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(Radiology. 2001;220:413-419.)
© RSNA, 2001

Experimental Studies

Delayed Muscle Injuries in Arterial Insufficiency: Contrast-enhanced MR Imaging and 31P Spectroscopy in Rats1

Roberto M. Asperio, DVM, PhD, Elena Nicolato, PhD, Pasquina Marzola, PhD, Paolo Farace, PhD, Ernesto Lunati, PhD, Andrea Sbarbati, MD, PhD and Francesco Osculati, MD

1 From the Department of Morphological-Biomedical Sciences, Institute of Anatomy and Histology, University of Verona, Medical Faculty, Strada Le Grazie 8, 37134 Verona, Italy. Received July 25, 2000; revision requested September 7; final revision received January 22, 2001; accepted February 12. F.O. supported by Ministero dell’Università e della Ricerca Scientifica e Tecnologica grant 9805406803. Address correspondence to A.B. (e-mail: sbarbati@borgoroma.univr.it).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
PURPOSE: To evaluate whether the vascular system resulting from an arterial lesion shows differences in permeability to a tracer with respect to the normal vascular system and whether eventual differences are maintained for long periods.

MATERIALS AND METHODS: Permanent ischemia was induced in rats with femoral arterial removal, and magnetic resonance (MR) imaging was performed after 1, 7, 14, and 90 days. Gadopentetate dimeglumine was injected, and the kinetics of its penetration in the leg were studied. Phosphorus 31 spectroscopy was performed to determine the bioenergetic characteristics of the gastrocnemius muscle at rest and stimulation. Ischemic muscles were then processed for electron microscopy.

RESULTS: After ischemia induction, a hyperintense area that progressively decreased was present on T2-weighted images. Gadopentetate dimeglumine improved the signal intensity of the area. Three months after arterial occlusion, the contrast-enhanced images still showed microvessels highly permeable to the tracers. Spectroscopic data revealed that 3 months after arterial removal, the bioenergetic reserve of the gastrocnemius muscle was reduced, suggesting that the contrast-enhanced MR imaging–visible area is functionally relevant. Ultrastructural examination revealed persistent muscle damage and signs of chronic microangiopathy.

CONCLUSION: After ischemia induction, the restitutio ad integrum is not complete, and delayed muscle injuries can result from arterial insufficiency.

Index terms: Animals • Magnetic resonance (MR), experimental studies • Magnetic resonance (MR), spectroscopy, 92.12945 • Magnetic resonance (MR), vascular studies, 92.12943, 92.12945, 92.12949 • Muscles, blood supply, 92.769 • Muscles, gastrocnemius, 45.91


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
After episodes of acute arterial insufficiency, the restoration of blood flow is due to the opening of preexistent collateral vessels and the development of new vessels (1,2). In the skeletal muscle of the limbs, this process leads to the formation of a rearranged vascular system.

The result of this process may have clinical relevance because the clinical outcome subsequent to acute ischemia is probably linked to the ability of the newly formed vascular system to adequately fulfill the metabolic needs of the muscle. In the past, investigators in some studies (3) quantified blood vessels in the newly formed bed with histologic and morphometric examination, but in skeletal muscle no data have been obtained for a well-recognized parameter of blood vessel function—permeability to intravascular tracers.

To date, technologic development has led to the feasibility of noninvasive in vivo studies of this parameter with high spatial (submillimetric) and temporal resolution. In particular, magnetic resonance (MR) imaging has the potential to depict characteristic properties of vascular beds by virtue of differential distribution of contrast media in normal and ischemic regions (4,5). Contrast material–enhanced MR imaging data have been obtained in acute models of muscle ischemia (6–8) and during postischemic reactive hyperthermia (9); however, this technique has not been applied to characterize the repair process of ischemic insult in skeletal muscle.

