Spin-Labeling MRI Detects Increased Myocardial BloodFlow After Endothelial Cell Transplantation in the
Infarcted HeartHualei Zhang, BS*; Hui Qiao, MD, PhD*; Rachel S. Frank, BS; Bin Huang, MD;
Kathleen J. Propert, PhD; Susan Margulies, PhD; Victor A. Ferrari, MD;Jonathan A. Epstein, MD; Rong Zhou, PhD
Background—We quantified absolute myocardial blood flow (MBF) using a spin-labeling MRI (SL-MRI) method aftertransplantation of endothelial cells (ECs) into the infarcted heart. Our aims were to study the temporal changes in MBFin response to EC transplantation and to compare regional MBF with contractile function (wall motion) andmicrovascular density.
Methods and Results—We first validated the SL-MRI method with the standard microsphere technique in normal rats. Wethen induced myocardial infarction in athymic rats and injected 5 million ECs (human umbilical vein endothelial cells)suspended in Matrigel or Matrigel alone (vehicle) along the border of the blanched infarcted area. At 2 weeks aftermyocardial infarction, MBF averaged over the entire slice (P�0.038) and in the infarcted region (P�0.0086) wassignificantly higher in EC versus vehicle group; the greater MBF was accompanied by an increase of microvasculaturedensity in the infarcted region (P�0.0105 versus vehicle). At 4 weeks after myocardial infarction, MBF in the remoteregion was significantly elevated in EC-treated hearts (P�0.0277); this was accompanied by increased wall motion inthis region assessed by circumferential strains. Intraclass correlation coefficients and Bland-Altman plot revealed a goodreproducibility of the SL-MRI method.
Conclusions—MBF in free-breathing rats measured by SL-MRI is validated by the standard color microsphere technique.SL-MRI allows quantification of temporal changes of regional MBF in response to EC treatment. The proof-of-principlestudy indicates that MBF is a unique and sensitive index to evaluate EC-mediated therapy for the infarcted heart. (CircCardiovasc Imaging. 2012;5:00-00.)
Quantification of absolute myocardial blood flow (MBF,in units of milliliters per minute per gram of tissue) is
important when evaluating stem cell mediated treatment ofmyocardial infarction (MI). First, the ability of local bloodsupply to match metabolic demand of the tissue will affect thesurvival of grafted cells. Second, new vasculature formed byeither transplanted cells or host cells will improve perfusion,which then facilitates graft survival and expansion. Becausean increase in capillary density does not necessarily lead to anincrease of blood flow in vivo,1 a noninvasive quantificationof MBF is desirable, and a measurable increase in MBF canbe a specific index to determine the success of therapeuticangiogenesis or vasculogenesis in the infarcted heart.
Clinical Perspective on p ●●●
In the REPAIR-AMI clinical trial,2 bone marrow–derivedprogenitor cells (BMCs) were infused in patients with reper-fused acute MI. By measuring blood flow using an intracoro-nary Doppler probe, the investigators found that the coronaryflow reserve (CFR, defined as the ratio of the maximal to theresting coronary blood flow) of infarct-related arteries recov-ered to a normal level in BMC-treated patients but not inplacebo controls. CFR thus provided direct evidence thatBMCs restored microvascular function of infarct-related ar-teries. Microvascular function, quantified by MBF, could bean important predictor of global functional recovery, whichwas achieved in some BMC trials3 but not in others.4,5
Received May 2, 2011; accepted January 31, 2012.From the Laboratories of Molecular Imaging, Department of Radiology (H.Z., H.Q., R.S.F., B.H., R.Z.), Department of Bioengineering (H.Z., S.M.,
R.Z.), Department of Medicine, Division of Cardiovascular Medicine (V.A.F., J.A.E.), and the Cardiovascular Institute (H.Z., H.Q., V.A.F., J.A.E.),Department of Biostatistics and Epidemiology (K.P.), and Department of Cell and Developmental Biology (J.A.E.), University of Pennsylvania,Philadelphia, PA.
*These authors contributed equally to this work.Correspondence to Rong Zhou, PhD, Laboratories of Molecular Imaging, Department of Radiology, University of Pennsylvania, B6 Blockley Hall, 422
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Whereas BMCs are a mixture of various types of cells, fullydifferentiated endothelial cells (ECs)6 or endothelial progen-itor cells7,8 represent a more purified cell population and havebeen suggested to form new vasculature, preserve left ven-tricular (LV) function, and inhibit apoptosis in the infarctedheart. However, mechanisms underlying the salutary effectsof these cells are not well understood: specifically, whether ornot neovascularization leads to improved regional MBF hasnot been studied extensively.
