Twelve week old male C57/BL6 mice were administered either MLI or sham surgery as previously described8
, to semi-quantitatively and quantitatively assess progressive injury-induced knee joint degeneration over a 20 week time period. There were 5 MLI and 5 sham surgery mice for each of 5 time points: 4, 8, 12, 16, & 20 weeks (post-surgery). Mouse surgical procedures were performed with the approval of the Institutional Animal Care & Use Committee at the University of Rochester Medical Center. Following anesthesia, a 5 mm incision was made on the medial aspect of the joint. The medial collateral ligament was transected, the joint space was opened slightly and a 25 gauge needle was used to detach the medial meniscus from its anterior tibial attachment. The sham group involved a similar incision, but tissues were not manipulated. The skin was closed with 4.0 silk sutures applied in an interrupted pattern. Post surgery, mice were provided analgesia (IP injection of 0.5 mg/kg buprenorphine) every 12 hrs for 72 hrs and the sutures were removed after 10 days.
A systematic approach to preparation, sectioning and visualizing articular cartilage was employed for all tissue-based assays13
. Mice were sacrificed via an AMVA-approved method and the surgically-manipulated knee joints were dissected with the femur and tibia intact to maintain the structural integrity of the joint. The tissue was fixed at 23°C in 10% neutral buffered formalin for 72 hours, de-calcified in 10% w/v EDTA for 21 days and embedded in paraffin. Tissue blocks were then serially sectioned in the midsagittal plane through the medial compartment of the joint. Sections (3 microns thick) were cut at 3 levels within the medial compartment, each level being 50 microns from the previous level (). These cut sections were mounted on positively-charged glass slides, baked at 60°C for 30 min, de-paraffinized in xylene and re-hydrated in decreasing concentrations of ethanol. Sections were stained with alcian blue/orange G for histologic grading and histomorphometry.
Schematic summarizing preparation of sections for histologic analysis of murine knee joints. Red lines represent serial midsagittal sections cut every 50 microns from the medial compartment of the knee joint, resulting in three sections per joint.
One stained section from each of the 3 histologic levels in each joint (described above) was assessed via histologic grading. Three sections (i.e. levels) per joint were evaluated to better visualize the OA status of the entire medial compartment. Grading of articular cartilage (including both the femoral chondyle and tibial plateau) was performed by two blinded observers based on two scoring systems. In the first system, described by Chambers et al9, 11
, 0 = normal cartilage, i.e. lack of superficial zone fibrillation or clefting; 1 = mild superficial fibrillation; 2 = fibrillation and/or clefting extending below the superficial zone; 3 = mild (<20%) loss of non-calcified cartilage; 4 = moderate (20%–80%) loss of non-calcified cartilage; 5 = loss of cartilage to the calcified zone; and 6 = Severe (>80%) loss of non-calcified cartilage. The second system that was employed was a minor modification of the scoring scheme described by Bendele et al6, 12
. Briefly, 0 = normal cartilage; 1 = minimal, superficial zone only; 2 = mild, extends into the upper middle zone; 3 = moderate, well into the middle zone; 4 = marked, into the deep zone but not reaching the tide mark; 5 = severe, full thickness degeneration to the tidemark. The assigned grade in this system is then multiplied by a weighting factor to account for the extent of the degeneration. Specifically, the score is multiplied by 1 if degeneration encompasses less than 1/3 of the articular surface, 2 if it encompasses between 1/3 and 2/3, and 3 if the degeneration encompasses more than 2/36, 12
. Agreement between the two blinded observers was confirmed for both scoring methods using simple and weighted kappa. The observer agreement for the Chambers scoring was 0.48 and 0.88 respectively. For the Bendele scoring, observer agreement was 0.48 and 0.93 respectively. The 2 observer scores were averaged for each section. These average scores from all sections in a given experimental group were then combined to calculate an overall average score. Significance between the injured and sham groups was assessed using the Mann-Whitney-Wilcoxon test, whereby a p
-value of less than 0.05 was taken to indicate significance.
