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A robust model of adjuvant-induced chronic unilateral arthritis in two mouse strains
Journal of Neuroscience Methods 139 (2004) 281–291
A robust model of adjuvant-induced chronic unilateral arthritis in two mouse strains Stephan D. Gauldie a,∗ , Daniel S. McQueen a , Christopher J. Clarke b , Iain P. Chessell c a
Department of Neuroscience, University of Edinburgh, 1 George Square, Edinburgh, EH8 9JZ, UK b Pathology Department, Safety Assessment, GlaxoSmithKline, Park Road, Ware, SG12 0DP, UK c Neurology CEDD, GlaxoSmithKline, Third Avenue, Harlow, CM19 5AW, UK Received 9 February 2004; received in revised form 7 May 2004; accepted 12 May 2004
Abstract We have developed a model of unilateral adjuvant-arthritis that is reproducible across two mouse strains. DBA/1 and C57BL/6 male mice were injected intra-articularly into a stifle joint with FCA (5 g in 5 l) once per week for 4 weeks. Measurements of joint diameter and joint histopathology were used to monitor the course of the arthritis. Inflammatory hyperalgesia was assessed as the pressure causing a limb withdrawal. FCA injection into the mouse stifle joint caused a pronounced increase in joint diameter when compared to the contralateral limb or vehicle controls. There was also a significant decrease in the pressure required to elicit a withdrawal of the injected limb. Histology showed arthritic changes, including synovial hypertrophy and polymorphonuclear neutrophil infiltration. In established chronic inflammation, seven days of treatment with either indomethacin (NSAID) or prednisolone (steroid) caused a significant decrease in joint inflammation and associated hyperalgesia. FCA induces a long-lasting joint inflammation in DBA/1 and C57BL/6 mice, which is restricted to the injected joint and exhibits some of the pathology associated with an arthritic condition. This model will be useful in examining the effect of joint inflammation on nociceptor sensitisation in both normal and transgenic mice. © 2004 Elsevier B.V. All rights reserved. Keywords: Arthritis; Nociception; Adjuvant; Mouse; Knee joint; Prednisolone; Indomethacin
1. Introduction Arthritis is an umbrella term describing more than 100 recognised conditions that, in the US alone, collectively affect approximately 70 million adults and 300,000 children (Arthritis Foundation, 2003). Rheumatoid arthritis (RA) is thought to affect approximately 1% of the population worldwide (Kelley et al., 1997), while osteoarthritis (OA) is the most common form of arthritis, affecting approximately 40 million in the US (Oddis, 1996). Although generally associated with the elderly, radiographic studies find 30–90% of American adults over the age of 75 years have osteoarthritis in at least one joint (Lawrence et al., 1998); arthritis in its various forms can affect individuals of any age. The common property grouping these 100-plus conditions is the targeting of the musculoskeletal system and specifically the joints. ∗ Corresponding author. Tel.: +44-131-650-3512; fax: +44-131-650-6530. E-mail address: [email protected] (S.D. Gauldie).
0165-0270/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2004.05.003
Arthritis-related joint problems include oedema, inflammation, and damage to the cartilage and surrounding structures. We are particularly interested in how these chronic insults affect sensory nerves innervating the arthritic joint and are studying how afferent sensitivity is altered in arthritis. To better investigate arthritic conditions, a number of animal models of chronic joint inflammation have been developed. Typically these models are based either on a loss of tolerance to a cartilage-specific auto-antigen or T-cell cross-reactivity to bacterial fragments by the systemic administration of collagen type II or Freund’s Complete Adjuvant (FCA, Mycobacterium tuberculosis). Either method produces polyarthritis with severe damage to many joints with great discomfort caused to the animals. In addition, using these systemic protocols in mice, there is a large genetic component to the inflammation and associated nociceptive responses that lead to susceptible and non-susceptible strains (Holmdahl et al., 1988; Mogil et al., 1998, 1999). This is of particular importance when examining transgenic lines that can be on varied genetic backgrounds.
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Less severe models of arthritis focus on the production of a joint specific, or unilateral, inflammation (Donaldson et al., 1993; Ferrell et al., 2003). Inflammation is induced by immunisation and challenge with an antigen such as bovine serum albumin (BSA) or the antigenic component of bacterial cell walls in existing models. Our laboratory has described a model of a unilateral arthritis using FCA injected directly into the stifle joint (Donaldson et al., 1993). The stifle joint is a focus for us because it permits electrophysiological recording from nociceptive afferents in the medial articular nerve that innervate the joint and surrounding tissue. This model permits the correlation of behavioural hyperalgesia data with changes in the sensory afferent sensitivity, and can provide valuable insight into the relationship between chronic inflammation and joint pain. This rat model, however, only enables us to use drugs to characterise pharmacological receptors involved in hyperalgesia. The aim of the present study was to produce a similar model in the mouse that would permit the use of transgenic animals to examine targets for which there are no pharmacological tools.
to a mild noxious stimulus (see below). Measurements were taken three times a week, and when they coincided with the injection of adjuvant, the measurements were completed prior to injection. The same experienced operator performed all measurements and used his experience to apply consistent stimuli that were subjectively assessed.
2. Methods
2.4. Drug treatment
Experiments were performed in accordance with Home Office regulations and within UK animal welfare guidelines, and received Local Ethics Committee approval. Male C57 Black 6 (C57BL/6; Charles River, UK) and DBA/1 (Harlan, UK) mice (8 weeks old; initial weight ranges: 17–29 g and 16–22 g, respectively) were used to establish a unilateral model of chronic joint inflammation. Animals were housed eight to a cage in a 12-h light/dark environment and were given free access to standard animal feed and water for the duration of the study.
Treatment of established arthritis was carried out using two standard drugs; the steroid prednisolone, and the non-steroidal anti-inflammatory drug indomethacin. Both prednisolone (1 mg kg−1 ) and indomethacin (1 mg kg−1 ) were administered daily by sub-cutaneous (s.c.) injection
2.1. Arthritis induction Animals were injected with either Freund’s Complete Adjuvant (FCA, 1 mg ml−1 Mycobacterium tuberculosis in paraffin oil, Sigma) or vehicle (heavy liquid paraffin oil; controls). Briefly, injections were carried out by transiently anaesthetising the animal (3% halothane in oxygen), and a small incision was made over the stifle joint to allow visual identification of the patella tendon. Using a 30-gauge needle mounted on a 50-l Hamilton syringe, FCA or vehicle (5 l) was injected under the patella tendon and directly into the synovial space of the stifle joint. To achieve a long-lasting inflammation, the injections were repeated four times, under general anaesthesia, at weekly intervals.
2.3. Hyperalgesia score Subjective measures were used to correlate an increased hyperalgesia score with an applied pressure to the joint induced by the injection of FCA. The scale was 0 (normal, high pressure applied) to 3 (very hyperalgesic, very low pressure applied) and was employed in the absence of any rapid, reproducible, and convenient qualitative index. Testing involved squeezing the joint between thumb and forefinger and determining the amount of pressure required for the animal to withdraw the limb (normal end-point) or vocalise (rare). In the majority of cases, in response to gradually increasing applied pressure, the animal withdrew the limb without vocalising.
