Histochem Cell Biol (2009) 131:605–614 DOI 10.1007/s00418-009-0569-1
ORIGINAL PAPER
The fate of chondrocytes during ageing of human thyroid cartilage Horst Claassen · Martin Schicht · Saadettin Sel · Jochen Werner · Friedrich Paulsen
Accepted: 29 January 2009 / Published online: 20 February 2009 © Springer-Verlag 2009
Abstract Human laryngeal cartilages, especially thyroid cartilage, exhibit gender-speciWc ageing. In contrast to male thyroid cartilages, the ventral half of the female thyroid cartilage plate remains unmineralized until advanced age. In cartilage specimens from laryngectomies and autopsies, apoptosis was studied immunohistochemically and the oxidative mitochondrial enzyme nicotinamide adenine dinucleotide hydride tetrazolium reductase (NADH-TR) was localized histochemically. In addition, very fresh specimens from laryngectomies were Wxed under addition of ruthenium hexamine trichloride or tannin to Wxation solution to study cell organelles of chondrocytes by electron microscopic methods. In general, apoptotic chondrocytes decreased in thyroid cartilages of both genders, especially after the second decade. In the age group 41–60 years, thyroid cartilage from male specimens revealed a signiWcantly higher percentage of apoptotic cells than did thyroid
cartilage from women (P = 0.004), whereas in the age groups 0–20 years and 61–79 years no statistically signiWcant gender diVerence was determined. In general, thyroid cartilage from women contained more living chondrocytes into advanced age than men. Chondrocytes adjacent to mineralized cartilage were partly positive for apoptosis and NADH-TR and partly negative. Apoptotic chondrocytes often were localized in areas of asbestoid Wbres where vascularization and mineralization took place Wrst. Electron microscopy revealed remnants of chondrocytes in asbestoid Wbres. Taken together, it can be assumed that some chondrocytes in thyroid cartilage die by apoptosis and that these chondrocytes are characterized by absent reactivity for the mitochondrial enzyme NADH-TR. A possible inXuence of sexual hormones on apoptotic death of thyroid cartilage cells requires further elucidation. Keywords Laryngeal cartilages · Ageing processes · Apoptosis · NADH-TR · Enzymes
H. Claassen (&) · M. Schicht · F. Paulsen Institute of Anatomy and Cell Biology, Martin Luther University of Halle-Wittenberg, Große Steinstraße 52, 06097 Halle (Saale), Germany e-mail:
[email protected] H. Claassen Institute of Anatomy, Christian Albrechts University of Kiel, Olshausenstraße 40, 24098 Kiel, Germany S. Sel Department of Ophthalmology, Martin Luther University of Halle-Wittenberg, Ernst-Grube-Straße 40, 06120 Halle (Saale), Germany J. Werner Department of Otorhinolaryngology, Head and Neck Surgery, Philipps University of Marburg, Deutschhausstraße 3, 35037 Marburg, Germany
Introduction Some parts of the human body, for example laryngeal cartilages, are well suited for study of ageing processes. The human laryngeal skeleton is characterized by gender-speciWc, progressive stiVening caused by cartilage mineralization with subsequent ossiWcation. In male thyroid cartilage, the mineralization and ossiWcation starting around puberty are nearly complete in advanced age. In females, by contrast, the ventral half of thyroid cartilage remains unmineralized and does not ossify. The ossiWcation of the laryngeal skeleton has consequences in terms of voice quality (Habermann 1996), but cannot be classiWed as pathological. The processes leading to ossiWcation are similar to those in
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enchondral ossiWcation of growth plates. Interestingly, the fate of hypertrophic chondrocytes can be studied much better in thyroid cartilage than in growth plates since the remnants of these cells are degraded much more slowly due the slow progress of vascularization in laryngeal cartilages. There is a growing body of evidence that death of chondrocytes, and chondrocyte apoptosis in particular, play an important role in cartilage development and ageing (Horton et al. 1998). Apoptosis is held to be the mechanism responsible for the death of chondrocytes during endochondral bone formation