HISTOCHEMISTRY OF GINGIVA

BY- DR RAJEEV RANJAN

CONTENTS:1.Introduction 2.Need for Histochemical techniques 3 Histochemical Techniques - Tissue processing - Specific histochemical methods - Immuno histochemistry 4. Histochemistry of Gingiva - Ground Substances - Enzymes 5. Clinical consideration 6. Conclusion 7. Reference

INTRODUCTION: Study of qualitative identification and quantitative assesment of chemical groupings within cells and tissues. Challenging due to presence of both hard and soft tissues in the oral cavity. . Histochemical techniques generally arise from precise chemical rationales for their ability to stain different biochemical substances.

NEED FOR HISTOCHEMISTRY:The microscopical identification - largely morphological. Histochemical techniques - chemical components, enzyme systems, physiologic processes, and the changes that occur in disease.

Histochemical techniques provide information regarding the chemical components and enzyme systems of normal gingiva that may assist in improving our understanding of physiologic processes in the gingiva and the changes that occur in disease.

Histochemical techniques necessitate using more stringent precautions to preserve the chemical integrity of tissue than, perhaps, are required in biochemical or an immunochemical assay.

Histochemical techniques provide in situ information that can not be obtained with biochemical methods.

HISTOCHEMICAL TECHNIQUES

FIXATION PROCEDURESIt is a complex series of chemical events and differs for the different groups of substances found in tissues. For histochemical study, a tissue block must be preserved in such a way that it causes minimal changes in the reactivity of cytoplasmic and extracellular macromolecules. This is accomplished using optimum osmotic condition, cold temp, controlled pH of the fixing solution and the minimal possible exposure to fixative

Aims The process of autolysins and bacterial attack should be prevented. Tissues which have been fixed should not change shape or volume during any of the subsequent procedures. They should be left in a condition with subsequently allows clear staining of section. Tissues should be as close of to their living state as possible.

Ideally, no small molecule should be lost. Fixtures used are :Aldehyde formaldehyde, Gluteraldehyde Oxidizing agents osmium tetroxide, potassium permanganate. Protein denaturing agents acetic acid, methyl alcohol, Ethyl alcohol. Cross linking agents carbodimides Physical heat, microwave.

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FORMALDEHYDE It one of the ideal fixative for enzymes and other

proteins. Because it s ability to react with major reactive groups of proteins to form polymeric or macromolecular networks, without affecting their native reactivity to histochemical procedure. Effective preservative for lipids by altering their relationships with proteins.

Use of electrolytes such as calcium or cadmium in formaldehyde prevents dissolution of phospholipids. Formaldehyde used as 10% solution buffered to Ph 7 at cold temperature in range 0-4 degree celsius. Other frequently used acroclein and glutaraldehyde. Conjunctive use of colloids such as sucrose, ficoll, polyvinylpyrrolidone, and dextrans in the fixing solutions is often made to prevent osmotic rupture of cell organelles. This helps to improve the in situ localization of the histochemical reactions.

Other fixatives: Rossman s fluid:- For visualization of glycogen, glycoproteins and proteioglycans. - Contains formaldehyde, alcohol, picric acid and acetic acid Carnoy s mixture:- used for histochemical staining of nucleic acids - composed of ethyl alcohol, acetic acid and chloroform

Feulgen's reaction -used for visualizing deoxyribonucleic acids (DNA), requires acid hydrolysis of the DNA polymers to expose the deoxyribose sugar residues of DNA molecules. -The aldehyde groups thus exposed (on the deoxyribose sugar residues) are then chemically reacted with leucofuchsin (Schiff's reagent) to form a reddish purple reaction product.

POSTFIXATION: Term postfixation specifically used when secondary fixation is done on lipid rich tissues and more appropriately for freeze-dried tissues. Require when primary fixation being not effective and has to be supplemented by another fixative to yield better staining quality.

e.g-Tissue post-fixed with imidizole-buffered osmium tetrachloride allows localization of lipids rich in unsaturated fatty acids. - Post-fixation with uranyl acetate preserves membrane phospholipids and dehydration with acetone minimizes the extraction of phospholipids during the study of ultrastructural element of cell membranes.

