Determination of fascicle length and pennation in a contracting human muscle in vivo

Fukunaga, Tetsuo, Yoshiho Ichinose, Masamitsu Ito, YasuoKawakami, and Senshi Fukashiro. Determination of fascicle lengthand pennation in a contracting human muscle in vivo.J. Appl. Physiol. 82(1): 354-358, 1997.We have developed a technique to determine fascicle length inhuman vastus lateralis muscle in vivo by using ultrasonography. Whenthe subjects had the knee fully extended passively from a position of110° flexion (relaxed condition), the fascicle length decreasedfrom 133 to 97 mm on average. During static contractions at 10% ofmaximal voluntary contraction strength (tensed condition), fascicleshortening was more pronounced (from 126 to 67 mm), especially when theknee was closer to full extension. Similarly, as the knee was extended, the angle of pennation (fascicle angle, defined as the angle between fascicles and aponeurosis) increased (relaxed, from 14 to 18°; tensed, from 14 to 21°), and a greater increase in the pennation angle was observed in the tensed than in the relaxed condition when theknee was close to extension (<40°). We conclude that there aredifferences in fascicle lengths and pennation angles when the muscle isin a relaxed and isometrically tensed conditions and that thedifferences are affected by joint angles, at least at thesubmaximal contraction level.

     

In vivo fascicle length measurements via B-mode ultrasound imaging with single vs dual transducer arrangements

Ultrasonography is a useful technique to study muscle contractions in vivo, however larger muscles like vastus lateralis may be difficult to visualise with smaller, commonly used transducers. Fascicle length is often estimated using linear trigonometry to extrapolate fascicle length to regions where the fascicle is not visible. However, this approach has not been compared to measurements made with a larger field of view for dynamic muscle contractions. Here we compared two different single-transducer extrapolation methods to measure VL muscle fascicle length to a direct measurement made using two synchronised, in-series transducers. The first method used pennation angle and muscle thickness to extrapolate fascicle length outside the image (extrapolate method). The second method determined fascicle length based on the extrapolated intercept between a fascicle and the aponeurosis (intercept method). Nine participants performed maximal effort, isometric, knee extension contractions on a dynamometer at 10° increments from 50 to 100° of knee flexion. Fascicle length and torque were simultaneously recorded for offline analysis. The dual transducer method showed similar patterns of fascicle length change (overall mean coefficient of multiple correlation was 0.76 and 0.71 compared to extrapolate and intercept methods respectively), but reached different absolute lengths during the contractions. This had the effect of producing force–length curves of the same shape, but each curve was shifted in terms of absolute length. We concluded that dual transducers are beneficial for studies that examine absolute fascicle lengths, whereas either of the single transducer methods may produce similar results for normalised length changes, and repeated measures experimental designs.     

In vivo assessment of subcutaneous fat in dogs by real-time ultrasonography and image analysis

Background

Systems for estimating body condition score (BCS) are currently used in canine practice to monitor fatness levels. These tools are cheap and easy to use but lack the necessary precision to monitor small changes in body fat, particularly during weight control treatments or in research. The present work aims to study the application of real-time ultrasonography (RTU) together with image analysis in the assessment of subcutaneous fat depots in dogs. Ultrasound images were collected from five anatomical locations (chest, flank, abdomen, thigh and lumbar) from 28 healthy dogs of different breeds and with a body weight (BW) ranging from 5.2 to 33.0 kg. BCS was collected by visual appraisal using a 5-point scale. Subcutaneous fat thickness (SFT) was estimated from RTU images, using the average of three measurements taken in fat deposits located above the muscles represented in each image. Correlations were established between SFT and BW or BCS as well as a classification of BCS-based fatness [overweight (BCS = 4), ideal (BCS = 3) and lean (BCS = 2)].

Results

SFT was found to differ between the five regions considered (P < 0.001). Abdomen and thigh were the areas displaying the widest variation for the different dogs included in the study and also those correlating most with BW, in contrast to the chest, which showed the least variation. Overall, a strong correlation was found between BCS and SFT. The highest correlations were established for the flank, abdomen and lumbar areas. In every anatomical area, a decrease in SFT was observed across all three BCS classes, ranging from 48 to 65 % among overweight and ideal dogs, and from 46 to 83 % among ideal and lean dogs.

