MUSCLE

Muscle is the third basic tissue to be studied. It is specialized for contraction and is characterized by having cells with contractile fibrils called myofibrils. The muscle cell is usually referred to as a fiber. There are three kinds of muscle in mammals: smooth, skeletal, and cardiac (See Figure 24 below). They are responsible for movements of the body and for changes in size and shape of the internal organs.

Figure 24: Schematic depiction of the three types of muscle found in the human body. Taken from Junqueira and Carneiro, Basic Histology, a text and atlas, p. 191, Figure 10-1.

Smooth muscle

Smooth muscle is the least specialized of the three varieties of muscle. It is nonstriated (lacks cross-banding pattern, or striations, found in skeletal muscle), involuntary (innervated by the autonomic nervous system and thus not under the control of the will), and functions largely as a regulator of the internal environment. It is found in the wall of hollow viscera (except the heart), such as in the gastrointestinal tract, uterus, urinary bladder, and blood vessels. It is also found in association with hair follicles, the dartos of the scrotum, and the eye (i.e.; the iris and ciliary body). Smooth muscle contracts slowly and tires slowly.

Smooth muscle fibers are spindle shaped and range from less than 1 μm in diameter at their ends to as much as 8 μm in diameter at their centers. They vary in length, depending upon the organ in which they are found. They are approximately 20 μm long in blood vessels, 200 μm long in the intestines, and as much as 500 μm long in the pregnant uterus. There is one nucleus in each fiber. It is elongated (ovoid) and centrally located in the widest portion of the fiber. The nucleus contains one or more small nucleoli. A small rim of chromatin is often condensed against the nuclear envelope, and small specks of chromatin are distributed in the nucleoplasm. Nuclei of fibers fixed in the contracted state appear wrinkled or corkscrew-shaped. The cytoplasm usually appears acidophilic and homogeneous after routine fixation and staining (H&E).

Since smooth muscle usually occurs in sheets or bands in which the individual fibers overlap, sections almost never show the full length or shape of the fibers. By teasing the fibers apart after treatment with nitric acid, the shape and individual lengths of the fibers can be studied. Teased preparations also show fine myofibrils which course lengthwise in the fiber.

Smooth muscle can be examined in a large variety of visceral organs, some of which have already been studied under epithelium or connective tissue.

Study smooth muscle in slide B51, small intestine, monkey (H&E) and slide A12, stomach, human (PAS). Also, study the smooth muscle in the walls of the vein and artery in slide A90, artery, vein, & nerve, monkey (H&E). Note that many nuclei of the smooth muscle fibers are folded or corkscrew-shaped, rather than elongated or ovoid. Shortening of the nuclei occurs when smooth muscle fibers contract to become shorter and thicker.

Study slide B40, esophagus-cardiac stomach junction, human (H&E). Note the outer longitudinal and inner circular muscular layers.

Drawing of smooth muscle

Draw an area from slide B51, small intestine, monkey, (H&E), showing eight to ten transversely cut smooth muscle fibers. Show the proximity of individual muscle fibers to each other and the accurate size of the fibers when sectioned through their tips, nuclei, or other regions.

Skeletal muscle

Skeletal muscle is highly specialized to provide rapid and powerful contraction for movement and locomotion. It is striated and voluntary (under conscious control). The fibers are cylindrical in shape and range in length from 1 to 40 mm. Short fibers are present in ocular muscles and long fibers are found in muscles of the extremities. Although fiber diameter varies from 10 to 100 μm and is influenced by such factors as age, testosterone, and exercise (exercise can increase the diameter by 25%) most fibers within the same muscle are about equal in diameter. The fibers are multinucleated (about 35 nuclei/mm of fiber) and the nuclei are usually located peripherally just beneath the sarcolemma. The nuclei are ovoid, oriented parallel with a long axis of the fiber, have 1 to 2 nucleoli and show a moderate amount of chromatin which is evenly distributed as fine granules throughout most of the nucleus. The cytoplasm (or sarcoplasm) of the muscle fiber can be seen between the groups of myofibrils.

