Name: a mini-invasive internal fixation technique for studying immobilization-induced knee flexion contracture in rats
Uploaded: 2019-05-20T13:00:00
Description: Watch this Scientific Journal Video about A Mini-Invasive Internal Fixation Technique for Studying Immobilization-Induced Knee Flexion Contracture in Rats at JoVE.com

This work was supported by grants from National Natural Science Foundation of China (No. 81772368), Natural Science Foundation of Guangdong Province (No. 2017A030313496), and Guangdong Provincial Science and Technology Plan Project (No. 2016A020215225; No. 2017B090912007). The authors thank Dr. Fei Zhang, M.D. from the Department of Orthopaedic Surgery, The Eighth Affiliated Hospital of Sun Yat-sen University for his technical assistance during modification.

All procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by The Third Affiliated Hospital of Sun Yat-sen University institutional animal care and use committee (permission number: 02-165-01). All the animal experiments were performed according to the ARRIVE guidelines.
1. Preoperative preparation
NOTE: Figure 1 shows the design of the surgical procedure.
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This study aimed to elucidate a step-by-step knee joint immobilization method using a mini-invasive technique that permits rapid postoperative rehabilitation in animals after surgery. Conventionally, the muscle-gap separation approach is thought to be a minimally invasive technique in orthopedic surgery. As expected, we found that rats can return to a normal diet and activities just one day postoperatively, which was consistent with the previous study. Moreover, no artery or nerve injury occurred after the surgery, evide...

Joint contractures are defined as a restriction in the passive range of motion (ROM) of a diarthrodial joint1,2. The current therapies aiming to prevent and treat joint contracture have achieved some success3,4. However, the underlying molecular mechanism of acquired joint contracture remains largely unknown5. The etiology of joint contractures in different social communities is highly diverse and includes genetic factors, posttraumatic states, chronic diseases, and prolonged immobility6. It is widely accepted that immobility is a critical issue in the development of acquired joint contracture7. People who suffer from major joint contracture may ultimately result in physical disability8. Thus, a stable and reproducible animal model is necessary for investigating the potential pathophysiological mechanisms of acquired joint contracture.
The currently built immobilization-induced knee joint contracture models are mostly achieved by utilizing non-invasive plaster casts, external fixations, and internal fixations. Watanabe et al. reported the possibility of the use of plaster cast immobilization on rat knee joints9. By wearing a special jacket, one side of the lower limb joint of the rat is immobilized by a cast. The rat knee joint can remain fully flexed without any surgical trauma10,11. However, both the hip and ankle joint movements are also affected by this form of immobilization, which may increase the degree of muscle atrophy in quadriceps femoris or gastrocnemius12. In addition, edema and congestion of the hind limbs must be avoided by replacing the cast at set time points, which may affect the continuity of immobility. Another accepted method for the establishment of a knee joint contracture model is using external surgical fixation. Nagai et al. combined Kirschner wire and steel wire into an external fixator, which immobilized the knee joint to approximately 140&#176; of flexion13. In this method, a resin is used to cover the surface to prevent skin scratches. Although external fixation immobilization is robust and reliable14,15, percutaneous Kirschner wire pin tracks may increase the risk of infection16. In our own experience, using the external fixation technique may reduce the daily activity of rats due to an increase in the conditioned lick behavior.
Alternatively, Trudel et al. described a well-accepted model of joint contracture in the rat knee joint based on a surgical internal fixation17 (this method was modified from the one used by Evans and colleagues18). Notably, this method highlights the importance of utilizing a mini-incision technique to minimize the surgical wounds. The efficient development of joint contracture has been proved in this model19. However, the protocol on how to perform a minimal dissection to expose the bone surface is still unclear20. Also, the precise position where the screw is drilling is not fully understood. The implantation of the internal fixation through a subcutaneous or submuscular way is still controversial21. To solve these problems, we have modified this method by including an appropriate muscle-gap separation modus, which allows a mini-invasive exposure of the bone surface and the placement of the implantation through a submuscular channel. This protocol led to rapid postoperative rehabilitation in rats after surgery. Animals developed a limited joint range of motion after joint immobilization, which was consistent with morphological changes of capsular adhesion obtained from the histological analysis. We also describe an exact possible location of the drilled screws as confirmed by X-ray analysis or micro-CT analysis. Thus, this study aimed to describe in detail a minimal-invasive technique in a knee joint contracture model that was established by a muscle-gap separation modus combined with a mini-incision method. We believe that minimally invasive techniques can both reduce animal trauma and effectively mimic the pathological process of joint flexion contracture.

