Polle Willemsen (filmer and producer) is acknowledged for excellent video recording and editing of the movie.

The protocol adheres to the guidelines established by our Institution&#39;s Human Research Ethics Committee.
1. Anesthesia
<ol>
	<li>Induce anesthesia according to the Institute&#39;s local protocol.</li>
</ol>
2. Set up
<ol>
	<li>Calculate the leg volume of the patient (1 day preoperative):
	<ol>
		<li>Lay the patient supine and draw a circular baseline starting in the groin. With 5 cm intervals, mark the patient&#39;s leg down to the ankle.</li>
		<li>If there is evidence of tumor load in the foot, include foot measurements by adding the length from the heel of the patient to the end of the first toe and the end of the fifth toe.</li>
		<li>Add all measurements in a spreadsheet with an underlying calculation model that summed the calculation of cones at the predefined levels of 5 cm.</li>
	</ol>
	</li>
	<li>Place a scintillation probe above the heart of the patient to enable capturing the percentage of leakage to the systemic circulation under the perfusion period.</li>
	<li>Inject a small dose of 10 MBq Tc99m marked albumin. To calculate the biological half-time, the activity is then measured for at least 20 min.</li>
</ol>
3. Femoral access
<ol>
	<li>Localize the superficial femoral or brachial vein using ultrasound guidance. Make a small 5 mm incision with a scalpel in the skin. Puncture the superficial artery first with a special needle (20 G, 12 cm) at approximately 15 cm below the inguinal ligament and place a 0.018&#34; introducer guidewire.</li>
	<li>Place a micro-introducer (4 French, 10 cm).</li>
	<li>Remove the 0.018&#34; introducer guidewire and the inner dilator.</li>
	<li>Introduce a 0.035&#34; exchange guidewire via the 4 French introducer and verify its position by fluoroscopy.</li>
	<li>Perform adequate predilation of the entry tract with several dilators over the exchange wire.</li>
	<li>After sufficient pre-dilatation, introduce a 12-14 Fr minimally invasive cannula and place it over the exchange wire into the vein. Flush the venous catheter with heparin saline. Then secure it with a skin suture.</li>
	<li>Cannulate slightly proximal to the venous level, in a similar fashion, the superficial femoral artery.</li>
	<li>Puncture the artery with a 20 G angiography needle and place a 0.018&#34; introducer guidewire.</li>
	<li>Remove the needle and place a 10 cm long 4 Fr mini access introducer over the wire. Remove both the guidewire and the inner dilator of the introducer.</li>
	<li>Confirm vascular access by aspirating blood via the introducer, as well as, hand injection of a small dose of iodinated contrast under fluoroscopy.</li>
	<li>Introduce a 0.035&#34; exchange wire via the 4 French introducer and verify its position by fluoroscopy. Remove the introducer.</li>
	<li>Place with a pre-closing technique two ProGlide sutures. Place the first one at the 10 o&#39;clock position and the other at the 2 o&#39;clock position. Do not tighten the sutures, but clamp the suture limbs slightly away from the puncture site.</li>
	<li>Place a 10-12 Fr minimal invasive cannula over the guidewire into the superficial femoral artery.</li>
</ol>
4. Isolating the leg
<ol>
	<li>Connect the arterial and venous catheters to the perfusion system, which consists of a heart-lung machine equipped with a pump, heat exchanger, oxygenator, and volume reservoir.</li>
	<li>Isolate the limb by applying two Esmarch tourniquets around the limb, proximal to the catheters.</li>
	<li>Remove the clamps from the arterial and venous cannulas to the shunt site. Start the perfusion and adjust the flow rate until a steady state level is reached, usually between 500 and 1,200 mL/min.</li>
	<li>Insert three thermistors for temperature monitoring at three different locations: ingoing blood cannula, subcutaneously 15 cm above the knee joint, and subcutaneously 15 cm below the knee joint.</li>
	<li>If the foot has no tumor load, isolate it by wrapping an Esmarch tourniquet around it.</li>
	<li>To maintain temperature, wrap a sterile green drape around the leg to maintain temperature. Aim for a temperature of 39 C for the ingoing blood.</li>
</ol>
5. The perfusion (Figure 1)
<ol>
	<li>Inject a high dose of 100 MBq Tc99m marked albumin in the perfusion system. The leakage rate is now calculated by measuring the transference from this radiotracer between the systemic circulation and the circulation in the isolated limb.</li>
	<li>When a steady-state of the flow is established and no leakage is present, infuse melphalan at a dose of 10 mg/L limb volume for lower limbs and 13 mg/L limb volume for upper limbs during 10 min.</li>
	<li>For patients with sarcoma, bulky tumors, or re-perfusions, give TNF-alpha (1-4 mg) as a bolus dose 10-15 min before starting the melphalan infusion.</li>
	<li>After melphalan infusion, continue the perfusion for another 45 min.</li>
</ol>
6. End of the perfusion and vessel closure
<ol>
	<li>After a total perfusion time of 55 min or 65 min (in those patients treated with TNF-alpha), rinse the leg with 3 L of Ringers Acetate and massage the leg to help empty the venous reservoir.</li>
	<li>Disconnect the arterial and venous catheter from the perfusion system and remove the temperature probes and the Esmarch tourniquets.</li>
	<li>Remove the arterial catheter first and use the suture-mediated closure device to close the vessel.</li>
	<li>Remove the venous catheter and apply pressure to the groin for 5-10 min.</li>
</ol>
7. Postoperative care
<ol>
	<li>Extubate the patient and transfer them to the post-operative care unit for 4-6 h.</li>
	<li>Give thrombosis prophylaxis with Xarelto 10 mg for 4 weeks.</li>
	<li>Follow the patient up at the surgical outpatient department at 1-, 3- and 12-months post-operative to assess toxicities (according to the Wieberdink classification scale) and response rates.</li>
</ol>

