OBJECTIVES:

1. Be able to describe the functions of cells commonly found in connective tissue and identify them.


2. Be able to recognize interstitial (fibrillar) collagens and elastic fibers at the light and electron microscopic levels.


3. Be able to distinguish between elastic, type I collagen, type III (reticular) collagen, and elastic  fibers when appropriately stained material is presented.


4. Be able to use knowledge about the physical characteristics of collagen and elastin in explaining the functions of tissue where these molecules occur in large quantities (e.g., coarse type I collagen fibrils present in dense connective tissue compared to more delicate type III fibers found closer to the interface of cells and the extracellular matrix).


5. Be able to recognize types of connective tissue (e.g., dense irregular, dense regular, loose, adipose) and provide examples where different types of connective tissue are found in the body.


6. Be able to recognize a basement membrane (or basal lamina) in sections or micrographs where the structure is conspicuously present and understand its functions.

NOTE:

1. A brief listing of some common stains is present at the end of this section. You should have a general familiarity with H&E (Hematoxylin and Eosin), Masson, PAS, and elastic stains.


2. Please read the descriptions of the connective tissue stains BEFORE you do your lab work.

I. LOOSE CONNECTIVE TISSUE
Slide 29 (small intestine, H&E) [WinLab] [Mac] [WinHome]

Look at the connective tissue in the submucosa which is the lighter staining area [example] between the intestinal epithelium and the smooth muscle layer (also see W pg. 274, Fig.14.16b).  In this area note the irregular, wavy collagen fibers arranged singly or in small groups. The collagen of fibrous supporting tissues, the dermis of the skin, tendon, ligaments and bone is type I collagen, which provides tensile strength (R. pg. 150-158, Figs. 6.4, .5, .7). The background will be clear, reflecting a "ground substance"-rich connective tissue.  Look for elongated nuclei, usually solitary, from which a modest amount of tapered cytoplasm extends from either one or both poles of the nucleus.  These are fibroblasts [example] (as opposed to clusters of similar appearing elongate nuclei that are usually smooth muscle cells or Schwann cells in a nerve that you will learn to recognize soon).  Note that the nucleoplasm of a fibroblast has a generally fine stippled (dot-like) chromatin pattern with occasional coarse chromatin clumps (heterochomatin) and one or two nucleoli.  Look for more examples of fibroblasts and note that you rarely see much cytoplasm and that the nuclei of these cells can be quite condensed and hyperchromatic depending upon the plane of section or the metabolic state.  You should be able to recognize a range of nuclear morphologies and be able to identify the cells as fibroblasts (R., pg. 167, fig. 6.19). Most of the rounded cells you may see in the submucosa are likely white blood cells (monocytes, macrophages, and occasional neutrophils) that have migrated out into the tissue.

Now look at the region immediately underneath the intestinal epithelium (Just for the sake of review, what type of epithelium is this? (CT1), which is called the lamina propria [example] (W pg. 277, Fig.14.20).  This region is also a loose, irregular connective tissue but can be so extensively infiltrated by white blood cells and plasma cells that the supporting fibers and ground substance are obscured.  Neutrophils and macrophages are also present and both are discussed below. 

A. Plasma Cells (W pg. 238, Figs. 12.6, 12.7; pg. 80, Fig. 4.19; R pg. 173, Fig. 6.25)
Slide 29 (small intestine, H&E) [WinLab] [Mac] [WinHome]
Slide 40 (trachea, H&E) [WinLab] [Mac] [Winhome]

Look for plasma cells within the lamina propria of slide 29 [example].  They are round to oval shaped cells with a distinct cell boundary and a nucleus set to one side.  Note the coarse chromatin clumps organzed as radial spokes in the round nuclei which is therefore often described as having a "clockface" or "wagon-wheel" appearance.  You should note that the cytoplasm is quite basophilic (i.e. "base loving" so it binds hematoxylin and stains dark blue/purple), and, in well fixed tissue, the cytoplasm in many of the plasma cells is frequently granular (the rough endoplasmic reticulum, really) rather than smooth or even in appearance.  Why is the rough ER basophilic? (CT2)   

Find the Golgi complex, a pale or slightly eosinophilic (=eosin "loving", an area rich in membranes containing basic amino acids, syn. = acidophilic) region adjacent to the cell nucleus.  The Golgi complex in these particular plasma cells is usually in the form of a fine crescent adjacent to the nucleus and it takes some practice to recognize.

