Connective Tissue Study Guide

Samantha Blum

Collagenous Fibers• Cellularity refers to the overall density of cells, and also to the ratio of cells to extracellular

components. Connective tissues vary in their degree of cellularity (cell density); note the cellularity in different areas of the slide.

• Connective tissue is less cellular than epithelial tissue, but it contains many extracellular fibers. You can distinguish extracellular fibers from cells because an extracellular fiber has no nucleus.

Elastic Fibers• Without the specific stain, you would not be able to distinguish elastic fibers from collagenous fibers. • Elastic fibers contain an amorphous core of elastin molecules that are deposited into a scaffold of fibrillin

microfibrils (EM). The elastin molecules are covalently linked to one another, forming a highly distensible (stretchable) network

• Variation in fiber size (thickness)• In an H&E-stained preparation, the elastic sheets are highly refractile; this means they bend light, and

therefore have a glassy appearance, but they are not colored (white arrows in rightmost picture)• In an elastic-stained preparation, these same elastic membranes are readily visible as dense black lines

Reticular Fibers• In many cases, these fibers are composed of Type III collagen.• A special, silver-based method (reticular stain) was used to stain the reticular fibers in E-44A

and B. Note how the reticular fibers provide a network (50X) in which cells are suspended. • Notice that the reticular fibers within the spleen merge with those of the capsule (10X),

forming a continuous, 3-dimensional structure that functions as the "skeleton" of the organ.

Ground Substance• Observe the spaces between cells, the ground substance (*, 20X; 50X), which is composed of

glycosaminoglycans and proteoglycans. If these molecules are sulfated, as are the chondroitin sulfate proteoglycans of cartilage, they stain with basic dyes and exhibit metachromasia (i.e. they look reddish or purple instead of blue). Ground substance also fills the spaces between mesenchymal cells.

Cells of Connective Tissue

Fibroblasts• In general, fibroblasts are flattened, spindle-shaped cells ((arrows, 100X; EM) vary in

morphology. • Note that the nucleus is the most visible part of the cell and can be flattened or rounded.• In H&E stain, nuclei are stained.• Many found in papillary dermis (most superficial layer, directly beneath epidermis)

Macrophages• These cells are amoeboid and phagocytic. They are quite variable in morphology, but

generally have large, oval, pale nuclei, a well-defined cell outline, and a well-stained, abundant cytoplasm. They are most easily recognized when they are filled with ingested material.

• Also, while they may not have obvious included particles, their cytoplasm often appears perforated and "moth-eaten"

Adipocytes• Look for the peripherally-located nuclei (100X) of adipocytes. • Compare the size of adipocytes to that of red blood cells within the same field of view. (RBCs

are about 7 microns in diameter). • Note that the adipocytes in these tissue sections have been stained with osmic acid, allowing

preservation of lipid in fat vacuoles.

Mast Cells• Note that the granules from some mast cells have been artifactually released into the

surrounding C.T. during preparation• Spot mast cells by looking for their dotted granules filling the cell

Plasma Cells• Look in the connective tissue underlying the mucosal epithelium to find plasma cells.• Examine the characteristic eccentric "clock face", or "cartwheel" nucleus of a plasma cell (

schematic; arrows, 100X, >>100X; EM). Also note that the Golgi apparatus does not stain with H&E, so it appears as a pale spot near the nucleus; this is a very recognizable characteristic of plasma cells.

Reticular Cells• Reticular cells synthesize the extracellular fibers that form the support structure for the

lymph node. These stellate (star-shaped) cells are surrounded by the reticular fibers, and by lymphocytes.

• Reticular fibers can only be seen when stained specifically (100X). The reticular cells themselves (100X) are easier to see in the H&E-stained slide, where their long cytoplasmic processes are visible (red arrows)

Reticular fibers can be seen stained black as well

Embryonic CT- Mesenchyme• Look for diffuse, stellate-shaped cells (100X) in a homogeneous ground substance. (LEFT)• Embryonic jaw and developing tooth• Observe that there are no extracellular fibers in this tissue. The long thin structures between

cells are processes of the mesenchymal cells (RIGHT)

Embryonic CT- Mucous CT• Note the presence of extracellular fibers. Examine the C.P. trichrome stained slide (50X) and

convince yourself that the fibers are not processes of the C.T. cells. • Mucous C.T. of umbilical cord is also called Wharton's Jelly. • Notice that the cells in this tissue are more widely-scattered than in mesenchymal C.T. • Umbilical cord

Adult CT- Areolar (Loose) CT•Areolar CT is “whispier” CT found in papillary region (directly under epithelial layer); areolar CT also found surrounding blood vessels and ducts and in pits/glands less dense= easier for transport cells and immune cells to travel through•More cells than Dense Irregular/Regular CT

Lamina Propria and Submucosa• Lamina propria (40X) is the layer of [areolar/loose] connective tissue that lies directly

under the epithelial layer. The epithelium and lamina propria, along with the muscularis mucosa (a layer of smooth muscle) comprise the mucosa.

