Blood Oxygen Level–Dependent MRI of Tissue Oxygenation
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动脉硬化血栓血管生物学 2005年第7期
From the Medical Faculty of the Charité (W.U., J.J., M.S., F.C.L., R.D., M.G.F.), Franz Volhard Klinik HELIOS Klinikum-Berlin at the Max Delbrück Center for Molecular Medicine, Berlin, Germany; GE Medical Systems (T.N.), Applied Science Laboratory, Boston, Mass; and Stephenson CHR Center, Departments of Cardiac Sciences and Radiology (M.G.F.), University of Calgary, Canada.
Correspondence to Matthias G. Friedrich, MD, FESC, Departments of Cardiac Sciences and Radiology, University of Calgary, 1403 29th St NW, Calgary, AB T2N 2T9, Canada. E-mail matthias.friedrich@ucalgary.ca
Abstract
Objectives— The contribution of endothelial function to tissue oxygenation is not well understood. Muscle blood oxygen level–dependent MRI (BOLD MRI) provides data largely dependent on hemoglobin (Hb) oxygenation. We used BOLD MRI to assess endothelium-dependent signal intensity (SI) changes.
Methods and Results— We investigated mean BOLD SI changes in the forearm musculature using a gradient-echo technique at 1.5 T in 9 healthy subjects who underwent a protocol of repeated acetylcholine infusions at 2 different doses (16 and 64 μg/min) and NG-monomethyl-L-arginine (L-NMMA; 5 mg/min) into the brachial artery. Sodium nitroprusside was used as a control substance. For additional correlation with standard methods, the same protocol was repeated, and forearm blood flow was measured by strain gauge plethysmography. We obtained a significant increase in BOLD SI during acetylcholine infusion (64 μg/min) and a significant decrease for L-NMMA infusion (P<0.005 for both). BOLD SI showed a different kinetic signal than did blood flow, particularly after intermittent ischemia and at high flow rates.
Conclusions— In standard endothelial function tests, BOLD MRI detects a dissociation of tissue Hb oxygenation from blood flow. BOLD MRI may be a useful adjunct in assessing endothelial function.
We used muscle blood oxygen level–dependent MRI (BOLD MRI) to study tissue Hb oxygenation in relation to postischemic hyperemia and endothelial stimulation. We found uncoupling of tissue Hb oxygenation from blood flow changes and conclude that BOLD MRI may provide additional information in assessing endothelial function.
Key Words: magnetic resonance imaging ? endothelial function ? BOLD ? plethysmography
Introduction
The vascular endothelium regulates tone and tissue perfusion through release of vasoactive substances such as NO. Numerous studies assessed the effect of endothelial function, and particularly endothelial dysfunction, in humans. However, the contribution of endothelial function to the regulation of tissue oxygenation in humans is poorly understood.1 Perfusion is not the sole mechanism that determines tissue oxygenation and metabolism. Therefore, blood flow measurements may have limited utility in this regard. Blood oxygen level–dependent MRI (BOLD MRI) reflects changes in the ratio of oxyhemoglobin to deoxyhemoglobin attributable to their different properties in a magnetic field.2,3 The BOLD technique is well established for functional brain MRI4 and has also been used to assess myocardial5–9 and skeletal muscle ischemia.10,11 More recently, the technique was applied to assess exercise-induced hyperemia in skeletal muscle.12,13 Available MRI systems are able to generate BOLD-sensitive small volume data sets in <1 s. Thus, BOLD MRI can noninvasively detect changes in intravascular tissue oxygenation with high spatial and temporal resolution. Furthermore, the tissue access is potentially unrestricted. We investigated changes of tissue oxygenation as defined by BOLD MRI signal intensity (SI) compared with blood flow changes after validated standard effectors on vascular tone and applied direct vasodilation and vasodilation attributable to stimulation of the endothelium.
Methods
Protocol
We studied 9 normal healthy men (25±4 years of age; 75±9 kg weight; 180±9 cm height). They received no medications. Our internal review board approved all studies, and written informed consent was obtained. All studies were conducted between 9 AM and noon after an overnight fast. A catheter (18G; Braun AG) was introduced into the brachial artery of the nondominant arm, followed by a resting period of 30 minutes. We compared BOLD SI changes with changes in blood flow determined by strain gauge plethysmography (SGP). Therefore, subjects underwent the following protocols twice within 2 hours: once in the MRI scanner and once while forearm blood flow (FBF) changes were assessed with SGP. Protocols were performed in a random order and with a 60-minute intermission between techniques.
We used the following known stimulators of vasodilation.
Reactive hyperemia after intermittent ischemia, which leads to vasodilation through regional metabolic changes as well as secondary endothelium-mediated smooth muscle cell relaxation.
Administration of sodium nitroprusside (SNP), which directly exposes smooth muscle cells to NO and leads to their relaxation without primary endothelium stimulation (endothelium-independent vasodilation).
Administration of acetylcholine (ACh), which is a physiological stimulus for endothelial cells to produce NO, which again relaxes vascular smooth muscles.
