Degenerative Joint Disease Induced by Repeated Intra-Articular Injections of Monosodium Urate Crystals in Rats as Investigated by Translational Imaging

doi:10.21203/rs.3.rs-758047/v1

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Research Article

Degenerative Joint Disease Induced by Repeated Intra-Articular Injections of Monosodium Urate Crystals in Rats as Investigated by Translational Imaging

https://doi.org/10.21203/rs.3.rs-758047/v1

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published 07 Jan, 2022

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Effects of repeated injection of monosodium urate (MSU) crystals, in combination with lipopolysaccharide (LPS), into rat knee joints every two weeks for a maximum of five administrations were investigated. Joint swelling, nociception and hard/soft tissue changes were assessed longitudinally by non-invasive imaging. MSU crystals induced joint swelling, synovial membrane thickening, fibrosis of the infrapatellar fat pad, tidemark breaching, and cartilage invasion by inflammatory cells. Several inflammatory proteins were present in synovial fluid. A higher sensitivity to mechanical stimulus was detected in paws of limbs receiving MSU/LPS compared to saline-injected limbs. In MSU/LPS-challenged joints, magnetic resonance imaging (MRI) revealed increased synovial fluid volume in the posterior region of the joint, alterations in the infrapatellar fat pad reflecting a progressive decrease of fat volume and fibrosis formation, and a significant increase in the relaxation time T₂ in femoral cartilage, consistent with a reduction of proteoglycan content. MRI also showed cyst formation in the tibia, femur remodeling, and T₂ reductions in extensor muscles consistent with fibrosis development. Repeated intra-articular MSU/LPS injections in the rat knee joint induced pathology in multiple tissues and may be a useful means to investigate the relationship between urate crystal deposition and the development of degenerative joint disease.

Rheumatology

Crystal

gout

infrapatellar fat pad

magnetic resonance imaging

monosodium urate

osteoarthritis

Gout represents a highly prevalent form of arthritis, characterized by recurrent episodes of painful acute inflammatory flares in response to monosodium urate (MSU) crystals that deposit predominantly in peripheral joints and surrounding tissues¹. Long-term deposition of MSU crystals can result in joint damage. Moreover, gout and osteoarthritis (OA) often occur concomitantly, and a positive correlation between synovial fluid uric acid and OA has been established². Nevertheless, it is presently not known whether and how these conditions are pathologically linked^3-5.

Recent advances in the understanding of crystal-induced inflammation provided further support for a shared inflammatory pathway between gout and OA. MSU crystals activate the macrophage innate immune response via the Nacht Domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NALP3) inflammasome, which is required for caspase-1 activation and subsequent interleukin (IL)-1β and IL-18 release⁶. IL-1β is considered to play a central role in OA^7,8, and synovial fluid levels of both IL-1β and IL-18 are strongly associated with OA severity².

Preclinical models play an important role in facilitating research in this area. Previous studies demonstrated that a single intra-articular injection of MSU crystals resulted in gouty-arthritis in mice and rats^9,10. However, these studies were limited to the acute phase of the response, with an observation time of up to 2 days only, following crystal administration.

In this work, we investigated the long-term effect of repeated injections of MSU crystals in combination with lipopolysaccharide (LPS) into the knee joints of rats every two weeks for a maximum of five administrations, with the aim to stimulate NLRP3 inflammasome activation as described previously^6,11. We assessed the swelling response after each injection, paw withdrawal sensitivity to mechanical stimuli using electric von Frey, as well as hard and soft tissue joint changes by magnetic resonance imaging (MRI) and micro-computerized tomography (micro-CT). Histology was performed at selected time points for the characterization of pathological features at the cellular level. The aim was to assess the consequences of repeated intra-articular crystal administration in order to simulate recurrent gout bouts in rats, using translational imaging to simultaneously detect and quantify injury in different areas of the knee joint.

Statement on animal welfare

In vivo experimental procedures followed the Swiss animal welfare regulations. The experimental protocols were approved by the Cantonal Veterinary Office of the City of Basel, Switzerland. The study was performed under the license number BS-1438, approved by the Cantonal Veterinary Office of the City of Basel. Authors complied with the ARRIVE guidelines for animal experimentation.

Animals

Female Lewis/OrlRj rats (n=25) from Janvier Laboratories (Le Genest-Saint-Isle, France), 150-180 g or eight weeks of age at the beginning of the study, were used. Rats were housed under standard conditions (12-hour light/dark cycle), with standard chow and water provided ad libitum. Upon arrival, rats were allowed two weeks of acclimatization before beginning the experiments.

Monosodium urate (MSU) crystals

MSU crystals were prepared according to the method originally described by Seegmiller et al¹².

