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Proliferative Activity of Epidermal Basal Cells after Wounding
Exp Toxic Pathol 2001; 53: 65–69 URBAN & FISCHER http://www.urbanfischer.de/journals/exptoxpath 1
Department of Neurosurgery and Department of Legal Medicine, University of Lübeck,Germany
Proliferative Activity of Epidermal Basal Cells after Wounding AgNOR Counts Compared with Bromodeoxyuridine Reactivity in Rats
A. P. REBOLLEDO GODOY1, M. REBOLLEDO GODOY2, C. MEISSNER, and M. OEHMICHEN With 2 figures Received: January 19, 2000; Revised: May 30, 2000; Accepted: August 10, 2000 Address for correspondence: Prof. Dr. med. M. OEHMICHEN, Department of Legal Medicine, University of Lübeck, Kahlhorststr. 31–35, D - 23562 Lübeck, Germany; fax: +49 (451) 500 2760. Key words: Epidermal basal cells; wound; proliferation; AgNOR; BrdU.
Summary Quantitative changes in nucleolus organizer regions (NORs) are known markers of proliferation that can be demonstrated by a specific silver staining technique on paraffin-embedded sections. Wounding of skin induces proliferation of basal epidermal cells at the wound margin. The degree of proliferation depends on the survival time and can be measured by morphometric assessment of argyrophilic NORs (AgNORs). Following incision wounding of the pinnae, rats were allowed to survive for different intervals (7 rats per interval) up to 120 hours. Before each sacrifice, biopsies were taken and incubated in a bromodeoxyuridine (BrdU) solution, embedded in paraffin, and stained with an antibody against BrdU. At the same time morphometric analysis of AgNOR counts was performed on sections made from the same material. BrdU incorporating nuclei were assessed by simple counting, whereas morphometric analysis of AgNOR counts was computer aided. Both methods revealed an increase in the number of proliferating cells, a plateau phase being reached after about 36 hours, followed by a decline after about 70 hours. Both methods thus allowed a reliable temporal classification of the skin injury according to survival time. The molecular background of the AgNOR changes in relation to the proliferation of cellular elements is discussed in detail.
Introduction Wound healing of the skin begins with proliferation of cells on the wound margin, in particular epidermal basal cells [30, 43]. These cells have a basic proliferative activity determined by the stable turnover of this cell population [3, 6]. Breaking the continuity of the epidermal
cell layer immediately triggers the process of wound healing, which leads among other things to stimulation of cell division processes, also in basal epidermal cells [7, 8], by serum factors such as complement proteins, cytokines and growth factors [16, 22, 49], and by mediators such as serotonin and histamine. Other factors may also be involved, including inhibitors of epidermal cell mitosis (chalones), which regulate „normal“ epidermal cell turnover and prevent overshooting cell growth [4]. DNA synthesis has been demonstrated by in vitro incubation of skin biopsies from wound margin using a BrdU solution [31, 48]. A time-dependent increase in the synthesis rate of the basal epidermal cells was traced, beginning after about 24 hours and continuing for at least 72 hours; during this period the synthesis rate increased approximately six-fold. However, the BrdU technique has the drawback of requiring in vitro incubation of living cells, i.e. biopsy material. Since living cells are not always available, there is a need for a method that achieves comparable results using a simple staining technique on paraffin sections. The AgNOR technique has obviously some advantages over other methods usually employed for the study of cell kinetics. This technique is not time-consuming (unlike the BrdU technique), can be carried out on routinely processed, paraffin-embedded sections without specific pretreatment (unlike the Ki67 immunoreactivity), avoids the use of radioactive substances (unlike the 3H-thymidine labeling), and is comparably simple and inexpensive. In the following we describe the computer-assisted morphometric image analysis [25, 46] of AgNORs on the borders of incision wounds in rats [9, 18, 38, 45] and compare the results with those obtained on the same material using the BrdU technique. 0940-2993/01/53/01-065 $ 15.00/0
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Materials and methods The experimental design is described in detail elsewhere [31, 32]. Briefly, a 6 mm long smooth-bordered linear scissor incision was made in both pinnae of each rat (Sprague Dawley rats). The animals were subsequently killed in groups of 7 at different intervals between 0 and 120 hours post-wounding. Before each sacrifice, two intravital needle biopsies were taken from the wound margin of the injured pinnae. The biopsies were immediately exposed in a BrdU solution. The incubation medium consisted of basal medium eagle (BME Earle, code no. 47350, Serva Feinbiochemica, Heidelberg), penicillin (10.000 E/ml), streptomycin (10 mg/ml), a glutamine solution (200 ml/l), and 5bromo-2’-deoxyuridine (code no. B5002, Sigma Deisenhofen) in a 10-3 M solution. Each biopsy was incubated for 1 hour at 37 °C under 2.2 atm. O2 pressure. Biopsies were washed 3 times and fixed in a 4% paraformaldehyde solution for 24 to 48 hours. The blocks were then embedded in paraffin according to routine methods.
