- Email: [email protected]
Effect of transcatheter endovascular radiation with holmium-166 on neointimal formation after balloon injury in porcine coronary artery
Effect of transcatheter endovascular radiation with holmium-166 on neointimal formation after balloon injury in porcine coronary artery Han-Soo Kim, MD,a Yo-Han Cho, MD,a Jung-Sun Kim, MD,b Young-Taek Oh, MD,c Hae-Jin Kang, MD,c Mi-Sun Chun, MD,c Chul-Woo Joh, MD,d Chan-Hee Park, MD,d Kyung-Bae Park, PhD,e Seung-Jea Tahk, MD,a and Byung-il William Choi, MDa Background. Neointimal formation in response to arterial injury is a major contributing element in restenosis after coronary balloon angioplasty and stenting. Endovascular irradiation has been reported to be effective in reducing restenosis. The purpose of this study was to investigate the effect of beta-emitting holmium-166 for the inhibition of neointimal formation in porcine coronary artery. Methods and Results. A total of 34 pigs weighing 25 to 30 kg underwent oversized balloon injury (balloon/artery ratio, 1.3:1.4) at the proximal portion of the left anterior descending and circumflex arteries. One artery was randomly assigned to receive radiation after injury. Ho-166 was left in the balloon within the delivery catheter for a period sufficient to deliver 9 Gy and 18 Gy to a depth of 1 mm from the surface of the balloon. Four weeks later, pigs were sacrificed and hearts were perfusion-fixed, followed by histopathologic analysis and planimetry for measurement of maximal intimal thickness, intimal area, and fracture length. The coronary segment of the pigs in the control group had neointimal area of 1.18 ± 0.55 mm2; the pigs in the 9-Gy group had neointimal area of 0.68 ± 0.40 mm2 (P < .05 vs control); and the pigs in the 18-Gy group had neointimal area of 0.29 ± 0.12 mm2 (P < .01 vs control). The maximal intimal thickness in the 18Gy group (0.14 ± 0.11 mm) was significantly reduced compared with the maximal intimal thickness in the control group (0.48 ± 0.13 mm) (P < .01). Conclusions. Intracoronary radiation with liquid Ho-166 contained in a perfusion balloon catheter is feasible and effective in reducing neointimal formation after coronary overstretch injury in pigs. Therefore intracoronary irradiation on the injured segment may further reduce restenosis after balloon injury. (J Nucl Cardiol 2000;7:478-83.) Key Words: Radioisotopes • radiation • restenosis • neointimal formation
After successful percutaneous transluminal coronary angioplasty (PTCA) or stenting, restenosis, which results from an interaction of early elastic recoil, arterial remodeling, and neointimal formation, remains the main limitation of this technique.1-4 In addition, vessel injury after PTCA may induce hyperplastic response of smooth muscle cells within the vessel wall characterized by migration and proliferation.5 From the Departments of Cardiology,a Pathology,b Radiation Oncology,c and Nuclear Medicine,d Ajou University of School of Medicine, Suwon, and Korea Atomic Energy Research Institute,e Taejeon, Korea. Reprint requests: Byung-il William Choi, MD, Department of Cardiology, Ajou University of School of Medicine, WonchonDong, Paldal-Gu, Suwon, South Korea 442-721; bchoi@madang. ajou.ac.kr. Copyright © 2000 by the American Society of Nuclear Cardiology. 1071-3581/2000/$12.00 + 0 43/1/107427 doi:10.1067/mnc.2000.107427 478
Radiation may provide a potential modality to reduce neointimal formation by causing cell death during mitosis and limiting proliferation by reducing the number of regenerating clonal progenitors.6-9 The use of radiation for the treatment of benign proliferative disorders has been well-established as shown in low-dose external beam irradiation to inhibit keloid formation after surgery10-12 and heterotopic ossification after total hip arthroplasty.13-15 Holmium-166 is mainly beta-emitting radionuclide in liquid form with a maximum electron energy of 1.86 MeV and a half-life of 26.8 hours. There is also a fraction of gamma photon of 0.08 MeV (5.4%), which interacts with potassium iodide crystals, offering a potential use of this isotope as an imaging agent. The purpose of this study was to determine whether irradiation with Ho-166 contained in a perfusion balloon could inhibit neointimal proliferation and restenosis after balloon injury on a segment of porcine coronary artery.
Journal of Nuclear Cardiology Volume 7, Number 5;478-83
Kim et al Effect of holmium radiation on neointimal formation
A
479
B
Figure 1. Penetration depth of radiation was recorded on exposed film with 240 mCi/mL for 5 minutes (A), and isodose line in scaled image was recorded on videodensitometer (B) from the phantom containing a perfusion balloon.
METHODS Holmium 166 Properties and Dosimetry Ho-166 was produced by neutron reaction (Ho-165 (n,γ Ho-166) of naturally abundant nonradioactive Ho-165 at a thermal flux of 4.2 × 1013 n cm–2 s–1 for 60 hours in a reactor at the Korean Atomic Energy Research Institute. Specific activity of Ho-166 (Ho-166[NO3]3,5H2O) is 200 mCi/mg. For Ho-166 dosimetry, A GAFCHROMIC film (MD-55, 941206, Nuclear Associates, NY) with a hole in the center was positioned between 2 tissue-equivalent solid phantoms that had a canal in the center with a diameter of 3 mm to simulate the balloon catheter, which was 20 mm long and 3 mm in diameter when inflated. The balloon filled with Ho-166 nitrate solution was then placed inside the hole of the GAFCHROMIC film sandwiched by the phantoms. The radiation-exposed film and isodose line of the scaled image with videodensitometer are shown in Figure 1. Radial distribution of radiation dose in relation to distance (mm) from the balloon surface is shown in Figure 2.
