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Histomorphometric assessment of bone changes in rats with type II collagen-induced arthritis
Bone Vol. 24, No. 5 May 1999:485– 490
Histomorphometric Assessment of Bone Changes in Rats With Type II Collagen-induced Arthritis T. HANYU,* T. CHOTANAPHUTI, K. ARAI, T. TANAKA, and H. E. TAKAHASHI Department of Orthopedic Surgery, Niigata University School of Medicine, Niigata, Japan
patients with rheumatoid arthritis, and the early stages of the process are difficult to study. Rats provide a useful model for osteoporosis, since in these animals bone remodeling occurs,2 the epiphyseal plate of long (axial) bones closes when it matures,29 and peak bone mass is found in aging.11 Numerous studies of immobilization-related bone loss in rats following tenotomy22,26 or nerve sectioning27,28 and of bone loss in ovariectomized rats29 –31 have been reported. On the other hand, there are several reports that bone metabolism is affected in rats during the inflammatory reaction following a local inflammatory stimulus, such as inflammation-mediated osteopenia.16 However, few histomorphometric reports have concerned bone alterations in rats with arthritis. We investigated bone loss in the proximal tibia and lumbar vertebra in rats with type II collagen-induced arthritis (CIA), an important experimental model of rheumatoid arthritis, using histomorphometric analysis. In this study, we aimed to clarify the changes occurring in the secondary spongiosa of rats with collagen-induced arthritis, including differences between the proximal tibia, near an arthritic joint, and the lumbar vertebra, an area not directly involved in this model of arthritis. Finally, in characterizing osteopenic changes as a function of time in arthritic rats, we compared these observations with reported changes in immobilized and inflammatory rats.
Numerous studies have demonstrated bone loss in rats following immobilization by tenotomy or nerve sectioning and following ovariectomy. However, few experiments have focused on bone change in rats with arthritis. We investigated bone loss in the proximal tibia and lumbar vertebra in rats with type II collagen-induced arthritis, an experimental model of rheumatoid arthritis, using histomorphometry. Bone loss in the early phase after immunization reflected a significant increase in numbers of osteoclasts and temporarily decreased bone formation. In the proximal tibia, near an arthritic joint, osteoclast numbers associated with bone trabeculae were increased four times over control numbers 4 weeks after immunization. In the lumbar vertebra, where arthritis was not shown, recruitment of osteoclasts occurred later than in the proximal tibia. With time, in both the proximal tibia and lumbar vertebra bone resorption normalized, but bone formation rate and double-label surface by tetracycline, a parameter reflecting bone formation, were increased above control values. We conclude that differences between the proximal tibia and lumbar vertebra probably reflected resumption of function as well as distance from areas of inflammation. These findings indicate that collageninduced arthritis in rats is a useful model not only of autoimmunity, but also of juxta-articular and generalized osteoporosis in rheumatoid arthritis. (Bone 24:485– 490; 1999) © 1999 by Elsevier Science Inc. All rights reserved.
Materials and Methods
Key Words: Bone formation; Bone resorption; Histomorphometry; Arthritis; Collagen.
Thirty-five virgin female Sprague-Dawley rats were purchased from Charles River Japan (Tokyo, Japan). All rats were 6 weeks old and weighed about 145 g at the beginning of the study. During the experimental period, they were housed in 20 3 15 3 45 cm cages and allowed free access to a commercially available pellet diet (MF; Oriental Yeast, Tokyo, Japan) and deionized water. CIA was induced using the method described by Trentham et al.24 Briefly, 20 rats were injected intradermally at the base of the tail with 1 mg of bovine type II collagen (Cosmo-Bio, Tokyo, Japan) including 0.2 mg of muramyl dipeptide dissolved in 0.5 mL of 0.05 mol/L cold acetic acid and emulsified in 0.5 mL of cold Freund’s incomplete adjuvant (Difco Laboratories, Detroit, MI). The remaining 15 rats were injected with 1 mL of 0.1 mol/L acetic acid as a vehicle control. Rats were observed daily for the onset of arthritis using a joint score based on grading severity for each limb from 0 to 4 according to degree of swelling and periarticular erythema, as described elsewhere.24 Rats with active CIA whose arthritic indices (sum of the score of two hindlimbs) were more than three were used. Animals from CIA and vehicle control groups were killed by cardiac puncture under
Introduction The occurrence of juxta-articular and generalized osteoporosis has been reported in patients with rheumatoid arthritis. Whether the osteoporosis results from the disease itself or from related factors such as steroid treatment and inactivity has been uncertain. An imbalance between bone absorption and formation results in osteopenia, but data have been conflicting regarding the contributions of these two processes in rheumatoid patients, whose osteoporosis has been attributed to increased bone absorption,8,21 decreased bone formation,6 or both.4 Multiple factors appear to be at work in the development of osteoporosis in
Address for correspondence and reprints: Tadamasa Hanyu, M.D., Department of Orthopedic Surgery, Niigata University School of Medicine, Asahimachi-dori 1, Niigata 951-8510, Japan. © 1999 by Elsevier Science Inc. All rights reserved.
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Figure 1. The region of histomorphometric analysis in the proximal tibia (A) and in the lumbar vertebra (B). Villanueva bone stain (original magnification 35).
ether anesthesia 4, 6, and 14 weeks after double labeling with tetracycline using a schedule of 1-2-1-1. The fifth lumbar vertebra and the both tibia were dissected, trimmed, and fixed in 70% alcohol for processing without decalcification. After fixation, the proximal two thirds of the tibia and the L-5 vertebra were immersed in Villanueva bone stain for 3 days. The specimens were dehydrated by sequential changes of ascending concentrations of ethanol and acetone, and then embedded in methyl methacrylate. Then, 5 mm frontal sections were cut with a Jung Model K (Reichert-Jung, Heidelberg, Germany) microtome. Histomorphometric analysis was performed in the secondary spongiosa extending 1.3–3.9 mm distally to the epiphyseal growth plate of the tibia, not including modeling bone (Figure 1a). A minimum of 15 adjacent fields were quantified at a magnification of 3160 in each section after processing by a semiautomatic digitizer (System Supply, Nagano, Japan). A similar histomorphometric analysis was carried out medially in the secondary spongiosa at a distance of 1.3 mm from the upper and lower epiphyseal plate and at a similar distance from the anterior and posterior cortex in the L-5 vertebra (Figure 1b). Bone histomorphometry was performed under a microscope using ultraviolet light (Optiphot, Nikon, Tokyo) to obtain static and dynamic parameters. Parameters for histomorphometry employed in this study, derived from Parfitt and colleagues, have been approved by an ASBMR committee.19 To assess bone structure, trabecular bone volume and osteoid volume were measured relative to total volume (BV/TV and OV/TV), and trabecular thickness (Tb.Th) and trabecular number (Tb.N) were recorded. To measure bone formation, osteoblast surface was quantified relative to bone surface (Ob.S/BS). To measure bone resorption, eroded surface, osteoclast surface, and osteoclast numbers were quantified relative to bone surface (ES/BS, Oc.S/BS, and N.Oc/BS). Cells with one or more nuclei that formed resorption lacunae at the surface of trabeculae were identified as osteoclasts. To study bone
formation, the following parameters were measured and calculated: double-label surface (dLS/BS); single-label surface (sLS/ BS); mineralizing surface (MS/BS0), equal to dLS/BS plus one half of sLS/BS; mineral apposition rate (MAR), calculated as the distance between double labels divided by interval labeling time and then multiplied by p/4; and formation rate per surface reference (BFR/BS0), equal to MS/BS0 times MAR. Results are shown as the mean 6 standard deviation of the mean. Data were compared by a two-factor factorial analysis of variance (two-way ANOVA) between the CIA and control groups. If the analysis showed a significant difference between the two groups, the difference between values at each time point was assessed by Student t-test. p values ,0.05 (two-tailed) were considered significant. Results Sign of arthritis appeared in the hindlimbs of all CIA rats used for further study within 10 –14 days after immunization. The incidence of arthritis was 85% (17 of 20 rats) at 14 days. The joint score was 5.2 6 1.1 (n 5 15) at 4 weeks, 6.0 6 1.5 (n 5 10) at 6 weeks, and 8.2 6 1.5 (n 5 5) at 14 weeks. The rats that developed apparent joint disease limped. Radiographic findings in the hindpaw of CIA rats at 14 weeks showed bone destruction with periosteal new bone formation, and in the two of these rats bony erosion of the forepaw was found. However, radiographic findings in the knee and lumbar vertebra did not show bone destruction (Figure 2). Body weight increased less rapidly in the CIA group than in controls, but no significant difference between the two groups was apparent by 14 weeks (Table 1). In the proximal tibia (Table 2), BV/TV was significantly diminished at 6 weeks in the CIA group, and changed little subsequently. The Tb.N showed changes similar to BV/TV in the CIA group, and the Tb.Th tended to be less than in controls. The OV/TV was significantly diminished at 4 and 6 weeks in the CIA group. Although the Ob.S/BS did not differ significantly at 4 and
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Figure 2. Radiographic findings in the CIA rats at 14 weeks after immunization.