Induction of gouty arthritis

Twenty five rats were used. On day 0, the right knee received 50 µl of a mixture containing 40 mg/ml MSU crystals and 0.1 µg/ml LPS from E. coli (0111:B4, Sigma L2630) in saline, while the left knee received 50 µl of saline. The intra-articular injections of MSU/LPS into the right and saline into the left knee were then repeated every two weeks (namely on days 0, 14, 28, 42 and 56). Saline was administered into the left knee to verify whether repeated intra-articular injection of fluid might elicit an inflammatory response in the knee joint. Rats were anesthetized with 3.5% isoflurane (Abbott, Cham, Switzerland)/air for each intra-articular injection. At each of the time points days 14, 28, 42, 56 and 70 five animals were culled for post-mortem analyses. The sacrificed rats had received the last MSU/LPS dose 14 days before being euthanized.

Exclusion criterion

A weight loss of more than 20% would lead to an exclusion and early euthanasia of an animal. However, no rat needed to be excluded from the study.

Knee swelling

Knee diameters were measured using calipers immediately before and again on days 1, 2, 3 and 7 after each intra-articular injection. Right and left knee diameters were determined in the medial-lateral direction, with the caliper positioned perpendicularly to the leg axis. Knee swelling was defined as the ratio between the knee diameter at a given time point and the mean knee diameter at baseline, before any injection.

Nociceptive test

Hind-paw sensitivity was evaluated by measuring the mechanical withdrawal threshold using a handheld electronic von Frey unit (Cat # 38450, Ugo Basile, Gemonio, Italy). An animal was placed in clear boxes on an elevated mesh screen (models BIO-STD EVF and BIO-PVF, Bioseb, Vitrolles, France), and allowed to habituate for 15 min before testing. A filament was applied to the plantar surface of each hind paw. The force was increased by increments of 0.1 g force units from zero until paw withdrawal. A transducer comprising a digital timer automatically recorded the force eliciting paw withdrawal and the corresponding response latency to the nearest 0.1 s. The filament was applied five times per paw, separated by a 5-min interval to prevent sensitization, and the threshold was defined as an average of the five withdrawals observed within the trials.

Imaging

During acquisitions animals were anesthetized with isoflurane 1.5-2% in air, administered via a nose cone.

MRI. Performed with a Pharmascan 7T scanner (Bruker, Etlingen, Germany). A T₂-weighted spin-echo sequence with the following parameters was applied: 16 echoes spaced by 11 ms, echo time (TE) from 11 to 176 ms, repetition time 2022 ms, pixel size 0.078x0.078 mm, slice thickness 0.48 mm, 8 slices, without and with fat suppression. A volume resonator (Model 1P-T11070V3, Bruker) with 72 mm inner diameter was used for transmission. A two-channel phased array receive-only mouse head surface coil (Model 1P-T11204V3, Bruker) was used for signal reception.

Relaxation time T₂ for cartilage, infrapatellar pad and muscle was determined by fitting with GraphPad Prism (version 8.1.2, GraphPad Software, San Diego, CA) the corresponding signals from regions-of-interest (ROIs) placed in these anatomical areas as function of TE. Volumes of effusion were determined by segmenting the corresponding signals by their intensity using a region grower algorithm available at the scanner software.

Micro-CT. Measurements were performed using a vivaCT-40 micro-CT system (Scanco Medical, Brüttisellen, Switzerland). The scan parameters were: voxel size 17.5x17.5x17.5 μm, 426 slices, integration time 130 ms, high resolution, 55 E(kVp), 145 μA, 8W mode, cone beam continuous rotation.

Ex vivo analyses

Two weeks after one or more MSU/LPS injections, rats were euthanized, and synovial fluid collected from the right and left knees. Synovial fluid supernatant stored at -20°C until further analysis. The skin was removed and knees were excised for histology.

Multiplex ELISA

Synovial fluid samples were analyzed using a multiplex enzyme-linked immunosorbent assay (ELISA) with rat specific reagents, following the manufacturer’s protocols (Bio-Rad Laboratories, Hercules, CA). The dedicated Bio-Plex Manager™ software running on a Bioplex 200 System array reader (Bio-Rad) was used to determine individual concentrations. Because of the small amount of synovial fluid drawn from each animal, data from different time points during the course of the study were pooled to allow statistical comparisons between saline- and MSU/LPS-injected joints.