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			<td height="62" style="height:63px;width:223px;">Anerdian</td>
			<td style="width:73px;">Shanghai Likang Ltd.</td>
			<td style="width:123px;">310173</td>
			<td style="width:172px;">antibacterial</td>
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			<td height="104" style="height:104px;width:223px;">Buprenorphine&#160;</td>
			<td style="width:73px;">Shanghai Shyndec Pharmaceutical Ltd.</td>
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			<td height="21" style="height:21px;width:223px;">Carprofen</td>
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			<td height="21" style="height:21px;width:223px;">Cross screwdriver</td>
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			<td height="62" style="height:63px;width:223px;">Electric drill</td>
			<td style="width:73px;">WEGO</td>
			<td style="width:123px;">185</td>
			<td style="width:172px;">drill hole(with stainless steel drill 0.9mm;1.0mm)</td>
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			<td height="42" style="height:42px;width:223px;">Microsurgical instruments</td>
			<td style="width:73px;">RWD</td>
			<td style="width:123px;">/</td>
			<td style="width:172px;">Orthopaedic surgical instruments for animals</td>
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			<td height="21" style="height:21px;width:223px;">Neomycin</td>
			<td style="width:73px;">Sigma</td>
			<td style="width:123px;">N6386</td>
			<td style="width:172px;">antibacterial</td>
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			<td height="21" style="height:21px;width:223px;">Sodium pentobarbital</td>
			<td style="width:73px;">Sigma</td>
			<td style="width:123px;">P3761</td>
			<td style="width:172px;">&#160;anaesthetize</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Stainless Steel screws</td>
			<td style="width:73px;">WEGO</td>
			<td style="width:123px;">m1.4*8; m1.2*6</td>
			<td style="width:172px;">screw(part of internal fixation)&#160;</td>
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			<td height="62" style="height:63px;width:223px;">Syringe&#160;</td>
			<td style="width:73px;">WEGO</td>
			<td style="width:123px;">3151474</td>
			<td style="width:172px;">use for plastic plate(part of internal fixation)&#160;</td>
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			<td height="21" style="height:21px;width:223px;">&#956;-CT&#160;</td>
			<td style="width:73px;">ALOKA</td>
			<td style="width:123px;">Latheta LCT-200</td>
			<td style="width:172px;">in vivo CT scan</td>
		</tr>
	</tbody>
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a mini-invasive internal fixation technique for studying immobilization-induced knee flexion contracture in rats

Joint contracture, resulting from a prolonged joint immobilization, is a common complication in orthopedics. Currently, utilizing an internal fixation to restrict knee joint mobility is a widely accepted model to generate experimental contracture. However, implanting application will inevitably cause surgical trauma to the animals. Aiming to develop a less invasive approach, we combined a muscle-gap separation modus with a previously reported mini-incision skill during the surgical procedure: Two mini skin incisions were made on the lateral thigh and leg, followed by performing muscle-gap separation to expose the bone surface. The rat knee joint was gradually immobilized by a preconstructed internal fixation at approximately 135&#176; knee flexion without interfering essential nerves or blood vessels. As expected, this simple technique permits rapid postoperative rehabilitation in animals. The correct position of the internal fixation was confirmed by an x-ray or micro-CT scanning analysis. The range of motion was significantly restricted in the immobilized knee joint than that observed in the contralateral knee joint demonstrating the effectiveness of this model. Besides, histological analysis revealed the development of fibrous deposition and adhesion in the posterior-superior knee joint capsule over time. Thus, this mini-invasive model may be suitable for mimicking the development of immobilized knee joint contracture.

We observed that rats received minimally invasive surgery can return to the regular diet just one day postoperatively. In particular, the surgical incision has scarred without exudate (Figure 5a). The swelling of the ankle and metacarpophalangeal joints in the operative hindlimb has almost wholly disappeared two days postoperatively (Figure 5b) when compared with the contralateral side (Figure 5c). N...