BrdU technique Five µm serial sections were cut perpendicular to the incision. Anti-BrdU antibody (code no. JO 216, BectonDickinson Heidelberg) diluted 1:100 served as the primary antibody, peroxidase-labeled, rabbit anti-mouse Ig (code no. P 260, Dakopatts Hamburg) diluted 1:60 as the secondary antibody. Staining was performed with diaminobenzidine. Nuclear fast red was used for nuclear staining.
AgNOR technique Three to five µm thick sections were cut with a rotation microtome and stained following the modified method for the detection of AgNORs established by KOREK et al [23]. This method combines the original silver staining [38] with a Feulgen staining of the same specimen. The staining solution consists of 50% aqueous silver nitrate and 2% gelatin in 1% aqueous formic acid and must be prepared immediately before use. The sections were treated for 32 minutes at room temperature in a dark place.
In each specimen, 40 basal cells were counted in each of 4 different sections beginning at the incision and proceeding outward, for a total of 120 cells. For each cell the size of the nucleus plus the number and size of the AgNORs were determined. The data were assessed according to the following parameters: 1) Total number of AgNORs* (NorNumb T) 2) Mean area of nucleus (µm2)* (Size) 3) Mean sum area of AgNORs in the (AreaSum) nucleus (µm2)* 4) Area of largest AgNOR (MaxNor) in the nucleus (µm2) 5) Mean AgNOR area / nucleus (µm2) (MeanArea) 6) Mean number of AgNORs (NorNumb) in the nucleus* 7) AreaSum/Size (Sizeratio) 8) MeanArea/Size (Sizeratiom) 9) Mean form factor of the nuclei (Form M) 10) Number of nuclei with (AnzNOR) 0 / 1 / 2 / 3 / 4 / 5 AgNORs 11) Coefficient of variation (Cv = AreaSum / Standard deviation)
Results The silver staining technique was able to demonstrate the presence of AgNORs in the epidermal basal cells (figure 1). Quantitative evaluation applying various assessment criteria arrived at consistent results: After wounding all values increased, peaking after no more than 60 hours survival time and then returning to normal values at 120 hours survival. figure 2 (a, b, c, d) shows an increase shortly after wounding in the total number of AgNORs (a), mean AgNOR number per nucleus (b), mean AgNOR sum area in the nucleus (c), and mean AgNOR area per nucleus (d). The BrdU results on material from the same animals are presented in figure 2e.
Discussion
BrdU technique: In each specimen, 150 basal cells were counted in each of 4 different sections beginning at the incision and proceeding outward, for a total of 600 cells. The labeling index was obtained by calculating the percentage of cells incorporating BrdU into their nucleus. In most specimens „labeled“ cells could be readily distinguished from „nonlabeled“ cells. Only basal cells in the epidermal layer, excluding hair follicles and excretory ducts of sweat glands, were counted.
Skin incision wounding triggers an immediate increase in the proliferative activity of basal epidermal cells up to a distance of 300–500 cells as measured outward from the wound margin [5, 26]. Four days posttrauma proliferative activity can only be demonstrated in a narrow seam along the wound margin [2]. Numerous experimental studies on various animal species [30] show that proliferation usually begins after 24 hours and peaks on the fourth day posttrauma. In our own studies using BrdU on the pinnae of rats, peak proliferation was attained after 36 hours and remained stable for up to 72 hours; by the eighth day posttrauma the proliferation rate had returned to normal [31].
