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Published online before print January 28, 2005, 10.1148/radiol.2343031440
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(Radiology 2005;234:833-841.)
© RSNA, 2005

Molecular Imaging

Leg Ischemia: Assessment with MR Angiography and Spectroscopy1

Iris Baumgartner, MD, Harriet C. Thoeny, MD, Oliver Kummer, MD, Christian Roefke, MD, Corinna Skjelsvik, MD, Chris Boesch, MD, PhD and Roland Kreis, PhD

1 From the Swiss Cardiovascular Center, Division of Angiology (I.B., O.K., C.S.) and Departments of Diagnostic, Interventional, and Pediatric Radiology (H.C.T., C.R.), University Hospital Bern, Freiburgstrasse 10, 3010 Bern, Switzerland; and Department for Clinical Research (MR Spectroscopy and Methodology), University of Bern, Bern, Switzerland (C.B., R.K.). Received September 8, 2003; revision requested November 20; final revision received June 23, 2004; accepted June 29. Supported by the Swiss National Science Foundation (4037–055161, 3100–065315, 3100059082). Address correspondence to H.C.T. (e-mail: harriet.thoeny@insel.ch).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
PURPOSE: To prospectively determine reproducibility of magnetic resonance (MR) angiography and MR spectroscopy of deoxymyoglobin in assessment of collateral vessels and tissue perfusion in patients with critical limb ischemia (CLI) and to follow changes in patients undergoing intramuscular vascular endothelial growth factor (pVEGF)-C gene therapy, percutaneous transluminal angioplasty, supervised exercise training, or no therapy.

MATERIALS AND METHODS: Study and gene therapy protocols were approved, and all patients gave written informed consent. To determine repeatability and reproducibility, seven patients underwent MR angiography and five underwent MR spectroscopy. The techniques were used to judge disease progress in 12 other patients with or without therapy: MR angiography to help determine change in visualization of collateral vessels and MR spectroscopy to help assess change in perfusion at proximal and distal calf levels. MR angiographic results were subjectively analyzed by three blinded readers. Intraobserver variability was expressed as 95% confidence interval (CI) (n = 7); interobserver variability, as {kappa} statistic (n = 15). Reexamination variability of MR spectroscopy was given as 95% CI for subsequent recovery times, and correlation with disease extent was calculated with Kendall {tau}b rank correlation. Fisher-Yates test was used to correlate changes with pressure measurements and clinical course.

RESULTS: Intraobserver and interobserver concordance was sensitive for detection of collateral vessels. Intraobserver agreement was 85.7% (95% CI: 42.1%, 99.6%). Interobserver agreement was high for small collateral vessels ({kappa} = 0.74, P < .001) and fair for large collateral vessels ({kappa} = 0.36, P = .002). MR spectroscopy was reproducible (95% CI: ±26 seconds for proximal, ±21 seconds for distal) and showed a correlation with disease extent (proximal calf, {tau}b = 0.84, P < .001; distal calf, {tau}b = 0.68, P = .04). Small collateral vessels increased over time (P = .04) but did not correlate with pressure measurements and clinical course. Recovery time correlated with clinical course (proximal calf, P = .03; distal calf, P = .005).

CONCLUSION: MR angiography and MR spectroscopy of deoxymyoglobin can help document changes in visualization of collateral vessels and tissue perfusion in patients with CLI.

© RSNA, 2005


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
Preclinical data on growth factor–augmented collateral vessel development (therapeutic neovascularization) has stimulated interest in therapy-related growth of collateral vessels and perfusion changes in patients with peripheral arterial occlusive disease (PAOD). Growth factor–induced neovascularization to promote reperfusion of ischemic but not yet necrotic tissue is certainly a promising but unproved therapeutic approach in patients with chronic critical leg ischemia (CLI) who are unsuitable for conventional revascularization (1–4).

