This study was supported by an unrestricted AUVA research grant to JH and CGT.

The experiments were approved by the institutional animal care and use committee at Innsbruck Medical University and by the Austrian ministry of science (BMWF-66.011/0110-V/3b/2019).
1. Induction of anesthesia and operational set-up
<ol>
	<li>Prepare a suitable environment for animal procedures: sterilize equipment, disinfect surfaces, make use of disposable masks, isolation gowns and gloves.</li>
	<li>Sedate a 18-12-weeks old mouse (strain and sex depending on the experimental setting) in a...

<ol>
	<li>Schaden, W., 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=Extracorporeal+shockwave+therapy+(ESWT)+-+First+choice+treatment+of+fracture+non-unions.">Extracorporeal shockwave therapy (ESWT) - First choice treatment of fracture non-unions.</a> International Journal of Surgery. 24, 179-183 (2015).</li><li>Xu, Z. H., 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=Extracorporeal+shock+wave+treatment+in+nonunions+of+long+bone+fractures.">Extracorporeal shock wave treatment in nonunions of long bone fractures.</a> International Orthopaedics. 33, 789-793 (2009).</li><li>Melegati, G., Tornese, D., Bandi, M., Rubini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+two+ultrasonographic+localization+techniques+for+the+treatment+of+lateral+epicondylitis+with+extracorporeal+shock+wave+therapy:+A+randomized+study.">Comparison of two ultrasonographic localization techniques for the treatment of lateral epicondylitis with extracorporeal shock wave therapy: A randomized study.</a> Clinical Rehabilitation. 18, 366-370 (2004).</li><li>Zhang, S., Li, H., Yao, W., Hua, Y., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+response+of+extracorporeal+shock+wave+therapy+for+insertional+achilles+tendinopathy+between+sports-active+and+nonsports-active+patients+with+5-year+follow-up.">Therapeutic response of extracorporeal shock wave therapy for insertional achilles tendinopathy between sports-active and nonsports-active patients with 5-year follow-up.</a> Orthopedic Journal of Sport Medicine. 8, 1-6 (2020).</li><li>Dedes, V., 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=Effectiveness+and+safety+of+shockwave+therapy+in+tendinopathies.">Effectiveness and safety of shockwave therapy in tendinopathies.</a> Materia Socio Medica. 30, 141 (2018).</li><li>Surace, S. J., Deitch, J., Johnston, R. V., Shock Buchbinder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=wave+therapy+for+rotator+cuff+disease+with+or+without+calcification.">wave therapy for rotator cuff disease with or without calcification.</a> Cochrane Database of Systematic Reviews. 3 (3), 008962 (2020).</li><li>Mittermayr, R., 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=Extracorporeal+shock+wave+therapy+(ESWT)+for+wound+healing:+Technology,+mechanisms,+and+clinical+efficacy.">Extracorporeal shock wave therapy (ESWT) for wound healing: Technology, mechanisms, and clinical efficacy.</a> Wound Repair Regeneration. 20, 456-465 (2012).</li><li>Fode, M., Hatzichristodoulou, G., Serefoglu, E. C., Verze, P., Albersen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-intensity+shockwave+therapy+for+erectile+dysfunction:+Is+the+evidence+strong+enough.">Low-intensity shockwave therapy for erectile dysfunction: Is the evidence strong enough.</a> Nature Reviews Urology. 14, 593-606 (2017).</li><li>Guo, P., 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=Positive+effects+of+extracorporeal+shock+wave+therapy+on+spasticity+in+poststroke+patients:+a+meta-analysis.">Positive effects of extracorporeal shock wave therapy on spasticity in poststroke patients: a meta-analysis.</a> Journal of Stroke and Cerebrovascular Diseases. 26 (11), 2470-2476 (2017).</li><li>Vardi, Y., Appel, B., Jacob, G., Massarwi, O., Gruenwald, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+low-intensity+extracorporeal+shockwave+therapy+improve+erectile+function?+A+6-month+follow-up+pilot+study+in+patients+with+organic+erectile+dysfunction.">Can low-intensity extracorporeal shockwave therapy improve erectile function? A 6-month follow-up pilot study in patients with organic erectile dysfunction.</a> European Urology. 58, 243-248 (2010).</li><li>Gollmann-Tepek&#246;yl&#252;, 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=miR-19a-3p+containing+exosomes+improve+function+of+ischaemic+myocardium+upon+shock+wave+therapy.">miR-19a-3p containing exosomes improve function of ischaemic myocardium upon shock wave therapy.</a> Cardiovascular Research. 116 (6), 1226-1236 (2019).</li><li>Otrock, Z. K., Mahfouz, R. A. R., Makarem, J. A., Shamseddine, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+biology+of+angiogenesis:+Review+of+the+most+important+molecular+mechanisms.">Understanding the biology of angiogenesis: Review of the most important molecular mechanisms.</a> Blood Cells, Molecules and Diseases. 39, 212-220 (2007).</li><li>Ahn, H., 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=A+murine+model+of+hind+limb+ischemia+to+study+angiogenesis+and+arteriogenesis.">A murine model of hind limb ischemia to study angiogenesis and arteriogenesis.</a> Physiology and Behavior. 176, 139-148 (2017).</li><li>P&#246;lzl, L., 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=Defining+a+therapeutic+range+for+regeneration+of+ischemic+myocardium+via+shock+waves.">Defining a therapeutic range for regeneration of ischemic myocardium via shock waves.</a> Science Reports. , 409 (2021).</li><li>Holfeld, J., 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=Low+energy+shock+wave+therapy+induces+angiogenesis+in+acute+hind-limb+ischemia+via+VEGF+receptor+2+phosphorylation.">Low energy shock wave therapy induces angiogenesis in acute hind-limb ischemia via VEGF receptor 2 phosphorylation.</a> PLoS One. 9, 1-7 (2014).</li><li>Theurl, 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=The+neuropeptide+catestatin+acts+as+a+novel+angiogenic+cytokine+via+a+basic+fibroblast+growth+factor-dependent+mechanism.">The neuropeptide catestatin acts as a novel angiogenic cytokine via a basic fibroblast growth factor-dependent mechanism.</a> Circulation Research. 107 (11), 1326-1335 (2010).</li><li>Noonan, D. M., De Lerma Barbaro, A., Vannini, N., Mortara, L., Albini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation,+inflammatory+cells+and+angiogenesis:+Decisions+and+indecisions.">Inflammation, inflammatory cells and angiogenesis: Decisions and indecisions.</a> Cancer Metastasis Reviews. 27, 31-40 (2008).</li><li>Aurora, A. B., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+modulation+of+stem+cells+and+regeneration.">Immune modulation of stem cells and regeneration.</a> Cell Stem Cell. 15, 14-25 (2014).</li></ol>