The purpose of this study was to evaluate whether the vascular system resulting from arterial ischemia shows differences in permeability to a tracer with respect to its normal counterpart and whether any differences are maintained for long periods.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
Animals
The care of the animals involved in this study complied with National Institutes of Health guidelines for the use of laboratory animals. Muscle hind limb ischemia was induced in a group (n = 8) of 4-month-old male Wistar rats (Harlan-Nossan; San Pietro al Natisone, Odine, Italy). The rats were anesthetized with intramuscularly injected xylazine (10 mg per kilogram of body weight of Rompun; Bayer, Leverkusen, Germany) and ketamine hydrochloride (75 mg/kg of Inoketam 500; Virbac, Carros, France). Surgery was performed in the medial part of the thigh. After the skin was prepared with antiseptic, a skin incision was made with a size-10 scalpel, and dissection with curved scissors was performed from the inguinal ligament to the patella. The neurovascular trunk of the thigh was well recognized after the subcutaneous adipose tissue was dissected. By means of microscopy, the femoral artery was then dissected with fine forceps from its proximal origin as a branch of the external iliac artery to the patella. Three arterial branch vessels (ie, the deep femoral, circumflexa femoris lateralis, and pudendo-epigastric artery) also were isolated. The femoral artery and its previously mentioned branch vessels were coagulated with a bipolar coagulator (Alsatom SU-50MB; Alsa, Bologna, Italy). The skin was then closed with interrupted vertical mattress stitches and 4/0 braided silk suture material (Ethicon; Johnson & Johnson, Dilbeek, Belgium).

An antibiotic (2.5 mg/kg of enrofloxacine, Baytril; Bayer) was subcutaneously injected after surgery; no postoperative restraints were used. All animals were fed ad libitum and checked frequently after ischemia induction.

MR Imaging
MR imaging was performed 24 hours, 7 days, 14 days, and 3 months after ischemic lesion induction. For MR imaging, the rats were anesthetized with an inhaled mixture of O2 and air containing 1%–2% halothane. This anesthetic was preferred because it is widely used in functional MR imaging experiments with rat microcirculation and MR spectroscopy (10–12). In addition, it is particularly useful when it is necessary to safely anesthetize animals several times within a short period.

All MR imaging experiments were performed by using a Biospec System (Bruker, Karlsruhe, Germany) equipped with a 4.7-T Oxford magnet with a 33-cm bore and a gradient insert (SMIS; Surrey Medical Imaging Systems, Guilford, England). A birdcage coil with a 72-mm internal diameter was used.

After a pilot scout image was acquired, coronal and transverse multisection T2-weighted spin-echo images were acquired by using the following parameters: repetition time, msec/echo time msec, 2,000/60; section thickness, 2 mm; matrix, 128 x 128; field of view, 8 x 8 cm2; and pixel size, 625 x 625 µm2. In this series, the best section on which to observe the affected area was localized.

Dynamic acquisition of a series of 20 coronal T1-weighted spin-echo images with a 1-second interval was started. During this dynamic acquisition, a bolus of 0.1 mmol/L/kg gadopentetate dimeglumine (Magnevist; Schering, Berlin, Germany) was injected via the tail vein. In this case, the imaging parameters were 100/10; section thickness, 2 mm; matrix size, 64 x 64; field of view, 8 x 8 cm2, and pixel size, 1.25 x 1.25 mm2, corresponding to a 6.4-second acquisition time.

The kinetics of contrast agent penetration in the leg were studied on serial images obtained by using the formula: {Delta}SI = SI(t) - SIpre on a pixel-by-pixel basis, where {Delta}SI is the signal intensity change. The signal intensity of an image obtained before contrast agent enhancement, SIpre, was subtracted from the signal intensity of images obtained at different times after contrast agent injection, SI(t). At different times, postcontrast images were quantitatively evaluated by calculating the average value for the parameter (SIpost - SIpre) for a region of interest selected in the affected area and for the contralateral leg; all regions of interest were selected by the same author (E.N.). The mean region of interest area was 0.141 cm2 ± 0.001. Values were expressed as means plus or minus standard errors of the mean (SEMs). Paired or unpaired t tests were performed to determine the significance of the difference between the signal intensities of the normal muscle and the muscle with lesion.

At MR imaging 3 months after ischemia induction, 12 additional images were acquired in approximately 50 minutes. In addition, in all rats, a T2 parametric map was obtained. This map was calculated by using a coronal T2-weighted multiecho single-section spin-echo image (1,200/20–120; matrix size, 128 x 128; field of view, 8 x 8 cm2; pixel size, 625 x 625 µm2; and six echoes).

