Name: a non-invasive method for generating the cyclic loading-induced intra-articular cartilage lesion model of the rat knee
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Description: Watch this Scientific Journal Video about A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee at JoVE.com

This study was supported in part by a JSPS KAKENHI grant (numbers JP18H03129 and JP18K19739). 
This research also received funding from the Alliance for Regenerative Rehabilitation Research &#38; Training (AR3T), which is supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institute of Neurological Disorders and Stroke (NINDS), and National Institute of Biomedical Imaging and Bioengineering (NIBIB) of the National Institutes of Health under Award Number P2CHD086843. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

The protocol was approved by the Animal Research Committee of Kyoto University (approval number: Med kyo 17616).
1. Perform in vivo cyclic compression on the rat knee
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	<li>Induce experimental animal anesthesia
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		<li>Induce anesthesia in a 12-week-old Wistar rat (256.8 &#177; 8.7 g) by inhalation of 5% isoflurane solution in the anesthesia box.</li>
		<li>Intraperitoneally inject a mixture of three anesthetic agents9, including medetomidine, midazol...

<ol>
	<li>De Souza, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+axial+loading+of+mouse+tibiae+increases+cortical+bone+formation+and+modifies+trabecular+organization:+a+new+model+to+study+cortical+and+cancellous+compartments+in+a+single+loaded+element.">Non-invasive axial loading of mouse tibiae increases cortical bone formation and modifies trabecular organization: a new model to study cortical and cancellous compartments in a single loaded element.</a> Bone. 37 (6), 810-818 (2005).</li><li>Poulet, B., Hamilton, R. W., Shefelbine, S., Pitsillides, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+a+novel+and+adjustable+noninvasive+murine+joint+loading+model.">Characterizing a novel and adjustable noninvasive murine joint loading model.</a> Arthritis and Rheumatism. 63 (1), 137-147 (2011).</li><li>Wu, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+response+of+mouse+joint+tissue+to+noninvasive+knee+injury+suggests+treatment+targets.">Early response of mouse joint tissue to noninvasive knee injury suggests treatment targets.</a> Arthritis and Rheumatism. 66 (5), 1256-1265 (2014).</li><li>Poulet, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermittent+applied+mechanical+loading+induces+subchondral+bone+thickening+that+may+be+intensified+locally+by+contiguous+articular+cartilage+lesions.">Intermittent applied mechanical loading induces subchondral bone thickening that may be intensified locally by contiguous articular cartilage lesions.</a> Osteoarthritis Cartilage. 23 (6), 940-948 (2015).</li><li>Ko, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+cell-mediated+changes+in+articular+cartilage+and+bone+in+mice+are+initiated+by+a+single+session+of+controlled+cyclic+compressive+loading.">Progressive cell-mediated changes in articular cartilage and bone in mice are initiated by a single session of controlled cyclic compressive loading.</a> Journal of Orthopaedic Research. 34 (11), 1941-1949 (2016).</li><li>Adebayo, O. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+subchondral+bone+properties+and+changes+in+development+of+load-induced+osteoarthritis+in+mice.">Role of subchondral bone properties and changes in development of load-induced osteoarthritis in mice.</a> Osteoarthritis Cartilage. 25 (12), 2108-2118 (2017).</li><li>Ko, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+cyclic+compression+causes+cartilage+degeneration+and+subchondral+bone+changes+in+mouse+tibiae.">In vivo cyclic compression causes cartilage degeneration and subchondral bone changes in mouse tibiae.</a> Arthritis and Rheumatism. 65 (6), 1569-1578 (2013).</li><li>Ji, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+in+vivo+cyclic+compressive+loading+on+the+distribution+of+local+Col2+and+superficial+lubricin+in+rat+knee+cartilage.">Effects of in vivo cyclic compressive loading on the distribution of local Col2 and superficial lubricin in rat knee cartilage.</a> Journal of Orthopaedic Research. 39 (3), 543-552 (2021).</li><li>Kawai, S., Takagi, Y., Kaneko, S., Kurosawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+three+types+of+mixed+anesthetic+agents+alternate+to+ketamine+in+mice.">Effect of three types of mixed anesthetic agents alternate to ketamine in mice.</a> Experimental Animals. 60 (5), 481-487 (2011).</li><li>Iijima, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Destabilization+of+the+medial+meniscus+leads+to+subchondral+bone+defects+and+site-specific+cartilage+degeneration+in+an+experimental+rat+model.">Destabilization of the medial meniscus leads to subchondral bone defects and site-specific cartilage degeneration in an experimental rat model.</a> Osteoarthritis Cartilage. 22 (7), 1036-1043 (2014).</li></ol>