Shockwave treatment shows promising results in several soft tissue regeneration settings. However, to further augment, improve or isolate these regenerative capability&#39;s, first the basics of SWT induced regeneration should be uncovered on a molecular level. Tissue regeneration is complex and involves many biological processes including, innate and acquired immunity, inflammation, cell cycle progression, apoptosis, cellular differentiation, angiogenesis and others17,<sup class="xref"...

Shockwave therapy (SWT) was first introduced in clinical practice as a means of disintegrating kidney stones via extracorporeal application. In the 1990&#180;s, an incidental finding of iliac crest thickening in X-ray recordings following repeated lithotripsy revealed a bone morphogenic effect of SWT1. This prompted a surge of new applications in orthopedic use. SWT, thereby evolved into an acknowledged treatment option for, long bone non-unions, lateral epicondylitis, as well as achilles tendonitis2,3,4,5. Recent evidence now again broadens the spectrum of appliances beyond orthopedics, into softer tissues and wound healing disorders6,7. Here studies could show effectiveness of SWT in a heterogeneous assembly of conditions including for example erectile dysfunction or spasticity after stroke8,9,10.
However, the molecular mechanisms underlying SWT are still not fully understood and require further research. With a focus on cardiovascular disease our previous work demonstrates a promising effect of SWT in a murine model of myocardial infarction. Thereby angiogenesis was discovered as a core driver of myocardial regeneration following SWT11.
Angiogenesis describes the development of new vessels through sprouting and splitting of preexisting vessels. In the case of injury these new vessels facilitate the restoration of blood flow to the damaged area and thereby regeneration12.
Angiogenesis, therefore, represents a hallmark of tissue regeneration and a potential explanation for SWT effects in softer tissues. However, regeneration is a complex process with numerous inductor and effector mechanisms. Albeit the possibility to investigate them in an isolated cell culture setting, animal models are best suited to emulate these complex processes. Hind limb ischemia is a well-established model to investigate angiogenesis and regeneration in vivo13. To support further research of the regenerative effect of SWT we hereby present a feasible, standardized, murine model of SWT in hind limb ischemia.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% Povidone</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 Nylon suture</td>
			<td>Ethicon Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>7-0 silk suture</td>
			<td>Ethicon Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cautery</td>
			<td>Martin</td>
			<td>ME-102</td>
			<td></td>
		</tr>
		<tr>
			<td>depilatory cream</td>
			<td>Nivea</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>Gazin</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heating Plate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride</td>
			<td></td>
			<td></td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>Laser Doppler</td>
			<td>Moor instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Tools</td>
			<td>Fine Science Tools</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine hydrochloride</td>
			<td></td>
			<td></td>
			<td>anesthesia</td>
		</tr>
	</tbody>
</table>

a standardized murine model of extracorporeal shockwave therapy induced soft tissue regeneration

Shockwave therapy (SWT) shows promising regenerative effects in several different tissues. However, the underlying molecular mechanisms are poorly understood. Angiogenesis, a process of new blood vessel formation is a leading driver of regeneration in softer tissues as well as a recently discovered effect of SWT. How the mechanical stimulus of SWT induces angiogenesis and regeneration and which pathways are involved is not fully understood. To further improve the clinical use of SWT and gain valuable information about how mechanical stimulation can affect tissue and tissue regeneration, a standardized model of SWT is needed. We, hereby, describe a standardized, easy to implement murine model of shockwave therapy induced regeneration, utilizing the hind-limb ischemia model.

Utilizing this protocol significant differences in hind limb perfusion can be observed and monitored after SWT intervention. Representative images show a marked difference in limbs treated with SWT (Figure 1B) compared to untreated control limbs (Figure 1A). Here, perfusion is portrayed via thermal flaring with cold colors representing low perfusion and warm colors representing high perfusion. Quantification of laser doppler rea...

Read this Scientific Article Protocol about A Standardized Murine Model of Extracorporeal Shockwave Therapy Induced Soft Tissue Regeneration at JoVE.com

A Standardized Murine Model of Extracorporeal Shockwave Therapy Induced Soft Tissue Regeneration

This article describes a standardized murine model of tissue regeneration via shockwave treatment.

A Standardized Murine Model of Extracorporeal Shockwave Therapy Induced Soft Tissue Regeneration

Abstract