Effects Of Long-term Simulated Weightlessness On Arterial Stiffness In Rats

Background:With the rapid development of domestic aerospace industry,astronauts will stay longer in space.The influence of long-term weightless on astronauts and the effective protection for their physical health are important research directions of aerospace medicine.The change in cardiovascular system induced by weightless is one of the many factors that affect the physical and mental health of astronauts.Recent study showed that astronauts’carotid artery stiffness increased,similar to 10²0 years of normal aging after a six-month spaceflight.Arterial stiffness（AS）is the earliest detectable index of structural and functional abnormalities in the arterial wall.The increase in arterial stiffness,as a predictive indicator and pathological basement of many cardiovascular diseases,suggests the occurrence of arteriosclerosis.A large number of studies have confirmed that arterial stiffness is an independent predictor of cardiovascular disease and all-cause mortality in different populations.Arterial stiffness is of great clinical value.Pulse wave velocity（PWV）is recognized as the most effective method for non-invasive measurement of arterial stiffness.Among them,the carotid-femoral PWV（cfPWV）has been used as the"gold standard"for measurement.Previous studies have shown that simulated weightlessness causes regional-specific arterial remodeling.Hypertrophic changes and increase in vascular reactivity occur in cerebral arteries of rats due to intracerebral hypertension while atrophic changes and decrease in vascular contraction reactivity occur in resistance arteries in hindlimb and visceral arteries.However,the effects of long-term simulated weightlessness on arterial stiffness in different regions of rats and the characteristics are not fully understood.At present,there are no unified evaluation method and index of rat arterial stiffness.Therefore,we aimed to establish a reliable method based on doppler ultrasound for evaluation of arterial stiffness in rats and study the effects of long-term simulated weightlessness on arterial stiffness.In addition,the rat model of simulated weightless is often established through tail suspension.The current classical method is invented by Morey-Holton et al and improved by Zhang et al.However,this classical method requires that researchers have rich experience in adjusting the tightness of the gauze wrapped outside the rat tail in the protocol of making the tail sheath.Otherwise the rat tail is prone to ischemia,necrosis or sheath detachment,causing failure in the model establishment.We modified the protocol of making tail sheath by a plastic net,which significantly increased the success rate of model establishment.Aims:1.To establish a reliable noninvasive method to evaluate the arterial stiffness in rats.2.To investigate the effects on arterial stiffness in different regions of rats induced by long-term simulated weightlessness.Methods:1.Two-dimensional and doppler ultrasonography were used to display and determine the sampling points of the pulse wave of the common carotid artery and femoral artery in rats.Doppler ultrasonography was used to obtain the blood flow velocity spectrum of the points.The slope method was used to determine the initial acceleration of the blood flow.The time interval as the pulse wave transit timeΔt was measured between the starting point and the apex of the R wave from the electrocardiogram.The distance from the tip of the nose to the root of the tail was measured as body length.Arteries were exposed from the aortic root to the right carotid artery and right femoral artery.The pulse wave transit distance D was accurately measured.The body length,body weight and age were taken as variables.The pulse wave transit distance was analyzed by multiple regression analysis.The calculation formula of the pulse wave transit distance D was obtained.According to PWV=D/Δt,the heart-femoral pulse wave velocity（hfPWV）,carotid-femoral pulse wave velocity（cfPWV）and heart-carotid pulse wave velocity（hcPWV）were calculated.2.The rat hindlimb was unload to establish the simulated weightlessness model by tail suspension.The classical protocol of making the tail sheath was modified.The success rate of model and the incidence of tail complications were compared in the classical and modified suspension groups.3.In the modified rat model of simulated weightlessness,the transit time was measured by two-dimensional and doppler ultrasound.The transit distance was calculated with the formulas.HcPWV,hfPWV and cfPWV were calculated on 0 day,3rd week and6th week after hindlimb reloading from 4-week simulated weightlessness.4.The specimens of the carotid artery,thoracic aorta and abdominal aorta were prepared.Weigert elastic fiber staining was performed to observe the arrangement and distribution of elastic