Architectural properties of the neuromuscular compartments in selected forearm skeletal muscles

Abstract

The purposes f this study were to (i) explore the possibility of splitting the selected forearm muscles into separate compartments in human subjects; (ii) quantify the architectural properties of each neuromuscular compartment; and (iii) discuss the implication of these properties in split tendon transfer procedures. Twenty upper limbs from 10 fresh human cadavers were used in this study. Ten limbs of five cadavers were used for intramuscular nerve study by modified Sihler's staining technique, which confirmed the neuromuscular compartments. The other 10 limbs were included for architectural analysis of neuromuscular compartments. The architectural features of the compartments including muscle weight, muscle length, fiber length, pennation angle, and sarcomere length were determined. Physiological cross-sectional area and fiber length/muscle length ratio were calculated. Five of the selected forearm muscles were ideal candidates for splitting, including flexor carpi ulnaris, flexor carpi radials, extensor carpi radialis brevis, extensor carpi ulnaris and pronator teres. The humeral head of pronator teres contained the longest fiber length (6.23 ± 0.31 cm), and the radial compartment of extensor carpi ulnaris contained the shortest (2.90 ± 0.28 cm). The ulnar compartment of flexor carpi ulnaris had the largest physiological cross-sectional area (5.17 ± 0.59 cm²), and the ulnar head of pronator teres had the smallest (0.67 ± 0.06 cm²). Fiber length/muscle length ratios of the neuromuscular compartments were relatively low (average 0.27 ± 0.09, range 0.18–0.39) except for the ulnar head of pronator teres, which had the highest one (0.72 ± 0.05). Using modified Sihler's technique, this research demonstrated that each compartment of these selected forearm muscles has its own neurovascular supply after being split along its central tendon. Data of the architectural properties of each neuromuscular compartment provide insight into the ‘design’ of their functional capability. In addition to improving our understanding of muscle anatomy and function, elucidation of forearm neuromuscular compartments architecture may ultimately provide information useful for selection of muscle subdivisions used in tendon transfer.

Introduction

Although muscle transplantation has been widely used for the functional reconstruction of forearms and hands (Liu et al. 2012), the paucity of available muscle units has posed a reconstructive challenge for surgeonswhen managing patients with severe crushed forearms, Volkmann's ischemic contracture, and partial brachial plexus lesions. One relatively new solution is to split a muscle into sub-regions with each region innervated by separate nerve branches (Hua et al. 1999; Lim et al. 1999, 2004). English and Letbetter have called these sub-regions neuromuscular compartments, and their researches have been used as a baseline for subsequent studies about neuromuscular partitioning and architectural design of skeletal muscles (English & Letbetter, 1982; English & Weeks, 1984; English et al. 1993). The neuromuscular compartment is defined as a muscle subvolumen supplied by a primary muscular nerve branch with homogeneous fibers and a characteristic architecture (English & Letbetter, 1982; English & Weeks, 1984; English et al. 1993). The important prerequisite for a functioning split muscle is that each compartment after splitting should have its own neurovascular supply (Segal et al. 2002; Pereira, 2004; Ravichandiran et al. 2012).

Based on the number of nerve branches entering the muscle belly, previous work has suggested that skeletal muscles with more than one branch entering the epimysium would possibly have more than one neuromuscular compartment or sub-region (Segal et al. 2002; Pereira, 2004; Ravichandiran et al. 2012). Recently, the anatomical basis for and the details of the partitioning of human forearm skeletal muscles have been investigated rigorously (Hua et al. 1999; Segal et al. 2002; Pereira, 2004; Ravichandiran et al. 2012; Lim et al. 1999, 2004, 2006; Chen et al. 2010). Our previous researches demonstrated that flexor carpi radialis (FCR), flexor carpi ulnaris (FCU), extensor carpi radialis brevis (ECRB), extensor carpi ulnaris (ECU) and pronator teres (PT) were the best candidate muscles for splitting (Hua et al. 1999; Chen et al. 2010).

