ARTICLE |
Correspondence to: Fatima PedrosaDomellöf, Dept. of Integrative Medical Biology, Section of Anatomy, Umeå University, SE-901 87 Umeå, Sweden. E-mail: Fatima.Pedrosa-Domellof@anatomy.umu.se
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Summary |
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Muscle spindle density is extremely high in the deep muscles of the human neck. However, there is a paucity of information regarding the morphology and immunoreactivity of these muscle spindles. The objective of this study was to investigate the intrafusal fiber content and to assess the myosin heavy chain (MyHC) composition of muscle spindles from human deep neck muscles. In addition to the conventional spindles containing bag1, bag2, and chain fibers (b1b2c spindle), we observed a number of spindles lacking bag1 (b2c spindle) or bag2 (b1c spindle) fibers. Both bag1 and bag2 fibers contained slow tonic MyHCs along their entire fiber length and MyHCI, MyHCIIa, embryonic, and -cardiac MyHC isoforms along a variable length of the fibers. Fetal MyHC was present in bag2 fibers but not in bag1 fibers. Nuclear chain fibers contained MyHCIIa, embryonic, and fetal isoforms with regional variations. We also compared the present data with our previous results obtained from muscle spindles in human biceps brachii and the first lumbrical muscles. The allotment of numbers of intrafusal fibers and the MyHC composition showed some muscle-related differences, suggesting functional specialization in the control of movement among different human muscles. (J Histochem Cytochem 51:175186, 2003)
Key Words: human, muscle spindle, deep neck muscles, mATPase, MyHC, intrafusal fibers
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Introduction |
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MUSCLE SPINDLES are important for the control of movement and posture in mammals. Mice lacking these receptors are unable to support their weight and have an abnormal posture (
Muscle spindles contain specialized fibers, called intrafusal fibers, innervated by both sensory neurons and -motor neurons. There are three types of intrafusal fibers, nuclear bag1, nuclear bag2, and nuclear chain fibers, classified according to their distinct myosin ATPase (mATPase) reactions (
In an attempt to correlate muscle spindle structure and function in human muscles, the fiber type content, general morphology, and MyHC composition of muscle spindles in the deep muscles of the human neck were examined and compared with the data available for other human muscles.
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Materials and Methods |
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Deep muscles of the neck (rectus capitis posterior major, rectus capitis posterior minor, obliquus capitis inferior and obliquus capitis superior) were collected at autopsy from two females, ages 26 and 17, three males, ages 55, 21, and unknown, without known muscle related disorders. The samples were collected according to the ethical recommendations of the Swedish Transplantation Law, with the approval of the Medical Ethical Committee, Umeå University.
The muscle samples were mounted, rapidly frozen in propane chilled with liquid nitrogen, and stored at -81C until sectioned. Frozen specimens were serially sectioned at -23C using a Reichert Jung cryostat (Leica; Nussloch, Germany). Sections to be used for demonstration of mATPase activity after preincubation at pH 10.4, 4.6, and 4.3 (
Antibodies and Labeling
Monoclonal antibodies specific for different MyHC isoforms were used (Table 1). The specificities of these antibodies have been carefully assessed for human muscles (
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Immunocytochemistry
Sections were processed for immunocytochemistry as previously described (
All sections were examined and photographed with a Nikon microscope (Eclipse, E800; Tokyo, Japan) equipped with a Spot camera (RT color; Diagnostic Instruments, Sterling Heights, MI).
Survey
Twenty-two muscle specimens were sectioned and stained. A total of 199 muscle spindles were examined, and 36 of these were studied in consecutive serial transverse sections covering a distance of approximately 2 mm. Three regions were examined: the A region, including equator and juxtaequatorial parts, containing the periaxial fluid space; the B region, extending from the end of the periaxial fluid space to the end of the capsule; and the C region, corresponding to the extracapsular portion of the spindle (
Statistical Analyses
StatView software (3rd ed; SAS Institute, Cary, NC) was used for statistical analysis. Statistic comparison of the intrafusal fiber numbers between different muscles was performed with an unpaired t-test. Differences were considered significant at p<0.05.
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Results |
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General Morphological Features
The 199 muscle spindles studied contained a total of 1039 intrafusal fibers. Two hundred and two fibers were classified as bag1 fibers, 185 as bag2, and 649 as chain fibers. The remaining three fibers were considered as exceptions because of their abnormal histochemical or immunocytochemical staining patterns (see below). The number of intrafusal fibers per spindle varied considerably between four and 15 (Table 2). On average, there were 7.2 (range 415) fibers per spindle and in each spindle there were 1.3 (range 03) bag1, 1.0 (range 04) bag2, and 4.9 (range 211) chain fibers.
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Although most spindles studied contained all three fiber types (b1b2c spindle), 13% of the spindles contained only one type of bag fiber (Fig 1); 10.4% lacked bag2 fibers (b1c spindle) whereas 2.6% lacked bag1 (b2c spindle). Two of 12 b1c spindles were part of two pairs of paired spindles, whereas the remaining were single receptors. These b1c spindles were found in three different subjects.
