US and NCS measurements of controls and patients with type 2 DM (unpublished data).
The World Health Organization estimates that more than 220 million people worldwide have diabetes mellitus (DM). This figure is estimated to more than double by 2030 (Wild et al., 2004). In Japan, the number of diabetic patients has increased to 8 million, and it is assumed that 35% to 45% of diabetic patients have diabetic symmetric polyneuropathy (DPN). Advanced DPN causes serious complications, such as diabetic foot ulcers, gangrene, and Charcot joint, all of which worsen the quality of life of diabetic patients (Ogawa et al., 2006). Therefore, early detection of nerve dysfunction is important to provide appropriate care for patients with DPN (Chudzik et al., 2007). The diagnosis of diabetic neuropathy is based primarily on characteristic symptoms and is confirmed with nerve conduction studies (NCS), which are time-consuming, slightly invasive, and occasionally not well tolerated for repeated evaluations (Colak et al., 2007). In contrast, ultrasonographic examinations can be performed to assess peripheral nerves with less discomfort and have already been used for the evaluation of several disorders of the peripheral nervous system such as carpal tunnel syndrome (Wiesler et al., 2006a; Abe et al., 2004; Jayaraman et al., 2004; Duncan et al., 1999; Lee et al., 1999), cubital tunnel syndrome (Okamoto et al., 2000; Wiesler et al., 2006b), and traumatic nerve lesions (Cartwright., 2007; Peer et al., 2001). High-resolution diagnostic ultrasonography (US) has improved greatly, allowing for evaluation of minute peripheral nerves (Fornage., 1993; Solbiati et al., 1985). We previously showed that the cross-sectional area (CSA) of the median nerve in the carpal tunnel of patients with DPN is greater than that of controls and correlates with NCS (Watanabe et al., 2009). Furthermore, it appears that the percentage of the hypoechoic area of the peripheral nerves was significantly greater in patients with lower motor nerve conduction velocity (MCV) and DM than in controls or patients with higher MCV and DM (Watanabe et al., 2010).
The purpose of this chapter is to review the current knowledge regarding the overview and diagnosis of the most common forms of neuropathy in type 2 DM. Furthermore, our current sonographic technique and preliminary studies are presented for some cases. In this chapter, we focus mainly on a simple and noninvasive approach to the evaluation of peripheral nerves in patients with type 2 DM. Although the exact mechanisms contributing to our study have not been clearly identified and are not the main focus of this chapter, they will be discussed in brief.
2. Clinical aspects of diabetic neuropathy
Diabetic neuropathy is a neuropathic disorder that is associated with DM. This condition is thought to result from both diabetic microvascular injury involving small blood vessels that supply nerves and macrovascular conditions that can culminate in DM. More than 80% of patients with clinical diabetic neuropathy have a distal, symmetrical form of the disorder (Said., 2007). In general, the symptoms included numbness, burning feet, pins-and-needles sensations, and lightning pains. These symptoms start in the feet go on to affect more proximal parts of the lower limbs, and eventually affect the distal parts of the upper limbs.
The unified clinical criteria and classification for DPN do not represent the international standard because the causes of peripheral nerve disorders associated with DM are complex and probably involve a variety of causative mechanisms. In this chapter, DPN is classified into 3 groups (hyperglycemic neuropathy, symmetric polyneuropathy, and focal and multifocal neuropathy) according to the classification of the Thomas et al (1997). This classfication is the easiest. The most common of these groups seen in the clinical setting is symmetric distal polyneuropathy.
3. Pathology of diabetic neuropathy
Abnormalities reported in diabetic neuropathy include axonal degeneration in nerve fibers, primary demyelination resulting from Schwann cell dysfunction, secondary segmental demyelination related to impairment of axonal control of myelination, remyelination, proliferation of Schwann cells, atrophy of denervated bands of Schwann cells, onion-bulb formations, and hypertrophy of the basal lamina. Early morphological changes include minimal alteration of myelinated and unmyelinated fibers and axonal regeneration (Yagihashi et al., 2007; Said et al., 2007).
