Rapid Scoring of Osteoarthritic Damage


Histology

Histomorphometry of the knee joint is the standard method for assessing osteoarthritis (OA) in experimental mice.

Fixation

For routine staining, joints are fixed in 10% formalin (NBF) for 24 hours then demineralized in 20% ethylenediaminetetraacetic acid (EDTA) for 7 days. This allows for paraffin embedded sectioning of the bone. It is also possible to decalcify with a 48 hour treatment in 10% formic acid, but this is generally avoided because of the higher risk of denaturing proteoglycans.

Bone Formation Rate

If the mice were injected with fluorochromes (e.g. calcein or alizarine red) to measure the rate of bone formation the histology protocol must be modified. Demineralization is not possible as it leaches out the fluorochromes along with the calcium. Such samples should be fixed in 10% NBF and transferred promptly to 70% ethanol for storage. Overfixation can cause the fluorochromes to fade.

To section non-decalcified bone, joints can be frozen and cut on a cryostat or embedded in methyl methacrylate resin (MMA) and cut on a motorized microtome.

Immunohistochemistry

For immunohistochemical staining, fixation in 4% paraformaldehyde may be preferred to maintain antigenicity.

Section Orientation

Regardless of the embedding protocol, the preferred sectioning orientation is frontal rather than sagittal because (1) frontal sectioning allows for the medial and lateral sides of the joint to be analyzed in the same image and (2) fewer sections are required to evaluate the entire joint.

Staining

Safranin-O & Fast-Green staining is most common, but Toluidine Blue & Fast-Green is also possible. Image analysis is generally more automated when using safranin-o staining due to the high contrast between the two colors. (Click image to enlarge.)

Safranin-O & Fast-Green staining is most common, but Toluidine Blue & Fast-Green is also possible. Image analysis is generally more automated when using safranin-o staining due to the high contrast between the two colors. (Click image to enlarge.)

Quantify Proteoglycan Loss in Cartilage

Click image to enlarge.

Click image to enlarge.

There are two techniques to quantify proteoglycan loss in histology. The most straight-forward method is to quantify the percent of the cartilage that is "strongly stained" for proteoglycans.

If additional care is taken, the quantity of stained proteoglycans can be quantified using optical density data of the safranin-o staining. 

Quantifying Damage to the Articular Surface

Click image to enlarge.

Click image to enlarge.

auto surfacE Tools

BIOQUANT has a specialized tool to rapidly quantify the surface length of structures. Structures can be defined automatically by color thresholding or manually with a brush.

Quantifying What's Lost

The basis for the Osteoarthritis Research Society International (OARSI ) score is the percentage of articular surface which is eroded or marked by clefts down to the calcified cartilage. The articular surface is, by definition, no longer present. Several strategies exist to address this issue. The most simple is for the technician to simply draw a line reconstructing the lost articular surface. Alternatively, the software can use the existing cartilage and reconstruct the likely articular surface.

Epiphyseal Bone Boundary

Click image to enlarge.

ROI Tools

BIOQUANT has several unique tools to help you define tissue volumes quickly and consistently. The Irregular TV tool makes it easy to define the outer “epiphyseal tissue volume” containing all the bone distal to the articular cartilage and proximal of the growth plate, including the cortical shell.

Irregular TV tool makes it easy to define the outer “epiphyseal tissue volume” containing all the bone distal to the articular cartilage and proximal of the growth plate, including the cortical shell

Epiphyseal Bone Structure

Click image to enlarge.

Referent Data tool 

With a single click, the Referent Data tool identifies and thresholds bone within the epiphyseal tissue volume, and automatically fills small holes and cracks while preserving actual voids in the bone. The Referent Data tool collects these data simultaneously:

  • Epiphyseal Tissue Volume (Ep.TV), Bone Volume (Ep.BV), Bone Surface (Ep.BS)
  • Ep.BV/Ep.TV, Ep.BS/Ep.BV
  • Epiphyseal Trabecular Number (Ep.Tb.N), Trabecular Diameter (Ep.Tb.Dm), Trabecular Separation (Ep.Tb.Sp)

Trabecular Bone Boundary

Click image to enlarge.

Click image to enlarge.

ROI TOOLS

BIOQUANT has several unique tools to help you define tissue volumes quickly and consistently. The Irregular TV tool makes it easy to define the outer “epiphyseal tissue volume” containing all the bone distal to the articular cartilage and proximal of the growth plate, including the cortical shell.

Irregular TV tool makes it easy to define the outer “epiphyseal tissue volume” containing all the bone distal to the articular cartilage and proximal of the growth plate, including the cortical shell

Trabecular Bone Structure

Click image to enlarge.

Click image to enlarge.

Referent Data tool

Second, a “trabecular tissue volume” is drawn that contains only epiphyseal trabecular bone, excluding the cortical shell, subchondral bone plate, and bone proximal to the growth plate.
Trabecular structure analysis

Next, BIOQUANT will re-use the threshold identification of the bone made in the previous
step. With the bone structure now restricted to the trabecular compartment, BIOQUANT collects these data simultaneously:

  • Tissue Volume (TV), Bone Volume (BV), Bone Surface (BS)
  • BV/TV, BS/BV
  • Trabecular Number (Tb.N), Trabecular Diameter (Tb.Dm), Trabecular Separation (Tb.Sp)  

Subchondral Bone Changes

Click image to enlarge.

Finally, BIOQUANT uses a specialized tool to measure the structure of the subchondral bone plate. Thickening of this plate is often associated with loss of articular cartilage.

  • Subchondral bone plate volume (Sb.BV)
  • Subchondral bone plate surface (Sb.BS)
  • Subchondral bone plate thickness (Sb.Th)

Sources

Afara I, Singh S, Moody H, Oloyede A. A Comparison of the Histochemical and Image-Derived Proteoglycan Content of Articular Cartilage. https://doi.org/10.4172/2161-0940.1000120

Glasson SS, Chambers MG, Van Den Berg WB, LIttle CB. The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the mouse. https://dx.doi.org/10.1016/j.joca.2010.05.025

Wikipedia. Beer-Lambert Law. https://en.wikipedia.org/wiki/Beer%E2%80%93Lambert_law