To evaluate EC-mediated or endothelial progenitor cell–mediated vascular repair over time and to be able to translateit to the clinic, noninvasive imaging–based methods for MBFestimation are desirable. At present, positron emission to-mography is the clinical standard for MBF measurement.9
First-pass contrast-enhanced MRI can be used to quantifyMBF only after extensive modeling of the kinetic data.10 Incontrast, spin-labeling–based MRI (SL-MRI) utilizes endog-enous water molecules, eliminating the need to inject anyexogenous tracers. First implemented on the heart by Belle etal11,12 on the rat in vivo, this technique has proven effectivefor mapping MBF in rapidly beating rodent hearts to studycardiovascular diseases in such models.13–17
In this study, we first validated the SL-MRI method with astandard microsphere technique; second, we used SL-MRI todetect changes of MBF over time in the infarcted, border, andremote regions in response to EC transplantation and dem-onstrated that the improved MBF is corroborated with in-creased wall motion and microvascular density in the corre-sponding regions.
Methods
Experimental Design and Study GroupsAll animal procedures were approved by the local InstitutionalAnimal Care and Use Committee. This study had 2 aims. Aim 1 wasto validate the SL-MRI–based MBF measurement; Aim 2 was toassess the utility of SL-MRI for detecting MBF changes mediated byEC transplantation in an MI model.
For Aim 1, 18 rats were used; 5 rats completed the study, whereas13 died during procedures (see section: Validation of SL-MRI–Based MBF With the Microsphere Method). For Aim 2, ECs in theform of human umbilical vein endothelial cells (HUVECs, ATCC,Manassas, VA) were expanded (�6–10 passages) and suspended ingrowth factor–reduced Matrigel (Collaborative Biomedical, Bed-ford, MA). MI was induced in male athymic nu/nu rats (6–8 weeksold; Frederick Cancer Center, Frederick, MD) by permanent ligationof the left anterior descending coronary artery.18 During the samesurgical session, 5 million ECs in 100 �L Matrigel, or Matrigel alone(vehicle) were injected in one spot in the border close to the blanchedarea. Initial infarct size (as a percentage of LV myocardial volume)was assessed at 1 day after MI, using late gadolinium enhancement(LGE), and rats having infarct size outside the range of [10%, 30%]were excluded as we described previously.18,19 A total of 51 rats (26were assigned to EC and 25 to vehicle group) were used in Aim 2;22 died as the result of surgery and 7 were excluded at day 1 due tounqualified infarct size; the remaining 22 rats (n�12 in EC andn�10 in the vehicle group) proceeded to studies at 2 weeks, and 4rats in EC and 3 in the vehicle group were euthanized for microvas-culature density (MVD) analysis; the remaining 15 animals wereimaged and euthanized at 4 weeks. As the result of instrumentdowntime, there are missing MBF data from unscanned rats at 1 dayand 2 weeks, whereas all rats were scanned at 4 weeks; the numberof rats scanned at each time point was specified in figures.
SL-MRI–Based MBF QuantificationMR experiments were performed on a 4.7-T horizontal bore magnetinterfaced to a Varian DirectDrive console. A combination of atransverse electromagnetic volume transmit coil and surface receivecoil (InsightMRI, Worcester, MA)18,19 was interfaced to a 12-cmgradient insert with maximum strength of 25 g/cm. The rat wasmaintained under anesthesia by 1.5% (unless indicated otherwise)isoflurane mixed with oxygen at a flow rate of 1 L/min through anose cone. ECG and respiration were monitored (SA Instruments,Stony Brook, NY), and core temperature was maintained at36.5�0.2°C by warm air. A gel phantom (with relaxation time,T1, slightly longer than normal myocardium) was fixed on theanimal holder.
Data AcquisitionWe modified the SL-MRI protocol by Kober et al,20 in which ECG-and respiration-gated gradient echo technique was used to achievehigh spatial resolution perfusion maps in free-breathing animals.Instead of referencing the phantom with a known T1 to derive tissueT1 values, our protocol directly calculates T1 from a series ofinversion recovery images using a least-squares fitting algo-rithm.21,22 Consequently, the mean perfusion value of the phantombeing close to 0 was used as a criterion to evaluate the quality of rawdata and T1 fitting.
