SHORT COMMUNICATION 
Annals of Nuclear Medicine Vol. 15, No.4, 397-401,2001
99mTc-sestamibi muscle scintigraphy to assess the response to neuromuscular electrical stimulation of normal quadriceps femoris muscle 
YeSim PEKINDIL,* Ali SARIKAYA,** Murat BIRTANE,* Gokhan PEKINDiL*** and Ahmet SALAN** 
*Department of Physical Medicine and Rehabilitation, Trakya University Hospital, Edirne, Turkey **Depart,nent of Nuclear Medicine, Trakya University Hospital, Edirne, Turkey ***Department of Radiology, Trakya University Hospital, Edirne, Turkey 
Objectives: Neuromuscular electrical stimulation (NMES) is widely used for improving muscle strength by simultaneous contraction in the prevention of muscle atrophy. Although there exist many clinical methods for evaluating the therapeutic response of muscles 99mTc-sestamibi which is a skeletal muscle perfusion and metabolism agent has not previously been used for this purpose. The aim of our work was to ascertain whether 99mTc-sestamibi muscle scintigraphy is useful in the monitoring of therapeutic response to NMES in healthy women. Methods: The study included 16 women aged betwecn 21 and 45, with a mean age of 32.7 +- 6.4. Both quadriceps femoris muscles (QFM) of each patient were studied. After randomization to remove the effect of the dominant side, one QFM of each patient was subjected to the NMES procedure for a period of 20 days. NMES was performed with an alternating biphasic rectangular current, from a computed electrical stimulator daily for 23 minutes. After measuremcnt of skinfold thickness over the thigh, pre- and post-NMES girth measurements were assessed in centimeters. Sixty minutes after injections of 555 MBq 99mTc-sestamibi, static images of the thigh were obtained for 5 minutes. The thigh-to-knee uptake ratio was calculated by semiquantitative analysis and normalized to body surface area (NUR = normalized uptake ratio). Results: The difference between the pre and post NMES NUR values was significant ( 1.76 +- 0.31 versus 2.25 +- 0.38, p = 0.0000). The percentage (%) increase in NUR values also well correlated with the % increase in thigh girth measurements (r = 0.89, p = 0.0000). Conclusion: These results indicated that 99mTc-sestamibi muscle scintigraphy as a new tool may be useful in evaluating therapeutic response to NMES. 
Key words: neuromuscular electrical stimulation 99mTc-sestamibi scintigraphy, muscle 
INTRODUCTION 
NEVROMUSCULAR ELECTRIC STIMULATION (NMES) has been widely used for many years in the rehabilitation of muscles.1 This training technique can increase the functional activity of muscles by improving their physiological, morphological, biochemical and motor performance properties 2 Chronic electrical stimulation has been reported to improve muscle strength and endurance,3-5 prevent denerva-
Received December 18, 2000, revision accepted May 15, 2001 . For reprint contact: Ali Sankaya. M.D., Trakya Universitesi Tip Fakultesi. Nnkleer Tip ABD 22030, Edirne, TORKIYE. E-mail : askaya@trakya.edu.tr 
tion atrophy,6 cause muscle hypertrophy,2 and increase capillary density in animal muscles,7 increase muscle cell mitochondrial fraction 8,9 succinic dehydrogenase10 and protein synthesis.11 The strengthening effects of NMES can be evaluated by many different evaluation methods. Isokinetic dynamometry (Cybex) can be used for the evaluation of the strength of muscles, EMG for the evaluation of motor unit potentials and basal metabolism measurements for the evaluation of the strength exerted. Manual muscle strength testing and some clinical scoring scales can also be used. But none of these techniques can assess the muscle from a perfusion and/or metabolism point of view. 99mTc-sestamibi is a lipophilic cation primarily used as a myocardial perfusion imaging agent in the assessment of coronary artery disease and it has also been employed in the evaluation of skeletal muscle perfusion and metabolism in patients with peripheral artery and other muscle diseases. 12-21 The aim of this study was to ascertain whether 99mTc-sestamibi muscle scintigraphy is useful in the monitoring of therapeutic response of muscles to NMES therapy in healthy women. 
