Name: the monoiodoacetate model of osteoarthritis pain in the mouse
Uploaded: 2016-05-16T13:00:00.000+00:00
Duration: 9 min 26 s
Description: Watch this Scientific Journal Video about The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse at JoVE.com

JSV is supported by a collaborative grant to MM by the European Commission (GAN 603191-PAINCAGE).

Procedures involving animal subjects have been approved by the Ethical Committee at King&#39;s College London and are in accordance with UK Home Office Regulations (Animals Scientific Procedures Act 1986).
1. Intra-articular Injection of Monoiodoacetate in the Knee
<ol>
	<li>House 8 - 10 week-old mice in groups of 5 under a 12 hr&#160;light/dark cycle (lights on at 7:00 AM) with food and water ad libitum. Let the mice acclimatize for 1 week prior to starting the experiment.</li>
	<li>Randomize and cage mice in groups of 5. Use animal numbers as codes to blind the experimenter to treatments. Use body weights as parameters for randomization.</li>
	<li>On the day of injection, freshly prepare the solution of monoiodoacetate in sterile saline (0.9% NaCl) at the desired concentrations. Use sterile saline for injections in a separate group of control mice. The highest recommend dose of MIA is 1 mg in 10 &#181;l. 
	Caution: Monoiodoacetate is very toxic. Therefore, it is recommended that gloves and mask are worn when handling the powder and preparing the solution. The solution should be sterile filtered with a 0.22&#160;&#956;m filter.&#160;</li>
	<li>Anesthetize mice using an anesthetic trolley by first placing them in a chamber delivering 2% isoflurane in O2 mixture (flow rate 1.5 L/min) and then transfer mice to the nose cone section, which also delivers the 2% isoflurane-O2 mixture, and as such, maintains anesthesia during injection. Place vet ointment on the eyes to avoid their drying out while&#160;under anesthesia. Wear surgical gown, gloves,&#160;and mask while performing injection procedure.</li>
	<li>Confirm anesthesia by checking the animal&#39;s lack of response to a pinch stimulus on the hind paws.</li>
	<li>Once the animal is under anesthesia, place it&#160;on its&#160;back. Trim and wipe the area surrounding the knee joint with alcohol. Povidone iodine or chlorhexidine can be used as well for disinfection.&#160;The patellar tendon (white line bellow the patella) will become visible.</li>
	<li>In order to stabilize the injection site, keep the knee still, in a bent&#160;position, by placing the&#160;index finger beneath the knee joint and the thumb above the anterior surface of the ankle joint. Joint preference is not required.</li>
	<li>To find the precise site of injection, run a 26 G&#160;needle attached to a syringe horizontally along the knee (so as&#160;not to&#160;pierce the skin with the tip)&#160;until it finds the gap beneath the patella. Apply gentle pressure to mark the area and then lift the needle and syringe vertically for the injection. Insert the needle in the marked area, through the patellar tendon, perpendicular to the tibia. No resistance should be felt.
	<ol>
		<li>Use thumb as a guide and inject superficial to the site of entry. After injection, massage the knee to ensure even distribution of the solution. Discard the needle immediately in the sharps bin.</li>
		<li>Place mice back into a clean home cage on a heated mat and allow them to recover. Keep constant vigilance on the animals until they regain suitable consciousness, which is measured by them regaining sternal recumbency. Once animals are recovered, return to their cage. 
		Note: It is suggested for best practice and training purposes that a dye is used and immediate post-mortem dissection performed to confirm correct localization of injection.</li>
	</ol>
	</li>
</ol>
2. Measurement of Mechanical Hypersensitivity (Allodynia)
Note: Static mechanical withdrawal thresholds are assessed by applying von Frey hairs to the plantar surface of the hind paw.&#160;
<ol>
	<li>Bring mice to the behavioral room and let unrestrained animals acclimatize in acrylic cubicles (8 cm x 5 cm x 10 cm) atop a wire mesh grid.
	<ol>
		<li>Train mice by handling and 2 hr&#160;habituation to the cubicles for two days prior to von Frey hair application in order to limit stress and ambulation during application of von Frey hairs. On test days, habituate animals to the cubicles for up to 60 min prior to testing. Wear gowns, gloves, and masks during all behavioral experiments.</li>