Table 1 Assessment of histopathological score in sections of mouse stifle jointa Pathology Stifle joint Synovium
Synovial inflammation
Mononuclear cells Polymorphonuclear neutrophils Fibrin/debris Fibroplasia/fibrosis
Cartilage
Pannus Erosion/destruction Cartilaginous hyperplasia
Bone
Resorption/destruction New bone formation Ankylosis
Periarticular tissue Muscle and ligaments
2.2. Assessment of arthritis Animals were weighed, the diameter across the joint (left and right), just below the level of the patella, was measured using hand-held micro-callipers (Mitutoyo, Japan; accurate to 0.01 mm), and scored for a hyperalgesic response
Synovial hypertrophy Oedema Vascularity
a
Mononuclear cells Polymorphonuclear neutrophils Fibrin/debris Fibroplasia/fibrosis
Criteria examined blind on histological sections to evaluate the extent of inflammation and joint destruction. Each variable was scored arbitrarily on a scale of 0 (no abnormality detected) to 5 (very marked). The histopathological score of a particular joint was given as the sum total of scores for each criterion.
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into the skin at the back of the neck. One treatment protocol began 7 days after the final injection of FCA on experimental day 28 and was continued for a period of 7 days. A second protocol examined the effect of prednisolone on the induction of monoarthritis by dosing animals 1 day before and 3 h before each FCA injection. Drugs were coded before the injections began, and testing was performed in a blinded manner. Each drug was tested alongside a corresponding vehicle control.
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0.6 M sodium formate) for 1 week. Samples were embedded in paraffin wax then cut (3-m sections) and stained with either haematoxylin/eosin or toluidine blue. The severity of the inflammation, joint damage, and characteristic markers of arthritis were arbitrarily and subjectively scored on an analogue scale (0 = no abnormality detected; 1 = very slight to maximum; and 5 = very marked), as detailed in Table 1, with all criteria given equal weighting. 2.6. Data analysis and statistics
2.5. Histopathology Animals were killed (cervical dislocation) on day 28 or 35 (following 7 days drug or vehicle treatment), and the left and right stifle joints surgically removed for histology. In some cases the ipsilateral ankle joint was also removed to check for spread of the inflammation. Joints, excised by cutting through both the femur and the tibia with the skin intact, were placed into screw-top vials containing 30 ml of 10% neutral buffered formalin (Sigma) where they were fixed for a minimum of 5 days at room temperature. Fixed tissue was decalcified in Kristenson’s Fluid (5.1 M formic acid,
Data were collected and analysed using Microsoft Excel, GraphPad Prism and GraphPad Instat software. Unpaired t-tests were used to analyse differences between the means of two normally distributed groups. When the sample size for each group was too small or the data was not normally distributed then the non-parametric Mann-Whitney U-test was used. For paired data, the Student’s paired t-test (parametric) or Wilcoxon U-test (non-parametric) tests were used. To determine differences between the means of more than two normally distributed groups a one-way analysis of variance (ANOVA) was done and a post-hoc test (Tukey’s
Fig. 1. Effect of repeated injection of FCA (at arrows; 5 g in 5 l, i.art.) on inflammation (joint diameter) and hyperalgesia (limb withdrawal to applied pressure) in the stifle joint of DBA/1 (A and B) and C57BL/6 (C and D) mice. Each data point represents the mean ± S.E.M. In both strains of mice, repeated injections of FCA produces a chronic inflammation in the injected stifle joint with a corresponding increase in hyperalgesic sensitivity to an applied pressure. The inflammation and hyperalgesia are not present in the contralateral limb of FCA-treated mice or in either limb of vehicle-treated controls.
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multiple comparison) was performed if the result was deemed to be significant. The medians of two or more groups of non-parametric data were analysed with a Kruskal-Wallis test, and post-hoc analysis was done using Dunn’s multiple comparison. Correlations between normally distributed
groups were determined using a linear (Pearson) correlation, and non-parametric data were compared using a Spearman Rank Correlation. In all cases, the null-hypothesis that the variance between groups could arrive from chance was rejected at the 0.05 level. Therefore, a P-value of less than
Fig. 2. Histology of midline sections from the injected stifle joint of C57BL/6 and DBA/1 mice, at day 28, after repeated injections of vehicle (a, b) or FCA (c–h). Large numbers of inflammatory cells have invaded the joint space and surrounding tissue (peri-articular inflammation; c, d: black arrows). Thickening of the synovial membrane (e, f: black arrows), new bone formation (f: white arrow) and/or bone erosion (h: white arrow), and cartilage destruction (g, h: black arrows). Sections stained with haematoxylin and eosin (b–f) or toluidine blue (a, g, h). Original magnification: (a, b) 40× and (c–h) 100×. JS, joint space; C, cartilage; F, femoral head; and S, synovial membrane.
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0.05 was considered significant, and where possible, the actual P-value was quoted to show proximity to the 0.05 limit.
3. Results 3.1. Unilateral arthritis with repeated injections of FCA in DBA/1 and C57BL/6 mice 3.1.1. Inflammation A single intra-articular injection of FCA into the stifle joint induced an inflammation in DBA/1 and C57BL/6 mice; however, the response was of short duration and resolved to baseline within 7 days (data not shown). To test the possibility that chronic inflammation may be induced by repeated intra-articular injection, as occurs with injection of streptococcal cell walls (Dr. Leo Joosten, personal communication), we examined the effect of repeated injections of FCA. It proved possible to induce chronic unilateral inflammation of the stifle joint in DBA/1 and C57BL/6 mice by repeated injections of FCA (5 g in 5 l i.art.; once per week for 4 weeks) as shown in Fig. 1. Examination of the mice throughout the experiment showed that 100% of animals receiving repeated injections of FCA showed some degree of inflammation, and that in every mouse the inflammation was restricted to the injected left stifle joint with no evidence of inflammation present in the contra-lateral limb (Fig. 1), which was not significantly different from uninjected controls (data not shown). Each subsequent injection produced an acute phase of inflammation that was significantly greater than the previous response, as judged from the diameter of the stifle joint. In addition, as the acute inflammation resolved, the diameter of the stifle joint remained increased above its starting size, indicating that residual chronic changes had occurred. Following four injections of FCA, the joint had swollen markedly and the accumulated changes were consistent with chronic inflammation. At day 28, the diameter of the left (injected) stifle joint was significantly increased in the group treated with FCA when compared to vehicle controls, in both DBA/1 (P = 0.005; unpaired t-test, Fig. 1A) and C57BL/6 mice (P = 0.0001, unpaired t-test, Fig. 1C). This increase in diameter was restricted to the injected joint, and there was no significant difference in the contra-lateral stifle joint between FCA and vehicle treated mice in either strain at day 28 (unpaired t-test). As the inflammation was restricted to the left (injected) stifle joint, the right stifle could be used as an internal control for examining the percent increase in the injected joint (left) when compared to the uninjected (right) joint. The percentage increase in diameter of the joint was greater in FCA-treated animals (16.7 ± 2.7% and 21.2 ± 2.0% in DBA/1 and C57BL/6, respectively) than in vehicle controls (2.9 ± 1.0% and 2.5 ± 1.1%; DBA/1 and C57BL/6, respectively). In both strains, the difference between FCA- and vehicle-treated mice was statistically
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significant (P = 0.001 for both, paired t-test). In addition, the amount of inflammation present at day 28 was equivalent as there was no significant difference between the two strains (unpaired t-test). The injection of FCA and the inflammation produced was well tolerated in both strains as the animals continued to feed and increase in weight normally with no significant difference in body weight between treatment and vehicle groups for either strain at experimental day 0, 14 or 28 (data not shown). 3.1.2. Hyperalgesia DBA/1 mice that received repeated intra-articular injections of FCA showed a decrease in the pressure required to
Fig. 3. Individual histology scores and median (thick line) from left (injected) or right (uninjected) stifle joints from (A) DBA/1 and (B) C57BL/6 mice 7 days after repeated injections (1 per week for 4 weeks) of FCA (5 l at 1 mg ml−1 ) or vehicle (5 l of heavy liquid paraffin). ∗∗ P < 0.01; ∗∗∗ P < 0.001. Repeated intra-articular injections of FCA induce a chronic inflammation with characteristic arthritic changes. The inflammation is restricted to the injected joint.