AgNOR technique: For microscopic evaluation computer-assisted image analysis was used to measure the AgNORs (see 43). The special software – SK-RM 1 and SKSTAT – was made by O. WEGNER, Adelbertstraße 25, D 24106 Kiel, Germany, according to our specifications.
*Although all of the parameters measured exhibited comparable distribution patterns, the study focussed on those 4 parameters allowing the most exact discrimination.
Quantitative evaluation
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Fig. 1. Demonstration of NORs by silver staining in combination with a Feulgen s staining according to KOREK et al (1991) (Magnification: × 1200).
Fig. 2. Quantitative assessment of AgNOR counts in epidermal basal cells on wound borders employing different morphometric parameters and in dependence on posttrauma survival time (a, b, c, d). Number of epidermal basal cells as detected by the BrdU technique in dependence on posttrauma survival time (e). Exp Toxic Pathol 53 (2001) 1
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Numerous growth and cellular adhesion factors are described in the literature [16, 22], some derived from serum, some secreted by local and/or immigrating cells. The transition through the cell cycle is regulated by the activities of cyclin dependent kinase (cdk) and their inhibitors [27]. In wound healing the absence of mitosis inhibiting factors, so-called chalones, also has an impact [19]. Roels [41] assumes that chalones function as pressors and operate on a specific gene. They are thought to be involved in cell cycle regulation and to release an antimitotic substance. In the cell cycle, cells are normally inhibited in the G1 phase about 9–12 hours before DNA synthesis begins and in the G2 phase immediately prior to the mitotic phase [42]. While this involves tissue specific inhibition of mitosis by signal transduction, chalones are not species specific, but they are organ specific [42]. The nucleolus plays an essential role in the regulation of proliferation and protein synthesis [12, 36, 47]. AgNORs are segments of DNA closely associated with nucleoli. They contain coding genes for ribosomal RNA and therefore play a major role in the regulation of protein synthesis [13]. AgNORs allow quantitative inferences regarding the cell’s rate of protein synthesis, and so cellular proliferation activity in general [11, 20, 46]. The individual phases of the proliferation cycle can be described as follows [17]: During the prophase, the nucleolus shrinks; in the meta- and anaphases the nucleolus is no longer visible. The nucleolus again becomes visible in the telophase. Field et al [15] showed that resting lymphocytes (G0 phase) exhibit only sparse AgNORs, while in the G1 phase the number of AgNORs grows [see also 37]; in the first generation this pattern remains unchanged in the S and G2 phases. In the second and third generations of mitosis numerous small granula appear. The AgNOR count has a linear relationship with the percentage of S-phase cells, as determined by DNA flow cytometry [10]. These findings are also supported by comparison of AgNOR counts and BrdU incorporation [33]. Furthermore, like the expression of Ki67 epitopes, AgNOR counts are also variably influenced by the proliferation activities of tumor cells, phases of the cell cycle, and cell differentiation [29, 44]. Today there is no doubt that AgNORs are a useful proliferation marker [1, 24, 33] that can aid, for example, in evaluating the proliferation rate of tumor cells [49]. However, AgNOR counts measure not only proliferation, but also differentiation, state and proliferation rate [14, 28, 40]. A basic criticism of the methods applied here was made by ÖFNER et al. [34, 35], who pointed out that staining artifacts can occur on routine paraffin-embedded tissues. In their opinion the number of AgNORs cannot be exactly determined since they cannot be clearly distinguished. A substantial improvement can be attained after wet autoclave pretreatment. Studies of the skin employing AgNOR have usually focused on skin tumors. Normal skin has only been examined by KANITAKES et al. [21] in 6 cases, revealing 68
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AgNOR counts per nucleus of 1.93 ± 0.14. Normal human mucous membranes have an AgNOR count of 4.51 (± 2,57). To our knowledge normal rat skin has never been studied, nor has rat skin after wounding. Our results demonstrate that on formalin-fixed, paraffin-embedded skin samples AgNOR morphometry can achieve results comparable to those of the BrdU technique. So both methods allow a reliable temporal classification of the skin injury according to survival time and are helpful in assessing wound age and vitality.
