Produção Nacional
Biological behavior of the gerbil ventral prostate in three phases of postnatal development
2006; Wiley; Volume: 288A; Issue: 7 Linguagem: Inglês
10.1002/ar.a.20347
ISSN1552-4892
AutoresSilvana G. P. Campos, Cristiani Zanetoni, Rejane M. Góes, Sebastião Roberto Taboga,
Tópico(s)Urological Disorders and Treatments
ResumoArticleFree Access Biological behavior of the gerbil ventral prostate in three phases of postnatal development Silvana Gisele Pegorin De Campos, Silvana Gisele Pegorin De Campos Department of Cell Biology, Institute of Biology, UNICAMP, São Paulo, Brazil The first two authors contributed equally to this work.Search for more papers by this authorCristiani Zanetoni, Cristiani Zanetoni Department of Cell Biology, Institute of Biology, UNICAMP, São Paulo, Brazil The first two authors contributed equally to this work.Search for more papers by this authorRejane Maira Góes, Rejane Maira Góes Laboratory of Microscopy and Microanalysis, Department of Biology, IBILCE/UNESP, São Paulo, BrazilSearch for more papers by this authorSebastião Roberto Taboga, Corresponding Author Sebastião Roberto Taboga taboga@ibilce.unesp.br Laboratory of Microscopy and Microanalysis, Department of Biology, IBILCE/UNESP, São Paulo, Brazil Fax: 55-17-32212390Departamento de Biologia, IBILCE/UNESP, Rua Cristóvão Colombo, 2265, Jardim Nazareth, São José do Rio Preto, SP, 15054-000, BrazilSearch for more papers by this author Silvana Gisele Pegorin De Campos, Silvana Gisele Pegorin De Campos Department of Cell Biology, Institute of Biology, UNICAMP, São Paulo, Brazil The first two authors contributed equally to this work.Search for more papers by this authorCristiani Zanetoni, Cristiani Zanetoni Department of Cell Biology, Institute of Biology, UNICAMP, São Paulo, Brazil The first two authors contributed equally to this work.Search for more papers by this authorRejane Maira Góes, Rejane Maira Góes Laboratory of Microscopy and Microanalysis, Department of Biology, IBILCE/UNESP, São Paulo, BrazilSearch for more papers by this authorSebastião Roberto Taboga, Corresponding Author Sebastião Roberto Taboga taboga@ibilce.unesp.br Laboratory of Microscopy and Microanalysis, Department of Biology, IBILCE/UNESP, São Paulo, Brazil Fax: 55-17-32212390Departamento de Biologia, IBILCE/UNESP, Rua Cristóvão Colombo, 2265, Jardim Nazareth, São José do Rio Preto, SP, 15054-000, BrazilSearch for more papers by this author First published: 15 June 2006 https://doi.org/10.1002/ar.a.20347Citations: 28 AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract In this study, we characterized the gerbil's ventral prostate histology ultrastructurally and quantitatively throughout three phases of postnatal development (young, adult, and old) in order to comprehend its biological behavior and propensity to developing spontaneous lesions with aging. The gerbil prostate is composed of alveoli and ducts immersed in a stroma composed of smooth muscle, fibroblasts, collagen and elastic fibers and vessels. The prostate tissue components present morphological and quantitative aspects that vary according to age. Young animals have an immature gland with modest secretory activity. Synthetic activity remained stable in adult and old gerbil. However, prostatic morphology was altered in the aging, showing an increased epithelium and stromal fibrosis. The nuclei of the secretory cells increased with aging, whereas nucleoli presented few alterations during postnatal development. The epithelial proliferation and stromal remodeling noted in this study indicate that the gerbil prostate may respond to the androgen declines typical of senescence through epithelial proliferation and stromal remodeling. Anat Rec, Part A, 288A:723–733, 2006. © 2006 Wiley-Liss, Inc. The prostate is a male accessory sex gland that, together with the seminal vesicle, produces the bulk of the seminal fluid. It is composed of two distinct compartments, the epithelial and the stromal or mesenquimal (Hayward et al.,1996). Prostatic morphogenesis is initiated in the fetal stage from the urogenital sinus and lasts until sexual maturity is fully obtained. The growth and development of the prostate are dependent on circulating androgens produced by the testes, and its homeostatic state during adult life is maintained by these steroid hormones, which act via stromal-epithelial interactions (Cunha et al.,1986; Marker et al.,2003). Thus, when the prostate attains adult size, a balance between cell proliferation and cell death