SYSTEMIC PATHOLOGY. Pathology of Muscle. Lecture 1. Paul Hanna Winter 2018

Transcription

1 SYSTEMIC PATHOLOGY Pathology of Muscle Lecture 1 Paul Hanna Winter 2018

2 Normal Skeletal Muscle [For Information only] Muscle Development Neural tube Dermis Muscles of the body develop from myotome which is a component of the somites (ie blocks of paraxial mesoderm) Vertebrae & ribs Tendons Muscles (back, body wall, limbs)

3 Muscle Development [For Information only] Lateral view of the myotome region of the somites Respecification of the sclerotome to form each vertebra. Note how spinal nerves innervate muscle early in development.

4 Muscle Development [For Information only] Most of the head musculature comes from the cranial paraxial mesoderm (not from somites) These cells originate adjacent to the sides of the brain, and they migrate into the center of the pharyngeal (branchial) arches

5 Muscle Development [For Information only] Fig 10 2 (Mescher). Development of skeletal muscle. Skeletal muscle begins to differentiate when mesenchymal cells called myoblasts align and fuse together to make longer, multinucleated tubes called myotubes. Myotubes synthesize the proteins to make up myofilaments and gradually begin to show cross striations by light microscopy. Myotubes continue differentiating to form functional myofilaments and the nuclei are displaced against the sarcolemma. Part of the myoblast population does not fuse and differentiate, but remains as a group of mesenchymal cells called muscle satellite cells located on the external surface of muscle fibers inside the developing external lamina. Satellite cells proliferate and produce new muscle fibers following muscle injury.

6 Muscle [For Information only] Connective tissue layers of muscle tissue: Fascia (or "deep fascia") covers the entire muscle and is located over the layer of epimysium. Epimysium - wraps the whole muscle. Perimysium - wraps fascicles (groups) of muscle cells. Endomysium - wraps each individual myofiber (muscle cell). Composed of an external lamina produced by the myofiber and ECM produced by fibroblasts Sarcolemma - cell membrane enclosing each myofiber. Figure 1-8 (Dyce) Osteofascial compartments, horse forearm. 1 - superficial fascia; 2 - cephalic vein; 3 - radius; 4 - septa of deep fascia enclosing individual muscles or groups of muscles; 5 - deep fascia.

7 Muscle [For Information only] Note: muscle cell = myocyte = muscle fiber = myofiber Figure 10 3 (Mescher). An entire skeletal muscle is enclosed within a thick layer of dense connective tissue called the epimysium that is continuous with fascia and the tendon binding muscle to bone. Large muscles contain several fascicles of muscle tissue, each wrapped in a thin but dense connective tissue layer called the perimysium. Within fascicles individual muscle fibers (elongated multinuclear cells) are surrounded by a delicate connective tissue layer, the endomysium.

8 Muscle [For Information only] a major role of this connective tissue arrangement is to transfer the mechanical force of the contracting muscle cells, because individual myofibers rarely extend the entire length of the muscle Fig 10 4 (Mescher). Connective tissue of skeletal muscle. (a): Micrograph shows a cross section of striated muscle demonstrating connective tissue and cell nuclei. The endomysium around individual muscle fibers is indicated by arrowheads. At left is a portion of the epimysium. All three of these tissues contain collagen types I and III (reticulin). X200. Fig (Robbin s) Relationship between the cell membrane (sarcolemma) and the sarcolemmal associated proteins.

9 Myofibers [For Information only] Fig 10-5 (Mescher). Capillaries of skeletal muscle. The blood vessels were injected with plastic polymer before the muscle was collected and sectioned longitudinally. A rich network of capillaries in endomysium surrounding muscle fibers is revealed by this method. X200. Giemsa with polarized light.

10 Myofibers [For Information only] Fig 3-9 (Maxie). Normal skeletal muscle in longitudinal section. Longitudinal sections reveal the striations characteristic of skeletal muscle.

