Research Of Molecular Nuclear Medicine For Atherosclerosis Imaging Study In Experimental Rabbits

Objective Targeting different aspects of cell proliferation, apoptosis and metabolism within the ahterosclerotic lesions, three kinds of molecular imaging methods, which included antisense imaging using ^99mTc-HYNIC-PCNA ASON SPECT, apoptosis imaging using ^99mTc-HYNIC-annexin V SPECT and metabolism imaging with ¹⁸F-FDG PET/CT, were performed respectively, to assess the feasibility, value and prospect of the application of the molecular nuclear medicine in the identification of the atherosclerotic plaquesMethods The experimental atherosclerotic rabbits were chosen as the object of the present study, using the combined method of vascular immunologic injury and high fat diet. ^99mTc-HYNIC-PCNA ASON SPECT, ^99mTc-HYNIC-annexin V SPECT and ¹⁸F-FDG PET/CT were performed respectively. Soon after the imaging, the atherosclerotic aorta were dissected for radioactivity detection, and pathological and immunohistochemical stainning. The relationship between the uptake of radiopharceuticals in the vascular segments and the content of the compound in the atherosclerotic lesions was evaluated by regression analysis.1.Induction and identification of Experimental Atherosclerosis in RabbitsMale Japanese white rabbits were obtained from the Experimental Animal Center in Tongji Medical College affiliated to Huazhong University of Science and Technology. They were fed with high-fat diet for about 12 weeks after the immunotic injury of the vascular by intravenous injection of bovine serum albumin via the ear vein (250mg/kg). The high-fat diet was prepared by adding 1% cholesterol, 5% grease, 5% yolk and 0.1% methylthiouracil. Total serum cholesterol levels were measured by standard enzymatic techniques at the beginning of the study and every 4 weeks from then on. Another group of 4 rabbits were fed with standard chow as normal control.2.Research on the scintigraphy of atherosclerotic lesions using ^99mTc-HYNIC-PCNA antisense oligonucleotide in experimental rabbitsTwelve male Japanese rabbits received immune injury of their arteries combined with about 12 weeks of high-fat diet underwent ^99mTc-HYNIC-PCNA oligonucleotide scintigraphy, including planar and tomographic images. Eight of them were injected ^99mTc-PCNA ASON (antisense oligonucleotide) and the rest were injected ^99mTc-PCNA SON (sense oligonucleotide) as control. In addition, four rabbits with normal chow were injected ^99mTc-ASON as normal control. Competitive blockage images were performed in 4 rabbits of ASON group that were injected unlabeled ASON 2h before the administration of radiolabeled ASON. The whole aortic arteries were dissected for ex vivo radioscan and were cut into segments for radioactivity detection. And the tissue sections were performed hematoxylin and eosin staining to demonstrate the general architecture and immunostaining of RAM11,α-SMA and PCNA to identify the burden of macrophage cells, VSMCs and proliferating cell nuclear antigen (PCNA) within the lesions. In addition, the expression of PCNA was detected by RT-PCR and Western Blotting. Finally, the relationship between the uptake of the radiolabled antisense probe and the content of PCNA, VSMCs and macrophages in the lesion was analyzed respectively.3.Research on the ^99mTc-HYNIC-annexin V imaging of atherosclerotic plaques in experimental rabbitsFive Japanese male rabbits, which received immune injury of arteries combined with 12 weeks of high-fat diet, underwent scintigraphy after intravenous injection of ^99mTc-HYNIC-annexin V (37MBq/kg). Another 5 rabbits without any manipulation were studied as controls. The aortas of the atherosclerotic rabbits were explanted for ex vivo imaging and then cut into segments for radioactivity detection and histological characterization of the lesions. 4.Imaging vulnerable atherosclerotic plaque with ¹⁸F-FDG PET/CT in experimental rabbitsThe subject concluded 5 male Japanese rabbits received immune injury of their arteries combined with about 12 weeks of high-fat diet and 5 rabbits dealed with normal chow as control. All the animals were performed ¹⁸F-FDG PET immediately after the CT scans, at the 1st, 2nd and 3rd hour after the injection of ¹⁸F-FDG. The parameter of CT scans were 80 kV,30mA,and 5mm-thick transaxial CT images were reconstructed at 4.25mm intervals to fuse with the transaxial PET images. Then, the whole aortic arteries were dissected and were cut into segments for radioactivity detection. In addition, the tissue sections were performed hematoxylin and eosin staining to demonstrate the general architecture and immunostaining of RAM11 to identify the content of macrophage cells within the plaques. Finally, the relationship between the uptake of ¹⁸F-FDG and the burden of macrophages within the lesion was analyzed by regression analysis.Results1. Identification of the experimetal atherosclerotic rabbitsThe blood cholesterol level of the rabbits increased gradually from the beginning of the study, to a high cholesterol level at the end of the 12th week after the high-fat diet respectively.Gross examinations revealed that white or light yellow lesions of various sizes and thickness located on the lining endothelium of the aortas, lamellar on the arch predominantly and scattered on the thoracic and upper abdominal aortic.Atherosclerotic lesions were characterized using a classification scheme based on the recommendations of the American Heart Association (AHA): AHA type I, early change with diffused distribution of the independent foam cells at the surface of intima. AHA type II, also referred to as fatty streaks or intimal xanthomas, were plaques consisting of macrophage-derived intimal foam cells with SMC and extracellular matrix. AHA type III lesions,or pathologic intimal thickening, were lesions with focal acellular areas containing extracellular lipid pools in between SMCs and proteoglycans. AHA type IV lesions, also