Subjects
A total of 111 subjects including 88 elderlies (age: 61~ 83 years) and 23 young (age: 19~ 26 years) participated in this study. The subjects living in a nursing home were included in the elderly group. The young subjects were male university students and not participated in any competitive sports events. Twenty-seven females were included in elderly group.
The subjects gave written informed consent for the study after receiving a detailed explanation of the purposes, potential benefits, and risks associated with participation. All procedures used in this study were approved by the Committee for Human Experimentation at the Graduate School of Human and Environmental Studies, Kyoto University and the Research Ethics Committee of Chukyo University, and were in accordance with the Declaration of Helsinki.
Experimental design
To familiarize themselves with the motor tasks used in the present study, all subjects came to the laboratory > 1 week before the experimental day. The subjects performed maximal voluntary contraction (MVC) and submaximal incremental ramp contraction during unilateral isometric knee extension. External force at the distal portion of the shank that fixed in a custom-made dynamometer (Takei Scientific Instruments Co., Ltd., Niigata, Japan) with a force transducer (LU-100KSE; Kyowa Electronic Instruments, Tokyo, Japan) was measured during isometric knee extension. During knee extension, both hip and knee joint angles were flexed at 90° (180° corresponds to full extension). Since European consensus on definition and diagnosis used this knee joint angle [6], the present study selected this knee joint angle.
The MVC trial consists of a gradual increase in knee extension force from baseline to maximum in 2–3 s and a plateau phase at maximal contraction for 2–3 s. The timing of the task was based on a verbal count given at 1-s intervals with vigorous encouragement from the investigators. After three submaximal trials at approximately 50, 70, and 90% of MVC as warming up, the subjects performed two MVC trials with ≥ 2 min rest in between. MVC force was mean value of 1 s, when the highest force was produced, during a plateau phase. MVC torque was calculated as the product of MVC force and the distance between the estimated knee joint center and center of force transducer at the distal portion of the shank. To normalize difference in body type among the subjects, MVC torque relative to body mass (MVC/BM) was also calculated and used for further analysis.
After MVC trials, the subjects performed submaximal ramp contractions from 0 to 70% MVC in 35 s (rate of force increase: 5% MVC/s) (Fig. 1). Target and performed forces were shown to the subjects on a monitor. Two submaximal contractions were performed with ≥ 2 min rest in between. Out of two trials, the one trial with the smaller error between the targeted and performed forces was selected with visual inspection and used for analysis.
Multi-channel surface EMG
Surface EMG signal was recorded from the VL muscle with a semi-disposable adhesive grid of 64 electrodes (ELSCH064R3S, OT Bioelectronica, Torino, Italy). The two-dimensional electrode grid is comprised of 13 rows and 5 columns of electrodes with 1 missing electrode in the upper left corner. Each electrode is 1-mm diameter and inter electrode distance was 8-mm inter-electrode distance in both directions (Fig. 1). Before attaching the electrode, the skin was cleaned with alcohol. The line between the head of the great trochanter and inferior lateral edge of the patella which was defined and used as reference line for positioning the electrode grid. The center of electrode grid was positioned at middle point of the reference line and columns of electrodes aligned with the reference line. The position of the missing electrode was located proximally. To assure proper electrode contact with the skin, conductive gel was inserted into the cavities of the grid electrode. The grid electrode was connected to the amplifier through 4 connectors which were fixed to the skin by elastic tape. A reference electrode (C-150, Nihon Kohden, Tokyo, Japan) was placed at the iliac crest.
Fifty-nine bipolar surface EMG signals along the columns were made from 64 electrodes. Root mean square (RMS) values were calculated from rom bipolar signal sampled over 1 s at 5% increment from 20 to 65% of MVC ramp contraction. Sampled signals were overlapped by 0.5 s between neighboring contraction levels, since performed ramp rate was 10% of the MVC force / 1 s.
We used correlation coefficients and modified entropy to compare spatial distribution pattern and to quantify a heterogeneity for RMS values of multi-channel surface EMG within a grid, respectively. Correlation coefficients were calculated from the 59 pairs of RMS values at the same regions between 20% of MVC and those of all other torque levels to compare the spatial EMG potential distribution pattern as used in our previous studies [40]. Decrease of correlation coefficient indicates change in spatial EMG potential distribution pattern. Modified entropy was calculated as done by Farina in a previous work [14] and our previous studies [39,40,41], modified entropy was defined as entropy of the signal power, that is.
$$ E=-{\sum}_{i=1}^{59}p{(i)}^2{\log}_2p{(i)}^2 $$
where p(i) is the square of the RMS value of channel i divided by the sum of the squares of all the 59 RMS values, at the given force level. Therefore p(i)2 represents the normalized power of each channel. It is E = 0 when all the p(i) are zero except one and is maximal and equal to log
2
59 = 5.884 when the p(i) values are identical and equal to 1/59 (all channels have the same energy). Decrease in modified entropy mean that increase of heterogeneity in spatial EMG potential distribution within an electrode grid.
At the center of electrode location, longitudinal ultrasonographic images (SSD-900, ALOKA, Tokyo, Japan, or Fazone CB, Fuji-film, Tokyo, Japan) were taken to measure the thickness of the subcutaneous tissue and VL muscle before attaching the electrode grid. On NIH ImageJ software, the vertical line of the image was defined on the center of horizontal axis for measurements. Distance between the intersections of that vertical line and deeper and superficial aponeurosis of the VL muscle were measured as muscle thickness of VL. Thickness of subcutaneous tissues was distance between the intersections of that vertical line and superficial aponeurosis of the VL muscle and skin surface.
Statistics
All data are provided as the mean and standard deviation. To test relationship between muscle strength and neuromuscular function, spearman’s rank correlation coefficient was calculated for MVC with multi-channel surface EMG variables and others for the elderly and young groups, respectively. We also performed a stepwise regression analysis for MVC in the elderly group. Nine independent variables, i.e., age, muscle thickness of VL, thickness of subcutaneous tissues, and six surface EMG variables, were entered the stepwise regression if they represented a significant contribution to the explained variance corresponding to an alpha level of p < 0.05. Six EMG variables were modified entropy, correlation coefficient value, mean RMS across all 59 channels, standard deviation of RMS across all 59 channels, mean RMS across all 59 channels normalized by those at 20% of MVC (normalized RMS), and standard deviation of mean RMS across all 59 channels normalized by those at 20% of MVC (normalized standard deviation) and these variables calculated at 65% of MVC were used for correlation analysis.
To compare neuromuscular function among the elderly with different muscle strength, 88 elderly subjects were divided into three groups based on MVC using cumulative frequency distribution, i.e., (Weak) < 33.3%, (Mid) 33.4~ 66.6%, and (Strong) > 66.7%, and the weak and strong groups were used for further analysis. Age, height, body mass, MVC, MVC/BM, muscle thickness of VL, and thickness of cutaneous tissues, and modified entropy, correlation coefficient value, and mean RMS across all 59 channels at from 20 to 65% of MVC were compared between the weak and strong strength groups by using Mann-Whitney test. EMG variables for the elderly groups with weak and strong strength were compared with young subjects by using Mann-Whitney test.
We also compared modified entropy, correlation coefficient value, and mean RMS across all 59 channels at from 20 to 65% of MVC between the elderly and young groups which are matched for MVC. MVC/BM, body mass, muscle thickness of VL, and thickness of cutaneous tissues were also matched between these groups.
The level of significance was set at p < 0.05. Statistical analysis was performed using SPSS (version 15.0, SPSS, Tokyo, Japan) and MATLAB (R2009b, MathWorks GK, Tokyo, Japan).