Animal models have shown that enhanced vascularity after growth factor application includes proliferation of medium-sized arteries, as revealed at premortem angiography, and increased capillary density, as demonstrated at postmortem histologic examination (5). Although experimental models have clearly defined the extent and anatomic type of enhanced vascularization (6,7), it is not known how well such animal models relate to theclinical scenario of patients whose limbs have failed to spontaneously develop a sufficient angiogenic response after decreased blood flow that lasts for weeks or months. In the absence of ideal clinical end points, surrogate end points are needed to measure the effects of therapy.

Intraarterial contrast material–enhanced angiography lacks the quantitative precision and reproducibility necessary for the study of small collateral vessels (8,9). A vast improvement in image quality was recently achieved with gadolinium-enhanced three-dimensional magnetic resonance (MR) angiography, which enabled detailed visualization of even small collateral vessels (10–12). MR angiography also allows three-dimensional postacquisition image reorientation for comparison of images at different time points. MR techniques can also be used to study tissue perfusion (13–16). One approach for quantification of relative differences in skeletal muscle perfusion is the measurement of ischemia-induced reoxygenation by using hydrogen 1 (1H) MR spectroscopy of deoxymyoglobin (16–19). It is important to note that although CLI is associated with severe ischemia at rest, differences between normal and severe PAOD remain small. 1H MR spectroscopy of deoxymyoglobin can help accurately measure perfusion changes in skeletal muscle whose resting blood flow is otherwise too low for accurate measurement, and findings have been shown to agree with those of other methods, with excellent reproducibility (19).

The purposes of the present study were to prospectively determine the reproducibility of MR angiography and 1H MR spectroscopy of deoxymyoglobin in the assessment of collateral vessels and tissue perfusion in patients with CLI and to follow changes in CLI patients undergoing intramuscular vascular endothelial growth factor (pVEGF)-C gene therapy, percutaneous transluminal angioplasty, supervised exercise training, or no therapy by using a 1.5-T MR imager.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
Patient Selection and Treatment Types
Patients with CLI, according to the Consensus Document of the European Working Group on Critical Leg Ischemia, were analyzed in a prospective consecutive manner (20). All patients with CLI unsuitable for revascularization who were seen between June 1999 and October 2001 and patients with CLI who underwent percutaneous transluminal angioplasty between June and July 2001 were selected. The first part of the study was designed to define intra- and interobserver variability of MR angiography and reproducibility of 1H MR spectroscopy. Patients scheduled to undergo MR angiography and 1H MR spectroscopy were evaluated in two separate groups not identical to each other to lower the burden of repetitive examination. The second part of the study was designed to determine the changes in visualization of collateral vessels and tissue perfusion in an identical group of patients over time. Patients examined with MR angiography and 1H MR spectroscopy were different from the patients examined in the first part of this study. Exclusion criteria were contraindication to MR imaging (eg, pacemaker), patient refusal or inability to understand study conditions, in-place vascular graft of the target limb, uncontrolled pain, or severe secondary infection. The Ethical Committee of the University Hospital Bern approved the study protocols. All patients gave written informed consent. Gene therapy protocols were approved by the Swiss Agency for the Environment, Forests, and Landscape and by the Swiss Federal Office of Public Health. The gene therapy protocol was open for randomization between 1999 and 2000.

Reproducibility.—Intra- and interobserver agreement for assessment of visualization of large and small collateral vessels at the calf level at MR angiography was evaluated in seven CLI patients (six men, one woman; mean age, 65 years ± 19 [standard deviation]) in two examinations performed a mean of 3 days ± 2 apart. Technical parameters, injection site, amount of contrast agent, medication, and positioning were kept identical for each patient. Blood pressure and heart rate were documented. Reexamination reproducibility of 1H MR spectroscopy of deoxymyoglobin in the assessment of lower limb perfusion was evaluated in another five CLI patients (four men and one woman; mean age, 60 years ± 16) in two independent examinations performed sequentially with a 10–20-minute break in between and complete repositioning of the patient and the radiofrequency coils. The analysis of the reliability of 1H MR spectroscopy in these five patients was reported previously (19).

