| Objective:On the basis of in vivo tracking the migration of neural stem cells (NSCs) labeled with superparamagnetic iron oxide particles (SPIO) in host rat brain successfully by MRI in previous researches, we applied manganese-enhanced MRI (ME-MRI) here to further evaluate the function of NSCs in local brain areas after transplantation non-invasively. Moreover, Proton nuclear magnetic resonance spectroscopy (H-NMR) was used to detect NSCs-specific metabolites and proton magnetic resonance spectroscopy (H-MRS) was applied to exploit endogenous NSCs in adult rat and human brain in vivo.Methods and Materials:1. Embryonic neural stem cells (NSCs) were isolated from the hippocampus and cortex of 14-16 day pregnant Sprague-Dawley rats, and cultured in vitro in non-serum medium DMEM/F12. The characteristics of NSCs were identified as the expression of Nestin with immunofluorescence staining, and its differential potential was also analyzed. The superparamagnetic iron oxide (SPIO) particles were used to label NSCs, which then examined by the Prussian blue staining. SPIO-labeled NSCs activity was valued by MTT method.2. Traumatic brain injury (TBI) model rats were made according to the Feeney's method, and SPIO-labeled NSCs were stereotactically transplanted around the lesion 1 week after brain trauma. T2-weighted GRE scanning was performed at day 1,7,14, 21 and 30, as well as 3 months later respectively after NSCs transplantation in order to track its migration and distribution in the host rat brain, and Prussian blue staining of brain section was conducted to confirm the existence of NSCs.3. Normal rats were performed ME-MRI scanning firstly to verify the effectiveness of its functional evaluation. MnCl2.4H2O was intravenously infused into rats, right blood brain barrier (BBB) was opened by injection of mannitol, left forepaw stimulation was induced, and Tl-weighted 3D-FSPGR scanning was performed finally. Rats in another group performed the same procedure, and diltiazem (a Ca2+channel blockage) was infused l0min before the electrical stimulation and continued during the entire stimulus period.4. About 2-4 weeks after NSCs transplantation, the SPIO-labeled NSCs could be found in the lesion sites by MRI tracking with hypointense signals and Prussian blue staining with positive signs. Then ME-MRI scanning was conducted in all TBI rats, and the procedure was same to the normal rats. Both the SPIO-MRI and ME-MRI were also performed in TBI rats without NSCs treatment as control group.5. H-NMR spectra of NSCs from embryonic mouse brain tissue cultivated as neurospheres in vitro with the spectra of cultured neurons, astrocytes, and oligodendrocytes were compared. The NSC spectra demonstrated a unique profile, including a prominent peak at the frequency of 1.28 parts per million (ppm). The presence of the 1.28-ppm biomarker correlates with the progenitor status of cells and neurogenesis. H-MRS was used to detect NSCs in live adult rat and human brain.Results:1. Embryonic neural stem cells (NSCs) were isolated and cultured from the hippocampus and cortex of 14-16 day pregnant Sprague-Dawley rats. Some neurospheres were formed and immunofluorescence studies revealed that these neurospheres become immunoreactive to the undifferentiated cell marker nestin. NSCs can differentiate to cells that expressed phenotypic marker for neurons and astrocytes by immunofluorescence assay. The SPIO could label NSCs, showing positive signs after Prussian blue staining of the labeled cells. Compared to the unlabeling NSCs, the activity of SPIO-labeled NSCs weren't affected, which was valued by MTT method.2. Serial MRI scans from TBI rats which received NSCs (labeled with SPIO) treatment were performed at day 1,7,14,21 and 30, as well as 3 months later respectively after NSCs transplantation, exibiting that NSCs had the ability of migrating to the injured brain areas from the injection sites. Hypointense signals were observed at the NSCs injection sites, the migration "stream" and around the lesion, a finding that was consistent with the presence of SPIO-labeled NSCs, which was revealed by Prussian blue staining of brain section after experiment.3. T1-weighted MRI images (3D-FSPGR) were acquired after MnCl2.4H2O infusion and electric stimulation of rat left forepaw in normal rats. Signal enhancement in the right forepaw, whisker somatosensory cortex, and lateral ventriclues can be readily detected. However, signal enhancement was disappeared in rats when diltiazem, a Ca2+ channel blockage was introduced. By comparison, enhancement in the right somatosensory forepaw and whisker cortex due to electric stimulation was "blocked". But the signal enhancement in the lateral ventricles was still existed, indicating that Mn2+ entered the exciting neural cells via Ca2+ channel, and ME-MRI could specially detect the local brain function.4. About 2-4 weeks after transplantation, NSCs can migrate to the injured regions which could be reflected by the hypointense signals around the lesion in MRI, and then ME-MRI scanning was performed. Hyperintense signals were finally detected around the lesion sites in TBI rats with NSCs treatment, which weren't presented in rats without NSCs implantation. However, the hyperintense signals were disappear when diltiazem, a Ca2+ channel blockage was introduced. All these data approved that transplanted NSCs not only could migrate to the injured brain areas, but also differentiate at local brain regions and participate into the local brain function. Consistent to this, the neurological behaviors of TBI rats which received NSCs treatment were better than that without NSCs therapy.5. The NSCs spectra demonstrated a unique profile, including a prominent peak at the frequency of 1.28 parts per million (ppm) by H-NMR, which was not observed in other neural cell types. The presence of the 1.28-ppm biomarker correlates with the progenitor status of cells and the amount of 1.28-ppm biomarker correlates with neurogenesis. Based on singular value decomposition (SVD) signal processing, we detected the 1.28-ppm biomarker in the adult rat and human hippocampus with MRS in vivo, indicating the possibility of the method in endogenous NSCs tracking.Conclusion:We have successfully labeled NSCs with SPIOs, transplanted them into rats' injured brain areas and revealed its migration in vivo by MRI. On the basis of it, we applied ME-MRI to map the NSCs function in animal injured brain areas, paving the way for further clinical researches. Furthermore, H-MRS was used to detect the endogenous NSC in live rat and human brain non-invasively. |
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