The pathophysiology of DPN is multifactorial and involves genetic, environmental, behavioral, metabolic, neurotrophic, and vascular factors (Vinik et al., 1999; Oates, 2002; Vincent, et al., 2002; Perkins, et al., 2003). The vascular concept of peripheral diabetic neuropathy implies that diabetes-induced endothelial dysfunction with a resultant decrease in nerve blood flow, vascular reactivity, and endoneurial hypoxia plays a key role in functional and morphological changes in the diabetic nerve (Cameron et al., 2001). Endothelial changes in the vasa nervorum have been attributed to multiple mechanisms, including increased aldose reductase activity, nonenzymatic glycation and glycoxidation, activation of protein kinase C, oxidative–nitrosative stress, and changes in arachidonic acid and prostaglandin metabolism (Cameron et al., 2001). The complex and interrelated effects of hyperglycemia include increased metabolic flux through the polyol pathway with consequent sorbitol and fructose accumulation and reduced sodium-potassium ATPase levels, altered fatty acid metabolism, alterations in the redox state, reduced myoinositol and sodium-potassium ATPase activity, accumulation of advanced glycated end products, accelerated neuronal apoptosis, immunological alterations, changes in blood flow, and increased oxidative stresses. The exact mechanisms of DPN are uncertain, but may involve activation of the polyol pathway due to hyperglycemia; the polyol pathway is considered to play a major role in diabetic neuropathy (Greene, et al., 1987). Excellent reviews of this information are available in previous publications.
Diagnosis of DPN on clinical grounds alone is not accurate, and it is difficult to detect small alterations in neuropathies (Feldman et al, 1994; Perkins et al, 2001). Therefore, as a surrogate measure, NCS are widely used as an evaluation of DPN. NCS measure the ability of peripheral nerves to conduct electrical signals, and this ability is impaired when pathological changes are present in the myelin, nodes of Ranvier, and axons. Routine NCS include evaluation of the motor function of the median, ulnar, peroneal, and tibial nerves, and evaluation of the sensory function of the median, ulnar, radial, and sural nerves. Velocities are universally reported in meters per second, motor amplitudes in millivolts, and sensory amplitudes in microvolts. These measurements of upper- and lower-limb motor and sensory nerve functions show the presence, distribution, and severity of peripheral nerve disease (Albers, et al., 1996).
The attribution of peripheral nerve dysfunction to either primary demyelination or primary axonal loss is usually based on nerve conduction velocity and action potential amplitude data. In general, demyelination is indicated by a decreased nerve conduction velocity, conduction block, or increased temporal dispersion, whereas axonal loss is indicated by a reduction in the amplitude or area of the sensory nerve action potential (SNAP) or compound muscle action potential (CMAP). However, there has been considerable disagreement in terms of the clinical and the electrophysiological criteria for the diagnosis of DPN.
Recently, various calculated indices such as the residual latency, terminal latency index, and modified F-wave ratio were introduced as more sensitive electrophysiological tools than a conventional NCS in patients with diverse types of peripheral neuropathies (Attarian et al, 2001; Kaplan, et al, 1978; Radziwill et al, 2003).
5.1. Sonographic features of normal peripheral nerves
US is a widely utilized diagnostic tool for gynecological purposes and examinations of the heart and intra-abdominal and superficial organs. With the advancement of sonographic resolution, normal peripheral nerves also can be clearly demonstrated. US is a useful technique for the investigation of a number of musculoskeletal disorders. Although US has the well-known advantages of low cost, accessibility, portability, noninvasiveness, and multiplanar imaging, one of its most important diagnostic advantages over other techniques is considered to be its real-time imaging capability, allowing for dynamic evaluation of the musculoskeletal field (Khoury et al., 2007).
