An Experimental Study On The Brain Blood Supply After Ligating The Left Subclavian Artery And Common Carotid Artery

The Aortic dissection is one of the common aortic diseases, and the blood penetrats vessel intima slit into the space between intima and ultima, resulting in the true and false lumen. The main treatments include medical therapy, endovascular stent-graft repair and surgery. Blood pressure control is the main method in medical therapy, and endovascular stent-graft repair has became an effective treatment, including security, effective and less complications, etc. Its exact effect and many other advantages, gradually broaden the scope of treatment from the drop department of aortic arch to the rise department. In the process of treatment, we often meet with a problem about how to deal with the left common carotid artery and the left subclavian artery. But blindly coverage may cause symptoms of cerebral ischemia, and excessively expanding branch bypass indications will increase surgical risk, so there is no uniform conclusion about whether carry out direct coverage or bypass surgery, and also have no systemic animal experimental study. Therefore, this research simulates completely covering the left subclavian artery and the left common carotid artery in clinic to set up a corresponding animal model for a question about the influence of blood supply to the brain and function after ligating the left subclavian artery and the common carotid artery, also observe expressions of nuclear factor-κB and hypoxia-inducible factor, and morphological changes. All that provide the experimental basis about how to deal with aortic arch branch to avoid blindly coverage the left subclavian artery and/or the left common carotid artery, also know well bypass indications among aortic arch branch.Part one The fabrication of an experimental animal modelObjectiveDissected the rabbit’s left subclavian artery and the left common carotid artery, and builded a model for further experimental study.Methods1. Experimental animalThirty healthy adult Japanese white rabbits, regardless of sex, weight in3～3.5KG, clean grade, were supplied by experimental animals cultivation company of Wanqianjiahe in Wuhan.2. Inclusion criteria of animalThe selective arteriography were carried out in all animals before surgery, and only the animals, with complete Willis ring, were fitted into our research.3. Fabrication ProcessArteriography process:Each rabbit was anesthetized by ear vein injection of30%solution of pentobarbital sodium, horizontal position, and the fluff on the chest, neck and inside of lower limbs was eliminated. Dissected the rabbit’s right femoral artery and carried out the arteriography process.Operation process:Kept the spontaneous breathing, and cut skin along midline of the sternum since the sternoclavicular joint to processus xiphoideus after disinfection. Cut open sternum from2cm above processus xiphoideus to a head, and were careful to protect the pleura, then small animal open device propped up chest to expose the left common carotid artery and left subclavian artery, set of3-0silk (Johnson medical equipment Co., LTD) for double ligation.ResultsThis experiment involved thirty health adult Japanese white rabbits, Willis ring complete revealed by DSA(Digital Subtraction Angiography), preliminary study included three cases, two sucsessfull, and one die of pneumothorax; model succeeded twenty-four cases, success rate88.9%, failure three animals including two dying of pneumothorax and one dying of uncontrolled hemorrhagic, randomly divided into three groups, and eight rabbits per group.ConclusionsThe research was based on clinical background which covered the left subclavian artery and even the left common carotid artery for expanding anchor area or repairing pathological changes in thoracic endovascular aortic repair. It was important to expose the left subclavian artery and the left common carotid artery, because we would ligate the left subclavian artery and the left common carotid artery in subsequent experiment.We chose rabbit as the research object, because its cerebrovascular was similar with the human cerebrovascular structure, the rabbit as a medium of experimental animals, easier feeding, reasonable cost, had became the first choice of cerebral ischemia model animals. And its pleural cavity which was divided into independent two parts, made the experiment easy for avoiding the use of breathing machine. Small animals such as rats and mice, were not suit for instrument testing for fine vessel, while, large animals such as dogs and pigs, costed too much, and were difficult to feed.Of course, this model had its shortcomings, because the rabbit left subclavian artery position was deeper, it was more difficult to ligate through the neck incision. We chose thoractomy method to build models, then increased the surgical trauma. Part two An Experimental Study on the Brain Blood Supply and the Function after Ligating