6 weeks, it was significantly increased at 14 weeks in the CIA group. The ES/BS was significantly increased about two times at 4 weeks, and Oc.S/BS and N.Oc/BS were significantly increased about four times at 4 weeks in the CIA group. At 6 weeks, however, no increase in osteoclasts was shown. The MAR and BFR/BS0 were significantly reduced in CIA rats at 4 weeks, but the dLS/BS and BFR/BS0 were relatively increased at 14 weeks compared with control values. In the L-5 vertebra (Table 3), BV/TV also was diminished at 6 weeks in the CIA groups but had recovered at 14 weeks. The values of Tb.Th, Tb.N, OV/TV, Ob.S/BS, ES/BS, and Oc.S/BS did not differ significantly from those in controls. The value of N.Oc/BS in L-5 vertebra was significantly increased in the CIA group at 6 weeks compared to controls. Osteoclasts recruitment occurred later in the vertebra than in the proximal tibia. The value of BFR/BS0 was significantly increased at 6 weeks, and the elevated value was maintained in later observations. The change in dLS/BS was similar to the change in the BFR/BS0. The MAR did not differ remarkably between CIA and control groups. Discussion Our results have shown a highly significant decrease in bone mass in growing rats with CIA relative to findings in controls of similar age, and provided the first unequivocal histomorphometric evidence of decreased BV/TV and increased N.Oc/BS in the proximal tibia and lumbar vertebra. Bone loss in the early phase of the proximal tibia was reflected by a significant increase in number of osteoclasts and in a temporary decrease in the bone Table 1. Body weight in vehicle control and collagen-induced arthritis (CIA) rats at 0, 4, 6, and 14 week after injection Week 0 4 6 14
Control
CIA
p value
163.2 6 12.1 (n 5 15) 271.8 6 28.0 (n 5 15) 292.0 6 23.8 (n 5 10) 323.7 6 24.2 (n 5 5)
161.1 6 8.8 (n 5 15) 230.9 6 20.2 (n 5 15) 263.3 6 20.7 (n 5 10) 314.7 6 28.2 (n 5 5)
n.s. ,0.001 ,0.05 n.s.
Data are expressed as the mean value 6 standard deviation. KEY: n.s., not significant.
formation rate. Bone loss in the secondary spongiosa of the proximal tibia continued for at least 14 weeks after immunization, and the percentage of the reduction measured by BV/TV became 67% at 14 weeks. At 14 weeks, although the Ob.S/BS, dLS/BS, and BFR/BS0 were relatively increased, the Tb.N was still decreased. The value of Tb.Th, however, was increased in the remaining trabecula. In the L-5 vertebra, the percentage reduction measured by BV/TV had reached 26% at 6 weeks, but no significant differences were noted between control and CIA rats by 14 weeks. Phenomena such as the inflammatory process leading to general bone loss have been described as inflammation-mediated osteopenia (IMO) by Minne et al.16 They used transplantation of the cotton wool16 or local subcutaneous injections of magnesium silicate20 to induce systemic inflammatory responses in rats. IMO during acute inflammation in rats is due to a transient inhibition of bone formation. Osteoblasts surface was strikingly reduced during the first week but normalized by the end of the third week. Bone loss in growing rat with CIA was accompanied by bone resorption, but in the IMO bone resorption was unchanged. Collagen-induced arthritis was induced within 10 –14 days after intradermal injection at the base of the tail with native type II collagen emulsified in Freund’s incomplete adjuvant. It has been reported that no swelling of the hindfeet was found in rats treated with type I collagen or denatured type II collagen.23–25 The mechanism by which arthritis is produced is related to humoral and cellular immunoresponses induced by type II collagen and its antibody. The differences of histomorphometric results between the IMO and CIA models may be brought by these immunoresponses. In growing Lewis rats with adjuvant induced arthritis and CIA, it has been reported that an elevation of interleukin (IL)-1 and IL-6 activity in the soluble component of bone marrow and an increase of the myeloid lineage in bone marrow began to rise before the onset of arthritis and increased in relation to the progression of arthritis. These bone marrow changes did not occur in rats immunized with type I collagen or denatured type II collagen.9 IL-1 produced several effects on bone metabolism, including inhibition of bone formation in vitro and stimulation of bone resorption and inhibition of bone formation in vivo.18 IL-6
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Table 2. Histomorphometry data in proximal tibia (n 5 10) 4 weeks Measurements BV/TV (%) Tb.Th (mm) Tb.N (no./mm) OV/TV (%) Ob.S/BS (%) ES/BS (%) Oc.S/BS (%) N.Oc/BS (no./mm) MAR (mm/day) dLS/BS (%) BFR/BS0 (mm3/ mm2/year)
6 weeks
14 weeks
Control
CIA
Control
CIA
Control
CIA
14.60 6 3.09 46.96 6 3.21 3.09 6 0.56 0.63 6 0.21 4.10 6 3.23 26.76 6 5.61 1.90 6 1.58 0.61 6 0.43 2.41 6 0.34 21.09 6 4.10 0.236 6 0.051
10.06 6 6.27 42.33 6 9.74 2.20 6 1.02a 0.30 6 0.17b 5.02 6 5.36 49.32 6 6.43c 8.61 6 2.22c 2.88 6 0.78c 1.54 6 0.42c 15.29 6 7.56 0.130 6 0.068b
20.15 6 7.67 54.61 6 8.19 3.60 6 1.15 0.43 6 0.18 1.40 6 2.05 30.46 6 5.78 2.28 6 1.30 0.81 6 0.42 1.98 6 0.30 14.00 6 5.58 0.137 6 0.054
10.50 6 7.59a 48.61 6 11.32 1.95 6 1.28b 0.19 6 0.12b 0.91 6 1.73 34.04 6 7.78 2.93 6 1.91 1.01 6 0.65 1.71 6 0.38 12.41 6 6.51 0.109 6 0.038
28.95 6 6.62 69.60 6 9.52 4.15 6 0.74 0.09 6 0.08 1.37 6 1.62 22.07 6 6.44 2.80 6 2.61 0.91 6 0.77 1.27 6 0.60 1.99 6 2.52 0.014 6 0.015
9.62 6 7.93c 52.82 6 17.00a 1.65 6 1.03c 0.11 6 0.12 11.49 6 8.16b 26.21 6 3.26 1.91 6 1.46 0.66 6 0.49 1.21 6 0.47 5.91 6 4.76a 0.042 6 0.035a
Data are expressed as the mean value 6 standard deviation. a p , 0.05; bp , 0.01; cp , 0.001, control versus collagen-induced arthritis (CIA). KEY: BV/TV, bone volume; Tb.Th, trabecular thickness; Tb.N, trabecular number; OV/TV, osteoid volume; Ob.S/BS, osteoblast surface; ES/BS, eroded surface; Oc.S/BS, osteoclast surface; N.Oc/BS, osteoclast number; MAR, mineral apposition rate; dLS/BS, double-labeled surface; BFR/BS0, bone formation rate per surface reference.