US can be used to determine the location, extent, type of lesion as well as the presence of nerve swelling and inflammation. Major peripheral nerves in the extremities, such as the median, ulnar, radial, sciatic, and posterior tibial nerves can be seen using conventional US performed with 5- to 12-MHz probes (Stokvis et al., 2009). In controls, peripheral nerves are seen as hypoechoic neuronal fascicles surrounded by echogenic connective tissue (Silvestri et al., 1995). The basic units of the peripheral nerve consist of a neural fiber embedded in the endoneurium. Because the endoneurium is too thin to reflect the sound beam, it is hypoechogenic on the US scan. The neural fascicle consists of several neural fibers and is embedded in a capsule called the perineurium. This capsule consists of connective tissue, vessels, and lymphatic ducts and is thick enough to reflect the sound beam, resulting in hyperechoic lines on the US scan. The trunk of the peripheral nerve consists of several neural fascicles and is embedded in a thicker membrane called the epineurium, which is seen as bold echogenic lines on the US. Therefore, a peripheral nerve is seen as several parallel hyperechoic lines and bold hypoechoic lines on longitudinal images and as a faveolate pattern on transverse images (Fig 1).
5.2. Sonographic features of the peripheral nerves in patients with type 2 DM
We previuosly reported that peripheral nerves in patients with low MCV and type 2 DM showed enlarged and hypoechoic patterns as compared with those of controls or patients with high MCV and type 2 DM. There are 2 sonographic methods of measuring nerve CSA: the indirect method (ellipsoid formula) and direct method (tracing). Recently, Alemán et al.(2008) reported that median nerve CSA measurements are reproducible by either the direct or indirect method when a standardized ultrasonographic examination protocol is applied. Sernik et al. (2008) also reported a high correlation (r = 0.99) between the areas calculated by the indirect and direct methods.
In patients with DPN, peripheral nerves showed enlarged and hypoechoic patterns. Figure 2 shows sonographic images of several nerves in the controls and in patients with type 2 DM. The CSAs of the median, ulnar, and tibial nerves in patients with DM were significantly larger than those in controls. It is likely that these findings reflect the pathological changes, although the pathogenesis of nerve enlargement and increased percentage of the hypoechoic area in peripheral nerves is uncertain because our study did not include histological evidence. In a 1H-nuclear magnetic resonance study, Suzuki et al. (1994) reported that sorbitol and the sodium accumulation caused by an increase in sorbitol may be major contributors to the increase in intracellular hydration. It has further been hypothesized that peripheral nerves are swollen in individuals with DM because of increased water content related to an increase in the aldose reductase-mediated conversion of glucose to sorbitol. We hypothesize that the increase in the hypoechoic area of peripheral nerves in diabetic patients may be because of increased water content, which is also a cause of enlargement of peripheral nerves.
5.3. Assessment of the internal echo of the peripheral nerves
The ultrasonographic images of the peripheral nerves were saved as JPEG files and transferred to a personal computer for analysis. The monochrome US image was quantized to 8 bits (i.e., 256 gray levels). The brightness of the pixels ranged from 0 (black) to 255 (white). Histogram analysis in US has been expected to offer an objective index for estimating the echo intensity, such as in the diagnosis of fatty liver or hepatitis (Lee et al., 2006; Osawa et al., 1996). The region of interest was set to cover the entire nerve, excluding its hyperechoic rim. We used the percentage of the hypoechoic area as the index after the effects of gain shift on echo intensity in the median nerve were confirmed (Fig. 3).
The normal appearance of a peripheral nerve should be readily recognized. Peripheral nerves consist of multiple hypoechoic bands corresponding to neuronal fascicles, which are separated by hyperechoic lines that correspond to the epineurium. Thus, the value obtained by the discriminant analysis method of Otsu was used as a threshold level for the analysis of the percentage of the hypoechoic area because the echogenicity of peripheral nerves was obtained as a graded echo density from black to white. Otsu’s method (1979), which selects a global threshold value by maximizing the separability of the classes in gray levels, is one of the better tequniques for image segmentation. Mathematically, this can be expressed as:
Assume that the image can be represented in L gray levels (1, 2,…, L). If the number of pixels at level i is denoted by fi, then the total number of pixels equals N = f1 + f2 + …+fL. If an image can be divided into 2 classes (C1 and C2) by a threshold at level t, where class C1 consists of gray levels from 0 to t, and class C2 contains the other gray levels with t + 1 to L, then the cumulative probabilities (w1 and w2) and mean levels (μ1 andμ2) for classes C1 and C2, respectively, are given by
Otsu selects an optimal threshold t* that maximizes the between-class variance σ2 in Eq. (6) based on the discriminant analysis, where μT is the mean intensity of the image.