<ol><li>Arnold, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+burden+of+cutaneous+melanoma+in+2020+and+projections+to+2040%5BTitle%5D)+AND+%22JAMA+Dermatol%22%5BJournal%5D)">Global burden of cutaneous melanoma in 2020 and projections to 2040.</a> JAMA Dermatol. 158 (5), 495-503 (2022).</li>
<li>Pawlik, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Predictors+and+natural+history+of+in-transit+melanoma+after+sentinel+lymphadenectomy%5BTitle%5D)+AND+%22Ann+Surg+Oncol%22%5BJournal%5D)">Predictors and natural history of in-transit melanoma after sentinel lymphadenectomy.</a> Ann Surg Oncol. 12 (8), 587-596 (2005).</li>
<li>Borgstein, P. J., Meijer, S., Van Diest, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Are+locoregional+cutaneous+metastases+in+melanoma+predictable%5BTitle%5D)+AND+%22Ann+Surg+Oncol%22%5BJournal%5D)">Are locoregional cutaneous metastases in melanoma predictable.</a> Ann Surg Oncol. 6 (3), 315-321 (1999).</li>
<li>Creech, O. Jr, Krementz, E. T., Ryan, R. F., Winblad, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemotherapy+of+cancer%3A+Regional+perfusion+utilizing+an+extracorporeal+circuit%5BTitle%5D)+AND+%22Ann+Surg%22%5BJournal%5D)">Chemotherapy of cancer: Regional perfusion utilizing an extracorporeal circuit.</a> Ann Surg. 148 (4), 616-632 (1958).</li>
<li>Olofsson, R., Mattsson, J., Lindner, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+follow-up+of+163+consecutive+patients+treated+with+isolated+limb+perfusion+for+in-transit+metastases+of+malignant+melanoma%5BTitle%5D)+AND+%22Int+J+Hyperthermia%22%5BJournal%5D)">Long-term follow-up of 163 consecutive patients treated with isolated limb perfusion for in-transit metastases of malignant melanoma.</a> Int J Hyperthermia. 29 (6), 551-557 (2013).</li>
<li>Raymond, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Current+trends+in+regional+therapy+for+melanoma%3A+Lessons+learned+from+225+regional+chemotherapy+treatments+between+1995+and+2010+at+a+single+institution%5BTitle%5D)+AND+%22J+Am+Coll+Surg%22%5BJournal%5D)">Current trends in regional therapy for melanoma: Lessons learned from 225 regional chemotherapy treatments between 1995 and 2010 at a single institution.</a> J Am Coll Surg. 213 (2), 306-316 (2011).</li>
<li>Beasley, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolated+limb+infusion+for+in-transit+malignant+melanoma+of+the+extremity%3A+A+well-tolerated+but+less+effective+alternative+to+hyperthermic+isolated+limb+perfusion%5BTitle%5D)+AND+%22Ann+Surg+Oncol%22%5BJournal%5D)">Isolated limb infusion for in-transit malignant melanoma of the extremity: A well-tolerated but less effective alternative to hyperthermic isolated limb perfusion.</a> Ann Surg Oncol. 15 (8), 2195-2205 (2008).</li>
<li>Kroon, H. M., Lin, D. Y., Kam, P. C., Thompson, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Safety+and+efficacy+of+isolated+limb+infusion+with+cytotoxic+drugs+in+elderly+patients+with+advanced+locoregional+melanoma%5BTitle%5D)+AND+%22Ann+Surg%22%5BJournal%5D)">Safety and efficacy of isolated limb infusion with cytotoxic drugs in elderly patients with advanced locoregional melanoma.</a> Ann Surg. 249 (6), 1008-1013 (2009).</li>
<li>Muilenburg, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Burden+of+disease+predicts+response+to+isolated+limb+infusion+with+melphalan+and+actinomycin+d+in+melanoma%5BTitle%5D)+AND+%22Ann+Surg+Oncol%22%5BJournal%5D)">Burden of disease predicts response to isolated limb infusion with melphalan and actinomycin d in melanoma.</a> Ann Surg Oncol. 22 (2), 482-488 (2015).</li>
<li>Huibers, A., Depalo, D. K., Perez, M. C., Zager, J. S., Olofsson Bagge, R. Isolated hyperthermic perfusions for cutaneous melanoma in-transit metastasis of the limb and uveal melanoma metastasis to the liver. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aHuibers%2C+A+%22Clin+Exp+Metastasis%22">Clin Exp Metastasis</a>. , (2023).</li>
<li>Olofsson Bagge, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Minimally+invasive+isolated+limb+perfusion+-+technical+details+and+initial+outcome+of+a+new+treatment+method+for+limb+malignancies%5BTitle%5D)+AND+%22Int+J+Hyperthermia%22%5BJournal%5D)">Minimally invasive isolated limb perfusion - technical details and initial outcome of a new treatment method for limb malignancies.</a> Int J Hyperthermia. 35 (1), 667-673 (2018).</li>
<li>Dossett, L. A., Ben-Shabat, I., Olofsson Bagge, R., Zager, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+response+and+regional+toxicity+following+isolated+limb+infusion+compared+with+isolated+limb+perfusion+for+in-transit+melanoma%5BTitle%5D)+AND+%22Ann+Surg+Oncol%22%5BJournal%5D)">Clinical response and regional toxicity following isolated limb infusion compared with isolated limb perfusion for in-transit melanoma.</a> Ann Surg Oncol. 23 (7), 2330-2335 (2016).</li>
</ol>

