Name: osteoarthritis pain model induced by intra-articular injection of mono-iodoacetate in rats
Uploaded: 2020-05-20T12:30:00
Description: Watch this Scientific Journal Video about Osteoarthritis Pain Model Induced by Intra-Articular Injection of Mono-Iodoacetate in Rats at JoVE.com

This study was funded by the Zhejiang Provincial Natural Science Foundation of China (Grant No: LY17H270016), the National Natural Science Foundation of China (Grant No: 81774331, 81873049, and 81673997), and the Zhejiang Provincial Science and Technology Project of Traditional Chinese Medicine of China (Grant No: 2013ZQ007 and 2016ZZ011).

Procedures involving animal subjects have been approved by the Medical Norms and Ethics Committee of Zhejiang Chinese Medical University and are in accordance with the China legislation on the use and care of laboratory animals.
1. Intra-articular injection of mono-iodoacetate in the knee
<ol>
	<li>After one-week of acclimation, randomly and equally divide 40 male Sprague-Dawley rats weighing 180&#8722;200 g (4&#8722;5 weeks old) into four groups (n = 10 rats/group). 
	NOTE: Rats in the...

<ol>
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Correlation of morphology with biochemical and metabolic data.</a> Journal of Bone and Joint Surgery. 53 (3), 523-537 (1971).</li><li>Yan, 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=Chondroprotective+effects+of+platelet+lysate+towards+monoiodoacetate-induced+arthritis+by+suppression+of+TNF-&#945;-induced+activation+of+NF-&#312;B+pathway+in+chondrocytes.">Chondroprotective effects of platelet lysate towards monoiodoacetate-induced arthritis by suppression of TNF-&#945;-induced activation of NF-&#312;B pathway in chondrocytes.</a> Aging. 11 (9), 2797-2811 (2019).</li><li>Yan, 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=Intra-Articular+Injection+of+Extract+Attenuates+Pain+Behavior+and+Cartilage+Degeneration+in+Mono-Iodoacetate+Induced+Osteoarthritic+Rats.">Intra-Articular Injection of Extract Attenuates Pain Behavior and Cartilage Degeneration in Mono-Iodoacetate Induced Osteoarthritic Rats.</a> Frontiers in Pharmacology. 9, 1360 (2018).</li><li>Wang, 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=Agkistrodon+ameliorates+pain+response+and+prevents+cartilage+degradation+in+monosodium+iodoacetate-induced+osteoarthritic+rats+by+inhibiting+chondrocyte+hypertrophy+and+apoptosis.">Agkistrodon ameliorates pain response and prevents cartilage degradation in monosodium iodoacetate-induced osteoarthritic rats by inhibiting chondrocyte hypertrophy and apoptosis.</a> Journal of Ethnopharmacology. 231, 545-554 (2019).</li><li>Yamada, E. F., 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=Evaluation+of+monosodium+iodoacetate+dosage+to+induce+knee+osteoarthritis:+Relation+with+oxidative+stress+and+pain.">Evaluation of monosodium iodoacetate dosage to induce knee osteoarthritis: Relation with oxidative stress and pain.</a> International Journal of Rheumatic Diseases. 22 (3), 399-410 (2019).</li><li>Schuelert, N., McDougall, J. 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The rat model of OA induced by MIA is a well-established, widely used model. Intra-articular injection of MIA initially causes severe and acute inflammation, which gives rise to the longer and degenerative phase of OA17,18. In this research, we measured nociceptive sensitivity by MWT and TWL, and assessed gait alterations with an imaging system. Previous reports found that the injection of MIA could raise the sensitivity of afferent knee joint fibers leading to n...