Conclusions

Preliminary data showed that within this population there was a strong correlation between BCS and SFT estimated from RTU images. It was also observed that RTU measurements for fat thickness differed among the anatomical points surveyed suggesting differences in their sensitivity to a change in BCS. The images displaying the best prediction value for fatness variations were those collected at the lumbar and abdomen areas.

     

Validity of fascicle length estimation in the vastus lateralis and vastus intermedius using ultrasonography

The purpose of this study was to determine the validity of fascicle length estimation in the vastus lateralis (VL) and vastus intermedius (VI) using ultrasonography. The fascicle lengths of the VL and VI muscles were measured directly (dFL) using calipers, and were estimated (estmFL) using ultrasonography, in 10 legs from five Thiel’s embalmed cadavers. To determine the validity of the estmFLs, FL was estimated using five previously published models and compared with dFL. The intraclass correlation coefficients (ICCs) of two of the five models were > 0.75, indicating that these estimates were valid. Both of these models combined measurement of the length of the visible part of the fascicle with linear extrapolation of the length of the part of the fascicle that was not visible on the sonographic image. The ICCs and absolute% difference were best in models that used appropriate pennation angles. These results suggest that two of the five previously published models are valid for obtaining estmFL of the VL and VI using ultrasonography.     

Relationship between sprint performance and muscle fascicle length in female sprinters

The purpose of this study was to investigate the relationship between sprint performance and architectural characteristics of leg muscles in 26 female 100-m sprinters. Pennation angle and muscle thickness of the vastus lateralis (VL) and gastrocnemius medialis (GM) and lateralis (GL) muscles were measured by B-mode ultrasonography, and fascicle length was estimated. Sprinters had a significantly lower VL pennation angle, but GM and GL pennation angle was similar between sprinters and female control subjects (N = 22). There was no significant correlation between pennation angle and 100-m personal best performance. Sprinters had significantly greater absolute fascicle length in VL and GL than controls, which significantly correlated to 100-m best-record (r = -0.51 and r = -0.44, respectively). Relative fascicle length (VL and GL) were also significantly greater in sprinters than controls. However, there were no significant correlation between relative fascicle length and 100-m best-record (r = -0.36 and r = -0.29, respectively). No relationship was found between the sprint performance and fat-free mass (r = -0.26) or body mass index (r = -0.03). However, there was a significant correlation between percent (%) body fat and 100-m best-record (r = 0.62, p < 0.01). Adjusting the confounding effect of % fat, significant correlations were seen between relative fascicle length and 100-m best-record (VL; r = -0.39 and GL; r = -0.40). Absolute and relative fascicle length were similar in elite female sprinters compared with previous reported values for elite male sprinters (Kumagai et al., 2000). It was concluded that longer fascicle length is associated with greater sprinting performance in sprinters, but there is no gender differences in fascicle length for elite sprinters.     

In vivo determination of fascicle curvature in contracting human skeletal muscles.

Fascicle curvature of human medial gastrocnemius muscle (MG) was determined in vivo by ultrasonography during isometric contractions at three (distal, central, and proximal) locations (n = 7) and at three ankle angles (n = 7). The curvature significantly (P < 0.05) increased from rest to maximum voluntary contraction (MVC) (0.4-5.2 m(-1)). In addition, the curvature at MVC became larger in the order dorsiflexed, neutral, plantar flexed (P < 0.05). Thus both contraction levels and muscle length affected the curvature. Intramuscular differences in neither the curvature nor the fascicle length were found. The direction of curving was consistent along the muscle: fascicles were concave in the proximal side. Fascicle length estimated from the pennation angle and muscle thickness, under the assumption that the fascicle was straight, was underestimated by ~6%. In addition, the curvature was significantly correlated to pennation angle and muscle thickness. These findings are particularly important for understanding the mechanical functions of human skeletal muscle in vivo.     

Computational methods for quantifying in vivo muscle fascicle curvature from ultrasound images

Muscle fascicles curve during contraction, and this has been seen using B-mode ultrasound. Curvature can vary along a fascicle, and amongst the fascicles within a muscle. The purpose of this study was to develop an automated method for quantifying curvature across the entirety of an imaged muscle, to test the accuracy of the method against synthetic images of known curvature and noise, and to test the sensitivity of the method to ultrasound probe placement. Both synthetic and ultrasound images were processed using multiscale vessel enhancement filtering to accentuate the muscle fascicles, wavelet-based methods were used to quantify fascicle orientations and curvature distribution grids were produced by quantifying local curvatures for each point within the image. Ultrasound images of ramped isometric contractions of the human medial gastrocnemius were acquired in a test–retest study.The methods enabled distinct curvatures to be determined in different regions of the muscle. The methods were sensitive to kernel sizes during image processing, noise within the image and the variability of probe placements during retesting. Across the physiological range of curvatures and noise, curvatures calculated from validation grids were quantified with a typical standard error of less than 0.026 m^?1, and this is about 1% of the maximum curvatures observed in fascicles of contracting muscle.     