In an initial series of experiments, we performed reactive hyperemia with 3 minutes of ischemia followed by a recovery period of 22 minutes and an incremental intra-arterial infusion of SNP (SNP2=2 μg/min; SNP4=4 μg/min; SNP6=6 μg/min for 5 minutes each) after a short break. Ischemia was induced by a rapid inflation of a cuff of a standard automatic blood pressure monitor (Omega; Saegeling Medizintechnik), with a complete cessation of blood flow within 2.5 s. On another day, endothelium-dependent perfusion changes were elicited by incremental infusions of ACh (ACh16=16 μg/min; ACh64=64 μg/min). Each infusion step lasted 4 minutes. After recovery, we infused the nonspecific NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) at a rate of 5 mg/min over 5 minutes. We used drug doses that are known to induce no systemic effects.14 The study drug concentrations were adjusted to achieve a constant intra-arterial infusion rate of 1.2 mL/min. In the course of our studies, all 8 data sets obtained during the reactive hyperemia/SNP experiments were suitable for evaluation. During the ACh/L-NMMA studies, significant motion artifacts occurred in 1 subject, making a reliable evaluation impossible.
Plethysmography
We used a Hokanson EC5R plethysmograph with mercury-in-silastic strain gauges that were wrapped around the forearm at its largest diameter. A wrist cuff was inflated to 50 mm Hg above systolic pressure 1 minute before the protocol started to exclude hand circulation. By intermittent application of a venous occlusion pressure of 50 mm Hg, the blood inflow into the forearm was measured every 15 s. Except for reactive hyperemia, flow values were calculated from 8 single measurements after flow reached a steady state in each section of the protocol.
Magnetic Resonance Imaging
We used a 1.5-T MRI system (Sigma CV/I; GE Medical Systems), equipped with a cardiovascular-optimized gradient system (maximum gradient strength 40 mT/m; slew rate 150 T/m per second). A quadrature head coil was used for excitation and reception to achieve a homogeneous radio frequency intensity profile. Subjects were asked to assume a prone "sphinx-like" position with their forearms in the head coil. A single cross-sectional slice was placed at the largest forearm diameter. We performed T2*-weighted single-shot, gradient-echo sequence, with an echo planar imaging readout using the following parameters: field of view 24x24 cm2; pixel size 1.87x1.87 mm2; slice thickness 10 mm; echo time (TE) 18.5 ms; and flip angle 20°. In the first series of experiments, we used a constant repetition time of 250 ms, and in the second set, we applied ECG triggering with a delay time of 200 ms to minimize the influence of flow-dependent saturation and T1 effects on the SI. We measured SI in a defined region of interest on the whole forearm musculature excluding large vessels and bones (Figure 1) using software designed for tracking the SI time course in functional MRI (Functool; GE Medical Systems). In addition, the images of the contralateral (noninfusion) forearm were processed to identify variations in the systemic circulation.
Figure 1. T2*-weighted MRI of human forearms during ischemia (left) and during hyperemia (right). The arm undergoing intermittent occlusion of circulation is depicted on the left side. In this arm, the flow-induced intensity increase after cuff release can be observed clearly in large vessels, whereas BOLD SI variation in the muscles is too small to be identified visually. Note the irregularly shaped region of interest as drawn manually.
Comparison With Theoretical Models
To assess the contributions of different compartments to BOLD SI in human skeletal muscle, we compared our data with predictions of theoretical models of the BOLD effect. For estimating extravascular BOLD effects, we adapted the model of Bauer et al3 to the skeletal muscle by using a typical set of tissue parameters (Table 1).For intravascular BOLD effects, we calculated BOLD SI changes on the basis of oxygenation-dependent changes of the blood relaxation rate as proposed by Silvennoinen et al.15 Similar to earlier simulations by Meyer et al,12 we used a constant relative blood volume of 3% and a muscle relaxation rate of 34/s.
TABLE 1. Tissue Parameters of Human Skeletal Muscle Used for Calculation of Extravascular BOLD Effects
Statistical Analysis
Descriptive statistics are expressed as mean±SEM. Statistical significance of BOLD SI changes between the sections of the protocol was assessed by ANOVA for repeated measurements. We used Bonferroni’s test to compare BOLD SI changes during infusion with mean control values. A P value <0.05 was considered significant.
Results
Postischemic Vasodilation
The SI in the T2*-weighted echo planar images yielded a signal-to-noise-ratio of 200 to 400. A low-frequency noise appeared synchronously on both forearms, resulting mainly from cardiac pulsation and breathing motion. Figure 1 shows representative axial BOLD images obtained during forearm ischemia and immediately after cuff release. The control forearm is depicted on the right side of each image. Reperfusion induced a strong SI increase in the luminal area of the visible vessels and, to a lesser extent, in the muscular tissue.
The time course of averaged BOLD SI and blood flow changes during the reactive hyperemia protocol are illustrated in Figure 2. Immediately after cuff inflation, the BOLD SI showed an exponential decay with a maximum decrease of 2.2±0.4% compared with baseline. After cuff release, the SI reached a maximum of 3.6±0.4% above baseline within 30 s and slowly returned to baseline values thereafter. The time course of the blood flow response was markedly different from that of the BOLD SI. The blood flow reached a maximum of 48.4±5.6 mL/minx100 mL tissue as early as 5 s after cuff release, with a rapid return to baseline (5.1±1.7 mL/minx100 mL tissue). The analysis of BOLD SI curves showed that the mean exponential time constant for the BOLD signal decrease during ischemia was 71±11 s, and the mean linear BOLD signal increase time (from 10% to 90% of the total increase after cuff release) was 9.5±1 s. The individually determined time constant for the decay in the BOLD SI after the maximum was 136±22 s, whereas the time constant for the blood flow decay subsequent to the blood flow overshoot was only 19.3±3.7 s.