Osteoarthritis (OA) is the most common joint disease in the world, affecting an estimated 10-12% populations in adults1. The most generally involved joint is the knee, and OA has a higher incidence in older adults, especially women2. As a chronic disease, OA develops progressively over decades into joint failure with symptoms such as cartilage loss, synovial inflammation, osteophytosis, decreased function, and chronic pain3. According to the World Health Organization (WHO), OA is the fourth most prevalent disease in females and the eighth most prevalent disease in males. By 2020, OA may become the fourth most disabling disease in humans4. However, currently available therapies of OA address only symptoms and extend the time until joint replacement surgery5.
The spontaneous OA in human patients often takes a long time to produce clinical symptoms such as joint related pain6. In the early stages of OA, pain is usually intermittent and becomes more frequent and severe as the disease progresses, making it the predominant complaint of patients7. Therefore, extensive animal models for OA pain have been developed over the past half century to promote pain relief therapy. OA models have classically been divided into spontaneous and induced models. Spontaneous models include naturally occurring models and genetically modified models, which can more closely simulate the course of primary OA in humans8. Induced models can generally be divided into two categories: 1) post-traumatic OA induced by surgery or other trauma; or 2) intra-articular injection of chondrotoxic or pro-inflammatory substances3. These models lay a foundation for the pathophysiological study of OA and contribute greatly to the development of drugs to reduce pain and increase function.
Recently, the most widely used inducer for OA modeling is mono-iodoacetate (MIA). MIA, an inhibitor of glyceraldehyde-3-phosphate dehydrogenase, can cause changes in cartilage matrix, degradation, loss of cartilage, synovitis and other changes, which are similar to the pathological changes of human osteoarthritis9. It has been noted that intra-articular injection of MIA induced ongoing pain at 28 days after MIA administration, indicating that the MIA model may be useful for investigating chronic nociceptive pain10,11,12. In this study, male Sprague-Dawley rats received intra-articular injections with 0.5, 1.5, or 3 mg of MIA in the knee joints. The severity of MIA-induced joint pain was measured by assessment of mechanical and thermal sensitivity at 1, 7, 14, 21, 28 and 35 days after injections. On this basis, 1.5 mg of MIA was selected as the final concentration to evaluate gait patterns and histological changes at 28 days after injections.

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	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Anti-Collagen II antibody</td>
			<td style="width:255px;">Abcam(UK)</td>
			<td style="width:115px;">34712</td>
			<td style="width:191px;">Primary antibody for immunohistochemistry (IHC)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Anti-Collagen X (Col10) antibody</td>
			<td style="width:255px;">Abcam(UK)</td>
			<td style="width:115px;">49945</td>
			<td style="width:191px;">Primary antibody for IHC</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">DigiGait Imaging System</td>
			<td style="width:255px;">Mouse Specifics (Boston, MA, USA)</td>
			<td style="width:115px;"></td>
			<td style="width:191px;">Equipment for gait patterns analyses</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Eosin</td>
			<td style="width:255px;">Sigma-Aldrich</td>
			<td style="width:115px;">861006</td>
			<td style="width:191px;">The dye for HE staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Fast Green FCF</td>
			<td style="width:255px;">Sigma-Aldrich</td>
			<td style="width:115px;">F7252</td>
			<td style="width:191px;">The dye for SO staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Goat anti-mouse antibody</td>
			<td style="width:255px;">ZSGQ-BIO (Beijing, China)</td>
			<td style="width:115px;">PV-9002</td>
			<td style="width:191px;">Secondary antibody for IHC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Goat anti-rabbit antibody</td>
			<td style="width:255px;">ZSGQ-BIO (Beijing, China)</td>
			<td style="width:115px;">PV-9001</td>
			<td style="width:191px;">Secondary antibody for IHC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Hematoxylin</td>
			<td style="width:255px;">Sigma-Aldrich</td>
			<td style="width:115px;">H3163</td>
			<td style="width:191px;">The dye for HE staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">MIA</td>
			<td style="width:255px;">Sigma-Aldrich</td>
			<td style="width:115px;">I4386-10G</td>
			<td style="width:191px;">powder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">MMP13</td>
			<td style="width:255px;">Cell Signaling Technology, Inc. (Danvers, MA, USA)</td>
			<td style="width:115px;">69926</td>
			<td style="width:191px;">Primary antibody for IHC</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Modular tissue embedding center</td>
			<td style="width:255px;">Thermo Fisher Scientific (USA)</td>
			<td style="width:115px;">EC 350</td>
			<td style="width:191px;">Produce paraffin blocks.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Plantar Test apparatus</td>
			<td style="width:255px;">UgoBasile (Italy)</td>
			<td style="width:115px;">37370</td>
			<td style="width:191px;">Equipment for TWL assay</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">PrimeScript RT reagent Kit (Perfect Real Time)</td>
			<td style="width:255px;">TaKaRa Biotechnology Co. Ltd. (Dalian, China)</td>
			<td style="width:115px;">RR037A</td>
			<td style="width:191px;">Extracte total RNA from cultured cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Rotary and Sliding Microtomes</td>
			<td style="width:255px;">Thermo Fisher Scientific (USA)</td>
			<td style="width:115px;">HM325</td>
			<td style="width:191px;">Precise paraffin sections.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Safranin-O</td>
			<td style="width:255px;">Sigma-Aldrich</td>
			<td style="width:115px;">S2255</td>
			<td style="width:191px;">The dye for SO staining</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Tissue-Tek VIP 5 Jr</td>
			<td style="width:255px;">Sakura (Japan)</td>
			<td style="width:115px;"></td>
			<td style="width:191px;">Vacuum Infiltration Processor</td>
		</tr>
	</tbody>
</table>

