Rat Adjuvant Methods

Rat Adjuvant Induced Arthritis (AIA) Histology/Histopathology Methods

 

Processing of Joints

Ankles from AIA rats are cut in half longitudinally and the two halves are embedded together in a paraffin block. A section is cut from each block and stained with toluidine blue (t. blue).


Evaluation of Joints

The following parameters are scored according to the indicated criteria.


Inflammation Score

0 = Normal (Dorsal to ventral paw thickness 80 to 100 units on 16x, 5040 to 6300 µm)

0.5 = Very minimal, generally focal infiltration of inflammatory cells in periarticular tissue (Paw thickness 80 t0 100 units, 5040 to 6300 µm)

1 = Minimal infiltration of inflammatory cells in periarticular tissue (Paw thickness 90 to 120 units, 5670 to 7560 µm)

2 = Mild infiltration of inflammatory cells in periarticular tissue (Paw thickness 90 to 120 units, 5670 to 7560 µm)

3 = Moderate infiltration with moderate edema (Paw thickness 121 to 130 units, 7523 to 8190 µm)

4 = Marked infiltration with marked edema (Paw thickness 131 to 140 units on 16x, 8253 to 8820 µm)

5 = Severe infiltration with severe edemamay have focal to multifocal microabscesses, periarticular tissue expansion (Paw thickness 141 to 150 units, 8883 to 9450 µm)

6 = Very Severe infiltration with severe edema, periarticular tissue expansion, may may have numerous microabscesses (Paw thickness 151 to 160 units, 9513 to 10080 µm)

7 = Very Severe infiltration with severe edema, periarticular tissue expansion and may have numerous microabscesses (Paw thickness >160 units, >10080 µm)


Pannus Score

0 = Normal

0.5 = Very minimal, focal or only few marginal zones affected

1 = Minimal infiltration of pannus in cartilage and subchondral bone, primarily affects marginal zones

2 = Mild infiltration (<1/4 width of tibia and/or most tarsals at marginal zones)

3 = Moderate infiltration (1/4 to 1/3 width of tibia and/or most small tarsals at marginal zones)

4 = Marked infiltration (1/2 to 3/4 width of tibia and/or most tarsals affected at marginal zones and extending to central areas)

5 = Severe infiltration (>3/4 width of tibia and/or most tarsals affected at marginal zones and extending to central areas)

6 = Very severe infiltration (>3/4 width of tibia and/or most tarsals affected at marginal zones and extending into central areas of bone, moderate distortion of overall architecture due to infiltration of pannus into bone with accompanying destruction)

7 = Severe infiltration (>3/4 width of tibia and/or tarsals affected at marginal zones and extending into central areas of bone, severe distortion of overall architecture due to infiltration of pannus into bone with accompanying destruction)


Cartilage Damage Score

0 = Normal

0.5 = Very minimal, focal or only few marginal zones affected

1 = Minimal multifocal, generally marginal zone proteoglycan loss

2 = Mild marginal zone proteoglycan loss with small areas of surface erosion

3 = Moderate, 1 cartilage surface/joint with up to 50% loss of matrix

4 = Marked, 2-3 cartilage surfaces/joints with at least 50 to 100% loss of matrix

5 = Severe, 4 cartilage surfaces/joints with at least 50 to 100% loss of matrix

6 = Very severe, 5 cartilage surfaces/joints with at least 50 to 100% loss of matrix,

7 = Near total destruction, most cartilage surfaces/joints have at least 75 to 100% loss of matrix


Bone Resorption Score

0 = Normal

0.5 = Very minimal, score reserved for those that are normal on low magnification but have the earliest hint of a few small areas of resorption in the metaphysis or in the tarsal bones.

1 = Minimal, small definite areas of resorption in distal tibial trabecular or cortical bone or in the tarsal bones apparent on low magnification. Rare osteoclasts.

2 = Mild, more numerous areas (≤25% loss of bone in growth plate/metaphyseal area) of resorption in distal tibial trabecular or cortical bone and tarsals apparent on low magnification, osteoclasts more numerous. If prior resorption has occurred, new medullary bone may have partially filled in areas of resorption.

3 = Moderate, obvious resorption of distal tibial medullary trabecular and some cortical bone without major defects in both distal tibial cortices. Loss of some medullary trabeculae with a possible 50 to 100% loss across growth plate/ metaphyseal area but very little effect on cortices, or 26 to 50% loss in some but not all tarsal bones. Lesion apparent on low magnification, osteoclasts more numerous. If prior resorption has occurred, new medullary bone may have partially filled in areas of resorption.

