MUSCLE

Objectives

1. To recognize skeletal, cardiac and smooth muscle in longitudinal and transverse section.

2  To recognize the following structure associated with:  
   
             A.  Skeletal muscle           B.  Cardiac muscle 
    skeletal muscle fiber    branching fibers
endomysium central nuclei
perimysium intercalated disks
fascicle    stained with PAS
myofibrils    stained with H&E
sarcomere 
relaxed band pattern 
contracted band pattern
A band, I band, Z line
muscle spindle
    intrafusal fibers
    extrafusal fibers 
 
 * * * * *

 

       

Blue Histology


Slides
D-12    Skeletal muscle stretched (H&E)    
D-13Muscle spindle, cross-section (H&E)
D-16Heart muscle (H&E)
D-17Cardiac muscle (PASH)
D-92Tongue (Masson)
D-101Esophagus (H&E)
D-114Colon (H&E)

 

Optional slides
Smooth muscle
 D-104Stomach monkey (PASH)
 D-109Duodenum rabbit (FeH)
  
Skeletal muscle
 D-11Abdominal wall, rat (Masson and PAS)
 D-95Tongue (H&E)
   

 

Slide descriptions

D-92, Tongue (Masson)

This cross-section through the tongue of a rabbit demonstrates skeletal muscle. It has been stained by the Masson technique to distinguish the muscle fibers from connective tissue. Hold the slide up and examine it with the naked eye. The tongue is surrounded by a layer of stratified squamous epithelium, stained orange. The green-blue material within is irregular connective tissue. It surrounds bundles of skeletal muscle fibers, stained red. The individual fibers are large enough to see easily with the 10X power objective (illustration). Compare their size with those of the other cells in the green connective tissue. The skeletal muscle fibers even dwarf fat cells. This is because the fibers are not true cells but "syncytia" formed by the fusion of hundreds of cells into a giant multinuclear structure surrounded by a continuous cell membrane.

The fibers are arranged very orderly in perpendicular directions so you an easily find examples cut transversely, longitudinally and obliquely. Exam each of these orientations at 430X (illustration). Especially, note the positions of the nuclei just under the cell membrane. In fibers running longitudinally you can see the cross striations. Transverse sections also show the fibrils within the fibers. These give the cross sections their grainy texture.

D-12 Skeletal muscle stretched (H&E)

Special precautions were taken to keep this muscle extended during fixation and dehydration. Thus, the fibers are straight and show the relaxed pattern cross banding, (illustration). The purpose of this slide is to show the details of the cross-bands. You should understand their appearance in terms of function. Three steps to this goal are:

1. Scan around with the 43X objective to find an area where the cross bands show up particularly well. This will probably be where the tissue is not too thick, and in fact, the best places to look may be near the frayed ends of the fibers where individual myofibrils are distinguishable.

2. Examine the cross-bands with oil immersion for maximum resolving power.

3. Read your textbook as you examine your slide to recognize areas where the banding pattern is relaxed. Even though individual myofilaments are visible only with the electron microscope, you should be able to tell from your text or the Australian Blue Histology program ( ) where the two types of myofilaments are located within the bands.

Having seen the pattern of banding in stretched skeletal muscle you can now look for the simpler pattern in contracted muscle (illustration). It should be possible to find local areas where the sarcomeres are shortened and the pattern of contracted muscle is obvious. If not, return to slide D-92.  In this slide the muscle was allowed to contract after death. In fact, it "super-contracted" in the fixative, bringing the z lines right up against the A bands, leaving no I band at all. Use the same steps mentioned above with the extra proviso of choosing to look only at fibers that have been cut perfectly longitudinally.

 

D-13 Muscle spindle, cross-section (H&E)

This cross section through a small skeletal muscle shows the relationship between the muscle fibers and connective tissue (illustration). The tissue has shrunk during fixation by opening up cracks in the connective tissue seams. The white spaces are purely artifactual but they make it easy to trace the locations of connective tissue. Thicker seams of perimysium divide the muscle into fascicles and provide a pathway for small arterioles, venules, and nerve bundles (illustration). Its Type I collagen fibers and the scattered fibroblasts are recognizable.  Remember, "fascicle" refers to a bundle and, as the slide shows, a muscle can have fascicles of fascicles each surrounded by a seam of perimysium. (This is different from lobules of a gland. There are not lobules within lobules.) Every individual muscle fiber is wrapped in its own very delicate connective tissue layer of endomysium. This layer contains many capillaries running in the direction of the muscle fiber and reticular fibers. You have to look carefully to find the capillaries but you will not see the fine reticular fibers no matter how closely you look. The epimysium is not really present on this slide, although we can pretend that the fairly thick layer of connective tissue along one corner of the tissue is this layer.