11 Myofibrils [For Information only] Fig 10-7b (Mescher). At higher magnification, each fiber can be seen to have three or four myofibrils, with their striations slightly out of alignment with one another. Myofibrils are cylindrical bundles of thick and thin myofilaments which fill most of each muscle fiber. The middle of each I band can be seen to have a darker Z line (or disk). X500. Giemsa. Fig 10-7c (Mescher). TEM showing the more electron dense A (anisotropic) bands bisected by a narrow, less electron dense region called the H zone and in the I (isotropic) bands the presence of sarcoplasm with mitochondria (M), glycogen granules (G), and small cisternae of SER around the Z line. X24,000.

12 Myofibrils [For Information only] Fig 10 8 (Mescher) Structure of a myofibril: a series of sarcomeres. Diagram indicates that each muscle fiber contains several parallel bundles called myofibrils. Thin filaments are actin filaments with one end bound to α actinin, the major protein of the Z disc. Thick filaments are bundles of myosin, which span the entire A band and are bound to proteins of the M line and to the Z disc across the I bands by a very large protein called titin, which has spring-like domains. The molecular organization of the sarcomeres has bands of greater and lesser protein density, resulting in staining differences that produce the dark and light staining bands seen by light microscopy & TEM. Fig 10 9 (Mescher). Molecules composing thin and thick filaments. The contractile proteins are the thin and thick myofilaments within myofibrils. (a): Each thin filament is composed of F actin, tropomyosin, and troponin complexes. (b): Each thick filament consists of many myosin heavy chain molecules bundled together along their rod like tails, with their heads exposed and directed toward neighboring thin filaments. (c): Besides interacting with the neighboring thin filaments, thick myofilament bundles are held in place by less well characterized myosin binding proteins within the M line.

13 Sarcoplasmic reticulum Blausen.com [For Information only] Sarcoplasmic reticulum and Transverse tubular system. The sarcoplasmic reticulum (SR) is specialized type of endoplasmic reticulum (ER) in myocytes. It consists of complex network of tubules, and cisternae that stores and pumps molecules, (esp calcium). - note how the terminal (lateral) cisterna of the SR comes in close contact with the T tubule. - also note, Cisterna (pl. cisternae) - from cistern, a receptacle for holding liquids, usually water

14 Neuromuscular junctions [For Information only] Fig (Mescher) The neuromuscular junction. (a) Silver staining reveals the nerve bundle (NB), the terminal axonal twigs, and the motor end plates (MEP) on striated muscle fibers (S). X1200. (b): A SEM shows the branching ends of a motor axon, each covered by an extension of the last Schwann cell and expanded terminally as a motor end plate embedded in a groove in the external lamina of the muscle fiber. (c): Diagram indicating key features of a typical neuromuscular junction: synaptic vesicles of acetylcholine (ACh), a synaptic cleft, and a postsynaptic membrane. This membrane, the sarcolemma, is highly folded to increase the number of Ach receptors at the NMJ. Receptor binding initiates muscle fiber depolarization, which is carried to the deeper myofibrils by the T tubules.

15 Muscle contraction [For Information only] Excitation-contraction coupling Somatic motor neuron releases Ach at the neuromuscular junction Net entry of Na + through Ach receptor channel initiates a muscle action potential Action potential in t-tubule alters conformation of DHP receptor (dihydropyridine L-type Ca 2+ channel) DHP receptor opens Ca 2+ release channels (= RyR = ryanodine receptor-channel) in sarcomplasmic reticulum and Ca 2+ enters cytoplasm Ca 2+ binds to troponin, allowing strong actin-myosin binding Myosin heads execute power stroke Actin filament slides toward center of sarcomere

16 Muscle contraction [For Information only]

17 Satellite cells [For Information only] myocytes are multinucleated with ~ nuclei on LM satellite cell nuclei cannot be distinguished from myocyte nuclei. in mature muscle about 3-5% of the myofiber nuclei are those of satellite cells. Electron micrograph of a typical myonucleus (A) and a muscle satellite cell (B). Muscle satellite cells (S) were identified by their location inside the basal lamina (arrowheads) and outside the sarcolemma (arrows) and an independent cytoplasm. In contrast, a myonucleus (M) is located inside the sarcolemma of the muscle fiber and does not contain an independent cytoplasm. Bar, 1 μm.