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Book reviews
Atlas of Immunology JULIUS M. CRUSE and ROBERT E. LEWIS 450 pages with numerous figures and tables. CRC Press LLC, Boca Raton USA and Springer Verlag GmbH, Heidelberg 1999. ISBN 3-540-64807-0 Immunology has rapidly developed over the last 20 years mainly due to new techniques in biological sciences which allowed a deeper insight into the structure and function of molecules and cells involved in immunological reactions in health and disease. This Atlas of Immunology is a compilation of the present knowledge in this field covering every important concept put torward over the last decades. The introductory History of Immunology describes briefly the curricula vitae of scientists who made the most important contributions to our understanding of immunology. This chapter gives the reader an idea of the long lasting process and the many steps in the accumulation of our present knowledge in immunology ranging from resistance against infectious diseases to MHC restriction. The first part of this atlas was devoted to the physiological processes of the immune response in human beings. Molecules, cells and tissues of the immune reactions were considered as well as the processes connected with antigen presentation, immunoglobulin synthesis and antigen-antibody interactions. Separate chapters give a solid overview of cytokines, the complement system, the types of hypersensitivity, immunoregulation, and immunohematology. In the second part the authors focused on immunological diseases and immunopathology. Appropriate emphasis was placed on congenital and acquired immunodeficiencies, in particular on AIDS. Immunosuppression, transplantation im-
munology, tumor immunology, and immunity against microorganisms were outlined in the following chapters. Immunologic methods were dealt with in the final chapter in which the basic techniques of measurement of antigens and antibodies were described. The atlas is an appealing reference book, comprehensive as can be expected from this type of a book. More than 1,000 illustrations illuminate the easy-to-read text. Unfortunately, all figures are given in black and white so that the information content of the numerous histological sections, organ preparations, and molecular structures of proteins is markedly restricted. On the other hand, the schematic drawings of molecules, cells, and reactions are highly informative. Particularly noteworthy is the basic concept of the textual description. All paragraphs start with a key word printed in bold letters so that the main topics are presented as in a dictionary. This might be useful for students and those readers who are not familiar with the principles of immunology. The disadvantage of such an approach is necessarily a lack of systematic presentation of general concepts. Thus, it might be difficult to gain an overview of the general ideas in the respective field put forward at present. Nevertheless, the authors did a good job in presenting this atlas so that it can be recommended to students, clinicians and scientists as a valuable source of information. R. DARGEL, Jena
Histological Typing of Lung and Pleural Tumours W. D. TRAVIS, T. V. COLBY, B. CORRIN, Y. SHIMOSATO, E. BRAMBILLA in collaboration with L. H. SOBIN and pathologists from 14 countries. 3rd ed. 162 pages with 150 figures and 7 tables. Springer Verlag, Berlin Heidelberg New York 1999. Softcover DM 129.00; öS 942.00; sFr 117.50; GBP 49.50; US $ 81.00. ISBN 3-540-65219-1 The first and the second editions of “Histological Typing of Lung and Pleural Tumours” were published in 1967 and 1981. A revised third edition has become necessary because over the last 10 to 15 years considerable progress has been made in understanding certain lung tumors. This gain in knowledge has resulted in some major and minor modifications of the classification. The concept of neuroendocrine tumors has been refined with inclusion of large cell neuroendocrine carcinoma and modification of the distinction between typical and atypical carcinoids. The histological heterogeneity of lung tumors is duly considered in the revised classification. More precise guidelines for grading squamous dysplasia and carcinoma in situ are provided and two categories of preinvasive lesions have been added: atypical adenomatous hyperplasia and diffuse idiopathic pulmonary neuroendocrine cell hyperplasia. In the introduction general principles of lung tumor typing and grading as well as molecular findings are explained concluding 70
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with the true neoplastic nature of chondroid hamartoma and sclerosing hemangioma. The definitions of tumor types are based primarily on light microscopic findings in surgical, fine-needle and autopsy material; cytologic specimens were dispensed with. The definitions and explanatory notes are concise and precise, the text is supplemented by excellent colored histological illustrations. As generally true for the WHO series “International Histological Classification of Tumours” the book is not intended to be a textbook, but is designed to provide uniform terminology as the basis for improved international communications. For this reason the reader will find the morphological codes for the International Classification of Diseases for Oncology (ICD-0) and the Systematized Nomenclature of Medicine (SNOMED). The book should not be lacking in the library of pathologists who are confronted with the histological examination of lung and pleural tumors. D. KATENKAMP, Jena
Department of Neurosurgery and Department of Legal Medicine, University of Lübeck,Germany
Proliferative Activity of Epidermal Basal Cells after Wounding AgNOR Counts Compared with Bromodeoxyuridine Reactivity in Rats
A. P. REBOLLEDO GODOY1, M. REBOLLEDO GODOY2, C. MEISSNER, and M. OEHMICHEN With 2 figures Received: January 19, 2000; Revised: May 30, 2000; Accepted: August 10, 2000 Address for correspondence: Prof. Dr. med. M. OEHMICHEN, Department of Legal Medicine, University of Lübeck, Kahlhorststr. 31–35, D - 23562 Lübeck, Germany; fax: +49 (451) 500 2760. Key words: Epidermal basal cells; wound; proliferation; AgNOR; BrdU.
