| Blood pressure(BP)is not constant,in which there exists a continuously spontaneous variation.This variation is defined as blood pressure variability(BPV).Chronic sinoaortic-denervated(SAD)rats exhibit an increased BPV with a normal BP level.It was found that SAD produced a remarkable left ventricular hypertrophy and damage in blood vessel,brain and kidney.Moreover,it is reported that target organ damage in hypertension was related to,and has been widely accepted that increased BPV results in target organ damage.Consequently,lessening organ damage by increased BPV is a major step to lessen organ damage in hypertension.Microcirculation is the place to exchange substances,energy and information.Normal vascular endothelial cell plays an important role in normal microcirculation. Microcirculation dysfunction leads to tissue and organ damage.It was documented that there are microcirculation dysfunction in hypertension.So,it was considered that microcirculation dysfunction perhaps participates in the mechanism of organ damage in high BPV,and speculated that microcirculation dysfunction may be one cause for organ damage by increased BPV.The present study aimed at the following purpose:1.The relation between left ventricular hypertrophy and microcirculation dysfunction was determined.2.Effects of pressure variability on platelet activation in vitro.The first part of experiment was performed in vivo.Chronic sinoaortic-denervated (SAD)rat is a BPV model without sustained hypertension.With microcirculation index of vascular endothelial cell damage,capillary density,blood viscosity and blood cell changes,we observed the microcirculation change with time course in left ventricle of SAD rats.Also the relation between the microcirculation change and left ventricular hypertrophy was studied.Then,the effects of nitrendipine on microcirculation in SAD rats were observed.The second part of experiment was performed in vitro.One reason for microcirculation dysfunction is the change of blood character,in which an important style is the abnormality of platelet adhesion and aggregation with activation.Not considering blood vessel regulation,increased BPV will lead to high blood flow fluctuation,which is a kind of machinery stress stimulation.Considering platelet activation with machinery stress stimulation,we propose that high blood flow fluctuation enable to activate platelet.Therefore,in this part,a high blood flow fluctuation model in vitro was made,in which effect of blood flow fluctuation on platelet function was observed.Furthermore,we investigated pressure variability in this model,and analyze the effect of increased pressure variability on platelet activation.The main methods were shown as follows:1.SAD was or sham-operation(Sham)performed in male Sprague-Dawley(SD) rats.Briefly,rats were anaesthetized and fixed in a supine position.The aortic baroreceptor denervation was carried out bilaterally by cutting the superior laryngeal nerves near the vagus,removing the superior cervical ganglia including a small section of the sympathetic trunk,and sectioning aortic depressor nerves.The carotid sinus baroreceptors were denervated bilaterally by stripping the carotid bifurcation and its branches followed by painting with 10%phenol in ethanol.In some rats,sham operation(Sham)was performed with the midline neck incision and bilateral isolation of the neck muscles.Hemodynamic parameters were continuously recorded in conscious unrestrained rats.BP,BPV,HR,HRV was calculated.All the studies were performed in SAD and Sham rats 2,4,8,16 weeks after operation.2.In another study,SAD or sham-operation(Sham)was performed in male SD rats. All the study was performed in SAD and Sham rats 2,4,8,16weeks after operation. Moreover,the SAD rats(group SAD-Nit)were treated with nitrendipine(10mg/kg/d, p.o.)for 16 weeks after operation.At corresponding time,weighing,taking blood, counting blood cells,detecting blood rheology,evaluation of platelet adhesion in a single-pass perfusion chamber and thrombus height with laser confocal technology, flow cytometric analysis of platelet surface P-selectin expression,detecting plasma (von Willebrand Factor,vWF),VEGF and nitric oxide(NO)with Elisa or nitrate reducase method,examining morphological changes of left ventricle by histopathological method and Van Gieson-stained,assessing capillary density using alkaline phosphatase method and detecting mRNA and protein production of VEGF, fms-like tyrosine kinasel(Flt-1),and fetal liver kinase-1(Flk-1)were performed in Sham and SAD rats.The relationships between left ventricular hypertrophy parameters and microcirculation dysfunction variables were analyzed by classic univariate correlation analysis.3.In a syringe pump flow system,blood was drawn directly through the perfusion chamber.A pulsatile flow model was made by periodic change flow rate of the syringe pump every 15 s,resulting in a periodic change from low blood flow(shear rate 300/s)to high one(shear rate 1300/s).Blood flow was monitored by laser Doppler flowmetry.Blood flow fluctuation was expressed with standard differentiation of blood flow during 5 min.Platelet adhesion on collagen typeⅢsurface was accessed with perfusion chamber in low blood flow,high one and pulsatile one.Expression of platelet surface P-selectin and PAC-1 was analyzed by flow cytometry.Pressure and pressure variability were calculated in low blood flow, high one and pulsatile one.Finally,the relationships between pressure and pressure variability and blood flow and blood flow fluctuation were analyzed.The main results are shown as follows:1.Characters of hemodynamic parameters and baroreceptor reflex function in SAD rats.In SAD rats 2,4,8 and 16 weeks after operation,SBP,DBP,MBP,HR and HRV level remained unchanged,while SBPV,DBPV and MBPV were significantly increased, when compared with time-matched sham-operated rats.Meanwhile,the response to phenylephrine was tested in conscious unstrained rats.Increased