Histopathologic Methods for Collagen Induced Arthritis (CIA) in Rat

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Processing of Joints for Collagen Induced Arthritis (CIA) in Rat

Ankles are cut in half longitudinally and the two halves are embedded together in a paraffin block. Knees are cut in half in the frontal plane and the two halves are embedded together. Sections are cut from each block and stained with toluidine blue (t. blue).

Evaluation of Joints

The following parameters are scored according to the indicated criteria. Unless otherwise indicated, criteria apply to both ankles and knees:

Inflammation Score

0 = Normal
0.5 = Minimal focal inflammation
1 = Minimal infiltration of inflammatory cells in synovium/periarticular tissue
2 = Mild infiltration
3 = Moderate infiltration with moderate edema
4 = Marked infiltration with marked edema
5 = Severe infiltration with severe edema

The inflammatory infiltrate in mice and rats with type II collagen arthritis consists of neutrophils and macrophages with smaller numbers of lymphocytes when the lesions are in the acute to subacute phase. Tissue edema and neutrophil exudates within the joint space are common in the acute to subacute phase. As the inflammation progresses to chronic, mononuclear inflammatory cells (monocytes, lymphocytes) predominate and fibroblast proliferation, often with deposition of metachromatic matrix, occurs in synovium and periarticular tissue. Exudate is less common in the joint space. Unless indicated in the comments area, the inflammation type is acute to subacute.

Pannus Score (Ankle)

0 = Normal
0.5 = Minimal infiltration of pannus in cartilage and subchondral bone, affects only marginal zones and affects only a few joints
1 = Minimal infiltration of pannus in cartilage and subchondral bone, primarily affects marginal zones
2 = Mild infiltration (<1/4 of tibia or tarsals at marginal zones)
3 = Moderate infiltration (1/4 to 1/3 of tibia or small tarsals affected at marginal zones)
4 = Marked infiltration (1/2 to 3/4 of tibia or tarsals affected at marginal zones)
5 = Severe infiltration (>3/4 of tibia or tarsals affected at marginal zones, severe distortion of overall architecture)

Pannus Score (Knee)

0 = Normal
0.5 = Minimal infiltration of pannus in cartilage and subchondral bone, affects only marginal zones and affects only a few joints
1 = Minimal infiltration of pannus in cartilage and subchondral bone, approximately 1 to 10% of cartilage surface or subchondral bone affected
2 = Mild infiltration (extends over up to 1/4 of surface or subchondral area of tibia or femur), approximately 11 to 25% of cartilage surface or subchondral bone affected
3 = Moderate infiltration (extends over >1/4 but < 1/2 of surface or subchondral area of tibia or femur), approximately 26 to 50% of cartilage surface or subchondral bone affected
4 = Marked infiltration (extends over 1/2 to 3/4 of tibial or femoral surface), approximately 51 to 75% of cartilage surface or subchondral bone affected
5 = Severe infiltration, approximately 76 to 100% of cartilage surface or subchondral bone affected

Cartilage Damage Score (Ankle, emphasis on small tarsals)

0 = Normal
0.5 = Minimal decrease in toluidine blue staining, affects only marginal zones and affects only a few joints
1 = Minimal to mild loss of t. blue staining with no obvious chondrocyte loss or collagen disruption
2 = Mild loss of t. blue staining with focal mild (superficial) chondrocyte loss and/or collagen disruption
3 = Moderate loss of t. blue staining with multifocal moderate (depth to middle zone) chondrocyte loss and/or collagen disruption, smaller tarsals affected to 1/2 to 3/4 depth with rare areas of full thickness loss
4 = Marked loss of t. blue staining with multifocal marked (depth to deep zone) chondrocyte loss and/or collagen disruption, one or two small tarsals surfaces have full thickness loss of cartilage
5 = Severe diffuse loss of t. blue staining with multifocal severe (depth to tide mark) chondrocyte loss and/or collagen disruption affecting more than two cartilage surfaces

Cartilage Damage Score (Knee)