31P in Vivo Localized Spectroscopy
31P spectroscopy was performed to determine the bioenergetic characteristics of the gastrocnemius muscle at rest and during electrical stimulation. In vivo 31P localized spectroscopy was performed in five of the original eight rats 3 months after ischemia induction and in five control rats. The rats were anesthetized with an inhaled mixture of O2 and air containing 1%–2% halothane. Stimulation electrodes were inserted into the proximal head of the gastrocnemius muscle and into the Achilles tendon and were connected to a stimulator (model S-48; Grass Instruments Manufacturing, Braintree, Mass). The gastrocnemius region was fitted on a home-built radio-frequency circular surface coil with a 3-cm diameter that was tuned to the 31P resonance. The foot of the stimulated leg was tied to a home-made force transducer connected to a 60-MHz oscilloscope (Tektronix 2215; Tektronix, Beaverton, Ore). Electrical stimulation was applied as a train of three 0.22-msec pulses separated by a 200-msec interval and was repeated every 2 seconds.

Each experiment required the acquisition of seven spectra: The first was acquired during rest; the second and the third were acquired during electrical stimulation of the muscle to induce an isometric contraction; and the remaining four were acquired during recovery. Spectra were obtained by using a 3-cm-diameter surface coil placed in direct contact with the rat limb. The space localization obtained with this coil was probed by acquiring 31P three-dimensional spin-echo images of a phantom containing phosphate buffer. The sensitive volume of the coil, delineated by halving the signal intensity, extended approximately 15 mm from the coil surface, thus including the gastrocnemius muscle and lesion. The acquisition sequence consisted of a single squared 90° radio-frequency pulse followed by an acquisition period. Localization has been achieved with the surface coil, according to Gowdak et al (13). No relevant changes in surface coil loading, receiver gain, or signal-to-noise ratio were observed during spectroscopy.

The 90° pulse length was 50 µsec; other parameters were TR, 3 sec; bandwidth, 10 KHz; and acquisition size, 2,048 points. Each spectrum was the average of 64 scans corresponding to a 192-second acquisition time. To better evaluate the bioenergetic characteristics of the gastrocnemius muscle during the repair process, 31P in vivo localized spectroscopy was performed in an additional group of six rats that were not part of the original eight and were examined 4 days, 21 days, and 50 days after ischemic lesion induction. The methods used in this group were similar to those described earlier.

31P spectra were analyzed in terms of the ratio between the intensity of the PCr and Pi peaks, PCr/(PCr + Pi) (the integral under the peaks was considered), which can be considered markers of the bioenergetic reserve of the muscle. All spectroscopic procedures were evaluated by one of the authors (P.M.).

Histologic and Ultrastructural Examination
After in vivo evaluations were completed 4–6 months after femoral arterial removal, four of the original eight rats were killed and underwent perfusion with intraaortic injection of 2.5% glutaraldehyde in Sorensen buffer at different times 4–6 months after femoral arterial occlusion. This long range was chosen to evaluate the persistence of lesions in the muscle. Ultrastructural examination also was performed in another group of four rats treated with similar surgical and radiologic methods at the same times. In all eight rats, the ischemic muscles were removed and fixed in the 2.5% glutaraldehyde in Sorensen buffer for 2 hours, postfixed in 1% osmium tetroxide for 1 hour, dehydrated in graded ethanols, embedded in Epon-araldite, and cut with an ultramicrotome (Ultracut E; Reichert, Vienna, Austria) to obtain sections of 70-nm thickness. The semithin sections were stained with toluidine blue. The ultrathin sections were stained with lead citrate and uranyl acetate and observed by using an electron microscope (model EM10; Zeiss, Oberkochen, West Germany). Nonischemic gastrocnemius muscle was used as a control. All histologic procedures were evaluated by the same author (A.S.).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
MR Imaging
Twenty-four hours after surgery, an area of hyperintensity localized mainly in the posterior and lateral calf muscles was clearly visible (Fig 1). In two rats, the hyperintensity on T2-weighted images involved the whole leg. On images acquired 7 and 14 days after muscle ischemia induction, the signal intensity of the hyperintense area was progressively reduced, as compared with that on the images acquired at 24 hours. In a single rat, a core of hypointense tissue was visible. Three months after ischemia induction, the area was still detectable on T2-weighted images; however, its signal intensity was reduced and its shape was barely identifiable.



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  		Figure 1. Coronal T2-weighted multisection spin-echo MR images (2,000/60) acquired in the fifth rat in our series during the repair period in the ischemic area (arrow). A, 1 day, B, 7 days, and C, 14 days, and D, 3 months. The lesion was still detectable 3 months after ischemia induction.

