The goal of this lab is to learn how to identify and describe the organization and key structural features of smooth and skeletal muscle in sections. A challenge is to be able to distinguish smooth muscles fibers from the collagen fibers of connective tissue.
I. Muscle Tissue
As you go through these slides, refer to this schematic drawing showing the key structural features and relative sizes of skeletal, smooth, and cardiac muscle as you would observe them with the highest objective setting.
1. Motor End Plate
Webslide 0102_N: skeletal muscle spread, acetylcholinesterase stain
Webslide #102 contains a whole mount of the motor end plate (MEP) region of several muscle fibers. Histochemical staining for cholinesterase lets you see the MEP (unfortunately, the staining also produces lots of fine, granular precipitate visible as little dark dots all over the tissue). The MEPs are hard to find in routine muscle sections, because there is only 1 per fiber. This prep was taken from the region of a muscle where most of the MEPs are concentrated, and they are visible as larger platelike structures found at the ends of the axons that appear as fine threads.
2. Sarcomere Organization in Slides
Webslide 0013_A: skeletal muscle, primate, c.s., H&E
At low magnification, note the range of fiber diameters and shapes; note the multiple, peripheral nuclei associated with each fiber (recall that each muscle fiber is a single cell) and numerous blood vessels in the connective tissue surrounding each fiber. At high magnification, note sizes and shapes of myofibrils within each muscle fiber. Within fibrils, electron microscopy would show an orderly hexagonal filament lattice filling out the many odd shapes. The perfect jigsaw-puzzle fit of polygonal fibrils to one another, and of fibers to one another, is characteristic of well-preserved and life-like muscle. Truly cylindrical fibrils are found only in text-book diagrams and some insect flight muscles.
Learn to distinguish the skeletal muscle fiber nuclei from the more elongated nuclei of capillaries and fibroblasts found in the surrounding connective tissue.
Webslide 0017_N: rabbit psoas muscle, stretched, long. sect. H&E.
Note fibers, nuclei, capillaries and connective tissue.
Observe myofibrils (staggering and longitudinal splitting of cross-banded substance) and try to determine the average sarcomere length (use the ruler tool and enter the data into this online calculator).
UMich slide 058thin: rabbit psoas muscle, long. sect., H&E.
Slide 058thin is similar to 0017_N above, but it was scanned with a higher power objective making it easier to see the fine structure of skeletal muscle such as Z lines (in the middle of the I bands) and, in some areas, H bands (in the middle of the A bands).
Webslide 0032_D: Ileum, monkey, H&E
Towards the top of the slide is a typical appearance of smooth muscle in H and E sections. This section from the gut shows the outer longitudinal layer in longitudinal section (LS) and the inner circular layer in cross-section (XS). In longitudinal section, note the serrated edges of the cells which represent cell-cell junctions. Note how clearly nuclei are seen within fibers, and note how in XS many fibers lack nuclei (remember why?). Collagen fibers, seen here in submucosa, stain more lightly. This makes the contrast with smooth muscle easier to notice. Note that collagen fibers appear looser-packed, and more varied in size and direction than smooth muscle fibers. Note how, among collagen fibers, nuclei are always fewer and lie external to fibers. Smooth muscle occurs in snug parallel bundles, with more nuclei and with nuclei all internal to fibers.
Webslide 0098_G: urinary bladder, H&E
The wall of the urinary bladder contains layers of smooth muscle interspersed with collagen. In this thin section, use the same criteria as described above for Webslide #32 to distinguish between collagen fibers and smooth muscle.
Webslide 0023_N: vena cava, monkey, plastic, c.s., H&E
Many longitudinal bundles of smooth muscle are cross sectioned in the outer wall of this large vein. Some bundles of smooth muscle look strange, almost nerve-like, because few or no nuclei appear at section level. At high power, many cell boundaries appear serrated like postage stamps. (In the EM, the bulges would show many subsurface vesicles and caveolae, while the grooves would locate dense adhesion plaques under the surface membrane that anchor the actin filament bundles.)
This is also a great slide for test your ability to distinguish smooth muscle from connective tissue.
