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Structure and composition of arytenoid cartilage of the bullfrog (Lithobates catesbeianus) during maturation and aging
Micron 77 (2015) 16–24
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Structure and composition of arytenoid cartilage of the bullfrog (Lithobates catesbeianus) during maturation and aging Priscila Eliane dos Santos Laureano a , Kris Daiana Silva Oliveira a , Andrea Aparecida de Aro b , Laurecir Gomes b , Edson Rosa Pimentel b , Marcelo Augusto Marretto Esquisatto a,∗ a
Programa de Pós-graduac¸ão em Ciências Biomédicas, Centro Universitário Hermínio Ometto, Av. Dr. Maximiliano Baruto, 500 Jd. Universitário, 13607-339, Araras, SP, Brazil b Departamento de Biologia Estrutural e Funcional, Instituto de Biologia, Universidade Estadual de Campinas, Rua Charles Darwin, s/n, CxP 6109, 13083-863, Campinas, SP, Brazil
a r t i c l e
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Article history: Received 13 April 2014 Received in revised form 24 May 2015 Accepted 28 May 2015 Available online 30 May 2015 Keywords: Cartilage Structure Larynx Frog Aging
a b s t r a c t The aging process induces progressive and irreversible changes in the structural and functional organization of animals. The objective of this study was to evaluate the effects of aging on the structure and composition of the extracellular matrix of the arytenoid cartilage found in the larynx of male bullfrogs (Lithobates catesbeianus) kept in captivity for commercial purposes. Animals at 7, 180 and 1080 days post-metamorphosis (n = 10/age) were euthanized and the cartilage was removed and processed for structural and biochemical analysis. For the structural analyses, cartilage sections were stained with picrosirius, toluidine blue, Weigert’s resorcin-fuchsin and Von Kossa stain. The sections were also submitted to immunohistochemistry for detection of collagen types I and II. Other samples were processed for the ultrastructural and cytochemical analysis of proteoglycans. Histological sections were used to chondrocyte count. The number of positive stainings for proteoglycans was quantified by ultrastructural analysis. For quantification and analysis of glycosaminoglycans were used the dimethyl methylene blue and agarose gel electrophoresis methods. The chloramine T method was used for hydroxyproline quantification. At 7 days, basophilia was observed in the pericellular and territorial matrix, which decreased in the latter over the period studied. Collagen fibers were arranged perpendicular to the major axis of the cartilaginous plate and were thicker in older animals. Few calcification areas were observed at the periphery of the cartilage specimens in 1080-day-old animals. Type II collagen was present throughout the stroma at the different ages. Elastic fibers were found in the stroma and perichondrium and increased with age in the two regions. Proteoglycan staining significantly increased from 7 to 180 days and reduced at 1080 days. The amount of total glycosaminoglycans was higher in 180-day-old animals compared to the other ages, with marked presence of chondroitin- and dermatan-sulfate especially in this age. The content of hydroxyproline, which infers the total collagen concentration, was higher in 1080-day-old animals compared to the other ages. The results demonstrated the elastic nature of the arytenoid cartilage of L. catesbeianus and the occurrence of age-related changes in the structural organization and composition of the extracellular matrix. These changes may contribute to alter the function of the larynx in the animal during aging. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction The aging process is affected by genetic, dietary, environmental and social factors. Morphological and physiological differences among the species of animals and the consequent changes in their
∗ Corresponding author. Tel.: +55 19 3543 1440; fax: +55 19 3543 1439. E-mail address: [email protected] (M.A.M. Esquisatto). http://dx.doi.org/10.1016/j.micron.2015.05.018 0968-4328/© 2015 Elsevier Ltd. All rights reserved.
life’s cycles during aging alter the structural and functional organization of cells and tissues and affect their homeostasis (Kirkwood, 2002, 2005; Teixeira and Guariento, 2010). In connective tissues, the age’s modifications are mainly related to alterations in the macromolecules that comprise the extracellular matrix (ECM) (Li et al., 2013). In cartilaginous tissue, the organization and relationship between ECM components become compromised with aging. Chondrocytes are widely distributed in tissues of young animals and exhibit great synthetic capacity,
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guaranteeing the functional competence of the ECM (Horton et al., 2006). Structurally, the ECM that surrounds the chondrocytes can be divided into a pericellular and a territorial matrix. The pericellular matrix, which is closer to the chondrocytes, is rich in proteoglycans (PGs) and it is in direct contact with the cells under normal physiological conditions. In contrast, the territorial matrix contains large amounts of fibrillar components such as type II collagen fibrils (Muir, 1995; Poole et al., 2001). In addition to these elements, the cartilaginous matrix contains elastic fibers (Kielty et al., 2002). In the respiratory system of most terrestrial vertebrates, cartilaginous tissue is found in the wall of the larynx, trachea, bronchi, and bronchioles. In the case of anuran amphibians, such as frogs, cartilage is only found in the larynx, which is situated between the oral cavity and the trachea. The larynx consists of two pairs of cartilaginous rings, divided into an anterior (arytenoid cartilage) and posterior pair (cricoid cartilage). The arytenoid cartilage forms the wall of the valve that permits the passage of air from the oral cavity to the trachea and vice-versa. On the other hand, the cricoid cartilage provides support to the membrane structures that form the vocal cords (Porter, 1972; Romer, 1985). Like all tissues, the cartilage found in the respiratory system also undergoes changes in the organization and composition of the ECM with aging. Age-related changes in the biosynthesis of glycosaminoglycans (GAGs) in the tracheal cartilage of mammals has been observed in adults and became more pronounced with advancing age (Lotz and Loeser, 2012), as well as a decrease in the number of PG sulfations. In addition, several authors reported important age-related structural changes in cartilage PGs in this tissue (Mallinger and Stockinger, 1987; Lotz and Loeser, 2012). Degenerative alterations in tracheal, thyroid and epiglottic cartilage have been described in detail in older adults, with focus on the process of calcification in these structures (Kusafuka et al., 2001; Cruz et al., 2003; Kano et al., 2005). Because of the amount and importance of the ECM in the organization of visceral cartilage isolated from the respiratory system of vertebrates (Vanperperstraete, 1973; Reid, 1976), the arytenoid ring become interesting for the study of the effect of aging on the cartilage matrix. Thus, the aim of the present study was to describe the changes that occur in the structure and composition of the ECM of the arytenoid cartilage found in the larynx of males in post-metamorphosis bullfrogs (Lithobates catesbeianus), during the process of maturation and aging.
