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Myology
Myology M. Navarro, J. Ruberte and A. Carretero
Muscles are organized in cellular structures that are capable of contraction and generation of strenght. Muscles are arranged into functional groups to enable either maintenance of body posture or produce a coordinated movement. Depending on their morphology and physiology, muscles are classified as smooth or striated. Smooth muscles are responsible for contraction of the viscera, glands and, blood and lymphatic vessels, whereas striated muscle forms the cardiac muscle and skeletal muscles. This chapter describes the form, structure and the most important functions of mouse skeletal muscles.
■ ■ FORM AND STRUCTURE OF MUSCLES In general, skeletal muscles are formed by a belly and two tendons. The contractile muscle fi bers are located in the muscle belly. The tendons attach the muscle to the skeleton, facilitating the transmission of forces originated in the muscle belly. Besides locomotion of certain body parts or the whole body, skeletal muscles also form the thoracic and abdominal wall and their contraction helps breathing, defecation and urination. Occasionally, muscles have two or more muscle bellies, named as digastric and polygastric muscles, such as the m. digastricus of the head and the m. rectus abdominis, respectively. Fusiform muscles are those in which all the muscle belly fibers are arranged parallel to each other. An example of the fusiform muscle is m. biceps brachii. Some muscles have tendinous tissue within the muscle belly and the muscle fi bers are inserted to the intramuscular tendons forming angles, which gives them a feather-like appearance, hence the name of penniform muscles. According to the arrangement of fi bers, penniform muscles can be classified as unipennate muscles, where muscle fi bers are orientated in a single direction (for example, the m. psoas minor); bipennate muscles, where muscle fibers are orientated in two different directions (for example, the m. extensor digitorum communis); and multipennate muscles, in which fi bers are orientated in different directions (for example, the m. subscapularis). Muscle fibers are coated by a series of connective tissue sheaths through which are distributed the blood vessels and nerves (Figs. 4-1 and 4-9). The epimysium is the densest connective tissue sheath which delimits the different muscles and facilitates their sliding movement between them. Every muscle is divided into fascicles by
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the perimysium, which groups together a variable number of muscle fibers. The perimysium is very important for fine control of muscle contraction. Finally, each muscle fiber is surrounded by a fine network of collagen called the endomysium. These connective tissue sheaths are continuous with the tendons which are very poorly vascularized and have a high tensile strength and tear resistance. Each muscle belly usually has one tendon of origin and one tendon of insertion. The tendon of origin correspond to the more medial or dorsal attachment in the trunk and the more proximal attachment in the muscles of the limbs. Muscles with two or three bellies, for example the m. biceps brachii and m. triceps brachii, have two or three tendons of origin. Bicaudal or polycaudal muscles, the muscles of the digits for example, have various insertion tendons. Tendons typically have the appearance of a cord of white connective tissue, but in wide muscles they may also take on the form of a broad sheet called aponeurosis, as in the m. latissimus dorsi. There are some exceptions in which muscles are not attached to bones, as in the case of cutaneous muscles and natural sphincter muscles. Except in these cases, the connective tissue fibers of the tendon are otherwise continuous with Sharpey’s fibers of the bone periosteum. Some muscles have in their tendons specialized structures that facilitate the sliding of muscles over joints, and these are: the synovial bursae, synovial tendon sheaths and sesamoid bones. The fasciae are also auxiliary structures that surround a group of muscles and can serve as a surface of origin or insertion for other muscles. Fasciae also cover the entire body of the mouse and are arranged in two layers, a loose superfi cial fascia which is located subcutaneously and a thicker deep fascia. In some regions of the body, the superficial fascia also includes cutaneous muscles. Tendinous retinacula are specialized thickenings of the fascia in certain locations, such as in the flexor or extensor articular surfaces, where they help tendons to keep in position.
■ ■ THE SKELETAL MUSCLE FIBER Skeletal muscle cells or fibers are highly elongated cells with a very elastic and resistant plasma membrane, called the sarcolemma. Fibers are characterized by the presence of numerous nuclei located at the periphery of the cell, hence muscle fibers are described as a syn63
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cytium. These cells present a large number of myofibrils (Figs. 4-2 and 4-3). Myofibrils are divided into contractile units, or sarcomeres, that are delimited by Z lines, giving the typical striated appearance of the muscle fiber. Within the sarcomeres there are thick myosin and thin actin myofilaments, which are responsible for muscle contraction (Fig. 4-3). Thin myofilaments consist mainly of F-actin (Fig. 4-2) and other associated proteins (troponin, tropomyosin) and are anchored in the Z line, which is rich in α-actin. Other proteins are also found in the Z line, such as desmin (Fig. 4-2), which helps maintain the structural and mechanical integrity of the cell, connecting the sarcomere to the sarcolemma and other subcellular structures. Each thick myofilament is formed by several myosin molecules (Figs. 4-2 and 4-3), each of which consists of two heavy chains in turn associated with two light chains. The myosin filaments are anchored in the center of the sarcomere at the M line. The central zone of the sarcomere (the A band), where the myosin is situated, is darker (electron-dense) in transmission electron microscopy. By contrast, the area which contains only actin (the I band), presents a more clear or electron-lucent appearance (Fig. 4-3). The H band is the area at the center of the A band where there is only myosin (Fig. 4-2). In the rest of the A band the actin and myosin filaments are intertwined (Fig. 4-3). In this zone, the movement of the myosin heads slides actin filaments towards the center of the sarcomere, thereby shortening the sarcomere and the muscle fiber to generate force. Depending on their rate of contraction, biochemistry and ultrastructure, two basic types of skeletal muscle fiber can be delineated: slow twitch fibers (type I) and fast twitch fibers (type II). Moreover, type II fibers can be subdivided into subtypes such as IIA, IIB and intermediates, depending on their content in myosin heavy chain isoforms. Type I fibers use oxidative phosphorylation as a source of energy and therefore have more mitochondria (Figs. 4-4 and 4-5). Muscles with type I fibers contract more slowly and are more resistant to fatigue. Slow-twitch fi bers are also more vascularized and store more lipids and myogloblin in the sarcoplasm. This, coupled with the relatively reduced density of myofi brils, gives a more reddish color to the muscle. By contrast, Type II fibers use in general, anaerobic metabolism to generate ATP. Ultrastructurally, type II fibers contain more glycogen granules and have less mitochondria and lipid droplets than type I fibers (Figs. 4-4 and 4-5). Therefore, muscles that contain mainly type II fibers have a whitish color. In fact, muscles are composed of a mixture of fiber types, being a mosaic of both type I and type II fi bers. The percentage of type I and II fi bers in the same muscle may vary over time, changing from slow to fast, or vice versa, depending on the degree of exercise. Anti-myosin slow or anti-myosin fast chain antibodies can be used to differentiate type I and type II fibers, respectively (Fig. 4-4). In addition, type I and II fibers can be distinguished by preincubation at acidic pH which inhibits the activity of myosin ATPase in the type II fibers (Fig. 4-4).
Succinate dehydrogenase (SDH) and the reduced form of nicotinamide adenine dinucleotide (NADH) can also be used to identify the oxidative potential of muscle fibers, which is higher in type I fibers (Fig. 4-5). These histochemical techniques mark mitochondria in the sarcoplasm of muscle fibers (Fig. 4-5). Mitochondria inside muscle fibers can also be visualized directly by transmission electron microscopy and by the use of fluorescent probes that accumulate in functional mitochondria (for example MitoTracker®) (Fig. 4-5). The glycolytic activity of muscle fi bers is easily identifi ed by visualizing the activity of glycerol-phosphate dehydrogenase (GPDH). Type II fibers not only have more GPDH activity, but also a greater accumulation of glycogen which can be visualized by PAS staining (Fig. 4-6). Muscle fibers are formed by the fusion of myoblasts, some of which remain in the mature muscle as undifferentiated cells known as satellite cells (Fig. 4-7). These cells are responsible for muscle repair and muscle development after birth. Satellite cells are located beneath the basal lamina, but overlying the muscle fibers, and are thus in direct contact with the sarcolemma of muscle fibers. Satellite cells have very little cytoplasm and a nucleus distinguished by the presence of abundant heterochromatin (Fig. 4-7). They express specific markers, such as the transcription factor Pax7, which are not expressed in the nuclei of mature muscle fibers (Fig. 4-7). To produce muscle fiber contraction, calcium needs to be released into the sarcoplasm. Calcium is stored in the terminal cisternae of the sarcoplasmic reticulum bound to the acidic protein calsequestrin (Fig. 4-8). Sarcoplasmic reticulum cisternae are in contact with invaginations of the sarcolemma called T tubules, where they form structures known as triads. These are located between the A and I bands of muscle fibers (Fig. 4-8). T tubules can be easily identified in transmission electron microscopy, or by using an anti-GLUT4 antibody, the most important glucose transporter in the muscle fiber (Fig. 4-8). Skeletal muscle plays a crucial role in maintaining blood glucose. Muscle uses glucose for energy during contractile activity and represents the most important tissue for glucose uptake and metabolism during the postprandial period. At rest, GLUT4 is stored in tubulo-vesicular structures located around the nucleus, mainly in the Golgi complex. When stimulated by muscle contractions and/or insulin, GLUT4 is translocated to the plasma membrane and T tubules (Fig. 4-8). Skeletal muscles are supplied by arteries and veins that enter and leave the muscle belly at the level of one or more hilum (plural: hila). Muscular arteries eventually form a capillary plexus, which surrounds each of the muscle fibers, although the distribution of the capillary plexus is not equal for each fiber forming the muscle, as capillary density depends on the muscle fi ber type (Fig. 4-9). Type I fibers are aerobic and are more vascularized than type II fibers, which are anaerobic. For visualization, the capillary endothelial cells of mouse muscle may be labeled with an anti-PECAM-1 (CD31) antibody.
