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 control group will be intra-articularly injected with 50 &#181;L of saline, while rats in the experimental groups will be treated with 0.5, 1.5 or 3 mg of MIA dissolved in 50 &#181;L of saline, respectively.</li>
	<li>On the day of injection, freshly prepare the solution of MIA in sterile saline (0.9% NaCl) at 15, 30 and 60 mg/mL concentration and 10% pentobarbital solution in sterile saline (0.9% NaCl). 
	CAUTION: MIA has an extremely destructive effect on the mucous membrane, the upper respiratory tract, the eye and the skin and other tissue. Therefore, mask and gloves are recommended when preparing a solution.</li>
	<li>Anesthetize rats by intraperitoneal injection of ketamine at 60 mg/kg and xylazine at 5 mg/kg (both prepared in&#160;saline). Gently clamp the toes of the rat with a tweezer to confirm anesthesia.</li>
	<li>Place the rat with its back facing down. Shave the knee and wipe the area surrounding the knee joint with alcohol.</li>
	<li>Keep the knee at a 90&#176; angle and reveal the white patellar tendon below the patella. Press the patellar tendon with the fingertip to find the gap beneath the patella.</li>
	<li>Choose the junction of the gap and the lateral patellar tendon as the injection site. Then, insert the 26 G needle vertically into the site about 5 mm. No resistance should be felt when the needle is in the articular space. 
	NOTE: It is important to keep the needle perpendicular to the injection site.</li>
	<li>Inject 50 &#181;L of saline or MIA solution into the joint cavity. Slowly pull out the needle and wrap a piece of gauze around the injection site to minimize reflux and leakage.&#160;After anesthesia recovery, remove the gauze.</li>
	<li>Test pain-related behavior at 1, 7, 14, 21, 28 and 35 days after injections as described in section 2.</li>
</ol>
2. Behavioral assessments
<ol>
	<li>Mechanical withdrawal threshold (MWT) 
	NOTE: The MWT was measured by the von Frey test13 and the observer was blinded to the injections that the animals had received.
	<ol>
		<li>Place a rat in an elevated plastic cage (17 cm x 11 cm x 13 cm) with a wire mesh base suspended 50 cm above a table. Ensure the testing environment is quiet and give the rat 30 min to adapt to the environment.</li>
		<li>Press the von Frey needle perpendicularly on the plantar surface of each rat&#8217;s hind paw. Increase the pressure gradually (approximately 20 g/s) and linearly until paw lifting or paw licking occurs.</li>
		<li>Use a force lower than the previous threshold to ensure that the threshold is the minimum withdrawal force. Test each rat more than three times, at least 3&#8722;5 min apart.</li>
		<li>Record the minimal force eliciting a paw withdrawal reflex. Average the data as the MWT of rats.</li>
	</ol>
	</li>
	<li>Thermal withdrawal latency (TWL) 
	NOTE: The TWL was measured using the plantar test apparatus (Table of Materials) and the observer was blinded to the injections that the animals had received.
	<ol>
		<li>Place a plexiglass box (60 cm x 20 cm x 14 cm, divided into 6 compartments) on a 3 mm thick glass plate, and put rats into the box (one in each compartment). Ensure the environment is quiet and the room temperature is constant.</li>
		<li>Give rats 30 min to adapt to the testing environment.</li>
		<li>Calibrate the thermal stimulus via the infrared radiometer. Set the desired infrared intensity at 70 units.</li>
		<li>Place the infrared emitter/detector on the container directly under the center of the paw being tested.</li>
		<li>Pressing the START button. The timer will start automatically. The controller will automatically turn off the infrared light and stop the timer altogether as soon as movement of the paw occurs.</li>
		<li>Record the reaction time when the appearance of paw withdrawal and paw licking. 
		NOTE: A positive response for the test is regarded as paw withdrawal and paw licking. If only paw withdrawal occurs, it should be regarded as a voluntary movement of the rat rather than a positive response.</li>
		<li>Repeat the test more than three times. Average the data as the TWL of rats. 
