Name: a reproducible cartilage impact model to generate post-traumatic osteoarthritis in the rabbit
Uploaded: 2023-11-21T12:20:00
Description: Watch this Scientific Journal Video about A Reproducible Cartilage Impact Model to Generate Post-Traumatic Osteoarthritis in the Rabbit at JoVE.com

This study was supported by DoD Peer Reviewed Medical Research Program - Investigator-Initiated Research Award W81XWH-20-1-0304 from the U.S. ARMY MEDICAL RESEARCH ACQUISITION ACTIVITY, by NIH NIAMS R01AR076477 and a Comprehensive Musculoskeletal T32 Training Program from the NIH (AR065971) and by NIH NIAMS Grant R01 AR069657. The authors would like to thank Kevin Carr for providing his expertise in machining and fabrication to this project, and Drew Brown and the Indiana Center for Musculoskeletal Health Bone Histology Core for aiding with histology.

The following procedure was performed with approval from the Indiana University School of Medicine Institutional Animal Care and Use Committee (IACUC). All survival surgeries were performed under sterile conditions, as outlined by the NIH guidelines. Pain and infection risks were managed with proper analgesics and antibiotics to optimize successful outcomes. Skeletally mature male New Zealand White rabbits, weighing 3.0-4.0 kg, were used for the present study.
1. Drop tower fabrication</...

This surgical procedure aims to generate consistent cartilage damage to the weight-bearing surface of the rabbit medial femoral condyle in a model of PTOA. An advantage of this procedure is that the posterior approach to the knee allows for direct visualization of the complete posterior medial femoral condyle, and it can be performed in approximately 37 min (Table 2). It should also be noted that this is an open injury model and may lead to acute inflammatory changes beyond just the impact due to potenti...