0 = Normal
0.5 = Minimal decrease in toluidine blue staining, affects only marginal zones
1 = Minimal to mild loss of t. blue staining with no obvious chondrocyte loss or collagen disruption
2 = Mild loss of t. blue staining with focal mild (superficial) chondrocyte loss and/or collagen disruption, may have few small areas of 50% depth of cartilage affected
3 = Moderate loss of t. blue staining with multifocal to diffuse moderate (depth to middle zone) chondrocyte loss and/or collagen disruption, may have one to two small areas of full thickness loss affecting less than ¼ of the total width of a surface and not more than 25% of the total width of all surfaces
4 = Marked loss of t. blue staining with multifocal to diffuse marked (depth to deep zone) chondrocyte loss and/or collagen disruption or one surface with near total loss and partial loss on others, total overall loss less than 50% of width of all surfaces combined
5 = Severe diffuse loss of t. blue staining with multifocal severe (depth to tide mark) chondrocyte loss and/or collagen disruption on both femurs and/or tibias, total overall loss greater than 50% of width of all surfaces combined

Bone Resorption Score (Ankle)

0 = Normal
0.5 = Minimal resorption affects only marginal zones and affects only a few joints
1 = Minimal: Small areas of resorption, not readily apparent on low magnification, rare osteoclasts
2 = Mild: More numerous areas of resorption, not readily apparent on low magnification, osteoclasts more numerous, <1/4 of tibia or tarsals at marginal zones resorbed
3 = Moderate: Obvious resorption of medullary trabecular and cortical bone without full thickness defects in cortex, loss of some medullary trabeculae, lesion apparent on low magnification, osteoclasts more numerous, 1/4 to 1/3 of tibia or tarsals affected at marginal zones
4 = Marked: Full thickness defects in cortical bone, often with distortion of profile of remaining cortical surface, marked loss of medullary bone, numerous osteoclasts, 1/2 to 3/4 of tibia or tarsals affected at marginal zones
5 = Severe: Full thickness defects in cortical bone, often with distortion of profile of remaining cortical surface, marked loss of medullary bone, numerous osteoclasts, >3/4 of tibia or tarsals affected at marginal zones, severe distortion of overall architecture

Bone Resorption Score (Knee)

0 = Normal
0.5 = Minimal resorption affects only marginal zones
1 = Minimal: Small areas of resorption, not readily apparent on low magnification, approximately 1 to 10% of total joint width of subchondral bone affected
2 = Mild: More numerous areas of resorption, definite loss of subchondral bone, approximately 11 to 25% of total joint width of subchondral bone affected
3 = Moderate: Obvious resorption of subchondral bone approximately 26 to 50% of total joint width of subchondral bone affected
4 = Marked: Obvious resorption of subchondral bone approximately 51 to 75% of total joint width of subchondral bone affected
5 = Severe: Distortion of entire joint due to destruction approximately 76 to 100% of total joint width of subchondral bone affected

Periarticular Matrix Deposition Score (Optional)

0 = Normal
1 = Faint, multi-focal metachromatic staining, no excessive expansion of periarticular tissue
2 = Darker, diffuse metachromatic staining, no excessive expansion of periarticular tissue
3 = Darker, diffuse metachromatic staining, mild expansion of periarticular tissue
4 = Darker, diffuse metachromatic staining, moderate expansion of periarticular tissue
5 = Darker, diffuse metachromatic staining, severe expansion of periarticular tissue

Periosteal New Bone Formation Score and Measurement (Ankles)

Studies that go beyond the acute inflammatory stage often show varying degrees of periosteal new bone formation. In order to convey the degree of periosteal new bone formation, the following scores are applied based on the distribution of periosteal bone proliferation and a measurement of width of periosteal new bone formation at the widest location.

0 = Normal, no periosteal proliferation
0.5 = Minimal focal or multifocal proliferation, measures less than 127 µm width (1 to 2 units at 16x) at any location
1 = Minimal multifocal proliferation, width at any location measures 127 µm to 252 µm (3 to 4 units at 16x)
2 = Mild multifocal on tarsals, diffuse in some locations, width at any location 253 µm to 441 µm (5 to 7 units at 16x)
3 = Moderate multifocal on tarsals, diffuse in most other locations, width at any location measures 442 µm to 630 µm (8 to 10 units at 16x)
4 = Marked multifocal on tarsals, diffuse at most other locations, width at any location measures 630 µm to 819 µm (11 to 13 units at 16x)
5 = Severe, multifocal on tarsals, diffuse at most other locations, width at any location measures >819 µm (>13 units at 16x)

Summed Score

A sum of the five histopathology scores is also calculated for each joint.