We have established a simple histomorphometric approach to quantify articular cartilage area using sections obtained from mouse knee joints13
. Using alcian blue/orange G stained sections, the Osteometrics system (OsteoMetrics) was utilized to quantify articular cartilage area on 1 tissue section at each of 3 levels (50 microns apart) in the medial compartment of every joint. Using the OsteoMetrics stylus, projected images of the articular cartilage, that were obtained using an Olympus microscope (4x objective) outfitted with a video camera, were outlined on the femoral condyle and tibial plateau in an area defined by the anterior and posterior margins of the meniscus (, green outline). Then, using an area-calculating algorithm in the Osteomeasure software, the area of collected regions of interest (ROI) was quantified for every section. Area values for every section from a given joint were then averaged. All histomorphometry data were normalized to the average of the sham-operated control joints from the 4 week time point and significant differences between groups were identified by ANOVA, with p
-values <0.05 taken as significant.
Figure 3 MLI-induced degeneration is significant based on histologic scoring and cartilage area determination. Three sections from each experimental joint (MLI or sham-operated) were analyzed by 2 histologic scoring methods (Chambers and Bendele). For grading, (more ...)
Prior to histologic processing, harvested knee joints were evaluated via microCT using a Scanco vivaCT40 scanner as we have previously described13
. Joints were scanned at a resolution of 12 μm with a slice increment of 10 μm from mid-femur to mid-tibia. Images from each group were reconstructed at identical thresholds and analysis of bone volume was performed on selected regions between the femoral and tibial physes. Significance differences between groups were identified by ANOVA, with p
-values <0.05 taken as significant.
There are numerous advantages to using a composite index14–16
, including increased statistical efficiency and a simplified analysis and interpretation. Combining several outcomes into a single composite index avoids the arbitrary selection of a single primary outcome variable, and it makes Type I error adjustments for multiple co-primary outcome variables unnecessary.
In order to create an arthritis index incorporating multiple outcome measures, each measurement must first be converted to a common scale. This can be accomplished by the use of t-score transformations17–18
, resulting in measurements on a unitless, standard deviation (SD) scale:
Here CA and CS represent an individual mouse’s cartilage area and cartilage score (Chambers or Bendele), mCA and mCS are the mean cartilage area and cartilage score over all sham-operated mice, and sCA and sCS are the SD of cartilage area and cartilage score over all sham-operated mice. As a consequence, these t-scores have a zero mean and a unit variance among sham-operated mice. In comparison, MLI mice tend to have smaller t-scores for cartilage area () and larger t-scores for either Chambers or Bendele cartilage score (, respectively).
Figure 5 Calculation of t-Chambers scores, t-Bendele scores and t-cartilage areas. The cartilage scores (Chambers and Bendele) and cartilage area data presented in were converted into t-scores and plotted individually as a prelude to calculation of the (more ...)
The simplest way to combine these t-scores into a single composite index is to average or sum them17–18
. However, an adverse effect is in opposite directions for cartilage area and the cartilage score. In order to prevent the t-scores from cancelling when summing, the sign of one of the t-scores must be reversed. Our simple index is then the difference in t-scores: tCS
Figure 6 An Arthritis Index as a quantitative measure of arthritic disease in the mouse knee. The t-Cartilage area () was subtracted from either the t-Chambers score () or the t-Bendele score () and the results are plotted in (A) and (more ...)
Ultimately our goal is to have a composite index to use as a primary outcome variable in a longitudinal study comparing treatments among MLI and sham-operated mice. An efficient statistical analysis in such a study would incorporate all measurements over time in comparing treatments. When progression over time is linear, comparing slopes (rates of change over time) is an efficient and valid approach to summarizing treatment efficacy. Combining the t-scores such that the linearity of the resulting index over time is maximal is therefore desirable.
We considered several alternatives to induce longitudinal linearity of our composite index, including linear and quadratic functions of the t-scores. Regression models were used to estimate coefficients for the t-scores in these functions. Coefficients were chosen such that the correlation of the composite index with time was maximized among the MLI mice. These coefficients were then normalized so that all indices have the same variance among sham-operated mice.