Correlation of the results with anatomic extent and level of PAOD complemented the analysis. The anatomic level and extent of arterial occlusive disease were graded as follows: score of 1 = foot artery occlusions; score of 2 = occlusion of one calf artery (plus foot artery occlusions); score of 3 = occlusion of two calf arteries; score of 4 = occlusion of all calf arteries; score of 5 = occlusion of calf and popliteal arteries; score of 6 = occlusion of calf, popliteal, and superficial femoral arteries.

Collateral vessel imaging and tissue perfusion.—Changes in the visualization of collateral vessels and tissue perfusion were studied in 12 additional CLI patients (eight men and four women; mean age, 63 years ± 23) undergoing intramuscular pVEGF-C gene therapy (n = 4), percutaneous transluminal angioplasty (n = 4), supervised exercise training (n = 2), or no therapy (n = 2). MR angiography and 1H MR spectroscopy of deoxymyoglobin were performed parallel at baseline and follow-up. Gene therapy was performed sequentially (single-dose followed 3 months later by multiple-dose pVEGF-C gene therapy). These patients had two follow-up assessments, and patients with other or no specific therapy had one follow-up assessment. Limb ischemia was documented with ankle-brachial and big toe–brachial indices. Arterial occlusions were documented with intraarterial contrast-enhanced angiography at baseline.

pVEGF-C gene therapy.—Patients were treated with a single-dose intramuscular gene therapy by using plasmid DNA encoding the human pVEGF-C gene transcriptionally regulated by a cytomegalovirus enhancer/respiratory syncytial virus promoter sequence (Vascular Genetics, Durham, NC) (21). Eight aliquots of 0.5 or 1 mg pVEGF-C (4 or 8 mg pVEGF-C per patient) were injected into calf muscles of the affected limb (1). Single-dose gene therapy was followed 3 months later by multiple-dose gene therapy (three times at 4-week intervals, 8 mg pVEGF-C per treatment).

Percutaneous transluminal angioplasty.—Percutaneous transluminal angioplasty was performed according to standard procedures described elsewhere (22,23).

Exercise training.—Patients performed a supervised exercise training for 1 hour twice a week for 3 months (24,25).

MR Angiography, 1H MR Spectroscopy, and Interpretation
MR angiography was performed with a clinical whole-body 1.5-T MR system (Vision; Siemens, Erlangen, Germany) by using an extremity coil. The area to be imaged extended from the proximal calf to the ankle (39-cm extremity coil). Coronal three-dimensional gradient-echo MR images were acquired with the following parameters: repetition time msec/echo time msec, 4.6/1.8; flip angle, 30°; field of view, 190 x 380 mm; slab thickness, 100 mm; one signal acquired. The matrix size of the raw data was 155 x 512 (pixel size, 1.23 x 0.74 mm). Zero padding to 256 x 512 yielded a pixel size of 0.74 x 0.74 mm. Forty sections were obtained by using three-fourths Fourier acquisition and were interpolated to 100 sections, with a resulting section thickness of 1 mm. Acquisition time was 28.5 seconds for each series. One precontrast and two consecutive postcontrast series were performed, with no interval between the postcontrast series.

The contrast agent was administered intravenously through a 20-gauge catheter that was placed in the antecubital fossa. A bolus of 30 mL of gadodiamide (Omniscan; Nycomed, Oslo, Norway) was injected with a power injector at 3 mL/sec, followed immediately by a 30-mL saline flush. Image acquisition commenced 10 seconds after the start of bolus injection. Subtraction images were processed by using a maximum intensity projection algorithm. Maximum intensity projections were reconstructed in 15° rotation steps, with four representative images documented on hard copies. Follow-up maximum intensity projections were reconstructed to mirror baseline rotational orientation. MR angiograms were reviewed by three independent readers (C.S. and O.K., with 1 year of experience each; H.C.T., with 5 years of experience) blinded to clinical data and using a predefined semiquantitative visual assessment protocol (unchanged, increased, or decreased visualization of large and small collateral vessels). Visualization was defined as a subjective impression of recognition of collateral vessels on an image. Arterial vessels were grouped into infrapopliteal main arteries (eg, anterior tibial artery), large collateral vessels, and small collateral vessels. Collateral vessels were categorized as large if they were visible for more than 25% of the imaged calf length and were visually estimated to be at least 50% as large as infrapopliteal main arteries (Fig 1). Since collateral vessels regress and vanish at angiography after revascularization, no MR angiographic follow-up was performed in patients with successful percutaneous transluminal angioplasty.



