Introduction
Gout is an inflammatory condition induced by the deposition of monosodium urate (MSU) crystals in joints and soft tissues that can produce an acute or chronic arthritis [
1]. The early phase of MSU-induced inflammation is associated with activation of the NACHT, LRR and PYD domain-containing protein 3 (NALP3) inflammasome or cryopyrin, triggering the release of IL-1β and IL-18 [
2], thus promoting cellular activation, cytokine and chemokine production, and infiltration of neutrophils, leading to an acute gouty attack [
3,
4]. Despite their similar morphology, allopurinol crystals have not been found to activate the NALP3 inflammasome [
5].
Several animal models of crystal-induced inflammation have been studied that involve direct injection of MSU crystals into both intra-articular and extra-articular locations. Extra-articular locations include: intradermal, subcutaneous, and intraperitoneal areas; however, only the intra-articular model reflects the true diarthrodial joint microenvironment, in which an acute gouty attack takes place [
6-
16].
Imaging techniques such as ultrasound (US) may reinforce the diagnosis, monitoring, and management of gout [
17-
19]. Several studies have shown its capability to detect both intra- and extra-articular abnormalities in patients with gout [
20-
28]. The high reflectivity of MSU-crystal aggregates, and the ability of US to detect even minimal MSU-crystal deposits explain the high sensitivity of US for revealing MSU deposits [
26]. To our knowledge no animal model has been used to sequentially evaluate the changes of MSU crystal-induced acute arthritis, using a multimodality approach including US as an imaging tool. Thus, the aim of this study was to assess the spatial and temporal joint changes in a rabbit model of acute MSU crystal-induced arthritis by US, synovial fluid (SF) cell counts, and histopathological analyses.
Discussion
This study sequentially evaluated the temporal and spatial ultrasonographic, inflammatory, and structural joint changes observed in an animal model of acute gouty attack, by applying a multimodality approach.
In the past, a variety of animal models have been used to study the acute inflammatory response in crystal-induced arthritis [
10-
13]. Faires and McCarty reproduced an acute gouty episode by injecting 20 mg of MSU crystals into their own knees and into a canine animal model [
14]. Subsequently, murine models of peritoneal [
7,
11] and subcutaneous air-pouch cavity of MSU-crystals have been utilized to study the inflammatory response [
7,
18]. Torres
et al. [
37] evaluated behavioral changes in mice after an intra-articular injection of MSU crystals and quantified systemic inflammatory biomarkers. An adult Japanese female rabbit model was used by Nishimura
et al. [
38] to evaluate the role of locally produced IL-8 in knee joints injected with MSU crystals.
On the other hand, contrast-enhanced US has been employed to assess synovial vascularity in a rabbit knee model of carrageenan-induced inflammatory arthritis [
39]. To the best of our knowledge there are no animal models that reported the use of US guidance to ensure proper needle placement in their very small joint cavities, allowing successful joint aspirations and injections, thus avoiding damage to adjacent anatomic structures.
Using a multimodality approach to monitor the temporal and spatial sonographic, inflammatory, and morphostructural changes observed in a rabbit knee model of acute gouty arthritis, we were able to reproduce two highly specific US signs of human gout: DCS and BSA [
17,
23,
27,
40]. Our results provide gross pathological evidence that the development of the characteristic sonographic features of gout, DCS and BSA after synthetic MSU-crystal injection, resulted from the precipitation of MSU crystals on the superficial layer of the articular cartilage and intra-articular soft tissues, respectively.
On the other hand, allopurinol crystals were used as a negative, microcrystalline, control group, because previous reports have shown the failure of these crystals to activate the NALP3 inflammasome [
5,
41]. US failed to demonstrate the presence of DCS and BSA in the allopurinol crystal-injected animal. However, our results at the joint level, showed a low-grade, limited, and transient inflammatory response demonstrated by mild intra-articular PD signal plus an inflammatory range of SF leukocyte cell counts.
By means of a multimodal approach, several temporal and spatial, early, MSU-deposition, gouty changes were observed in the rabbit knee model. DCS appeared in one half of MSU-injected joints at day 1, and increased up to 75% at days 3 and 7, indicating that these crystals tend to deposit very early in the initial acute, inflammatory stages of disease, and not only in its late, chronic stages, as previously proposed [
1]. As expected, from the synovial vascularization perspective, PD signals appeared as early as day 1, revealing hyperemia of synovial tissues in acutely inflamed rabbit joints, followed by a gradual reduction in signal intensity at days 3 and 7, respectively. These findings are consistent with the human counterpart; as atypical acute gouty attack is self-limiting, it peaks at 24 to 48 h after symptoms appear, and lasts 5 to 10 days [
4]. US evaluations in the rabbit model failed to show additional, previously reported, sonographic findings in gouty patients, such as tophus, hyperechoic cloudy areas, and/or erosions [
27]. The absence of these findings in our acute inflammatory animal model suggests that an extended time period with MSU crystals may be required to produce and display these elementary lesions as seen in gout on US.
