| Objective: Through the establishment of chick embryo yolk sac membrane(Sac)vascular injury model,to investigate the effect of Direct Current Electric Field(DCEF)effect on blood vessel growth model,which lays a foundation for further study of exogenous DC electric field on microstructure to ischemic injury repair.Methods:The fertilized chicken embryos(Roman chicken embryo)were sterilized with 75% alcohol solution and incubated in incubator.The temperature was 37.5 ℃ and the humidity was >70%.After incubated for 72 h,they were used to construct the chick embryo model(outshell model),five of them were observed under the anatomical microscope and recorded the blood flow of the Sac membrane of the chicken embryo.According to the blood flow,the Photoshop software was used to create a layer to simulate the Sac membrane blood vessel simulation.Then the outshell model of chicken embryo(40 in total)were randomly divided into 4 groups,each group of 10.group A as the control group,did not deal with;group B was ligation group,ligation of right main abdominal vessels only;group C without ligation of the right abdominal main blood vessels,but the loading of DCEF 40 m V/mm,which left of the embryo was positive,right was the negative;group D was performed with ligation of the right abdominal aorta vessels,and loading the DCEF 40 m V/mm,which left of the embryo was positive,the right was negative.groups A,B,C,D were placed in a constant temperature and humidity box to hatch,37.5 ℃,humidity >70%,and connected to the computer for real-time dynamic shooting based on CANON EOS Unitity software,30 min interval shooting a total of shooting 24 h.group A continued incubating until the embryos died.Record the chicken embryo survival timein vitro.After finishing the captured images,0h,6h,12 h,18h,and 24 h chick embryo vascular pattern were obtained respectively.Gray scale and brightness on the image value dealed with Photoshop software,Image J software used to analyse the changes of vascular length,angle and density.Group B,D needed to record the time and direction of the right main blood vessels after ligation.Result: 1.The model of chick embryo Sac membrane was established,and the chicken embryo could survive and continue to develop in the incubation chamber with temperature of 37.5 ℃and humidity >70%,with an average survival time of 82.65+ 14.07h(n=10).2.Through the observation of chick embryo Sac membrane blood flows(n=5),chick sac membrane contained a total of two sets of symmetrical growth of blood vessels:(1)abdominal aorta and vena comitans and dorsal aorta and vena comitans.Abdominal vessels located in the right of the embryo,dorsal vessels located in left side.this set of blood vessels during normal development were not across the embryo axis;(2)The blood vessels located in the upper and lower body of the embryo were the superior and the inferior veins.3.After ligation of the main vessels of the right side of the abdomen,the growth rate and the growth angle of the left side blood vessels were changed,and the left blood vessels could be circled with the right regenerating vessels.4.(1)The rate of vascular growth: the growth rate of the left terminal branches was relevant with the ligation of right abdomen main vessels,DCEF loading and DCEF loading direction: 1)Cosα0>0,The angle between the electric field direction and vascular growth direction <90°,ligation of right abdominal aorta blood vessel and DCEF would promote the growth of blood vessels,between groups during the same period were D>B>C>A(P<0.01),the difference was statistically significant;but the same stimuli,and the growth rate of the vascular changes were not(incubation time 24h),group at different time growth rate had no significant difference(P>0.05).2)Cosα0<0,The angle between the electric field direction and vascular growth direction>90°Ligation of the right side of the abdomenmain vessels promoted the growth of blood vessels,while DCEF would inhibit the growth rate of blood vessels with the incubation increasing.There was significant difference in B,D>A,C(P<0.01)(6h-0h);while there was no significant difference between group A and group C;group B and group D(P>0.05).As the incubation time increased to 6h,DCEF had a certain inhibitory effect on the vascular growth,12h-6h,18h-12 h,24h-18 h group: B,D>A>C(P<0.05),the difference was statistically significant.In the same group at different period,C group: 6h-0h group >12h-6h group >18h-12 h group,24h-18 h group(P<0.05),the growth rate will be inhibited with the increase of electric time increased,the difference was statistically significant;in D group,24h-18 h group,the slower growth than the first three hours group(P<0.05),there was statistical significance the difference.3)DCEF loading direction and the direction of vascular growth,the same group of the same period of time,in group C and group D,Cosα0>0 group vascular growth rate were greater than Cosα0<0 group(P<0.05),the difference was statistically significant.(2)Changes in vascular deflection angle:The changes of the left side of the terminal branch vascular growth deflection angle was related to the ligation of the main vessels and the DCEF loading.The effect of the ligation of right side abdominal aorta was greater than that of DCEF loading.The same period of time were: D>C>B>A(P<0.01),the difference was statistically significant.In the same group,in the group B,C,D respectively,in the rest of the time,the changes of vascular deflection angle were greater than those of the 24h-18 h group vascular deflection angle(P<0.05),the difference was statistically significant.(3)The change of vascular density was mainly related to the incubation time and the ligation of the main vessels of the right abdomen,which was not related to the DCEF loading.Different groups at the same time(24h),B,D group(ligation group)>A,C group(without ligation)(P<0.05),the difference was statistically significan.There was no significant difference between the groups at the rest time points.In the same group,the density of B group and D group at 18 h and 24 h were increased compared with 0h(P<0.05).(4)Vascular regeneration: In group B,the time of regeneration of the main blood vessels was later than that of D group,and the difference was statistically significant(P<0.05);The angle between the direction of growth of the main blood vessel and the horizontal direction:group B>group D,the difference was significant(P<0.05),that was,the angle between the direction of the growth of the main blood vessels and the horizontal direction(electric field direction)of D group was smaller.Conclusion: 1.The model of chick embryo Sac cultured in vitro and the model of vascular ischemia were successfully prepared,and it was proved that it was feasible to regulate the chicken embryo Sac membrane.2.The study methods of the growth rate,growth angle and the density of the Sac membrane of chick embryo were established.3(1)the growth rate of blood vessels was related to the DCEF loading,the electric field direction after DCEF loading,and the ischemia and hypoxia induced by the ligation of the blood vessels.When the direction of the vascular growth was consistent with the electric field,DCEF promoted the growth of blood vessels.When the direction of vascular growth was opposite to the electric field,DCEF inhibited the growth of blood vessels.The effect of ischemia and hypoxia on the vascular growth rate was greater than the effect of DCEF on the vascular growth rate.When the direction of vascular growth was consistent with the electric field,the two had synergistic effect.(2)the left terminal blood vessel growth angle was relevant with DCEF loading and local tissue ischemia and hypoxia,blood vessels would move toward the direction of the electric field and the deflection of hypoxic ischemic site.The effects of local tissue ischemia and hypoxia on the left end vessel deflection angle is greater than the effect of DCEF on vascular deflection angle.(3)The density changes of left vessels mainly related to vascular tissue ischemia,hypoxia and incubator time factors.While tissue was ischemia and hypoxia,in order to meet the needs of the body,the chick embryo would promote the regeneration of the blood vessels.(4)Angiogenesis was related to the local tissue ischemia and hypoxia in the ischemic model of chick embryo Sac membrane.DCEF could promote the regeneration of blood vessels to some degree,and the direction of vascular regeneration was affected by the electric field direction after DCEF loading. |
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