Post-traumatic osteoarthritis (PTOA) is a leading cause of disability worldwide, and accounts for 12%-16% of symptomatic osteoarthritis (OA)1. The current gold standard for end-stage OA management is total knee and hip arthroplasty2 or arthrodesis, as in the case of end-stage tibiotalar or subtalar arthritis. Although largely successful, arthroplasty can have costly and morbid complications3. In addition, arthroplasty is less desirable in patients under 50 years, given the low revision-free implant survivorship of 77%-83%4,5. Currently, there are no FDA-approved treatments to prevent or mitigate the progression of PTOA.
PTOA affects the whole joint, including the synovial tissue, subchondral bone, and articular cartilage. It is characterized by articular cartilage degeneration, synovial inflammation, subchondral bone remodeling, and osteophyte formation6,7. The phenotype of PTOA develops via a complex process of interplay between cartilage, synovium, and subchondral bone. The current understanding is that cartilage injury leads to the liberation of extra-cellular matrix (ECM) components such as type 2 collagen (COL2) and aggrecan (ACAN). These ECM component fragments are pro-inflammatory and cause increased production of IL-6, IL-1&#946;, and reactive oxygen species. These mediators act on chondrocytes, causing upregulation of matrix metalloproteinases (MMPs), such as MMP-13, which degrade articular cartilage while also decreasing matrix synthesis, leading to an overall catabolic environment for the articular cartilage8. In addition, there is evidence of increased chondrocyte apoptosis in primary osteoarthritis and PTOA9,10. Mitochondrial dysfunction occurs after supraphysiological loading of cartilage11,12,13,14, which can lead to increased chondrocyte apoptosis12,15. Enhanced chondrocyte apoptosis has been associated with increased proteoglycan depletion and cartilage catabolism and has been shown to precede changes in cartilage and subchondral bone remodeling16,17,18.
As with most human diseases, reliable and translational models of PTOA are needed to further understand the pathophysiology of the disease and test novel therapeutics. Large animals such as swine and canines have been used in intra-articular fracture and impact models of PTOA17,19, but they are costly. Smaller animal models, such as mice, rats, and rabbits&#160;are less expensive and are used to study PTOA generated through joint destabilization, which typically involves surgical transection of the anterior cruciate ligament (ACL) and/or disruption of the medial meniscus20,21,22,23,24,25. Although joint trauma can lead to various consequences, including ligamentous injury26, mechanical overload of the cartilage occurs in nearly all cases.
There is emerging evidence that the pathology behind the development of PTOA after ligamentous instability (as in ACL transection) and acute chondral injury is due to distinct mechanisms27. Therefore, developing models of direct injury to cartilage is important. There are currently a limited number of impact models generating osteochondral or chondral injury in rats and mice28,29. However, murine cartilage is not well-suited for generating isolated chondral defects. This is because murine articular cartilage is only 3-5 cell layers thick and lacks organized superficial, radial, and transitional cartilage zones, as well as the thick calcified cartilage layer found in humans and larger animals. Murine models also display spontaneous resolution of partial cartilage defects30,31. Hence, we chose the rabbit for this impact model as its cartilage thickness and organization are similar to those of humans, and it is the smallest animal model that will allow for the delivery of a consistent chondral impact that results in PTOA. Prior open surgical models of femoral condyle impact in the rabbit have employed a pendulum32, a hand-held spring-loaded cartilage impaction device33, and a drop tower that allowed rabbit-specific impactor creation34. However, these studies lacked in vivo data. Others have reported in vivo data with pendulum-based35, pneumatic36, and spring-loaded37 impact devices10, and these studies show a high rate of variability in peak stress and loading rates between the methods. Still, the field lacks a consistent approach to reliably model acute cartilage trauma in vivo.