	</ol>
	</li>
	<li>Apply calibrated von Frey hairs (flexible nylon fibers of increasing diameter that exert defined levels of force as calibrated by the manufacturing company and expressed as grams (g)) to the plantar surface of the hind paw until the fiber bends. Use 0.008, 0.02, 0.04, 0.07, 0.16, 0.4, 0.6, and 1.0 g fibers during testing.
	<ol>
		<li>Hold each hair in place for 3 sec&#160;or until the paw is withdrawn, the latter defining a positive response. Starting with a stimulus strength of 0.07 g, apply hairs according to the &#34;up-down method&#34;21: mark as X a withdrawal response and O an absence of response. Apply in ascending order of force, up to 1 g (cut-off force),&#160;until a response is detected.</li>
		<li>Re-test the paw by repeating step 2.2.1, starting with the filament that&#160;exerts a force&#160;below the one that produced a withdrawal.</li>
		<li>Then,&#160;apply the remaining filaments sequentially, by&#160;descending force, until no withdrawal occurs. Re-apply filaments in ascending order until a response is observed. Continue until a sequence of six responses is obtained (e.g., OXOXOX), in order to obtain the &#39;k&#39; value by referring to tabular values 21.</li>
		<li>Express paw withdrawal values as 50% paw withdrawal thresholds in grams. Use the formula (10[Xr + K&#948;])/10,000&#160;where Xr = value of last von Frey filament used in the sequence (in log units), k = tabular value, and &#948; = mean difference in forces between fibers. Where no response is detected, use the maximal response of 1 g 21,22.</li>
	</ol>
	</li>
	<li>Following the procedure described above (2.2.1-2.2.4), assess mechanical thresholds of both hind paws before MIA injection as baseline values. After injection, assess thresholds of the ipsilateral and contralateral paws at regular day intervals for several weeks after MIA to ascertain the development of mechanical allodynia. 
	Note: For example, we report thresholds measured 0, 3, 5, 7, 10, 14, 21, and 28 days after MIA injection. Animals are considered allodynic when they display a response to 0.1 g or less. Normal responses fall within 0.6 - 1 g range.</li>
</ol>
3. Measurement of Weight Bearing Deficit
Note: Changes in weight bearing are measured using a weight incapacitance tester.
<ol>
	<li>Train each mouse to walk into a Plexiglass chamber on the apparatus and sit in the holding box. Place the mouse in front of the holding box, lift the entrance up 45&#176;, and allow the mouse to walk in and close the box. Allow the animals to move freely until they adopt a sitting posture. This training takes at least two days and guarantees that the animal is still and not leaning on either side&#160;of the chamber. Calibrate the instrument before use with a 100 g check weight (or according to equipment instruction).
	<ol>
		<li>Make sure that each hind paw is placed on the appropriate recording pad&#160;11. The duration of each measurement takes 1 sec, as per the manufacturer&#39;s instructions.</li>
	</ol>
	</li>
	<li>Collect three measurements of the weight borne on each hind paw from the recording pad for each recording session and use the mean value to calculate the difference in weight borne by ipsilateral and contralateral paws. Express values as the difference between contralateral and ipsilateral paws in grams.</li>
	<li>Assess weight bearing changes before MIA injection as baseline values. Then, repeat assessments at regular intervals over several weeks to ascertain the development of gate changes. For example, we report thresholds measured on 0, 3, 5, 7, 10, 14, 21, and 28 days after MIA injection. 
	Note: A normal weight&#160;bearing value of 50% represents an equal weight distribution across ipsilateral and contralateral hindlimb. Animals considered hypersensitive display a weight bearing change of approximately 45%. Measurements of mechanical thresholds and weight bearing deficits can be performed in the same mice, as neither end point affects the other. For pharmacological assessment, each group of animals should be tested at set times after dosing in line with the pharmacokinetic profile of the compound used.</li>
</ol>

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None of the authors have competing interests or conflicting interests.