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.

With this methodology, we established an OA pain model in the rat and detected the resulting changes. MWT and TWL reflected mechanical allodynia and thermal hyperalgesia, respectively. As shown in Figure 1, MIA induced mechanical allodynia and thermal hyperalgesia present in a dose-dependent manner. Remarkably, the decrease of MWT reached a peak from 21 days to 28 days, and then rebounded, suggesting that joint repair may occur at this stage, but MWT of 3 mg MIA group was still at a low leve...

Watch this Scientific Journal Video about Osteoarthritis Pain Model Induced by Intra-Articular Injection of Mono-Iodoacetate in Rats at JoVE.com

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

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 ...

Intra-Articular MI injection causes measurable changes in pain related behavior, which plays an important role in evaluating the success of modeling The procedure reproducible and it's time efficient. In addition, the dose accelerated are contra label falls small as well as light mammals. And demonstrating the procedure will be Zuxiang Chen, a grad student from our laboratory.Place the anesthetized rat in the supine position and remove the fur from one knee. Disinfect the exposed skin with alcohol and position the knee at a 90 degree angle to reveal the white patella tendon below the patella. Apply pressure to the patellar tendon with a fingertip to find the gap beneath the patella.And holding the needle perpendicular to the joint, insert a 26 gauge needle into the junction of the gap and the lateral patellar tendon. When the needle is in place, inject 50 microliters of MIA solution into the joint cavity before slowly retracting the needle. Then wrap a piece of gauze around the injection site to minimize reflux and leakage.At 1, 7, 14, 21, 28, and 35 days after injection place one injected rat in an elevated 17 by 11 by 13 centimeter plastic with a wire mesh base suspended 50 centimeters above a table, in a quiet environment Give the rat 30 minutes to adapt to the environment before pressing a Von Frey Needle perpendicular on the planter's surface of one hind paw. Increasing the pressure gradually and linearly until paw lifting or paw licking occurs. When paw withdrawal occurs, use a force lower than the previous threshold to ensure that the threshold is the minimum withdrawal force.When each rat has been tested at least four times, at least three to five minutes apart, record the minimal force eliciting a paw withdrawal reflex and average the data as the MWT of the rats. To test the TWL place one injected rat into each compartment of a 60 by 20 by 14 centimeter plexiglass box. Set down to a three millimeter thick glass plate in a quiet room with a constant temperature and give the rats 30 minutes to adapt to the testing environment.At the end of the orientation period calibrate the thermal stimulus via an infrared radiometer and set the desired infrared intensity to 70 units. Place the infrared emitter detector on the container directly under the center of the paw being tested, and press start. The timer will start automatically.As soon as movement of the paw occurs the controller will automatically turn off the infrared light and stop the timer. Record the reaction time when paw withdrawal and paw licking is observed. After repeating the test at least four times for each rat average the data as the TWL of the rats.To assess the gate pattern of the MIA treated rats, first use the knobs at both ends of a walking compartment to adjust the length of the compartment to 61 centimeters. Next, place a rat into the walking compartment and train the rat to make uninterrupted runs for at least five step cycles at a speed of 18 centimeters per second. When the rat has been trained, capture at least five second videos of continuous movement of each rat with a high speed digital video camera mounted below the transparent treadmill belt.After capturing at least three uninterrupted runs five minutes apart, draw a bounding box to define the boundary of the animal walking image in the imaging software and enter the run speed for each video. Then start the automatic analysis by the software. After processing, the software will output several spreadsheets reporting