In vivo MRI detection of atherosclerosis in ApoE-deficient mice by using tenascin-C-targeted USPIO.

BACKGROUND: Atherosclerosis is the main cause of cardiovascular and cerebrovascular diseases. Non-invasive molecular imaging to detect and characterize the plaques is essential for reducing life-threatening cardiovascular events. PURPOSE: To investigate the possibility of the anti-tenascin-C-USPIO specific probe as a molecular marker of atherosclerotic plaques detected by 7.0-T magnetic resonance imaging (MRI). MATERIAL AND METHODS: Twenty ApoE-/- mice fed with a high fat diet were used for detecting the aorta arch atherosclerotic plaques by 7.0-T MRI at 16 and 24 weeks. Ten mice in the targeted group were injected with anti-tenascin-C-USPIO and another ten in the control group were injected with pure USPIO (n = 5 each time point in each group). Histopathologic examination was used to evaluate the plaques and immunohistochemistry analysis was used to compare tenascin-C expression. RESULTS: The relative signal intensity (rSI) changes of the targeted group decreased more than those of the control group (16 weeks: -15.65 ± 0.78% vs. -3.43 ± 2.57%; 24 weeks: -26.38 ± 1.54% vs. -11.12 ± 1.60%, respectively; P < 0.05). Histopathological analyses demonstrated visible atherosclerotic plaques formation and development over time from 16 weeks to 24 weeks. Tenascin-C expression of the plaques at 24 weeks was higher than that at 16 weeks (0.22 ± 0.04 vs. 0.13 ± 0.02, P < 0.05). The MR images correlated well with the progression of atherosclerotic plaques. CONCLUSION: Tenascin-C expression increased with the progression of atherosclerosis. Anti-tenascin-C-USPIO could provide a useful molecular imaging tool for detecting and monitoring atherosclerotic plaques by MRI.

Bifunctional staining for ex vivo determination of area at risk in rabbits with reperfused myocardial infarction.

AIM: To develop a method for studying myocardial area at risk (AAR) in ischemic heart disease in correlation with cardiac magnetic resonance imaging (cMRI). METHODS: Nine rabbits were anesthetized, intubated and subjected to occlusion and reperfusion of the left circumflex coronary artery (LCx) to induce myocardial infarction (MI). ECG-triggered cMRI with delayed enhancement was performed at 3.0 T. After euthanasia, the heart was excised with the LCx re-ligated. Bifunctional staining was performed by perfusing the aorta with a homemade red-iodized-oil (RIO) dye. The heart was then agar-embedded for ex vivo magnetic resonance imaging and sliced into 3 mm-sections. The AAR was defined by RIO-staining and digital radiography (DR). The perfusion density rate (PDR) was derived from DR for the AAR and normal myocardium. The MI was measured by in vivo delayed enhancement (iDE) and ex vivo delayed enhancement (eDE) cMRI. The AAR and MI were compared to validate the bifunctional straining for cardiac imaging research. Linear regression with Bland-Altman agreement, one way-ANOVA with Bonferroni's multiple comparison, and paired t tests were applied for statistics. RESULTS: All rabbits tolerated well the surgical procedure and subsequent cMRI sessions. The open-chest occlusion and close-chest reperfusion of the LCx, double suture method and bifunctional staining were successfully applied in all animals. The percentage MI volumes globally (n = 6) and by slice (n = 25) were 36.59% ± 13.68% and 32.88% ± 12.38% on iDE, and 35.41% ± 12.25% and 32.40% ± 12.34% on eDE. There were no significant differences for MI determination with excellent linear regression correspondence (r global = 0.89; r slice = 0.9) between iDE and eDE. The percentage AAR volumes globally (n = 6) and by slice (n = 25) were 44.82% ± 15.18% and 40.04% ± 13.64% with RIO-staining, and 44.74% ± 15.98% and 40.48% ± 13.26% by DR showing high correlation in linear regression analysis (r global = 0.99; r slice = 1.0). The mean differences of the two AAR measurements on Bland-Altman were almost zero, indicating RIO-staining and DR were essentially equivalent or inter-replaceable. The AAR was significantly larger than MI both globally and slice-by-slice (P < 0.01). After correction with the background and the blank heart without bifunctional staining (n = 3), the PDR for the AAR and normal myocardium was 32% ± 15% and 35.5% ± 35%, respectively, which is significantly different (P < 0.001), suggesting that blood perfusion to the AAR probably by collateral circulation was only less than 10% of that in the normal myocardium. CONCLUSION: The myocardial area at risk in ischemic heart disease could be accurately determined postmortem by this novel bifunctional staining, which may substantially contribute to translational cardiac imaging research.