4. Myology
Skeletal muscle is innervated by motor neurons which originate in the brain (cranial nerves) and the spinal cord (spinal nerves). Each muscle fiber is innervated by at least one motor neuron axon. The site of contact between the muscle fi ber and the axon is a specialized synaptic junction called the motor end-plate, which is responsible for the release of the neurotransmitter acetylcholine (Fig. 4-9). Each motor axon branches before reaching the end-plate contact with each muscle fi ber, thereby forming several coordinated axon terminals on adjacent muscle fibers (Fig. 4-9). This set of muscle fi bers that are innervated by a single axon is called a motor unit. Muscle fibers act according to the law of «all or nothing», that is they are contracted or relaxed, with no intermediate states between contraction or relaxation. Therefore, the degree of contraction of a muscle depends on the number of muscle fibers that are simultaneously contracted, that is, the number of motor units that are activated.
■ ■ COMPARATIVE ANATOMY OF MOUSE MUSCULATURE Depending on the effect produced by a muscle on a joint, the muscles of the mouse can be classified as extensors (increase the angle of the joint), flexors (decrease the angle of the joint), adductors (limb approximators), abductors (limb separators), rotators (within this class are included the supinator and pronator muscles, which orientate the palm in a dorsal or ventral direction, respectively) and elevators. The movements of a particular joint are produced by the coordinated action of several muscles, either simultaneously or one after another. The muscle
with the most strength and the most suitable insertion for a particular movement is known as the main motor muscle. The action of this principal muscle is modulated by agonistic and synergistic muscles which help the movement, whereas antagonistic muscles counteract movement. Overall, the mouse has a similar musculature, not only to other rodents, but also in comparison to other domestic mammals and man. Despite this similarity, several differences can be found in the muscles of the head. Firstly, being a rodent, the mouse has very developed pterygoid muscles. In particular, the m. pterygoideus lateralis, which brings the mandible forward to gnaw is very large. However, the mouse not only gnaws, but it chews, necessitating a m. masseter that is also very well developed (Fig. 4-10). The m. temporalis in mouse is small compared to the m. masseter. Unlike men, the auricular muscles and the muscles responsible for opening the nasal orifices (nares) are well developed (Fig. 4-10). Mice have a well-developed clavicle, similarly to what is found in man (Figs. 2-28, 4-11 to 4-13), and therefore neck muscles which in other animals are fused and form part of the m. brachiocephalicus are separated in mice. For example, the m. cleidocephalicus, the m. cleidomastoideus and the clavicular part of the m. deltoideus are all independent muscles in the mouse. The m. rhomboideus capitis is also present in mice and is more developed than in carnivores. Mice have the m. omohyoideus which is also present in humans and other domestic mammals, except carnivores (Figs. 4-13 and 4-15). Other important distinctive features of the mouse cervical muscles are that the m. sternomastoideus, is divided into two parts (Fig. 4-13), and that is also possible to distinguish two scalene
SUPERFICIAL MUSCLES OF THE MOUSE M. trapezius M. obliquus externus abdominis
M. levator nasolabialis
M. tensor fasciae latae
M. biceps femoris
Muscles of the tail
M. sternomastoideus M. deltoideus M. triceps brachii M. gastrocnemius M. extensor digitorum communis
M. flexor carpi ulnaris
M. tibialis cranialis
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muscles, the middle and dorsal (Fig. 4-11), and only two pectoral muscles, the superficial and deep (Fig. 4-13). Thoracic limb (forelimb) muscles are very similar to those of the carnivores and indeed, most pentadactyl animals. One fundamental difference with other animals, however, is that the mouse m. biceps brachii has two heads, the long and short (Figs. 4-14 and 4-15), as occurs in humans. The trunk muscles are morphologically similar to those of other mammals that have a well developed tail (Fig. 4-20). There are three major muscle groups that form the epaxial muscles or the erector muscles of the vertebral column: spinal and semispinal muscles, longissimus muscles and iliocostalis muscles (Figs. 4-18 and 4-20). The diaphragm also has a morphology similar to that of domestic mammals and man, although with a less developed central tendon, and in the mouse it is a mixed muscle with a preponderance of type II fibers (Fig. 4-19). The abdominal muscles of the mouse, the m. obliquus externus abdominis, m. obliquus internus abdominis and the m. transversus abdominis, are extremely thin and almost transparent, and consequently very diffi cult to separate from each other during dissection (Fig. 4-20). There are three major muscles in the sublumbar region of the mouse, the m. iliacus, m. psoas major and m. psoas minor, which together with the m. quadratus lumborum, form roof wall of the abdominal cavity. These three muscles are considered pelvic limb muscles as they are inserted into the lesser trochanter of the femur, similar to what is found in domestic mammals and man (Figs. 4-18 and 4-20).
In general, the musculature of the pelvic limb (hindlimb) is also similar to that of carnivores (Figs. 4-21 to 4-28). Notable features are that the mouse has highly developed tensor fasciae latae and adductor muscles. The m. biceps femoris, m. semimembranosus and the m. gracilis are all divided and have two heads (Figs. 4-21 and 4-22). Mice, like humans, also have well-developed m. soleus (Figs. 4-23 and 4-24) and m. flexor digitorum brevis in the hindpaw (Figs. 4-27 and 4-28). The main extensor muscles of the digits are the m. extensor digitorum longus, which divides in tendons for all digits, and the m. extensor digitorum lateralis, which tendons ends in digits II-V only (Fig. 4-25). The major flexor muscles are the m. flexor digitorum profundus, which has three heads and five tendons extending to all digits, and the m. flexor digitorum superficialis which inserts on digits II-V only (Figs. 4-23, 4-27 and 4-28). The m. flexor digitorum superficialis forms part of the common calcaneal tendon with the m. triceps surae, which is itself formed by the two heads of m. gastrocnemius (the medial and lateral) and the m. soleus (Figs. 4-23 and 4-24). The leg muscles are frequently analyzed when studying muscle phenotypes in mutant mice. The m. gastrocnemius is composed of a mixture of fiber types, with around 85-90% being fast twitch fibers. Slow twitch fibers are usually grouped in specific regions of the muscle (Fig. 4-24). The soleus muscle is also mixed, although in this case most of its fibers are slow type I fibers (Fig. 4-24). The percentage of muscle fibers and fast-slow ratio may vary depending on the strain of mouse tested.
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Figure 4-1. Connective tissue in skeletal muscle. A and B) M. quadriceps femoris. Masson’s trichrome stain (20X and 400X, respectively). C) Confocal laser microscopy image of endomysium. Immunodetection of laminin (green). Nuclei counterstained with TO-PRO 3 (blue) (400X). 1: Epimysium; 2: M. rectus femoris; 3: Vastus medialis; 4: Vastus lateralis; 5: Perimysium; 6: Muscle fascicle; 7: Endomysium; 8: Muscle fiber (or myofiber); 9: Femur.
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Figure 4-2. Muscle fiber (1,000X). A) Hematoxylin-eosin stain. B, C and D) Confocal laser microscopy images. B: Desmin (orange). C: Phalloidin (red). D: Slow myosin (green). Nuclei counterstained with TO-PRO 3 (blue). 1: Nucleus; 2: Z Line; 3: H band; 4: A band; 5: I band.
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Figure 4-3. Myofibril structure. Transmission electron microscopy images (50,000X). A) Longitudinal section. B) Transverse section through A-band. 1: Sarcomere; 2: Z line; 3: M line; 4: A band; 5: I band; 6: H band; 7: Myosin myofilaments (thick filaments); 8: Actin myofilaments (thin filaments); 9: Sarcoplasmic reticulum.
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Figure 4-4. Type I and type II muscle fibers. A) Myosin ATPase activity (preincubation at pH 4.6) (400X). B) Immunodetection of fast myosin (red). Confocal laser microscopy image. Nuclei counterstained with TO-PRO 3 (blue) (400X). C) Immunodetection of slow myosin revealed with DAB (brown) (400X). D) Transmission electron microscopy image (3,000X). 1: Type I muscle fiber; 2: Type II muscle fiber; 3: Nuclei; 4: Mitochondria; 5: Capillary.