Figure 2. BOLD SI changes (line) and FBF in a reactive hyperemia experiment. FBF and tissue oxygenation return to resting values on a different time scale. Squares indicate rest.
Intra-Arterial Infusions
SNP and ACh led to a dose-dependent BOLD SI increase, reflecting increased oxygenation attributable to increased blood flow. With both substances, blood flow as measured by SGP remained high throughout the infusion period, whereas the BOLD SI decreased before the end of infusion. Figure 3a shows a representative recording of BOLD SI and flow from a subject during incremental intra-arterial SNP infusions. Compared with baseline, the BOLD SI increases were 2.0±0.3%, 2.4±0.3%, and 2.6±0.3% during SNP infusions at 2, 4, and 6 μg/min, respectively (P<0.001). The corresponding FBF values were 17±2, 24±2, and 29±3 mL/minx100 mL tissue. Whereas FBF stabilized within 2 minutes at each infusion step, the BOLD SI did not reach a steady state. We induced endothelium-dependent vasodilatation with incremental intra-arterial ACh infusion. The BOLD SI and FBF changes in a representative subject are illustrated in Figure 3b. Whereas FBF rapidly approached steady state at each infusion step, BOLD SI decreased at each ACh infusion step early after reaching a maximum, despite persisting high blood flow. During ACh infusion at the high dose (ACh64), the maximal BOLD SI increase was 3.6±0.5% (P<0.005), corresponding to 8.4-fold FBF increase, whereas it was not significant during low-dose ACh16 (1.9±0.6, P>0.05; FBF increase 4.2-fold, P=0.38). Table 2 summarizes the results.
Figure 3. BOLD SI (oscillating line) and flow (solid line) changes during intra-arterial infusion of SNP (a) and incremental infusion of ACh, recovery, and L-NMMA (b).
TABLE 2. Observed Changes of Forearm BOLD SI and Blood Flow in the Settings of Reactive Hyperemia, Nitroprusside Administration, and Endothelial Stimulation
FBF and maximum BOLD SI values encountered in each section of the protocol are depicted in a scatter plot in Figure 4a. Although there was an almost-linear increase in FBF with incremental SNP doses, the BOLD SI increases reached saturation with increasing SNP dose. During intra-arterial L-NMMA, BOLD SI and FBF decreased markedly (change –1.6±0.2% [P<0.005] and –35%, respectively). Figure 4b shows the blood flow values obtained during L-NMMA and ACh infusions plotted against the BOLD SI values. The BOLD SI curve shows a steep increase at lower blood flow values and saturation at higher blood flow values. A 35% blood flow decrease during L-NMMA infusion roughly induced the same amount of BOLD SI change as the 4-fold blood flow increase during ACh16 infusion. BOLD SI at similar perfusion changes did not differ between endothelial-dependent and endothelial-independent studies (P=0.3).
Figure 4. Left, Scatter plot of FBF and BOLD SI changes during endothelium-independent blood flow changes after SNP (2 of fit=0.00007). Right, Similar data comparing endothelium-dependent blood flow changes induced by incremental ACh and by L-NMMA (2 of fit=0.00016). The BOLD SI saturates for increasing FBF because of saturating tissue oxygenation.
Comparison With Theoretical Models
For an estimation of the extracapillary BOLD effects, we inserted the measured FBF data at rest and after L-NMMA, ACh64, and SNP6 infusion, respectively, into Equations 1, 3, and 48, as described in Bauer et al.3 As an approximation for a small intracapillary blood volume, we applied Equation 40 from the same publication. Compared with resting values, the calculations resulted in relative muscle relaxation rate changes (R2*) of –0.013, 0.009, and 0.009 per second for L-NMMA, ACh64, and SNP6, respectively. The relative BOLD SI change was calculated by the relationship TExR2*, resulting in an absolute extravascular contribution of –0.024%, 0.016%, and 0.016% for L-NMMA, ACh64, and SNP6, respectively. These values are in the range of only 1% of the measured BOLD SI changes.
Using Equation 48 from Bauer et al to determine the flow-dependent hemoglobin (Hb) saturation and Equation 1 from Meyer et al to determine the relaxation rate of blood in the microcirculation, we could estimate intravascular BOLD effects.3,15 Relative BOLD SI changes yielded –0.22, 0.21, and 0.20% for L-NMMA, ACh64, and SNP6, respectively, 10% of the observed total BOLD SI change. However, when we accounted for the relative blood volume expansion during hyperperfusion an increase in the relative blood volume from 3% to 4% resulted in a relative BOLD SI change of 1.8% for SNP6.16,17
Discussion
To our knowledge, our study is the first to systematically correlate BOLD SI changes to endothelial-mediated blood flow changes in the human forearm. With increasing blood flow, we observed an increase in BOLD SI, with saturation at high blood flow levels. However, after intermittent ischemia, BOLD SI remained elevated even after normalization of blood flow. Because O2 consumption is constant for PO2 levels of 1 mm Hg and thus does not account for the observed dissociation, the decay time constant for the BOLD MRI signal may indicate the presence of microvascular oxygen reserve.