With this methodology, we describe a preferred method for inducing OA-like pain in the mouse by an intra-articular injection of MIA in a knee joint and assessment of sensitivity to non-noxious and noxious stimuli in the hind limbs. MIA injection is associated with persistent pain behavior, namely altered hind limb weight bearing and development of referred mechanical hypersensitivity (allodynia). Such static measurements can be complemented by gait analysis on a treadmill or by catwalk analysis in freely moving animals. MIA models are responsive to conventional pain-relieving therapies&#160;24, indicating&#160;that they&#160;may be useful for discerning therapeutic approaches. While the injection of MIA is not technically difficult, the joint capsule can be pierced during the injection, resulting in leakage of MIA outside the capsule, and subsequent failure to induce toxicity of chondrocytes. Indeed, systemic injection of MIA can be fatal in rodents and possible effects of MIA on tissues and cells other than chondrocytes may confound results, besides being undesirable. As such, it must be stressed that great care needs to be given to the injection of the MIA, as it is a critical component of the model, and confidence needs to be given that the injection occurs into the articular space.&#160;This protocol helps to achieve that.
The protocols described here aim to ensure the animals provide consistent pain-like responses throughout the test period. Also, they allow adjustment of disease severity by altering the dose of MIA used to induce the pathology&#160;15,23. The rapid induction of both disease state and pain-like behavior allow timely evaluation of pain-modifying compounds. This is advantageous over existing surgical and spontaneously developing models of OA, which can take a longer period of time to develop hypersensitivity. Also, particularly for the spontaneous models, the disease pathology does not manifest in all animals (approximately 20 - 80%&#160;7), whereas the MIA model is associated with significant incidence of responders. Furthermore, spontaneous models are not suitable for measurements of changes in&#160;weight&#160;bearing, as OA develops in both knees. When considering behavioral measurements, the animals need to be kept calm and relaxed during the assessments. This is achieved, as detailed in the protocol, by early training before recording measurements and by repetitive handling, which allows animals to become familiar with the experimenter. A key point to reduce stress is to use the same experimenter for the behavioral test throughout, as constant changing will induce the issues previously mentioned. Like any model, the MIA model of OA bears limitations, such as the rapidity of joint disruption, which does not resemble the slow development of OA pathology in patients. One way to overcome this issue would be to complement this model with a surgical model of OA. The use of the MIA chemical model in compound development allows for the use of preventative and therapeutic protocols over the development and maintenance of OA-like pain. Finally, the MIA model would complement studies of phenotypical traits of knock-out mice, helping to further understand the OA disease.

Clinically, osteoarthritis (OA), or degenerative joint disease, is a painful and debilitating condition characterized by a progressive loss of articular cartilage, mild inflammation of the tissues in and around the joints, and sometimes formation of osteophytes and bone cysts. Patients with OA report persistent pain 1 and display increased sensitivity to pressure and noxious stimuli in the arthritic joint 2-4. At present, there is no cure for OA with available therapeutic approaches and analgesics are prescribed to alleviate the pain associated with this condition, with some degree of success5. However, OA pain remains a clinical issue and animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and disclose novel targets for therapy.
There are several animal models of OA available with different characteristics 6. Surgical methods, such as anterior cruciate ligament transection, can be utilized. However, they involve skillful surgical intervention and are primarily performed in the rat, while&#160;destabilization of medial meniscus (DMM) is used in the mouse. Spontaneous development of OA occurs in guinea pig and spontaneous joint degeneration has been reported in C57 black mice from 3 to 16 months of age 7,8. Spontaneous OA models do not involve any intervention to induce the condition, but they have inherent variability, and as such, incur greater numbers and cost 9,10. Chemically induced models, on the other hand, require much less invasive procedures than surgical models, and as such, are easier to implement and permit the study of OA lesions at different stages. These models include single injections in the knee of inflammatory agents, immunotoxins, collagenase, papain, or monoiodoacetate, which can be toxic if they escape the joint space. Of all chemical models of OA, MIA is the one most often used, particularly to test the efficacy of pharmacologic agents to treat pain, as this model generates a reproducible, robust, and rapid pain-like phenotype that can be graded by altering MIA dosage 11-15.
Intra-articular injection of MIA in rodents reproduces OA-like lesions and functional impairment that can be analyzed and quantified. MIA is an inhibitor of glyceraldehyde-3-phosphatase, disrupting cellular glycolysis and eventually resulting in cell death 16,17. Intra-articular injection of MIA causes chondrocyte cell death, leading to cartilage degeneration and subsequent subchondral bone alterations such as appearance of bone osteophytes 18,19.
As the utility of MIA in the rat has been described before 20, in this paper we focus on the methodology of MIA-induced OA in mice as this model is being increasingly used with the availability of knock-out mice. We describe a procedure for the injection of very small volumes into the knee and methods for measuring sensitivity to noxious and non-noxious stimuli in the hind limbs.
The breakdown of the methodology will help to reduce variability, and as such, refine the model and reduce the number of animals needed for study.