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Figure 4-5. Oxidative activity in type I and type II muscle fibers. A and C) SDH histochemistry (dark blue) (400X and 1,000X, respectively). B) NADH histochemistry (darck blue) (400X). D) MitoTracker® (green). Confocal laser microscopy image. M. quadriceps femoris. E and F) Transmission electron microscopy images of type I and type II muscle fibers, respectively. Transverse sections (30,000X). 1: Type I muscle fiber; 2: Type II muscle fiber; 3: Mitochondria.
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Figure 4-6. Glycolytic activity in type I and type II muscle fibers (400X). A) GPDH histochemistry (dark blue). B) PAS-hematoxylin stain. 1: Type I muscle fiber; 2: Type II muscle fiber; 3: Nerve; 4: Capillary (in the endomysium).
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Figure 4-7. Satellite cell. A) Pax7 positive nucleus (red). Transmission confocal laser microscopy image. Nuclei counterstained with TO-PRO 3 (blue). B) Transmission electron microscopy image (8,000X). 1: Nucleus of satellite cell; 2: Muscle fiber; 3: Nucleus of muscle fiber; 4: Sarcolemma; 5: Capillary (in the endomysium).
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Figure 4-8. Triad. A) Transmission electron microscopy image (50,000X). B) Confocal laser microscopy image. Immunodetection of calsequestrin (green). C) Immunodetection of GLUT4 (green), staining with phalloidin (red) and merged. Nuclei counterstained with TO-PRO 3 (blue). 1: A band; 2: I band; 3: Z line; 4: Nucleus; 5: T tubules (or transverse tubules); 6: Terminal cisternae (sarcoplasmic reticulum); 7: Mitochondria; 8: Glycogen.
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Figure 4-9. Vascularization and innervation of muscle fibers (400X). A) Immunodetection of PECAM-1 (brown). B) Neuromuscular junction. Immunodetection of acetylcholinesterase (brown). 1: Muscle fiber; 2: Capillaries; 3: Axon (motor neuron); 4: Axon terminal; 5: Motor end plate.
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Figure 4-10. Muscles of head. A) Muscles of head. Lateral view. B) Muscles of external ear. Dorsal view. 1: External nose; 2: Eyeball; 3: Auricle; 4: Anular cartilage; 5: Parietal bone; 6: Mandible; 7: M. levator nasolabialis; 8: M. buccinator; 9: M. temporalis; 10: M. masseter; 11: M. orbicularis oculi; 12: M. cleidocephalicus; 13: M. sternomastoideus; 14: M. omotransversarius; 15: M. sternohyoideus; 16: M. digastricus (caudal belly); 17: M. trapezius (cervical part); 18: M. cervicoauricularis rostralis; 19: M. cervicoauricularis caudalis; 20: Median fibrous raphe of the neck; 21: Facial nerve (VII); 22: Dorsal buccal branch (facial nerve); 23: Ventral buccal branch (facial nerve); 24: Auriculopalpebral nerve (facial nerve); 25: Lacrimal gland of third eyelid (harderian gland).
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Figure 4-11. Muscles of neck, shoulder, dorsum and thorax. Lateral view. A) Superficial muscles. Auricula and muscles of external ear were removed. B) M. trapezius was removed. 1: External acoustic meatus; 2: M. temporalis; 3: M. masseter; 4: M. cleidocephalicus; 5: M. sternohyoideus; 6: M. sternomastoideus; 7: M. trapezius (cervical part); 8: M. trapezius (thoracic part); 9: M. omotransversarius; 10: M. deltoideus (scapular part); 11: M. deltoideus (clavicular part); 12: M. latissimus dorsi; 13: M. supraspinatus; 14: M. infraspinatus; 15: M. triceps brachii (long head); 16: M. triceps brachii (lateral head); 17: M. teres major; 18: M. rhomboideus capitis; 19: M. rhomboideus cervicis; 20: M. rhomboideus thoracis; 21: M. serratus dorsalis cranialis; 22: M. pectoralis profundus; 23: M. splenius; 24: M. longissimus thoracis.
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Figure 4-12. Muscles of neck, shoulder, dorsum and thorax. Lateral view. A) M. rhomboideus and m. latissimus dorsi were removed. B) Deep muscles. Left thoracic limb, clavicle and pectoralis, splenius, omotransversarius, cleidocephalicus, cleidomastoideus and sternomastoideus muscles were removed. M. serratus dorsalis cranialis and m. serratus ventralis were sectioned (the dotted line indicates the limit of the muscles sectioned). 1: M. cleidocephalicus; 2: M. sternomastoideus; 3: M. sternohyoideus; 4: M. deltoideus (scapular part); 5: M. deltoideus (clavicular part); 6: M. supraspinatus; 7: M. infraspinatus; 8: M. teres major; 9: M. serratus ventralis cervicis; 10: M. serratus ventralis thoracis; 11: M. serratus dorsalis cranialis; 12: M. pectoralis profundus; 13: M. splenius; 14: M. biventer cervicis (m. semispinalis capitis); 15: M. complexus (m. semispinalis capitis); 16: M. spinalis et semispinalis thoracis; 17: M. longissimus capitis; 18: M. longissimus thoracis; 19: M. iliocostalis thoracis; 20: M. longus capitis; 21: M. obliquus capitis cranialis; 22: M. scalenus medius; 23: M. scalenus dorsalis; 24: M. rectus thoracis; 25: M. digastricus (caudal belly); 26: Facial nerve (VII); 27: Cervical nerves C2 and C3; 28: Roots of brachial plexus; 29: Seventh rib; 30: Intercostal nerve (ventral branch of thoracic nerve); 31: Long thoracic nerve.
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Figure 4-13. Muscles of neck and thorax. Ventral view. A) Superficial muscles. B) Right sternohyoideus and sternomastoideus muscles were removed. C) Deep muscles of neck. Right cleidocephalicus and left sternohyoideus and sternomastoideus (superficial part) muscles were removed. 1: M. masseter; 2: M. digastricus (rostral belly); 3: M. digastricus (caudal belly); 4: M. mylohyoideus; 5: M. sternohyoideus; 6: M. sternomastoideus (superficial part); 7: M. cleidocephalicus; 8: M. latissimus dorsi; 9: M. pectoralis superficialis; 10: M. pectoralis profundus; 11: M. biceps brachii; 12: M. triceps brachii; 13: M. omotransversarius; 14: M. deltoideus (clavicular part); 15: M. sternothyroideus; 16: M. omohyoideus; 17: M. sternomastoideus (deep part); 18: M. cleidomastoideus; 19: M. longus capitis; 20: Xiphoid cartilage; 21: Axillary lymph node; 22: Auricle; 23: Anular cartilage; 24: Trachea; 25: Hyoid bone; 26: Thyroid cartilage; 27: Cricoid cartilage; 28: Facial nerve (VII).
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Figure 4-14. Muscles of shoulder and arm. Isolated left thoracic limb. A) Lateral view. B) Medial view. C and D) Magnetic resonance images. Sagittal sections laterally and medially to the shoulder joint, respectively. 1: Clavicle (sectioned); 2: Spine of scapula; 3: Acromion; 4: Head of humerus; 5: Deltoid tuberosity; 6: Articular cavity (shoulder joint); 7: M. supraspinatus; 8: M. infraspinatus; 9: M. teres major; 10: M. subscapularis; 11: M. latissimus dorsi (sectioned); 12: M. triceps brachii (lateral head); 13: M. triceps brachii (long head); 14: M. triceps brachii (medial head); 15: M. tensor fasciae antebrachii; 16: M. biceps brachii (long head); 17: M. biceps brachii (short head); 18: M. brachialis; 19: M. deltoideus (clavicular part).
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Figure 4-16. Muscles of forearm and forepaw. Isolated left thoracic limb. M. triceps brachii was removed. A) Lateral view. B) Medial view. C) Magnetic resonance image. Sagittal section. D) Palmar view of the forepaw. 1: First digit; 2: Deltoid tuberosity; 3: Olecranon (ulna); 4: M. brachialis; 5: Fifth digit; 6: M. biceps brachii (long head); 7: M. biceps brachii (short head); 8: M. anconeus; 9: Radial nerve; 10: Median nerve; 11: Ulnar nerve; 12: M. triceps brachii; 13: M. extensor carpi radialis; 14: M. extensor digitorum communis (medially lies the m. extensor digitorum lateralis); 15: M: extensor carpi ulnaris; 16: M. abductor digiti I longus (or m. extensor carpi obliquus); 17: M. adductor digiti V; 18: M. pronator teres; 19: M. flexor carpi radialis; 20: M. flexor digitorum profundus; 21: M. flexor digitorum superficialis; 22: M. flexor carpi ulnaris.