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  		Figure 1. Patient 2. Coronal maximum intensity projection from contrast-enhanced three-dimensional gradient-echo MR angiography (4.6/1.8) at calf level in a 47-year-old man (reliability study) shows large collateral vessels (arrowhead), small collateral vessels (arrow), and occlusion of anterior tibial artery (*).

 
Deoxygenation and reoxygenation of myoglobin, as indicated by changes in the deoxymyoglobin signal at 1H MR spectroscopy, were measured by using a clinical 1.5-T MR system (Signa, system software 5.8; GE Medical Systems, Milwaukee, Wis). In resting muscle, deoxymyoglobin signal levels were below the detection sensitivity of 1H MR spectroscopy. Myoglobin of calf muscles was completely converted to deoxymyoglobin by thigh air-cuff–induced arterial occlusion. Assessment of the relative skeletal muscle perfusion by monitoring the disappearance of the ischemia-induced deoxymyoglobin signal (recovery time) is described in detail elsewhere (19).

In brief, a selective pulse (inherent effective dead time of 0.44 msec) was used for the excitation of the deoxymyoglobin signal at 78 ppm, sampled with a repetition time of 110 msec. Data processing included a moving average of 64 acquisitions. The time course of the deoxymyoglobin signal was evaluated by using a time resolution of 3.5 seconds, which was obtained by averaging 32 images. The body coil was used for transmission, and two independent surface coils were used for signal reception. Reproducible positioning of the subjects was achieved with a specially tailored cushion and was controlled with transverse and coronal gradient-echo MR imaging (150/10, 30° flip angle).

To observe ischemia-induced deoxymyoglobin, the signal acquisition was started as soon as the pressure cuff on the thigh was inflated to 220 mm Hg. Ischemia was maintained for 7 minutes until instantaneous release of the pressure. Signal acquisition was continued for another 8 minutes. The observed signal originated from a volume of interest of approximately 50 cm3, 7 cm (distal calf) and 22 cm (proximal calf) above the medial malleolus. 1H MR spectroscopy was performed by one of two operators (R.K. and another operator with 10 and 2 years of experience, respectively, in medical MR spectroscopy) blinded to angiographic and clinical data. Signal analysis of 1H MR spectroscopy and time-course evaluations were performed automatically without operator input. Temporal signal analysis was performed by using computer software (Excel 5.0; Microsoft, Redmond, Wash) (19).

Statistical Analysis
Statistical analysis was performed with software packages (Excel 97, Microsoft; SPSS version 5.1, SPSS, Chicago, Ill; and StatView 4.0, SAS Institute, Cary, NC). Numeric data were reported as means ± standard deviation. For MR angiography, the intraobserver variability of collateral vessel visualization in repeat examinations (n = 7) was expressed as percentage of agreement and its 95% confidence interval (CI). Interobserver agreement among the three readers was expressed as percentage of agreement and was evaluated with {kappa} statistic for all available independent comparisons (n = 15) (26). Reexamination variability of 1H MR spectroscopy was evaluated by comparing the values from the two repeat measurements at proximal and distal levels of the calf. The 95% CI for the difference between the two subsequent examinations was determined for both locations according to the method of Bland and Altman (27).