Sprint performance is related to muscle fascicle length in male 100-m sprinters.

The purpose of this study was to investigate the relationship between muscle fascicle length and sprint running performance in 37 male 100-m sprinters. The sample was divided into two performance groups by the personal-best 100-m time: 10.00-10.90 s (S10; n = 22) and 11.00-11.70 s (S11; n = 15). Muscle thickness and fascicle pennation angle of the vastus lateralis and gastrocnemius medialis and lateralis muscles were measured by B-mode ultrasonography, and fascicle length was estimated. Standing height, body weight, and leg length were similar between groups. Muscle thickness was similar between groups for vastus lateralis and gastrocnemius medialis, but S10 had a significantly greater gastrocnemius lateralis muscle thickness. S10 also had a greater muscle thickness in the upper portion of the thigh, which, given similar limb lengths, demonstrates an altered "muscle shape." Pennation angle was always less in S10 than in S11. In all muscles, S10 had significantly greater fascicle length than did S11, which significantly correlated with 100-m best performance (r values from -0.40 to -0.57). It is concluded that longer fascicle length is associated with greater sprinting performance.     

In vivo operational fascicle lengths of vastus lateralis during sub-maximal and maximal cycling

Instantaneous contractile characteristics of skeletal muscle, during movement tasks, can be determined and related to steady state mechanical properties such as the force–length relationship with the use of ultrasound imaging. A previous investigation into the contractile characteristics of the vastus lateralis (VL) during cycling has shown that fascicles operate on the “weak” descending limb of the force–length relationship, thus not taking advantage of the “strong” plateau region. The purpose of this study was to investigate if VL fascicle lengths change from sub-maximal to maximal cycling conditions, and if maximal cycling results in VL fascicle lengths which operate across the plateau of the force–length relationship. Fifteen healthy male subjects (age 20.9±1.8 yr, wt. 67.0±6.3 kg, ht. 176.7±7.2 cm) were tested to establish the maximal force–length relationship for the VL through ten maximal isometric contractions at various knee angles. Subjects then cycled on an SRM cycle ergometer at cadences of 50 and 80 revolutions per minute at 100 W, 250 W, and maximal effort. Fascicle lengths were determined at crank angles of 0, 90, and 180°. Fascicles operated at or near the plateau of the maximal force–length relationship for maximal cycling, while operating on the descending limb during sub-maximal conditions for both cadences. However, when comparing the fascicle operating range for the sub-maximal cycling conditions to the corresponding sub-maximal force–length relationships, the VL now also operated across the plateau region. We concluded from these results that regardless of cycling effort, the VL operated through the ideal plateau region of the corresponding force–length relationship, hence always working optimally. We hypothesize that this phenomenon is due to the coupling of series elastic compliance and length dependent calcium sensitivity in the VL.     

Changes in fascicle length from rest to maximal voluntary contraction affect the assessment of voluntary activation

The purpose of this study was to investigate the effect of the differences between the actual fascicle length during a voluntary contraction and the fascicle length at rest of the triceps surae muscle on the determination of the voluntary activation (VA) by using the interpolated twitch technique. Twelve participants performed isometric voluntary maximal (MVC) and submaximal (20%, 40%, 60% and 80% MVC) contractions at two different ankle angles (75 degrees and 90 degrees ) under application of the interpolated twitch technique. Two ultrasound probes were used to determine the fascicle length of soleus, gastrocnemius medialis and gastrocnemius lateralis muscles. Further, the MVCs and the twitches were repeated for six more ankle angles (85 degrees , 95 degrees , 100 degrees , 105 degrees , 110 degrees and 115 degrees ). The VA of the triceps surae muscle were calculated (a) using the rest twitch force (RTF) measured during the same trial as the interpolated twitch force (ITF; traditional method) and (b) using the RTF at an ankle angle where the fascicle length showed similar values between ITF and RTF (fascicle length consideration method). The continuous changes in fascicle length from rest to MVC affect the accuracy of the assessment of the VA. The traditional method overestimates the assessment of the VA on average 4% to 12%, especially at 90 degrees ankle angle (i.e. short muscle length). The reason for this influence is the unequal force-length potential of the muscle at twitch application by the measure of ITF and RTF. These findings provide evidence that the fascicle length consideration method permits a more precise prediction (an improvement of 4-12%) of the voluntary contraction compared to the traditional method.     