2. Materials and methods 2.1. Animals and sample collection Thirty male bullfrogs (L. catesbeianus) were obtained from a commercial breeder in Araras, São Paulo, Brazil. The arytenoid cartilage found in the larynx of animals at 7, 180 and 1080 days after the end of metamorphosis (n = 10/age) was analyzed. These ages were chosen according to the different phases of sexual maturation of the animal in captivity (immaturity, maturity, and decline in fertility) (Porter, 1972; Romer, 1985). The end of metamorphosis was defined as the complete disappearance of the tail. In bullfrogs, as in most other anurans, only males ever give an advertisement call. In relation to this, there are significant structural differences between male and female larynges in many anuran amphibian species. In this context, the skeletal cartilage of larynx presents significant alterations during sexual maturation and aging (Boyd et al., 1999). Samples were collected from male animals at each age. For cartilage removal, the animals were kept in cold water at 4 ◦ C for 1 h and then sacrificed by spinal cord transection. Samples destined for biochemical analysis (5/age) were frozen at −20 ◦ C or immediately
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used for the tests. Samples destined for structural analysis (5/age) were fixed according to the protocol established for each technique. The study protocol was approved by the Ethics Committee on Animal Use of the institution (FHO/UNIARARAS, CONCEA/MCTi/Brazil) (Permit No. 081/2005). 2.2. Morphological analysis 2.2.1. Processing for histological and histochemical analysis After dissection, the cartilage samples were fixed in 10% formalin in Millonig buffer, pH 7.4, for 24 h at room temperature. Next, the specimens were washed in buffer and processed for embedding in Paraplast® (Merck, Darmstadt-Germany) using standard procedures. Longitudinal sections (6-m) were submitted to the following histochemical techniques: picrosirius-hematoxylin for visualization of collagen fibers under polarized light; toluidine blue in McIlvaine buffer, pH 4.0, for the evaluation of tissue basophilia and chondrocyte count; Weigert’s resorcin-fuchsin method for analysis of the elastic fiber system, and Von Kossa staining for the detection of calcium deposits. For observations in polarization microscopy, the sections were positioned so that the longest axis of the cartilage was at 45◦ in relation to the polarized light plane, as described by Vidal (1986). The preparations were examined and documented with a Leica DM-2000 photomicroscope. 2.2.2. Morphometric analysis The number of chondrocytes (No. in 104 m2 ) was quantified using a 40× objective in five fields of each of three sections obtained from the arytenoid cartilage of each animal at each age. All images were captured and digitized with a Leica DM-2000 photomicroscope. The measurements and counts were performed on the digitized images using the Sigma Scan Pro 6.0® software. 2.2.3. Immunohistochemical analysis Three 6-m sections obtained from each animal at each age were transferred to cuvettes and incubated with a solution of 0.01 M sodium citrate buffer, pH 6.0, and 0.05% Tween-20 (v/v) in a microwave (high power, 5 min + 5 min), alternating with an ice bath (5 min + 5 min). The sections were then immediately cooled in a water bath for 30 min at room temperature. Next, the slides were washed three times in PBS (0.01 M, pH 7.4). The sections were immersed in methanol containing 0.3% (v/v) hydrogen peroxide for 30 min to block endogenous peroxidase activity (∼5 mL per assay) and washed three times in PBS (0.01 M, pH 7.4). For the blockade of nonspecific sites (0.5 mL per slide), the sections were immersed in fetal bovine serum for 20 min (∼5 mL per assay), followed by three washes in PBS (0.01 M, pH 7.4). The slides were treated with the primary antibody diluted in 0.01 M PBS plus 1% bovine serum albumin (BSA) for 18 h at 4 ◦ C. The following primary antibodies were used: rabbit polyclonal anticollagen I (diluted 1:100, Rockland Gilbertsville, PA, USA), mixing 2 L of the stock solution + 98 L 0.01 M PBS plus 1% BSA (∼2.5 mL per assay); rabbit polyclonal anti- collagen II (diluted 1:100, Rockland, USA), mixing 2 L of the stock solution + 98 L 0.01 M PBS plus 1% BSA (∼2.5 mL per assay). The slides were then washed three times in PBS (0.01 M, pH 7.4) and incubated with the secondary antibody diluted in 0.01 M PBS plus 1% BSA for 30 min at room temperature. Sections in which the primary antibodies were omitted served as negative control. Peroxidase-conjugated anti-rabbit IgG was used as the secondary antibody (diluted 1:500, Rockland, USA), mixing 2 L of the stock solution + 498 L 0.01 M PBS plus 1% BSA (∼3 mL per assay). For the detection of peroxidase activity, the slides were washed in 0.05 M Tris–HCl, pH 7.4, and incubated with a solution of 0.05% (w/v) diaminobenzidine in 0.05 M Tris–HCl, pH 7.4, containing 0.03% (v/v) hydrogen peroxide for 5 min at room
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temperature. The reaction was stopped by washing the slides in distilled water. 2.2.4. Processing for ultrastructural and cytochemical analysis The arytenoid cartilage samples obtained at different ages were fixed in a solution of 2% glutaraldehyde and 0.1% tannic acid dissolved in 0.1 M sodium cacodylate buffer, pH 7.3, for 2 h at room temperature. Next, the material was washed in buffer and postfixed in 1% osmium tetroxide for 1 h at 4 ◦ C. After this step, the fragments were washed in saline-glucose and treated with 1% uranyl acetate for 18 h at 4 ◦ C, followed by washing in saline-glucose and dehydration. Fragments were also treated with cuprolinic blue for the ultrastructural detection and counting of PG staining (Scott et al., 1989). After fixation in the different procedures, all specimens were dehydrated in an increasing ethanol series and double passage through propylene oxide. Next, the specimens were embedded in mixtures of propylene oxide/Epon resin (1:1, 1:2 and pure) and, finally, placed in plastic molds in an oven. Sections were cut with a glass and diamond knife in an Ultracut UCT ultramicrotome (Leica) and counterstained with 2% uranyl acetate in water and 0.2% lead citrate in 0.1 N NaOH. The sections were examined under an LEO 906 transmission electron microscope (Leica, LME, UNICAMP, Brazil) and documented. The images of cross-sections of the cartilage samples obtained at different ages were digitized and analyzed with the Sigma Scan Pro 6.0® software for determination of the number of PG stainings (in 25 m2 ). 2.3. Biochemical analysis 2.3.1. Quantification and analysis of GAGs For the removal of GAGs, the fragments of territorial matrix of arytenoid cartilage were chopped with a scalpel blade and digested with trypsin for 24 h at 37 ◦ C. Undigested material was removed by centrifugation at 1000 × g for 10 min and the soluble material in the supernatant was precipitated with two volumes of absolute ethanol for 18 h at 4 ◦ C. The precipitate was resuspended in water and quantified by the dimethyl methylene blue method (Farndale et al., 1986). The resuspended samples were also analyzed by electrophoresis on 0.5% agarose gel in 0.05 M propylenediamine at 0.1 mA for 45 min. The agarose gels were fixed in Cetavlon and stained with toluidine blue. The gels were washed with a solution of 50% ethanol and 1% acetic acid for observation of the bands. GAGs were identified by comparison with the standard containing chondroitin, dermatan and heparan sulfate (Sigma, São Paulo, Brazil). 2.3.2. Quantification of hydroxyproline Cartilage fragments were immersed in acetone for 48 h and then in chloroform:ethanol (2:1) for an additional 48 h. The fragments
were hydrolyzed in 6N HCl (1 mL per 10 mg tissue) for 16 h at 110 ◦ C. The hydrolysate was neutralized with 6N NaOH and hydroxyproline was quantified using the chloramine T method, as described by Stegemann and Stalder (1967), with some modifications. Concentrations of hydroxyproline of 0.2–6 mg/mL were used for construction of the standard curve. 2.4. Statistical analysis The mean quantitative results were entered into Excel spreadsheets program (2007 version – Microsoft® ) and analyzed by ANOVA and the Tukey post-test. A level of significance of 5% (p < 0.05) was adopted. 3. Results 3.1. Morphological analysis Analysis of toluidine blue-stained sections of arytenoid cartilage obtained at different ages (Fig. 1A–C) showed a predominance of spherical cells with abundant cytoplasm and round nuclei which were surrounded by a basophilic ECM. Two distinct regions were noted in the latter, one close to the cells (pericellular) and the other among groups of cells (territorial). The cells were distributed throughout the cartilaginous structure and were arranged perpendicularly to its major axis. Basophilia was more homogenous in animals at 7 days of age (Fig. 1A). In contrast, at 180 days of age basophilia was found preferentially in the pericellular region and in areas near the perichondrium (Fig. 1B). In older animals (Fig. 1C), basophilia was reduced in the tissue and predominated in the pericellular region. Quantitative analysis of the number of chondrocytes revealed a significant difference between ages. The number of these cells was significantly higher in 7-day-old animals compared to 180and 1080-day-old animals. However, there was a significant difference between the last two groups, with the observation of a smaller number of chondrocytes in older animals (Table 1). Collagen fibers predominated in the territorial matrix of arytenoid cartilage and exhibited a complex organization at the different ages. These fibers were arranged perpendicular to the major axis of the structure and parallel at the margins near the perichondrium. The fibrillar elements were arranged in the same way as the cell groups and the bundles parallel to the perichondrium were thicker than on the outer surface of the structure (Fig. 2A–C). Analysis of picrosirius-stained sections under polarized light (Fig. 2D–F) showed thick collagen fibers in the territorial matrix in the 1080-days-old animals. In addition, a peculiar arrangement of the birefringent fibers involving isogenous groups
Fig. 1. Cross-sections of arytenoid cartilage obtained from bullfrogs at 7 (A), 180 (B) and 1080 (C) days post-metamorphosis. The sections were stained with toluidine blue. Pericellular matrix (arrowhead); territorial matrix (arrow); perichondrium (P). Bar: 50 m.
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Fig. 2. Cross-sections of arytenoid cartilage obtained from bullfrogs at 7 (A and D), 180 (B and E) and 1080 (C and F) days post-metamorphosis. The sections were stained with picrosirius-hematoxylin for analysis of collagen fiber arrangement and distribution. In A–C, the sections were documented under bright-field illumination. In D–F, the sections were photographed under polarized light. Territorial matrix (*); collagen fibers (arrow); perichondrium (P). Bar: 50 m. Table 1 Morphometric and biochemical parameters of the arytenoid cartilage of bullfrogs at different post-metamorphosis ages. Age (days) Parameter
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Number of chondrocytes (No. in 104 m2 ) PGs markings (No. in 25 m2 ) GAGs content (mg/g fresh tissue) Hydroxyproline content(g/mg dry tissue)
141.5 ± 22.4* 18.2 ± 4.1 31.8 ± 5.7a 64.6 ± 7.2
p = 0,044
180
1080
89.4 ± 14.8 37.8 ± 6.3* p = 0.047 44.3 ± 4.8* p = 0.046 88.3 ± 9.2
45.7 ± 14.8 22.4 ± 3.7a 34.2 ± 3.6 116.3 ± 10.2*
p = 0.043
Values are the mean and standard deviation obtained from each age (n = 5/age) and were compared by ANOVA and the Tukey post-test (p < 0.05). * Significant difference (p < 0.05). a Values do not differ between 7- and 1080- day old animals.
of cells could be noted at all ages. This arrangement seemed to be stable during aging. Weigert’s resorcin-fuchsin staining was used for the detection and analysis of the elastic fibers at the different ages (Fig. 3A–C). Elastic fibers were detected in the territorial and pericellular matrix of arytenoid cartilage and in the region of the perichondrium. No difference in the distribution of these elements was observed between the different cartilage regions. However, the detection of elastic fibers was higher in 1080-day-old animals, but no difference
was observed between younger animals. In the perichondrium, the distribution of these fibers accompanied the data observed for collagen fibers, with a predominance of these elements on the outer surface of cartilage. The arytenoid cartilage samples obtained at the different ages were treated by the Von Kossa method for the detection of calcium deposits. Calcification points were detected in the territorial matrix only in 1080-day-old animals (Fig. 4A–C).