Roger Olofsson Bagge has received institutional research grants from Bristol-Myers Squibb (BMS), Endomagnetics Ltd (Endomag), SkyLineDx and NeraCare GmbH, speaker honorarium from Roche, Pfizer and Pierre-Fabre, and has served on advisory boards for Amgen, BD/BARD, Bristol-Myers Squibb (BMS), Cansr.com, Merck Sharp &#38; Dohme (MSD), Novartis, Roche and Sanofi Genzyme, and is a shareholder in SATMEG Ventures AB.

Patients with ITM have a poor prognosis and their treatment remains challenging. A key factor in treating these patients is aiming for local control. In a minority of these patients, surgical excision can be used when the tumor load is limited. When patients have more extensive disease or when ITMs are rapidly recurring, surgical resection is not possible. In these patients, isolated limb perfusion is an effective treatment option, with a high overall response rate of around 80%10. Despite its high response rates, ILP necessitates invasive open surgery for vascular access, whereas ILI offers a minimally invasive alternative, with catheter placement performed percutaneously. A study comparing the two procedures across two high-volume centers showed a significantly higher overall response rate (ORR) and complete response rate (CRR) for ILP compared to ILI, respectively (80% vs 53%) and (60% vs 29%)12. However, no randomized trials currently exist to definitively establish superiority between the two techniques.
One of the advantages of ILI is that it is done percutaneously, potentially associated with fewer adverse events related to the open procedure. Furthermore, ILI obviates the need for an extracorporeal oxygenator. The benefits of the ILP, which could potentially explain the higher response rates, are the higher flow rate achieved and the possibility of performing the perfusion during a longer time due to oxygenation. In this paper we showed the feasibility and technical details of a procedure that combined the benefits of both ILP and ILI (MI-ILP). The procedure involves four sequential and critical steps 1). Minimally invasive isolation of the limb from the rest of the systemic circulation 2). Delivery of a high dose chemotherapy to the limb during a hyperthermic oxygenated perfusion. 3). Washout of the chemotherapy 4). Re-establishing the normal circulation to the limb. In all these steps, multidisciplinary teamwork from various disciplines (surgeons, interventional radiologists, perfusionists, anesthetists, and nurses) is essential to safely perform the treatment.

