In Vivo MRI Tracking Of Bone Marrow Mesenchymal Stem Cells Labeled With Dual Modal Contrast Agent-Molday ION^TM EverGreen For Treating The Hepatic Ischemia-Reperfusion Injury In Rats

Introduction:At this stage,the most effective and commonly treatment method for end-stage liver disease is liver resection and liver transplantation,and liver ischemia-reperfusion injury is common and unavoidable during liver resection and liver transplantation.Stem cells have a wide range of applications in the experimental study of ischemia-reperfusion injury due to their strong self-replication ability and multi-directional differentiation potential in vitro.Previous studies have confirmed that mammalian bone marrow-derived stem cells have strong migration and differentiation ability under physiological or pathological conditions,and can be differentiated into multiple germ layer-derived cells in vitro under specific conditions,such as hepatocyte-like cells,neuroblasts,adipocytes,osteoblasts,etc.,and stem cell transplantation treatment is beneficial to tissue repair after injury and functional recovery.It is an ideal seed cell for tissue repair engineering and a clinically promising therapeutic strategy.But how can we monitor the stem cells transplanted into the liver with ischemia-reperfusion injury?The answer is that we need the support of molecular imaging technology.Advances in imaging technology and innovation in imaging reagents have led to rapid development of molecular imaging technology.In the field of molecular imaging,the combination of molecular biology and imaging technology has led to a rapid increase in the ability to conduct research and diagnostics at the molecular or cellular level.Medical molecular imaging technology has many imaging devices,such as US,CT,MRI,OI,PET,SPECT,etc.These molecular imaging devices have their own advantages and disadvantages in terms of imaging sensitivity,spatial resolution and time resolution.These imaging technologies can providing clinicians and basic researchers with information at the cellular and even molecular levels,can also provide detailed information on morphological structure and functional metabolism.The combined use of these imaging techniques can compensate for the deficiencies of using one imaging method alone.For example,in animal experiments,a variety of molecular imaging devices(such as MRI)can achieve long-term non-invasive observation of animals without the need to sacrifice experimental animals,but MRI spatial resolution and signal-to-noise ratio(SNR)has certain disadvantages.Therefore,it is often desirable to improve spatial resolution and SNR by using contrast or molecular imaging probes,or in combination with other imaging methods for dual/multimodal imaging.In theory,bimodal and multimodal medical examinations require only one type of probe,and more accurate and complete information can be obtained through this multimodal imaging,helping clinicians detect disease early,improve diagnosis,and evaluate efficacy.At present,bimodal and multimodal medical imaging methods are increasingly applied to molecular imaging studies in small animals,and bimodal and multimodal contrast agents are also widely used in animal markers for labeling transplanted cells.In this study,rat bone marrow-derived mesenchymal stem cells were labeled with fluorescent and MR bimodal contrast agent-Molday IONTM EverGreen.The T2WI sequence in the MR test sequence can trace the paramagnetic substance-labeled cells in vivo,showing the low signal region,therefore,Molday IONTM EverGreen-labeled rat bone marrow-derived mesenchymal stem cells can be traced in vitro and in vivo,and the effect of rat bone marrow mesenchymal stem cell transplantation on hepatic ischemia-reperfusion injury in rats can be studied,that is,anti-injury,anti-apoptosis,and promote repair.At present,with the development of medical image post-processing technology,magnetic resonance imaging(MRI)imaging technology can not only observe the migration and proliferation of cells in living tissue by MR non-invasive method,but also can be quantitative or semi-quantitative.The quantitative post-processing analysis software evaluates the organ function and hemodynamic changes after cell transplantation,which is of great significance for evaluating the effect of stem cell transplantation.This study uses dynamic contrast-enhanced(DCE)MRI scan combined with quantitative analysis software,the hemodynamic parameters of hepatic microvessels in the rat liver ischemia-reperfusion injury area were analyzed,which provided help for stem cell transplantation in vivo for hepatic ischemia-reperfusion injury.The research is divided into the following two parts.The First Part In Vitro MR Imaging of Bone Marrow Mesenchymal Stem Cells of Rats Labeled With Dual Modal Contrast Agent Molday IONTM EverG reenObjective:Bone marrow mesenchymal stem cells have the ability to multi-differentiate under certain conditions in vitro,and therefore become more and more widely used in tissue engineering research.If we want to study the distribution and proliferation of BMSCs in vivo and their effects on organ function by means of medical imaging equipment,we must first solve the problem of how to safely and efficiently use image developer to label BMSCs.In this paper,BMSCs were labeled with novel dual modal contrast agent Molday IONTM EverGreen to investigate the appropriate labeling concentration of Molday IONTM EverGreen;and the viability and labeling rate of BMSCs at different time points within 6 weeks after labeled with BMSCs;the feasibility of in vitro MR imaging of Molday IONTM EverGreen-labeled BMSCs was explored;which provided experimental basis for further study of live tracer of BMSCs after transplantation.Methods:After isolation,culture and passage of rat BMSCs,the third generation BMSCs were fluorescently and magnetically double-labeled with three concentrations of Molday IONTM EverGreen(iron concentration of 10,20,50 ?g/ml).The iron labeling rate of cells was observed by Prussian blue staining.The fluorescence labeling rate of cells was observed by fluoroscopy,and the positive labeling rate of three concentrations of Molday IONTM EverGreen-labeled BMSCs was compared.Observation of the fluorescence labeling rate of BMSCs at 1 day,1 week,2 weeks,4 weeks,and 6 weeks after labeled with 20 ?g/ml Molday IONTM EverGreen;Detection of BMSCs' viability at 1 day,1 week,2 weeks,3 weeks,4 weeks,5 weeks,and 6 weeks after labeled with 20 ?g/ml Molday IONTM EverGreen by trypan blue exclusion assay;survival rate of different concentrations of Molday IONTM EverGreen-labeled BMSCs was observed by fluorescent dye HOECHST33342/PI double-stained nuclear under confocal microscopy scanning;the T1 and T2 sequences were scanned by different concentrations of labeled cells using 3.0T MR.Combine the above methods to determine the optimal labeling concentration of Molday IONTM EverGreen.Results:The primary cells of BMSCs were round,triangular and polygonal in shape,and a few were spindle-shaped.Most of the primary cells were fusiform in the late stage.In vitro,after 1 week of the second generation cell culture,about 80%of the cells were fused and form a uniform long fusiform shape;the third generation BMSCs were substantially purified,and the cell surface markers were identified by immunofluorescence assay:CD29(+),CD34(-),CD44(+),CD45(-).After labeling BMSCs with three concentrations of Molday IONTM EverGreen(iron concentration of 10,20,50?g/ml),the labeling rate by Prussian blue staining and fluoroscopy was close to 100%,while the control group(without Molday IONTM EverGreen labeling)was zero.Prussian blue staining showed blue-stained iron particles in the cytoplasm of Molday IONTM EverGreen-BMSCs,and with the increase of the concentration of Molday IONTM EverGreen,the blue-stained particles increased and the color deepened.The fluorescence labeling rates of the three concentrations of Molday IONTM EverGreen were close to 100%,but the fluorescence intensity of 10 ?g/ml Molday IONTM EverGreen-labeled BMSCs was poor,while the adherent cells of 50 ?g/ml Molday IONTM EverGreen-labeled BMSCs which cultured for 16 hours were decrease.After 6 weeks of 20 ?g/ml Molday IONTM EverGreen-labeled BMSCs,although the green fluorescence intensity decreased to some extent,the fluorescence labeling rate was still 93%.The results of trypan blue exclusion assay(cell viability assay)showed that there was no significant difference in cell viability between the labeled group and the unlabeled group after 20 ?g/ml Molday IONTM EverGreen-labeled BMSCs for 1 day and 1,2,3,4,5,and 6 weeks(p>0.05).The cell death rate of Molday IONTM EverGreen-labeled BMSCs which labeled with 0 ?g/ml,10 ?g/ml,20 ?g/ml,and 50?g/ml Molday IONTM EverGreen was observed to be 4%,5%,5%,and 8%by the method of double staining the nucleus with the fluorescent dye HOECHST33342/PI.The results of in vitro MR scan showed that there was no significant difference in T1 signal intensity of different concentrations of Molday IONTM EverGreen-labeled third-generation BMSCs(cell number 1×106/ml);the signal of Molday IONTM EverGreen-labeled cells decreased on T2WI.The higher the concentration of Molday IONTM EverGreen,the lower the T2WI signal.Conclusion:Using this experimental method,rat BMSCs can be isolated and cultured convenientTMly and rapidly,and can be stably passaged,and can be used for further animal studies in vivo.BMSCs labeled with bimodal modality agent-Molday ION EverGreen at a suitable concentration(20 ?g/ml)were highly efficient,and the labeling rate was 93%for a longer period of time(6 weeks),and had no effect on cell viability and survival rate of BMSCs.In vitro MR scanning T2WI sequence can sensitively detect changes in signal intensity after different concentrations of Molday IONTM EverGreen-labeled BMSCs.The experimental basis was established for MR tracing after transplantation of BMSCs in vivo.The Second Part In Vivo MRI Tracking of Allogeneic Bone Marrow Mesenchymal Stem Cells Labeled With Molday IONTM EverGreen For Treating The Rat Hepatic Ischemia-Reperfusion Injury Grafted Via Hepatic Portal VeinObjective:Rat models of right outer lobe and triangular lobe ischemia-reperfusion injury were made.After the bimodal