Recall that the primary function of plasma cells is antibody secretion, so they are a prominent constituent of loose connective tissue wherever antigens may enter the body, such as the gastrointestinal, urogenital, and respiratory tracts. Plasma cells may also be found within the connective tissue of many of the glands that secrete into these regions. An excellent example of this is slide 40 from the trachea (part of the respiratory tract). Look at the areas outlined in the orientation diagram of the trachea and locate the loose, cellular connective tissue within the glands (the "glands" are coiled tubes of columnar epithelial cells; some the epithelial cells are tall and eosinophilic, whereas others are shorter and more basophilic). In addition to some fibroblasts and a few delicate collagen fibers, you should see quite a few plasma cells [example] amongst the epithelial tubes.

Slide 40 is also a very good speciment to examine the pseudostratified, ciliated columnar epithelium of the trachea.   Note also that the basement membrane underlying this particular epithelium is especially prominent. Type IV collagen, which does not form fibrils, but rather a fine meshwork, is present in all basement membranes. The epithelium is anchored to the basal lamina by fine fibrils of type VII collagen (you obviously can't tell this looking at it in the light microscope, but you should recall this from lecture).

B. Neutrophils
Slide 29 (small intestine, H&E) [WinLab] [Mac] [WinHome]

Look in the lamina propria amongst the plasma cells and you will find neutrophils [example] that have emigrated from the bloodstream into the tissue space as part of the immune response.  Neutrophils can be identified by their granular cytoplasm and their multilobular, condensed nuclei (W pg. 50, fig. 3.4c).  Because of their nuclear morphology, they are frequently also called “polymorphonuclear leukocytes” (aka "PMNs" or “polys”).  Neutrophils generally enter tissues in large numbers only in response to a disease stimulus.   However, as seen in this slide, it is quite normal to find them in tissues such as the gut where foreign substances frequently invoke an inflammatory response.  You will study neutrophils in much greater detail in other sequences and in your histopathology course, but it is useful for now to at least be able to recognize them in various tissues and organs.

C. Macrophages
Slide 26 (lymph node, H&E) [WinLab] [Mac] [WinHome]

With low power, locate the medulla (the interior) of the lymph node (W pg. 219-20, Figs. 11.8-9).  Look for a region characterized by interlacing cords of cells (label MC in W pg. 220, Fig. 11.9).  Macrophages [example] are the biggest, rounded cells that are floating free in the spaces between the cords of cells.  Many of the free cells in these medullary sinuses cannot be identified; however, the large rounded cells, with eccentrically placed, vesicular nuclei are the ones you should try to find.

Many of these macrophages contain phagocytosed red blood cells or the brownish breakdown pigment, hemosiderin (which is the result of lysosomal action on the ingested red blood cells.).  Be sure you can identify a macrophage and not just a bunch of cells superimposed upon one another.  Macrophages can be seen also in the subcapsular sinus [example] (lighter staining area just under the capsule at the periphery of the lymph node, labeled "S" in W pg. 221-2, Figs. 11.10b and 11.11, see also schematic diagram in fig. 11.10a).

The "mononuclear phagocyte system" (also called the "reticuloendothelial system" for historic reasons) consists of free and fixed macrophages throughout the body.  These cells are important in removing all kinds of debris from the body as well as playing a major role in the immune response.  Review this macrophage system in Wheater, p. 78 and Ross and Pawlina, pgs. 167-8 and p. 170, Box 6.3.