and it is also claimed that apoptosis of the chondrocytes is age-related (Ploumis et al. 2004). Many of the maturing hypertrophic chondrocytes in growth plates undergo programmed cell death with plasma membrane breaks, hydropic swelling and cell fragmentation, as demonstrated by immunohistochemical and electron microscopic studies (Hatori et al. 1995; Adams and Shapiro 2002; Garimella et al. 2004; Shapiro et al. 2005). Furthermore, the endochondral ossiWcation process mediated by chondrocyte apoptosis may include intracellular calcium accumulation and release (Gibson 1998), and cell death caused by apoptosis is often mediated by endocrinological inXuences (Wyllie 1992). Even though cartilage is mainly anaerobic, oxidative enzymes as nicotinamide adenine dinucleotide hydride tetrazolium reductase (NADH-TR), succinate dehydrogenase (SDH), malate dehydrogenase (MDH) and cytochrome oxidase are involved in its metabolic pathways (Person and Fine 1959). Especially cartilage regions undergoing mineralization are equipped with all the enzymes required for an active aerobic metabolism (Whitehead and Weidmann 1959). A dramatic increase of reduced pyridine nucleotide (NADH) and an increased NADH/NAD ratio was found in the hypertrophic zone of growth plates while NADH was decreased in the proliferating and calcifying cartilage (Shapiro et al. 1982; Kakuta et al. 1986). The Wnding that NADH is accumulated in the tissue zone associated with earliest deposition of bone mineral supports the hypothesis that a change in the redox state initiates tissue mineralization (Shapiro et al. 1982). In broiler chickens, MDH was present in normal physeal cartilage but not in dyschondroplastic cartilage, reXecting a reduction in the activity of energetic metabolism of the latter (Zhang et al. 1997). We recently showed that the ventral half of male thyroid cartilages contained fewer chondrocytes with a territorial rim of glycosaminoglycans, probably preventing mineralization, compared with the ventral half of age-matched female thyroid cartilage (Claassen and Werner 2004). The reduced number of chondrocytes is likely related to the death of cells. Using immunohistochemical and electron microscopic methods, we tried to answer the question of whether the death of thyroid cartilage cells is caused by apoptosis or necrosis. Additionally, we localized NADH-TR
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Histochem Cell Biol (2009) 131:605–614
Fig. 1 Left side of a thyroid cartilage. The diagram shows the sections into which each cartilage plate was dissected for investigation of unmineralized and mineralized cartilage areas
as the main oxidative mitochondrial enzyme histochemically in thyroid cartilages of diVerent age and sex. Our aim was to elucidate the fate of chondrocytes, especially hypertrophic chondrocytes, during ageing of thyroid cartilage.
Materials and methods Investigations involving bioptic material from laryngectomies were only performed if the patients had given their informed consent. The Ethics Commission, Kiel, approved the study. Sources of material and tissue preparation The larynges for immunohistochemical staining of apoptotic chondrocytes and enzyme histochemical localization of the oxidative mitochondrial enzyme NADH-TR were obtained from autopsies and laryngectomies as follows: 1 male foetus (23 weeks of gestation), 4 male children (5 months and 3, 6, and 8 years of age), 1 female child (2 months of age), 2 male adolescents (17 and 20 years of age), 1 female adolescent (16 years of age), 14 male adults (30, 49, 50, 50, 52, 52, 57, 58, 59, 71, 73, 74, 79 and 82 years of age) and 8 female adults (21, 31, 34, 36, 44, 53, 57 and 73 years of age). In all cases, thyroid cartilage was removed from the bisected larynges and X-rayed to determine the extent of cartilage mineralization and ossiWcation. In radiographs of thyroid cartilage, spotted and opaque cartilage mineralization was clearly distinguishable from ossiWcation characterized by a Wligree trabecular architecture. Finally, each cartilage specimen was dissected into eight deWned transverse segments (see Fig. 1), which were frozen in liquid nitrogen or embedded in paraYn.