For the first time, the current protocol shows how to establish a model of loading-induced cartilage lesion on the lateral femoral condyle in rats, similar to the intra-articular damage model in smaller rodents such as the mouse2. However, the loading protocol in mice caused severe osteophyte formation and cruciate ligament lesions, which was not ideal for evaluating the effects of cyclic compression. The current protocol created a focal cartilage lesion in rats with a much lower loading force. Co...

Traditionally, anterior cruciate ligament (ACL) transection or destabilization of the medial meniscus has been considered optimal for investigating post-traumatic osteoarthritis (PTOA) in small animals. In recent years, non-invasive cyclic compression models have been used to study PTOA. This model was originally designed to investigate the cancellous bone response to mechanical loading1 and was then modified as a non-surgical animal model for PTOA studies2,3,4,5,6. The rationale is to collide&#160;the articular cartilage by applying a periodic external force, which triggers a series of inflammatory responses. However, this model has only been applied to mice, and the appropriate magnitude of loading on larger animals has not been discussed.
Another problem with the previous model is that the high-volume protocol included too many cycles, which caused excessive thickening of the subchondral bone, an unwanted side effect, in several samples7. Therefore, a novel method of cyclic compression with the appropriate magnitude for large animals and a lower loading side effect was developed8. The overall goal of the current article is to describe the protocol of the non-invasive cyclic compression model in rats and observe the representative results of cartilage degeneration. The current protocol would help readers interested in the application of the non-invasive cyclic compression model on rats.

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		<tr>
			<td>Anesthetic Apparatus for Small Animals</td>
			<td>SHINANO MFG CO.,LTD.</td>
			<td>SN-487-0T</td>
			<td></td>
		</tr>
		<tr>
			<td>Autograph AG-X</td>
			<td>Shimadzu Corp</td>
			<td>N.A.</td>
			<td>Precision Universal / Tensile Tester</td>
		</tr>
		<tr>
			<td>Fluoview FV10i microscope</td>
			<td>Olympus Corp</td>
			<td>N.A.</td>
			<td>A fully automated confocal laser-scanning microscope</td>
		</tr>
		<tr>
			<td>ISOFLURANE Inhalation Solution</td>
			<td>Pfizer Japan Inc.</td>
			<td>(01)14987114133400</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEA Viability/Cytotoxicity Kit</td>
			<td>Thermo Fisher Scientific Japan Inc</td>
			<td>L3224</td>
			<td>A quick and easy two-color assay to determine viability of cells</td>
		</tr>
		<tr>
			<td>TRAPEZIUM X Software</td>
			<td>Shimadzu Corp</td>
			<td>N.A.</td>
			<td>Data processing software for Autograph AG-X</td>
		</tr>
	</tbody>
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a non-invasive method for generating the cyclic loading-induced intra-articular cartilage lesion model of the rat knee

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely correlated with a history of articular cartilage damage and ligament avulsion. Surgical animal models of OA of the knee play an important role in understanding the onset and progression of post-traumatic OA and aid in the development of novel therapies for this disease. However, non-surgical models have been recently considered to avoid traumatic inflammation that could affect the evaluation of the intervention. 
In this study, an intra-articular cartilage lesion rat model induced by in vivo cyclic compressive loading was developed, which allowed researchers to (1) determine the optimal magnitude, speed, and duration of load that could cause focal cartilage damage; (2) assess post-traumatic spatiotemporal pathological changes in chondrocyte vitality; and (3) evaluate the histological expression of destructive or protective molecules that are involved in the adaptation and repair mechanisms against joint compressive loads. This report describes the experimental protocol for this novel cartilage lesion in a rat model.

