| 1 Experiment ObjectiveSort the Cause of death of disease in the world's population by order,the cardiovascular and cerebrovascular diseases pathologically based on atherosclerosis(Atherosclerosis,As)are the first one,and we should take the study of the formation mechanism of As and the study of preventive measures based on the study as a primary focus in our research work.AS only occurs in the large-sized and medium-sized arteries with nutrient system composed of vasa vasorum,nutrient lymphatic vessels,rather than the small arteries absence of nutrient system,what occur in the small artery is fibrosclerosis of artery.So far,the medical hypothesis of the formation of As are:thrombosis theory(1841,Rokitansky),inflammation theory(1856,Virchow),lipid infiltration theory(1863,Virchow),monoclonal theory(1973,Benditt),injury response theory(1976,Ross),oxidation theory(1983,Steinberg),etc.,but they are not perfect,and the hypothesis system they composed is not perfect either.These theories can not explain many phenomenons and facts in the the formation and development of As,such as:①why coronary atherosclerosis only occurs in the epicardial segment of coronary artery absence of nourish lymphatic system,rather than occurs in the coronary artery within the myocardial wall;②why the coronary artery within the myocardial wall of transplanted heart prone to As,but the coronary artery within the myocardial wall of normal heart does not occur As;③why A S occurs in the lower extremities with poor lymphatic and blood circulation early and seriously,but seldom occurs in the upper limbs with better lymphatic and blood circulation;④why As only occurs in the large-sized and medium-sized arteries contain vasa vasorum,nutrient lymphatic vessels,but not in the small arteries without vasa vasorum,nutrient lymphatic vessels;⑤why early pathological changes of As are segmental distribution rather than a continuous distribution.Most of these problems are closely related with the anatomical features of lymphatic of the large-sized artery system.Which described above show that the hypothesis system composed by all of the current hypothesis about the exact development mechanism of As does not seize the initiating factors of the formation process of As,and the hypothesis system of As need to be complemented and perfected.A large number of studies have shown that impaired lymphatic drainage have an important impact on the course of natural or experimental atherosclerosis,lymphatic obstruction may result in histological damages in the artery wall similar as As.In the acquaintance of As in the traditional Chinese medicine,we believe that the the cause of As is deficiency generate phlegm,phlegm generate stasis,phlegm stasis transforming into poison.The basic pathological mechanism is deficiency-phlegm-stasis-poison,while the main pathological process is:Qi deficiency and yang deficiency lead to failure of movement and transformation results in rich food and damp turbidity accumulate and transport into phlegm,abundant phlegm further obstructing the collaterals,and condenses into subcutaneous nodule,obstructing the collaterals,then the large vessel(blood vessel) nourished by the collaterals occurs qi and blood stagnation,and lead to the formation of micro-subcutaneous nodule and micro-gynecological mass,which block the blood vessel causing the blood occlusion,and the stasis of the blood circulation,the the phlegm and the stasis snarl together to generating poison which damage the blood vessel,and finally cause the disease.The location of As is large vessels(blood vessels).Our subject seeks to bring up the hypothesis of nourishing lymph microcirculation disturbance of adventitia about the formation of As after it has been silented for more than a decade and stressed its importance in the formation of As,then to establish an animal model of As caused by nourishing lymph microcirculation disturbance and a study platform of As based on the principle of anatomy and pathology lymphatic.Subjects of this study have certain innovation,application and application value,and may create considerable economic and social benefits in the future.2 Experiment Methods64 Wistar rats were randomly divided into four time groups(3 days group,7 days group,14 days group,28 days group,each time group 16),and each time group was divided into normal group and model group(n=8).The left limbs of rats of model group are operated by surgical ligation of their lymphatic around lymph nodes of the popliteal,inguinal,axillary and removal of all of these lymph nodes in order to the establish a model of lymphatic microcirculation disturbance,while the right limbs of rats of model groups and both limbs of rats of normal group are not done with any operation.After 3 days,7 days,14 days,28 days of modeling,rats of model groups and normal groups each 8 respectively were anaesthetized,taked blood,perfusion and fixation,draw materials.Detect Blood Routine,blood biochemistry and blood hemorrheology.Organizations which were taked are the left and right femoral artery of the rats,which are isolated fixed,embedded,sliced and HE staining,Masson staining.Slices under the microscope were taked photograph by digital imaging system and observed their microstructure,then the photos were analyzed by image analysis system,measuring the values of thickness of the intima,thickness of the media,the proportion of the collagen per unit area in the membrane,number of small blood vessels per unit area in the adventitia,and taking statistics comparison analyzing to the measurement results.3 Experiment Results3.1 Blood tests:Comparation of the indicators of Blood Routine and blood biochemical of the model groups and normal groups of time periods of 3 days,7 days,14 days,28 days show no difference with statistically significant(P＞0.05).In the indicators of blood hemorrheology,the comparations of the model groups and normal groups of the periods of 3 days,7 days,28 days show no difference with statistically significant(P＞0.05);while the comparation of the model groups and normal groups of the periods of 14 days show that whole blood viscosity,the reduced viscosity,plasma viscosity were significantly higher in model groups,and hematocrit were significantly lower in modes groups,and the difference is significant with statistical significance(P＜0.05),while other items show no difference with significant difference(P＞0.05).3.2 The results of pathological changes under light microscopeThe femoral artery of normal groups of time periods of 3 days,7 days,14 days,28 days show integral and smooth intimal,integral endothelial cells,and show no cells collection in the lower under the endothelial cells,no intimal thickening;and