Summary Quantitative changes in nucleolus organizer regions (NORs) are known markers of proliferation that can be demonstrated by a specific silver staining technique on paraffin-embedded sections. Wounding of skin induces proliferation of basal epidermal cells at the wound margin. The degree of proliferation depends on the survival time and can be measured by morphometric assessment of argyrophilic NORs (AgNORs). Following incision wounding of the pinnae, rats were allowed to survive for different intervals (7 rats per interval) up to 120 hours. Before each sacrifice, biopsies were taken and incubated in a bromodeoxyuridine (BrdU) solution, embedded in paraffin, and stained with an antibody against BrdU. At the same time morphometric analysis of AgNOR counts was performed on sections made from the same material. BrdU incorporating nuclei were assessed by simple counting, whereas morphometric analysis of AgNOR counts was computer aided. Both methods revealed an increase in the number of proliferating cells, a plateau phase being reached after about 36 hours, followed by a decline after about 70 hours. Both methods thus allowed a reliable temporal classification of the skin injury according to survival time. The molecular background of the AgNOR changes in relation to the proliferation of cellular elements is discussed in detail.
Introduction Wound healing of the skin begins with proliferation of cells on the wound margin, in particular epidermal basal cells [30, 43]. These cells have a basic proliferative activity determined by the stable turnover of this cell population [3, 6]. Breaking the continuity of the epidermal
cell layer immediately triggers the process of wound healing, which leads among other things to stimulation of cell division processes, also in basal epidermal cells [7, 8], by serum factors such as complement proteins, cytokines and growth factors [16, 22, 49], and by mediators such as serotonin and histamine. Other factors may also be involved, including inhibitors of epidermal cell mitosis (chalones), which regulate „normal“ epidermal cell turnover and prevent overshooting cell growth [4]. DNA synthesis has been demonstrated by in vitro incubation of skin biopsies from wound margin using a BrdU solution [31, 48]. A time-dependent increase in the synthesis rate of the basal epidermal cells was traced, beginning after about 24 hours and continuing for at least 72 hours; during this period the synthesis rate increased approximately six-fold. However, the BrdU technique has the drawback of requiring in vitro incubation of living cells, i.e. biopsy material. Since living cells are not always available, there is a need for a method that achieves comparable results using a simple staining technique on paraffin sections. The AgNOR technique has obviously some advantages over other methods usually employed for the study of cell kinetics. This technique is not time-consuming (unlike the BrdU technique), can be carried out on routinely processed, paraffin-embedded sections without specific pretreatment (unlike the Ki67 immunoreactivity), avoids the use of radioactive substances (unlike the 3H-thymidine labeling), and is comparably simple and inexpensive. In the following we describe the computer-assisted morphometric image analysis [25, 46] of AgNORs on the borders of incision wounds in rats [9, 18, 38, 45] and compare the results with those obtained on the same material using the BrdU technique. 0940-2993/01/53/01-065 $ 15.00/0
65
Materials and methods The experimental design is described in detail elsewhere [31, 32]. Briefly, a 6 mm long smooth-bordered linear scissor incision was made in both pinnae of each rat (Sprague Dawley rats). The animals were subsequently killed in groups of 7 at different intervals between 0 and 120 hours post-wounding. Before each sacrifice, two intravital needle biopsies were taken from the wound margin of the injured pinnae. The biopsies were immediately exposed in a BrdU solution. The incubation medium consisted of basal medium eagle (BME Earle, code no. 47350, Serva Feinbiochemica, Heidelberg), penicillin (10.000 E/ml), streptomycin (10 mg/ml), a glutamine solution (200 ml/l), and 5bromo-2’-deoxyuridine (code no. B5002, Sigma Deisenhofen) in a 10-3 M solution. Each biopsy was incubated for 1 hour at 37 °C under 2.2 atm. O2 pressure. Biopsies were washed 3 times and fixed in a 4% paraformaldehyde solution for 24 to 48 hours. The blocks were then embedded in paraffin according to routine methods.