MBF was measured from a 3-mm, short-axis slice at mid LV. Tomap T1 corresponding to non–slice-selective and slice-selectiveinversion of the magnetization, a modified TOMROP sequence21,23
was used, which consists of an inversion pulse (followed by crushergradients) and a gradient echo module that samples the samephase-encoding line multiple times along the magnetization recov-ery. A hyperbolic secant adiabatic pulse24 lasting 6–7 ms (permittedby RF coil power limit) was used for the inversion. The slicethickness of the inversion pulse was set to a large value (3�105 mm)in the case of global inversion and to 2.5� of the imaging slicethickness in the case of slice-selective inversion. The ratio of 2.5 wasto compensate the imperfect matching of the inversion and excitationpulse profiles and was determined using the agarose gel phantom (ie,under the no-flow condition) by stepwise increasing the ratio ofinversion to excitation pulse slice width as 1, 1.5, 2, 2.5, 3, and 3.5and identifying the smallest inversion pulse slice width that canachieve the “zero” flow results in the phantom.
Immediately after the inversion pulse and under ECG-gating, aseries of images was acquired for at least 7 seconds, followed by a4-second delay to ensure that the spins in myocardium wererecovered to �99% of the equilibrium magnetization before the nextinversion pulse. The respiratory waveform and k-space acquisitionswere simultaneously recorded (SA Instrument, Stony Brook, NY) forretrospective elimination of images acquired outside the quiescentphase of expiration. The following parameters were used: field ofview (FOV)�35�35 mm2, acquisition matrix�192�80, TE�2.19ms, bandwidth�96 kHz, inversion time spacing�2 heart beats,gaussian excitation pulse of 800 æs and 10° flip angle; each pair ofT1 measurements took about 25 minutes, 2 signal averages.
Data ProcessingA 3-parameter fitting algorithm21,22 was used to calculate pixel-wiseT1 values under non–slice-selective and slice-selective inversion,designated as T1ns and T1ss, respectively. The pixel-wise blood flowwas quantified using the formula below.11
(1)MBF
��
T1ns
T1b� 1
T1SS�
1
T1ns�,
where ��quantity of water per gram of tissue
quantity of water per gram of bloodis the tissue-blood
partition coefficient of water and was set to 0.83 mL/g for ratmyocardium. T1b is the blood T1 under global inversion and was setto 1.6 seconds at 200 MHz.11 The phantom was included for flowcalculation (Figure 1A), and an MBF measurement was accepted ifthe phantom had a mean flow value within �0.5 mL/min/g; if
rejected, the data were reacquired within 2 days of the 2-week and4-week time points in the EC study or considered as missing.
Validation of SL-MRI–Based MBF With theMicrosphere MethodBefore the SL-MRI session, the carotid artery (for direct injection ofmicrospheres into LV) and femoral artery (for withdrawal ofreference blood) were cannulated.25 Placement of the catheter insideLV was verified by proper length of the inserted catheter and rapidblood pulsations in the catheter.
Immediately after SL-MRI, 200 000 fluorescent microspheres(FMs) of 15-�m diameter (Dye-Trak, Triton Technology, SanDiego, CA) in 200 �L saline was infused in 10 seconds, followed by500 �L saline flush for 20 seconds. Meanwhile, reference blood waswithdrawn through the femoral artery catheter at 330 �L/minthrough a syringe pump (Harvard Apparatus, Boston, MA) starting30 seconds before the FM injection and lasting for 180 seconds. Oncompletion of blood sampling, the heart was harvested and embed-ded in 1% agarose gel.
