SLIDES OF MUSCLES

There are three major types of muscle, and their structure reflects their function.  Skeletal and cardiac muscle cells are called striated muscle because of the very regular arrangement of their intracellular contractile units, sarcomeres, at the light microscope (LM) and electron microscope (EM) levels.  This regular arrangement imparts a cross-striated (or striped) appearance.  Such an arrangement is not seen in smooth muscle cells.  Skeletal muscle is also called voluntary muscle, because its contraction is under conscious neural control.  In contrast, cardiac and smooth muscles are called involuntary muscles because their contractions are either spontaneously generated or are under the control of the autonomic nervous system.

Each of these three types of muscle has a characteristic appearance in both cross and longitudinal sections.  You should be able to recognize each type of muscle in both planes of section.

SKELETAL MUSCLE

A. Cytology of skeletal muscle cells

• 058L skeletal muscle H&E longitudinal Webscope 
• 058Lex Skeletal muscle H&E longitudinal Webscope 
• 058Thin skeletal muscle H&E longitudinal Webscope 
• 058T Skeletal muscle H&E cross Webscope 

In longitudinal sections of skeletal muscle (Slide 58, odd-numbered slide boxes), observe the following :

1. Nonbranching, cylindrical shape of the cell (also referred as muscle fiber). These cells are very long; you cannot see their ends.
2. Be sure you can identify the borders of the muscle cell. You might see occasional nuclei which appear to be centrally located, but aren’t. Why?  Again the answer to the question is in the plane of section. Cells with peripheral nuclei that are cut at an angle can appear to have centrally located nuclei. This is why it is important to look at other features of the cell to determine what exactly it is.
3. Peripheral position of the elongate nuclei just inside of the sarcolemma (plasma membrane). Note that each cell contains large numbers of nuclei.

4. Cross striations can be seen, and are due to the structure of the sarcomere. A sarcomere consists of the structures between two Z lines. You should recognize: (Using your microscope and glass slides may help to see these fine structures)

   1. The dark A band
   2. The lighter H zone which bisects the A band
   3. The light I band d. The dark Z line which bisects the I band.

   It is admittedly difficult to see the Z line and especially the H zone with the light microscope. However, they can be seen clearly on some areas of almost all slides, and it is just necessary to do some looking around for a favorable area on your slide. Here are some good examples showing cross striations: #058L   Webscope #058L    Webscope 

5. A less regular longitudinal striping can sometimes be seen within the muscle fibers and is due to the bundling together of the thick and thin filaments into myofibrils, which are arranged parallel to the long axis of the cell. Note that sometimes the cross striations are not aligned all the way across the cell. This is because different myofibrils may not be aligned.In transverse sections of skeletal muscle cells (slide 58, even slide boxes), observe thecylindrical shape of the cells (fibers) and the peripherally-located nuclei. Note also the the cytoplasm of the muscle cells has a stippled, punctate appearance which is due to the bundling of thick and thin filaments into myofibrils as mentioned above. What exactly is a muscle cell and what’s the difference between a muscle fiber and a muscle fibril? The basic units of muscle are the contractile proteins actin and myosin arranged in sarcomeres.  Placed end to end, these sarcomeres form long bands called myofibrils.  Within a skeletal muscle cell, the numerous myofibrils are separated by glycogen, mitochondria, and muscle triads (two terminal cisternae and a T tubule) and other organelles.  Chances are, if you are looking at an electron micrograph of muscle tissue, you are looking at myofibrils.  Groups of these fibrils form a muscle fiber, which is surrounded by endomysium.  Muscle fiber and muscle cell are synonymous.  Groups of individual cells that are surrounded by perimysium are known as fascicles and groups of these fascicles, surrounded by epimysium make up a muscle.