This section also cuts transversely through a muscle spindle. Muscle spindles are a part of the proprioceptive system, supplying sensory information to the nervous system concerning how extended the muscle is. Locate the cross-section of the one spindle on this slide by finding the area indicated in the accompanying diagram (either by naked eye or under lowest power magnification). Do not mistake the sections of a nerve bundle, artery and vein running nearby in the perimysium for the spindle (illustration). Now examine the components of the muscle spindle with your 43X or 100X objective. A spindle includes a few almost vestigial skeletal muscle fibers as well as a complex of nerve endings wrapped up in a connective sheath. Muscle spindles are spindle shaped (no surprise) but sometimes they bifurcate at the ends.  For this reason, your section may appear to show two or even three sections individually surrounded by a C.T. sheath. The red circles inside are "intrafusal" skeletal muscle fibers (can you see peripheral nuclei?). They are much smaller than the regular "extrafusal" fibers outside of the spindle.

D-17, Cardiac muscle (PASH)

Much of this cardiac muscle is sectioned in the longitudinal plane.  Observe the characteristic features that distinguish it from skeletal muscle (low mag., high mag.).

         1.  The nuclei are large, pale and in the center of the fibers.
2.The fibers branch.
3.Connective tissue is abundant between the fibers.
4.The fibers have intercalated disks.

PAS stains the intercalated disks particularly well . Note that they often have a stepped appearance across the fibers. The stain also brings out the cross banding pattern (illustration). This pattern similar to that of skeletal muscle, but characteristically does not show up quite as well.  Although heart muscle usually contracts as it is fixed (as in the above image), in this particular slide many of the myofibrils do show the pattern of "relaxed" muscle (illustration). The degree of contraction varies abruptly so that neighboring cells may allow a direct comparison between relaxed and contracted muscle. Observe also the connective tissue spaces and the extent of the vascular bed.

D-16 Heart muscle (H&E)

Slide D-l6 shows cardiac muscle sectioned in various orientations and stained with (H&E). Find an area of transverse section (illustration). Note how irregular the individual muscle cells are in size and shape. This is in part because cardiac muscle cells branch. The myofibrils are readily apparent. They are ribbon-shaped instead of round rods, as is the case for skeletal muscle. The nuclei of cardiac muscle cells are large and pale and located in the middle of the cells. Quite a few other smaller nuclei lie in the connective tissue between the muscle cells. Most of these belong to endothelial cells, and fibroblasts. Be sure that you do not mistake these nuclei around the periphery of muscle fibers for muscle cell nuclei. You should be able to see the great abundance of capillaries, which outnumber the cardiac fibers. Thicker seams of connective tissue dissect the muscle into fascicles and provide a pathway for arterioles and venules.

After assuring yourself that you can distinguish transversely sectioned cardiac muscle from skeletal muscle, scan at low power for an area of longitudinally presented fibers. You may have to hunt around a bit. Observe that the large pale nuclei lie in the center of the cells, again making sure not to be misled by the numerous endothelial cell nuclei. This time the capillaries are sectioned longitudinally (illustration). Can you find examples with erythrocytes scattered along them? Cross bands show up well, most in a contracted pattern. In contrast, intercalated disks are difficult to see except at high power (illustration). It is true that intercalated disks are absolutely diagnostic for cardiac muscle but it is equally true that they are not stained well by H&E. They show up more as discontinuities or irregularities across the fibers than as stained structures like they did with PAS staining. (Talk over why PAS stains intercalated disks but H&E does not). Find a super good example of a cardiac muscle fiber branching (illustration). The branches do not occur only at the intercalated disks (i.e. the ends of the cells), right? Right!

Finally, look at an area of muscle cut obliquely, since this is the typical way that most will be sectioned (illustration). The myofibers will look elongated and messy, and neither the cross striations nor intercalated disks will show up well. The irregularity of cell shape should still be striking and, of course, the scattered nuclei will be large, pale, irregular in outline and located in the middle of the muscle cells.

Optional

If you scan around very carefully you will see that the nuclei occupy spindle shaped holes with a tapering space at either end. The space is filled with droplets of yellowish material (illustration). This is lipofuscin. It is a waste product that slowly accumulates over the years. Cardiac muscle cells do not divide and the cells you see were present since birth. Like all cells, those of cardiac muscle gradually turn over their components with endosomes. The indigestible molecules, mainly of a lipid nature, accumulate as lipofuscin. Now that you have examined these deposits here you may recall seeing them in the cross section (illustration). When you looked for nuclei in the centers of these cells you may have been puzzled that in the middle of many cells you saw a bit of yellow instead of a nucleus. Those sections were cut through the cell right next to the nucleus where the lipofuscin was stored.