Fig. 3. Cross-sections of arytenoid cartilage obtained from bullfrogs at 7 (A), 180 (B) and 1080 (C) days post-metamorphosis. The sections were stained with Weigert’s resorcin-fuchsin for the detection of elastic system fibers. Elastic fibers (arrowhead); perichondrium (P). Bar: 50 m.
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Fig. 4. Cross-sections of arytenoid cartilage obtained from bullfrogs at 7 (A), 180 (B) and 1080 (C) days post-metamorphosis. The sections were treated by the Von Kossa method for the detection of calcium deposits. Positive reaction (arrowhead); perichondrium (P). Bar: 50 m.
Immunohistochemical analysis was performed on arytenoid cartilage samples obtained from animals at 7, 180 and 1080 days of age. Negative staining for type I collagen was observed in the cartilage matrix at all ages (Fig. 5A–C). In addition, staining for type II collagen was detected in the territorial and pericellular matrix and its intensity was similar at all ages (Fig. 5D–F). Ultrastructural analysis showed similar cellular characteristics of arytenoid cartilage at the ages studied (Fig. 6A–C). The cells had a round shape and contained a spherical nucleus, with predominance of euchromatin. The cytoplasm exhibited large amounts of rough endoplasmic reticulum, several secretory vesicles, and a large number of cytoplasmic extensions. However, few vesicles and less developed rough endoplasmic reticulum were observed in older animals. The pericellular matrix
exhibits fine granular and fibrillar elements distributed in all directions. No difference in the region between the ages was observed. Analysis of cuprolinic blue-stained sections (Fig. 7A–C) showed the presence of a large number of rod-shaped PG precipitates. In animals at 7 days of age, these molecules had a similar size and were uniformly distributed throughout the stroma. The number of staining increased in 180-day-old animals with predominance in the pericellular matrix. However, in the different regions of the cartilaginous matrix it was reduced in animals at 1080 days of age. The quantity of PG staining obtained by ultrastructural analysis is summarized in Table 1. Significantly higher values were observed in animals with 180 days of age, whereas no significant difference was detected between 7- and 1080-day-old animals.
Fig. 5. Immunohistochemical detection of collagen type I (A–C) and type II (D–F) in sections of arytenoid cartilage obtained from bullfrogs at 7 (A and D), 180 (B and E), and 1080 (C and F) days post-metamorphosis. Observe the negative staining for type I collagen and the positive staining for type II collagen (arrowhead) in the territorial and pericellular matrix at all ages. Chondrocytes (*). Bars: 75 m.
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Fig. 6. Transmission electron photomicrographs of bullfrog arytenoid cartilage at 7 (A), 180 (B) and 1080 (C) days post-metamorphosis. The samples were treated by a conventional method for the analysis of cells and fibrillar elements in the pericellular matrix. Cell projections (arrow); Endoplasmic reticulum (arrowhead); vesicles (*). Bars: 1 m.
Fig. 7. Transmission electron photomicrographs of bullfrog arytenoid cartilage at 7 (A), 180 (B) and 1080 (C) days post-metamorphosis. The samples were stained with cuprolinic blue for ultrastructural staining of proteoglycans. Chondrocyte (Ch); territorial matrix (*); positive staining in the pericellular matrix (arrowhead). Bars: A = 0.5 m; B and C = 0.25 m.
4. Discussion
Fig. 8. Agarose gel electrophoresis for the analysis of glycosaminoglycans. The standard (S) contains chondroitin (CS), dermatan (DS) and heparan sulfate (HS). Note that all samples contain CS (arrow) and DS (arrowhead), with a more prominent CS band in animals at 180 days of age.
3.2. Biochemical analysis The total amount of sulfated GAGs and hydroxyproline in the ECM of arytenoid cartilage at each age are shown in Table 1. The amount of sulfated GAGs was higher in 180-day-old animals compared to the other ages. No difference was observed between animals at 7 and 1080 days of age. Analysis of agarose gels (Fig. 8) showed the marked presence of chondroitin sulfate and, to a lesser extent, of dermatan sulfate at all ages, especially in 180-day-old animals. Hydroxyproline content was higher in 1080-day-old animals compared to the other ages. However, a significant increase in the amount of this amino acid was observed in animals from 7 to 180 days of age.
Age-associated alterations in cartilaginous tissue are intimately related to changes in ECM macromolecules. Qualitative and quantitative changes in PGs and collagen fibers induce alterations in the physicochemical properties of the tissue that consequently affect the physiology of the organ (Sokoloff, 1983, 1987; Buckwalter et al., 1993). One of the most important changes that cartilage undergoes due to aging is a reduction in the synthetic activity of chondrocytes. According to Roughley (2001), this factor probably contributes most to the changes in this tissue. In addition to reduced synthetic activity, the total number of cells in vertebrate cartilage decreases with age (Hall et al., 2005; Hermette et al., 2006), as observed in the present study. The number of chondrocytes in the arytenoid cartilage of the bullfrog larynx is much more representative in younger animals than older ones. In addition to chondrocytes, another tissue component affected by aging is the ECM. PGs of the cartilaginous matrix undergo qualitative and quantitative changes with age. These alterations directly affect tissue homeostasis since they modify the transport of water and metabolites in the tissue (DeGroot et al., 1999; Loeser, 2000). The present results demonstrated that tissue basophilia, which indicates the presence of PGs, decreases progressively with increasing age. However, this reduction occurs uniformly across the different matrix regions. PGs are uniformly distributed in the ECM of younger animals, whereas in older animals these molecules predominate in the pericellular matrix of chondrocytes and in the
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territorial matrix close to the perichondrium. There are important areas in the territorial matrix of the cartilage specimen where basophilia is absent. Furthermore, a significant increase in the amount of GAGs in the cartilaginous matrix was observed in animals from 7 to 180 days of age and a reduction at 1080 days. Qualitative analysis of the type of GAG revealed the presence of dermatan and chondroitin sulfate at all ages, with a predominance of the latter. A reduction in PG content during aging of the ECM has been described for different cartilages in different animals (Lotz and Loeser, 2012). This finding is mainly the result of changes in the biosynthetic profile of chondrocytes, in conjunction with a reduction in the number and length of GAG side chains associated with these molecules (Inerot and Heinergård, 1983; Roberts and Pare, 1991; DeGroot et al., 1999; Loeser, 2000; Huldelmaier et al., 2001). In addition, the progressive reduction in the number of chondrocytes can also explain the overall reduction of PGs in the cartilaginous matrix (DeGroot et al., 1999; Bobacz et al., 2004). Furthermore, alterations in the activity of some matrix metalloproteinases increase the degradation of PGs with age and consequently reduce intermolecular interactions, hydration and the transport of metabolites, leading to tissue degeneration (Chambers et al., 2001; Gepstein et al., 2003; Forsyth et al., 2005). Studies indicate that the collagen content of tissues increases with age (Robert and Labat-Robert, 2000). The collagen fibrils and fibers become densely compacted due to the increase in the number of intermolecular and interfibrillar cross-links (Scott and Parry, 1992). However, these events vary between tissues of the same animal and among different species (Martin and Buckwalter, 2002). Another factor that leads to important functional changes in cartilage tissue is the presence of collagen XI in the collagen II fibril, as well as the organization and size of fibrils and the frequency of intermolecular and interfibrillar cross-links and interactions with PGs (Grodzinky, 1983; Blaschke et al., 2000). Furthermore, the synthesis and deposition of collagen molecules (Floridi et al., 1981; Robert and Labat-Robert, 2000), as well as their degradation (Niedermüller et al., 1977), are more intense in younger animals, suggesting different densities of fiber compaction. However, aging collagen molecules undergo intense compaction and the fibrils and fibers become crystalline structures (Baer et al., 1988). In the arytenoid cartilage found in the bullfrog larynx, collagen fibers predominate in the perichondrium and territorial matrix, as observed in other types of visceral cartilage. Fibers arranged perpendicular to the perichondrium were detected in the territorial matrix. In animals with 1080 days of age, these fibers were more birefringent when observed under polarized light. These data agree with literature reports suggesting that, with the greater accumulation of organized collagen fibers with increased caliber, the fibers become more birefringent (Martin and Buckwalter, 2002). Also in 1080-day-old animals, fibers in the same areas were arranged in a three-dimensional network resembling a multi-grid lattice. This arrangement was also reported by Zambrano et al. (1982) for different visceral cartilages of different mammalian organs. Also in 1080-day-old animals, the hydroxyproline quantification support the data of birefringence, since a higher collagen amount was observed in animals with 1080 days compared to younger animals. The organizational changes of collagen fibers in the tissue framework also result in gradual alterations in the mechanical properties of cartilage and consequent adaptations with age (Roberts et al., 1998; Huldelmaier et al., 2001). In the specific case of the frog larynx, opening and closing movements of the laryngeal ostium and vocalization may become compromised (Porter, 1972; Romer, 1985). Immunohistochemistry confirmed the predominance of type II collagen fibers found in the cartilage ECM. This distribution has also been reported for analogous cartilages of other animals (Evans et al., 1983; Eyre et al., 1991; Morrison et al., 1996).
