Connective Tissue

York College CUNYHPMT 332

Professor EmsleySpring 2014

Definition of Connective Tissue

• Connective tissue may be defined as an animal tissue that supports, connects, and surrounds organs and other body parts and consists mainly of collagen, elastic fibers, fatty tissue, cartilage or bone.

Collagen Fibers• Collagen-are the main protein of

connective tissue in animals and the most abundant protein in mammals making up about 25% of the total protein content.

• Collagen fibers provide strength; the more collagen present, the stronger the tissue.

• A dense regular arrangement of collagen fibers is found in tendons, organ capsules, and the dermis.

Collagen contd.• Collagen is eosinophillic and readily visible

with light microscopy.

• When we demonstrate connective tissue element in histology we focus on the fibers or cells of connective tissue proper.

Special Stains used to demonstrate Collagen

• Trichrome stains are commonly used to differentiate between collagen and smooth muscle in tumors, and to identify increases in collagenous tissue in diseases such as cirrhosis of the liver.

• Trichrome procedures are so named because 3 dyes which may or may not include the nuclear stain, are used.

Masson Trichrome Technique• Sections are first stained with an acid

dye such as Biebrich’s Scarlet ; all acidophilic tissue elements, such as cytoplasm, muscle, and collagen, will bind the acid dyes.

• Next the sections are treated with phosphotungstic and /or phosphomolybdic acid.

Masson Trichrome Technique contd.

• Because cytoplasm is much less permeable than collagen, phosphotungstic and phosphomolybdic acids cause the Biebrich scarlet to diffuse out of the collagen but not out of the cytoplasm.

• Controls are used during all special staining technique.

• The control tests, that the stain is working.

Masson Trichrome Technique contd.

• Results:– Collagen, cartilage - blue– Cytoplasm, muscle fibers- red– Blood vessels – yellow, orange

Reticular Fibers

• Reticular fibers -are identified as one type of collagen.

• Routine H&E stain doesn’t demonstrate the fibers , but may be demonstrated with an argyrophilic reaction, as they have the ability to adsorb silver from solution.

• The silver may then be reduced to its visible metallic form.

• Reticular fibers form delicate networks and are much smaller than collagen fibers.

Reticular Fibers contd.• Clean glassware has to be used because

silver is very peculiar.

• The Gomori’s technique is used for demonstrating reticular fibers and is important in diagnosing certain types of tumors.

• A change in the normal reticular fiber pattern gives an indication in the case of some liver diseases.

Gomori’s Technique

• The tissue is first oxidized using potassium permanganate to enhance subsequent staining of the reticular fibers, and the excess potassium permanganate is removed with potassium metabisulfite.

• Ferric ammonium sulfate acts as the sensitizer and is subsequently replaced by silver from the diamino silver solution.

Gomori’s Technique contd.

• Following impregnation, formalin is used to reduce the silver to its visible metallic form.

• Toning with gold chloride and removal of unreacted silver with sodium thiosulfate follow.

• Counterstain with nuclear fast red.

• The glassware has to be chemically cleaned because silver is very peculiar

Gomori’s Technique contd.

• Results:– Reticular fibers- black

Elastic Fibers• Elastic fibers - are present in most

fibrous connective tissues, but are most plentiful in tissue requiring flexibility as the elastic fibers allow tissues to stretch as in the aorta, largest artery.

• The fibers are not usually seen in routine H&E and require special stain such as Verhoeff’s van Gieson’s elastic technique.

• Elastic fiber techniques are used for the demonstration of pathologic changes in elastic fibers.

Gomoris One Step Trichrome

• Purpose: To identify an increase in collagenous connective tissue fibers or differentiation between collagen and smooth muscle fibers.

• Principle: plasma stain ( chromotrope 2R) and connective fiber stain ( fast green or aniline blue) are combined in a solution of phosphotungstic acid to which glacial acetic acid has been added. Phosphotungstic acid favors the red staining of muscle and cytoplasm. The tungstate ion is specifically taken up by collagen, and the connective tissue fiber stain is subsequently bound to this complex.

Staining Technique for One step Trichrome

• 1 Deparaffinise and hydrate slides• 2 Mordant in Bouins for 30 mins.• 3 Wash well in water for 2 mins• 4 Stain in hematoxylin for 2 mins• 5 Rinse in water• 6 Dip in ammonia for 10 secs• 7 Stain in Gomoris 1-step trichrome for 15 mins• 8 Rinse• 9 Dehydrate 95 alc. Absolute, xylene then coverslip• Result: nuclei – black, cytoplasm, muscle fibers- red,

collagen and mucin – green for fast green or blue for aniline blue

Verhoeff’s van Gieson technique

• The tissue is overstained with a soluble lake of hematoxylin-ferric chloride-iodine.

• Both ferric chloride and iodine served as mordants, but they also have an oxidizing function that assists in converting hematoxylin to hematein.

• The sections are overstained and then differentiated, so it is regressive.

Elastic Fibers contd.

• These include atrophy of the elastic tissue, thinning or loss that may result from arteriosclerotic changes and reduplication, breaks or splitting that may be used to demonstrate normal elastic tissue, as in the identification of veins and arteries, and to determine whether or not blood vessels have been invaded by tumor.

Verhoeff’s van Gieson technique contd.

• Differentiation is accomplished by using excess mordant, or ferric chloride, to break the tissue-mordant-dye complex.

• The dye will be attracted to the larger amount of mordant in the differentiating solution and will be removed from the tissue.

• The elastic tissue has the strongest affinity for the iron-hematoxylin complex and will retain the dye longer than other tissue elements.

Verhoeff’s van Gieson technique contd.

• This allows other elements to be decolorized and the elastic fibers to remain stained.

• Sodium thiosulfate is used to remove excess iodine.

• Van Gieson’s solution is the most commonly used counterstain, but others may be used.

Verhoeff’s van Gieson technique contd.

• Results:– Elastic fibers-blue-black, black– Collagen- Red– Nuclei-blue or black– Other elements- yellow

Connective Tissue Proper

• Cells found in connective tissue proper- fibroblasts- most common cell in connective tissue which produce the connective fibers( extracellular, non living elements)

• Mesenchymal cells-• Adipose or fat cells- synthesize and store lipid.• Mast cells – contain secretory granules, histamine and

heparin• Macrophages- “ big eaters” or scavenger cells, found

also in tissues eg. Liver, myeloid, and lymphatic tissues. Play important role in immune mechanism.

• Plasma cells- derived from B lymphocytes, produce immunoglobulins.

• Blood cells- of all types

Muscle

• 1. Skeletal muscle- may also be classified as (a). striated- has the characteristic dark (A) and light (I) bands seen microscopically, and (b) voluntary- contraction can be brought about at will.

• 2. Cardiac muscle- striated but involuntary type of muscle, has specialized intercellular junctions called intercalated discs.

• Refer to figs. (i8.1 Skeletal muscle and i8.2 Cardiac muscle