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.
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.
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 conditions1,3,4,5. 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 stairs6,7.
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.
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
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.
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.
4. Knee ROM Measurement Analysis
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 manner that reduces bias in the data. In the example provided, we evaluated the maximum knee extension following 16 weeks of immobilization for 7 rats, comparing the immobilized (experimental) limb to the non-immobilized contralateral limb. The limb chosen for immobilization alternated from one rat to the next (e.g., rat 1 had the right knee immobilized, rat 2 the left). The investigator measuring the angles was blinded to which side was immobilized during measurements. The results are presented in Figure 3. For the immobilized knee, the ability for maximum extension was reduced compared to the contralateral. Division of transarticular muscles eliminates the myogenic component of the flexion contracture. Following myotomy, the maximal extension capability for the experimental and contralateral knee increased; however, the experimental knee continued to demonstrate a flexion contracture (Figure 3).
Figure 1: Rat knee arthrometer. (A) Entire apparatus (B) Representative image of animal within arthrometer (C) Electronics display and clamps for arthrometer. (D) Enlarged image of electronics display. Numbers indicate the various torques being applied: torque 1 = 2.53 N-cm, torque 2 = 7.53 N-cm, Torque 3 = 12.53 N-cm, torque 4 = 17.53 N-cm. (E) Enlarged image of the femoral clamp and tibial pushing apparatus. The femoral clamp is fixed to the stage. The tibial moving arm has 2 upright posts that fix the distal lower extremity and move the knee into extension. Arrowhead and chevron indicate the femur clamp and the moveable arm, respectively. Please click here to view a larger version of this figure.
Figure 2: Arthrometer and experimental rat for evaluation. (A) Knee extension of the posterior limb is measured using the tibial line drawn from the lateral femoral condyle to the lateral malleolus and the femoral line drawn from the lateral condyle to the middle of the femur clamp. Arrowhead and chevron indicate the femur clamp and the moveable arm, respectively. (B) High magnification images of the femoral condyle and (C) High magnification of lateral malleolus.
Figure 3: Immobilized and contralateral rat knee range of motion following 16 weeks immobilization. For both knees, after testing knee extension with all articular structures intact (n=7), a myotomy of the trans-articular muscles was performed to determine the arthrogenic limitation on ROM. Data are presented as mean degrees from full extension (using the convention 0° = full extension) with error bars representing standard deviation. * represents p < 0.01 using the independent-samples T-test. Please click here to view a larger version of this figure.
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, following testing of the fully intact joint, the posterior transarticular knee muscles could be divided and arthrometer testing repeated in order to determine the arthrogenic contribution to extension limitation11.
Specific mechanical features of the arthrometer that optimize measurement accuracy and precision include the grooved clamp, which is designed to prevent rotation of the femur during testing (Figure 1). The distal two metal upright posts engage the leg posteriorly, pushing the knee into extension in a clockwise direction, and minimize the risk of posterior dislocation of the tibia on the femur during testing (Figure 1). The height of the posts and anterior overhang of the upper upright link ensure that the tibia does not slip off the posts. The ability of the upright posts to rotate and maintain their position on the tibia just proximal to the calcaneus ensures constant torque. Four torques are tested in sequence: 2.53 N-cm, 7.53 N-cm, 12.53 N-cm, and 17.53 N-cm. The highest torque level was determined to be the amount of force that led to capsular failure in normal (unoperated) rat knee joints (i.e., extension surpassing 0°) after division of all transarticular muscle. The lowest torque was the point of resistance to angular motion just above measurable amounts on normal rat knee joints. The middle two torques were set to be approximately midway between the highest and lowest torques.
Other methods for measuring joint ROM at specific torques have been described for both rat and other animal models12,13,14,15,16. Some of the advantages of our model over these other systems include a convenient size that allows benchtop placement of the device without the need for special facilities. Other models may also require disarticulation of the limb being studied while the model presented here does not. Mechanically, the arcing pathway of the post applies the extension force to the distal leg, following the angular progression of the knee, thus maintaining a consistent angle of force application. The stage of the arthrometer allows the placement of the entire rat on the measuring tool, allowing the testing of all in situ articular structures that may contribute to lost ROM without violating joint anatomy. While the ethics protocol for our lab excludes testing on live animals, this would theoretically be possible with appropriate analgesia and post-test sacrificing protocols.
The arthrometer does present some disadvantages. The device was sized to the adult rat knee to ensure that the moveable arm was at a length to ensure it would not slip off the leg and the leg would not slip off the arm. Younger rats, or smaller and larger species would benefit from appropriately-sized components. Additionally, a reprogramming of the optimal moment arm and force (torque) would be required. If, for example a larger or smaller animal was used, adjusting the length of the moment arm and/or amount of force applied to reach optimal torque may be necessary. Other parts of the arthrometer might also need to be resized, according to the size of the animal. While the movements of the arthrometer are user-independent, joint measurement using ImageJ may be subject to human error. We have found however that, using the methods presented here, there is high intra-rater and inter-rater reliability with intraclass correlation coefficients of 0.987 and 0.903, respectively. Because the highest torque often damages the articular structures during testing, a valid inter-rater reliability for the animal placement and activation of the arthrometer is difficult to determine. To avoid measurement error that might be associated with having more than one investigator perform this part of the protocol, we recommend having the same investigator secure the rats to the arthrometer for the duration of a study so that any bias is consistent between experimental and control knees. Because the hamstrings cross both the knee and hip joints, retroversion of the pelvis may occur during testing at torques 1 and 2 prior to myotomy. This may contribute to increases in knee extension for both experimental and control knees at these torques. Finally, results may vary from true in vivo ROM as the protocol was developed to test euthanized rather than live animals.
While we described the use of a device for evaluating the effects of immobility on the rat knee joint, other conditions affecting joint ROM could also be studied. There are numerous examples, some of which include effects of trauma, increased muscular tone secondary to central nervous system insult, or genetic modifications relating to neuromuscular disease. Respective interventions such as stem cell application to the knee joint, neuromuscular junction blockade, or gene therapy treatments that help discover new treatment options could also be assessed using the device.
The authors have nothing to disclose.
The authors would like to thank Joao Tomas for his technical assistance with the device and Khaoula Louati for assistance in developing the image analysis methods.
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. (40mm) 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. (15cm) |
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 |