Histology

Knee joints were fixed in 10% neutral buffered formalin for three days and then placed in a decalcification solution (ImmunoCal Cat # 1440, Decal Chemical Corp, Suffern, NY) for 5 days. On the fourth day, knees were trimmed along the sagittal axis approximately in the middle of the joint and decalcified to completion. After sample dehydration and paraffin embedding, 5-µm-thick sections were cut and stained: hematoxylin and eosin (H&E) for general observation, and proteoglycan-containing cartilage identified by Safranin O/Fast green using a procedure adapted from Lu et al¹³. Joint pathology was scored according to the Mankin system¹⁴, considering cartilage surface integrity (0-6), proteoglycan loss (0-4), chondrocyte morphology (0-3), fibrovascular replacement of subchondral marrow fat spaces (0-1), synovitis (0-3) and tidemark breaching (0-1). Macrophages and osteoclasts were detected with an anti-CD68 antibody (MCA341R, Serotec, Puchheim, Germany) applied on paraffin sections as described by Damoiseaux et al¹⁵.

Statistics

Multiplex assay data from synovial fluid samples were analyzed using Student’s t-tests (Origin 2021, OriginLab Corporation, Northampton, MA, USA). Paw withdrawal threshold and MRI data were analyzed using ANOVA with random effects (Systat version 13; Systat Software Inc., San Jose, California, USA) to take into account the longitudinal structure of the data. A value of p < 0.05 was considered statistically significant.

Repeated injection of MSU/LPS every two weeks into the knee joint for a maximum of 5 times induced marked swelling of the injected joint, joint inflammation, synovial membrane thickening, fibrosis of the infrapatellar fat pad, proliferation of the synovial membrane, breaching of the tidemark and cartilage invasion by inflammatory cells. Principal actors of the crystal-induced response were neutrophils, mast cells and macrophages (supplementary figure 1). All these features are present in human gout pathology¹⁶.

Knee swelling and inflammatory markers in synovial fluid

Intra-articular administration of saline led to a small increase in left knee diameter compared to baseline, reaching a maximum of 7% after the fifth injection (figure 1a). Swelling was maximal at day 1 after each MSU/LPS injection (figure 1a), attaining joint diameter increase of 35% and 62% from the first to the fifth challenge. Knees remained slightly swollen just before the next administration of crystals, and this slight additional swelling increased up to the fourth challenge. The maximum (peak) swelling increased after each injection until the third MSU/LPS administration (figure 1b). Subsequent injections did not increase the maximum knee swelling response further.

Figure 2 summarizes a number of markers in the synovial fluid that were increased in MSU/LPS compared to saline-injected joints. Because of the limited sample volume per joint at each individual time point, data were pooled for the statistical analysis. The levels of IL-1a, IL-1b, KC/GRO (the IL-8 related protein in rodents¹⁷), MIP-1a, MCP-1 and VEGF were higher in the synovium of MSU/LPS-treated joints.

Paw sensitivity to mechanical stimulus

The nociceptive test revealed that the peak force applied for left hind limb withdrawal was unchanged during the course of the study. By contrast, the peak force applied for right hind limb withdrawal was decreased from the first injection of MSU/LPS onwards (figure 3a). Moreover, starting from day 25 (11 days after the second MSU/LPS injection), the withdrawal latency was significantly shorter in MSU/LPS-treated right hind limbs as compared to saline-treated left hind limbs (figure 3b). These data indicated a higher paw sensitivity to mechanical stimulus on the right side, corresponding to the knee which received the injections of MSU crystals.

In vivo imaging and histology

A significant and sustained increase of the synovial fluid volume in the posterior joint region was determined by MRI in joints treated with MSU/LPS (figure 4a). No inflammation was observed in the synovial membrane from a saline-injected joint (figure 4b). In contrast, synovial inflammation was confirmed by histology, which identified a higher cellular density within the thickened membrane 14 days after a single MSU/LPS injection (figure 4c). This feature was accentuated after further MSU/LPS injections until lymphoid infiltration in the synovial subintimal layer (figure 4d).

In the Hoffa’s infrapatellar fat pad area, non-fat suppressed MRI revealed a significant decrease of fat volume starting at day 11 after the first MSU/LPS injection (figure 5a,b). Additional analyses of fat-suppressed images showed a significant T₂ reduction over the experimental time (figure 5c). At day 69 (day 13 after the fifth administration of crystals), T₂ in the infrapatellar fat pad (21.0±2.7 ms) was significantly lower by 46% (p=0.0001) compared to baseline (38.8±5.2 ms). Moreover, it was significantly lower by 45% (p=0.0006) with respect to infrapatellar fat pad T₂ values of saline-injected joints at the same time point (38.2±6.0 ms). The T₂ decrease in the infrapatellar fat pad was consistent with fat reduction and/or extracellular matrix remodeling (fibrosis) revealed by histology. Two weeks after the second MSU/LPS injection, numerous cells had already invaded the infrapatellar fat pad (figure 5d). On day 52, i.e. 10 days after the fourth injection of crystals, histology demonstrated significant fat reduction and fibrosis in this region (figure 5d).

