Study The Formation Of Carotid Athero-sclerosis In Rabbits Which Were Dieted With High Lipid And Alcohol

ObjectiveSet up carotid atherosclerosis (CAS) medels of rabbits, and discuss the influence upon the happening and developing of rabbit's CAS by heavy alcohol purely intake or by high fat plus high cholesteroldiet accompanying with different dose alcohol, and deeply exploring whether such disposal can postpone or accelerate the progression of CAS, what relationship between this disposal and the dynamic changes of rabbit's serum lipid markers, carotid artery vascular remodeling markers, and the expression of marix metalloproteinase-2 (MMP-2) in carotid atherosclerostic plaques.Matherials and Methods1. Animal groups and raiseIn this study, we used healthy male New Zealand rabbits which weighted 2.00±0.15kg, and were 3-4 months old. Sixty rabbits were divided into six groups randomly, each one had ten. (A) Control, which was fed normal chow; (B) Model, which was fed high lipid diet chow served as CAS control;(C) Alcohol, which was fed normal chow and heavy alcohol intake with 1.5g/kg·d; (D) Model plus Alcohol, which was fed high lipid diet chow and low alcohol intake with 0.3g/kg·d; (E) Model plus Alcohol, which was fed high lipid diet chow and moderate alcohol intake with 0.6 g/kg·d; (F) Model plus Alcohol, which was fed high lipid diet chow and heavy alcohol intake with 1.5g/kg·d.The high lipid diet chow was composed od different propotion of cholesterol, yolk, lard and normal chow. The rabbits which needed alcohol consumption adopted 38 degree white wine by poured into stomach everyday. Each rabbit had his cage, were given 120g forage and drinked water freely everyday. All animals were killed at the end of 12 weeks. 2. The serum lipid markers and glucose assayThe blood was drawn from the ear edge vein to assay some serum cholesterol, lipoproteins and glucose at the prior experiment and the end of 4 weeks,8 weeks and 12 weeks. The markers included total cholesterol (TCH), triglycerides (TG), high density lipoprotein (HDL), low density lipoprotein (LDL) and glucose (GLU).3. Vascular remodeling (VR) markers assayWe separated carotid artery specimens and took section cut straight through carotid artery which was 1mm near to the crotch, then embeded the section side and got 5μm slice each sheet, stained it with hematoxylin and eosin (HE). All markers of vascular remodeling were assayed in Image-Pro-Plus 4.0 analysis system, including elastic circumference, external elastic circumference, plaque area (PA), internal elastic laminal area (IELA), external elastic laminal area (EELA), medial area (MA), lumen area (LA), and the carotid artery stenotic degree was also calculated by PA and IELA datas.4. Analysis of optical density of MMP-2A 4μm paraff section was made, then we assayed the expression of MMP-2 in carotid atherosclerotic plaques by using immunohistochemical method. The positive expression of MMP-2 was judged as buffy particles presenc in plaques. Eight typical sections were selected from each groups, five integrital and un-overlapping visual fields were selected at randomly in each section at high power objective, then we estimated the OD tite of MMP-2 in each plaque in Image-Pro-Plus 4.0 analysis system.5. Statistic disposalAll datas were analyzed by SAS 6.12 statistic software and statistical significance was set at P<0.05.Results1. The markers of serum lipidTCH:There were no differences between group A and group C. The datas of all models increased fiercely at the end of 4 weeks and decreased lightly at the end of 8 weeks, reached their peaks uitil 12 weeks. The minimum is 35.10±3.06mmol/L in group E, compared with group B, difference had a statistical significance (P=0.0137).TG:All groups had no differences before 4 weeks. But all models'datas showed a linear increasing tendency. At the end of 8 and 12 weeks, group E had two less datas, compared with the other two high lipid and alcohol using groups.That were 1.44±0.42 and 1.56±0.66mmol/L, and compared with group B, the P were 0.0103 and 0.0145.HDL:Group D's data reached its peak at the end of 4weeks, and group E's at the end of 8 weeks, reached its peak (8.21±0.60mmol/L, compared with group B, P=0.0008). At the end of 12 weeks, the maximum data still was in group E (compared with group B, P=0.0409), followed by group D.LDL:All model groups'datas increased notably at the last of 4 weeks, then decreased at the end of 