91
J. Anat. (1976), 122, 1, pp. 91-112 With 23 figures Printed in Great Britain
Changes in articular cartilage following intraarticular injection of tritiated glyceryl trioleate R. SPRINZ AND R. A. STOCKWELL
Departnment of Anatomy, University Medical School, Teviot Place, Edinburgh EH8 9AG (Accepted 5 August 1975) INTRODUCTION
Lipid droplets are a normal and constant inclusion of chondrocytes (Putschar, 1931), but it is not known if their presence is of particular significance. If the lipid is an energy reserve then it would appear to be in too insignificant a quantity to contribute much to the needs of the whole organism and, as has been remarked, in too great a quantity for the needs of the tissue itself (Collins, Ghadially & Meachim, 1965). There is less fat in articular chondrocytes than in hyaline cartilage elsewhere (Sacerdotti, 1900). In the rabbit, intracellular fat increases during maturation and ageing, little neutral fat being detectable in the superficial regions of the cartilage before the animals are about three months old (Stockwell, 1967). Recent investigations of experimental lipoarthrosis in rabbits have shown that there is a marked increase in the number and size of lipid droplets within articular chondrocytes 2-4 days following an injection of lipid into the synovial cavity (Ghadially, Mehta & Kirkaldy-Willis, 1970; Mehta & Ghadially, 1973). Degeneration of the cells and roughening of the articular surface follow intracellular lipid accumulation. However, there is some doubt as to how the fat deposition takes place and even whether the fat originates from the joint space. There is little evidence of fat droplets in the matrix and there seems to be no diminishing concentration gradient of extracellular fat in the cartilage from the articular surface inwards as might be expected if fat were diffusing in from the joint space. In this study, tritiated glyceryl trioleate, labelled either in the fatty acid or in the glycerol moieties of the molecule, has been employed as a radioactive marker to obtain more data concerning the origin of the intracellular lipid. In addition, effects of experimental lipoarthrosis on the enzyme activity and on the ultrastructure of the cartilage have been observed. MATERIALS AND METHODS
Rabbits of mixed breed weighing between 1 6 and 3-3 kg were used. Under intravenous Nembutal anaesthesia (approx. 36 mg per kg body weight in a solution of 20 mg/ml) and with surgically aseptic conditions, the capsule of the right mandibular joint was exposed and the lower joint cavity injected with up to 0 1 ml glyceryl trioleate. Similarly, the right knee joint received about 0-25 ml trioleate into its synovial cavity. Animals were killed as a rule on the fourth post-operative
92 R. SPRINZ AND R. A. STOCKWELL day with an overdose of intravenous Nembutal. Thirty animals were used in the experiment. Two were subjected to sham operations (joint exposure with needle puncture of the joint capsule, but with no injection of fat) and five were given intraarticular non-radioactive ('cold') trioleate. One group of eight animals received glyceryl-2-H3 trioleate and a second similar group received glyceryl tri(oleate-9,l0-H3), both isotopes having an activity of approximately 1 mC/ml trioleate. However, the surgical approach to the mandibular joint was technically more difficult than to the knee joint and a number of attempts failed to achieve success in the case of glycerol-labelled trioleate. Of the remaining seven animals, five received injections with radioactive levels which proved to be too low for autoradiographic purposes and two were killed less than 4 days after injection with glycerol-labelled trioleate (one animal at 1 day, one animal at 2 days). Light microscopy Several adjacent blocks of articular cartilage were excised from one area of the mandibular and medial femoral condyles of both sides, the left (uninjected) control side being sampled first. In the knee joints, synovial membrane from the suprapatellar region was also taken. One specimen from each site was fixed in Baker's formalcalcium for 18 hours and frozen (cryostat) sections 10 ,um thick were cut normal to the articular surface. Sections were treated as follows: (i) Some sections were coloured with Sudan black B in propylene glycol (Pearse, 1960) for total lipid while others were stained with Nile blue sulphate for neutral and acidic lipid. Pyridine-extracted