Dehydration: e ove ater from specimen wet fixed tiss e can not be directly infiltrated. done with a series of alcohol ascendin grade of alcohol( 7 ,8 ,9 ,95 and absol te alcohol

Clearing: removal of alcohol, by xylene Toluene( more tolerant but expensive) and chloroform other option.

Infiltration: Tissue imersed in melted paraffin( mp-5 -60 c) done in paraffin wax bath for 2 hours

Embedding: done in paraffin two L shaped metal pieces are used to prepare rectangular block.

Sectioning:1) Paraffin wax sections To permit the microscopic examination of cells in their anatomical relationship with one another, thin (ex: one cell thickness) slices at sections of tissues are required. To facilitate this, the tissue must be solidified and supported. Infiltration of the tissue by molten paraffin was helps to ensure that this tissue is supported both internally and externally. Other materials are Water-soluble waxes and Resin embedding

Sectioning with microtome: Microtome equipment that holds a knife with a mechanism for advancing paraffin block. Sharp knife require desired thickness is approx 5 micron

Picking the sections: nce sections are c t, floated on a warm water bath to remove wrinkles. then picked up on a glass microscopic slide, coated with egg albumin. hen placed glass slide in warm oven for about 15 minutes to help section adhere to the glass slide.

2) Frozen sections Some enzyme systems, such as Cytochrome oxidases, are highly labile and therefore cannot be preserved by chemical fixation. Visualization of such enzymes is performed on fresh frozen (cryostat) sections. to prevent diffusion and to preserve the in vivo status of the tissue macromolecules, one must fix the tissue blocks by a freeze-drying procedure. However,

Tissues frozen rapidly at very low temperatures ( usually in liq. Nitrogen) Placed in refrigerated vaccum chamber

Removal of ice formed in tissues by process of sublimation

After dehydration in vaccum , tissue blocks are embedded in paraffin and sectioned routinely with a microtome

Advantages of freeze- dried: Exhibit optimal enzyme activity Show excellent histologic characteristics do not show any shrinkage artifacts that are seen with routine fixation Precise localisation of otherwise diffusible inorganic ions

Specific Histochemical methods:Histochemical techniques primarily used in the study of oral tissues may he categorized as:(1) glycogen, glycoprotein, and proteoglycan methods; (2) Protein and lipid methods (3) Enzyme Methods. They are all characterized by a direct staining reaction or by the formation of an insoluble dye or precipitate at the reactive sites.

Glycogen, Glycoprotein, and Proteoglycan methods :-

The best-known and most frequently used technique for detection of carbohydrate groupings is the periodic acidSchiff (PAS) technique. The chemical basis of this method lies in the fact that periodic acid oxidizes the glycol groups to aldehyde and these in turn are revealed as a reddish purple dye product on treatment with Leucofuchsin (Schiff s reagent).

Use of fluorescent reagent Anthracene-9Carboxyaldehye Carbohydrozone as a substitute for Schiff's reagent has been made by Cotelli and Livingston

Proteoglycans demonstrated by Thiazinc dyes such as Toluidine Blue, Azure A, and Alcian Blue. Toluidine blue produces a Metachromatic reaction ranging from purple to a red reaction product.

This change of color (Metachromasia) from the original (Orthochromatic) blue color of the monomeric form of Toluidine blue reflects the extent of polymerization of the dye molecules as they tag onto the anionic residues on the Glycosaminoglycans molecule

Alcian blue staining has been used to considerable advantage in characterizing the specific types of acid radicals present within Proteoglycans.

When used at pH 2 to 2.8, Alcian blue stains weakly acid sulfated Proteoglycans. However, when it is used at pH 1 to 1.2, Alcian blue binds to highly sulfated Proteoglycans.

The high iron Diamine Thiocarbohydrazide-silver Proteinate method of Spicer provides high specificity for sulfated glycoconjugates.