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Figure 4-17. Muscles of thoracic wall. A) Roof of thoracic cavity. Ventral view. B) Histological section of mm. intercostales. Hematoxylin-eosin stain (20X). C) Floor of the thoracic cavity. Dorsal view. 1: M. longus colli; 2: M. longus capitis; 3: Mm. intercostales interni; 4: Mm. intercostales externi; 5: M. transversus thoracis; 6: M. omotransversarius; 7: M. omohyoideus; 8: M. subscapularis; 9: M. serratus ventralis thoracis; 10: M. latissimus dorsi; 11: M. sternohyoideus; 12: Diaphragm (sternal part); 13: Sixth thoracic vertebra; 14: Rib; 15: Sixth costal cartilage; 16: Fifth sternebra; 17: Xiphoid cartilage; 18: Suprascapular nerve; 19: Subscapular nerve; 20: Musculocutaneous nerve; 21: Axillary nerve; 22: Dorsal intercostal arteries and veins; 23: Intercostal nerves (ventral branches of thoracic nerves); 24: Internal thoracic artery; 25: Internal thoracic vein; 26: Ventral intercostal artery and vein.
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Figure 4-19. Diaphragm. A) Cranial view. B) Magnetic resonance image. Transverse section. C) Caudal view. D) Immunodetection of fast myosin (brown) (200X). E) Immunodetection of slow myosin (brown) (200X). 1: Spinal cord; 2: Aorta; 3: Caudal vena cava; 4: Esophagus; 5: Sternal part; 6: Costal part; 7: Central tendon; 8: Right crus (lumbar part); 9: Left crus (lumbar part); 10: Aortic hiatus; 11: Esophageal hiatus; 12: Lumbocostal arch; 13: Left phrenic nerve; 14: Right phrenic nerve; 15: Left cranial phrenic vein; 16: Right cranial phrenic vein; 17: Left lung; 18: Right lung; 19: Accessory lobe (right lung); 20: Type I muscle fiber; 21: Type II muscle fiber.
4. Myology
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Figure 4-20. Muscles of trunk. A) Epaxial muscles. Dorsal view. B) Muscles of abdominal wall. Ventral view. C) Sublumbar muscles. Ventral view. D) Muscles of tail. Dorsal view. E) Magnetic resonance image. Horizontal section. F) Muscles of tail. Ventral view. 1: M. spinalis et semispinalis thoracis; 2: M. longissimus thoracis; 3: M. longissimus lumborum; 4: Interscapular adipose tissue; 5: M. latissimus dorsi; 6: M. obliquus externus abdominis; 7: M. rectus abdominis; 8: M. pectoralis profundus; 9: M. rectus femoris (m. quadriceps femoris); 10: Femoral artery and vein; 11: M. transversus abdominis; 12: M. quadratus lumborum; 13: M. psoas minor; 14: M. psoas major; 15: M. iliacus; 16: M. gracilis; 17: Intercostal nerves (ventral branches of thoracic nerves); 18: Costoabdominal nerve (thirteenth intercostal nerve); 19: Femoral nerve; 20: Saphenous nerve; 21: Cranial pelvic aperture; 22: M. sacrocaudalis ventralis lateralis; 23: M. sacrocaudalis dorsalis lateralis; 24: M. sacrocaudalis dorsalis medialis; 25: Mm. intertransversarii dorsales caudae; 26: M. sacrocaudalis ventralis medialis; 27: Mm. intertransversarii ventrales caudae; 28: M. coccygeus; 29: Liver; 30: Stomach; 31: Right kidney; 32: Left kidney; 33: Penis; 34: Testicle; 35: M. bulbospongiosus; 36: Bulbourethral gland; 37: Pancreas; 38: Spleen; 39: Ilium; 40: Spinous process (caudal vertebrae).
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Figure 4-21. Muscles of hip joint and thigh. Left lateral view. A) Superficial muscles. B) M. tensor fasciae latae and m. biceps femoris were removed. 1: M. tensor fasciae latae; 2: M. biceps femoris; 3: M. semitendinosus; 4: M. gluteus medius; 5: M. gluteus superficialis; 6: M. semimembranosus (cranial part); 7: M. semimembranosus (caudal part); 8: M. coccygeus; 9: M. sacrocaudalis dorsalis lateralis; 10: M. quadratus femoris; 11: M. adductor magnus; 12: M. vastus lateralis (m. quadriceps femoris); 13: Greater trochanter (femur); 14: Third trochanter (femur); 15: Sciatic nerve; 16: Tibial nerve; 17: Common peroneal nerve; 18: Patellar ligament; 19: M. tibialis cranialis; 20: M. gastrocnemius (lateral head).
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Figure 4-22. Muscles of hip joint and thigh. Left medial view. A) Superficial muscles. B) M. gracilis was removed. 1: M. semitendinosus; 2: M. gracilis; 3: M. pectineus; 4: M. adductor magnus; 5: M. adductor longus; 6: M. semimembranosus (caudal part); 7: M. rectus femoris (m. quadriceps femoris); 8: M. vastus medialis (m. quadriceps femoris); 9: Mm. abdominis (sectioned); 10: M. coccygeus; 11: M. sacrocaudalis ventralis lateralis; 12: M. sacrocaudalis ventralis medialis; 13: M. iliacus; 14: M. psoas minor; 15: M. psoas major; 16: Femoral nerve; 17: Saphenous nerve; 18: Patellar ligament; 19: Pelvic symphysis.
4. Myology
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1: M. tibialis cranialis; 2: M. peroneus longus; 3: M. gastrocnemius (lateral head); 4: M. extensor digitorum longus; 5: M. extensor digitorum lateralis; 6: M. flexor digitorum superficialis; 7: M. soleus; 8: M. flexor digitorum lateralis (m. flexor digitorum profundus); 9: M. flexor digitorum medialis (m. flexor digitorum profundus); 10: M. tibialis caudalis (m. flexor digitorum profundus); 11: M. gastrocnemius (medial head); 12: Common calcaneal tendon; 13: M. vastus lateralis (m. quadriceps femoris); 14: M. semimembranosus (cranial part); 15: M. semimembranosus (caudal part); 16: Sciatic nerve; 17: Common peroneal nerve; 18: Tibial nerve; 19: Caudal sural cutaneous nerve (tibial nerve); 20: M. interosseous; 21: Cranial border (or tibial crest).
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Figure 4-24. Sural muscles. A) Left leg. Lateral view. Sural muscle insertions were cut. B) Isolated sural muscles. Cranial view. C, D and E) Immunodetection of fast myosin (red) in m. gastrocnemius and m. soleus (200X). Nuclei counterstained with SYTOX Green. 1: M. gastrocnemius (lateral head); 2: M. gastrocnemius (medial head); 3: M. flexor digitorum superficialis; 4: M. soleus; 5: Common calcaneal tendon; 6: Calcaneus; 7: M. tibialis cranialis.
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Figure 4-27. Muscles of hindpaw. Plantar view of left hindpaw.
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Muscles are organized in cellular structures that are capable of contraction and generation of strenght. Muscles are arranged into functional groups to enable either maintenance of body posture or produce a coordinated movement. Depending on their morphology and physiology, muscles are classified as smooth or striated. Smooth muscles are responsible for contraction of the viscera, glands and, blood and lymphatic vessels, whereas striated muscle forms the cardiac muscle and skeletal muscles. This chapter describes the form, structure and the most important functions of mouse skeletal muscles.