18 Myofiber Types common classification based on three major physiologic features: (1) rates of contraction (slow or fast) (2) rates of fatigue (resistant or sensitive) (3) types of metabolism (oxidative, glycolytic, or intermediate) two main types: Type 1 fibers ( red muscle ) slow twitch fatigue resistant aerobic/oxidative metabolism ( myoglobin/mitochondria) Type 2 fibers ( white muscle ) fast twitch fatigue sensitive anaerobic/glycolytic metabolism ( myoglobin/mitochondria; glycogen) in many species type 2 can be further divided into Type 2a and Type 2b Type 2a (intermediate oxidative-glycolytic) fibers are fast twitch but fatigue resistant. Type 2b (glycolytic) fibers are as described above for classic type 2 fibers.

19 Myofiber Types Fig (Zachary) Muscle fiber typing, myofibrillar adenosine triphosphatase (ATPase) reaction, normal skeletal muscle, transverse section. A, Dog. Type 1 (light) and type 2A (dark) fibers are arranged in a mosaic pattern. Frozen section, ATPase ph 10.0 (alkaline preincubation). A A (note reversal of dark / light staining and fiber type at alkaline ph above and acid ph below) B, Horse. type 1 (dark), type 2A (intermediate = gray), type 2B (light), Frozen section, ATPase ph 4.35 (acid preincubation). B

20 Innervation of Myofibers [For Information only] Fig (Zachary) Schematic diagram of motor units of a muscle. Each motor unit consists of a motor neuron within the central nervous system and all the myofibers (muscle cells) supplied by the neuron and its axon branches. All fibers in a single motor units are of the same type (ie either type I or II).

21 Examination of Muscles Gross examination check color, volume and texture compare muscles with those of the opposite side or animals of the same age & breed make several slices into the muscles to look for internal lesions

22 Histopathologic examination most muscle diseases require microscopic examination for definitive DX good fixation requires a 10:1 volume ratio (formalin to tissue) take small (1 x 1 x 3 cm) slices of muscle from several sites Fig (McGavin) Technique for collection of a muscle sample for histologic examination. Pinning strips of muscle onto a rigid surface (eg piece of tongue depressor) before immersion in 10% neutral-buffered formalin, will minimize fixation artifacts.

23 Postmortem changes 1) Rigor mortis = postmortem contraction of skeletal muscles results in the fixation of joints starts ~ 2 to 4 hours after death; persists for 24 to 48 hours pathogenesis: after death, circulation of blood / O 2 ceases muscle cells resort to anaerobic glycolysis glycogen stores run out & ATP becomes depleted (~2-4 hrs) Ca 2+ floods into sarcoplasm muscles contract (rigor) further depleting ATP rigor mortis gradually dissipates with autolysis of muscle proteins (24-48 hrs) presence and intensity of rigor mortis depends on several factors such as: - body condition / glycogen stores (eg rapid when low glycogen stores) - external / internal temperatures (eg rigor mortis accelerated by heat)

24 Disturbances of Growth and Postmortem Alterations Muscle Atrophy reduction of muscle size is mostly due to decreased myofiber size, not cell loss reversible providing the source of injury is removed in relatively short time interval histologically, see reduction in myofiber diameters with an unchanged amount of CT types of muscle atrophy include: a) Denervation atrophy b) Disuse atrophy c) Atrophy of malnutrition / cachexia / senility d) Atrophy of endocrine disease

25 Denervation Atrophy myofibers that lose tonic stimulation due to nerve damage undergo atrophy can be rapid & severe, eg > 50% of muscle mass can lost in a few weeks histo: see atrophy of usually both type 1 and 2 myofibers

26 Equine Laryngeal Hemiplegia ("roarers ) due to axonal degeneration of long recurrent laryngeal nerves, esp left side current hypothesis a dying-back neuropathy caused by an inability of cell bodies in the nucleus ambiguus to maintain the integrity of long motor neurons A. Trachea B. Cartilage C. Vocal cord D. Epiglottis Normal Endoscopic view of larynx with left laryngeal paralysis Recurrent laryngeal nerves branch off of vagus nerves and wraps under the subclavian (on the right) and the aortic arch (on the left) to reach the laryngeal intrinsic muscles:

27 Equine Laryngeal Hemiplegia ("roarers ) Veterinary Neuroanatomy and Clinical Neurology, 4th ed. Veterinary Neuroanatomy and Clinical Neurology, 4th ed. FIG 6-52 & 53 Craniocaudal (left) and dorsal (right) view of the larynx of a horse with a left-side paralysis that resulted in severe denervation atrophy of all the intrinsic muscles of the larynx except for the cricothyroid. This is especially evident here in the cricoarytenoideus dorsalis muscle.

