The basic goal of these two labs is to learn the microscopic anatomy of the alimentary tract. The labs involve examining a number of slides from different regions of the tract, so there is a lot of information to assimilate. Here are a few tips that should be helpful in processing this potentially confusing material:
First, always scan each section at low power. Look for obvious junctions, for example between the esophagus and stomach.
Next, identify the four layers characteristic of the G.I. tract: mucosa, submucosa, muscularis externa, adventitia/serosa. If these 4 layers are present you must be viewing part of the alimentary tube.
Once you have identified the 4 layers of the G.I. tract, look for the key structural features within each layer for each specific region of the tract. For example, stratified squamous epithelium lines the esophagus and anal canal, whereas simple columnar epithelium lines the stomach, small intestine, and large intestine. However, these GI organs with simple columnar epithelium can be distinguished by several criteria, most importantly by differences in mucosal folding. There are pits leading into coiled glands in the stomach, villi and straight glands (crypts) in the small intestine, and straight glands (crypts) in the large intestine. Thus villi are unique to the small intestine. TO HELP YOU KEEP TRACK OF CHANGES IN THE VARIOUS LAYERS, CLICK HERE FOR AN EXCEL SPREADSHEET THAT YOU CAN FILL IN AS YOU WORK THROUGH THESE SLIDES (click here to see a filled in version to check your work).
Cross-sectioned villi and glands will both appear as circular structures. However, in villi the surface epithelium will be seen enclosing cellular connective tissue (lamina propria), whereas in glands the epithelium will ring open spaces--the lumens of the glands.
At the end of this section of the lab manual you were issued, you will find a Review Exercise Chart (click here for a PDF version of the chart) that you should fill in to remind you of the key features of each region. As a study aid, relate structure with function. For example, absorptive cells with microvillus brush borders are found in the small and large intestine where most absorption takes place.
Use the orientation of the muscle layers of the muscularis externa to determine the plane of section for each slide. A cross-section of the tube will show longitudinally-sectioned muscle in the inner layer and cross-sectioned muscle fibers in the outer layer, whereas these relationships are reversed in a longitudinal section. (An oblique section of the tube cuts both muscle layers obliquely).
The textbook, lecture Powerpoints, and lecture notes should be helpful during slide examinations.
This slide shows all of the major components of the generalized alimentary tube wall. Can you find mucous glands in the submucosa? Use the orientations of the two muscle layers in the muscularis externa to determine the plane of section. Based on the type(s) of muscle present in the muscularis externa, was this section taken from the upper, middle, or lower region of the esophagus?
Identify the four layers characteristic of the GI tract. What sort of muscle do you find in the muscularis externa? From what portion of the esophagus is this section taken? Do you understand the plane of section?
Scan the slide at low power and note the change from esophagus (right-hand side of slide) to cardiac stomach (left hand side). Examine the esophagus at higher powers and determine the type of muscle present in the muscularis externa. At the junction observe the abrupt change in epithelium from stratified squamous to simple columnar.
In the cardiac stomach region, note the following:
Relatively short gastric pits leading into cardiac glands.
Disorderly arrangement of the glands, cut transversely and obliquely. What does this indicate?
As is characteristic of the cardiac stomach, all surface and glandular epithelial cells secrete mucus. What is the shape of these cells?
After identifying the 4 layers of the GI tract, focus on the gastric pits extending down to tubular gastric glands. PAS avidly stains carbohydrates, including those of mucous glycoproteins. This explains the intense dark staining of the apical regions of the mucous surface and neck cells.
Also apparent are the pale, round parietal cells in the middle of the gland and the granular chief cells near the base of each gland. Remember that parietal and chief cells are found in the fundus and corpus of the stomach, but not in the cardiac stomach (previous Webslide 0054).
This is an important slide, allowing a detailed examination of the different cells of the mucosa. Find the four layers of the GI tract, especially noting the mesothelium of the serosa on the outside of the stomach. Based on the orientation of the muscularis externa this is a longitudinal section. Find the muscularis mucosae and observe the folding of the mucosa into rugae. How does the shape and disposition of the lamina propria in the stomach differ from what you observed in the esophagus? What cell types do you observe here?
