morphometric analysis. However, in a recent study using a porcine model, although the overall rate of thrombosis increased dose-dependently from 0 to 18 Gy, the area of thrombus also decreased with increasing radiation dose. The data analysis suggested that intravascular brachytherapy could induce nonocclusive thrombotic foci [32].

The difficulty with intravascular irradiation is that it requires dosimetry and radioprotection. In the case of irradiation by stent, the duration of dose delivery theoretically is unlimited. It has not been clearly established whether permanent irradiation from a radio-active stent is damaging to the reendothelialization process, an essential condition for stent patency [33]. Moreover, stent deployment may induce edge failure or a “geographic miss” by barotrauma [34]. Thus, if ¹⁹⁸Au were used in stent form for irradiation, its half-life of 2.7 days could be safe in terms of vascular patency. Conversely, in-stent absorption caused by the reabsorption of charged beta particles by the radiation source may be found to be a limitation of radioactive stents with low radioactivity. Recent reports calculating dose distributions around ¹⁹⁸Au stents have emphasized that careful consideration should be given to the dramatic dose fall-off due to in-stent absorption when determining the initial activity level [11]. However, in vivo experimental evaluation of ¹⁹⁸Au stents is still required, and the potential dose fall-off can be eliminated by use of temporary wires, the initial activity of which is considerably higher than that of stents.

Two-centimeter-thick lead screens were initially used, because ¹⁹⁸Au is also a gammaray emitter. However, lead screens finally were found to be unnecessary because the gamma radiation dose was assessed as negligible. Surgical forceps were an essential part of operator safety because they increased the source-to-operator distance as did the acrylic screens, which increased the absorption of beta particles. Apart from providing all the advantages of beta irradiation [9, 13, 28], the use of a ¹⁹⁸Au wire provides an operator dose lower than that delivered by fluoroscopy. In our experience, the dose delivered by radioactive gold wire (0.0183 mSv) at 30 cm represents 72% of the dose delivered by fluoroscopy. Five procedures per day for an annual activity of 220 days would therefore deliver a maximal annual dose of approximately 48 mSv to the first operator, who is closer to the radiation source. The practical use of a predominantly beta-ray-emitting source does not require a bunker as gammaray emitters with high dose rates do. Gold-198 is actually a beta- and gamma-ray emitter with a low-energy gamma-ray-emitting component. Its use as a high-dose-rate beta-ray source in conventional catheterization facilities allows the irradiation dose to be delivered in approximately 10 min.

Even though the model mimics a renal stenotic artery feeding an ischemic kidney [17], it remains an incomplete treatment approach to such disease because of the variability of responses among species to angioplasty, procedure-related complications, and prevention methods. Nonatheromatous rabbits were used to test the efficacy of endovascular brachytherapy in a mechanically induced single-parameter model because intravascular radiotherapy has been shown to interfere with cholesterol metabolism [35]. Although the morphology of the lesions created in the rabbits differs from what is normally observed in human renal artery stenosis with regard to cholesterol overload, this nonatherosclerotic model eliminates the potential for heterogeneous dose distribution through calcified plaque. The difference between irradiated and nonirradiated arteries was assessed with only a 25-Gy dose delivered at a radial 2-mm depth in the irregular but thin walls of rabbit renal arteries. If targeting of the adventitial layer—which is the layer assumed to be responsible for late-stage constriction of the arterial wall [36]—is necessary in endovascular brachytherapy, no technical solution to improve centering is likely to completely prevent asymmetrical irradiation of grossly irregular artery tunics. Because of the small size of the artery, no centering was necessary in our study to show an antirestenosis effect. Therefore, initial activity should be adapted in human renal artery applications. The maximal inflammation of the arterial wall is reached 3 weeks after percutaneous transluminal renal angioplasty, so the end point we chose seems to be a reasonable period from which to draw short-term conclusions. Further studies should be planned to evaluate these experimental irradiated restenotic lesions with long-term follow-up.

The main objective of our experimental study was to compare stenotic renal arteries treated by percutaneous transluminal renal angioplasty and beta irradiation and arteries treated by percutaneous transluminal renal angioplasty alone. Our data show that ¹⁹⁸Au endovascular brachytherapy may offer a 33% reduction of renal artery restenosis in a rabbit model under extremely safe conditions of radiation protection. If these preliminary data could be extrapolated to, for example, patients with genetic risk factors for restenosis [37], renal artery ¹⁹⁸Au endovascular brachytherapy would probably constitute an attractive restenosis prevention technique.