A fluorescent plate/slide imaging protocol26 was used to quantifythe number of FMs. The heart was sectioned into �100-�m-thicksections on a vibratom (Leica VT1000S, Leica Microsystems GmbH,Germany). Sections corresponding to the imaging slice were deter-mined by their positions relative to the apex and mounted onmicroscope slides. FMs in the reference blood were retrieved bymembrane filters, which were also mounted on slides. The slideswere then scanned on a fluorescent plate reader (Alpha Innotech,CA). The number of microspheres in slides was counted by a customprogram written in MATLAB (Mathworks, Natick, MA). MBF wasestimated on the basis of the amount of FMs in the tissue andreference blood as well as the reference blood withdrawal rate.25
Cardiac Function After EC TransplantationEvaluated by MRIMBF was measured at 1 day, 2 weeks, and 4 weeks after MI; infarctsize and LV ejection fraction (LVEF)27 were estimated at 1 day and
4 weeks. Regional wall motion was measured at 4 weeks only, fromthe same slice position as MBF but with 1.5-mm thickness. The MRIprotocol for wall motion, which was detailed previously,19 wasupdated to a tag spacing of 0.9 mm (ke�1.11 cycle/mm) and 10cardiac frames (15 ms per frame). To obtain regional MBF and wallmotion, LV myocardium was segmented into I-B-R, where infarcted(I) region was defined on the LGE image (at the same position asMBF slice), border region (B) as two 60° sectors on each side of theinfarcted segment, and the remote region (R) encompassed theremaining myocardium.
MVD and Incorporation of Grafted ECs Intothe VasculatureMVD was estimated at 2 and 4 weeks. On euthanasia, a 5-mm-thickslab centered at the midventricular level was embedded in OptimalCutting Temperature media and cut in short-axis orientation. After1 mm was trimmed off the top layer, 3 sections (each 10 �m thick)were cut at 3 levels with 1-mm spacing. Sections were immuno-stained using the following antibodies: (1) rabbit polyclonal anti–vonWillebrand Factor (vWF) antibody (Sigma, St Louis, MO) andFITC-conjugated goat anti-rabbit secondary antibody and (2) mousemonoclonal antihuman CD34 antibody (Abcam, Cambridge, MA)and Cy3-conjugated goat anti-mouse secondary antibody. One sec-tion at each level was stained with hematoxylin and eosin.
To estimate regional MVD, each section was segmented into theI-B-R region under 2� objective lens: the infarcted region wasidentified on the adjacent hematoxylin and eosin section; the borderand remote regions were defined in the same way as on MRI. Then,under 10�, at least 6 FOVs (each covering 1.1 mm2) were capturedincluding 3 in the Remote, 2 in the Border, and 1–3 in the Infarctedregion, depending on the infarct size. Clustered cells or continuousbranching structures with positive vWF staining were counted as 1capillary. To visualize engrafted ECs, double immunostaining forhuman CD34 and vWF was performed.
Figure 1. A through H, Validation of spin-labeling MRI (SL-MRI)–based myocardial blood flow (MBF) with color microsphere technique.Non–slice-selective T1 map (T1ns, B), slice-selective T1 map (T1ss, C), and MBF map (D) are calculated from the area enclosed in theblue box on the corresponding GRE image (A). White arrows point to the reference phantom; yellow arrows point to the catheterinside the left ventricle (LV). Distribution of fluorescent microspheres (FMs) in a section is shown (E). Correlation of MBF was measuredby SL-MRI versus FM technique: average from entire slice (F) or segments (G). Myocardium wall on MBF maps and on micrographs oftissue sections was divided into 3 segments (septal, lateral, and combined anterior/posterior), resulting in 3 data points per animal �5animals�15 points in G. For Bland-Altman analysis (H), the difference of MBF, (SL minus FM) was plotted against the average of MBF(SL plus FM)/2; mean difference (bias) and the 95% limits of agreement defined as bias �1.96 standard deviation (STD�0.767 mL/minper gram) were computed.
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Zhang et al EC-Mediated Myocardial Blood Flow Increase 3
Statistical AnalysesData are presented as mean (�SD) in text and figures. TheBland-Altman plot and intraclass correlation coefficients (ICC,MedCalc 11.6, Mariakerke, Belgium), calculated from segmentaland sliced-averaged MBF, were used to evaluate the agreementbetween MRI- and FM-based MBF measurements. The same ap-proaches were applied to 12 animals (6 in EC and 6 in the vehiclegroup), each having 2 serial scans in the same imaging session at 2weeks after MI, for assessing the reproducibility of the SL-MRImethod. To assess the treatment effect (EC versus the vehicle group),a mixed-effect model with repeated measures (PROC MIXED, SASInstitute Inc, Cary, NC) was applied. This model allows inclusion ofdata from all time points under data missing and evaluates thetreatment effect between groups (subjects) and regions (withinsubject) over time; P�0.05 is considered to be statisticallysignificant.
