| Background and ObjectiveRenal allograft acute rejection, a common complication after kidney transplantation, most likely leads to the lost of the early function of the transplanted kidney, and thus it becomes one of the main obstacles to the survival and function-maintenance of the transplanted renal. Studies have shown that the first sign of renal allograft acute rejection (AR) is the renal micro-vascular endothelial injury. The injury is characterized by the inflammatory and rejection-related molecular markers such as intercellular adhesion molecule, vascular cell adhesion molecule, which are expressed or secreted by the endothelial cells. These markers can promote the adhesion, aggregation and leakage of the platelets and neutrophilic granulocytes along the endothelium. What's worse, the neutrophilic granulocytes can result in inflammatory reaction and release several inflammatory mediators or pro-inflammatory cytokines into the renal interstitial tissues, such as lymphocytes, tumor necrosis factor-a, interleukin-1, thromboxane A2 and so on, which then lead to the injury of renal capillary vessel and cells. Therefore, to the extent that vascular endothelial injury is an important feature at the early stage of renal transplant rejection, we can presume the presence of AR considering the specific molecular markers such as ICAM-1 expressed by vascular endothelial. In fact, scholars have made long researches and conducted a variety of studies on the diagnosis of renal transplant rejection, especially on the diagnosis of acute rejection, in order to make an accurate diagnosis and treatment before irreversible changes occur in the organization. Many researchers have carried out large numbers of explorations and studies from the aspect of the immunological monitoring,renal imaging and pathology testing and so on. However, different means and methods of examination have their own limitations.For example, renal scintigraphy can evaluate the ischemic injury, but it can not identify the acute rejection, acute tubular interstitial nephritis and cyclosporine toxicity at the early stage after the renal transplantation, and only has certain value for the recovery and short-term or long-term of graft survival. Other non-targeted radiologic imaging methods, including MRI and PET, can provide valuable information for the assessment of morphological and metabolic changes respectively associated with rejection. Despite the advantages, these methods have some disadvantages which can not be solved. For example, instruments with high requirements, not the bedside examination, the lack of stability of the radioactive tracer, and the radioactive contamination have made certain restrictions in the clinical applications.Nowadays, histopathological examination is still the most accurate method for the diagnosis of AR. However, due to its injury to the kidney, bleeding and other serious complications, it can not be used as routine monitoring tools. Thus, it is in great clinical significance to search for a safe, noninvasive, sensitive monitoring method for early diagnosis of AR.In recent year, with the appearance of targeted ultrasound contrast agents, targeted ultrasound molecular imaging gradually becomes a reality, which has greatly expanded the scope of application of ultrasound. Its role as an intravascular tracer in being detected while adhering to endothelial surface makes it possible to apply the molecular lever ultrasound technology in the early diagnosis of various diseases. Currently, targeted ultrasound molecular imaging has successfully evaluated in the areas of vascular endothelial inflammation/angiogenesis of kidneys, limbs, tumor, and it has also conducted a preliminary exploration to the heart transplant rejection. But its targeted imaging research makes no progress, because renal transplantation models are difficult and fussy to build. After many years of exploration and technical improvements, our laboratory has made considerable achievements in building targeted ultrasound micro bubbles, the size, concentration, stability and antibody rate of which have reached the international equivalent or higher level. And we have successfully assessed the endothelial inflammatory response in the kidney/heart and other parts by targeting intercellular adhesion molecule (ICAM-1). On the basis of the preliminary study, this study tries to combine targeted micro bubbles and Ultrasound contrast to evaluate and diagnose of AR early, which will undoubtedly have great clinical significance.In conclusion, on the basis of common lipid micro bubbles, we have constructed the micro bubbles targeted to ICAM-1 by combining the anti-rat-ICAM-lmonoclone antibodies to the shell of general lipid microbubbles via "avidin-biotin" bridging chemistry. By using this micro bubbles targeted to ICAM-1 (MBI) and the control microbubbles (MB), the VI (video intensity) of renal were measured separately by CEU. We hypothesized that the AR could be accurately evaluated with microbubbles targeted to ICAM-1 by using CEU.Methods1.Microbubbles preparationGeneral lipid microbubbles (MB) and lipid microbubbles with biotin were prepared by sonication of perfluorocarbon gas (C3H8) with aqueous dispersion of several lipids in determinate ratio. After being washed (1×) to remove excess free unincorporated lipid, streptavidin in determinate ratio were added to the lipid microbubbles with biotin, then washed (1×) to removed excess free unincorporated streptavidin and the biotin conjugated mouse-anti rat ICAM-1 monoclone antibodies and isotype control antibodies in determinate ratio were added to complete the preparation of MBI and MB. At last, the MBI and MB were washed (1×) to remove excess free unincorporated antibodies. Both MB and MBI were storaged in refrigerator at 4℃.2. Evaluation of microbubble in vitro:2.1 The mean diameter and density in both MB and MBI were measured by coulter counter. Using green fluorescent-labeled antibody for anti rat ICAM-1 monoclone antibody to identify the linking antibodies on the MBI.2.2 Assessment of microbubbles targeted to ICAM-1 with Parallel plate flow chamberThe binding and retention of targeted microbubbles to ICAM-1 immobilized on a culture dish were assessed in a parallel-plate flow chamber. Targeted