Our reactive hyperemia results are consistent with the magnitude and dynamics of BOLD SI–related changes reported previously.10,11 Very recent data by Towse et al also provided evidence for a dissociation of flow and BOLD SI.18 Persisting high values of BOLD SI after blood flow normalization are met by postischemic and even posthyperemic elevations of oxygen saturation of venous blood and tissue oxygen tension.19–21 Intermittent recruitment of capillaries after ischemia increases oxygenated blood content and leads to a more uniform and efficient O2 exchange between capillary blood and surrounding tissues.16,22 Shortly after hyperemia, elevated tissue O2 tension may cause an intermittent decrease in O2 extraction until the preischemic equilibrium is reinstalled. The mechanism is amplified by flow-mediated NO release.
Endothelial NO mediates exercise-related responses23,24 and reactive hyperemia.14,25 Jordan et al recently showed that the prolonged postexercise tissue oxygenation and increased BOLD SI observed in wild-type mice was absent in endothelial NO synthase gene-disrupted mice.13 Postexercise blood flow rapidly returned to baseline in both groups. This state of affairs may be explained by the fact that endothelial NO not only regulates vascular tone and, thus, O2 supply, but also O2 consumption. Exogenous as well as endogenous NO decreases mitochondrial O2 consumption, whereas endothelial NO synthase inhibition increases O2 uptake.26–29 Thus, limitations in endothelial NO production may contribute to ischemia through a combination of limited O2 supply and failure to shut off O2 consumption.
Infusion of endothelium-dependent or endothelium-independent vasodilators did not lead to a stable BOLD SI plateau, whereas blood flow rapidly approached a steady state. Besides the mentioned impact of NO, this phenomenon may be explained in part by a tissue oxygen tension–dependent redistribution of blood volume into regions of lower O2 tension. In a previous study, regional blood flow and functional capillary density were inversely related to tissue O2 tension.17
BOLD SI in our study approached saturation with increasing FBF. The response to endothelial-dependent and endothelial-independent vasodilatation was similar. The saturation of the BOLD SI suggests that microvascular Hb oxygenation gradually approached arterial Hb oxygenation as blood flow increased. Theoretically, changes in myoglobin oxygenation may also induce a BOLD effect. However, even with the L-NMMA–induced reduced blood flow, the myoglobin oxygen saturation remained 100% in our study.11 Furthermore, myoglobin is fully reoxygenated within 15 s after reperfusion. Thus, we do not believe that myoglobin oxygenation saturation affected our results.
BOLD SI is affected by blood volume, blood flow, and tissue oxygenation; thus, the observed BOLD SI changes could have been the result of factors other than oxygenation. However, studies of Li et al have shown that changes of BOLD SI in vivo are mainly attributable to changes of tissue oxygenation.5 Contributions to the BOLD signal originate from intravascular and extravascular compartments. However, Meyer et al12 showed that intravascular rather than extravascular effects mainly explain BOLD SI changes in the skeletal muscle. Variations in microvascular blood volume or in the mean angle between muscle fibers and the main field axis may alter the magnitude of calculated BOLD SI. A wide range of values has been reported particularly for capillary density, which is known to vary with the physical training level.30 Furthermore, functional capillary density is dependent of tissue O2 tension and precapillary pressure. These variables may have altered significantly during the course of our experiments.
We could not measure FBF and BOLD SI simultaneously because SGP could not be performed within the MRI scanner. However, we standardized test conditions during both measurements. We maintained the conditions of SGP and BOLD MRI as similarly as possible. We did not perform simultaneous metabolic studies, for instance with magnetic resonance spectroscopy of myoglobin. Thus, we cannot comment on metabolic aspects of the observed changes; however, coupling magnetic resonance spectroscopy with microdialysis techniques might provide interesting future perspectives.
The potential impact of our results is manifold.
The new technique allows for directly assessing oxygen availability as the regulated vascular metabolic variable itself instead of using surrogate markers such as blood flow or vessel diameters.
Mechanisms of regional oxygenation regulation in the tissue other than endothelium-dependent blood flow regulation can be investigated. Candidates could be precapillary shunts, new tissue regulators of oxygen consumption, or modifiers of Hb deoxygenation.
These mechanisms may be future targets for therapeutic approaches addressing tissue oxygenation.
BOLD MRI may provide an accurate, stable, and reproducible tool to assess endothelial function as an early hallmark of hypertension and atherosclerosis and their precursors diabetes, obesity, physical inactivity, lipid disorders, or systemic inflammation. Applied in research trials, it may overcome known limitations of established modalities.
Because BOLD MRI does not need local manipulations such as strain gauges and is largely independent from the location of the tissue to be studied, other areas such as intra-abdominal organs and the heart are within reach.