The current protocol employs a drop-tower-based system to deliver a consistent impact to the posterior medial condyle of the rabbit knee. A posterior approach to the knee is employed to expose the posterior medial femoral condyle. A Steinman pin is then placed across the femoral condyles from medial to lateral in line with the joint surface and secured to the platform. Once secured, a load is delivered to the posterior medial femoral condyle. This method allows for consistent cartilage damage to be delivered to the weight-bearing surface of the rabbit distal&#160;femur.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
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			<td>Flat head screw</td>
			<td>McMaster-Carr</td>
			<td>92210A194</td>
			<td>Stainless steel hex drive flat head screw, 8-32, 1/2&#34;</td>
		</tr>
		<tr>
			<td>#15 scalpel blades</td>
			<td>McKesson</td>
			<td>1029066</td>
			<td>Scalpel McKesson No. 15 Stainless Steel / Plastic Classic Grip Handle Sterile Disposable</td>
		</tr>
		<tr>
			<td>1/2&#8221;-20 threaded rod</td>
			<td>McMaster-Carr</td>
			<td>99065A120</td>
			<td>1/2&#8221;-20 threaded rod</td>
		</tr>
		<tr>
			<td>10 mL syringe</td>
			<td>McKesson</td>
			<td>1031801</td>
			<td>For irrigation; General Purpose Syringe McKesson 10 mL Blister Pack Luer Lock Tip Without Safety</td>
		</tr>
		<tr>
			<td>3 mL syringe</td>
			<td>McKesson</td>
			<td>1031804</td>
			<td>For lidocaine/bupiviacaine injection; General Purpose Syringe McKesson 3 mL Blister Pack Luer Lock Tip Without Safety.</td>
		</tr>
		<tr>
			<td>3-0 polysorb</td>
			<td>Ethicon</td>
			<td>J332H</td>
			<td>3-0 Vircryl, CT-2, 1/2 circle, 26 mm, tapered</td>
		</tr>
		<tr>
			<td>4-0 monosorb</td>
			<td>Ethicon</td>
			<td>Z397H</td>
			<td>4-0 PDS 2, FS-2, 3/8 circle, 19mm, cutting edge</td>
		</tr>
		<tr>
			<td>5-0 polysorb</td>
			<td>Med Vet International</td>
			<td>NC9335902</td>
			<td>Med Vet International 5-0 ETHICON COATED VICRYL C-3</td>
		</tr>
		<tr>
			<td>Accelerometer</td>
			<td>Kistler</td>
			<td>8743A5</td>
			<td>Accelerometer</td>
		</tr>
		<tr>
			<td>Adson-Browns Forceps</td>
			<td>World precision tools</td>
			<td>500177</td>
			<td>Adson-Brown Forceps, 12 cm, Straight, TC Jaws, 7 x 7 Teeth</td>
		</tr>
		<tr>
			<td>Alfaxalone</td>
			<td>Jurox</td>
			<td>49480-002-01</td>
			<td>Alfaxan Multidose by Jurox : 10 mg/mL</td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Par Pharmaceuticals</td>
			<td>42023-0179-05</td>
			<td>Buprenorphine HCL injection: 0.3 mg/mL</td>
		</tr>
		<tr>
			<td>Butorphanol&#160;</td>
			<td>Zoetis</td>
			<td>54771-2033</td>
			<td>Butorphanol tartrate&#160;10mg/ml by Zoetis</td>
		</tr>
		<tr>
			<td>Chlorhexidine Hand Scrub</td>
			<td>BD</td>
			<td>371073</td>
			<td>BD E-Z Scrub 107 Surgical Scrub Brush/Sponge, 4% CHG, Red</td>
		</tr>
		<tr>
			<td>Collet</td>
			<td>STRYKER</td>
			<td>14023</td>
			<td>Stryker 4100-62 wire Collet 0.28-0.71&#39;&#39;</td>
		</tr>
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			<td>Cordless Driver handpiece</td>
			<td>STRYKER</td>
			<td>OR-S4300</td>
			<td>Stryker 4300 CD3 Cordless Driver 3 handpiece</td>
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		<tr>
			<td>Cricket Retractors</td>
			<td>Novosurgical</td>
			<td>G3510 21</td>
			<td>2x Heiss (Holzheimer) Cross Action Retractor</td>
		</tr>
		<tr>
			<td>Dissector Scissors</td>
			<td>Jorvet labs</td>
			<td>J0662</td>
			<td>Aesculap AG, Metzenbaum, Scissors, Straight 5 3/4&#8243;</td>
		</tr>
		<tr>
			<td>Elizabethian Collar</td>
			<td>ElizaSoft</td>
			<td>62054</td>
			<td>ElizaSoft Elizabethan Recovery Collar</td>
		</tr>
		<tr>
			<td>Enrofloxacin</td>
			<td>Custom Meds</td>
			<td></td>
			<td>Enrofloxacin compounded by Custom Meds</td>
		</tr>
		<tr>
			<td>Eye Ointment</td>
			<td>Pivetal&#160;</td>
			<td>46066-753-55</td>
			<td>Pivetal Articifical Tears- recently recalled</td>
		</tr>
		<tr>
			<td>Face-mount shaft collar</td>
			<td>McMaster-Carr</td>
			<td>5631T11</td>
			<td>Face-mount shaft collar</td>
		</tr>
		<tr>
			<td>Fast green</td>
			<td>Millipore Sigma</td>
			<td>F7258</td>
			<td>Fast green</td>
		</tr>
		<tr>
			<td>Freer</td>
			<td>Jorvet labs</td>
			<td>J0226Q</td>
			<td>Freer elevator</td>