A representative result of the short-term changes (1 h and 12 h) in chondrocyte viability in samples subjected to 20 N cyclic loading was obtained. As shown in Figure 3, the number of dead chondrocytes (red fluorescence) increased at 12 h post-trauma. Conversely, the number of living chondrocytes (green fluorescence) continued to decrease, with some samples containing no live chondrocytes in the affected area.
Histology showed that the articular cartilage of the r...

Watch this Scientific Journal Video about A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee at JoVE.com

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely ...

This protocol shows that it is possible to cause cartilage damage in animals, larger animals without any surgical intervention or structured destruction of the animal knee. The biggest advantage is that the proves to be repetitive in operators even with no experience, which is difficult to achieve with any surgical method. Begin by mounting the anesthetized rat on the fixation device.Place the anesthetized rat lying on its belly on the base plate with its right knee attached to a small piece of resin with a concave groove. Place the right hind limb in the hip extension, knee flexion, and ankle extension positions with the knee flexed at approximately 140 degrees. Accommodate the heel of the rat on the wedge-shaped groove on the movable fixture.Move the fixation device to the stress and tensile testing instrument and ensure that there is no contact with the load cell. Open the stress and tensile testing instrument control software and click on the calibration button. After calibration, carefully attach the top of the frame to the load cell.Turn on the rotary knob on the movable main operational panel slowly until the preload reaches five Newtons to keep the knee joint closely attached to the frame. On the main menu, click on create a new method and system label. Set the test mode to cycle and the test type to compression.Click on the sensor label and select the test tab to check that the limit is within 60 Newtons. Then select the stroke tab and check that the limit is within 500 millimeters. Under the testing control label, select origin of growth to start the main program with 0.3%or full scale.Set the stroke speed in control in the first and third sections to one millimeter per second, then set the maximum testing force in the second section to 20 Newtons and the minimum testing force in the fourth section to five Newtons. Set the duration of whole to 0.5 seconds for the peak load and 10 seconds for the minimum load. In the preload tab at the bottom of the page, ensure that on is checked, the speed of deflection removal is set to 100 millimeters per minute, and the maximum force is five Newtons.Set the material as metal in the specimen label. In the main menu under the select method and test section, select the method that was just built and click on start to begin the test. Set the number of cycles to 60.Return the rat to its cage after loading and maintain a 12-12 hour light dark schedule in a cage with sufficient space and food ad libitum until it is time for analysis. The short term changes in chondrocyte viability in samples subjected to 20 Newtons of cyclic loading was studied. The number of dead chondrocytes increased at 12 hours post-trauma.Conversely, the number of living chondrocytes continued to decrease. The articular cartilage of the rat knees that underwent 20 Newtons of dynamic loading was damaged and one focal lesion zone was confirmed in the lateral femoral condyle in all the samples. The lesion size did not progressively increase during the eight week observational period.The border corresponding to the interface of the lesion and the unaffected cartilage could be observed in the affected area. The most important thing is the proper settings of the testing force and the number of cycles. With this technique, one can control mechanical loading to the articular coverage of blood, paving the way to explore how mechanical loading affect the development or prevention of osteoarthritis.

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Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

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Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

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Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

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Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

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

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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.

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 ...

The Lower Body Positive Pressure Treadmill for Knee Osteoarthritis Rehabilitation

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Here, based on a clinician&#8217;s point-of-view, we propose a two-model lower body positive pressure (LBPP) protocol (walking and squatting models) in addition to a clinical, functional assessment methodology, including details for further encouragement of the development of non-drug surgical intervention strategies in knee osteoarthritis patients. However, we only present the effect of LBPP training in improvement of pain and knee function in one patient through three-dimensional gait analysis. The exact, long-term effects of this approach should be explored in future studies.