In vivo serial MR imaging of magnetically labeled endothelial progenitor cells homing to the endothelium injured artery in mice.

BACKGROUND: Emerging evidence of histopathological analyses suggests that endothelial progenitor cells (EPCs) play an important role in vascular diseases. Neointimal hyperplasia can be reduced by intravenous transfusion of EPCs after vascular injury in mice. Therefore, it would be advantageous to develop an in vivo technique that can explore the temporal and spatial migration of EPCs homing to the damaged endothelium noninvasively. METHODOLOGY/PRINCIPAL FINDINGS: The left carotid common artery (LCCA) was injured by removal of endothelium with a flexible wire in Kunming mice. EPCs were collected by in vitro culture of spleen-derived mouse mononuclear cells (MNCs). EPCs labeling was carried out in vitro using Fe2O3-poly-L-lysine (Fe2O3-PLL). In vivo serial MR imaging was performed to follow-up the injured artery at different time points after intravenous transfusion of EPCs. Vessel wall areas of injured artery were computed on T2WI. Larger MR signal voids of vessel wall on T2WI was revealed in all 6 mice of the labeled EPC transfusion group 15 days after LCCA injury, and it was found only in 1 mouse in the unlabeled EPC transfusion group (p = 0.015). Quantitative analyses of vessel wall areas on T2WI showed that the vessel wall areas of labeled EPC transfusion group were less than those of unlabeled EPC transfusion group and control group fifteen days after artery injury (p<0.05). Histopathological analyses confirmed accumulation and distribution of transfused EPCs at the injury site of LCCA. CONCLUSIONS/SIGNIFICANCE: These data indicate that MR imaging might be used as an in vivo method for the tracking of EPCs homing to the endothelium injured artery.

Resting brain connectivity: changes during the progress of Alzheimer disease.

PURPOSE: To investigate alterations in functional connectivity in the resting brain networks in healthy elderly volunteers and patients with mild, moderate, or severe Alzheimer Disease (AD). MATERIALS AND METHODS: This study was approved by the institutional ethics committee, and informed consent was obtained. Forty-six patients with AD and 16 healthy elderly volunteers were prospectively examined. Resting-state functional magnetic resonance imaging was used to detect alterations in posterior cingulate cortex (PCC) functional connectivity through a comparison of the healthy control group with three separate AD groups-mild, moderate, and severe AD. A temporal correlation method was used to obtain PCC connectivity maps. RESULTS: Dissociated functional connectivity between the PCC and a set of regions, including the visual cortices bilaterally, the inferior temporal cortex, the hippocampus, and especially the medial prefrontal cortex and the precuneus and/or cuneus, was observed in all AD groups. The disruption of connectivity intensified as the stage of AD progression increased. There were also regions that exhibited increased connectivity; these regions extended from left lateralized frontoparietal regions and spread to bilateral frontoparietal regions along with AD progression. CONCLUSION: Changes in PCC functional connectivity comprised bidirectional alterations in the resting networks in AD-affected brains, and the impaired resting functional connectivity seemed to change with AD progression. Therefore, alterations in functional connectivity in the default mode network might play a role in the progression of AD.

Assessments of proliferation capacity and viability of New Zealand rabbit peripheral blood endothelial progenitor cells labeled with superparamagnetic particles.

Magnetic resonance imaging (MRI) has proven to be effective in tracking the distribution of transplanted stem cells to target organs by way of labeling cells with superparamagnetic iron oxide particles (SPIO). However, the effect of SPIO upon labeled cells is still unclear on a cellular level. With this study, the proliferation and viability of New Zealand rabbit peripheral blood endothelial progenitor cells (EPCs) labeled with SPIO were evaluated and in vitro images were obtained using a 1.5 T MR scanner. Mononuclear cells (MNCs) were isolated from peripheral blood of the adult New Zealand rabbit and cultured in fibronectin-coated culture flasks, in which EPCs were identified from cell morphology, outgrowth characteristics, and internalization of DiI-Ac-LDL and binding to FITC-UEA I. EPCs were incubated with the self-synthesized poly-L-lysine-conjugated SPIO (PLL-SPIO) particles in a range of concentrations. The prevalence of iron-containing vesicles or endosomes in the cytoplasm of labeled cells was confirmed with Prussian blue staining and transmission electron microscopy. Tetrazolium salt (MTT) assay, cell apoptosis, and cycle detection were assessed to evaluate proliferation and function of various concentrations, magnetically labeled EPCs. The quantity of iron per cell was determined by atomic absorption spectrometry. The cells underwent MRI with different sequences. The result showed that rabbit EPCs were efficiently labeled with the home synthesized PLL-SPIO. There was found to be no statistically significant difference in the MTT values of light absorption measured on the third and fifth days. Between labeled and unlabeled cells, there were also no aberrations found in the cell cycles, apoptosis, or growth curves. The atomic absorption spectrophotometer showed that the intracellular content of Fe decreased as more time elapsed after labeling. The labeled EPCs demonstrated a loss of MRI signal intensity (SI) when compared with the SI of unlabeled cells. These signal changes (ASI) were visible when cells were labeled with more than 5 x 104/ml of SPIO. The change in SI corresponded to the amount of iron in the EPCs, which reached a maximum at T2*WI. These data demonstrate that EPCs from the peripheral blood of the New Zealand rabbit can be effectively labeled with self-synthesized PLL-SPIO with minimal effects on cell proliferation and activity. Magnetically labeled EPCs can be imaged at 1.5 T MR and can therefore be used as an MR tracker of implanted EPCs.