In addition to collagen fibers, elements of the elastic system are important components of the arytenoid cartilage found in the bullfrog larynx, especially once the animals have reached maturity (180 days). Likewise, the cartilages of the respiratory system of mammals contain large amounts of elastic fibers (Zielinski, 2001; Kielty et al., 2002). However, there are few studies in the literature evaluating the age-related changes in these components. Cox and Peacock (1977) described a progressive increase in the deposition of elastic fibers in the auricular cartilage of rabbits with age. Similar descriptions were made by Isamu et al. (2001) for the auricular cartilage of humans and by Kano et al. (2005) for epiglottic cartilage. The deposition of thick fibers with a similar diameter was observed in these cases. The present findings are similar to those reported in the literature and suggest that an important percentage of the tissue mass responsible for the development of cartilage is related to the deposition of elastic fibers (Watanabe et al., 1982). Calcification is the most common event observed during the aging of visceral cartilages (Robert and Labat-Robert, 2000; Martin and Buckwalter, 2002). Studies in the literature have described calcium deposits widely distributed in laryngeal cartilage of mammals after maturity (Kusafuka et al., 2001; Claassen and Werner, 2004; Kano et al., 2005). In contrast to these reports, in the arytenoid cartilage of the bullfrog larynx, small calcium deposits were only detected in the territorial matrix of 1080-day-old animals, despite a reduction in the number of chondrocytes accompanied by a reduction in GAG content and an increase in the deposition and thickness of collagen fibers in the cartilaginous stroma. This finding might be explained by the fact that the age limit studied is still beyond that at which, proportionally, calcifications were described in other vertebrates, or by the existence of another still unknown factor that regulates this process and delays the onset of calcification. The latter might be related to the action of regulatory molecules on cartilage metabolism which induce the production of local modulators that reduce the velocity of calcium deposition in the matrix (Suzuki, 1996). Analysis of structural modifications of arytenoid cartilage showed changes in the ultrastructural organization of chondrocytes and ECM in animals at 180 and 1080 days of age. The cells exhibited a marked reduction in rough endoplasmic reticulum. This organelle is related to the synthesis of matrix macromolecules (Martin and Buckwalter, 2006; Freemont and Hoyland, 2007). Furthermore, Ghadially et al. (1974), in humans, and Bhatnagar et al. (1981), in swine, described an increase in the number of lysosomes and vacuoles containing lipids and particles of matrix proteoglycan aggregates in the chondrocyte cytoplasm. Similarly, Yamamoto et al. (2005) observed a significant decrease, with the age, in plasma membrane extensions in mice. According to these authors, the membrane extensions still present in older individuals were mainly related to the phenomenon of phagocytosis. Taken together, the results indicate that the cellular alterations described for the bullfrog are promoted by the process of physiological aging of cartilage, similar to the descriptions for other vertebrates. However, the dimensions of the cell populations seem to be maintained in this tissue with advancing age, in contrast to what is observed in most of the species studied so far (Hall, 2005). In addition to chondrocytes, the ECM is markedly affected by aging. PGs of the cartilaginous matrix undergo qualitative and quantitative changes with age as discussed earlier (DeGroot et al., 1999; Loeser, 2000). Cuprolinic blue staining permitted inferences on the dimensions of the PG aggregates and on the number of elements distributed in the ECM. The number of stainings agrees with the basophilia and GAG quantification results. PG molecules that are uniformly distributed throughout the ECM and present similar dimensions start to predominate in the pericellular matrix with advancing age. Their diameter is increased and smaller quantities are subsequently observed in the different regions of the
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cartilaginous matrix. However, the diameter of the precipitates indicates a predominance of high molecular weight PGs. The present results agree with those reported by Li et al. (1994) for the trachea of mice. Nevertheless, a reduction in PG content with the age has been described for different cartilages obtained from different animals and is mainly due to a decrease in the number and length of the side chains of GAGs associated with these molecules (Inerot and Heinergård, 1983; Roberts and Pare, 1991; DeGroot et al., 1999; Loeser, 2000; Huldelmaier et al., 2001). The progressive reduction in the biosynthesis of matrix macromolecules by the remaining cells may also explain the reduction in the dimensions of PGs in the cartilaginous matrix (DeGroot et al., 1999; Bobacz et al., 2004), and due to the increase in age, the activity of some matrix metalloproteinases that degrade PGs reduces the quantity and dimensions of these molecules (Chambers et al., 2001; Gepstein et al., 2003; Forsyth et al., 2005). In contrast to PGs, no important age-related change was observed in the ultrastructure of collagen fibrils in the pericellular matrix. These findings are supported by studies in the literature involving other vertebrates (Ottani et al., 2001; Hall, 2005). However, as described in this study, collagen fibers in the territorial matrix increased in thickness with age and contributed to the agerelated organizational changes in the tissue framework. In conclusion, the aging process acts on the arytenoid cartilage of the bullfrog larynx, which is elastic, causing changes in the quantity and structure of chondrocytes, as well as in the synthesis and distribution of PGs, elastic fibers and collagens of the ECM. These changes, in turn, lead to functional adaptations in these animals similar to the pattern observed in studies on mammals in an equivalent phase of life. Acknowledgments We thank FAPESP (Fundac¸ão de Amparo a Pesquisa do Estado de São Paulo) and Fundac¸ão Hermínio Ometto for financial support and declare that have no conflict of interest in this study. References Baer, E., Cassidy, J.J., Hiltner, A., 1988. Hierarchical structure of collagen and its relationship to the physical properties of tendon. In: Nimni, M.E. (Ed.), Collagen: Biochemistry and Biomechanics, vol. II. CRC Press, Boca Raton, Los Angeles, USA. Bhatnagar, R., Christian, R.G., Nakano, T., Aherne, F.X., Thompson, J.R., 1981. Age related changes and osteochondrosis in swine articular and epiphyseal cartilage: light and electron microscopy. Can. J. Comp. Med. 45, 188–195. Blaschke, U.K., Eikenberry, E.F., Hulmes, D.J., Galla, H.J., Bruckner, P., 2000. Collagen XI nucleates self-assembly and limits lateral growth of cartilage fibrils. J. Biol. Chem. 275, 10370–10378. Bobacz, K., Erlacher, L., Smolen, J., Soleiman, A., Graninger, W.B., 2004. Chondrocyte number and proteoglycan synthesis in the aging and osteoarthritic human articular cartilage. Ann. Rheum. Dis. 63, 1618–1622. Boyd, S.K., Wissing, K.D., Heinsz, J.E., Prins, G.S., 1999. Androgen receptors and sexual dimorphisms in the larynx of the bullfrog. General Comp. Endocr. 113, 59–68. Buckwalter, J.A., Woo, S., Goldberg, V., Hadley, E., Booth, F., Oegema, T., 1993. Soft-tissue aging. J. Bone Joint Surg. 75, 1533–1548. Chambers, M.G., Cox, L., Chong, L., Suri, N., Cover, P., Bayliss, M.T., Mason, R.M., 2001. Matrix metalloproteinases and aggrecanases cleave aggrecan in different zones of normal cartilage but colocalize in the development of osteoarthritic lesions in str/ort mice. Arth. Rheum. 44, 1455–1465. Claassen, H., Werner, J., 2004. Gender-specific distribution of glycosaminoglycans during cartilage mineralization of human thyroid cartilage. J. Anat. 205, 371–380. Cox, R.W., Peacock, M.A., 1977. The fine structure of developing elastic cartilage. J. Anat. 123, 283–296. Cruz, W.P., Rogério, A., Dedivitis, A., Sementilli, A., 2003. Histologic study of ossification of the thyroid cartilage. Rev. Bras. Otorrinol. 69, 87–92. DeGroot, J., Verzijl, N., Bank, R.A., Labefer, F.P., Bijlmsa, J.W., Tekoppele, J.M., 1999. Age-related decrease in proteoglycan synthesis of human articular chondrocytes. Arth. Rheum. 42, 1003–1009. Evans, H.B., Ayad, S., Abedin, M.Z., Hopkins, S., Morgan, K., Walton, K.W., Weiss, J.B., Holt, P.J., 1983. Localisation of collagen types and fibronectin in cartilage by immunofluorescence. Ann. Rheum. Dis. 42, 575–581.