D. Fat Cells
Slide 152 (pharynx, H&E) [WinLab] [Mac] [WinHome] (W pg. 75-6, Figs. 4.14-15; R pg. 242, Fig. 9.2)
Slide 30 (mesentery, H&E) [WinLab] [Mac] [WinHome]
Slide H2 (fetal thorax, H&E) [WinLab] [Mac] [WinHome]

Slide 152 is a section from the pharynx. What type of lining epithelium is present?  (CT3)   Locate the large clear circles [example] in the connective tissue that sits beneath the epithelium.  These are fat cells (or adipocytes). Can you see a nucleus in each cell?  (CT4)  In white or unilocular adipose tissue, lipids are stored as a single, non-membrane bound droplet in these cells.  A fatty tissue called brown or multilocular fat, produced during fetal development, has adipocytes that contain multiple fat droplets (W pg. 76, Figs. 4.16-17; R. pg. 244, Fig. 9.4). Brown fat is important for thermoregulation in newborns and hibernating mammals. In humans, brown fat is widely distributed throughout the body in the first decade of life, but it then disappears except for regions around the kidney, suprarenal glands, aorta, neck and mediastinum. None of our slides of adult tissue shows any brown fat, however this rather unique tissue can be seen in slide H2 [example], which is from a developing fetus. 

Look for adipose tissue in Slide #30 which is taken from abdominal mesentery (the connective tissue that suspends the viscera within the abdominal cavity). Some of the individual fat cells are often broken during tissue preparation, but the overall impression of what the tissue looks like is the important point.

E. Mast Cells (W pg. 79-80, Fig. 4.19-19; R pg. 169-172, Fig. 6.23)
Slide 160 (stomach, PAS & Azure II) [WinLab] [Mac] [WinHome] --you may find it helpful to view these cells in a "real" microscope.

Mast cells can only be definitively recognized with special stains such as PAS (periodic acid-Schiff) or Azure II that identify the heparin storage granules (Azure metachromatically stains the heparin purple). Mast cells are most abundant in the connective tissue associated with the lining of the digestive and respiratory systems, and your collection just so happens to contain a tissue section from the stomach that has been stained with PAS and Azure II. As you look at this section, you will see a very obvious layer of mucous epithelial cells (PAS also reacts with the carbohydrate-rich mucin). However, to see the mast cells, you will need to look deeper in the submucosa where you should find small, ovoid cells amongst the collagen fibers with spherical, eccentric nuclei and intensely basophilic (dark purple to black) granules [example]. The granules are often so dark that they obscure the nucleus. What do these cells do? (CT5)

II. Reticular tissue (W pg. 220-1, Figs. 11.9-10; R pg. 159, Fig. 6.12)
Slide 27 (lymph node, H&E) [WinLab] [Mac] [WinHome]
Slide 28 (lymph node, silver stain) [WinLab] [Mac] [WinHome]

The fine collagenous network that provides support in the bone marrow, lymphatic organs, around individual smooth muscle cells, and beneath most epithelia is composed mainly of Type III collagen. The collagen has an associated carbohydrate moiety (uncharacterized) that can reduce Ag+ to metallic Ag revealing a network (reticulum) of fine, black fibrils. These are termed reticular (or rarely, argyrophilic= "silver loving") fibers. On slide #27 look at the accumulations of darkly stained cells (lymphocytes in a lymph node). Note at high power that fibrils or fibers of any type cannot be readily observed. Now with slide #28 (make sure your slide is stained with silver; it should say “Ag” on the label!), note how a network of fine black fibrils is present in this same tissue following silver staining. These are reticular fibers, found in skin, muscle and blood vessels. The reticular fibers provide physical support for all the cells present in tissues subject to stretching. You are not responsible for recognizing reticular fibers unless a silver stain is used.

Please remember that virtually all cells (except for those in the brain and spinal cord) are provided with some degree of support by collagen (reticular fibers) even though that may not be apparent with H&E staining. (Masson Trichrome and silver staining are frequently used in pathology to determine if connective tissue has proliferated--a sign of damage and attempted repair--in the liver, kidney and lung.)