Experimental Protocol This investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Thirty-four juvenile domestic pigs weighing 25 to 30 kg were given 200 mg of aspirin 1 day before and on the day of the procedure. The animals were sedated with a combination of ketamine (25 mg/kg) and xylazine hydrochloride (4 mg/kg) and atropine (0.6 mg/kg) by intramuscular injection. After an intravenous line at the posterior auricular vein was estabilished, general anesthesia was maintained with electrocardiographic monitoring by using an intravenous ketamine hydrochloride and midazolam infusion at a rate of 1 mg/kg and 1.5 mg/kg per hour, respectively. Each animal received a single dose of heparin (200 U/kg) after an 8-F sheath was placed retrograde in the right carotid artery. Under fluoroscopic guidance, an 8-F hockey-stick–guid-
ing catheter was positioned in the left coronary ostium. Coronary angiography with 45° left anterior oblique and 45° right anterior oblique views was performed after intracoronary administration of nitroglycerine (200 µg). Coronary overstretch injury was conducted with an angioplasty balloon (Scimed, Quantum Ranger, 15 mm in length) 30% larger than the reference vessel diameter, which was positioned in the proximal segments of the artery, inflated at 10 atm for 30 seconds 3 times, and interrupted by 1-minute deflation to restore coronary perfusion. The angioplasty balloon was withdrawn after the third inflation and additional nitroglycerin (200 µg) was administered to limit coronary spasm and was followed by the final angiogram. After injury, a coronary artery was randomly assigned to undergo radiation in either a segment of the left anterior descending or left circumflex artery with the other used as a control. For radiation treatment, a perfusion balloon catheter (ACS, Rx Flowtack, 20 mm in length) was positioned over a flexible wire to the injured artery by using the side branches as a landmark as a bolus of Ho-166 solution (~0.3 ml) containing a small amount of contrast was injected into the lumen of the perfusion catheter and inflated at 2 atm, enough to maintain the shape of balloon after cinefluoroscopic confirmation of the balloon catheter position. Liquid Ho-166 was left in the perfusion balloon for a period sufficient to deliver 9 Gy and 18 Gy to 1-mm depth from the surface of the balloon calculated by the dosimetry curve designed and validated in our laboratory (Figure 2). Dwelling time of the isotope was 160 ± 15 seconds for 9 Gy and 218 ± 16 seconds for 18 Gy. The dosage on the external surface of balloon contacting endothelium was around 27 Gy in the 9-Gy group and 54 Gy in the 18-Gy group, and the dosage for the external elastic lamina was around 15.3 Gy in the 9-Gy group and 30.6 Gy in the 18-Gy group. The other artery was assigned as a control and underwent only balloon injury. After balloon withdrawal, a coronary angiogram was performed in identical left anterior oblique and right anterior oblique views after 200 µg of nitroglycerin administration. The guiding catheter with a sheath was removed, the cutdown injury was coapted with stitches, and the animals were allowed to recover in the laboratory.
480
Kim et al Effect of holmium radiation on neointimal formation
Figure 2. Radial distribution of absorbed radiation and distance from the surface of the balloon containing Ho-166 with 3.0 mm in diameter and 20 mm in length.
Journal of Nuclear Cardiology September/October 2000
Morphometric analysis was performed on the section of each artery at the largest neointimal lesion by using a computerized image analyzing system (Leitz ASM68K). The maximal intimal thickness was determined by a line between the lumen and outermost point of the neointima. The arc length of the medial fracture was measured in the neointima from one dissected medial end to the other. Area measurements were obtained by tracing the lumen perimeter for luminal area (mm2), external elastic lamina for vessel area (mm2), and neointimal perimeter for intimal area (IA, mm2), which was defined by the borders of the internal elastic lamina, lumen, media, and external elastic lamina. The ratio of IA to arc length of the medial fracture was calculated to correct the extent of injury.
Statistical Analysis The animals underwent a follow-up coronary angiogram after 4 weeks from the initial overstretch injury in 45° left anterior oblique and right anterior oblique views similar to the initial study. After 200 U/kg of heparin infusion, the animals were sacrificed by an injection of concentrated potassium chloride. The heart was taken out immediately through left thoracotomy. The left main coronary artery was connected to a perfusion cannula, and the left coronary system was perfusion-fixed at 100 mm Hg, with 10% buffered formalin for 15 minutes.
Radiation Safety To minimize radiation exposure to the operator, the syringes used for radioisotope were placed in an acrylic betashielding syringe holder. This device shielded >90% of the dose to the fingers of the operator. All laboratory personnel wore 2 sets of disposable shoe covers, and physicians handling the isotope used double gloves. The syringe holding the radioisotope was attached to a luer-lock 3-way stopcock in the radiopharmacy to minimize possible ex vivo radioisotope leakage. Before opening the 3-way stopcock to the perfusion balloon, the inflation port of the balloon was attached to the stopcock and a vacuum was established in the balloon with the available free port. The entire perfusion inflation port, stopcock, and radioisotope syringe were placed in a sterile, shielding box. After the procedure, the balloon and guide wire were quickly withdrawn as a unit and placed directly into the radioactive waste box.