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Structure and composition of arytenoid cartilage of the bullfrog (Lithobates catesbeianus) during maturation and aging Priscila Eliane dos Santos Laureano a , Kris Daiana Silva Oliveira a , Andrea Aparecida de Aro b , Laurecir Gomes b , Edson Rosa Pimentel b , Marcelo Augusto Marretto Esquisatto a,∗ a
Programa de Pós-graduac¸ão em Ciências Biomédicas, Centro Universitário Hermínio Ometto, Av. Dr. Maximiliano Baruto, 500 Jd. Universitário, 13607-339, Araras, SP, Brazil b Departamento de Biologia Estrutural e Funcional, Instituto de Biologia, Universidade Estadual de Campinas, Rua Charles Darwin, s/n, CxP 6109, 13083-863, Campinas, SP, Brazil
a r t i c l e
i n f o
Article history: Received 13 April 2014 Received in revised form 24 May 2015 Accepted 28 May 2015 Available online 30 May 2015 Keywords: Cartilage Structure Larynx Frog Aging
a b s t r a c t The aging process induces progressive and irreversible changes in the structural and functional organization of animals. The objective of this study was to evaluate the effects of aging on the structure and composition of the extracellular matrix of the arytenoid cartilage found in the larynx of male bullfrogs (Lithobates catesbeianus) kept in captivity for commercial purposes. Animals at 7, 180 and 1080 days post-metamorphosis (n = 10/age) were euthanized and the cartilage was removed and processed for structural and biochemical analysis. For the structural analyses, cartilage sections were stained with picrosirius, toluidine blue, Weigert’s resorcin-fuchsin and Von Kossa stain. The sections were also submitted to immunohistochemistry for detection of collagen types I and II. Other samples were processed for the ultrastructural and cytochemical analysis of proteoglycans. Histological sections were used to chondrocyte count. The number of positive stainings for proteoglycans was quantified by ultrastructural analysis. For quantification and analysis of glycosaminoglycans were used the dimethyl methylene blue and agarose gel electrophoresis methods. The chloramine T method was used for hydroxyproline quantification. At 7 days, basophilia was observed in the pericellular and territorial matrix, which decreased in the latter over the period studied. Collagen fibers were arranged perpendicular to the major axis of the cartilaginous plate and were thicker in older animals. Few calcification areas were observed at the periphery of the cartilage specimens in 1080-day-old animals. Type II collagen was present throughout the stroma at the different ages. Elastic fibers were found in the stroma and perichondrium and increased with age in the two regions. Proteoglycan staining significantly increased from 7 to 180 days and reduced at 1080 days. The amount of total glycosaminoglycans was higher in 180-day-old animals compared to the other ages, with marked presence of chondroitin- and dermatan-sulfate especially in this age. The content of hydroxyproline, which infers the total collagen concentration, was higher in 1080-day-old animals compared to the other ages. The results demonstrated the elastic nature of the arytenoid cartilage of L. catesbeianus and the occurrence of age-related changes in the structural organization and composition of the extracellular matrix. These changes may contribute to alter the function of the larynx in the animal during aging. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction The aging process is affected by genetic, dietary, environmental and social factors. Morphological and physiological differences among the species of animals and the consequent changes in their
∗ Corresponding author. Tel.: +55 19 3543 1440; fax: +55 19 3543 1439. E-mail address: [email protected] (M.A.M. Esquisatto). http://dx.doi.org/10.1016/j.micron.2015.05.018 0968-4328/© 2015 Elsevier Ltd. All rights reserved.
life’s cycles during aging alter the structural and functional organization of cells and tissues and affect their homeostasis (Kirkwood, 2002, 2005; Teixeira and Guariento, 2010). In connective tissues, the age’s modifications are mainly related to alterations in the macromolecules that comprise the extracellular matrix (ECM) (Li et al., 2013). In cartilaginous tissue, the organization and relationship between ECM components become compromised with aging. Chondrocytes are widely distributed in tissues of young animals and exhibit great synthetic capacity,
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guaranteeing the functional competence of the ECM (Horton et al., 2006). Structurally, the ECM that surrounds the chondrocytes can be divided into a pericellular and a territorial matrix. The pericellular matrix, which is closer to the chondrocytes, is rich in proteoglycans (PGs) and it is in direct contact with the cells under normal physiological conditions. In contrast, the territorial matrix contains large amounts of fibrillar components such as type II collagen fibrils (Muir, 1995; Poole et al., 2001). In addition to these elements, the cartilaginous matrix contains elastic fibers (Kielty et al., 2002). In the respiratory system of most terrestrial vertebrates, cartilaginous tissue is found in the wall of the larynx, trachea, bronchi, and bronchioles. In the case of anuran amphibians, such as frogs, cartilage is only found in the larynx, which is situated between the oral cavity and the trachea. The larynx consists of two pairs of cartilaginous rings, divided into an anterior (arytenoid cartilage) and posterior pair (cricoid cartilage). The arytenoid cartilage forms the wall of the valve that permits the passage of air from the oral cavity to the trachea and vice-versa. On the other hand, the cricoid cartilage provides support to the membrane structures that form the vocal cords (Porter, 1972; Romer, 1985). Like all tissues, the cartilage found in the respiratory system also undergoes changes in the organization and composition of the ECM with aging. Age-related changes in the biosynthesis of glycosaminoglycans (GAGs) in the tracheal cartilage of mammals has been observed in adults and became more pronounced with advancing age (Lotz and Loeser, 2012), as well as a decrease in the number of PG sulfations. In addition, several authors reported important age-related structural changes in cartilage PGs in this tissue (Mallinger and Stockinger, 1987; Lotz and Loeser, 2012). Degenerative alterations in tracheal, thyroid and epiglottic cartilage have been described in detail in older adults, with focus on the process of calcification in these structures (Kusafuka et al., 2001; Cruz et al., 2003; Kano et al., 2005). Because of the amount and importance of the ECM in the organization of visceral cartilage isolated from the respiratory system of vertebrates (Vanperperstraete, 1973; Reid, 1976), the arytenoid ring become interesting for the study of the effect of aging on the cartilage matrix. Thus, the aim of the present study was to describe the changes that occur in the structure and composition of the ECM of the arytenoid cartilage found in the larynx of males in post-metamorphosis bullfrogs (Lithobates catesbeianus), during the process of maturation and aging.