Correlation of the recovery times at 1H MR spectroscopy of deoxymyoglobin at the proximal and distal calf levels with the anatomic level and the extent of arterial occlusive disease (six-point scoring system) were estimated by using the Kendall {tau}b rank correlation. A two-sided Fisher-Yates test using nominal values was applied to correlate changes in visualization of collateral vessels on MR angiograms compared with hard copies of repeat examinations and to correlate changes in visualization of collateral vessels and tissue perfusion with pressure measurements and clinical course. Analysis was performed for visualization of large and small collateral vessels, as well as for recovery times at 1H MR spectroscopy of deoxymyoglobin at the proximal and distal calf levels. A change in the ankle-brachial index or toe-brachial index of more than 0.1 and at least one clinical category (Rutherford categories, 0–6) were defined as clinically relevant (28). P < .05 was considered to indicate a statistically significant difference.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
Reproducibility
All patients included in this part of the study had cross-sectional occlusion at the calf or ankle and/or foot level, five patients had additional critical (>80%) stenoses or occlusion at the thigh level (including popliteal artery), and no patient had greater than 50% stenosis or occlusion at the abdominal level.

Repeated imaging with MR angiography was satisfactorily reproducible for documenting large and small collateral vessels, as evaluated by three blinded independent readers. The intraobserver concordance for visualization of collateral vessels is given in Table 1A. Intraobserver agreement was 85.7% (95% CI: 42.1%, 99.6%). Interobserver agreement showed a particularly high grade of conformity for perception of the visualization of small collateral vessels (percentage of agreement, 87%; {kappa} = 0.74; P < .001; Table 1B), while concordance was fair for large collateral vessels (percentage of agreement, 78%; {kappa} = 0.36; P = .002; Table 1B). Reexamination reproducibility and interobserver agreement indicated that MR angiography is sensitive in detection of large and small collateral vessels, with an acceptable degree of variability. Blood pressure variability was 14% ± 10 (median, 14%; 20 mm Hg ± 8, median of 17 mm Hg), and heart rate variability was 15% ± 11 (median, 11%; 11 beats per minute ± 8, median of 9 beats per minute).


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  		TABLE 1. Intraobserver and Interobserver Concordance for Visualization of Collateral Vessels on Three-dimensional Contrast-enhanced MR Angiograms in Patients with Chronic CLI

 
Myoglobin reoxygenation (recovery time in seconds) within a volume of interest at the proximal and distal calf levels with 1H MR spectroscopy of deoxymyoglobin was well reproducible. The 95% CI for the difference in recovery time obtained from repeat examinations for the distal and proximal locations was found to be 21 and 26 seconds, respectively. Recovery times were 4–86 seconds in the reproducibility study and 5–350 seconds in the therapy study. Recovery times were in agreement with the anatomic level and extent of PAOD at the proximal ({tau}b = 0.84, P < .001) and distal ({tau}b = 0.68, P = .04) (Fig 2) calf levels. Individual results of 1H MR spectroscopy of deoxymyoglobin revealed almost normal recovery times, which previously were shown to range around 10 seconds (19), in patients with arterial occlusions limited to the ankle and/or foot level. Recovery times in patients with proximal or multilevel arterial obstructions were usually more than 10 times longer.



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  		Figure 2. Graph depicts recovery times (reoxygenation) of ischemia-induced deoxymyoglobin at 1H MR spectroscopy at proximal and distal calf correlated with anatomic extent and level of arterial occlusive disease, as graded with the six-point anatomic score.

 
Collateral Vessel Imaging and Tissue Perfusion
Patient characteristics and the anatomic level and extent of occlusion are shown in Table 2. With the exception of patients 6, 7, and 8, the remaining patients had arterial obstructions that were considered to be occlusions. Patients 7 and 8 had infrapopliteal critical stenoses, and patient 6 had critical superficial femoral artery stenosis and infrapopliteal occlusions. Follow-up schedule for MR angiographic and 1H MR spectroscopic assessment is listed in Table 3. Individual comparison of MR angiography, 1H MR spectroscopy, pressure measurements, and the clinical course in 12 patients followed up for changes over time are shown in Table 4. Results of MR angiography are presented as a consensus among three blinded readers.