A temporal correlation between SF cell counts and US findings was found at all experimental study time points. In contrast, histological findings were delayed in time relative to the SF inflammatory changes. Total SF cell counts from the MSU crystal group reached an inflammatory range that peaked at day 1. Despite the lack of standard normal values reported for rabbit SF, the control group displayed cell counts <2,000 cells/mm
3 that in its human counterpart are considered non-inflammatory [
42]. At day 1 pleomorphic cells, consistent with macrophages, were observed. These resident macrophages and infiltrating monocytes are responsible for initiating and driving the early inflammatory phase of the acute gout attack [
7,
43,
44]. Histological assessment revealed marked synovial lining hyperplasia following injection of MSU crystals at days 3 and 7. Remarkably, only scattered inflammatory cells were observed infiltrating the synovium of the MSU crystal group at day 1; notwithstanding this, the number of inflammatory cells in the SF reached its peak. However, intense infiltration of the synovial membrane by polymorphonuclear leukocytes occurred only until day 3.
Interestingly, the observation of an intense inflammatory reaction spatially coincides with the detection of giant macrophages in the SF. At day 7 histologic evaluation revealed only focal neutrophilic infiltrates of the synovial membrane that are consistent with mild and focal synovitis, which correlates with the non-inflammatory SF white-cell count observed at this time point. These findings suggest that deposition of MSU crystal in the joint space and synovial membrane elicits a strong inflammatory reaction that promotes recruitment of a large number of polymorphonuclear leukocytes to the SF within 24 h. As the inflammatory response temporarily progresses, the synovial membrane becomes hyperplastic and thickened by widespread inflammatory cell infiltrates, resident macrophages in the SF become active and start to phagocyte MSU crystals, and polymorphonuclear leukocytes segregate within the synovial membrane. This reaction is self-limited and by day 7 the majority of MSU crystal deposits have been phagocytized, the SF becomes non-inflammatory, and few leukocytes can be found within the synovial membrane, with only mild synovitis. Visualization of MSU crystals over the cartilage surface was accomplished by gross examination of the articular cartilage but was not shown histologically. This discrepancy can be explained by mechanical loss of the crystals by surgical removal of the extremity, vibration of the band saw and mechanical washing process resulting in MSU-crystal loss [
35].
Animal models of human illness provide invaluable tools to understand the basic biological mechanisms for identifying and validating novel molecular targets and pathways involved in the pathogenesis of the disease, and to identify potential therapeutic and preventive agents [
45]. The fact that the rabbit knees injected with MSU crystals were the only ones to produce a severe inflammatory synovial response and reproduced the typical US findings of human gout, supports both the internal and external validity of our animal model. The concurrent validity of the animal model was established by demonstrating US-detected abnormalities and the presence of MSU crystals by both polarized light microscopy and histological analysis as the gold standard.
Our study has several limitations. First, in contrast to the systemic and local inflammatory process triggered by the in-situ re-crystallization of endogenous MSU crystals in the synovial membrane, our model responded with a local inflammatory reaction after inoculation of exogenous synthetic MSU crystals. Second, clinical parameters such as pain, temperature, swelling, behavioral testing, and disability were not evaluated. Third, we only evaluated intra-articular changes due to MSU-crystal injections within a limited time period (7 days); it would be appropriate to monitor the intra-articular changes for a longer period of time and/or recurrent MSU-crystal injections.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
CP participated in the conception of study, interpretation of data, drafted the manuscript, and gave final approval of the version of the paper to be published. AJFG participated in the conception of study, perfomed the statistical analysis, and was involved in drafting the manuscript. YZC carried out the chemical synthesis and characterization of uric acid and allopurinol crystals, sterility test of crystals, histological cuts, and helped to draft the manuscript. JFT performed the synovial fluid analysis, participated in the interpretation of data, and drafted the manuscript. CHD performed ultrasound studies of rabbit knees, ultrasound data interpretation, and helped to draft the manuscript. ALM participated in the acquisition and interpretation of histological images, electron microscopy, and helped to draft the manuscript. IAS participated in the surgical removal of rabbit knees, identification of macroscopic findings of joints, performed arthrocentesis, and helped to revise the manuscript. JCG participated in the identification of macroscopic findings of joints, surgical removal of rabbit knees, and helped to revise the manuscript. LV performed ultrasound studies of rabbit knees, ultrasound data interpretation, and helped to draft the manuscript. FGV carried out histological staining process, gave interpretation of results, and helped to revise the manuscript. LEGQ made substantial contributions to the conception and design of the study, participated in the interpretation of data, and helped to draft the manuscript. MCGR made substantial contributions to the conception and design of the study, participated in the acquisition of data, and helped to draft the manuscript. MG participated in the acquisition of data, ultrasound data interpretation, drafted the manuscript, and gave final approval of the version to be published. AMR made substantial contributions to the conception and design of the study, participated in the acquisition of data, helped to draft the manuscript, and gave final approval of the version to be published. ALR participated in the conception of study and interpretation of data, was involved in drafting the manuscript, and gave final approval of the final version manuscript, had full access to of the all data in the study, and takes full responsibility for the integrity of the data and the accuracy of the data analysis. All authors read and approved the final version of the manuscript.
AMR work was supported in part by grant P20GM104937 from the NIH.