2. Materials and methods 2.1. Animals and sample collection Thirty male bullfrogs (L. catesbeianus) were obtained from a commercial breeder in Araras, São Paulo, Brazil. The arytenoid cartilage found in the larynx of animals at 7, 180 and 1080 days after the end of metamorphosis (n = 10/age) was analyzed. These ages were chosen according to the different phases of sexual maturation of the animal in captivity (immaturity, maturity, and decline in fertility) (Porter, 1972; Romer, 1985). The end of metamorphosis was defined as the complete disappearance of the tail. In bullfrogs, as in most other anurans, only males ever give an advertisement call. In relation to this, there are significant structural differences between male and female larynges in many anuran amphibian species. In this context, the skeletal cartilage of larynx presents significant alterations during sexual maturation and aging (Boyd et al., 1999). Samples were collected from male animals at each age. For cartilage removal, the animals were kept in cold water at 4 ◦ C for 1 h and then sacrificed by spinal cord transection. Samples destined for biochemical analysis (5/age) were frozen at −20 ◦ C or immediately
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used for the tests. Samples destined for structural analysis (5/age) were fixed according to the protocol established for each technique. The study protocol was approved by the Ethics Committee on Animal Use of the institution (FHO/UNIARARAS, CONCEA/MCTi/Brazil) (Permit No. 081/2005). 2.2. Morphological analysis 2.2.1. Processing for histological and histochemical analysis After dissection, the cartilage samples were fixed in 10% formalin in Millonig buffer, pH 7.4, for 24 h at room temperature. Next, the specimens were washed in buffer and processed for embedding in Paraplast® (Merck, Darmstadt-Germany) using standard procedures. Longitudinal sections (6-m) were submitted to the following histochemical techniques: picrosirius-hematoxylin for visualization of collagen fibers under polarized light; toluidine blue in McIlvaine buffer, pH 4.0, for the evaluation of tissue basophilia and chondrocyte count; Weigert’s resorcin-fuchsin method for analysis of the elastic fiber system, and Von Kossa staining for the detection of calcium deposits. For observations in polarization microscopy, the sections were positioned so that the longest axis of the cartilage was at 45◦ in relation to the polarized light plane, as described by Vidal (1986). The preparations were examined and documented with a Leica DM-2000 photomicroscope. 2.2.2. Morphometric analysis The number of chondrocytes (No. in 104 m2 ) was quantified using a 40× objective in five fields of each of three sections obtained from the arytenoid cartilage of each animal at each age. All images were captured and digitized with a Leica DM-2000 photomicroscope. The measurements and counts were performed on the digitized images using the Sigma Scan Pro 6.0® software. 2.2.3. Immunohistochemical analysis Three 6-m sections obtained from each animal at each age were transferred to cuvettes and incubated with a solution of 0.01 M sodium citrate buffer, pH 6.0, and 0.05% Tween-20 (v/v) in a microwave (high power, 5 min + 5 min), alternating with an ice bath (5 min + 5 min). The sections were then immediately cooled in a water bath for 30 min at room temperature. Next, the slides were washed three times in PBS (0.01 M, pH 7.4). The sections were immersed in methanol containing 0.3% (v/v) hydrogen peroxide for 30 min to block endogenous peroxidase activity (∼5 mL per assay) and washed three times in PBS (0.01 M, pH 7.4). For the blockade of nonspecific sites (0.5 mL per slide), the sections were immersed in fetal bovine serum for 20 min (∼5 mL per assay), followed by three washes in PBS (0.01 M, pH 7.4). The slides were treated with the primary antibody diluted in 0.01 M PBS plus 1% bovine serum albumin (BSA) for 18 h at 4 ◦ C. The following primary antibodies were used: rabbit polyclonal anticollagen I (diluted 1:100, Rockland Gilbertsville, PA, USA), mixing 2 L of the stock solution + 98 L 0.01 M PBS plus 1% BSA (∼2.5 mL per assay); rabbit polyclonal anti- collagen II (diluted 1:100, Rockland, USA), mixing 2 L of the stock solution + 98 L 0.01 M PBS plus 1% BSA (∼2.5 mL per assay). The slides were then washed three times in PBS (0.01 M, pH 7.4) and incubated with the secondary antibody diluted in 0.01 M PBS plus 1% BSA for 30 min at room temperature. Sections in which the primary antibodies were omitted served as negative control. Peroxidase-conjugated anti-rabbit IgG was used as the secondary antibody (diluted 1:500, Rockland, USA), mixing 2 L of the stock solution + 498 L 0.01 M PBS plus 1% BSA (∼3 mL per assay). For the detection of peroxidase activity, the slides were washed in 0.05 M Tris–HCl, pH 7.4, and incubated with a solution of 0.05% (w/v) diaminobenzidine in 0.05 M Tris–HCl, pH 7.4, containing 0.03% (v/v) hydrogen peroxide for 5 min at room
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temperature. The reaction was stopped by washing the slides in distilled water. 2.2.4. Processing for ultrastructural and cytochemical analysis The arytenoid cartilage samples obtained at different ages were fixed in a solution of 2% glutaraldehyde and 0.1% tannic acid dissolved in 0.1 M sodium cacodylate buffer, pH 7.3, for 2 h at room temperature. Next, the material was washed in buffer and postfixed in 1% osmium tetroxide for 1 h at 4 ◦ C. After this step, the fragments were washed in saline-glucose and treated with 1% uranyl acetate for 18 h at 4 ◦ C, followed by washing in saline-glucose and dehydration. Fragments were also treated with cuprolinic blue for the ultrastructural detection and counting of PG staining (Scott et al., 1989). After fixation in the different procedures, all specimens were dehydrated in an increasing ethanol series and double passage through propylene oxide. Next, the specimens were embedded in mixtures of propylene oxide/Epon resin (1:1, 1:2 and pure) and, finally, placed in plastic molds in an oven. Sections were cut with a glass and diamond knife in an Ultracut UCT ultramicrotome (Leica) and counterstained with 2% uranyl acetate in water and 0.2% lead citrate in 0.1 N NaOH. The sections were examined under an LEO 906 transmission electron microscope (Leica, LME, UNICAMP, Brazil) and documented. The images of cross-sections of the cartilage samples obtained at different ages were digitized and analyzed with the Sigma Scan Pro 6.0® software for determination of the number of PG stainings (in 25 m2 ). 2.3. Biochemical analysis 2.3.1. Quantification and analysis of GAGs For the removal of GAGs, the fragments of territorial matrix of arytenoid cartilage were chopped with a scalpel blade and digested with trypsin for 24 h at 37 ◦ C. Undigested material was removed by centrifugation at 1000 × g for 10 min and the soluble material in the supernatant was precipitated with two volumes of absolute ethanol for 18 h at 4 ◦ C. The precipitate was resuspended in water and quantified by the dimethyl methylene blue method (Farndale et al., 1986). The resuspended samples were also analyzed by electrophoresis on 0.5% agarose gel in 0.05 M propylenediamine at 0.1 mA for 45 min. The agarose gels were fixed in Cetavlon and stained with toluidine blue. The gels were washed with a solution of 50% ethanol and 1% acetic acid for observation of the bands. GAGs were identified by comparison with the standard containing chondroitin, dermatan and heparan sulfate (Sigma, São Paulo, Brazil). 2.3.2. Quantification of hydroxyproline Cartilage fragments were immersed in acetone for 48 h and then in chloroform:ethanol (2:1) for an additional 48 h. The fragments
were hydrolyzed in 6N HCl (1 mL per 10 mg tissue) for 16 h at 110 ◦ C. The hydrolysate was neutralized with 6N NaOH and hydroxyproline was quantified using the chloramine T method, as described by Stegemann and Stalder (1967), with some modifications. Concentrations of hydroxyproline of 0.2–6 mg/mL were used for construction of the standard curve. 2.4. Statistical analysis The mean quantitative results were entered into Excel spreadsheets program (2007 version – Microsoft® ) and analyzed by ANOVA and the Tukey post-test. A level of significance of 5% (p < 0.05) was adopted. 3. Results 3.1. Morphological analysis Analysis of toluidine blue-stained sections of arytenoid cartilage obtained at different ages (Fig. 1A–C) showed a predominance of spherical cells with abundant cytoplasm and round nuclei which were surrounded by a basophilic ECM. Two distinct regions were noted in the latter, one close to the cells (pericellular) and the other among groups of cells (territorial). The cells were distributed throughout the cartilaginous structure and were arranged perpendicularly to its major axis. Basophilia was more homogenous in animals at 7 days of age (Fig. 1A). In contrast, at 180 days of age basophilia was found preferentially in the pericellular region and in areas near the perichondrium (Fig. 1B). In older animals (Fig. 1C), basophilia was reduced in the tissue and predominated in the pericellular region. Quantitative analysis of the number of chondrocytes revealed a significant difference between ages. The number of these cells was significantly higher in 7-day-old animals compared to 180and 1080-day-old animals. However, there was a significant difference between the last two groups, with the observation of a smaller number of chondrocytes in older animals (Table 1). Collagen fibers predominated in the territorial matrix of arytenoid cartilage and exhibited a complex organization at the different ages. These fibers were arranged perpendicular to the major axis of the structure and parallel at the margins near the perichondrium. The fibrillar elements were arranged in the same way as the cell groups and the bundles parallel to the perichondrium were thicker than on the outer surface of the structure (Fig. 2A–C). Analysis of picrosirius-stained sections under polarized light (Fig. 2D–F) showed thick collagen fibers in the territorial matrix in the 1080-days-old animals. In addition, a peculiar arrangement of the birefringent fibers involving isogenous groups
Fig. 1. Cross-sections of arytenoid cartilage obtained from bullfrogs at 7 (A), 180 (B) and 1080 (C) days post-metamorphosis. The sections were stained with toluidine blue. Pericellular matrix (arrowhead); territorial matrix (arrow); perichondrium (P). Bar: 50 m.
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Fig. 2. Cross-sections of arytenoid cartilage obtained from bullfrogs at 7 (A and D), 180 (B and E) and 1080 (C and F) days post-metamorphosis. The sections were stained with picrosirius-hematoxylin for analysis of collagen fiber arrangement and distribution. In A–C, the sections were documented under bright-field illumination. In D–F, the sections were photographed under polarized light. Territorial matrix (*); collagen fibers (arrow); perichondrium (P). Bar: 50 m. Table 1 Morphometric and biochemical parameters of the arytenoid cartilage of bullfrogs at different post-metamorphosis ages. Age (days) Parameter
7
Number of chondrocytes (No. in 104 m2 ) PGs markings (No. in 25 m2 ) GAGs content (mg/g fresh tissue) Hydroxyproline content(g/mg dry tissue)
141.5 ± 22.4* 18.2 ± 4.1 31.8 ± 5.7a 64.6 ± 7.2
p = 0,044
180
1080
89.4 ± 14.8 37.8 ± 6.3* p = 0.047 44.3 ± 4.8* p = 0.046 88.3 ± 9.2
45.7 ± 14.8 22.4 ± 3.7a 34.2 ± 3.6 116.3 ± 10.2*
p = 0.043
Values are the mean and standard deviation obtained from each age (n = 5/age) and were compared by ANOVA and the Tukey post-test (p < 0.05). * Significant difference (p < 0.05). a Values do not differ between 7- and 1080- day old animals.
of cells could be noted at all ages. This arrangement seemed to be stable during aging. Weigert’s resorcin-fuchsin staining was used for the detection and analysis of the elastic fibers at the different ages (Fig. 3A–C). Elastic fibers were detected in the territorial and pericellular matrix of arytenoid cartilage and in the region of the perichondrium. No difference in the distribution of these elements was observed between the different cartilage regions. However, the detection of elastic fibers was higher in 1080-day-old animals, but no difference
was observed between younger animals. In the perichondrium, the distribution of these fibers accompanied the data observed for collagen fibers, with a predominance of these elements on the outer surface of cartilage. The arytenoid cartilage samples obtained at the different ages were treated by the Von Kossa method for the detection of calcium deposits. Calcification points were detected in the territorial matrix only in 1080-day-old animals (Fig. 4A–C).