		</tr>
		<tr>
			<td>Head screw -1</td>
			<td>McMaster-Carr</td>
			<td>91251A197</td>
			<td>Black-oxide alloy steel socket head screw, 8-32, 3/4&#34;</td>
		</tr>
		<tr>
			<td>Head screw -2</td>
			<td>McMaster-Carr</td>
			<td>92196A194</td>
			<td>Stainless steel socket head screw, 8-32, 1/2&#34;</td>
		</tr>
		<tr>
			<td>Head screw -3</td>
			<td>McMaster-Carr</td>
			<td>92196A146</td>
			<td>Stainless steel socket head screw, 8-32, 1/2&#34;</td>
		</tr>
		<tr>
			<td>Head screw -4</td>
			<td>McMaster-Carr</td>
			<td>92196A151</td>
			<td>Stainless steel socket head screw, 6-32, 3/4&#34;</td>
		</tr>
		<tr>
			<td>Hematoxylin Solution, Gill No. 1</td>
			<td>Millipore Sigma</td>
			<td>GHS132-1L</td>
			<td>Hematoxylin Solution, Gill No. 1</td>
		</tr>
		<tr>
			<td>Hex nut</td>
			<td>McMaster-Carr</td>
			<td>91841A007</td>
			<td>Stainless steel hex nut, 6-32</td>
		</tr>
		<tr>
			<td>Hold-down toggle clamp</td>
			<td>McMaster-Carr</td>
			<td>5126A71</td>
			<td>Hold-down toggle clamp</td>
		</tr>
		<tr>
			<td>Impact device</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>custom made</td>
		</tr>
		<tr>
			<td>Impact platform</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>custom made</td>
		</tr>
		<tr>
			<td>K-wires</td>
			<td>Jorvet Labs</td>
			<td>J0250A</td>
			<td>JorVet Intramedullary Steinman Pins, Trocar-Trocar 1/16&#34; x 7&#34;</td>
		</tr>
		<tr>
			<td>Lab View</td>
			<td>National Instruments</td>
			<td>n/a</td>
			<td>n/a</td>
		</tr>
		<tr>
			<td>Load cell</td>
			<td>Kistler</td>
			<td>9712B5000</td>
			<td>Load cell</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>The MathWorks Inc.</td>
			<td>n/a</td>
			<td>n/a</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica</td>
			<td>DMi-8</td>
			<td>Leica DMi8 microscope with LAS-X software</td>
		</tr>
		<tr>
			<td>Midazolam</td>
			<td>Almaject</td>
			<td>72611-749-10</td>
			<td>Midazolam Hydrochloride injection: 5mg/ml by Almaject</td>
		</tr>
		<tr>
			<td>milling machine depth stops</td>
			<td>McMaster-Carr</td>
			<td>2949A71</td>
			<td>Clamp-on milling machine depth stops</td>
		</tr>
		<tr>
			<td>Mobile C-arm</td>
			<td>Philips</td>
			<td>718095</td>
			<td>BV&#160;Pulsera, Mobile C-arm</td>
		</tr>
		<tr>
			<td>Mounted linear ball bearing</td>
			<td>McMaster-Carr</td>
			<td>9338T7</td>
			<td>Mounted linear ball bearing</td>
		</tr>
		<tr>
			<td>Needle Driver</td>
			<td>A2Z Scilab</td>
			<td>A2ZTCIN39</td>
			<td>TC Webster Needle Holder Smooth Jaws 5&#34;, Premium</td>
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		<tr>
			<td>Pentobarbital</td>
			<td>Vortech</td>
			<td>0298-9373-68</td>
			<td>Pentobarbital 390 mg/mL by Vortech</td>
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		<tr>
			<td>Safranin O</td>
			<td>Millipore Sigma</td>
			<td>HT90432</td>
			<td>Safranin O</td>
		</tr>
		<tr>
			<td>Small Battery pack</td>
			<td>STRYKER</td>
			<td>NS014036</td>
			<td>6212 Small Battery pack- 9.6 V</td>
		</tr>
		<tr>
			<td>Steel rod, 2&#8217;</td>
			<td>McMaster-Carr</td>
			<td>89535K25</td>
			<td>Steel rod, 2&#8217;</td>
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		<tr>
			<td>Sterile Saline</td>
			<td>ICU Medical</td>
			<td>6139-22</td>
			<td>AquaLite Solution Pour Bottles, 250 mL</td>
		</tr>
		<tr>
			<td>Stryker 6110-120 System 6 Battery Charger</td>
			<td>STRYKER</td>
			<td>OR-S6110-120</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical gloves</td>
			<td>McKesson</td>
			<td>1044729</td>
			<td>Surgical Glove McKesson Perry Size 6.5 Sterile Pair Latex Extended Cuff Length Smooth Brown Not Chemo Approved</td>
		</tr>
		<tr>
			<td>Surgical gown</td>
			<td>McKesson</td>
			<td>1104452</td>
			<td>Non-Reinforced Surgical Gown with Towel McKesson Large Blue Sterile AAMI Level 3 Disposable</td>
		</tr>
		<tr>
			<td>Suture scissors</td>
			<td>Jorvet Labs</td>
			<td>J0910SA</td>
			<td>Super Cut Scissors, Mayo, Straight, 5 1/2&#8243;</td>
		</tr>
		<tr>
			<td>TUNEL staining kit</td>
			<td>ABP Bioscience</td>
			<td>A049</td>
			<td>TUNEL Chromogenic Apoptosis Detection Kit</td>
		</tr>
		<tr>
			<td>Weitlaner Retractors</td>
			<td>Fine Science Tools</td>
			<td>17012-11</td>
			<td>2x Weitlaner-Locktite Retractors</td>
		</tr>
	</tbody>
</table>

