Name: effects of allogeneic platelet-rich plasma (prp) on the healing process of sectioned achilles tendons of rats: a methodological description
Uploaded: 2018-03-19T12:30:00
Description: Watch this Scientific Journal Video about Effects of Allogeneic Platelet-Rich Plasma PRP on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description at JoVE.com

This study was supported by Standard de Li&#232;ge and Lejeune- Lechien grants of the Leon Fr&#233;d&#233;ricq Funds.

Care and handling of the animals were performed in accordance with the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (USA). European and national legislation were followed carefully.
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). Based on Dell<s...

<ol>
	<li>Kaux, J., Degrave, N., Crielaard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+rich+plasma+traitement+des+tendinopathies+chroniques?+Revue+de+la+litt&#233;rature.+Platelet+rich+plasma+treatment+of+chronic+tendinopathies?+Review+of+literature.">Platelet rich plasma traitement des tendinopathies chroniques? Revue de la litt&#233;rature. Platelet rich plasma treatment of chronic tendinopathies? Review of literature.</a> J. Traumatol. du Sport. 24, 99-102 (2007).</li><li>Anitua, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+preparations+rich+in+growth+factors+promote+proliferation+and+induce+VEGF+and+HGF+production+by+human+tendon+cells+in+culture.">Autologous preparations rich in growth factors promote proliferation and induce VEGF and HGF production by human tendon cells in culture.</a> J. Orthop. Res. 23, 281-286 (2005).</li><li>Bosch, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+platelet-rich+plasma+on+the+quality+of+repair+of+mechanically+induced+core+lesions+in+equine+superficial+digital+flexor+tendons:+A+placebo-controlled+experimental+study.">Effects of platelet-rich plasma on the quality of repair of mechanically induced core lesions in equine superficial digital flexor tendons: A placebo-controlled experimental study.</a> J. Orthop. Res. 28, 211-217 (2010).</li><li>Kaux, J. F., Drion, P., Croisier, J. L., Crielaard, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tendinopathies+and+platelet-rich+plasma+(PRP):+From+pre-clinical+experiments+to+therapeutic+use.">Tendinopathies and platelet-rich plasma (PRP): From pre-clinical experiments to therapeutic use.</a> J. Stem Cells Regen. Med. 11, P7-P17 (2015).</li><li>Maffulli, N., Wong, J., Almekinders, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Types+and+epidemiology+of+tendinopathy.">Types and epidemiology of tendinopathy.</a> Clin. Sports Med. 22, 675-692 (2003).</li><li>Molloy, T., Wang, Y., Murrell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+growth+factors+in+tendon+and+ligament+healing.">The roles of growth factors in tendon and ligament healing.</a> Sports Med. 33, 381-394 (2003).</li><li>Lyras, D. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+platelet-rich+plasma+gel+in+the+early+phase+of+patellar+tendon+healing.">The effect of platelet-rich plasma gel in the early phase of patellar tendon healing.</a> Arch. Orthop. Trauma Surg. 129, 1577-1582 (2009).</li><li>Aspenberg, P., Virchenko, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+concentrate+injection+improves+Achilles+tendon+repair+in+rats.">Platelet concentrate injection improves Achilles tendon repair in rats.</a> Acta Orthop. Scand. 75, 93-99 (2004).</li><li>Visser, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+Factor-Rich+Plasma+Increases+Tendon+Cell+Proliferation+and+Matrix+Synthesis+on+a+Synthetic+Scaffold:+An+In+Vitro+Study.">Growth Factor-Rich Plasma Increases Tendon Cell Proliferation and Matrix Synthesis on a Synthetic Scaffold: An In Vitro Study.</a> Tissue Eng. Part A. 16, 1021-1029 (2010).</li><li>Zhang, J., Wang, J. H. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-Rich+Plasma+Releasate+Promotes+Differentiation+of+Tendon+Stem+Cells+Into+Active+Tenocytes.">Platelet-Rich Plasma Releasate Promotes Differentiation of Tendon Stem Cells Into Active Tenocytes.</a> Am. J. Sports Med. 38, 2477-2486 (2010).</li><li>Kajikawa, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-rich+plasma+enhances+the+initial+mobilization+of+circulation-derived+cells+for+tendon+healing.">Platelet-rich plasma enhances the initial mobilization of circulation-derived cells for tendon healing.</a> J. Cell. Physiol. 215, 837-845 (2008).</li><li>Pasternak, B., Aspenberg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metalloproteinases+and+their+inhibitors-diagnostic+and+therapeutic+opportunities+in+orthopedics.">Metalloproteinases and their inhibitors-diagnostic and therapeutic opportunities in orthopedics.