A N N A L E S H I S T O R E C O - N A T U R A L E S M U S E I N A T I O N A L I S H U N G A R I C I Tomus 8 7 . Budapest , 1 9 9 5 p. 1 3 7 - 1 4 6

Histochemical and immunohistochemical analysis of mummy skin*

by L . J Ó Z S A & I . PAP, Budapest

JÓZSA, L . & PAP, I . (1995): Histochemical and immunohistochemical analysis of mummy skin. -Annls hist.-nat. Mus. natn. hung. 87: 137-146.

Abstract - Two Egyptian mummies' facial skin fragments were analysed by histochemical and immunohistochemical methods. The melanin contents of epithelium and the glycoproteins and proteoglycans of corium were clearly presented. Glycosaminoglycans were only faintly tintable. The peroxidase activity of red blood cells could be indicated by benzidine reaction. Immunohistochemical analysis made it possible to detect collagen Types I , I I I and IV, laminin, fibronectin and desmin. Epithelium contained cytokeratin and epithelial membrane antigen. With 12 figures and 2 tables.

I N T R O D U C T I O N

Microscopic examination of mummified tissue was initiated at the middle of the last century. JÁNOS C Z E R M Á K , professor of the university of Pest, was the first ( 1 8 5 2 ) who published his

data of observations. A t the beginning o f this century an increasing number of histological reports was published ( S M I T H 1908 , RUFFER 1911 ) . Microscopic analysis gave a new dimension to the identification and certain diagnosis of a growing number of diseases. Not only arteriosclerosis but pneumonia, plague, nephritis and some diseases o f the liver were also possible to detect. The ultrastructural level demonstrated reasonable preservation of cellular organelles ( L E W I N 1 9 6 7 ) . Sometimes even pathogenic agents (Koch bacillus, fungi, worm eggs) became recognizable (RUFFER 1 9 1 0 , A L L I S O N et al. 1979 , J Ó Z S A 1 9 6 6 , W E I 1 9 7 3 ) .

Electron microscopy of mummified tissues made it possible to demonstrate smallpox-like viral particles ( L E W I N 1 9 8 4 , 1 9 9 1 , F O R N A C I A R I & M A C H E T T I 1986 ) .

Histochemistry (the study of the distribution of substances in tissues) ( H A L E & M A R G H A M 1 9 8 8 ) is based on well-known chemical reactions and physico-chemical characteristics, it makes tissue components and/or materials accumulating under pathological conditions visible. The composition of fatty tissue ingredients, polysaccharides of connective tissue and chondral matrix, urate crystals of gouty concretions as well as a number of other substances were established by histochemical analysis (BORN 1959, SANDISON 1955, 1959 , L E E & S T E N N 1978, S T E N N 1981) .

Enzymatic actions of cells and the localization of the different enzymes are also feasible in recent material. However, it is still beyond the limitations of our methods to identify their presence in mummy tissues as far as we know it.

Immunohistochemistry reveals the presence of all materials and l iving organisms (bacteria and viruses) initiating antibody production ( Y A Z D I & D A R D I C K 1 9 9 1 ) . The high sensibility of immune reactions makes it possible to detect even matters of just a few molecules. Their specificity is 9 9 - 1 0 0 % . Aspecific reactions fall under two types. There is a type where antigen-antibody reaction is not generic. For example human collagen starts the production of antibodies that react to the collagen proteins of other species as well. The other type of

This study was supported by the Hungarian National Scientific Research Fund (OTKA No. 3161 and 3162).

antibody reacts not only to a specific antigen but to other, chemically similar proteins, too. Antigens can be detected with great certainty by immunohistochemical methods thanks to the extraordinary specificity of these methods.

Immunohistochemical investigations were started with blood group characteristics o f ancient material. First ABO, and later Rh, M N and P blood groups became detectable. The two later ones were identified only in mummy tissues, but the others in skeletalized material, too ( L E N G Y E L 1970, C R A I N I C et al. 1989). The number and variety of detectable substances and pathogens is constantly growing. More than one thousand various antigens (proteins, cellular markers, receptors, etc.) are recognisable on recent material.

Not a fraction of these was described for mummy tissue and immunohistochemistry was only scarcely i f ever utilized for paleohistology.

Our purpose was to determine the tissue components and cell markers that can be detected in mummy tissues.

MATERIAL AND METHODS

We analysed the scalps of two Egyptian mummies from the 1st century A. D. housed in the Department of Anthropology of the Hungarian Natural History Museum, Budapest. Their detailed paleopathological examination was carried out by MÉREI & NEMESKÉRI (1958).

