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

Summary

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.

Abstract

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° 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.

Introduction

Joint contractures are defined as a restriction in the passive range of motion (ROM) of a diarthrodial joint^1,2. The current therapies aiming to prevent and treat joint contracture have achieved some success^3,4. 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 trauma^10,11. 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° of flexion¹³. In this method, a resin is used to cover the surface to prevent skin scratches. Although external fixation immobilization is robust and reliable^14,15, 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.

Protocol

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.

1. Rigidly immobilize the knee joint with a plastic plate and two metal screws at approximately 135° flexion.
   NOTE: Perform the surgery at the proximal femur and the distal tibia without violating the joint component.
2. Prepare materials and instruments for internal fixation.
   1. Construct a medical grade polypropylene plastic plates by cutting a 5 mL syringe (Figure 2a) using a surgical scissor to fit the following dimensions: length, 25 mm; width, 10 mm; thickness, 1 mm (Figure 2b). Smooth the perimeter of the plate with a scalpel vertically. Rinse the plate with sterile saline to wash off the debris by three times.
      1. Sterilize with 75% ethanol for 4 h followed by irradiating with ultraviolet light for 3 h.
   2. Pre-drilling holes in the plastic plate: Prepare a hand-held low-speed electric drill with a speed of about 0-4000 rpm (Figure 2c). Drill two holes at both ends of the plate, diameters are 1 mm and 0.9 mm, respectively (Figure 2d). Match both ends of the plate with M 1.4 mm x 8 mm and M 1.2 mm x 6 mm steel screws, respectively (Figure 2e).
      1. Wipe with 75% ethanol and sterilize with UV light for 3 h before use.
3. Prepare surgical instruments: 1 straight Mosquito-Type hemostatic clamp, 1 smooth curved forceps, 2 eyelid retractors, 1 needle-holder, 1 tissue forceps, 1 suture scissor, 1 micro tissue scissor and 1 scalpel (Figure 2f). Sterilize the surgical instruments by autoclaving at 121.3 °C for 20 min and drying.
4. Experimental animals
   1. Use Specific Pathogen Free (SPF) grade skeletally mature male Sprague-Dawley (or Wistar) rats, weighing between 250 - 350 g in the experiment.
      NOTE: Choose either female or male rats for the experiment.
   2. Place the rats in cages and keep in a 12 h light/12 h dark cycle-controlled laboratory room. Provide adequate food and water.

2. Surgery

1. Adjust the temperature. Place a warming pad on a surgical platform in a thermostatic operating room.
2. Anesthesia and skin preparation
   1. Weigh the rat with an electronic scale and record.
   2. Restrain the rat and perform an intraperitoneal injection of sodium pentobarbital (30 mg/kg) to induced anesthesia. Assessing that the animal is sufficiently anesthetized using the toe pinch²². Administer the eyes with lubricant to protect the cornea from drying during surgery.
   3. Shave the lower body of the rat including the two hind limbs with an electric clipper and disinfect with a tincture of povidone iodine twice and 75% ethanol three times.
   4. Place the rat laterally, and cover with the surgical drape exposing one side hind leg and hip.
   5. Disinfect the surgical area again with povidone iodine.
3. Immobilize the knee joint with internal fixation using a mini-invasive technique.
   NOTE: Keep the incision properly moist with sterile saline during the operation. The surgery usually requires two surgeons.
   1. Mark the direction of skin incision. 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 (Figure 3a). Incise the epidermis skin along the drawing line approximate 1.5 cm (Figure 3b).
   2. Bluntly dissect the muscle gap between vastus lateralis and biceps femoris with a tissue forceps until the femoral shaft is exposed approximately 1 cm in length (Figure 3c). Use the retractor to facilitate continuous separation of the muscle gap.
   3. Incise the epidermis skin approximate 1 cm along the body surface projection of the muscle gap between the tibialis anterior and fibularis longus on the distal lower extremity (Figure 3d). Bluntly dissect the muscle gap until the tibia is exposed approximately 1 cm in length (Figure 3e).
   4. Separate the soft tissues by the retractor and the smooth forceps, keep perpendicular and drill one 1.0 mm diameter hole into the femoral shaft at a speed of 1,500 rpm using an electric drill (Figure 3f). The proper drilling position is approximate 8 mm below the lower edge of the greater trochanter. Quickly press the wound to stop bleeding.
      NOTE: Proper drilling diameter can avoid intraoperative fractures.
   5. Drill one 0.9 mm diameter hole into the tibia approximate 4 mm below the edge of the tibiofibular fusion (Figure 4a). Perform the drilling carefully to prevent the crushing of muscles or tendons.
   6. Use the straight Mosquito-Type hemostatic clamp to form a submuscular course from the tibia hole to femur hole. The submuscular tunnel passes below the gastrocnemius in the tibia end and above the gluteus medius, below the biceps femoris in the femur end.
   7. Use one M 1.4 mm x 8 mm steel screw to secure one end of the plastic plate (with the 1.0 mm diameter hole) in the proximal femur (Figure 4b). Use one M 1.2 mm x 6 mm steel screw to secure another end of the plastic plate (with the 0.9 mm diameter hole) in the distal tibia (Figure 4c). Ensure the knee joint without varus deformity.
4. Close the wound: Suture the myofascia, deep fasciae, and subcutaneous tissue using 4-0 absorbable sutures (Figure 4d). Close the skin with Polyamide sutures (Figure 4f).