Tissue Preparation and Analysis The left anterior descending and left circumflex coronary arteries with 1 to 2 cm of normal segments just proximal and distal to the injured part were dissected for serial cross sections at every 2- to 3-mm length of the injured segment. The sections (4 µm thick) of coronary arterial wall were stained with hematoxylin-eosin and Verhoeff-van Gieson. Each specimen was evaluated for the presence of intimal proliferation, luminal encroachment, medial dissection, and alteration of the internal and external elastic lamina with a grading system based on criteria outlined by Schneider et al.16
All the data were expressed as mean ± standard deviation. A one-way analysis of variance was used to test for simultaneous comparisons of baseline, angiographic, and histopathologic characteristics between the control and 2 radiated groups. Morphometric measurements of the control and radiated groups were compared with unpaired Student t tests. A value of P < .05 was considered statistically significant.
RESULTS Balloon-induced injury of the coronary artery was carried out in 34 pigs. There were no significant differences in weight between the groups at the time of balloon injury. There was no procedural death or complication after the procedure. Histopathologic Analysis In injured segments of both control and irradiated arteries, the rupture of the internal elastic lamina with neointimal growth replacing the disrupted media was observed at 4 weeks after injury. The neointima was formed with stellate and spindle-shaped cells, which were modified smooth muscle cells, and loose extracellular matrix (Figure 3). The neointimal area from the irradiated arteries was markedly smaller in size than the controls. In some samples of the 18-Gy treated group, there was almost no neointima (Figure 3). There were no radiation-associated changes such as endothelial proliferations in small vessels or atypical fibroblasts in the adjacent adventitia, epicardial fat, or myocardium. There was no evidence of malignant and premalignant transformation in any cell types or infarction in the myocardial bed perfused by the treated vessel. Dissection was present in 30 (88%) of 34 vessels. The degree of injury assessed by Schneider’s histopathologic grading system16 did not differ between groups (P = not significant) (Table 1).
Journal of Nuclear Cardiology Volume 7, Number 5;478-83
Kim et al Effect of holmium radiation on neointimal formation
481
A
C
B
Figure 3. Representative micrographs (40 × instrument magnification) of histologic sections of pig coronary arteries with elastin staining at 4 weeks after injury. Samples from control group (A), 9 Gy-treated group (B), and 18 Gy-treated group (C).
Morphometry The extent of neointimal formation was determined in each group to find whether holmium radiation altered the response to injury. Pigs receiving 9 Gy and 18 Gy of radiation had less neointimal area as shown by the absolute IA than the control group (1.18 ± 0.55 mm2 in control, 0.68 ± 0.40 mm2 in 9-Gy group; P < .05 vs control; 0.29 ± 0.12 mm2 in 18-Gy group; P < .01 vs control). Maximal intimal thickness was significantly reduced in the 18-Gy group with 0.14 ± 0.11 mm compared with the control group with 0.48 ± 0.13 mm (P < .01) (Table 1). Thus holmium radiation caused 42% and 75% reductions in IA in the 9-Gy and 18-Gy groups, respectively. There was a significant reduction of IA (P < .015) and IA/fracture length (P < .007) in the treated group. However, no significant changes in vessel area of the groups were observed.
DISCUSSION This study indicates that intravascular irradiation with Ho-166 reduced the extent of intimal proliferation
after oversized balloon injury in a porcine model of coronary restenosis. The effect of holmium radiation was significant in reducing the neointimal area in the 9-Gy and 18-Gy treated group compared with the untreated control animals. A clear trend was noted in reducing the neointimal thickness in the 18-Gy group. Several groups with catheter-based systems have reported consistent findings on the efficacy of radiation in preventing restenosis. 17-21 Waksman et al17 demonstrated that both 7 and 14 Gy at 2-mm depth with iridium 192 showed a significant reduction of neointimal formation compared with the control group at 2 weeks after the injury. Verin et al18 delivered intraarterial radiation of yttrium 90 in an atherosclerotic rabbit model and demonstrated reduction in neointimal cells with 12 and 18 Gy. Gamma emitters can deliver a uniform dose to the arterial wall deeper than beta emitters. However, higher radiation exposure to both the patient and staff may require a specialized laboratory. Beta particles have a limited effective range in tissue, and 95% of them are absorbed within 3 to 4 mm of the tissue. Thus beta sources have an advantage in terms of less radiation
482
Kim et al Effect of holmium radiation on neointimal formation
Journal of Nuclear Cardiology September/October 2000
Table 1. Baseline characteristics and histomorphometric analysis of arteries harvested from pigs in the control and the radiation-treated groups
Control No. of arteries Balloon/artery ratio Injury score Vessel area (mm2) Maximal intimal thickness (mm) Intimal area (mm2) Fracture length (mm) Intimal area/fracture length ratio
1.35 1.75 3.85 0.48 1.18 2.50 0.48
34 ± 0.12 ± 0.13 ± 1.25 ± 0.13 ± 0.55 ± 1.25 ± 0.25
9 Gy
1.40 1.69 3.78 0.42 0.68 2.06 0.28
17 ± 0.09 ± 0.12 ± 1.20 ± 0.10 ± 0.40‡ ± 0.98 ± 0.19‡
18 Gy
1.38 1.79 4.25 0.14 0.29 2.34 0.12
17 ± 0.11 ± 0.15 ± 1.39 ± 0.11*† ± 0.12*† ± 1.34 ± 0.16*§
Values are mean ± standard deviation. *P < .01 vs control. †P < .01 vs 9 Gy. ‡P < .05 vs control. §P < .05 vs 9 Gy.
