Foot Small Muscle Atrophy Is Present Before the Detection of Clinical Neuropathy

The study cohort included 12 neuropathic diabetic, 9 nonneuropathic diabetic, and 12 healthy nondiabetic subjects. A diagnosis of type 1 or type 2 diabetes was established according to the recommendations of the American Diabetes Association (12). Subjects with open lesions on their feet, peripheral vascular disease (symptoms of claudication and absence of peripheral pulses, ankle brachial index [ABI] < 0.7]), any other serious chronic diseases requiring active treatment, or a contraindication to MRI examination were excluded from the study. The study protocol was approved by the Institutional Review Board of Beth Israel Deaconess Medical Center and all participants gave written informed consent.

Patients’ medical history, including history of alcohol consumption, type and duration of diabetes, and the presence of other micro- or macrovascular complications, was obtained. Patient characteristics, including age, sex, weight, height, and BMI were also evaluated.

The presence of diabetic peripheral neuropathy was defined according to the principles of the San Antonio Consensus criteria (13). For this, the Neuropathy Symptom Score and the Neuropathy Disability Score (NDS) were evaluated as previously described (14). The vibration perception threshold (VPT) was evaluated using a biothesiometer (Biomedical Instruments, Newbury, OH). The cutaneous pressure perception threshold was evaluated using a set of eight Semmes-Weinstein monofilaments that apply a 1- to 100-g pressure. Details of these techniques have been described elsewhere (14). Neuropathy was diagnosed when two or more measurements were abnormal.

³¹P RARE and ¹H MRI were performed on all study subjects using a whole-body 3.0T MR Scanner (General Electric Medical Systems, Milwaukee, WI). A “birdcage” radio frequency coil designed specifically to acquire both ³¹P and ¹H MRI data (15,16) was constructed at our facility. Thus ³¹P and high-resolution ¹H registration images could be acquired without moving the subject’s foot, which resulted in improved registration of the ³¹P and ¹H images. A reference standard containing an aqueous solution of 220 mmol/l inorganic phosphate was rigidly attached to the radio frequency coil. The standard was present during all MRI procedures and served two purposes: 1) the measurement of the ³¹P concentration and 2) as a fiduciary marker for the co-registration of ³¹P and ¹H images. All of the acquisitions were performed in the axial plane. The subjects were placed on the scanning bed in the supine position with their knees elevated ∼30 cm using a foam cushion. One foot was placed into the birdcage radio frequency coil so that the metatarsal heads were at the center of the coil.

To acquire detailed images of the foot anatomy and delineate regions of edema, fatty infiltration, and scar tissue, T2-weighted ¹H spin-echo imaging was first performed on all subjects (repetition time [TR] = 1.5 s, echo time [TE] = 30 ms, field of view [FOV] = 15 cm, matrix = 256 × 256, slice thickness = 2.5 mm, scan time = 6 min 24 s). Images oriented in the axial plane were acquired at each of 10 contiguous locations. The 10 slice locations of the ¹H acquisition were prescribed so that the maximum extent of coverage in the axial direction was identical to that of the single-slice ³¹P RARE image acquisition, described below.

Axial ³¹P image data were acquired using the RARE pulse sequence (two echo trains, 32 echoes per echo train, receiver bandwidth = 4 kHz, TR = 12 s, FOV = 30 cm, matrix = 64 × 64, slice thickness = 25 mm, 10 signal averages, scan time = 4 min, voxel volume = 0.55 cm³). In the RARE pulse sequence, the time spacing between the echoes can be adjusted to facilitate constructive or destructive interference of the three ATP resonances (10,17). The echo spacing and effective TE were selected to maximize the destructive interference of the ATP resonances. This substantially suppressed the ATP contribution to the ³¹P images. The resulting image intensities were proportional to the combined concentrations of the inorganic phosphate and phosphocreatine cellular metabolites.

Image data were analyzed using Interactive Data Language software (Research Systems, Boulder, CO). The background noise of each ³¹P image was measured and a threshold technique was used to eliminate it, leaving only the ³¹P signal from muscle and the reference standard. An outline of the muscle tissue and the reference standard was created using a contour mapping function set to a single level that was equal to the noise threshold value. The ³¹P muscle outline was registered to one of the central ¹H T2-weighted images, using the reference standard as a fiduciary marker. The average ³¹P concentration values were measured and calculated using the ³¹P reference standard and a previously described method (10).

The in-plane area across the entire axial view of the foot was calculated for each subject. The 10 ¹H T2-weighted images were added together to create a single image that represented the range covered by the ³¹P imaging. The in-plane (cross-sectional) area of each pixel was calculated from knowledge of the image FOV and the number of pixels in the image (matrix size). The pixels that represented a signal from the foot tissues were then counted and the resulting number was multiplied by the pixel size. The cross-sectional area of the muscle tissue was calculated by counting the number of pixels in the ³¹P images in the same manner.

