Effects of Mild and Moderate Monoclonal Antibody Dose on Inflammation, Bone Loss, and Activation of the Central Nervous System in a Female Collagen Antibody-induced Arthritis Mouse Model

Abstract

Induction of severe inflammatory arthritis in the collagen antibody-induced arthritis (CAIA) murine model causes extensive joint damage and pain-like behavior compromising analysis. While mild models are less severe, their reduced, variable penetrance makes assessment of treatment efficacy difficult. This study aimed to compare macroscopic and microscopic changes in the paws, along with central nervous system activation between a mild and moderate CAIA model. Balb/c mice (n=18) were allocated to control, mild, and moderate CAIA groups. Paw inflammation, bone volume (BV), and paw volume (PV) were assessed. Histologically, the front paws were assessed for joint inflammation, cartilage damage, and pre/osteoclast-like cells and the lumbar spinal cord and the periaqueductal gray (PAG) region of the brain for glial reactivity. A moderate CAIA dose induced (1) significantly greater local paw inflammation, inflammatory cell infiltration, and PV; (2) significantly more osteoclast-like cells on the bone surface and within the surrounding soft tissue; and (3) significantly greater glial reactivity within the PAG compared with the mild CAIA model. These findings support the use of a moderate CAIA model (higher dose of monoclonal antibodies with low-dose lipopolysaccharide) to induce more consistent histopathological features, without excessive joint destruction.

Keywords: bone; glial cells; inflammation; inflammatory arthritis; micro-computed tomography; osteoclast.

Figure 1.

Evaluation of local inflammation in front and hind paws. (A) Representative macroscopic appearance of representative front paws at day 7 post-arthritis induction. (B) Daily average paw scores of each group throughout the 10-day model. Control mice had paw score 0 at each time point. Error bars represent SEM (n=5 mice per control and mild CAIA groups, n=8 mice per moderate CAIA group; ****p<0.0001 vs. control, ^*p<0.05 mild CAIA vs. moderate CAIA). Abbreviations: CAIA, collagen antibody-induced arthritis; SEM, standard error of the mean.

Figure 2.

Bone volume and paw volume of the front and hind paws assessed by high-resolution micro-CT. (A) Representative 3D micro-CT models of the right radiocarpal joint (white) and surrounding soft tissue (red). Arrows represent the width of the soft tissue, white arrows for control mice, compared with yellow arrows for disease groups. Mean BV and PV in the radiocarpal joint (B and D, respectively) and hind paws (C and E, respectively) expressed in mm³. Error bars represent SEM, n=10 paws (left and right paws taken together) per control and mild CAIA groups, n=16 (left and right paws taken together) per moderate CAIA group, one front paw was removed from the control group due to movement artifacts (****p<0.0001 vs. control, ^#p<0.01 vs. mild CAIA). Scale bar represents 1 mm and refers to (B) and (D). Abbreviations: BV, bone volume; CAIA, collagen antibody-induced arthritis; CT, computer tomography; PV, paw volume; SEM, standard error of the mean. *p<0.01, **p<0.003, ***p<0.0003.

Figure 3.

Representative 3D models of the right hind paw showing bone resorption pits. (A) Superior view of the right hind paw from control, mild CAIA, and moderate CAIA mice (3D micro-CT rendering). Orange boxes identify the navicular which is presented at greater magnification (B) to highlight the pitting present in this bone. (C) Lateral view of the right hind paw from control, mild CAIA, and moderate CAIA mice (3D micro-CT rendering). Orange boxes identify the cuboid which is presented at greater magnification (D) to show the pitting present in this bone. Red arrows represent bone resorption pits observed in the talus (A) and calcaneus (C). Scale bar represents 1 mm. Abbreviations: CAIA, collagen antibody-induced arthritis; CT, computer tomography.

Figure 4.

Histological assessment of inflammation, cartilage and bone destruction, and osteoclast-like cells in the radiocarpal joint. Representative H&E (A; 20× magnification) and TRAP (B; 40× magnification) stained tissue from mice radiocarpal joints. Blue arrows represent increased inflammatory cell infiltrate, pannus invasion, and subsequent cartilage/bone destruction. Yellow arrows represent multinucleated TRAP positive cells. C. Histological scores of H&E stained sagittal sections of the radiocarpal joint. D. Average values of TRAP positive multinucleated cells on the bone surface. Error bars represent SEM, n=10 paws (left and right together) per control and mild CAIA groups and n=15 paws (left and right together) per moderate CAIA group for histological analysis; n=10 paws (left and right together) per control and mild CAIA groups and n=14 (left and right together) paws per moderate CAIA group for TRAP analysis (*p<0.03, **p<0.0005, ***p<0.0001 vs. control, ^#p=0.02 vs. mild CAIA). Scale bars represent 100 µm for 20× magnification and 50 µm for 40× magnification. Abbreviations: CAIA, collagen antibody-induced arthritis; H&E, haematoxylin and eosin; SEM, standard error of the mean; TRAP, tartrate-resistant acid phosphatase.

Figure 5.

Histological assessment of GFAP-positive cells in the lumbar spinal cord and PAG region of the brain. Representative GFAP-stained tissue (40× magnification) from the lumbar spinal cord (A) and PAG region of the brain (B). Arrows represent GFAP-positive cells. (C) Area under the curve analysis of the average number of GFAP-positive cells in the lumbar spinal cord. (D) Average number of GFAP-positive cells in the PAG region of the brain. Error bars represent SEM (n=5 mice per control and mild CAIA groups, n=8 mice per moderate CAIA groups; *p=0.0134, ****p<0.0001 vs. control, ^#p=0.0179 vs. mild CAIA). Scale bars represent 50 µm. Abbreviations: CAIA, collagen antibody-induced arthritis; GFAP, glial fibrillary acidic protein; PAG, periaqueductal gray; SEM, standard error of the mean.

Figure 6.

Histological assessment of IBA1-positive cells in the lumbar spinal cord and PAG region. Representative IBA1-stained tissue (40× magnification) of the lumbar spinal cord (A) and PAG region of the brain (B). Arrows represent IBA-1 positive cells. (C) Area under the curve analysis of the average number of IBA1-positive cells in the lumbar spinal cord. (D) Average number of IBA1-positive cells in the PAG region of the brain. Error bars represent SEM (n=5 mice per control and mild CAIA groups, n=8 mice per moderate CAIA groups; *p=0.0381, ****p<0.0001 vs. control, ^#p=0.0144 vs. mild CAIA). Scale bars represent 50 µm. Abbreviations: CAIA, collagen antibody-induced arthritis; PAG, periaqueductal gray; SEM, standard error of the mean.