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evoke a withdrawal (increased hyperalgesia score) at day 28, when compared with the uninjected, contra-lateral joint (P = 0.004; Mann-Whitney) or the vehicle-treated controls (P = 0.004; Mann-Whitney; see Fig. 1B). The same effect was obtained in C57BL/6 mice, in which the hyperalgesia score for the injected limb was significantly greater than that of the contralateral limb (P = 0.0002; Mann-Whitney) or vehicle controls (P = 0.002; Mann-Whitney, Fig. 1D). There was no significant difference between DBA/1 and C57BL/6 strains in their increased responsiveness to applied noxious pressure at day 28 (Mann-Whitney). Overall, there was a significant correlation between the hyperalgesia score and the percentage increase in the diameter of the injected stifle in both DBA/1 (ρ2 = 0.59, P < 0.0001, Spearman Rank Correlation) and C57BL/6 mice (ρ2 = 0.17, P = 0.0003, Spearman Rank Correlation), as shown in Fig. 4A and B.
3.1.3. Histology Pathological changes resulting from repeated injections of FCA or vehicle into the stifle joint of DBA/1 and C57BL/6 mice on day 28 are shown in Fig. 2. In the majority of joints, the uninjected (right) stifle joint showed no changes. In a few instances, mild pathology was noted in the absence of either joint swelling or hyperalgesia, likely the result of injury during injection. All FCA-treated animals had pathological changes in the injected (left) stifle joint. In most cases, there was moderate hypertrophy of the synovial membrane and the infiltration of a large number of polymorphonuclear neutrophils (PMN) and macrophages into the joint space. There was also extensive inflammation in tissues outside the joint (peri-articular inflammation) including tendons, ligaments and surrounding muscle. In many, small amounts of pannus were observed associated with mild erosion of the articular cartilage and erosion of
Fig. 4. Correlation between hyperalgesia score and the percent increase in the injected limb (FCA or vehicle), at day 28, in C57BL/6 (A) and DBA/1 (B) mice. Also shown are the histology scores in relation to the stifle diameter (C and D, respectively), and the histology score related to the hyperalgesia score (E and F, respectively). Data points represent individual values. In C57BL/6 and DBA/1 mice an increase in stifle joint diameter induced by repeated injections of FCA is significantly correlated with an increase in hyperalgesia to an applied pressure and joint pathology.
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bone. Examples of new bone formation were also observed on the head of the tibia. In vehicle-treated mice, some animals showed mild thickening of the synovial membrane, PMN infiltration and peri-articular inflammation. Histological grading of the pathology gave an individual histological score for each animal. In both C57BL/6 and DBA/1 mice treated with FCA, the median histological score was significantly higher for the left (injected) stifle joint when compared to the right (uninjected) stifle (P = 0.0006 for both; Mann-Whitney; see Fig. 3). Also, the median histological score was significantly greater in FCA-injected joints when compared to vehicle controls for both strains (P = 0.002 and P = 0.006 for DBA/1 and C57BL/6, respectively; Mann-Whitney). Comparison of the histological scores showed that there was no significant difference between DBA/1 and C57BL/6 in terms of the overall severity of the pathological changes induced by the intra-articular injections of FCA (Mann-Whitney). In both strains there was a significant correlation between the histology score and (i) the percentage increase in the diameter of the injected stifle (C57BL/6: ρ2 = 0.38, P = 0.01; DBA/1: ρ2 = 0.32, P = 0.04, Spearman Rank Correlation; Fig. 4C and D, respectively) and (ii) the hyperalgesia score (C57BL/6: ρ2 = 0.53, P = 0.001; DBA/1: ρ2 = 0.46, P = 0.01, Spearman Rank Correlation; Fig. 4E and F, respectively). 3.2. Effect of indomethacin and prednisolone on FCA-induced unilateral arthritis in C57BL/6 mice In order to validate this model of murine FCA-induced unilateral arthritis, two standard drugs used for treating arthritis were tested: indomethacin (NSAID) and prednisolone (steroid). 3.3. Indomethacin treatment in established arthritis Chronic unilateral arthritis was induced in two groups (n = 8) and treatment with indomethacin or vehicle began on day 28, 7 days after the final injection of FCA. The level of inflammation present before treatment was similar for both treatment groups with no significant difference in the diameter of the injected joint between groups (unpaired t-test; see Fig. 5A). Seven days of treatment with indomethacin (1 mg kg−1 , s.c. daily) caused a significant decrease in the diameter of the FCA-injected stifle joint when compared to vehicle controls (P = 0.007, unpaired t-test). Similarly, there was a significant decrease in the hyperalgesia score after 7 days of treatment (day 35) with indomethacin when compared to pre-treatment values at day 28 (median: 0 and 2, respectively; n = 8; P = 0.007, Mann-Whitney) and vehicle controls at day 35 (P = 0.049, Mann-Whitney; Fig. 5B). Following indomethacin treatment, stifle joints were removed to examine the pathology. Although the treatment had reduced both the joint diameter and hyperalgesia, joint pathology in the treated group was not found to be
Fig. 5. Effect of indomethacin (1 mg kg−1 , s.c. daily; rectangle) on repeated injections of FCA (at arrows, 5 g in 5 l, i.art. into left stifle joint) in established (A) inflammation (percentage increase in the injected limb), (B) hyperalgesia (limb withdrawal from applied pressure at day 35), and (C) joint pathology (histology score of joint damage at day 35) in C57BL/6 mice. Data points represent the mean ± S.E.M. (A), or individual values (B, C). ∗∗∗ P < 0.001, ∗∗ P < 0.01, ∗ P < 0.05. Indomethacin decreased the joint inflammation and hyperalgesia associated with chronic unilateral arthritis. A decrease in the joint pathology after 7 days of treatment was not statistically significant.
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Fig. 6. Effect of prednisolone (1 mg kg−1 , s.c. daily; rectangle) on repeated injections of FCA (at arrows, 5 g in 5 l, i.art. into left stifle joint) in established (A) inflammation (percentage increase in the injected limb), (B) hyperalgesia (limb withdrawal from applied pressure at day 35), and (C) joint pathology (histology score of joint damage at day 35). (D) Effect of prednisolone (1 mg kg−1 , s.c. daily; rectangle) on the induction of joint inflammation in C57BL/6 mice. Data points represent the mean ± S.E.M. (A, D), or individual values (B, C). ∗∗∗ P < 0.001, ∗∗ P < 0.01, ∗ P < 0.05. Prednisolone decreases the joint inflammation and hyperalgesia associated with chronic unilateral arthritis. Although there is a decrease in the joint pathology in prednisolone-treated animals, it was not found to be statistically significant. Prednisolone also inhibits the ability to induce unilateral arthritis.
statistically significant when compared to vehicle controls (Mann-Whitney; Fig. 5C). 3.4. Prednisolone treatment on established arthritis Chronic arthritis was established in two groups (n = 8) and treatment with prednisolone (1 mg kg−1 , s.c. daily) or vehicle began on day 28, 7 days after the final injection of FCA (Fig. 6A). By day 35, after 7 days of treatment, there was a significant decrease in the diameter of the injected stifle joint when compared to vehicle controls (P = 0.002; unpaired t-test). Once treatment was stopped, the inflammation returned, and 14 days after prednisolone, the joint inflammation had returned to the level of the vehicle-treated group. Fig. 6B shows that following prednisolone treatment there was a significant decrease in the hyperalgesia score of the corresponding vehicle controls at day 35 (P = 0.007, Mann-Whitney) and similarly, there was a significant
decrease when compared to pre-treatment values (median scores: 0 and 1.5, respectively; P = 0.007, MannWhitney). In stifle joints collected following 7 days of treatment with prednisolone, there was no significant difference in the histological score when compared to vehicle controls, (median scores: 7 and 10, respectively; Mann-Whitney; see Fig. 6C). 3.5. Prednisolone treatment on arthritis induction A second experiment looked at the effect of prednisolone on the induction of FCA unilateral arthritis. Prednisolone (1 mg kg−1 , s.c. daily) was given 1 day before, and 3 h before, each injection of FCA. Fig. 6D shows that there was a significant decrease in the diameter of the injected stifle following each treatment with prednisolone when compared to controls; however, overall, on day 28, seven days after the treatment was stopped, the level of inflammation returned to
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control level as there was no significant difference between groups (unpaired t-test).