• Notice the presence of lymphocytes within the fibrous matrix of the lamina propria; these have dark, round nuclei and almost no visible cytoplasm (40X). Given that lymphocytes function to defend against invading microbes, why are they present in this particular spot? because it’s easier for the lymphocytes to travel through and chase the microbes…

Adult CT- Dense Irregular CT• Look in the reticular layer of the dermis for this tissue type. • Note the density, thickness and disarray of the collagenous fibers (arrows, 50x). • Note that cells are relatively sparse in this tissue. • Skin of axilla, spleen, long bone• The covering of bone (periosteum) has an inner, cellular layer that acts as a reservoir for new

bone-producing cells, and an outer fibrous layer made up of dense irregular collagen fibers

Adult CT- Dense Regular CT• Dense regular CT found in tendons: have strength for stress in a single direction• The connective tissue fibers and cells are organized into bundles called subfascicles (5X).

Groups of these, in turn, are collected into fascicles. Dense, irregular connective tissue (endotenon) surrounds the subfascicles and fascicles. A thicker C.T. layer, the epitenon, surrounds the entire tendon

Longitudinal section

Adult CT- Adipose Tissue• Adipose tissue is atypical among the connective tissues proper in that it contains more cells

and less extracellular matrix. • All three fiber types are present: collagenous, elastic and reticular. • Notice the typical "chicken-wire" appearance of the adipocytes ( red arrows) when reticular

stain used.

WHITE FAT BROWN FAT

Adult CT- Reticular Tissue• The reticular cells are more visible in the H&E-stained slide; look for flattened, pale-staining

reticular cells among rounded lymphocytes (red arrows, 50X). The reticular fibers are much more visible in the slide with a reticular stain (40X).

• Spleen, lymph nodes

Adult CT- Elastic CT

Clinical Correlation: Connective Tissue Pathology

• A lipoma is a benign tumor of adipose tissue.• A liposarcoma is a malignant (cancerous)

tumor of adipose tissue.• A fibroasarcoma is a malignant tumor of

connective tissue.

Muscle Study Guide

Samantha Blum

Skeletal Muscle: Myofibers• Note the dimensions of these very large cells. The cells are multinucleated . • Look along the edges of several cells to convince yourself that nuclei are peripherally located.• For skeletal muscle, find an area containing cross-sectioned cells and note the peripheral

location of the nuclei, and their flattened shape (RIGHT IMAGE, arrows, 100X). In longitudinal sections of skeletal muscle, the nuclei appear elongated. This characteristic is helpful for distinguishing skeletal muscle from cardiac muscle.

Skeletal Muscle: Fibroblasts In Between

• Identify fibroblasts (arrows, 100x) between the muscle fibers. Fibroblast nuclei are flattened, and are well-stained in H&E preparations, while their cytoplasm is lightly stained and therefore difficult to see.

Skeletal Muscles: Fascicles and CT• Endomysium is connective tissue that surrounds each myofiber. It is a fine network of

collagenous and reticular fibers with varying amounts of elastic fibers. Some connective tissue (CT) cells, fine capillaries and nerves are also present.

• Look for an area where individual myofibers have been separated somewhat by tissue preparation. It will be easier to see endomysium in these areas (40X).

• Study the intercellular spaces for evidence of elastic and collagenous fibers. • Identify fibroblasts in this area. These cells are responsible for producing all three C.T. fiber

types.

Skeletal Muscles: Fascicles and CT• Perimysium bundles several myofibers into groups called fascicles. It is a loose, irregularly

arranged, predominantly collagenous CT. The CT fibers are considerably coarser than in endomysium. Blood vessels and nerves are distributed throughout the muscle body in the perimysium.

Fascicles circled

Skeletal Muscles: Fascicles and CT• Epimysium is a dense CT sheath that binds fascicles together to form the muscle body. This is

termed deep fascia by gross anatomists. The major blood vessels and nerves penetrate through the epimysium.

Skeletal Muscles: Fascicles and CT

Note how myofibers are grouped into fascicles that run in a variety of directions (1-2-3, 20x).