<table><tbody><tr><td>Monoiodoacetate</td><td>Sigma-Aldrich</td><td>I-2512-25G</td><td>&amp;#8805; 98% purity</td></tr><tr><td>0.9% Saline</td><td>Mini-Plasco basic</td><td>365 4840</td><td></td></tr><tr><td>Isoflurane</td><td>Merial</td><td>DNI 4090/1</td><td></td></tr><tr><td>26 G Needle</td><td>Fisher Scientific</td><td>12947606</td><td></td></tr><tr><td>50 &amp;#956;l Hamilton Syringe</td><td>Sigma-Aldrich</td><td>20701</td><td></td></tr><tr><td>Von Frey Hairs</td><td>Linton Instruments</td><td>NC 122775-99</td><td></td></tr><tr><td>Incapacitance tester</td><td>Linton Instruments</td><td></td><td>Delivery on Request</td></tr><tr><td>Testing Cage Rack&amp;nbsp;</td><td>Ugo Basile</td><td>37450</td><td></td></tr><tr><td>Compact Anesthetic system</td><td>Vet-Tech</td><td>AN001B</td><td></td></tr><tr><td>Medical O&lt;sub&gt;2&lt;/sub&gt;</td><td>BOC</td><td>101-F</td><td></td></tr><tr><td>Aldasorbers</td><td></td><td>Vet -Tech</td><td>AN006A</td></tr></tbody></table>

the monoiodoacetate model of osteoarthritis pain in the mouse

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The current treatments for OA pain such as NSAIDS or opiates are neither sufficiently effective nor devoid of detrimental side effects. Animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and define novel pharmacological targets for therapy. Currently available models of OA in rodents include surgical and chemical interventions into one knee joint. The monoiodoacetate (MIA) model has become a standard for modelling joint disruption in OA in both rats and mice. The model, which is easier to perform in the rat, involves injection of MIA into a knee joint that induces rapid pain-like responses in the ipsilateral limb, the level of which can be controlled by injection of different doses. Intra-articular injection of MIA disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase and results in chondrocyte death, neovascularization, subchondral bone necrosis and collapse, as well as inflammation. The morphological changes of the articular cartilage and bone disruption are reflective of some aspects of patient pathology. Along with joint damage, MIA injection induces referred mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that are measurable and quantifiable. These behavioral changes resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain.
The aim of this article is to describe the methodology of intra-articular injections of MIA and the behavioral recordings of the associated development of hypersensitivity with a mind to highlight the necessary steps to give consistent and reliable recordings.