the gate indices, including the stance, swing, breaking, propulsion, cadence and step sequence.MIA induces mechanical allodynia and thermal hyperalgesia in a dose-dependent manner. Remarkably, the decrease of MWT reaches a peak from 21 days to 28 days before rebounding suggesting that joint repair may occur at this stage. Note that the MWT of the three milligram MIA group is still at a low level at these time points.The change of TWL is roughly consistent with that observed for the MWT. At 1.5 milligrams of MIA, the levels of the gait parameters including the total paw area and the unit stride length are significantly reduced in the MIA group. After 28 days suggesting that MIA induces osteoarthritis related joint pain in rats.Histopathological analysis by Makin Scoring reveals clear cartilage degeneration collagen disruption and matrix disorganization in the MIA group. In addition, treatment with 1.5 milligrams of MIA induces a significant up regulation of MMP 13 and collagen type 10 and significant downregulation of type two collagen. It's important to find the right junction.No resistance should be filled when the needle reaches atricular space. This technique facilitated as a production of a stable and reproducible OA pain model making an important contribution to the development of drugs for reducing pain and increasing function. Another side to MI has an extremely objective effect on the hail membrane and there for mask and the gloves should always be worn when preparing this solution.

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Medicine

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present here a protocol for elevating intraocular pressure (IOP) in the rat, by injecting magnetic microspheres into the anterior chamber of the eye. The use of magnetic particles allows the user to manipulate the beads into the iridocorneal angle, thus providing a very effective blockade of fluid outflow from the trabecular meshwork. This leads to long-lasting IOP rises, and eventually neuronal death in the ganglion cell layer (GCL) as well as optic nerve pathology, as seen in patients with the disease. This method is simple to perform, as it does not require machinery, specialist surgical skills, or many hours of practice to perfect. Furthermore, the pressure elevations are very robust, and reinjection of the magnetic microspheres is not usually required unlike in some other models using plastic beads. Additionally, we believe this method is suitable for adaptation for the mouse eye.

We present a method for inducing elevated intraocular pressure (IOP), by injecting magnetic microspheres into the rat eye, to model glaucoma. This leads to strong pressure rises, and extensive neuronal death. This protocol is easy to perform, does not require repeat injections, and produces stable long-lasting IOP rises.

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present ...

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.

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 ...

A Model of Glaucoma Induced by Circumlimbal Suture in Rats and Mice

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known risk factor for glaucoma. This protocol demonstrates a step-by-step guide on this technique in Long Evans rats and C57BL/6 mice. Under general anesthesia, a "purse-string" suture is applied on the conjunctiva, around the equator and behind the limbus of the eye. The fellow eye serves as an untreated control. Over the duration of our study, which was a period of 8 weeks for rats and 12 weeks for mice, IOP remained elevated, as measured regularly by rebound tonometry in conscious animals without topical anesthesia. In both species, the sutured eyes showed electroretinogram features consistent with preferential inner retinal dysfunction. Optical coherence tomography showed selective thinning of the retinal nerve fiber layer. Histology of the rat retina in cross-section found reduced cell density in the ganglion cell layer, but no change in other cellular layers. Staining of flat-mounted mouse retinae with a ganglion cell specific marker (RBPMS) confirmed ganglion cell loss. The circumlimbal suture is a simple, minimally invasive and cost-effective way to induce ocular hypertension that leads to ganglion cell injury in both rats and mice.