4. Conclusions The main finding in this study is that it is possible to induce a chronic unilateral arthritis in both DBA/1 and C57BL/6 mice using repeated intra-articular injections of FCA into the stifle joint. The development of this murine model progressed from the rat FCA model of unilateral arthritis previously developed in this laboratory (Donaldson et al., 1993). The DBA/1 and C57BL/6 strains were chosen because they have previously been used in the study of experimentally induced arthritis. DBA/1 mice are the only strain that responds to collagen-induced arthritis (Wooley et al., 1981; Joosten et al., 1996; Joosten et al., 1999; Joosten et al., 2000; Lubberts et al., 2000) and in some cases in aged animals can spontaneously develop an RA-like condition (Holmdahl et al., 1992; Nordling et al., 1992b; Nordling et al., 1992a). C57BL/6 mice have been primarily used in the development of antigen-induced arthritis (Brackertz et al., 1977a; Brackertz et al., 1977b; van de Putte et al., 1983; van Meurs et al., 1999; van Lent et al., 2000). In addition, C57BL/6 mice are widely used as a transgenic background and therefore are ideally suited for the examination of a genetic manipulation on the induction and maintenance of experimental arthritis. As the results show, four injections of 5 g of FCA in a volume of 5 l given at weekly intervals proved able to evoke chronic unilateral arthritis in both DBA/1 and C57BL/6 mice, with the progression of the inflammation and hyperalgesia being broadly similar in both strains. In the initial phase, acute inflammation occurred after each injection; this flared and then resolved within 7 days, as seen with the single intra-articular injection. However, with each subsequent injection, the acute phase response was greater and the resolution of the inflammation was less such that 1 week after the final injection, chronic inflammation was established. The inflammation was restricted to the injected joint, and with the doses of FCA used, there was no swelling in the contra-lateral leg, such as can be induced with a sufficiently high dose of FCA in the rat (Donaldson et al., 1993). Further studies are required to determine whether it is possible to produce the cross-over of inflammation in mice by increasing the dose of FCA. Hyperalgesia induced by inflammation was assessed in terms of the sensitisation of the stifle joint to a noxious stimulus, in this case squeezing the joint between thumb and forefinger. Again due to the very small size of mouse joints, it was not feasible to develop a quantitative measuring device for hyperalgesia as has been previously used in our laboratory in rats. Measurements therefore were based on subjective scoring and conducted blind. The results show that in both DBA/1 and C57BL/6 mice, there is a sensitisation that is restricted to the injected limb, permitting an
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internal control for hyperalgesia. Future work is planned to provide a quantitative measure of the hyperalgesia in the joint to allow the model to be used to examine the effect of genetic alterations in transgenic animals on inflammatory hyperalgesia. The behavioural changes in the response to pain as well as the gross changes in the inflamed joint were correlated with the pathology associated with FCA-treated joints. Overall, the extent of the inflammatory changes in both DBA/1 and C57BL/6 mice was consistent with a mild to moderate arthritis that was restricted to the injected limb. Lack of any pathology in the contra-lateral limb or the ipsilateral ankle (data not shown) meant that the inflammation was indeed isolated to the injected stifle joint. The results show that the primary feature of the histology is the presence of extensive peri-articular inflammation—inflammation of the tissue surrounding the joint capsule and including the muscle, tendons and ligaments. The smaller amount of fibrosis present in peri-articular inflammation, in comparison with the inflammation within the joint, is possibly a sign that the inflammation began within the joint and later moved to surrounding tissues. Large amounts of swelling in the joint, particularly prior to the third and fourth FCA injections, meant that it was not always possible to identify the patellar tendon to ensure that the injection was made directly into the joint space, and it is possible that the later injections were made into the surrounding tissue where they would produce the extra-articular inflammation. In both DBA/1 and C57BL/6 strains, there was a significant correlation between the inflammation induced and the pressure required to evoke a limb withdrawal. Similarly, there was a correlation between the pathology seen in joint and (i) the joint inflammation as determined by stifle diameter, and (ii) the hyperalgesia. These correlations indicate that drugs that decrease the amount of inflammation should also have an effect on the hyperalgesia arising from inflammation of the joint. Proving the model with two “gold standards” further supported this. The “gold standards” used in our study were a steroid, prednisolone, and a non-steroidal anti-inflammatory drug (NSAID), indomethacin, both capable of reducing inflammation and pain in a number of animal models and in clinical situations (Kelley et al., 1997). Both prednisolone and indomethacin at the doses given significantly reduced the inflammation in the injected limb in established arthritis, and prednisolone was also shown to inhibit the induction of experimental arthritis. In all cases, the animals treated with either prednisolone or indomethacin showed a decrease in hyperalgesia when compared to pre-treatment values, and were also significantly decreased when compared to vehicle controls. Although the drugs were able to reduce inflammation and hyperalgesia, the decrease in the histopathology score for indomethacin- and prednisolone-treated groups was not found to be statistically significant when compared to vehicle controls. This is consistent with short-duration
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treatment in an established arthritis, and significant reversal of pathology would not be expected. It has been shown that prednisolone given for an extended period is capable of ameliorating joint damage in both collagen- (Paska et al., 1986; Geiger et al., 1994) and antigen-induced (van den Berg et al., 1991) murine arthritis. Conversely, it has been shown that in these same models, indomethacin does not reverse pathological changes in the joint, even following long-term treatment (de Vries et al., 1988; Cannon et al., 1990; Malfait et al., 2001). Further work will be required to determine if long-term steroid administration can significantly reduce the pathological changes associated with FCA-induced arthritis. The reduction of inflammation and hyperalgesia by the two drugs in the established arthritis means that the FCA-induced unilateral arthritis model could be useful in predicting effective drugs for the treatment of arthritis and chronically inflammatory hyperalgesia. In addition, the model has been used to examine nociceptor sensitisation in an arthritic joint by electrophysiologically recording evoked responses from the medial articular nerve innervating the inflamed joint capsule (manuscript in preparation). In conclusion, these studies show it is possible to induce a chronic, unilateral joint inflammation in both DBA/1 and C57BL/6 mice by repeated intra-articular injections of FCA. The pathology present in these joints is consistent with a mild to moderate arthritis, and it responds well to both prednisolone and indomethacin. With the development of an objective measure of nociception, this model should prove valuable for the examination of chronic inflammatory conditions, the relationship between inflammation and nociception, and how these are altered in transgenic mice.
Acknowledgements We would like to thank Susan Bond for her contribution to animal measurements in this project and Barry Reed for processing the joint tissues for histology. Work supported by a grant from GlaxoSmithKline, UK.
