| Coronary atherosclerotic herat disease is the main disease which threateninghuman health severely. An unstable plaque rupture is the main reason of themostly acute coronary syndrome (ACS) , which including the acute myocardialinfarction, it threatened the human life severely. Revent studies have shown thatthe size of lipid core, the thickness of fibrous cap and the situation ofinflammatory cell infiltration, rather than plaque size and the results of the bloodvessel cavity stenosis degree,determine atherosclerotic plaque stability. Theeffective methods to prevente unstable plaque rupture are timely examination andthe prompt intervention. The atheromatous plaque diagnostic imaging techniquesinvolve intravascular unltrasound, the high definition contrast nuclear magneticresonance magnetic, optical coherence tomography, Raman spectroscopyexamination and so on. Raman spectroscopy has molecular sensitive specificreaction, it can detect plaque stability by quantitative analysis atherosclerosisplaque chemical composition such as cholesterol, collagen fibrin, phospholipid,calcium mineralization. Because Raman spectroscopy may quantitatively examinemany kinds of compositions, it can sensitively discriminate the plaque fibrous cap,the lipid core, the inflammation and the calcification, it also hasn't destructivenessfor the organization, so Raman spectroscopy has unique superiority inatherosclerotic plaque detection.The experiment took atherosclerotic rabbits as the animal model, it explainedthe degree of arteriosclerosis and the correlated stablity information, throughresearch at the relation of the endothelial cell function and the MMPs level withatherosclerosis plaque formation and development. Atherosclerotic plaqueorganization was carried on Micro-Raman spectroscopy detection, and the parallelpathology examination. This experiment confirmed that Raman spectroscopy wasaccuracy in Atherosclerotic plaque detection. It maked preclinical phasefoundation work for Raman spectroscopy becomes an effective detectiontechnology for Atherosclerotic vascular disease. Empirical method: Thirty rabbitswere divided into 3 groups at random: control group(n=10);atherogenic dietgroup(n=10);balloon-injury + atherogenic diet group(n=10). 3 months later thethree groups underwent pharmacological triggering with Chinese Russell's vipervenom and histamine. The information of blood fat (TC, TG, LDL, HDL) andweight were monitored at beginning and each month. The levels of nitrogenmonoxidum (NO), nitric oxide synthase (NOS), hydroxyl (OH) and Matrixmetalloproteinases (MMPs) in the serum were obtained in the three months.Balloon-injury + atherogenic diet group's carotid balloon dilatation process asfollows: Rabbits were anesthetized with 3% pentobarbitol sodium 30mg/Kg withan ear vein injection. After conventional disinfection, the right carotid arterieswere exposed by careful dissection via a midline incision.The distal end ofcarotid artery was ligated, the proximal part blood stream was blocked. To openthe artery anterior wall, the balloon catheter and guide wire were inserted andpushed into the proximal part ( the balloon inside diameter is 3.0mm,Long 20mm).The balloon was posted in the right carotid artery approximately 10cm, then theballoon was inflated to a pressure of 4 atm and drawn to the near insertion place.This procedure was repeated 3 times. Then the carotid artery was ligated at nearincision site. After sprinkling cidomycin at the operating field, the conventionalsaturation was performed. The procedure of pharmacological triggering: ChineseRussell's viper venom (CRVV,0.15mg/kg,intraperitoneal) followed 30 minuteslater by histamine(0.02mg/Kg, intravenous) were injected twice at 48 and 24hours before euthanasia. The levels of blood fat, nitrogen monoxidum (NO), nitricoxide synthase (NOS), hydroxyl (OH) in the serum were examinated by thecorresponding kits, the operations were according to instruction strictly. Thelevels of serum MMP-2 and MMP-9 were detected by SDS-PAGE enzymograph.Sample processing: After feeded 3 months, these animals were killed by excessiveanaesthesia with pentobarbitol sodium, heparin (10u/Kg) was injected to veinbefore execute. Opened the chest and abdomen to obtain the aortae, the arterieswere opened longitudinally, and the plaques in them were observed. The arterysegments were rinsed with phosphate buffered solution (PBS, PH=7.4)snap-frozen in liquid nitrogen, and stored at -80℃ centi-degree refrigerator untilthe Raman spectroscopy detection. Micro-Raman spectroscopy detection:Beforeuse, specimens were allowed to reach room temperature. The specimens wereilluminated in a 25-μm-diameter location with 300 mW of 488nm laser. TheRaman shift range was 500 to 2000 cm-1. The spectra were collected and rectifiedin 15 to 60 seconds. The Raman spectra of arteries tissue with different stages ofpathological change were obtained. After Raman spectroscopy examination, thesamples were fixed, then stained with hematoxylin and eosin, and carried onpathology examination. The relative areas under the Raman spectra characteristicband ( 1450 cm-1 and 1660 cm-1)were calculated by Origin6.0 software.Measurement data statistics used SPSS11.5 statistical package, data analysis wascarried out by t test .A level of P less than 0.05 was considered significant.Experimental result: In the control group, there were not differences in allobserved items at all stages. In atherogenic diet group and balloon-injury +atherogenic diet group, the levels of NO hadn't difference at the beginning andatherogenic diet later, but it reduced after pharmacological triggering. The serumlevels of NOS and OH were higher after given atherogenic diet, which obviouslyreduced after pharmacological triggering too. In atherogenic diet group theactivity of serum MMP-9 obviously markedly increased after pharmacologicaltriggering. In balloon-injury + atherogenic diet group the activity of serumMMP-2 and MMP-9 markedly increased after pharmacological triggering. Thearteries were classified roughly by visual pathology inspection, we selected 52specimens detected spots to carry out Raman spectroscopy detection fromatherosclerotic arteries (n=29) and some normal arteries. In obviousatherosclerotic plaques, the spectra existed obvious characteristic bands at 1450cm-1 and 1660 cm-1. In slight atherosclerotic plaques, the spectra existed indefinitecharacteristic bands at 1450 cm-1 and 1660 cm-1. In normal arteries,the spectrawas smooth and didn't existe obvious characteristic bands. In thromb plaques, thespectra existed strong fluorescence signal, characteristic bands couldn't beobserved. The statistics results of the relative areas under the Raman spectracharacteristic band ( 1450 cm-1 and 1660 cm-1): this relative areas were higherin obvious atherosclerotic plaques ( 5.80 × 10-3 ± 3.51 × 10-3 ) than slightatherosclerotic plaques (2.01×10-3±1.49×10-3)and normal arteries(1.01×10-3±0.94×10-3), P < 0. 05. Pathology results: After pharmacological triggering,there were 2 thrombus in distended carotids among 18 atherosclerotic plaques inballoon-injury + atherogenic diet group, there wasn't thromb among 11atherosclerotic plaques in atherogenic diet group. There wasn't atheroscleroticplaque in control group. Conclusions: In this animal model, there were theincrease of MMPs and endothelium dysfunction when the atherosclerotic plaqueswere unstable. Micro-Raman spectroscopy could detect atherosclerotic plaqueswith high identification ability. It was a feasible method to detect atheroscleroticplaques. With the technology of detecting atherosclerotic plaques by Ramanspectroscopy is matured further, this technology would be used to diagnosisatheromatous vascular diseases in Clinical. |
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