sections were also treated with Sudan black B. (ii) For autoradiography, unstained sections (serial to those used for lipid histochemistry) were washed in water and mounted on gelatin-coated glass slides. Autoradiographs were prepared according to the methods described by Rogers (1969), using a dipping film technique with Ilford K2 emulsion. The preparations were developed after exposure for two weeks and the unstained sections mounted in glycerine jelly. In eight animals receiving triolein injections, a second block of femoral condylar cartilage was fixed in cold neutral formalin and passed through gum sucrose for 24 hours (Pearse, 1968). The following enzyme techniques were carried out on 10 ,um sections: (i) Acid phosphatase (Pearse, 1968), using Fast Garnet GBC and z-naphthyl phosphate as substrate with an incubation time of 30 minutes at room temperature. (ii) /3-glucosaminidase (Pearse, 1972), using Fast Garnet GBC and naphthol ASBI N-acetyl /-glucosaminide with an incubation time of 20 minutes at 37 'C. A third block of tissue was frozen to -79 'C at excision. Fresh frozen sections of this were used for respiratory enzyme histochemical techniques (Pearse, 1972) carried out at 37 'C using the appropriate substrate and coenzymes, with nitro-blue tetrazolium salt as the electron acceptor. Enzymes studied and incubation times were as follows: (i) Nicotinamide-adenine dinucleotide-cytochrome C reductase (NADH diaphorase), with an incubation time of 5 minutes. (ii) Lactic dehydrogenase (LDH), using lithium lactate as substrate, incubated for 20 minutes.
Lipid in articular cartilage
93
(iii) x-glyczrophosphate (NAD-linked) dehydrogenase (GPDH), incubated for 20 minutes. (iv) Uridinc diphosphoglucose dehydrogenase (UDPGDH), incubated for 60 minutes. In all the enzyme studies, control sections were incubated in media from which substrates had been omitted. Electron microscopy Further blocks were fixed in osmium tetroxide and embedded in Araldite. Thick sections (l,am) were stained with toluidine blue-pyronin Y (Ito & Winchester, 1963); thin sections stained with uranyl acetate and lead citrate were viewed in an EM-6 electron microscope at 75 kV. Analysis of autoradiographs Autoradiographs were examined both by transmitted and incident light, using a Leitz Ortholux microscope fitted with 'Ultrapak' equipment. Grain counting was carried out at x 275 magnification on three regions in the specimens viewed in transmitted and incident light simultaneously, namely: (a) In the superficial and upper middle zones, where chondrocyte fat content increased following experimental lipoarthrosis; at least 20 lacunae were counted in each section. (b) In the deep zone adjacent to the calcified layers where no change in fat content was observed; the lacunae served as a 'tissue' background control. (c) In areas of the glass slide adjacent to the superficial (joint surface) and deep (osseous surface) boundaries of the cartilage sections, to provide 'slide' background controls. Estimate of intracellular lipid In the knee joint an estimate of the amount of intracellular lipid in at least 20 cells in the superficial cartilage of each section was obtained by counting the numbers of intracellular lipid globules coloured with Sudan black and measuring their diameters with a stepped eyepiece graticule. Specimens from both injected and control joints were assessed in this way to provide an index of the mean content of lipid per cell which could be related to the results of autoradiography. RESULTS
Post-operatively the animals appeared to eat normally and no abnormal incisor occlusion was observed, a feature associated with mandibular joint imbalance in rabbits (Sprinz, 1954). The animals did not lose weight significantly throughout the duration of the experiment but the operated limb was not used so actively as the control. On examination of the joints 4 days after operation there was usually little or no evidence of injected material and Gram stains of swabs taken from the synovial cavities revealed no organisms. In the mandibular joint the articular cartilage was noticeably thinner on the injected than on the normal side. In the knee joint there was usually no macroscopic difference between the two sides, though sometimes the cartilage of the injected joint was slightly more yellow. In only one specimen (a knee joint) was an arthritic erosion of the cartilage observed.
