Assessing the Effects of Parthenolide on Inflammation, Bone Loss, and Glial Cells within a Collagen Antibody-Induced Arthritis Mouse Model

Abstract

Rheumatoid arthritis is characterised by a chronic inflammatory response resulting in destruction of the joint and significant pain. Although a range of treatments are available to control disease activity in RA, bone destruction and joint pain exist despite suppression of inflammation. This study is aimed at assessing the effects of parthenolide (PAR) on paw inflammation, bone destruction, and pain-like behaviour in a mild collagen antibody-induced arthritis (CAIA) mouse model. CAIA was induced in BALB/c mice and treated daily with 1 mg/kg or 4 mg/kg PAR. Clinical paw inflammation was scored daily, and mechanical hypersensitivity was assessed on alternate days. At end point, bone volume and swelling in the paws were assessed using micro-CT. Paw tissue sections were assessed for inflammation and pre-/osteoclast-like cells. The lumbar spinal cord and the periaqueductal grey (PAG) and rostral ventromedulla (RVM) regions of the brain were stained for glial fibrillary acidic protein (GFAP) and ionised calcium-binding adaptor molecule 1 (IBA1) to assess for glial reactivity. Paw scores increased in CAIA mice from days 5-10 and were reduced with 1 mg/kg and 4 mg/kg PAR on days 8-10. Osteoclast-like cells on the bone surface of the radiocarpal joint and within the soft tissue of the hind paw were significantly lower following PAR treatment (p < 0.005). GFAP- and IBA1-positive cells in the PAG and RVM were significantly lower following treatment with 1 mg/kg (p < 0.0001 and p = 0.0004, respectively) and 4 mg/kg PAR (p < 0.0001 and p = 0.001, respectively). In the lumbar spinal cord, IBA1-positive cells were significantly lower in CAIA mice treated with 4 mg/kg PAR (p = 0.001). The findings indicate a suppressive effect of both low- and moderate-dose PAR on paw inflammation, osteoclast presence, and glial cell reactivity in a mild CAIA mouse model.

Figure 1

Evaluation of local inflammation and mechanical hypersensitivity of mouse paws: (a) representative macroscopic appearance of the front paws at day 7 postarthritis induction; (b) average paw scores of each group over the 10-day model. Control mice had a paw score of 0 at each time point; (c) mean tactile paw withdrawal threshold of each group on alternate days throughout the 10-day model. Error bars represent SEM (n = 8 mice per control, CAIA+PAR 1 mg/kg, and CAIA+PAR 4 mg/kg; n = 6 mice per CAIA; ^∗p < 0.05).

Figure 2

Assessment of bone volume (BV) and paw volume (PV) in mouse paws by high-resolution micro-CT: (a) three-dimensional micro-CT models of the radiocarpal joint and surrounding soft tissue (indicated in red) in the right paw. White arrows represent soft tissue volume for control. Yellow arrows represent the soft tissue volume for each disease group and highlight the difference in soft tissue and volume observed. Mean BV in the radiocarpal joint and hind paw (b and c, respectively) and mean PV in the radiocarpal joint and hind paw (d and e, respectively) expressed in mm³, as assessed by micro-CT analysis at day 11. Error bars represent SEM (n = 16 paws per control, CAIA+PAR 1 mg/kg, and CAIA+PAR 4 mg/kg; n = 12 paws per CAIA; ^∗p < 0.05, ^∗∗∗p = 0.0001, ^∗∗∗∗p < 0.0001 compared to control; ^##p = 0.04 compared to CAIA).

Figure 3

Representative three-dimensional micro-CT models of the hind paw showing pitting in the navicular: (a) superior view of left hind paws from the control (first column), CAIA (second column), CAIA+PAR 1 mg/kg (third column), and CAIA+PAR 4 mg/kg groups (fourth column). Orange boxes identify the navicular which is presented at greater magnification (b) to emphasise the pitting present in this bone.

Figure 4

Histological assessment of the radiocarpal joint and hind paws: (a) representative haematoxylin and eosin- (H&E-) stained images of the radiocarpal joint (20x magnification; scale bars represent 100 μm). Semiquantitative analysis of inflammatory cell infiltration, cartilage and bone degradation, and pannus formation in H&E-stained sagittal sections of the radiocarpal (b) and hind paws (c). Error bars represent SEM (n = 16 paws per control, CAIA+PAR 1 mg/kg, and CAIA+PAR 4 mg/kg, n = 12 paws per CAIA; ^∗∗∗p < 0.0005 and ^∗∗∗∗p < 0.0001 compared to control).

Figure 5

Histological assessment of osteoclast-like cells in the radiocarpal joint and hind paws: (a) representative TRAP-stained images of the radiocarpal joint (20x magnification; scale bars represent 100 μm). Average values of TRAP-positive multinucleated cells on the bone surface and within the surrounding soft tissue in the radiocarpal joint (b and c, respectively) and the hind paws (d and e, respectively). Error bars represent SEM (n = 16 paws per control, CAIA+PAR 1 mg/kg, and CAIA+PAR 4 mg/kg, n = 12 paws per CAIA; ^∗∗∗∗p < 0.0001 compared to control; ^#p < 0.05 compared to CAIA; ^##p = 0.006 compared to CAIA).

Figure 6

Histological assessment of GFAP-positive cells in the lumbar spinal cord region, PAG, and RVM: (a) Representative GFAP-stained image of the spinal cord (40x magnification; scale bars represent 50 μm); (b) representative GFAP-stained image of the PAG (40x magnification; scale bars represent 50 μm); (c) area under the curve analysis of the average number of GFAP-positive cells in serial sections within the lumbar region of the spinal cord. Average number of GFAP-positive cells in the PAG and RVM of the brain (d and e, respectively). Error bars represent SEM (n = 8 animals per control, CAIA+PAR 1 mg/kg, and CAIA+PAR 4 mg/kg, n = 6 animals per CAIA), ^∗∗∗∗p < 0.00001, ^∗∗p < 0.002, ^∗p < 0.03.

Figure 7

Histological assessment of IBA1-positive cells in the lumbar spinal cord region, PAG, and RVM: (a) representative IBA1-stained image of the spinal cord (40x magnification; scale bars represent 50 μm); (b) representative IBA1-stained image of the PAG (40x magnification; scale bars represent 50 μm); (c) area under the curve analysis of the average number of IBA1-positive cells on serial sections in the lumbar region of the spinal cord. Average number of IBA1-positive cells in the PAG and RVM of the brain (d and e, respectively). Error bars represent SEM (n = 8 animals per control, CAIA+PAR 1 mg/kg, and CAIA+PAR 4 mg/kg, n = 6 animals per CAIA), ^∗∗∗∗p < 0.00001, ^∗∗p < 0.002, ^∗p < 0.03.