</a> Acta Orthop. 80, 693-703 (2009).</li><li>Dell, R. B., Holleran, S., Ramakrishnan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+size+determination.">Sample size determination.</a> ILAR J. 43, 207-213 (2002).</li><li>M&#228;hler Convenor, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FELASA+recommendations+for+the+health+monitoring+of+mouse,+rat,+hamster,+guinea+pig+and+rabbit+colonies+in+breeding+and+experimental+units.">FELASA recommendations for the health monitoring of mouse, rat, hamster, guinea pig and rabbit colonies in breeding and experimental units.</a> Lab. Anim. 48, 178-192 (2014).</li><li>Kaux, J. -. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#201;tude+comparative+de+cinq+techniques+de+pr&#233;paration+plaquettaire+(platelet-rich+plasma).">&#201;tude comparative de cinq techniques de pr&#233;paration plaquettaire (platelet-rich plasma).</a> Pathol. Biol. 59, 157-160 (2011).</li><li>Wieloch, P., Buchmann, G., Roth, W., Rickert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cryo-jaw+designed+for+in+vitro+tensile+testing+of+the+healing+Achilles+tendons+in+rats.">A cryo-jaw designed for in vitro tensile testing of the healing Achilles tendons in rats.</a> J. Biomech. 37, 1719-1722 (2004).</li><li>Kaux, J. -. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+Endothelial+Growth+Factor-111+(VEGF-111)+and+tendon+healing:+preliminary+results+in+a+rat+model+of+tendon+injury.">Vascular Endothelial Growth Factor-111 (VEGF-111) and tendon healing: preliminary results in a rat model of tendon injury.</a> Muscles. Ligaments Tendons J. 4, 24-28 (2014).</li><li>Docheva, D., Hunziker, E. B., F&#228;ssler, R., Brandau, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tenomodulin+is+necessary+for+tenocyte+proliferation+and+tendon+maturation.">Tenomodulin is necessary for tenocyte proliferation and tendon maturation.</a> Mol. Cell. Biol. 25, 699-705 (2005).</li><li>Lambert, C. A., Colige, A. C., Munaut, C., Lapi&#232;re, C. M., Nusgens, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+pathways+in+the+over-expression+of+matrix+metalloproteinases+in+human+fibroblasts+by+relaxation+of+mechanical+tension.">Distinct pathways in the over-expression of matrix metalloproteinases in human fibroblasts by relaxation of mechanical tension.</a> Matrix Biol. 20, 397-408 (2001).</li><li>Nurden, A. T., Nurden, P., Sanchez, M., Andia, I., Anitua, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelets+and+wound+healing.">Platelets and wound healing.</a> Front. Biosci. 13, 3532-3548 (2008).</li><li>Woodall, J., Tucci, M., Mishra, A., Benghuzzi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+effects+of+platelet+rich+plasma:+a+study+on+HL-60+macrophage-like+cells.">Cellular effects of platelet rich plasma: a study on HL-60 macrophage-like cells.</a> Biomed. Sci. Instrum. 43, 266-271 (2007).</li><li>Taylor, D. W., Petrera, M., Hendry, M., Theodoropoulos, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+review+of+the+use+of+platelet-rich+plasma+in+sports+medicine+as+a+new+treatment+for+tendon+and+ligament+injuries.">A systematic review of the use of platelet-rich plasma in sports medicine as a new treatment for tendon and ligament injuries.</a> Clin. J. Sport Med. 21, 344-352 (2011).</li><li>Mazzocca, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+positive+effects+of+different+platelet-rich+plasma+methods+on+human+muscle,+bone,+and+tendon+cells.">The positive effects of different platelet-rich plasma methods on human muscle, bone, and tendon cells.</a> Am. J. Sports Med. 40, 1742-1749 (2012).</li><li>McCarrel, T. M., Minas, T., Fortier, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+leukocyte+concentration+in+platelet-rich+plasma+for+the+treatment+of+tendinopathy.">Optimization of leukocyte concentration in platelet-rich plasma for the treatment of tendinopathy.</a> J. Bone Joint Surg. Am. 94 (1-8), e143 (2012).</li><li>Boswell, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+platelet+concentrations+in+leukocyte-reduced+platelet-rich+plasma+decrease+collagen+gene+synthesis+in+tendons.">Increasing platelet concentrations in leukocyte-reduced platelet-rich plasma decrease collagen gene synthesis in tendons.</a> Am. J. Sports Med. 42, 42-49 (2014).</li><li>Virchenko, O., Aspenberg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+can+one+platelet+injection+after+tendon+injury+lead+to+a+stronger+tendon+after+4+weeks?:+Interplay+between+early+regeneration+and+mechanical+stimulation.">How can one platelet injection after tendon injury lead to a stronger tendon after 4 weeks?: Interplay between early regeneration and mechanical stimulation.</a> Acta Orthop. 77, 806-812 (2006).</li></ol>