is established so that no further growth occurs in the gland (Banerjee et al.,2000, Banerjee et al.,2001). However, during aging in man and several other species, including the dog and some strains of rodent, cellular hyperplasias may occur despite a decrease in the production of sex hormones such as testosterone, generating age-dependent prostatic hyperplasias (Bonkhoff and Remberger,1998; Banerjee et al.,2001; Leav et al.,2001). These alterations may evolve into prostate cancer, a disease that affects men throughout the world, which takes the form of a lesion with heterogeneous behavior that is still poorly understood. For this reason, recent decades have witnessed a growth in interest in both the morphology and behavior of the prostate during its normal development and in different disorders that target this gland during senescence. A number of animal models, particularly murines, have been used for prostate study. These studies have examined significant similarities and differences in the gross and microscopic anatomy between the rat, mouse, and human prostate and have analyzed the potential of these models to recapitulate human prostatic disease (Huss et al.,2001; Roy-Burman, et al.,2004). The rodent prostate complex is composed of four distinct paired lobes, the ventral, lateral, dorsal, and anterior, each of which serves a particular function in relation to histology and secretory protein production (Sugimura et al.,1986). The morphology of the human prostate is more compact, without distinct lobes, and is divided into three zones: central, transitional, and peripheral (McNeal,1983). Some murine species of the subfamily Gerbillinae have been utilized as models for studies of the male reproductive tract. Among them stand out Praomys natalensis (Gross and Didio,1987), Psammomys obesus (Sprando et al.,1999), and Meriones unguiculatus (Williams,1974), the last species being known as the Mongolian gerbil. The gerbil's prostate has a similar morphology to that of the human gland with respect to the fusion of its lobes in a compact structure, unlike the prostate of mice and rats. However, more specific data on the prostate of these murines have not been found in current literature. It is therefore of interest to know more about the prostate structure of this animal, especially since, although several rodent models exist for the study of the normal morphological aspects and proliferative alterations in the prostate, at this moment, none is completely effective in representing aspects of human prostate cancer (Bostwick et al.,2000; Shirai et al.,2000; Wang et al.,2001). In the present study, we characterized the histological, ultrastructural, and quantitative behavior of the gerbil ventral prostate in three phases of postnatal development (young, adult, and old). Our goal was to improve our understanding of prostate biology in this animal species besides its propensity to develop spontaneous lesions with aging. MATERIALS AND METHODS Animal and Sample Preparations Male gerbils, being 10 young (with 48 ± 15.9 days of mean age), 10 adults (112 ± 27.7 days), and 10 old (18 ± 5.4 months), were housed under conventional conditions (25°C, 40–70% relative humidity, 12-hr light/12-hr dark) and allowed access to chow and water ad libitum. The animals were placed in a chamber with CO2 gas prior to sacrifice. After this, the entire prostatic complex was excised and the ventral prostate removed and cut into fragments. For light microscopy, the samples were fixed for 24 hr in Karnovsky solution (0.1 M Sörensen phosphate buffer, pH 7.2, containing 5% paraformaldehyde and 2.5% glutaraldehyde). After fixation, the material was dehydrated in graded ethanol series and the embedding was performed in glycol methacrylate resin (Leica historesin embedding kit). Structural Analysis The 3 μm histological sections were submitted to hematoxylin-eosin (H&E) staining for general morphological analysis, Gömöris reticulin staining for collagen and reticular fibers, Feulgen reaction for nuclear phenotypes, and AgNOR for nucleolus study. The microscopy analyses were performed with Zeiss-Jenaval or Olympus photomicroscopes. The Image-Pro Plus software version 4.5 for Windows software was used to digitalize the images of each histological section. Transmission Electron Microscopy (TEM) Ventral prostatic fragments were fixed by immersion with 3% glutaraldehyde plus 0.25% tannic acid solution in Millonig's buffer, pH 7.3, containing 0.54% glucose for 24 hr (Cotta-Pererira et