a platelet-rich plasma injection into the sectioned achilles tendon of a rat

A Platelet-Rich Plasma Injection into the Sectioned Achilles Tendon of a Rat

Begin by weighing and ear-tagging the rat. After confirming the appropriate level of sedation by toe pinch, apply water drops onto the animal&#39;s corneas and remove the hair from the left hind limb. Disinfect the exposed skin with a 1-to-10 dilution of Iso-betadine solution, and place the animal on a 20 degrees Celsius heating pad, under a dissecting microscope.
In the lateral debits 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 transversally 5 millimeters proximal to its calcaneal insertion, and remove a 5-millimeter long portion. Then, use resorbable yarn in continuous 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 x 106 platelets per microliter. Next, gently resuspend 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 1 hour at room temperature. Then load the platelets into a 1-milliliter syringe equipped with a 21-gauge needle, and inject 50 microliters of the platelets directly into the suture site of each animal.

a-platelet-rich-plasma-injection-into-sectioned-achilles-tendon

Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Model Development and Disease Study

EoE Sub Articles

eoe sub articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Animal Preparation
<ol>
	<li>Use 132 2-month-old male Sprague-Dawley rats weighing 320-450 g (120 rats for experimentation and 12 rats for blood sampling, Figure 1). Fifteen rats for each group was enough for a power of 0.8 (80% chance of finding statistical significance if the specified effect exists).</li>
	<li>House all rats in classical cages under isolation conditions in a conventional facility equipped with a changing station. Instigate breeding in an IVC (individually ventilated cage) rack, with SPF (specific pathogen-free) rats being specifically purchased.</li>
	<li>Use the following housing conditions: temperature range from 19-24 &#176;C; relative humidity: 40-60%; ventilation: frequency of air renewal: 10-15 per h; light/dark cycle: 12 h-12 h.</li>
	<li>Sterilize all caging equipment and use irradiated bedding. Systemically implement cage enrichment by placing sterile cardboard trays and towels inside the cages.</li>
	<li>Allow access to the zone under restricted conditions: new lab coat, mask, gloves, footwear, and hair bonnet at the entrance.</li>
	<li>Carry out colony health monitoring on sentinel animals housed on dirty bedding annually, according to the Felasa recommendations for conventional facilities. Keep four to five animals per cage with the appropriate irradiated diet and provide acidified water ad libitum. Monitor daily.</li>
	<li>Transfer rats elected for surgical intervention in filtering top cages to the surgical rooms 2 h before surgery.</li>
</ol>
2. Surgical Procedure
<ol>
	<li>Prepare the surgical instruments: fine scissors, a clamp, two hemostatic clamps, and a needle holder. During all the procedures, wear gloves, a mask, a hood, and a lab coat.</li>
	<li>Randomly divide the rats into two groups (PRP and saline solution).</li>
	<li>Take the rat out of its cage and weigh it. Use numbered ear tags to identify the rats. To avoid desiccation of the eyes, place waterdrops on the cornea.</li>
	<li>Anesthetize the rat intraperitoneally with xylazine (10 mg/kg of body weight) and ketamine (80 mg/kg of body weight). Place the animals under cardio-respiratory monitoring to confirm anesthetization and check for eye reflexes.</li>
	<li>Inject 50 &#181;L of buprenorphine subcutaneously (0.01-0.05 mg/kg every 8-12 h) in the neck area as a painkiller.</li>
	<li>Shave the left hind limb with an electric shaver, disinfect it with 3 alternating scrubs of iso-betadine/alcohol (diluted 1:10) solution, and place the rat on a warm pad (20 &#176;C) under a dissecting microscope. Place the rat on lateral decubitus with the leg to be operated on in a superior position. Hold the paw with surgical forceps.</li>
	<li>Using the scissors, make a small lateral incision (20-25 mm) in the skin surrounding the left Achilles tendon, and dissect the fascia using fine scissors to expose the Achilles tendon complex (Figure 2a).</li>
	<li>Remove the plantaris tendon with scissors (Figure 2b).</li>
	<li>Cut the Achilles tendon transversally 5 mm proximal to its calcaneal insertion and remove a 5-mm long portion using scissors. Do not suture the tendon (Figure 2c, 2d).</li>
	<li>Suture the fascia and the skin with resorbable yarn, doing an overjet suture (continuous suture). Monofilament sutures can also prevent wicking from the skin into the surgical incision.</li>
	<li>Place the rat under a heating lamp until awake, and then place the rat back in a cage (58 x 38 cm) (Nota bene: No immobilization is imposed).</li>
</ol>
3. PRP Preparation
<ol>
	<li>Anesthetize the donor rats with xylazine (10 mg/kg of body weight) and ketamine (80 mg/kg of body weight) by intraperitoneal injection. The PRP injection is carried out 2 h postoperatively without any re-anesthetic.</li>