exposure that makes it easier to handle in a conventional catheterization laboratory without converting into a radiation therapy room. Several investigators have reported reduction in neointima formation after the use of radioactive liquidfilled balloons22-24 filled with rhenium 188, which is primarily a beta-emitter with a maximum energy of 2.12 MeV with a half-life of 16.9 hours, whereas maximum energy of Ho-166 is 1.86 MeV. Therefore the effective range of Re-188 is deeper than that of Ho-166. Weinberger et al22 studied the response to Re-188 with 25 Gy to a target point 0.5 mm in the vessel wall in a balloon overstretch model showing almost complete suppression of neointimal growth. Recently, copper 62, which has short half-life of 9.74 minutes and higher maximum energy of 2.93 MeV, was tested with successful inhibition of neointima.24 There are a number of safety issues regarding the liquid radioisotope system. To minimize the likelihood of ex vivo radioisotope leakage, careful liquid handling techniques should be used as described in the methods section. However, the mean energy level of exposure to the operators was less than 5 mrem per treatment, which is a tolerable range. Another potential issue would be intracoronary isotope leakage from possible balloon rupture, although it may not likely happen at 2 atm of intraballoon pressure. Based on the manufacturer’s specification, the expected balloon rupture rate may be less than 1 in 10,000 treatments, with nominal pressure of 6 to 10 atm. Our group used only minimal pressure of 2 atm, enough to maintain the shape of balloon. In addition, modification of the perfusion balloon by adding 1 or more layers would further protect the leakage of the isotope from possible rupture. Furthermore, chelation of isotope with
diethylenetriaminepentaacetic acid might be helpful to facilitate rapid renal excretion of radioisotope in the event of possible balloon rupture as a means of minimizing untoward radiation hazard. We thank Ms. Joo Eun Song for her editorial assistance.
References 1. Holmes DR Jr, Vlietstra RE, Smith HC, Vetrotec GW, Kent KM, Cowely MJ, et al. Restenosis after percutaneous transluminal coronary angioplasty (PTCA): a report from the PTCA registry of the National Heart, Lung, and Blood Institute. Am J Cardiol 1984;53:77C-81C. 2. Austin GE, Ratliff MB, Hollman J. Intimal proliferation of smooth muscle cells as an explanation for recurrent coronary artery stenosis after percutaneous transluminal coronary angioplasty. J Am Coll Cardiol 1985;6:369-75. 3. Liu MW, Roubin GS, King SB III. Restenosis after coronary angioplasty. Potential biologic determinants and role of intimal hyperplasia. Circulation 1989;79:1374-87. 4. Schwartz RS, Holmes DR, Topol EJ. The restenosis paradigm revisited: an alternative proposal for cellular mechanisms. J Am Coll Cardiol 1992;20:1284-93. 5. Forrester JS, Fishbein M, Helfant R, Fagin J. A paradigm for restenosis based on cell biology: clues for the development of new preventive therapies. J Am Coll Cardiol 1991;17:758-69. 6. Puck TT, Morkovin D, Marcus PI. Action of x-rays on mammalian cells, II: survival curves of cells from normal human tissues. J Exp Med 1957;106:485-500. 7. Sinclair W. Cyclic x-ray response in mammalian cells in vitro. Radiat Res 1968;63;620-43. 8. Fischer-Dzoga K, Dimitrievich GS, Griem ML. Differential radiosensitivity of aortic cells in vitro. Radiat Res 1984;99:536-46. 9. Fischer-Dzoga K, Dimitrievich GS, Schaffner T. Effect of hyperlipemic serum and irradiation on wound healing in primary quiescent cultures of vascular cells. Exp Mol Pathol 1989;52:1-12. 10. Enhamre A, Hammer H. Treatment of keloids with excision and postoperative x-ray irradiation. Dermatologica 1983;167:90-3.
Journal of Nuclear Cardiology Volume 7, Number 5;478-83
11. Kovalic JJ, Perez CA. Radiation therapy following keloidectomy: a 20year experience. Int J Radiat Oncol Biol Phys 1989;17:77-80. 12. Levy DS, Salter MM, Roth RE. Postoperative irradiation in the prevention of keloids. Am J Roentgenol 1976;127:509-10. 13. Ayers DC, Evarts CM, Parkinson JR. The prevention of heterotopic ossification in high-risk patients by low-dose radiation therapy after total hip arthroplasty. J Bone Joint Surg Am 1986;68:1423-30. 14. Anthony P, Keys H, Evarts CM. Prevention of heterotopic bone formation with early postoperative irradiation in high risk patients undergoing total hip arthroplasty: comparison of 10 Gy vs. 20 Gy schedules. Int J Radiat Oncol Biol Phys 1986;13:365-9. 15. Sylvester JE, Greenberg P, Selch MT. The use of postoperative irradiation for the prevention of heterotopic bone formation after total hip replacement. Int J Radiat Oncol Biol Phys 1988;14:471-6. 16. Schneider JE, Berk BC, Gravanis MB, Santoian EC, Cipolla GD, Tarazona N, et al. Probucol decreases neointimal formation in a swine model of coronary artery balloon injury. Circulation 1993;88:628-37. 17. Waksman R, Robinson KA, Crocker IR, Gravanis MB, Cipolla GD, King SB III. Endovascular low-dose irradiation inhibits neointima formation after coronary artery balloon injury in swine: a possible role for radiation therapy in restenosis prevention. Circulation 1995;91:1533-9. 18. Verin V, Popowski Y, Urban P, Belenger J, Redard M, Costa M, et al. Intra-arterial β-irradiation prevents neointimal hyperplasia in a hyper-
Kim et al Effect of holmium radiation on neointimal formation
19.
20.
21.
22.
23.
24.