Samples of approximately 10x6 mm were taken from the preauricular regions (regio parotidea) of the mummies' scalps. The skin pieces were fixed in Ruffer solution (formalin, water, sodium carbonate). They were softened and rehydrated for three days, then they were washed in tap water, and they were subjected to the routine histological paraffin embedding utilizing a Sandon-Elliot automatic processor. The material was oriented and cut in a way that the full thickness of epithelium and corium layers was contained within the specimen.

Histological methods: Haematoxilin-eosin (HE), picrosyrius, Gömöri's reticulin staining, Mallory-trichrom, Mas-son-trichrom staining were carried out for histological evaluation. Melanin pigment was indicated by the method of Masson-Fontana. Myelinated neurofibrils were indicated by Luxol-Fasl blue staining. Elastic fibres were stained by orcein and resorcin fuchsin. All sections were analysed and photographed by light and polar microscopes.

Histochemical methods: Proteins with aldohexose contents (glycoproteins, proteoglycans) were indicated by periodic Schiff reaction (PAS), while glycosaminoglycans by alcian blue (pH 0.5 pH 2.5) and alcian blue PAS reactions by toluidin blue staining. Prussian blue reaction was used in an attempt to indicate siderous materials.

Immunohistochemical methods: Sections were digested in 37 °C, 0.1% pepsin solution (Merck, Darmstadt) for two hours. Recent technology dictates a hydrogen peroxide solution to inhibit tissue peroxidase as a next step. As we supposed that there were no active enzymes in mummy tissues, first we passed over this step. We were astonished to find a large proportion of red blood cells with various strengths of peroxidase activity. This accident led us to detect enzymes in mummy tissue for the first time. Observing this fact we stopped to pass over hydrogen peroxide treatment before immunohistochemical reactions in all other analysis processes. Antigen-antibody reaction took 18 hours in a wet chamber at room temperature. The location of the reaction was marked by ovidin-biotin-peroxidase (Vector Laboratories Burlingames, USA) reaction. The antibodies and dilutions applied were summarized in Table 1.

Table 1. Immunohistochemical reagents and solutions applied

Antibody Manufacturer Solution

Fibronectin Dako/Copenhagen 1 500 Laminin Sigma/ST Louis 1 500 Collagen Type I Biogenesis/Bourhemouth 1 1500 Collagen Type III Biogenesis/Bourhe mouth 1 500 Collagen Type IV Biogenesis/Bourhemouth 1 200 Tenascin Telios/San Diego 1 300 Thrombospondin Oncogene/Manasset 1 1000 Vitronectin Chemicon/Temecula 1 250 EMA (E 29) Dako/Copenhagen 1 200 Cytokeratin (LP 34) Dako/Copenhagen 1 250 Vimentin (V.9) Dako/Copenhagen 1 200 Desmin (D33) Dako/Copenhagen 1 200

Figs 1-3. Fig. I . The collagen fibres show strong anisotropy. Picrosyrius staining + polar microscope. Magnification 100 x. - Fig. 2. Gömöri's reticulin staining. Magnification 200 x. - Fig. 3. The myelin sheath is

well visible by Luxol Fast-blue staining. Magnification 100 x

Figs 4-6. Fig. 4. Both in the Malpighi-layer and hair follicles marked melanin contents could be demonstrated. Masson-Fontana staining. Magnification 200 x. - Fig. 5. Hyperpigmented basal layer in the epithelium. HE

staining. Magnification 300 x. - Fig. 6. Moderate PAS positivity is visible on the basal mebranes, as well as in the luminal ends of the epithelium of the sweat glands. PAS staining. Magnification 200 x

Figs 7-9. Fig. 7. The thick collagen fibers showed moderate staining by Type I collagen. ABP immunohistochemical reaction. Magnification 200 x. - Fig. 8. The thin collagen fibers of the dermis were

positively staining with Type III collagen ABP immunohistochemical reaction. No cross reaction between Type I and Type I I I collagens could be observed. Magnification 200 x. - Fig. 9. Mild fibronectin reactivity (arrow) could

be demonstrated in the arterial wall. Fibronectin ABP immunohistochemical reaction. Magnification 400 x

Figs 10-12. Fig. 10. Cytokeratin could be demonstrated in some areas of epithelium and in the cells of the hair follicle. CK ABP immunohistochemical reaction. Magnification 200 x. - Fig. 11. Diffuse EMA reactivity was seen both in the epithelium of the skin, hair follicle and sweat glands. EMA ABP immunohistochemical

reaction. Magnification 100 x. - Fig. 12. The erythrocytes showed peroxidase activity. Benzidine reaction. Magnification 400 x