III. DENSE CONNECTIVE TISSSUE

A. DENSE IRREGULAR CONNECTIVE TISSUE (W pg. 168, Fig. 9.1; pg. 181, Fig. 9.12; pg. 73, Fig. 4.12; R pg. 63, Fig. 6.3; pgs. 176-7. Plate 4; pgs. 180-1, Plate 6, Figs. 1-2).
Slide 33 (skin, Verhoeff stain) [WinLab] [Mac] [WinHome] 
Slide 250-1 (vagina, H&E) [WinLab] [Mac] [WinHome]
Slide 250-2 (vagina, trichrome) [WinLab] [Mac] [WinHome]

The area beneath the stratified squamous epithelium shown in slide 33 is the dermis, which is composed of dense irregular connective tissue.  In this section, the fibers clearly predominate.  This slide has been stained with iron hematoxylin and eosin so you can see collagen fibers (orange) as well as elastic fibers (purple/black) in the dermis [example]. Note how the diameter of the fibers varies with location.  In the region immediately beneath the epidermis you can see how the elastic fibers are interconnected forming an elaborate, delicate net of fibers [example] amongst thin strands of collagen. However, deeper in the dermis, the collagen and elastic fibers are much thicker.

Slide 250 which you used to look at stratified squamous non-keratinizing epithelium is also useful for the study of connective tissue (we will also use this slide to study smooth muscle and peripheral ganglia).  Be sure you look at both the H&E and Masson trichrome-stained slides as they provide an excellent opportunity to see how collagen stains in connective tissue when either stain is applied.  When we study smooth muscle and peripheral nerve tissue we will come back to this slide to try and distinguish between collagen fibers and fascicles of smooth muscle and/or nerve fibers and ganglia.

NOTE:  Slide 250 illustrates a point about the limits of classification schemes. Even though we try to set up rigid categories (e.g. "loose" versus "dense" connective tissue), sometimes it is not always possible to classify connective tissue in a given section; it may be a little loose, a little dense, a little fatty, etc. In these particular instances, don't worry so much about trying to exactly classify the tissue per se, but at least try to identify the cellular and extracelluar components that you can (also bear in mind that you can't always definitely identify every cell) and think how its overall appearance reflects its function.

B. DENSE REGULAR CONNECTIVE TISSUE

1. Collagenous (W. pg. 206, Figs. 10.31-32; R. pgs. 178-9)
Slide 106 (plantar skin, H&E) [WinLab] [Mac] [WinHome]
Slide 112 (plantar skin, H&E) [WinLab [Mac] [WinHome]

Slide 106 and 112 have bits of well preserved flexor tendon at the top of the section (the tissue at the very top of slide 112 is actually skeletal muscle -which you'll study in the next lab; the tendon is just below it).  Note the regular orientation of the collagen fibers (there's a bit a "waviness" but you should get the idea). You should also observe that there aren't a lot of cells, a characteristic of "dense" connective tissue. The very small cracks between the fibers are just artifacts of shrinkage that occured during tissue preparation. Of course, there are some places where there are breaks in the dense regular connective tissue of the tendon containing loose connective tissue associated with nerves and blood vessels or the occasional bit of adipose tissue.

2. Elastic tissue (W pg. 157, Fig. 8.9; pg. 68, Fig. 4.6a,b; R. pgs. 180-1, Plate 6, Fig 3)
slide 36 (Aorta, aldehyde fuchsin) [WinLab] [Mac] [WinHome]
slide 88 (Aorta, H&E) [WinLab] [Mac] [WinHome] 

These slides are examples of regularly arranged sheets (lamellae) of elastin.  Slide 36 is stained with aldehyde fuchsin and Masson trichrome (Aldehyde Fuchsin, Fe. Hem. & Mass.), so the elastic lamellae are purple. Slide 88 is stained with H&E where the concentric rings of elastic lamellae are intensely stained with eosin giving a glassy red appearance [example] (one of the few places where elastin is easy to recognize in H&E sections). 

Two other locations where elastic fibers can be readily seen in H&E sections are in the lamina propria of the pharynx (slide 152 [WinLab] [Mac] [WinHome]) and the trachea (slide 40 [WinLab] [Mac] [WinHome]). Unlike the tissue of the aorta which would be classified as "regular," these tissues are obviously irregular but they're mentioned here for the purpose of illustrating how aggregates of elastic fibers appear in H&E-stained sections. Just as in the H&E-stained aorta, the elastic fibers in the pharynx and trachea are glassy and orange-red --they appear as stippled dots because they’ve been cut in cross section.