483
cholesterolemic rabbit restenosis model. Circulation 1995;92: 2284-90. Waksman R, Robinson KA, Crocker IR, Wang C, Gravanis MB, Cipolla GD, et al. Intracoronary low-dose β-irradiation inhibits neointimal formation after coronary artery balloon injury in the swine restenosis model. Circulation 1995;92:3025-31. Wiederman JG, Marboe C, Schwartz A, Amols H, Weinberger J. Intracoronary irradiation markedly reduces restenosis after balloon angioplasty in a porcine model. J Am Coll Cardiol 1994;23:1491-8. Mazur W, Ali MN, Dabaghi SF, Cristead C, Abukhalil J, Paradise P, et al High dose rate intracoronary radiation suppresses neointimal proliferation in the stented and ballooned model of porcine restenosis. Circulation 1994;90(Suppl I):I-652. Weinberger J, Amols H, Ennis RD. Intracoronary irradiation: Dose response for the prevention of restenosis in swine. Int J Radiat Oncol Biol Phys 1996;36:767-75 Makkar R, Whiting J, Li A, Cordero H, Honda H, Frimerman A, et al. A beta-emitting liquid isotope filled balloon markedly inhibits restenosis in stented porcine coronary arteries [abstract]. J Am Coll Cardiol 1998;31:350. Waksman R, Chan R, Vodovotz Y, Bhargave B, Collins S, Cates PC, et al. A Cu-62 liquid filled balloon system inhibits neointima formation after balloon overstretch injury in porcine coronary arteries. Circulation 1999;100:I-517.
After successful percutaneous transluminal coronary angioplasty (PTCA) or stenting, restenosis, which results from an interaction of early elastic recoil, arterial remodeling, and neointimal formation, remains the main limitation of this technique.1-4 In addition, vessel injury after PTCA may induce hyperplastic response of smooth muscle cells within the vessel wall characterized by migration and proliferation.5 From the Departments of Cardiology,a Pathology,b Radiation Oncology,c and Nuclear Medicine,d Ajou University of School of Medicine, Suwon, and Korea Atomic Energy Research Institute,e Taejeon, Korea. Reprint requests: Byung-il William Choi, MD, Department of Cardiology, Ajou University of School of Medicine, WonchonDong, Paldal-Gu, Suwon, South Korea 442-721; bchoi@madang. ajou.ac.kr. Copyright © 2000 by the American Society of Nuclear Cardiology. 1071-3581/2000/$12.00 + 0 43/1/107427 doi:10.1067/mnc.2000.107427 478
Radiation may provide a potential modality to reduce neointimal formation by causing cell death during mitosis and limiting proliferation by reducing the number of regenerating clonal progenitors.6-9 The use of radiation for the treatment of benign proliferative disorders has been well-established as shown in low-dose external beam irradiation to inhibit keloid formation after surgery10-12 and heterotopic ossification after total hip arthroplasty.13-15 Holmium-166 is mainly beta-emitting radionuclide in liquid form with a maximum electron energy of 1.86 MeV and a half-life of 26.8 hours. There is also a fraction of gamma photon of 0.08 MeV (5.4%), which interacts with potassium iodide crystals, offering a potential use of this isotope as an imaging agent. The purpose of this study was to determine whether irradiation with Ho-166 contained in a perfusion balloon could inhibit neointimal proliferation and restenosis after balloon injury on a segment of porcine coronary artery.
Journal of Nuclear Cardiology Volume 7, Number 5;478-83
Kim et al Effect of holmium radiation on neointimal formation
A
479
B
Figure 1. Penetration depth of radiation was recorded on exposed film with 240 mCi/mL for 5 minutes (A), and isodose line in scaled image was recorded on videodensitometer (B) from the phantom containing a perfusion balloon.
METHODS Holmium 166 Properties and Dosimetry Ho-166 was produced by neutron reaction (Ho-165 (n,γ Ho-166) of naturally abundant nonradioactive Ho-165 at a thermal flux of 4.2 × 1013 n cm–2 s–1 for 60 hours in a reactor at the Korean Atomic Energy Research Institute. Specific activity of Ho-166 (Ho-166[NO3]3,5H2O) is 200 mCi/mg. For Ho-166 dosimetry, A GAFCHROMIC film (MD-55, 941206, Nuclear Associates, NY) with a hole in the center was positioned between 2 tissue-equivalent solid phantoms that had a canal in the center with a diameter of 3 mm to simulate the balloon catheter, which was 20 mm long and 3 mm in diameter when inflated. The balloon filled with Ho-166 nitrate solution was then placed inside the hole of the GAFCHROMIC film sandwiched by the phantoms. The radiation-exposed film and isodose line of the scaled image with videodensitometer are shown in Figure 1. Radial distribution of radiation dose in relation to distance (mm) from the balloon surface is shown in Figure 2.