Selection of a muscle for surgical transfer is based on its excursion and tension generating capacity (Lieber et al. 1990), which are determined, respectively, by the muscle fiber length and physiological cross-sectional area (two main parameters for muscle architecture). Muscle architecture was defined as the arrangement of the fibers in a muscle and its values, including muscle mass and weight, muscle length, fiber length, pennation angle and physiological cross-sectional area. Although other physiological parameters such as muscle volume and other metabolic parameters such as fiber type distribution substantially influence contractile properties, none predicts muscle function as well as muscle architecture (Lieber & Friden, 2000, 2001). Numerous upper-limb skeletal muscles have been studied to identify their architectural properties, such five human wrist flexor and extensor muscles (Lieber et al. 1990), some selected human arm and forearm muscles (Lieber et al. 1992), and even the neurovascular compartments of Macaca forearm muscles (Pereira, 2004). However, few focused on the architectural properties of these forearm neuromuscular compartments.

The objective of this preliminary study, therefore, was to (i) explore the possibility of splitting the selected forearm muscles into separate compartments in human subjects; (ii) quantify the architectural properties of each neuromuscular compartment; and (iii) discuss the implication of these properties in split tendon transfer procedures.

Materials and methods

Twenty upper limbs from 10 fresh human cadavers were used in this study. Of these 20 upper limbs, 10 limbs of five cadavers were used for intramuscular nerve staining; the other 10 limbs were included for architectural analysis of neuromuscular compartments. Subjects were six males and four females ranging in age from 26 to 49 years (average 39.4 ± 7.3 years) at the time of death. They were donated voluntarily to the Department of Anatomy, Second Military Medical University, Shanghai, China, for research and education. The study protocol was approved by the local institutional review board and was in accordance with guidelines regarding the use of human cadavers for research at the authors' institution.

Intramuscular nerve distribution of selected forearm muscles

According to the protocol of modified Sihler's technique (Chen et al. 2010; Liu et al. 2010), the specimens were fixed via perfusion of 10% formalin into the brachial artery for 4 weeks. Selected muscles (FCR, FCU, ECRB, ECU and PT) together with their neurovascular bundles were excised from 10 upper limbs of five cadavers (three males and two females). Each muscle was labeled to identify its orientation properly, and then additional fixation was performed ex vivo for 8–12 weeks to obtain better penetration. After depigmentation, decalcification, staining, destaining, neutralization and clearing, the transparent specimens were photographed with a digital camera to observe and record orientation and distribution of the intramuscular nerves.

Architectural analysis of neuromuscular compartments

The other 10 forearms were included in this study (three males and two females). After removing the surrounding fascia and fat tissues, the muscles were harvested intact (from the most proximal origin to the most distal tendon attachment) and stored in 1× phosphate-buffered saline for 24–48 h before architectural measurements.

According to the observed intramuscular nerve distribution, muscle compartments of each selected forearm muscles were obtained by splitting the muscle distally-proximally along the central tendon. Muscle architecture was measured according to the methods developed by Sacks and Roy (Lieber et al. 1990, 1992) for upper extremity muscles.

Briefly, muscle specimens were removed from buffer, gently blotted dry, and weighed. Muscle mass or wet muscle weight (MW) was not corrected for formaldehyde fixation, but external tendons, connective tissue and fat were removed before weighing. Muscle length (ML) was defined as the distance from the origin of the most proximal fibers to insertion of the most distal fibers.

One difficulty in obtaining accurate muscle fiber length (FL) values is compensating for the natural fiber length variation that occurs simply because muscles are fixed at different joint angles; the other is the natural variation in fiber length that occurs within muscles, making it difficult to compare either between muscles or between treatments (Felder et al. 2005). The procedure typically used to standardize FL values and compensate for such variation is to measure sarcomere length within a specimen, select a standard sarcomere length, and then normalize all raw fiber lengths using the equation (Lieber et al. 1990, 1992):

where FL is the normalized fiber length, FL′ is the experimentally measured (raw) fiber length, SL is a standard sarcomere length (2.7 μm) and SL′ is the experimentally measured sarcomere length at the experimentally measured fiber length. This commonly used equation assumes that sarcomere length and fiber length are linearly related. Muscle fiber bundles were isolated from proximal, middle and distal muscle regions, and then raw (measured) fiber length (FL′) was measured with a digital caliper (accuracy, 0.01 cm).