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A robust model of adjuvant-induced chronic unilateral arthritis in two mouse strains Stephan D. Gauldie a,∗ , Daniel S. McQueen a , Christopher J. Clarke b , Iain P. Chessell c a
Department of Neuroscience, University of Edinburgh, 1 George Square, Edinburgh, EH8 9JZ, UK b Pathology Department, Safety Assessment, GlaxoSmithKline, Park Road, Ware, SG12 0DP, UK c Neurology CEDD, GlaxoSmithKline, Third Avenue, Harlow, CM19 5AW, UK Received 9 February 2004; received in revised form 7 May 2004; accepted 12 May 2004
Abstract We have developed a model of unilateral adjuvant-arthritis that is reproducible across two mouse strains. DBA/1 and C57BL/6 male mice were injected intra-articularly into a stifle joint with FCA (5 g in 5 l) once per week for 4 weeks. Measurements of joint diameter and joint histopathology were used to monitor the course of the arthritis. Inflammatory hyperalgesia was assessed as the pressure causing a limb withdrawal. FCA injection into the mouse stifle joint caused a pronounced increase in joint diameter when compared to the contralateral limb or vehicle controls. There was also a significant decrease in the pressure required to elicit a withdrawal of the injected limb. Histology showed arthritic changes, including synovial hypertrophy and polymorphonuclear neutrophil infiltration. In established chronic inflammation, seven days of treatment with either indomethacin (NSAID) or prednisolone (steroid) caused a significant decrease in joint inflammation and associated hyperalgesia. FCA induces a long-lasting joint inflammation in DBA/1 and C57BL/6 mice, which is restricted to the injected joint and exhibits some of the pathology associated with an arthritic condition. This model will be useful in examining the effect of joint inflammation on nociceptor sensitisation in both normal and transgenic mice. © 2004 Elsevier B.V. All rights reserved. Keywords: Arthritis; Nociception; Adjuvant; Mouse; Knee joint; Prednisolone; Indomethacin
1. Introduction Arthritis is an umbrella term describing more than 100 recognised conditions that, in the US alone, collectively affect approximately 70 million adults and 300,000 children (Arthritis Foundation, 2003). Rheumatoid arthritis (RA) is thought to affect approximately 1% of the population worldwide (Kelley et al., 1997), while osteoarthritis (OA) is the most common form of arthritis, affecting approximately 40 million in the US (Oddis, 1996). Although generally associated with the elderly, radiographic studies find 30–90% of American adults over the age of 75 years have osteoarthritis in at least one joint (Lawrence et al., 1998); arthritis in its various forms can affect individuals of any age. The common property grouping these 100-plus conditions is the targeting of the musculoskeletal system and specifically the joints. ∗ Corresponding author. Tel.: +44-131-650-3512; fax: +44-131-650-6530. E-mail address: [email protected] (S.D. Gauldie).
0165-0270/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2004.05.003
Arthritis-related joint problems include oedema, inflammation, and damage to the cartilage and surrounding structures. We are particularly interested in how these chronic insults affect sensory nerves innervating the arthritic joint and are studying how afferent sensitivity is altered in arthritis. To better investigate arthritic conditions, a number of animal models of chronic joint inflammation have been developed. Typically these models are based either on a loss of tolerance to a cartilage-specific auto-antigen or T-cell cross-reactivity to bacterial fragments by the systemic administration of collagen type II or Freund’s Complete Adjuvant (FCA, Mycobacterium tuberculosis). Either method produces polyarthritis with severe damage to many joints with great discomfort caused to the animals. In addition, using these systemic protocols in mice, there is a large genetic component to the inflammation and associated nociceptive responses that lead to susceptible and non-susceptible strains (Holmdahl et al., 1988; Mogil et al., 1998, 1999). This is of particular importance when examining transgenic lines that can be on varied genetic backgrounds.
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Less severe models of arthritis focus on the production of a joint specific, or unilateral, inflammation (Donaldson et al., 1993; Ferrell et al., 2003). Inflammation is induced by immunisation and challenge with an antigen such as bovine serum albumin (BSA) or the antigenic component of bacterial cell walls in existing models. Our laboratory has described a model of a unilateral arthritis using FCA injected directly into the stifle joint (Donaldson et al., 1993). The stifle joint is a focus for us because it permits electrophysiological recording from nociceptive afferents in the medial articular nerve that innervate the joint and surrounding tissue. This model permits the correlation of behavioural hyperalgesia data with changes in the sensory afferent sensitivity, and can provide valuable insight into the relationship between chronic inflammation and joint pain. This rat model, however, only enables us to use drugs to characterise pharmacological receptors involved in hyperalgesia. The aim of the present study was to produce a similar model in the mouse that would permit the use of transgenic animals to examine targets for which there are no pharmacological tools.
to a mild noxious stimulus (see below). Measurements were taken three times a week, and when they coincided with the injection of adjuvant, the measurements were completed prior to injection. The same experienced operator performed all measurements and used his experience to apply consistent stimuli that were subjectively assessed.
2. Methods
2.4. Drug treatment
Experiments were performed in accordance with Home Office regulations and within UK animal welfare guidelines, and received Local Ethics Committee approval. Male C57 Black 6 (C57BL/6; Charles River, UK) and DBA/1 (Harlan, UK) mice (8 weeks old; initial weight ranges: 17–29 g and 16–22 g, respectively) were used to establish a unilateral model of chronic joint inflammation. Animals were housed eight to a cage in a 12-h light/dark environment and were given free access to standard animal feed and water for the duration of the study.
Treatment of established arthritis was carried out using two standard drugs; the steroid prednisolone, and the non-steroidal anti-inflammatory drug indomethacin. Both prednisolone (1 mg kg−1 ) and indomethacin (1 mg kg−1 ) were administered daily by sub-cutaneous (s.c.) injection
2.1. Arthritis induction Animals were injected with either Freund’s Complete Adjuvant (FCA, 1 mg ml−1 Mycobacterium tuberculosis in paraffin oil, Sigma) or vehicle (heavy liquid paraffin oil; controls). Briefly, injections were carried out by transiently anaesthetising the animal (3% halothane in oxygen), and a small incision was made over the stifle joint to allow visual identification of the patella tendon. Using a 30-gauge needle mounted on a 50-l Hamilton syringe, FCA or vehicle (5 l) was injected under the patella tendon and directly into the synovial space of the stifle joint. To achieve a long-lasting inflammation, the injections were repeated four times, under general anaesthesia, at weekly intervals.
2.3. Hyperalgesia score Subjective measures were used to correlate an increased hyperalgesia score with an applied pressure to the joint induced by the injection of FCA. The scale was 0 (normal, high pressure applied) to 3 (very hyperalgesic, very low pressure applied) and was employed in the absence of any rapid, reproducible, and convenient qualitative index. Testing involved squeezing the joint between thumb and forefinger and determining the amount of pressure required for the animal to withdraw the limb (normal end-point) or vocalise (rare). In the majority of cases, in response to gradually increasing applied pressure, the animal withdrew the limb without vocalising.