has many biologic activities and can induce synovial hyperplasia, joint cartilage destruction, osteoporosis, and anemia.12 Close relationships between IL-6 and osteoclasts have been also reported.15 Therefore, bone loss with osteoclast recruitment in our results may be connected with the elevated activities of these cytokines which are found in the bone marrow of animals with arthritis. On the other hand, immobilization for pain and swelling in these rats should be considered, since a long-term period such as 4 –14 weeks was needed to evaluate bone turnover. Unilateral hindlimb tenotomy22,26 or sciatic neurotomy27,28 has been used for many years in the rat as an immobilization model which results in considerable osteopenia. Weinreb et al.28 showed that in the immobilized legs of growing rats, bone loss displays a rapid phase reflected in a significant increase in N.Oc/BS at 30 and 72 h, but not at later times. This model also shows a sustained decrease in MAR and BFR (MAR divided by the number of osteoblasts per millimeter of osteoid surface) throughout the 6 week immobilization period, suggesting reduced osteoblastic activity. In vivo studies have implicated parathyroid hormone (PTH) in the development of immobilization osteope-
nia, since removal of total parathyroid gland alleviates immobilization osteopenia.5,14 However, in our study, increased osteoclast recruitment was prominent not only in the proximal tibia near an arthritic joint, but also in the lumbar vertebra where arthritis had not occurred. The N.Oc/BS was higher and more persistent in our study of CIA than in previously reported immobilization experiments. Parameters of bone formation showed a temporary depression which was not sustained. Since the osteopenia induced by CIA differs from the one in immobilization models, we support the findings that these bone changes are induced not by immobilization due to pain, but by immune reaction. Tissue damage, autoimmune reactions, and endotoxin shock have been associated with a variety of endocrine and metabolic changes, such as activation of the pituitary-adrenal system and altered carbohydrate metabolism. Several cytokines, especially IL-1, IL-6, and tumor necrosis factor-a, act at the level of the hypothalamus, pituitary, and adrenal. Infection and inflammation sometimes are accompanied by an inhibition of reproductive function, such as a decreased level of estrogen.10 In our study, stimulation of osteoclast recruitment was evident in the lumbar
Table 3. Histomorphometry data in lumbar vertebra (n 5 5) 4 weeks Measurement BV/TV (%) Tb.Th (mm) Tb.N (no./mm) OV/TV (%) Ob.S/BS (%) ES/BS (%) Oc.S/BS (%) N.Oc/BS (no./mm) MAR (mm/day) dLS/BS (%) BFR/BS0 (mm3/mm2/year)
6 weeks
14 weeks
Control
CIA
Control
CIA
Control
CIA
30.83 6 3.42 82.97 6 12.77 3.76 6 0.54 0.35 6 0.10 0.29 6 0.41 28.37 6 5.71 2.86 6 0.40 0.89 6 0.19 1.23 6 0.69 8.12 6 3.37 0.048 6 0.032
23.66 6 10.54 69.24 6 17.35 3.30 6 0.67 0.32 6 0.25 0.74 6 0.66 34.26 6 7.00 2.45 6 0.81 0.80 6 0.28 1.28 6 0.38 7.09 6 6.62 0.054 6 0.050
31.58 6 5.61 90.57 6 13.78 3.49 6 0.43 0.11 6 0.04 0.34 6 0.77 27.54 6 2.75 1.95 6 1.21 0.68 6 0.35 1.27 6 0.15 3.56 6 1.46 0.024 6 0.009
23.30 6 0.68a 77.52 6 7.86 3.03 6 0.37 0.32 6 0.20 0.17 6 0.37 27.41 6 4.00 3.08 6 1.10 1.29 6 0.42a 1.36 6 0.12 8.50 6 3.88a 0.062 6 0.030a
30.38 6 3.17 83.77 6 8.13 3.63 6 0.22 0.14 6 0.09 1.60 6 0.73 24.41 6 8.74 0.78 6 0.87 0.24 6 0.28 1.14 6 0.67 2.30 6 0.75 0.014 6 0.011
29.95 6 6.60 86.07 6 11.12 3.48 6 0.60 0.34 6 0.28 4.80 6 3.66 20.24 6 2.92 1.19 6 0.74 0.41 6 0.31 1.46 6 0.21 6.63 6 3.95a 0.053 6 0.035a
Data are expressed as the mean value 6 standard deviation. a p , 0.05, control versus collagen-induced arthritis (CIA). KEY: BV/TV, bone volume; Tb.Th, trabecular thickness; Tb.N, trabecular number; OV/TV, osteoid volume; Ob.S/BS, osteoblast surface; ES/BS, eroded surface; Oc.S/BS, osteoclast surface; N.Oc/BS, osteoclast number; MAR, mineral apposition rate; dLS/BS, double-labeled surface; BFR/BS0, bone formation rate per surface reference.
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vertebra at 6 weeks in CIA rats, and it occurred later in the vertebra than in the proximal tibia. However, while at 14 weeks of CIA bone turnover was increased in CIA rats, cancellous bone loss had been recovered in the lumbar vertebra. We speculate that the stimulation of osteoclast recruitment also brings about a relation to systemic immune reaction, where arthritis is not shown in this vertebra. Furthermore, we speculate that these differences reflect a distance from areas of inflammation. Bonnet et al.3 reported a decrease in bone formation that appears in part responsible for the osteopenia observed in the noninjected site 2 weeks after a single injection of Freund’s complete adjuvant in rats. In their study, acute inflammation occurred around days 4 –5 at the right hindpaw of injection site, followed by a later chronic polyarthritis, which started around day 11 and was well established on day 14. On the other hand, in Langman’s report,13 in which histomorphometry was evaluated at 2 weeks in caudal vertebrae located in the vicinity of the adjuvant injection site, increased bone resorption was described. Bonnet et al. showed that the contrast in these results appears to occur by the difference of the adjuvant injection site, the duration for the evaluation of the disease, and the localization of the bone sample. To explain why the bone formation in vertebra and tibia in the CIA groups at 14 weeks after immunization increased more significantly than in those in the control groups, the following reports are helpful. Bab et al.1 proposed a generalized enhancement of bone formation, since they observed increased formation in rat mandibular condyles during postablation regeneration of tibial bone marrow. The rate of remodeling in the region of a bone defect exceeds the ordinary tissue activity to potentiate tissue healing and local tissue defense reactions. Frost described this reaction as a regional acceleratory phenomenon (RAP).7 Mueller et al.17 investigated the local healing process with rats with a burr hole defect in the left tibia to determine whether this regional enhancement of bone formation would result in a systemic impact on bone metabolism. Both tibiae and the fourth lumbar vertebra showed a significant increase in mineralizing surface, MAR, and bone formation rate. Based on these results, they concluded that a systemic acceleratory phenomenon (SAP) accompanies the RAP. Threntham et al.24 also reported that the radiographic and pathological findings in the hindpaw of CIA rats mirrored the progression from soft-tissue swelling, bone destruction, and prominent periosteal new bone formation, to bony ankylosis. In our study, we speculate that the RAP by these findings in the region of joint destruction would result in a systemic impact on bone metabolism, so that the relatively enhancement of the parameter for bone formation was found in both tibiae and vertebra as mention above, at 14 weeks after immunization. Finally, with histomorphometric analysis of the proximal tibia in the growing rat, the timing and degree of osteoclast recruitment in CIA differed from findings in the inflammatory or neurectomy immobilization model. However, we have not yet studied animals earlier than 4 weeks after immunization, in terms of the onset of osteoclast recruitment. Furthermore, the parameters of bone formation, including the osteoblast surface, might be depressed at the earlier period. On the other hand, both the bone formation rate and double-label surface were significantly increased at later times, without persistence of the increase in the osteoclast number. It will be necessary to study CIA rats longer than 14 weeks after immunization. However, our results suggest that the generalized osteoporosis seen in patients with rheumatoid arthritis reflects not only immobilization due to pain or treatment, but also the disease itself. Collagen-induced arthritis in rats is a unique model not only of autoimmunity, but also of juxta-articular and generalized osteoporosis in rheumatoid arthritis.