Starting at day 52, MRI additionally revealed a significant increase in cartilage T₂ in the medial femoral condyle with respect to baseline values, whereas tibial cartilage T₂ values remained constant (figure 6). At day 69, the femoral cartilage T₂ of joints receiving MSU/LPS (44.5±7.3 ms) was significantly increased by 68% (p=0.0002) with respect to baseline (26.5±2.5 ms). It was also significantly increased by 74% (p=0.001) with respect to T₂ values in saline-injected contralateral joints (25.6±3.5 ms). The T₂ increase in femoral cartilage was consistent with a reduction in proteoglycan staining detected by histology predominantly in front of the patella (figure 6). Histology of cartilage also revealed fibrillation and detachment of the superficial layer, an increased number of chondrocyte clusters and the presence of hypertrophic chondrocytes in the middle zone (supplementary figure 2). Moreover, we observed tidemark breaching and cellular infiltration in the cartilage associated to the presence of osteoclasts. Cartilage OA Mankin scores and representative histological images are presented in supplementary figure 2.

Synovial membrane thickening and proliferation were detected by histology at days 52 and 69 (day 10 after the fourth and day 13 after the fifth crystal administration, respectively) in the anterior portion of the joint (figure 7). Macrophage proliferation was observed in the hypertrophic synovial membrane (supplementary figure 1). These findings might explain the large increase in T₂ observed in and around the femoral cartilage close to the meniscus (figure 7).

Changes in bone structure were also observed. Fifty percent of the rats injected with MSU/LPS presented with cyst formation in the tibia, and bone remodeling in the femur, with the development of a woven bone structure as determined by MRI and histology (figure 8a). Moreover, chondrophytes appeared at the margins of cartilage, as seen in all coronal sections of animals injected four or five times with MSU/LPS (figure 8b). These chondrophytes, which were not detectable by micro-CT (supplementary figure 3), formed in the periosteum at the junction between cartilage and bone. Finally, a reduction of T₂ was detected in a region comprising the knee extensor muscles (figure 8c). This reduction was consistent with fibrosis revealed by histology in the same area.

In the present study, we utilized longitudinal and endpoint measurements to characterize a rat model of urate crystal-induced joint injury. Our findings demonstrate that an early osteoarthritic-like phenotype can be generated following multiple intra-articular challenges with MSU/LPS, thereby lending further support to the concept of a link between gout and OA.

Mast cells, macrophages and polymorphonuclear cells are considered the main drivers of urate crystal-induced arthritis¹⁶. We were able to observe polymorphonuclear cells appearing in the bone marrow and to detect tissue invasion by mast cells and macrophages in rat knee joints during the early time points following MSU/LPS challenge. Similarly, we also observed increases in several soluble pro-inflammatory proteins. IL-1b synovial fluid levels were upregulated at several time points, with a significant increase reported overall. This was expected, as IL-1b is produced as a direct consequence of inflammasome activation with MSU crystals following LPS priming⁶. The characteristic influx of neutrophils and macrophages during a gout flare is coordinated by cytokines/chemokines including IL-1b, IL-8 (CXCL8), MCP-1 (CCL2) and MIP1 (CCL3)^18,19. Upregulation of these mediators was observed in our model following single or repeated intra-articular challenge with MSU/LPS. Synovial inflammation with cell influx and proliferation is also associated with the production of new blood vessels via the process of angiogenesis. Hypoxia within the proliferating synovial tissue contributes to this process with the production of vascular endothelial growth factor (VEGF), mainly in rheumatoid arthritis, but also in OA joint disease²⁰. VEGF was increased in the synovial tissue from all MSU/LPS injected joints at each of the five MSU injection time points, suggesting that angiogenesis was induced throughout the development of this gouty arthritis model. This inflammatory reaction further translated into knee swelling peaking in the days immediately after the MSU/LPS injection. The peak swelling ratio increased up to the third MSU/LPS injection, and remained constant following subsequent challenges. This was suggestive of a habituation of the biological system to the inflammatory stimuli elicited by the crystals, in agreement with other inflammatory models. For instance, repeated ovalbumin administration in rats was found to reduce its capacity to induce lung inflammation²¹. Further evaluation using non-invasive MRI identified synovial effusion within MSU/LPS-treated joints, peaking at day 11.