28 Equine Suprascapular Neuropathy ( Sweeney ) atrophy of the infraspinatus and supraspinatus muscles due to damage to the suprascapular nerve caused by sudden trauma (eg horses colliding with stall doors, trees, etc) or chronic low-grade trauma (eg poor fitting collar in a work horse, called sweeney ) Fig 23-6B (Dyce) Muscles associated with shoulder & elbow joints; lateral view. 7, supraspinatus muscle 8, infraspinatus muscle

29 Equine Suprascapular Neuropathy ( Sweeney ) injury to the suprascapular nerve occurs where it crosses the scapular notch on the cranial surface of the neck of the scapula Fig (Dyce) Distribution of the nerves in the right forelimb; medial view. The axillary artery at the shoulder joint is stippled. 2, suprascapular n.

30 Radial Nerve Paralysis Fig 5-10 A mixed-breed dog with a distal fracture of the left humerus and suspected radial nerve paralysis. Handbook of Veterinary Neurology, 5th Edition. Fig Distribution of radial nerve, right forelimb, schematic lateral view. Guide to the Dissection of the Dog, 7th Ed

31 Disuse Atrophy innervation is intact but there is reduced movement, eg pain, bone fracture, ankylosis, limb immobilization / cast, etc lesions are localized to affected groups of muscles & is mostly type 2 myofibers Horse with severe muscular atrophy (white asterisk) of the left leg. Muscle atrophy was localized to this limb and was caused by lack of movement (disuse atrophy) as a result of a chronic joint injury resulting in partial ankylosis. Web Figure 15-4 (Zachary). Disuse atrophy, dog. A, Transverse section of normal biceps femoris muscle. B, Same muscle, same magnification, 60 days after disuse. Both type 1 (light) & type 2 (dark) fibers are atrophic, but type 2 fibers are more severely affected. Frozen section, ATPase ph 9.8.

32 Atrophy of malnutrition / cachexia / senility progressive muscle atrophy occurs in malnutrition, cachexia & senility note, 1 to 5% of muscle protein is turned over each day in malnutrition: muscle becomes the source of nutrients atrophy can start within 24 hours following starvation type 2 fibers affected more than type 1

33 Malnutrition Carcass of dog showing severe muscle atrophy due to starvation (owner was charged by the police). Note the extensive atrophy of scapular muscles (arrow) and intercostal muscles (asterisks). Intercostal muscles are so atrophic that the lungs can be seen right through them.

34 Atrophy of malnutrition / cachexia / senility cachexia: = muscle wasting, weight loss, general debility can occur during a chronic disease occurs with certain neoplasms and chronic inflammatory diseases (eg TB) due to cytokines (eg TNF = cachectin ) from tumor cells or macrophages

35 Atrophy of Endocrine Disease neuromuscular weakness & muscle atrophy is seen in some endocrine disorders, eg hypothyroidism and hyperadrenocorticism

36 Muscle Hypertrophy response of muscle to increased demand; either physiologic (response to increased workload) or compensatory (unaffected myofibers adjacent to weak / dysfunctioning myofibers) muscular hypertrophy can also be enhanced by trophic stimulation, eg anabolic steroids! increase in the size / diameter (but not in the number) of myofibers

37 Degeneration And Repair Of Muscle Muscle Degeneration and Necrosis (rhabdomyolysis) degeneration is a common sequel to myofiber injury regardless of its cause myofiber degeneration can be reversible, but can progress to irreverible injury necrosis muscle degeneration & necrosis can only be detected grossly in severe lesions; typically appears pale calcification of degenerate / necrotic myofibers is common in many muscle disease; enhanced pallor and when severe see chalky white foci or streaks. red discoloration may be present when degenerated muscle coexists with hemorrhage or with extensive release of myoglobin into ECF from myocyte necrosis

38 Muscle Degeneration and Necrosis (rhabdomyolysis) This photo shows the pale degenerate muscle (d) in a foal with white muscle disease. Normal muscle (n) is shown for comparison.

