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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+burden+of+cutaneous+melanoma+in+2020+and+projections+to+2040.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predictors+and+natural+history+of+in-transit+melanoma+after+sentinel+lymphadenectomy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+locoregional+cutaneous+metastases+in+melanoma+predictable.">Are locoregional cutaneous metastases in melanoma predictable.</a> Ann Surg Oncol. 6 (3), 315-321 (1999).</li><li>Creech, O., Krementz, E. T., Ryan, R. F., Winblad, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapy+of+cancer:+Regional+perfusion+utilizing+an+extracorporeal+circuit.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+follow-up+of+163+consecutive+patients+treated+with+isolated+limb+perfusion+for+in-transit+metastases+of+malignant+melanoma.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+trends+in+regional+therapy+for+melanoma:+Lessons+learned+from+225+regional+chemotherapy+treatments+between+1995+and+2010+at+a+single+institution.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+limb+infusion+for+in-transit+malignant+melanoma+of+the+extremity:+A+well-tolerated+but+less+effective+alternative+to+hyperthermic+isolated+limb+perfusion.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+efficacy+of+isolated+limb+infusion+with+cytotoxic+drugs+in+elderly+patients+with+advanced+locoregional+melanoma.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burden+of+disease+predicts+response+to+isolated+limb+infusion+with+melphalan+and+actinomycin+d+in+melanoma.">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. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+hyperthermic+perfusions+for+cutaneous+melanoma+in-transit+metastasis+of+the+limb+and+uveal+melanoma+metastasis+to+the+liver.">Isolated hyperthermic perfusions for cutaneous melanoma in-transit metastasis of the limb and uveal melanoma metastasis to the liver.</a> Clin Exp Metastasis. , (2023).</li><li>Olofsson Bagge, 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=Minimally+invasive+isolated+limb+perfusion+-+technical+details+and+initial+outcome+of+a+new+treatment+method+for+limb+malignancies.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+response+and+regional+toxicity+following+isolated+limb+infusion+compared+with+isolated+limb+perfusion+for+in-transit+melanoma.">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 hig...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.018&#8221; introducer guidewire&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.035&#8221; exchange guidewire&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10-12Fr BioMedicus Nextgen Pediatric Arterial cannulae.&#160;</td>
			<td>Medtronic, Minneapolis, MN, USA</td>
			<td>96820-110 (10Fr); 96820-112 (12Fr)</td>
			<td></td>
		</tr>
		<tr>
			<td>12-14 Fr BioMedicus&#160; NextGen Pediatric Venous Cannulae&#160;</td>
			<td>Medtronic, Minneapolis, MN, USA</td>
			<td>96830-112 (12Fr); 96830-114 (14Fr)</td>
			<td></td>
		</tr>
		<tr>
			<td>Esmarch tourniquets&#160;</td>
			<td>McKesson&#160;</td>
			<td>16-50409</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-introducer, 4 Fr, 10 cm&#160;</td>
			<td>MAK, Merit Medical, USA&#160;</td>
			<td>MAK401&#160;</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&#160;</td>
			<td>12773-02</td>
			<td>Includes:&#160; (1)Perclose Suture-Mediated Closure and repair device; (2) suture trimmer; (3) snared knot pusher&#160;</td>
		</tr>
		<tr>
			<td>Special Needle (20G, 12 cm)&#160;</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 percutane...