fibers.HE staining was performed to measure the intima-media thickness and cross-sectional area.The content of collagen fibers was determined by masson staining.The expression levels of Collagen I,Collagen III and elastin mRNA in rat carotid arteries were detected by qRT-PCR.5.Vasodilation induced by ACh and SNP was assessed on the isolated carotid artery rings.6.The miRNA profiling assays of the carotid arteries in HU and control groups were performed with Illumina Hiseq 2500 high throughput sequencing.Expression of candidate miRNA selected by bioinformatics analysis was further detected by qRT-PCR.7.Statistical analysis was performed using GraphPad Prism version 6.0 software.Sample means were analysed by t-test in two groups and by one-way ANOVA in three groups.Sample rates were analysed by Chi-square test.P<0.05 was considered statistically significant.Results:1.Compared with the control group,the ratio of right soleus wet weight to body weight was significantly decreased in both the classical suspension group and the modified suspension group.The incidence of tail ischemia and necrosis（13.3%vs.40.0%in the classical suspension group）and the incidence of sheath detachment from tail（3.3%vs.26.7%in the classical suspension group）were significantly lower whereas the success rates of model（83.3%vs.33.3%in classical suspension group）was significantly higher in the modified suspension group（n=30,P<0.05）.2.There was a good linear correlation between the invasively measured heart-femoral transit distance and the body length of rats（n=200,P<0.0001）,with a high determination coefficient（R²=0.9858）and a concise regression equation(D_hf=0.6086×L-1.6523 mm).When adding variables including the age and body weight to the analyses,the R² had a very slight increase,but the equations became more complex and lengthy.Based on this principle,D_cf can be calculated as 0.4614×L+1.8335 mm,and D_hc can be completed as0.1472×L-3.4858 mm.These formulas for calculating the accurate pulse wave transit distance of rats noninvasively provide a basis for measuring the pulse wave velocity of rats.3.After 4-week simulated weightlessness,the hcPWV in HU group significantly increased compared to control（n=8,P<0.05）.However,there was no significant difference between HU and control groups in hfPWV and cfPWV（n=8,P>0.05）.On 3rd week after reloading,hcPWV in HU group was still significantly higher（n=8,P<0.01）;On 6th week after reloading,no significance was found in hcPWV between HU and control groups（n=8,P>0.05）.4.Compared with the control group,the intima-media thickness（IMT）and cross-sectional area（CSA）of the carotid artery in HU group significantly increased（n=8,P<0.01）.However,IMT and CSA of the thoracic aorta and abdominal aorta showed no significant difference between the two groups.According to the weigert staining,the distribution of elastic fibers of the carotid artery,thoracic aorta and abdominal aorta in the two groups were regular and orderly.Masson staining showed that compared with the control group,content of collagen fibers increased in the carotid artery in HU group（n=8,P<0.01）while no significant difference of collagen content was found in thoracic aorta and abdominal aorta（n=8,P>0.05）;RT-PCR showed that the expression of Collagen I and Collagen III mRNA in the carotid artery of the HU group significantly increased（n=8,P<0.01）,but there was no significant difference in expression of elastin mRNA.5.Compared with the control group,the endothelium-dependent vasodilation induced by acetylcholine（ACh）was significantly attenuated in the carotid artery of the HU group,while the endothelium-independent vasodilation induced by sodium nitroprusside（SNP）was not significantly different between the two groups.6.Twelve differentially expressed candidate miRNAs were screened from the carotid arteries of HU and control groups by high-throughput sequencing.Two of them were further identified by qRT-PCR:miR-582-5p level was increased and miR-380-3p level was decreased by long-term simulated weightlessness in the rat carotid arteries.MiR-582-5p increased the expression of collagen in vascular smooth muscle cells.Conclusions:1.Formulas of the pulse wave transit distance(D_hc,D_hf and D_cf)were proposed for the non-invasive measurement of PWV in the rat models,providing a methodological basis for the non-invasive evaluation of arterial stiffness.2.Long-term simulated weightlessness has discrepant influences on arterial stiffness in different regions.The stiffness of arteries above heart including the carotid artery increased,accompanied by thickening of vascular walls,increase in collagen synthesis and attenuation of endothelium-dependent vasodilation.There was no significant change in arterial stiffness below heart including thoracic and abdominal aorta.The increase of arterial stiffness induced by long-term simulated weightlessness was reversible.