called fibrous cap atheromas, were plaques containing a necrotic core with prominent cholesterol clefts and overlying fibrous cap; occasionally the core region showed calcification within the deep intimal layers. AHA type V lesions were charcterized by large lipid core and serious fibrous, sometimes with calcification of various degree and it were divided into three subtypes according to the major ingredient of the plaques: Va(fiber and lipid), Vb(calcification) and Vc (collagen). AHA type IV lesions were complex lesions with decreased cell desity, some debris and lipid. It were also divided into three subtypes including rupture or ulcer of plaques (VIa), intrmural hematoma or bleeding of plaques (VIb) and thrombosis (VIc).In the present study, the histopathological results exhibited thickening intima of the vessel wall with the accumulation of foam cells, hyperplastic smooth muscle cells, some lip pools and even fibrous cap, without any bleeding or thrombi, predominantly of American Heart Association class II^IV. No bleeding, rupture or thrombosis of the atherosclerotic plaques were observed in this study.Immunohistochemical analysis demonstrated thatα-SMA positive VSMCs occupied the peripheral part and a large number of RAM11 positive macrophages located mostly in the shoulder and base region of the plaques. PCNA immunostaining showed focal proliferating cells both in the macrophage-rich area and VSMCs. And TUNEL results suggested almost all of the positive cells distributed in the plaque tissues.2. ^99mTc-HYNIC-PCNA ASON imaging resultsThe ^99mTc radiolabeling efficiency of the ASON and SON were (64.13±4.06)% and (63.87±4.26)%, respectively. And both acquired high radiochemical purity over 90%. Only the ^99mTc-HYNIC-PCNA ASON images of the rabbits in ASON group showed distinct focal uptake of the radionuclide in the experimental atherosclerotic aorta 2h after the injection, and no uptake was observed in the SON group, normal group and the competitive blockage images. The ex vivo imaging of the arteries revealed a good consistency with the tracer accumulation in vivo. The percentage of injected dose per gram of the vascular segments in ASON group (0.033±0.011) was much higher than the control groups(SON group, 0.013±0.004,F=106.38,P<0.001; normal group, 0.014±0.004,F=96.91,P<0.001). The biodistribution of the radiotracer in the non-target organs at 2h showed maximum radioactivity in the kidney (0.047±0.006), followed by the liver (0.023±0.003)and the spleen(0.019±0.003). The uptake of the tracer was correlated significantly with the PCNA content, by both PCNA index (r=0.69, P<0.001) and its protein density (r=0.78,P<0.001). Moreover, the uptake of the radiotracer showed an excellent correlation with the macrophage content (r= 0.59, P <0.001) and well correlation with the burden of VSMCs within the plaques (r=0.54, P<0.001) by regression analysis.3. ^99mTc-HYNIC-annexin V imaging resultsThe ^99mTc radiolabeling efficiency of HYNIC-annexin V was (96.32±2.08)% and high radiochemical purity over 90% were acquired There was intense uptake of the radionuclide in the lesions of atherosclerotic rabbits while no uptake was seen in the controls at 2h after injection of the radiopharmaceutical, and the count ratio of the target to non-target was significantly higher than that of the controls (2.70±0.26 vs 1.30±0.13, P < 0.001). The ex-vivo imaging of the arteries revealed good correlation of the tracer accumulation. Quantitative uptake of the tracer in lesions was (2.55±0.69)-fold of non-plaque areas. The percentage of injected dose per gram (%ID/g) of the lesions were significantly higher than those of the non-plaque vessel segments (0.058±0.014 vs 0.020±0.012, P<0.001). And their %ID/g correlated significantly with the apoptotic index (r=0.82, P< 0.001)and the burden of the macrophages in the lesions (r=0.75, P< 0.001)by regression analysis, but the relationship between the uptake and the content of the vascular smooth cells in the lesions was not high(r=0.34, P> 0.05).4. ¹⁸F-FDG PET/CT resultsAbnormal focal uptake of ¹⁸F-FDG could be observed in the atherosclerotic thoracic aorta of the experimental rabbits at each imaging point, and the standardized uptake value (SUV)were 1.34±0.17, 1.45±0.18 and 1.41±0.20. No uptake was observed in the control group, and the mean SUV (1.07±0.11) was much lower than the AS group (F =65.43,P<0.001). Both PET positive and CT positive lesions could be invisible in the thoracic aorta of the atheroclerotic rabbits, including 20 lesions with positive uptake of ¹⁸F-FDG and 23 lesions with increased density in CT scans. DUR (differential uptake ratio) of the atherosclerotic segments (1.42±0.38) was much higher than DUR of the non-plaque segments (0.55±0.36,F=68.9,P<0.001). And the uptake of ¹⁸F-FDG of the vessel segments was correlated significantly with the macrophage content (r= 0.84, P<0.001) by regression analysis.Conclusion: Positive results were obtained using three different imaging technology in the experimental atherosclerotic rabbits. ^99mTc-HYNIC- PCNA ASON scintigraphy could identify the abnormal proliferation of macrophages and VSMCs in the rabbit model. It is a promising novel antisense probe to diagnose atherosclerotic plaque nonivasively at the early stage. ^99mTc-HYNIC-annexin V scintigraphy could identify the apoptosis of atherosclerotic plaques in the rabbit model. It is a promising noninvasive method to diagnose unstable atherosclerotic plaques. ¹⁸F-FDG PET/CT could identify the atherosclerotic lesions of the aorta in the experimental rabbits accurately and it might be able to appraise the stability of the plaques. In summary, the molecular imaging technologies of nuclear medicine targeting various targest within the atherosclerotic lesions with the different radiophamarceuticals as molecular probe, could be used to noninvasively image the plaques at the early stage and evaluate their stability, which showed important value and wide prospect.