The method may be useful in monitoring changes during therapeutic interventions, particularly in areas inaccessible to other techniques.
Acknowledgments
We are thankful to Todd Anderson for his excellent revision of the manuscript and helpful discussions. We are furthermore indebted to the technical assistance of Kerstin Kretschel, Evelyn Polzin, and Ursula Wagner.
References
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Correspondence to Matthias G. Friedrich, MD, FESC, Departments of Cardiac Sciences and Radiology, University of Calgary, 1403 29th St NW, Calgary, AB T2N 2T9, Canada. E-mail matthias.friedrich@ucalgary.ca
Abstract
Objectives— The contribution of endothelial function to tissue oxygenation is not well understood. Muscle blood oxygen level–dependent MRI (BOLD MRI) provides data largely dependent on hemoglobin (Hb) oxygenation. We used BOLD MRI to assess endothelium-dependent signal intensity (SI) changes.
Methods and Results— We investigated mean BOLD SI changes in the forearm musculature using a gradient-echo technique at 1.5 T in 9 healthy subjects who underwent a protocol of repeated acetylcholine infusions at 2 different doses (16 and 64 μg/min) and NG-monomethyl-L-arginine (L-NMMA; 5 mg/min) into the brachial artery. Sodium nitroprusside was used as a control substance. For additional correlation with standard methods, the same protocol was repeated, and forearm blood flow was measured by strain gauge plethysmography. We obtained a significant increase in BOLD SI during acetylcholine infusion (64 μg/min) and a significant decrease for L-NMMA infusion (P<0.005 for both). BOLD SI showed a different kinetic signal than did blood flow, particularly after intermittent ischemia and at high flow rates.
Conclusions— In standard endothelial function tests, BOLD MRI detects a dissociation of tissue Hb oxygenation from blood flow. BOLD MRI may be a useful adjunct in assessing endothelial function.
We used muscle blood oxygen level–dependent MRI (BOLD MRI) to study tissue Hb oxygenation in relation to postischemic hyperemia and endothelial stimulation. We found uncoupling of tissue Hb oxygenation from blood flow changes and conclude that BOLD MRI may provide additional information in assessing endothelial function.
Key Words: magnetic resonance imaging ? endothelial function ? BOLD ? plethysmography
Introduction
The vascular endothelium regulates tone and tissue perfusion through release of vasoactive substances such as NO. Numerous studies assessed the effect of endothelial function, and particularly endothelial dysfunction, in humans. However, the contribution of endothelial function to the regulation of tissue oxygenation in humans is poorly understood.1 Perfusion is not the sole mechanism that determines tissue oxygenation and metabolism. Therefore, blood flow measurements may have limited utility in this regard. Blood oxygen level–dependent MRI (BOLD MRI) reflects changes in the ratio of oxyhemoglobin to deoxyhemoglobin attributable to their different properties in a magnetic field.2,3 The BOLD technique is well established for functional brain MRI4 and has also been used to assess myocardial5–9 and skeletal muscle ischemia.10,11 More recently, the technique was applied to assess exercise-induced hyperemia in skeletal muscle.12,13 Available MRI systems are able to generate BOLD-sensitive small volume data sets in <1 s. Thus, BOLD MRI can noninvasively detect changes in intravascular tissue oxygenation with high spatial and temporal resolution. Furthermore, the tissue access is potentially unrestricted. We investigated changes of tissue oxygenation as defined by BOLD MRI signal intensity (SI) compared with blood flow changes after validated standard effectors on vascular tone and applied direct vasodilation and vasodilation attributable to stimulation of the endothelium.
Methods
Protocol
We studied 9 normal healthy men (25±4 years of age; 75±9 kg weight; 180±9 cm height). They received no medications. Our internal review board approved all studies, and written informed consent was obtained. All studies were conducted between 9 AM and noon after an overnight fast. A catheter (18G; Braun AG) was introduced into the brachial artery of the nondominant arm, followed by a resting period of 30 minutes. We compared BOLD SI changes with changes in blood flow determined by strain gauge plethysmography (SGP). Therefore, subjects underwent the following protocols twice within 2 hours: once in the MRI scanner and once while forearm blood flow (FBF) changes were assessed with SGP. Protocols were performed in a random order and with a 60-minute intermission between techniques.
We used the following known stimulators of vasodilation.
Reactive hyperemia after intermittent ischemia, which leads to vasodilation through regional metabolic changes as well as secondary endothelium-mediated smooth muscle cell relaxation.
Administration of sodium nitroprusside (SNP), which directly exposes smooth muscle cells to NO and leads to their relaxation without primary endothelium stimulation (endothelium-independent vasodilation).
Administration of acetylcholine (ACh), which is a physiological stimulus for endothelial cells to produce NO, which again relaxes vascular smooth muscles.