Detection of PCC functional connectivity characteristics in resting-state fMRI in mild Alzheimer's disease.

Resting-state networks dissociate in the early stage of Alzheimer's disease (AD). The posterior cingulate cortex (PCC) in AD brain is vulnerable to isolation from the rest of brain. However, it remains unclear how this functional connectivity is related to PCC changes. We employed resting-state functional MRI (fMRI) to examine brain regions with a functional connection to PCC in a mild AD group compared with matched control subjects. PCC connectivity was gathered by investigating synchronic low frequency fMRI signal fluctuations with a temporal correlation method. We found asymmetric PCC-left hippocampus, right dorsal-lateral prefrontal cortex and right thalamus connectivity disruption. In addition, some other regions such as the bilateral visual cortex, the infero-temporal cortex, the posterior orbital frontal cortex, the ventral medial prefrontal cortex and the precuneus showed decreased functional connectivity to the PCC. There were also some regions, primarily in the left frontal-parietal cortices, that showed increased connectivity. These regions included the medial prefrontal cortex, bilateral dorsal-lateral prefrontal cortex, the left basal ganglia and the left primary motor cortex. Impairments to memory, high vision-related functions and olfaction in AD can be explained by a disruption to the functional connection of resting-state networks. The results of increased connectivity may support the compensatory-recruitment hypothesis. Our findings suggest that the characteristics of resting-state functional connectivity could plausibly provide an early imaging biomarker for AD.

Inhibited atherosclerotic plaque formation by local administration of magnetically labeled endothelial progenitor cells (EPCs) in a rabbit model.

PURPOSE: To investigate whether atherosclerosis can be prevented by magnetically labeled endothelial progenitor cells (EPCs) in rabbits. MATERIALS AND METHODS: EPCs derived from rabbit periphery blood were labeled with a superparamagnetic iron oxide (SPIO) agent Fe(2)O(3)-poly-L-lysine (Fe(2)O(3)-PLL). Rabbit atherosclerosis was induced by high-cholesterol-diet following balloon injury via catheterization of right common carotid artery (RCCA). Fe(2)O(3)-PLL labeled EPCs (2 x 10(6)) and media were allowed to interact with the RCCA for 25 min in EPC-treated rabbits (n=14) and control rabbits (n=7) animals respectively. MRI was performed with a 1.5T-magnet to measure RCCA signal intensity (SI) and caliber at week 1, 2, 3, 6, 12, and 15 with animals euthanized in groups for histopathology. RESULTS: In EPC-treated rabbits, T(2)(*)-weighted MRI showed SI loss in RCCA at week 1 and 2 followed by normalization after week 3. MRI outcomes corresponded well to findings of Prussian blue staining. MRI at week 6, 12 and 15 showed little stenosis of RCCA in EPC-treated rabbits, but moderate to severe stenoses in control rabbits. Histology at week 15 revealed significantly thinner RCCA wall (277.62 microm vs. 382.95 microm, P=0.026), greater internal diameter (913.33 microm vs. 789.64 microm, P=0.037) and smaller plaque (398.60mm(2) vs. 597.70 mm(2), P=0.047) in EPC-treated rabbits relative to control rabbits. CONCLUSION: Atherosclerosis at RCCA was inhibited by SPIO-labeled EPCs, which was depicted with a clinical MRI scanner over 2 weeks after cell administration, suggesting that EPCs may play a role in restoration of endothelial injury and prevention of atherosclerosis.

[Magnetic resonance imaging of mesenchymal stem cells transplanted intravascularly in treatment of acute renal failure:in vivo experiment with rats].

