Histology laboratory manual

Histology laboratory manual

Topic 6

Muscle

Test questions: 1. Structure and function of skeletal muscle2. Structure and function of smooth muscle 3. Structure and function of cardiac muscle.

Introduction. Muscle tissue is classified on the basis of the appearance of its contractile cells. Two major types are recognized: striated muscle, in which the cells exhibit a cross-striation pattern when observed at the light microscope level; and smooth muscle, in which the cells lack striations. Striated muscle is further subclassified based on location, namely, skeletal muscle, visceral striated muscle, and cardiac muscle. Skeletal muscle is attached to bone and is responsible for movement of the axial and appendicular skeleton and for maintenance of body position and posture. Visceral striated muscle is morphologically identical but is restricted to soft tissues, including the tongue, pharynx, upper part of the esophagus, and the diaphragm. Cardiac muscle is a type of striated muscle found in the heart and the base of the large veins that empty into the heart.

The cross-striations in striated muscle are due to the organization of the contractile elements that occur in the muscle cell, namely, thin filaments composed largely of the protein actin and thick filaments composed of the protein myosin II. The two types of myofilaments occupy bulk of the cytoplasm. The skeletal and visceral striated muscle cells, more commonly called fibers, are a multinucleated syncytium formed during development by the fusion of individual small muscle cells called myoblasts.

Surrounding each fiber is a delicate mesh of collagen fibrils referred to as endomysium. In turn, bundles of muscle fibers that form functional units within a muscle are surrounded by a thicker connective tissue layer. This connective tissue is referred to as perimysium. Lastly, a sheath of dense connective tissue that surrounds the muscle is referred to as epimysium. The force generated by individual muscle fibers is transferred to the collagenous elements of each of these connective tissue elements to end in a tendon.

1. Using 15x40, study and draw a longitudinal section (A) and cross section (B) of striated muscle. The muscle tissue within the muscle is arranged in series of fascicles (F). The individual muscle fibers within a fascicle are in close proximity to one another but are not individually discernable. However, the small blue dot-like structures are nuclei of the fibers. Between the fascicles, although difficult to see at this magnification, is connective tissue, the perimysium (P). Also evident in the micrograph is a nerve (Nv). The cross-banding pattern is just perceptible. With few exceptions, the nuclei (N), which tend to run in linear arrays, belong to individual muscle fibers. Also evident in this micrograph is a small blood vessel (BV). The inset, taken from a glutaraldehyde-fi xed, plastic-embedded specimen, is a much higher magnification of a portion of two muscle fibers. The major bands are readily identifiable at this magnification and degree of specimen preservation. The thick, darkstained band is the A band. Between A bands is a lightly stained area, the I band, which is bisected by the Z line. The two elongate nuclei (N) belong to the muscle fibers. Below them are a capillary (C) and a portion of an endothelial cell nucleus (End). At this higher magnification, the endothelial nuclei, as well as the nuclei of the fibroblasts, can be distinguished from the muscle cell nuclei by their smaller size and heterochromatin, giving them a dark stain. The muscle cell nuclei (N)exhibit more euchromatin with a speckling of heterochromatin, thus giving them a lighter staining appearance.

2. Study the structure of skeletal muscle which was got using electron microscope.

The myofibril is the structural and functional subunit of a muscle fiber containing sarcomeres. Myofibrils are best seen at higher magnification in the light microscope in a cross-section of the cell where they appear as dot-like structures. The overall effect is a stippled, appearance of the cytoplasm. Each myofibril is composed of two types of myofilaments arranged in sarcomeres. One type is the, myosin II thick filament. The other is actin and its associated proteins that make up the thin filaments. It is the arrangement of the thick and thin filaments that produce density differences that in turn create the cross-striations of the myofibril when viewed in longitudinal section. The site of overlap of thin and thick filaments produces the dark A band. The light-appearing I band contains the thin filaments. Careful examination of the A band reveals a light-staining area in the middle of the A band. This is referred to as the H band, which is occupied by thick filaments and is devoid of thin filaments. At the middle of each I band is the thin, dense Z line to which the thin filaments are attached. The distance between Z lines is referred to as a sarcomere. When a muscle contracts, the sarcomere and I band shorten. The filaments, however, maintain a constant length, thus, the contraction is produced by an increase in the overlap between the two filament types.