al.,1976). After washing with the same buffer, they were postfixed with 1% osmium tetroxide for 2 hr, washed again, dehydrated in graded acetone series, and embedded in Araldite resin. Ultrathin sections were cut using a diamond knife and contrasted with 2% alcoholic uranil acetate for 30 min, followed by 2% lead citrate in sodium hydroxide solution for 10 min. The samples were observed and evaluated with a LEO-Zeiss 906 transmission electron microscope. Quantitative Analysis Thirty random prostatic areas of each of the three phases of postnatal development staining by H&E sections were analyzed. The morphometric-stereological analyses were carried out using Weibel's multipurpose graticulate with 120 points and 60 test lines (Weibel,1978) to compare the relative proportion (%) of the gland portions (glandular and stromal) and each prostatic tissue constituent (epithelium, lumen, nonmuscular interacinar stroma, smooth muscle cell, and collagen subepithelial layer). For the morphometric analyses, 200 random measurements of the epithelium height and the thickness of the smooth muscle layer that surrounds each acinum were also obtained. Caryometric evaluation was carried out using Feulgen reaction-stained sections. Nuclear areas, perimeters, and the form factor [= 4π × nuclear area/(nuclear perimeter)2] parameter were determined for 200 nuclei of the epithelial secretory cells in each phase. The form factor parameter measures nuclear roundness in such a way that values < 1 are associated with nuclei that are more rounded (Taboga et al.,2003). Nuclei with the following numbers of nucleoli—zero (not observed), one, two, and more than two—were counted in 25 fields selected by group of animals. The absolute values found were converted into percentages. The nucleolus number/nucleus age ratio was calculated and 30 area and perimeter measurements of nuclei and nucleoli simultaneously in each group were also taken. This parameter was adopted in order to verify whether the quantitative alterations suffered by the areas and nucleolar perimeters of the secretory cells were proportional to those suffered by the nuclei. All measurements were obtained using the Image-Pro Plus program version 4.5 for Windows. Serum Testosterone Concentration Blood samples were collected from each animal group. The serum was separated by centrifuge at 3,000 rpm and stored until assayed. Testosterone concentration was determined by automatic equipment (VITROS ECi-Johnson and Johnson Ultra-Sensitive Quimioluminescent analysis) in a renowned clinical analysis laboratory using specific reagents supplied by Johnson and Johnson Orthoclinical. The sensitivity of this assay was 0.1–150.0 ng/ml. Statistical Analysis All statistical analyses were performed with Statistica 6.0 software (StatSoft). The ANOVA and Tukey honest significant difference (HSD) tests were applied and P ≤ 0.05 was considered statistically significant. RESULTS Prostate Structure and Ultrastructure In postnatal phases, the gerbil prostate develops a branched alveoli and ducts resembling a tubuloacinar gland. The stroma was composed of smooth muscle cells (SMCs), collagen, and elastic fibers and blood vessels (Fig. 1A, D, and G). Figure 1Open in figure viewerPowerPoint A, B, D, E, G–J: Hematoxylin-eosin stain. A–C: Young group. A: General vision of the ventral prostate of the young gerbil constituted by the epithelial (ep) and stromal compartments (S) organized in small acini (a) surrounded by a thick smooth muscle (sm). Arrowhead indicates vessel. B: Detail of acini with epithelial cells exhibiting voluminous nuclei (arrowhead). C: Dense arrangement of thin reticular fibers (white arrow) in base of epithelium and interspersed with SMC (arrows). D–F: Adult group. D: General aspect of acinus with wide acinar lumen active in secretion production (arrow). E: Secretory epithelium composed of columnar cells surrounded by compacted smooth muscle layer. Note collagen deposit at the epithelium base (arrow). F: Disposal of thin reticular fibers in a more densely packed arrangement than that of young animals (arrows). Collagen deposit (white arrow) at the base of epithelial structure. G–L: Old group. G: Decreased interacinar spaces; some acini with evident secretory activity (asterisk) intercalated by acini with larger collagen accumulation (arrows) in the subepithelial region and allocated irregularly smooth muscle. H: Denser and undulated epithelium, increased density of subepithelial collagen (arrowhead) and thicker SMC. Arrow indicates