Mouse Collagen Methods

Collagen Induced Arthritis in Mice (CIA) Histology/Histopathology Methods

 

Processing of Joints

Paws are embedded in paraffin in the frontal plane and knees are embedded with the patella facing down. Ankles, if left attached to the hind paw, are also embedded in the frontal plane but may be detached and sectioned in the sagittal plane for special purposes. Left/right pairs are typically embedded in the same block.  Sections are cut and stained with toluidine blue.


Evaluation of Joints

When scoring paws or ankles from mice with lesions of type II collagen arthritis, severity of changes as well as number of individual joints affected must be considered. When only one to three joints of the paws or ankles out of a possibility of numerous metacarpal/metatarsal/digit or tarsal/tibiotarsal joints are affected, an arbitrary assignment of a maximum score of 0.5, 1, 2 or 3 for parameters below is given depending on severity of changes. If more than three joints are involved, the criteria below are applied to the most severely affected/majority of joints.  In the case of knees, severity of changes in medial and lateral, as well as femoropatellar spaces, are considered.

The following parameters are scored according to the indicated criteria. Unless otherwise indicated, criteria apply to both paws and knees.  Mean values for each parameter are determined separately for the paws, knees, and the entire animal (if applicable):


Inflammation Score (Paw)

0 = Normal

0.5 = Very minimal, affects only one joint or minimal multifocal periarticular infiltration of inflammatory cells

1 = Minimal infiltration of inflammatory cells in synovium and periarticular tissue of affected joints

2 = Mild infiltration of inflammatory cells. If referring to paws, generally restricted to affected joints (1 to 3 affected)

3 = Moderate infiltration with moderate edema. If referring to paws, restricted to affected joints, generally three to four joints and the wrist or ankle are affected

4 = Marked infiltration affecting most areas with marked edema, one or two unaffected joints may be present

5 = Severe diffuse infiltration with severe edema affecting all joints (to some extent) and periarticular tissues


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 synovial areas

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

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.

DBA mice have an increased incidence of dactylitis and onchyoperiostitis affecting the nail bed and distal phalynx [1].  These lesions are recorded in the comments section but are not included in the inflammation score.


Pannus Score

0 = Normal

0.5 = Very minimal.  If paws, affects only one joint at marginal zone

1 = Minimal infiltration of pannus in cartilage and subchondral bone, marginal zones.  If paws, affects two or more joints

2 = Mild infiltration with marginal zone destruction of hard tissue in affected joints

3 = Moderate infiltration with moderate hard tissue destruction in affected joints

4 = Marked infiltration with marked destruction of joint architecture, affecting most joints

5 = Severe infiltration associated with total or near total destruction of joint architecture, affects all joints


Cartilage Damage Score

0 = Normal

0.5 = Very minimal, affects marginal zones only of one to several areas (knees) or joints (paws)

1 = Minimal: Generally minimal to mild loss of toluidine blue staining (proteoglycan) with no obvious chondrocyte loss or collagen disruption in affected joints/areas

2 = Mild: Generally mild loss of t. blue staining (proteoglycan) with focal areas of chondrocyte loss and/or collagen disruption in some affected joints/areas. Paws may have one or two digit joints with near total to total loss of cartilage

3 = Moderate: Generally moderate loss of t. blue staining (proteoglycan) with multifocal chondrocyte loss and/or collagen disruption in affected joints/areas. Paws may have three or four joints with near total or total loss. In the knee, some matrix remains on any affected surface with areas of severe matrix loss

4 = Marked: Marked loss of t. blue staining (proteoglycan) with multifocal marked (depth to deep zone or tidemark) chondrocyte loss and/or collagen disruption in most joints with a few unaffected or mildly affected. In the knee, one surface with total to near total cartilage loss

5 = Severe: Severe diffuse loss of t. blue staining (proteoglycan) with severe (depth to tide mark) chondrocyte loss and/or collagen disruption in most or all joints.  In the knee, two or more surfaces with total to near total cartilage loss


Bone Resorption Score (Paw)

0 = Normal

0.5 = Very minimal, affects only one joint or is restricted to cortical/subperiosteal areas

1 = Minimal: Small/few areas of definite resorption, not readily apparent on low magnification, rare osteoclasts in affected joints, restricted to marginal zones

2 = Mild: More numerous/larger areas of resorption, osteoclasts more numerous in affected joints, mainly in marginal zones but some extension to load bearing areas, may have endosteal proliferation in areas of resorption

3 = Moderate: Obvious resorption of medullary trabecular and cortical bone without widespread full thickness defects in cortex, loss of medullary trabeculae, lesion apparent on low magnification, osteoclasts more numerous in affected joints, may have endosteal proliferation in areas of resorption

4 = Marked: Full thickness defects in cortical bone, often with distortion of profile of remaining cortical surface, marked loss of medullary bone, numerous osteoclasts, affects most joints, may have endosteal proliferation in areas of resorption

5 = Severe: Full thickness defects in cortical bone and destruction of joint architecture of all joints, may have endosteal proliferation in areas of resorption


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


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 less than 40 µm width (<1 unit on 25x)

1 = Minimal multifocal early proliferation, measures 40 to 119 µm width (1 to 2 units on 25x)

2 = Mild multifocal to diffuse with widths that measure 120 to 239 µm (3 to 5 units on 25x)

3 = Moderate diffuse with widths that measure 240 to 319 µm (6 to 7 units on 25x)

4 = Marked diffuse with widths that measure 320 to 439 µm  (8 to 10 units on 25x)

5 = Severe, diffuse with widths that measure greater than 439 µm (>10 units on 25x)


Summed Score

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


 

References
1. Lories RJU, Matthys P, de Vlam K, Derese I, Luyten FP.  Ankylosing enthesitis, dctylitis, and oncychoperiostitis in male DBA/1 mice: a model of psoriatic arthritis.  Ann Rheum Dis 2004;63l595-598.

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