first year laboratory: connective tissue (proper)

Connective Tissue (Proper) Lab October 14th, 2014

First Year Laboratory: Connective Tissue (Proper) Histology Laboratory Guide

Dr. K.E. Pinder ([email protected])

Objectives: The main objectives of today’s lecture/lab combination are to describe and identify the different types and cells of connective tissue (CT) proper and to explain the relationship between the structural classifications of connective tissue (for example, loosely vs. densely arranged collagen) and the related functions (using the same example, binding of tissues vs. tensile strength). You will accomplish this using a combination of light microscope (LM) slides and electron micrograph (EM) images; you should work to integrate information from both sources. At the end of the connective tissue lecture/lab session you should be able to: 1. Name the four types of connective tissue (cartilage, bone, blood, connective tissue proper). 2. Explain the cellular and fibre (extra-cellular (ECM)) compositions of connective tissue (CT) proper. 3. Explain the classifications of connective tissue proper, based on the major fibre types (for example: loose CT

versus dense irregular CT versus elastic CT). In routine histological sections, identify the major fibre types (collagen (reticular) and elastic) of connective tissue. Identify the cells and composition of the specialized loose CT, adipose tissue.

4. Name, and be able to identify in routine histological sections/images, the five major cell types (fibroblasts, adipocytes, macrophages, plasma cells and mast cells) of connective tissue proper. Understand the relationship between the structure and the function of each cell.

Slides for Study: (Please see individual images/sections of these lab notes for slide attributions.) Fibres of connective tissue:

Ø LM #3: Areolar Tissue (loose connective tissue) Ø LM #4: Tendon (dense connective tissue) Ø EM: Collagen fibrils beside fibroblast Ø LM #44: Thick skin (diversity of density of connective tissues) Ø LM #52: Aorta (elastin fibres) Ø LM #18: Lymph node (reticular fibres highlighted with silver impregnation)

Cells of connective tissue:

Ø LM #4: Tendon (fibroblasts) Ø LM #44: Thick skin (adipocytes) Ø EM: Adipocyte Ø LM #113: Liver (macrophages highlighted with India ink) Ø EM: Macrophage Ø LM #20: Tonsil (plasma cells) Ø LM #103: Stomach (mast cells) Ø EM: Mast cell in loose connective tissue

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Part I: Classification of Connective Tissue - the Fibres You will recall from lecture that connective tissue proper is classified in a number of different ways. Let’s take a quick review of the three types of fibres: collagen, elastin and reticular.

A. Collagen Fibres: Loose, Dense & Everything In-between LM #3: Areolar Tissue (H&E) (UBC CPS Histology Virtual Slide #3, Areolar Tissue). The main purpose of slide #3 is

to identify loose, irregular CT. This tissue is classified as ‘loose’ because the fibres are spaced relatively far apart with lots of intervening spaces. It is classified as ‘irregular’ because the fibres are not arranged in any particular orientation. Loose CT is composed mainly of cells and ground substance. That is, compared to other tissues, there are relatively few fibres in loose CT. This type of CT won’t be able to impart any significant strength to tissues, but rather acts as a binding tissue and a tissue through which nutrients and waste products may easily diffuse.

LM #4: Tendon (H&E) (UBC CPS Histology Virtual Slide #4, Tendon). At the other end of the spectrum: Contrast the above loose and irregular arrangement of fibres with the collagen fibres in slide #4 … dense, regular CT (this CT type forms tendons and ligaments). The collagen fibres are grouped in dense and regularly arranged parallel bundles. It is easy to appreciate the tensile strength that such an arrangement will impart on the tissue. You should also look at this slide under higher magnification to find one of the five major cell types of connective tissue proper: fibroblasts. Look between the rows of collagen fibres to find the flattened nuclei of these cells. Recall that it is fibroblasts that are responsible for ECM synthesis and maintenance in this tissue. EM: Collagen fibrils beside fibroblast (EM on MEDICOL (Histology section) and in Section 3.7, page 58, Ovalle and Nahirney, Netter’s Essential Histology, 2nd Edition. Used with permission. Copyright © 2013, 2008 Elsevier Inc. All rights reserved). Now that you are comfortable with the appearance of collagen fibres stained in routine H&E (LM) sections, let’s explore these fibres at higher magnification with an EM. This EM illustrates the ultra-structure of collagen fibrils located beside a fibroblast: in the bottom right lower portion of the image is part of the cell body of a fibroblast. (Are you able to identify the RER and a mitochondrion in the cell cytoplasm?) In the upper left hand side of the image are extra-cellular collagen fibres. Do you see the banding pattern? This banding pattern is typical of collagen fibres under the EM and is indicative of the way that collagen fibrils are arranged. (You will learn more about collagen synthesis elsewhere in the curriculum; if you wish to learn more now I will refer you to Figure 6.7 – and the pages surrounding it – of Ross and Pawlina, Histology A Text and Atlas, 6th Edition. © 2011 Lippincott Williams & Wilkins.)

