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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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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	</colgroup>
	<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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JoVE Journal

Medicine

7.6K vistas.  Zhejiang Chinese Medical University. 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 ...