| Objective:Use Contrast-enhanced Ultrasound (CEUS) and quantitative analysis technology to assess of renal ischemia-reperfusion injury in rabbits and compared the correlation of each parameter of time-intensity curves (TIC) with its renal Intercellular Adhesion Molecule-1 (ICAM-1) of renal cortical, To evaluate the diagnostic value of CEUS and quantitative analysis technology in the rabbit renal ischemia-reperfusion injury (RIRI). Methods:24 healthy New Zealand rabbits were randomly divided into normal control group and ischemia-reperfusion group, according to the different time points, ischemia-reperfusion group were also divided into 24h,3d,5d 3 subgroups(n=6).Cut hair of rabbit kidney area preoperative, use two-dimensional gray ultrasound to mark the location of rabbit kidney, then, inject Xylazine Hydroch injection (SuMinXing II) at the most abundant muscle in rabbit leg for general anesthesia. After the success of anesthesia, try to exposed skin for operation, Disinfect local skin with alcohol cotton balls for the renal area. Control groups only removed the right kidney, left kidney not be processed, the abdomen was closed. Then be inspected by ultrasonic. Ischemia-reperfusion groups (I24h, I3d, I5d 3 groups, n=6) the right kidney of rabbits were removed, the left kidneys were isolated by non-invasive blood vessel clip,the kidneys were observed changing rapidly from bright red to dark red, It indicated that the lack of blood was successful,It was the occlusion of the left renal ischemia, started to keep ischemia time, released non-invasive artery clip after an hour, kidneys changed from dark to red, It indicated that reperfusion was successful and started to keep ischemia time immediately. At last, the abdomen of rabbits were closed, feed them at clean and tidy room. Use two-dimensional gray ultrasound(B),Color Doppler flow imaging(CDFI),Power Doppler imaging(PDI) and Contrast-enhanced Ultrasound(CEUS) to check at different time points (24h,3d,5d). Compared perfusion of renal with different group。Analyzed time- intensity curves (TIC) of renal cortex of different parts of state patterns by quantitative ultrasound analysis software (wash in/wash out). It have many Parameters, including AT (the time began to increase,Arrival Time, AT), TTP (perfusion peak time,time to peak, TTP), A (perfusion peak intensity difference between B and the intensity at t=0, A), Area (area under the curve, Area), Grad (curve ascending branch slope,gradient between start frame to peak frame, Grad). Each rabbit was killed immediately after examination by CEUS at low temperature, a portion of renal cortical tissue was biopsied for section of pathology, another part of renal cortical tissue was frozen at -60℃.Measured the expression of CIAM-1 of rabbit renal cortical tissue by ELISA based on strictly double antibody sandwich technique, and analyzed the relationship of TIC curve relevant parameters and its expression of CIAM-1.The parameters of different groups of TIC curve were undertaking ANOVA analysis (P<0.05 was regarded as statistically significance); The relationship between AT,PPT,A,Area,Grad of each group and the value of ICAM-1 were analyzed by linear correlation,calculate the correlation coefficient r value (P<0.05 was regarded as statistically significant). Results:Anesthesia was markedly and modeling was good smoothly in all rabbits,They were performed operation in the fully awake state. (1) Conventional two-dimensional gray ultrasound cleared rabbit kidney depth of about 1.3-1.8cm rules from the surface, the size of (2.85±1.03) x (2.23±0.67) cm2, Envelope is Complete, real echo is uniform, CDFI and PDI can display the Perfusion of vascular, and CDFI can detect blood flow velocity of rabbit renal artery, but it does not clearly show the Arcuate artery and its less arterial perfusion. PDI can show the capsule of blood perfusion, but no quantitative scale indicators. (2) The results of Harmonic Ultrasound:①Control group:The contrast agent reached quickly from the renal sinus to renal capsule after rabbit's ear vein was injected.(3-8s) SonoVon Enhance order of the renal segmental arteries, interlobar artery, arcuate artery, renal cortex, renal medulla, renal sinus. At last, the left kidney is a "fireball-like". After about 35-50s the contrast agent began to subside, the same order with order of enhance, but the renal cortex is latest subsided.