We have recently reported that the injection of 0.5 - 1 mg MIA in the mouse knee joint induces referred mechanical hypersensitivity (allodynia) in the ipsilateral hind paw and weight bearing deficits for up to 4 weeks, although onsets are dose-dependent 23.
The data reported in Figure 1 constitute an example of the time course of MIA-induced mechanical hypersensitivity in the ipsilateral hind paws following a range of doses injected in the knee. Specifically, the lowest dose of MIA (0.5 mg/mouse) induced a 50% decrease of thresholds compared to the injection of saline on day 10, and thresholds decreased to 70% of those of&#160;saline controls by day 28 after&#160;injection. The intermediate dose of 0.75 mg of MIA resulted in a gradual decrease in&#160;thresholds that were 80% lower than saline control thresholds on day 10 and remained low up to day 28. The highest dose of 1 mg MIA was associated with a significant drop in&#160;threshold&#160;on day 5 and a further decrease on day 10, which was sustained up to day 28.
The data reported in Figure 2 provide examples of weight bearing changes that are associated with MIA injection in the knee joints. In this set of experiments, while&#160;the 0.5 mg MIA dose did not induce&#160;significant changes in weight bearing throughout the 28 day&#160;duration of the study, the 0.75 mg MIA dose resulted in a significant reduction in the weight borne by&#160;the ipsilateral paw from day 10 onwards. Notably, weight bearing asymmetry associated with 0.75 mg of MIA may produce variable and inconsistent results between studies 23. Instead, the dose of 1 mg MIA generally induces reproducible weight bearing asymmetry and the data in Figure 2 demonstrate significant reduction of weight borne on the ipsilateral hind paw from day 3 until the end of the observation period. As expected, saline-treated animals showed no weight bearing changes.
<img alt="Figure 1" src="/files/ftp_upload/53746/53746fig1.jpg" /> 
Figure 1. Development of Mechanical Allodynia Post MIA Injection. Paw withdrawal thresholds of the ipsilateral and contralateral hind paws were assessed before and after injection of MIA (0.5, 0.75, and 1mg/mouse) and saline (0.9% NaCl), n = 8 - 10 mice/group. *P&#60;0.05, **P&#60;0.01, ***P&#60;0.001&#160;versus saline-treated group; Two-way repeated measurements ANOVA followed by Student Newman-Keuls post hoc test. <a href="https://www.jove.com/files/ftp_upload/53746/53746fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53746/53746fig2.jpg" /> 
Figure 2. Development of Weight Bearing Deficits Post MIA Injection. Changes in body weight distribution between the two hind limbs were calculated as [(weight borne on ipsilateral paw / sum of the weight borne on the ipsilateral and contralateral paws)*100] were assessed before and after&#160;injection of MIA (0.5, 0.75, and 1&#160;mg/mouse) and saline (0.9% NaCl), n = 8 - 10 mice/group. *P&#60;0.05, **P&#60;0.01, ***P&#60;0.001&#160;versus saline-treated group. Two-way repeated measurements ANOVA followed by Student Newman-Keuls post hoc test. <a href="https://www.jove.com/files/ftp_upload/53746/53746fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse at JoVE.com

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The ...

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Research

JoVE Journal

Medicine

34.9K Views.  King's College London. Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

Video: The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

Inflammatory TMJ Pain Mouse Model: Bite Force and Von Frey Filament Assessments

Temporomandibular Joint Pain Measurement by Bite Force and Von Frey Filament Assays in Mice

Temporomandibular joint (TMJ) pain and osteoarthritis (OA) are common and debilitating disorders that impair patients' quality of life. The mechanisms driving diseases-related pain are poorly understood, and current treatments fail to provide effective and long-term therapeutic effects. Additionally, pain assessment in research, particularly orofacial pain, poses several challenges that complicate studies in both clinical and basic science settings. Therefore, we have established an inflammatory TMJ pain mouse model via intra-articular injection of CFA (Complete Freund's Adjuvant) and evaluated pain behaviors by bite force measurement and the von Frey filament test. Mice with CFA injection exhibited orofacial pain behaviors compared to PBS injection, including reduced bite force and head withdrawal threshold in the von Frey filament test. These methods are relatively easy to execute to have reproducible results and can be potentially extended to pain studies for other disease models related to TMJ disorders. Together, bite force, and the von Frey filament tests are reliable in measuring orofacial pain, as demonstrated in CFA injection-induced painful TMJOA mouse models.

This protocol describes how measuring bite force and head withdrawal threshold can capture pain behavior in Complete Freund's Adjuvant (CFA)-induced inflammatory temporomandibular joint (TMJ) pain.

Temporomandibular joint (TMJ) pain and osteoarthritis (OA) are common and debilitating disorders that impair patients' quality of life. The mechanisms ...