■ ■ FORM AND STRUCTURE OF MUSCLES In general, skeletal muscles are formed by a belly and two tendons. The contractile muscle fi bers are located in the muscle belly. The tendons attach the muscle to the skeleton, facilitating the transmission of forces originated in the muscle belly. Besides locomotion of certain body parts or the whole body, skeletal muscles also form the thoracic and abdominal wall and their contraction helps breathing, defecation and urination. Occasionally, muscles have two or more muscle bellies, named as digastric and polygastric muscles, such as the m. digastricus of the head and the m. rectus abdominis, respectively. Fusiform muscles are those in which all the muscle belly fibers are arranged parallel to each other. An example of the fusiform muscle is m. biceps brachii. Some muscles have tendinous tissue within the muscle belly and the muscle fi bers are inserted to the intramuscular tendons forming angles, which gives them a feather-like appearance, hence the name of penniform muscles. According to the arrangement of fi bers, penniform muscles can be classified as unipennate muscles, where muscle fi bers are orientated in a single direction (for example, the m. psoas minor); bipennate muscles, where muscle fibers are orientated in two different directions (for example, the m. extensor digitorum communis); and multipennate muscles, in which fi bers are orientated in different directions (for example, the m. subscapularis). Muscle fibers are coated by a series of connective tissue sheaths through which are distributed the blood vessels and nerves (Figs. 4-1 and 4-9). The epimysium is the densest connective tissue sheath which delimits the different muscles and facilitates their sliding movement between them. Every muscle is divided into fascicles by
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the perimysium, which groups together a variable number of muscle fibers. The perimysium is very important for fine control of muscle contraction. Finally, each muscle fiber is surrounded by a fine network of collagen called the endomysium. These connective tissue sheaths are continuous with the tendons which are very poorly vascularized and have a high tensile strength and tear resistance. Each muscle belly usually has one tendon of origin and one tendon of insertion. The tendon of origin correspond to the more medial or dorsal attachment in the trunk and the more proximal attachment in the muscles of the limbs. Muscles with two or three bellies, for example the m. biceps brachii and m. triceps brachii, have two or three tendons of origin. Bicaudal or polycaudal muscles, the muscles of the digits for example, have various insertion tendons. Tendons typically have the appearance of a cord of white connective tissue, but in wide muscles they may also take on the form of a broad sheet called aponeurosis, as in the m. latissimus dorsi. There are some exceptions in which muscles are not attached to bones, as in the case of cutaneous muscles and natural sphincter muscles. Except in these cases, the connective tissue fibers of the tendon are otherwise continuous with Sharpey’s fibers of the bone periosteum. Some muscles have in their tendons specialized structures that facilitate the sliding of muscles over joints, and these are: the synovial bursae, synovial tendon sheaths and sesamoid bones. The fasciae are also auxiliary structures that surround a group of muscles and can serve as a surface of origin or insertion for other muscles. Fasciae also cover the entire body of the mouse and are arranged in two layers, a loose superfi cial fascia which is located subcutaneously and a thicker deep fascia. In some regions of the body, the superficial fascia also includes cutaneous muscles. Tendinous retinacula are specialized thickenings of the fascia in certain locations, such as in the flexor or extensor articular surfaces, where they help tendons to keep in position.
■ ■ THE SKELETAL MUSCLE FIBER Skeletal muscle cells or fibers are highly elongated cells with a very elastic and resistant plasma membrane, called the sarcolemma. Fibers are characterized by the presence of numerous nuclei located at the periphery of the cell, hence muscle fibers are described as a syn63
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cytium. These cells present a large number of myofibrils (Figs. 4-2 and 4-3). Myofibrils are divided into contractile units, or sarcomeres, that are delimited by Z lines, giving the typical striated appearance of the muscle fiber. Within the sarcomeres there are thick myosin and thin actin myofilaments, which are responsible for muscle contraction (Fig. 4-3). Thin myofilaments consist mainly of F-actin (Fig. 4-2) and other associated proteins (troponin, tropomyosin) and are anchored in the Z line, which is rich in α-actin. Other proteins are also found in the Z line, such as desmin (Fig. 4-2), which helps maintain the structural and mechanical integrity of the cell, connecting the sarcomere to the sarcolemma and other subcellular structures. Each thick myofilament is formed by several myosin molecules (Figs. 4-2 and 4-3), each of which consists of two heavy chains in turn associated with two light chains. The myosin filaments are anchored in the center of the sarcomere at the M line. The central zone of the sarcomere (the A band), where the myosin is situated, is darker (electron-dense) in transmission electron microscopy. By contrast, the area which contains only actin (the I band), presents a more clear or electron-lucent appearance (Fig. 4-3). The H band is the area at the center of the A band where there is only myosin (Fig. 4-2). In the rest of the A band the actin and myosin filaments are intertwined (Fig. 4-3). In this zone, the movement of the myosin heads slides actin filaments towards the center of the sarcomere, thereby shortening the sarcomere and the muscle fiber to generate force. Depending on their rate of contraction, biochemistry and ultrastructure, two basic types of skeletal muscle fiber can be delineated: slow twitch fibers (type I) and fast twitch fibers (type II). Moreover, type II fibers can be subdivided into subtypes such as IIA, IIB and intermediates, depending on their content in myosin heavy chain isoforms. Type I fibers use oxidative phosphorylation as a source of energy and therefore have more mitochondria (Figs. 4-4 and 4-5). Muscles with type I fibers contract more slowly and are more resistant to fatigue. Slow-twitch fi bers are also more vascularized and store more lipids and myogloblin in the sarcoplasm. This, coupled with the relatively reduced density of myofi brils, gives a more reddish color to the muscle. By contrast, Type II fibers use in general, anaerobic metabolism to generate ATP. Ultrastructurally, type II fibers contain more glycogen granules and have less mitochondria and lipid droplets than type I fibers (Figs. 4-4 and 4-5). Therefore, muscles that contain mainly type II fibers have a whitish color. In fact, muscles are composed of a mixture of fiber types, being a mosaic of both type I and type II fi bers. The percentage of type I and II fi bers in the same muscle may vary over time, changing from slow to fast, or vice versa, depending on the degree of exercise. Anti-myosin slow or anti-myosin fast chain antibodies can be used to differentiate type I and type II fibers, respectively (Fig. 4-4). In addition, type I and II fibers can be distinguished by preincubation at acidic pH which inhibits the activity of myosin ATPase in the type II fibers (Fig. 4-4).
Succinate dehydrogenase (SDH) and the reduced form of nicotinamide adenine dinucleotide (NADH) can also be used to identify the oxidative potential of muscle fibers, which is higher in type I fibers (Fig. 4-5). These histochemical techniques mark mitochondria in the sarcoplasm of muscle fibers (Fig. 4-5). Mitochondria inside muscle fibers can also be visualized directly by transmission electron microscopy and by the use of fluorescent probes that accumulate in functional mitochondria (for example MitoTracker®) (Fig. 4-5). The glycolytic activity of muscle fi bers is easily identifi ed by visualizing the activity of glycerol-phosphate dehydrogenase (GPDH). Type II fibers not only have more GPDH activity, but also a greater accumulation of glycogen which can be visualized by PAS staining (Fig. 4-6). Muscle fibers are formed by the fusion of myoblasts, some of which remain in the mature muscle as undifferentiated cells known as satellite cells (Fig. 4-7). These cells are responsible for muscle repair and muscle development after birth. Satellite cells are located beneath the basal lamina, but overlying the muscle fibers, and are thus in direct contact with the sarcolemma of muscle fibers. Satellite cells have very little cytoplasm and a nucleus distinguished by the presence of abundant heterochromatin (Fig. 4-7). They express specific markers, such as the transcription factor Pax7, which are not expressed in the nuclei of mature muscle fibers (Fig. 4-7). To produce muscle fiber contraction, calcium needs to be released into the sarcoplasm. Calcium is stored in the terminal cisternae of the sarcoplasmic reticulum bound to the acidic protein calsequestrin (Fig. 4-8). Sarcoplasmic reticulum cisternae are in contact with invaginations of the sarcolemma called T tubules, where they form structures known as triads. These are located between the A and I bands of muscle fibers (Fig. 4-8). T tubules can be easily identified in transmission electron microscopy, or by using an anti-GLUT4 antibody, the most important glucose transporter in the muscle fiber (Fig. 4-8). Skeletal muscle plays a crucial role in maintaining blood glucose. Muscle uses glucose for energy during contractile activity and represents the most important tissue for glucose uptake and metabolism during the postprandial period. At rest, GLUT4 is stored in tubulo-vesicular structures located around the nucleus, mainly in the Golgi complex. When stimulated by muscle contractions and/or insulin, GLUT4 is translocated to the plasma membrane and T tubules (Fig. 4-8). Skeletal muscles are supplied by arteries and veins that enter and leave the muscle belly at the level of one or more hilum (plural: hila). Muscular arteries eventually form a capillary plexus, which surrounds each of the muscle fibers, although the distribution of the capillary plexus is not equal for each fiber forming the muscle, as capillary density depends on the muscle fi ber type (Fig. 4-9). Type I fibers are aerobic and are more vascularized than type II fibers, which are anaerobic. For visualization, the capillary endothelial cells of mouse muscle may be labeled with an anti-PECAM-1 (CD31) antibody.
4. Myology
Skeletal muscle is innervated by motor neurons which originate in the brain (cranial nerves) and the spinal cord (spinal nerves). Each muscle fiber is innervated by at least one motor neuron axon. The site of contact between the muscle fi ber and the axon is a specialized synaptic junction called the motor end-plate, which is responsible for the release of the neurotransmitter acetylcholine (Fig. 4-9). Each motor axon branches before reaching the end-plate contact with each muscle fi ber, thereby forming several coordinated axon terminals on adjacent muscle fibers (Fig. 4-9). This set of muscle fi bers that are innervated by a single axon is called a motor unit. Muscle fibers act according to the law of «all or nothing», that is they are contracted or relaxed, with no intermediate states between contraction or relaxation. Therefore, the degree of contraction of a muscle depends on the number of muscle fibers that are simultaneously contracted, that is, the number of motor units that are activated.