Platelets are essential for the early inflammatory phase of the tendon healing process. When these platelets are exposed to binding tissue or factors that induce coagulation, they will release growth factors that are stocked in &#945; granules. Due to this interaction, extracellular matrix macromolecules are synthetized and mesenchymal cells proliferate. Platelets also have a chemotactic activity on progenitor cells in the blood circulation, enhancing angiogenesis and stimulating cellular differentiation<sup class="xref"...

Coagulation, inflammatory processes, and the immunity modulation roles of platelets are well known1. More recently, it has been demonstrated that they also have restorative properties2,3. Indeed, various cytokines and growth factors (VEGF, PDGF, TGF-B, IGF-I, and HGF) are released by platelets during degranulation. These growth factors promote angiogenesis, tissue remodeling, and wound healing (bone, skin, muscle, tendon)2. Centrifuging autologous blood produces platelet rich plasma (PRP) which contains high platelet concentrations depending on the isolation method (between 3 and 10 times blood baseline concentrations). Indeed, various PRP preparation techniques cannot provide an identical final product. Up to now, there has been no international general agreement on this issue. Overall, PRP could be an attractive therapeutic option for treating chronic musculoskeletal conditions, such as tendinopathy, plantar fasciitis, osteoarthritis, and nonunion4. It was used for the first time in oral surgery and implantology4 to improve and accelerate bone healing after placing a dental implant. In this study, we describe a reproducible method that allows the acquisition of PRP for animal experimentation4.
Since lesions of tendons are frequently observed in sportsmen and physical workers, enhancing the healing process and thus reducing the time for recovery are of great interest5. New treatment methods that are developing often involve the use of growth factors, and the administration of PRP is a simple and minimally invasive way to deliver a blend of endogenous growth factors4.
Several in vitro or animal studies have demonstrated that the administration of plasma containing a high level of platelets, by releasing biological mediators, can stimulate tendon and ligament repair by releasing biological mediators6,7,8,9. Furthermore, other studies have shown that PRP can stimulate type I and III collagen synthesis in tendon cells9,10,11. It has also been suggested that PRP can decrease the activation of matrix metalloproteinases (MMPs) and therefore reduce the degradation of the matrix. Cells that are involved in the inflammation process can produce MMP-9, which plays a role in tissue remodeling (physiologic and pathologic) induced by inflammation12.
Based on this information, we hypothesized that a single PRP injection into the sectioned Achilles tendons of rats could improve the recovery process and the mechanical strength of the repaired tissue. This is tested by measuring the biomechanical properties of the healing tendons during the recovery process and by performing histological and molecular analyses to evaluate collagen remodeling in the newly formed tissue. The aim of the study was to observe if a single injection of allogeneic PRP could influence the healing of sectioned Achilles tendons.

<table>
	<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>20,922,364</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 95%</td>
			<td>VWR</td>
			<td>20,823,362</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>
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</table>

effects of allogeneic platelet-rich plasma (prp) on the healing process of sectioned achilles tendons of rats: a methodological description

This article describes the experimental procedures used to observe if PRP can positively affect tendon healing. There are 4 main steps to follow: induce a lesion in the Achilles tendon; prepare PRP and inject it (or the saline solution); remove the tendon; and perform biomechanical, molecular, and histological evaluations. At each step, all the procedures and methods are described in detail, so they can be reproduced easily.
Achilles tendons have been surgically sectioned (removal of a 5-mm long section). Afterwards, PRP or saline solution was injected to study whether PRP has a positive effect on the healing of the tendon. Three groups of 40 animals (a total of 120 rats were used in this study) were subdivided into 2 subgroups: PRP injection group and a saline injection control group. Rats were sacrificed at increasing time points (Group A: 5 days; Group B: 15 days; Group C: 30 days) and tendons were removed. 90 tendons underwent biomechanical testing before performing transcriptomic analysis and the 30 remaining tendons were submitted to histological analysis.

