# Rat MIA Methods

### Rat mono-iodoacetate induced mono-arthritis Histology/Histopathology Methods

**Processing of Joints **

Knees are cut in half in the frontal plane and both halves are embedded together in paraffin. Sections are cut and stained with toluidine blue (t. blue).

Scores and measurements are determined for the knee half with the most severe lesions in the central load bearing location.

Percent loss and bone resorption are evaluated for each of the four surfaces (MTP, MFC, LTP, and LFC), and the mean values for the entire joint are also calculated. Other parameters represent the joint as a whole.

**Percent Loss of Cells**

Cell loss is determined by estimating the area of cartilage in which there are no viable-appearing chondrocytes. This is expressed as a percentage of the total cartilage area.

**Percent Loss of PG**

Proteoglycan (PG) loss was determined by estimating differences in intensity of t. blue matrix staining. If there is total loss of matrix, PG loss is given a value of 100%. If matrix loss is near total with minor staining in damaged, mostly acellular matrix, then a value of 90 to 99% is given. If most of the collagen matrix is intact and there is faint loss in surfaces with near normal cellularity, then a value of 1 to 10% is given. Otherwise, a value of 5% less than the cell loss is used.

**Percent Loss of Matrix**

Collagen matrix loss is an indication of disruption and loss of type II collagen. A value of 5% is given if there is definite collapse and 10% if there is a focal cleft. Otherwise, the thickness of the cartilage decrease is estimated using a micrometer and expressed as a percentage of the total original cartilage thickness.

**Mean Percent Loss**

A mean of the cell, PG, and matrix loss parameters is calculated for each surface.

**Cartilage Damage Score**

The cartilage damage score is calculated from the mean of all cartilage loss as follows:

0 = Normal

1 = 1 to 20% damage

2 = 21 to 40% damage

3 = 41 to 60% damage

4 = 61 to 80% damage

5 = 81 to 100% damage

**Bone Resorption Percent**

Bone resorption is indicative of resorption of bone and calcified cartilage often with collapse of cartilage into the epiphysis, with and without chondrogenesis in the area of collapse. In focal or diffuse areas, cartilage necrosis extended through the articular-epiphyseal (AE) growth plate (tidemark) and resulted in areas of subchondral bone resorption and/or thickened areas of proliferated new cartilage, which often extended into the subjacent bone marrow. Affected areas with subchondral bone resorption had replacement of hematopoietic marrow with fibrous tissue and occasionally chondrogenesis was evident. The percent of the total bone that is affected (by linear estimate across the surface, not depth) is recorded.

**Bone Resorption Score**

A score for bone resorption is calculated from the mean (all four surfaces) percent as follows:

0 = None

1 = ≤10%

2 = 10 to 30%

3 = 31 to 60%

4 = 61 to 90%

5 = >90%

**Subchondral Bone Sclerosis Score**

Subchondral bone sclerosis is scored as follows:

0 = None

1 = Minimal: One surface affected, usually MTP, less than 30% of total area of epiphysis affected

2 = Mild: One to three surfaces affected, usually MTP and LTP, at least 2 have 30% or 1 has 50% of epiphysis affected and others have less than 30% of epiphysis affected

3 = Moderate: Two to four surfaces affected, usually MTP, LTP and LFC, at least one has 50 to 100% of total epiphysis area affected and others have lesser amounts

4 = Marked: Three to four surfaces affected, at least two have 50 to 100% of total epiphysis area affected and others have lesser amounts

5 = Severe: All surfaces affected; at least three have 50 to 100% of total epiphysis area affected with lesser effects in 4^{th}

**Osteophyte Score and Measurement**

Osteophyte/chondrophyte proliferation in the marginal zones was measured using an ocular micrometer. The largest/greatest area of proliferation (worst case scenario) was used to obtain a single measurement. In addition, a score was calculated based on this measurement as follows:

0 = None

1 = 200 to 399 µm

2 = 400 to 599 µm

3 = 600 to 799 µm

4 = 800 to 999 µm

5 = 1000 or greater µm

**Total Joint Score**

The total joint score is the sum of the cartilage damage, bone resorption, subchondral bone sclerosis, and osteophyte scores. A second score incorporating all but the cartilage score was also calculated.

**Synovitis and Fibrosis Scores**

Synovial inflammation (synovitis) and synovial fibrosis are given scores as follows:

0 = None

0.5 = Very Minimal

1 = Minimal

2 = Mild

3 = Moderate

4 = Marked

5 = Severe