the Left Subclavian Artery and Common Carotid Artery in Rabbit ModelObjectiveTo explore the effect on the cerebral blood supply and function after ligating the left subclavian artery, the left subclavian artery plus the left common carotid artery.Methods1. Grouping and experimental treatmentTwenty-four rabbits, which were prepared in former part, were randomly divided into three groups (8rabbits per group), ligating the left subclavian artery group, ligating the left subclavian artery plus the left common carotid artery group and normal control group without ligation, respectively.2. Digital subtraction angiographyThe digital subtraction angiography was carried out in group1and2after ligation to observe the result of ligation and collateral circulation.3. Data acquisition and computeAfter the digital subtraction angiography, the ultrasonic testing was carried out immediately, the7.5MHz ultrasonic transducer was set both neck, to observe the change of blood flow, including vessel diameter, peak systolic velocity, mean blood flow velocity, and resistive index about both carotid artery and vertebral in level of cervical vertebra3and4. All of the parameters were measured for four cardiac cycle, and took the average, then computed blood flow volume in formula [BFV (blood flow volume)=Vm×(DIA./2)2×π×60] and total blood flow into cranial (TBFIC)(TBFIC=BFVLCCA+BFVRCCA+BFVLSA+BFVRSA). And we would observe the collateral circulation pathway by angiogram, including evaluation of the brain function. In the process, we observed animal life signs, the sports ability, and gave a score about brain function influence; took the scores>2points as ischemia injury.4. Data statisticsAll the data were analysised in SPSS13.0statical analysis software, the continuous variables were presented as means±standard deviation for normally distributed data. The differences between groups were examined by least significance difference (LSD) compared to one way analysis of variance (ANOVA) among groups, respectively. A p value of less than0.05was considered.Results1. Collateral circulation inspectionThe aortic arch angiography showed no forward blood imaging in the left subclavian artery and the left common carotid artery after ligating the the left subclavian artery and the left common carotid artery. The digital subtraction angiography of group1and group2showed that the Willis ring was more obvious than before ligating, the collateral opened, and the right brain blood flow through the collateral circulation supplied the left.2. Changes of total blood volume into brainFirst group blood flow volume was58.40ml/min, and second group blood flow volume was50.55ml/min, compared with control group of57.16ml/min. Although the average blood flow volume of second group had decreased than control group, and the first group slightly increased, but the difference among three groups was not statistically significant. It suggested that some arteries play compensatory mechanism, which enabled the total blood volume into brain to be more stable.3. Changes of the right vertebral artery blood flow dynamic (table1)The difference of the vertebral artery diameter among three groups was not statistically significant. But the difference of RI among three groups,was significant(P=0.030,0.000,0.003), control group was highest than the other groups, while group2was the last. Although PSV, BFV, and Vm of group1were higher or lower greatly than control group, but had no statistical significance, and the second was higher than the other groups, following statistical significance (P=0.000,0.000,0.000), the same as comparison of the two experiment groups(P=0.000,0.001,0.000). It suggested that only ligating the left subclavian artery had no significant effect on PSV、BFV and Vm in contralateral vertebral artery, except RI decreased; while ligating the left subclavian artery and the left common carotid artery played the significant compensation, RI reduced, PSV speeded, and BFV increased, in order to ensure less effect on the cerebrum blood.4. Changes of the right common carotid artery blood flow dynamic (table2)The difference of the vertebral artery diameter among the three groups was not statistically significant. The RI of experiment groups was less than normal control group, the difference was significant (P=0.005,0.007), but the difference between experences groups was no significant. The PSV, Vm, and BFV of the right common carotid artery in group2were higher than that in normal control group with significant difference(P=0.000,0.000,0.000); the PSV in group1was lower than normal control group, but the Vm and BFV were higher, the difference between group1and control group was not significant. Compared with group2, the difference of PSV, Vm and BFV in experiment groups was significant (P=0.000,0.000,0.001). The results showed that group1could meet the brain blood needs during anesthesia by decreasing the blood flow resistance of the right common carotid arterial; however, the experimental group2still needed the right carotid artery to play more compensation, not only blood flow resistance decreased, but also the flow velocity speeded up and BFV increased. 