C, capillaries E, endomysium ECN, endothelial cell nuclei FN, fibroblast nuclei MF, muscle fiber MFN, muscle fiber nuclei My, myofibril N, nucleus

3. Using 15x40, study and draw the specimen of cardiac muscle. Cardiac muscle consists of fibers that possess the same arrangement of contractile filaments and thus the same cross-banding patterns that are present in striated skeletal and visceral muscle. Although cardiac muscle is, therefore, also striated, it differs in many significant respects from skeletal and striated visceral muscle. Cardiac muscle consists of individual cells that are joined by complex cell-to-cell junctions to form a functional unit (fiber). The histologically obvious differences between cardiac and the other striated muscle fibers are the presence in cardiac muscle of intercalated discs (the light microscopic representation of the cell-to-cell junctions), the location of cardiac muscle cell nuclei in the center of the fiber, and the branching of the cardiac muscle fibers. All of these characteristics are evident in a well-prepared longitudinal section of the muscle.

The myofibrils separate to bypass the nuclei, and in doing so, they delineate a perinuclear region of cytoplasm that is free of myofibrils and their cross-striations. These perinuclear cytoplasmic areas (asterisks) contain the cytoplasmic organelles that are not directly involved in the contractile process. Many cardiac muscle cells are binucleate; both nuclei typically occupy the myofibril free region of cytoplasm, as shown in the cell marked by the asterisks. The third nucleus in this region appears to belong to the connective tissue either above or below the “in-focus” plane of section. Often, the staining of muscle cell nuclei in a specific specimen is very characteristic, especially when seen in face view as here. Notice, in the nucleus between the asterisks, the well-stained nucleolus and the delicate pattern of the remainder of the nucleus. Once such features have been characterized for a particular specimen, it becomes easy to identify nuclei with similar staining characteristics throughout the specimen. For example, survey the field in figure on the left for nuclei with similar features. Having done this, it is substantially easier to identify nuclei of connective tissue cells (CT), which display different staining properties and are not positioned in the same relationship to the muscle cells.

Cardiac musclecells possess the ability for spontaneous rhythmic contractions. The contraction or beat of the heart is regulated and coordinated by specialized and modified cardiac muscle cells that are found in nodes and muscle bundles. The beat of the heart is initiated at the sinoatrial (SA) node, which consists of a group of specialized cardiac muscle cells located at the junction of the superior vena cava in the right atrium. The impulse spreads from this node along the cardiac muscle fibers of the atria. The impulse is then received at the atrioventricular (AV) node, which is located on the inner or medial wall of the right ventricle adjacent to the tricuspid valve. Specialized cardiac muscle cells then conduct impulses from the AV node along the ventricular septum and into the ventricular walls. Within the ventricular septum, the specialized cells are grouped into a bundle, the AV bundle (of His). This bundle then divides into two main branches, a left and right bundle branch, the former going to the left ventricle and the latter to the right ventricle. The specialized conducting fibers carry the impulse at a rate that is approximately four times faster than the cardiac muscle fibers. They are responsible for the final distribution of the electrical stimulus to the myocardium. While the sinoatrial node on its own exhibits a constant or inherent rhythm, it is modulated by the autonomic nervous system. Thus, the rate of the heartbeat can be decreased by parasympathetic fibers from the vagus nerve or increased by fibers from sympathetic ganglia. The specialized conducting cells within the ventricles are referred to as Purkinje fibers. The cells that make up the Purkinje fibers differ from cardiac muscle cells in that they are larger and have their myofibrils located mostly at the periphery of the cell. Their nuclei are also larger. The cytoplasm between the nucleus and the peripherally located myofibrils stains poorly, a reflection, in part, of the large amount of glycogen present in this part of the cell.