GENERAL AND CONNECTIVE TISSUE STAINS

Hematoxylin and Eosin

Hematoxylin is the most commonly used nuclear stain in histology and pathology although, despite its long use and honorable history, the chemistry of the dye is still not fully understood. Essentially, hematoxylin is a basic dye and complexes with nucleic acids (DNA and RNA in the nucleus; RNA in the cytoplasm) or other negatively charged molecules (such as sulfate groups). Structures that bind hematoxylin are therefore termed "basophilic" (base loving). Cells actively secreting proteins exhibit basophilic cytoplasm. Why? (CT6)

Eosin is an acidic dye and the basic structures it stains are termed "eosinophilic" or less commonly "acidophilic" (acid loving). It stains membranes and most proteins. Cells that have large quantities of folded membranes stain intensely with eosin, because of basic amino acids in the membranes (e.g. macrophages contain lots of membrane in the form of phagocytic vesicles as well as basic lysosomal enzymes within those vesicles that stain with eosin). Collagen is generally stained some shade of red/orange whereas actin (such as in smooth muscle cells) is a bit more pink. Elastin, when present in relatively large amounts (such in the walls of blood vessels, in elastic cartilage, and in the esophagus and trachea), will appear glassy red.

A note about acids/bases and their charges: It always seems to a point of confusion as to how it is that an acid such as DNA can have a negative charge when we generally think of something that is acidic as being positively charged (i.e. a solution with lots of H+ ions is "acidic"). However, the better way to think of acids is as proton donors --in solution, an acid such as DNA donates H+ protons (which makes the solution acidic). Upon donating protons, the DNA therefore becomes negatively charged and it is in this state that it binds hematoxylin.

Masson Triple Stain (or "Trichrome")

This dye combination stains mucus as well as collagenous and reticular fibers blue (aniline blue) or green (fast green) depending on the mixes of dyes used; muscle red; nuclei red (they are black if preceded by an iron hematoxylin). This is a commonly used connective tissue stain in both histology and pathology. On your slides the stain is designated "Masson" or "Mass"; but the blue or green collagen is the tip-off.

Elastic stains

• Aldehyde fuchsin
  Aldehyde Fuchsin is a deep purple dye. It stains elastic fibers and granules of beta cells in the islets of Langerhans, cartilage matrix, and stored neurosecretory product in the hypophyseal pars nervosa, among other things. In some of your slides, it is the only stain and therefore only elastin is demonstrated. Other times it is combined with Masson's trichrome.
  
  
• Weigert's stain
  Uses a different kind of fuchsin (basic fuchsin), but the result is similar: elastic fibers stain a deep purple color.
  
  
• Verhoeff/van Gieson elastic tissue stain
  Verhoeff's hematoxylin contains ferric chloride and iodide which causes it to stain elastic fibers deep purple/black. Frequently counterstained van Gieson's solution with which stains collagen red/orange and cytoskeletal elements (such as actin) yellow-brown.

Silver Stain

In this case silver nitrate is reduced to metallic (black) silver. The process of development and fixation is similar to developing a photograph (stains reticular fibers).

Periodic Acid Schiff (PAS)

This is an extremely useful technique for demonstrating glycoproteins, mucins and some proteoglycans -anything that contains a relatively high amount of sugar groups. It involves the generation of dialdehydes from hexoses (present as the carbohydrate portion of the aforementioned compounds. One of its main uses is the demonstration of basement membranes, especially in the kidney, and/or in sections with epithelia atypia, where breech of the basement membrane is suspected in early carcinomas. An excellent example is slide 210 from the kidney [WinLab] [Mac] [WinHome] where PAS staining demonstrates the basement membranes (pink lines) of the simple cuboidal epithelium lining the tubules and squamous epithelium in the glomeruli (the round tangles of cells). Note that PAS staining also shows the glycocalyx associated with microvilli (appears as a fuzzy pink border) on epithelia lining some of the tubules.

