ARTICLE |
Correspondence to: Juan B. Kourí, Centro de Investigación y de Estudios Avanzados (CINVESTAV-IPN), Ave. Politécnico Nacional 2508, Col. San Pedro de Zacatenco, Apdo. Postal 14-740, México DF, CP 07300. E-mail: bkouri@enigma.red.cinvestav.mx
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Summary |
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The status of the Golgi complex in normal vs osteoarthrotic (OA) cartilage has not yet been studied. A monoclonal antibody, MAb 58-K-9, allowed scoring of Golgi labeling intensity. In addition, ultrastructural assessment enabled us to focus on the distribution and relation between the endoplasmic reticulum (ER) and Golgi membranes. The study was performed in both normal and partially menisectomized OA-induced rat cartilage 20 and 45 days after surgery. Comparing Golgi immunolabeling intensities (mean ± SEM) revealed a highly significant difference between normal (9.98 ± 1.25), 20-day (2.49 ± 0.34), and 45-day (0.82 ± 0.22) cartilage. Moreover, chondrocytes from normal cartilage displayed 71.18% of labeling intensity in contrast to OA cartilage, in which chondrocyte labeling intensities were 24.95% (20 days) and 8.11% (45 days). OA chondrocytes appeared to display an overall reduction in Golgi labeling intensity, suggesting disruption of this organelle as the OA damage progressed. Interestingly, many 20-day OA-induced chondrocytes exhibited bubble-like Golgi immunolabeling compartmentalizing the cytoplasm, concomitant with putative apoptotic nuclear changes. At the same time, OA chondrocytes with a typical ultrastructural apoptotic pattern revealed a prominent ER gathered together with Golgi vesicles and saccules, also appearing to compartmentalize chondrocyte cytoplasm. We speculate about the role of Golgi modifications and apoptosis in OA pathogenesis.
(J Histochem Cytochem 50:13331339, 2002)
Key Words: osteoarthrosis, Golgi, cartilage, chondrocytes, immunohistochemistry, ultrastructure
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Introduction |
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The Golgi complex plays a pivotal role in the synthesis of cells and displays a linked stack of cisterns, often located in the juxtanuclear region of the cell (
Changes in Golgi articular cartilage have been reported in connection with the secretion of extracellular matrix components (
In this study we assessed the distribution of Golgi components in chondrocytes from normal cartilage and in an OA-induced rat model (
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Materials and Methods |
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Tissue Sampling
Knee condyles from three normal Wistar rats (approximately 150 g) and six rats with OA induced by partially menisectomy (20 and 45 days after surgery) were included in this study. The surgical procedure for inducing OA cartilage changes has been described elsewhere (
Full-thickness cartilages were fixed with 4% PBSparaformaldehyde at 4C from 4 to 12 hr, cryosectioned (Leica cryostat CM 1100; Heerbrugg, Switzerland), and mounted on gelatin-coated slides. Then they were hydrated for 10 min in PBS and treated with 0.2% Triton X-100 in PBS (PBS-T) for 10 more min. Pre-incubation was achieved with 0.2% IgG-free bovine albumin serum (Sigma Chemical; St Louis, MO) for 20 min at room temperature (RT). Sections were then incubated overnight at 4C with an anti-Golgi MAb 58-K-9 (1:200; Sigma Chemical), followed by FITC-tagged anti-mouse IgG (1:250; Vector Laboratories, Burlingame, CA) for 1 hr at RT. Two negative controls were performed, one consisting of the omission of the primary MAb and the other by the use of an anti-Tau MAb 423 (1:60; provided by C.M. Wischk, Brain's Bank, Cambridge, UK). This marker is found only in the nervous system. No labels were observed in the two negative controls. Positive controls were performed on rat pancreatic acinar tissue. To avoid propidium iodide cytoplasmic RNA staining, 1-hr 4% paraformaldehydePBS-fixed tissues were pretreated with ammonium chloride (50 mM) for 10 min and hydrochloride acid (1 M) for 2 hr. Some samples did not receive any previous treatment. In all samples, nuclei were counterstained with propidium iodide for 510 min (1:1000; Vector Laboratories) and coverslipped in Vectashield mounting medium (Vector Laboratories). The double-labeled sections were viewed with a x60 (NA 1.4) oil immersion objective on a Nikon microscope with attached confocal system (Bio-Rad MRC-60; Watford, UK). Samples were excited with a krytonargon laser in green (blue exciter filter, 418 nm) and red (green exciter filter, 514 nm). From each area, seven to15 serial optical sections (0.51 µm thick) were collected using the dual-channel image system. Projected images were stored on a compact disk. For MRC/Cas statistical analysis, micrographs were observed with a x20 objective lens, always with the same setting.
Ultrastructural study of Golgi components was accomplished in at least three thin sections obtained from three 2.5% glutaraldehyde-cacodylate fixed samples of OA cartilage. The samples were processed, mounted on a formvar-coated one-hole grid, stained with uranyl acetate and lead citrate as described elsewhere (
Statistics
Semi-quantitative analysis.
To determine Golgi immunolabeling in the different zones of the cartilage, confocal micrographs were used to count the number of immunolabeled chondrocytes in each cartilage zone (superficial, middle, deep). The analysis was performed in cartilage from nine different animals (three normal, three OA 25 days, and three OA 45 days). Three sections from each animal were obtained and three different fields from each section were photographed. Golgi-labeled chondrocytes were scored on micrographs with the same magnification. The tissues were divided into three previously defined zones as follows: superficial (approximately 12 mm from the surface of the cartilage), deep zone (approximately 24 mm from the subchondral bone), and middle zone, located between the preceding ones. Two different staff members carried out the semi-quantitative procedure and the results were then compared. The labeling ranking used to score the number of Golgi labeled chondrocytes is shown in Table 1.