a reproducible cartilage impact model to generate post-traumatic osteoarthritis in the rabbit

Post-traumatic osteoarthritis (PTOA) is responsible for 12% of all osteoarthritis cases in the United States. PTOA can be initiated by a single traumatic event, such as a high-impact load acting on articular cartilage, or by joint instability, as occurs with anterior cruciate ligament rupture. There are no effective therapeutics to prevent PTOA currently. Developing a reliable animal model of PTOA is necessary to better understand the mechanisms by which cartilage damage proceeds and to investigate novel treatment strategies to alleviate or prevent the progression of PTOA. This protocol describes an open, drop tower-based rabbit femoral condyle impact model to induce cartilage damage. This model delivered peak loads of 579.1 &plusmn; 71.1 N, and peak stresses of 81.9 &plusmn; 10.1 MPa with a time-to-peak load of 2.4 &plusmn; 0.5 ms. Articular cartilage from impacted medial femoral condyles (MFCs) had higher rates of apoptotic cells (p = 0.0058) and possessed higher Osteoarthritis Research Society International (OARSI) scores of 3.38 &plusmn; 1.43 compared to the non-impacted contralateral MFCs (0.56 &plusmn; 0.42), and other cartilage surfaces of the impacted knee (p &lt; 0.0001). No differences in OARSI scores were detected among the non-impacted articular surfaces (p &gt; 0.05).

