The Effect Of Rotatory Manipulation On The Stability Of Early Atherosclerotic Plaques

This research is a funded project for the National Natural Science Foundation of China (81273871)BACKGROUND:Carotid atherosclerotic plaque is harmful condition caused by depositing of a fat, blood clots, connective tissue and calciums in the carotid artery, is arterial vascular disease pathological caused by a variety of risk factors, often lead cerebral infarction and other serious consequences caused by ischemia. With the improvement of living standards, hyperlipidemia, hypertension, diabetes, smoking, obesity increasing, particularly in the mostly middle-aged, these are important risk factors caused atherosclerosis currently. At the same time, elderly patients also are the high risk group of neck disorders, including common cervical spondylosis, cervical disc herniation, cervical joint disorders. Because of the significant effect about giving the ease of manipulation therapy of neck pain or discomfort, and patient concerns about the side effects of oral drugs, the patients often make the cervical rotatory manipulation and other physical therapy as the preferred way. According to statistics, about 8 million people suffer from neck pain treatment each year in the United States, of which 1 million people will choose massage therapy, but followed the one hand the efficacy of recognition while on the other hand adverse events reported on neck disorders caused by improper manual therapy. Data show that in 1995 the United States due to neck massage cerebral vascular damage caused by accidents accounted for 6.2% of all accidents massage. The endless stream of adverse events (including stroke, cerebral infarction, etc.) and its high mortality rate caused inevitably relevant experts and clinicians attention, the effect of neck manipulation on the patients with carotid atherosclerotic also become the focus of attention of Related discipline specialists. The main mechanism of cerebral ischemia is carotid artery bureaucratic stenosis, hemodynamic changes caused by carotid atherosclerotic plaque and emboli formation caused by plaque off. Among them, the most patients with ischemic stroke or cerebral infarction closely related to stability of carotid plaque. According to pathological changes of arterial plaques, which can be divided into two categories: the stability of plaque and unstable plaque, the latter is often easier off than the former, thrombosis, caused by a higher risk of cerebrovascular disease, so the effect of neck manipulation on the instability atherosclerotic plaque has undoubtedly become the focus of current research in related fields. However, carotid atherosclerosis is a progressive disease, with the progress of the disease and stimulation of a variety of factors, unstable plaque is likely to develop into plaque instability. So, the key issue of this study is that whether neck manipulation is an important factor to stimulate the development of stability plaque to plaque instability.PURPOSES:This study includes two aspects:1, by comparing with the fracture pressure values of carotid artery among the cervical rotation group and pushing carotid arteries, to study whether neck manipulation can cause plaque physical structural changes to reduce stability; 2, by comparing the micro-Raman spectroscopy among three groups (control group, the treatment group rotatory manipulation, fat diet group) rabbits of carotid atherosclerosis plaque to study whether arterial plaque characteristics and Raman spectra neck manipulation will change plaque lipid composition to affect the stability of arterial plaque.METHODS:The establishment of a rabbit atherosclerotic plaque stability model and groups: General level of health purebred male adult New Zealand rabbits 40 was bought, with normal diet for 15 days, the rabbits were divided into two groups, in which randomly selected 10 as the normal diet group, and continue to be normal diet to 18 weeks. The remaining 30 regarded as a high-fat diet A group and was in favor of a high-fat diet for 14 weeks. After 14 weeks, and then the 30 rabbits from high-fat diet A group were randomly divided into three groups, namely:the cervical rotatory manipulation group, pushing carotid arteries group and high-fat diet group B, and continue to be fed with high-fat diet to 18 weeks. Finally, experimental rabbits with intervention techniques and the results included in the analysis were divided into four groups:normal diet group, cervical rotatory manipulation group, pushing carotid arteries group and high-fat diet group B.2, The assessment of rabbit stability of atherosclerotic plaques modelFirstly, the rabbit blood lipid testingRespectively, in six weeks,10 weeks,14 weeks, eight rabbits were randomly selected from the high-fat diet fed rabbits every time to make blood biochemical tests. The rabbits prepared for blood were fasting at one day before the afternoon, the next morning (fasting more than 12h) 2.5mL of blood collected from the rabbit ear vein. Poured into an ordinary test tube containing an anticoagulant, allowed to stand for 1h, the tube was placed in a centrifuge and centrifuged 10min, speed of 1500r/min. Extracting the upper serum, sent to Nanfang Hospital laboratory, testing the triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL), high density lipoprotein (HDL) and very low density lipoprotein (VLDL) content in serum by automatic biochemical analyzer.Secondly, pathology observedRespectively, in six weeks,10 weeks,14 weeks, one rabbit selected randomly from high fat diet A group was killed, a total length of carotid artery, longitudinal incision, which was placed in 4% formaldehyde solution, fixed, progressively dehydrated, embedded in paraffin, continuous sections, HE staining pathological changes.Thirdly, statistical methodsSPSS 13.0 software for general use and lipid biochemistry test data obtained rabbit for statistical analysis. Two sets of data are presented as mean ± standard deviation. Two sets of data using two independent sample t test. P<0.05 was considered significant difference.3, Manipulation interventionFor rabbits of cervical rotatory manipulation group and push carotid arteries group corresponding manipulation intervention at the time 14 weeks, respectively. Each