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Acknowledgments: The authors thank H. Akazawa and A. Ito for their excellent technical assistance. The study was partially supported by the Niigata Orthopedic International Exchange Fund.
References 1. Bab, I., Gazit, D., Massarawa, J., and Sela, J. Removal of tibial marrow induces increased formation of bone and cartilage in rat mandibular condyle. Calcif Tissue Int 37:551–555; 1985. 2. Baron, R., Tross, R., and Vignery, A. Evidence of sequential remodeling in rat trabecular bone: Morphology, dynamic histomorphometry, and changes during skeletal maturation. Anat Rec 208:137–145; 1984. 3. Bonnet, J., Zerath, E., Picaud, N., Lesur, C., Mattio, A., Tordjman, C., Hott, M., and Marie, P. J. Bone morphometric changes in adjuvant-induced polyarthritic osteopenia in rats: Evidence for an early bone formation defect. J Bone Miner Res 8:659 – 668; 1993. 4. Bressot, C., Meunier, P. J., Chapuy, M. C., Lejeune, E., Edouard, C., and Darby, A. J. Histomorphometric profile, pathophysiology and reversibility of corticosteroid-induced osteoporosis. Metab Bone Dis Rel Res 1:303–311; 1979. 5. Burkhart, J. M. and Jowsey, J. Parathyroid and thyroid hormones in the development of immobilization osteoporosis. Endocrinology 81:1053–1062; 1967. 6. Compston, J. E., Vedi, S., Mellish, R. W. E., Croucher, P., and O’Sullivan, M. M. Reduced bone formation in non-steroid treated patients with rheumatoid arthritis. Ann Rheum Dis 48:483– 487; 1989. 7. Frost, H. M. The regional acceleratory phenomenon: A review. Henry Ford Hosp Med J 31:3–9; 1983. 8. Gough, A. K., Peel, N. F., Eastell, R., Holder, R. L., Lilley, J., and Emery, P. Excretion of pyridinium crosslinks correlates with disease activity and appendicular bone loss in early rheumatoid arthritis. Annu Rheum Dis 53:14 –17; 1994. 9. Hayashida, K., Ochi, T., Fujimoto, M., Owaki, H., Shimaoka, Y., and Ono, K. Bone marrow changes in adjuvant-induced and collagen-induced arthritis: Interleukin-1 and interleukin-6 activity and abnormal myelopoiesis. Arthritis Rheum 35:241–245; 1992. 10. Imura, H., Fukata, J., and Mori, T. Cytokines and endocrine function: An interaction between the immune and neuroendocrine systems. Clin Endocrinol 35:107–115; 1991. 11. Kalu, D. N., Hardin, R. H., Cockerham, R., and Yu, B. P. Aging and dietary modulation of rat skeleton and parathyroid hormone. Endocrinology 115: 1239 –1247; 1984. 12. Kishimoto, T. and Hirano, T. Molecular regulation of B lymphocyte response. Annu Rev Immunol 6:485–512; 1988. 13. Langman, C. B., Ford, K. K., Pachman, L. M., and Glorieux, F. Vitamin D metabolism in rats with adjuvant-induced arthritis. J Bone Miner Res 5:905– 913; 1990. 14. Lindgren, U. The effect of thyroparathyroidectomy on the development of disuse osteoporosis in adult rats. Clin Orthop 118:251–256; 1975. 15. Michalevicz, R., Lifshitz, D., and Revel, M. Interferon beta 2/interleukin-6 and interleukin-3 synergize in stimulating proliferation of human early hematopoietic progenitor cells. Scann Microsc 3:1143–1150; 1989. 16. Minne, H. W., Pfeilschifter, J., Scharla, S., Mutschelknauss, S., Schwartz, A., Krempien, B., and Ziegler, R. Inflammation-mediated osteopenia in the rat: A new animal model for pathological loss of bone mass. Endocrinology 115:50 – 54; 1984. 17. Mueller, M., Schilling, T., Minne, H. W., and Ziegler, R. A systemic acceleratory phenomenon (SAP) accompanies the regional acceleratory phenomenon (RAP) during healing of a bone defect in the rat. J Bone Miner Res 6:401– 410; 1991. 18. Nguyen, L., Dewhirst, F. E., Hauschka, P. V., and Stashenko, P. Interleukin-1 beta stimulates bone resorption and inhibits bone formation in vivo. Lymphokine Cytokine Res 10:15–21; 1991. 19. Parfitt, A. M., Drezner, M. K., Glorieux, F. H., Kanis, J. A., Malluche, H., Meunier, P. J., Ott, S. M., and Recker, R. R. Bone histomorphometry: Standardization of nomenclature, symbols and units. J Bone Miner Res 2:596 – 610; 1987. 20. Pfeilschifter, J., Wu¨ster, C., Vogel, M., Enderes, B., Ziegler, R., and Minne, H. W. Inflammation-mediated osteopenia (IMO) during acute inflammation in rats is due to a transient inhibition of bone formation. Calcif Tissue Int 41:321–325; 1987.
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T. Hanyu et al. Histomorphometry in rats with arthritis
21. Shimizu, S., Shiozawa, S., Shiozawa, K., Imura, S., and Fujita, T. Quantitative histologic studies on the pathogenesis of periarticular osteoporosis in rheumatoid arthritis. Arthritis Rheum 28:25–31; 1985. 22. Thompson, D. D. and Rodan, G. A. Indomethacin inhibition of tenotomyinduced bone resorption in rats. J Bone Miner Res 3:409 – 414; 1988. 23. Trentham, D. E., Dynesius, R. A., and David, J. R. Passive transfer by cells of type II collagen-induced arthritis in rats. J Clin Invest 62:359 –366; 1978. 24. Trentham, D. E., Townes, A. S., and Kang, A. H. Autoimmunity to type II collagen: An experimental model of arthritis. J Exp Med 146:857– 868; 1977. 25. Trentham, D. E., Townes, A. S., Kang, A. H., and David, J. R. Humoral and cellular sensitivity to collagen in type II collagen-induced arthritis in rats. J Clin Invest 61:89 –96; 1978. 26. Turner, R. T. and Bell, N. H. The effects of immobilization on bone histomorphometry in rats. J Bone Miner Res 1:399 – 407; 1986. 27. Tuukkanen, J., Wallmark, B., Jalovaara, P., Takala, T., Sjo¨rgen, S., and Va¨a¨na¨nen, K. Changes induced in growing rat bone by immobilization and remobilization. Bone 12:113–118; 1991. 28. Weinreb, M., Rodan, G. A., and Thompson, D. D. Osteopenia in the immobi-
Bone Vol. 24, No. 5 May 1999:485– 490 lized rat hind limb is associated with increased bone resorption and decreased bone formation. Bone 10:187–194; 1989. 29. Wronski, T. J., Dann, L. M., and Horner, S. L. Time course of vertebral osteopenia in ovariectomized rats. Bone 10:295–301; 1989. 30. Wronski, T. J., Walsh, C. C., and Ignaszewski, L. A. Histologic evidence for osteopenia and increased bone turnover in ovariectomized rats. Bone 7:119 – 123; 1986. 31. Yamamura, M., Nakamura, T., Tsurukami, H., Hijioka, A., Narusawa, K., Ohnishi, H., Ohta, T., and Hosada, K. Local bone turnover in the metaphysis of the proximal tibia and the lumbar vertebra during the early periods after ovariectomy in rats. Calcif Tissue Int 58:52–59; 1996.
