Research On The Regulatory Role Of N-acetyl-L-cysteine In Vascular Remodeling Of Aortic Dissection And The Involved Mechanisms

PART ? Application of contrast-enhanced aorta angiography in vivo observation of mouse models with aortic dissection by using micro-CTBackground Mouse models with aortic dissection are the commonly used animal models with aortic dissection at present.The current methods of judging aortic lesions mainly depend on the results of pathology due to the lack of corresponding imaging technology and the animals must be executed in this process.It is impossible to dynamically observe the formation and progress of aortic dissection,which has caused some limitations to the research.Therefore,an imaging method is urgently needed to observe aortic lesions in vivo.Objective The aim of this study is to obtain the optimal imaging scheme for mouse models with aortic dissection by using micro-CT and contrast agent,and to realize the observation of aortic lesions in the mouse models with aortic dissection in vivo,so as to apply it to the basic research of aorta diseases represented by aortic dissection.To investigate the effects of contrast agent types,scanning time and dosage on the imaging effect of contrast-enhanced aorta angiography in mice,summarizing the optimal scheme for aorta imaging.To establish mouse models with aortic dissection and verify the feasibility and validity of the scanning scheme.Methods Two commonly used small animal contrast agents,ExiTron nano 12000 and eXIA 160XL,were selected for our research.Six C57BL/6 wild-type normal mice were randomly divided into two groups,group I(ExiTron nano 12000 group)and group II(eXIA 160XL group).Two contrast agents were injected into mice through tail vein respectively.The contrast-enhanced effect of the two contrast agents was observed by micro-CT scanning and the optimal contrast agent for aorta imaging was selected.Three C57BL/6 wild-type normal mice were scanned by micro-CT immediately,1h,2h,3h,6h and 12h after injection of the optimal contrast agent based on the above experiments.The optimal scanning time was obtained by observing the scanning results.Twenty-one mice with aortic dissection were randomly divided into three groups by using the optimal contrast agent and scanning time obtained by the above experiments.The three groups were injected with different doses of contrast agent,group I(70 ml/25g),group II(100ml/25g)and group III(150 ml/25g).The optimal contrast agent dose for aorta imaging was obtained by comparing imaging results.The mice were anatomized and the aorta specimens were collected and stained with hematoxylin-eosin(H&E).Measuring the aorta diameter of mice by NDP.view 2 software.The aorta diameter measured by micro-CT was compared with which obtained by H&E,verifying the feasibility and validity of the scanning scheme.Results The contrast-enhanced aorta angiography showed that the peak CT value of ExiTron nano 12000 was 610 HU,and the retention time in vivo was about 12 hours.While the peak CT value of eXIA 160XL was 235 HU,and the retention time in vivo was about 6 hours.ExiTron nano 12000 is better than eXIA 160XL in imaging effect and was selected for the following experiment.Micro-CT scanning was performed immediately,1 h,2 h,3 h,6 h and 12 h after the injection of ExiTron nano 12000.The results showed that the CT value of aorta reached the highest level at 2 h after the injection of contrast agent and the imaging effect of aorta was the optimal.The aorta imaging effect of mouse models with aortic dissection injected with different doses of ExiTron nano 12000 was compared.The results showed that the aorta structure and the true or false lumen could be observed when the dosage was 100?l/25g.The aorta diameter obtained by micro-CT was basically the same with which obtained by H&E staining(R~2=0.96).Conclusions Combined Application of Micro-CT and ExiTron nano 12000 could be used to observe the morphology of aorta in mice in vivo dynamically,and the optimal scanning time is 2 hours after injection,and the optimal contrast dosage of contrast agent is100?l/25g.The scanning scheme can be used for contrast-enhanced imaging of aorta in mice and dynamic observation of mouse models with aortic dissection.The scanning scheme is suitable for the basic experimental study of aorta diseases.PART ? Research on the regulatory role of N-acetyl-L-cysteine in vascular remodeling of aortic dissection and the involved mechanismsBackground Aortic dissection is a dangerous and lethal aortic disease.With the aging of population structure,the incidence of aortic dissection,as the representative of aortic dilatation,is increasing year by year.Our previous studies found that the level of L-cysteine in serum of patients with aortic dissection was significantly lower than that of healthy volunteers.Adding L-cysteine diet during the construction of mouse models with aortic dissection can significantly reduce the incidence of aortic dissection,indicating that L-cysteine can affect the occurrence of aortic dissection.N-acetyl-L-cysteine(NAC)is a water-soluble precursor of cysteine,which can be directly converted into L-cysteine in vivo.Previous studies have shown that L-cysteine can regulate cell metabolic activity,reduce DNA damage,and has certain antioxidant and anti-inflammatory effects.Therefore,this study is to observe whether L-cysteine can affect vascular remodeling in mouse models with aortic dissection,thereby affecting the outcome of aortic dissection,and to provide new targets and new ideas for intervention strategies for aortic dissection.Objective After successful establishment of the mouse models with aortic dissection,NAC was added to the diet of mice to interfere with the content of L-cysteine in the mouse models with aortic dissection,and to observe whether NAC could reverse the degree of dialation and severity of aortic lesions and participate in vascular remodeling,thereby affecting the outcome of aortic dissection.Methods Three-week-old C57BL/6 wild-type mice were randomly selected and fed with ?