RESULTS

Histological examinations

H E stained sections presented the silhouettes of skin components but there was no nuclear staining. Epithelium, connective tissue o f corium, sebaceous and perspiratory glands were clearly discernible. Sections with picrosyrius staining presented not only anisotropy of cori-um's collagen fibre, but anisotropy o f epithelium's keratin, too, when observed by polar microscope (Fig. 1). Glandular epithelium's nuclei were possible to record beyond reticular fibre and basal membranes applying Gömóri ' s reticulin staining, but no connective tissue nuclei (Fig. 2). Nuclei were not possible to indicate with any other process in mummified tissues. Epithelium was orange red, collagen was blue or bluish-grey, red blood cells were yellow and erector muscle was bluish-pink in Mallory- and Masson-trichrom stained sections. Masson-trichrom staining produced bright red parts that normally turn blue in collagen fibre. This is a marker of the denaturation o f collagen protein. Nerves and blood vessels became recognizable by these methods, but we did not manage to indicate tactile corpuscles or other neurosensory end-organs. Luxol-Fast blue stained specimens presented clearly visible myelin sheath of peripherial nerves (Fig. 3).

A large quantity o f melanin was present not only in the epithelium but in the cells of hair follicles (Figs 4-5). No sign of marked pathological alteration could be seen and the skin pieces had regular structure. A lesser interstitial haemorrhage could be seen in the skin of one of the mummies. It must have occurred a short time before the individual's death (within 24-48 hours). No red blood cell solution and ferrous pigment (hemosiderin) release could be identified. Elastic fibres were easy to stain with both methods.

Histochemical analysis

The glycoproteins and proteoglycans o f interstitial matrix produced a weak PAS positivity. Epithelium's glycogen was not possible to record. Glycosaminoglycans produced no meta-chromasy with toluidin blue but they were slightly coloured with alcian blue and alcian blue PAS staining. Basal membranes had moderate PAS positivity as well as the lumen side parts of epithelial cells of perspiratory glands (Fig. 6).

Immunohistochemical analysis

Immunohistochemical reactions of thick connective tissue fibre containing Type I collagen were of moderate intensity while thin fibre with Type I I I collagen molecules were extremely intensive (Figs 7-8). Non-fibre forming Type TV collagen could not be detected. Fibronectin and laminin could be found in vessel walls, and laminin could be met with sporadically in basal membranes, too (Fig. 9). Tenascin, trombospondin and vitronectin could not be recognized.

As far as cell markers were concerned, muscle specific desmin's presence was detected in certain cells of erector muscle, but there was no staining in others. Vimentin, the marker o f connective tissue cells could not be traced though we did not manage to point out fibrocytes (neither with immunohistochemical nor with histological methods). We examined two types of epithelial cell markers, cytokeratin (CK) and epithelial membrane antigen ( E M A ) . CK could be found here and there in the deeper layers of epithelium and almost everywhere in hair follicle and sebaceous gland cells. It had the correct pattern of localization, too (Fig. 10). E M A produced powerful reactions in tectorial and glandular epithelium and also in hair follicle cells

but epithelial cells produced diffuse staining and localization was not correct (Fig. 11). Table 2 shows materials detectable by immunohistochemistry.

Though it is not an immunohistochemical process, red blood cells' peroxidase enzyme could be identified by benzidine reaction as a side product of the analysis outlined (Fig. 12).

Table 2. Immunohistochemical and histochemical results

Tissue component Detection method Result

Glycoprotein PAS Moderate positivity Proteoglycan PAS Moderate positivity Glycosaminoglycan Alcian blue Moderate positivity Melanin Masson Montana Marked positivity Medullary sheath Luxol-Fast-Blue Medium positivity Collagen Type I ABP Medium positivity Collagen Type I I I ABP Marked positivity Collagen Type IV ABP Moderate positivity Fibronectin ABP Moderate positivity Laminin ABP Seldom positivity Tenascin ABP Negative Thrombospondin ABP Negative Vitronectin ABP Negative Desmin ABP Moderate positivity Vimentin ABP Negative EMA ABP Marked positivity CK ABP Medium positivity Peroxidase enzyme Benzidine Medium positivity

DISCUSSION

Skeletalized material preserved a considerable proportion of its organic material contents in osseous tissues. Collagen, polysaccharides, some lipids survived not only in ancient remains but in 5 0 - 7 0 million year old dinosaur fossils and they can be detected by comparatively simple methods ( P A W L I C K I 1 9 8 3 ) . Cholesterine, haemosiderin and calcium contents o f arteriosclerotic arteriae were the first to be discovered in human remains and polysaccharides, bile pigments, urate crystals, etc. were identified much later. Histochemistry was booming in the fifties but paleohistology was slow to utilize these technical possibilities ( S A N D I S O N 1 9 6 2 ) . As far as we know it, enzyme activities were never detected in mummy tissue before. We think enzymes have various levels of survivability and resistance and therefore some of them had to be able to survive mummy making processes as well as dehydration. There is a possibility that not only peroxidase but other enzymes are also detectable in mummified tissue.