Chronic ocular hypertension is induced by applying a circumlimbal suture in rats and mice, leading to functional and structural deterioration of the retinal ganglion cells consistent with glaucoma.

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known ...

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.

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 ...

Protocol for Developing a Femur Osteotomy Model in Wistar Albino Rats

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. Osteoanabolic drugs have the potential to augment the repair of fractures but have constraints like high costs or undesirable side effects. The bone healing potential of a drug can initially be determined by in vitro studies, but in vivo studies are needed for the final proof of concept. Our objective was to develop a femur osteotomy rodent model that could help researchers understand the development of callus formation following fracture of the shaft of the femur and that could help establish whether a potential drug has bone healing properties. Adult male Wistar albino rats were used after Institutional Animal Ethics Committee clearance. The rodents were anesthetized, and under aseptic conditions, complete transverse fractures at the middle one-third of the shafts of the femurs were created using open osteotomy. The fractures were reduced and internally fixed using intramedullary K-wires, and secondary fracture healing was allowed to take place. After surgery, intraperitoneal analgesics and antibiotics were given for 5 days. Sequential weekly x-rays assessed callus formation. The rats were sacrificed based on radiologically pre-determined time points, and the development of the fracture callus was analyzed radiologically and using immunohistochemistry.

Here, we present a protocol to iatrogenically fracture the shaft of the femur of Wistar albino rats and follow up on the development of the callus. This femur osteotomy model can help researchers evaluate the process of fracture healing and to study how a drug could influence fracture healing.

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. ...

FSN Therapy for Knee Osteoarthritis Pain Management

Fu's Subcutaneous Needling for Knee Osteoarthritis Pain

Fu's subcutaneous needling (FSN) is a new acupuncture and dry needling technique based on traditional Chinese medicine. It rapidly produces long-lasting effects in soft tissue injuries, particularly in painful musculoskeletal conditions, by providing stimulation primarily in the subcutaneous area. Osteoarthritis (OA) is the most common joint disease in adults worldwide and is often accompanied by a painful syndrome of structural changes in the peripheral joints of the knee. However, the etiology of OA pain is not fully understood, though myofascial trigger points (MTrPs) are commonly found in the lower limb muscles (so-called "tightened muscles") of patients with knee OA. 
FSN has been used in many fields for the treatment of acute pain problems and can relieve muscle contraction from MTrPs, thereby improving the local circulation. This study recruited patients with pain from knee OA into an FSN group or a transcutaneous electrical nerve stimulation (TENS) group with three treatment sessions and a follow-up over the course of 2 weeks. The results showed that FSN was effective in treating soft tissue pain around the knee with OA. This study aimed to establish and visualize three key technical indicators during FSN therapy, including the FSN needle insertion point and layer; the frequency and duration of the swaying movement; and the manipulation of the reperfusion approach. These findings have great potential for future applications in myofascial pain treatment, especially for pain management. Following this protocol could enhance FSN skills.

Author Spotlight: Fu's Subcutaneous Needling for Knee Osteoarthritis Pain  

We present a protocol for using Fu's subcutaneous needling for knee osteoarthritis pain, which combines swaying movement and reperfusion approach techniques. This protocol has great potential for future applications in myofascial pain treatment and could enhance Fu's subcutaneous needling (FSN) manipulation skills.

Fu's subcutaneous needling (FSN) is a new acupuncture and dry needling technique based on traditional Chinese medicine. It rapidly produces long-lasting ...