		NOTE: Each exposure of radiant heat should not exceed 20 s. Infrared stimulations on the same hind paw should be at least 3&#8722;5 min apart.</li>
	</ol>
	</li>
	<li>Gait pattern analyses 
	NOTE: The gait pattern analyses were measured using an imaging system (Table of Materials) and the observer was blinded to the injections that the animals had received.
	<ol>
		<li>Adjust the length of the walking compartment by using the knobs at both ends of the walking compartment to suitable length for rats (e.g., 61 cm).</li>
		<li>Place a rat into the walking compartment and train rats to make uninterrupted runs for at least 5 step-cycles at a speed of at 18 cm/s before the formal experiment. 
		NOTE: When a rat is first placed in the walking compartment, the speed can be set to about 20 cm/s and shut off when running around 2 s. Then set the speed to 18 cm/s. Gently tap the back of the rat with the partition, if the rat pauses or retreats during walking. Try to reduce the speed gradually from 18 cm/s to 10 cm/s. If the rat eventually fails to perform the test, it is acceptable to choose another rat for the test.</li>
		<li>Slowly increase the speed of the treadmill until it reaches the target speed (18 cm/s). Capture at least 5 s video of continuous movement of the rat with the high-speed digital video camera mounted below the transparent treadmill belt.</li>
		<li>Test each rat at least 5 min apart to obtain at least three uninterrupted runs.</li>
		<li>Draw a &#8220;bounding box&#8221; to define the boundary of animal walking image in the imaging software and enter the run speed for each video. Select videos as a group and process videos automatically by the software. After processing videos, the software will output several spreadsheets to report gait indices, including stance, swing, braking, propulsion, cadence, step sequence, etc.</li>
		<li>Calculate total paw area (cm2), average stride length (cm), and unite stride length. 
		NOTE: Total paw area is the mean of the total area of four paws of each group of rats and paw area is defined as the maximal paw area in contact with the treadmill during the stance phase of the step cycle. Stride length is the distance between initial contacts of the same paw in one complete stride. Unit stride length = average stride length (cm)/body length (cm).&#160;</li>
	</ol>
	</li>
</ol>
3. Histopathological and immunohistochemical analyses
<ol>
	<li>Euthanize rats by intraperitoneal injection of pentobarbital sodium at 150 mg/kg (prepared in saline). Resect the&#160;knee joints immediately for the histologic analyses.</li>
	<li>Fix the joints in 10% formalin for 24 h, decalcify with 10% EDTA in phosphate buffered saline (PBS) for 8 weeks, and then embed joints in paraffin. 
	CAUTION: Formalin can cause eye, skin, and respiratory tract irritation. It should be handled in a hood.</li>
	<li>Section the paraffin embedded joints at 3 mm thickness with a microtome and float in a 40 &#176;C water bath containing distilled water.</li>
	<li>Transfer sections onto glass slides. Dry slides overnight and store slides at room temperature (RT) to continue the following staining.</li>
	<li>Place slides in a 60 &#176;C oven for 4 h to deparaffinize.</li>
	<li>Immerse the slides successively in xylene, xylene, 100% ethanol, 100% ethanol, 95% ethanol, 80% ethanol and 75% ethanol for 5 min, respectively, at RT.</li>
	<li>Stain sections with hematoxylin and eosin (H&#38;E), safranin-O (SO) and Alcian blue hematoxylin (ABH) as well as antibodies against rat type II collagen (Col2), type X collagen (Col10) and matrix metalloproteinase 13 (MMP13), as described in steps 3.8&#8722;3.12.</li>
	<li>H&#38;E staining
	<ol>
		<li>Rinse slides with deionized H2O for 3 min. Stain slides with hematoxylin for 3&#8722;5 min.</li>
		<li>Rinse slides with deionized H2O 3x, 1 min each, until no hematoxylin remains on the surface.</li>
		<li>Stain slides with eosin for 30 s. Then, rinse slides with deionized H2O 3x, 1 min each, until no eosin remains on the surface.</li>