The percentage of the hypoechoic area was studied using computer analysis. Using ImageJ software, the amount of the hypoechoic area falling below the threshold echo intensity was calculated. In our computerized method, the flowchart of the echo intensity evaluation process is demonstrated in Figure 4.
Representative sonographic images and three-dimensional graphic using ImageJ software of diabetic patients and controls are shown in Fig 5.
6. Sonography and NCS in patients with type 2 DM
6.1. Relationship between sonography and NCS
We studied 144 peripheral nerves (40 median, 40 ulnar, 40 tibial, and 24 sural nerves) of 40 subjects who underwent both US and NCS (unpublished data). Overall, 20 type 2 diabetes patients [10 men and 10 women; age range, 50-88 years (mean, 68.5 ± 10.7 years)] and 20 healthy volunteers [12 men and 8 women; age range, 26-59 years (mean, 40.0 ± 12.3 years)] were enrolled in this study. All participants whose wrists had symptoms of carpal tunnel syndrome were excluded from the study. This study was approved by the Institutional Review Board of Gifu University Hospital, and informed consent was obtained from all participants.
Ultrasonographic examination was performed by a board-certified sonographer who was blinded to the knowledge of the electrodiagnostic results. A 6.0- to 14.0-MHz linear array probe was used (portable real-time apparatus: Aplio XG; Toshiba Medical Systems, Japan). All subjects were seated on the examination table with their arms on a pillow and fingers semi-extended during examination of the median or ulnar nerves, and in the prone position during examination of the tibial and sural nerves. The CSA of the median nerve was measured at the carpal tunnel (MA) and at 5 cm proximal to the wrist (MB). The CSA of the ulnar nerve was measured at 5 cm proximal to the wrist (UA). The CSA of the tibial nerve was measured at the posterior medial malleolus (TA). The CSA of the sural nerve was measured at the lower third of the crus (SA). US images were quantitatively analyzed using the ImageJ software (National Institutes of Health, USA). We evaluated the relationship between the US and NCS results.
Routine NCS were performed using conventional procedures and standard electromyography (Neuropack MEB-2200; Nihon Kohden Corp., Japan). All examinations were performed in a room with an ambient temperature of 25°C. The skin surface temperature in all cases was 31°C to 34°C. Mann-Whitney U test was used to compare data between the 2 groups. Pearson’s correlation coefficients were used to investigate the correlation of CSA and the percentage of the hypoechoic area with several NCS parameters. Results are given as mean ± SD, and statistical significance was assessed at P < 0.05.
The US and NCS results of diabetic patients and controls are shown in Table 1. The CSAs in the patients with DM group were 0.13 ± 0.05 cm2 in the MA, 0.09 ± 0.03 cm2 in the MB, 0.0.7 ± 0.02 cm2 in the UA, 0.18 ± 0.04 cm2 in the TA, and 0.05 ± 0.01 cm2 in the SA. The CSAs in the controls were 0.08 ± 0.02 cm2 in the MA, 0.07 ± 0.02 cm2 in the MB, 0.04 ± 0.01 cm2 in the UA, 0.10 ± 0.03 cm2 in the TA, and 0.04 ± 0.01 cm2 in the SA. The CSAs was a significant increase in the diabetic patients compared with that in the controls, with the exception of the sural nerve. The percentage of the hypoechoic area was significantly increased in the diabetic patients compared with that in the controls (P < 0.05). The MCV and sensory nerve conduction velocity of all nerves in the diabetic patients showed a significant decrease compared with those in the controls (P < 0.001). The CMAP and SNAP of all nerves in the diabetic patients showed a significant decrease compared with those in the controls, with exception of the CMAPs of both the median and tibial nerves (P < 0.001). On the other hand, distal latency (DL) was significantly lesser in the diabetic patients than in the controls, with the exception of the sensory ulnar and motor tibial nerves.
