Evaluation of antigen-induced synovitis in a porcine model: Immunological, arthroscopic and kinetic studies

Abstract

Background: Synovitis is an inflammation-related disease linked to rheumatoid arthritis, osteoarthritis, infections and trauma. This inflammation is accompanied by immune cells infiltration which initiates an inflammatory response causing pain, discomfort and affecting the normal joint function. The treatment of synovitis is based on the administration of anti-inflammatory drugs or biological agents such as platelet rich plasma and mesenchymal stem cells. However, the evaluation and validation of more effective therapies of synovitis requires the establishment of clinically relevant animal models.

Results: In this study, Large White pigs were pre-immunized to evaluate an antigen-induced synovitis. The immune monitoring of synovial fluids in this model allowed us the identification of IL-12p40 and T cell subsets as immune biomarkers. Moreover, the evolution of synovitis was performed by arthroscopic procedures and kinetic analysis. In summary, this paper describes an animal model of antigen-induced synovitis to be used in the evaluation of anti-inflammatory therapies.

Conclusions: The novelty of this paper lies in the development of a clinically relevant model of synovitis which permits the simultaneous evaluation of synovitis from an immunological, surgical and kinetic point of view.

Keywords: Animal model; Inflammation; Synovitis.

Fig. 1

Temporal scheme of the immunization protocol and monitoring. Subcutaneous BSA injections (black arrows), intra-articular BSA injection (grey arrow), blood sampling (triangles), synovial fluid sampling (squares), pressure platform gait analysis (rhombus) and the arthroscopic surgery (circle) are shown

Fig. 2

Lymphocyte subsets distribution in peripheral blood cells from BSA-immunized animals. Peripheral blood lymphocytes were weekly collected for flow cytometry analysis. Black arrows indicate the subcutaneous BSA injections and the grey arrow indicates the intra-articular injection. The graphic shows the percentage of CD4+ T cells, CD8+ T cells and their ratio. The lower boundary of the box indicates the 25th percentile and the upper boundary the 75^thpercentile. Bars above and below the box indicate the 90th and 10^th percentiles. The line within the box marks the median (n = 4). No statistically significant differences were found between groups

Fig. 3

Humoral response to bovine serum albumin in immunized animals. Plasma samples were weekly collected and anti-BSA IgG levels were quantified by ELISA immunoassay. In the graphic, black arrows indicate the subcutaneous BSA injections and the grey arrow indicates the intra-articular injection. Values show the mean ± SD (n = 4). *Statistically significant difference (p ≤ 0.05) compared to basal level

Fig. 4

Distribution of synovial lymphocyte subsets. Synovial fluid lymphocytes were collected for flow cytometric analysis just before intra-articular injection (basal) and 7 days after. The graphic shows the percentage of CD4+ T cells (a) CD8+ T cells (b) and their ratio c. Values show the mean ± SD (n = 3). *Statistically significant difference (p ≤ 0.05) compared to basal level

Fig. 5

Quantification of IL-12p40 levels in synovial fluid. Synovial fluid was collected at day 7, 14 and 21 after intra-articular injection of PBS and BSA. Cytokine levels were determined by Luminex xMAP technology. The lower boundary of the box indicates the 25th percentile and the upper boundary the 75th percentile. Bars above and below the box indicate the 90^th and 10^th percentiles. The line within the box marks the median (n = 4). Dot line indicates the basal levels (just before intra-articular injection). *Statistically significant difference (p ≤ 0.05) compared to basal level

Fig. 6

Surgical approach and arthroscopic analysis. Two weeks after intra-articular injection of PBS or BSA, an arthroscopic evaluation was performed. Figure shows the access to the articular cavity a, the arthroscopic procedure b representative image of arthroscopy in the control joint c and representative image of arthroscopy in the BSA-injected joint d. Synovial fluid classification according to nucleated cells/mm³ e. Synovial fluid is classified as “normal” if it contains less than 180 nucleated cells/mm³ or “non-inflammatory” when synovial fluid contains less than 2000 cells/mm³. On the contrary, when synovial fluid contains 2000–50,000 cells/mm³ it is classified as “inflammatory” [19]

Fig. 7

Pressure platform gait analysis. Seven days after intra-articular injection of PBS or BSA, a pressure platform gait analysis was performed to evaluate plantar pressure distributions. a Above, a representative image of the gait analysis (LF: left forelimb; LH: left hind limb; RF: right forelimb; RH: right hind limb) is represented. Below, the pressure of each limb is shown. The legend on the right shows the equivalence between numeric and colorimetric values. Maximum forces b and impulses c in control and BSA-injected limbs (n = 4). The lower boundary of the box indicates the 25^th percentile and the upper boundary the 75th percentile. Bars above and below the box indicate the 90^th and 10^th percentiles. The line within the box marks the median. No significant differences were found between PBS and BSA injected joints