3. Postoperative management

1. Apply postoperative analgesia through subcutaneous injection of Buprenorphine (0.03 mg/mL) at 0.05 mg/kg . Add 5 mg/mL Neomycin into drinking water for 5 days after the surgery.
2. Inject the analgesia mixture (Buprenorphine and Carprofen) respectively at 0.05mg/kg and 5 mg/kg subcutaneously twice a day for at least 72 hours post-operation.
3. Check whether the hind limb had over-edema in case of vascular injury. Made sure that the rats can walk normally in the case of nerve injury during surgery.

4. Postoperative examination

1. Observe the healing of the surgical incision and physically examine the knee joint to evaluate early signs of infection every other day postoperatively. Check the degree of swelling of the ankle and metacarpophalangeal joint in case of continuous edema.
   NOTE: Early postoperative infection can cause wound exudate, leg swelling, and delayed wound healing.
2. Perform X-ray imaging of the hindlimb to ensure that correctly placed the screws on the first postoperative day.
   NOTE: A Micro-CT scan analysis is another alternative option to display the proper location and the direction of the steel screws.
3. Measure the passive range of motion (ROM) to evaluate the development of contracture. Take a knee joint ROM measurement at different time cohorts postoperatively as described previously²⁰.
   1. In brief, euthanize the rats and skin the hindlimbs. Remove the immobilizer and measure the knee joint angle using a mechanical arthrometer at two torques (667 or 1,060 g/cm)²³.
   2. Calculate the ROM as a result of the total contracture, the myogenic contracture, and the arthrogenic contracture separately based on the investigation objectives²⁴.
      NOTE: Set different time cohorts (i.e., 1, 2, 4, 8, 16, and 32 weeks) according to the research objectives. The contralateral knee joint (non-operative or sham-operated) can serve as a control².
4. Histological analysis of the posterior knee joint capsules.
   1. Prepare the joint tissues. Dissect the knee joint tissue and fix it with 4% paraformaldehyde. Decalcify and embed it in paraffin as previously reported²⁵. Cut the sections (5 μm) at the medial midcondylar level in the sagittal plane.
      NOTE: Choose to perform different evaluating staining including HE, aldehyde-fuchsin-Masson Goldner (AFMG), Elastica–Masson, or Immunohistochemistry staining for histological study in the joint capsule based on your study objectives^15,26.
   2. Observe histomorphometric changes in the posterior knee joint capsules. Photograph the posterior region of the knee joint. Observe fibrous deposition and adhesion changes between the diaphysis-synovium junction and the meniscus⁶.
      NOTE: Pathological changes of joint capsule are considered to be a pathogenic factor for knee joint contracture. Measure the length, the thickness, and the capsular areas of the posterior capsule as previously described according to the research content²⁷.

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anerdian	Shanghai Likang Ltd.	310173	antibacterial
Buprenorphine	Shanghai Shyndec Pharmaceutical Ltd.	/	analgesia
Carprofen	MCE	HY-B1227	analgesia
Cross screwdriver	STANLEY	PH0*125mm	tighten the screws
Electric drill	WEGO	185	drill hole(with stainless steel drill 0.9mm;1.0mm)
Microsurgical instruments	RWD	/	Orthopaedic surgical instruments for animals
Neomycin	Sigma	N6386	antibacterial
Sodium pentobarbital	Sigma	P3761	anaesthetize
Stainless Steel screws	WEGO	m1.4*8; m1.2*6	screw(part of internal fixation)
Syringe	WEGO	3151474	use for plastic plate(part of internal fixation)
μ-CT	ALOKA	Latheta LCT-200	in vivo CT scan