The Minitab statistical package (Minitab, State College, PA) for personal computers was used for statistical analysis. The ANOVA test, followed by the Fisher’s test, was used to identify differences among the various groups. Correlation between variables was tested using Pearson correlation analysis. All of the tests were two-tailed with α = 0.05. Data are presented as means ± SD.

The clinical characteristics of the study groups are shown in Table 1. A history of foot ulceration was present in five (50%) of the neuropathic diabetic patients, and in none of the participants in the other two groups. No ulcers were present in any participant at the time of the study.

The ³¹P and ¹H MRI images obtained using the 3.0T whole-body MR scanner from a healthy, nondiabetic subject and a nonneuropathic diabetic patient are shown in Fig. 1. As described in the figure legend, despite the fact that no clinical neuropathy was present, changes can be clearly seen in both the ³¹P and the ¹H MRI images that were acquired from the diabetic patient, indicating that these changes preceded the development of clinical neuropathy.

The results of the muscle area−to−total area ratio calculation differed among all three groups: 0.55 ± 0.04 vs. 0.44 ± 0.05 vs. 0.06 ± 0.06 for the control, nonneuropathic, and neuropathic groups, respectively (P < 0.0001) (Fig. 2A). Average ³¹P concentrations also differed among the three groups: 27.7 ± 3.8 vs. 21.7 ± 4.8 vs. 7.9 ± 8.8 mmol/l for the control, nonneuropathic, and neuropathic groups (P < 0.0001) (Fig. 2A). No differences were observed between the measurements in male and female subjects (data not shown).

When all subjects were considered as one group, the muscle area−to−total area ratios strongly correlated with clinical measurements of the severity of neuropathy, including the NDS (r = −0.83, P < 0.0001) (Fig. 3), VPT (r = −0.79, P < 0.0001), and SWM (r = −0.87, P < 0.0001) scores. It also correlated with diabetes duration (r = −0.56, P < 0.05) and ABI (r = 0.75, P < 0.0001), whereas no correlations were observed with age or BMI. Multivariate regression analysis showed that only NDS was an independent factor.

The ³¹P concentration also correlated with the NDS (r = −0.73, P < 0.001), VPT (r = −0.69, P < 0.05), SWM (r = −0.75, P < 0.01), and ABI (r = 0.73, P < 0.001) scores. Multivariate regression analysis showed that only SWM was an independent factor.

In the present study, ³¹P RARE and ¹H T2-weighted spin-echo MRI were performed on the feet of neuropathic and nonneuropathic patients and normal control subjects. A reduced ratio of viable muscle tissue to all other types of tissue was observed in the feet of both nonneuropathic and neuropathic diabetic patients as compared with normal control subjects. Similarly, a reduction in the average ³¹P concentration in both the nonneuropathic and neuropathic diabetic patients was observed when compared with the healthy, nondiabetic control subjects. Furthermore, the observed changes strongly correlated with clinical measurements of peripheral nerve function, indicating an association between muscle atrophy and diabetic neuropathy. Our results indicate that muscle changes in the diabetic foot begin during the subclinical stage of diabetic neuropathy and before the development of clinical signs of the disease.

Small muscle atrophy is a well-known process in the diabetic foot and is related to the development of diabetic motor neuropathy. However, given the paucity of techniques that can quantify muscle atrophy, these changes have not been adequately studied. Instead, motor neuropathy has been evaluated mainly by measuring the motor peroneal conduction velocity and sensory neuropathy has been evaluated through quantitative sensory testing (18). Because small muscle atrophy is the main process that leads to anatomical foot changes, such as clawing of the toes and prominent metatarsal heads, that are directly related to the development of foot ulceration, the direct evaluation of muscle changes rather than nerve function may prove more helpful in clinical research studies. More specifically, evaluating changes in the ratio of viable muscle tissue to all other types of foot tissues using ¹H MRI and images based on the ³¹P cellular metabolite activity of muscle potentially allows clinicians to detect anatomic and metabolic changes and relate these changes to the development of foot ulceration and wound healing potential.

All previously published studies of ¹H MRI have agreed about the existence of small muscle atrophy in neuropathic diabetic patients (6,7,19,20). However, there have been no reports of atrophy in nonneuropathic patients. We believe that the difference between our results and those of previous studies is related to the specific techniques used. Previous studies have used T1-weighted (20) or T2-weighted imaging methods (6) and T2 mapping techniques (6) to evaluate the cross-sectional area of muscle tissue. However, as muscle atrophy occurs, the T1 and T2 relaxation rates in muscle-containing regions increase, causing the various tissue types to become nearly isointense with each other. This obscures the tissue boundaries, thus making it difficult or impossible to segment or determine the threshold of muscle tissue from adjacent tissues, as acknowledged by the authors of one study (6). In contrast to segmentation and thresholding methods, the distinction between muscle tissue and other tissues in the foot using ³¹P RARE MRI is straight forward because muscle is the only tissue of the foot that has ³¹P concentrations that are detectable by MRI. Therefore, the ³¹P RARE MRI method offers a more accurate evaluation of viable muscle tissue; in our study, this method allowed us to identify relatively small changes in the nonneuropathic patients in comparison with the status of healthy nondiabetic subjects.