 
Gadopentetate dimeglumine strongly enhanced the signal intensity of the affected area (Fig 2) on T1-weighted images. The contrast with the surrounding region increased, and shape evaluation was facilitated, as compared with that on the T2-weighted images. The utility of gadopentetate dimeglumine in detection was particularly clear in the animals that underwent MR imaging 2 weeks or more after ischemia induction. At these intervals, the T2-weighted images showed poor contrast in comparison with the gadolinium-enhanced images.



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  		Figure 2. Coronal pre- and postcontrast (*) T1-weighted spin-echo MR images (100/10) acquired 24 hours (A, A*), 7 days (B, B*), 14 days (C, C*), and 3 months (D, D*) after muscle ischemia induction. T1-weighted images were acquired on the same sections as the T2-weighted images in Figure 1 before and approximately 2 minutes after contrast material administration. Gadopentetate dimeglumine strongly enhances the signal intensity of the affected area (arrows).

 
The kinetics of contrast agent penetration in the legs 3 months after arterial occlusion is shown on a series of images in Figure 3. Signal-intensity enhancement starts to become relevant approximately 30 seconds after injection and reaches a plateau after 60 seconds. A more detailed analysis of the process was possible with quantitative evaluation of kinetic curves. Figure 4 shows signal-intensity enhancement from contrast medium at different times after arterial occlusion. At 1 day, 7 days, and 14 days, the enhancement was substantially higher than that in the contralateral leg (Fig 4). At all times studied, the curves obtained in the affected areas showed rapid enhancement, and a plateau was reached after approximately 60 seconds, confirming the data suggested at visual inspection. In the contralateral nonischemic leg, the trend was clearly different, showing slower enhancement.



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  		Figure 3. Coronal dynamic contrast-enhanced T1-weighted spin-echo MR images (100/10) of the ischemic leg and the normal leg 3 months after ischemia induction. The first image was obtained before contrast material injection; the last image, after contrast material injection. The other images were obtained pixel by pixel with the formula: {Delta}SI = SI(t) - SIpre. Signal intensity enhancement started to become relevant approximately 30 seconds after injection and reached a plateau after 60 seconds. Arrows = affected area.

 


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  		Figure 4. Graph shows time dependence of (SIpost - SIpre) in the ischemic leg and the normal leg at different times after ischemia induction (mean ± SEM). Data were obtained in the ischemic leg 1 day ({blacklozenge}), 7 days ({blacksquare}), and 14 days ({blacktriangleup}) after ischemia induction and in the contralateral nonischemic leg ({bullet}). {ddagger} = 1 day was significantly different from the control (unpaired t test; P < .05). * = 1, 7, and 14 days were significantly different from the control (unpaired t test, P < .05). The curves obtained in the affected areas showed rapid enhancement; this was followed by a plateau after approximately 60 seconds.

 
Three months after arterial occlusion (Fig 5), enhancement was still substantially higher in the affected area, as compared with that in the contralateral leg. Moreover, the enhancement values observed in the contralateral leg were higher than those observed in the same leg at earlier times (Fig 4). Figure 6 shows the wash-out data, which occurred on a slower time scale, as compared with the wash-in data.



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  		Figure 5. Graph shows time dependence of (SIpost - SIpre) in the ischemic leg ({blacklozenge}) and the normal leg ({blacksquare}) 3 months after ischemia induction (mean ± SEM). The time range was 0-128 seconds. {ddagger} = Three months was significantly different from the control (paired t test; P < .05). * = Three months was significantly different from the control (paired t test; P < .01). The enhancement was still substantially higher in the affected area, as compared with that in the contralateral leg.

 


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  		Figure 6. Graph shows time dependence of (SIpost - SIpre) in the ischemic leg ({blacklozenge}) and the normal leg ({blacksquare}) 3 months after ischemia induction (mean ± SEM). The time range was 2.8-71.8 minutes. * = Three months was significantly different from the control (unpaired t test; P < .05). # = Three months was significantly different from the control (unpaired t test; P < .01). The affected area showed enhancement substantially higher than that in the contralateral leg.