4. Study and draw the Purkinje fibers of a heart. This micrograph shows the area in the rectangle of the orientation micrograph. At this site, the endocardium (Ec) occupies the upper three-quarters of the micrograph. It consists of the endothelium (Et) that lines the ventricle but is barely detectable at this magnification. Beneath the endothelium is the subendothelial layer of dense connective tissue (SELCT), in which elastic fibers are present as well as some smooth muscle cells. The deeper layer is called the subendocardial layer of the endocardium (SELE); it contains bundles of Purkinje fibers (bundle of His) (PF) coursing along the ventricle wall. The deeper part of subendocardial layer (SELE) consists of more irregularly arranged connective tissue (DICT) with blood vessels and occasional adipocytes separating the Purkinje fibers from the myocardium (My) at the bottom of the micrograph. Note how darkly stained the cardiac muscle fibers are compared to those of the Purkinje fibers.

5. Study the electron micrograph of cardiac cells. Portion of two cardiac muscle cells joined by intercalated disc. The parts of those include the transverse components (fascia adherens and macula adherens (FA) and lateral components (gap junctions (GJ) and macula adherens (MA). Other parts of cardiac muscle cells are also present: mitochondria (M)< sarcoplasmic reticulum (SR), Z-line (Z), M line (M).

6. Using 15x40, study and draw the specimen of smooth muscle. Those cells are elongate and have tapering ends. Nuclei appear elongate and also exhibit tapering ends.

7. Answer the questions.

Q1. Smooth Muscle is not cross striated because: 1) Myosin and actin in the myofibril. 2) Myofibrils are in register with each other 3) Myofibrils are not in register with each other 4) It has gap junctions 5) It is surrounded by a basal lamina.

Q2. What is the connective tissue covering of a muscle fascicle? 1) Sarcolemma 2) Endomysium 3) Epimysium 4) Perimysium 5) Sarcoplasm

Q3. What is actin? 1) Myofilament 2) Myosin 3) Muscle fibers 4) Myofibrils 5) Myocardium

Q4. Which of the following is composed of smooth muscle? 1) Upper esophagus 2) Heart 3) Tongue 4) Biceps muscle 5) Walls of the visceral organs.
Q 5. What is line that bisects the dark band in muscle? 1) A band 2) I band 3) Z line 4) H band 5) M-line.

Q6. When Skeletal muscle contracts an arrangement of the alternating light and dark bands traversing each skeletal muscle cell changes. Which of the following statements is not correct. 1) The dark A bands will remain a constant length 2) The z lines come closer together 3) The space occupied by the H zone will not change 4) The light I bands will shorten 5) The I band consists solely of thin actin filaments.

Q7. Function of intercalated disc: 1) Site of attachment between Cardiac muscle cells 2) Region where blood vessels enter cells 3) Region where electrical impulses stimulate 4) All of the above 5) None of the above

Q8. Which of following statements is false? 1) Endomysium surrounding smooth muscle is very rich in reticular fibres 2) There are no tendons in smooth muscle 3) The nuclei of smooth muscle cells are heterochromatic 4) Smooth muscle specialises for continuous contractions of relatively low force resulting in contraction of the whole muscle 5) Smooth muscle is responsible for the movement of the skeleton.

Q9. Insert the terms: 1) Unit of skeletal muscle is _________________. 2) The I band consists of ____________filaments. 3) Hollow organ muscle wall consists of _____________ muscle. 4) Myosin molecule heavy chain consists of tail and _______________. 5) Thin myofibrils consist of actin, __________________ and troponin. 6) Rhithmic contraction of heart depend on cells of __________________ node.

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