The global incidence of melanoma is increasing and is particularly pronounced in the Western population, where one out of every 50 individuals will develop melanoma1. Patients with cutaneous melanoma can develop in-transit metastasis (ITM), most often localized to the limb2. These metastatic deposits appear as discrete tumor nodules within the subcutaneous or cutaneous tissue and arise between the primary melanoma site and the nearest regional lymph node basin. A compelling hypothesis implies that ITM develops after the entrapment and subsequent proliferation of tumor cell emboli in dermal lymphatic vessels between the primary tumor location and regional lymph nodules3. Surgical resection is an option for those patients with limited ITM, but when the tumor load is more extensive or when it is rapidly recurring, isolated limb perfusion (ILP) is an option.
ILP is an extremely effective regional treatment for advanced tumors but likely has no influence on the development of metastatic disease outside of the perfused region. The introduction of this technique dates back to 1958, when Creech and Krementz first described its principles4. The procedure consists of the isolation of the limb from the systemic circulation, achieved through the application of a pneumatic tourniquet or an Esmarch bandage, followed by its connection to an extracorporeal heart-lung machine. This allows for regional administration of heated chemotherapy (melphalan) at concentrations that would not be possible to give systemically5.
A minimally invasive counterpart is isolated limb infusion (ILI), where access is gained via percutaneous insertion of arterial and venous catheters via the contralateral groin. Subsequently, chemotherapy is then infused under tourniquet isolation in an ischemic environment for a duration of 20-30 min. Across various studies, overall tumor response rates for ILI and its counterpart ILP, have ranged between 40%-90%, whereas ILP generally yields higher response rates6,7,8,9,10. ILI offers several advantages, including the possibility of repeating it more easily, its percutaneous vascular access, and, therefore, potentially fewer adverse events, such as wound complications. Furthermore, it does not necessitate the use of an extracorporeal oxygenator.
In 2016, Sahlgrenska University Hospital developed a technique that combined both methods, minimally-invasive limb perfusion (MI-ILP), with the goal of performing isolated limb perfusion, but with the advantage of doing this in a minimally invasive setting. In this procedure, the femoral vessels are percutaneously accessed in a similar way as in ILP. In this paper, we present the technical details of the procedure, which is of special interest to clinicians who treat melanoma ITM and sarcoma.

<table><tbody><tr><td>0.018&amp;rdquo; introducer guidewire&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>0.035&amp;rdquo; exchange guidewire&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>10-12Fr BioMedicus Nextgen Pediatric Arterial cannulae.&amp;nbsp;</td><td>Medtronic, Minneapolis, MN, USA</td><td>96820-110 (10Fr); 96820-112 (12Fr)</td><td></td></tr><tr><td>12-14 Fr BioMedicus&amp;nbsp; NextGen Pediatric Venous Cannulae&amp;nbsp;</td><td>Medtronic, Minneapolis, MN, USA</td><td>96830-112 (12Fr); 96830-114 (14Fr)</td><td></td></tr><tr><td>Esmarch tourniquets&amp;nbsp;</td><td>McKesson&amp;nbsp;</td><td>16-50409</td><td></td></tr><tr><td>Micro-introducer, 4 Fr, 10 cm&amp;nbsp;</td><td>MAK, Merit Medical, USA&amp;nbsp;</td><td>MAK401&amp;nbsp;</td><td>Includes: (1) Co-axial introducer/dilator pair; (2) 21G Needle; (3) Guide wire</td></tr><tr><td>PerClose ProGlide closure devices</td><td>Abbott&amp;nbsp;</td><td>12773-02</td><td>Includes:&amp;nbsp; (1)Perclose Suture-Mediated Closure and repair device; (2) suture trimmer; (3) snared knot pusher&amp;nbsp;</td></tr><tr><td>Special Needle (20G, 12 cm)&amp;nbsp;</td><td>Mediplast AB, Sweden</td><td>662620091</td><td></td></tr></tbody></table>

minimally invasive isolated limb perfusion (mi-ilp) for locally advanced melanomas and sarcomas of the extremity