The success of this procedure was monitored immediately after impact by visualization of the condyle by the surgeon (Figure 4A) and by radiography to ensure no fracture occurred (Figure 4B). There is a risk of impact failure leading to an intra-operative fracture of the condyle. This was typically due to improper Steinman pin placement (Figure 5). Using this model, there was a fracture failure rate secondary to intra-operative fract...

Watch this Scientific Journal Video about A Reproducible Cartilage Impact Model to Generate Post-Traumatic Osteoarthritis in the Rabbit at JoVE.com

A Reproducible Cartilage Impact Model to Generate Post-Traumatic Osteoarthritis in the Rabbit

The open medial femoral condyle impact model in rabbits is reliable for studying post-traumatic osteoarthritis (PTOA) and novel therapeutic strategies to mitigate PTOA progression. This protocol generates an isolated cartilage defect of the posterior medial femoral condyle in rabbits using a carriage-based drop tower with an impactor head.

Post-traumatic osteoarthritis (PTOA) is responsible for 12% of all osteoarthritis cases in the United States. PTOA can be initiated by a single traumatic ...

This method causes acute cartilage damage to the weight-bearing surface of the rabbit knee and reliably induces post-traumatic osteoarthritis. This model mirrors joint trauma that is relevant to clinical practice. It will allow for the evaluation of novel therapeutics to mitigate post-traumatic arthritis.The main technical hurdle is placement of the Steinmann pin into the femoral shaft. Misplacement will result in femoral shaft fracture. We recommend practicing this model first on rabbit cadavers.To begin, place the stainless steel front leg block under the end of the impact platform and cover the platform with a heating pad. Place the anesthetized rabbit in the prone position on the heating pad. Place a padded bump under the contralateral hip.Using silk tape, gently retract the tail superior and contralateral to the operative extremity. Clean the surgical site with chlorhexidine and 70%alcohol-soaked sterile gauze, starting from the posterior knee with a circular sweep outward. Place a sterile glove over the operative foot up to the ankle and wrap it with a sterile cohesive wrap.Drape the surgical site using three drapes by placing one directly under the operative extremity and the other two covering the rest of the body. Secure the drapes with towel clamps. Palpate the position of the patella anteriorly to estimate the position of the knee joint.Using a 15 blade, make a three to four-centimeter incision along the posterior aspect of the extended knee from the level of the superior pole of the patella distally. Perform blunt and sharp dissections through the underlying superficial fascia. After developing the interval between the skin medially and the medial gastrocnemius laterally, place a self retaining Weitlaner retractor in this interval.Then dissect laterally to the saphenous and retract the vasculature medially and the gastrosoleus complex laterally. Continue dissecting distally until a small mobile fabella is identified over the posteromedial femoral condyle. Perform an arthrotomy demobilize the fabella superolateral, exposing the underlying medial femoral condyle.Retract the soft tissue. While maintaining the condyle exposed, place a 0.062-inch Steinmann pin at the superior aspect of the medial femoral condyle and centered in the anterior posterior direction of the medial femoral condyle, roughly five millimeters from the posterior aspect of the condyle. Next, using a battery-powered Steinmann pin driver, drive the pin laterally through the bone and lateral skin parallel to the joint surface.Remove the retractors and close the skin incision with a 3-0 Polysorb suture in a running fashion. Cover the incision with sterile gauze. Remove the drape under the operative limb.Adjust the heights of the lower aspects of the Steinmann pin clamps so that they match the height of the pin. Secure the Steinmann pin on both sides of the knee by attaching the top aspects of the clamps and tightening the screws. Remove the suture and reopen the incision.Use self-retaining Weitlaner and cricket retractors to expose the medial femoral condyle. Attach the sterile three-millimeter impacter head to the drop tower carriage. Bring the drop tower over the operative extremity and place its base underneath the impacting platform.Gently lower the impacter onto the center of the posteromedial femoral condyle. Move the rabbit or tower to ensure the impacter head is centered over the medial femoral condyle. Once appropriate trajectory is ensured, clamp the tower onto the platform with the toggle clamps.Next, set the impacter on the drop tower at seven centimeters above the medial femoral condyle. In the lab view data acquisition software, click on the start button just before freeing the spindle stop to release the carriage and allow it to drop under the force of gravity. Perform visualization of the cartilage surface to determine whether appropriate cartilage damage has occurred.To analyze the data using the MATLAB code, move the data file from the sensors to the same folder as MATLAB and run the code. Inspect the load time curve and ensure that it is smooth, indicating that no fracture has occurred. Remove the drop tower from over the operative extremity, placing all used surgical tools aside.After wearing the new sterile gloves, reapply a sterile drape to the lower extremity. Then re-expose the medial femoral condyle. Thoroughly rinse the surgical site with 50 to 60 milliliters of sterile saline.Close the posterior capsule using a 5-0 Polysorb suture, followed by skin closure with a 4-0 Monosorb suture. Remove the height adjustable screw mechanism securing the Steinmann pin. Remove the Steinmann pin from the femur with the powered wire driver.Dress the wound with a non-adhesive dressing, followed by adhesive tape. Perform an X-ray of the operative extremity to ensure no fracture occurred and appropriate pin placement. The success of the surgical procedure was monitored through visualization of the condyle and radiography to confirm no fracture.Improper Steinmann pin placement resulted in an osteochondral fracture on impact. In this model, the average peak impact stress was 82 megapascals, and the average loading rate was 37 megapascals per millisecond. Cartilage damage was not observed in the contralateral uninjured femoral condyle and was mainly localized to the impact site.Impacted 16-week medial femoral condyles, or MFCs, had higher OARSI scores compared to the control MFCs. Further, impacted knee MFCs also displayed higher OARSI scores compared to the MTP, LTP, and LFC. In contrast, no differences in OARSI scores were observed among the MFC, LTP, MTP, and LFC compartments of the contralateral non-impacted knee.There were no significant differences between the impacted and non impacted LFC, MTP, and LTP joint surfaces. Articular cartilage from impacted MFC had higher levels of tunnel positivity, indicating increased chondrocyte apoptosis compared to non-impacted MFCs. At the end of the procedure, the pain behavior of the rabbits can be tracked.After the study, the development of post-traumatic osteoarthritis will be evaluated using imaging techniques, histology, and other methods. We're using this model to test therapeutics and devices that aim to mitigate post-traumatic osteoarthritis. The model may also help us to better understand the progression of this disease.