Histochem Cell Biol (2009) 131:605–614
To achieve adequate tissue conservation, the larynges for electron microscopic investigations were obtained from laryngectomies as follows: 7 male adults (49, 52, 57, 58, 71, 73, and 82 years of age) and one female adult (36 years of age). Sections of 1–2 mm in size were dissected out of unmineralized and mineralized or ossiWed areas of thyroid cartilages and Wxed in two diVerent solutions within 2 h postoperatively. Solution 1 containing ruthenium-hexamine-trichloride (RHT) was used for the study of cell morphology. Specimens were Wxed in 2% glutaraldehyde in 0.05 M sodium cacodylate buVer, pH 7.4, with addition of 0.7% RHT, osmolarity adjusted to 330 mosmol/l with NaCl (Hunziker et al. 1982). Fixation was continued for 3 to 5 to 8 h depending on specimen size. The specimens were washed 3 times in 0.1 M sodium cacodylate buVer, pH 7.4, 300 mosmol/l, for 20 min each. PostWxation in 1% osmium tetroxide in 0.1 M sodium cacodylate buVer, pH 7.4, with addition of 0.7% RHT, osmolarity adjusted to 330 mosmol/ l with NaCl, lasted from 3 to 5 to 8 h. Solution 2 containing tannin was used for the study of membrane-surrounded cell organelles and collagen Wbrils of extracellular matrix. Specimens were Wxed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buVer, pH 7.4, with addition of 2% tannin, osmolarity adjusted to 280 mosmol/l with NaCl, for 12 h (Eggli et al. 1985). Three washing procedures were performed in 0.1 M sodium cacodylate buVer, pH 7.4, 280 mosmol, for 20 min each. PostWxation in 1% osmium tetroxide in 0.1 M sodium cacodylate buVer, pH 7.4, osmolarity adjusted to 280 mosmol/l, continued for 12 h. PostWxation was washed out in the respective washing buVers, 3 times for 20 min each, and the specimens were transferred to diVerent concentrations of ethanol solution (70, 80, 90, 96, 100%) 4 times for 20 min each. After additional dehydration in propylenoxide 4 times for 20 min each, specimens were transferred to Spurr:propylenoxide 1:1 for 12 h, Spurr:propylenoxide 2:1 for 12 h, and freshly mixed pure Spurr for 24 h. Specimens were embedded in freshly mixed pure Spurr in a plastic vessel and polymerization was started at 50°C for 2 h in a vacuum exsiccator. Polymerization was continued at 70°C for 8 h and afterwards at 67°C for an additional 12 h. Semi-thin and ultra-thin sections were cut on a Reichert Jung Ultracut E. Ultra-thin sections were placed on coated grids (Veco 841180 R150A). Immunohistochemistry for detection of apoptotic chondrocytes The detection of apoptotic chondrocytes was performed according to the protocol of the apoptosis detection kit (Oncor S 7100). BrieXy, freshly cut cryostat sections of
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thyroid cartilage were Wxed with cold acetone (¡10 to ¡25° C) and rinsed in PBS buVer, pH 7.4, for 5 min each. Endogenous peroxidases were blocked with 5% H2O2 methanol for 5 min. After additional washing in PBS for 3 £ 5 min, sections were incubated with the enzyme terminal deoxynucleotidyl transferase (TdT) and digoxigeninlabelled deoxyribonucleotide adenosine triphosphate (dATP) as substrate. By this procedure, free 3⬘-OH termini of double and single DNA strand breaks were marked. Specimens of growth plate and thymus were used as control because apoptosis in these tissues occurs reliably. A section covered with PBS buVer instead of the enzyme TdT served as the negative control. The immunoreaction was developed with the peroxidase-anti-peroxidase (PAP) method. Sections were counterstained with methyl green and covered with Aquatex. Enzyme histochemistry for NADH-TR As the key enzyme of the oxidative metabolism, NADHTR was localized in cryostat sections of thyroid cartilage (Lojda et al. 1976). Nicotinamide adenine dinucleotide served as the substrate. The reaction was developed by the tetrazolium salt technique. Cryostat sections of the cat soleus muscle, which contains a high number of oxidative NADH-TR-positive muscle Wbres (Claassen and Werner 1992), served as the positive control. Transmission electron microscopy Ultra-thin specimen sections Wxed by addition of RHT and tannin were contrasted using uranyl acetate and lead citrate and investigated in a Zeiss EM 900. Documentation was done on Agfa Scientia EM Wlms. Statistical analysis Apoptotic chondrocytes were counted and computed as a percentage of all chondrocytes in Wve neighbouring Welds of vision (0.325 mm2). Since thyroid cartilage mineralizes by enchondral ossiWcation, we generally restricted counting to cartilage segments 2, 4 and 5 (see Fig. 1), which contained parts of unmineralized cartilage until advanced age. The percentage of apoptotic cells was tested for statistical signiWcance between age-matched specimens of thyroid cartilages from men and women. Data were analyzed using the Mann–Whitney U test. For this purpose, we deWned four age groups: (1) 0–20 years, (2) 21–40 years, (3) 41–60 years, (4) 61–79 years for male specimens and 61–73 years for female specimens. A P value of