rabbit was intervened with cervical rotatory manipulation group around the neck of rotatory (approach to rabbit neck rotation to the limit last),1/3 days, a total of 15 days, a total of five times; pushing carotid arteries along the left and right carotid bow group, each pushed 20 times,1 time/day, a total of 15 days, a total of 15 times.4, Vitro stability analysis of neck manipulation on rabbit carotid atherosclerotic plaqueFirstly, a pressure recording device (SMC PRESSURE, ZSE30-01-65M) analyzer, a 150ml syringe, a tee, three pressure-transfer tubes, a 7 needles, one pair and one 9V battery battery clip. Use tee and pressure transfer tube connected respectively the syringe, pressure recording device, the 7th needle. Battery clamp was connected to power lines of pressure recording device, positive and negative battery terminals were connected respectively. Gradually pressurized by syringe, the needle at the end of pressure transmission channel was passed into the plaque, the device recorded the maximum pressure when plaque rupture by pressure recorded.Secondly, the vitro stability analysis of rabbit carotid artery atherosclerotic plaqueeight fresh plaques projected obviously into the lumen with visually observed were chosen in three carotid artery groups, vascular wall and outer membrane about 30°irection of the needle, according to the resistance change and the naked eye of the needle to judge whether the needle got into the site of intimal plaque, the plaque determined the tip into the intima, with its 15ml/s push syringe uniform pressure, recorded plaque pressure upon the collapse.Thirdly, statistical methodsEach set of data were expressed as mean ± standard deviation. Number of groups were compared using ANOVA, the mean between pairwise comparisons using LSD-t test. Using SPSS 13.0 software was used for statistical analysis. In sided a=0.05 as statistically significant difference.5, The effect of Raman spectroscopy Characteristics of cervical rotatory manipulation on carotid atherosclerosisFirstly, the sample processingAnimals were sacrificed at the end of experiment, freed carotid artery, longitudinal incision, then the artery was placed into PBS (pH7.4) solution at -80℃ refrigerator to prepare for Raman spectroscopy.Secondly, Raman SpectroscopyThawed samples prior to testing, six arteries plaques were chosen from the two groups of arteries (cervical rotatory manipulation group and high-fat diet group B), six arteries were randomly selected from normal diet group, washed with NS,633nm excitation as the excitation light source, the laser output power:10.4mw, spot diameter:1μm; Raman shift range of the spectrum of each substance rotate 1100- 1900cm-1, and the correction signals are collected, the signal collection time is 15-60s.Thirdly, Statistical Analysis of the relative intensity of the characteristic peaksAdopt a zero as the base, the relative strength of each feature peak (1450cm-1 and 1660cm-1) was calculated of the signal by using Origin6.0 software. Raman spectroscopy strength of cervical rotatory manipulation and high-fat diet group B was measured by using statistical software SPSS13.0, processed data to ± S., T-test was used to compare between each two. In P<0.05 statistically significant.RESULTS:1, Number of animals died40 adult male New Zealand white rabbits were Included,10 died during feeding. Among them, five rabbits of 30 rabbits in the high-fat diet A group were died, five of 10 rabbits died in Normal diet group. Experimental modeling processing, the overall survival rate of 66.7%. Final test packet analysis included a total of 30 rabbits.2, Changes of carotid atherosclerosis lipid levels RabbitsAt 6 weeks,10 weeks,14 weeks, total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL) and very low density lipoprotein (VLDL) levels of 30 rabbits serum from high-fat diet A group were gradually increased, high density lipoprotein (HDL) levels were gradually reduced, the indicators between the two groups was not statistically significant (P> 0.05).3, Pathological observationsThe first six weeks, the carotid artery biopsy showed:endometrial complete and smooth, the smooth muscle Traveling clear, no hyperplasia; the first 10 weeks, the carotid artery biopsy showed:arterial endothelial damage hyperplasia, protruding into the lumen, foam cell formation; the first 14 weeks, the carotid artery biopsy showed: significant foam cell accumulation visible lumen showed obvious red thrombus formation, in line with atherosclerotic lesions characteristic of early atherosclerotic plaque stability.4, Burst pressure test resultsPlaque ruptured under pressure, the visible portion of plaque when the pressurized plaque, the projection disappeared with convexly continuously pressurized to a predetermined value, while reducing the pressure gauge value, the recording of the maximum instantaneous shown plaque rupture pressure. There was no significant difference (P> 0.05) Cervical rotatory manipulation group, pushing carotid arteries and high-fat diet group B.5, Raman Spectroscopy between rotatory manipulation group and high fat diet group BFirstly, Detecting the Raman Spectroscopy between rotatory manipulation group and high fat diet group BIn the normal diet group, at 1450cm"1 and 1660cm"1 of Raman spectra had no obvious peaks lipid profile; in the cervical rotatory manipulation group, at 1450cm"1 and 1660cm"1 of Raman spectra, significant lipid peaks had been found; in the high fat diet group B, at 1450cm-1 and 1660cm-1 of Raman spectra, distinct lipid peaks can be seen.Secondly, result of relative intensity measurements of characteristic peaksRead spectral line of cervical rotatory manipulation group and high-fat diet group B, Raman peak and the corresponding wavelength (relative intensity) were performed T test with SPSS 13.0 software, relative intensity of lipid peak of two specimens at 1450cm-1and 1660cm-1 have no statistically significant(P> 0.05).CONCLUSIONS:1, In the early carotid atherosclerosis, rotatory manipulation and pushing carotid arteries have no obvious influence on the physical structure of early atherosclerotic plaque.2, In the early carotid atherosclerosis, the lipid content of atherosclerotic plaques will not increase in the short term with rotatory manipulation.