Table 1 Assessment of histopathological score in sections of mouse stifle jointa Pathology Stifle joint Synovium
Synovial inflammation
Mononuclear cells Polymorphonuclear neutrophils Fibrin/debris Fibroplasia/fibrosis
Cartilage
Pannus Erosion/destruction Cartilaginous hyperplasia
Bone
Resorption/destruction New bone formation Ankylosis
Periarticular tissue Muscle and ligaments
2.2. Assessment of arthritis Animals were weighed, the diameter across the joint (left and right), just below the level of the patella, was measured using hand-held micro-callipers (Mitutoyo, Japan; accurate to 0.01 mm), and scored for a hyperalgesic response
Synovial hypertrophy Oedema Vascularity
a
Mononuclear cells Polymorphonuclear neutrophils Fibrin/debris Fibroplasia/fibrosis
Criteria examined blind on histological sections to evaluate the extent of inflammation and joint destruction. Each variable was scored arbitrarily on a scale of 0 (no abnormality detected) to 5 (very marked). The histopathological score of a particular joint was given as the sum total of scores for each criterion.
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into the skin at the back of the neck. One treatment protocol began 7 days after the final injection of FCA on experimental day 28 and was continued for a period of 7 days. A second protocol examined the effect of prednisolone on the induction of monoarthritis by dosing animals 1 day before and 3 h before each FCA injection. Drugs were coded before the injections began, and testing was performed in a blinded manner. Each drug was tested alongside a corresponding vehicle control.
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0.6 M sodium formate) for 1 week. Samples were embedded in paraffin wax then cut (3-m sections) and stained with either haematoxylin/eosin or toluidine blue. The severity of the inflammation, joint damage, and characteristic markers of arthritis were arbitrarily and subjectively scored on an analogue scale (0 = no abnormality detected; 1 = very slight to maximum; and 5 = very marked), as detailed in Table 1, with all criteria given equal weighting. 2.6. Data analysis and statistics
2.5. Histopathology Animals were killed (cervical dislocation) on day 28 or 35 (following 7 days drug or vehicle treatment), and the left and right stifle joints surgically removed for histology. In some cases the ipsilateral ankle joint was also removed to check for spread of the inflammation. Joints, excised by cutting through both the femur and the tibia with the skin intact, were placed into screw-top vials containing 30 ml of 10% neutral buffered formalin (Sigma) where they were fixed for a minimum of 5 days at room temperature. Fixed tissue was decalcified in Kristenson’s Fluid (5.1 M formic acid,
Data were collected and analysed using Microsoft Excel, GraphPad Prism and GraphPad Instat software. Unpaired t-tests were used to analyse differences between the means of two normally distributed groups. When the sample size for each group was too small or the data was not normally distributed then the non-parametric Mann-Whitney U-test was used. For paired data, the Student’s paired t-test (parametric) or Wilcoxon U-test (non-parametric) tests were used. To determine differences between the means of more than two normally distributed groups a one-way analysis of variance (ANOVA) was done and a post-hoc test (Tukey’s
Fig. 1. Effect of repeated injection of FCA (at arrows; 5 g in 5 l, i.art.) on inflammation (joint diameter) and hyperalgesia (limb withdrawal to applied pressure) in the stifle joint of DBA/1 (A and B) and C57BL/6 (C and D) mice. Each data point represents the mean ± S.E.M. In both strains of mice, repeated injections of FCA produces a chronic inflammation in the injected stifle joint with a corresponding increase in hyperalgesic sensitivity to an applied pressure. The inflammation and hyperalgesia are not present in the contralateral limb of FCA-treated mice or in either limb of vehicle-treated controls.
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multiple comparison) was performed if the result was deemed to be significant. The medians of two or more groups of non-parametric data were analysed with a Kruskal-Wallis test, and post-hoc analysis was done using Dunn’s multiple comparison. Correlations between normally distributed
groups were determined using a linear (Pearson) correlation, and non-parametric data were compared using a Spearman Rank Correlation. In all cases, the null-hypothesis that the variance between groups could arrive from chance was rejected at the 0.05 level. Therefore, a P-value of less than
Fig. 2. Histology of midline sections from the injected stifle joint of C57BL/6 and DBA/1 mice, at day 28, after repeated injections of vehicle (a, b) or FCA (c–h). Large numbers of inflammatory cells have invaded the joint space and surrounding tissue (peri-articular inflammation; c, d: black arrows). Thickening of the synovial membrane (e, f: black arrows), new bone formation (f: white arrow) and/or bone erosion (h: white arrow), and cartilage destruction (g, h: black arrows). Sections stained with haematoxylin and eosin (b–f) or toluidine blue (a, g, h). Original magnification: (a, b) 40× and (c–h) 100×. JS, joint space; C, cartilage; F, femoral head; and S, synovial membrane.
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0.05 was considered significant, and where possible, the actual P-value was quoted to show proximity to the 0.05 limit.
3. Results 3.1. Unilateral arthritis with repeated injections of FCA in DBA/1 and C57BL/6 mice 3.1.1. Inflammation A single intra-articular injection of FCA into the stifle joint induced an inflammation in DBA/1 and C57BL/6 mice; however, the response was of short duration and resolved to baseline within 7 days (data not shown). To test the possibility that chronic inflammation may be induced by repeated intra-articular injection, as occurs with injection of streptococcal cell walls (Dr. Leo Joosten, personal communication), we examined the effect of repeated injections of FCA. It proved possible to induce chronic unilateral inflammation of the stifle joint in DBA/1 and C57BL/6 mice by repeated injections of FCA (5 g in 5 l i.art.; once per week for 4 weeks) as shown in Fig. 1. Examination of the mice throughout the experiment showed that 100% of animals receiving repeated injections of FCA showed some degree of inflammation, and that in every mouse the inflammation was restricted to the injected left stifle joint with no evidence of inflammation present in the contra-lateral limb (Fig. 1), which was not significantly different from uninjected controls (data not shown). Each subsequent injection produced an acute phase of inflammation that was significantly greater than the previous response, as judged from the diameter of the stifle joint. In addition, as the acute inflammation resolved, the diameter of the stifle joint remained increased above its starting size, indicating that residual chronic changes had occurred. Following four injections of FCA, the joint had swollen markedly and the accumulated changes were consistent with chronic inflammation. At day 28, the diameter of the left (injected) stifle joint was significantly increased in the group treated with FCA when compared to vehicle controls, in both DBA/1 (P = 0.005; unpaired t-test, Fig. 1A) and C57BL/6 mice (P = 0.0001, unpaired t-test, Fig. 1C). This increase in diameter was restricted to the injected joint, and there was no significant difference in the contra-lateral stifle joint between FCA and vehicle treated mice in either strain at day 28 (unpaired t-test). As the inflammation was restricted to the left (injected) stifle joint, the right stifle could be used as an internal control for examining the percent increase in the injected joint (left) when compared to the uninjected (right) joint. The percentage increase in diameter of the joint was greater in FCA-treated animals (16.7 ± 2.7% and 21.2 ± 2.0% in DBA/1 and C57BL/6, respectively) than in vehicle controls (2.9 ± 1.0% and 2.5 ± 1.1%; DBA/1 and C57BL/6, respectively). In both strains, the difference between FCA- and vehicle-treated mice was statistically
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significant (P = 0.001 for both, paired t-test). In addition, the amount of inflammation present at day 28 was equivalent as there was no significant difference between the two strains (unpaired t-test). The injection of FCA and the inflammation produced was well tolerated in both strains as the animals continued to feed and increase in weight normally with no significant difference in body weight between treatment and vehicle groups for either strain at experimental day 0, 14 or 28 (data not shown). 3.1.2. Hyperalgesia DBA/1 mice that received repeated intra-articular injections of FCA showed a decrease in the pressure required to
Fig. 3. Individual histology scores and median (thick line) from left (injected) or right (uninjected) stifle joints from (A) DBA/1 and (B) C57BL/6 mice 7 days after repeated injections (1 per week for 4 weeks) of FCA (5 l at 1 mg ml−1 ) or vehicle (5 l of heavy liquid paraffin). ∗∗ P < 0.01; ∗∗∗ P < 0.001. Repeated intra-articular injections of FCA induce a chronic inflammation with characteristic arthritic changes. The inflammation is restricted to the injected joint.