Behavior

Spontaneous and Evoked Measures of Pain in Murine Models of Monoarticular Knee Pain

Pain is the main cause of disability from arthritis. There is currently an unmet need for adequate treatments for arthritis pain. Pre-clinical models are necessary and useful for studying the mechanisms of pain and for evaluating efficacy of arthritis therapies. Measuring pain in animal models of arthritis is challenging. We have developed methods for measuring evoked and spontaneous pain in three models of murine arthritis. We quantitate the evoked pain responses of mice subjected to firm palpation of a painful knee. We also evaluate spontaneous pain by the proportion of weight and the amount of time placed on each of their 4 limbs after induction of arthritis pain in one knee. Joint pain in these mouse models produces a significant increase in evoked pain responses and an alteration in weight bearing. Since mice are quadrupeds, they offload the painful limb to the contralateral limb, to the forelimbs, or some combination. These methods are simple, require minimal equipment, and are reproducible and sensitive for detecting pain. They are useful for studying both disease-modifying arthritis treatments and analgesics in mice.

We have developed an evoked measure of arthritis pain and coupled it with a standardized method for measuring spontaneous pain in different murine models of chemically induced arthritis. These measures are sensitive and reproducible for different types of joint pain.

Pain is the main cause of disability from arthritis. There is currently an unmet need for adequate treatments for arthritis pain. Pre-clinical models are ...

Osteoarthritis Pain Model Induced by Intra-Articular Injection of Mono-Iodoacetate in Rats

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 ...

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent ...

Software-Assisted Quantitative Measurement of Osteoarthritic Subchondral Bone Thickness

Subchondral bone thickening and sclerosis are the major hallmarks of osteoarthritis (OA), both in animal models and in humans. Currently, the severity of the histologic subchondral bone thickening is mostly determined by visual estimation based semi-quantitative grading systems. This article presents a reproducible and easily executed protocol to quantitatively measure subchondral bone thickness in a mouse model of knee OA induced by destabilization of the medial meniscus (DMM). This protocol utilized ImageJ software to quantify subchondral bone thickness on histologic images after defining a region of interest in the medial femoral condyle and the medical tibial plateau where subchondral bone thickening usually occurs in DMM-induced knee OA. Histologic images from knee joints with a sham procedure were used as controls. Statistical analysis indicated that the newly developed quantitative subchondral bone measurement system was highly reproducible with low intra- and inter-observer variabilities. The results suggest that the new protocol is more sensitive to subtle or mild subchondral bone thickening than the widely used visual grading systems. This protocol is suitable for detecting both early and progressing osteoarthritic subchondral bone changes and for assessing in vivo efficacy of OA treatments in concert with OA cartilage grading.

This methodology article presents a software-assisted quantitative measurement protocol to quantify histologic subchondral bone thickness in murine osteoarthritic knee joints and normal knee joints as controls. This protocol is highly sensitive to subtle thickening and is suitable for detecting early osteoarthritic subchondral bone changes.

Subchondral bone thickening and sclerosis are the major hallmarks of osteoarthritis (OA), both in animal models and in humans. Currently, the severity of ...

Destabilization of the Medial Meniscus and Cartilage Scratch Murine Model of Accelerated Osteoarthritis

Osteoarthritis is the most prevalent musculoskeletal disease in people over 45, leading to an increasing economic and societal cost. Animal models are used to mimic many aspects of the disease. The present protocol describes the destabilization and cartilage scratch model (DCS) of post-traumatic osteoarthritis. Based on the widely used destabilization of the medial meniscus (DMM) model, DCS introduces three scratches on the cartilage surface. The current article highlights the steps to destabilize the knee by transecting the medial meniscotibial ligament followed by three intentional superficial scratches on the articular cartilage. The possible analysis methods by dynamic weight-bearing, microcomputed tomography, and histology are also demonstrated. While the DCS model is not recommended for studies that focus on the effect of osteoarthritis on the cartilage, it enables the study of osteoarthritis development in a shorter time window, with special focus on (1) osteophyte formation, (2) osteoarthritic and injury pain, and (3) the effect of cartilage damage in the whole joint.

The present protocol describes the controlled microblade scratches on the surface of the articular cartilage after destabilizing the mouse knee by cutting the medial miniscotibial ligament. This animal model presents an accelerated form of osteoarthritis (OA) suitable for studying osteophyte formation, osteosclerosis, and early-stage pain.