Surface pennation angle (PA) varied along the length of the muscle and therefore, measurements were obtained with a standard goniometer from the proximal, middle and distal muscle portions and averaged to yield one value per muscle. Firstly, a 1.0 ×1.0 × 0.5 cm muscle specimen was incised parallel to the long axis of the muscle fiber from the center of every muscle compartment. Then it was, in sequence, fixed by neutral formaldehyde, stained with Mallory phosphotungstic acid-hematoxylin solution, dehydrated with ethanol and washed with xylene. Finally, an image analyzer was used to measure the length of 20 sarcomeres from 10 muscle fibers after the slides were sealed, and sarcomere length (SL) was obtained.

In addition to the measured parameters, the fiber length to muscle length ratio (FL/ML ratio) was calculated by dividing normalized fiber length by normalized muscle length. The physiological cross-sectional area (PCSA) was calculated using the following equation (Sacks & Roy, 1982):

where θ is surface pennation angle and ρ is muscle density (1.056 g cm⁻³) (Ward & Lieber, 2005).

Statistical analysis

All values were reported as mean ± SD. Comparisons of muscle weight, muscle fiber length, and PCSA were made with one-way analyses of variance (anovas). All analyses were performed using spss software (Version 16.0; SPSS Inc, Chicago, IL, USA), and the significance level was set at P < 0.05.

Results

The intramuscular nerve distribution of selected forearm muscles by modified Sihler's technique

A total of 50 specimens were processed by the Sihler's staining technique. The intramuscular nerves of all the specimens had been successfully stained, and the 3-dimensional distribution could be clearly viewed. Then, according to their intramuscular nerve distribution, all the specimens were split into two compartments. In bipennate spindle-shaped muscles (Class IIb), the extended aponeurosis of the distal tendon separated the muscle into two compartments, both with their independent innervated nerves, which were the secondary branches of the primary intramuscular nerve of the muscle. In our study, the flexor carpi ulnaris (n = 10), the flexor carpi radialis (n = 10), extensor carpi ulnaris (n = 10), and extensor carpi radialis brevis (n = 10) (Fig. 1) belonged to this kind of muscle. Similarly, pronator teres could also be divided into two compartments with independent innervations, although it is a multi-heads muscle (Class III, n = 10). Its superficial humeral head and deep ulnar head obtained their intramuscular innervations from the medial nerve.

Fig. 1.

(A) Sihler's staining result of ERCB, muscles appeared semi-transparent with their shapes intact and an intramuscular nerve branch stained purple-black. (B) One ERCB was split into two compartments according to Sihler's staining. The radial nerve (RN) is shown contributing two intramuscular branches (n) to either side of the tendon (T) in ECRB, and each splitting compartment received independent innervations. Intramuscular nerve distribution of other selected muscles are available in our previous study (Chen et al. 2010).

Properties of the two neuromuscular compartments in candidate muscles

Data of architectural properties of all 100 neuromuscular compartments were acquired from 50 muscles from 10 forearms. Physiological cross-sectional areas (PCSA) and FL/ML ratios were calculated according to the equations (Table 1, Fig. 2).

Table 1.

Architectural properties of each compartments of selected forearm muscles ( ± S,n = 10,P < 0.05)