Perimuscular Connective Tissue

• 059-3 forearm muscle Masson cross fetal Webscope 
• 059-1 forearm muscle H&E cross fetal Webscope 

In slide 59, stained with trichrome (even slide boxes) or H&E (odd slide boxes), three layers of connective tissue sheaths are visible. This is a transverse section of an entire fetal forearm, and contains many elements in addition to the muscle, such as nerves and tendons, both of which are, unfortunately, similarly bundled into fascicles and therefore easily confused with muscle. So, make sure you’re looking at the muscles Image  Image

1. Endomysium – thin connective tissue sheath, consisting of basal lamina, some reticular fibers and capillaries around each muscle fiber. In fetal muscle, endomysium is not always clearly defined, so it is best to use the trichrome-stained slide (slide 59-3) to this –it is the connective tissue immediately surrounding the individual cells and very delicate perhaps visible only as a bluish tinge.
2. Perimysium – loose connective tissue sheath consisting of type I collagen fibers found around fascicles (bundles of muscle fibers). Note that the tissue in these specimens underwent some shrinkage during preparation so the separation between fascicles is somewhat exaggerated. Similarly, much of the perimysium has been damaged; however there are still some areas where it can be clearly seen (e.g. around many of the blood vessels found within the belly of the muscles).
3. Epimysium – the dense irregular connective tissue sheath around the entire muscle = deep fascia in gross anatomy; contains even larger blood vessels and nerves.

Three layers can be seen in #059-3   Webscope  Image

CARDIAC MUSCLE

• 057 ventricle H&E Webscope 
• 098-1 Heart ventricle H&E Webscope 
• 098N right wall Masson Webscope 
• 305 heart ventricle H&E Webscope (note: this slide NOT in glass slide collection)

Cardiac muscle will be studied in the wall of the ventricle of the heart. In comparison with skeletal muscle, note the following differences. Cardiac muscle cells branch and form a three-dimensional network. These branch points can sometimes be seen in your sections, and you should also note that the muscle fibers are less parallel than in skeletal muscle.

A. In longitudinal sections, observe:

1. Intercalated discs which are dark lines which cross the cell transversely. Look around in slide 57 #057  Webscope or slide 305 INDO 390 for lightly stained regions of the slide. There are artifactual transverse breaks in some of the muscle fibers, which are NOT intercalated discs. Intercalated discs are also fairly easy to find in the areas where muscle fibers are longitudinally oriented in slide 98-1 #098-1  Webscope and/or slide 98-N#098N   Webscope (be sure to look at both H&E and trichrome-stained sections).
2. The nucleus is centrally located in a fiber.
3. Longitudinal striping due to myofibrils can sometimes be seen.

B. In transverse sections, observe:

1. The myofibrils are coarse and give rise to the nonhomogeneous, punctate appearance of the sarcoplasm.
2. The nuclei are centrally-located. You won’t see a nucleus in every fiber cross-section.
3. The extensive network of capillaries in the endomysium –heart muscle is ALWAYS beating and therefore always in demand of oxygen and nutrients delivered via the blood.

MAKE SURE YOU CAN DIFFERENTIATE BETWEEN CARDIAC AND SKELETAL MUSCLE IN BOTH LONGITUDINAL AND TRANSVERSE SECTIONS!

SMOOTH MUSCLE

• 029-1 Small Intestine (simple columnar epithelium, simple squamous epithelium) H&E crossWebscope 
• 169 jejunum H&E cross Webscope 
• 155 gastro-esophageal junction H&E longitudinal Webscope 
• 250-2 vagina Masson Webscope 
• 250-1 vagina H&E Webscope 

Smooth muscle may be studied using slide #29 #029-1 smooth muscle  Webscope or slide #169, #169   Webscope,  both in the intestine.  To find the muscle layer, look at the at slide at the lowest power (this is about the same as looking at the glass slide with the naked eye).  The purple layer is largely the epithelium and the lamina propria filled with plasma cell and lymphocytes.  Next to that you see a lighter region of connective tissue (the submucosa you looked at to see loose connective tissue and fibroblasts), then a darker pink region which is made up of the two layers of smooth muscle you want to look at.  Slide 29 is a cross section of the intestine, so the inner, circular layer of muscle will have cells oriented longitudinally (or, in places, the cells may appear to be oriented more obliquely).  Move further out to see the outer sheet of smooth muscle, which runs longitudinally along the intestine, and will therefore be seen in cross section.