Webslide 0093_J: recto-anal junction, mammal, H&E
As always, start with lowest magnification to get an overall idea how tissues are distributed. This well-stained slide gives excellent and easy practice in distinguishing between 3 types of fibrous tissue, namely collagen, smooth muscle and skeletal muscle. (Why is it not enough just to call it striated muscle?) For now examine only the muscle and connective tissue that is towards the right-hand side of the slide. In the review portion of this lab we will consider other tissues. Note typical fiber diameters, textures, and placement of nuclei. Smooth muscle nuclei in LS appear less elongated than in previous slides, evidently shortened here by contraction.
II. Pathology Correlate
Slide 33 [DigitalScope]
This slide contains a biopsy of the uterus from a 38-year-old who presented with irregular menstruation and intense, episodic uterine cramping. Grossly, the muscle layer in the wall of the uterus (i.e., the "myometrium") was distorted by numerous circumscribed nodules ranging in size from 2 to 6 centimeters. As you inspect this slide at low magnification, you will note a roughly circular area in the center of the tissue section [EXAMPLE] that stains more deeply than the surrounding myometrium. This area are not truly encapsulated, but it looks different, and appears to be separated from the adjacent myometrium along at least part of its circumference and thus has the appearance of a nodule, which is commonly called a "fibroid."
Note that this circumscribed area is more densely cellular, with a higher concentration of nuclei and a bluer color.
Note the structure of the surrounding typical myometrium -- interlacing bundles of spindle-shaped, smooth muscle cells that run in fascicles. Some of these fascicles are cut longitudinally, and others in cross-section.
You will see that the neoplasm is also composed of interlacing bundles of similary spindle-shaped, smooth muscle cells. While not actually encapsulated, the nodule is easily distinguished from its surroundings by its denser cellularity and darker color.
Compare the nuclei in the neoplasm with those in the normal myometrium and note that they are essentially identical (although more closely packed), thus indicating this is a BENIGN tumor of smooth muscle, also known as "leiomyoma."
Extra slides to study muscle tissue:
alternate UMich slide 155: esophagus-stomach junction, human, l.s., H&E (compare to WebSlide 96 above)
The alternate UMich slide 155 features very well-preserved and stained tissue and provides another opportunity to observe smooth muscle in several different planes of section. Refer to this orientation image to find the various layers of smooth muscle that can be seen.
In the esophagus:
- muscularis mucosae (shown here in longitudinal section) -this is a strip of smooth muscle adjacent to the epithelium of the esophagus.
- inner circular layer (shown here in cross section).
- outer longitudinal layer (shown here in longitudinal section).
In the stomach:
- innermost "oblique" layer (mostly in longitudinal section here) -this layer is thin and poorly organized, so it tends to appear more as strips of differently oriented smooth muscle next to the prominent middle circular layer.
- middle circular layer (in cross section here) -this is the most prominent muscle layer in the stomach.
- outer "longitudinal" layer (some parts are in longitudinal section; others are in cross section) -this layer is also rather thin and poorly organized in the stomach.
UMich slide 155 is also an excellent specimen to test your ability to differentiate smooth muscle from nearby connective tissue and peripheral nerve fibers.
Slide UMich 71-1B: Muscle and muscle spindle, c.s., H&E)
Neuromuscular spindles are stretch receptor organs that regulate muscle tone via the spinal stretch reflex. Look at slide #71-1B and identify the neuromuscular spindle in the within the perimysium between fascicles in the belly of the muscle [example]. In this preparation, the sensory nerve fibers of the spindle are NOT visible, but the modified skeletal muscle fibers (intrafusal fibers), which are smaller than the muscle fibers proper (extrafusal fibers), are easily visualized -- 2 to 10 fibers are contained in a fluid-filled space within a discrete, external connective tissue capsule. Note the intrafusal fibers are bundled together by a delicate internal capsule that is not so evident in these sections. The sensory receptors (nerve endings) are activated by stretching of the intrafusal fibers, which opens mechanically-gated ion channels in the nerve endings. This sensory input may evoke a reflex contraction of the extrafusal fibers that is driven by large (alpha) somatic motor neurons (located in the ventral horn) in a two-neuron spinal reflex arc.
It is worth noting that, in addition to being stretch receptors, the intrafusal fibers are functional, contractile muscle cells. They are innervated by special (gamma) motor neurons that set the tone of the intrafusal fibers thus modulating sensitivity of the stretch receptor (contraction of the spindle cells makes them taut and therefore even more sensitive to stretch). This also allows the spindle cells to contract in concert with the extrafusal fibers thus maintaining sensitivity to stretch over the muscle's full range of motion [see explanatory figure].