The Study Of Animal Experiment On Ultrasound For The Diagnosis Of Acute Renal Allograft Rejection

ObjectivesAcute rejection(AR) after renal transplantation is the most frequent cause ofearly graft failure, so early, accurate diagnosis of AR and institution of proper therapies are crucial to preserve the function of the transplanted kidney. There is no doubt that Doppler sonography has proven to be an important tool in monitoring postoperative complications of renal allografts over the past decade because of its convenience, noninvasiveness, repeatability, and relatively low cost nature. However, the value of Doppler sonography in diagnosis of AR is still a matter of debate. Therefore, based on color Doppler flow imaging(CDFI) and color Doppler energy(CDE), we aim to explore the value of using ultraharmonic contrast imaging(UHCI) and acoustic densitometry(AD) technique to quantify cortex blood perfusion during different periods of acute renal allograft rejection in this study; and to investigate the relationship between each perfusion parameter of renal cortex and biochemical indices, in order to find more objective and sensitive index of detection and provide valuable information for the early diagnosis of AR after renaltransplantation. Materials and Methods1. The models of acute renal allograft rejection were established. Ten maledogs(between 10 to 15 kg body weight) were divided into 5 groups at random, tow dogs each group. After induction of anesthesia with 3% sodium pcntobarbital(30mg/kg), they underwent renal allograft transplantation with unilateral native kidney nephrectomy. The donor renal was located in the right iliac fossa.2. The examination of ultrasound. The equipment used was HP Sonos 5500 color Doppler ultrasound with L7540(frequency 4¹0MHz) and S3(frequency 1-3 MHz) transducer. The routine examinations of renal allograft were performed every other day after transplantation(T₁, T₃,T₅……) until the animal dead, and the levels of serum creatinine(SCr) and endogenous creatinine clearing(CCr) were measured simultaneously.(1) High frequency and Doppler ultrasound. The transplanted kidney was first scanned in gray-scale mode, and then the equipment was switched to CDFI and CDE mode. The cortical blood perfusion in renal allograft was showed and CDE maps were divided into four grades according to the vascular image level. The spectral Doppler waveforms were obtained from the interlobular arteries and some hemodynamic parameters-the 'peak systolic velocity(PSV), the end diastolic velocity(EDV), resistive index(RI) were measured. Three measurements of these parameters for each dog, and the mean values of them were recorded.(2)Contrast ultrasound. Triggered ultraharmonic imaging was performed after administration of contrast agent "Quanfuxian", using transmit and receive frequencies of 1.3 and 3.6MHz, respectively. The interval of trigger was 3000ms, the mechanical index was 1.5, and all equipment settings were kept constant for each study. When maximum bipolar length of transplanted kidney was obtained, fixuped probe and startuped T-INT condition. Intravenous bolus injection of contrast agent, followed by immediate flushing with 2 mL saline. The dosage of contrast agent was 0.03ml/kg, and repeated injections were given to one animal with an interval of more than 15 min between injections. The all courses of contrast were recorded and stored in MO disk for later analysis.(3)Acoustic densitometry(AD) analysis. To analyse the saved images with AD software. The lunate sampling frame(region-of-interest, 21^21 pixel) was placedover the cortex of transplanted kidney(3mm within envelope), some parameters related to perfusion such as peak intensity(PI), the area under the curve(AUC), the half time of descending(HT) and the mean transit time(MTT) were acquired. Then wash-out 50% slope was calculated as the ratio: (peak signal intensity-50% peak signal intensity)/time interval from the peak to the 50% peak intensity. The above parameters were measured three times and recorded by the mean values.3. Pathological examination. The graft biopsies were done after ultrasound examination every time or necropsy were performed until the animal dead. The samples were placed in 10% formalin and stained with hematoxylin and eosin(HE) and periodic-acid Schiff (PAS) for histopathologic evaluation.4. Statistical analysis. Data were expressed as mean ± standard deviation. The changes of each parameter(PSV, EDV, RL PI> AUC> HT, MTT> wash-out 50% slope n SCr> CCr) at different time point were evaluated statistically using Bonferroni analysis. The correlation analysis was estimated by Pearson correlation coefficient. AP value below 0.05 was considered statistically significant. Results1. The graft biopsies demonstrated that the structure of transplanted kidney wasnormal on Ti and T3, while the renal allografts were beginning to occur AR that became more and more severe from T?to Tn. In view of this, the developmental course of AR was divided into four phases: pre-AR(Ti and T3), the early AR period(T5), the medium ARperiod(T7)r the terminal ARperiod(T9 and Tn).2. CDFI showed that a progressive decrease in PSV, EDV, and a progressive increase in RI with the severity of AR. The changes of PS V, EDV, and RI appeared earlier than the change of SO, so they were the quite good parameters for the diagnosis of AR. But the value of RI might also show normal in the early AR period, it was no specificity in diagnosis of AR.3. CDE showed that it could give more information about the kidney blood supply compared with CDFI in the same of scanning plane and at the same time of AR. We also found that CDE grading of renal allograft was correlated with the degreeof AR. The graded image of cortex obtained by CDE could evaluate the blood perfusion of transplanted kidney well, however the diagnostic sensitivity was high and the diagnostic specificity was not accurate.4. The gray-scale mode ultrasound images after contrast agent administration suggested a distinct difference in pre and post-AR. A marked homogeneous enhancement within the renal allograft cortex was seen before AR, however the cortex of renal allograft with acute rejection showed nonenhancing defects after contrast agent injection.5. Acoustic densitometry analysis indicated that from T5 to Tn after renal transplantation, the PI, AUC, HT, MTT of renal cortex were increasingly decreased, and the 50% wash-out slope was increasingly increased with the severity of AR; furthermore, there were significant differences compared to those on Ti(P<0.05).6. The correlation analysis proved that the PI, AUC, HT, and MTT in renal cortex had a negative correlation'to SCr level and had a positive correlation to CCr level from Ti to Tn. But the tendency of change was consistent between all of the perfusion parameters and CCr level, which exhibited that both of them had a more close correlation. 'Conclusion1. Using high frequency ultrasound, CDFI and CDE can see clearly the distal vessels under renal envelope, and is therefore a good method for assessing cortex blood perfusion of the AR in renal allograft. But CDFI or CDE alone is unsatisfactory to detect AR, while they could improve the diagnostic accuracy of AR and would has a better predictive value of histology than serum creatinine values alone if coupled with CDFI and CDE.2. Combination of UHCI and AD technology can offer more valuable and accurate information regarding renal tissue perfusion than conventional Doppler flow imaging, so it is a ideal, safe, and efficient technique of monitoring changes in cortical perfusion in renal allografts. On the other hand, the PI, AUC, HT, and MTT,obtained by AD analysis, have an excellent correlation with renal cortical blood flow and are shown to be objective and sensitive parameters for determining the status of renal allograft perfusion in our this study. Consequently, these perfusion parameters are expected to make further application in clinic for the early diagnosis of AR after renal transplantation.