In vivo assessment of human skeletal muscle mitochondria respiration in health and disease

One of many problems to be faced when assessing in vivo human muscle mitochondria respiration by phosphorus magnetic resonance spectroscopy (³¹P-MRS) is the definition of the correct reference population and the values of reference range. To take into account most factors that influence muscle activity as age, sex, physical activity; nutritional state etc., an exceedingly high number of different reference groups are needed. To overcome this problem we developed specific tests to assess separately in vivo the activity and the functionality of muscle mitochondria by ³¹P-MRS in clinical settings. By activity we refer to muscle whole metabolic activity, i.e. the total oxidative capacity of muscle mitochondria which is influenced by many factors (age, sex, physical activity, nutritional state etc.). By functionality we refer to the qualitative aspects of mitochondrial respiration which depends on the integrity of mitochondrial multienzyme systems and on substrate availability. Our tests ha ve been experienced on some 1200 patients and are currently used to detect deficits of mitochondrial respiration and ion transport in patients with suspected primary or secondary muscle mitochondrial malfunctioning. (Mol Cell Biochem 174: 11–15, 1997)     

Optimal muscle fascicle length and tendon stiffness for maximising gastrocnemius efficiency during human walking and running

Muscles generate force to resist gravitational and inertial forces and/or to undertake work, e.g. on the centre of mass. A trade-off in muscle architecture exists in muscles that do both; the fibres should be as short as possible to minimise activation cost but long enough to maintain an appropriate shortening velocity. Energetic cost is also influenced by tendon compliance which modulates the timecourse of muscle mechanical work. Here we use a Hill-type muscle model of the human medial gastrocnemius to determine the muscle fascicle length and Achilles tendon compliance that maximise efficiency during the stance phase of walking (1.2 m/s) and running (3.2 and 3.9 m/s). A broad range of muscle fascicle lengths (ranging from 45 to 70 mm) and tendon stiffness values (150-500 N/mm) can achieve close to optimal efficiency at each speed of locomotion; however, efficient walking requires shorter muscle fascicles and a more compliant tendon than running. The values that maximise efficiency are within the range measured in normal populations. A non-linear toe-region region of the tendon force-length properties may further influence the optimal values, requiring a stiffer tendon with slightly longer muscle fascicles; however, it does not alter the main results. We conclude that muscle fibre length and tendon compliance combinations may be tuned to maximise efficiency under a given gait condition. Efficiency is maximised when the required volume of muscle is minimised, which may also help reduce limb inertia and basal metabolic costs.     

In vivo moment arm determination using B-mode ultrasonography

The tendon excursion of the tibialis anterior (TA) muscle was measured in vivo using B-mode ultrasonography in seven subjects under three force levels (0, 30 and 60% maximal voluntary contraction, MVC). For each force level, the TA moment arm (m) was determined by calculating the derivative of the tendon excursion relative to the ankle angle (a). A dynamometer controlled the ankle angle while force levels were monitored. The parametric model proposed by Miller and Dennis (1996), m = R sin(a + delta), where R is the largest moment arm and delta represents the offset angle of R from 90 degrees, was used in a least-squares fit of the relationship between moment arm and ankle angle. The R values at 0% MVC were significantly smaller than those at 30 and 60% MVC. The values of calculated moment arm at 0% MVC were not considered adequate estimates of the TA moment arm because of the possible confounding effect of the slackness of the relaxed muscle-tendon unit in more dorsiflexed positions. The moment arm values at 30 and 60% MVC were believed to provide reliable estimates of those of TA since the application of tension probably reduced the effects of the slackness of the muscle-tendon unit and tendon elongation on tendon excursion measurement at these force levels. Since the ultrasonographic technique is an in vivo application of the tendon excursion technique and therefore takes the functional meaning into consideration, it can yield more significant moment arms than other in vivo or cadaver techniques.     