LM #44: Thick Skin (H&E) (UBC CPS Histology Virtual Slide #44, Thick Skin). This is a section of thick skin, which is found in locations such as the palm of the hand and is well adapted to withstand constant abrasion and desiccation. You’ve seen this slide a few times already J. Slide #44 is useful for identifying a number of features. Let’s start by scanning the slide at low magnification and classifying the organization of the connective tissue fibres.

What type of CT is found in this section? Not so straight forward, is it? Do you agree that in some sections of this slide the connective tissue appears quite loose and irregular while in other sections it appears denser and more regularly arranged? This is because this slide is especially useful at indicating a key concept in the classification of


CT: connective tissues exhibit such a diversity of density and regularity that rigid descriptions such as ‘dense irregular CT’ may not always be appropriate. Clinical relevance: Heritable connective tissue disorders are serious illnesses associated with decreased life spans. What information are you able to find about some of these disorders (some examples are Marfan syndrome, Ehlers-Danlos syndrome, Osteogenesis imperfecta and Stickler syndrome)? To this point in the lab we have been classifying the organization of collagen fibres. But remember that there are two additional ways in which we can classify CT: by the presence of elastin and/or reticular fibres.

B. Elastin Fibres

LM #52 – Aorta of a 25 Year Old (H&E) (UBC CPS Histology Virtual Slide #52, Aorta). Elastin fibres are physiological elastic bands. These fibres are found in locations that are subject to normal physiological deformation. A good example of such a location is the aorta. In particular, this aorta from a relatively young person contains a large amount of elastin fibres. Look under higher magnification at the left-hand side of this slide. The deeply eosinophilic structure is called the tunica media (you will learn more about this in your Cardiovascular block next term). Within this tunica media are thick squiggly eosinophilic bands. These are the elastin fibres that allow for enormous stretch and recoil of the wall of the aorta;

imagine these fibres stretching out like elastic bands as blood pressure increases in the aorta and then recoiling as the blood pressure drops in the aorta – and they do this with each and every heartbeat!

C. Reticular Fibres LM #18 – Lymph Node (Silver Impregnation) (UBC CPS Histology Virtual Slide #18, Lymph Node). Reticular fibres are very clearly seen in your lymph node slide and the main purpose of slide #18 is to identify these fibres. Reticular fibres act as a meshwork of support for highly cellular organs (such as lymph nodes) … that is, these fibres provide a framework for the cells of the tissue to rest upon. This slide has been stained with the silver impregnation method and three key features should be noted: (i) the reticular fibres themselves, which appear as a meshwork of black fibres (not unlike a spider web), (ii) the cells embedded/supported in the meshwork of reticular fibres; these are lymphoid cells and (iii) the fact that reticulin is also a major component of the walls of small blood vessels. What type of collagen are reticular fibres composed of?

Part II: The Cells of Connective Tissue Proper

In amongst the fibres of CT proper (in tissues and organs throughout the human body) the five main cells of CT proper will be found. These cells may be viewed under the light microscope (LM) and/or under the electron microscope (EM). The cells are: fibroblasts, adipocytes, macrophages, plasma cells and mast cells. 1. Fibroblasts

LM #4: Tendon (H&E) (UBC CPS Histology Virtual Slide #4, Tendon). As introduced earlier, fibroblasts are readily located in-between the tendon fibres in slide #4. You will see fibroblasts time and time again in many of the LM slides that you examine in histology. If you haven’t already located these cells then please return to slide #4 and do so. In this slide of a tendon, the nuclei of the fibroblasts are thin and elongated, indicating that the cells are relatively inactive (in contrast to their plump shape when they are active).

Clinical relevance: Fibroblasts are crucial in wound healing. For example: following a surgical incision, the fibroblasts in the area will multiply and change morphology, becoming plump and actively secreting the components


of collagen, thereby aiding in the repair of the incision. Scars are due to the activity of fibroblasts – they do not lay down collagen perfectly during wound repair and the results are scars. 2. Adipocytes

LM # 44: Thick Skin (H&E) (UBC CPS Histology Virtual Slide #44, Thick Skin). If we return to slide #44 we can identify adipocytes. There are two types of adipose tissue – white adipose and brown adipose. Brown adipose tissue is a highly specialised type of fat storage/fuel generally only found in newborn mammals and some hibernating animals, while white fat is found in adult humans and functions in fat storage and metabolism.