MATERIALS AND METHODS 
Patients This study was approved by the local ethical committee and written informed consent was obtained from all volunteers. Sixteen healthy women aged 21-45 years (32.7 +- 6.4) were included in the study. Patients with systemic diseases, such as generalized muscle disease, diabetes mellitus, lupus erythematosus, polyarteritis nodosa, systemic sclerosis, amyloidosis, carcinomatosis, renal failure and cardlovascular disease were excluded from the study. Also patients with painful knee joints and those performing regular exercises were not included in the study. We selected only female patients to study for two reasons: to eliminate the effect of sex differences and because of the lack of individual fibril type variations in female patients.22,23 
NMES application
After randomization to remove the effect of the dominant side, the selected QFM of the patients (right or left) were subjected to the NMES procedure with a COMPEX PM 
device, a computed electrostimulator. The time and the modulation of stimulation procedures were directed by stimulation programs preserved in the hard disc of the device. Electrical stimulation was applied through three channels by a monopolar, large negative electrode and four small, positive electrodes on the motor stimulation points of the quadriceps muscles. These electrodes were applied with gels to increase the impulse transmission. They were fixed with velcro tapes to the thigh. NMES was applied to the patient in the sitting position near the examination table and the thigh of the patient was fixed manually to the table by the physician in order to prevent knee movement during strong contractions. Motor unit stimulation and strong, tetanic isometric muscle contractions were formed by bipolar and symmetrical square waves. Programs with frequencies of 75 and 85 hertz were selected to obtain maximal contractions in fast muscle fibrils. The intensity was increased by verbal motivation of the patient, after every three or four contractions according to the compliance and tolerance of the patient. Impulses of 20-25 amperes were used for the beginning and these values were increased to 50-55 amperes until the end of the training set. The programs specific for strengthening of large muscle groups were selected in the first training week. Programs with higher impulse frequencies and intensities were applied in the second and third weeks. Each training session consisted of three parts. The first part was for warming and lasted for four minutes. The second part was for training and lasted for 15 minutes. The third part was a four minute period for relaxation. 
99mTc-sestamibi imaging
All study patients were fasted and were maintained at rest for at least 30 min before the intravenous administration of 99mTc-sestamibi [555 MBq]. Scintigraphic images were obtained approximately 60 min later. Twenty days after NMES, second scintigraphic images were performed. Studies were performed with a large field of view gamma camera (PHILIPS diagnost tomo) equipped with a low-energy, parallel-hole, high-resolution collimator, and a 20% energy window centered at 140 keV. Images were acquired in a 128 x 128 pixel matrix until 400,000 counts/view were obtained or for 5 min. The semiquantitative analysis was performed in a blinded fashion 1 week later. Symmetrical rectangular regions of interest (ROIs) for both thighs were drawn around muscular [thigh (T)] and nonmuscular [knee (K)] areas (Fig. 1a). The uptake ratios (T/K) were computed by dividing the mean counts in the muscular ROI by mean counts in the nonmuscular ROI.20,24 The contralateral thigh on which NMES was not performed was used as a control. Because these ratios could be affected by increases in muscle mass during the NMES therapy period, the normalized uptake ratio (NUR) was calculated by dividing these ratios by the patient's body surface area.25 Intraobserver variations were assessed by reanalyzing all data 1 week later. 
Thigh girth measurements
Thigh girths were measured in centimeters, around the thigh region localized 10 centimeters above the apical tip point of the patella. Then the thigh skinfold measurement (mm) was performed and used for determining the fatty free girth of the thigh (fatty free thigh girth (cm) = thigh girth - (pi x thigh skinfold value/10). 
Statistical analysis
Dependent Student's t-test was used to analyze differences between pre- and post-NMES thigh NURs. Comparisons between NMES performed and contralateral thighs were made using independent Student's t-test. Data are the expressed as mean +- standard deviation. Correlations between parameters were calculated by means of Pearson's correlation coefficient. A p value below 0.05 was considered significant. 
RESULTS 
The pre- and post-NMES theraphy NURs, girth, skinfold and % uptake, % girth increases are shown in Table 1. No significant difference was seen between the two pre-NMS thigh NURS (1.76 +- 0.31 vs. 1.73 +- 0.27, p = 0.58). Statistical analysis of the pre- and post-NMES NURS showed a significant increase with respect to the NMES performed thigh (1.76 +- 0.31 vs. 2.25 +- 0.38, p = 0.0000) (Fig. 1a, b). A significant difference was also seen between the post-NMES and contralateral thigh NURS (2.25 +- 0.38 vs. 1.74 +- 0.29, p = 0.0000). A good positive correlation was found between % girth increase and % uptake increase (r = 0.89, p = 0.0000). The mean of post-NMES girth values was also higher than the pre-NMES value, even though statistical significance was not reached (37.41 +- 9.27 vs. 39.88 +- 5.64, p = 0.21). There was no difference between pre- and post-NMES thigh skinfold values (24.75 +- 14.64 vs. 24.56 +- 15.03, p = 0.53). 