BrdU technique Five µm serial sections were cut perpendicular to the incision. Anti-BrdU antibody (code no. JO 216, BectonDickinson Heidelberg) diluted 1:100 served as the primary antibody, peroxidase-labeled, rabbit anti-mouse Ig (code no. P 260, Dakopatts Hamburg) diluted 1:60 as the secondary antibody. Staining was performed with diaminobenzidine. Nuclear fast red was used for nuclear staining.
AgNOR technique Three to five µm thick sections were cut with a rotation microtome and stained following the modified method for the detection of AgNORs established by KOREK et al [23]. This method combines the original silver staining [38] with a Feulgen staining of the same specimen. The staining solution consists of 50% aqueous silver nitrate and 2% gelatin in 1% aqueous formic acid and must be prepared immediately before use. The sections were treated for 32 minutes at room temperature in a dark place.
In each specimen, 40 basal cells were counted in each of 4 different sections beginning at the incision and proceeding outward, for a total of 120 cells. For each cell the size of the nucleus plus the number and size of the AgNORs were determined. The data were assessed according to the following parameters: 1) Total number of AgNORs* (NorNumb T) 2) Mean area of nucleus (µm2)* (Size) 3) Mean sum area of AgNORs in the (AreaSum) nucleus (µm2)* 4) Area of largest AgNOR (MaxNor) in the nucleus (µm2) 5) Mean AgNOR area / nucleus (µm2) (MeanArea) 6) Mean number of AgNORs (NorNumb) in the nucleus* 7) AreaSum/Size (Sizeratio) 8) MeanArea/Size (Sizeratiom) 9) Mean form factor of the nuclei (Form M) 10) Number of nuclei with (AnzNOR) 0 / 1 / 2 / 3 / 4 / 5 AgNORs 11) Coefficient of variation (Cv = AreaSum / Standard deviation)
Results The silver staining technique was able to demonstrate the presence of AgNORs in the epidermal basal cells (figure 1). Quantitative evaluation applying various assessment criteria arrived at consistent results: After wounding all values increased, peaking after no more than 60 hours survival time and then returning to normal values at 120 hours survival. figure 2 (a, b, c, d) shows an increase shortly after wounding in the total number of AgNORs (a), mean AgNOR number per nucleus (b), mean AgNOR sum area in the nucleus (c), and mean AgNOR area per nucleus (d). The BrdU results on material from the same animals are presented in figure 2e.
Discussion
BrdU technique: In each specimen, 150 basal cells were counted in each of 4 different sections beginning at the incision and proceeding outward, for a total of 600 cells. The labeling index was obtained by calculating the percentage of cells incorporating BrdU into their nucleus. In most specimens „labeled“ cells could be readily distinguished from „nonlabeled“ cells. Only basal cells in the epidermal layer, excluding hair follicles and excretory ducts of sweat glands, were counted.
Skin incision wounding triggers an immediate increase in the proliferative activity of basal epidermal cells up to a distance of 300–500 cells as measured outward from the wound margin [5, 26]. Four days posttrauma proliferative activity can only be demonstrated in a narrow seam along the wound margin [2]. Numerous experimental studies on various animal species [30] show that proliferation usually begins after 24 hours and peaks on the fourth day posttrauma. In our own studies using BrdU on the pinnae of rats, peak proliferation was attained after 36 hours and remained stable for up to 72 hours; by the eighth day posttrauma the proliferation rate had returned to normal [31].
AgNOR technique: For microscopic evaluation computer-assisted image analysis was used to measure the AgNORs (see 43). The special software – SK-RM 1 and SKSTAT – was made by O. WEGNER, Adelbertstraße 25, D 24106 Kiel, Germany, according to our specifications.