Compartments	MW (g)	ML (cm)	FL (cm)	SL (µm)	PA (°)	PCSA (cm2)	FL /ML
FCR-r	14.26 ± 1.53	19.70 ± 0.95	5.30 ± 0.34	2.33 ± 0.11	13.25 ± 0.68	2.48 ± 0.28	0.27 ± 0.01
FCR-u	8.79 ± 0.85	18.45 ± 0.59	4.98 ± 0.21	2.37 ± 0.11	6.80 ± 0.54	1.66 ± 0.19	0.27 ± 0.01
FCU-r	10.82 ± 1.12	23.37 ± 0.62	5.39 ± 0.44	2.42 ± 0.11	13.50 ± 0.71	1.85 ± 0.15	0.23 ± 0.02
FCU-u	20.68 ± 1.44	25.24 ± 1.56	4.47 ± 0.26	2.55 ± 0.21	16.75 ± 1.21	4.21 ± 0.48	0.18 ± 0.00
ECRB-r	18.47 ± 1.01	19.20 ± 1.21	6.37 ± 0.36	2.24 ± 0.11	10.00 ± 0.82	2.72 ± 0.25	0.33 ± 0.01
ECRB-u	10.33 ± 1.13	16.20 ± 0.60	6.03 ± 0.31	2.24 ± 0.11	8.05 ± 0.98	1.60 ± 0.13	0.37 ± 0.01
ECU-r	9.82 ± 1.12	17.94 ± 0.67	3.56 ± 0.35	2.78 ± 0.15	7.80 ± 0.79	2.58 ± 0.13	0.20 ± 0.02
ECU-u	13.29 ± 1.09	22.82 ± 1.39	4.35 ± 0.24	2.52 ± 0.21	10.20 ± 1.01	2.86 ± 0.36	0.19 ± 0.00
PT-h	23.69 ± 0.51	19.62 ± 0.89	7.65 ± 0.38	2.21 ± 0.08	11.95 ± 1.09	2.87 ± 0.15	0.39 ± 0.01
PT-u	3.29 ± 0.15	7.83 ± 0.43	5.66 ± 0.43	2.16 ± 0.09	9.85 ± 0.75	0.55 ± 0.05	0.72 ± 0.05

Values are expressed as mean ± SD. Muscle length and fiber length have been normalized to a sarcomere length of 2.7 μm. Abbreviations are the same as in the text.

PSCA, physiological cross-sectional area; r, radial compartment; u, ulnar compartment.

Fig. 2.

Bar graph of normalized fiber length, physiological cross-sectional area, and FL/ML ratios from the neuromuscular compartments examined. Each bar represents mean ± SEM. Abbreviations are the same as in the text.

The largest neuromuscular compartments, by far, were the PT-h (MW = 23.69 ± 0.51 g) and FCU-u (20.68 ± 1.44 g). Sarcomere length varied greatly from (2.16 ± 0.09) μm to (2.78 ± 0.15) μm among different compartments. There are no differences between the two compartments of the same muscle (P > 0.05) except for the ECU-r and ECU-u (P < 0.05). In terms of muscle fiber length, after being normalized to an optimal sarcomere length of 2.7 μm, the humeral head of pronator teres was the longest (7.65 ± 0.38 cm), followed by ECRB-r (6.37 ± 0.36) and ECRB-u (6.03 ± 0.31 cm), and the radial compartment of extensor carpi ulnaris was the shortest (3.56 ± 0.35 cm).

Physiological cross-sectional area varied greatly between the neuromuscular compartments. FCU-u (4.21 ± 0.48 cm²) had the largest PCSA, followed by PT-h (2.86 ± 0.36 cm²) and ECU-u (2.87 ± 0.15 cm²); PT-u (0.55 ± 0.05 cm²) had the smallest PCSA. There was a significant difference in PCSA between the two compartments of the same muscles (P < 0.05). The compartments followed the classic tradeoff between PCSA and fiber length; large PCSA correlates with short fibers (Fig. 3). And, neuromuscular compartments that clustered together in this graph were architecturally similar.

Fig. 3.

Scatter plots of normalized fiber length vs. PCSA for the compartments in selected human forearm muscles. Neuromuscular compartments that cluster together in this graph are architecturally similar.

FL/ML ratios of the neuromuscular compartments in these five selected forearm muscles were relatively low (0.27 ± 0.08, range from 0.18 to 0.39) except for the PT-u, which had the highest one (0.72 ± 0.05). The ratios also differed significantly between the two compartments in the same muscles (P < 0.05) except for FCR-r and FCR-u.