Minimally invasive isolated limb perfusion (MI-ILP) is a treatment option for patients with locally advanced melanomas and sarcomas of the extremities. Briefly, the procedure starts with percutaneous access of the femoral or brachial vessels in the diseased extremity. It is then isolated from the rest of the body with a tourniquet. The catheters are connected to a heart-lung machine, and the extremity is perfused with a high dose of melphalan. In the literature, the reported overall and complete response rates for ILP are approximately 80% and 60%, respectively. Our previously reported results using MI-ILP, showed similar response rates. The objective of this manuscript is to provide a step-by-step guide on how to perform an MI-ILP. The purpose of this protocol is to enable local perfusion of the extremities with a high dose of chemotherapy without systemic leakage in a minimally invasive manner.

We have previously published the results of the first six patients treated with MI-ILP (five femoral and one brachial) between June 2016 and March 2017 at Sahlgrenska University Hospital11. Patients who were 18 years or older with a locally advanced tumor of the extremity that had an indication for ILP were included in the study. Patients were excluded if they had a previous lymph node dissection, current lymph node disease, or severe atherosclerosis, as this could potentially complicate percutaneously vascular access. Indications for MI-ILP were: ITM of malignant melanoma in five patients and recurrent squamous cell carcinoma in one patient. The median age was 60 years (range 48-87). Patients had, on average, 11 ITMs (range 1-50), and the average size of their largest tumor was 30 mm (range 8-110). At the time of treatment, none of the patients had general metastases. All patients underwent the procedure without the need for conversion to an open procedure. The blood flow rates within the perfused extremity in those patients undergoing femoral perfusion ranged between 270-711 ml/min, with a median flow rate of 610 ml/min. For the patient treated with brachial perfusion, a flow rate of 110 ml/min was reached. The median leakage rate was 0.1% (range 0.0-3.2). Response rates at 3 months were prospectively recorded. Four out of six patients (67%), had a complete response rate, and two patients (33%) had stable disease.
Local toxicity was described as the worst reaction up to 3 months according to the Wieberdink classification. Four out of six patients (67%) had a Wieberdink grade II reaction, and two patients (33%) had a Wieberdink grade III reaction. There was no evidence of systemic toxicity during the post-operative period. In two patients, Clavien-Dindo grade II postoperative complications were described. The first patient had an infusion-related erysipelas at the hand and was treated with intravenous antibiotics. The second patient, who had an 11 cm large melanoma, developed postoperative tumor necrosis-associated fever, which was treated with intravenous antibiotics. Within this limited series of patients, leakage rates, flow rates, and clinical outcomes are comparable with historical data on ILP11,12 (Table 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66876/66876fig1.jpg" /> 
Figure 1: MI-ILP (Minimally Invasive Isolated Limb Perfusion). In this case, the femoral vessels are percutaneously accessed in the diseased extremity, which is then isolated from the rest of the body, with a tourniquet cranial to the catheterized vessels. The patient is then connected to a heart lung machine and the extremity is perfused with a high dose of melphalan (10 mg/L limb volume) for 55 minutes. This figure has been modified from11. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66876/66876fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Isolated Limb Perfusion 
			(ILP)&#160;&#160;</td>
			<td>Isolated Limb Infusion 
			(ILI)&#160;</td>
			<td>Minimal-Invasive Isolated 
			&#160;Limb Perfusion 
			(MI-ILP)</td>
		</tr>
		<tr>
			<td>Technique</td>
			<td>open surgical exposure of vessels</td>
			<td>percutaneous catheterization</td>
			<td>percutaneous catheterization&#160;</td>
		</tr>
		<tr>
			<td>Duration&#160;</td>
			<td>approximately 3 hours</td>
			<td>approximately 1.5 hours</td>
			<td>approximately 2 hours</td>
		</tr>
		<tr>
			<td>Fluoroscopy</td>
			<td>no fluoroscopy needed</td>
			<td>fluoroscopy needed&#160;</td>
			<td>fluoroscopy needed&#160;</td>
		</tr>
		<tr>
			<td>Overall response rate</td>
			<td>80% - 81%&#160;</td>
			<td>43%-53%</td>
			<td>87%</td>
		</tr>
		<tr>
			<td>Complete response rate&#160;</td>
			<td>55% - 57%&#160;</td>
			<td>24%-50%</td>
			<td>56%</td>
		</tr>
	</tbody>
</table>
Table 1.Comparison of technique and outcome of ILP (Isolated Limb Perfusion), ILI (Isolated Limb Infusion), and MI-ILP (Minimally Invasive Isolated Limb Perfusion) for melanoma in-transit metastases11,12.