D-114 Colon (H&E)

Hold this slide up and examine it with the naked eye to get oriented. Lining the scalloped lumen is a purplish stained mucosa which consists of simple columnar epithelium and a connective tissue bed filled with tubular glands. Under it is loose to moderately dense connective tissue, the submucosa. The next layer out consists of a band of circularly arranged smooth muscle with the cells cut in the plane of section. The outermost layer appears as three larger bumps called tenia coli. These are longitudinally running bands of smooth muscle cut in cross-section. Now confirm these identifications with the l0X objective. Next, examine the layers of smooth muscle with the 43X objective. Look for typical characteristics of longitudinally and transversely sectioned smooth muscle as described in the lecture (illustration). Can you distinguish this tissue in both orientations from connective tissue? Take a look at tendon (slide D-5) for comparison. How do you tell the two apart? Where else in the body will you find smooth muscle? What is its general function?

 

D-101 Esophagus (H&E)

The esophagus is interesting in that in the upper portion the external muscle layer is made up of skeletal muscle while the lower portion has only smooth muscle. This allows you to start a swallow intentionally, but once it gets underway you lose control over it. (Treat yourself to a moments Rec break to start to swallow and see if you can stop it. Ignore your lab partner's blather if he claims that he can.) In the middle region of the esophagus the two types of muscle intermingle. This makes the esophagus a great place for comparing and sorting out smooth from skeletal muscle. The muscle is interwoven with a lot of connective tissue inviting a three-way comparison (illustration). Would you say that skeletal or smooth muscle predominates on your slide? Does this agree with the label on the slide that the tissue came from the upper portion of the esophagus?

In general, skeletal muscle moves joints or organs such as the tongue voluntarily, while smooth muscle is involuntary and located in visceral organs. There are just enough exceptions to show that these are not iron clad rules, but few enough to be "exceptions that prove the rules" instead of invalidating them.


Optional slides

D-104 Stomach monkey (PASH)

Find the external smooth muscle layers of the stomach as you did earlier for the colon. The muscle layers are not as orderly in the stomach as they are in the intestines so look around for transverse and longitudinal profiles of smooth muscle. PAS is an interesting stain for this tissue because it stains the bits of carbohydrate on the cell surface but not the cytoplasm (illustration). Thus you can get a good feel for how big and closely packed the cells are. This is another case where it pays to snoop around for the best field.

The "H" in PASH stands for hematoxylin to stain the nuclei. Thus, you can see how wide the diameters of the nuclei are relative to the diameter of the cells (would you say about half the width?) Try to estimate how much longer the cell is than its nucleus (how would you do this?) The smooth muscle cells are quite long in some tissues, such as the gut, while stubby in others, such as those wrapped around tiny arterioles. Small smooth muscle cells with lesser amounts of actin and myosin are sometimes called myofibroblasts. The name is a warning that poorly differentiated smooth muscle cells can resemble fibroblasts. Embryonically, both cell types trace back to mesenchyme

D-109 Duodenum rabbit (FeH)

The section of duodenum on this slide has been cut especially thin and stained with iron hematoxylin to show cellular detail.  It offers an excellent view of smooth muscle.  Find a place where the outer longitudinal layer of muscle is cut exactly longitudinally, and enjoy (illustration).

D-11 Abdominal wall, rat, (Masson and PAS)

This is a section through the entire body wall of a rat including the skin. Examine it with your naked eye. The very dark, almost purple, boarder consists of stratified squamous epithelium. Sandwiched between it and the reddish stained skeletal muscle is a layer of (bluish) dense irregular connective tissue -- the dermis of skin. The body wall has several layers of skeletal muscle oriented in different directions. One layer is actually in the dermis. (Humans do not have skeletal muscle in any of their skin, what do you think it function is in animals?) The other layers of muscle are below the skin. Confirm these statements under the lowest power objective (illustration). Now examine the layer of skeletal muscle with the 10X objective. You can observe nice examples of fibers cut transversely and obliquely but few or none run in the plane of section. Of course in each muscle bundle the fibers run exactly parallel with their neighbors. Note the seams of connective tissue separating the muscles from each another and the numerous small blood vessels in them. Skeletal muscle is well vascularized.

The complex stain for this slide allows you to distinguish between the red and white muscle fibers. Find an area where the fibers are cut transversely. White and red fibers are intermingled; the red ones being grayish and small the white ones larger and bright red (illustration).  If you have not yet covered the difference between them in your physiology course, keep in mind what you see here until you do.

If you use oil immersion (which you may not want to bother with) you can see the myofibrils very well .  It will be obvious that these are round in cross section and very regular in size.  You can even see that they are not dispersed uniformly in the fiber, but instead are arranged in clusters separated by extra amounts of cytoplasm. Please forget that these clusters are called Cohnheim's fields. (Remember the child's game that Dostoevski described: Young Russian kids stand in a corner with their eyes closed trying not to think of a white bear. Now you can play the Cohnheim's fields game.)

D-95 Tongue (H&E)

This slide is comparable to D-92, except that it has been stained with H&E. Follow the guide to that slide for this one as well. the description of D-92 .