In 1 9 1 4 U H L E N H U T H & W E I D O N Z discovered that bones preserved generic immunologic characteristics. The U H L E N H U T H reaction was based on this. Though it is obsolete today it was used to determine the human origin of bone remains for a long time. Twenty years after the discovery of U H L E N H U T H & W E I D O N Z it was established that polysaccharides containing proteins with A B O blood group characteristics were also preserved in skeletalized material and mummy tissue. For a long time blood groups were determined by immunochemical processes. A microscopic method that was able to decide the group and to localize blood group material was established only thirty years ago. This way immunohistochemistry became a part of paleohistology ( L E N G Y E L 1 9 7 5 ) . Recently A S C E N Z I et al. ( 1 9 8 5 ) detected haemoglobin in bones and they managed to separate normal haemoglobin and the S-haemoglobin occurring in sickle cell anaemia. L E W I N ( 1 9 8 4 ) demonstrated smallpox-like viral agents from the mummy

of Rhamses the Fifth. A n Italian team identified smallpox virus particles by immune electron microscopic analysis in a 16th century mummified child's skin alteration ( F O R N A C I A R I & M A R C H E T T I 1986).

Immunohistochemistry is starting to gain ground in the analysis of skeletalized material. This started us to examine the detectability o f certain mummy tissue components. Contemporary pathology utilizes collagen and non-collagen matrix proteins o f skin, their localization and the changes of their proportion in several disease entities. We utilized recent material's distribution and detectability as a control to compare tissue proteins identifiable in mummy skin. Several matrix proteins detectable in recent skin, tenascin, vitronectin, thrombospondin, etc. are permanent components of intact skin. Laminin and Type I V collagen are present in all blood vessels and basal membranes and in the basal membrane between epithelium and cori-um. No matrix proteins could be detected with the exception of laminin and fibronectin in mummy skin. These later were detected in traces. Fibronectin could be found in vessel walls of mummy skin. As it could be expected fibre forming collagens kept not only their staining and refraction characteristics but their immunological ones as well . The detectability of Type I and I I I collagens by immunohistochemical methods provides hope to recognize other diseases by analysing mummy skin (and other tissues) where pathological alterations o f collagens are present. We do not know any paleopathological works that presented collagen diseases (lupus erythematosus, scleroderma, dermatomyositis, etc.). The typical inflammatory reactions of these diseases are impossible to detect in mummy tissues therefore the detection o f pathological alterations of collagens could result in a viable paleopathological diagnosis.

The other group of our immunohistochemical examinations was centred on the detectability of cell markers. The identification of cell markers mentioned above is far from having only theoretical significance. The very detectability of CK and E M A characteristic for epithelial cells in mummy skin opened up the prospect to uncover the histogenesis of benign and malign tumours - not at all infrequent in mummies. According to our results, it is well worth the effort - what is more, it is necessary to detect cell markers to establish the epithelial, interstitial or muscular origin of a tumour. It is often not easy to establish the nature of any tumour (cancer, sarcoma or melanoma) in recent material. Deprived of the possibility to observe the morphology of cells and nucleus karyokinesis, etc. the paleopathologist is facing a much more difficult task. These are the cases when immunohistochemistry can be of great use and sometimes it can provide the only means of analysis.

Our results prove the long term survivability of several tissue and cell proteins. These materials are well able to survive post mortal processes as well as the techniques of mummification. It indicates the practicability of recent pathohistologic methods in mummy tissue and skeletalized material analysis. The more methods are applied the more complete picture can be obtained.

S U M M A R Y

We analysed two Egyptian mummies' facial skin fragments by histochemical and immunohistochemical methods. The melanin contents of epithelium and the glycoproteins and proteoglycans of corium were clearly presented. The tinction of glycosaminoglycans were only faintly recognizable. The peroxidase activity of red blood cells could be indicated by benzidine reaction. Immunohistochemical analysis made it possible to detect collagen Types I , I I I and IV, laminin, fibronectin and desmin. Epithelium contained cy to keratin and epithelial membrane antigen.

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Authors' addresses: Dr. ILDIKÓ PAP Dr. LÁSZLÓ JÓZSA

Department of Anthropology Hungarian Natural History Museum H-1062 Budapest, Bajza u. 39. Hungary

Department of Morphology National Institute of Traumatology H-1430 Budapest, P.O.B. 21 Hungary