Using a Knee Arthrometer to Evaluate Tissue-specific Contributions to Knee Flexion Contracture in the Rat

Summary

The goal of the protocol is to measure the extension range of motion of the rat knee. The effects of various diseases that increase the stiffness of the knee joint and the effectiveness of treatments can be quantified.

Abstract

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ROM is called a joint contracture and results in increased morbidity. Due to the difficulty of reversing established knee contractures, early detection is important, and hence, knowing risk factors for their development is essential. The rat represents a good model with which the effect of an intervention can be studied due to the similarity of rat knee anatomy to that of humans, the rat's ability to tolerate long durations of knee immobilization in flexion, and because mechanical data can be correlated with histologic and biochemical analysis of knee tissue.

Using an automated arthrometer, we demonstrate a validated, precise, reproducible, user-independent method of measuring the extension ROM of the rat knee joint at specific torques. This arthrometer can be used to determine the effects of interventions on knee joint ROM in the rat.

Introduction

Having full range of motion (ROM) of the joints is critical for health and well-being¹. A loss in joint passive ROM is called a contracture². Joint contractures may arise from numerous conditions, including prolonged bedrest, paralysis, joint arthroplasty, burns, infection, and neurologic conditions^1,3,4,5. A contracture of the knee can be disabling as it accelerates joint degeneration, increases the risk of falls and detrimentally affects a person's ability to perform basic functional tasks including walking, sitting, and climbing stairs^6,7.

Once established, contractures of the knee are difficult to treat, and therefore determining which patients are at the highest risk of developing this condition is essential for prevention and avoidance of contracture-associated morbidity⁸. Experiments are designed to evaluate 1) the conditions causing or influencing knee joint contractures, 2) the severity of contractures, 3) their temporal progression, 4) the tissues involved in the contracture, 5) their reversibility as well as 6) the usefulness of various preventive and curative interventions on knee joint ROM. For all of these experiments, a valid, objective, precise and reproducible method for measuring the ROM is critical. Other ancillary measures (energy expenditure, histomorphometry, gene expression and protein content) are useful markers to understand the pathophysiology of joint contractures, but the mechanical limitation is what limits the patient and leads to disability. Some of the challenges in this area of research includes the heterogeneous methods by which knee ROM may be tested experimentally, as well as a lack of quantitative data⁹. The use of a variety of different experimental methods leads to results that are not comparable from laboratory to laboratory. This has led to controversy regarding the conditions (such as immobilization or joint arthroplasty) that cause joint contractures¹⁰. An automated method of experimentally measuring joint ROM following an intervention is therefore needed.

Here, we describe a user-independent, valid, precise and reproducible protocol for evaluating the rat knee ROM using a custom-built arthrometer linked to a digital camera to precisely measure the knee ROM in extension. We tested the effect of various periods of immobilization on knee ROM. We then describe the methods for measuring ROM at pre-specified torques on the resulting digital images using fixed bony landmarks. Overall, these methods reliably measure rat knee ROM and provide quantitative data.

Protocol

The rat knee immobilization model used has been approved by the University of Ottawa Animal Care and Veterinary Service and the local ethics committee.

1. Animal Preparation

1. At the end of the predetermined immobilization period, euthanize the rats by administration of carbon dioxide.
   NOTE: Here we used an immobilization model with a plate and 2 screws (one inserted in the proximal femur and the other in the distal tibia), which avoids violation of any knee joint structures, and maintains a knee-flexed position of 135° as described previously⁶. Over a period of time, this produces a knee flexion contracture¹¹.
2. Cover the area both on and around the surface that the arthrometer will be placed upon with absorbent, water-proof protection pads. Wear gloves, lab coat, and eye protection, while completing the experiment.
3. Using a scalpel, divide the skin to expose the plate and screws (see the note following step 1.1); insert the more proximal screw in the proximal femur and insert the more distal screw in the distal tibia. Palpate to locate the screws. Once the screw heads are accessible, remove the screw using a screwdriver.
   NOTE: During the period of immobilization, the heads of the screws may become covered by soft tissue. If this occurs, use the scalpel to gently remove the tissue and uncover the screw heads.
4. Once the screws are removed, remove the plate manually or using forceps from a dissection kit.
5. Using scissors and forceps, deglove the lower extremity to remove skin from underlying fascia.

2. Animal Positioning on the Motor-driven Arthrometer

NOTE: All testing should be performed at room temperature. Here the arthrometer is powered by a standard North American 120 V input. The adapter output is 12 V and 500 mA.