DISCUSSION 
Neuromuscular electric stimulation has been used for many years in the rehabilitation of muscles.1 Many reports have supported the physiological, morphological, biochemical and muscle force improving effect of NMES on muscles.2 This method is also well known for its denervation atrophy prevention effect as well as its muscle strength and endurance improvement effect.3-6 There are many studies in the literature which demonstrate the underlying mechanisms responsible for these effects. It has been suggested that capillary blood flow and oxygen delivery to tissues increase during contractions with the application of NMES.26 Blood flow was observed to increase by 20%, according to the results of a study in which the flow in gastrochinemius muscle during NMES 27 application was measured by Doppler ultrasonography. And, intermittent electrical stimulation with high frequency waves has been shown to cause intensitive capillary growth prominently in the glycolytic fibrils of muscle7 beginning from the seventh day of the intervention. Twenty-one days of NMES application to triceps surae muscle resulted in increases in capillary number and density, as well as the capillary/fibril ratio in another previous study.28 The increase in blood flow after NMES seems to be related with this capillary growth in muscles. Other studies also confirm that the onset of hemodynamic and oxidative-glycolytic enzyme profile changes with NMES usually occur between the 4th and 7th days of the application 7 At the end of 2.5 weeks of NMES application to rabbit tibialis anterior muscle, oxidative enzymes such as citrate hexokinase and 3-oxoacid coatransferase were found to increase between two and ten folds. It was shown in the same study that the NMES could alter the mitochondrial content of a muscle cell.29 The reports of another study suggesting increases in nucleus size and number, nuclear DNA content and fibril size as well as increases in the mitochodrial fraction in type 2 fibrils, after 19-21 days of NMES application to the gastrochinemius muscle in healthy population confirmed the results of previous studies 8,9 Moreover NMES has been shown to increase succinic dehydrogenase activities and muscle protein synthesis.11,30 We applied 15 sessions of NMES in our study, and that seemed to be sufficient to bring out these changes. Recently, 99mTc-sestamibi has been found to be an appropriate radiopharmaceutical for noninvasive imaging of the perfusion of the lower extremities. Cellular uptake and trapping of 99mTc-sestamibi are related not only to regional blood flow but also to mitochondrial metabolic conditions and viability.31 The tracer is driven across both plasma and mitochondrial membranes in response to progressively larger negative transmembrane potentials, and trapped within the mitochondrial layer.32 According to our previously studies 99mTc-sestamibi scintigraphy can assess the perfusion and viability of muscle tissue, and the changes in skeletal muscle me-tabollsm induced by therapy can also be tracked non-invasively.19-21 Using treadmill stress testing with an injection of 99mTc-sestamibi at peak exercise in a normal person, it was shown that the muscular 99mTc-sestamibi uptake in extremity muscle increased significantly from rest to exercise.12,13 Therefore, exercise 99mTc-sestamibi leg scintigraphy has been found to be useful for assessment of ischemic extremities 12-15,18 In our study, a significant difference was seen between pre- and post-NMES uptake values, and also the % uptake increase correlated closely with the % girth increase. Muscle cell hypertrophy and consequent extremity diameter increases have been re-ported after NMES application 2 Treadmill exercises acutely increase the blood flow in muscle tissue by leading to vasodilation, and so 99mTc-sestamibi uptake increases, but in our study NMES therapy increased 99mTc-sestamibi uptake in a long time period of three weeks, by increasing the metabolism and consequently the mass of muscle tissue. Since NMES helps strengthen or maintain mucle mass, maintain or increase the range of motion, facilitate voluntary motor control and reduce the effects of spasticity,33 it can be used in the period of rehabilitation of traumas, such as sport injuries, surgical operations, osteoarthritis of joints, structural abnormalities such as scoliosis, paresis and paralysis observed in upper and lower motor neuron lesions and rheumatological diseases such as rheumatoid arthritis and ankylosing spondylitis. Thus, the clinical significance of our findings must nevertheless be confirmed in further studies to assess the influence of NMES treatment. 
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