Fig. 3. Cross-sections of arytenoid cartilage obtained from bullfrogs at 7 (A), 180 (B) and 1080 (C) days post-metamorphosis. The sections were stained with Weigert’s resorcin-fuchsin for the detection of elastic system fibers. Elastic fibers (arrowhead); perichondrium (P). Bar: 50 m.
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Fig. 4. Cross-sections of arytenoid cartilage obtained from bullfrogs at 7 (A), 180 (B) and 1080 (C) days post-metamorphosis. The sections were treated by the Von Kossa method for the detection of calcium deposits. Positive reaction (arrowhead); perichondrium (P). Bar: 50 m.
Immunohistochemical analysis was performed on arytenoid cartilage samples obtained from animals at 7, 180 and 1080 days of age. Negative staining for type I collagen was observed in the cartilage matrix at all ages (Fig. 5A–C). In addition, staining for type II collagen was detected in the territorial and pericellular matrix and its intensity was similar at all ages (Fig. 5D–F). Ultrastructural analysis showed similar cellular characteristics of arytenoid cartilage at the ages studied (Fig. 6A–C). The cells had a round shape and contained a spherical nucleus, with predominance of euchromatin. The cytoplasm exhibited large amounts of rough endoplasmic reticulum, several secretory vesicles, and a large number of cytoplasmic extensions. However, few vesicles and less developed rough endoplasmic reticulum were observed in older animals. The pericellular matrix
exhibits fine granular and fibrillar elements distributed in all directions. No difference in the region between the ages was observed. Analysis of cuprolinic blue-stained sections (Fig. 7A–C) showed the presence of a large number of rod-shaped PG precipitates. In animals at 7 days of age, these molecules had a similar size and were uniformly distributed throughout the stroma. The number of staining increased in 180-day-old animals with predominance in the pericellular matrix. However, in the different regions of the cartilaginous matrix it was reduced in animals at 1080 days of age. The quantity of PG staining obtained by ultrastructural analysis is summarized in Table 1. Significantly higher values were observed in animals with 180 days of age, whereas no significant difference was detected between 7- and 1080-day-old animals.
Fig. 5. Immunohistochemical detection of collagen type I (A–C) and type II (D–F) in sections of arytenoid cartilage obtained from bullfrogs at 7 (A and D), 180 (B and E), and 1080 (C and F) days post-metamorphosis. Observe the negative staining for type I collagen and the positive staining for type II collagen (arrowhead) in the territorial and pericellular matrix at all ages. Chondrocytes (*). Bars: 75 m.
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Fig. 6. Transmission electron photomicrographs of bullfrog arytenoid cartilage at 7 (A), 180 (B) and 1080 (C) days post-metamorphosis. The samples were treated by a conventional method for the analysis of cells and fibrillar elements in the pericellular matrix. Cell projections (arrow); Endoplasmic reticulum (arrowhead); vesicles (*). Bars: 1 m.
Fig. 7. Transmission electron photomicrographs of bullfrog arytenoid cartilage at 7 (A), 180 (B) and 1080 (C) days post-metamorphosis. The samples were stained with cuprolinic blue for ultrastructural staining of proteoglycans. Chondrocyte (Ch); territorial matrix (*); positive staining in the pericellular matrix (arrowhead). Bars: A = 0.5 m; B and C = 0.25 m.
4. Discussion
Fig. 8. Agarose gel electrophoresis for the analysis of glycosaminoglycans. The standard (S) contains chondroitin (CS), dermatan (DS) and heparan sulfate (HS). Note that all samples contain CS (arrow) and DS (arrowhead), with a more prominent CS band in animals at 180 days of age.
3.2. Biochemical analysis The total amount of sulfated GAGs and hydroxyproline in the ECM of arytenoid cartilage at each age are shown in Table 1. The amount of sulfated GAGs was higher in 180-day-old animals compared to the other ages. No difference was observed between animals at 7 and 1080 days of age. Analysis of agarose gels (Fig. 8) showed the marked presence of chondroitin sulfate and, to a lesser extent, of dermatan sulfate at all ages, especially in 180-day-old animals. Hydroxyproline content was higher in 1080-day-old animals compared to the other ages. However, a significant increase in the amount of this amino acid was observed in animals from 7 to 180 days of age.
Age-associated alterations in cartilaginous tissue are intimately related to changes in ECM macromolecules. Qualitative and quantitative changes in PGs and collagen fibers induce alterations in the physicochemical properties of the tissue that consequently affect the physiology of the organ (Sokoloff, 1983, 1987; Buckwalter et al., 1993). One of the most important changes that cartilage undergoes due to aging is a reduction in the synthetic activity of chondrocytes. According to Roughley (2001), this factor probably contributes most to the changes in this tissue. In addition to reduced synthetic activity, the total number of cells in vertebrate cartilage decreases with age (Hall et al., 2005; Hermette et al., 2006), as observed in the present study. The number of chondrocytes in the arytenoid cartilage of the bullfrog larynx is much more representative in younger animals than older ones. In addition to chondrocytes, another tissue component affected by aging is the ECM. PGs of the cartilaginous matrix undergo qualitative and quantitative changes with age. These alterations directly affect tissue homeostasis since they modify the transport of water and metabolites in the tissue (DeGroot et al., 1999; Loeser, 2000). The present results demonstrated that tissue basophilia, which indicates the presence of PGs, decreases progressively with increasing age. However, this reduction occurs uniformly across the different matrix regions. PGs are uniformly distributed in the ECM of younger animals, whereas in older animals these molecules predominate in the pericellular matrix of chondrocytes and in the
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territorial matrix close to the perichondrium. There are important areas in the territorial matrix of the cartilage specimen where basophilia is absent. Furthermore, a significant increase in the amount of GAGs in the cartilaginous matrix was observed in animals from 7 to 180 days of age and a reduction at 1080 days. Qualitative analysis of the type of GAG revealed the presence of dermatan and chondroitin sulfate at all ages, with a predominance of the latter. A reduction in PG content during aging of the ECM has been described for different cartilages in different animals (Lotz and Loeser, 2012). This finding is mainly the result of changes in the biosynthetic profile of chondrocytes, in conjunction with a reduction in the number and length of GAG side chains associated with these molecules (Inerot and Heinergård, 1983; Roberts and Pare, 1991; DeGroot et al., 1999; Loeser, 2000; Huldelmaier et al., 2001). In addition, the progressive reduction in the number of chondrocytes can also explain the overall reduction of PGs in the cartilaginous matrix (DeGroot et al., 1999; Bobacz et al., 2004). Furthermore, alterations in the activity of some matrix metalloproteinases increase the degradation of PGs with age and consequently reduce intermolecular interactions, hydration and the transport of metabolites, leading to tissue degeneration (Chambers et al., 2001; Gepstein et al., 2003; Forsyth et al., 2005). Studies indicate that the collagen content of tissues increases with age (Robert and Labat-Robert, 2000). The collagen fibrils and fibers become densely compacted due to the increase in the number of intermolecular and interfibrillar cross-links (Scott and Parry, 1992). However, these events vary between tissues of the same animal and among different species (Martin and Buckwalter, 2002). Another factor that leads to important functional changes in cartilage tissue is the presence of collagen XI in the collagen II fibril, as well as the organization and size of fibrils and the frequency of intermolecular and interfibrillar cross-links and interactions with PGs (Grodzinky, 1983; Blaschke et al., 2000). Furthermore, the synthesis and deposition of collagen molecules (Floridi et al., 1981; Robert and Labat-Robert, 2000), as well as their degradation (Niedermüller et al., 1977), are more intense in younger animals, suggesting different densities of fiber compaction. However, aging collagen molecules undergo intense compaction and the fibrils and fibers become crystalline structures (Baer et al., 1988). In the arytenoid cartilage found in the bullfrog larynx, collagen fibers predominate in the perichondrium and territorial matrix, as observed in other types of visceral cartilage. Fibers arranged perpendicular to the perichondrium were detected in the territorial matrix. In animals with 1080 days of age, these fibers were more birefringent when observed under polarized light. These data agree with literature reports suggesting that, with the greater accumulation of organized collagen fibers with increased caliber, the fibers become more birefringent (Martin and Buckwalter, 2002). Also in 1080-day-old animals, fibers in the same areas were arranged in a three-dimensional network resembling a multi-grid lattice. This arrangement was also reported by Zambrano et al. (1982) for different visceral cartilages of different mammalian organs. Also in 1080-day-old animals, the hydroxyproline quantification support the data of birefringence, since a higher collagen amount was observed in animals with 1080 days compared to younger animals. The organizational changes of collagen fibers in the tissue framework also result in gradual alterations in the mechanical properties of cartilage and consequent adaptations with age (Roberts et al., 1998; Huldelmaier et al., 2001). In the specific case of the frog larynx, opening and closing movements of the laryngeal ostium and vocalization may become compromised (Porter, 1972; Romer, 1985). Immunohistochemistry confirmed the predominance of type II collagen fibers found in the cartilage ECM. This distribution has also been reported for analogous cartilages of other animals (Evans et al., 1983; Eyre et al., 1991; Morrison et al., 1996).