ResultsValidation of SL-MRI by the Microsphere MethodTo validate SL-MRI as a method to accurately measure MBFin living animals, we directly compared SL-MRI measure-ments with the standard microsphere method in 5 animals: wecannulated the carotid and femoral arteries right before theSL-MRI session and injected FMs immediately after SL-MRI. MR images were first cropped, and the region definedby the blue box (Figure 1A) was processed to derive T1 mapscorresponding to non–slice-selective (T1ns, Figure 1B) andslice-selective inversion (T1ss, Figure 1C). Pixel-wise MBFwas calculated by Equation 1 (Figure 1D). MBF was alsoestimated by counting the number of FMs in myocardium (1section is shown in Figure 1E). A strong correlation betweenSL-MRI and FM technique (R�0.972) was revealed for MBFaveraged from the entire slice; a slope close to unity suggestsan excellent agreement between the 2 techniques (Figure 1F).To obtain regional MBF measurements, the LV wall on theMBF map and corresponding micrographs were divided intoseptal, lateral, anterior, and posterior quadrants. The anteriorand posterior quadrants were combined into 1 segmentbecause they were not distinguishable on tissue sections.Averaged MBF in the septal, lateral, and combined anterior/posterior segments obtained by the 2 methods remain wellcorrelated, with an R value of 0.753 (Figure 1G). A goodagreement between the 2 methods was also demonstrated bythe ICC derived from slice-averaged MBF (Table 1) and byBland-Altman plot (Figure 1H): it revealed a bias of �0.065mL/min per gram, which is not statistically significant fromzero (P�0.897), with 95% limits of agreements including allbut 1 data point. To facilitate correlation studies, the percent-age of isoflurane applied to individual animals was variedfrom 1–2.5% because MBF is shown to be regulated by levelsof anesthesia.13 For each animal, however, the percentage ofisoflurane was kept the same. The heart and respiration ratewere stably maintained during the experiments (Table 2).
Changes in Regional MBF in Response toEC TransplantationTo establish the utility of SL-MRI to measure MBF in the settingof EC therapy, we injected ECs or vehicle into the border zoneof infarcted hearts and performed serial evaluations over theensuing 4 weeks. At 1 day after MI, a relatively uniform infarctsize distribution in the 2 groups was obtained: 18�4.7% in theEC (n�12) versus 17�5.6% in vehicle (n�10) group (Table 3,P�0.553), which facilitated a fair comparison for EC-mediatedeffects. At 1 day after MI, shown in the inset of Figure 2A,regional MBF was depressed in the infarcted versus remoteregion. In comparison of slice-averaged MBF over time in ECversus the vehicle group, the MBF is significantly higher in theEC group at 2 weeks (P�0.0380). A significant treatment effecton regional MBF was revealed at 2 weeks across regions(P�0.0057) and in the remote region across time points(P�0.0402). When the analysis was refined to specific region ortime point, a significant treatment effect in the infarcted region at2 weeks (P�0.0086) and in the remote region at 4 weeks(P�0.0277) were obtained. Representative MBF maps at 2weeks indeed show higher MBF in the infarcted segment in theEC-treated heart, which had similar infarct size as the vehicle-treated one (Figure 2B through 2E). More capillaries in theinfarcted region in EC-treated hearts were revealed by vWFimmunostaining (Figure 2F and 2G); quantitative analysis con-firmed a significantly higher MVD in the infarcted region(P�0.0105 EC versus vehicle group, Figure 2J). Furthermore,double staining for vWF and human CD34 demonstrated incor-poration of ECs into capillaries in the infarcted (Figure 2H) andborder region (Figure 2I). Taken together, these data provideconvincing evidence that EC engraftment enhanced new vesselformation, leading to improved perfusion in the infarcted regiondetected by SL-MRI.
At 4 weeks after MI, whereas MBF in the infarcted regionwas no longer different between the 2 groups, an elevatedMBF in the remote region was detected in EC-treated heartsas visualized in MBF maps from individual rats (Figure 3A
through 3D) and confirmed by statistical analysis (P�0.0277,Figure 3E). Interestingly, elevated MBF was matched by asignificant increase (greater absolute values) in circumferen-tial strains (Ecc) in the remote region (P�0.0075) as well asEcc averaged over the entire slice (P�0.0123, Figure 3G).The enhanced MBF and Ecc, however, did not lead to anincrease in LVEF in EC group (Table 3), suggesting thatLVEF is not sensitive to detect local changes in perfusion andwall motion. Although MVD was greater in all regions ofEC-treated hearts, statistical significance was not obtained inany region (Figure 3F). Compared with 1 day after MI, infarctsize decreased in both EC and vehicle groups (Table 3),probably as the result of wall thinning in the infarcted regionobserved frequently in both groups. Compared with 2 weeksafter MI, CD34-positive cells were rarely found in sections
(data not shown), suggesting that the vast majority of trans-planted cells were dead or removed.