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Research

JoVE Journal

Medicine

Transthoracic Echocardiographic Examination in the Rabbit Model

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to that of humans and that of other large animal models such as dogs and pigs. However, the rabbit model has the additional advantage of lower maintenance costs compared to other large animal models. The longitudinal evaluation of cardiac function using echocardiography, when appropriately implemented, is a useful methodology for preclinical assessment of novel therapies for heart failure with reduced ejection fraction (e.g. cardiac regeneration). The correct use of this non-invasive tool requires the implementation of a standardized examination protocol following international guidelines. Here we describe, step by step, a detailed protocol supervised by veterinary cardiologists for performing echocardiography in the rabbit model, and demonstrate how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Here we describe, step by step, a detailed protocol for performing echocardiography in the rabbit model. We show how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to ...

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 ...

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

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

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

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

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 ...

Impact of Non-Invasive ACL Injury and Surgery on Protease Activity in Mice

Non-Invasive Compression-Induced Anterior Cruciate Ligament (ACL) Injury and In Vivo Imaging of Protease Activity in Mice

Traumatic joint injuries such as anterior cruciate ligament (ACL) rupture or meniscus tears commonly lead to post-traumatic osteoarthritis (PTOA) within 10-20 years following injury. Understanding the early biological processes initiated by joint injuries (e.g., inflammation, matrix metalloproteinases (MMPs), cathepsin proteases, bone resorption) is crucial for understanding the etiology of PTOA. However, there are few options for in vivo measurement of these biological processes, and the early biological responses may be confounded if invasive surgical techniques or injections are used to initiate OA. In our studies of PTOA, we have used commercially available near-infrared protease activatable probes combined with fluorescence reflectance imaging (FRI) to quantify protease activity in vivo following non-invasive compression-induced ACL injury in mice. This non-invasive ACL injury method closely recapitulates clinically relevant injury conditions and is completely aseptic since it does not involve disrupting the skin or the joint capsule. The combination of these injury and imaging methods allows us to study the time course of protease activity at multiple time points following a traumatic joint injury.

Author Spotlight: Investigating Early Events and Long-Term Effects of ACL Injuries for Osteoarthritis Progression

Non-invasive ACL injury is a reliable and clinically relevant method for initiating post-traumatic osteoarthritis (PTOA) in mice. This injury method also allows for in vivo quantification of protease activity in the joint at early time points post-injury using protease-activatable near infrared probes and fluorescence reflectance imaging.

Traumatic joint injuries such as anterior cruciate ligament (ACL) rupture or meniscus tears commonly lead to post-traumatic osteoarthritis (PTOA) within ...

