Experimental Research Of Monitoring Of Rabbit's Renal Ischemic Reperfusion Injury Under The Intervention From The Use Of Root Of Red-rooted Salvia By Contrast-enhanced Ultrasound

Objective:Monitoring of the characteristics for the renal cortex blood perfusion in case of rabbit's renal ischemic reperfusion injury(hereinafter referred to as "RIRI")before and after the intervention from the use of root of red-rooted salvia by contrast-enhance ultrasound(hereinafter referred to as "CEUS"),thus to discuss the application value for CEUS in diagnosing RIRI,as well as in evaluating curative effect for RIRI under the intervention from the use of root of red-rooted salvia.Methods:Select 63 New Zealand Rabbits that are weighted from 2.2kg to 2.6kg,and randomly divide respectively 9 rabbits into sham-operated group(R0),2 hours of perfusion after 60 min of post-operation ischemia group(R1),16 hours of perfusion after 60 min of post-operation ischemia group(R2),24 hours of perfusion after 60 min of post-operation ischemia group(R3),2 hours of perfusion after 60 min of ischemia under intervention from the use of root of red-rooted salvia in 2 days before operation(R4),16 hours of perfusion after 60 min of ischemia under intervention from the use of root of red-rooted salvia in 2 days before operation(R5),24 hours of perfusion after 60 min of ischemia under intervention from the use of root of red-rooted salvia in 2 days before operation(R6).All 63 experimental rabbits should be fasted for 12 hours and be deprived of water for 4 hours prior to the operation.Inject Xylazine Hydrochloride(Sumianxin II)at a dose of 0.2-0.3ml/kg to the experimental rabbit's rich leg muscle area for general anesthesia.Mark out the position of rabbit's kidney before operation using two-dimensional ultrasound.Cut off the hair located in the kidney area and perform aseptic operation after such area is sterilized using alcohol.For group R0,only cut out the right kidney and free left kidney without doing nothing more,then close the abdominal cavity and eat and drink as usual.For 54 rabbits in group R1?R6 their right kidney are cut off and left kidney are separated.Ureters are carefully protected during separation.The artery of left kidney is clipped for 60min using non-invasive arterial clip to cause ischemia.An indication of a successful operation for causing ischemia in Rabbit's left kidney is that the color of left kidney has turned from bright red into dark purplish-red.Then remove the arterial clip to cause a re-perfusion.An indication of a successful re-perfusion of left kidney is that the color of left kidney has turned from dark purplish-red into bright red.The re-perfusion time is thereby calculated.Close the abdominal cavity and start normal eating and drinking once the incision is examined with no active bleeding found.For rabbits in R4,R5 or group,intramuscular injection of root of red-rooted salvia at a dose of 1.5g/Kg 2 days before establishing RIRI model.Intramuscular injection of NS simultaneously at a dose of 1.5g/kg to rabbits in group R0?R3 2 days before establishing RIRI model.Monitor the renal blood perfusion for rabbits in each group at multiple time points using CEUS and save 6min of dynamic images output from CEUS.Through the quantitative analysis software(wash in/wash out)installed on CEUS to analyze the time intensity curve(hereinafter referred to as "TIC"),and parameters include "time to peak"(hereinafter referred to as "TTP"),(time taken for contrast agent instilled to renal cortex to reach peak intensity)and peak intensity(hereinafter referred to as "PI")(the gray-scale value of echo intensity when TIC reaches its peak),which shows the maximum dosage of contrast agent that reaches such section during an unit time interval.Such parameter relates to the perfusion amount for a tissue.There is a certain linear correlation between area under curve(hereinafter referred to as "Area")and blood flow within renal tissue.The gradient between start frame and peak frame(hereinafter referred to as "Grad")reflects the perfusion speed for local tissue.Inject 30ml of air into the vein of Rabbit's ear to kill the rabbit when finishing CEUS examination of each group.Take a pathological section from the left renal cortical tissue for HE staining,and regard the result of pathological section as a reference to analyze and judge the characteristics of renal cortex blood perfusion,and its relation to the renal pathological damage for the group R0?R6 monitored by CEUS.Results:The anesthesia effect for experimental rabbits is good,and model is established as a result.(1)Analyze of TIC parameters:?Compare group R1,R2 or R3 with group R0,"TTP" and "Area" increases while "Grad" decreases(P<0.05),PI value of group R3 is decreased,comparing it with that of group R0(P<0.05),the difference of above which are of statistical significance.PI value of group R1 and group R2 are decreased,comparing it with that of group R0(P>0.05),the difference of above which are of no statistical significance.Differences of TTP,Grad and Area among group of R1,R2 and R3 are of statistical significance(P<0.05),in which,TTP of group R3 is increased,and the decrease of Grad and increase of Area are the most evident.PI differences among group of R1,R2,R3 are of no statistical significance(P>0.05).?If comparing group R4,R5,R6 with group R0,Grad decreases and Area and TTP increases(P<0.05).The comparison of PI value of group R4,R5,R6 with that of group R0 and that of corresponding group R1?R2?R3 are of no statistical significance(P>0.05).After intervention by the medicine,TTP is decreased while Grad is increased,which are of statistical significance if compared with groups without intervention(P<0.05).The Area value of group R4 are of statistical significance if compared with group R1(P<0.05).The Area value of group R5?R6 are of no statistical significance if compared with that of group R2?R3 at identical time points(P>0.05).(2)Pathological results:?There is no evident change of the shape of kidney in group R0 if observed by naked eyes.While under microscopic observation,the renal tubules are in nice order,there is no sign of any necrosis or peeling off of renal tubular epithelial cell,no hyperemia and edema occurred to renal interstitial and no exudation of inflammatory cells;?Pathologic change of IRI group is aggravated as a result of the prolonged time and corticomedullary swells more or less as observed by naked eyes.Under microscopic observation,the proximal renal tubules are arranged disorderly,lumens are expanded,and vesicular degeneration,patchy necrosis and karyopyknosis,dark stained nucleus appears in epithelial cell can be found in more seriously damaged cases,or even cellular casts peel off into the lumens,interstitial inflammatory cell infiltration and hyperemia and edema can be seen as well.However,there is no evident change of glomerulus,either before or after RIRI.? For group R4,R5,R6,which is the RIRI under intervention from the use of the root of red-rooted salvia,we can see by naked eyes that the extent of swelling for corticomedullary is alleviated somehow if compared with identical time point of RIRI.Under microscopic observation,only little epithelial cell is found with Degeneration and necrosis,part of the renal tubular epithelial cells regenerate,only a few cellular casts can be found in lumens,the nucleus increases,chromatin condensation,interstitial edema,hyperemia and edema occurred to renal interstitial are alleviated and inflammatory cell infiltration such as a little neutrophile granulocyte,mononuclear cell,etc.are seen as well.Conclusions:(1)CEUS is capable of monitoring the change of Rabbit's renal blood perfusion under the Intervention from the use of root of red-rooted salvia,which can be used to reflect the curative treatment to some extent,thus to provide a new method for clinical assessment of the curative effect of a drug.(2)CEUS is able to observe the blood perfusion characteristics for RIRI in a dynamic,non-invasive and real-timely manner,and can prompt the progress of RIRI accurately at an earlier stage so that it can offer timely guidance to clinician on the execution of a treatment plan.(3)The parameters acquired from quantitative analysis of TIC curve are of reference value to the assessment of the characteristics of the local microcirculation perfusion.(4)the CEUS quantitative analysis technology features safe,convenient,high accuracy,and an improved capability for displaying blood flow with low flow rate and low flow speed,as a result,it is expected to be a safe,fast and effective testing method for the check of renal functions.