The results are expressed as the mean &#177; standard deviation of the mean and were compared with analysis of variance (ANOVA). A two-way ANOVA and post hoc test de Scheff&#233;, which is a parametric test, were used.
The ultimate tensile strength (UTS) required to provoke a rupture of the non-injured Achilles tendons of rats was 42.0 &#177; 5.7 N (n = 10). The tensile strength increased significantly (p&#60;0.0001) in...

Watch this Scientific Journal Video about Effects of Allogeneic Platelet-Rich Plasma PRP on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description at JoVE.com

Effects of Allogeneic Platelet-Rich Plasma PRP on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description

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.

Effects of Allogeneic Platelet-Rich Plasma (PRP) on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description

This article describes the experimental procedures used to observe if PRP can positively affect tendon healing. There are 4 main steps to follow: induce a ...

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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Medicine

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform PrPSc 1. PrPC is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrPSc forms detergent-insoluble aggregates and is partially resistant to PK2-6. The conversion of PrPC to PrPSc is known to involve a conformational transition of &alpha;-helical to &beta;-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrPSc, intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain7.
Using a combination of biophysical and biochemical approaches, we identified insoluble PrPC aggregates (designated iPrPC) from uninfected mammalian brains and cultured neuronal cells8, 9. Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms10, and PK-treatment. The combination of these approaches isolates not only insoluble PrPSc and PrPC aggregates but also soluble PrPC oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrPSc from infected brains and iPrPC from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrPC are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrPSc that shares the immunoreactive behavior and fragmentation with iPrPC 11, 12. Moreover, we recently demonstrated that iPrPC is the main species that interacts with amyloid-&beta; protein in Alzheimer disease13. In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer's disease13, suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders.

A new species of cellular prion protein (PrPC) has recently been identified in uninfected human brains using the methods described here. These methods can be used to isolate various PrP species, while some of them are also useful in isolating other misfolded protein aggregates from human brains.

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform ...

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause severe problems, such as abdominal cramping, bloody diarrhea, and weight loss, in affected individuals. Unfortunately, there is no cure yet, and treatments only aim to alleviate symptoms. Current treatments include anti-inflammatory and immunosuppressive drugs that may cause severe side effects. This warrants the search for alternative treatment options, such as nutritional supplements, that do not cause side effects. Before their application in clinical studies, such compounds must be rigorously tested for effectiveness and security in animal models. A reliable experimental model is the dextran sulfate sodium (DSS) colitis model in mice, which reproduces many of the clinical signs of ulcerative colitis in humans. We recently applied this model to test the beneficial effects of a nutritional supplement containing vitamins C and E, L-arginine, and &#969;3-polyunsaturated fatty acids (PUFA). We analyzed various disease parameters and found that this supplement was able to ameliorate edema formation, tissue damage, leukocyte infiltration, oxidative stress, and the production of pro-inflammatory cytokines, leading to an overall improvement in the disease activity index. In this article, we explain in detail the correct application of nutritional supplements using the DSS colitis model in C57Bl/6 mice, as well as how disease parameters such as histology, oxidative stress, and inflammation are assessed. Analyzing the beneficial effects of different diet supplements may then eventually open new avenues for the development of alternative treatment strategies that alleviate IBD symptoms and/or that prolong the phases of remission without causing severe side effects.

Current treatments of inflammatory bowel diseases (IBD) aim at alleviating disease symptoms but may cause severe side effects. Thus, alternative strategies are being investigated in animal colitis models. Here, we explain how the beneficial effects of diet supplements on clinical IBD signs are analyzed in such a colitis model.

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause ...