OBJECTIVE: To evaluate the efficacy of in vivo magnetic resonance imaging (MRI) of mesenchymal stem cells (MSCs) injected intravascularly in treatment of acute renal failure (ARF) , and to investigate the changes of renal function and pathology of ARF after MSC transplantation. METHODS: Rat MSCs were isolated and labeled with Fe2O3-PLL in vitro. Thirty SD rats underwent intramuscular injection of glycerol so as to establish ARF models and then randomly divided into 3 equal groups: Group I undergoing injection of labeled MSCs into abdominal aorta via transcatheter, Group II injected with unlabelled MSCs, and Group III injected with normal saline as controls. MRI of kidney was conducted before injection, and 0.5 h, and 1, 2, and 5 days after injection. One and 2 days after the transplantation 3 rats from each group underwent MRI and extraction of blood samples from the abdominal aorta and then killed with their kidneys taken out, and 5 days after the rest rats were all killed after MRI with their kidneys taken out. Serum creatinine (Scr) and blood urea nitrogen (BUN) were examined so as to evaluate the renal function. Microscopy was conducted to observe the pathological changes. Prussian blue + CD68 antibody staining was performed to identify the labeled MSCs. RESULTS: MRI showed decrease of signal intensity in renal cortex on the T2 *-weighted MR images up to 5 days after transplantation. Histological analysis showed that most Prussian blue-positive cells were in the glomerular capillaries, corresponding to the areas where signal intensity decrease was observed by MRI. The Scr and BUN levels 2 and 5 days after the implantation of Group I were both lower than those of the control group, and there were not significant differences in the Scr and BUN levels between Groups I and II. Renal tubular injury scoring showed that the renal tubular injury was significantly lighter than that of the control group. CONCLUSION: 1.5-T MRI seems a good in vivo technique to monitor the magnetically labeled MSCs administered into the abdominal aorta of ARF animals, which are distributed in the glomerular capillaries in the early stage after transplantation. MSCs may promote the recovery of ARF.

MR tracking of magnetically labeled mesenchymal stem cells in rat kidneys with acute renal failure.

Stem cell transplantation is emerging as a potential treatment option for acute renal failure (ARF) because of its capability to regenerate tissues and organs. To better understand the mechanism of cell therapy, in vivo tracking cellular dynamics of the transplanted stem cells is needed. In the present study, in vivo monitored magnetically labeled mesenchymal stem cells (MSCs) were transplanted intravascularly into an ARF rat model using a conventional magnetic resonance imaging (MRI) system. Rat bone marrow MSCs were labeled with home synthesized Fe2O3-PLL, and labeled (n = 6) or unlabeled MSCs (n = 6) were injected into the renal arteries of the rats with ARF induced by the intramuscular injection of glycerol. Using the same technique, labeled MSCs were also injected into the rats assigned to a control group (n = 8). MR images of kidneys were obtained before injection of MSCs as well as immediately, 1, 3, 5, and 8 days afterwards. MR findings were analyzed and compared with histopathological and immunohistochemical results. These results showed that the rat MSCs were successfully labeled with the home synthesized Fe2O3-PLL. In both renal failure and intact rat models, the labeled MSCs demonstrated a loss of signal intensity in the renal cortex on T2*-weighted MR images, which was visible up to 8 days after transplantation. Histological analyses showed that most of the labeled MSCs that tested positive for Prussian blue staining were in glomerular capillaries, corresponding to the areas where a loss in signal intensity was observed in the MRI. A similar signal intensity decrease was not detected in the rats with unlabeled cells. These data demonstrate that the magnetically labeled MSCs in the rat model of ARF were successfully evaluated in vivo by a 1.5 T MRI system, showing that the mechanisms of stem cell therapy have great potential for future ARF treatment recipients.

A rabbit model of atherosclerosis at carotid artery: MRI visualization and histopathological characterization.

To induce a rabbit model of atherosclerosis at carotid artery, to visualize the lesion evolution with magnetic resonance imaging (MRI), and to characterize the lesion types by histopathology. Atherosclerosis at the right common carotid artery (RCCA) was induced in 23 rabbits by high-lipid diet following balloon catheter injury to the endothelium. The rabbits were examined in vivo with a 1.5-T MRI and randomly divided into three groups of 6 weeks (n=6), 12 weeks (n=8) and 15 weeks (n=9) for postmortem histopathology. The lesions on both MRI and histology were categorized according to the American Heart Association (AHA) classifications of atherosclerosis. Type I and type II of atherosclerotic changes were detected at week 6, i.e., nearly normal signal intensity (SI) of the injured RCCA wall without stenosis on MRI, but with subendothelial inflammatory infiltration and proliferation of smooth muscle cells on histopathology. At week 12, 75.0% and 62.5% of type III changes were encountered on MRI and histopathology respectively with thicker injured RCCA wall of increased SI on T(1)-weighted and proton density (PD)-weighted MRI and microscopically a higher degree of plaque formation. At week 15, carotid atherosclerosis became more advanced, i.e., type IV and type V in 55.6% and 22.2% of the lesions with MRI and 55.6% and 33.3% of the lesions with histopathology, respectively. Statistical analysis revealed a significant agreement (p<0.05) between the MRI and histological findings for lesion classification (r=0.96). A rabbit model of carotid artery atherosclerosis has been successfully induced and noninvasively visualized. The atherosclerotic plaque formation evolved from type I to type V with time, which could be monitored with 1.5-T MRI and confirmed with histomorphology. This experimental setting can be applied in preclinical research on atherosclerosis.