Proteins and lipids:This histochemistry of proteins is based on classic reactions of protein chemistry involving various amino acid groups, that is, Amino, Imino, Carboxyl Disulfide, and Sulfhydryl Groups. Reagents such as Dinitro Fluorbenzene, Ninhydrin, or Ferric Ferricyanide are utilized to give insoluble colored reaction products. Histochemical study of lipids frequently implies use of frozen or freeze-dried sections.

Total lipids are studied by using fat colorant dyes such as Sudan dyes. Chromation of formol calcium fixed tissues and their subsequent staining with Sudan black has been employed for the identification of. phospholipids. .

Enzymes:The enzyme techniques utilize many different principles. The Gomori method for phosphatases uses phosphoric esters of glycerol, glucose, or adenosine.

The enzymatically liberated phosphate ion is converted into an insoluble salt, which can be visualized by polarized light or phase contrast, or the salt can be transformed into a cobalt or lead compound, which is black. Riboflavin 5'-phosphate has been used as substrate, which at the site of phosphatase activity results in the formation of a fluorescent precipitate employs a Naphthol Phosphate or other type of ester.

IMMUNOHISTOCHEMISTRY:Precise localization of specific biologic molecules in different intra cellular compartments, on cell surfaces, or in extra cellular matrices is now made possible by the application of some basic principles of immunochemistry. The immunohistochemical techniques are based on the premise that protein-based antigens or immunogens bind avidly to their specific antibodies. Antibodies to specific antigens can be prepared by injecting the known antigen into an animal in order to provoke an immune response.

This response results in the production of antibody immunoglobins, and these can be isolated from the serum of the injected animal. When a solution containing an antibody or an antiserum is directly applied to a tissue section containing the antigen, the antibody binds specifically to that antigen. This antigen- antibody complex is subsequently attached to a second antibody, which is conjugated either to a fluorescent dye, like Rhodamine or FITC (fluorescien isothiocynate), or to an enzyme conjugated to its antibody, like peroxidaseantiperoxidase (PAP).

Direct method

Indirect method

The antigen-antibody complexes bound to a fluorescent dye are examined in a fluorescence microscope. The antigenic sites fluoresce against a dark background and are immediately photographed on high speed film.

The enzyme-bound antigen- antibody complexes are further developed histochemistry by exposure to an enzyme substrate. This results in the development of a dark brown to a black color, which allows examination of the antigenic sites by light or electron microscopy

Gingiva, direct immunofluorescence with FITC antihuman IgG (scale in micrometers).

HISTOCHEMICAL ASPECTS OF NORMAL GINGIVA

NORMAL GINGIVA:-

A) Polysaccharides, proteins and mucins:Polysaccharides: Dye carmine- often used to demonstrate glycogen but it is not as specific as PAS method. Nonkeratinized alveolar mucosa constant levels of glycogen. Attached human gingiva- shows variation in extent of keratinization varition in glycogen content Epithelial glycogen increases during inflammation and repair.

Proteoglycans with chondroitin sulfate and hyaluronic acid-a major intercellular component of human gingival epithelium. Proteoglycans (PGs) are extracellular matrix and cellsurface associated macromolecules that vary in form and function (Hardingham and Fosang, 1 2). Proteoglycans (PGs) function in regulating aspects of cell behavior, such as proliferation, adhesion, and migration.

Heparan sulphate PGs (HSPGs), CD44 is known to function in cell adhesion, in lymphocyte homing, and in Binding:- hyaluronan (HA) (Aruffo et al., 1 0), -collagen (Carter and Wayner, 1 ), and -fibronectin (Jalkanen and Jalkanen, 1 2). Syndecan-1 is another integral cell membrane PG that binds:-collagen (Rapraeger et al., 1 5), -fibronectin (Saunders and Bernfield, 1 ), and -thrombospondin (Sun et al., 1 ).