The Rabbit Model of Accelerated Atherosclerosis: A Methodological Perspective of the Iliac Artery Balloon Injury

Acute coronary syndrome resulting from coronary occlusion following atherosclerotic plaque development and rupture is the leading cause of death in the industrialized world. New Zealand White (NZW) rabbits are widely used as an animal model for the study of atherosclerosis. They develop spontaneous lesions when fed with atherogenic diet; however, this requires long time of 4 - 8 months. To further enhance and accelerate atherogenesis, a combination of atherogenic diet and mechanical endothelial injury is often employed. The presented procedure for inducing atherosclerotic plaques in rabbits uses a balloon catheter to disrupt the endothelium in the left iliac artery of NZW rabbits fed with atherogenic diet. Such mechanical damage caused by the balloon catheter induces a chain of inflammatory reactions initiating neointimal lipid accumulation in a time dependent fashion. Atherosclerotic plaque following balloon injury show neointimal thickening with extensive lipid infiltration, high smooth muscle cell content and presence of macrophage derived foam cells. This technique is simple, reproducible and produces plaque of controlled length within the iliac artery. The whole procedure is completed within 20 - 30 min. The procedure is safe with low mortality and also offers high success in obtaining substantial intimal lesions. The procedure of balloon catheter induced arterial injury results in atherosclerosis within two weeks. This model can be used for investigating the disease pathology, diagnostic imaging and to evaluate new therapeutic strategies.

Animal models of atherosclerosis are essential to understand the mechanism and to investigate newer approaches to prevent plaque development or rupture, a leading cause of death in the industrialized world. This protocol uses a combination of balloon injury and cholesterol rich diet to induce atherosclerotic plaques in rabbit iliac artery.

Acute coronary syndrome resulting from coronary occlusion following atherosclerotic plaque development and rupture is the leading cause of death in the ...

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.

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

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a nonmammalian animal model that is widely used as an assay system due to its short lifespan, transparency, cost-effectiveness, and lack of animal ethics issues. In this study, a method was developed to investigate the effects of different bacteria and 3,3'-diindolylmethane (DIM) on the intestinal permeability of C. elegans with a high-throughput image analysis system. The worms were infected with different gut bacteria or cotreated with DIM for 48 h and fed with fluorescein isothiocyanate (FITC)-dextran overnight. Then, the intestinal permeability was examined by comparing the fluorescence images and the fluorescence intensity inside the worm bodies. This method may also have the potential to identify probiotic and pathogenic intestinal bacteria that affect intestinal permeability in the animal model and is effective for examining the effects of harmful or health-promoting chemicals on intestinal permeability and intestinal health. However, this protocol also has some considerable limitations at the genetic level, especially for determining which genes are altered to control illness, because this method is mostly used for phenotypic determination. In addition, this method is limited to determining exactly which pathogenic substrates cause inflammation or increase the permeability of the worms' intestines during infection. Therefore, further in-depth studies, including investigation of the molecular genetic mechanism using mutant bacteria and nematodes as well as chemical component analysis of bacteria, are required to fully evaluate the function of bacteria and chemicals in determining intestinal permeability.

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

This protocol describes how to measure intestinal permeability of Caenorhabditis elegans. This method is helpful for basic biological research on intestinal health related to the interaction between intestinal bacteria and their host and for screening to identify probiotic and chemical agents to cure leaky gut syndrome and inflammatory bowel diseases.

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a ...

Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface glomeruli provide a unique opportunity to investigate physiological and pathophysiological processes using intravital 2-photon microscopy. Quantification of glomerular capillary blood flow and vasoconstriction and dilatation in response to drugs, permeability, and inflammation are just some of the processes that can be studied. In addition, transgenic rats, i.e., podocytes labeled with fluorescent dyes and other molecular biomarker approaches, provide increased resolution to directly monitor and quantify protein-protein interactions and the effects of specific molecular alterations.
In mice, which lack surface glomeruli after four weeks of age, unilateral ureteral obstruction (UUO) for several weeks has been used to induce surface glomeruli. As this induction model does not allow for baseline studies, we quantified the effects of UUO on glomerular processes in the UUO model in Munich Wistar Fr&#246;mter (MWF) rats, which have surface glomeruli under physiologic conditions. The UUO model for five weeks or more induced significant alterations to gross renal morphology, the peritubular and glomerular microvasculature, as well as the structure and function of tubular epithelia. Glomerular and peritubular red blood cell (RBC) flow decreased significantly (p &#60; 0.01), probably due to the significant increase in the adherence of white blood cells (WBCs) within glomerular and peritubular capillaries. The glomerular sieving coefficient of albumin increased from 0.015 &#177; 0.002 in untreated MWFs to 0.045 &#177; 0.05 in 5-week-old UUO MWF rats. Twelve weeks of UUO resulted in further increases in surface glomerular density and glomerular sieving coefficient (GSC) for albumin. Fluorescent albumin filtered across the glomeruli was not reabsorbed by the proximal tubules. These data suggest that using UUO to induce surface glomeruli limits the ability to study and interpret normal glomerular processes and disease alterations.