Comparison between muscle activation measured by electromyography and muscle thickness measured using ultrasonography for effective muscle assessment

In this study, we aimed to compare the intrarater reliability and validity of muscle thickness measured using ultrasonography (US) and muscle activity via electromyography (EMG) during manual muscle testing (MMT) of the external oblique (EO) and lumbar multifidus (MF) muscles. The study subjects were 30 healthy individuals who underwent MMT at different grades. EMG was used to measure the muscle activity in terms of ratio to maximum voluntary contraction (MVC) and root mean square (RMS) metrics. US was used to measure the raw muscle thickness, the ratio of muscle thickness at MVC, and the ratio of muscle thickness at rest. One examiner performed measurements on each subject in 3 trials. The intrarater reliabilities of the % MVC RMS and raw RMS metrics for EMG and the % MVC thickness metrics for US were excellent (ICC = 0.81–0.98). There was a significant difference between all the grades measured using the % MVC thickness metric (p < 0.01). Further, this % MVC thickness metric of US showed a significantly higher correlation with the EMG measurement methods than with the others (r = 0.51–0.61). Our findings suggest that the % MVC thickness determined by US was the most sensitive of all methods for assessing the MMT grade.     

Relationship between short latency stretch reflex and fascicle behavior in the soleus muscle in vivo

Objectives:Stretch reflex responses were considered to be affected by the velocity of muscle fiber lengthening and angular velocity. However, the results of previous studies in vivo and in vitro are inconsistent in this regard. The purpose of the present study was to investigate the effects of the velocity of fascicle lengthening on the amplitude of the stretch reflex for each trial with a high angular velocity and wide range of motion.Methods:Thirteen healthy men volunteered for this study. While the ankle was passively moved from 100 to 80 deg at five different angular velocities (100, 200, 300, 500, and 600 deg⋅s^-1), the velocity of fascicle lengthening in the soleus muscle was measured using ultrasonography. In addition, the amplitude of the short latency stretch reflex in the soleus muscle was also measured.Results:As angular velocity increased, the amplitude of the stretch reflex and velocity of fascicle lengthening significantly increased (both p<0.001). For each trial in all subjects, the amplitude of the stretch reflex was not correlated with the velocity of fascicle lengthening at any of the angular velocities.Conclusion:In conclusion, the stretch reflex size is not related to the fascicle behavior in each trial.     

In vivo DNA electrotransfer into muscle

Naked plasmid DNA injected into skeletal muscle is taken up by muscle cells and the genes in the plasmid are expressed. Among the non-viral techniques for gene transfer in vivo , this method is especially simple, inexpensive, and safe. However, the relatively low expression levels attained by this method have limited its applications for uses other than as a DNA vaccine. We and other groups investigated the applicability of in vivo electroporation for gene transfer into muscle, using plasmid DNA vector. The results demonstrated that gene transfer into muscle by in vivo electroporation is far more efficient than simple intramuscular DNA injection and provides a potential approach to systemically delivering cytokines, growth factors, and other serum proteins for basic research and human gene therapy.     

Automatic tracking of medial gastrocnemius fascicle length during human locomotion

During human locomotion lower extremity muscle-tendon units undergo cyclic length changes that were previously assumed to be representative of muscle fascicle length changes. Measurements in cats and humans have since revealed that muscle fascicle length changes can be uncoupled from those of the muscle-tendon unit. Ultrasonography is frequently used to estimate fascicle length changes during human locomotion. Fascicle length analysis requires time consuming manual methods that are prone to human error and experimenter bias. To bypass these limitations, we have developed an automatic fascicle tracking method based on the Lucas-Kanade optical flow algorithm with an affine optic flow extension. The aims of this study were to compare gastrocnemius fascicle length changes during locomotion using the automated and manual approaches and to determine the repeatability of the automated approach. Ultrasound was used to examine gastrocnemius fascicle lengths in eight participants walking at 4, 5, 6, and 7 km/h and jogging at 7 km/h on a treadmill. Ground reaction forces and three dimensional kinematics were recorded simultaneously. The level of agreement between methods and the repeatability of the automated method were quantified using the coefficient of multiple correlation (CMC). Regardless of speed, the level of agreement between methods was high, with overall CMC values of 0.90 ± 0.09 (95% CI: 0.86-0.95). Repeatability of the algorithm was also high, with an overall CMC of 0.88 ± 0.08 (95% CI: 0.79-0.96). The automated fascicle tracking method presented here is a robust, reliable, and time-efficient alternative to the manual analysis of muscle fascicle length during gait.     

In vivo micro-circulation measurement in skeletal muscle by intra-vital microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman''s method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized, ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT & CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.Download video file.^{(92M, mpg)}