		<li>Immerse slides in 0.1% ammonia for 10&#8722;20 s. Then, rinse slides with deionized H2O 3x, 1 min each.</li>
		<li>Immerse slides successively in 95% ethanol, 100% ethanol, xylene, xylene and xylene for 1 min, respectively.</li>
		<li>Coverslip the slides by neutral resin.</li>
	</ol>
	</li>
	<li>SO staining
	<ol>
		<li>Rinse slides with deionized H2O for 3 min. Stain slides with hematoxylin for 3&#8722;5 min.</li>
		<li>Differentiate quickly in 1% acid alcohol (about 3 s). Then, rinse slides with deionized H2O 3x, 1 min each, until no hematoxylin remains on the surface.</li>
		<li>Stain slides with Fast Green (FCF) solution for 5 min. Then, rinse slides with deionized H2O 3x, 1 min each.</li>
		<li>Stain slides with SO for 1&#8722;2 min. Then, rinse slides with deionized H2O 3x, 1 min each.</li>
		<li>Rinse slides with 1% acetic acid for 1&#8722;2 min to remove the residual FCF. Rinse slides with deionized H2O for 1 min.</li>
		<li>Immerse slides successively in 95% ethanol, 100% ethanol, xylene, xylene and xylene for 1 min, respectively.</li>
		<li>Coverslip the slides by neutral resin.</li>
	</ol>
	</li>
	<li>ABH staining
	<ol>
		<li>Rinse slides with deionized H2O for 3 min. Immerse slides in 1% acid alcohol for 30 s and drain briefly on a paper towel (do not rinse).</li>
		<li>Immerse slides in ABH for 1 h. Then, rinse slides with deionized H2O 3x, 1 min each, until no ABH remains on the surface.</li>
		<li>Differentiate quickly in 1% acid alcohol (about 3 s). Rinse slides with deionized H2O 3x, 1 min each.</li>
		<li>Immerse slides in 0.5% ammonium water for 15 s. Then, rinse slides with deionized H2O 3x, 1 min each.</li>
		<li>Immerse slides in 95% ethanol for 1 min. Immerse slides in eosin/orange G solution for 1.5 min.</li>
		<li>Immerse slides successively in 95% ethanol, 100% ethanol, xylene, xylene and xylene for 1 min, respectively.</li>
		<li>Coverslip the slides by neutral resin.</li>
	</ol>
	</li>
	<li>Examine all slides under a microscope and statistically grade on a scale of 0&#8722;13 by double-blind observation, according to Mankin&#8217;s scoring system14.</li>
	<li>Immunohistochemistry
	<ol>
		<li>Rinse slides with deionized H2O for 3 min.</li>
		<li>Immerse slides in 0.1 M sodium citrate and place slides in a 60 &#176;C oven for 4 h to retrieve the antigen.</li>
		<li>Immerse slides in 0.3% Triton X-100 in PBS for 10 min. Then, rinse slides with PBS 2x, 3 min each.</li>
		<li>Incubate sections in 3% H2O2 solution in methanol at RT for 10 min to block endogenous peroxidase activity. Rinse slides with PBS 2x, 3 min each.</li>
		<li>Incubate sections with 5% goat serum in PBS for 30 min at RT to block any non-specific binding. Rinse slides with PBS 2x, 3 min each.</li>
		<li>Incubate sections overnight at 4 &#176;C with 100 &#181;L of PBS-diluted (1:1,000) primary antibodies against rat type II collagen (Col2), type X collagen (Col10) and matrix metalloproteinase 13 (MMP13). Rinse slides with PBS 2x, 3 min each.</li>
		<li>Incubate sections with 100 &#181;L of PBS-diluted (1:1,000) secondary antibody (goat anti-mouse secondary or goat anti-mouse secondary) for 20 min at RT. Rinse slides with PBS 2x, 3 min each.</li>
		<li>Incubate sections with 100 &#181;L of 3,3&#8242;-diaminobenzidine (DAB) working solution. Monitor the reaction as the chromogenic reaction turns the epitope sites brown. 
		CAUTION: DAB is a carcinogen. Always wear gloves and work in a hood when working with DAB. 
		NOTE: The time of color development may vary from a few seconds to 10 min.</li>
		<li>As soon as a brown color develops on the sections, rinse slides with deionized H2O 2x, 3 min each.</li>
		<li>Immerse slides in hematoxylin for 1&#8722;2 min to counterstain slides. Then, rinse slides with deionized H2O 3x, 1 min each.</li>
		<li>Immerse slides successively in 95% ethanol, 100% ethanol, xylene, xylene and xylene for 1 min, respectively.</li>
		<li>Coverslip the slides by neutral resin.</li>
		<li>Observe the color of the antibody staining in the sections under a microscope.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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 ...