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Only 20 of 116 muscle spindles examined in the A and/or B regions, where the full complement of intrafusal fibers is present, presented a unique allotment of numbers of bag1, bag2, and chain fibers. Twenty-one muscle spindles had exactly the same intrafusal fiber content: one bag1, one bag2, and four chain fibers.
In transverse sections, nuclear bag fibers were in general much larger than nuclear chain fibers. The diameter of the two types of bag fibers was most often quite similar and rather constant along their length. However, some fibers exhibited dramatic changes in diameter along their length (Fig 2). The nuclear bag fibers usually extended beyond the capsule, whereas the chain fibers usually ended within the capsule, yet some exceptions were observed. In seven spindles, bag2 fibers terminated surprisingly in the A region at least in one pole, whereas the bag1 and chain fibers continued further along the spindle. In one spindle, we observed nine chain fibers together with one bag1 and one bag2 fiber in the C region.
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Most spindles appeared as single isolated receptors, but 22% of the total number of muscle spindles and 36% among those followed along serial consecutive sections occurred in different forms of linkage. The linked forms observed in the present study generally fitted the previous descriptions of parallel and paired muscle spindles in cat neck muscles (
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In two spindles, at the end of the polar region, bag1 fibers adhered to the capsular wall, exhibiting no myofibrils for a short distance, and then parted from the wall to continue further outside the capsule (Fig 4).
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mATPase Activity
The mATPase staining features of nuclear bag1, bag2, and chain fibers at different pH values were generally in accordance with our earlier report in human biceps brachii muscle (
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One bag fiber with unusual mATPase staining was encountered. This bag fiber displayed exactly the same staining features as bag1 fibers in the intracapsular region (Fig 5A, Fig 5B, Fig 5D, Fig 5E, Fig 5G, and Fig 5H). In the extracapsular region, however, this fiber stained like a bag2 fiber (Fig 5C, Fig 5F, and Fig 5I) and it was therefore called "mixed bag1/bag2" fiber.
Immunoreactivity
The staining patterns observed in the A, B, and C regions of nuclear bag1, bag2, and chain fibers with a battery of nine antibodies are summarized in Fig 6.
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Nuclear Bag1 Fibers
All nuclear bag1 fibers were strongly stained with anti-MyHCsto (Fig 5J5L) and showed no staining with anti-MyHCfet (Fig 7M7O) along their entire length. The vast majority of bag1 fibers showed similar staining patterns with the two anti-MyHCI antibodies. Their staining intensity with both anti-MyHCI/1st and anti-MyHCI/2nd MAbs gradually increased along the fiber length from the A to the C region (Fig 7A7F). Bag1 fibers were weakly stained with anti-MyHCI/3rd+IIa* only in the outer B and C regions (Fig 7G7I). Clearly, bag1 fibers reacted with anti-MyHCIIa antibody (Fig 7J7L). The number of positive fibers was much higher in the A and B regions than that in the C region, in which only three of 31 bag1 fibers were stained. Bag1 fibers usually showed no staining with anti-MyHC"all except IIx" in the equatorial region, and they were either unstained or weakly stained in the juxtequatorial region. Their staining intensity gradually increased along the fiber length from the B into the C region (Fig 5M5O). The majority of the bag1 fibers showed weak to moderate staining with anti-MyHCemb in the A region, fewer fibers were stained in the B region, and only 29% of the fibers were stained in the C region (Fig 5P5R). Approximately one half of the bag1 fibers in the A region and 16% in the B region were labeled by anti-MyHC-c, whereas they showed no staining in the C region (Fig 7P7R).
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Nuclear Bag2 Fibers
Nuclear bag2 fibers were strongly and evenly stained with anti-MyHCsto along their entire fiber length (Fig 5J5L) except for three fibers that showed less reactivity at their tapering ending in the C region. Bag2 fibers were strongly and homogeneously stained with anti-MyHCI/1st and anti-MyHCI/2nd in both the C and B regions (Fig 7B, Fig 7C, Fig 7E, and Fig 7F). In the A region, 97% and 94% of the bag2 fibers were stained with anti-MyHCI/1st (Fig 7A) and anti-MyHCI/2nd (Fig 7D), respectively. In general, bag2 fibers were unlabeled by anti-MyHCI/3rd+IIa* in the inner A region and weakly labeled along the remaining of their length (Fig 7G7I). Although bag2 fibers mostly did not react with anti-MyHCIIa, a number of fibers were weakly or very weakly stained in the A and B regions (Fig 7J7L). The staining intensity of bag2 fibers with anti-MyHC"all except IIx" gradually increased along the fiber length from the A to the C regions (Fig 5M5O). Bag2 fibers did not react with anti-MyHCfet in the C region but 15% of these fibers were stained in both the A and B regions (Fig 7M7O). The majority of bag2 fibers in the A and B regions were weakly or moderately stained with anti-MyHCemb (Fig 5P5R). Bag2 fibers generally showed a higher level of reactivity with anti-MyHC-c than with bag1 fibers in the same region (Fig 7P7R).