	<li>Inject 50 &#181;L of buprenorphine subcutaneously as a painkiller.</li>
	<li>Collect whole blood (20 mL per rat, final bleeding) by cardiac puncture into sterile tubes containing 3.2% buffered sodium citrate (0.109 M, anticoagulant). Then, euthanize the rat by intracardiac injection.</li>
	<li>To obtain PRP, centrifuge the blood for 10 min at 150 x g and room temperature. This initial low-speed centrifugation step yields two distinct phases: a lower phase consisting of red blood cells (RBCs) that accounts for about 80-90% of the total volume and an upper phase composed of platelet-rich plasma (PRP) (typically 10-20% of the blood sample).</li>
	<li>Collect the PRP (upper phase) in a secondary plastic tube using a plastic transfer pipette. As the interface between PRP and red blood cells is very loose, do not pipette too close to the interface. When handled properly, contaminating RBCs in PRP should be below 0.05 x 103 cells/&#181;L. Discard the remaining lower phase and the primary blood collection tube.</li>
	<li>Determine the volume of collected PRP and measure the platelet count on a hematology analyzer. These values are required for calculations to adjust platelet concentration in the next step. A blood cell count is also helpful to evaluate potential contamination with RBCs and WBCs. Platelet concentration in PRP at this stage is typically 1 &#8211; 1.5 x106/&#181;L.</li>
	<li>Perform a second centrifugation of the PRP for 10 min at 1,000 x g and room temperature. This high-speed centrifugation step yields a loose platelet pellet and a supernatant of autologous platelet-poor plasma (PPP). Using the values determined in step 3.6, calculate the volume of supernatant to be discarded to concentrate the PRP and reach a final concentration of 2.5 x106/&#181;L.</li>
	<li>Gently collect the supernatant (PPP) in a secondary plastic tube using a plastic transfer pipette, leaving about two-thirds of the final volume calculated in step 3.7. As the pellet is loose, a significant quantity of platelets is lost when PPP is discarded, potentially reducing the desired concentration.</li>
	<li>Carefully resuspend the platelet pellet with the remaining supernatant by gentle repeated pipetting. Measure the platelet count on this concentrated PRP. If required, add the appropriate volume of autologous PPP to reach the final target concentration. 
	NOTE: Use the PRP within 3 h of preparation.</li>
	<li>Add 50 &#181;L of CaCl2 (11 mEq 10 mL) per mL of PRP to activate the platelets.</li>
	<li>Inject 50 &#181;L of fresh PRP or saline solution with a 21G needle directly into the sutured operation site about 1 h after preparation. During this time, keep PRP at room temperature.</li>
	<li>Monitor the functional recovery of the rats closely during the days after surgery and before the removal of the Achilles tendon.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Xylazine (Xyl-M)</td>
			<td>VMD</td>
			<td>none</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Ketamin (J&#233;tamine 1000 CEVA)</td>
			<td>CEVA Sant&#233; Animale</td>
			<td>none</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Buprenorphin (Veterg&#233;sic Multidosis)</td>
			<td>ALSTOE</td>
			<td>none</td>
			<td>Painkiller</td>
		</tr>
		<tr>
			<td>iso-Betadine</td>
			<td>MEDA-Pharma</td>
			<td>none</td>
			<td>Desinfectant</td>
		</tr>
		<tr>
			<td>Resorbable yarn Vicryl 6/0</td>
			<td>Johnson &#38; Johnson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nembutal</td>
			<td>CEVA Sant&#233; Animale</td>
			<td>none</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>Preserves structure of the tissue</td>
		</tr>
		<tr>
			<td>Isopropanol 100%</td>
			<td>VWR</td>
			<td>2,09,22,364</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 95%</td>
			<td>VWR</td>
			<td>2,08,23,362</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>VWR</td>
			<td>28973.363</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>VWR</td>
			<td>LEIC3950.1006</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Millipore</td>
			<td>1.15938.0025</td>
			<td>Colorant</td>
		</tr>
		<tr>
			<td>Eosin</td>
			<td>Millipore</td>
			<td>1.15935.0100</td>
			<td>Colorant</td>
		</tr>
		<tr>
			<td>Eukitt</td>
			<td>Sigma-Aldrich</td>
			<td>3989</td>
			<td>Mounting Medium</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Greimers, L., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/55759">Effects of Allogeneic Platelet-Rich Plasma (PRP) on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description.</a> J. Vis. Exp. (2018).
This video demonstrates a technique for injecting platelet-rich plasma (PRP) into the sectioned Achilles tendon of rats. A small segment is excised from the Achilles tendon of a recipient rat, the site is sutured, and the rat is allowed to recover. Concurrently, whole blood is collected from a donor rat and centrifuged to obtain PRP, which is then activated and injected into the sutured site of the recipient rat to induce tendon repair.