secretion, blebs. I: Acinus with high epithelial proliferation characterizing a PIN. J: Detail of cells related to the proliferative aggregate exhibiting altered phenotypes and pale basophilic cytoplasm (arrow). L: Increased denser reticular fibers (arrows) disposed in loose network accompanying SMC distribution. C, F, and L: Gömöri's reticulin method. In the young animal, the ventral prostate is an immature gland (Fig. 1A and B) composed of some acini still in the process of glandular modeling (Fig. 1B); the majority of the acini with defined ductal canalization and epithelial layer are arranged in such a way as to surround the narrow lumen (Fig. 1A). The epithelium is composed of voluminous cells with prominent nuclei occupying large portions of the cell (Fig. 1B and 2A). Ultrastructurally, the nuclear chromatin remains descondensed, suggesting synthetic activity. In the cytoplasm, a relative scarcity of synthesis organelles confirms the generally undifferentiated state of the epithelial cells (Fig. 2B). Specializations of the membrane, such as adherent junctions, may be found uniting such cells and a basement membrane separates the epithelium from the stromal compartment (Fig. 2C and D). Figure 2Open in figure viewerPowerPoint Ultrastructure of the young ventral prostate. A: Epithelium (ep) composed of immature epithelial cells with copious nuclei (N) and in the stroma (S), largely well-spaced undifferentiated SMCs. The fibroblasts (arrows) emit cytoplasmic projections throughout stroma. Arrowhead, collagen fibrils; bm, basement membrane. Magnification: 3,597×. B: Immature epithelial cell with voluminous nucleus and descondensed chromatin. mt, mitochondrias. Collagen fibrils (arrowhead) distributed in different directions in the interface between epithelium and stroma associated with fibroblast projection (arrow) and basement membrane. Magnification: 7,750×. C: Detail of the adherent junctions uniting two epithelial cells (arrowheads). mt, large mitochondrias. Magnification: 35,970×. D: Collagen fibrils (co) transversally arranged below the epithelium intimately associated with basement membrane. Magnification: 16,700×. E: Fibroblast with prominent nucleus and in association with elastic fibers (arrowhead). Magnification: 7,750×. F: SMCs are extensive, poor in contractile element and are not densely packed. Collagen fibrils (arrows) and fibroblast process (arrowhead) are inserted between them. Magnification: 7,750×. G: More electrondense SMCs, and caveolae (arrow) in the cell membrane, indicating advanced differentiation process. Magnification: 12,930×. H: Detail of SMCs showing elastic fibers inside their cytoplasmic process (arrowhead). Arrows indicate caveolae. The stroma of the young animal was composed of two cellular types: SMC and fibroblasts (Fig. 2A). Among the fibrilar elements of the extracellular matrix, collagen and reticular fibers are found in large quantities, forming a sustentation net for the prostate gland (Fig. 1C). Ultrastructurally, the collagen fibrils are accumulated at the epithelium base and dispersed throughout the stroma, including between SMC, being disposed in different directions (Fig. 2D, E, F, and H). The SMCs are arranged in an expansive layer around each acinus (Fig. 1A and B) and they also join in a differentiation process with a nucleus and a dilated cytoplasm with low electrondensity and poor contractile elements (Fig. 2F and G). The fibroblasts have a functional phenotype distributed at the base of the epithelium and among the muscle cells (Fig. 2A and E). Few elastic fibers were found to be associated with the cell surface of the fibroblasts (Fig. 2E) and/or involved in the SMC cytoplasmic process (Fig. 2H). In the adult animal, the ventral prostate was recognized as a completely developed gland, with a wide acinar lumen, which indicates intense secretory activity, and with secretory epithelial cells (Fig. 1D) disposed in a simple columnar pattern (Fig. 1E). The epithelium, at the ultrastructural level, is composed of two cellular lineages, secretory and basal cells (Fig. 3A and B). The secretory cells remain in contact with the glandular lumen and have a cytoplasm with a well-developed rough endoplasmic reticulum, Golgi apparatus, mitochondria, and secretion vesicles at the apical pole (Fig. 3A, C, and D). Their nuclei are bulky, with uniform outlines and deposits of condensed chromatin in the peripheral region (Fig. 3B and D). The inconspicuous basal cells localized along the basement membrane