Histopathologic Methods for Mouse Antibody Arthritis Model

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Antibody Induced Arthritis in Mice Histology/Histopathology Methods

Processing of Joints

Decalcified knees are embedded with the patella facing down (frontal plane). Sections are cut and stained with toluidine blue.

Evaluation of Joints

Antigen arthritis knees are scored for multiple pathology parameters using the criteria delineated below and expressed as individual values as well as summed (total joint score) values.

Inflammation Score (Knee)

0 = Normal
0.5 = Very minimal, affects only one area of the synovium or minimal multifocal periarticular infiltration of inflammatory cells
1 = Minimal infiltration of inflammatory cells in synovium and periarticular tissue of affected joints
2 = Mild diffuse infiltration of inflammatory cells
3 = Moderate diffuse infiltration of inflammatory cells
4 = Marked diffuse infiltration of inflammatory cells
5 = Severe diffuse infiltration of inflammatory cells

Pannus Score

0 = Normal
0.5 = Very minimal, affects only one small area at marginal zone
1 = Minimal infiltration with marginal zone destruction and slight extension into subchondral bone of 1 surface
2 = Mild infiltration with marginal zone destruction and slight extension into subchondral bone of 2 or more surfaces
3 = Moderate infiltration with marginal zone destruction and moderate extension (1/3 of subchondral plate) into subchondral bone of 1 surface, minor at others
4 = Marked infiltration with marked destruction of subchondral bone (greater than 1/2 of total width) of at least one surface and less destruction at others
5 = Severe infiltration associated with severe cartilage and bone loss

Cartilage Damage Score

0 = Normal
0.5 = Very minimal, affects marginal zones, proteoglycan loss mainly.
1 = Minimal, generally minimal to mild diffuse or multifocal loss of toluidine blue staining (proteoglycan) with no obvious chondrocyte loss or collagen disruption in affected areas. Matrix or chondrocyte loss 5-10% of total cartilage area.
2 = Mild, generally mild loss of t. blue staining (proteoglycan) with focal areas of chondrocyte loss and/or collagen disruption in some affected areas. Matrix or chondrocyte loss 11-25% of total cartilage area.
3 = Moderate, generally moderate loss of t. blue staining (proteoglycan) with multifocal chondrocyte loss and/or collagen disruption in affected joints/areas. Matrix or chondrocyte loss 26-50% of total cartilage area.
4 = Marked, marked loss of t. blue staining (proteoglycan) with multifocal marked (depth to deep zone or tidemark) chondrocyte loss and/or collagen disruption. Matrix or chondrocyte loss 51-75% of total cartilage area.
5 = Severe, severe diffuse loss of t. blue staining (proteoglycan) with severe (depth to tidemark) chondrocyte loss and/or collagen disruption in most or all joints. Matrix or chondrocyte loss 76-100% of total cartilage area.

Bone Resorption Score (Knee)

0 = Normal
0.5 = Minimal resorption affects only marginal zones
1 = Minimal, small areas of resorption, approximately 5-10% of total joint width of subchondral bone affected
2 = Mild, more numerous areas of resorption, definite loss of subchondral bone, approximately 11-25% of total joint width of subchondral bone affected
3 = Moderate, obvious resorption of subchondral bone, approximately 26-50% of total joint width of subchondral bone affected
4 = Marked, obvious resorption of subchondral bone, approximately 51-75% of total joint width of subchondral bone affected
5 = Severe, distortion of entire joint due to destruction, approximately 76-100% of total joint width of subchondral bone affected

Periosteal New Bone Formation Score and Measurement

Studies that go beyond the acute inflammatory stage often show varying degrees of periosteal new bone formation. The width of the largest area of new bone formation in a non-tangential section is measured and used to determine a score based on the following criteria:

0 = Normal, no periosteal proliferation
0.5 = Minimal focal or multifocal early proliferation, measures 40 µm width or less (1-2 units on 50x)
1 = Minimal multifocal early proliferation, measures 40-80 µm width (3-4 units on 50x)
2 = Mild multifocal to diffuse with widths that measure approximately 100-140 µm (5-7 units on 50x)
3 = Moderate diffuse with widths that measure approximately 160-220 µm (8-11 units on 50x)
4 = Marked diffuse with widths that measure approximately 240-300 µm (12-15 units on 50x)
5 = Severe, diffuse with widths that measure greater than 300 µm (>15 units on 25x)

Subchondral Bone Sclerosis Score

0 = Normal
0.5 = Very minimal focal sclerosis, approximate 1-5% increase in overall subchondral/epiphyseal bone density
1 = Minimal, approximate 6-10% increase in overall subchondral/epiphyseal bone density
2 = Mild, approximate 11-25% increase in overall subchondral/epiphyseal bone density
3 = Moderate, approximate 26-50% increase in overall subchondral/epiphyseal bone density
4 = Marked, approximate 51-75% increase in overall subchondral/epiphyseal bone density
5 = Severe, approximate 76-100% increase in overall subchondral/epiphyseal bone density

Summed Score

A sum of the five histopathology scores (subchondral sclerosis exclude from total) is calculated for each joint.

References

1. Baddack U, Hartmann S, Bang H, Grobe J, Loddenkemper C, Lipp M, Muller G. A chronic model of arthritis supported by a strain-specific periarticular lymph node in BALB/c mice. Nature Communications. DOI: 10.1038/ncomms2625.