Cartilage damage is often observed in joints affected by advanced gout²². Chhana et al.²³ demonstrated that MSU crystals contribute to cartilage damage in gout through a reduction in chondrocyte viability and function, and increased catabolic activity within cartilage. Here, MSU/LPS challenge also led to biochemical and structural changes in articular cartilage, as evidenced by MRI and histology. The collagen-proteoglycan matrix limits the mobility of water protons, thus promoting relaxation, leading to low T₂’s in hyaline cartilage. At baseline, cartilage T₂ (26.5±1.5 and 17.6±1.6 ms for femoral and tibial cartilage, respectively) was in good agreement with literature values measured at 7 Tesla in healthy humans (24.9±1.3 ms for femoral and 19.2±1.0 ms for tibial cartilage)²⁴ and rats^25,26. Because of its sensitivity to changes in collagen content and orientation²⁷, as well as in proteoglycan content²⁸, MRI T₂ mapping can identify abnormalities of the cartilage extracellular matrix and probe early stages of cartilage degeneration occurring prior to macroscopic cartilage defects and thinning. Increase in cartilage T₂ relaxation time in the absence of erosion thus reflects proteoglycan depletion and/or changes in the collagen organization^29-31. Here, T₂ increased primarily in femoral cartilage, close to the patellar region, by 21% and 68% compared to baseline at days 52 and 69, respectively. Histology revealed proteoglycan reduction predominantly in the femoral cartilage at these time points, in areas displaying increased T₂. Other rat models of cartilage injury showed comparable T₂ increases. For instance, an increase by approximately 40% and 50% was detected three weeks after meniscectomy³² or anterior cruciate ligament transection²⁶. Immobilization of the knee joint for two weeks led to cartilage T₂ increase by approximately 60%²⁵. The regional distribution of crystal-induced cartilage degeneration was consistent with earlier work demonstrating proteoglycan degradation predominantly in the anterior femoral cartilage in front of the patella following intra-articular injection of papain³³ or other chemicals³⁴. Finally, proliferation of the synovial membrane with a large increase in relaxation time T₂ was also detected in the present model. Synovitis comprising the influx of inflammatory cells (lymphocytes, macrophages, neutrophils) and the proliferation of fibroblast-like synoviocytes, forming pannus and leading to cartilage and ultimately bone destruction, is an important feature of gout² and OA³⁵.

Along with cartilage, menisci, ligaments, and synovium, fat pads are major constituents of the knee joint. The infrapatellar Hoffa’s pad, one of the largest fat pads, plays a role in facilitating the distribution of synovial fluid and mechanical forces throughout the joint. This fat pad, which produces several pro-inflammatory adipokines and cytokines, such as, TNF-a, IL-6, and leptin, as well as VEGF, can influence inflammatory processes in the knee³⁶. Hoffa’s disease is characterized by inflammation, hypertrophy, and fibrosis of the pad in response to repetitive trauma³⁷. Moreover, the infrapatellar fat pad and synovial membranes in OA were shown to be more inflamed, vascularized and fibrous compared with those of healthy individuals³⁸. Here, MRI revealed significant decrease of the relaxation time T₂ at the level of the infrapatellar fat pad during the course of the experiment. Since water molecules interacting with collagen and other macromolecules have very short T₂’s³⁹, collagen production and tissue remodeling are expected to lead to a reduction in T₂. The decreased T₂ observed here was thus consistent with fat decrease and fibrosis development revealed by histology. In clinics, a shortening of T₂ was reported for the infrapatellar fat pad chronically after arthroscopy surgery indicating tissue fibrosis⁴⁰. Moreover, larger infrapatellar fat pad volume has been associated with greater knee cartilage volume and fewer structural abnormalities, suggesting a protective role of infrapatellar fat pad size in knee OA⁴¹. The reduction in fat pad volume detected by MRI and confirmed by histology would therefore be in accordance with the development of relevant knee joint pathology in our model.

The infrapatellar fat pad is richly innervated⁴², being one source of anterior knee pain⁴³. Either acute or chronic trauma can lead to local bleeding, inflammation and eventually to fibrotic lesions, all of which are causes of infrapatellar fat pad-related pain³⁷. Preclinically, irreversible structural changes in the infrapatellar fat pad, such as extensive fibrosis, were described to occur prior to persistent pain development in Wistar rats receiving a single intra-articular injection of monoiodoacetic acid⁴⁴. In our study, a positive correlation was found between the T₂ values of the infrapatellar fat pad in fat-suppressed images and the withdrawal latency time of the paw upon von Frey filament stimulation. Despite this positive correlation, a more detailed investigation would be necessary to verify whether T₂ assessments of the infrapatellar fat pad could serve as an objective surrogate measure of increased paw sensitivity in the present crystal-induced knee joint injury model. Of note, a significant decrease of infrapatellar fat pad T₂ was detected at day 11 in fat-suppressed images only, suggesting that changes in fat pad at this early time point were primarily driven by cell invasion/tissue remodeling as confirmed by histology. At later time points, T₂ reductions in non-suppressed and fat-suppressed images were consistent with fat reduction and tissue remodeling/fibrotic lesion development evidenced histologically. Concerning the nociceptive test, while the withdrawal latency was significantly shorter in the MSU-crystal-injected leg, there was also a shorter withdrawal latency in the contralateral leg. Since the animals were growing, the higher sensitivity could be a natural weight compensation. More weight balanced on the non-injured leg might have affected the sensitivity in this leg. In order to properly evaluate this possibility, a naive control group without knee injections would be required in future experiments.