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  		TABLE 2. Baseline Characteristics of 12 Patients with Chronic CLI Followed Up for Changes in Visualization of Collateral Vessels and Tissue Perfusion Over Time

 

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  		TABLE 3. Time Schedule of Assessment

 

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  		TABLE 4. Results of Different Therapies in 12 Patients with CLI Followed Up for Changes Over Time

 
There were no relevant changes in visualization of infrapopliteal main arteries and large collateral vessels, whereas small collateral vessels increased over time. Changes in visualization of small collateral vessels were significant compared with those on hard-copy sets of repeat examinations shown in the first part of the study (P = .04). Despite interobserver agreement between 78% and 87% (Table 1B), the correlations of changes on MR angiograms with pressure measurements and clinical course were poor: For large collateral vessels, agreement was 83% (10 of 12, not significant) and 50% (six of 12, not significant), respectively, and for small collateral vessels it was 50% (six of 12, not significant) and 67% (eight of 12, not significant), respectively. Overall agreement was 67% (16 of 24, not significant) and 58% (14 of 24, not significant), respectively. In patients with proximal or multilevel occlusions, all three blinded readers ranked baseline examinations lowest with regard to visualization of collateral vessels (Fig 3, Table 4). Correlation between 1H MR spectroscopy and pressure measurement was poor, whereas it was significant with the clinical course. At the proximal level, agreement with pressure measurement and clinical course was 60% (nine of 15, not significant) and 80% (12 of 15, P = .03), respectively. At the distal level, the respective agreements were 57% (eight of 14, not significant) and 93% (13 of 14, P = .005). Overall agreements were 59% (17 of 29, not significant) and 86% (25 of 29, P < .001), respectively.



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  		Figure 3a. Patient 3. Coronal maximum intensity projections from contrast-enhanced three-dimensional gradient-echo MR angiography (4.6/1.8) at the level of calf in an 84-year-old man show increased visualization of collateral vessels after pVEGF-C gene therapy. (a) Baseline, (b) 3 months after single-dose gene therapy, and (c) 9 months after multiple-dose gene therapy.

 


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  		Figure 3b. Patient 3. Coronal maximum intensity projections from contrast-enhanced three-dimensional gradient-echo MR angiography (4.6/1.8) at the level of calf in an 84-year-old man show increased visualization of collateral vessels after pVEGF-C gene therapy. (a) Baseline, (b) 3 months after single-dose gene therapy, and (c) 9 months after multiple-dose gene therapy.

 


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  		Figure 3c. Patient 3. Coronal maximum intensity projections from contrast-enhanced three-dimensional gradient-echo MR angiography (4.6/1.8) at the level of calf in an 84-year-old man show increased visualization of collateral vessels after pVEGF-C gene therapy. (a) Baseline, (b) 3 months after single-dose gene therapy, and (c) 9 months after multiple-dose gene therapy.

 
For example, patient 3 experienced profound clinical deterioration, developing multiple new ischemic lesions within 2 months after single-dose gene therapy (Table 4). Recovery time deteriorated by 142% (142.8–348.1 seconds) at the proximal calf and 62% (143.0–232 seconds) at the distal calf, which is consistent with the clinical course. After multiple-dose gene therapy, lesions healed and rest pain resolved in parallel with an impressive improvement in recovery times (Figs 4, 5). Ankle- and toe-brachial indices remained unchanged at critically low levels, although MR angiography demonstrated an increase in vascularity. Patient 8 underwent balloon angioplasty of the popliteal artery that was complicated by distal embolization. 1H MR spectroscopy of deoxymyoglobin helped confirm improved perfusion at the proximal calf level owing to a patent popliteal artery but a brisk deterioration at the distal calf level owing to diffuse embolic occlusions in accordance with the clinical course.