Skeletal Muscles: Fascicles and CT• Identify the specific points at which the muscle fibers insert into the tendon, and compare

the tendon C.T. (dense regular) and the skeletal muscle (5X, 40X; 1X, 20X).• At the precise point of contact between muscle and tendon, the muscle membrane is

organized into a series of folds and ridges, providing a larger contact area to withstand the forces exerted on the junction by muscular contraction (40X; *, EM).

Skeletal Muscle: Nervous Innervation and Blood Supply

• Note that nerves and blood vessels (10X) are located predominantly in the loose areolar connective tissue of the perimysium. Identify encapsulated nerve fascicles both in longitudinal (40X) and cross section (40X).

• Look for nerve branches (arrows, 40X) entering muscle fascicles. • Identify smaller blood vessels (arrows) within fascicles (100X). (YELLOW ARROWS)• Note the vascularity of the tissue (40X).

Skeletal Muscle: Neuromuscular Junction

• Nerves control the contraction of skeletal muscle. The point of communication is the neuromuscular junction (myoneural junction, endplate); here, the nerve makes a synaptic connection onto the membrane of the muscle fiber. Muscles that require precise control (e.g. oculomotor muscles) have a nerve:myofiber ratio of 1; where precise control is not needed (e.g. latissimus), one nerve fiber will branch to innervate many myofibers.

The mammalian neuromuscular junction uses acetylcholine (ACh) as its neurotransmitter. The postsynaptic cell (a skeletal muscle fiber) limits the action of the transmitter by hydrolysing it with acetylcholinesterase (AChE), an enzyme embedded in the muscle membrane. AChE can be visualized with a specific cytochemical stain that produces a red precipitate where the enzyme is located

(Silver stain)

Skeletal Muscle: Muscle Spindles• Muscle spindles are stretch receptor organs within skeletal muscles that regulate muscle

tone. Spindles are encapsulated, lymph-filled, fusiform structures that lie parallel to the skeletal myofibers in connective tissue between bundles of myofibers. They contain two to ten modified muscle fibers, called intrafusal fibers. When a muscle is stretched, the sensory fiber receptors are distorted and stimulated, and the relay of this information leads to muscle contraction.

Stretch receptor organs

Note the intrafusal fibers (40X) bounded by a discrete connective tissue capsule. Compare this structure with a nerve fasicle.

Cardiac Muscle: Myofibers• Note that nuclei (arrows, 40X) are centrally located (unlike nuclei in skeletal myofibers, which

are periphera), and that each cell has only one or two nuclei.• Follow individual cardiac myofibers to identify points at which they branch • Identify intercalated discs (arrows, 40X, 100X), which join cells together to form a functional

syncytium. (YELLOW ARROWS)

Cardiac Muscle: Myofibers• Identify cross-striations (100X). (YELLOW CIRCLES)• Look near the nuclei for cytoplasm that is free of myofibrils ( 100X); this feature is also

characteristic of skeletal muscle. (YELLOW ARROWS)

Cardiac Muscle: Purkinje Fibers• These modified myofibers are easily found because they contain fewer intracellular filaments

than normal cardiac muscle cells do. They function primarily to conduct impulses through the heart muscle, rather than to generate mechanical force.

Cardiac Muscle: Contractile Units and CT

• Endomysium- fine CT fibers between cells

Fibroblasts that have manufactured CT fibers of endomysium

Cardiac Muscle: Contractile Units and CT

• Perimysium:• Identify irregular cardiac fascicles (10X), bound together by collagenous fibers of the

perimysium. • Note that the perimysium (40X) is very dense in certain areas, adding to the overall

architecture of the heart.

Cardiac Muscle: Contractile Units and CT

• Epimysium:• Identify the epimysium at both endocardial (40X) and epicardial (40X) surfaces of the heart.

Cardiac Muscle: Innervation and Blood Supply

• Note that the myocardium has an abundant supply of blood vessels (arrows, 10X). • Note the abundance of capillaries (arrows, 40X, 100X) among myofibers. (BLUE ARROWS)

100X40X

Smooth Muscle: Myofibers• The myofilaments are not arranged into sarcomeres and myofibrils, and cannot be seen in the light

microscope. A single, central nucleus causes the cell to bulge in the middle and take on a fusiform appearance.

• Smooth muscle cytoplasm contains numerous dense bodies, which are fusiform densities in the sarcoplasm that contain alpha-actinin and that are analogous to Z lines of striated myofibers. These structures serve as anchorage sites for actin myofilaments.