②Ischemia-reperfusion groups:enhanced mode is the same with the control group, enhancing and fading time is extent by the naked eye, but there is little difference. (3) Parameters of TIC curve①Io group:TIC curves of renal cortex showed increased rapidly and decreased rapidly after the peak, the parameters are as follows:A (15.027±13.488dB), AT(8.628±1.546s), TTP (4.671±0.853s), Area (630.573±137.793dBs), Grad (4.176±0.2911 dB/s);②I24h group:TIC curves of renal cortex showed slow rise, slow decline, The parameters are as follows:A (16.626±15.271dB), AT (12.806±4.011s), TTP (18.187±4.172s), Area (971.799±206.877dBs), Grad (1.498±1.460 dB/s);③I3d group:TIC curves of renal cortex showed slower rise, fall more slowly, the parameters are as follows:A (15.846±16.233dB), AT (13.492±4.978s), TTP (23.692±7.232s), Area (939.190±393.443dBs), Grad (1.196±0.977 dB/s);④I5d group:TIC curves of renal cortex showed it is faster than I24h group and I3d group, but less than Io group, the rate of decline is the same. Parameters are as follows:A (18.655±5.835dB), AT (10.567±3.849s), TTP (13.313±3.913s), Area (964.248±300.197dBs), Grad (2.014±0.474 dB/s). Compare with two groups, it found that:①The values of TTP and AT are the same trend between the two groups:The values of TTP and AT of the group is longer than the control group, it is the crease tends between reperfusion 24h and reperfusion 3d, it is the peak at the third day after reperfusion, it is decrease tend from reperfusion 3d to reperfusion 5d, but the time is longer than the normal control group, the difference was statistically significant (P<0.05).②Values of Grad among groups, the difference was significant (P<0.01):ischemia reperfusion group compared with the control group were significantly lower slope, and the differences between reperfusion group is also very significant. TIC curve of reperfusion 3d is the slowest, TIC of reperfusion 5d pick up, but still slower than the normal control group;③The value of Area of ischemia-reperfusion group was significantly higher than the control group(P<0.01). No significant difference with each ischemia-reperfusion group(P>0.05).④The values of A is not statistically significant among groups (P>0.05). (4) According to the standard curve by calculating standard concentration and the corresponding value of OD and the values of OD samples calculated the sample concentration in the regression equations. The value of ICAM-1 expression in renal cortex content was as follows:I0 group (29.272±5.257ng/ml), I24h group (58.770±6.599ng/ml), I3d group (80.120±10.539ng/ml), I5d group (50.676±6.319ng/ml). Ischemia-reperfusion groups were significantly higher than the control group, P<0.05, the difference was statistically significant. I3d group was the highest, I5d group decreased, although still higher than I24h group, and the difference between two groups were statistically significantly (P<0.01). (5)The results of pathology:normal control group showed clearly renal units, renal tubular and glomerular were neat; I24h group, the epithelial cells of tubular of rabbit renal increased in size, mainly by ballooning degeneration. Significant increase in interstitial, inflammatory infiltration; I3d group, bureaucratic expansion of rabbit kidney tubules, The nuclear of some epithelial cells showed condensation, fragmentation and dissolution, was coagulation necrosis, while necrosis occurred around congestion and inflammatory response with interstitial edema, showing a large number of lymphocytes; I5d group, bureaucratic expansion of rabbit kidney tubules, control-type cells were seen in less bureaucratic, only a few epithelial cells were seen the degeneration and necrosis, Increase in the number of new capillaries, interstitial edema, showed lymphocytic. (6) Compare with between parameters of TIC curve each group and renal cortical expression of ICAM-1 by levels of linear correlation analysis:The value of Grad and ICAM-1 expression was negatively correlated (r=-0.923, P<0.01), The value of AT, TTP and renal cortical ICAM-1 expression was positively correlated (r=0.697, r=0.892, P<0.01), The value of A, Area and ICAM-1 expression have no correlation (r=0.108, r=0.295,P>0.05). Conclusion:(1) CEUS imaging technique is more sensitive than conventional ultrasound on reflecting the artery perfusion of rabbit kidney after ischemia-reperfusion injury; (2) TIC curve can dynamically reflect the change of contrast agent concentration with the change of blood, Quantitative parameters obtained is speed of filling the micro-bubble and sound intensity after balance, which accurately reflect the flow of red blood cell velocity in capillaries and tissue blood volume; (3) A large number of Neutrophils was adhered to endothelial cells and tissue space of rabbit renal ischemia-reperfusion injury within three days. Resulting is severe inflammation while a large number of necrotic-material blocking vascular bureaucratic and kidney function is the most serious injury. Through the "dangerous period "of renal cell function can be partially or fully restored. It is described neutrophil-mediated ischemia-reperfusion injury further. (4) The various parameters of TIC curve and ICAM-1 expression of renal showed that the correlation between the detection of CEUS imaging technology not only quantitative evaluation of the renal perfusion, but also reflects the non-invasive degree of kidney inflammation. (5) CEUS is a kind of non-invasive, safe and reliable method of examination, It can be quantitatively assess the local and global renal perfusion,is expected to be a simple, noninvasive, sensitive examination to response renal ischemia reperfusion injury and reaction nephritis patients. It is new method and idea to detection early, diagnosis early, prognosis and effective evaluation of renal ischemia-reperfusion injury disease for clinical.
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