The cross striations of skeletal muscle are due to alternating light and dark bands crossing the myofibrils of a fiber and can be studied under oil immersion in well fixed and suitably stained longitudinal sections. The A (dark band), I (light band), and Z (dark band bisecting the I) bands are usually visible, but H and M bands are seen infrequently. The portion of a fiber between two successive Z-bands is a sarcomere and is the unit of muscle contraction.

Microscopic study of skeletal muscle

1. Connective tissue coverings. Examine slide A42, muscle-tendon intersection,

human (H&E). Note the yellow collagen fibers of the tendon interact exclusively with the red muscle, both at the tip of the muscle and the continuation of the tendon along the lateral borders of the muscle. With the low power objective, the boundary between muscle and tendon is seen to have a zigzag or interdigitating appearance. Using the high power objective, note that the striations of the muscle become less distinct as it nears the tendon, so that it is difficult to find the exact point where muscle ends and tendon begins. However, there is not direct continuity of muscle fibers with the collagenous fibers of the tendon, but instead there is a merging of the connective tissue covering (epi-, peri-, and endomysium) of the muscle with the connective tissue sheaths (epi-, peri-, and endotendineum) of the tendon. Electron micrographs also show that the sarcolemma at the ends of the muscle fibers is deeply invaginated and thickened. The tendon fibers insert into the invaginations of the sarcolemma.

Within the longitudinally cut muscle, look for peripherally located nuclei. Some sections show part of the epimysium surrounding the periphery of the muscle. See Figure 25 below as a reference. It consists mostly of coarse collagenous and elastic fibers and is the deep fascia seen in gross anatomy. The perimysium is formed by connective tissue septa, which extend inward from the epimysium and divide the muscle into fascicles or bundles. The endomysium is a sheath of delicate tissue surrounding an individual muscle fiber. In fixed preparations, it is sometimes pulled away from the muscle fiber.

Figure 25: Organization of muscle fibers and surrounding connective tissue elements. Taken from: Stevens and Lowe, Color Histology, p. 228, Figure 13.3, left photo; Junqueira and Carneiro, Basic Histology, a text and atlas, p. 193, Figure 10-3, right photo..

The arrangement of the connective tissue can also be appreciated in slide A45, longitudinal section of skeletal muscle, monkey, plastic, (H&E). In this slide, identify the sarcolemma, nuclei, myofibrils, and cross-striations. Can you detect both light and dark bands? Appreciate the shape and location of these multi-nucleated cells.

2. Cross section of skeletal muscle. Study the transverse sections of muscle fibers in slide A44, transverse section of skeletal muscle, monkey (H&E). Compare the size of these fibers with the smooth muscle studied previously. Note that nuclei of the muscle fiber are located just beneath the sarcolemma (not to be confused with the endomysium). Other nuclei occur between the fibers and most of them belong to connective tissue cells. Can you locate the epimysium, perimysium, and endomysium?

Slide A48, upper to middle part of esophagus, human (H&E) shows both skeletal and smooth muscle. Can you distinguish between these two types?

3. Sensory innervation of muscles. Examine slide A47, muscle spindles, monkey

(H&E). Encapsulated sensory receptors in muscles and tendons that provide information on the degree of stretch (or tension) in the muscle are called muscle spindles. These specialized receptor units are comprised of two types of modified muscle fibers (nuclear bag fibers and a nuclear chain fibers) and a neuron terminals that are separated by a fluid-filled space and surrounded by a capsule. Figure 26 below depicts a muscle spindle between skeletal muscle fibers. In the right photograph, the muscle spindle is cut in cross section and resembles the structures that you are looking for in slide A47.