Date Received: March 11, 1998 Date Revised: November 25, 1998 Date Accepted: December 22, 1998
Histomorphometric Assessment of Bone Changes in Rats With Type II Collagen-induced Arthritis T. HANYU,* T. CHOTANAPHUTI, K. ARAI, T. TANAKA, and H. E. TAKAHASHI Department of Orthopedic Surgery, Niigata University School of Medicine, Niigata, Japan
patients with rheumatoid arthritis, and the early stages of the process are difficult to study. Rats provide a useful model for osteoporosis, since in these animals bone remodeling occurs,2 the epiphyseal plate of long (axial) bones closes when it matures,29 and peak bone mass is found in aging.11 Numerous studies of immobilization-related bone loss in rats following tenotomy22,26 or nerve sectioning27,28 and of bone loss in ovariectomized rats29 –31 have been reported. On the other hand, there are several reports that bone metabolism is affected in rats during the inflammatory reaction following a local inflammatory stimulus, such as inflammation-mediated osteopenia.16 However, few histomorphometric reports have concerned bone alterations in rats with arthritis. We investigated bone loss in the proximal tibia and lumbar vertebra in rats with type II collagen-induced arthritis (CIA), an important experimental model of rheumatoid arthritis, using histomorphometric analysis. In this study, we aimed to clarify the changes occurring in the secondary spongiosa of rats with collagen-induced arthritis, including differences between the proximal tibia, near an arthritic joint, and the lumbar vertebra, an area not directly involved in this model of arthritis. Finally, in characterizing osteopenic changes as a function of time in arthritic rats, we compared these observations with reported changes in immobilized and inflammatory rats.
Numerous studies have demonstrated bone loss in rats following immobilization by tenotomy or nerve sectioning and following ovariectomy. However, few experiments have focused on bone change in rats with arthritis. We investigated bone loss in the proximal tibia and lumbar vertebra in rats with type II collagen-induced arthritis, an experimental model of rheumatoid arthritis, using histomorphometry. Bone loss in the early phase after immunization reflected a significant increase in numbers of osteoclasts and temporarily decreased bone formation. In the proximal tibia, near an arthritic joint, osteoclast numbers associated with bone trabeculae were increased four times over control numbers 4 weeks after immunization. In the lumbar vertebra, where arthritis was not shown, recruitment of osteoclasts occurred later than in the proximal tibia. With time, in both the proximal tibia and lumbar vertebra bone resorption normalized, but bone formation rate and double-label surface by tetracycline, a parameter reflecting bone formation, were increased above control values. We conclude that differences between the proximal tibia and lumbar vertebra probably reflected resumption of function as well as distance from areas of inflammation. These findings indicate that collageninduced arthritis in rats is a useful model not only of autoimmunity, but also of juxta-articular and generalized osteoporosis in rheumatoid arthritis. (Bone 24:485– 490; 1999) © 1999 by Elsevier Science Inc. All rights reserved.
Materials and Methods
Key Words: Bone formation; Bone resorption; Histomorphometry; Arthritis; Collagen.
Thirty-five virgin female Sprague-Dawley rats were purchased from Charles River Japan (Tokyo, Japan). All rats were 6 weeks old and weighed about 145 g at the beginning of the study. During the experimental period, they were housed in 20 3 15 3 45 cm cages and allowed free access to a commercially available pellet diet (MF; Oriental Yeast, Tokyo, Japan) and deionized water. CIA was induced using the method described by Trentham et al.24 Briefly, 20 rats were injected intradermally at the base of the tail with 1 mg of bovine type II collagen (Cosmo-Bio, Tokyo, Japan) including 0.2 mg of muramyl dipeptide dissolved in 0.5 mL of 0.05 mol/L cold acetic acid and emulsified in 0.5 mL of cold Freund’s incomplete adjuvant (Difco Laboratories, Detroit, MI). The remaining 15 rats were injected with 1 mL of 0.1 mol/L acetic acid as a vehicle control. Rats were observed daily for the onset of arthritis using a joint score based on grading severity for each limb from 0 to 4 according to degree of swelling and periarticular erythema, as described elsewhere.24 Rats with active CIA whose arthritic indices (sum of the score of two hindlimbs) were more than three were used. Animals from CIA and vehicle control groups were killed by cardiac puncture under
Introduction The occurrence of juxta-articular and generalized osteoporosis has been reported in patients with rheumatoid arthritis. Whether the osteoporosis results from the disease itself or from related factors such as steroid treatment and inactivity has been uncertain. An imbalance between bone absorption and formation results in osteopenia, but data have been conflicting regarding the contributions of these two processes in rheumatoid patients, whose osteoporosis has been attributed to increased bone absorption,8,21 decreased bone formation,6 or both.4 Multiple factors appear to be at work in the development of osteoporosis in
Address for correspondence and reprints: Tadamasa Hanyu, M.D., Department of Orthopedic Surgery, Niigata University School of Medicine, Asahimachi-dori 1, Niigata 951-8510, Japan. © 1999 by Elsevier Science Inc. All rights reserved.
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Figure 1. The region of histomorphometric analysis in the proximal tibia (A) and in the lumbar vertebra (B). Villanueva bone stain (original magnification 35).
ether anesthesia 4, 6, and 14 weeks after double labeling with tetracycline using a schedule of 1-2-1-1. The fifth lumbar vertebra and the both tibia were dissected, trimmed, and fixed in 70% alcohol for processing without decalcification. After fixation, the proximal two thirds of the tibia and the L-5 vertebra were immersed in Villanueva bone stain for 3 days. The specimens were dehydrated by sequential changes of ascending concentrations of ethanol and acetone, and then embedded in methyl methacrylate. Then, 5 mm frontal sections were cut with a Jung Model K (Reichert-Jung, Heidelberg, Germany) microtome. Histomorphometric analysis was performed in the secondary spongiosa extending 1.3–3.9 mm distally to the epiphyseal growth plate of the tibia, not including modeling bone (Figure 1a). A minimum of 15 adjacent fields were quantified at a magnification of 3160 in each section after processing by a semiautomatic digitizer (System Supply, Nagano, Japan). A similar histomorphometric analysis was carried out medially in the secondary spongiosa at a distance of 1.3 mm from the upper and lower epiphyseal plate and at a similar distance from the anterior and posterior cortex in the L-5 vertebra (Figure 1b). Bone histomorphometry was performed under a microscope using ultraviolet light (Optiphot, Nikon, Tokyo) to obtain static and dynamic parameters. Parameters for histomorphometry employed in this study, derived from Parfitt and colleagues, have been approved by an ASBMR committee.19 To assess bone structure, trabecular bone volume and osteoid volume were measured relative to total volume (BV/TV and OV/TV), and trabecular thickness (Tb.Th) and trabecular number (Tb.N) were recorded. To measure bone formation, osteoblast surface was quantified relative to bone surface (Ob.S/BS). To measure bone resorption, eroded surface, osteoclast surface, and osteoclast numbers were quantified relative to bone surface (ES/BS, Oc.S/BS, and N.Oc/BS). Cells with one or more nuclei that formed resorption lacunae at the surface of trabeculae were identified as osteoclasts. To study bone
formation, the following parameters were measured and calculated: double-label surface (dLS/BS); single-label surface (sLS/ BS); mineralizing surface (MS/BS0), equal to dLS/BS plus one half of sLS/BS; mineral apposition rate (MAR), calculated as the distance between double labels divided by interval labeling time and then multiplied by p/4; and formation rate per surface reference (BFR/BS0), equal to MS/BS0 times MAR. Results are shown as the mean 6 standard deviation of the mean. Data were compared by a two-factor factorial analysis of variance (two-way ANOVA) between the CIA and control groups. If the analysis showed a significant difference between the two groups, the difference between values at each time point was assessed by Student t-test. p values ,0.05 (two-tailed) were considered significant. Results Sign of arthritis appeared in the hindlimbs of all CIA rats used for further study within 10 –14 days after immunization. The incidence of arthritis was 85% (17 of 20 rats) at 14 days. The joint score was 5.2 6 1.1 (n 5 15) at 4 weeks, 6.0 6 1.5 (n 5 10) at 6 weeks, and 8.2 6 1.5 (n 5 5) at 14 weeks. The rats that developed apparent joint disease limped. Radiographic findings in the hindpaw of CIA rats at 14 weeks showed bone destruction with periosteal new bone formation, and in the two of these rats bony erosion of the forepaw was found. However, radiographic findings in the knee and lumbar vertebra did not show bone destruction (Figure 2). Body weight increased less rapidly in the CIA group than in controls, but no significant difference between the two groups was apparent by 14 weeks (Table 1). In the proximal tibia (Table 2), BV/TV was significantly diminished at 6 weeks in the CIA group, and changed little subsequently. The Tb.N showed changes similar to BV/TV in the CIA group, and the Tb.Th tended to be less than in controls. The OV/TV was significantly diminished at 4 and 6 weeks in the CIA group. Although the Ob.S/BS did not differ significantly at 4 and
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Figure 2. Radiographic findings in the CIA rats at 14 weeks after immunization.