5. Changes of the left vertebral artery blood flow dynamic (table3)The difference of the left vertebral artery diameter among the three groups was not statistically significant. The reverse flow was seen in the left vertebral artery,and RI,PSV,Vm and BFV were lower than control group, the difference had statistical significance(P=0.004,0.000,0.002,0.002; P=0.013,0.000,0.005,0.025); the differences between group1and group2was not significant. The above results showed that the cerebrovascular collateral circulation played its compensation function, after ligating the left subclavian artery and the left common carotid artery, that maked the right blood supply to the left, to prevent the afterbrain and left upper limb ischemia.6. Changes of the left common carotid artery blood flow dynamicThe difference of the left common carotid artery diameter among the three groups was not statistically significant. The difference of the left common carotid artery RI, PSV, Vm and BFV between group1and control group was not statistically significant. It didn’t detect signal of blood in the left common carotid artery after ligating left subclavian artery plus left common carotid artery. The results showed that only ligating the left subclavian artery had no obvious influence on the left common carotid artery blood flow dynamic, and the reverse blood flow wasn’t detected in the left common carotid artery after ligating left subclavian artery plus left common carotid artery.7. Brain function evaluationDuring eight hours for experimental observation, animal vital signs was normal, the limb didn’t moved disorders, and could quickly respond to the stimulation in the three groups, brain function scores were all less than2points, the difference of each other was not statistically significant. The results suggested that it didn’t find some difference for performance and brain function scores in seconds between only ligating the left subclavian artery and ligating both the left subclavian artery and the left common carotid artery, effect on the brain significant difference, withal, we still needed further research.Conclusions The research showed that the total blood flow volume to brain always not significantly changed after ligating the left subclavian artery and the common carotid artery under the condition of complete Willis ring. The reverse flow were seen in the left vertebral artery with reduced blood volume, and supplied to the left upper limb through the axillary arterial. RI of the common carotid artery and right vertebral artery decreased, but no effect on PSV and BFV, after only ligating the left vertebral artery, it suggested that the Willis system still had potential compensatory ability to meet the needs of cerebral blood increased; the right carotid artery and the right side vertebral artery played great compensatory function, after ligating the left subclavian artery and the left common carotid artery, its vascular resistance dropped, blood velocity and volume increased, just it made the total blood flow into the cranial less decline in the anesthetized state. It suggested that it had no obvious effect to brain supply only covering the left subclavian artery under the condition of complete Willis ring, but covering the left subclavian artery and the left common carotid artery at the same time could lead to imblances between blood supply and demand, although the opposite common carotid artery, vertebral artery and Willis system played its compensatory.We couldn’t have a sign of cerebral ischemia after ligating the left vertebral artery or the vertebral artery plus the left common carotid artery, for the compensatory of the opposite common carotid artery and vertebral artery, and we would carry out a further study on differences of both.Part three An Experimental Study on Histopathological Changes and the Expression of Nuclear Factor-KB and Hypoxia-Inducible Factor after Ligating the Left Subclavian Artery and Common Carotid Artery in Rabbit ModelObjective To study whether lead to ischemic damage after ligating the left subclavian artery and the left subclavian artery plus the left common carotid artery in rabbit model.Methods1. Experimental animal selection and grouping were the same as part two;2. Experimental treatments were the same as part two;3. Methods and principle3.1TTC(2,3,5-triphenyltetrazolium chloride) stainingTTC dyeing could make the normal tissue bright red, and infarction area a pale colour. After8hours ligating the rabbit left subclavian artery and the left common carotid artery, formaldehyde perfusion and drawing materials, the first four brain tissue was kept in the below20℃refrigerator for30minutes, then all the tissue which transformed into histotomy, every piece of thickness kept in5mm, would