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10067 - Guinea Pig, Joint Damage, Toluidine blue+Safron

INTRODUCTION

Surgically-induced knee joint instability in rats by transection of the Anterior Cruciate Ligament (ACLT), resection of the medial meniscus (tMx) or a combination of both (ACLT and tMx), is one of the most commonly used experimental animal models of osteoarthritis (OA) [See REFERENCES: 1, 2].The model is suitable for studying basic pathophysiological aspects of OA and for the pharmaceutical development of disease-modifying drugs.

The severity of joint destruction can best be demonstrated in coronal histological sections of the whole knee joint stained with Toluidine Blue -Safron du Gatinais (or with other combinations of staining [see Related APP: 10026, 10027 and 10061]). The most important histopathological hallmarks are surface fibrillation and erosion of the articular cartilage, decrease in chondrocyte number, loss of proteoglycan staining and a sclerosis (thickening) of the subchondral bone plate.

Traditionally, the evaluation of the severity of joint damage is done by subjective semi-quantitative grading of the different histopathological features using defined scoring systems [See REFERENCES: 3]. However, even with “blinding' of the observers and sample randomization, this methodology is prone to bias, suffers from low sensitivity to change and requires considerable experience in histopathology. On the other hand, quantitative digital histomorphometry of cartilage destruction and subchondral bone sclerosis can offer an objective, less time consuming and more sensitive assessment of OA histopathology [See REFERENCES: 4].

This APP can be used for quantifying a number of parameters relating to joint damage: cartilage surface length, cartilage surface fibrillation, and solid bone percent.

KEYWORDS

Osteoarthritis, Joint, knee, Toluidine blue, Safron du Gatinais, Rat, Cartilage, Fibrillation, Bone sclerosis, quantitative, digital pathology, image analysis.

METHODS

This APP can be used on virtual slides, where an Analysis Box with a predefined size is placed manually at a tibia or femur cartilage region, including potential lesion area and excluding osteophytes.

The APP will delineate the residual cartilage, bone, and joint space within the Analysis Box.

On the cartilage surface towards the joint space, the surface length and Euclidean length between measurement points can be obtained for calculation of the Fibrillation Index.

After automatic analysis a review step can be performed, where manual corrections can be made to the tissue classification and a calculation protocol is applied to update the results.

QUANTITATIVE OUTPUT VARIABLES

The output variables obtained from this APP include:

- Cartilage Surface Length = distance between ends of cartilage along cartilage surface

- Fibrillation Index = Relation between Euclidean distance between ends of cartilage surface towards femur and distance between ends of cartilage along cartilage surface

- Bone Density = Part of bone area consisting of solid bone


AUXILIARY APPS (included)

Auxiliary APPs are used for additional process steps, e.g. finding Region of Interest (ROI).

This APP takes advantage of prior knowledge about the morphology of the knee joint. To classify and measure correctly there are 4 protocols available. 

QUANTIFICATION

01 - QUANTIFICATION - Left
For classification and measurement on cartilage regions on the left side of a joint space.

02 - QUANTIFICATION - Right
For classification and measurement on cartilage regions on the right side of a joint space.

RECALCULATION

Aux 01 - RECALCULATE - Left
For recalculation of quantitative output variables after manual adjustments of tissue classification, on cartilage regions on the left side of a joint space.

Aux 02 - RECALCULATE - Right
For recalculation of quantitative output variables after manual adjustments of tissue classification, on cartilage regions on the right side of a joint space.


STAINING PROTOCOL

There is no staining protocol available.


ADDITIONAL INFORMATION

The APP utilizes the EngineTM and Viewer software modules, where EngineTM offers an execution platform to expand processing capability and speed of image analysis. Viewer gives a fast review together with several types of image adjustment properties ex. outlining of regions, annotations and direct measures of distance, curve length, radius, etc. 
By adding the AuthorTM module the APP can be customized to fit other purposes. AuthorTM offers a comprehensive and dedicated set of tools for creating new fit-for-purpose analysis APPs, and no programming experience is required.

WORKFLOW

Step 1: Place an analysis box on a relevant cartilage area with the region drawing tool.

Step 2: Load the APP for tissue component detection and quantification “01 - QUANTIFICATION - Left' (for cartilage regions on the left side of a joint space in the scanned image) or “02 - QUANTIFICATION - Right' (for cartilage regions on the right side of a joint space in the scanned image). Click the save button to transfer the results to the database.
The APP can also be executed in a batch process on multiple images at once.

Step 3: If necessary perform a manual review step and adjust tissue classification. Load the APP for recalculation of outputs “Aux 01 - RECALCULATION - Left' (for cartilage regions on the left side of a joint space in the scanned image) or “Aux 02 - RECALCULATION - Right' (for cartilage regions on the right side of a joint space in the scanned image). Click the save button to transfer the results to the database.



REFERENCES

USER

This APP was developed in corporation with and verified by, Dr. Sven Lindemann and Donata Harazin, Merck KGaA, Germany, Osteoarthritis Research & Early Clinical Development.

LITERATURE

1. Gerwin, N. et al. Osteoarthritis & Cartilage 18 (S3): 24-34, 2010

2. Hayami, T. et al. Bone 38:234-243, 2006

3. Pritzker KP. et al. Osteoarthritis Cartilage 14:13-29, 2006

4. Rudolphi K. Et al. Osteoarthritits Cartilage in press for OARSI, 2012

RUO
FIGURE 1
FIGURE 1
Coronal histological section of guinea pig knee joint, stained with Toluidine blue and Safron. Analysis box placed at tibia cartilage.
FIGURE 2
FIGURE 2
Close-up on tibia cartilage with Analysis box.
FIGURE 3
FIGURE 3
Tissue classified in cartilage, bone, bone lacunae, and joint space. Fibrillation Index measures marked as rulers on cartilage surface.