dense,full internal elastic plate which have smooth circle distribution;and show media containing smooth muscle layer with regular oval-shaped nucleus whose longitudinal direction is the same as the running direction of the smooth muscles,and levels of dense regular elastic fibers which have concentric-shaped distribution;and show a small amount of light green collagen fibers scattered within the media by Masson staining;and show compact,complete external elastic plate which have smooth-shaped distribution of concentric circles;and show adventitia loose connective tissue which scattered by nutrient vessels,lymphatic vessels and nerve fiber with luminal patency without occlusion and the phenomenon of expansion and tortuous.Compared with the normal groups,the non-operated side(right)of femoral artery of rats of model groups of time periods of 3 days,7 days,14 days,28 days have no obvious changes.Compared with the normal group,operated side femoral arteries of rats of 3 days model group show:mild intimal thickening;nucleus of media smooth muscle layer was no longer rules oval,large and round cells markedly increased,and the longitudinal direction no longer be the same direction as the smooth muscle,and Masson staining show the area of collagen fibers are slight increased;and vasa vasorum in the adventitial began to appear wall cells hyperplasia phenomenon,and the nourish lymphatics are in state of expansion and tortuous,and the number of nutrient vessels in the adventitia increases.Compared with 3 days model group,operated side femoral arteries of rats of 7 days model group show similar changs in intimal and media,while show more serious state of the expansion tortuous lymphatic vessels and the number of small nutrient vessels increases.Compared with 7 days model group,operated side femoral arteries of rats of 14 days model group show:intimal thickening is more obvious,cells in the smooth muscle layers become lager and rounder,with the direction of slightly disordered;Masson staining showed the collagen membrane fiber content increased significantly;external elastic plate turn loose,and less smooth,occasionally partial fracture;the wall of vasa vasorums in adventitia show hyperplasia phenomenon,the nourish lymphatic vessels show serious expansion and tortuous,the number of small nourish vessels obviously increased and have cluster distribution.Compared with 14 days model group,operated side femoral arteries of rats of 28 days model group show:more apparent intimal thickening;smooth muscle layer turn larger,more rounded cells,with a more disordered arry,Masson staining shows the plastic fibrils of the media are more obvious increased;external elastic plate are more loose,visible fracture,nourish lymphatic vessels in the adventitia have more obvious phenomenon of expansion and tortuous,and the number of small nourish vessels increases more significant.3.3 Analysis of intimal thickness show:(1)compared with the normal groups,the the operated side femoral arteries of the model groups of each time period show intimal absolute thickness markly increases,the difference was significant with statistical significance(P＜0.05);(2)intimal/medial thickness ratio of the operated side femoral arteries of the model groups of 3 days increases compared with nomal groups,the difference was significant with statistical significance(P＜0.05),while this ratio does not increase compared with the non-operated side femoral arteries,the difference was not significant with statistically significant(P＞0.05).Compared with normal groups,the operated side femoral arteries of model groups of other time periods show significantly increased intimal/medial thickness ratio,the difference was significant with statistical significance(P＜0.05).3.4 Analysis of the proportion of collagen per unit area in the media show:compared with the normal groups and the non-operated side of model groups,the proportion collagen per unit area in the media of the the operated side femoral arteries of the model groups of 3 days show no difference with statistically significant(P＞0.05),while the proportion collagen per unit area in the media of the the operated side femoral arteries of the model groups of other three time periods show obvious increases,and the difference was significant with statistical significance(P＜0.05).3.5 Analasis of the number of small blood vessels per unit area in the adventitia show:compared with the normal groups and the non-operated side of model groups,the number of small blood vessels per unit area in the adventitia of the the operated side femoral arteries of the model groups of four time period were significantly increased,and the difference was significant with statistical significance(P＜0.05).4 Experiment Conclusions4.1 Operation method of lymphatic ligation of this experiment does not prone to cause chronic inflammatory response,and there are no high-sugar,high fat factors in the diet,do not the formate hyperglycemia and hyperlipidemia;4.2 14 days after lymphatic ligation,whole blood viscosity,reduced viscosity,plasma viscosity indices were significantly higher,and hematocrit was lower,suggesting that 2 weeks lymphatic microcirculation disturbance can lead to changes in blood rheology of rats,However,28 days after the operation,the hemorrheology returned to normal may be caused by the role of self-regulation of rats;4.3 femoral arteries of the model groups of four time period occur varying degrees of pathological changes after lymphatic ligation,indicatsthat lymphatic microcirculation disturbance can lead to structure reconstruction in layers of the femoral artery of rats,and the extremely expantion of lymphatics in the adventitia shows the mode operation is successful;4.4 3 days,7 days,14 days,28 days after lymphatic ligation,the number of small nourish vessels in the adventitia increased and gather into a film,which may be a compensatory response of the vasa vasorum microcirculation system occur after nourish lymphatic microcirculation system become disturbance.4.5 3 days,7 days,14 days,28 days after lymphatic ligation,the femoral arteries of rats most have intimal thickening of varying degrees,may be caused by the failure of reflex of the metabolism substances in the intima of the femoral arteries of rats and accumulation took place after lymphatic microcirculation disturbancelead;4.6 3 days,7 days,14 days,28 days after lymphatic ligation,proportion of collagen per unit area in the media of the femoral artery of rats increase,can be regarded as a material reconstruction of organization,and be recognized as an early pathological manifestations of atherosclerosis according to the literature. |
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