In an initial series of experiments, we performed reactive hyperemia with 3 minutes of ischemia followed by a recovery period of 22 minutes and an incremental intra-arterial infusion of SNP (SNP2=2 μg/min; SNP4=4 μg/min; SNP6=6 μg/min for 5 minutes each) after a short break. Ischemia was induced by a rapid inflation of a cuff of a standard automatic blood pressure monitor (Omega; Saegeling Medizintechnik), with a complete cessation of blood flow within 2.5 s. On another day, endothelium-dependent perfusion changes were elicited by incremental infusions of ACh (ACh16=16 μg/min; ACh64=64 μg/min). Each infusion step lasted 4 minutes. After recovery, we infused the nonspecific NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) at a rate of 5 mg/min over 5 minutes. We used drug doses that are known to induce no systemic effects.14 The study drug concentrations were adjusted to achieve a constant intra-arterial infusion rate of 1.2 mL/min. In the course of our studies, all 8 data sets obtained during the reactive hyperemia/SNP experiments were suitable for evaluation. During the ACh/L-NMMA studies, significant motion artifacts occurred in 1 subject, making a reliable evaluation impossible.
Plethysmography
We used a Hokanson EC5R plethysmograph with mercury-in-silastic strain gauges that were wrapped around the forearm at its largest diameter. A wrist cuff was inflated to 50 mm Hg above systolic pressure 1 minute before the protocol started to exclude hand circulation. By intermittent application of a venous occlusion pressure of 50 mm Hg, the blood inflow into the forearm was measured every 15 s. Except for reactive hyperemia, flow values were calculated from 8 single measurements after flow reached a steady state in each section of the protocol.
Magnetic Resonance Imaging
We used a 1.5-T MRI system (Sigma CV/I; GE Medical Systems), equipped with a cardiovascular-optimized gradient system (maximum gradient strength 40 mT/m; slew rate 150 T/m per second). A quadrature head coil was used for excitation and reception to achieve a homogeneous radio frequency intensity profile. Subjects were asked to assume a prone "sphinx-like" position with their forearms in the head coil. A single cross-sectional slice was placed at the largest forearm diameter. We performed T2*-weighted single-shot, gradient-echo sequence, with an echo planar imaging readout using the following parameters: field of view 24x24 cm2; pixel size 1.87x1.87 mm2; slice thickness 10 mm; echo time (TE) 18.5 ms; and flip angle 20°. In the first series of experiments, we used a constant repetition time of 250 ms, and in the second set, we applied ECG triggering with a delay time of 200 ms to minimize the influence of flow-dependent saturation and T1 effects on the SI. We measured SI in a defined region of interest on the whole forearm musculature excluding large vessels and bones (Figure 1) using software designed for tracking the SI time course in functional MRI (Functool; GE Medical Systems). In addition, the images of the contralateral (noninfusion) forearm were processed to identify variations in the systemic circulation.
Figure 1. T2*-weighted MRI of human forearms during ischemia (left) and during hyperemia (right). The arm undergoing intermittent occlusion of circulation is depicted on the left side. In this arm, the flow-induced intensity increase after cuff release can be observed clearly in large vessels, whereas BOLD SI variation in the muscles is too small to be identified visually. Note the irregularly shaped region of interest as drawn manually.
Comparison With Theoretical Models
To assess the contributions of different compartments to BOLD SI in human skeletal muscle, we compared our data with predictions of theoretical models of the BOLD effect. For estimating extravascular BOLD effects, we adapted the model of Bauer et al3 to the skeletal muscle by using a typical set of tissue parameters (Table 1).For intravascular BOLD effects, we calculated BOLD SI changes on the basis of oxygenation-dependent changes of the blood relaxation rate as proposed by Silvennoinen et al.15 Similar to earlier simulations by Meyer et al,12 we used a constant relative blood volume of 3% and a muscle relaxation rate of 34/s.
TABLE 1. Tissue Parameters of Human Skeletal Muscle Used for Calculation of Extravascular BOLD Effects
Statistical Analysis
Descriptive statistics are expressed as mean±SEM. Statistical significance of BOLD SI changes between the sections of the protocol was assessed by ANOVA for repeated measurements. We used Bonferroni’s test to compare BOLD SI changes during infusion with mean control values. A P value <0.05 was considered significant.
Results
Postischemic Vasodilation
The SI in the T2*-weighted echo planar images yielded a signal-to-noise-ratio of 200 to 400. A low-frequency noise appeared synchronously on both forearms, resulting mainly from cardiac pulsation and breathing motion. Figure 1 shows representative axial BOLD images obtained during forearm ischemia and immediately after cuff release. The control forearm is depicted on the right side of each image. Reperfusion induced a strong SI increase in the luminal area of the visible vessels and, to a lesser extent, in the muscular tissue.
The time course of averaged BOLD SI and blood flow changes during the reactive hyperemia protocol are illustrated in Figure 2. Immediately after cuff inflation, the BOLD SI showed an exponential decay with a maximum decrease of 2.2±0.4% compared with baseline. After cuff release, the SI reached a maximum of 3.6±0.4% above baseline within 30 s and slowly returned to baseline values thereafter. The time course of the blood flow response was markedly different from that of the BOLD SI. The blood flow reached a maximum of 48.4±5.6 mL/minx100 mL tissue as early as 5 s after cuff release, with a rapid return to baseline (5.1±1.7 mL/minx100 mL tissue). The analysis of BOLD SI curves showed that the mean exponential time constant for the BOLD signal decrease during ischemia was 71±11 s, and the mean linear BOLD signal increase time (from 10% to 90% of the total increase after cuff release) was 9.5±1 s. The individually determined time constant for the decay in the BOLD SI after the maximum was 136±22 s, whereas the time constant for the blood flow decay subsequent to the blood flow overshoot was only 19.3±3.7 s.