Nuclear Chain Fibers
The nuclear chain fibers showed no staining with anti-MyHCsto, anti-MyHCI/1st, anti-MyHCI/2nd, and anti-MyHC-c but were strongly stained with anti-MyHC- "all except IIx" (Fig 5 and Fig 7). Almost all chain fibers showed strong staining with anti-MyHCI/3rd+IIa* (Fig 7G7I) and anti-MyHCIIa (Fig 7J7L). The chain fibers that were less stained with anti-MyHCI/3rd+IIa* showed also absent or weak staining with anti-MyHCIIa, except in one spindle where two chain fibers were weakly stained by anti-MyHCI/3rd+IIa* but strongly stained by anti-MyHCIIa. Nineteen percent of chain fibers in the A region, 47% in the B region and 64% in the C region showed no staining with anti-MyHCfet, whereas the remainder generally showed decreasing levels of staining intensity from the A to C regions (Fig 7M7O). Chain fibers showed much higher staining intensity to anti-MyHCemb than bag fibers, although a few of chain fibers in all three regions were negative (Fig 5P5R). As a general rule, fibers negative to anti-MyHCemb were also negative to anti-MyHCfet.
Exceptions
Although most intrafusal fibers clearly fitted into one of the three fiber types with the general staining patterns described above, two atypical intrafusal fibers were encountered in one spindle. These two fibers, seen in the A region, were similar to chain fibers in diameter and showed low mATPase activity at pH 10.4 and high activity at pH 4.3. They showed no staining with anti-MyHCfet, weak staining with anti-MyHCsto, and moderate to strong staining by all the other MAbs used in the present study (Fig 8).
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Discussion |
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The major highlights provided by the present study regarding the spindles in human deep neck muscles can be summarized as follows: (a) some spindles lacked bag2 fibers; (b) there was a low incidence of spindles with a unique allotment of intrafusal fiber types; (c) there was a low number of intrafusal fibers per spindle; (d) the content of MyHCemb and MyHCfet varied along the length of nuclear chain fibers; and (e) there were wide variations in MyHC composition among intrafusal fibers.
One-bag-fiber Spindles
Three categories of muscle spindles, b1b2c, b1c, and b2c, were identified. Our findings show that human neck muscle spindles can lack either bag1 or bag2 fibers. Moreover, 10 of 12 b1c spindles existed as single isolated receptors, indicating that the b1c spindles in human neck muscles are in fact rather common and are not likely to represent a developmental deviation. The ability to distinguish between b1c and b2c spindles relies on the precise classification of the two types of bag fibers. In the outer A and B regions, the bag2 fiber can be clearly distinguished from the bag1 fiber by its higher staining intensity after preincubation at pH 4.6 and pH 4.3 (Fig 1). Moreover, immunocytochemistry can also be used as an additional way of identifying intrafusal fiber types, and therefore the use of both these criteria in the present study allowed a reliable classification of nuclear bag1 and bag2 fibers.
The absence of bag2 fibers has previously been reported in a small number of muscle spindles in human biceps brachii muscle (
One-bag-fiber spindles have been described in the cat (
Intrafusal Fiber Type and Number
We have previously shown that each muscle spindle in the human biceps brachii has a unique identity, revealed in part by a unique allotment of numbers of bag1, bag2, and chain fibers (
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MyHC Composition in Intrafusal Fibers
This is, to the best of our knowledge, the first investigation to examine the MyHC composition of intrafusal fibers in the deep muscles of the human neck. The immunocytochemical staining profiles of the intrafusal fibers reported here generally paralleled those of human biceps brachii (
The most striking difference was the variation in MyHCemb and MyHCfet content of nuclear chain fibers. The distribution of the embryonic and fetal myosin varied between the chain fibers from different spindles, and along the length of a given chain fiber. These variations seemed to correlate with the variability in mATPase activity noted along the length of individual nuclear chain fibers in the neck muscle spindles. Nuclear chain fibers usually have uniform mATPase activity and a homogeneous MyHC composition along their entire length in muscle spindles of human biceps brachii (
There were wide variations in MyHC composition among the intrafusal fibers studied. In fact, we found more variation in the staining patterns along the intrafusal fibers in the deep neck muscles than in the biceps brachii, as shown in Fig 6. For example, staining with MAb A4.840 did generally increase along the length of nuclear bag1 fibers, but the transition in staining level was spread along the A and B regions. The widely variable staining features revealed in the present study are likely to reflect a wider range of contractile properties of the intrafusal fibers and the more complex architecture and functions of these small deep neck muscles. The deep neck muscles have limited lever action because of their small size (
The high muscle spindle density and the special features of the muscle spindles in the deep neck muscles allow not only great precision of movement but also adequate proprioceptive information needed both for control of head position and movements and for eye/head movement coordination (
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Acknowledgments |
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Supported by grants from the Medical Faculty of Umeå University and by The Swedish Research Council.
We thank Drs N. Rubinstein, D.A. Fischman, G.S. ButlerBrowne, J.J. Leger, and S. Schiaffino for kindly providing antibodies. The excellent technical assistance of Margaretha Enerstedt is also acknowledged.
Received for publication May 17, 2002; accepted October 2, 2002.
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