<img alt="Figure 1" src="/files/ftp_upload/22265/22265_Figure1.jpg" /> 
Figure 1: Experimental design of the study.
<img alt="Figure 2" src="/files/ftp_upload/22265/22265_Figure2.jpg" /> 
Figure 2: Surgical Procedure.
(a) The tendon complex after removal of the surrounding fascia.
(b) Removal of the planta...

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Animal ...

Place an anesthetized recipient rat with a shaved hind limb on a warm pad under a dissecting microscope.
Incise the skin and dissect the fascia, exposing the Achilles tendon complex.
Remove the plantaris tendon and excise a small segment from the Achilles tendon, creating a lesion.
Suture the incision and allow the rat to recover.
Collect whole blood from a genetically different donor rat, mixed with an anticoagulant.
Centrifuge to obtain a lower phase of red blood cells and an upper phase of platelet-rich plasma, or PRP.
Transfer the PRP to a tube and spin down the platelets. Partially remove the supernatant and resuspend the platelets to obtain concentrated PRP.
Add calcium chloride and incubate to induce platelet activation. Then, inject the activated platelets into the sutured site of the recipient.
Activated platelets release specific cytokines and growth factors that induce tendon repair at the lesion site.

19 ビュー. ラットの切片アキレス腱への多血小板血漿注入

ビデオ: ラットの切片アキレス腱への多血小板血漿注入 - 実験

Production of Autologous Platelet-Rich Plasma for Boosting In Vitro Human Fibroblast Expansion

There is currently great clinical interest in the use of autologous fibroblasts for skin repair. In most cases, culture of skin cells in vitro is required. However, cell culture using xenogenic or allogenic culture media has some disadvantages (i.e., risk of infectious agent transmission or slow cell expansion). Here, an autologous culture system is developed for the expansion of human skin fibroblast cells in vitro using a patient&#8217;s own platelet-rich plasma (PRP). Human dermal fibroblasts are isolated from the patient while undergoing abdominoplasty. Cultures are followed for up to 7 days using a medium supplemented with either fetal bovine serum (FBS) or PRP. Blood cell content in PRP preparations, proliferation, and fibroblast differentiation are assessed. This protocol describes the method for obtaining a standardized, non-activated preparation of PRP using a dedicated medical device. The preparation requires only a medical device (CuteCell-PRP) and centrifuge. This device is suitable under sufficient medical practice conditions and is a one-step, apyrogenic, and sterile closed system that requires a single, soft spin centrifugation of 1,500 x g for 5 min. After centrifugation, the blood components are separated, and the platelet-rich plasma is easily collected. This device allows a quick, consistent, and standardized preparation of PRP that can be used as a cell culture supplement for in vitro expansion of human cells. The PRP obtained here contains a 1.5-fold platelet concentration compared to whole blood together, with a preferential removal of red and white blood cells. It is shown that PRP presents a boosting effect in cell proliferation compared to FBS (7.7x) and that fibroblasts are activated upon PRP treatment.