Experimental Protocol This investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Thirty-four juvenile domestic pigs weighing 25 to 30 kg were given 200 mg of aspirin 1 day before and on the day of the procedure. The animals were sedated with a combination of ketamine (25 mg/kg) and xylazine hydrochloride (4 mg/kg) and atropine (0.6 mg/kg) by intramuscular injection. After an intravenous line at the posterior auricular vein was estabilished, general anesthesia was maintained with electrocardiographic monitoring by using an intravenous ketamine hydrochloride and midazolam infusion at a rate of 1 mg/kg and 1.5 mg/kg per hour, respectively. Each animal received a single dose of heparin (200 U/kg) after an 8-F sheath was placed retrograde in the right carotid artery. Under fluoroscopic guidance, an 8-F hockey-stick–guid-
ing catheter was positioned in the left coronary ostium. Coronary angiography with 45° left anterior oblique and 45° right anterior oblique views was performed after intracoronary administration of nitroglycerine (200 µg). Coronary overstretch injury was conducted with an angioplasty balloon (Scimed, Quantum Ranger, 15 mm in length) 30% larger than the reference vessel diameter, which was positioned in the proximal segments of the artery, inflated at 10 atm for 30 seconds 3 times, and interrupted by 1-minute deflation to restore coronary perfusion. The angioplasty balloon was withdrawn after the third inflation and additional nitroglycerin (200 µg) was administered to limit coronary spasm and was followed by the final angiogram. After injury, a coronary artery was randomly assigned to undergo radiation in either a segment of the left anterior descending or left circumflex artery with the other used as a control. For radiation treatment, a perfusion balloon catheter (ACS, Rx Flowtack, 20 mm in length) was positioned over a flexible wire to the injured artery by using the side branches as a landmark as a bolus of Ho-166 solution (~0.3 ml) containing a small amount of contrast was injected into the lumen of the perfusion catheter and inflated at 2 atm, enough to maintain the shape of balloon after cinefluoroscopic confirmation of the balloon catheter position. Liquid Ho-166 was left in the perfusion balloon for a period sufficient to deliver 9 Gy and 18 Gy to 1-mm depth from the surface of the balloon calculated by the dosimetry curve designed and validated in our laboratory (Figure 2). Dwelling time of the isotope was 160 ± 15 seconds for 9 Gy and 218 ± 16 seconds for 18 Gy. The dosage on the external surface of balloon contacting endothelium was around 27 Gy in the 9-Gy group and 54 Gy in the 18-Gy group, and the dosage for the external elastic lamina was around 15.3 Gy in the 9-Gy group and 30.6 Gy in the 18-Gy group. The other artery was assigned as a control and underwent only balloon injury. After balloon withdrawal, a coronary angiogram was performed in identical left anterior oblique and right anterior oblique views after 200 µg of nitroglycerin administration. The guiding catheter with a sheath was removed, the cutdown injury was coapted with stitches, and the animals were allowed to recover in the laboratory.
480
Kim et al Effect of holmium radiation on neointimal formation
Figure 2. Radial distribution of absorbed radiation and distance from the surface of the balloon containing Ho-166 with 3.0 mm in diameter and 20 mm in length.
Journal of Nuclear Cardiology September/October 2000
Morphometric analysis was performed on the section of each artery at the largest neointimal lesion by using a computerized image analyzing system (Leitz ASM68K). The maximal intimal thickness was determined by a line between the lumen and outermost point of the neointima. The arc length of the medial fracture was measured in the neointima from one dissected medial end to the other. Area measurements were obtained by tracing the lumen perimeter for luminal area (mm2), external elastic lamina for vessel area (mm2), and neointimal perimeter for intimal area (IA, mm2), which was defined by the borders of the internal elastic lamina, lumen, media, and external elastic lamina. The ratio of IA to arc length of the medial fracture was calculated to correct the extent of injury.
Statistical Analysis The animals underwent a follow-up coronary angiogram after 4 weeks from the initial overstretch injury in 45° left anterior oblique and right anterior oblique views similar to the initial study. After 200 U/kg of heparin infusion, the animals were sacrificed by an injection of concentrated potassium chloride. The heart was taken out immediately through left thoracotomy. The left main coronary artery was connected to a perfusion cannula, and the left coronary system was perfusion-fixed at 100 mm Hg, with 10% buffered formalin for 15 minutes.
Radiation Safety To minimize radiation exposure to the operator, the syringes used for radioisotope were placed in an acrylic betashielding syringe holder. This device shielded >90% of the dose to the fingers of the operator. All laboratory personnel wore 2 sets of disposable shoe covers, and physicians handling the isotope used double gloves. The syringe holding the radioisotope was attached to a luer-lock 3-way stopcock in the radiopharmacy to minimize possible ex vivo radioisotope leakage. Before opening the 3-way stopcock to the perfusion balloon, the inflation port of the balloon was attached to the stopcock and a vacuum was established in the balloon with the available free port. The entire perfusion inflation port, stopcock, and radioisotope syringe were placed in a sterile, shielding box. After the procedure, the balloon and guide wire were quickly withdrawn as a unit and placed directly into the radioactive waste box.
Tissue Preparation and Analysis The left anterior descending and left circumflex coronary arteries with 1 to 2 cm of normal segments just proximal and distal to the injured part were dissected for serial cross sections at every 2- to 3-mm length of the injured segment. The sections (4 µm thick) of coronary arterial wall were stained with hematoxylin-eosin and Verhoeff-van Gieson. Each specimen was evaluated for the presence of intimal proliferation, luminal encroachment, medial dissection, and alteration of the internal and external elastic lamina with a grading system based on criteria outlined by Schneider et al.16
All the data were expressed as mean ± standard deviation. A one-way analysis of variance was used to test for simultaneous comparisons of baseline, angiographic, and histopathologic characteristics between the control and 2 radiated groups. Morphometric measurements of the control and radiated groups were compared with unpaired Student t tests. A value of P < .05 was considered statistically significant.
RESULTS Balloon-induced injury of the coronary artery was carried out in 34 pigs. There were no significant differences in weight between the groups at the time of balloon injury. There was no procedural death or complication after the procedure. Histopathologic Analysis In injured segments of both control and irradiated arteries, the rupture of the internal elastic lamina with neointimal growth replacing the disrupted media was observed at 4 weeks after injury. The neointima was formed with stellate and spindle-shaped cells, which were modified smooth muscle cells, and loose extracellular matrix (Figure 3). The neointimal area from the irradiated arteries was markedly smaller in size than the controls. In some samples of the 18-Gy treated group, there was almost no neointima (Figure 3). There were no radiation-associated changes such as endothelial proliferations in small vessels or atypical fibroblasts in the adjacent adventitia, epicardial fat, or myocardium. There was no evidence of malignant and premalignant transformation in any cell types or infarction in the myocardial bed perfused by the treated vessel. Dissection was present in 30 (88%) of 34 vessels. The degree of injury assessed by Schneider’s histopathologic grading system16 did not differ between groups (P = not significant) (Table 1).