Discussions

At present, muscle transplantation is still an effective method for rebuilding the motor function impairment of face and limbs (Liu et al. 2012). The neuromuscular compartment concept, first proposed by English and colleagues, has provided broad application prospects in muscle transplantation (English & Letbetter, 1982; English & Weeks, 1984; English et al. 1993).

Successful blood and nerve transfers are the two main goals in muscle transplantation. Meanwhile, it is also significant to provide the best match of properties of muscle architecture between the transplanted muscle (or neuromuscular compartments) and disused muscle (or neuromuscular compartments) for ideal function reconstruction. Muscle architectural values are the best predictors of muscle function, and the most important architectural properties are muscle fiber length and PCSA, because muscle excursion and velocity are directly proportional to muscle fiber length while isometric muscle force is directly proportional to muscle PCSA (Lieber et al. 1990, 1992; Pereira, 2004).

Scholars all over the world have focused on the research of the architectural properties of these muscle compartments in the past two decades. Lieber et al. (1990) studied five human wrist flexor and extensor muscles in 1990, focusing on the properties of the entire muscle1990. Hua et al. (1999) and Lim et al. (1999) separately clarified the architectural properties of neuromuscular compartments of human FCU and FCR in 1999. Pereira et al. (2004) studied the properties of the neuromuscular compartments of FCU in four adult Macaca fascicularis monkeys in 2004. Our previous researches demonstrated that FCR, FCU, ECRB, ECU and PT might be ideal candidate muscles for splitting (Hua et al. 1999; Chen et al. 2010). However, according to our knowledge, few studies have demonstrated the properties of neuromuscular compartments of these muscles. This is the reason that we launched this study.

Using modified Sihler's nerve staining technique, our research demonstrated that each compartment of these selected forearm muscles (FCR, FCU, ECRB, ECU and PT) has its own neurovascular supply after being split along its central tendon. Our data agree qualitatively with the data of other researchers (Hua et al. 1999; Lim et al. 1999, 2004, 2006; Abrams et al. 2005), that is: (i) there exists a significant difference in PCSA between the two compartments or heads of the same muscles; (ii) neuromuscular compartments achieve large PCSAs (force-generating capacity) by adding muscle weight; and (iii) the compartments follow the classic tradeoff between PCSA and fiber length (large PCSA correlates with short fibers). At the quantitative level, we differ with others in the absolute size, both FL and PCSA. These differences mainly result from the different age of the human specimens. For example, the cadavers used for the study of PT had an average age of 79 (Abrams et al. 2005), whereas the average age in our group was 39.4 ± 7.3 years.

Of the muscles researched in our study, PCSA varied greatly between the neuromuscular compartments. FCU-u (4.21 ± 0.48 cm²) had the largest PCSA, and PT-u (0.55 ± 0.05 cm²) the smallest. There was a significant difference in PCSA between the two compartments of the same muscles (P < 0.05). In terms of muscle fiber length, the humeral head of pronator teres was the longest (7.65 ± 0.38 cm), and the radial compartment of ECU the shortest (3.56 ± 0.35 cm). It is noteworthy that muscle length does not always correlate with fiber length. For example, the FL of PT-h was the longest but the ML of PT-h was not, and the FL of ECU-r was the shortest but the ML of ECU-r was not. In stating that velocity is proportional to fiber length, it is implicit that the total excursion (active range) of a muscle is also proportional to fiber length (Lieber, 1993). Thus, increasing fiber length results in both increased muscle velocity and increased excursion. Also, the compartments followed the classic tradeoff between PCSA and fiber length; large PCSA correlates with short fibers (Fig. 3). In addition, neuromuscular compartments that clustered together in this graph were architecturally similar.

The FL/ML ratio could be seen as a relative measure of design preference for excursion (high ratio) or force production (low ratio). For example, if a muscle contains fibers that span the entire muscle length (FL/ML ratio = 1), it is more proper for excursion compared with a muscle that has fibers spanning only half the muscle length (FL/ML ratio = 0.5). The relatively low FL/ML ratios of the neuromuscular compartments (0.27 ± 0.08, range 0.18–0.39) in these five selected forearm muscles demonstrated that these compartments would be considered capable of force production preference in terms of design. This ratio is useful because it is independent of the absolute magnitude of muscle fiber length and permits design comparisons across muscles.