The relationships between US findings and NCS parameters are shown in Figure 6. The motor DL period of the median nerve was divided into 3 groups: DL of <3.5 ms; DL of 3.5 to 4.0 ms; and DL of >4.0 ms. The MCV of the median nerve was also divided into 3 groups: MCV of <50 m/s; MCV = 50 to 55 m/s; and MCV of >55 m/s. The categorization of subjects into tertiles of DL yielded 3 separate groups. Compared with the first tertile, the CSAs of the median nerve increased significantly with each tertile. Moreover, after combining tertiles of DL and MCV, even more comprehensive CSA stratification was possible, with all-cause CSA ranging from 0.07 cm2 in subjects in the lowest tertile of both parameters to 0.17 cm2 in subjects in the highest tertile (Fig. 6a). The hypoechoic area of the median nerve also increased significantly with each tertile. The hypoechoic area of the median nerve stratification was 52.5% in subjects in the highest tertile of both parameters (Fig. 6b). These results correlated with the electrophysiological severity.
|Parameters||Nerve||Controls||Patients with type 2 DM|
|Sonographic measurements (CSA)|
|MA (cm2)||Median||0.08 ± 0.02||0.13 ± 0.05***|
|MB (cm2)||Median||0.07 ± 0.02||0.09 ± 0.03**|
|UA (cm2)||Ulnar||0.04 ± 0.01||0.07 ± 0.02***|
|TA (cm2)||Tibial||0.10 ± 0.03||0.18 ± 0.04***|
|SA (cm2)||Sural||0.04 ± 0.01||0.05 ± 0.01|
|MB (%)||Median||43.7 ± 5.1||50.0 ± 9.3*|
|UA (%)||Ulnar||43.6 ± 4.1||49.6 ± 10.6*|
|TA (%)||Tibial||44.8 ± 4.5||51.4 ± 7.5*|
|SA (%)||Sural||40.4 ±7.1||48.8 ± 3.7*|
|MCV (m/s)||Median||59.9 ± 4.4||50.7 ± 5.7***|
|DL (ms)||Median||3.3 ± 0.5||4.5 ± 1.3***|
|CMAP (mV)||Median||11.2 ± 3.7||11.6 ± 4.4|
|SCV (m/s)||Median||69.1 ± 8.6||57.3 ± 5.7***|
|DL (ms)||Median||2.7 ± 0.4||3.3 ± 0.4***|
|SNAP (uV)||Median||38.3 ±11.7||12.5 ± 8.3***|
|MCV (m/s)||Ulnar||64.7± 6.1||52.1± 6.7***|
|DL (ms)||Ulnar||2.5 ± 0.4||2.9 ± 0.5*|
|CMAP (mV)||Ulnar||14.9 ± 3.8||9.4 ± 2.5***|
|SCV (m/s)||Ulnar||72.0 ± 5.4||61.7 ± 10.5***|
|DL (ms)||Ulnar||3.3 ± 0.7||3.9 ± 0.8|
|SNAP (uV)||Ulnar||20.4 ± 10.0||6.3 ± 2.9***|
|MCV (m/s)||Tibial||49.4 ± 2.4||40.3 ± 5.1***|
|DL (ms)||Tibial||3.9 ± 0.5||4.4 ± 0.9|
|CMAP (mV)||Tibial||16.1 ± 5.3||13.1 ± 6.9|
|SCV (m/s)||Sural||55.7± 2.5||49.1± 5.3***|
|DL (ms)||Sural||2.5 ± 0.1||2.9 ± 0.3***|
|SNAP (uV)||Sural||10.3± 4.1||3.6 ± 2.5***|
Mann-Whitney U test: *P<0.05 versus controls; **P<0.01 versus controls; ***P<0.001 versus controls.
6.2. Comparison of diagnostic ability between US and NCS parameters
NCS are widely used for the diagnosis of DPN. The examinations are deemed to be objective, reliable, and sensitive, and can be used as statistical instruments or surrogate endpoints for neuropathy in large clinical investigations of DPN (Diabetes Control and Complications Trial Research Group., 1995; Cornblath et al., 1999; Dyck et al., 1991). NCS have also been recommended in the medical literature as the “gold standard” with which to evaluate and validate other screening tests that are used to diagnose peripheral neuropathy. We aimed to determine the best diagnostic criterion for diagnosing DPN by US and NCS. A receiver operating characteristic (ROC) curve was generated for each parameter in the US and NCS examinations, and areas under the curve (AUC) were determined. The ROC curves are plots of the true-positive rate (sensitivity) against the false-positive rate (1.0 – specificity) for the different possible cutoff points of a diagnostic test. To determine the accuracy of detection of DPN, we calculated and compared the sensitivity and specificity of both US and NCS.