This protocol presents a device that produces PRP to boost the in vitro expansion of cells in a 100% autologous fibroblast culture system.

in vitroヒト線維芽細胞増殖を促進するための自己多血小板血漿の産生

There is currently great clinical interest in the use of autologous fibroblasts for skin repair. In most cases, culture of skin cells in vitro is ...

JoVE Journal

生物工学

Preparation, Procedures and Evaluation of Platelet-Rich Plasma Injection in the Treatment of Knee Osteoarthritis

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department. Existing non-surgical treatments have a limited effect on the repair of cartilage and on bone regeneration. Platelet-rich plasma (PRP) is an autologous bioactive substance that can repair cartilage injury and accelerate bone regeneration effectively. However, reporting of PRP preparation protocols in clinical studies is highly inconsistent, with the majority of studies providing insufficient information to allow the protocol to be reproduced. We describe a repeatable method of preparing PRP visually, the treatment of KOA using PRP intra-articular injection, and methods of evaluating the outcome. PRP was prepared using manual double centrifugation. The PRP layer was extracted from peripheral blood and used for knee joint cavity injection. Evaluations included assessments of blood platelet concentrations and clinical outcomes. Preparation of PRP by manual centrifugation requires less apparatus and is less costly than plasma filtration or centrifugation using equipment. The centrifugation time of our double centrifugation method was 6 and 5 minutes for the respective centrifugations at forces of 800 and 1400 x g, respectively, to allow for the consistent preparation of standardized PRP. However, a manual method is susceptible to operator error, and PRP batch preparation is not available. Intra-articular injection of PRP proved to be an effective treatment for knee osteoarthritis. The entire treatment procedure took less than 30 minutes, the blood platelet concentration of PRP could be standardized, and treatment was proven to be effective when evaluated by follow-up.

Knee osteoarthritis is frequently seen in the orthopedic department. We introduce in detail the entire knee osteoarthritis treatment process with platelet-rich plasma injection, including preparation, procedures, and evaluation.

準備・手順・膝変形性関節症の治療における多血小板血漿注入の評価

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department. Existing non-surgical treatments have a limited ...

医学

Platelet-Rich Plasma Lysate for Treatment of Eye Surface Diseases

Various ocular surface diseases are treated with blood-derived eye drops. Their use has been introduced in clinical practice because of their metabolite and growth factor content, which promotes eye surface regeneration. Blood-based eye drops can be prepared from different sources (i.e., whole blood or platelet apheresis donation), as well as with different protocols (e.g., different dilutions and freeze/thaw cycles). This variability hampers the standardization of clinical protocols and, consequently, the evaluation of their clinical efficacy. Detailing and sharing the methodological procedures may contribute to defining common guidelines. Over the last years, allogenic products have been diffusing as an alternative to the autologous treatments since they guarantee higher efficacy standards; among them, the platelet-rich plasma lysate (PRP-L) eye drops are prepared with simple manufacturing procedures. In the transfusion medicine unit at AUSL-IRCCS di Reggio Emilia, Italy, PRP-L is obtained from platelet-apheresis donation. This product is initially diluted to 0.3 x 109 platelets/mL (starting from an average concentration of 1 x 109 platelets/mL) in 0.9% NaCl. Diluted platelets are frozen/thawed and, subsequently, centrifuged to eliminate debris. The final volume is split into 1.45 mL aliquots and stored at &#8722;80 &#176;C. Before being dispensed to patients, eye drops are tested for sterility. Patients may store platelet lysates at &#8722;15 &#176;C for up to 1 month. The growth factor composition is also assessed from randomly selected aliquots, and the mean values are reported here.

Platelet lysates represent an emerging tool for the treatment of ocular surface diseases. Here, we propose a method for the preparation, dispensation, storage, and characterization of platelet lysate collected from platelet donors.

眼表面疾患の治療のための多血小板血漿ライセート

Various ocular surface diseases are treated with blood-derived eye drops. Their use has been introduced in clinical practice because of their metabolite ...

A Platelet-Rich Plasma Injection into the Sectioned Achilles Tendon of a Rat

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A Platelet-Rich Plasma Injection into the Sectioned Achilles Tendon of a Rat