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Quantitative Score of Labeling Intensity. To quantify Golgi labeling (FITC) and nuclear staining (propidium iodide) intensities, we scanned, always using the same magnification, three randomly picked microscopic fields from three different sections from cartilage of three different normal rats and three OA-induced rats (20 and 45 days after surgery). A total of three sections and roughly nine microscopic fields were scored per animal. Immunofluorescence intensity scoring was possible by means of MRC/Cas software used with the confocal microscope. Only average nuclear red fluorescence intensity (R) and green Golgi immunofluorescence (G) were considered. The mean ± SEM from all the measurements was calculated.
Statistical Analysis
The statistical analysis was performed using the TukeyKramer multiple comparison test. The percentage of Golgi immunolabeling was also determined, considering the nuclear staining intensity as the total number of chondrocytes in each field and the green labeling intensity as Golgi. A simple equation was applied: G/R x 100 = %.
In addition, we confirmed the possible nuclear changes using an alternative statistical analysis. For this purpose, long axes from 20 propidium-iodide stained nuclei from three different sections were measured. Only normal and 45-day OA-induced chondrocytes were included. The mean and SEM were calculated and statistical analysis was performed using an unpaired t-test.
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Results |
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Immunohistochemistry
Throughout all experiments, Golgi labeling obtained from normal cartilage was homogeneous and was more abundant in the deep zone of the tissue (Table 1). In the OA cartilage, the reduction of Golgi labeling correlated with the progression of the induced damage (Table 2 and Table 3). Statistical analysis showed an overall reduction in Golgi labeling of OA chondrocytes compared with normal cartilage (Table 3). The percentage of Golgi labeling intensity in normal chondrocytes was 71.18%, whereas in 20- and 45-day OA-induced cartilage it was 24.95% and 8.11%, respectively (Table 2). Furthermore, the assessment of Golgi labeling intensity between normal and OA-induced cartilage showed a highly significant statistical difference, whereas nuclear staining intensities showed significant statistical difference between normal and 45-day OA-induced cartilage (Table 3). In addition, statistical analysis of the nuclear diameter corroborated the previous test. A significant statistical difference was found between normal and 45-day OA-induced cartilage (not shown).
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Positive Control
Pancreatic acinar cells showed a well-defined juxtanuclear apical labeling (Fig 1A and Fig B).
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Normal Cartilage
Faded juxtanuclear Golgi immunolabeling was observed in most of the chondrocyte cytoplasm. Normal chondrocyte nuclei showed typical morphology, elongated in the superficial zone and rounded in the middle zone of the cartilage (Fig 1C1E).
Twenty-day OA-induced Cartilage
The immunolabeling distribution was completely different from that of normal cartilage (Fig 1F1I). Chondrocytes from certain locations displayed intense immunolabeling, which concentrated in several cytoplasmic domains, frequently observed to form bubble-like structures. These structures compartmentalized the cytoplasm. Concomitantly, chondrocytes with bubble-like structures showed nuclei particularly irregular and sometimes fragmented.
Forty-five-day OA-induced Cartilage
Most chondrocytes did not display Golgi labeling. However, some showed sparse dotted labeling distributed within the cell cytoplasm, and very few exhibited intense perinuclear labeling. Most nuclei were irregular and contracted (Fig 1J1K).
Ultrastructure
The ultrastructure of individual chondrocytes from 20-day OA-induced cartilage showed a remarkable Golgi development. The organelles' saccules and vesicles were located in several cytoplasmic domains. Some cells exhibited Golgi components running in several intracytoplasmic directions, flowing concomitantly with a prominent RER with large cisternae in succession and forming a kind of compartment. Another characteristic of these cells was their nuclei, which showed remarkable differences from normal chondrocytes, being pyknotic and frequently condensed and irregular (Fig 2).
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Discussion |
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To the best of our knowledge, this is the first time that the Golgi complex from normal chondrocytes and chondrocytes from an OA experimental model has been described and statistically assessed. The MAb 58-K-9 has demonstrated good specificity as an immunohistochemical marker for Golgi membranes (
Our results showed that OA rat cartilage displayed an overall reduction of Golgi labeling intensity correlated with the progression of tissue damage. Comparison of percentages of Golgi labeling intensities from normal and OA-induced cartilage (20 and 45 days after surgery) showed a four- and 12-fold reduction, respectively. This reduction may well match with cartilage damage described 20 and 45 days after experimental OA induction documented in a previous report (
In addition, we recently described the kinetics of chondrocyte ultrastructural changes in our OA model, 5, 20, 45, and 60 days after OA induction (
Here we demonstrated that Golgi immunolabeling and the ultrastructural pattern described above, may well correspond because, using two different approaches, membranes were observed in diverse cytoplasmic domains, appearing to compartmentalize the cytoplasm. These changes, which were observed in most but not all chondrocytes, might reflect an increase in Golgi activity in individual chondrocytes, followed by loss of cell function and subsequent death. These observations might explain the apparent contradiction that Golgi labeling diminished in the cartilage as damage progressed, although some chondrocytes displayed increased Golgi labeling. Cartilage damage evolution does not presume synchronic chondrocytes regarding a metabolic, functional, and pathological state. Therefore, it is possible to have an increase of Golgi activity in individual chondrocytes within an overall decrease of the organelle function in the cartilage.
It has been reported that OA chondrocytes, in contrast to normal chondrocytes, increase metalloproteinase synthesis over its inhibitors (
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Acknowledgments |
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Supported in part by CONACYT grant #28100M.
We are grateful to Dr Leopoldo Flores and Dr Carlos Lavalle from our department for critical reading of the manuscript and to Ms Irma Miranda for assistance in preparing the manuscript.
Received for publication July 16, 2001; accepted May 15, 2002.
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