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Acute Compartment Syndrome is a devastating consequence of musculoskeletal trauma. Currently the diagnosis is based on clinical signs and symptoms, and while adjuncts such as invasive intra-compartmental pressure measurements are often used to corroborate the physical exam findings, there remains no reliable objective test to aid in the decision to perform a decompressive fasciotomy. In a cadaver model of compartment syndrome, an ultrasound (US) based method has been shown to be a reliable measurement of increased intra-compartmental pressure. An absolute pressure of &#62;100 mbar or a difference of 50 mbar in the CFFP between the legs can be considered pathologic. Using an ultrasound transducer, coupled with a pressure sensor, the pressure needed to flatten the superficial fascia of the anterior compartment of lower legs (Compartment Fascia Flattening Pressure [CFFP]) can be measured. The CFFP of the injured leg is compared to the CFFP of the uninjured leg. This US measured index can then serve as an adjunct to the physical exam in evaluating injured lower extremities and assessing the need for decompressive fasciotomy. The advantages of this protocol include: being a non-invasive method and an easily reproducible technique.

An ultrasound probe coupled with a pressure sensor is used to assess the intra-compartmental pressure of the leg by directly measuring the compartment fascia flattening pressure (CFFP). This non-invasive protocol will provide reliable assessment of the pressure inside the anterior muscular compartment of the lower leg.

Acute Compartment Syndrome is a devastating consequence of musculoskeletal trauma. Currently the diagnosis is based on clinical signs and symptoms, and ...

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The current animal models of osteoarthritis (OA) can be divided into spontaneous models and induced models, both of which aim to simulate the pathophysiological changes of human OA. However, as the main symptom in the late stage of OA, pain affects the patients&#39; daily life, and there are not many available models. The mono-iodoacetate (MIA)-induced model is the most widely used OA pain model, mainly used in rodents. MIA is an inhibitor of glyceraldehyde-3-phosphate dehydrogenase, which causes chondrocyte death, cartilage degeneration, osteophyte, and measurable changes in animal behavior. Besides, expression changes of matrix metalloproteinase (MMP) and pro-inflammatory cytokines (IL1 &#946; and TNF &#945;) can be detected in the MIA-induced model. Those changes are consistent with OA pathophysiological conditions in humans, indicating that MIA can induce a measurable and successful OA pain model. This study aims to describe the methodology of intra-articular injection of MIA in rats and discuss the resulting pain-related behaviors and histopathological changes.

This study describes the method of intra-articular injection of mono-iodoacetate in rats and discusses the resulting pain-related behaviors and histopathological changes, which provide references for future applications.

The current animal models of osteoarthritis (OA) can be divided into spontaneous models and induced models, both of which aim to simulate the ...

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To date, the development of parathyroid gland (PG) identification methods is limited by the lack of animal models in preclinical research. Here, we establish a simple and effective rat model for intraoperative PG imaging and evaluate its effectiveness by using iron oxide nanoparticles as a novel PG contrast agent.

Parathyroid gland (PG) identification is a critical unmet need in thyroidectomy. The identification of the PG is challenging in thyroid surgery as it is ...

Development and Evaluation of a Rat Model of Full-Thickness Cartilage Defects

Cartilage defects of the knee joint caused by trauma are a common sports joint injury in the clinic, and these defects result in joint pain, impaired movement, and eventually, knee osteoarthritis (kOA). However, there is little effective treatment for cartilage defects or even kOA. Animal models are important for developing therapeutic drugs, but the existing models for cartilage defects are unsatisfactory. This work established a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats, and the subsequent pain behavior and histopathological changes were used as readout experiments. After surgery, the mechanical withdrawal threshold was decreased, chondrocytes at the injured site were lost, matrix metalloproteinase MMP13 expression was increased, and type II collagen expression decreased, consistent with the pathological changes observed in human cartilage defects. This methodology is easy and simple to perform and enables gross observation immediately after the injury. Furthermore, this model can successfully mimic clinical cartilage defects, thus providing a platform for studying the pathological process of cartilage defects and developing corresponding therapeutic drugs.

This protocol establishes a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats and measuring the subsequent pain behavior and histopathological changes.

Cartilage defects of the knee joint caused by trauma are a common sports joint injury in the clinic, and these defects result in joint pain, impaired ...