contrast agent Molday IONTM EverGreen labeled bone marrow mesenehymal stem cells(BMSCs),they were transplanted into allogeneic liver ischemia-reperfusion injury rats,and then MR scan was performed.The effects of cell transplantation on liver function,pathological changes and hepatocyte apoptosis in rats were studied.Dynamic contrast enhanced(DCE)magnetic resonance imaging(MRI)technique was used to study the dynamic changes of microvascular permeability parameters in rat liver ischemia-reperfusion injury model and the effect of Molday IONTM EverGreen-BMSCs transplantation on it.Methods:Healthy male SD rats of the same age and quality were selected,and bone marrow mesenchymal stem cells were isolated,cultured and passaged in vitro.The third-generation BMSCs were marked with the dual-mode contrast agent Molday IONTM EverGreen before transplantation.The blood vessels of the right outer lobe and the triangular lobe of the rat liver were ligated,that is,the right branch of the hepatic artery and the right branch of the portal vein.After 45 minutes of ligation,the vessels were reperfused and a rat model of hepatic ischemia-reperfusion injury was established.Rats were randomly divided into three groups:sham operation group(after the abdominal wall was opened for 1 hour,the abdominal wall was sutured,no ischemia reperfusion surgery),Molday IONTM EverGreen-labeled BMSCs transplantation group(BMSCs were injected through the right branch of the portal vein,temporarily clamping the left liver pedicle during implantation to ensure that enough cells were transplanted into the damaged liver vessels)and the ischemia-reperfusion group(the same amount of normal saline was injected through the right branch of the portal vein,temporarily clamp the left liver pedicle when injecting),Each group underwent DCE-MRI at 24 hours,2 days,3 days,4 days,5 days,6 days,and 7 days after reperfusion.The obtained images were processed by the full-quantitative analysis and processing software package Omnikinetics software to measure the hepatic microvascular permeability parameters of the operation area:Ktrans,Ve and Kep,and the three parameters reflected the changes of microvascular permeability;after MRI scan,serum and liver tissue samples were taken for liver function and pathology analysis:serum aspartate aminotransferase(AST)and alanine aminotransferase(ALT)levels were measured by inferior vena cava blood collection;Hepatic tissue was stained with HE staining to observe the pathological changes.The frozen section of the liver tissue was analyzed by laser confocal microscopy and tissue section Prussian blue staining,both methods were used to observe the distribution of Molday IONTM EverGreen-BMSCs in the liver tissue of the operation area after transplantation.The apoptotic index(AI)of damaged liver tissue cells was detected by TUNEL method.Results:The stable model of right hepatic lobe and triangular lobe ischemia-reperfusion injury was successfully established.The morphology and color of ischemia-reperfusion injury liver lobe returned to normal 7 days after operation.DCE-MR scan showed 24 h after ischemia-reperfusion injury in rats;in the I/R group,the liver tissue of the transplanted group of Molday IONTM EverGreen-BMSCs showed uneven T1 and long T2 signal,DWI showed a high signal,and the dynamic enhanced scan area of the liver tissue enhancement increased compared with sham group.Delayed scanning(5 minutes and 40 seconds after injection of contrast agent)showed a non-uniform high signal,confirming the success of the ischemia-reperfusion injury model.At 24 h after operation,the long signal range of T2WI in the transplantation group of Molday IONTM EverGreen-BMSCs was slightly smaller than that in the I/R group,and the long signal level of T2WI was slightly lighter than that in the I/R group.The difference between the two groups was not significant in the other sequences(T1WI,DWI,DCE-MRI and delayed phase scan).At each time point(1 d,2 d,3 d,4 d,5 d,6 d,7 d),the T2 signal intensity of the injured liver region transplanted by Molday IONTM EverGreen-BMSCs was lower than that of the liver without Molday IONTM EverGreen-BMSCs transplantation,and the difference in signal changes was most obvious on the 3-5th day after surgery.Changes in liver microvascular permeability parameters with different recovery time:1 day after ischemia-reperfusion,Ktrans and Ve increased,Kep decreased,2 days after ischemia-reperfusion,Ktrans and Ve reached the highest level,Kep reached the lowest level,the Ktrans,Ve,Kep values in the cell transplantation group were not significantly different from those in the I/R group.At 3,4,5,and 6 days after ischemia-reperfusion,the Ktrans and Ve values in the cell transplantation group were lower than those in the I/R group,and the difference was statistically significant(p<0.05).At 3,4,and 5 days after ischemia-reperfusion,the Kep value of the cell transplantation group was higher than that of the I/R group,the difference was statistically significant(p<0.05).At 6 days after operation,the Kep value of the cell transplantation group was higher than I/R group,but the difference was