Standardized Tuina Manipulation for Knee Osteoarthritis in Rats

Tuina Manipulation to Reduce Inflammation and Cartilage Loss in Knee Osteoarthritis Rats

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its mechanism. Therefore, the manipulation of animal models of knee osteoarthritis is critical. This protocol provides a standard process for Tuina manipulation on KOA rats and a preliminary exploration of the mechanism of Tuina for KOA. The press and kneading manipulation method (a kind of Tuina manipulation that refers to pressing and kneading the specific area of the body surface) is applied on 5 acupoints around the knee joint of rats. The force and frequency of the manipulation were standardized by finger pressure recordings, and the position of the rat during manipulation is described in detail in the protocol. The effect of manipulation can be measured by pain behavior tests and microscopic findings in synovial and cartilage. KOA rats showed significant improvement in pain behavior. The synovial tissue inflammatory infiltration was reduced in the Tuina group, and the expression of tumor necrosis factor (TNF)-&#945; was significantly lower. Compared to the control group, chondrocyte apoptosis was less in the Tuina group. This study provides a standardized protocol for Tuina manipulation on KOA rats and preliminary proof that the therapeutic effects of Tuina may be related to reducing synovial inflammation and delayed chondrocyte apoptosis.

Author Spotlight: Understanding the Mechanism of Tuina Manipulation in Rats 

We present a protocol for Tuina manipulation performed on plaster-immobilized-induced knee osteoarthritis rats. Based on the preliminary results, it suggested that the efficacy of the method relied on the reduction of inflammation and cartilage loss.

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its ...

Treating Knee Osteoarthritis with Tuina in a Rabbit Model

Tuina Intervention in Rabbit Model of Knee Osteoarthritis

Knee osteoarthritis (KOA) is mainly characterized by degenerative changes in the knee joint's cartilage and surrounding soft tissues. The efficacy of Tuina in treating KOA has been confirmed, but the underlying mechanism needs to be investigated. This study aims to establish a scientifically feasible KOA rabbit model treated with Tuina to reveal the underlying mechanisms. For this, 18, 6-month-old normal-grade male New Zealand rabbits were randomly divided into sham, model, and Tuina groups, with 6 rabbits in each group. The KOA model was established by injecting 4% papain solution into the knee joint cavity. The Tuina group was intervened with Tuina combined with the knee joint rotary correction method for 4 weeks. Only the standard grasping and fixation were performed in sham and model groups. At the end of the 1-week intervention, the knee joint range of motion (ROM) was observed, and cartilage hematoxylin-eosin (HE) staining was done. The study shows that Tuina could inhibit chondrocyte apoptosis, repair cartilage tissue, and restore knee joint ROM. In conclusion, this study demonstrates the scientific feasibility of Tuina treatment for KOA model rabbits, highlighting its potential application in the study of KOA and similar knee joint-related conditions.

Author Spotlight: Using a Rabbit Model to Explore the Efficacy of Tuina in Treating Knee Osteoarthritis 

The protocol describes a method for Tuina intervention in a rabbit model of knee osteoarthritis.

Knee osteoarthritis (KOA) is mainly characterized by degenerative changes in the knee joint's cartilage and surrounding soft tissues. The efficacy of ...

Experimental Modeling of Knee Osteoarthritis in Rats

After anesthetizing the rat, use a shaving machine to remove the hair of the right hind limb. Then, place a medical cotton pad between the right ankle and hip joint. Fix the right knee joint at 180 degree extension using five to six layers of wet plaster bandage evenly.Spiral wrap the plaster bandage starting from the ankle and covering one-third of the previous one. Use a hairdryer to dry and harden the plaster. After the plaster bandage dries and hardens, mix the denture base materials to make it sticky.And externally wrap the plaster with denture based materials to fix and prevent it from gnawing. Once the mixture hardens, turn off the anesthesia machine and supervise the blood circulation in the terminal limb. Then, wait for the rat to wake up naturally.After three weeks of continuous immobilization, using surgical scissors, cut off the denture base material outside and plaster bandage. Rinse the lower limb of the rat with saline and dry it with gauze.