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  		Figure 4. Patient 3. Graphs in a patient who underwent 1H MR spectroscopy of deoxymyoglobin and pVEGF-C gene therapy. Data points for the second examination (top row) were recorded 4 months after single-dose pVEGF-C gene therapy, with clinical deterioration and development of new ischemic lesions. Data points for the third examination (bottom row) were recorded after additional multiple-dose pVEGF-C gene therapy, with complete healing of lesions. In distal and proximal locations, third examination revealed clear shortening of the recovery time due to improved perfusion compared with that in second examination. Mb = myoglobin.

 


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  		Figure 5a. Patient 3. (a) Summed 1H MR spectrum (2.2-7.6 minutes after application of the cuff) and (b) 1H MR spectra of deoxymyoglobin as function of time demonstrate the spectral quality of the deoxymyoglobin signal at 78 ppm and its temporal course. The deoxymyoglobin signal arises during application of the pressure cuff for the first 7 minutes. Each spectrum corresponds to the average of 128 acquisitions, while each data point in Figure 4 represents the average of 64 acquisitions. The spectra correspond to baseline measurements in the proximal location (recovery time, 2.4 minutes). The small signals near 120 ppm are the infolded residual peaks of water and fat.

 


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  		Figure 5b. Patient 3. (a) Summed 1H MR spectrum (2.2-7.6 minutes after application of the cuff) and (b) 1H MR spectra of deoxymyoglobin as function of time demonstrate the spectral quality of the deoxymyoglobin signal at 78 ppm and its temporal course. The deoxymyoglobin signal arises during application of the pressure cuff for the first 7 minutes. Each spectrum corresponds to the average of 128 acquisitions, while each data point in Figure 4 represents the average of 64 acquisitions. The spectra correspond to baseline measurements in the proximal location (recovery time, 2.4 minutes). The small signals near 120 ppm are the infolded residual peaks of water and fat.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
 
In the present study, contrast-enhanced high-spatial-resolution MR angiography was found to be a reproducible and robust modality for detecting different sizes of collateral vessels at the calf level and to allow good perception of changes over time. MR angiographic findings, however, correlated rather poorly with clinical course, giving an agreement in less than two-thirds of evaluations. By contrast, recovery time of ischemia-induced deoxymyoglobin signal at 1H MR spectroscopy showed good reproducibility for determining tissue perfusion at the calf level and helped quantify changes over time in accordance with clinical course (86%). The results suggest that MR angiography (visualization of collateral vessels) and 1H MR spectroscopy of deoxymyoglobin (index of effective tissue perfusion) can be used to complement one another in the assessment of the effect of angiogenic protocols undergoing clinical testing, with the advantage that both can be performed with a routine MR imager.

PAOD is characterized by preexisting major collateral vessels interconnecting main arteries and an abundance of smaller arterial networks (middle zone collaterals), which join de novo developing vessels (angiogenesis). We demonstrate in the present study that MR angiography is sufficiently sensitive in helping to recognize various sizes of collateral vessels and changes over time, with good reproducibility and interobserver agreement. Although this would appear to indicate that MR angiography is an appropriate technique for following collateral development, its findings correlated poorly with the clinical course. A number of reasons may explain this discrepancy. First, experimental data show that the spatial resolution of MR angiography allows depiction of only a fraction of the total number of collateral vessels. Vessel proliferation, though, is greatest among collateral vessels with the smallest diameters, and collateral-dependent flow typically improves owing to the proliferation of vessels of less than 180 µm in diameter (6,29,30). Second, the clinical course of CLI is influenced by factors independent of collateral-dependent flow (eg, local infection, heart failure, blood pressure). Third, the temporal development of angiographically visible collateral-dependent flow may not be accompanied by a parallel increase in nutritive flow essential for lesion healing.