-aminopropionitrile(1 g/kg/d)in drinking water for 4 weeks.Then,1 ?g/kg/min angiotensin II was continuously infused subcutaneously for 3 days by using a micro-osmotic mini pump to establish the mouse models with aortic dissection.At the same time,three-week-old C57BL/6 wild-type mice were fed with water,and then the mice were anesthetized and implanted with a micro-osmotic mini pump(0.9% Na Cl)on their back for 3 days,which was the sham operation group.Forty-nine mouse models with aortic dissection were randomly divided into two groups,and were intervened by NAC(model+NAC group)and saline(model control group).Once the mice died in the course of the study,the time of death was recorded immediately.After 28 days of intervention,the survival curve was drawn according to the recorded results.After 28 days of intervention,the ejection fraction(EF)and the fraction shortening(FS)of the mice were measured by small animal ultrasound to eliminate the effect of NAC on the cardiac function of the mice.At the 1st,14 th and 28 th day after NAC intervention,the changes of aortic lesions in mouse models with aortic dissection were dynamically observed by micro-CT.After 28 days of intervention,mice were sacrificed and the aortas were dissected and separated.The aorta was taken from the root of aorta to the common iliac artery for H&E staining,and the severity of aorta lesions in each tissue sample was assessed according to the criteria for evaluating the severity of aorta lesions.The diameter of each aorta segment(ascending aorta,aortic arch,thoracic descending aorta,suprarenal aorta)was measured.Masson staining was used to detect the collagen volume fraction in the aorta of mice,and to analyze the degradation degree of collagen fibers;EVG staining was used to analyze the degradation degree of elastic fibers;Immunohistochemical method was used to detect the expression of neutrophil specific marker Ly6G+ in the aorta,and to quantitatively analyze the difference of neutrophil content in the aorta of mice in each group.Results The results of survival curve showed that there was no significant difference in survival rate between model+NAC group(23/25,92%)and model control group(18/24,75%),P=0.09.There was no significant difference in ejection fraction(EF)and fraction shortening(FS)between model+NAC group and model control group(EF,P=0.43;FS,P=0.07).The results of micro-CT imaging showed that the difference value of diameter of ascending aorta(-0.087±0.064 mm VS 0.075±0.055 mm,P<0.001)and aortic arch(-0.084±0.066 mm VS 0.066±0.055 mm,P<0.001)in model+NAC group and model control group were statistically significant,while there was no significant difference in the difference value of diameter of descending aorta(0.012±0.036 mm VS 0.02±0.03 mm,P=0.428)and suprarenal aorta(-0.007±0.036 mm VS 0.01±0.021 mm,P=0.055)between model+NAC group and model control group.The incidence of severe aortic dissection(dissection and rupture)of mice in model+NAC group(36%)was significantly lower than that of model control group(66.67%),and there was significant difference between the two groups(P=0.03).The results of H&E staining showed that the diameter of ascending aorta(1.52±0.35 mm VS 1.77±0.43 mm,P=0.027)and aortic arch(1.34±0.18 mm VS 1.47±0.26 mm,P=0.039)in model+NAC group was significantly lower than those in model control group,while there was no significant difference in the diameter of descending aorta(1.16±0.12 mm VS 1.20±0.29 mm,P=0.54)and suprarenal aorta(0.97±0.08 mm VS 0.99±0.08 mm,P=0.45)between model+NAC group and model control group.There was no significant difference in the degradation of collagen fibers between model control group(3.00±0.78)and model+NAC group(2.64±0.86),P=0.09.The collagen volume fraction of mouse aorta in model control group(43.83±6.39%)and model+NAC group(54.37±6.59%)was significantly higher than that in sham operation group(20.60±3.08%).The collagen volume fraction of mouse aorta in model+NAC group(54.37±6.59%)was significantly higher than that of model control group(43.83±6.39%),and there was significant difference between the two groups(P<0.01).There was no significant difference in the degradation of elastic fibers between model+NAC group(2.96±0.81)and model control group(2.76±0.88),P=0.37.The content of neutrophils in mouse aorta in model+NAC group(7.72±3.95%)was significantly lower than that of model control group(10.04±4.43%),and there was a significant difference between the two groups(P=0.037).Conclusions NAC could reduce the degree of aorta dilatation and severity in mouse models with aortic dissection and the incidence of severe aortic dissection(dissection and rupture);NAC could promote the deposition of collagen in the aortic media,and reduce the infiltration and aggregation of neutrophils into the aortic lesions,promoting the forward vascular remodeling of aorta in mouse models with aortic dissection and affecting the outcome of aortic dissection.