Look at slide #155, which is a longitudinal section of the GI tract at the gastro-esophageal junction, to see more smooth muscle in various planes of section.  The smooth muscle in the esophagus (the part lined with a stratified, non-keratinizing squamous epithelium) #155  Webscope is organized in the “classic” inner circular and outer longitudinal arrangement.  However, the stomach (the part lined by a columnar epithelium) #155 Webscope  has an inner oblique layer (seen mostly as longitudinal here), a very prominent middle circular layer, and a sometimes less obvious outer longitudinal layer. Don’t worry knowing about the specific layers or being able to tell esophagus from stomach. However, you should definitely be able to identify smooth muscle in any plane of section (tranverse, longitudinal, or even oblique).   In this particular slide, both the hematoxylin and eosin staining are quite intense, which should help you to see the cytoplasm more clearly, especially when the muscle is cut in cross section.

In longitudinally cut smooth muscle cells, observe the following points:

1. Cells are small and spindle-shaped (fusiform); this may be hard to appreciate because the cell membrane is indistinct.
2. Myofibirils and cross striation cannot be seen.
3. Nuclei are narrow, elongated and sometimes kinked or spiraling. They are centrally located.

In transversely cut smooth muscle cells, observe the following points:

1. The cell has a small diameter.
2. The nucleus is located centrally, but will not be seen in every cross section.
3. Myofibrils cannot be seen.
4. Cross-sectional diameters vary due to the spindle shape of the cells.

Now, look at slide #250 and see if you can distinguish between small fascicles of smooth muscle and collagen fibers in the lamina propria (this task will be easier if you look first at the trichrome-stained section, which stains the muscle pink(ish) and the collagen blue) #250-2  Webscope .  It’s more challenging to make this distinction in the H&E-stained section #250-1  Webscope.  You should note that smooth muscle is pink, wheras collagen is a bit more orange-red.  Also, smooth muscle tissue is mostly cellular (and therefore more nuclei are present), whereas the connective tissue is mostly extracellular collagen fibers with fewer cells. The table below compares the differences in the morphology of the three types of muscle.

1. Why do some skeletal muscle cells seem wider than others?
   Answer:The plane of section is generally responsible for making some cells look larger than others.  Some skeletal muscle cells on your slide may be sliced through the middle and appear quite large, while as other cells may just graze the section and appear much smaller.  Also, some muscle cells are simply smaller or larger than others.  They can range in size from 10 to 100 mm in width.
2. You might see occasional nuclei which appear to be centrally located, but aren’t. Why?
   Answer:Again the answer to the question is in the plane of section. Cells with peripheral nuclei that are cut at an angle can appear to have centrally located nuclei. This is why it is important to look at other features of the cell to determine what exactly it is.
3. So what exactly is a muscle cell and what’s the difference between a muscle fiber and a muscle fibril?
   Answer:The basic units of muscle are the contractile proteins actin and myosin arranged in sarcomeres. Placed end to end, these sarcomeres form long bands called myofibrils. Within a skeletal muscle cell, the numerous myofibrils are separated by glycogen, mitochondria, and muscle triads (two terminal cisternae and a T tubule) and other organelles. Chances are, if you are looking at an electron micrograph of muscle tissue, you are looking at myofibrils. Groups of these fibrils form a muscle fiber, which is surrounded by endomysium. Muscle fiber and muscle cell are synonymous. Groups of individual cells that are surrounded by perimysium are known as fascicles and groups of these fascicles, surrounded by epimysium make up a muscle.