put in the TTC dye liquor, dyeing30minutes for avoiding light, calculated infarction area percentage with image analysis software (Image Pro Plus Version4.5, the American Media Cybernatics company).3.2HE staining and SABC (Strept Avidin-Biotin Complex) methodBrain tissue was paraffined and transformed into paraffin slice, part of the slice dewaxed and gradient dehydrated, then HE dyed; Part of the slice were inactivated endogenous peroxidase of tissue in3%H2O2role20minutes; Distilled water to wash, microwave repaired antigen, added the NF-κ Bp65or HIF-1a polyclonal antibodies,37℃for30minutes,4℃for the night; PBS washed three times, added the second antibody,37℃for30min, then PBS washed, added SABC complex,37℃reaction30minutes; after PBS and distilled water to wash, added DAB reagent, controlled showing color time under a microscope. PBS instead of the first antibody as blank control, taken known as the positive slice specimens for control positive, observed the expression of NF-κB and HIF-1a in brain tissue.4. Data statisticsAll the data were analysised in SPSS13.0statical analysis software, the continuous variables were presented as means±standard deviation for normally distributed data. The differences between groups were examined by least significance difference (LSD) compared to one way analysis of variance (ANOVA) among groups, respectively. Ap value of less than0.05was considered.Results1.TTC stainingThe infarction area showed pale after TTC staining in contrast with bright red in the non-infarction area. The normal control brain stained bright red, and tissue also showed bright red after ligating the left subclavian artery without obvious pale infarction area. After ligating the left subclavian artery and the left common carotid artery, only one animal(12.5%) part of left brain stained pale, infarction area percentage for15±5%. The results showed that ischemic damage wouldn’t come out after ligating the left subclavian artery, while ligating the left common carotid artery at the same time, cerebral ischemic damage obvious, cerebral infarction area had appeared in some animals.2. Pathological ObservationAt the end of experiment, we took the brain tissue into pathology examination using optical microscope(×200), and the results indicated that the control group brain’s structure was clear, cells were arranged in order, and the nucleus was clear, the glial cells’structure was normal; but when we ligated the left subclavian artery, the cells was in order, a neuron structure was normal, part of the brain tissue was mild edema, a few inflammatory was oozy, compared with the control group, the difference of half quantitative analysis results was not statistically significant; but in group2animals, part of the brain edema was obvious, neurons shrinked, and inflammatory exudate increased, compared with the control, the difference of half quantitative analysis results was statistically significant (P=0.000), all the index were higher than group1(P=0.000). The results indicated that ligating the left subclavian artery had less effect on blood supply to the brain, and lead to less obvious organization ischemic injury;but part of the brain edema was obvious, neurons shrinked, and inflammatory exudate increased, that resulted from ligating the left subclavian artery and the left common carotid artery.3. The Expression of NF-κB and HIF-1αIn this study, the normal control group and the experimental group1were occasionally expressed, and had no significant difference in two groups. After ligating the left subclavian artery and the left common carotid artery, both expression increased significantly, some cells were stained tan, compared with the control, difference was statistically significant (P=0.000,0.000; P=0.000,0.000), and all the results were the same as comparation between the experiment groups(P=0.000,0.000; P=0.000,0.000). The results showed that only ligating the left subclavian artery leaded to less obvious ischemic damage on the brain, While the brain tissue ischemic changes became obvious and inflammatory reaction aggravated after ligating the left common carotid artery plus the left subclavian artery, which induced the high expression of NF-κB and HIF-1α.ConclusionsBrain morphology had no obvious changes and the expression of NF-κB and HIF-1α hadn’t greatly increased, it suggested that ligating the left subclavian artery caused obvious ischemia pathological changes and biochemical indicators increased significantly under the condition of complete Willis ring; even under the condition of complete Willis ring, ischemia pathological changes would become obvious and the NF-κB, HIF-1α expressed greatly after ligating the left common carotid artery at the same time. All those results further supplied experiment basis for the feasibility of covering the left subclavian artery forever and the necessity of bypass surgery after covering the left subclavian artery plus the left common carotid artery.