Changes in knee loading are considered to be an important contributor to the development of knee OA in humans. It has been found that the quadriceps muscle, which plays a central role in modulating loads across the knee joint, may be up to 40% weaker in OA patients than in healthy individuals⁴⁵. Such weakness contributes not only to pain and loss of function in knee OA, but is observed early in the disease process, often preceding disease onset⁴⁵. Lower quadriceps function in moderate OA has been associated with extracellular matrix (ECM) expansion in muscle⁴⁶. In addition, the quadriceps muscle displayed atrophy, decreased satellite cell number, and fibrosis following anterior cruciate ligament injury⁴⁷. Here, we observed a reduction of the T₂ relaxation time in muscle, consistent with fibrosis observed by histology. Of note, a trend towards reduced T₂ appeared on day 25 and the effect became highly significant at later time points, while femoral cartilage damage became apparent at days 53 and 69 only. These results suggest that ECM remodeling in muscle described for knee OA patients was recapitulated in the present animal model. Although not carried out here, non-invasive assessment of muscle atrophy as described by Giorgetti et al.⁴⁸ will be performed in future studies.

Cartilage outgrowths, or chondrophytes, were observed in our model. These undergo endochondral ossification during gout⁴⁹ or OA progression⁵⁰. However, we did not detect osteophytes per se neither by micro-CT nor by histology. An increased study duration might have provided the opportunity for the detection of osteophytes.

Taken together, our findings indicate that the repeated injection of MSU crystals, in combination with LPS, into the knee joints of rats every two weeks leads to distinctive pathological changes reflecting degenerative joint disease. This model provides the opportunity to simulate repeated bouts of gouts in a small rodent, and to study possible consequences of them. The changes detected were multifactorial, with cartilage and bone alterations coexisting with pathological features of inflammation and hyperplasia. Finally, the results reported here serve to illustrate the power of non-invasive translational imaging to longitudinally quantify pathology in multiple tissues of the knee joint.

CT computerized tomography; ECM extracellular matrix; ELISA enzyme-linked immunosorbent assay; KC/GRO keratinocyte chemoattractant/growth-regulated oncogene (IL-8); IL-1b interleukin-1b; IL-18 interleukin-18 ; LPS lipopolysaccharide ; MCP-1 monocyte chemoattractant protein-1; micro-CT micro-computerized tomography; MIP-1a macrophage inflammatory protein-1a; MRI magnetic resonance imaging; MSU monosodium urate; NLRP3 NACHT, LRR and pyrin domains-containing protein 3; OA osteoarthritis; ROI region-of-interest; sd standard deviation; sem standard error of the means; TE echo time; TR repetition time; VEGF vascular endothelial growth factor

Acknowledgement

The support of Anja Scheidegger and of the Comparative Medicine Group at Novartis during the execution of this study is gratefully acknowledged.

Contributions

All authors contributed to the study design, analyzed the data, provided critical revisions for important intellectual content, and approved the final version of the manuscript. J.D., N.A., P.R. and N.B. wrote the first draft of the manuscript. M.O., C.L., J.K. and M.F. acquired the data for the study.

Funding source

This work was funded by Novartis Pharma AG, Basel, Switzerland. The funding source played no role in designing this study, analyzing the data, writing the manuscript, or submitting the manuscript for publication.

Competing interest statement

Authors are employed by and/or own stocks from Novartis Pharma AG.