■ ■ COMPARATIVE ANATOMY OF MOUSE MUSCULATURE Depending on the effect produced by a muscle on a joint, the muscles of the mouse can be classified as extensors (increase the angle of the joint), flexors (decrease the angle of the joint), adductors (limb approximators), abductors (limb separators), rotators (within this class are included the supinator and pronator muscles, which orientate the palm in a dorsal or ventral direction, respectively) and elevators. The movements of a particular joint are produced by the coordinated action of several muscles, either simultaneously or one after another. The muscle
with the most strength and the most suitable insertion for a particular movement is known as the main motor muscle. The action of this principal muscle is modulated by agonistic and synergistic muscles which help the movement, whereas antagonistic muscles counteract movement. Overall, the mouse has a similar musculature, not only to other rodents, but also in comparison to other domestic mammals and man. Despite this similarity, several differences can be found in the muscles of the head. Firstly, being a rodent, the mouse has very developed pterygoid muscles. In particular, the m. pterygoideus lateralis, which brings the mandible forward to gnaw is very large. However, the mouse not only gnaws, but it chews, necessitating a m. masseter that is also very well developed (Fig. 4-10). The m. temporalis in mouse is small compared to the m. masseter. Unlike men, the auricular muscles and the muscles responsible for opening the nasal orifices (nares) are well developed (Fig. 4-10). Mice have a well-developed clavicle, similarly to what is found in man (Figs. 2-28, 4-11 to 4-13), and therefore neck muscles which in other animals are fused and form part of the m. brachiocephalicus are separated in mice. For example, the m. cleidocephalicus, the m. cleidomastoideus and the clavicular part of the m. deltoideus are all independent muscles in the mouse. The m. rhomboideus capitis is also present in mice and is more developed than in carnivores. Mice have the m. omohyoideus which is also present in humans and other domestic mammals, except carnivores (Figs. 4-13 and 4-15). Other important distinctive features of the mouse cervical muscles are that the m. sternomastoideus, is divided into two parts (Fig. 4-13), and that is also possible to distinguish two scalene
SUPERFICIAL MUSCLES OF THE MOUSE M. trapezius M. obliquus externus abdominis
M. levator nasolabialis
M. tensor fasciae latae
M. biceps femoris
Muscles of the tail
M. sternomastoideus M. deltoideus M. triceps brachii M. gastrocnemius M. extensor digitorum communis
M. flexor carpi ulnaris
M. tibialis cranialis
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muscles, the middle and dorsal (Fig. 4-11), and only two pectoral muscles, the superficial and deep (Fig. 4-13). Thoracic limb (forelimb) muscles are very similar to those of the carnivores and indeed, most pentadactyl animals. One fundamental difference with other animals, however, is that the mouse m. biceps brachii has two heads, the long and short (Figs. 4-14 and 4-15), as occurs in humans. The trunk muscles are morphologically similar to those of other mammals that have a well developed tail (Fig. 4-20). There are three major muscle groups that form the epaxial muscles or the erector muscles of the vertebral column: spinal and semispinal muscles, longissimus muscles and iliocostalis muscles (Figs. 4-18 and 4-20). The diaphragm also has a morphology similar to that of domestic mammals and man, although with a less developed central tendon, and in the mouse it is a mixed muscle with a preponderance of type II fibers (Fig. 4-19). The abdominal muscles of the mouse, the m. obliquus externus abdominis, m. obliquus internus abdominis and the m. transversus abdominis, are extremely thin and almost transparent, and consequently very diffi cult to separate from each other during dissection (Fig. 4-20). There are three major muscles in the sublumbar region of the mouse, the m. iliacus, m. psoas major and m. psoas minor, which together with the m. quadratus lumborum, form roof wall of the abdominal cavity. These three muscles are considered pelvic limb muscles as they are inserted into the lesser trochanter of the femur, similar to what is found in domestic mammals and man (Figs. 4-18 and 4-20).
In general, the musculature of the pelvic limb (hindlimb) is also similar to that of carnivores (Figs. 4-21 to 4-28). Notable features are that the mouse has highly developed tensor fasciae latae and adductor muscles. The m. biceps femoris, m. semimembranosus and the m. gracilis are all divided and have two heads (Figs. 4-21 and 4-22). Mice, like humans, also have well-developed m. soleus (Figs. 4-23 and 4-24) and m. flexor digitorum brevis in the hindpaw (Figs. 4-27 and 4-28). The main extensor muscles of the digits are the m. extensor digitorum longus, which divides in tendons for all digits, and the m. extensor digitorum lateralis, which tendons ends in digits II-V only (Fig. 4-25). The major flexor muscles are the m. flexor digitorum profundus, which has three heads and five tendons extending to all digits, and the m. flexor digitorum superficialis which inserts on digits II-V only (Figs. 4-23, 4-27 and 4-28). The m. flexor digitorum superficialis forms part of the common calcaneal tendon with the m. triceps surae, which is itself formed by the two heads of m. gastrocnemius (the medial and lateral) and the m. soleus (Figs. 4-23 and 4-24). The leg muscles are frequently analyzed when studying muscle phenotypes in mutant mice. The m. gastrocnemius is composed of a mixture of fiber types, with around 85-90% being fast twitch fibers. Slow twitch fibers are usually grouped in specific regions of the muscle (Fig. 4-24). The soleus muscle is also mixed, although in this case most of its fibers are slow type I fibers (Fig. 4-24). The percentage of muscle fibers and fast-slow ratio may vary depending on the strain of mouse tested.
4. Myology
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Figure 4-1. Connective tissue in skeletal muscle. A and B) M. quadriceps femoris. Masson’s trichrome stain (20X and 400X, respectively). C) Confocal laser microscopy image of endomysium. Immunodetection of laminin (green). Nuclei counterstained with TO-PRO 3 (blue) (400X). 1: Epimysium; 2: M. rectus femoris; 3: Vastus medialis; 4: Vastus lateralis; 5: Perimysium; 6: Muscle fascicle; 7: Endomysium; 8: Muscle fiber (or myofiber); 9: Femur.
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Figure 4-2. Muscle fiber (1,000X). A) Hematoxylin-eosin stain. B, C and D) Confocal laser microscopy images. B: Desmin (orange). C: Phalloidin (red). D: Slow myosin (green). Nuclei counterstained with TO-PRO 3 (blue). 1: Nucleus; 2: Z Line; 3: H band; 4: A band; 5: I band.
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Figure 4-3. Myofibril structure. Transmission electron microscopy images (50,000X). A) Longitudinal section. B) Transverse section through A-band. 1: Sarcomere; 2: Z line; 3: M line; 4: A band; 5: I band; 6: H band; 7: Myosin myofilaments (thick filaments); 8: Actin myofilaments (thin filaments); 9: Sarcoplasmic reticulum.
4. Myology
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Figure 4-4. Type I and type II muscle fibers. A) Myosin ATPase activity (preincubation at pH 4.6) (400X). B) Immunodetection of fast myosin (red). Confocal laser microscopy image. Nuclei counterstained with TO-PRO 3 (blue) (400X). C) Immunodetection of slow myosin revealed with DAB (brown) (400X). D) Transmission electron microscopy image (3,000X). 1: Type I muscle fiber; 2: Type II muscle fiber; 3: Nuclei; 4: Mitochondria; 5: Capillary.
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Morphological Mouse Phenotyping: Anatomy, Histology and Imaging
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Figure 4-5. Oxidative activity in type I and type II muscle fibers. A and C) SDH histochemistry (dark blue) (400X and 1,000X, respectively). B) NADH histochemistry (darck blue) (400X). D) MitoTracker® (green). Confocal laser microscopy image. M. quadriceps femoris. E and F) Transmission electron microscopy images of type I and type II muscle fibers, respectively. Transverse sections (30,000X). 1: Type I muscle fiber; 2: Type II muscle fiber; 3: Mitochondria.
4. Myology
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Figure 4-6. Glycolytic activity in type I and type II muscle fibers (400X). A) GPDH histochemistry (dark blue). B) PAS-hematoxylin stain. 1: Type I muscle fiber; 2: Type II muscle fiber; 3: Nerve; 4: Capillary (in the endomysium).
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Figure 4-7. Satellite cell. A) Pax7 positive nucleus (red). Transmission confocal laser microscopy image. Nuclei counterstained with TO-PRO 3 (blue). B) Transmission electron microscopy image (8,000X). 1: Nucleus of satellite cell; 2: Muscle fiber; 3: Nucleus of muscle fiber; 4: Sarcolemma; 5: Capillary (in the endomysium).
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Morphological Mouse Phenotyping: Anatomy, Histology and Imaging
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Figure 4-8. Triad. A) Transmission electron microscopy image (50,000X). B) Confocal laser microscopy image. Immunodetection of calsequestrin (green). C) Immunodetection of GLUT4 (green), staining with phalloidin (red) and merged. Nuclei counterstained with TO-PRO 3 (blue). 1: A band; 2: I band; 3: Z line; 4: Nucleus; 5: T tubules (or transverse tubules); 6: Terminal cisternae (sarcoplasmic reticulum); 7: Mitochondria; 8: Glycogen.