Journal of Nuclear Cardiology Volume 7, Number 5;478-83
Kim et al Effect of holmium radiation on neointimal formation
481
A
C
B
Figure 3. Representative micrographs (40 × instrument magnification) of histologic sections of pig coronary arteries with elastin staining at 4 weeks after injury. Samples from control group (A), 9 Gy-treated group (B), and 18 Gy-treated group (C).
Morphometry The extent of neointimal formation was determined in each group to find whether holmium radiation altered the response to injury. Pigs receiving 9 Gy and 18 Gy of radiation had less neointimal area as shown by the absolute IA than the control group (1.18 ± 0.55 mm2 in control, 0.68 ± 0.40 mm2 in 9-Gy group; P < .05 vs control; 0.29 ± 0.12 mm2 in 18-Gy group; P < .01 vs control). Maximal intimal thickness was significantly reduced in the 18-Gy group with 0.14 ± 0.11 mm compared with the control group with 0.48 ± 0.13 mm (P < .01) (Table 1). Thus holmium radiation caused 42% and 75% reductions in IA in the 9-Gy and 18-Gy groups, respectively. There was a significant reduction of IA (P < .015) and IA/fracture length (P < .007) in the treated group. However, no significant changes in vessel area of the groups were observed.
DISCUSSION This study indicates that intravascular irradiation with Ho-166 reduced the extent of intimal proliferation
after oversized balloon injury in a porcine model of coronary restenosis. The effect of holmium radiation was significant in reducing the neointimal area in the 9-Gy and 18-Gy treated group compared with the untreated control animals. A clear trend was noted in reducing the neointimal thickness in the 18-Gy group. Several groups with catheter-based systems have reported consistent findings on the efficacy of radiation in preventing restenosis. 17-21 Waksman et al17 demonstrated that both 7 and 14 Gy at 2-mm depth with iridium 192 showed a significant reduction of neointimal formation compared with the control group at 2 weeks after the injury. Verin et al18 delivered intraarterial radiation of yttrium 90 in an atherosclerotic rabbit model and demonstrated reduction in neointimal cells with 12 and 18 Gy. Gamma emitters can deliver a uniform dose to the arterial wall deeper than beta emitters. However, higher radiation exposure to both the patient and staff may require a specialized laboratory. Beta particles have a limited effective range in tissue, and 95% of them are absorbed within 3 to 4 mm of the tissue. Thus beta sources have an advantage in terms of less radiation
482
Kim et al Effect of holmium radiation on neointimal formation
Journal of Nuclear Cardiology September/October 2000
Table 1. Baseline characteristics and histomorphometric analysis of arteries harvested from pigs in the control and the radiation-treated groups
Control No. of arteries Balloon/artery ratio Injury score Vessel area (mm2) Maximal intimal thickness (mm) Intimal area (mm2) Fracture length (mm) Intimal area/fracture length ratio
1.35 1.75 3.85 0.48 1.18 2.50 0.48
34 ± 0.12 ± 0.13 ± 1.25 ± 0.13 ± 0.55 ± 1.25 ± 0.25
9 Gy
1.40 1.69 3.78 0.42 0.68 2.06 0.28
17 ± 0.09 ± 0.12 ± 1.20 ± 0.10 ± 0.40‡ ± 0.98 ± 0.19‡
18 Gy
1.38 1.79 4.25 0.14 0.29 2.34 0.12
17 ± 0.11 ± 0.15 ± 1.39 ± 0.11*† ± 0.12*† ± 1.34 ± 0.16*§
Values are mean ± standard deviation. *P < .01 vs control. †P < .01 vs 9 Gy. ‡P < .05 vs control. §P < .05 vs 9 Gy.
exposure that makes it easier to handle in a conventional catheterization laboratory without converting into a radiation therapy room. Several investigators have reported reduction in neointima formation after the use of radioactive liquidfilled balloons22-24 filled with rhenium 188, which is primarily a beta-emitter with a maximum energy of 2.12 MeV with a half-life of 16.9 hours, whereas maximum energy of Ho-166 is 1.86 MeV. Therefore the effective range of Re-188 is deeper than that of Ho-166. Weinberger et al22 studied the response to Re-188 with 25 Gy to a target point 0.5 mm in the vessel wall in a balloon overstretch model showing almost complete suppression of neointimal growth. Recently, copper 62, which has short half-life of 9.74 minutes and higher maximum energy of 2.93 MeV, was tested with successful inhibition of neointima.24 There are a number of safety issues regarding the liquid radioisotope system. To minimize the likelihood of ex vivo radioisotope leakage, careful liquid handling techniques should be used as described in the methods section. However, the mean energy level of exposure to the operators was less than 5 mrem per treatment, which is a tolerable range. Another potential issue would be intracoronary isotope leakage from possible balloon rupture, although it may not likely happen at 2 atm of intraballoon pressure. Based on the manufacturer’s specification, the expected balloon rupture rate may be less than 1 in 10,000 treatments, with nominal pressure of 6 to 10 atm. Our group used only minimal pressure of 2 atm, enough to maintain the shape of balloon. In addition, modification of the perfusion balloon by adding 1 or more layers would further protect the leakage of the isotope from possible rupture. Furthermore, chelation of isotope with
diethylenetriaminepentaacetic acid might be helpful to facilitate rapid renal excretion of radioisotope in the event of possible balloon rupture as a means of minimizing untoward radiation hazard. We thank Ms. Joo Eun Song for her editorial assistance.