Decorin and biglycan represent connective-tissue associated small dermatan/chondroitin sulphate PGs (DS/CSPGs) that are widely distributed in various soft connective tissues including human periodontium (Larjava et al., 1 2; Hakkinen et al., 1 3; Oksala et al., 1 5). Fibromodulin, biglycan and lumican were indentified in basal epithelial cells. Mast cells become visible with metacromatic dye like toludine blue in loose connective tissue. Lack of mast cells in acute necrotizing gingivitis is significant.

Frozen sections were analyzed by immunofluorescence microscopy for heparan sulphate PG, CD44, syndecan-1 decorin and biglycan). Oksala et al.( 1997): BM heparan sulphate PG showed reduced immunostaining in subepithelial and subendothelial basement membrane. Loss of CD44 and syndecan-1 was common in epithelial cells of inflamed periodontal tissue. decorin and biglycan, were also present in markedly reduced amounts in the periodontal connective tissue in chronic inflammation.

Immunolocalization of basement membrane heparan sulphate PG (HSPG) in chronically inflamed human periodontium. Frozen sections were analyzed by immunofluorescence. OE, oral epithelium, PE, pocket epithelium. Short arrows point to the presence of HSPG at the basement membrane zone in the relativelv non-inflamed area. Long arrows point to the reduced and discontiniued immunoreactivitv against HSPG in the inflamed area.

Glycosaminoglycans and phenytoin indiced gingival overgrowth Overgrowth due to accumulation of connective tissue substances in gingival propria( Hassell and Lindhe-1 7 ) fibroblasts of PHT- treated person,characterised by marked increase in protein synthesis in vitro(Hassell, Naranyanan and Copper 1 76) increased accumulation of sulfated glycosaminoglycans as intra- and extra-cellular material in PHT- treated person(Kantor & Hassell-1 3)

CANCELLARO, KLINGSBERG, AND BUTCHER(1961) conducted study on O.M.M of rats and hamster to check concentration of sulfhydryl, disulfide, and total protein DNA and RNA not vary with age staining reaction of RNA decreases in intensity from basal to corneum layer

PROTEINS AND PROTEINS GROUP:Keratinization is imporotant characteristic of epidermis. Although normally it occurs only in some areas of oral epi. But in pathologic condition it occurs anywhere in mouth. Sulfhydryl groups are demonstrated histochemically by ferric ferricyanide method in which this compound is reduced to prussian blue by these protein groups. Thus the extent of blue reaction product reflects the degree of keratinization..

Changes to the gingival tissues with onset of inflammation:EPITHELIAL CHANGES

CONNECTIVE TISSUE CHANGESA, B. Foci of infiltrating leukocytes at medium post-capillary and high magnifications (H&E).

C. Alcian blue staining demonstrating an increase of Alcian blue positive matrix material associated with localized foci of inflammation. D. Masson s trichrome demonstrating loss of collagenous material at inflammatory foci.

ENZYME HISTOCHEMISTRY:A) ALKALINE PHOSPHATESE: Mostly present in capillary endothelium of lamina propria. Reaction product observed in gingival epithelium and collagen fibers. Salivary gland acini in basement membrane exhibit high alkaline phosphates activity

Alkaline phosphatase is a lysosomal enzyme found in osteoblasts, fibroblasts, neutrophils, and bacteria. It is found in serum and in gingival crevicular fluid. Increased levels of alkaline phosphatase have been noted in experimental gingivitis studies and at periodontitis sites (Ishikawa and Cimasoni 1970 Binder et al 1987)

Alkaline phosphatase in lamina propria of human gingiva revealed by ultraviolet fluorescence( 0 x)

ACID PHOSPHATASE: Related with degree ofkeratinization in gingiva Seen high in zone of keratinizaton and low in non keratinization.

That pattern observed in skin epidermis.Acid phosphatase in gingival immunocytes (scale in micrometers).

ESTERASE: Little information on the esterse activity of human gingiva. Superficial layers, including the keratinizing zone, show the presence of some esterase activity. High esterase activity in salivary gland duct also in the serous demilunes of the sublingual gland.