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J. Anat. (1976), 122, 1, pp. 91-112 With 23 figures Printed in Great Britain
Changes in articular cartilage following intraarticular injection of tritiated glyceryl trioleate R. SPRINZ AND R. A. STOCKWELL
Departnment of Anatomy, University Medical School, Teviot Place, Edinburgh EH8 9AG (Accepted 5 August 1975) INTRODUCTION
Lipid droplets are a normal and constant inclusion of chondrocytes (Putschar, 1931), but it is not known if their presence is of particular significance. If the lipid is an energy reserve then it would appear to be in too insignificant a quantity to contribute much to the needs of the whole organism and, as has been remarked, in too great a quantity for the needs of the tissue itself (Collins, Ghadially & Meachim, 1965). There is less fat in articular chondrocytes than in hyaline cartilage elsewhere (Sacerdotti, 1900). In the rabbit, intracellular fat increases during maturation and ageing, little neutral fat being detectable in the superficial regions of the cartilage before the animals are about three months old (Stockwell, 1967). Recent investigations of experimental lipoarthrosis in rabbits have shown that there is a marked increase in the number and size of lipid droplets within articular chondrocytes 2-4 days following an injection of lipid into the synovial cavity (Ghadially, Mehta & Kirkaldy-Willis, 1970; Mehta & Ghadially, 1973). Degeneration of the cells and roughening of the articular surface follow intracellular lipid accumulation. However, there is some doubt as to how the fat deposition takes place and even whether the fat originates from the joint space. There is little evidence of fat droplets in the matrix and there seems to be no diminishing concentration gradient of extracellular fat in the cartilage from the articular surface inwards as might be expected if fat were diffusing in from the joint space. In this study, tritiated glyceryl trioleate, labelled either in the fatty acid or in the glycerol moieties of the molecule, has been employed as a radioactive marker to obtain more data concerning the origin of the intracellular lipid. In addition, effects of experimental lipoarthrosis on the enzyme activity and on the ultrastructure of the cartilage have been observed. MATERIALS AND METHODS
Rabbits of mixed breed weighing between 1 6 and 3-3 kg were used. Under intravenous Nembutal anaesthesia (approx. 36 mg per kg body weight in a solution of 20 mg/ml) and with surgically aseptic conditions, the capsule of the right mandibular joint was exposed and the lower joint cavity injected with up to 0 1 ml glyceryl trioleate. Similarly, the right knee joint received about 0-25 ml trioleate into its synovial cavity. Animals were killed as a rule on the fourth post-operative
92 R. SPRINZ AND R. A. STOCKWELL day with an overdose of intravenous Nembutal. Thirty animals were used in the experiment. Two were subjected to sham operations (joint exposure with needle puncture of the joint capsule, but with no injection of fat) and five were given intraarticular non-radioactive ('cold') trioleate. One group of eight animals received glyceryl-2-H3 trioleate and a second similar group received glyceryl tri(oleate-9,l0-H3), both isotopes having an activity of approximately 1 mC/ml trioleate. However, the surgical approach to the mandibular joint was technically more difficult than to the knee joint and a number of attempts failed to achieve success in the case of glycerol-labelled trioleate. Of the remaining seven animals, five received injections with radioactive levels which proved to be too low for autoradiographic purposes and two were killed less than 4 days after injection with glycerol-labelled trioleate (one animal at 1 day, one animal at 2 days). Light microscopy Several adjacent blocks of articular cartilage were excised from one area of the mandibular and medial femoral condyles of both sides, the left (uninjected) control side being sampled first. In the knee joints, synovial membrane from the suprapatellar region was also taken. One specimen from each site was fixed in Baker's formalcalcium for 18 hours and frozen (cryostat) sections 10 ,um thick were cut normal to the articular surface. Sections were treated as follows: (i) Some sections were coloured with Sudan black B in propylene glycol (Pearse, 1960) for total lipid while others were stained with Nile blue sulphate for neutral and acidic lipid. Pyridine-extracted