In our preliminary study, the CSA at TA in the tibial nerve had the best diagnostic accuracy for DPN of all the sonographic examinations. The ROC curves of the CSA at TA revealed that the AUC was 0.919 (P < 0.001) with an optimal cutoff value of 0.145 cm2, yielding 80% sensitivity and 94% specificity. For the NCS, the SNAPs had the best diagnostic accuracy for DPN; each nerve had an extremely high AUC (median nerve, 0.971; ulnar nerve, 0.944; and sural nerve, 0.938; P < 0.001). These cutoffs also yielded very good sensitivity (93% - 94%) and specificity (80% to 92%). Some investigators have reported that sural nerve dysfunction is the most common indicator of peripheral nerve dysfunction, is the first to be affected, and correlates most closely with the neuropathological findings (Dyck et al., 1985; Dyck., 1988; Redmond et al., 1992). Dyck et al. (1985) found that the peroneal motor nerve had the highest degree of abnormality, followed by the sural, median sensory, and median motor nerves. Karsidag et al. (2005) also reported that the most affected nerves were the sural sensory, peroneal motor, posterior tibial motor, median motor, ulnar motor, median sensory, and ulnar sensory nerves. In our study, the sural nerve had a high AUG, as reported in previous reports. Furthermore, the CSA at TA showed the most effective parameter in the US examinations; it was suggested that the most useful and practical nerves for electrophysiological and sonographical studies in diabetic patients are the lower extremity nerves.
According to the ROC curve analysis, to investigate whether the use of US and NCS could accurately determine the presence of DPN, we compared the sensitivity and specificity of different parameters. Both sensitivity and specificity were higher in NCS than in US. These results were consistent with the current status that NCS is widely accepted as the more sensitive system of evaluation of polyneuropathy. Although sonographic measurements have insufficient sensitivity and specificity compared with those of the NCS, the ROC curves showed that AUCs were as high as 0.681 to 0.919, yielding 43% to 94% sensitivity and 50% to 94 % specificity. We promote the possibility of using sonography to diagnose DPN.
In this chapter, we have reviewed the current knowledge of neuropathy in type 2 DM and have introduced a sonographical examination for DPN. Based on our present data, it appears that both size and hypoechoic area of nerves were increased in patients with type 2 DM compared with controls. US is a noninvasive method that can be used to evaluate detailed nerve structures. The results from this preliminary study indicate that US might be considered as a valuable tool for the evaluation of DPN. In this work, we focused on the development of an objective method of quantitative analysis of echogenicity changes in peripheral nerves over a clarification of the mechanism. Some limitations of our study should be mentioned. First, a relatively small number of participants were studied and no adjustments were made for age differences. Second, our study was an ultrasonographic examination only; therefore, exactly what causes an increased hypoechoic area or CSA remains unknown. Furthermore, we must describe the property of sound waves. The property depends on both the object and the matrix in which it occurs, in that it relates to changes in acoustic impedance between 2 abutting structures. It is generally known that most nerves, including the median nerve, are surrounded by hyperechoic structures such as the tendons and that these hyperechoic structures may affect their appearance. Further investigation is required to clarify these findings in larger groups of diabetic patients using other modalities, such as magnetic resonance imaging.
Finally, there is little doubt that NCS are widely accepted as more sensitive than US in the evaluation of peripheral nerve disorders. However, US is able to directly show morphological changes in the peripheral nerves. Compared with NUS, US caused less discomfort to patients and was less time-consuming in our study. For these reasons, we promote the possibility of using this technique for the diagnosis of DPN.
The support of the clinical laboratory staff at the Gifu University Hospital is gratefully acknowledged. Authors also gratefully thank Dr. D. Fukuoka for helpful analysis of the echogenisity in the nerve.