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

Research

JoVE Journal

Medicine

347 Views.  Sahlgrenska University Hospital. 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.

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

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

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 ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

Minimally Invasive Transverse Aortic Constriction in Mice

Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard open-chest transverse aortic constriction (TAC). Although transverse aortic constriction is a highly functional method for the induction of high pressure in the left ventricle, it is a more difficult and lengthy procedure due to its use of artificial ventilation with tracheal intubation. TAC is oftentimes also less survivable, as the newer method, MTAC, neither requires the cutting of the ribs and intercostal muscles nor tracheal intubation with a ventilation setup. In MTAC, as opposed to a thoracotomy to access to the chest cavity, the aortic arch is reached through a midline incision in the anterior neck. The thyroid is pulled back to reveal the sternal notch. The sternum is subsequently cut down to the second rib level, and the aortic arch is reached simply by separating the connective tissues and thymus. From there, a suture can be wrapped around the arch and tied with a spacer, and then the sternal cut and skin can be closed. MTAC is a much faster and less invasive way to induce left ventricular hypertension and enables the possibility for high-throughput studies. The success of the constriction can be verified using high-frequency trans-thoracic echocardiography, particularly color Doppler and pulsed-wave Doppler, to determine the flow velocities of the aortic arch and left and right carotid arteries, the dimension of the blood vessels, and the left ventricular function and morphology. A successful constriction will also trigger significant histopathological changes, such as cardiac muscle cell hypertrophy with interstitial and perivascular fibrosis. Here, the procedure of MTAC is described, demonstrating how the resulting flow changes in the carotid arteries can be examined with echocardiography, gross morphology, and histopathological changes in the heart.

Minimally invasive transverse aortic constriction (MTAC) conserves the essentials of regular transverse aortic constriction (TAC) while eliminating the use of a ventilator with tracheal intubation. It proves to be a highly desirable method for high-throughput studies on left ventricular overload, particularly in translational studies.

Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard ...

Electromagnetic Navigation Transthoracic Nodule Localization for Minimally Invasive Thoracic Surgery

The increased use of chest computed tomography (CT) has led to an increased detection of pulmonary nodules requiring diagnostic evaluation and/or excision. Many of these nodules are identified and excised via minimally invasive thoracic surgery; however, subcentimeter and subsolid nodules are frequently difficult to identify intra-operatively. This can be mitigated by the use of electromagnetic transthoracic needle localization. This protocol delineates the step-by-step process of electromagnetic localization from the pre-operative period to the postoperative period and is an adaptation of the electromagnetically guided percutaneous biopsy previously described by Arias et al. Pre-operative steps include obtaining a same day CT followed by the generation of a three-dimensional virtual map of the lung. From this map, the target lesion(s) and an entry site are chosen. In the operating room, the virtual reconstruction of the lung is then calibrated with the patient and the electromagnetic navigation platform. The patient is then sedated, intubated, and placed in the lateral decubitus position. Using a sterile technique and visualization from multiple views, the needle is inserted into the chest wall at the prechosen skin entry site and driven down to the target lesion. Dye is then injected into the lesion and, then, continuously during needle withdrawal, creating a tract for visualization intra-operatively. This method has many potential benefits when compared to the CT-guided localization, including a decreased radiation exposure and decreased time between the dye injection and the surgery. Dye diffusion from the pathway occurs over time, thereby limiting intra-operative nodule identification. By decreasing the time to surgery, there is a decrease in wait time for the patient, and less time for dye diffusion to occur, resulting in an improvement in nodule localization. When compared to electromagnetic bronchoscopy, airway architecture is no longer a limitation as the target nodule is accessed via a transparenchymal approach. Details of this procedure are described in a step-by-step fashion.

Presented here is a protocol for lung nodule localization using dye marking via electromagnetically navigated transthoracic needle access. The technique described here can be accomplished in the peri-operative period to optimize nodule localization and to successful resection when performing minimally invasive thoracic surgery.

The increased use of chest computed tomography (CT) has led to an increased detection of pulmonary nodules requiring diagnostic evaluation and/or ...