Now concentrate on the cells and folding of the mucosal epithelium:
Surface epithelium: The surface of the stomach is lined by simple columnar cells that secrete mucus. Unlike the goblet cells found in the trachea and intestines, these stomach cells appear rectangular in longitudinal section, with the top third of the cell filled with mucous secretion droplets
Pits: The wide-bore, funnel shaped pits are also lined by mucus-secreting cells which become shorter as one progresses deeper into the pit.
Gastric glands: Each pit leads to several branches of the gastric glands. Because the tubes are so tortuous, it is difficult to find a good longitudinal section of a gland for any length, but short segments are numerous. The lumens of these gastric glands are quite small in diameter compared to the pits. Cell types present in the glandular epithelium include:
Mucous neck cells: Pyramid-shaped cells with narrow bases containing nuclei and wider apices containing mucus droplets
Parietal (oxyntic) cells: Round, pale staining cells with round, centrally located nuclei.
Chief cells: Located at the base of the glands near the submucosa, the zymogen-secreting chief cells contain pale basophilic apical secretion granules. Parietal and chief cells are characteristic of the fundus and corpus of the stomach.
Undifferentiated cells: Since the epithelium of the stomach is renewed every several days, you might observe undifferentiated cells near the base of the gland that will eventually differentiate into one of the other cell types.
Endocrine cells: These cells, squeezed between the parietal and chief cells and the basal lamina, are pale with round nuclei and masses of small acidophilic granules located near the base of the cell. (Why are the granules located in this position?) Endocrine cells are rather infrequent and you may not find any: don't spend a lot of time looking for them.
After defining the four layers and deciding what the plane of section is, note the following:
Mucosa with mucous-secreting surface cells. Where pits are cut tangentially, note the close packing of these cells (cross-sectioned). Gastric pits are nearly equal in length to glands. Cells of neck and base of glands are similar to surface cells. Some of the slides may contain a few chief cells and/or parietal cells, perhaps indicating that the section comes from the region of the corpus-pylorus transition.
Scanty lamina propria. The section contains a couple of mucosal lymph nodules. Often, you can discern lymphocytes between the cells of the epithelium, especially in the pits.
Muscularis mucosae and a very robust muscularis externa.
Nerve plexuses (autonomic) in the submucosa (Meissner’s) and between the layers of the muscularis externa (Auerbach’s).
Slide 10 has been stained with silver to show basal laminae, resulting in clear outlines of the various layers. Note the basal laminae underneath the surface epithelium, submucosal glands, the endothelium of capillaries, smooth muscle cells, and cells of the myenteric plexus. However, with this stain the cytoplasms and nuclei of the cells are quite pale. To get a complete picture of the organization of the duodenum, combine in your mind the images of cell structure from the last slide (Webslide 0081) with the images here of the underlying basal laminae.
Villi: In longitudinal sections of villi, note connective tissue of the lamina propria, blood vessels, lymphatics (lacteals), and smooth muscle extending from muscularis mucosae. (What is the difference between basal lamina and lamina propria?)
Intestinal glands (crypts): Find one cut longitudinally for a short distance to observe goblet and absorptive cells.
Submucosal (Brunner’s) glands: Composed entirely of mucus secreting cells with silver-stained mucus droplets. It may be possible to find penetration of a duct from these glands through the muscularis into the base of a crypt.
In the small intestine villi and intestinal crypts in the mucosa are common to all regions, whereas submucosal glands are found only in the duodenum.
Webslide 0032 was taken from a portion of the ileum in which the lymphoid tissue is organized into closely packed lymphocytes along the boundary of the lamina propria and submucosa. Distinct nodules are rare in this slide.
Note the lacteals in the lamina propria of many villi. These blind-ended lymphatic vessels empty into a lymphatic network in the submucosa. The lymphatic vessels can be distinguished from veins in the submucosa by their lighter-staining lymph and the absence of red blood cells.
Find a crypt cut in longitudinal section and observe the presence of the various cell types you saw in the duodenum and jejunum. Of particular interest in this slide is the intact serosa continuous with the mesentery. Note the simple squamous epithelium, connective tissue, and vasculature of this supporting layer.
Although the villi are not well preserved here, this slide can be used to review quickly the differences among the segments of the small intestine. Which section is duodenum, jejunum, and ileum? Why? Pay particular attention to the M cells in the ileum.
