Molecular Imaging Of Activated Fibroblast After Myocardial Infarction In Rats Using A ⁶⁸Ga-Labeled Fibroblast Activation Protein Inhibitor 04

Objective: Noninvasively monitoring the activated fibroblasts is of great value for studying myocardial fibrosis after myocardial infarction.The main purpose of this study was to construct a myocardial infarction model in rats and perform ⁶⁸Ga-labeled FAP inhibitor 04(⁶⁸Ga-DOTA-FAPI-04)Positron Emission Tomography/Computed Tomography（PET/CT）dynamic imaging to evaluate the feasibility of monitoring the activated fibroblasts and reactive fibrosis after myocardial infarction.Methods: The myocardial infarction model of rats was constructed by permanent ligation of the coronary artery.Then we used the electrocardiogram,hematoxylin and eosin（HE）staining of the heart section and 18F-fluorodeoxyglucose（18F-FDG）PET/CT to determine whether the myocardial infarction was constructed.Preparing ⁶⁸Ga-DOTA-FAPI-04(⁶⁸Ga-FAPI-04)and performing quality control.This study was divided into experimental group,sham-operated group and imaging blocking group.Sham-operated animals underwent the same surgical procedure except the ligation.The animals of the experimental group（n=16）and sham-operated group（n=17）were performing ⁶⁸Ga-FAPI-04 PET/CT imaging on day 1,3,6,9,12,15,18,21,28,35 after MI,and the area of interest was delineated for quantitative analysis.As for imaging blocking experiment,the MI rats that underwent ⁶⁸Ga-FAPI-04 PET/CT imaging on the 6th day were injected with ⁶⁸Ga-FAPI-04 and FAPI-04（200 times）via the tail vein on the 7th day after MI to verify the specificity of this probe.As for autoradiography,the animals of the experimental group and sham-operated group were injected with ⁶⁸GaFAPI-04 via the tail vein on the 6th day after MI,then the rats were sacrificed and the hearts were prepared into serial sections to perform autoradiography.After that,the HE staining and immunofluorescence staining were preformed.After the rats in the experimental group and the sham operation group were sacrificed at different times,heart sections were prepared and used for HE staining,Masson’s trichrome staining and immunofluorescence staining.Finally,sections that were prepared from skin,lungs,liver and intercostal muscles at the trauma site of thoracotomy were used for immunofluorescence staining.Results: Myocardial infarction was confirmed by electrocardiogram（ECG）,HE staining and 18F-FDG PET/CT imaging.Compared with the pre-operative ECG,the ECG of Wistar rats after coronary artery ligation showed that the ST-segment elevation in lead II was more than 0.2 m V,and the infarct area could be observed by the corresponding HE staining of the heart sections,while the rats in the sham-operated group couldn’t observe the infarct area.In addition,18F-FDG PET/CT imaging of the rats of experimental group on the first day after MI showed obvious metabolic reduction or defect areas.The tracer was radiolabeled with 68 Ga at a molar activity of 9.0-11.0 MBq/nmol.The labeling rate of ⁶⁸Ga-FAPI-04 was 72.8% ± 3.7%,the radiochemical purity after purification was greater than 90%.The corresponding area that 18F-FDG PET/CT imaging showed decreased or defective myocardial metabolism uptook ⁶⁸GaFAPI-04 in the rats of the experimental group on the 3rd-18 th day after MI.Quantitative analysis showed that the probe uptake of the rats of experimental group and shamoperated group were statistically different from day 1 to 28 after MI,and the rats of experimental group showed the highest uptake on the 6th day after MI（The maximum uptake value of the experimental group and the sham-operated group were 0.806 ± 0.257 %ID/cc vs.0.199 ± 0.012 %ID/cc,P<0.05）,the maximum %ID/cc of the myocardial infarction area of experimental group and sham-operated group/the mean %ID/cc of liver were 8.781 ± 2.822 vs.3.635 ± 0.421,P<0.05.The blocking experiment showed that the uptake at the site of myocardial infarction in the experimental group was significantly reduced after blocking,and the physiological uptake at the trauma site of thoracotomy and the skin were also significantly reduced.Autoradiography showed that the infarcted area of the rats of experimental group which was confirmed by HE staining showed obvious uptake,and immunofluorescence staining showed that there were many Fibroblast activation protein（FAP）+ cells in this area.The HE staining and Masson’s trichrome staining of the heart sections of the experimental group rats at different times showed the presence of myocardial infarction and myocardial fibrosis.The immunofluorescence staining showed that there were many FAP+ cells in the myocardial infarction area of the heart sections on the 3rd,6th and 15 th day after MI,a small amount of FAP+ cells in the heart sections on the 21 st and 28 th day,and few FAP+ cells on the 35 th day.The immunofluorescence staining of other organs such as the lung and liver showed that there was few FAP expression in the lung and liver,a small amount of FAP expression in skin,and more FAP expression in the intercostal muscles at the trauma site of thoracotomy（on the 6th day after MI）.Conclusions: This study preliminarily confirmed that ⁶⁸Ga-DOTA-FAPI-04 PET imaging can non-invasively monitor the activation of fibroblasts in the early stage of acute myocardial infarction,and confirmed that reparative fibrosis occured on the first day after MI,reached the peak on the sixth day after MI and then decreased after that.It existed until the 28 th day after MI.However,this study failed to detect reactive fibrosis of the myocardium remote to the infarction by using ⁶⁸Ga-DOTA-FAPI-04.In short,this study suggests that ⁶⁸Ga-DOTA-FAPI-04 PET imaging will help to understand the degree of reparative fibrosis in the early stage of myocardial infarction,but further research is still needed to confirm whether it can monitor reactive fibrosis.