Figure 2. BOLD SI changes (line) and FBF in a reactive hyperemia experiment. FBF and tissue oxygenation return to resting values on a different time scale. Squares indicate rest.
Intra-Arterial Infusions
SNP and ACh led to a dose-dependent BOLD SI increase, reflecting increased oxygenation attributable to increased blood flow. With both substances, blood flow as measured by SGP remained high throughout the infusion period, whereas the BOLD SI decreased before the end of infusion. Figure 3a shows a representative recording of BOLD SI and flow from a subject during incremental intra-arterial SNP infusions. Compared with baseline, the BOLD SI increases were 2.0±0.3%, 2.4±0.3%, and 2.6±0.3% during SNP infusions at 2, 4, and 6 μg/min, respectively (P<0.001). The corresponding FBF values were 17±2, 24±2, and 29±3 mL/minx100 mL tissue. Whereas FBF stabilized within 2 minutes at each infusion step, the BOLD SI did not reach a steady state. We induced endothelium-dependent vasodilatation with incremental intra-arterial ACh infusion. The BOLD SI and FBF changes in a representative subject are illustrated in Figure 3b. Whereas FBF rapidly approached steady state at each infusion step, BOLD SI decreased at each ACh infusion step early after reaching a maximum, despite persisting high blood flow. During ACh infusion at the high dose (ACh64), the maximal BOLD SI increase was 3.6±0.5% (P<0.005), corresponding to 8.4-fold FBF increase, whereas it was not significant during low-dose ACh16 (1.9±0.6, P>0.05; FBF increase 4.2-fold, P=0.38). Table 2 summarizes the results.
Figure 3. BOLD SI (oscillating line) and flow (solid line) changes during intra-arterial infusion of SNP (a) and incremental infusion of ACh, recovery, and L-NMMA (b).
TABLE 2. Observed Changes of Forearm BOLD SI and Blood Flow in the Settings of Reactive Hyperemia, Nitroprusside Administration, and Endothelial Stimulation
FBF and maximum BOLD SI values encountered in each section of the protocol are depicted in a scatter plot in Figure 4a. Although there was an almost-linear increase in FBF with incremental SNP doses, the BOLD SI increases reached saturation with increasing SNP dose. During intra-arterial L-NMMA, BOLD SI and FBF decreased markedly (change –1.6±0.2% [P<0.005] and –35%, respectively). Figure 4b shows the blood flow values obtained during L-NMMA and ACh infusions plotted against the BOLD SI values. The BOLD SI curve shows a steep increase at lower blood flow values and saturation at higher blood flow values. A 35% blood flow decrease during L-NMMA infusion roughly induced the same amount of BOLD SI change as the 4-fold blood flow increase during ACh16 infusion. BOLD SI at similar perfusion changes did not differ between endothelial-dependent and endothelial-independent studies (P=0.3).
Figure 4. Left, Scatter plot of FBF and BOLD SI changes during endothelium-independent blood flow changes after SNP (2 of fit=0.00007). Right, Similar data comparing endothelium-dependent blood flow changes induced by incremental ACh and by L-NMMA (2 of fit=0.00016). The BOLD SI saturates for increasing FBF because of saturating tissue oxygenation.
Comparison With Theoretical Models
For an estimation of the extracapillary BOLD effects, we inserted the measured FBF data at rest and after L-NMMA, ACh64, and SNP6 infusion, respectively, into Equations 1, 3, and 48, as described in Bauer et al.3 As an approximation for a small intracapillary blood volume, we applied Equation 40 from the same publication. Compared with resting values, the calculations resulted in relative muscle relaxation rate changes (R2*) of –0.013, 0.009, and 0.009 per second for L-NMMA, ACh64, and SNP6, respectively. The relative BOLD SI change was calculated by the relationship TExR2*, resulting in an absolute extravascular contribution of –0.024%, 0.016%, and 0.016% for L-NMMA, ACh64, and SNP6, respectively. These values are in the range of only 1% of the measured BOLD SI changes.
Using Equation 48 from Bauer et al to determine the flow-dependent hemoglobin (Hb) saturation and Equation 1 from Meyer et al to determine the relaxation rate of blood in the microcirculation, we could estimate intravascular BOLD effects.3,15 Relative BOLD SI changes yielded –0.22, 0.21, and 0.20% for L-NMMA, ACh64, and SNP6, respectively, 10% of the observed total BOLD SI change. However, when we accounted for the relative blood volume expansion during hyperperfusion an increase in the relative blood volume from 3% to 4% resulted in a relative BOLD SI change of 1.8% for SNP6.16,17
Discussion
To our knowledge, our study is the first to systematically correlate BOLD SI changes to endothelial-mediated blood flow changes in the human forearm. With increasing blood flow, we observed an increase in BOLD SI, with saturation at high blood flow levels. However, after intermittent ischemia, BOLD SI remained elevated even after normalization of blood flow. Because O2 consumption is constant for PO2 levels of 1 mm Hg and thus does not account for the observed dissociation, the decay time constant for the BOLD MRI signal may indicate the presence of microvascular oxygen reserve.