Our work offers data representative of the functional capability of the neuromuscular compartments of the five selected forearm muscles based on gross and microscopic measurements. In addition to improving our understanding of muscle anatomy and function, elucidation of forearm neuromuscular compartments architecture may ultimately provide information useful for selection of muscle subdivisions used in tendon transfer. To substitute a lost muscle function, it would seem reasonable to select a donor muscle with similar architectural properties as the original muscle, although numerous other factors may influence the donor selection, including donor muscle availability, donor muscle morbidity, preoperative strength, integrity, expendability, synergism, transfer route and direction, as well as surgeon experience and preference (Lieber & Friden, 2000, 2001). In addition, muscle force is always expressed externally as a joint torque, the product of muscle force and moment arm (Lieber, 1993). Lim et al. (1999, 2006) clarified the architectural properties of FCU neuromuscular compartments (listed in Table 2),and then described the clinical use of a pedicled or free split FCU transfer to provide independent finger and thumb extension based on these values, for which they felt the match of donor and recipient muscle was suitable. We also demonstrated the possibility and efficacy of splitting FCR transfer to restore independent finger and thumb extension thumb and finger extension (Fig. 4).

Table 2.

Muscle fiber length and PCSA of FCR from other studies ( ± S)

Compartments	FL (cm)	PCSA (cm2)	Authors
FCR	5.21 ± 0.20	1.90 ± 0.30	Hua et al. (1999) (n = 6)
FCR-r	5.27 ± 0.21	1.28 ± 0.15
FCR-u	5.14 ± 0.19	0.61 ± 0.19
FCU	4.23 ± 0.05	3.36 ± 0.02	Lim et al. 1999a, b; (n = 3)
FCU-r	4.29 ± 0.08	1.65 ± 0.05
FCU-u	4.18 ± 0.02	1.71 ± 0.04
ECRB	6.17 ± 0.27	3.3 ± 0.3	Abrams et al. 2005 (n = 10)
ECRB-r	–	–
ECRB-u	–	–
ECU	5.07 ± 0.25	2.60 ± 0.71	Lieber et al. 1992 (n = 5)
ECU-r	–	–
ECU-u	–	–
PT	7.02 ± 0.49	3.5 ± 0.4	Abrams et al. 2005 (n = 10)
Humeral head	7.19 ± 0.52	3.3 ± 0.3
Ulnar head	4.14 ± 0.25	0.4 ± 0.1

Values are expressed as mean ± SD. Muscle length and fiber length have been normalized to a sarcomere length of 2.7 μm. Abbreviations are the same as in the text.

PSCA, physiological cross-sectional area; r, radial compartment; u, ulnar compartment.

Fig. 4.

Preoperative and postoperative figures of reconstructing the extension of thumb and digit by split flexor carpi radialis tendon transfer. The FCR tendon was split into ulnar and radial compartments along the central tendon up to the proximal dominant vascular pedicle in the proximal quarter of the muscle belly. The radial head was woven to the four extensor digitorum communis tendons and the ulnar head was transferred extensor pollicis longus tendon. (A) Preoperative extension of the digits (A), extension of the digits 3 years postoperatively (C) and extension of the thumb 3 years postoperatively (D). Good function with no morbidity of the donor site 3 years postoperatively.

Conclusions

Using modified Sihler's technique, this research demonstrated that each compartment of these selected forearm muscles has its own neurovascular supply after being split along its central tendon. Data of the architectural properties of each neuromuscular compartment provide insight into the ‘design’ of their functional capability. In addition to improving our understanding of muscle anatomy and function, elucidation of forearm neuromuscular compartments architecture may ultimately provide information useful for selection of muscle subdivisions used in tendon transfer.

Disclosures

None of the authors has a financial interest in any of the products, devices or drugs mentioned in this article.