4 = Marked, full or partial thickness defects in one or both distal tibial cortices, often with distortion of profile of remaining cortical surface. Marked loss of medullary bone of distal tibia (50 to 100% loss across growth plate/metaphyseal area and cortices and may have minor loss in small tarsals) but good retention of architecture or, if little effect on distal tibia, then 50 to 75% loss of cortical and medullary bone in tarsals. Numerous osteoclasts. If prior resorption has occurred, new medullary bone may have partially filled in areas of resorption.

5 = Severe, full thickness defects in both distal tibial cortices with >75% loss across growth plate/metaphyseal area and up to 50% loss in some tarsals. Severe loss of medullary bone of distal tibia but overall architecture is still evident. Numerous osteoclasts. If prior resorption has occurred, new medullary bone may have partially filled in areas of resorption.

6 = Very Severe, full thickness defects in both distal tibial cortices and total loss in growth plate/metaphyseal area and in most tarsal bones. Distortion of the profile of the ankle, but the basic structure is identifiable. Marked loss of medullary bone of distal tibia and numerous osteoclasts. If prior resorption has occurred, new medullary bone may have partially filled in areas of resorption.

7 = Near Total Destruction, full thickness defects in both distal tibial cortices with total loss across growth plate/metaphyseal area and in most tarsal bones. Severe distortion of the profile of the ankle – basic structure is difficult to identify. Numerous osteoclasts. If prior resorption has occurred, new medullary bone may have partially filled in areas of resorption.


Periosteal New Bone Formation Score and Measurement

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 (1 to 2 units, 63 to 126 µm at any location)

1 = Minimal multifocal proliferation, width at any location (3 to 4 units, 127 to 252 µm)

2 = Mild multifocal on tarsals, diffuse in some locations, width at any location (5 to 6 units, 253 to 378 µm)

3 = Moderate multifocal on tarsals, diffuse in most other locations (7 to 8 units, 379 to 504 µm)

4 = Marked multifocal on tarsals, diffuse at most other locations (9 to 10 units, 505 to 630 µm)

5 = Severe, multifocal on tarsals, diffuse at most other locations (11 to 12 units, 631 to 756 µm)

6 = Very Severe, multifocal on tarsals, diffuse at most other locations (13 to 15 units, 757 to 945 µm)

7 = Very Severe, multifocal on tarsals, diffuse at most other locations (>15 units, >945 µm)


Summed Score

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


Paw Thickness

Measurements are taken from the dorsal skin surface (in flexion angle) to ventral skin surface (across the tarsal joints) in an attempt to quantitate the inflammatory edema. Corrections are made for any artifactual tissue separation.

Spleen Histopathology (optional)

Spleens are embedded in the cross-section with multiple spleens per block. Sections are cut and stained with hematoxylin and eosin (H&E). The following parameters are scored according to the indicated criteria.


Inflammation Score

0 = None

0.5 = Mild increase in neutrophils in red pulp

1 = Minor in capsule/subcasule with no distinct foci in red pulp, mild diffuse increase in neutrophils in red pulp

2 = Capsular/subcapsular inflammation plus small distinct foci in red pulp, diffuse increase in neutrophils in red pulp

3 = Multifocal areas of pyogranulomatous infiltrate in capsule and red pulp occupying less than 1/4 of the total red pulp, diffuse increase in neutrophils in red pulp

4 = Multifocal areas of pyogranulomatous infiltrate in capsule and red pulp occupying less than 1/2 but greater than 1/4 of the total red pulp, diffuse increase in neutrophils in red pulp

5 = Multifocal areas of pyogranulomatous infiltrate in capsule and red pulp occupying greater than 1/2 of the total red pulp, diffuse increase in neutrophils in red pulp


Lymphoid Atrophy Score

0 = None

1 = Minimal loss of marginal zone but no decrease in periarteriolar T cells

2 = Mild loss of marginal zone with minimal to no decrease in periarteriolar T cells

3 = Moderate loss of marginal zone with mild decrease in periarteriolar T cells

4 = Marked (near total) loss of marginal zone with mild to moderate decrease in periarteriolar T cells

5 = Severe (near total) loss of marginal zone with marked to severe decrease in periarteriolar T cells


Extramedullary Hematopoiesis (EMH) Score

0 = None present

0.5 = Small, multifocal areas of megakaryocytes and erythroid/myeloid precursors in red pulp (normal in male rats), not visible on low magnification

1 = Small, multifocal areas of megakaryocytes and erythroid/myeloid precursors in red pulp, visible on low magnification

2 = Mild increase in multifocal areas of megakaryocytes and erythroid/myeloid precursors in red pulp, visible on low magnification

3 = Moderate increase in multifocal areas of megakaryocytes and erythroid/myeloid precursors in red pulp, visible on low magnification

4 = Marked increase in diffuse areas of megakaryocytes and erythroid/myeloid precursors in red pulp, visible on low magnification

5 = Severe diffuse areas of megakaryocytes and erythroid/myeloid precursors in red pulp-dominates splenic morphology, visible on low magnification

 

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