¹⁸F-FDG PET/CT Imaging In Anti-inflammatory Therapy For Acute Myocardial Infarction

【Background】:Acute myocardial infarction（AMI）is a disease that seriously endangers human health and life.The infarcted myocardium can trigger a strong aseptic inflammatory response.The dysregulated inflammatory response causes the infarct area to expand through paracrine pathways and cascade amplification effects,leading to adverse ventricular remodeling,which eventually results in chronic heart failure and death.Therefore,reducing the inflammatory response via anti-inflammatory therapy is expected to reduce the infarct size,improve poor ventricular remodeling.The NOD-like receptor protein 3（NLRP3）inflammasome is a macromolecular protein complex that are widely present in cells and initiate inflammatory responses.Damage signals induce IL-1βand IL-18 production by activating NLRP3.In recent years,relevant studies have successively confirmed that the selective NLRP3 inhibitor MCC950 can reduce inflammatory damage by inhibiting NLRP3 inflammasome and suppressing inflammatory storms.Therefore,the application of MCC950 in anti-inflammatory treatment of myocardial infarction is expected to improve cardiac function and decrease mortality of AMI.Appropriate methods for monitoring cardiac response to MCC950’s anti-inflammatory therapy are still lacking,which impede its translational application in clinical trials and practice.In previous AMI anti-inflammatory studies,the changes in inflammatory response were assessed mainly by blood cell counts,inflammatory or myocardial injury biomarkers measuring,and the changes in viable myocardium were commonly evaluated by Evans/2,3,5-triphenyltetrazolium chloride（TTC）staining.However,serological tests do not provide information about the spatial location and activity of post-MI inflammation,and they are susceptible to interference from potential inflammatory foci of other tissues and organs,causing inaccuracies.And also,Evans/TTC staining of cardiac tissue is not suitable for clinical research and practice.Hence,novel monitoring methods,with advantages such as in vivo application,non-invasiveness and precise quantification of inflammatory response and infarct size changes,are urgently needed.Molecular imaging has the characteristics of living,non-invasive,dynamic,quantitative and high resolution.Among them,PET/CT imaging is one of the most efficient molecular imaging methods as to clinical application.¹⁸F-FDG（2-fluoro-2-deoxy-D-glucose）can be taken up by inflammatory cells and cardiomyocytes due to their high metabolic state.Therefore,the study proposed a Dual ¹⁸F-FDG PET/CT strategy,aimed at exploring whether¹⁸F-FDG PET/CT（inflammation）imaging and ¹⁸F-FDG PET/CT（viability）imaging could noninvasively and quantitatively monitor the changes in inflammatory response and viable myocardium during MCC950’s anti-inflammatory therapy in AMI,via adjusting the metabolic status of myocardium.The final purpose of this study is to promote the clinical translation of MCC950 anti-inflammatory therapy in AMI,and to achieve the purpose of molecular imaging guiding the precise treatment of AMI.【Objectives】:1.To compare the effects of multiple independent methods on the suppression of physi-ologically myocardial uptake of ¹⁸F-FDG,and explore the feasibility of combined methods to improve the suppression effect in ¹⁸F-FDG PET/CT（inflammation）imaging;2.To explore whether the Dual ¹⁸F-FDG PET/CT strategy can be used for in vivo moni-toring of inflammatory response and viable myocardium changes during MCC950,a novel NLRP3 inflammasome inhibitor,treatment in AMI;3.To investigate the relationship between ¹⁸F-FDG PET/CT（inflammation）imaging in the acute phase and ¹⁸F-FDG PET/CT（viability）imaging in the chronic phase,and with cardiac function by ultrasound;To observe the long-term effects of MCC950 on ventricular remodeling,heart failure and survival rate.【Methods】:1.¹⁸F-FDG PET/CT imaging was performed on healthy/AMI model mice among groups of fasting,ketamine-xylazine,heparin,low-carbohydrate,high-fat diet and pentobarbital respectively.The suppressive effects of each method were compared qualitatively and quantitatively.In addition,two combined methods:Fasting+heparin+Ketamine-xylazine（FHKX）;High Fat,Low Carbohydrate Diet+Pentobarbital sodium（HFLCP）were also compared in ¹⁸F-FDG PET/CT inflammation imaging using similar method;2.