*Although all of the parameters measured exhibited comparable distribution patterns, the study focussed on those 4 parameters allowing the most exact discrimination.
Quantitative evaluation
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Fig. 1. Demonstration of NORs by silver staining in combination with a Feulgen s staining according to KOREK et al (1991) (Magnification: × 1200).
Fig. 2. Quantitative assessment of AgNOR counts in epidermal basal cells on wound borders employing different morphometric parameters and in dependence on posttrauma survival time (a, b, c, d). Number of epidermal basal cells as detected by the BrdU technique in dependence on posttrauma survival time (e). Exp Toxic Pathol 53 (2001) 1
67
Numerous growth and cellular adhesion factors are described in the literature [16, 22], some derived from serum, some secreted by local and/or immigrating cells. The transition through the cell cycle is regulated by the activities of cyclin dependent kinase (cdk) and their inhibitors [27]. In wound healing the absence of mitosis inhibiting factors, so-called chalones, also has an impact [19]. Roels [41] assumes that chalones function as pressors and operate on a specific gene. They are thought to be involved in cell cycle regulation and to release an antimitotic substance. In the cell cycle, cells are normally inhibited in the G1 phase about 9–12 hours before DNA synthesis begins and in the G2 phase immediately prior to the mitotic phase [42]. While this involves tissue specific inhibition of mitosis by signal transduction, chalones are not species specific, but they are organ specific [42]. The nucleolus plays an essential role in the regulation of proliferation and protein synthesis [12, 36, 47]. AgNORs are segments of DNA closely associated with nucleoli. They contain coding genes for ribosomal RNA and therefore play a major role in the regulation of protein synthesis [13]. AgNORs allow quantitative inferences regarding the cell’s rate of protein synthesis, and so cellular proliferation activity in general [11, 20, 46]. The individual phases of the proliferation cycle can be described as follows [17]: During the prophase, the nucleolus shrinks; in the meta- and anaphases the nucleolus is no longer visible. The nucleolus again becomes visible in the telophase. Field et al [15] showed that resting lymphocytes (G0 phase) exhibit only sparse AgNORs, while in the G1 phase the number of AgNORs grows [see also 37]; in the first generation this pattern remains unchanged in the S and G2 phases. In the second and third generations of mitosis numerous small granula appear. The AgNOR count has a linear relationship with the percentage of S-phase cells, as determined by DNA flow cytometry [10]. These findings are also supported by comparison of AgNOR counts and BrdU incorporation [33]. Furthermore, like the expression of Ki67 epitopes, AgNOR counts are also variably influenced by the proliferation activities of tumor cells, phases of the cell cycle, and cell differentiation [29, 44]. Today there is no doubt that AgNORs are a useful proliferation marker [1, 24, 33] that can aid, for example, in evaluating the proliferation rate of tumor cells [49]. However, AgNOR counts measure not only proliferation, but also differentiation, state and proliferation rate [14, 28, 40]. A basic criticism of the methods applied here was made by ÖFNER et al. [34, 35], who pointed out that staining artifacts can occur on routine paraffin-embedded tissues. In their opinion the number of AgNORs cannot be exactly determined since they cannot be clearly distinguished. A substantial improvement can be attained after wet autoclave pretreatment. Studies of the skin employing AgNOR have usually focused on skin tumors. Normal skin has only been examined by KANITAKES et al. [21] in 6 cases, revealing 68
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AgNOR counts per nucleus of 1.93 ± 0.14. Normal human mucous membranes have an AgNOR count of 4.51 (± 2,57). To our knowledge normal rat skin has never been studied, nor has rat skin after wounding. Our results demonstrate that on formalin-fixed, paraffin-embedded skin samples AgNOR morphometry can achieve results comparable to those of the BrdU technique. So both methods allow a reliable temporal classification of the skin injury according to survival time and are helpful in assessing wound age and vitality.
