Medicine
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Effects of Allogeneic Platelet-Rich Plasma (PRP) on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description
Chapters
Summary March 19th, 2018
This protocol describes the evaluation process of healing tendons in rats that have been injected with allogeneic platelet rich plasma (PRP) or saline solution after removing part of the Achilles tendon. The progress of tendon healing is evaluated at several time points using different types of analyses.
Transcript
The overall goal of this procedure is to observe the effects of a single injection of Platelet-Rich Plasma, or PRP, on the resistance and healing process of surgically-sectioned rat Achilles tendons at various time points. This method can help answer quick questions on musculoskeletal field about tendon lesions. The main advantage of this technique is that the surgical, biomechanical, and PRP progression procedures are well-standardized.
The implications of these techniques extend toward the use of PRP in humans, because these types of lesion and the tendon healing process are similar between rats and human. Though this method can provide insight into PRP preparation for musculoskeletal injuries, this injection technique can also be applied to other systems, such as heart or skin disease. Visual demonstration of the method is very important, because surgical and biomechanical steps require consistent reproducibility in order to obtain optimal results.
Begin by weighing and ear-tagging the rat. After confirming the appropriate level of sedation by toe pinch, apply water drops onto the animal's corneas and remove the hair from the left hind limb. Disinfect the exposed skin with a one-to-10 dilution of iso-Betadine solution and place the animal on a 20-degree Celsius heating pad under a dissecting microscope in the lateral decubitus position, with the left hind limb in the superior position.
Grasp the left hind limb paw with forceps and make a 20 to 25 millimeter lateral skin incision around the Achilles tendon. Using fine scissors, dissect the fascia to expose the Achilles tendon complex and remove the plantaris tendon. Cut the Achilles tendon transversely, five millimeters proximal to its calcaneal insertion, and remove a five-millimeter long portion.
Then use resorbable yarn and continue with sutures to close the fascia and the skin, and place the rat under a heat source with monitoring until full recovery. While the recipient animals are recovering, collect 20 milliliters of whole blood from one donor rat heart per experimental animal into individual tubes containing 3.2%buffered sodium citrate. To obtain the platelet-rich plasma, centrifuge the blood and use a plastic transfer pipette to carefully harvest the upper PRP-containing phase into a second plastic tube.
Determine the volume of collected PRP and measure the platelet count on a hematology analyzer. Centrifuge the PRP again and carefully transfer the supernatant into a new plastic tube. The remaining volume in the PRP tube should be around 2/3 of the volume required to achieve 2.5 times 10 to the sixth platelets per microliter.
Next, gently re-suspend the platelet pellet with pipetting. Measure the platelet count of this concentrated platelet-rich plasma, adding back the appropriate volume of autologous platelet-poor plasma to reach the target concentration as necessary. Activate the platelets with 50 microliters of calcium chloride per milliliter of PRP for about one hour at room temperature.
Then load the platelets into a one-milliliter syringe equipped with a 21-gauge needle and inject 50 microliters of the platelets directly into the suture site of each animal. At the appropriate experimental endpoint, shave the left hind limb of the anesthetized experimental animal, and position the rat under a dissecting microscope as just demonstrated. Holding the paw with fingers, make a 10-millimeter incision in the surgical site to expose the triceps suralis.
To remove the Achilles tendon, cut the calcaneal bone transversally, five to 10 millimeters distal to its attachment and dissect a piece of the triceps suralis large enough to fit into the cryo-jaw. Use forceps to place the sample immediately into the upper jaw of the cryo-jaw and close the instrument. Place the cryo-jaw vertically into a universal testing machine, and fix the calcaneal bone between the lower clamps.
To calculate the cross-sectional area, place cameras perpendicular to each other. Add liquid nitrogen into both of the upper jaw basins to freeze the muscle to an order of magnitude stiffer than the tendon, so that it does not deform during the tensile test. When the freezing zone reaches the metal clamp border, set the displacement rate of the machine at a constant speed of one millimeter per second until rupture, and begin the tensile test, using a computer to record the ultimate tensile strength in Newtons.
To account for differences in the cross-sectional areas of the healing tendons, normalize the ultimate tensile strength to a unit area that represents the mechanical stress experienced by the tissue. Then when the tendon ruptures, snap freeze the ruptured tendon samples in liquid nitrogen and store them at negative 80 degrees Celsius until RNA extraction and RTPCR analysis. In this representative experiment, a greater force was required to induce tendon rupture in the PRP-injected group than in the control saline-treated group at every time point tested, suggesting a more robust healing process or better mechanical properties within the PRP-treated animals.
Although the PRP group demonstrated a higher intra-group scatter than did the saline group, the cross-sectional area of the tendons was determined to be larger in the PRP-injected group at days 15 and 30 after lesion induction. Further, Masson's trichrome staining revealed a higher presence fibrillar collagen, a key component in tendon morphology and strength, in PRP-treated animals on days five and 15, with a similar intensity of staining observed in both groups by day 30 after injection. After watching this video, you should have a good understanding of how to induce a rat standard lesions, and to sample the tendon to evaluate their healing process after a PRP injection.
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