6 weeks, it was significantly increased at 14 weeks in the CIA group. The ES/BS was significantly increased about two times at 4 weeks, and Oc.S/BS and N.Oc/BS were significantly increased about four times at 4 weeks in the CIA group. At 6 weeks, however, no increase in osteoclasts was shown. The MAR and BFR/BS0 were significantly reduced in CIA rats at 4 weeks, but the dLS/BS and BFR/BS0 were relatively increased at 14 weeks compared with control values. In the L-5 vertebra (Table 3), BV/TV also was diminished at 6 weeks in the CIA groups but had recovered at 14 weeks. The values of Tb.Th, Tb.N, OV/TV, Ob.S/BS, ES/BS, and Oc.S/BS did not differ significantly from those in controls. The value of N.Oc/BS in L-5 vertebra was significantly increased in the CIA group at 6 weeks compared to controls. Osteoclasts recruitment occurred later in the vertebra than in the proximal tibia. The value of BFR/BS0 was significantly increased at 6 weeks, and the elevated value was maintained in later observations. The change in dLS/BS was similar to the change in the BFR/BS0. The MAR did not differ remarkably between CIA and control groups. Discussion Our results have shown a highly significant decrease in bone mass in growing rats with CIA relative to findings in controls of similar age, and provided the first unequivocal histomorphometric evidence of decreased BV/TV and increased N.Oc/BS in the proximal tibia and lumbar vertebra. Bone loss in the early phase of the proximal tibia was reflected by a significant increase in number of osteoclasts and in a temporary decrease in the bone Table 1. Body weight in vehicle control and collagen-induced arthritis (CIA) rats at 0, 4, 6, and 14 week after injection Week 0 4 6 14
Control
CIA
p value
163.2 6 12.1 (n 5 15) 271.8 6 28.0 (n 5 15) 292.0 6 23.8 (n 5 10) 323.7 6 24.2 (n 5 5)
161.1 6 8.8 (n 5 15) 230.9 6 20.2 (n 5 15) 263.3 6 20.7 (n 5 10) 314.7 6 28.2 (n 5 5)
n.s. ,0.001 ,0.05 n.s.
Data are expressed as the mean value 6 standard deviation. KEY: n.s., not significant.
formation rate. Bone loss in the secondary spongiosa of the proximal tibia continued for at least 14 weeks after immunization, and the percentage of the reduction measured by BV/TV became 67% at 14 weeks. At 14 weeks, although the Ob.S/BS, dLS/BS, and BFR/BS0 were relatively increased, the Tb.N was still decreased. The value of Tb.Th, however, was increased in the remaining trabecula. In the L-5 vertebra, the percentage reduction measured by BV/TV had reached 26% at 6 weeks, but no significant differences were noted between control and CIA rats by 14 weeks. Phenomena such as the inflammatory process leading to general bone loss have been described as inflammation-mediated osteopenia (IMO) by Minne et al.16 They used transplantation of the cotton wool16 or local subcutaneous injections of magnesium silicate20 to induce systemic inflammatory responses in rats. IMO during acute inflammation in rats is due to a transient inhibition of bone formation. Osteoblasts surface was strikingly reduced during the first week but normalized by the end of the third week. Bone loss in growing rat with CIA was accompanied by bone resorption, but in the IMO bone resorption was unchanged. Collagen-induced arthritis was induced within 10 –14 days after intradermal injection at the base of the tail with native type II collagen emulsified in Freund’s incomplete adjuvant. It has been reported that no swelling of the hindfeet was found in rats treated with type I collagen or denatured type II collagen.23–25 The mechanism by which arthritis is produced is related to humoral and cellular immunoresponses induced by type II collagen and its antibody. The differences of histomorphometric results between the IMO and CIA models may be brought by these immunoresponses. In growing Lewis rats with adjuvant induced arthritis and CIA, it has been reported that an elevation of interleukin (IL)-1 and IL-6 activity in the soluble component of bone marrow and an increase of the myeloid lineage in bone marrow began to rise before the onset of arthritis and increased in relation to the progression of arthritis. These bone marrow changes did not occur in rats immunized with type I collagen or denatured type II collagen.9 IL-1 produced several effects on bone metabolism, including inhibition of bone formation in vitro and stimulation of bone resorption and inhibition of bone formation in vivo.18 IL-6
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Table 2. Histomorphometry data in proximal tibia (n 5 10) 4 weeks Measurements BV/TV (%) Tb.Th (mm) Tb.N (no./mm) OV/TV (%) Ob.S/BS (%) ES/BS (%) Oc.S/BS (%) N.Oc/BS (no./mm) MAR (mm/day) dLS/BS (%) BFR/BS0 (mm3/ mm2/year)
6 weeks
14 weeks
Control
CIA
Control
CIA
Control
CIA
14.60 6 3.09 46.96 6 3.21 3.09 6 0.56 0.63 6 0.21 4.10 6 3.23 26.76 6 5.61 1.90 6 1.58 0.61 6 0.43 2.41 6 0.34 21.09 6 4.10 0.236 6 0.051
10.06 6 6.27 42.33 6 9.74 2.20 6 1.02a 0.30 6 0.17b 5.02 6 5.36 49.32 6 6.43c 8.61 6 2.22c 2.88 6 0.78c 1.54 6 0.42c 15.29 6 7.56 0.130 6 0.068b
20.15 6 7.67 54.61 6 8.19 3.60 6 1.15 0.43 6 0.18 1.40 6 2.05 30.46 6 5.78 2.28 6 1.30 0.81 6 0.42 1.98 6 0.30 14.00 6 5.58 0.137 6 0.054
10.50 6 7.59a 48.61 6 11.32 1.95 6 1.28b 0.19 6 0.12b 0.91 6 1.73 34.04 6 7.78 2.93 6 1.91 1.01 6 0.65 1.71 6 0.38 12.41 6 6.51 0.109 6 0.038
28.95 6 6.62 69.60 6 9.52 4.15 6 0.74 0.09 6 0.08 1.37 6 1.62 22.07 6 6.44 2.80 6 2.61 0.91 6 0.77 1.27 6 0.60 1.99 6 2.52 0.014 6 0.015
9.62 6 7.93c 52.82 6 17.00a 1.65 6 1.03c 0.11 6 0.12 11.49 6 8.16b 26.21 6 3.26 1.91 6 1.46 0.66 6 0.49 1.21 6 0.47 5.91 6 4.76a 0.042 6 0.035a
Data are expressed as the mean value 6 standard deviation. a p , 0.05; bp , 0.01; cp , 0.001, control versus collagen-induced arthritis (CIA). KEY: BV/TV, bone volume; Tb.Th, trabecular thickness; Tb.N, trabecular number; OV/TV, osteoid volume; Ob.S/BS, osteoblast surface; ES/BS, eroded surface; Oc.S/BS, osteoclast surface; N.Oc/BS, osteoclast number; MAR, mineral apposition rate; dLS/BS, double-labeled surface; BFR/BS0, bone formation rate per surface reference.