have a largely undifferentiated phenotype with no secretory bleb production (Fig. 3B). Figure 3Open in figure viewerPowerPoint Ultrastructure of the adult ventral prostate. A: Epithelial cells (ep) with numerous secretory vesicles (sv) in the cytoplasm. S, stroma. Magnification: 3,597×. B: Two cell types form the prostate epithelium, the secretory (sc) and basal cells (white arrow). RER, endoplasmic reticulum cisternae (black arrow). The latter are smaller, have fewer synthesis organelles, and the nucleus occupies most of the cell. Arrowhead indicates basement membrane. Magnification: 10,000×. C: Abundant Golgi stacks (G) and secretory vesicle in cytoplasm of secretory cell. Magnification: 16,700×. D: Endoplasmic reticulum cisternae in the perinuclear region. Deposit of condensed chromatin in nuclear periphery (arrow). Magnification: 27,800×. E: Collagen (arrow) and elastic fibers (arrowhead) associated with basal membrane (bm) in the interface between epithelium and stroma. F: Stroma composed of fibroblasts (F) localized below glandular epithelium and packed SMC layer with reduced spaces between the cells, occupied by collagen fibrils (arrowhead). Basement membrane (arrow). Magnification: 6,000×. G: Differentiated SMCs exhibiting abundant contractile elements (asterisks) surrounded by collagen fibrils disposed in several directions (arrow). Magnification: 7,750×. H: Fusiform SMCs with large nucleus accompanying cell shape and surrounded by fibroblast process (arrow). co, collagen fibrils; mt, mitochondrias. Magnification: 10,000×. I: Detail of SMCs in close contact with collagen (co) and elastic fibers (arrowhead). Magnification: 12,930×. The SMCs acquire a fusiform phenotype shape, forming concentric layers and strongly packed layers around the acini with small intercellular spaces (Figs. 1D and E and 3F), which were occupied by collagen fibrils (Fig. 3F, H, and I). The nuclei of the SMCs usually accompany the shape of the cell and the cytoplasms are abundant in contractile filaments (Fig. 3G). The fibroblasts are located preferentially at the base of the epithelium (Fig. 3F) and, in some areas, their projections establish a close contact with SMCs (Fig. 3H). The reticular fibers diminish accompanying the more slender pattern of the smooth muscle layer, and collagen fibers were found to form an approximately concentric subepthelial layer, as verified by the Gömöri method (Fig. 1F). Elastic fibers were observed to be bare and organized as short fibers associated with the collagen fibrils at the base of the epithelium (Fig. 3E) and/or deposited in the thicker layer around the muscle cells (Fig. 3I), maintaining contact with their membranes. The prostate gland of the old animals was characterized by morphologically heterogeneous areas. In a single gland, there were sites with a secretory epithelium functionally (Figs. 1G and H and 4A) similar to that found in adult animals, with some secretory cells maintaining marked secretion liberation in their apex (Figs. 1H and 4C), while in others, there were altered sites with a hyperplastic increase in epithelial cells (Fig. 1I). In the latter case, infoldings of the epithelium into the luminal spaces could be observed, as well as cells with phenotypic alterations (Fig. 1J) characterizing prostatic intraepithelial neoplasia (PIN; including pale cytoplasm and nuclear pleomorphism). In addition, secretory cells were observed to have fatty deposits distributed by the whole cytoplasm (Fig. 4B) and osmiophylic deposits resembling lipofuscin, indicative of cellular aging (Fig. 4C). The basal cells maintained their morphological characteristics and they also had lipofuscin-like and ceramide-like deposits (Fig. 4D). In senescence, the main alteration in the prostatic stroma was the fibrilar component remodeling of the extracellular matrix. This process involved a marked increase in the number of collagen fibers, which formed a wide subepithelial accumulation, including infoldings (Fig. 1H and L) and an increase in reticular fibers. This loose rearrangement contributed an alteration in the distribution of the muscular layer around many acini (Fig. 1L), but the majority of the SMCs maintained their fusiform shapes (Fig. 1H). Some exhibited an irregular external outline, nuclear shrinkage, and great mitochondria in the perinuclear area (Fig. 1F). The increase in the number and thickness of collagen fibrils among the SMCs also contributed to a larger separation between them (Fig. 4E and F). Figure 4Open in figure