In Vivo MRI Tracking of Endothelial Progenitor Cells Labeled with Ultrasmall Superparamagnetic Iron Oxide in Rat Model of Cerebral Ischemia
|School||Anhui Medical University,|
|Course||Medical Imaging and Nuclear Medicine|
|Keywords||Endothelial Progenitor Cells Angiogenesis Ultrasmall Superparamagnetic Iron OxideMagnetic resonance imaging Cerebral ischemic|
BACKGROUNDS AND OBJRCTIVESStroke is the leading cause of morbidity and mortality in adults worldwide. Currently,the effective treatment for stroke is the use of thrombolytic drugs to restore brainperfusion during the hyper-acute phase (<6h). However, this treatment is given to fewstroke patients due to its narrow therapeutic window and it may increase the risk ofintracranial hemorrhage. In fact, angiogenesis and vascular remodeling are basis of strokerecovery, via generating new blood vessels during the sub-acute phase, andconsequently improving functional recovery and promoting neurogenesis. Therefore,the therapeutic angiogenesis provides a new ideal for stroke treatment. Endothelialprogenitor cells (EPCs) primarily are located at the bone marrow after birth. In somecases they could be mobilized into the peripheral blood, and then homed to targettissues involving in physiological or/and pathological angiogenesis. The purpose of thisresearch is to detect the distribution of magnetically labeled EPCs by MRI and explorethe role of EPCs in angiogenesis in the damage area of ischemic rat brain.METHODSSprague–Dawley (SD) rats weighing from90g to110g were collected, bilateral femurs and tibias were taken off for marrow collection. Lymphocyte separation medium wasused to isolate the mononuclear cells, and the attachment growth method was used topurify EPCs. Cells were identified by immunohistochemisty and immunofluorescentstaining with surface marker CD133, VEGFR-2and CD34, Dil-labeled acetylatedlow-density lipoprotein (DiI-ac-LDL) and FITC-labeled ulex europaeus agglutinin-1(FITC-UEA-1). Poly-lysine (PLL) was used as the transfection agent (TA) to inductultrasmall superparamagnetic iron oxide(USPIO) particles with the concentration of25ug/ml enter into EPCs in vitro, and the ratio of USPIO to PLL was1:0.03. Transientmiddle cerebral arterial occlusion(tMCAO) was successfully performed in20adult SDrats weighing from250g to300g. Magnetically labeled cells(the experimental group)and PBS(the control group) were injected intravenously into the tMCAO rats throughthe tail vein, neurologial severity scoring(NSS) and MR imaging was performed at24hours,3days,5days and7days after that. Rat brain tissues were obtained and examinedby Prussian blue staining and immunohistochemical techniques for the expression ofendothelial cell markers using anti-mouse CD34、anti-mouse CD68and KDR1(VEGFreceptor2) antibodies. The microvessel density (MVD) was measured, the NSS, relativeinfarct volume of models and the MVD in two groups were recorded and compared.RESULTSMononuclear cells could be induced and differentiated into EPCs in vitro. USPIO-PLLas an intracellular contrast agent could label EPCs successfully, and the labeling ratereached as high as98%after3days. Prussian blue staining showed that the ironparticles were deposited in the cytoplasma, and the magnetic tag had no significanteffect on the biological characteristics of cells. MR imaging showed significant lowsignal intensity at the outer boundary of ischemic area both on T2WI and T2*mapsequences, especially on T2*map sequence. The ischemic area contained iron-positive cells showed on Prussian blue staining was corresponding to that of low signal intensityon T2WI and T2*map sequences. Immunohistochemical results suggested thatmagnetically labeled EPCs could migrate to the damage zone, and improveneovasculature appearance as transform into endothelial cells. The NSS and MVD inexperimental group7days after cells transplanted were significantly higher than those incontrol group(P<0.05). The change of the relative infarct volume showed no significantdifferences between two groups in7days after transplant (P>0.05).CONCLUSIONSMRI is feasible for in vivo tracking of EPCs labled with USPIO in tMCAO rats, andEPCs could be incorporated into the neovasculature to participate in angiogenesis ofischemic area and improve neurogenesis functional recovery.