Kuo, C.F., Grainge, M.J., Zhang, W. & Doherty, M. Global epidemiology of gout: prevalence, incidence and risk factors. Nat. Rev. Rheumatol. 11, 649-662 (2015).
Denoble, A.E. et al. Uric acid is a danger signal of increasing risk for osteoarthritis through inflammasome activation. Proc. Natl. Acad. Sci. U.S.A. 108, 2088-2093 (2011).
Roddy, E. & Doherty, M. Gout and osteoarthritis: a pathogenetic link? Joint Bone Spine 79, 425-427 (2012).
Ma, C.A. & Leung, Y.Y. Exploring the Link between Uric Acid and Osteoarthritis. Front. Med. (Lausanne) 4, 225, doi: 10.3389/fmed.2017.00225 (2017).
Neogi, T., Krasnokutsky, S. & Pillinger, M.H. Urate and osteoarthritis: Evidence for a reciprocal relationship. Joint Bone Spine 86, 576-582 (2019).
Martinon, F., Petrilli, V., Mayor, A., Tardivel, A. & Tschopp, J. Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature 440, 237-241 (2006).
Kapoor, M., Martel-Pelletier, J., Lajeunesse, D., Pelletier, J.P. & Fahmi, H. Role of proinflammatory cytokines in the pathophysiology of osteoarthritis. Nat. Rev. Rheumatol. 7, 33-42 (2011).
Jenei-Lanzl, Z., Meurer, A. & Zaucke, F. Interleukin-1β signaling in osteoarthritis - chondrocytes in focus. Cell Signal 53:212-223 (2019).
Marcotti, A. et al. Joint nociceptor nerve activity and pain in an animal model of acute gout and its modulation by intra-articular hyaluronan. Pain 159, 739-748 (2018).
Raucci, F. et al. IL-17A neutralizing antibody regulates monosodium urate crystal-induced gouty inflammation. Pharmacol. Res. 147, 104351, doi: 10.1016/j.phrs.2019.104351 (2019).
Ghonime, M.G. et al. Inflammasome Priming by Lipopolysaccharide Is Dependent upon ERK Signaling and Proteasome Function. J. Immunol. 192, 3881-3888 (2014).
Seegmiller, J.E., Howell, R. & Malawista, S.E. The Inflammatory Reaction to Sodium Urate: Its Possible Relationship to the Genesis of Acute Gouty Arthritis. JAMA J. Am. Med. Assoc. 180, 469-475 (1962).
Lu, Y. et al. Effect of bipolar radiofrequency energy on human articular cartilage. Comparison of confocal laser microscopy and light microscopy. Arthroscopy 17, 117-123 (2001).
Rutgers, M., van Pelt, M.J.P., Dhert, W.J.A., Creemers, L.B. & Saris, D.B.F. Evaluation of histological scoring systems for tissue-engineered, repaired and osteoarthritic cartilage. Osteoarthritis Cartilage 18, 12-23 (2010).
Damoiseaux, J.G. et al. Rat macrophage lysosomal membrane antigen recognized by monoclonal antibody ED1. Immunology 83, 140-147 (1994).
Desai, J., Steiger, S. & Anders, H.J. Molecular Pathophysiology of Gout. Trends Mol. Med. 23, 756-768 (2017).
Bozic, C.R. et al. The murine interleukin 8 type B receptor homologue and its ligands. Expression and biological characterization. J. Biol. Chem. 269, 29355-29358 (1994).
Scanu, A. et al. Synovial fluid proteins are required for the induction of interleukin-1β production by monosodium urate crystals. Scand. J. Rheumatol. 45, 384-393 (2016).
Amezcua-Castillo, L.M., Juárez-Vicuña, Y., Márquez-Velasco, R. & Amezcua-Guerra, L.M. Activation Status of NLRP3 Inflammasome in Peripheral Blood Mononuclear Cells From Patients With Gout Flare. J. Clin. Rheumatol. 26(7S Suppl 2), S208-S212 (2020).
Haywood, L. et al. Inflammation and angiogenesis in osteoarthritis. Arthritis Rheum. 48, 2173-2177 (2003).
Tigani, B. et al. Lung Inflammation and Vascular Remodeling After Repeated Allergen Challenge Detected Noninvasively by MRI. Am. J. Physiol. Lung Cell. Mol. Physiol. 292, L644-L653 (2007).
Dalbeth, N., Clark, B., McQueen, F., Doyle, A. & Taylor, W. Validation of a radiographic damage index in chronic gout. Arthritis Rheum. 57, 1067-1073 (2007).
Chhana, A. et al. The Effects of Monosodium Urate Monohydrate Crystals on Chondrocyte Viability and Function: Implications for Development of Cartilage Damage in Gout. J. Rheumatol. 40, 2067-2074 (2013).
Juras, V. et al. The compositional difference between ankle and knee cartilage demonstrated by T2 mapping at 7 Tesla MR. Eur. J. Radiol. 85, 771-777 (2016).
Gu, Q. et al. Effects of Nicotine on a Rat Model of Early Stage Osteoarthritis. Int. J. Clin. Exp. Pathol. 8, 3602-3612 (2015).
Mohan, G. et al. Kartogenin Treatment Prevented Joint Degeneration in a Rodent Model of Osteoarthritis: A Pilot Study. J. Orthop. Res. 34, 1780-1789 (2016).
Dunn, T.C., Lu, Y., Jin, H., Ries, M.D. & Majumdar, S. T2 relaxation time of cartilage at MR imaging: comparison with severity of knee osteoarthritis. Radiology 232, 592-598 (2004).