This protocol presents a step-by-step guide for performing minimally invasive isolated limb perfusion (MI-ILP), a treatment option for patients with locally advanced melanomas and sarcomas of the extremities.

Minimally Invasive Isolated Limb Perfusion (MI-ILP) for Locally Advanced Melanomas and Sarcomas of the Extremity

Minimally invasive isolated limb perfusion (MI-ILP) is a treatment option for patients with locally advanced melanomas and sarcomas of the extremities. ...

minimally-invasive-isolated-limb-perfusion-mi-ilp-for-locally

Nanyang Technological University

Research

JoVE Journal

Medicine

383 Views.  Sahlgrenska University Hospital. Minimaal invasieve geïsoleerde ledemaatperfusie voor lokaal gevorderde melanomen en sarcomen van de extremiteit. Melanoom in-transit metastase zal zich ontwikkelen bij ongeveer 3-5% van de patiënten met melanoom met een hoog risico in een vroeg stadium. Ze verschijnen als tumorafzettingen in het dermale of onderhuidse weefsel.De hypothese is dat ITG afkomstig is van tumorcelembolieën die gevangen zitten in dermale lymfevaten, maar de onderliggende p...

Video: Minimaal invasieve geïsoleerde ledemaatperfusie (MI-ILP) voor lokaal gevorderde melanomen en sarcomen van de extremiteit 

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal hyperplasia (IH) that eventually leads to vessel occlusion and graft failure. Mechanical forces, particularly low shear stress and high wall tension, are thought to initiate and to sustain these cellular and molecular changes, but their exact contribution remains to be unraveled. To selectively evaluate the role of pressure and shear stress on the biology of IH, an ex&#160;vivo perfusion system (EVPS) was created to perfuse segments of human saphenous veins under arterial regimen (high shear stress and high pressure). Further technical innovations allowed the simultaneous perfusion of two segments from the same vein, one reinforced with an external mesh. Veins were harvested using a no-touch technique and immediately transferred to the laboratory for assembly in the EVPS. One segment of the freshly isolated vein was not perfused (control, day 0). The two others segments were perfused for up to 7 days, one being completely sheltered with a 4 mm (diameter) external mesh. The pressure, flow velocity, and pulse rate were continuously monitored and adjusted to mimic the hemodynamic conditions prevailing in the femoral artery. Upon completion of the perfusion, veins were dismounted and used for histological and molecular analysis. Under ex&#160;vivo conditions, high pressure perfusion (arterial, mean = 100 mm Hg) is sufficient to generate IH and remodeling of human veins. These alterations are reduced in the presence of an external polyester mesh.

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mechanisms leading to the development of intimal hyperplasia (IH) and vein graft failure are still poorly understood. This study describes an ex&#160;vivo system to perfuse human veins under controlled flow and pressure. Furthermore the efficiency of external mesh reinforcement to limit the development of IH was evaluated.

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal ...

Remote Limb Ischemic Preconditioning:  A Neuroprotective Technique in Rodents

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. Sublethal ischemia has also been shown to upregulate protective mechanisms in remote tissues. A brief period of ischemia (5-10 min) in the hind limb of mammals induces self-protective responses in the brain, lung, heart and retina. The effect is known as remote ischemic preconditioning (RIP). It is a therapeutically promising way of protecting vital organs, and is already under clinical trials for heart and brain injuries. This publication demonstrates a controlled, minimally invasive method of making a limb &#8211; specifically the hind limb of a rat &#8211; ischemic. A blood pressure cuff developed for use in human neonates is connected to a manual sphygmomanometer and used to apply 160 mmHg pressure around the upper part of the hind limb. A probe designed to detect skin temperature is used to verify the ischemia, by recording the drop in skin temperature caused by pressure-induced occlusion of the leg arteries, and the rise in temperature which follows release of the cuff. This method of RIP affords protection to the rat retina against bright light-induced damage and degeneration.

