| Aims:Ischemic heart disease causes 7 million deaths every year around the world, accounting for 12.9% of the total deaths by disease. When acute myocardial infarction occurs, revascularization is the main effort to protect the cardiomyocyte, but restored blood supply brings reperfusion injury as well. Animal experiments and clinical research indicates that reperfusion causes lethal injury to cardiomyocyte, accounting for 50% of total ischemia induced infarction. 25% of the patients who receives PCI treatments ended up with bigger infarct size. In this context, the recently introduced incretin hormone glucagon-like peptide-1(GLP-1) may hold greater promise. As a new class of anti-diabetic drug, they have demonstrate potentially cardiopotective effects in both animal models and early clinical studies, thus become hot field in ischemia/ reperfusion(I/R) injury research.The incretin hormone GLP-1 is derived from a proglucagon precursor and secreted by intestinal L-cells in response to oral nutrient ingestion. GLP-1 receptors belong to G protein coupled receptor(GPCR),they exert their physiologic effect by stimulating GLP-1R and corresponding intracellular signal path. Researchers found that GLP-1R expresses in many of tissue. GLP-1R exerts different physiologic effect in different tissue, among which the cardioprotective effect of GLP-1R is of great concern. GLP-1R exerts anti-apoptosis effect through activation of c AMP/PI-3K pathway. In myocardium suffered from I/R injury, GLP-1 can remarkably reduce the size of infarct area. By locating GLP-1R expression at the early stage of reperfusion injury and exercising regulation, we can effectively limit the infarct procedure after I/R injury. But traditional test cannot recognize and quantify GLP-1R in vivo. Our research group synthesis a new radiolabeled probe 18F-Al F-NOTA-MAL-Cys40-Exendin-4, preliminary experiment shows that it can image GLP-1R in ischemic myocardium in vivo. Our research group manages to utilize this probe to perform high specific, high sensitive, noninvasive in vivo imaging of GLP-1R in mouse heart and confirms the GLP-1R expression with molecular methods. Thus through visualizable, quantifiable imaging of radiolabeled probe, we provide new methods to recognize receptor expression after I/R injury in vivo and show new target spot for intervening I/R injury. Methods:1. Preparation of rat I/R models: Male SD rats weighted 200-250 g were chose and divided into five groups which are sham group, reperfusion 8h group, reperfusion 1d group, reperfusion 3d group, reperfusion 7d group. I/R procedure performed after intraperitoneal injection of 0.3% pentobarbital sodium by ligation of left anterior descending branch of coronary artery with width 2-3mm and depth 1-2mm. Ischemia persisted for 30 min. Sham group manage to put suture under left anterior descending branch of coronary artery but do not perform ligation.2. Verification of I/R models: Cardiac oscilloscope was performed during the I/R procedure to monitor ECG changes. Left ventricular ejection fraction(LVEF) was calculated by color Doppler ultrasound one day after the I/R procedure. In the same day, PET scan of 18F-FDG was performed to evaluate the vitality of rat myocardium.3. Synthesis of radiolabeled tracer 18F-Al F-NOTA-MAL-Cys40-Exendin-4: 18F-Al F-NOTA-MAL-Cys40-Exendin-4 was synthesized using one-step method by introducing 18F-Al F to NOTA-MAL-Cys40-Exendin-4. Analytical high performance liquid chromatography(HPLC) was performed to obtain chromatogram and preparative HPLC was performed to purify the tracer.4. PET scanning of 18F-Al F-NOTA-MAL-Cys40-Exendin-4 in rat heart: At 8h, 1d, 3d,7d after I/R, PET were performed. Animals were anesthetized with isoflurane, and approximately 200μCi/100 g of 18F-Al F-NOTA-MAL-Cys40-Exendin-4 was administered via tail vein injection. One hour later, 10-min static PET images were acquired. The images were reconstructed using a 2-dimensional ordered-subset expectation maximum algorithm. In addition, to access the specificity of tracer uptake, 8mg/kg of unlabeled exendin-4 were injected 10 min before 18F-Al F-NOTA-MALCys40-Exendin-4 administration in rats at 3d after I/R. After the 18F-Al F-NOTA-MALCys40-Exendin-4 scan, animals were injected with 200μCi/100 g of 18F-FDG. One hour afterward, 10-min static images were acquired, with the mid thorax in the center of the field of view, and images were reconstructed and analyzed with the same procedure as described above. For each scan, radiation dose before injection, injection time, remaining radiation dose after injection, scanning time are recorded to access the %ID/g.5. Western Blot analysis and immunohistochemistry staining of GLP-1R: Rats were sacrificed after PET scanning, and hearts were harvested. Western blot was performed to semi-quantitative analyze GLP-1R expression in infarcted area. Immunohistochemistry staining was performed to visually show the GLP-1R expression in rats myocardium. Results:1. Success in establishing rats myocardial I/R model: Cardiac oscillograph monitoring during ischemia showed that ST-T segment elevated after the occlusion of left anterior descending branch of coronary artery. At 1d after myocardial I/R, akinesis of the anterolateral wall with a significant decrease in cardiac function was identified by M mode high-resolution ultrasound. In addition, an obvious uptake defect in the anterolateral wall of the left ventricle was observed with 18F-FDG PET.2. PET image of GLP-1R in rats with 18F-Al F-NOTA-MAL-Cys40-Exendin-4:Observed through sagittal plane, coronal plane and transaxial plane, there shows high uptake of 18F-Al F-NOTA-MAL-Cys40-Exendin-4 in lung, kidney, bladder and pancreas. In normal hearts, the tracer accumulation is subtle because of low GLP-1R expression. After myocardial I/R injury, tracer uptake in ischemic area increases significantly. Fusion image of 18F-Al F-NOTA-MAL-Cys40-Exendin-4 and 18F-FDG indicates that up-regulation of GLP-1R occurs in area where experience ischemia. Blocking experiment shows defect in tracer uptake in both lung and heart, verifying the specificity of Exentin-4 combining to GLP-1R.3. GLP-1R expression in rats after myocardial I/R: Fusion image of 18F-FDG and 18F-Al F-NOTA-MAL-Cys40-Exendin-4 at 1d after myocardial I/R provides evidence to locate GLP-1R up-regulation point. PET image corrected by %ID/g shows that tracer uptake increases after myocardial I/R, it reaches a peak at 8h after myocardial I/R and recovers to normal at 7d after myocardial I/R. In vitro experiment shows similar changes in GLP-1R expression. Conclusion:1. Radiotracer 18F-Al F-NOTA-MAL-Cys40-Exendin-4 is synthesized using one-step method. This method is time saving and highly efficient and can be achieved even outside the lab. The radiotracer 18F-Al F-NOTA-MAL-Cys40-Exendin-4 performs well in tracing GLP-1R in rats, thus has great promise in recognizing early stage of I/R injury and guiding drug usage.2. I/R injury up-regulates the expression of GLP-1R in rats. The expression of GLP-1R in rats increases before it recovers to normal. It reaches a peak at 8h after myocardial I/R and recovers to normal at 7d after myocardial I/R. |
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