 
31P in Vivo Localized Spectroscopy
Figure 7 shows the time dependence of the ratio PCr/(PCr + Pi) for the ischemic muscle 3 months after ischemia induction and for the normal muscle. The ischemic calf at rest, the first point in Figure 7a, was not significantly different from the normal calf. However, when the leg was maintained at exercise induced with electrical stimulation, the ratio PCr/(PCr + Pi) decreased to a significantly more pronounced degree with respect to the normal hind limb. A significant difference in the PCr/(PCr + Pi) ratio between the control and treated rats was present after 10 minutes of stimulation(Fig 7a). Figure 7b shows that a low PCr/(PCr + Pi) ratio, 0.3–0.4:0.6 in normal calf, was present within the 1st days after ischemia and remained throughout the experimental period.



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  		Figure 7a. (a) Graph shows time dependence of the ratio (PCr)/(PCr + Pi) in ischemic legs ({circ}) and in normal legs ({square}) 3 months after ischemia induction. Values are expressed as mean ± SEM for five rats. In each curve, the first point was acquired at rest. The second and third points were acquired at electrical stimulation of the muscle to induce an isometric contraction. The remaining four points were acquired during recovery. The marked (*) ratio (PCr)/(PCr + Pi) at 3 months was significantly different from the control (unpaired t test; P < .01). (b) Graph shows the mean values of the ratio (PCr)/(PCr + Pi) for six rats at 4 days ({lozenge}, dotted line), 21 days ({square}, dashed line) and 50 days ({triangledown}, solid line) after ischemia induction. At the marked time (*), the values at 50 and 21 days were significantly different (paired t test; P < .05).

 


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  		Figure 7b. (a) Graph shows time dependence of the ratio (PCr)/(PCr + Pi) in ischemic legs ({circ}) and in normal legs ({square}) 3 months after ischemia induction. Values are expressed as mean ± SEM for five rats. In each curve, the first point was acquired at rest. The second and third points were acquired at electrical stimulation of the muscle to induce an isometric contraction. The remaining four points were acquired during recovery. The marked (*) ratio (PCr)/(PCr + Pi) at 3 months was significantly different from the control (unpaired t test; P < .01). (b) Graph shows the mean values of the ratio (PCr)/(PCr + Pi) for six rats at 4 days ({lozenge}, dotted line), 21 days ({square}, dashed line) and 50 days ({triangledown}, solid line) after ischemia induction. At the marked time (*), the values at 50 and 21 days were significantly different (paired t test; P < .05).

 
Histologic and Ultrastructural Examination
In all rats, histologic analysis of the gastrocnemius muscle after femoral arterial removal showed moderate intercellular fibrosis. At ultrastructural examination, in all rats there was a loss of myofilaments with enlargement of the intermyofibrillary spaces containing clusters of hyperplastic mitochondria; this loss was most prominent in the subsarcolemmal areas. Many myofibers displayed irregular expansions of the sarcolemma (Fig 8). Thickening of the basal lamina was observed in numerous endomysial capillaries.



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  		Figure 8. Photomicrograph shows the ultrastructure of the gastrocnemius muscle after femoral arterial removal. Myofibers display irregular expansions of the sarcolemma (large asterisks). Basal laminar thickening is observed in the endomysial capillaries (small asterisk). Collagen fibers (F) are abundant in the interstitial space. (Lead citrate and uranyl acetate stain; original magnification, x12,500.)

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
 
Arterial occlusion and the resultant acute ischemia represent a major cause of morbidity and mortality (14). Promising approaches have been developed to therapeutically induce angiogenesis and in particular to treat myocardial and limb ischemia due to atherosclerosis (15–19). New methods are necessary to evaluate the effectiveness of these treatments and follow the time course of angiogenetic processes, also considering that, to our knowledge, a reference standard to document angiogenesis in humans is not currently available.

Dynamic contrast-enhanced MR imaging has well-recognized value in investigations of the musculoskeletal system, in which it has been applied to evaluate inflammatory and tumoral lesions (20–22). The tissue accumulation of contrast media and, ultimately, the induced changes in tissue signal intensity, depend on the complex interplay of multiple variables, including perfusion, transendothelial diffusion, and blood clearance; however, it has been demonstrated that this technique can provide diagnostically useful information regarding the microvascular bed (23–25).

In animal models, femoral arterial ablation causes substantial reduction in resting and stimulated blood flow (26). Similar findings have been described in cardiac muscle, in which severe blood flow reduction results in reduced contrast agent flow (27).