MR spectroscopy for clinical in vivo assessment of the skeletal muscle system focuses mainly on high-energy phosphates and intracellular pH using phosphorus 31 (31P) MR spectroscopy (31,32). One study attempted to use 31P MR spectroscopy of the foot to evaluate peripheral vascular disease, but without success, for individual disease assessment (33). In contrast to levels of high-energy phosphates and intracellular pH, which have complex perfusion and metabolism-dependent regulation mechanisms (34), reoxygenation of myoglobin (16–19) is primarily a measure of reperfusion efficiency. Brillault-Salvat et al (18) showed that after 7 minutes of air-cuff–induced ischemia, a significant correlation exists between myoglobin reoxygenation and tissue reperfusion, perfusion being the rate-limiting factor for myoglobin reoxygenation.

Peripheral pressure measurements in patients with CLI have well-known limitations (35,36), allowing perfusion changes to sometimes elude detection. In the present study, we show that even modest changes in perfusion can be more sensitively, and probably more reliably, detected with measurement of reoxygenation by using 1H MR spectroscopy of deoxymyoglobin (recovery time) than with peripheral pressure measurement. Despite the presence of CLI, no quantifiable deoxymyoglobin signal was detected in patients at rest. Evaluation of exercise-induced deoxymyoglobin would be very hard to standardize and reproduce in this patient population in general and after therapy in particular. Risk of harm with use of the occlusion-reperfusion approach is theoretically possible and was stated in the written patient information. Clinically relevant deterioration of PAOD and reperfusion edema were not seen in any of the patients repeatedly tested. Although our series is small, it seems that the occlusion-reperfusion approach is safe in patients with severe end-stage PAOD.

Our study had certain limitations. We did not include MR imaging at the foot level, which would have allowed better assessment of the effectiveness of the different therapeutic protocols, particularly in patients with distal arterial occlusions. Technical problems prevented reliable documentation of small foot arteries with MR angiography, while 1H MR spectroscopy of deoxymyoglobin was not possible in the foot and the very distal part of the lower leg, owing partly to disease-related muscle atrophy. The main limitation was the small number of patients studied, a problem common to clinical trials enrolling patients with CLI unsuitable for conventional revascularization. Further, since there is no validated reliable tool to follow de novo developing vessels in patients with PAOD, a standard of reference is lacking against which MR angiography and 1H MR spectroscopy could be compared, which is reflected by the presentation of correlations instead of calculation of predictive values and accuracy of the test. Peripheral pressure measurements show considerable variability, and clinical course can be influenced by cofactors such as blood pressure, heart rate, oxygen saturation, and infection (37). Therefore, validation of MR angiography and 1H MR spectroscopy of deoxymyoglobin based on peripheral pressure measurements and clinical end points remains problematic. Finally, it needs to be stressed that high-quality MR angiography involves time-consuming postprocessing and that 1H MR spectroscopy of deoxymyoglobin is compromised by its reliance on pressure-cuff–induced ischemia, which may not be well tolerated by some patients.

In summary, in the lower limb contrast-enhanced high-spatial-resolution MR angiography represents a robust modality to observe collateral vessels, and 1H MR spectroscopy of deoxymyoglobin is reliable to quantify changes in tissue perfusion. Thus, they appear well suited to assess the effect of angiogenic protocols undergoing clinical testing with the advantage that both can be performed with a routine MR imager.


   ACKNOWLEDGMENTS
 
We thank I. R. Haubitz, PhD, for statistical support.


   FOOTNOTES
 
Abbreviations: CI = confidence interval, CLI = critical limb ischemia, PAOD = peripheral arterial occlusive disease, pVEGF = vascular endothelial growth factor

Authors stated no financial relationship to disclose.

Author contributions: Guarantors of integrity of entire study, I.B., R.K., H.C.T.; study concepts, I.B., R.K., C.B.; study design, I.B., R.K.; literature research, C.S.; clinical studies, C.S., O.K., C.R.; data acquisition, O.K., C.S., R.K.; data analysis/interpretation, I.B., R.K., O.K., H.C.T., C.S.; statistical analysis, I.B., R.K.; manuscript preparation and revision/review, I.B., H.C.T., R.K.; manuscript definition of intellectual content, I.B., R.K., C.B.; manuscript editing, R.K., C.B.; manuscript final version approval, I.B., R.K.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 

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