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Figure 4-9. Vascularization and innervation of muscle fibers (400X). A) Immunodetection of PECAM-1 (brown). B) Neuromuscular junction. Immunodetection of acetylcholinesterase (brown). 1: Muscle fiber; 2: Capillaries; 3: Axon (motor neuron); 4: Axon terminal; 5: Motor end plate.
4. Myology
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Figure 4-10. Muscles of head. A) Muscles of head. Lateral view. B) Muscles of external ear. Dorsal view. 1: External nose; 2: Eyeball; 3: Auricle; 4: Anular cartilage; 5: Parietal bone; 6: Mandible; 7: M. levator nasolabialis; 8: M. buccinator; 9: M. temporalis; 10: M. masseter; 11: M. orbicularis oculi; 12: M. cleidocephalicus; 13: M. sternomastoideus; 14: M. omotransversarius; 15: M. sternohyoideus; 16: M. digastricus (caudal belly); 17: M. trapezius (cervical part); 18: M. cervicoauricularis rostralis; 19: M. cervicoauricularis caudalis; 20: Median fibrous raphe of the neck; 21: Facial nerve (VII); 22: Dorsal buccal branch (facial nerve); 23: Ventral buccal branch (facial nerve); 24: Auriculopalpebral nerve (facial nerve); 25: Lacrimal gland of third eyelid (harderian gland).
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Figure 4-11. Muscles of neck, shoulder, dorsum and thorax. Lateral view. A) Superficial muscles. Auricula and muscles of external ear were removed. B) M. trapezius was removed. 1: External acoustic meatus; 2: M. temporalis; 3: M. masseter; 4: M. cleidocephalicus; 5: M. sternohyoideus; 6: M. sternomastoideus; 7: M. trapezius (cervical part); 8: M. trapezius (thoracic part); 9: M. omotransversarius; 10: M. deltoideus (scapular part); 11: M. deltoideus (clavicular part); 12: M. latissimus dorsi; 13: M. supraspinatus; 14: M. infraspinatus; 15: M. triceps brachii (long head); 16: M. triceps brachii (lateral head); 17: M. teres major; 18: M. rhomboideus capitis; 19: M. rhomboideus cervicis; 20: M. rhomboideus thoracis; 21: M. serratus dorsalis cranialis; 22: M. pectoralis profundus; 23: M. splenius; 24: M. longissimus thoracis.
4. Myology
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Figure 4-12. Muscles of neck, shoulder, dorsum and thorax. Lateral view. A) M. rhomboideus and m. latissimus dorsi were removed. B) Deep muscles. Left thoracic limb, clavicle and pectoralis, splenius, omotransversarius, cleidocephalicus, cleidomastoideus and sternomastoideus muscles were removed. M. serratus dorsalis cranialis and m. serratus ventralis were sectioned (the dotted line indicates the limit of the muscles sectioned). 1: M. cleidocephalicus; 2: M. sternomastoideus; 3: M. sternohyoideus; 4: M. deltoideus (scapular part); 5: M. deltoideus (clavicular part); 6: M. supraspinatus; 7: M. infraspinatus; 8: M. teres major; 9: M. serratus ventralis cervicis; 10: M. serratus ventralis thoracis; 11: M. serratus dorsalis cranialis; 12: M. pectoralis profundus; 13: M. splenius; 14: M. biventer cervicis (m. semispinalis capitis); 15: M. complexus (m. semispinalis capitis); 16: M. spinalis et semispinalis thoracis; 17: M. longissimus capitis; 18: M. longissimus thoracis; 19: M. iliocostalis thoracis; 20: M. longus capitis; 21: M. obliquus capitis cranialis; 22: M. scalenus medius; 23: M. scalenus dorsalis; 24: M. rectus thoracis; 25: M. digastricus (caudal belly); 26: Facial nerve (VII); 27: Cervical nerves C2 and C3; 28: Roots of brachial plexus; 29: Seventh rib; 30: Intercostal nerve (ventral branch of thoracic nerve); 31: Long thoracic nerve.
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Morphological Mouse Phenotyping: Anatomy, Histology and Imaging
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Figure 4-13. Muscles of neck and thorax. Ventral view. A) Superficial muscles. B) Right sternohyoideus and sternomastoideus muscles were removed. C) Deep muscles of neck. Right cleidocephalicus and left sternohyoideus and sternomastoideus (superficial part) muscles were removed. 1: M. masseter; 2: M. digastricus (rostral belly); 3: M. digastricus (caudal belly); 4: M. mylohyoideus; 5: M. sternohyoideus; 6: M. sternomastoideus (superficial part); 7: M. cleidocephalicus; 8: M. latissimus dorsi; 9: M. pectoralis superficialis; 10: M. pectoralis profundus; 11: M. biceps brachii; 12: M. triceps brachii; 13: M. omotransversarius; 14: M. deltoideus (clavicular part); 15: M. sternothyroideus; 16: M. omohyoideus; 17: M. sternomastoideus (deep part); 18: M. cleidomastoideus; 19: M. longus capitis; 20: Xiphoid cartilage; 21: Axillary lymph node; 22: Auricle; 23: Anular cartilage; 24: Trachea; 25: Hyoid bone; 26: Thyroid cartilage; 27: Cricoid cartilage; 28: Facial nerve (VII).
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Figure 4-14. Muscles of shoulder and arm. Isolated left thoracic limb. A) Lateral view. B) Medial view. C and D) Magnetic resonance images. Sagittal sections laterally and medially to the shoulder joint, respectively. 1: Clavicle (sectioned); 2: Spine of scapula; 3: Acromion; 4: Head of humerus; 5: Deltoid tuberosity; 6: Articular cavity (shoulder joint); 7: M. supraspinatus; 8: M. infraspinatus; 9: M. teres major; 10: M. subscapularis; 11: M. latissimus dorsi (sectioned); 12: M. triceps brachii (lateral head); 13: M. triceps brachii (long head); 14: M. triceps brachii (medial head); 15: M. tensor fasciae antebrachii; 16: M. biceps brachii (long head); 17: M. biceps brachii (short head); 18: M. brachialis; 19: M. deltoideus (clavicular part).
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Figure 4-15. Brachial plexus. Ventral view. A) Ventral muscles of neck and pectoral muscles were removed. B) Deep dissection. Clavicle and m. deltoideus (clavicular part) were removed.
2
1: Clavicle; 2: M. deltoideus (clavicular part); 3: M. omohyoideus; 4: M. omotransversarius; 5: M. longus capitis; 6: M. biceps brachii (long head); 7: M. biceps brachii (short head); 8: M. triceps brachii (medial head); 9: M. triceps brachii (long head); 10: M. subscapularis; 11: M. latissimus dorsi; 12: M. rectus thoracis; 13: M. scalenus medius; 14: M. scalenus dorsalis; 15: Suprascapular nerve; 16: Axillary nerve; 17: Musculocutaneous nerve; 18: Radial nerve; 19: Median nerve; 20: Ulnar nerve; 21: Accessory nerve (XI); 22: Thyroid cartilage; 23: Trachea; 24: Sternum; 25: Xyphoid process.
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Figure 4-16. Muscles of forearm and forepaw. Isolated left thoracic limb. M. triceps brachii was removed. A) Lateral view. B) Medial view. C) Magnetic resonance image. Sagittal section. D) Palmar view of the forepaw. 1: First digit; 2: Deltoid tuberosity; 3: Olecranon (ulna); 4: M. brachialis; 5: Fifth digit; 6: M. biceps brachii (long head); 7: M. biceps brachii (short head); 8: M. anconeus; 9: Radial nerve; 10: Median nerve; 11: Ulnar nerve; 12: M. triceps brachii; 13: M. extensor carpi radialis; 14: M. extensor digitorum communis (medially lies the m. extensor digitorum lateralis); 15: M: extensor carpi ulnaris; 16: M. abductor digiti I longus (or m. extensor carpi obliquus); 17: M. adductor digiti V; 18: M. pronator teres; 19: M. flexor carpi radialis; 20: M. flexor digitorum profundus; 21: M. flexor digitorum superficialis; 22: M. flexor carpi ulnaris.
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Figure 4-17. Muscles of thoracic wall. A) Roof of thoracic cavity. Ventral view. B) Histological section of mm. intercostales. Hematoxylin-eosin stain (20X). C) Floor of the thoracic cavity. Dorsal view. 1: M. longus colli; 2: M. longus capitis; 3: Mm. intercostales interni; 4: Mm. intercostales externi; 5: M. transversus thoracis; 6: M. omotransversarius; 7: M. omohyoideus; 8: M. subscapularis; 9: M. serratus ventralis thoracis; 10: M. latissimus dorsi; 11: M. sternohyoideus; 12: Diaphragm (sternal part); 13: Sixth thoracic vertebra; 14: Rib; 15: Sixth costal cartilage; 16: Fifth sternebra; 17: Xiphoid cartilage; 18: Suprascapular nerve; 19: Subscapular nerve; 20: Musculocutaneous nerve; 21: Axillary nerve; 22: Dorsal intercostal arteries and veins; 23: Intercostal nerves (ventral branches of thoracic nerves); 24: Internal thoracic artery; 25: Internal thoracic vein; 26: Ventral intercostal artery and vein.