Electron Micrograph Wall Charts

#21 DENSE CONNECTIVE TISSUE [WinLab] [Mac] [WinHome]

Note the alternating layers of fibroblasts and collagenous fiber bundles. Make sure you can see the difference between cross sectioned and longitudinally sectioned collagenous fibrils. In dense connective tissue, which type of cell is most common? (CT7)

#22 LOOSE & DENSE CONNECTIVE TISSUE [WinLab] [Mac] [WinHome]

This is an excellent low power example of the roles connective tissue serve. Observe how the connective issue fibers and cells "embed" other tissues, such as nerves, smooth muscle, and serve as base for epithelium. Blood vessels are also encircled by connective tissue, and an extension of the epithelium -- the glands. Where are the nuclei of the fat cells? (CT8)

#23 FIBROBLASTS [WinLab] [Mac] [WinHome]

Observe the large amount of rough endoplasmic reticulum (ER) in these cells. Is this an indication of an active or inactive cell? (CT9) Test your ability to identify different organelles at this magnification!

#24 COLLAGEN & ELASTIN (CROSS SECTION) [WinLab] [Mac] [WinHome]

In this cross section of connective tissue fibers, note the rather equal diameter of all collagenous fibrils. Observe the two components of the elastic fiber.

#25 COLLAGEN & ELASTIN ( LONGITUDINAL SECTION) [WinLab] [Mac] [WinHome]

Observe the branching nature of the elastic fiber and the "mantle" of elastic microfibrils. The cross banding of the collagenous fibrils is easily observed.

#26 CONNECTIVE TISSUE CELLS [WinLab] [Mac] [WinHome]

In this micrograph of loose connective tissue (core of intestinal villus, the lamina propria) numerous (labeled) cells of the connective tissue are present. Note the relative size of the different cell types, their shapes, amount of rough ER and variously sized granules and inclusions. Then use your text and atlas to review the diagnostic features of each connective tissue cell present in the micrograph. Note the paucity of collagen fibrils. What was present in the "empty" looking intercellular space? (CT10)

#27 MAST CELL [WinLab] [Mac] [WinHome]

Mast cell granules exhibit ultrastructural differences that are species specific. The human mast cell contains secretory granules which have the specific ultrastructure illustrated, the significance of which is unknown. What are the secretory products of the mast cell? (CT11)

#28 FAT CELLS [WinLab] [Mac] [WinHome]

Study the formation of fat droplets. Note that each fat cell is enclosed by a thin basal lamina.

Review Question Answers

CT1: What type of epithelium lines the luminal surface of the intestine?

answer

CT2: Why is rough endoplasmic reticulum basophilic?

answer

CT3: What type of lining epithelium is present in the pharynx?

answer

CT4: Can you see a nucleus in each fat cell?

answer

CT5: What do mast cells do?

answer

CT6: Why do cells actively secreting proteins exhibit basophilic cytoplasm?

answer

CT7: In dense connective tissue, which type of cell is most common?

answer

CT8: Where are the nuclei of fat cells?

answer

CT9: Is rough ER an indication of an active or an inactive cell?

answer

CT10: What was present in the "empty" looking intercellular space?

answer

CT11: What are the secretory products of the mast cell?

answer

Practice Questions

Click either on the [Mac] or [PC] link to open the virtual slide.

1. One of the major functions of the cells indicated is:

1. collagen production.
2. triglyceride (fat) storage.
3. antibody production.
4. release of histamine during allergic reactions.
5. phagocytosis of cell debris and/or foreign material.

ANSWER

Click here to view image

2. The tissue shown is:

1. stratified squamous keratinized epithelium
2. stratified squamous non-keratinized epithelium
3. dense regular connective tissue
4. dense irregular connective tissue
5. loose connective tissue

ANSWER

Click here to view image

1. pro-alpha collagen chain
2. procollagen triple helix
3. tropocollagen triple helix
4. collagen fibril
5. collagen fiber

Click here to view image

1. comprised primarily of type I collagen.
2. comprised primarily of type II collagen.
3. produced by plasma cells.
4. significantly weaker in individuals with Marfan syndrome.
5. significantly weaker in individuals with type IV Ehlers-Danlos syndrome.

ANSWER