Remote ischemic preconditioning (RIP) is a method of conditioning tissues against damaging stress. We have established a method of remote ischemia at the hind limb, by inflating a sphygmomanometer cuff for 5-10 min. The neuroprotective capabilities of RIP have been demonstrated in a model of retinal degeneration in rodents.

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. ...

Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic agent melphalan. Briefly, the procedure starts with the setup of a percutaneous veno-venous bypass from the femoral vein to the external jugular vein. Via a laparotomy, catheters are then inserted into the proper hepatic artery and the caval vein. The portal vein and the caval vein, both supra- and infrahepatically, are then clamped. The arterial and venous catheters are connected to a heart lung machine and the liver is perfused with melphalan (1 mg/kg body weight) for 60 min. This way it is possible to locally perfuse the liver with a high dose of a chemotherapeutic agent, without leakage to the systemic circulation.
In previous studies including patients with isolated liver metastases of uveal melanoma, an overall response rate of 33-100% and a median survival between 9 and 13 months, have been reported. The aim of this protocol is to give a clear description of how to perform the procedure and to discuss IHP as a treatment option for liver metastases of uveal melanoma.

Here, we present a protocol how to perform an isolated liver perfusion (IHP) with melphalan and we also discuss IHP as a treatment option for liver metastases of uveal melanoma.

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic ...

Percutaneous Hepatic Perfusion (PHP) with Melphalan as a Treatment for Unresectable Metastases Confined to the Liver

Unresectable liver metastases of colorectal cancer can be treated with systemic chemotherapy, aiming to limit the disease, extend survival or turn unresectable metastases into resectable ones. Some patients however, suffer from side effects or progression under systemic treatment. For patients with metastasized uveal melanoma there are no standard systemic therapy options. For patients without extrahepatic disease, isolated liver perfusion (IHP) may enable local disease control with limited systemic side effects. Previously, this was performed during open surgery with satisfying results, but morbidity and mortality related to the open procedure, prohibited a widespread application. Therefore, percutaneous hepatic perfusion (PHP) with simultaneous chemofiltration was developed. Besides decreasing morbidity and mortality, this procedure can be repeated, hopefully leading to a higher response rate and improved survival (by local control of disease). During PHP, catheters are placed in the proper hepatic artery, to infuse the chemotherapeutic agent, and in the inferior caval vein to aspirate the chemosaturated blood returning through the hepatic veins. The caval vein catheter is a double balloon catheter that prohibits leakage into the systemic circulation. The blood returning from the hepatic veins is aspirated through the catheter fenestrations and then perfused through an extra-corporeal filtration system. After filtration, the blood is returned to the patient by a third catheter in the right internal jugular vein. During PHP a high dose of melphalan is infused into the liver, which is toxic and would lead to life threatening complications when administered systemically. Because of the significant hemodynamic instability resulting from the combination of caval vein occlusion and chemofiltration, hemodynamic monitoring and hemodynamic support is of paramount importance during this complex procedure.

Percutaneous Hepatic Perfusion PHP with Melphalan as a Treatment for Unresectable Metastases Confined to the Liver

In this manuscript, we describe percutaneous isolated hepatic perfusion with simultaneous chemofiltration as treatment for unresectable liver metastases. This procedure is performed under general anaesthesia in the angiosuite by an experienced team, consisting of an interventional radiologist, a clinical perfusionist and anaesthesiologist.

Unresectable liver metastases of colorectal cancer can be treated with systemic chemotherapy, aiming to limit the disease, extend survival or turn ...

Methods for Acute and Subacute Murine Hindlimb Ischemia

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries, and animal models that reliably reproduce the human disease are necessary to develop new therapies for this disease. The mouse hindlimb ischemia model has been widely used for this purpose, but the standard practice of inducing acute limb ischemia by ligation of the femoral artery can result in substantial tissue necrosis, compromising investigators&#39; ability to study the vascular and skeletal muscle tissue responses to ischemia. An alternative approach to femoral artery ligation is the induction of gradual femoral artery occlusion through the use of ameroid constrictors. When placed around the femoral artery in the same or different locations as the sites of femoral artery ligation, these devices occlude the artery over 1 - 3 days, resulting in more gradual, subacute ischemia. This results in less substantial skeletal muscle tissue necrosis, which may more closely mimic the responses seen in human PAD. Because genetic background influences outcomes in both the acute and subacute ischemia models, consideration of the mouse strain being studied is important in choosing the best model. This paper describes the proper procedure and anatomical placement of ligatures or ameroid constrictors on the mouse femoral artery to induce subacute or acute hindlimb ischemia in the mouse.