39 Muscle Degeneration and Necrosis (rhabdomyolysis) Fig (Zachary) Localized pallor, necrosis, injection site of an irritant substance, semitendinosus muscle, cow. The irritant was injected just under the perimysium and caused necrosis and disruption of the myofibers. Photo showing muscle necrosis at an injection site with seepage of the medicant along fascial planes.

40 Muscle Degeneration and Necrosis (rhabdomyolysis) In contrast to previous slide, degenerate / necrotic muscle can appear darker than normal. The darker color occurs when degenerated muscle coexists with hemorrhage or with extensive release of myoglobin from necrotic myofibers.

41 Muscle Degeneration and Necrosis (rhabdomyolysis) Histo: vacuoles & loss of striations followed by swelling hypereosinophilia & glassy appearance (so-called hyaline or Zenker's degeneration) +/- calcification segmental rupture of fibers and formation of retraction caps Figure (Zachary) Myofiber necrosis, skeletal muscle. A, Hypercontraction, transverse section. Large, deeply stained fibers ( large dark fibers ) are hypercontracted segments of a myofiber, the initial stage of necrosis. Note the rounded outline of these myofibers compared with the polygonal outlines of normal myofibers. Formalin fixation, H&E. B, Segmental necrosis, monensin toxicosis, longitudinal section, horse. Segments of the myofibers have undergone hypercontraction, and the remaining cytoplasm is fragmented. Formalin fixation, H&E stain.

42 Note, ruptured fibers typically produce the formation of so-called retraction caps. Retraction caps appear as concavities at the free end of ruptured myofiber fragments (arrows) * * Note fragmented myofibers with swelling, hyalinization and loss of striations in some areas (asterisks) and extensive blue-purple granularity, representing calcification (arrows), in other areas. H&E stain.

43 Regeneration and Repair of Muscle skeletal muscle has remarkable ability to regenerate integrity of the BL / endomysium (ie sarcolemmal tube / scaffold) is critical intact BL keeps satellite cells, myonuclei and myoblasts inside & fibroblasts outside, but allows easy entry and exit of phagocytes. macrophages and neutrophils clean cell debris within hours of necrosis. satellite cells initially round-up within hours & proliferate produce myoblasts that fuse within the sarcolemmal tube and produces new sarcomeres myoblasts eventually unite (bridge) with remaining viable segments of the original myofiber. if sarcolemmal tubes are disrupted (trauma, infarction, infection), partial regeneration can occur, but is generally complicated with fibrosis (scarring).

44 Regeneration and Repair of Muscle FIGURE 3-26 (Maxie) Segmental necrosis. A muscle fiber (top) is intact on the left but has undergone lysis on the right, leaving an empty tube formed by the basal lamina. Some macrophages are present in the tube. There is myofibrillar disarray in the junctional area between intact and lysed segments. Below there is another tube densely infiltrated by macrophages and lined by plump activated satellite cells. One satellite cell is in mitosis (arrow). Fig (Zachary) Schematic diagram of segmental myofiber necrosis and regeneration. A, Myofiber, longitudinal section. B, Segmental coagulation necrosis. C, The necrotic segment of the myofiber has become floccular and detached from the adjacent viable portion of the myofiber. The satellite cells are enlarging. D, The necrotic segment of the myofiber has been invaded by macrophages, and satellite cells are migrating to the center. The latter will develop into myoblasts. The plasmalemma of the necrotic segment has disappeared. E, Myoblasts have formed a myotube, which has produced sarcoplasm. This extends out to meet the viable ends of the myofiber. The integrity of the myofiber is maintained by the sarcolemmal tube formed by the basal lamina & endomysium F, Regenerating myofiber. There is a reduction in myofiber diameter with central rowing of nuclei. There is early formation of sarcomeres (cross striations), and the plasmalemma has re-formed. Such fibers stain basophilically with H&E.

45 Regeneration and Repair of Muscle FIG 3-32 (Maxie) Muscle necrosis and regeneration showing areas from the muscle of a cat with ischemic myopathy, (A) an acutely necrotic fiber (coagulative necrosis) (B) another fiber infiltrated by macrophages (C) basophilic regenerating fibers with chains of central nuclei.