4. Myology
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Figure 4-18. Muscles of trunk. A) Sagittal section of trunk. B) Magnetic resonance image. Sagittal section. C) Right half of trunk without viscera. Medial view. 1: M. longissimus thoracis; 2: M. longissimus lumborum; 3: M. quadratus lumborum; 4: M. psoas major; 5: M. psoas minor; 6: M. iliacus; 7: M. sacrocaudalis ventralis lateralis; 8: M. sacrocaudalis ventralis medialis; 9: Mm. abdominis; 10: M. transversus abdominis; 11: M. rectus abdominis; 12: Diaphragm; 13: Spinal cord; 14: Heart; 15: Lung (accessory lobe of right lung); 16: Liver; 17: Stomach; 18: Sternum; 19: Ilium; 20: Pelvic symphysis; 21: Interscapular adipose tissue.
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Figure 4-19. Diaphragm. A) Cranial view. B) Magnetic resonance image. Transverse section. C) Caudal view. D) Immunodetection of fast myosin (brown) (200X). E) Immunodetection of slow myosin (brown) (200X). 1: Spinal cord; 2: Aorta; 3: Caudal vena cava; 4: Esophagus; 5: Sternal part; 6: Costal part; 7: Central tendon; 8: Right crus (lumbar part); 9: Left crus (lumbar part); 10: Aortic hiatus; 11: Esophageal hiatus; 12: Lumbocostal arch; 13: Left phrenic nerve; 14: Right phrenic nerve; 15: Left cranial phrenic vein; 16: Right cranial phrenic vein; 17: Left lung; 18: Right lung; 19: Accessory lobe (right lung); 20: Type I muscle fiber; 21: Type II muscle fiber.
4. Myology
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Figure 4-20. Muscles of trunk. A) Epaxial muscles. Dorsal view. B) Muscles of abdominal wall. Ventral view. C) Sublumbar muscles. Ventral view. D) Muscles of tail. Dorsal view. E) Magnetic resonance image. Horizontal section. F) Muscles of tail. Ventral view. 1: M. spinalis et semispinalis thoracis; 2: M. longissimus thoracis; 3: M. longissimus lumborum; 4: Interscapular adipose tissue; 5: M. latissimus dorsi; 6: M. obliquus externus abdominis; 7: M. rectus abdominis; 8: M. pectoralis profundus; 9: M. rectus femoris (m. quadriceps femoris); 10: Femoral artery and vein; 11: M. transversus abdominis; 12: M. quadratus lumborum; 13: M. psoas minor; 14: M. psoas major; 15: M. iliacus; 16: M. gracilis; 17: Intercostal nerves (ventral branches of thoracic nerves); 18: Costoabdominal nerve (thirteenth intercostal nerve); 19: Femoral nerve; 20: Saphenous nerve; 21: Cranial pelvic aperture; 22: M. sacrocaudalis ventralis lateralis; 23: M. sacrocaudalis dorsalis lateralis; 24: M. sacrocaudalis dorsalis medialis; 25: Mm. intertransversarii dorsales caudae; 26: M. sacrocaudalis ventralis medialis; 27: Mm. intertransversarii ventrales caudae; 28: M. coccygeus; 29: Liver; 30: Stomach; 31: Right kidney; 32: Left kidney; 33: Penis; 34: Testicle; 35: M. bulbospongiosus; 36: Bulbourethral gland; 37: Pancreas; 38: Spleen; 39: Ilium; 40: Spinous process (caudal vertebrae).
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Figure 4-21. Muscles of hip joint and thigh. Left lateral view. A) Superficial muscles. B) M. tensor fasciae latae and m. biceps femoris were removed. 1: M. tensor fasciae latae; 2: M. biceps femoris; 3: M. semitendinosus; 4: M. gluteus medius; 5: M. gluteus superficialis; 6: M. semimembranosus (cranial part); 7: M. semimembranosus (caudal part); 8: M. coccygeus; 9: M. sacrocaudalis dorsalis lateralis; 10: M. quadratus femoris; 11: M. adductor magnus; 12: M. vastus lateralis (m. quadriceps femoris); 13: Greater trochanter (femur); 14: Third trochanter (femur); 15: Sciatic nerve; 16: Tibial nerve; 17: Common peroneal nerve; 18: Patellar ligament; 19: M. tibialis cranialis; 20: M. gastrocnemius (lateral head).
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Figure 4-22. Muscles of hip joint and thigh. Left medial view. A) Superficial muscles. B) M. gracilis was removed. 1: M. semitendinosus; 2: M. gracilis; 3: M. pectineus; 4: M. adductor magnus; 5: M. adductor longus; 6: M. semimembranosus (caudal part); 7: M. rectus femoris (m. quadriceps femoris); 8: M. vastus medialis (m. quadriceps femoris); 9: Mm. abdominis (sectioned); 10: M. coccygeus; 11: M. sacrocaudalis ventralis lateralis; 12: M. sacrocaudalis ventralis medialis; 13: M. iliacus; 14: M. psoas minor; 15: M. psoas major; 16: Femoral nerve; 17: Saphenous nerve; 18: Patellar ligament; 19: Pelvic symphysis.
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Figure 4-23. Muscles of leg. A) Left lateral view. Superficial muscles after remove the m. biceps femoris. B) M. tibialis cranialis and a portion of the m. gastrocnemius (lateral head) were removed. C) Medial view of left leg. M. semitendinosus was removed. 20
1: M. tibialis cranialis; 2: M. peroneus longus; 3: M. gastrocnemius (lateral head); 4: M. extensor digitorum longus; 5: M. extensor digitorum lateralis; 6: M. flexor digitorum superficialis; 7: M. soleus; 8: M. flexor digitorum lateralis (m. flexor digitorum profundus); 9: M. flexor digitorum medialis (m. flexor digitorum profundus); 10: M. tibialis caudalis (m. flexor digitorum profundus); 11: M. gastrocnemius (medial head); 12: Common calcaneal tendon; 13: M. vastus lateralis (m. quadriceps femoris); 14: M. semimembranosus (cranial part); 15: M. semimembranosus (caudal part); 16: Sciatic nerve; 17: Common peroneal nerve; 18: Tibial nerve; 19: Caudal sural cutaneous nerve (tibial nerve); 20: M. interosseous; 21: Cranial border (or tibial crest).
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Figure 4-24. Sural muscles. A) Left leg. Lateral view. Sural muscle insertions were cut. B) Isolated sural muscles. Cranial view. C, D and E) Immunodetection of fast myosin (red) in m. gastrocnemius and m. soleus (200X). Nuclei counterstained with SYTOX Green. 1: M. gastrocnemius (lateral head); 2: M. gastrocnemius (medial head); 3: M. flexor digitorum superficialis; 4: M. soleus; 5: Common calcaneal tendon; 6: Calcaneus; 7: M. tibialis cranialis.
4. Myology
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Figure 4-25. Muscles of hindpaw. Dorsal view of left hindpaw.
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Figure 4-26. Nerves of hindpaw. Transverse histological section of left hindpaw through the base of first metatarsal bone. Hematoxylin-eosin stain (20X). Nerves immunodetected with S-100 (green). Confocal laser microscopy images. 1, 3, 4, 5 and 8: Dorsal common digital nerves (superficial peroneal nerve); 2: Dorsal metatarsal nerve (deep peroneal nerve); 6: Plantar common digital nerves (medial plantar nerve branch of the tibial nerve); 7: Plantar metatarsal nerves (lateral plantar nerve branch of the tibial nerve); 9-13: First to fifth metatarsal bones; 14: Mm. interossei; 15: M. flexor digitorum brevis.
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Figure 4-27. Muscles of hindpaw. Plantar view of left hindpaw.
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Figure 4-28. Blood vessels of hindpaw. Transverse histological section of left hindpaw through the base of first metatarsal bone. Masson’s trichrome stain (20X). Immunodetection of vessels with collagen IV (red). Confocal laser microscopy images. 1: Lateral saphenous vein; 2: Dorsal metatarsal veins; 3-5: Medial and lateral plantar arteries (saphenous artery); 6-10: First to fifth metatarsal bones; 11: Mm. interossei; 12: M. flexor digitorum brevis; 13: Tendons of m. extensor digitorum longus; 14: Tendons of m. extensor digitorum lateralis; 15: Tendon of m. flexor digitorum superficialis.