¹⁸F-FDG PET/CT（inflammation）and ¹⁸F-FDG PET/CT（viability）imaging were performed respectively on the AMI model without MCC950 administration,and the feasibility of the Dual ¹⁸F-FDG PET/CT strategy was verified by overlay the polar maps of the two kinds of imaging;The possible effect of MCC950 on the distribution of ¹⁸F-FDG was observed after 7 days’MCC950 administration;¹⁸F-FDG PET/CT（inflammation）imaging was performed on the day 3 and day 5 post-MI to observe the changes in inflammatory among groups;Immunohistochemical staining and Western blot Validated changes in inflammatory cells and NLRP3/IL-1βsignaling pathways,and further confirm¹⁸F-FDG PET/CT（inflammation）imaging results;¹⁸F-FDG PET/CT（viability）imaging was undertake before surgery,after surgery,and on day 7 post-MI to monitor the changes of viable myocardium bring by MCC950 treatment.M-mode echocardiography was used to detect changes in cardiac function;TUNEL staining was used to observe myocardial apoptosis.3.On the 28th day post-MI,¹⁸F-FDG PET/CT（viability）imaging and M-mode echocardiography were performed respectively.¹⁸F-FDG PET（inflammation）signals on day 3 were correlated with survival defect area in ¹⁸F-FDG PET（viability）imaging,and also with changes of left ventricular ejection fraction（LVEF）;Survival analysis was performed during the 28-days.Adverse remodeling was determined by Wheat Germ Agglutinin（WGA）and Masson trichrome staining.The expression of heart failure markers was tested by Western blot on day 28.【Results】:1.Five independent methods have different suppression effects in healthy and AMI mice;As to AMI mice,heparin can’t suppress the myocardium uptake of ¹⁸F-FDG independently,but it can reduce the uptake（Efficient/Inefficient:0/8;8.62±0.56 vs.10.43±1.28%ID/g,p<0.05 vs.Control）.Fasting has suppression effect but unstable（3/5;5.49±1.05%ID/g）;ketamine-xylazine,low-carb and high-fat diet and pentobarbital sodium exhibit significant suppression effect（K-X,6/2;4.09±1.03%ID/g;HFLC,7/1;3.26±0.75%ID/g;Pento,5/3;4.11±1.14%ID/g）;Both combined methods,FHKX and HFLCP,can improve the success rate and effect of suppression in ¹⁸F-FDG PET/CT（inflammation）imaging（FHKX,8/0;2.34±0.77%ID/g;HFLCP,8/0;2.11±0.66%ID/g）.2.In AMI model,the area where ¹⁸F-FDG accumulated in the ¹⁸F-FDG PET/CT（inflammation）imaging,approximately overlapped with the defect region in the ¹⁸F-FDG PET/CT（viability）imaging;7 consecutive days MCC950 treatment did not significantly affect the distribution of ¹⁸F-FDG;¹⁸F-FDG PET/CT（inflammation）imaging revealed that MCC950 treatment led to significant reductions of accumulated ¹⁸F-FDG on day 3 and 5when compared to the MI group（day 3:5.96±0.98 vs.8.01±1.15%ID/g,-25.6%,p<0.05;day 5:5.14±0.91 vs.7.45±0.88%ID/g,-31.0%,p<0.05）;The increased infiltration of CD86⁺M1 macrophages and Ly-6G⁺neutrophils on day 3 were significantly prevented by MCC950 post-MI;The expression of target proteins including NLRP3,cleaved-caspase-1 and cleaved-IL-1βwere down-regulated in AMI mice;¹⁸F-FDG PET/CT（viability）imaging demonstrated that MCC950 resulted in a lower viability defect on day 7 when compared to MI group（31.90±1.60%vs.36.30±3.31%,-12.1%,p<0.05）;M-mode echocardiography showed that the reductions of LVEF and LVFS were significantly attenuated by MCC950 treatment when compared to that in the MI group（EF:51.26±3.56%vs.43.89±2.98%,p<0.05;FS:24.77±2.74%vs.19.89±2.10%,p<0.05）;Cardiomyocytes apoptosis was assessed using TUNEL staining,which revealed that the apoptotic index of the MI+MCC950 group was significantly reduced when compared to MI group（11.85±2.43%vs.22.47±3.62%,p<0.05）.3.MCC950 significantly decreased viability defect（33.05±2.18%vs.37.50±2.04%,-11.86%,p<0.05）and improved cardiac function（LVEF:48.72±3.86%vs.39.64±2.85%,p<0.05;LVFS:22.93±2.58%vs.17.54±1.91%,p<0.05;LVEDV:70.91±3.31 vs.82.40±5.30 ul,p<0.05;LVESV:38.95±4.38 vs.50.29±3.76 ul,p<0.05）on day 28post-MI;¹⁸F-FDG PET/CT（Inflammation）imaging was closely related with ¹⁸F-FDG PET/CT（Viability）,the inflammatory ¹⁸F-FDG uptake on day 3 was positively correlated with viability defect on day 28（r=0.78,p<0.01）and also with viability defect changes_△d28-post（r=0.69,p<0.05）;Furthermore,the inflammatory ¹⁸F-FDG uptake also positively correlated with the reduction in LVEF_{（d28-post）}（r=0.79;p<0.01）;Masson’s trichrome staining and WGA staining revealed that the administration of MCC950 ameliorates collagen deposition at the interstitial and peri-vascular regions of MI mice（interstitial:9.62±2.35%vs.17.01±3.14%,peri-vascular:1.59±0.92%vs.3.58±1.06%,p<0.05）,and decreased cardiomyocyte transverse cross-sectional area（278.30±21.29 vs.342.60±18.60μm²,p<0.05）;In addition,MCC950 reduced mortality and heart failure risk.【Conclusion】:1.Two combined suppression methods:Fasting+heparin+Ketamine-xylazine（FHKX）;High Fat,Low Carbohydrate Diet+Pentobarbital sodium（HFLCP）can improve the ¹⁸F-FDG PET/CT inflammation imaging success rate and myocardial inhibition effect;2.Dual ¹⁸F-FDG PET/CT imaging strategy can be used for non-invasive,living,quantitative monitoring of the changes in inflammatory response and viable myocardium during MCC950 treatment in AMI;3.The ¹⁸F-FDG PET（inflammation）signal in the acute phase has predictive value for late viable myocardium and changes of cardiac function;MCC950 can improve long-term ventricular remodeling in AMI and reduce mortality and risk of heart failure.