Osteoarthritis is the most prevalent musculoskeletal disease in people over 45, leading to an increasing economic and societal cost. Animal models are ...

Dual Hind Paw Injection for Enhanced Analgesic Testing

A Modified Inflammatory Pain Model to Study the Analgesic Effect in Mice

The hot plate test is widely used to evaluate analgesic effects on inflammatory pain in mice. A commonly used model of inflammatory pain was induced with an intraplantar injection of carrageenan in one hind paw. However, the findings from our laboratory showed that mice with a single-hind-paw injection of carrageenan lifted their paws to avoid thermal nociception during the hot plate test. Because of this response,&#160;previous injection method cannot accurately reflect the thermal pain threshold. Thus, we investigated a new method to avoid this issue. In the present study, we modified the previous method by injecting carrageenan into both hind paws to establish the model of inflammatory pain. The results demonstrated that both-hind-paw injection with carrageenan was sensitive and a better method to induce inflammatory pain when using the hot plate test than single-hind-paw injection. On the basis of these findings, we designed further experiments in which mice with either both-hind-paw or single-hind-paw injection of carrageenan were treated intragastrically with celecoxib (30 mg/kg). The results of the hot plate test showed that celecoxib augmented the thermal pain threshold in mice with both-hind-paw injection of carrageenan but not in mice with single-hind-paw injection of carrageenan. In summary, we developed a superior method to induce a model of inflammatory pain to evaluate analgesic effect.

Author Spotlight: Enhanced Method for Evaluating Analgesic Effects &#8212; Dual Hind Paw Carrageenan Injection in Mice

Here, we present a modified inflammatory pain model with the both-hind-paw carrageenan injection to evaluate the analgesic effect.

The hot plate test is widely used to evaluate analgesic effects on inflammatory pain in mice. A commonly used model of inflammatory pain was induced with ...

Neuroscience

Therapeutic Potential of Tuina Massage for Knee Osteoarthritis

Tuina Intervention in Sodium Monoiodoacetate Injection-Induced Rat Model of Knee Osteoarthritis

Knee osteoarthritis (KOA), a common degenerative joint disorder, is characterized by chronic pain and disability, which can progress to irreparable structural damage of the joint. Investigations into the link between articular cartilage, muscles, synovium, and other tissues surrounding the knee joint in KOA are of great importance. Currently, managing KOA includes lifestyle modifications, exercise, medication, and surgical interventions; however, the elucidation of the intricate mechanisms underlying KOA-related pain is still lacking. Consequently, KOA pain remains a key clinical challenge and a therapeutic priority. Tuina has been found to have a regulatory effect on the motor, immune, and endocrine systems, prompting the exploration of whether Tuina could alleviate KOA symptoms, caused by the upregulation of inflammatory factors, and further, if the inflammatory factors in skeletal muscle can augment the progression of KOA.
We randomized 32 male Sprague Dawley (SD) rats (180-220 g) into four groups of eight animals each: antiPD-L1+Tuina (group A), model (group B), Tuina (group C), and sham surgery (group D). For groups A, B, and C, we injected 25 &#181;L of sodium monoiodoacetate (MIA) solution (4 mg MIA diluted in 25 &#181;L of sterile saline solution) into the right knee joint cavity, and for group D, the same amount of sterile physiological saline was injected. All the groups were evaluated using the least to most stressful tests (paw mechanical withdrawal threshold, paw withdrawal thermal latency, swelling of the right knee joint, Lequesne MG score, skin temperature) before injection and 2, 9, and 16 days after injection.

Author Spotlight: Treating Knee Osteoarthritis with Tuina - A New Perspective 

This protocol describes the methods of Tuina intervention in sodium monoiodoacetate injection-induced rat model of knee osteoarthritis (KOA), which provides a reference for the application of Tuina in KOA animal models. This protocol also studies the effective mechanism of Tuina for KOA, and the results will help promote its application.

Knee osteoarthritis (KOA), a common degenerative joint disorder, is characterized by chronic pain and disability, which can progress to irreparable ...