1. Position the animal to be tested on its side with the experimental leg (the leg to be tested) facing upwards (Figure 2).
   1. Secure the femur in the grooved metal clamp that is integrated into the mounting stage of the arthrometer. Punch holes through the muscle using a precision screwdriver to place the clamp distal to the greater trochanter and secure the femur. Adjust the lateral femoral condyle over the center of rotation of the arthrometer (Figure 1, 2).
   2. Position the movable arm with two upright posts behind the leg, just superior to the calcaneus, to push the knee into passive extension once the electric motor is activated.
   3. Tighten the femur clamp at its base using a hex key until it is secured.
2. Ensure the camera is correctly mounted on the arthrometer using a screwdriver and is on Manual Focus. Focus the camera on the femoral condyle.
3. Select the direction setting on the arthrometer (clockwise or counterclockwise) depending on the direction of the knee ROM being tested and the position of the rat.
4. Activate the arthrometer motor by simultaneously pushing the Power and Start button.
   NOTE: The necessity of pushing the power and start button simultaneously is a safety feature of the device, which prevents accidental activation.
   1. Observe that the arthrometer motor will move at a speed of 6.6 RPM and then stop for 2.1 s upon reaching the first pre-set torque.
   2. NOTE: that when the first torque is reached, the corresponding LED will light up and the digital camera will take a picture of the knee automatically.
      NOTE: Once the picture is taken, the arthrometer will continue to the next, higher preset torque. Once the four torques have been applied, the arthrometer will stop. Once the rat is positioned on the arthrometer and testing is initiated, the total time for testing one knee is approximately 18.8 s. Times may vary slightly depending on the condition of the joint contracture. The images taken are used to measure the extension at each torque.

3. Capturing the Angle of Knee Extension Using the Motor-driven Arthrometer

NOTE: Once the motor has stopped at each applied torque, a digital camera is triggered to take a picture. The camera is positioned on the frame such that it is directly above the knee joint being tested and focused on the femoral condyle.

1. Continue testing with the same knee from the same animal but in a different situation, e.g., after a myotomy of the posterior transarticular muscles is performed to isolate the arthrogenic (non-muscular) component of a contracture, or with a knee from another animal.
   1. When completing the myotomy, dissect the muscle proximal enough to the knee joint to ensure that the capsule is not cut.
      NOTE: It is easiest to complete the myotomy when the leg is in extension, following application of torque setting 4 (17.53 N-cm). Then, repeat steps 2.1 through 3.1.
2. Once both legs have been tested in all conditions (e.g., before and after myotomy), dispose of the animal carcass and all biohazardous materials following institutional protocol, and clean the arthrometer.

4. Knee ROM Measurement Analysis

1. Analyze ROM using ImageJ.
   NOTE: Here version 1.45s was used.
2. Open the file containing the digital image taken by the camera mounted on the rat arthrometer.
   NOTE: The person performing the analysis should be blinded to the experimental grouping of the animal (e.g., immobilized versus control).
3. Select the Angle tool from the main toolbar and trace the femorotibial angle by drawing a femoral line from the middle of the femur clamp to the lateral condyle (aligned with the femoral diaphysis, Figure 2), and a tibial line from the lateral femoral condyle to the lateral malleolus (Figure 2).
   NOTE: The femoro-tibial angle corresponds to the maximal angle of knee extension reached at each preset torque.
4. Use the measuring tool by clicking Analyze| Measure to show the calculated angle produced by the 2 lines drawn above. Use the convention of 0° to mean full extension.

Results

The amount of knee extension determined for various periods of immobility are summarized for increasing durations of immobility and show that more severe contractures were produced following increasing lengths of immobilization. Representative results using ImageJ are shown in Figure 3.

The ability to measure maximum extension of rat knees in a valid, precise and reproducible, user-independent manne...

Discussion

The rat knee arthrometer was developed to reproducibly and reliably determine the maximum extension of the rat knee following an intervention. Advantages of this device include the consistent generation of torque across the knee joint with a constant arm length and extension force. Another advantage includes the ability to set the torque at a level that allows repetitive testing on the same joint to evaluate the influence of different articular structures on knee ROM, such as muscle, capsule, or ligament. For example, fo...

Materials

Name	Company	Catalog Number	Comments
Arthrometer	The Ottawa Hospital Rehabilitation Centre - Rehabilitation Engineering	N/A
Camera	Canon	EOS-500D	Commonly known as EOS Rebel T1i
ImageJ	National Institutes of Health	Version 1.45s
Absotbent Underpads	VWR	820202-845
Dissection Kit	Fisher	08-855	Kit Includes:  Forceps: medium points, nickel-plated  Scissors: 1.5 in. (40 mm) blades, stainless steel  Dissecting knife handle: nickel-plated  Knife blades: stainless steel, pack of 3  Dropping pipet: glass  Bent dissecting needle: stainless steel with plastic handle  Straight dissecting needle: stainless steel with plastic handle Vinylite Ruler 6 in. (15 cm)
Precision Screw Driver	Mastercraft	057-3505-8
Scalpel Blades - #10	Fine Science Tools	10010-00
Screwdriver	Stanley	057-3558-2
Hex Keys	Mastercraft	058-9684-2
Universal AC to DC powder adapter	RCA	108004951