Figure 26: Left photo is a schematic diagram of a muscle spindle between skeletal muscle fibers. Taken from Junqueira and Carneiro, Basic Histology, a text and atlas, p. 204, Figure 10-19. Right photo is a micrograph showing a muscle spindle in cross section. There are two bundles of spindle cells in the fluid-filled capsule. Taken from Ross et al, Histology, a text and atlas, 4^th edition, p. 260, Figure 10.11b.

4. Blood vessels in skeletal muscle. Skeletal muscle has a rich blood supply. Arteries in the epimysium course with the epimysium to enter the septa between the muscle fascicles. They branch in the perimysium and give rise to arterioles, which, in turn, give rise to capillaries that run in the endomysium to supply the muscle fibers. Most capillaries run parallel with the muscle fibers, but some give off branches that course at right angles to the fibers. Lymphatic vessels of skeletal muscle are confined almost entirely to the epimysium and perimysium.

Drawing of skeletal muscle fibers

Using slides A44 and A45, (H&E), draw in detail a short extent of a skeletal muscle fiber cut longitudinally, and a few skeletal muscle fibers cut in cross section. This drawing should show the diameter of the fibers, the appearance of the cross striations, and the position of the nuclei.

Cardiac muscle

Cardiac muscle comprises the myocardium of the heart and resembles skeletal muscle in that it is striated. Unlike skeletal muscle, it is involuntary, consists of a network of branching fibers with centrally located nuclei, has cross striations which are closer together and less prominent than those in skeletal muscle, and possesses intercalated disks. An intercalated disk is a 0.5 to 1 μm thick, undulating or steplike band, which crosses the fiber at the level of the Z-band. It has been shown by electron microscopy to be the point of junction between two cardiac muscle fibers. The fact that one fiber ends and another begins at the intercalated disk proves that cardiac muscle is not a syncytium (unbroken chain of fibers) as was once believed. Functionally, the disks not only hold the fibers together, but they constitute areas of low electrical resistance which permit the rapid spread of impulses from fiber to fiber and allow the muscle fibers to behave as though they were a syncytium. H&E preparations do not show the disks as clearly as sections stained with iron hematoxylin or phosphotungstic acid hematoxylin. See Figure 27 below for a schematic diagram of cardiac muscle.

Figure 27: Schematic drawing of cardiac muscle. Note the intercalated disks, centrally located nuclei and striations. Taken from Junqueira and Carneiro, Basic Histology, a text and atlas, p. 206, Figure 10-22.

Cross sections of cardiac muscle can be distinguished from transverse -sections of smooth muscle because the fibers of cardiac muscle are (1) larger than those of smooth muscle, (2) may show branching, (3) have distinct myofibrils, and (4) contain numerous capillaries between the fibers.

Microscopic study of cardiac muscle

Study longitudinal, cross and oblique sections of cardiac muscle under different powers

of the microscope using slide A49, cardiac muscle, human, (Trichrome). Identify the branching fibers, intercalated discs, centrally positioned nuclei, myofibrils, and capillaries.

Study slide A50, cardiac muscle, (Fe-Hematoxylin). Iron hematoxylin staining of cardiac

muscle highlights the cross striations and intercalated discs. Focusing up and down on the discs shows their jagged or stair-step structure.

Study slide A78, aortic valve, human, (Trichrome). Nice profile of the valve leaflet flopped back against the aortic wall. The stain is unusual. Most fibers (myofibrils, collagen, etc.) are blue, nuclei are very pale often appearing as ghosts or negative images. Intercalated disks of ventricular myocardial cells are very prominent. RBC's are red-orange and enable visualization of the extensive plexus of capillaries in the myocardium.

Drawing of cardiac muscle fibers

From slide A50, draw a few cardiac muscle fibers cut longitudinally, and a few cut in cross section.

Tabulation of differences between the types of muscle and tendon

Prepare a list of the criteria you have observed which will serve in identifying smooth muscle, cardiac muscle, skeletal muscle, and tendon. The list should indicate the shape of the fiber (longitudinal and cross section); the size of the fiber (length and diameter); location and number of nuclei per fiber; and the appearance of the fibrils in the fiber.