Nishioka, H. et al. T1r and T2 Mapping Reveal the In Vivo Extracellular Matrix of Articular Cartilage. J. Magn. Reson. Imaging 35, 147-155 (2012).
David-Vaudey, E., Ghosh, S., Ries, M. & Majumdar, S. T2 relaxation time measurements in osteoarthritis. Magn. Reson. Imaging 22, 673-682 (2004).
Watrin-Pinzano, A. et al. Effect of Proteoglycan Depletion on T2 Mapping in Rat Patellar Cartilage. Radiology 234, 162-170 (2005).
Eckstein, F., Kwo,h, C.K., Link, T.M.; O.A.I. investigators. Imaging research results from the osteoarthritis initiative (OAI): a review and lessons learned 10 years after start of enrolment. Ann. Rheum. Dis. 73, 1289-1300 (2014).
Spandonis, Y., Heese, F.P. & Hall, L.D. High Resolution MRI Relaxation Measurements of Water in the Articular Cartilage of the Meniscectomized Rat Knee at 4.7 T. Magn. Reson. Imaging 22, 943-951 (2004).
Grevenstein, J., Michiels, I., Arens-Corell, M. & Stofft, E. Cartilage Changes in Rats Induced by Papain and the Influence of Treatment with N-Acetylglucosamine. Acta Orthop. Belg. 57, 157-161 (1991).
Takada, S., Kato, M. & Takayama, S. Comparison of Lesions Induced by Intra-Articular Injections of Quinolones and Compounds Damaging Cartilage Components in Rat Femoral Condyles. J. Toxicol. Environ. Health 42, 73-88 (1994).
Scanzello, C.R. & Goldring, S.R. The role of synovitis in osteoarthritis pathogenesis. Bone 51, 249-257 (2012).
Clockaerts, S. et al. The infrapatellar fat pad should be considered as an active osteoarthritic joint tissue: a narrative review. Osteoarthritis Cartilage 18, 876-882 (2010).
Eymard, F. & Chevalier, X. Inflammation of the infrapatellar fat pad. Joint Bone Spine 83, 389-393 (2016).
Favero, M. et al. Infrapatellar fat pad features in osteoarthritis: a histopathological and molecular study. Rheumatology (Oxford) 56, 1784-1793 (2017).
Siu, A.G. et al. Characterization of the ultrashort-TE (UTE) MR collagen signal. NMR Biomed. 28, 1236-1244 (2015).
Torriani, M. et al. T2 Relaxometry of the Infrapatellar Fat Pad After Arthroscopic Surgery. Skeletal Radiol. 43, 315-321 (2014).
Cai, J. et al. Association Between Infrapatellar Fat Pad Volume and Knee Structural Changes in Patients With Knee Osteoarthritis. J. Rheumatol. 42, 1878-1884 (2015).
Lehner, B. et al. Preponderance of sensory versus sympathetic nerve fibers and increased cellularity in the infrapatellar fat pad in anterior knee pain patients after primary arthroplasty. J. Orthop. Res. 26, 342-350 (2008).
Belluzzi, E. et al. Contribution of Infrapatellar Fat Pad and Synovial Membrane to Knee Osteoarthritis Pain. Biomed. Res. Int. 6390182, doi: 10.1155/2019/6390182 (2019).
Clements, K.M. et al. Cellular and histopathological changes in the infrapatellar fat pad in the monoiodoacetate model of osteoarthritis pain. Osteoarthritis Cartilage 17, 805-812 (2009).
Bennell, K.L., Hunt, M.A., Wrigley, T.V., Lim, B.W. & Hinman, R.S. Role of muscle in the genesis and management of knee osteoarthritis. Rheum. Dis. Clin. North Am. 34, 731-754 (2008).
Noehren, B. et al. Alterations in quadriceps muscle cellular and molecular properties in adults with moderate knee osteoarthritis. Osteoarthritis Cartilage 26, 1359-1368 (2018).
Fry, C.S., Johnson, D.L., Ireland, M.L. & Noehren, B. ACL injury reduces satellite cell abundance and promotes fibrogenic cell expansion within skeletal muscle. J. Orthop. Res. 35, 1876-1885 (2017).
Giorgetti, E. et al. Magnetic Resonance Imaging as a Biomarker in Rodent Peripheral Nerve Injury Models Reveals an Age-Related Impairment of Nerve Regeneration. Sci. Rep. 9, 13508, doi: 10.1038/s41598-019-49850-2 (2019).
Dalbeth, N., Milligan, A., Doyle, A.J., Clark, B. & McQueen, F.M. Characterization of new bone formation in gout: a quantitative site-by-site analysis using plain radiography and computed tomography. Arthritis Res. Ther. 14, R165, doi: 10.1186/ar3913 (2012).
50. Gelse, K., Söder, S., Eger, W., Diemtar, T. & Aigner, T. Osteophyte development‐molecular characterization of differentiation stages. Osteoarthritis Cartilage11, 141-148 (2003).

Competing interest reported. Authors are employed by and/or own stocks from Novartis Pharma AG. However, the company had no influence on the decision to submit these results for publication.

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Degenerative Joint Disease Induced by Repeated Intra-Articular Injections of Monosodium Urate Crystals in Rats as Investigated by Translational Imaging

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