has many biologic activities and can induce synovial hyperplasia, joint cartilage destruction, osteoporosis, and anemia.12 Close relationships between IL-6 and osteoclasts have been also reported.15 Therefore, bone loss with osteoclast recruitment in our results may be connected with the elevated activities of these cytokines which are found in the bone marrow of animals with arthritis. On the other hand, immobilization for pain and swelling in these rats should be considered, since a long-term period such as 4 –14 weeks was needed to evaluate bone turnover. Unilateral hindlimb tenotomy22,26 or sciatic neurotomy27,28 has been used for many years in the rat as an immobilization model which results in considerable osteopenia. Weinreb et al.28 showed that in the immobilized legs of growing rats, bone loss displays a rapid phase reflected in a significant increase in N.Oc/BS at 30 and 72 h, but not at later times. This model also shows a sustained decrease in MAR and BFR (MAR divided by the number of osteoblasts per millimeter of osteoid surface) throughout the 6 week immobilization period, suggesting reduced osteoblastic activity. In vivo studies have implicated parathyroid hormone (PTH) in the development of immobilization osteope-
nia, since removal of total parathyroid gland alleviates immobilization osteopenia.5,14 However, in our study, increased osteoclast recruitment was prominent not only in the proximal tibia near an arthritic joint, but also in the lumbar vertebra where arthritis had not occurred. The N.Oc/BS was higher and more persistent in our study of CIA than in previously reported immobilization experiments. Parameters of bone formation showed a temporary depression which was not sustained. Since the osteopenia induced by CIA differs from the one in immobilization models, we support the findings that these bone changes are induced not by immobilization due to pain, but by immune reaction. Tissue damage, autoimmune reactions, and endotoxin shock have been associated with a variety of endocrine and metabolic changes, such as activation of the pituitary-adrenal system and altered carbohydrate metabolism. Several cytokines, especially IL-1, IL-6, and tumor necrosis factor-a, act at the level of the hypothalamus, pituitary, and adrenal. Infection and inflammation sometimes are accompanied by an inhibition of reproductive function, such as a decreased level of estrogen.10 In our study, stimulation of osteoclast recruitment was evident in the lumbar
Table 3. Histomorphometry data in lumbar vertebra (n 5 5) 4 weeks Measurement BV/TV (%) Tb.Th (mm) Tb.N (no./mm) OV/TV (%) Ob.S/BS (%) ES/BS (%) Oc.S/BS (%) N.Oc/BS (no./mm) MAR (mm/day) dLS/BS (%) BFR/BS0 (mm3/mm2/year)
6 weeks
14 weeks
Control
CIA
Control
CIA
Control
CIA
30.83 6 3.42 82.97 6 12.77 3.76 6 0.54 0.35 6 0.10 0.29 6 0.41 28.37 6 5.71 2.86 6 0.40 0.89 6 0.19 1.23 6 0.69 8.12 6 3.37 0.048 6 0.032
23.66 6 10.54 69.24 6 17.35 3.30 6 0.67 0.32 6 0.25 0.74 6 0.66 34.26 6 7.00 2.45 6 0.81 0.80 6 0.28 1.28 6 0.38 7.09 6 6.62 0.054 6 0.050
31.58 6 5.61 90.57 6 13.78 3.49 6 0.43 0.11 6 0.04 0.34 6 0.77 27.54 6 2.75 1.95 6 1.21 0.68 6 0.35 1.27 6 0.15 3.56 6 1.46 0.024 6 0.009
23.30 6 0.68a 77.52 6 7.86 3.03 6 0.37 0.32 6 0.20 0.17 6 0.37 27.41 6 4.00 3.08 6 1.10 1.29 6 0.42a 1.36 6 0.12 8.50 6 3.88a 0.062 6 0.030a
30.38 6 3.17 83.77 6 8.13 3.63 6 0.22 0.14 6 0.09 1.60 6 0.73 24.41 6 8.74 0.78 6 0.87 0.24 6 0.28 1.14 6 0.67 2.30 6 0.75 0.014 6 0.011
29.95 6 6.60 86.07 6 11.12 3.48 6 0.60 0.34 6 0.28 4.80 6 3.66 20.24 6 2.92 1.19 6 0.74 0.41 6 0.31 1.46 6 0.21 6.63 6 3.95a 0.053 6 0.035a
Data are expressed as the mean value 6 standard deviation. a p , 0.05, control versus collagen-induced arthritis (CIA). KEY: BV/TV, bone volume; Tb.Th, trabecular thickness; Tb.N, trabecular number; OV/TV, osteoid volume; Ob.S/BS, osteoblast surface; ES/BS, eroded surface; Oc.S/BS, osteoclast surface; N.Oc/BS, osteoclast number; MAR, mineral apposition rate; dLS/BS, double-labeled surface; BFR/BS0, bone formation rate per surface reference.
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vertebra at 6 weeks in CIA rats, and it occurred later in the vertebra than in the proximal tibia. However, while at 14 weeks of CIA bone turnover was increased in CIA rats, cancellous bone loss had been recovered in the lumbar vertebra. We speculate that the stimulation of osteoclast recruitment also brings about a relation to systemic immune reaction, where arthritis is not shown in this vertebra. Furthermore, we speculate that these differences reflect a distance from areas of inflammation. Bonnet et al.3 reported a decrease in bone formation that appears in part responsible for the osteopenia observed in the noninjected site 2 weeks after a single injection of Freund’s complete adjuvant in rats. In their study, acute inflammation occurred around days 4 –5 at the right hindpaw of injection site, followed by a later chronic polyarthritis, which started around day 11 and was well established on day 14. On the other hand, in Langman’s report,13 in which histomorphometry was evaluated at 2 weeks in caudal vertebrae located in the vicinity of the adjuvant injection site, increased bone resorption was described. Bonnet et al. showed that the contrast in these results appears to occur by the difference of the adjuvant injection site, the duration for the evaluation of the disease, and the localization of the bone sample. To explain why the bone formation in vertebra and tibia in the CIA groups at 14 weeks after immunization increased more significantly than in those in the control groups, the following reports are helpful. Bab et al.1 proposed a generalized enhancement of bone formation, since they observed increased formation in rat mandibular condyles during postablation regeneration of tibial bone marrow. The rate of remodeling in the region of a bone defect exceeds the ordinary tissue activity to potentiate tissue healing and local tissue defense reactions. Frost described this reaction as a regional acceleratory phenomenon (RAP).7 Mueller et al.17 investigated the local healing process with rats with a burr hole defect in the left tibia to determine whether this regional enhancement of bone formation would result in a systemic impact on bone metabolism. Both tibiae and the fourth lumbar vertebra showed a significant increase in mineralizing surface, MAR, and bone formation rate. Based on these results, they concluded that a systemic acceleratory phenomenon (SAP) accompanies the RAP. Threntham et al.24 also reported that the radiographic and pathological findings in the hindpaw of CIA rats mirrored the progression from soft-tissue swelling, bone destruction, and prominent periosteal new bone formation, to bony ankylosis. In our study, we speculate that the RAP by these findings in the region of joint destruction would result in a systemic impact on bone metabolism, so that the relatively enhancement of the parameter for bone formation was found in both tibiae and vertebra as mention above, at 14 weeks after immunization. Finally, with histomorphometric analysis of the proximal tibia in the growing rat, the timing and degree of osteoclast recruitment in CIA differed from findings in the inflammatory or neurectomy immobilization model. However, we have not yet studied animals earlier than 4 weeks after immunization, in terms of the onset of osteoclast recruitment. Furthermore, the parameters of bone formation, including the osteoblast surface, might be depressed at the earlier period. On the other hand, both the bone formation rate and double-label surface were significantly increased at later times, without persistence of the increase in the osteoclast number. It will be necessary to study CIA rats longer than 14 weeks after immunization. However, our results suggest that the generalized osteoporosis seen in patients with rheumatoid arthritis reflects not only immobilization due to pain or treatment, but also the disease itself. Collagen-induced arthritis in rats is a unique model not only of autoimmunity, but also of juxta-articular and generalized osteoporosis in rheumatoid arthritis.