In addition to collagen fibers, elements of the elastic system are important components of the arytenoid cartilage found in the bullfrog larynx, especially once the animals have reached maturity (180 days). Likewise, the cartilages of the respiratory system of mammals contain large amounts of elastic fibers (Zielinski, 2001; Kielty et al., 2002). However, there are few studies in the literature evaluating the age-related changes in these components. Cox and Peacock (1977) described a progressive increase in the deposition of elastic fibers in the auricular cartilage of rabbits with age. Similar descriptions were made by Isamu et al. (2001) for the auricular cartilage of humans and by Kano et al. (2005) for epiglottic cartilage. The deposition of thick fibers with a similar diameter was observed in these cases. The present findings are similar to those reported in the literature and suggest that an important percentage of the tissue mass responsible for the development of cartilage is related to the deposition of elastic fibers (Watanabe et al., 1982). Calcification is the most common event observed during the aging of visceral cartilages (Robert and Labat-Robert, 2000; Martin and Buckwalter, 2002). Studies in the literature have described calcium deposits widely distributed in laryngeal cartilage of mammals after maturity (Kusafuka et al., 2001; Claassen and Werner, 2004; Kano et al., 2005). In contrast to these reports, in the arytenoid cartilage of the bullfrog larynx, small calcium deposits were only detected in the territorial matrix of 1080-day-old animals, despite a reduction in the number of chondrocytes accompanied by a reduction in GAG content and an increase in the deposition and thickness of collagen fibers in the cartilaginous stroma. This finding might be explained by the fact that the age limit studied is still beyond that at which, proportionally, calcifications were described in other vertebrates, or by the existence of another still unknown factor that regulates this process and delays the onset of calcification. The latter might be related to the action of regulatory molecules on cartilage metabolism which induce the production of local modulators that reduce the velocity of calcium deposition in the matrix (Suzuki, 1996). Analysis of structural modifications of arytenoid cartilage showed changes in the ultrastructural organization of chondrocytes and ECM in animals at 180 and 1080 days of age. The cells exhibited a marked reduction in rough endoplasmic reticulum. This organelle is related to the synthesis of matrix macromolecules (Martin and Buckwalter, 2006; Freemont and Hoyland, 2007). Furthermore, Ghadially et al. (1974), in humans, and Bhatnagar et al. (1981), in swine, described an increase in the number of lysosomes and vacuoles containing lipids and particles of matrix proteoglycan aggregates in the chondrocyte cytoplasm. Similarly, Yamamoto et al. (2005) observed a significant decrease, with the age, in plasma membrane extensions in mice. According to these authors, the membrane extensions still present in older individuals were mainly related to the phenomenon of phagocytosis. Taken together, the results indicate that the cellular alterations described for the bullfrog are promoted by the process of physiological aging of cartilage, similar to the descriptions for other vertebrates. However, the dimensions of the cell populations seem to be maintained in this tissue with advancing age, in contrast to what is observed in most of the species studied so far (Hall, 2005). In addition to chondrocytes, the ECM is markedly affected by aging. PGs of the cartilaginous matrix undergo qualitative and quantitative changes with age as discussed earlier (DeGroot et al., 1999; Loeser, 2000). Cuprolinic blue staining permitted inferences on the dimensions of the PG aggregates and on the number of elements distributed in the ECM. The number of stainings agrees with the basophilia and GAG quantification results. PG molecules that are uniformly distributed throughout the ECM and present similar dimensions start to predominate in the pericellular matrix with advancing age. Their diameter is increased and smaller quantities are subsequently observed in the different regions of the
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cartilaginous matrix. However, the diameter of the precipitates indicates a predominance of high molecular weight PGs. The present results agree with those reported by Li et al. (1994) for the trachea of mice. Nevertheless, a reduction in PG content with the age has been described for different cartilages obtained from different animals and is mainly due to a decrease in the number and length of the side chains of GAGs associated with these molecules (Inerot and Heinergård, 1983; Roberts and Pare, 1991; DeGroot et al., 1999; Loeser, 2000; Huldelmaier et al., 2001). The progressive reduction in the biosynthesis of matrix macromolecules by the remaining cells may also explain the reduction in the dimensions of PGs in the cartilaginous matrix (DeGroot et al., 1999; Bobacz et al., 2004), and due to the increase in age, the activity of some matrix metalloproteinases that degrade PGs reduces the quantity and dimensions of these molecules (Chambers et al., 2001; Gepstein et al., 2003; Forsyth et al., 2005). In contrast to PGs, no important age-related change was observed in the ultrastructure of collagen fibrils in the pericellular matrix. These findings are supported by studies in the literature involving other vertebrates (Ottani et al., 2001; Hall, 2005). However, as described in this study, collagen fibers in the territorial matrix increased in thickness with age and contributed to the agerelated organizational changes in the tissue framework. In conclusion, the aging process acts on the arytenoid cartilage of the bullfrog larynx, which is elastic, causing changes in the quantity and structure of chondrocytes, as well as in the synthesis and distribution of PGs, elastic fibers and collagens of the ECM. These changes, in turn, lead to functional adaptations in these animals similar to the pattern observed in studies on mammals in an equivalent phase of life. Acknowledgments We thank FAPESP (Fundac¸ão de Amparo a Pesquisa do Estado de São Paulo) and Fundac¸ão Hermínio Ometto for financial support and declare that have no conflict of interest in this study. References Baer, E., Cassidy, J.J., Hiltner, A., 1988. Hierarchical structure of collagen and its relationship to the physical properties of tendon. In: Nimni, M.E. (Ed.), Collagen: Biochemistry and Biomechanics, vol. II. CRC Press, Boca Raton, Los Angeles, USA. Bhatnagar, R., Christian, R.G., Nakano, T., Aherne, F.X., Thompson, J.R., 1981. Age related changes and osteochondrosis in swine articular and epiphyseal cartilage: light and electron microscopy. Can. J. Comp. Med. 45, 188–195. Blaschke, U.K., Eikenberry, E.F., Hulmes, D.J., Galla, H.J., Bruckner, P., 2000. Collagen XI nucleates self-assembly and limits lateral growth of cartilage fibrils. J. Biol. Chem. 275, 10370–10378. Bobacz, K., Erlacher, L., Smolen, J., Soleiman, A., Graninger, W.B., 2004. Chondrocyte number and proteoglycan synthesis in the aging and osteoarthritic human articular cartilage. Ann. Rheum. Dis. 63, 1618–1622. Boyd, S.K., Wissing, K.D., Heinsz, J.E., Prins, G.S., 1999. Androgen receptors and sexual dimorphisms in the larynx of the bullfrog. General Comp. Endocr. 113, 59–68. Buckwalter, J.A., Woo, S., Goldberg, V., Hadley, E., Booth, F., Oegema, T., 1993. Soft-tissue aging. J. Bone Joint Surg. 75, 1533–1548. Chambers, M.G., Cox, L., Chong, L., Suri, N., Cover, P., Bayliss, M.T., Mason, R.M., 2001. Matrix metalloproteinases and aggrecanases cleave aggrecan in different zones of normal cartilage but colocalize in the development of osteoarthritic lesions in str/ort mice. Arth. Rheum. 44, 1455–1465. Claassen, H., Werner, J., 2004. Gender-specific distribution of glycosaminoglycans during cartilage mineralization of human thyroid cartilage. J. Anat. 205, 371–380. Cox, R.W., Peacock, M.A., 1977. The fine structure of developing elastic cartilage. J. Anat. 123, 283–296. Cruz, W.P., Rogério, A., Dedivitis, A., Sementilli, A., 2003. Histologic study of ossification of the thyroid cartilage. Rev. Bras. Otorrinol. 69, 87–92. DeGroot, J., Verzijl, N., Bank, R.A., Labefer, F.P., Bijlmsa, J.W., Tekoppele, J.M., 1999. Age-related decrease in proteoglycan synthesis of human articular chondrocytes. Arth. Rheum. 42, 1003–1009. Evans, H.B., Ayad, S., Abedin, M.Z., Hopkins, S., Morgan, K., Walton, K.W., Weiss, J.B., Holt, P.J., 1983. Localisation of collagen types and fibronectin in cartilage by immunofluorescence. Ann. Rheum. Dis. 42, 575–581.
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