After finding the 4 layers of the tract, carefully examine the folding of the mucosal epithelium into straight, uniform diameter tubular glands. Notice the appearance of these “test-tube” shaped glands in both cross and longitudinal section, and appreciate how this folding can be distinguished from the pits and coiled glands of the stomach and villi of the small intestine (previous slides). Simple columnar epithelium with goblet and absorptive cells lines the glands of the colon and rectum. The brush borders of the absorptive cell are much easier to see in Webslide 0093 below.
Identify the 4 layers of the tract and notice the folding of the pale-staining mucosa. Of particular interest in this section is the muscularis externa, where the outermost longitudinal layer is divided into three prominent bands, the taeniae coli (observed by scanning the outer regions of the tube at low power).
This longitudinal section includes the recto-anal junction, with the rectum on the right hand side of the slide. First examine the rectum, noting the four layers, the mucosal folding, and the characteristic goblet and absorptive cells in the surface epithelium. A thin brush border characterizes the absorptive cells. Find the junction and note how the muscularis mucosae decreases in thickness near the junction. Is the stratified squamous epithelium of the anal canal keratinized distally or proximally? Note the striated muscle bundles below the muscularis externa. What is their function? Find the circumanal (apocrine sweat) glands and other epidermal structures nearby.
For the purpose of histological descriptions, the esophagus is subdivided into upper (entirely skeletal muscle in the muscularis externa), middle (mixed smooth and skeletal muscle) and lower (entirely smooth muscle) portions. Slide UCSF 226 is from the upper 1/3; slides 126 and 153 are from the middle 1/3; and slide 155 is from the lower 1/3 (at the esophageal-cardiac junction). The esophageal epithelium[example] is the non-keratinized stratified squamous type and is supported by a connective tissue lamina propria. Note the presence of isolated lymphoid nodules[example] and scattered leukocytes in the lamina propria. A rather thick layer of longitudinally arranged smooth muscle fibers form the muscularis mucosae [example]. The connective tissue of the submucosa consists of mostly collagenous fibers with some elastic fibers and varying amounts of fat as well as submucosal sero-mucous glands which can be readily observed in both slide 126 [example] and slide 153 [example] (those in slide 155 are not very well preserved).
In the upper esophagus, as shown in Slide UCSF 226, the muscularis externa consists of both inner and outer layers of skeletal muscle only. In the middle esophagus, the muscularis externa contains a mixture of skeletal and smooth muscle as seen in slide 126[example], whereas in the lower esophagus only smooth muscle is found as seen in slide 155[example]. Present in all regions of the esophagus (upper, mid, and lower) is the myenteric (Auerbach’s) plexus[example] between the two layers of the muscularis externa. For most of its extent, the esophagus is retroperitoneal, so its outermost layer consists of a connective tissue adventitia which merges with the adjacent connective tissue associated with nearby structures (such as the trachea as shown in slide 126). Below the diaphragm, however, the esophagus is suspended within abdominal cavity and is therefore covered by a connective tissue serosa as shown in slide 155.
A. Cardiac glands (cardiac region of stomach) Slide UMich 155 40x (gastro-esophageal junct, H&E) WebScopeImageScope
These mucosal glands of the stomach are composed mainly of mucous cells with pale staining cytoplasm and basally located nuclei. Present, but not seen, are stem cells and endocrine cells. In slide 155, locate the cardio-esophageal junction[example]. Note the abrupt transition from the mucosa of the esophagus with its stratified squamous epithelium to the glandular mucosa of the stomach. The cardiac gastric glands[example] are present only in a very small segment of the stomach mucosa adjacent to this junction. They are mucous glands comprised of a HOMOGENEOUS population of pale-staining columnar cells with a “bubbly” supranuclear cytoplasm. As you move further into the stomach, the cardiac glands are very quickly replaced by gastric proper (or fundic) glands which, as described below, consist of a notably HETEROGENEOUS mixture of basophilic chief cells and eosinophilic parietal cells.
“Gastric” or “fundic” glands are characteristic of the mucosa throughout the fundus and corpus regions of the stomach. At low magnification, notice that the gastric pits are relatively more shallow here [example] and the tubular gastric or fundic glands are relatively much longer than those in the cardia or pylorus. Observe that the tall columnar cells lining the luminal surface and pits have basally located nuclei and lightly staining cytoplasm. Most of these cells secrete mucus, that is stored in the apical cytoplasm. Also identifiable are lighter-staining “mucous neck cells” present in the neck region of the gastric glands and mucus-secreting cells of the cardiac glands found near the gastro-espophageal junction.