Finally, the SL-MRI method is shown to have a highdegree of reproducibility by ICC (Table 1) and by theBland-Altman plot of regional MBF, which revealed auniform distribution of differences in MBF (scan 2, scan 1)around a bias very close to zero (Figure 4). For reproducibil-ity as well as validation study, segment-specific ICC valuesare relatively low in 1 or 2 segments, suggesting a decreasedagreement in these segments.
DiscussionHUVECs are commercially available and can be expandedto a large number in vitro, therefore providing a convenientEC source for this proof-of-principle study to examine
Figure 2. A through J, Regional myocardial blood flow (MBF) estimated by in vivo MRI and microvasculature density (MVD) at 2 weeks aftermyocardial infarction (MI). Slice average and regional MBF at 1 day (inset of A) and 2 weeks after MI (A) are shown. The MBF maps and cor-responding late gadolinium enhancement (LGE) images are shown for a representative heart from the endothelial cell (EC) group (B and C,arrows pointing to the phantom) and the vehicle group (D and E). Immunostaining for von Willebrand Factor (vWF) from an EC-treated (F) orvehicle-treated heart (G) is shown. Double immunostaining of CD34, which reacts only to human tissue, and vWF antibody, which reacts torat and human antigen, revealed incorporation of ECs into capillaries in the infarcted (H) and border (I) regions. Scale bars�100 �m. MVD�thenumber of capillaries per field of 1.1 mm2. *Statistically significant comparing the EC versus the vehicle group (A and J).
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Zhang et al EC-Mediated Myocardial Blood Flow Increase 5
whether or not SL-MRI is able to detect temporal changesof regional MBF in response to EC engraftment in theinfarcted heart. Our results suggest that EC transplantationinduces a strong neovascularization response in the in-farcted region detectable at 2 weeks after injection, leadingto a substantial increase of regional blood flow andcapillary density accompanied by incorporation of graftedECs into capillaries in the infarcted and border regions
(Figure 2). The localized responses, although strong,appear to be short-termed and transit to a prominentincrease of MBF in the remote territory (Figure 3E). Thisfinding is intriguing because it is generally expected thatonly infarcted and border zones would benefit from ECengraftment. However, there is compelling evidence thatthe remote region is affected during unfavorable post-MIremodeling,28 and stem cells may partially rescue/stabilize
Figure 3. A through G, Regional myocardial blood flow (MBF) and wall motion estimated by MRI and microvasculature density (MVD) at4 weeks after myocardial infarction. MBF maps and corresponding late gadolinium enhancement (LGE) images are shown for a repre-sentative heart from the endothelial cell (EC) group (A and B) and the vehicle group (C and D). Regional MBF (E), MVD (F), and Ecc (G)from the EC and vehicle groups is shown. *Statistically significant comparing the EC group versus the vehicle group (E and G).
Figure 4. Bland-Altman plot demonstrating thetest-retest reproducibility of spin-labeling MRImethod. Average myocardial blood flow (MBF)was obtained from the septal, anterior, poste-rior, and lateral segments for each animal. Thebias between the retest and test scan is 0.088mL/min per gram, with standard deviation(STD)�0.533 mL/min per gram.
that region.29 Consistently, an increase in MBF wasaccompanied by a recovery of wall motion (Ecc) in theremote region.
SL-MRI (also called arterial spin-labeling) has achieved agreat success in measuring blood flow in the brain.30 Inconventional brain SL, the RF inversion pulse is typicallyintroduced at an upstream location proximal to the tissue ofinterest. For MBF measurement, however, on-slice SL,namely, the flow-sensitive alternating inversion recovery31
technique, can minimize the magnetization transfer artifactand the underestimation of flow when feeding arteries havetortuous paths. SL-based MBF in small rodents is based onT1 mapping, and the arterial transit time (ATT) is ignored.This approach might be justified by the fact that small rodentshave much higher MBF (3–5 mL/min per gram11,20,32) thanhumans (0.7–1 mL/min per gram33) and are studied at higherfield strength than clinical scanners, leading to prolongedblood T1. Therefore, the ATT of uninverted blood spins ismuch shorter than blood T1, which allows a measurableinflow effect in slice-selective T1 values.12 ATT of humanheart was estimated at 1.5 T recently.34 Although furtherstudy is necessary to evaluate the effect of ATT on MBFquantification in small rodents, our validation study suggeststhe T1 mapping approach is in excellent agreement withstandard microsphere method.