Surgical induction of hindlimb ischemia in the mouse is useful to examine angiogenesis, however this is compromised in certain inbred mouse strains that display marked ischemia-induced tissue necrosis. Methods are described to induce subacute limb ischemia using ameroid constrictors to circumvent this problem through the induction of gradual arterial occlusion.

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries, and animal models that reliably ...

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.

Here, a critical hindlimb ischemia experimental model is presented followed by a battery of functional, histologic and molecular tests&#160;to assess the effectiveness of angiogenic therapies.&#8203;

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard ...

Establishing In Situ Closed Circuit Perfusion of Lower Abdominal Organs and Hind Limbs in Mice

Ex vivo perfusion is an important physiological tool to study the function of isolated organs (e.g. liver, kidneys). At the same time, due to the small size of mouse organs, ex vivo perfusion of bone, bladder, skin, prostate, and reproductive organs is challenging or not feasible. Here, we report for the first time an in situ lower body perfusion circuit in mice that includes the above tissues, but bypasses the main clearance organs (kidney, liver, and spleen). The circuit is established by cannulating the abdominal aorta and inferior vena cava above the iliac artery and vein and cauterizing peripheral blood vessels. Perfusion is performed via a peristaltic pump with perfusate flow maintained for up to 2 h. In situ staining with fluorescent lectin and Hoechst solution confirmed that the microvasculature was successfully perfused. This mouse model can be a very useful tool for studying pathological processes as well as mechanisms of drug delivery, migration/metastasis of circulating tumor cells into/from the tumor, and interactions of immune system with perfused organs and tissues.

A protocol is described for in situ perfusion of the mouse lower body, including the bladder, the prostate, sex organs, bone, muscle and foot skin.

Ex vivo perfusion is an important physiological tool to study the function of isolated organs (e.g. liver, kidneys). At the same time, due to the small ...

Predicting Amputation using Local Circulating Mononuclear Progenitor Cells in Angioplasty-treated Patients with Critical Limb Ischemia

Critical limb ischemia (CLI) represents an advanced stage of the peripheral arterial disease. Angioplasty improves the blood flow to the lower limb; however, some patients irreversibly progress to limb amputation. The extent of vascular damage and the mechanisms of vascular repair are factors affecting post-angioplasty outcome. Mononuclear Progenitor Cells (MPCs) are reactive to vascular damage and repair, with the ability to reflect vascular diseases. The present protocol describes quantification of MPCs obtained from blood circulation from vessel close to the angioplasty site, as well as its relationship with endothelial dysfunction and its predictive ability for limb amputation in the next 30 days after angioplasty in patients with CLI.

Lower limb amputation may occur even after angioplasty of obstructed vessels in Critical Limb Ischemia (CLI). Mononuclear Progenitor Cells (MPCs) reflect vascular repair. The present protocol describes the quantification of MPCs from circulation close to angioplasty, and its relationship with endothelial dysfunction and prediction of lower limb amputation.

Critical limb ischemia (CLI) represents an advanced stage of the peripheral arterial disease. Angioplasty improves the blood flow to the lower limb; ...

Laser Doppler Perfusion Imaging in the Mouse Hindlimb

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is a common, noninvasive, repeatable method for assessing blood flow recovery. The technique calculates overall blood flow in the sampled tissue from the Doppler shift in frequency caused when a laser hits moving red blood cells. Measurements are expressed in arbitrary perfusion units, so the contralateral non-intervened upon leg is usually used to help control measurements. Measurement depth is in the range of 0.3-1 mm; for hindlimb ischemia, this means that dermal perfusion is assessed. Dermal perfusion is dependent on several factors&#8212;most importantly skin temperature and anesthetic agent, which must be carefully controlled to result in reliable readings. Furthermore, hair and skin pigmentation can alter the ability of the laser to either reach or penetrate to the dermis. This article demonstrates the technique of LDPI in the mouse hindlimb.

Here, we present a protocol that demonstrates the technique and necessary controls for Laser Doppler perfusion imaging to measure blood flow in the mouse hindlimb.

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is ...

Minimally Invasive Isolated Limb Perfusion (MI-ILP) for Locally Advanced Melanomas and Sarcomas of the Extremity

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