Here, we present a protocol using 2-photon microscopy in Munich Wistar Fromter rats with surface glomeruli to quantifythe effects of prolonged ureteral obstruction on glomerular dynamics and function.

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface ...

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.

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

Ultrasound Evaluation of Achilles Tendon Morphology

Measurement of Healthy and Injured Triceps Surae Morphology

Achilles tendon injuries occur throughout the lifespan and can negatively affect quality of life and overall health. Achilles tendinopathy is generally classified as an overuse injury associated with fusiform tendon thickening, neovascularization, and interstitial tendon degeneration. Current literature suggests these structural changes are associated with&#160;symptoms and lower physical activity levels, as well as&#160;symptoms and lower extremity function in the long term. Surgically and non-surgically managed Achilles tendon ruptures result in increased tendon cross-sectional area (CSA) and a lengthened Achilles tendon. Both structural outcomes have clinical implications, as larger CSA positively predicts function, whereas increased tendon lengthening predicts reduced function after Achilles tendon rupture. Given the relationship between structural changes associated with Achilles tendon injuries for both injury severity and injury recovery, it is critical to be able to quantify Achilles tendon structure reliably and accurately. Silbernagel&#39;s group has established a valid and reliable method for efficiently evaluating triceps surae muscle and tendon structure. In this protocol, B-mode musculoskeletal ultrasound imaging is used to measure triceps surae structure, including Achilles tendon thickness and CSA, soleus thickness, and the presence of additional findings (calcifications and bursitis). B-mode extended field-of-view is used to measure Achilles tendon length and gastrocnemius anatomical CSA. Finally, power Doppler is used to identify intratendinous neovascularization. Quantification of triceps surae structure allows for comparison between limbs as well as longitudinal changes in response to exercise and treatment for healthy individuals and those with Achilles tendon injuries. This protocol has been used in many research studies to date and proves valuable in understanding the relationship between tendon structure and injury development, severity, and recovery. As ultrasound devices are becoming more affordable and portable, this protocol proves promising as a clinical tool, given its quick and efficient methods.

Author Spotlight: Exploring the Complexities of Achilles Tendon Injuries &#8212; Research and Future Directions

Ultrasound imaging is becoming more accessible in clinical and research settings, and a consistent protocol will be beneficial for comparison between studies and for clinical interpretations. This protocol for ultrasound evaluation is a valid and reliable method to evaluate Achilles tendon morphology in healthy, tendinopathic, and ruptured tendons.

Achilles tendon injuries occur throughout the lifespan and can negatively affect quality of life and overall health. Achilles tendinopathy is generally ...

Establishing and Validating a Chronic Unpredictable Mild Stress Rat Model

Chronic Unpredictable Mild Stress in Rats based on the Mongolian medicine

Depression is a prevalent affective disorder and constitutes a leading cause of global disability. The limitations of current pharmacological interventions contribute to the substantial health burden attributed to this condition. There is a pressing need for a deeper understanding of the underlying mechanisms of depression, making pre-clinical models with translational potential highly valuable. Mongolian medicine, a subset of traditional medicine, posits that disease occurrence is closely tied to the equilibrium of wind, bile, and Phlegm. In this study, we introduce a protocol for the chronic unpredictable mild stress (CUMS) method in rats. Within this framework, rats are subjected to a series of fluctuating, mild stressors to induce a depression-like phenotype, mimicking the pathogenesis of human depression. Behavioral assays employed in this protocol include the sucrose preference test (SPT), indicative of anhedonia-a core symptom of depression; the open field test (OFT), which measures anxiety levels; and the Morris water maze test (MWM), which evaluates spatial memory and learning abilities. The CUMS method demonstrates the capability to induce anhedonia and to cause long-term behavioral deficits. Furthermore, this protocol is more aligned with Mongolian medical theory than other animal models designed to elicit depression-like behavior. The development of this animal model and subsequent research provide a robust foundation for future innovative studies in the realm of Mongolian medicine.

Author Spotlight: Establishing a Rodent Model for Investigating Depression Factors in Traditional Mongolian Medicine

This protocol outlines a chronic unpredictable mild stress (CUMS) model for depression based on Mongolian medical theory, along with methods for validating behavioral tests.