Our reactive hyperemia results are consistent with the magnitude and dynamics of BOLD SI–related changes reported previously.10,11 Very recent data by Towse et al also provided evidence for a dissociation of flow and BOLD SI.18 Persisting high values of BOLD SI after blood flow normalization are met by postischemic and even posthyperemic elevations of oxygen saturation of venous blood and tissue oxygen tension.19–21 Intermittent recruitment of capillaries after ischemia increases oxygenated blood content and leads to a more uniform and efficient O2 exchange between capillary blood and surrounding tissues.16,22 Shortly after hyperemia, elevated tissue O2 tension may cause an intermittent decrease in O2 extraction until the preischemic equilibrium is reinstalled. The mechanism is amplified by flow-mediated NO release.
Endothelial NO mediates exercise-related responses23,24 and reactive hyperemia.14,25 Jordan et al recently showed that the prolonged postexercise tissue oxygenation and increased BOLD SI observed in wild-type mice was absent in endothelial NO synthase gene-disrupted mice.13 Postexercise blood flow rapidly returned to baseline in both groups. This state of affairs may be explained by the fact that endothelial NO not only regulates vascular tone and, thus, O2 supply, but also O2 consumption. Exogenous as well as endogenous NO decreases mitochondrial O2 consumption, whereas endothelial NO synthase inhibition increases O2 uptake.26–29 Thus, limitations in endothelial NO production may contribute to ischemia through a combination of limited O2 supply and failure to shut off O2 consumption.
Infusion of endothelium-dependent or endothelium-independent vasodilators did not lead to a stable BOLD SI plateau, whereas blood flow rapidly approached a steady state. Besides the mentioned impact of NO, this phenomenon may be explained in part by a tissue oxygen tension–dependent redistribution of blood volume into regions of lower O2 tension. In a previous study, regional blood flow and functional capillary density were inversely related to tissue O2 tension.17
BOLD SI in our study approached saturation with increasing FBF. The response to endothelial-dependent and endothelial-independent vasodilatation was similar. The saturation of the BOLD SI suggests that microvascular Hb oxygenation gradually approached arterial Hb oxygenation as blood flow increased. Theoretically, changes in myoglobin oxygenation may also induce a BOLD effect. However, even with the L-NMMA–induced reduced blood flow, the myoglobin oxygen saturation remained 100% in our study.11 Furthermore, myoglobin is fully reoxygenated within 15 s after reperfusion. Thus, we do not believe that myoglobin oxygenation saturation affected our results.
BOLD SI is affected by blood volume, blood flow, and tissue oxygenation; thus, the observed BOLD SI changes could have been the result of factors other than oxygenation. However, studies of Li et al have shown that changes of BOLD SI in vivo are mainly attributable to changes of tissue oxygenation.5 Contributions to the BOLD signal originate from intravascular and extravascular compartments. However, Meyer et al12 showed that intravascular rather than extravascular effects mainly explain BOLD SI changes in the skeletal muscle. Variations in microvascular blood volume or in the mean angle between muscle fibers and the main field axis may alter the magnitude of calculated BOLD SI. A wide range of values has been reported particularly for capillary density, which is known to vary with the physical training level.30 Furthermore, functional capillary density is dependent of tissue O2 tension and precapillary pressure. These variables may have altered significantly during the course of our experiments.
We could not measure FBF and BOLD SI simultaneously because SGP could not be performed within the MRI scanner. However, we standardized test conditions during both measurements. We maintained the conditions of SGP and BOLD MRI as similarly as possible. We did not perform simultaneous metabolic studies, for instance with magnetic resonance spectroscopy of myoglobin. Thus, we cannot comment on metabolic aspects of the observed changes; however, coupling magnetic resonance spectroscopy with microdialysis techniques might provide interesting future perspectives.
The potential impact of our results is manifold.
The new technique allows for directly assessing oxygen availability as the regulated vascular metabolic variable itself instead of using surrogate markers such as blood flow or vessel diameters.
Mechanisms of regional oxygenation regulation in the tissue other than endothelium-dependent blood flow regulation can be investigated. Candidates could be precapillary shunts, new tissue regulators of oxygen consumption, or modifiers of Hb deoxygenation.
These mechanisms may be future targets for therapeutic approaches addressing tissue oxygenation.
BOLD MRI may provide an accurate, stable, and reproducible tool to assess endothelial function as an early hallmark of hypertension and atherosclerosis and their precursors diabetes, obesity, physical inactivity, lipid disorders, or systemic inflammation. Applied in research trials, it may overcome known limitations of established modalities.
Because BOLD MRI does not need local manipulations such as strain gauges and is largely independent from the location of the tissue to be studied, other areas such as intra-abdominal organs and the heart are within reach.
The method may be useful in monitoring changes during therapeutic interventions, particularly in areas inaccessible to other techniques.
Acknowledgments
We are thankful to Todd Anderson for his excellent revision of the manuscript and helpful discussions. We are furthermore indebted to the technical assistance of Kerstin Kretschel, Evelyn Polzin, and Ursula Wagner.
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