Ultrasound Molecular Imaging Of Cardiac Acute Rejection With Lymphocyte-microbubble Complexes

Part 1.The test of lymphocyte chemotaxis in cardiac acute rejection and the fabrication of lymphocyte-microbubble complexesBackground:Acute rejection?AR?is one of the main complications after heart transplantation.Lymphocytes are the dominant reactive cells mediating AR.The study aims to fabricate ultrasound contrast agents with lymphocytes to target cardiac AR.Methods:Rat heart transplantation models were established.In the allogeneic group,Brown Norway rats were donors and Lewis rats were recipients.In the syngeneic group,Lewis rats were both donors and recipients.At 2 h after transfusion of DiR-labeled lymphocytes,main organs of heart recipients were obtained for ex vivo fluorescence imaging.At 15 min post injection of DiI-labeled lymphocytes,cardiac allografts were retrieved for fluorescence microscopy.Anti-CD4 antibody-conjugated microbubbles(MB_CD4)were obtained and incubated with lymphocytes to fabricate lymphocyte-microbubble complexes?cell-MBs?.Results:At 2 h post DiR-labeled lymphocyte transfusion,the fluorescence signals in allografts were significantly higher than those in isografts.At 15 min post injection of DiI-labeled lymphocytes,lymphocyte adhesion with allograft endothelia and infiltration in myocardial interstitium were observed.Microscopic images illustrated the binding of lymphocytes with microbubbles,and cell-MBs were successfully fabricated.The diameters of cell-MBs detected by Optical Particle Sizer were 3.16±0.2?m.Conclusion:Exogenous lymphocytes could be chemoattracted to cardiac allografts and bind with cardiac allograft endothelia in 15 min.Cell-MBs might be potential ultrasound contrast agents targeting cardiac acute rejection.Part 2.Ultrasound molecular imaging of cardiac acute rejectionBackground: Until now,electrocardiography,conventional echocardiogpaphy,or cardiac magnetic resonance could not detect cardiac acute rejection specifically.Endomyocardial biopsy is the “gold standard” for diagnosing acute rejection,but it is invasive and had many serious complications.Ultrasound molecular imaging was performed with cell-MBs to noninvasively detect cardiac acute rejection.Methods: Rat heart transplantation models were established.In the untreated allogeneic group,Brown Norway rats were donors and Lewis rats were recipients.In the cyclosporin A?Cs A?-treated group,the donors and recipients were as in the untreated allogeneic group while the recipients were treated with Cs A 7.5 mg/kg/d.In the syngeneic group,Lewis rats were both donors and recipients.In the three groups,ultrasound molecular imaging was performed on cardiac grafts with MBs,MBCD4,and cell-MBs.Histology was used to assess rejection grades.Results: In the untreated allogeneic group,ultrasound molecular imaging signal of cell-MBs was significantly higher than that of MBCD4 or MBs?p < 0.05?.And the signal of MBCD4 was significantly higher than that of MBs.In the Cs A-treated group,the signal of cell-MBs was significantly higher than that of MBCD4 or MBs as well?p < 0.05?.But the signal of MBCD4 was comparable with that of MBs.The signal of cell-MBs in the untreated allogeneic group was higher than that in the Cs A-treated group?p < 0.05?,and the signal in the Cs A-treated group was higher than that in the syngeneic group?p < 0.05?.The signal of MBCD4 in the untreated allogeneic group was higher than that in the Cs A-treated group?p < 0.05?,but the signal in the Cs A-treated group was comparable with that in the syngeneic group.Histology confirmed 3R rejection in the untreated allogeneic group,2R rejection in the Cs A-treated group,and no rejection in the syngeneic group.Conclusion: Ultrasound molecular imaging with cell-MBs might be a new method for noninvasively detecting cardiac acute rejection.Part 3.Chemokines and adhesion molecules in ultrasound molecular imaging of cardiac acute rejection with cell-MBsBackground: Chemokines and adhesion molecules were important molecules mediating lymphocyte chemotaxis to rejecting sites and cell adhesion with vascular endothelia.Whether chemokines and adhesion molecules contributed to the ultrasound molecular signal of cell-MBs was studied.Methods: Rat heart transplantation models were established.In the allogeneic group,Brown Norway rats were donors and Lewis rats were recipients.In the syngeneic group,Lewis rats were both donors and recipients.On post-transplantation day 3,cardiac grafts were obtained for RT-PCR to detect m RNA expression of CXCL9,CXCL10,CCL2,CCL4,and CCL5,and also for immunohistochemistry to assess the expression of intercellular adhesion molecules 1?ICAM-1?and vascular cell adhesion molecule 1?VCAM-1?.On post-transplantation day 3,ultrasound molecular imaging with cell-MBs was performed on allografts before and after CXCL9,ICAM-1,or VCAM-1 block.Results: CXCL9 m RNA expression was significantly higher in cardiac allografts than in isografts?p < 0.05?.m RNA expression of CXCL10,CCL2,CCL4,and CCL5 were comparable in allografts and isografts.Either ICAM-1 or VCAM-1 expression was much higher in allografts than in isografts by immunohistochemistry?p < 0.05?.CXCL9 block significantly reduced the ultrasound molecular imaging signal of cell-MBs in allografts?p < 0.05?.In contrast,nither ICAM-1 nor VCAM-1 block significantly reduced the signal of cell-MBs in allografts?p > 0.05?.Conclusion: CXCL9 was crucial in ultrasound molecular imaging of cardiac acute rejection with cell-MBs.