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Book reviews
Atlas of Immunology JULIUS M. CRUSE and ROBERT E. LEWIS 450 pages with numerous figures and tables. CRC Press LLC, Boca Raton USA and Springer Verlag GmbH, Heidelberg 1999. ISBN 3-540-64807-0 Immunology has rapidly developed over the last 20 years mainly due to new techniques in biological sciences which allowed a deeper insight into the structure and function of molecules and cells involved in immunological reactions in health and disease. This Atlas of Immunology is a compilation of the present knowledge in this field covering every important concept put torward over the last decades. The introductory History of Immunology describes briefly the curricula vitae of scientists who made the most important contributions to our understanding of immunology. This chapter gives the reader an idea of the long lasting process and the many steps in the accumulation of our present knowledge in immunology ranging from resistance against infectious diseases to MHC restriction. The first part of this atlas was devoted to the physiological processes of the immune response in human beings. Molecules, cells and tissues of the immune reactions were considered as well as the processes connected with antigen presentation, immunoglobulin synthesis and antigen-antibody interactions. Separate chapters give a solid overview of cytokines, the complement system, the types of hypersensitivity, immunoregulation, and immunohematology. In the second part the authors focused on immunological diseases and immunopathology. Appropriate emphasis was placed on congenital and acquired immunodeficiencies, in particular on AIDS. Immunosuppression, transplantation im-
munology, tumor immunology, and immunity against microorganisms were outlined in the following chapters. Immunologic methods were dealt with in the final chapter in which the basic techniques of measurement of antigens and antibodies were described. The atlas is an appealing reference book, comprehensive as can be expected from this type of a book. More than 1,000 illustrations illuminate the easy-to-read text. Unfortunately, all figures are given in black and white so that the information content of the numerous histological sections, organ preparations, and molecular structures of proteins is markedly restricted. On the other hand, the schematic drawings of molecules, cells, and reactions are highly informative. Particularly noteworthy is the basic concept of the textual description. All paragraphs start with a key word printed in bold letters so that the main topics are presented as in a dictionary. This might be useful for students and those readers who are not familiar with the principles of immunology. The disadvantage of such an approach is necessarily a lack of systematic presentation of general concepts. Thus, it might be difficult to gain an overview of the general ideas in the respective field put forward at present. Nevertheless, the authors did a good job in presenting this atlas so that it can be recommended to students, clinicians and scientists as a valuable source of information. R. DARGEL, Jena
Histological Typing of Lung and Pleural Tumours W. D. TRAVIS, T. V. COLBY, B. CORRIN, Y. SHIMOSATO, E. BRAMBILLA in collaboration with L. H. SOBIN and pathologists from 14 countries. 3rd ed. 162 pages with 150 figures and 7 tables. Springer Verlag, Berlin Heidelberg New York 1999. Softcover DM 129.00; öS 942.00; sFr 117.50; GBP 49.50; US $ 81.00. ISBN 3-540-65219-1 The first and the second editions of “Histological Typing of Lung and Pleural Tumours” were published in 1967 and 1981. A revised third edition has become necessary because over the last 10 to 15 years considerable progress has been made in understanding certain lung tumors. This gain in knowledge has resulted in some major and minor modifications of the classification. The concept of neuroendocrine tumors has been refined with inclusion of large cell neuroendocrine carcinoma and modification of the distinction between typical and atypical carcinoids. The histological heterogeneity of lung tumors is duly considered in the revised classification. More precise guidelines for grading squamous dysplasia and carcinoma in situ are provided and two categories of preinvasive lesions have been added: atypical adenomatous hyperplasia and diffuse idiopathic pulmonary neuroendocrine cell hyperplasia. In the introduction general principles of lung tumor typing and grading as well as molecular findings are explained concluding 70
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with the true neoplastic nature of chondroid hamartoma and sclerosing hemangioma. The definitions of tumor types are based primarily on light microscopic findings in surgical, fine-needle and autopsy material; cytologic specimens were dispensed with. The definitions and explanatory notes are concise and precise, the text is supplemented by excellent colored histological illustrations. As generally true for the WHO series “International Histological Classification of Tumours” the book is not intended to be a textbook, but is designed to provide uniform terminology as the basis for improved international communications. For this reason the reader will find the morphological codes for the International Classification of Diseases for Oncology (ICD-0) and the Systematized Nomenclature of Medicine (SNOMED). The book should not be lacking in the library of pathologists who are confronted with the histological examination of lung and pleural tumors. D. KATENKAMP, Jena