Structural and Biochemical Changes in Osteoarthritis (Histological Scoring)

Introduction

Methods that allow the visualisation of the destruction of joint tissues are important in evaluating degenerative joint diseases such as osteoarthritis. In humans, osteoarthritis is diagnosed by evidence of ‘joint space narrowing’ on x-rays, where loss of the articular cartilage which covers the ends of the bones in a joint is visualized by the bones becoming closer together as the cartilage is lost. As the articular cartilage is invisible on x-rays, Magnetic Resonance Imaging (MRI) is also commonly used to reveal perturbations in the cartilage surface and damage to, or inflammation of other soft tissues in the joint such as the synovium, meniscus and ligaments. Arthroscopy, where a camera is inserted into the joint, also allows analysis of structural damage.

If the joint tissue is removed after arthroscopy or surgery, more detailed analysis of the extent of destruction is possible by sectioning the joint and visualizing the sections under a microscope. Histochemical methods have been developed that use specific chemicals to selectively stain the joint matrix and cells. Common histologic stains for joint tissues include hematoxylin and eosin (H&E) used to visualize the cells and matrix, toluidine blue and safranin O to quantify proteoglycan content and picrosirius red and Goldner’s trichrome to reveal collagen content.

Animal models of osteoarthritis are used to define the factors that cause the joint to degenerate enabling new treatments to be identified and tested. Scoring systems have been designed to quantify the extent of joint damage in histologically stained sections. This allows assessment of the progression of joint degeneration over time in an animal model of osteoarthritis and allows a researcher to test whether a new treatment is effective. Immunohistochemical methods can be used on adjacent tissue sections to determine the role of specific protein or matrix components in the degenerative process.

All materials for this practical are provided on Blackboard Ultra in 23/24- BI2332 Concepts of Disease.  

Navigate to Practicals and open the Histological Scoring Folder

Aims: 

1. To watch the video on ‘histological sample preparation for light microscopy’ and read the protocol below that describes how toluidine blue is used to stain sections of mouse stifle joint.
2. To score the images from 2 mice (left and right joints) provided digitally and determine which, if any, are arthritic.

Toluidine Blue Staining 

• Materials: Two square glass staining dishes, containing either tap water or 0.1% Toluidine Blue, 1 slide rack, 1 dewaxed paraffin section of mouse stifle joint on microscope slide, 50mm coverslips, Aqueous Mounting Medium.
• Method: Place slide with section into the slide rack and dip into the toluidine blue solution for 1 minute. Wash for 5 minutes in tap water, replacing the water regularly until it is clear. Dab the slide dry with tissue, being careful not to touch the section and place into 37°C incubator for 1hr to dry. Add a drop of mountant over the section, gently lower the coverslip and leave for 2 minutes to stabilize. 
• Microscopy: These sections have been examined under the microscope and a digital camera used to obtain one image of the entire joint at low power (1.6x), and one image of each quadrant (medial femoral condyle, lateral femoral condyle, medial tibial plateau, lateral tibial plateau) at higher power (x10).

Histological scoring of joint degeneration

1. Open the file named ‘BI2332 sections for scoring’ and identify the numbers of the two mice assigned to you on the table in the first slide. Use the drawing on the 2nd slide to familiarise yourself with the anatomical orientation.
2. Score all 4 joint quadrants and both left and right legs from the digital images of the two assigned mice. Score for OA damage, proteoglycan loss, bone changes and osteophytes using the scoring system below. Examples of scored images in file named ‘BI2332 Scoring examples’ may help.
3. Record your data in the results table below indicating which, if any, of the knees that you have analysed are osteoarthritic?
4. Draw a graph to compare the total scores of the assigned sections, ie. left leg and right leg of 2 mice.

Scoring system

Osteoarthritis damage
Grade	Description
0	Normal
0.5	Loss of Toluidine blue with no structural damage
1	Small fibrillations without loss of cartilage
2	Vertical clefts down to immediately below the superficial layer and some loss of surface lamina
3	Vertical clefts/erosion down to calcified cartilage extending to less than 25% of articular surface
4	Vertical clefts/erosion down to calcified cartilage extending to less than 25% -50% of articular surface
5	Vertical clefts/erosion down to calcified cartilage extending to less than 50%-75% of articular surface
6	Vertical clefts/erosion down to calcified cartilage extending to more than 75% of articular surface
Loss of cartilage proteoglycan
Grade	Description
0	Normal staining of non calcified cartilage
1	Decreased but not complete loss of toluiding blue staining over 1-100% of articular surface
2	Complete loss of the toluidine blue staining in the non calcified cartilage extending to <25% of the articular surface
3	Complete loss of the toluidine blue staining in the non calcified cartilage extending to 25% – 50% of the articular surface
4	Complete loss of the toluidine blue staining in the non calcified cartilage extending to 50% – 75% of the articular surface
5	Complete loss of the toluidine blue staining in the non calcified cartilage extending to > 75% of the articular surface
Subchondral bone changes
Grade	Description
0	normal
1	mild
2	moderate
3	severe
Osteophytes
Grade	Description
0	absent
1	present

Results table

Animal number	Left / Right	Medial/lateral	Femoral condyle/tibial plateau	Scores					Healthy (Y/N)	Arthritic (Y/N)
				OA  damage	Proteoglycan loss	Subchondral bone	Osteophyte	Total
	R	M	FC
	R	L	FC
	R	M	TP
	R	L	TP
TOTAL SCORE FOR 1st DIGITAL SECTION RIGHT LEG (MFC+LFC+MTP+LTP) =
	L	M	FC
	L	L	FC
	L	M	TP
	L	L	TP
TOTAL SCORE FOR 1st DIGITAL SECTION LEFT LEG (MFC+LFC+MTP+LTP) =
	R	M	FC
	R	L	FC
	R	M	TP
	R	L	TP
TOTAL SCORE FOR 2ND DIGITAL SECTION RIGHT LEG (MFC+LFC+MTP+LTP) =
	L	M	FC
	L	L	FC
	L	M	TP
	L	L	TP
TOTAL SCORE FOR 2ND DIGITAL SECTION LEFT LEG (MFC+LFC+MTP+LTP) =