Establishment of the KOA Model in Rabbits Using the Modified Videman Method

Application of Acupotomy in a Knee Osteoarthritis Model in Rabbit

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department, which seriously reduces the quality of life of people with KOA. Among several pathogenic factors, the biomechanical imbalance of the knee joint is one of the main causes of KOA. Acupotomology believes that restoring the mechanical balance of the knee joint is the key to treating KOA. Clinical studies have shown that acupotomy can effectively reduce pain and improve knee mobility by reducing adhesion, contracture of soft tissues, and stress concentration points in muscles and tendons around the knee joint. 
In this protocol, we used the modified Videman method to establish a KOA model by immobilizing the left hindlimb in a straight position. We have outlined the method of operation and the precautions related to acupotomy in detail and evaluated the efficacy of acupotomy in conjunction with the theory of "Modulating Muscles and Tendons to Treat Bone Disorders" through the detection of the mechanical properties of quadriceps femoris and tendon, as well as cartilage mechanics and morphology. The results show that acupotomy has a protective effect on cartilage by adjusting the mechanical properties of the soft tissues around the knee joint, improving the cartilage stress environment, and delaying cartilage degeneration.

Author Spotlight: Investigating the Mechanism of Action of Acupotomy in Treating Knee Osteoarthritis

In this protocol, a knee osteoarthritis model was prepared using the modified Videman method, and the operation procedures and precautions of acupotomy are detailed. The effectiveness of acupotomy has been demonstrated by testing the mechanical properties of quadriceps femoris and tendon and the mechanical and morphological properties of cartilage.

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department, which seriously reduces the quality of life of ...

Therapeutic Potential of Tuina Massage for Knee Osteoarthritis

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

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

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

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

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

Training Prior to Executing Tuina on Knee Osteoarthritis Rabbit Model

Initiate training using the Tuina Technique Parameter Determination Tool prior to executing the Tuina manipulation. To do so, on the Tuina Manipulation Simulation Platform, perform the rotary kneading and pressing method using the thumb. At the same time, in the Tuina Manipulation Parameter Processing Software observe the magnitude, frequency, and duration of action of the applied force across the x, y, and z-axes.Standardize the training in an applied force of five newtons and a frequency of 60 times per minute. Maintain the standardized rotary kneading and pressing continuously for an operation time of 10 minutes.

Performing Tuina on Knee Osteoarthritis (KOA) Rabbit Model

To begin Tuina intervention on the model rabbit with osteoarthritis established in its left knee, place the animal in the rabbit fixation box, and sooth it by gently stroking it for about 10 seconds. Perform the rotary kneading method using the thumb on the rabbit's left patella and the left peri-knee having muscle stiffness and tendon knots. Maintaining a force of 5 Newtons, perform an up and down, round tip manipulation at a frequency of 60 times per minute for five minutes.Then proceed to perform Tuina on the acupoints listed on the screen. Using the thumb end, press onto each acupoint, and maintaining the same force and frequency as demonstrated previously, operate on each point for 30 seconds. To perform rotary correction on the rabbit knee joint, hold the femur with one hand while positioning the other hand behind the knee joint.Fix the lateral and medial tibial condyles using the thumb and ring fingers respectively. Secure the popliteal fossa with the index and middle fingers. Using the fingers as demonstrated, hold the skin in place, avoiding friction between the skin and fingers.Then keeping the direction of traction force parallel to the long axis of the tibia, apply traction and twisting force. Secure the femur again with one hand. Establish a grip on the medial and lateral edges of the patella using the thumb and little fingers of the other hand while securing the base of the patella with the index, middle, and ring fingers.Then, keeping the direction of the torsion force in line with the direction of the lower Xiyan, apply a torsion force. Estimating the left knee ROM in the different groups of rabbits revealed that the Tuina group showed significant improvement in knee joint mobility after receiving the Tuina intervention from week seven. However, the model group that did not receive Tuina intervention showed no significant increase in ROM.