8 weeks significantly, re-increased at the end of 12 weeks, but alldatas didn't exceed those of 4 weeks. The tendency of LDL'concentration dynamic change was coincidence with serum TCH. Group D had the lest data from the beginning to the end. At the end of 12 weeks, moderate alcohol intake group's data was also less than group B (P=0.0112).2. The reasults of glucoseThe glucose concentration at the end of 4 weeks were approached to the end of 8 weeks'in group B, C, D and E, except for group F. All experimental groups'glucose data increased at the end of 12 weeks besides group D. There was a obverse correlated relation between glucose concentration and alcohol consumption(R=0.61306, P=0.0024). The linear regression equation was Y(glucose concentration)=7.912621+3.203883X (alcohol consumption).3. The results of pathomophologyThere were no carotid atherosclerostic plaues in group A and C. The plaques were easily found in the other four groups. In these groups, there were lots of SMCs and macrophages aggregating in plaques. Some SMCs migrated under the endothelium, swallowed lipid and became typical foam cells. Some blood vessels showed lumen stenosis. The gentlest lesions in carotid artery were in group E. The most serious lesions were found in group F, even some cholesterol crystals could be found in those plaques.4. The markers of vascular remodelingMost markers of VR in group B were smaller than control group. However, the group C had high datas in every markers of. VR than control group, displaying a vascular dilatation and thinner tunica media. In groups which were fed with high lipid diet chow and different dose alcohol, the group E had ideal datas, its PA was the smallest (compared with group B, D, F, P=0.0172,0.2304,0:0112). Among the three groups which were given not only lipid but alcohol, group E had the smallest blood vessel constriction rate (compared with group D, F, P=0.0979,0.0145). The correlation analysis showed that PA and LA were both correlated to the IELA and EELA, especially the IELA and EELA, these two markers'correlation were significant, and R=0.96385, P<0.001.5. The results of immunohistochemistryIn this study,we found the expression of MMP-2 existed only in plaques of CAS. So there were no MMP-2 expression in group A and C because there were no plaques formed in these two groups. The expression of MMP-2 was weakly found in SMCs in tunica media. The expression of MMP-2 was the strongest in plaques of group F and the weakest in group E's plaques.Conclusions1. The rabbit's CAS models could be made by given lipid diet chow, or at this foundation given different dose alcohol. Such method was safer, more scientific and exacter than damaging endothelium. The most important was this method was in line with CAS's natural process.2. Only using alcohol could not result in CAS of rabbits which reversely proved the role of lipid invasion. Moderate alcohol intake had anti-atherosclerosis effects, some mechanisms could be come true by rasing HDL's concentration, reducing TCH, LDL and TG's concentration.3. The CAS plaques formed easily at carotid artery crotch. The proliferation and migration of SMCs acted as an important role in occurrence and development of CAS. Macrophages and lymphocytes also took part in this process. There were many foam cells which came from SMCs and macrophages in plaques. The PA in group E was smallest and only displayed as fatty streak period of atherosclerosis.4. The carotid artery displayed a restriction remodeling in group B. The alcohol purely intake group took on a dilataltion change in carotid artery. In group E, the carotid artery stenosis was the lightest, and the markers like PA, IELA, EELA in this group were also the smallest in those groups which were fed high lipid and alcohol.5. Giving 0.6k/kg everyday could not increase the expression of MMP-2, which might have an important protective action in postponing the development of plaques in CAS. The different expression of MMP-2 in those lipid and alcohol consumption groups showed a J-shaped association accompanying with alcohol dosage. It was also suggested that the expression of MMP-2 might be one of the mechanisms between the relation of alcohol intake and CAS.6. Moderate alcohol intake could increase serum HDL's concentration, decrease TCH and LDL concentration notably, inhibit the expression of MMP-2, these effects could do important roles in deferring the progression of CAS.