By performing dynamic contrast-enhanced MR imaging, we have found new information about the consequences of arterial insufficiency of muscle: Delayed muscle injuries that may be functionally relevant and detected in vivo can result.

A further finding was hyperintense lesion development in specific areas of the ischemic leg. Localization of the lesions mainly in the gastrocnemius and tibialis anterior muscles was probably due to the richness of fast-twitch type II and III fibers in these muscles, which is in accordance with previous findings (7,8). Our data demonstrate that the muscle lesion remains visible for at least 2 weeks on T2-weighted images. This finding is in agreement with that of Thompson et al (28), who described how, in cardiac muscle, signal intensity changes in the infarcted region continued over 3 months.

Muscle T2 changes are nonspecific and may be due to different factors (ie, edema, changes in fiber size, or vascular modifications). Hyperintensity on T2-weighted images can be induced in skeletal muscle with infarct (29) or simply with exercise (30–34). Ischemic exercise probably modifies T2, inducing a water increase in the muscle (31). Signal intensity increases caused by exercise are brief and limited to the muscles that are active during exercise (35).

However, eccentric muscular actions may cause T2 increases that are indicative of muscle injury, as demonstrated by Shellock et al (36). In their study, MR imaging depicted subclinical abnormalities that lasted as long as 75 days after the disappearance of symptoms.

The major finding of our work was that 3 months after arterial removal, contrast-enhanced images showed microvessels that were highly permeable to the tracers. The data obtained with contrast-enhanced MR imaging were confirmed with histologic and ultrastructural data, which revealed the persistence of muscle damage and signs of chronic microangiopathy. In accordance with these findings, spectroscopic data revealed that 3 months after arterial removal, the bioenergetic reserve of the gastrocnemius muscle was reduced, suggesting that the contrast-enhanced MR imaging–visible lesion was functionally relevant.

Data from several reports suggest that the aforementioned event may help to increase the understanding of human muscle reactivity. It is interesting to note the similarity of our findings to data describing the dramatic recovery delay in human muscle after injury. In sports-related muscle injury, an area of hyperintensity on T2-weighted images may last longer than symptoms by up to 3 weeks; this indicates that MR imaging is sensitive to tissue alteration (ie, edema, cellular infiltrates, phagocytosis, degeneration, and regeneration) that is not clinically apparent (37). In addition, in healthy volunteers, delayed reversible MR imaging signal intensity increase is visible on T2-weighted images from the 6th to 9th days after eccentric exercise (38). Results of the current study demonstrate that delayed muscle changes can be induced in a reproducible animal model and can be localized in vivo with contrast-enhanced MR imaging or MR spectroscopy.

We have shown that, at long-term follow-up after arterial occlusion, the restitutio ad integrum is not complete, and microcirculatory and functional impairments are still present, as demonstrated with gadopentetate dimeglumine kinetics and in vivo spectroscopy. The increased permeability that we have demonstrated could be related to the expression of chemical mediators such as vascular endothelial growth factor, which is potentiated in response to hypoxia and increases blood vessel permeability (39). Authors of one study (40) described professional athletes’ muscle injuries that were occult at conventional MR imaging but depicted as areas of localized enhancement at contrast-enhanced imaging. Results of the current study experimentally demonstrate that similar findings could be a long-term consequence of arterial insufficiency, such as was recently demonstrated in another organ by using methods of regional blood volume mapping (41).

Practical application: The results of our experimental model seem relevant for clinical radiologists, since the results suggest that similar findings may also be present in patients with chronic arterial insufficiency of the lower limbs, in which muscle damage has been previously demonstrated (42).


   ACKNOWLEDGMENTS
 
The authors are grateful to Roberta Sala, DVM, and Barbara Rogers for their technical assistance.


   FOOTNOTES
 
Abbreviations: {Delta}SI = signal intensity change, SEM = standard error of the mean

Author contributions: Guarantor of integrity of entire study, F.O.; study concepts, F.O.; study design, A.S.; literature research, A.S.; experimental studies, R.M.A.; data acquisition, E.N.; data analysis/interpretation, E.L.; statistical analysis, E.L.; manuscript preparation, R.M.A.; manuscript definition of intellectual content, A.S.; manuscript editing, P.F.; manuscript revision/review, P.M.; manuscript final version approval, F.O.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 

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