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evoke a withdrawal (increased hyperalgesia score) at day 28, when compared with the uninjected, contra-lateral joint (P = 0.004; Mann-Whitney) or the vehicle-treated controls (P = 0.004; Mann-Whitney; see Fig. 1B). The same effect was obtained in C57BL/6 mice, in which the hyperalgesia score for the injected limb was significantly greater than that of the contralateral limb (P = 0.0002; Mann-Whitney) or vehicle controls (P = 0.002; Mann-Whitney, Fig. 1D). There was no significant difference between DBA/1 and C57BL/6 strains in their increased responsiveness to applied noxious pressure at day 28 (Mann-Whitney). Overall, there was a significant correlation between the hyperalgesia score and the percentage increase in the diameter of the injected stifle in both DBA/1 (ρ2 = 0.59, P < 0.0001, Spearman Rank Correlation) and C57BL/6 mice (ρ2 = 0.17, P = 0.0003, Spearman Rank Correlation), as shown in Fig. 4A and B.
3.1.3. Histology Pathological changes resulting from repeated injections of FCA or vehicle into the stifle joint of DBA/1 and C57BL/6 mice on day 28 are shown in Fig. 2. In the majority of joints, the uninjected (right) stifle joint showed no changes. In a few instances, mild pathology was noted in the absence of either joint swelling or hyperalgesia, likely the result of injury during injection. All FCA-treated animals had pathological changes in the injected (left) stifle joint. In most cases, there was moderate hypertrophy of the synovial membrane and the infiltration of a large number of polymorphonuclear neutrophils (PMN) and macrophages into the joint space. There was also extensive inflammation in tissues outside the joint (peri-articular inflammation) including tendons, ligaments and surrounding muscle. In many, small amounts of pannus were observed associated with mild erosion of the articular cartilage and erosion of
Fig. 4. Correlation between hyperalgesia score and the percent increase in the injected limb (FCA or vehicle), at day 28, in C57BL/6 (A) and DBA/1 (B) mice. Also shown are the histology scores in relation to the stifle diameter (C and D, respectively), and the histology score related to the hyperalgesia score (E and F, respectively). Data points represent individual values. In C57BL/6 and DBA/1 mice an increase in stifle joint diameter induced by repeated injections of FCA is significantly correlated with an increase in hyperalgesia to an applied pressure and joint pathology.
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bone. Examples of new bone formation were also observed on the head of the tibia. In vehicle-treated mice, some animals showed mild thickening of the synovial membrane, PMN infiltration and peri-articular inflammation. Histological grading of the pathology gave an individual histological score for each animal. In both C57BL/6 and DBA/1 mice treated with FCA, the median histological score was significantly higher for the left (injected) stifle joint when compared to the right (uninjected) stifle (P = 0.0006 for both; Mann-Whitney; see Fig. 3). Also, the median histological score was significantly greater in FCA-injected joints when compared to vehicle controls for both strains (P = 0.002 and P = 0.006 for DBA/1 and C57BL/6, respectively; Mann-Whitney). Comparison of the histological scores showed that there was no significant difference between DBA/1 and C57BL/6 in terms of the overall severity of the pathological changes induced by the intra-articular injections of FCA (Mann-Whitney). In both strains there was a significant correlation between the histology score and (i) the percentage increase in the diameter of the injected stifle (C57BL/6: ρ2 = 0.38, P = 0.01; DBA/1: ρ2 = 0.32, P = 0.04, Spearman Rank Correlation; Fig. 4C and D, respectively) and (ii) the hyperalgesia score (C57BL/6: ρ2 = 0.53, P = 0.001; DBA/1: ρ2 = 0.46, P = 0.01, Spearman Rank Correlation; Fig. 4E and F, respectively). 3.2. Effect of indomethacin and prednisolone on FCA-induced unilateral arthritis in C57BL/6 mice In order to validate this model of murine FCA-induced unilateral arthritis, two standard drugs used for treating arthritis were tested: indomethacin (NSAID) and prednisolone (steroid). 3.3. Indomethacin treatment in established arthritis Chronic unilateral arthritis was induced in two groups (n = 8) and treatment with indomethacin or vehicle began on day 28, 7 days after the final injection of FCA. The level of inflammation present before treatment was similar for both treatment groups with no significant difference in the diameter of the injected joint between groups (unpaired t-test; see Fig. 5A). Seven days of treatment with indomethacin (1 mg kg−1 , s.c. daily) caused a significant decrease in the diameter of the FCA-injected stifle joint when compared to vehicle controls (P = 0.007, unpaired t-test). Similarly, there was a significant decrease in the hyperalgesia score after 7 days of treatment (day 35) with indomethacin when compared to pre-treatment values at day 28 (median: 0 and 2, respectively; n = 8; P = 0.007, Mann-Whitney) and vehicle controls at day 35 (P = 0.049, Mann-Whitney; Fig. 5B). Following indomethacin treatment, stifle joints were removed to examine the pathology. Although the treatment had reduced both the joint diameter and hyperalgesia, joint pathology in the treated group was not found to be
Fig. 5. Effect of indomethacin (1 mg kg−1 , s.c. daily; rectangle) on repeated injections of FCA (at arrows, 5 g in 5 l, i.art. into left stifle joint) in established (A) inflammation (percentage increase in the injected limb), (B) hyperalgesia (limb withdrawal from applied pressure at day 35), and (C) joint pathology (histology score of joint damage at day 35) in C57BL/6 mice. Data points represent the mean ± S.E.M. (A), or individual values (B, C). ∗∗∗ P < 0.001, ∗∗ P < 0.01, ∗ P < 0.05. Indomethacin decreased the joint inflammation and hyperalgesia associated with chronic unilateral arthritis. A decrease in the joint pathology after 7 days of treatment was not statistically significant.
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Fig. 6. Effect of prednisolone (1 mg kg−1 , s.c. daily; rectangle) on repeated injections of FCA (at arrows, 5 g in 5 l, i.art. into left stifle joint) in established (A) inflammation (percentage increase in the injected limb), (B) hyperalgesia (limb withdrawal from applied pressure at day 35), and (C) joint pathology (histology score of joint damage at day 35). (D) Effect of prednisolone (1 mg kg−1 , s.c. daily; rectangle) on the induction of joint inflammation in C57BL/6 mice. Data points represent the mean ± S.E.M. (A, D), or individual values (B, C). ∗∗∗ P < 0.001, ∗∗ P < 0.01, ∗ P < 0.05. Prednisolone decreases the joint inflammation and hyperalgesia associated with chronic unilateral arthritis. Although there is a decrease in the joint pathology in prednisolone-treated animals, it was not found to be statistically significant. Prednisolone also inhibits the ability to induce unilateral arthritis.
statistically significant when compared to vehicle controls (Mann-Whitney; Fig. 5C). 3.4. Prednisolone treatment on established arthritis Chronic arthritis was established in two groups (n = 8) and treatment with prednisolone (1 mg kg−1 , s.c. daily) or vehicle began on day 28, 7 days after the final injection of FCA (Fig. 6A). By day 35, after 7 days of treatment, there was a significant decrease in the diameter of the injected stifle joint when compared to vehicle controls (P = 0.002; unpaired t-test). Once treatment was stopped, the inflammation returned, and 14 days after prednisolone, the joint inflammation had returned to the level of the vehicle-treated group. Fig. 6B shows that following prednisolone treatment there was a significant decrease in the hyperalgesia score of the corresponding vehicle controls at day 35 (P = 0.007, Mann-Whitney) and similarly, there was a significant
decrease when compared to pre-treatment values (median scores: 0 and 1.5, respectively; P = 0.007, MannWhitney). In stifle joints collected following 7 days of treatment with prednisolone, there was no significant difference in the histological score when compared to vehicle controls, (median scores: 7 and 10, respectively; Mann-Whitney; see Fig. 6C). 3.5. Prednisolone treatment on arthritis induction A second experiment looked at the effect of prednisolone on the induction of FCA unilateral arthritis. Prednisolone (1 mg kg−1 , s.c. daily) was given 1 day before, and 3 h before, each injection of FCA. Fig. 6D shows that there was a significant decrease in the diameter of the injected stifle following each treatment with prednisolone when compared to controls; however, overall, on day 28, seven days after the treatment was stopped, the level of inflammation returned to
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control level as there was no significant difference between groups (unpaired t-test).