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Acknowledgments: The authors thank H. Akazawa and A. Ito for their excellent technical assistance. The study was partially supported by the Niigata Orthopedic International Exchange Fund.
References 1. Bab, I., Gazit, D., Massarawa, J., and Sela, J. Removal of tibial marrow induces increased formation of bone and cartilage in rat mandibular condyle. Calcif Tissue Int 37:551–555; 1985. 2. Baron, R., Tross, R., and Vignery, A. Evidence of sequential remodeling in rat trabecular bone: Morphology, dynamic histomorphometry, and changes during skeletal maturation. Anat Rec 208:137–145; 1984. 3. Bonnet, J., Zerath, E., Picaud, N., Lesur, C., Mattio, A., Tordjman, C., Hott, M., and Marie, P. J. Bone morphometric changes in adjuvant-induced polyarthritic osteopenia in rats: Evidence for an early bone formation defect. J Bone Miner Res 8:659 – 668; 1993. 4. Bressot, C., Meunier, P. J., Chapuy, M. C., Lejeune, E., Edouard, C., and Darby, A. J. Histomorphometric profile, pathophysiology and reversibility of corticosteroid-induced osteoporosis. Metab Bone Dis Rel Res 1:303–311; 1979. 5. Burkhart, J. M. and Jowsey, J. Parathyroid and thyroid hormones in the development of immobilization osteoporosis. Endocrinology 81:1053–1062; 1967. 6. Compston, J. E., Vedi, S., Mellish, R. W. E., Croucher, P., and O’Sullivan, M. M. Reduced bone formation in non-steroid treated patients with rheumatoid arthritis. Ann Rheum Dis 48:483– 487; 1989. 7. Frost, H. M. The regional acceleratory phenomenon: A review. Henry Ford Hosp Med J 31:3–9; 1983. 8. Gough, A. K., Peel, N. F., Eastell, R., Holder, R. L., Lilley, J., and Emery, P. Excretion of pyridinium crosslinks correlates with disease activity and appendicular bone loss in early rheumatoid arthritis. Annu Rheum Dis 53:14 –17; 1994. 9. Hayashida, K., Ochi, T., Fujimoto, M., Owaki, H., Shimaoka, Y., and Ono, K. Bone marrow changes in adjuvant-induced and collagen-induced arthritis: Interleukin-1 and interleukin-6 activity and abnormal myelopoiesis. Arthritis Rheum 35:241–245; 1992. 10. Imura, H., Fukata, J., and Mori, T. Cytokines and endocrine function: An interaction between the immune and neuroendocrine systems. Clin Endocrinol 35:107–115; 1991. 11. Kalu, D. N., Hardin, R. H., Cockerham, R., and Yu, B. P. Aging and dietary modulation of rat skeleton and parathyroid hormone. Endocrinology 115: 1239 –1247; 1984. 12. Kishimoto, T. and Hirano, T. Molecular regulation of B lymphocyte response. Annu Rev Immunol 6:485–512; 1988. 13. Langman, C. B., Ford, K. K., Pachman, L. M., and Glorieux, F. Vitamin D metabolism in rats with adjuvant-induced arthritis. J Bone Miner Res 5:905– 913; 1990. 14. Lindgren, U. The effect of thyroparathyroidectomy on the development of disuse osteoporosis in adult rats. Clin Orthop 118:251–256; 1975. 15. Michalevicz, R., Lifshitz, D., and Revel, M. Interferon beta 2/interleukin-6 and interleukin-3 synergize in stimulating proliferation of human early hematopoietic progenitor cells. Scann Microsc 3:1143–1150; 1989. 16. Minne, H. W., Pfeilschifter, J., Scharla, S., Mutschelknauss, S., Schwartz, A., Krempien, B., and Ziegler, R. Inflammation-mediated osteopenia in the rat: A new animal model for pathological loss of bone mass. Endocrinology 115:50 – 54; 1984. 17. Mueller, M., Schilling, T., Minne, H. W., and Ziegler, R. A systemic acceleratory phenomenon (SAP) accompanies the regional acceleratory phenomenon (RAP) during healing of a bone defect in the rat. J Bone Miner Res 6:401– 410; 1991. 18. Nguyen, L., Dewhirst, F. E., Hauschka, P. V., and Stashenko, P. Interleukin-1 beta stimulates bone resorption and inhibits bone formation in vivo. Lymphokine Cytokine Res 10:15–21; 1991. 19. Parfitt, A. M., Drezner, M. K., Glorieux, F. H., Kanis, J. A., Malluche, H., Meunier, P. J., Ott, S. M., and Recker, R. R. Bone histomorphometry: Standardization of nomenclature, symbols and units. J Bone Miner Res 2:596 – 610; 1987. 20. Pfeilschifter, J., Wu¨ster, C., Vogel, M., Enderes, B., Ziegler, R., and Minne, H. W. Inflammation-mediated osteopenia (IMO) during acute inflammation in rats is due to a transient inhibition of bone formation. Calcif Tissue Int 41:321–325; 1987.
490
T. Hanyu et al. Histomorphometry in rats with arthritis
21. Shimizu, S., Shiozawa, S., Shiozawa, K., Imura, S., and Fujita, T. Quantitative histologic studies on the pathogenesis of periarticular osteoporosis in rheumatoid arthritis. Arthritis Rheum 28:25–31; 1985. 22. Thompson, D. D. and Rodan, G. A. Indomethacin inhibition of tenotomyinduced bone resorption in rats. J Bone Miner Res 3:409 – 414; 1988. 23. Trentham, D. E., Dynesius, R. A., and David, J. R. Passive transfer by cells of type II collagen-induced arthritis in rats. J Clin Invest 62:359 –366; 1978. 24. Trentham, D. E., Townes, A. S., and Kang, A. H. Autoimmunity to type II collagen: An experimental model of arthritis. J Exp Med 146:857– 868; 1977. 25. Trentham, D. E., Townes, A. S., Kang, A. H., and David, J. R. Humoral and cellular sensitivity to collagen in type II collagen-induced arthritis in rats. J Clin Invest 61:89 –96; 1978. 26. Turner, R. T. and Bell, N. H. The effects of immobilization on bone histomorphometry in rats. J Bone Miner Res 1:399 – 407; 1986. 27. Tuukkanen, J., Wallmark, B., Jalovaara, P., Takala, T., Sjo¨rgen, S., and Va¨a¨na¨nen, K. Changes induced in growing rat bone by immobilization and remobilization. Bone 12:113–118; 1991. 28. Weinreb, M., Rodan, G. A., and Thompson, D. D. Osteopenia in the immobi-
Bone Vol. 24, No. 5 May 1999:485– 490 lized rat hind limb is associated with increased bone resorption and decreased bone formation. Bone 10:187–194; 1989. 29. Wronski, T. J., Dann, L. M., and Horner, S. L. Time course of vertebral osteopenia in ovariectomized rats. Bone 10:295–301; 1989. 30. Wronski, T. J., Walsh, C. C., and Ignaszewski, L. A. Histologic evidence for osteopenia and increased bone turnover in ovariectomized rats. Bone 7:119 – 123; 1986. 31. Yamamura, M., Nakamura, T., Tsurukami, H., Hijioka, A., Narusawa, K., Ohnishi, H., Ohta, T., and Hosada, K. Local bone turnover in the metaphysis of the proximal tibia and the lumbar vertebra during the early periods after ovariectomy in rats. Calcif Tissue Int 58:52–59; 1996.
Date Received: March 11, 1998 Date Revised: November 25, 1998 Date Accepted: December 22, 1998