osteoarthritis-pain-model-induced-intra-articular-injection-mono

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JoVE Journal

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8.0K Views.  Zhejiang Chinese Medical University. 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.

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

Chronic Constriction of the Sciatic Nerve and Pain Hypersensitivity Testing in Rats

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of the population1,2. Symptoms include spontaneous (tingling, burning, electric-shock like) pain, dysaesthesia, paraesthesia, allodynia (pain resulting from normally non-painful stimuli) and hyperalgesia (an increased response to painful stimuli). The sensory symptoms are co-morbid with behavioural disabilities, such as insomnia and depression. To study chronic neuropathic pain several animal models mimicking peripheral nerve injury have been developed, one of the most widely used is Bennett and Xie's (1988) unilateral sciatic nerve chronic constriction injury (CCI)3 (Figure 1). Here we present a method for performing CCI and testing pain hypersensitivity.
CCI is performed under anaesthesia, with the sciatic nerve on one side exposed by making a skin incision, and cutting through the connective tissue between the gluteus superficialis and biceps femoris muscles. Four chromic gut ligatures are tied loosely around the sciatic nerve at 1 mm intervals, to just occlude but not arrest epineural blood flow. The wound is closed with sutures in the muscle and staples in the skin. The animal is then allowed to recover from surgery for 24 hrs before pain hypersensitivity testing begins.
For behavioural testing, rats are placed into the testing apparatus and are allowed to habituate to the testing procedure. The area tested is the mid-plantar surface of the hindpaw (Figure 2), which falls within the sciatic nerve distribution. Mechanical withdrawal threshold is assessed by mechanically stimulating both injured and uninjured hindpaws using an electronic dynamic plantar von Frey aesthesiometer or manual von Frey hairs4. The mechanical withdrawal threshold is the maximum pressure exerted (in grams) that triggers paw withdrawal. For measurement of thermal withdrawal latency, first described by Hargreaves et al (1988), the hindpaw is exposed to a beam of radiant heat through a transparent glass surface using a plantar analgesia meter5,6. The withdrawal latency to the heat stimulus is recorded as the time for paw withdrawal in both injured and uninjured hindpaws. Following CCI, mechanical withdrawal threshold, as well as thermal withdrawal latency in the injured paw are both significantly reduced, compared to baseline measurements and the uninjured paw (Figure 3). The CCI model of peripheral nerve injury combined with pain hypersensitivity testing provides a model system to investigate the effectiveness of potential therapeutic agents to modify chronic neuropathic pain. In our laboratory, we utilise CCI alongside thermal and mechanical sensitivity of the hindpaws to investigate the role of neuro-immune interactions in the pathogenesis and treatment of neuropathic pain.

Due to the simplicity of surgery and the robust behavioural outcome, chronic constriction of the sciatic nerve is one of the pre-eminent animal models of neuropathic pain. Within 24 hrs following surgery, pain hypersensitivity is established and can be quantitatively measured using a von Frey aesthesiometer (mechanical test) and plantar analgesia meter (thermal test).

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of ...

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 ...

The Lower Body Positive Pressure Treadmill for Knee Osteoarthritis Rehabilitation

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

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

Here, based on a clinician&rsquo;s point-of-view, we propose a two-model lower body positive pressure (LBPP) protocol (walking and squatting models) in ...

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, ...

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 ...