Moving into the body of a fundic gland, identify the parietal and chief cells in slides155[example][ORIENTATION], slide 156[example][ORIENTATION], and slide 157 [example] [ORIENTATION]. Parietal cells are large, ovoid to pyramidal shaped cells with their broad side adjacent to the basement membrane. Each cell contains a round, centrally located nucleus and reveals a lightly eosinophilic cytoplasm that appears granular due to the presence of many mitochondria. The chief cells are present in the lower one-third to one-half of the gastric glands. The apical cytoplasm of these cells may appear granular due to the presence of zymogen granules, that may stain bright red in H&E preparations (as in slide 157). However, in other preparations (slides 155 and 156), the pepsinogen has been extracted and the empty secretory granules resemble many glass beads in the supranuclear cytoplasm. The base of the cytoplasm, on the other hand, is distinctly basophilic.
C. Pyloric glands (pylorus or antrum of stomach) Slide 162 40x (gastro-duodenal junct, H&E) WebScopeImageScope
In slide 162, you can see the transition from pylorus of the stomach to duodenum of the small intestine. The pyloric region of the stomach is characterized by a thick wall due to the presence of the pyloric sphincter muscle[example], which is comprised primarily of the inner circular layer of the muscularis externa. Compare its wall thickness with that of the adjacent duodenum (W pg 273, 14.15). The pyloric glands[example] at the base of each gastric pit [ORIENTATION] are also composed again of a mostly HOMOGENEOUS population of mucous cells that are similar in appearance to those in cardiac glands although the pits are much deeper compared to cardiac glands. Present, but not seen, are stem cells and endocrine cells. An occasional parietal cell may be also found. Note that the bases of the pyloric glands abut the muscularis mucosae whereas in the duodenum, you will see abundant glands (Brunner’s glands) DEEP to the muscularis mucosae (i.e. in the SUBMUCOSA).
Just in case you are worried about identifying cardiac glands versus pyloric glands, it is admittedly very difficult to tell these two apart based only on high-mag views, but you can always use contextual information to help you out: cardiac glands will be right near the gastro-esophageal junction whereas pyloric glands are at the gastro-duodenal junction.
Look at slide UCSF 242 first. Locate the duodenal portion in this slide and notice the presence of submucosal mucous glands (Brunner’s glands). Observe that the ducts of these glands (and, occasionally, some acini) penetrate the muscularis mucosae and open into a crypt of Lieberkühn. After viewing slide 242, move to slides 162 and 161 and try to find the duodenal region in these tissue sections.
View these sections with the low power objective and identify the mucosa, submucosa and the muscularis externa. Note that the mucosa consists of three sub-layers:
lamina propria (or lamina propria mucosa –”propria” means “belonging to”
muscularis mucosae (or lamina muscularis mucosae –”mucosae” here is not plural, but genitive, so this literally means “muscular layer of the mucosa”)
The mucosa, which is clearly demarcated from the submucosa by the prominent muscularis mucosae layer, frequently shows heavy lymphocytic infiltration in the lamina propria.
The appearance of the submucosa layer is a bit variable, but, in general, it’s best considered as irregular connective tissue: in slide 29 the submucosa appears more “loose” whereas in slides 168 and 170 it is more dense, and, in slide 169, here the submucosa is edematous and exhibits unusually dilated blood vessels. You can see the intestinal villi and intestinal glands (crypts of Lieberkühn). Examine the villi at a higher magnification and note that the lining epithelium consists of simple columnar cells (aka enterocytes) with a brush border and interspersed goblet cells, particularly well-demonstrated in slide 168.
The epithelium lining the villi continues into the intestinal glands. Examine several of these glands in slides 169 and 169 and note that goblet cells and enterocytes similar to those lining the villi, cover the upper portions of the gland. Also, notice that there are many mitotic figures[example] near the base of the crypt consistent with the location of the stem cell population for the mucosa. You may also be able to see the enteroendocrine cells[example] in this region. These are the cells with spherical nuclei and clear cytoplasm –the secretory granules of these cells are not always stained very well, but, if they are, you should note that the granules are oriented basally.