viewerPowerPoint Ultrastructure of old ventral prostate. A: Site of normal secretory epithelial cells (ep) with secretory vesicle in the apex (arrow). Magnification: 7,750×. B: Site of epithelial cells with marked accumulation of lipid droplets (ld), a frequent event during gerbil aging. Magnification: 4,646×. C: Apical portion of a secretory cell with bleb (apocrine secretion, arrow), integrated lipid droplets (asterisk), and lipofuscin-like structure (arrowhead). Magnification: 16,700×. D: Basal cell (bc) with large nuclei and few synthesis organelles, in the nuclear periphery ceramide-like (arrowhead) and lipofuscin-like deposits (double arrowhead), surrounded by secretory cells full of secretory vesicles (arrow). A thick basement membrane (bm) delimits the epithelium of the stroma, below which is a larger deposit of collagen fibrils (co), another mark of the aging process. Magnification: 10,000×. E: Stroma (S) showing increase of collagen fibrils in subepithelial area (arrows) and between SMCs, which maintain condensed cytoplasm. F, fibroblast. Magnification: 12,930×. F: SMCs with irregular and compressed nucleus in the nuclear periphery. Lipofuscin-like structure (arrowhead) and remarkably extensive mitochondrias (mt). Arrow indicates collagen fibrils. Magnification: 16,700×. Quantitative Analysis The data regarding quantitative analysis are set out in Tables 1 and 2. As regards stereological parameters (Table 1), the proportions of the epithelial and stromal compartments of the ventral prostate in the young group were different from those of the other groups, because the glandular lumens were not so significant. At the same time, the proportion of the stromal compartment was greater due to the larger percentage of the nonmuscular interacinar stroma and SMCs. In the adult and senile phases, the prostate epithelial and stromal compartments were of similar stereological proportions. However, the percentages of epithelial tissue and the nonmuscular subepithelial stroma (collagen layer) increased significantly with aging while the nonmuscular interacinar stroma decreased. The thickness of the epithelium and the smooth muscular layer varied significantly during development, the lowest measurements being found in the adult stage. The significant increase in these measurements during aging confirms the occurrence of glandular hyperplasia during this phase, a process verified by histochemical evaluation. Table 1. Values of the morphometric-stereological data in the gerbil ventral prostate in three phases of postnatal development Prostatic Measurements Groups Young (48 ± 15.9 days) Adult (112 ± 27.7 days) Old (18 ± 5.4 months) Testosterone levels (ng/mL)** p ≤ 0.01 3.95 ± 0.57aa, b, c Different superindices indicate inter-group significant differences. 4.82 ± 0.33ba, b, c Different superindices indicate inter-group significant differences. 2.80 ± 0.23ca, b, c Different superindices indicate inter-group significant differences. Density of Compartment (%) Glandular Epithelial ****** p ≤ 0.0001. 34.64 ± 13.10aa, b, c Different superindices indicate inter-group significant differences. 56.33 ± 11.24ba, b, c Different superindices indicate inter-group significant differences. 60.95 ± 12.14ba, b, c Different superindices indicate inter-group significant differences. Stromal****** p ≤ 0.0001. 65.36 ± 13.10aa, b, c Different superindices indicate inter-group significant differences. 43.67 ± 11.24ba, b, c Different superindices indicate inter-group significant differences. 39.05 ± 12.14ba, b, c Different superindices indicate inter-group significant differences. Volume of tissue components (%) Epithelium**** p ≤ 0.001 22.00 ± 6.60aa, b, c Different superindices indicate inter-group significant differences. 14.77 ± 4.76ba, b, c Different superindices indicate inter-group significant differences. 22.20 ± 7.04 aa, b, c Different superindices indicate inter-group significant differences. Lumen****** p ≤ 0.0001. 12.64 ± 9.87aa, b, c Different superindices indicate inter-group significant differences. 41.56 ± 11.99ba, b, c Different superindices indicate inter-group significant differences. 38.74 ± 13.96ba, b, c Different superindices indicate inter-group significant differences. Smooth muscle cells****** p ≤ 0.0001. 27.72 ± 7.82aa, b, c Different superindices indicate inter-group significant differences. 16.18 ± 6.42ba, b, c Different superindices indicate inter-group significant differences. 14.23 ± 7.74ba, b, c
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