not statistically significant;7 days after ischemia-reperfusion,the Ktrans,Ve,and Kep values of the two groups returned to normal,and the difference was not statistically significant.One day after ischemia-reperfusion,there was no significant difference in ALT and AST between I/R group and Molday IONTM EverGreen-BMSCs transplantation group,which was higher than that of sham operation group and reached the highest value;2 days and 3 days after operation,ALT The AST values were gradually decreased.The ALT and AST of the Molday IONTM EverGreen-BMSCs transplantation group were lower than those of the I/R group.The difference between the two groups was statistically significant(p<0.05).4 days,5 days,6 days after operation,the ALT and AST decreased gradually,and returned to normal on days 5-6 in the two groups,there was no significant difference between the two groups.At 7 days after operation,the ALT and AST values of the I/R group and the Molday IONTM EverGreen-BMSCs transplantation group were similar to those of the sham operation group,the difference in data between the groups was not statistically significant.After transplantation of Molday IONTM EverGreen-BMSCs into the right branch of rat portal vein,frozen sections of damaged liver tissue were made at 24 h,2 d,3 d,4 d,5 d,6 d,7 d after reperfusion,and directly observed the colonization of BMSCs under the confocal microscope.It can be seen that the early distribution of green fluorescent staining cells is sparse,but the fluorescence is strong.With the prolongation of postoperative recovery time,the fluorescence intensity gradually decreases and the range gradually expands.Prussian blue staining of liver tissue in the operation area:1 day after cell transplantation in the transplantation group of Molday IONTM EverGreen-BMSCs,a few blue deep-dyed iron particles in the liver tissue of the operation area were located in the hepatic sinusoid and a few hepatocytes.With the prolongation of time after surgery,cells with blue-stained iron particles in the cytoplasm appeared in the injured area of the liver tissue.Part of the blue stained granules are located in the hepatic sinus,the portal area and the sinusoids.The area containing blue-stained iron particles in the liver tissue damage area gradually expanded,and the liver cells containing blue-stained particles gradually increased,but the staining gradually became lighter.HE staining showed that the hepatic lobule and hepatocyte structure of Sham group were normal;the hepatic lobule structure of I/R group and the Molday IONTM EverGreen-BMSCs transplantation group were disordered,a large number of inflammatory cells were scattered,hepatocytes were obviously swollen,vacuolar degeneration,hepatic sinus stenosis,and a large number of red blood cells in part of liver Sinus.The pathology scores of the I/R group and the Molday IONTM EverGreen-BMSCs transplantation group were compared according to Suzuki's liver damage histopathology scoring criteria.The injury scores of I/R group and Molday IONTM EverGreen-BMSCs transplantation group reached the highest one day after operation,but the difference between the two groups was not statistically significant.The degree of injury(score)of Molday IONTM EverGreen-BMSCs transplantation group was lighter than I/R group from 2nd to 5th day after operation,the difference of injury scores between the two groups was statistically significant(p<0.05).There was no significant difference in liver injury score between the two groups at 6 days after operation,but higher than the sham operation group;7 d after surgery,the injury scores of both groups were similar to those of the sham operation group,and there was no significant difference in the injury scores between the two groups.The TUNEL staining of hepatocyte apoptosis showed that the apoptosis of Molday IONTM EverGreen-BMSCs transplantation group and I/R group was the most serious at 24 h after operation.The apoptotic cells gradually decreased on the 2nd,3rd,and 4th postoperative day.On day 5,it returned to normal;apoptotic cells were more prominent around the central vein;the apoptotic index(AI)of the I/R group was higher than that of the Molday IONTM EverGreen-BMSCs transplantation group at 1-4 days after ischemia-reperfusion,the difference was statistically significant(p<0.05);the apoptotic index of the two groups was similar and normal at 5-7 days after operation,which was similar to the control group,and there was no significant difference in the apoptotic index between the two groups.Conclusion:Molday IONTM EverGreen-labeled BMSCs can be successfully transplanted into the rat model of hepatic ischemia-reperfusion injury through the right portal vein injection,and can be traced by T2WI.The signal changes are most obvious 3-5 days after transplantation.Ttransplantation of Molday IONTM EverGreen-BMSCs to the liver can reduce the effects of hepatic ischemia-reperfusion on liver function,hepatocyte pathological changes and impaired liver microvascular permeability,inhibit hepatocyte apoptosis after hepatic ischemia-reperfusion injury,and protect rat liver ischemia reperfusion inj ury.