In a recently published study, Andersen et al. (20) calculated the total volume of foot muscles from ¹H MRI by segmenting the muscle tissue using an image-intensity threshold method. Although the total muscle volume was significantly reduced in neuropathic diabetic patients compared with in control subjects, no difference was found between the nonneuropathic diabetic patients and control subjects. Because the total muscle volume is related to the foot size, the results of the Andersen et al. study (20) may have been influenced by differences in foot size among the various groups. In the present study, we compared the ratio of the cross-sectional muscle area to the total cross-sectional area at the same location in the foot; this ratio gave us a better indication of the amount of muscle loss by eliminating differences in foot size among population groups. In addition, we used ³¹P RARE MRI, which provided a direct measurement of the amount of muscle tissue. This different methodology resulted in a sensitive technique that enabled us to more accurately detect differences in muscle atrophy among various groups. We believe that this was the main reason for the observed differences between our results and the results of the previous study.

MRS was used in one previous study that examined changes between diabetic patients and healthy control subjects (5). The results of that study demonstrated changes in the ³¹P metabolites and an increased fat-to-water ratio in diabetic patients with superficial ulcers compared with diabetic patients without ulcers and healthy subjects, whereas no differences were observed between diabetic patients without foot ulceration and healthy control subjects. As with the previous studies, we believe that the differences between our findings and the results of the MRS study are related to the techniques used. The ³¹P MRS method used in the previous study (5) resulted in ³¹P data sets with a spatial resolution of 11.25 cm³ (requiring a scan time of >50 min), whereas in the present study the ³¹P images had a spatial resolution of 0.55 cm³ (requiring a scan time of 4 min). The improved spatial resolution of the ³¹P RARE method resulted in better identification of focal regions of reduced ³¹P concentration when compared with the previous MRS methods. In addition, the higher spatial resolution of the ³¹P RARE MRI method allowed a more accurate delineation of the boundaries and contours of the muscle tissue, thus providing better registration with ¹H anatomical images and a more accurate measurement of the muscle cross-sectional area.

The concentration of various ³¹P metabolites is also influenced by the presence of ischemia. Thus, when muscle is ischemic, the concentration of phosphocreatine decreases from its normal value as the concentration of inorganic phosphate increases (3,4). Preliminary ongoing studies at our facility indicate that the grading of the degree of ischemia is feasible and can be achieved using ³¹P RARE MRI to create separate phosphocreatine and inorganic phosphate images in the diabetic foot. However, in the present study we used ³¹P RARE MRI to acquire data that reflected the combined concentrations of phosphocreatine and inorganic phosphate, as we aimed to identify metabolically active muscle tissue and not to grade the degree of muscle ischemia.

In the present study, in agreement with previous studies, we observed strong correlations between quantitative clinical measurements of the severity of neuropathy and the muscle area−to−total area ratio or ³¹P measurements (5,20). In addition, strong correlations were observed between the muscle area−to−total area ratio or ³¹P concentration measurements and the duration of diabetes and the ABI. However, as the multiple regression analysis excluded these parameters as independent factors, it can be concluded that the severity of neuropathy was the only factor that influenced these measurements. Finally, there were no differences between male and female subjects in the muscle area−to−total area ratio or ³¹P measurements, and no correlations were noticed between BMI and the above measurements. These findings further indicate that the size of the foot did not influence the observed results.

A word of caution should be added at this point. The term “nonneuropathic diabetic patient” indicates a patient in whom there is no evidence of clinical neuropathy as determined using standard, widely used clinical techniques. However, the existence of subclinical neuropathy in these patients cannot be excluded. Because previous studies using sophisticated experimental techniques have shown subclinical nerve function abnormalities in most diabetic patients, it seems reasonable to suspect that such changes were also present in the nonneuropathic patients who participated in this study. Despite this, we believe that the results of our study are still interesting and the conclusions are still valid, as they indicate that changes in foot muscles occur before changes in nerve function can be detected using standard techniques available to the practicing clinician.

In summary, we have shown that small muscle atrophy is present in diabetes before clinical peripheral neuropathy can be detected using standard techniques available in clinical practice. The ³¹P RARE MRI method can evaluate the severity of muscle atrophy, even in the early stages when clinically detectable neuropathy is absent. Furthermore, the muscle tissue in patients can be noninvasively evaluated within a short time using this MRI method compared with existing localized MRS methods. The ³¹P RARE MRI method may prove to be a useful diagnostic tool that can facilitate the implementation of clinical trials in the early stages of diabetic foot problems as well as monitor the response to treatment.

The study was supported in part by research grants from the Juvenile Diabetes Research Foundation International (JDRF 5-2002-329) and the National Institutes of Health (R01-HL0-75678-01 to A.V., R21-DK-58651 to R.L.G., and RR-01032 to the Beth Israel Deaconess Medical Center General Clinical Research Center).