• Note that cells are spindle-shaped (40X), and have one centrally located nucleus. • No cross striations or myofibrils are present.

• The nuclei of contracted cells appear crinkled or spiral-shaped (40X), due to contraction of the cells post mortem ("after death")

Smooth Muscle: Myofibers• Note that the smooth muscle cells (40X) are more densely arranged here. • Note the very elongated cells and nuclei (40X).

Smooth Muscle vs. CT• Smooth muscle (10X) is sometimes confused with connective tissue. In this slide, compare (

40X) smooth muscle with adjacent, coarse collagenous connective tissue of the submucosa. • Note that the smooth muscle bundles contain many more nuclei than the connective tissue,

because the c.t. fibers are extracellular, while the muscle fibers are cells. • Compare smooth muscle cells with the characteristic wavy fibers of irregularly arranged

connective tissue.

Smooth Muscle: Contractile Units and CT

• Wall of blood vessels• Note the circular sheet-like arrangement of smooth muscle cells (1X, 5X, 40X); note that their

nuclei are very visible, but their cytoplasmic processes are difficult to see.

Smooth Muscle: Contractile Units and CT

• Wall of ductus deferens and ureter• Note that both circular and longitudinal layers are present in the walls of both the ductus

deferens (10X, 40X) and the ureter (10X, 40X)

Smooth Muscle: Contractile Units and CT

• Wall of bladder• Note that myofibers are arranged here in anastomosing bundles

Smooth Muscle: Contractile Units and CT

• Wall of small intestine• Identify inner circular and outer longitudinal layers

Smooth Muscle: Contractile Units and CT

• Wall of uterus• The outer wall of the uterus (myometrium) is a thick layer of smooth muscle. In this layer,

note that bundles of myofibers in various orientations are interspersed with connective tissue

Smooth Muscle: Contractile Units and CT

Arector pili muscles in scalp Bundles of smooth muscle myofibers in nipple

Scattered smooth muscles in spleen

Distinguishing Muscle Types

Distinguishing Muscle Types:CROSS-SECTION

• 1. Skeletal muscle: Look for large-diameter muscle fibers, connective tissue, and peripherally-placed nuclei, with several nuclei per fiber (cell).

2. Smooth muscle: Individual muscle fibers are usually not visible. If smooth muscle cells are present as bundles or sheets, very little intercellular C.T. is seen. Bundles of cells may have the appearance of "naked" nuclei crowded together, since cells are small and cytoplasm is not readily seen. Since each cell is a fiber, you should see one nucleus per fiber (cell), although plane of section doesn't always permit this.

3. Cardiac muscle: Look for irregularly-sized fibers, smaller than skeletal muscle. Individual muscle fibers may be seen, and may appear vacuolated due to perinuclear cytoplasm. Each nucleus lies in the middle of the fiber (cell). Connective tissue is readily visible.

Distinguishing Muscle Types:LONGITUDINAL

• 1. Cardiac muscle: Look for intercalated discs, longitudinal striations, cross striations, and some connective tissue.

2. Skeletal muscle: Look for prominent cross-striations but no intercalated discs, for nuclei which appear to be on the periphery of the fibers, and for appreciable connective tissue.

3. Smooth muscle: Look for the absence of a pronounced connective tissue sheath. Also, no striations or intercalated discs are present.

Distinguishing Smooth Muscle, CT, and Peripheral Nerve

• 1. Look at the number of nuclei. If you see very few nuclei, then you are looking at connective tissue (i.e. few cells, lots of extracellular fibers).

2. Look at homogeneity of nuclei. If they are all the same, then you are probably looking at smooth muscle. If several different nuclear types are present, then you are probably looking at peripheral nerve.

3. Peripheral nerves have a distinct C.T. sheath (although the sheath may not be in the field of view if you are looking at a photograph). Also, with H&E, peripheral nerve looks ragged or moth-eaten, because holes appear wherever myelin had been located (myelin is not preserved during preparation of H&E-stained sections).

Clinical Correlation: Muscle Pathology

• Muscular dystrophy is a congenital disease in which the skeletal muscle degenerates due to a lack of the protein dystrophin.

• Denervation atrophy refers to the deterioration of skeletal muscle that occurs when it is deprived of its normal nervous innervation.

• Poliomyelitisis a condition in which the polio virus infects the spinal cord, causing destruction of motorneurons, and causing degeneration of the skeletal muscle innervated by those motorneurons.

• Trichinosis is a condition in which skeletal muscle degenerates due to an infection by a parasite found in under-cooked pork.