sections were also treated with Sudan black B. (ii) For autoradiography, unstained sections (serial to those used for lipid histochemistry) were washed in water and mounted on gelatin-coated glass slides. Autoradiographs were prepared according to the methods described by Rogers (1969), using a dipping film technique with Ilford K2 emulsion. The preparations were developed after exposure for two weeks and the unstained sections mounted in glycerine jelly. In eight animals receiving triolein injections, a second block of femoral condylar cartilage was fixed in cold neutral formalin and passed through gum sucrose for 24 hours (Pearse, 1968). The following enzyme techniques were carried out on 10 ,um sections: (i) Acid phosphatase (Pearse, 1968), using Fast Garnet GBC and z-naphthyl phosphate as substrate with an incubation time of 30 minutes at room temperature. (ii) /3-glucosaminidase (Pearse, 1972), using Fast Garnet GBC and naphthol ASBI N-acetyl /-glucosaminide with an incubation time of 20 minutes at 37 'C. A third block of tissue was frozen to -79 'C at excision. Fresh frozen sections of this were used for respiratory enzyme histochemical techniques (Pearse, 1972) carried out at 37 'C using the appropriate substrate and coenzymes, with nitro-blue tetrazolium salt as the electron acceptor. Enzymes studied and incubation times were as follows: (i) Nicotinamide-adenine dinucleotide-cytochrome C reductase (NADH diaphorase), with an incubation time of 5 minutes. (ii) Lactic dehydrogenase (LDH), using lithium lactate as substrate, incubated for 20 minutes.
Lipid in articular cartilage
93
(iii) x-glyczrophosphate (NAD-linked) dehydrogenase (GPDH), incubated for 20 minutes. (iv) Uridinc diphosphoglucose dehydrogenase (UDPGDH), incubated for 60 minutes. In all the enzyme studies, control sections were incubated in media from which substrates had been omitted. Electron microscopy Further blocks were fixed in osmium tetroxide and embedded in Araldite. Thick sections (l,am) were stained with toluidine blue-pyronin Y (Ito & Winchester, 1963); thin sections stained with uranyl acetate and lead citrate were viewed in an EM-6 electron microscope at 75 kV. Analysis of autoradiographs Autoradiographs were examined both by transmitted and incident light, using a Leitz Ortholux microscope fitted with 'Ultrapak' equipment. Grain counting was carried out at x 275 magnification on three regions in the specimens viewed in transmitted and incident light simultaneously, namely: (a) In the superficial and upper middle zones, where chondrocyte fat content increased following experimental lipoarthrosis; at least 20 lacunae were counted in each section. (b) In the deep zone adjacent to the calcified layers where no change in fat content was observed; the lacunae served as a 'tissue' background control. (c) In areas of the glass slide adjacent to the superficial (joint surface) and deep (osseous surface) boundaries of the cartilage sections, to provide 'slide' background controls. Estimate of intracellular lipid In the knee joint an estimate of the amount of intracellular lipid in at least 20 cells in the superficial cartilage of each section was obtained by counting the numbers of intracellular lipid globules coloured with Sudan black and measuring their diameters with a stepped eyepiece graticule. Specimens from both injected and control joints were assessed in this way to provide an index of the mean content of lipid per cell which could be related to the results of autoradiography. RESULTS
Post-operatively the animals appeared to eat normally and no abnormal incisor occlusion was observed, a feature associated with mandibular joint imbalance in rabbits (Sprinz, 1954). The animals did not lose weight significantly throughout the duration of the experiment but the operated limb was not used so actively as the control. On examination of the joints 4 days after operation there was usually little or no evidence of injected material and Gram stains of swabs taken from the synovial cavities revealed no organisms. In the mandibular joint the articular cartilage was noticeably thinner on the injected than on the normal side. In the knee joint there was usually no macroscopic difference between the two sides, though sometimes the cartilage of the injected joint was slightly more yellow. In only one specimen (a knee joint) was an arthritic erosion of the cartilage observed.
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