References 1. Holmes DR Jr, Vlietstra RE, Smith HC, Vetrotec GW, Kent KM, Cowely MJ, et al. Restenosis after percutaneous transluminal coronary angioplasty (PTCA): a report from the PTCA registry of the National Heart, Lung, and Blood Institute. Am J Cardiol 1984;53:77C-81C. 2. Austin GE, Ratliff MB, Hollman J. Intimal proliferation of smooth muscle cells as an explanation for recurrent coronary artery stenosis after percutaneous transluminal coronary angioplasty. J Am Coll Cardiol 1985;6:369-75. 3. Liu MW, Roubin GS, King SB III. Restenosis after coronary angioplasty. Potential biologic determinants and role of intimal hyperplasia. Circulation 1989;79:1374-87. 4. Schwartz RS, Holmes DR, Topol EJ. The restenosis paradigm revisited: an alternative proposal for cellular mechanisms. J Am Coll Cardiol 1992;20:1284-93. 5. Forrester JS, Fishbein M, Helfant R, Fagin J. A paradigm for restenosis based on cell biology: clues for the development of new preventive therapies. J Am Coll Cardiol 1991;17:758-69. 6. Puck TT, Morkovin D, Marcus PI. Action of x-rays on mammalian cells, II: survival curves of cells from normal human tissues. J Exp Med 1957;106:485-500. 7. Sinclair W. Cyclic x-ray response in mammalian cells in vitro. Radiat Res 1968;63;620-43. 8. Fischer-Dzoga K, Dimitrievich GS, Griem ML. Differential radiosensitivity of aortic cells in vitro. Radiat Res 1984;99:536-46. 9. Fischer-Dzoga K, Dimitrievich GS, Schaffner T. Effect of hyperlipemic serum and irradiation on wound healing in primary quiescent cultures of vascular cells. Exp Mol Pathol 1989;52:1-12. 10. Enhamre A, Hammer H. Treatment of keloids with excision and postoperative x-ray irradiation. Dermatologica 1983;167:90-3.
Journal of Nuclear Cardiology Volume 7, Number 5;478-83
11. Kovalic JJ, Perez CA. Radiation therapy following keloidectomy: a 20year experience. Int J Radiat Oncol Biol Phys 1989;17:77-80. 12. Levy DS, Salter MM, Roth RE. Postoperative irradiation in the prevention of keloids. Am J Roentgenol 1976;127:509-10. 13. Ayers DC, Evarts CM, Parkinson JR. The prevention of heterotopic ossification in high-risk patients by low-dose radiation therapy after total hip arthroplasty. J Bone Joint Surg Am 1986;68:1423-30. 14. Anthony P, Keys H, Evarts CM. Prevention of heterotopic bone formation with early postoperative irradiation in high risk patients undergoing total hip arthroplasty: comparison of 10 Gy vs. 20 Gy schedules. Int J Radiat Oncol Biol Phys 1986;13:365-9. 15. Sylvester JE, Greenberg P, Selch MT. The use of postoperative irradiation for the prevention of heterotopic bone formation after total hip replacement. Int J Radiat Oncol Biol Phys 1988;14:471-6. 16. Schneider JE, Berk BC, Gravanis MB, Santoian EC, Cipolla GD, Tarazona N, et al. Probucol decreases neointimal formation in a swine model of coronary artery balloon injury. Circulation 1993;88:628-37. 17. Waksman R, Robinson KA, Crocker IR, Gravanis MB, Cipolla GD, King SB III. Endovascular low-dose irradiation inhibits neointima formation after coronary artery balloon injury in swine: a possible role for radiation therapy in restenosis prevention. Circulation 1995;91:1533-9. 18. Verin V, Popowski Y, Urban P, Belenger J, Redard M, Costa M, et al. Intra-arterial β-irradiation prevents neointimal hyperplasia in a hyper-
Kim et al Effect of holmium radiation on neointimal formation
19.
20.
21.
22.
23.
24.
483
cholesterolemic rabbit restenosis model. Circulation 1995;92: 2284-90. Waksman R, Robinson KA, Crocker IR, Wang C, Gravanis MB, Cipolla GD, et al. Intracoronary low-dose β-irradiation inhibits neointimal formation after coronary artery balloon injury in the swine restenosis model. Circulation 1995;92:3025-31. Wiederman JG, Marboe C, Schwartz A, Amols H, Weinberger J. Intracoronary irradiation markedly reduces restenosis after balloon angioplasty in a porcine model. J Am Coll Cardiol 1994;23:1491-8. Mazur W, Ali MN, Dabaghi SF, Cristead C, Abukhalil J, Paradise P, et al High dose rate intracoronary radiation suppresses neointimal proliferation in the stented and ballooned model of porcine restenosis. Circulation 1994;90(Suppl I):I-652. Weinberger J, Amols H, Ennis RD. Intracoronary irradiation: Dose response for the prevention of restenosis in swine. Int J Radiat Oncol Biol Phys 1996;36:767-75 Makkar R, Whiting J, Li A, Cordero H, Honda H, Frimerman A, et al. A beta-emitting liquid isotope filled balloon markedly inhibits restenosis in stented porcine coronary arteries [abstract]. J Am Coll Cardiol 1998;31:350. Waksman R, Chan R, Vodovotz Y, Bhargave B, Collins S, Cates PC, et al. A Cu-62 liquid filled balloon system inhibits neointima formation after balloon overstretch injury in porcine coronary arteries. Circulation 1999;100:I-517.