Watch this Scientific Journal Video about A Mini-Invasive Internal Fixation Technique for Studying Immobilization-Induced Knee Flexion Contracture in Rats at JoVE.com

A Mini-Invasive Internal Fixation Technique for Studying Immobilization-Induced Knee Flexion Contracture in Rats

Here, we present a protocol to describe a minimally invasive technique for knee joint immobilization in a rat model. This reproducible protocol, basing on muscle-gap separation modus and the mini-incision skill, is suitable for studying the underlying molecular mechanism of acquired joint contracture.

Joint contracture, resulting from a prolonged joint immobilization, is a common complication in orthopedics. Currently, utilizing an internal fixation to ...

This simple and effective muscle-gap separation model technique combine the ways of mini-invasion method is the best internal fixation in our rat knee joint contraction model. The main advantage of this technique is that it's process are reproducible and the minimally invasive alternative to inducing contracture of the knee joint in animals. Demonstrating the procedure will be Shihai Jiang and Fei Zhang, graduate student from my laboratory.After confirming a lack of response to toe-pinch, shave the lower body of the anesthetized rat, including the two hind limbs. Disinfect the exposed skin with tincture of povidone iodine two times and 75%ethanol three times. Apply ointment to the animal's eyes and place the rat laterally under a surgical drape with one hind limb and hip exposed.Disinfect the surgical area with additional povidone iodine. At the distal end of the femur greater trochanter, draw a line along the body surface projection of the muscle gap between the vastus lateralis and biceps femoris to mark the direction of the skin incision. Next, make a 1.5 centimeter incision in the epidermis along the line.Use tissue forceps to bluntly dissect the muscle gap between the vastus lateralis and biceps femoris until approximately one centimeter of the femoral shaft is exposed. Use a retractor to facilitate a continuous separation of the muscle gap. Make a one centimeter incision in the epidermis along the body surface projection of the muscle gap between the tibialis anterior and fibularis longus on the distal lower extremity.Bluntly dissect the muscle gap until approximately one centimeter of the tibia is exposed. Use the retractor and smooth forceps to separate the soft tissues. Holding a drill perpendicular to the bone, drill a one millimeter diameter hole into the femoral shaft at 1, 500 rotations per minute approximately eight millimeters below the lower edge of the greater trochanter.Quickly press the wound to stop the bleeding and drill a 0.9 millimeter diameter hole into the tibia approximately four millimeters below the edge of the tibiofibular fusion. Use a straight mosquito-type hemostatic clamp to form a submuscular course from the tibia hole to the femur hole, passing below the gastrocnemius in the tibia end and above the gluteus medius below the biceps femoris in the femur end. Then use one m1.4 by eight millimeter steel screw to secure one end of a plastic plate in the proximal femur.Use one m1.2 by six millimeter steel screw to secure another end of the plastic plate in the distal tibia. When the plate has been secured, use 4-0 absorbable sutures to close the myofascia, deep fascia, and subcutaneous tissue. Close the skin with silk sutures.X-ray imaging reveals a correct placement of the steel screws in the femur or the tibia. Three-dimensional reconstruction analysis after high-resolution microcomputed tomography scanning demonstrates the lateral placement of the screws approximately eight millimeters below the lower edge of the greater trochanter at the proximal femur and about four millimeters below the edge of the tibiofibular fusion at the distal tibia. The arthrogenic deficits in the the range of motion increase during immobilization in a time-dependent manner.As demonstrated by the higher average arthrogenic deficits measured for the immobilized knee joint compared to the control knee joint at 56 days of immobilization. On day one of the immobilization, no adhesion is observed by elastic staining of the posterior-superior knee joint capsule in the joint space between the posterior-superior joint capsule in the femur in the immobilized or the contralateral side knee joint. By 28 days of immobilization however, fibroadipose tissue deposits and adhesion develop in the joint space of the immobilized knee.After 56 days of immobilization, the fibrous tissues are partially replaced by the deposition while this type of adhesion is not observed in the contralateral knee joint. This method is modified from that used by Professor Trudel and colleagues from the University of Ottawa. It is key to identify and separate the muscle gaps during the surgery.Following different immobilization techniques other experimental process such as the culture of the knee joint capsule can be performed to study the underlying molecular mechanisms.

Research

JoVE Journal

Developmental Biology

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