4. Conclusions The main finding in this study is that it is possible to induce a chronic unilateral arthritis in both DBA/1 and C57BL/6 mice using repeated intra-articular injections of FCA into the stifle joint. The development of this murine model progressed from the rat FCA model of unilateral arthritis previously developed in this laboratory (Donaldson et al., 1993). The DBA/1 and C57BL/6 strains were chosen because they have previously been used in the study of experimentally induced arthritis. DBA/1 mice are the only strain that responds to collagen-induced arthritis (Wooley et al., 1981; Joosten et al., 1996; Joosten et al., 1999; Joosten et al., 2000; Lubberts et al., 2000) and in some cases in aged animals can spontaneously develop an RA-like condition (Holmdahl et al., 1992; Nordling et al., 1992b; Nordling et al., 1992a). C57BL/6 mice have been primarily used in the development of antigen-induced arthritis (Brackertz et al., 1977a; Brackertz et al., 1977b; van de Putte et al., 1983; van Meurs et al., 1999; van Lent et al., 2000). In addition, C57BL/6 mice are widely used as a transgenic background and therefore are ideally suited for the examination of a genetic manipulation on the induction and maintenance of experimental arthritis. As the results show, four injections of 5 g of FCA in a volume of 5 l given at weekly intervals proved able to evoke chronic unilateral arthritis in both DBA/1 and C57BL/6 mice, with the progression of the inflammation and hyperalgesia being broadly similar in both strains. In the initial phase, acute inflammation occurred after each injection; this flared and then resolved within 7 days, as seen with the single intra-articular injection. However, with each subsequent injection, the acute phase response was greater and the resolution of the inflammation was less such that 1 week after the final injection, chronic inflammation was established. The inflammation was restricted to the injected joint, and with the doses of FCA used, there was no swelling in the contra-lateral leg, such as can be induced with a sufficiently high dose of FCA in the rat (Donaldson et al., 1993). Further studies are required to determine whether it is possible to produce the cross-over of inflammation in mice by increasing the dose of FCA. Hyperalgesia induced by inflammation was assessed in terms of the sensitisation of the stifle joint to a noxious stimulus, in this case squeezing the joint between thumb and forefinger. Again due to the very small size of mouse joints, it was not feasible to develop a quantitative measuring device for hyperalgesia as has been previously used in our laboratory in rats. Measurements therefore were based on subjective scoring and conducted blind. The results show that in both DBA/1 and C57BL/6 mice, there is a sensitisation that is restricted to the injected limb, permitting an
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internal control for hyperalgesia. Future work is planned to provide a quantitative measure of the hyperalgesia in the joint to allow the model to be used to examine the effect of genetic alterations in transgenic animals on inflammatory hyperalgesia. The behavioural changes in the response to pain as well as the gross changes in the inflamed joint were correlated with the pathology associated with FCA-treated joints. Overall, the extent of the inflammatory changes in both DBA/1 and C57BL/6 mice was consistent with a mild to moderate arthritis that was restricted to the injected limb. Lack of any pathology in the contra-lateral limb or the ipsilateral ankle (data not shown) meant that the inflammation was indeed isolated to the injected stifle joint. The results show that the primary feature of the histology is the presence of extensive peri-articular inflammation—inflammation of the tissue surrounding the joint capsule and including the muscle, tendons and ligaments. The smaller amount of fibrosis present in peri-articular inflammation, in comparison with the inflammation within the joint, is possibly a sign that the inflammation began within the joint and later moved to surrounding tissues. Large amounts of swelling in the joint, particularly prior to the third and fourth FCA injections, meant that it was not always possible to identify the patellar tendon to ensure that the injection was made directly into the joint space, and it is possible that the later injections were made into the surrounding tissue where they would produce the extra-articular inflammation. In both DBA/1 and C57BL/6 strains, there was a significant correlation between the inflammation induced and the pressure required to evoke a limb withdrawal. Similarly, there was a correlation between the pathology seen in joint and (i) the joint inflammation as determined by stifle diameter, and (ii) the hyperalgesia. These correlations indicate that drugs that decrease the amount of inflammation should also have an effect on the hyperalgesia arising from inflammation of the joint. Proving the model with two “gold standards” further supported this. The “gold standards” used in our study were a steroid, prednisolone, and a non-steroidal anti-inflammatory drug (NSAID), indomethacin, both capable of reducing inflammation and pain in a number of animal models and in clinical situations (Kelley et al., 1997). Both prednisolone and indomethacin at the doses given significantly reduced the inflammation in the injected limb in established arthritis, and prednisolone was also shown to inhibit the induction of experimental arthritis. In all cases, the animals treated with either prednisolone or indomethacin showed a decrease in hyperalgesia when compared to pre-treatment values, and were also significantly decreased when compared to vehicle controls. Although the drugs were able to reduce inflammation and hyperalgesia, the decrease in the histopathology score for indomethacin- and prednisolone-treated groups was not found to be statistically significant when compared to vehicle controls. This is consistent with short-duration
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treatment in an established arthritis, and significant reversal of pathology would not be expected. It has been shown that prednisolone given for an extended period is capable of ameliorating joint damage in both collagen- (Paska et al., 1986; Geiger et al., 1994) and antigen-induced (van den Berg et al., 1991) murine arthritis. Conversely, it has been shown that in these same models, indomethacin does not reverse pathological changes in the joint, even following long-term treatment (de Vries et al., 1988; Cannon et al., 1990; Malfait et al., 2001). Further work will be required to determine if long-term steroid administration can significantly reduce the pathological changes associated with FCA-induced arthritis. The reduction of inflammation and hyperalgesia by the two drugs in the established arthritis means that the FCA-induced unilateral arthritis model could be useful in predicting effective drugs for the treatment of arthritis and chronically inflammatory hyperalgesia. In addition, the model has been used to examine nociceptor sensitisation in an arthritic joint by electrophysiologically recording evoked responses from the medial articular nerve innervating the inflamed joint capsule (manuscript in preparation). In conclusion, these studies show it is possible to induce a chronic, unilateral joint inflammation in both DBA/1 and C57BL/6 mice by repeated intra-articular injections of FCA. The pathology present in these joints is consistent with a mild to moderate arthritis, and it responds well to both prednisolone and indomethacin. With the development of an objective measure of nociception, this model should prove valuable for the examination of chronic inflammatory conditions, the relationship between inflammation and nociception, and how these are altered in transgenic mice.
Acknowledgements We would like to thank Susan Bond for her contribution to animal measurements in this project and Barry Reed for processing the joint tissues for histology. Work supported by a grant from GlaxoSmithKline, UK.
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