microbubbles drawn through the flow chamber coated with ICAM-1 (1000ng/ml) at a shear stress of 0.5dyn/cm2. All groups use 3 samples according to the recommended protocol. The adhesion abilities of control microbubbles with isotype antibodies were tested as control groups. Quantitative analysis of microbubble accumulation was performed by counting the number of microbubbles adhered in the observed area and a graph of microbubble accumulation with time was plotted.3. Rat model for acute transplant rejectionThe 10 male Wistar rats and SD rats respectively(250-300g) used in this study were fasted 24 hours before surgery, but free of water. Wistar rats were anesthetized by intraperitoneal injection of Pentobarbital (1g/kg). The skin was incised by a longitudinal cut in the middle of abdomen. Left ureter, left renal artery and vein,vena cava and abdominal aorta were separated,4-0 silks were placed in the upper and lower of inferior vena cava and abdominal aorta. A small hole was cut between the traction line of the inferior vena cava to do as a perfusate outflow tract.At the same time, Cut a hole in the abdominal aorta anterior to prime heparin Ringer's solution(4C,25u/ml), Until the color of kidneys paled and outflow of liquid of inferior vena cava turned clear, The amount of perfusate was 5-10ml. After removing the left renal artery and vein, ureter,the left kidney was placed in the small cap filled with Ringer's lactate(0-4℃).After abdominal incision, SD rats were ligated renal artery and vein and ureter near the left renal hilum, and left kidney was removed. the same name of renal arterial and venous of the kidney of donor and recipient were anastomosed in turn. The kidney perfused well, turned pink very quickly, and functioned immediately. Rat model established successfully.4.CEU imaging:Ten SD rats with renal allograftt acute rejection (AR) and ten normal SD rats were performed with CEU respectively by using MBI and MB, the intravenous injection of 1×109 microbubbles were made in random order with 30 minutes interval. After Three minutes of intravenous injection, microbubbles in the circulation were eliminated, the ultrasound signal (video intensity, VI) from MB and MBI were measured by second harmonic CEU imaging with pulsing interval time (PI) of ten seconds and a mechanical index (MI) of 0.2, transmission frequency of 7.0 MHz. After the first picture of CEU imaging being taken, the microbubbles were destroyed by two to three seconds of continuous imaging with a high MI of 1.9 and the background subtracted VI of Renal were measured.5.Examination of pathology and immunohistochemisty:After CEU imaging, all kidney of experimental rats were cutted for the examination of pathology and immunohistochemisty.6.Statistical analysis:Data are expressed as mean±SD. Experiment one: Comparisons between different groups were made with repeated measures; comparison between the two groups within each time point using One-way ANOVA to analyze; Experiment two:Comparisons between different groups were made with independent t-test, Interval comparisons were made with two stages cross-over design. Differences were considered significant at a value of P<0.05(2-sided).Results1.Results for microbubble preparation:The density of MBI and MB is about1.5×109/ml and 1.7×109/ml separately, the mean sizes for MBI and MB were about 2.71μm and 2.86μm respectively.2.Results for evaluation of microbubble in vitro:The anti-Rat ICAM-1 monoclone antibodies linked well to the surface of microbubbles, which were observed with fluorescence microscopy, shell of ultrasound microbubbles showed obvious green fluorescence, That shows targeted microbubbles constructed successfully.3.Results for evaluation of MBI using parallel plate flow chamber:Keeping the shear stress and microbubble concentration constant at 0.5dyn/cm2 and 2×108/ml, respectively, the microbubble accumulation over a 6-min interval was assessed. Numbers of microbubbles increased gradually with the increase of time. But there was only minimal and no significant adhesion in the control group.there was obvious differences(P=0.000).4.Results for CEU imaging:A significant enhancement in ultrasound was observed in transplant kidney of MBI-group. Increase in VI value of transplant kidney region in MBI-group was great and it amounted to 27.0±7.4. However, increase in VI value of transplant kidney in MB-group was minor, it was just 10.2±2.4. Difference was evident in transplant kidney between of the two groups (P=0.000). The VI in transplant kidney of MBI-group is 3.7±1.3 times to the MBI-group of normal kidney (7.7±2.2) and 4.2±1.2times to the MB-group of normal kidney (6.5±1.0). There was obvious difference between the two groups (P=0.000) The VI in transplant kidney of MB-group is 1.4±0.4 times to the MBI-group of normal kidney and 1.6±0.6times to the MB-group of normal kidney. There was obvious difference between the two groups (P<0.05)5. Results for examination of pathology and immunohistochemisty:Pathological examination showed that renal tubules arranged well and there was no congestion, interstitial edema in the normal kidney; We observed tubular epithelial cell becomed swelling, degeneration, and necrosis. Renal interstitial edema and neutrophil increased was also observed in transplant kidney. It was indicated by immunohistochemisty that the expression of endothelial ICAM-1 increased in transplant kidney compared to normal kidney.Conclusions1. Parallel plate flow chamber experiments show that targeted microbubbles can be successfully constructed via "avidin-biotin" bridging chemistry. The adhesion performance of targeted microbubble was very well, which indicated targeted microbubbles can be Possiblely used to evaluate the severity of lesions.2. Anastomosis the same name of renal arterial and venous of donor and recipient, which reduced the impact on the circulatory system, ensuring a good postoperative survival, mading a solid foundation for further the experiments.3.Microbubbles targeted to ICAM-1 (MBI) and CEU that create "active targeted CEU imaging" can effectively evaluate the acute renal allograft rejection injury in rat, and may be used to evaluate the microvascular inflammation and other endothelial responses. |
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