AMINOPEPTIDASELow activity seen in gingiva normally. Localised primarily in the basal layer of the epihelium and in the underlying connective tissue of gingiva. An increased activity seen in gingival inflammation and hyperplasia caused by drug phenytoin.

BETA-GLUCURONIDASE:-

involved in conjugation of steroidhormones and in hydrolysis of conjugated glucuronides and play a role in cell proliferation.

Seen in basal cell layers of oral epithelium in humanb-Glucuronidase activity in chronically inflamed human gingiva. Note intense activity in connective tissues underlying crevicular epithelium (CE) (scale in micrometers).

CYTOCROME OXIDASE: Low level of cytochrome oxidase activity in human gingiva Localized in basal cell layers of free and attached gingiva, crevicular epi. and epi. attachment. Increased in inflammatory condition in both epithelium as well as connective tissue.

ATP, chronically inflamed human gingiva. Note intense reaction in connective tissue underlying the crevicular epithelium (CE). M, gingival margin; ge, gingival epithelium (scale in micrometers).

Cytochrome oxidase activity in gingival immunocytes (scale in micrometers ).

SUCCINATE DEHYDROGENASE AND GLUCOSE 6-PHOSPHATE DEHYDROGANASE:Present in basal cell layers of gingival epi. and duct of salivary glands. Distribution pattern of succinate dehydrogenase is similar to that of cytochrome oxidase. G6-PD present in significant quantities of oral mucosal epithelium. Increased in malignant dyslastic lesions of oral mucosa. So, assist in the diagnosis of oral cancer.

COLLAGENASEis produced in epithelium and connective tissue of normal gingiva as well as in the periodontal ligament and alveolar bone. it may be derived from host cells including PMN, macrophages, fibroblasts, keratinocytes and osteoclasts as well as bacteria Since it appears that the collagenase secreted from PMN S is the most important in the degradation of periodontal collagens( Overall And Sodek 1987)

CLINICAL CONSIDERATION

A) Tissue biopsy materials are usually stained with H and E stain.

B) Varity of fungi that infect human contains mucopolysaccharides that can correctly diagnosed by special histochemical stains.

C).Diagnosis of Histoplasmosis, Actinomycosis, Blastomycosis, and Coccidioidomycosis can make only after special histochemical stains . D) For correct diagnosis of tumour that arises from fat cells. E) Important aid in identification of vesicles that may appear in tumours cell of various benign and malignant tumour.

CONCLUSION

Histochemistry has been advantageously used with increasing frequency in the diagnosis of disease and in studying changes in metabolic pathways of tissues under normal and altered physiologic environments.

Most histochemical techniques have generally been used for qualitative analysis of chemical substances in cells and tissues. However, many sophisticated techniques have been devised recently for quantitative analysis of histochemical reactions.

REFERENCES: Orban s- Oral histolgy and embryology, 12th edition, 322-344 Ten Cate s- Oral histology: Development, structure and function, 6th edition, 32 -37 Anil ghom and Shubhangi Mhaske- Textbook of oral pathology, 1st edition, 1 -37 Geoffrey C. Cowley. Enzyme Activity in Gingival Immunocytes. J Dent Res 1972; 51: 284-294 Kantor M. L and Hassell T. M. Increased Accumulation of Sulfated Glycosaminoglycans in Culturesof Human Fibroblasts from Phenytoin-induced Gingival Overgrowth. J Dent Res 1 3;62(3):3 3-3 7

Oksala O, Haapasalmi K, H'akkinen L, Uitto V. J, and Larjava H. Expression of Heparan Sulphate and Small Dermatan/Chondroitin SulphateProteoglycans in Chronically Inflamed Human Periodontium. J Dent Res 1 7; 76(6): 1250-125 Sanjay saraf- Text book of oral pathology, 1st edition, 511-517 Louis A. Cancellaro, Jules Klingsberg and Earl O. Butcher, Histochemistry of Oral Mucous Membrane: Total Protein, Sulfhydryls, Disulfides, Ribonucleic Acid, and Desoxyribonucleic Acid. J DENT RES 1961 40: 436