Slides UCSF 246and UCSF 247 have some excellent examples of enteroendocrine cells [example]. Again, the enteroendocrine cells have a clear cytoplasm and, if visible, basally-oriented granules. Slide 247 in particular has been stained with ammonium silver nitrate to demonstrate a subset of enteroendocrine cells called “argentaffin” cells [example] because silver salts specifically stain their granules (the latin word for silver is 'argentum').
Paneth cells[example] occupy the base of the intestinal cypts/crypts of Lieberkuhn. They are fairly well preserved in slides 168 and 29. These cells are pyramidal shaped with round nuclei located near their base. They contain brightly eosinophilic (almost orange) secretory granules in the apical cytoplasm. In slide 168, the secretory granules in the Paneth cells stain a refractory brown or green.
Just under the mucosal epithelium is the lamina propria (or lamina propria mucosa), which consists of loose connective tissuethat fills the spaces between the intestinal glands and forms the cores of the intestinal villi. Within the core of each villus is a central lacteal, capillaries, and delicate wisps of smooth muscle that extend from the muscularis mucosae below. However, in some regions, the lamina propria may be so packed with a heavy infiltration of lymphoid cells that these finer structures may not be visible. You may hear the term “Peyer’s patches” used to describe such regions in the GI tract. However, technically, Peyer’s patches are found ONLY in the ileum and they are big enough to be visible with the naked eye.
The muscularis mucosae (or laminae muscularis mucosae) consists of smooth muscle fibers. Observe that strands of smooth muscle fibers from the muscularis mucosae extend into the cores of the intestinal villi along the central axis. Contractions of this muscle layer are controlled by ganglion cells and nerve fibers of the submucosal (Meissner’s) plexus[example] located in the submucosa, (W pg 266, 14.4a). The muscularis externa consists of two layers of smooth muscle: inner circular and outer longitudinal. Observe the ganglion cells and nerve fibers of the myenteric (Auerbach’s) plexus[example] located between the two muscle layers.
The mucosa of the colon is lined by a simple columnar epithelium with a thin brush border and numerous goblet cells. Note that there are no plicae or villi. The crypts of Lieberkühn are straight and unbranched and lined largely with goblet cells. In many regions the mucus is partially preserved and stains with hematoxylin. At the base of the crypts, undifferentiated cells and endocrine cells are present; however, Paneth cells are not usually present. The appearance of the lamina propria is essentially the same as in the small intestine: Leukocytes are abundant and the isolated lymphoid nodules present in this tissue extend into the submucosal layer. The muscularis mucosae is a bit more prominent compared to the small intestine, and consists of distinct inner circular and outer longitudinal layers. The submucosa of this specimen is particularly well fixed such that you may better appreciate the mixture of irregular connective and adipose tissue, numerous blood vessels, and several excellent examples of ganglion cells and nerves of the submucosal plexus. The muscularis externa of the large intestine is different from that of the small intestine in that the outer longitudinal layer of smooth muscle varies in thichness and forms three thick longitudinal bands, the taeniae coli (taenia = worm). This section happened to be cut such that a piece of one of these longitudinal bands may be seen.
Look at these slides (especially slide 177-3) at low magnification first to locate the recto-anal junction. Here you will observe a narrow zone of transition from the simple columnar epithelium of the intestine to the keratinized stratified squamous epithelium of skin. Within the transition zone, you may find stratified columnar (or sometimes cuboidal) epithelium followed by nonkeratinized stratified squamous epithelium. While looking, move the image from the colon toward the direction of the recto-anal junction. Observe that the crypts become shorter and shorter, eventually disappearing near the junction. Also observe that the muscularis mucosae becomes tattered and disappears, allowing the lamina propria merge with the underlying submucosa in this area. Note the presence of a large number of submucosal veins [example]. When these veins become dilated and varicose, they cause the mucosa to bulge and create the condition commonly known as hemorrhoids. Examine the skin lining the anal region and observe sebaceous and sweat glands, hair follicles, etc., particularly evident in slide 177-2[example]. Also, note the massive amount of smooth and skeletal muscle that form the internal[example] and external anal sphincters [example], respectively. The primate specimen (slide 177) shows these muscles quite well.