[In vitro MR imaging of Fe(2)O(3)-PLL labelled rabbit peripheral blood endothelial progenitor cells].

OBJECTIVE: To perform in vitro magnetic resonance imaging on magnetic iron oxide (Fe(2)O(3)-PLL) labeled rabbit peripheral blood endothelial progenitor cells (EPCs). METHODS: Fe(2)O(3) was incubated with PLL for 2 hours to form Fe(2)O(3)-PLL. Rabbit peripheral blood mononuclear cells (MNCs) were isolated and EPCs were selected by adherence method, expanded and incubated with Fe(2)O(3)-PLL. Intracellular iron was detected by Prussian blue stain and under electron microscope. MTT assay was used to evaluate cell survival and proliferation of Fe(2)O(3)-PLL labeled EPCs. Flow cytometry was used to analysis cell cycle and apoptosis. The cells underwent in vitro MR imaging with various sequences. RESULTS: Iron-containing intracytoplasmatic vesicles could be observed clearly with Prussian blue staining and electron microscope observation. Survival, life cycle and apoptosis values obtained by MTT and flow cytometry analysis were similar among unlabelled EPCs and EPCs labeled with various concentrations Fe(2)O(3)-PLL. The signal intensity on MRI was significantly decreased in labeled cells compared with that in unlabeled cells. The percentage change in signal intensity (DeltaSI) was most significant on T(2)*WI and DeltaSI was significantly lower in cells labeled for 7 days than that labeled for 1 day. CONCLUSIONS: The rabbit peripheral blood EPCs can be labeled with Fe(2)O(3)-PLL without significant change in viability and proliferation. The labeled EPCs can be imaged with standard 1.5 T MR equipment. The degree of MR signal decreasing may indirectly reflect the cells count, growth state and division.

[Magnetic resonance imaging of magnetically labeled endothelial progenitor cells homing to the injured endothelium].

OBJECTIVE: Accumulating evidence suggests that the endothelial progenitor cells (EPCs) can reendothelialization the injured endothelium. Superparamagnetic iron-oxide particles are being used for intracellular magnetic labeling of cells and in vivo cells tracking. The aim of this study was to investigate the possibility of depict and track magnetically labeled EPCs in vivo for carotid artery endothelium injured New Zealand White rabbit model by 1.5 T magnetic resonance imaging system after EPCs transplantation. METHODS: The EPCs of New Zealand White rabbit were isolated, confirmed, expanded and then incubated with home synthesized Fe2O3-PLL for 24 hours, Prussian blue stain was performed for showing intracellular irons. The model of carotid arterial injury was performed by 2.5 F balloons. The group A of 5 rabbits were transplanted with magnetically labeled EPCs, the group B of 5 rabbits were received fluorescent-labeled EPCs, and 5 rabbits of the group C were received the same volume of saline injection immediately after the carotid artery endothelium was injured. The transfused EPCs were strictly restricted to the injury site. MR imaging and histology were performed and compared 7 days late. RESULTS: The Epcs labeling efficiency of Fe2O3-PLL was more than 95% identified by Prussian blue stain. Seven days after the transplanted of EPCs, only in group A, the injured endothelium of carotid artery wall had the signal intensity loss in T(2)WI MRI, which corresponding to the injured endothelium where the most Prussian blue staining-positive cells were in histopathological analyses. While histopathological slides showed that the fluorescence-positive cells in the injured endothelium which had been transplanted Dil labeled EPCs also. The group C was either in negative. CONCLUSION: The rabbits endothelial progenitor cells can be effectively labeled with Fe2O3-PLL, 1.5 T magnetic resonance imaging system could depict and monitor the magnetically labeled endothelial progenitor cells homing to the injured endothelium of the artery, which may have much more potential values for studying the engraftment of EPCs in cardiovascular disease.