Although the goals of this study have been fulfilled, severallimitations should be discussed. First, compared with theresting MBF, CFR might be more representative of coronarymicrovascular function than the resting MBF.35 CFR mea-surement can be implemented by means of pharmacologicalstimulation in SL-MRI protocol for future studies. Second,single-slice MBF was obtained in the current protocol due torelatively lengthy acquisition time (�25 minutes), whichcould lead to concerns that MBF may vary during acquisition.Inversion recovery–based T1 mapping, while being the mostrobust method, is inherently time-consuming. Noncartesiank-space trajectory such as spiral16 or radial36 imaging tech-niques can reduce acquisition time and resist respiratorymotion. Third, due to inflammatory reactions induced byacute MI and/or the use of human cells in a rat model (albeitimmune-compromised), injection of ECs during MI surgerymight have a negative impact on EC survival. Therefore,better EC engraftment is expected if they are injected 7 daysafter MI, as we have shown with embryonic stem cell–derived cardiomyocytes.19 Finally, the high postsurgery mor-tality rate and the exclusion of 7 rats from the Aim 2 analysisbecause of unqualified infarct size raise the possibility ofselection bias in addition to impacting statistical power.
In summary, our results indicate excellent agreement ofMBF in free-breathing rats measured by SL-MRI and thestandard color microsphere technique. Noninvasive SL-MRIallows serial assessments of regional MBF in response to ECtreatment. The presented method offers a promising frame-work to quantify MBF as a specific and sensitive index toevaluate EC-mediated therapy for the infarcted heart.
AcknowledgmentsWe are grateful to Xiaoling Hou, MS, Saran Vardhanabhuti, PhD,and Amy Praestgaard, MS, for performing statistical analyses.
Special thanks to Dr Ronald Morris from SA Instruments formodifications of breakout module to allow recordings of respirationwaveform with gate bits. We are indebted to Drs Stephanie Euckerand William M. Armstead for help with FM protocol. We thank DrJifu Yang for tissue sectioning and the Small Animal ImagingFacility for technical support.
Sources of FundingThis work was funded by National Institutes of Health grantsR01-HL081185A1 and R01-HL081185S1 (Dr Zou).
DisclosuresNone.
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CLINICAL PERSPECTIVEEndothelial cells (ECs) and endothelial progenitor cells (EPCs) have been isolated or derived from various sourcesincluding blood, bone marrow, embryonic stem cells, and induced pluripotent stem cells. ECs and EPCs have beenpostulated to form new vasculature, preserve cardiac function, and inhibit apoptosis in the infarcted heart. However,mechanisms underlying the salutary effects of these cells are not well understood: in fact, whether neovascularization leadsto improved regional myocardial blood flow (MBF) has yet to be clearly demonstrated. To evaluate EC- or EPC-mediatedcardiovascular repair over time and to improve clinical translation for cell-based therapies, noninvasive imaging methodsfor estimating MBF are desirable. At present, positron emission tomography is the clinical standard for MBF measurement.However, radioactive perfusion tracers such as N-13 ammonia, O-15 water, or Rb-82 have short half-lives and hencerequire an on-site cyclotron or Rubidium generator. Optimally, a method that permits serial MBF evaluations withoutaccruing radiation exposure/risk is most useful. In the present report, we first validate a spin-labeling MRI (SL-MRI)technique for quantification of MBF; we then demonstrate that this technique is highly reproducible and sensitive fordetecting regional MBF changes in response to EC engraftment in a rat model of myocardial infarction. Because SL-MRIutilizes endogenous blood (water) as a perfusion tracer, it eliminates the need to inject exogenous tracers. The clinicalfeasibility of SL-MRI–based MBF measurement has recently been demonstrated in humans. Our study provides evidencethat MBF is a unique and sensitive index to evaluate the impact of EC-mediated therapy on regional vascularization afterinfarction.
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