blood pressure induced by phenylephrine in SAD group was not different from that in Sham group, but heart rate decrease induced by phenylephrine in SAD group was obviously attenuated,suggesting impairment of arterial baroreceptor reflex function in SAD rats.2.Morphometry of left ventricle in SAD rats At 4,8 and 16 weeks after SAD,compared with age-matched sham-operated rats, SAD rats exhibited a significant loss in body weight,while heart weight,heart weight index(heart weight/body weight),left ventricular weight,left ventricular weight index(left ventricular weight/body weight)and left ventricular wall thickness increased significantly.Histopathological observation found that,in left ventricular, there were swelling,hypertrophy,breaking and necrosis.Morphometric measurement showed that the left ventricular collagen volume fraction(CVF)and myocardium cross-section area(MCA)increased.All these indicated that left ventricular hypertrophy existed in SAD rats.3.Microcirculation dysfunction in SAD ratsAt 2,4,8 and 16 weeks after SAD,while compared with Sham rats,the plasmatic concentrations of von Willebrand factor(vWF)and level of VEGF mRNA and protein in left ventricle were higher,while the level of Nitric oxide(NO)was lower,indicating blood vessel endothelium dysfunction in SAD rats.At 4,8 and 16 except 2 weeks after SAD,compared with age-matched sham-operated rats,there was obvious decrease in capillary density in left ventricle,From 2 to 16 weeks after SAD, expression of Flk-1 mRNA and protein in left ventricle were unregulated with no change with the expression of Flt-1.Additionally,red blood cell(RBC),white blood cell(WBC)and platelet count were unchanged in SAD rats.But mean platelet volume (MPV),platelet large cell ratio(PLCR)and platelet distribution width(PDW)were increased in SAD rats,suggesting platelet disorder.Moreover,in SAD rats,there were obvious increase in whole blood viscosity(WBV)at shear rate 1 s-1,30 s-1and 200 s-1 as well as increased plasma viscosity(PV).Platelet adhesion and thrombus height at shear rate 300 s-1were obvious increased in SAD rats.P-selectin positive platelets in SAD rats were significant higher.All these indicated that there were blood ingredient and character and platelet activation dysfunction in SAD rats.4.Effect of nitrendipine on left ventricular hypertrophy and microcirculationCompared with 16-week SAD rats,in SAD-Nit rats,heart weight index,left ventricular weight index and left ventricular wall thickness,left ventricular CVF and left ventricular MCA was decreased.Histopathological observation found that,there were less swelling,hypertrophy,breaking and necrosis in left ventricle of SAD-Nit rats.Meanwhile,capillary density in left ventricle were increased.The plasmatic concentrations of vWF and VEGF were decreased.On the contrary,the NO level was increased.Additionally,nitrendipine reduced MPV,PLCR,PDW,WBV,and PV, platelet adhesion,thrombus height and P-selectin positive platelets.Moreover, nitrendipine obviously down regulated VEGF and Flk-1 level in left ventricle while Flt-1 level unchanged.5.Relation between left ventricular hypertrophy and microcirculation dysfunctionLeft ventricular weight,left ventricular weight index,left ventricular CVF and MCA, as the indexes of left ventricular hypertrophy were all negatively related to plasma NO level and capillary density in left ventricle,and positively related to plasma vWF and VEGF level,left ventricular VEGF level,positive percentage of P-selectin and blood viscosity in SAD rats and SAD rats treated with nitrendipine.6.Effect of fluctuated blood on platelet activation in vitroThere was more blood flow in state of pulsatile blood flow than in that of low blood flow(58μl/min,shear rate 300s-1),less than in that of high blood flow(252μl/min, shear rate 1300s-1).But blood flow fluctuation in pulsatile blood flow was higher than in both low flow and high flow.Platelet adhesion to collagen and vWF increased more significant in high blood flow than in low flow,while there were more platelet adhesion to collagen and vWF in pulsatile blood flow than in both high flow and low flow.Compared with pre-perfusion state,expression of P-selectin on platelet surface increased obviously in pulsatile blood flow after perfusion over collagen and vWF surface,with no changes in both high flow and low flow.In all three blood flow state, expression of PAC-1 on platelet surface remained unchanged.Pressure in pulsatile blood flow increased more significantly than that in low flow and decreased more obviously than that in high flow.However,pressure variability in pulsatile blood flow was higher than in both low flow and high flow.In addition,in perfusion chamber, pressure was positively correlated with blood flow,while pressure variability was positively correlated with blood flow variability,indicating that pressure variability is reflected with pulsatile blood flow model in vitro.So,from a pressure point of view,it suggested that increased pressure variability activate platelet.The results suggest that:1.Increased BPV may produce microcirculation dysfunction that characterized with vascular endothelium damage,increase in blood viscosity and decrease in capillary density are early changes,which left ventricular hypertrophy lag behind.Once these changes occur,they are persistent. 2.Pressure variability may directly activate platelet by machinery stimulation,which plays an important role in microcirculation dysfunction induced by increased BPV.3.Microcirculation dysfunction is one cause of left ventricular hypertrophy induced by increased BPV.4.Nitrendipine has organ protection from increased BPV,which may relate to improved microcirculation dysfunction.These studies may be helpful to further elucidate the mechanisms involved in organ damages with increased BPV,which emphasizes the way to treat cardiovascular disease with improvement of microcirculation.
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