The adipose found in LM #44 is white adipose tissue. You should identify the single droplet of lipid (washed out, but use your imagination) with the thin rim of cytoplasm surrounding it and the single nucleus of each adipocyte pushed to the periphery (the lipid droplet pushes the nucleus to the periphery). Now move to the electron microscope to have a closer look at a white adipocyte. EM: Adipocyte (EM on MEDICOL (Histology section)). This is an EM of one white adipocyte. In the middle is a large, single, spherical lipid droplet. To the right of the lipid droplet is a nucleus; take note how the lipid droplet has displaced the nucleus to one side of the cell. Surrounding the lipid droplet is a thin rim of cytoplasm. What structures are you able to identify within the cytoplasm? 3. Macrophages LM # 113: Liver (H&E) (UBC CPS Histology Virtual Slide #113, Liver). Depending on their state of activity and tissue location, the morphology of macrophages varies widely and this can make them difficult to accurately identify in routine H&E histological sections. However, if we inject the animal with India ink prior to retrieval of organs then one can readily identify the macrophages – the cells ingest the ink and appear as large black spots, as seen in this slide of the liver. Can you spot them? In the liver, macrophages are given a special name: Kuppfer cells.

EM: Macrophage (EM on MEDICOL (Histology section) and in Section 3.15, page 66, Ovalle and Nahirney, Netter’s Essential Histology, 2nd Edition. Used with permission. Copyright © 2013, 2008 Elsevier Inc. All rights reserved). Electron microscopy reveals further details about macrophages. The structural details of macrophages reflect their main role: phagocytosis (aided by their ability to move/crawl both within and between tissues). First, are you able to identify the basic features of this cell under the EM: the nucleus, the chromatin, the cytoplasm, mitochondria?

Now take a closer look at the contents of the cytoplasm. You should observe a number of non-descript structures; these are lysosomes in various stages of digestion of the substances that have been phagocytised by the macrophage. Recall that you learned about lysosomes in a previous lab (Cell Cytoplasm, Organelles, Inclusions & Apical Specializations). Finally, have a look at the shape of the outer limit of the cell. Do you see anything different? The cell membrane has irregular projections from it that look like fingers poking out at odd angles from the macrophage. These are called pseudopodia and they function to allow the macrophage to move, or crawl, through tissues. Clinical relevance: “Inhaled particles in cigarette smoke are phagocytosed by alveolar macrophages (AM) and lead to characteristic morphologic changes. AM from cigarette smokers are more numerous, larger in size, frequently multinucleated, and contain characteristic dark basophilic smoker's inclusion bodies within the cytoplasm as a result


of ingestion of inhaled smoke particulates.” (Marques et al., 1997. Smoker’s Lung Transplanted to a Nonsmoker. American Journal of Respiratory and Critical Care Medicine, Vol. 156, No. 5 (1997), pp. 1700-1702.) If you wish, you may read more at: http://www.atsjournals.org/doi/full/10.1164/ajrccm.156.5.9611052 4. Plasma Cells

LM # 20: Tonsil (H&E) (UBC CPS Histology Virtual Slide #20, Tonsil). Despite the distinct morphology of plasma cells, they can be difficult to locate in routine histological sections. The main purpose of slide #20 is to assist you in training your eyes for the morphology of these cells. Plasma cells can be identified in your slide #20 of the tonsil by looking at higher magnification in the loose CT that surrounds the glands, which are located in the lower middle part of this slide. Be sure that you

can locate them. If you are really lucky, you will spot one with a juxtanuclear halo, which is how a very large Golgi apparatus appears under the LM. 5. Mast Cells

LM # 103: Stomach (UBC CPS Histology Virtual Slide #103, Stomach). Mast cells are found in all connective tissues but can be difficult to identify without special staining. This is because the cytoplasm of mast cells is filled with secretory granules (do you know what these granules contain?) that are water soluble and are therefore washed out during routine histological staining. However, when using specialised stains such as toludine blue the granules stain basophilic, as do the nuclei. Mast cells stained this way therefore appear as large basophilic blobs in histological sections. Are you able to locate them in the submucosa (the region underlying the epithelium) on slide #103?

EM: Mast cell in loose connective tissue (EM on MEDICOL (Histology section) and in Section 3.11, page 62, Ovalle and Nahirney, Netter’s Essential Histology, 2nd Edition. Used with permission. Copyright © 2013, 2008 Elsevier Inc. All rights reserved). This EM was taken through a section of a mast cell that contains so many granules that the entire cytoplasm appears full of them (the nucleus is obscured by them). What this EM nicely demonstrates is the heterogeneity of the granules: they vary widely in their sizes, densities and shapes.

Clinical relevance: Mast cell degranulation! Can you imagine the result of all of those granules de-granulating and releasing their contents? It’s easy to understand what a major role these cells play in allergies and anaphylaxis, isn’t it?

Mast cells are really great for demonstrating the power of the LM oil immersion lens. • Shown at left is a text-book image taken under oil immersion – you can clearly see

the granules of the mast cell (Fig. 6.23, Ross & Pawlina, Histology: A Text and Atlas, 6th Edition. Used with permission. Copyright © 2011 Lippincott Williams & Wilkins. All rights reserved).

• In contrast, our slide #103 is not scanned in under oil immersion and you can clearly see the difference in the image at right: we aren’t under a high enough magnification to as clearly see the granules of the mast cell. If you squint you may be able to make out granules but comparatively speaking, the cells just appear as basophilic blobs under lower magnification.

first year laboratory: connective tissue (proper)

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