Absorbent Microbiopsy Sampling and RNA Extraction for Minimally Invasive, Simultaneous Blood and Skin Analysis

Conventional skin biopsy limits the clinical research that involves cosmetically sensitive areas or pediatric applications due to its invasiveness. Here, we describe the protocol for using&#160;an absorbent microneedle-based device, absorbent microbiopsy, for minimally invasive sampling of skin and blood mixture. Our goal is to help facilitate rapid progress in clinical research, the establishment of biomarkers for skin disease and reducing the risk for clinical research participants. In contrast to conventional skin biopsy techniques, the absorbent microbiopsy can be performed within seconds and does not require intensive training due to its simple design. In this report, we describe the use of absorbent microbiopsy, including loading and application, on a&#160;volunteer. Then, we show how to isolate RNA from the absorbed sample. Finally, we demonstrate the use of quantitative reverse transcription PCR (RT-qPCR) to quantify mRNA expression levels of both blood (CD3E and CD19) and skin (KRT14 and TYR). The methods that we describe utilize off the shelf kits and reagents. This protocol offers a minimally invasive approach for simultaneous sampling of skin and blood within the same absorbent microbiopsy matrix. We have found human ethics committees, clinicians and volunteers to be supportive of this approach to dermatological research.

In this article, we demonstrate how the absorbent microbiopsy technique is performed and how the sample can be used for RNA extraction for simple and simultaneous sampling of skin and blood in a minimally invasive manner.

Conventional skin biopsy limits the clinical research that involves cosmetically sensitive areas or pediatric applications due to its invasiveness. Here, ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Evaluation of Capnography Sampling Line Compatibility and Accuracy when Used with a Portable Capnography Monitor

Capnography is commonly used to monitor patient&#8217;s ventilatory status. While sidestream capnography has been shown to provide a reliable assessment of end-tidal CO2 (ETCO2), its accuracy is commonly validated using commercial kits composed of a capnography monitor and its matching disposable nasal cannula sampling lines. The purpose of this study was to assess the compatibility and accuracy of cross-paired capnography sampling lines with a single portable bedside capnography monitor. A series of 4 bench tests were performed to evaluate the tensile strength, rise time, ETCO2 accuracy as a function of respiratory rate, and ETCO2 accuracy in the presence of supplemental O2. Each bench test was performed using specialized, validated equipment to allow for a full evaluation of sampling line performance. The 4 bench tests successfully differentiated between sampling lines from different commercial sources and suggested that due to increased rise time and decreased ETCO2 accuracy, not all nasal cannula sampling lines provide reliable clinical data when cross-paired with a commercial capnography monitor. Care should be taken to ensure that any cross-pairing of capnography monitors and disposable sampling lines is fully validated for use across respiratory rates and supplemental O2 flow rates commonly encountered in clinical settings.

The goal of this study was to evaluate the accuracy of capnography sampling lines used in conjunction with a portable bedside capnography monitor. Sampling lines from 7 manufacturers were evaluated for tensile strength, rise time, and ETCO2 accuracy as a function of respiratory rate or supplemental oxygen flow rate.

Capnography is commonly used to monitor patient&#8217;s ventilatory status. While sidestream capnography has been shown to provide a reliable assessment ...

Isolated Lung Perfusion System in the Rabbit Model

The isolated lung perfusion system has been widely used in pulmonary research, contributing to elucidate the lungs&#39; inner workings, both micro and macroscopically. This technique is useful in the characterization of pulmonary physiology and pathology by measuring metabolic activities and respiratory functions, including interactions between circulatory substances and the effects of inhaled or perfused substances, as in drug testing. While in vitro methods involve the slicing and culturing of tissues, the isolated ex vivo lung perfusion system allows to work with a complete functional organ making possible the study of a continuous physiological function while recreating ventilation and perfusion. However, it should be noted that the effects of the absence of central innervation and lymphatic drainage still have to be fully assessed. This protocol aims to describe the assembly of the isolated lung apparatus, followed by the surgical extraction and cannulation of lungs and heart from experimental lab animals, as well as to display the perfusion technique and signal processing of data. The average viability of the isolated lung ranges between 5-8 h; during this period, the pulmonary capillary permeability increases, causing edema and lung injury. The functionality of preserved pulmonary tissue is measured by the capillary filtration coefficient (Kfc), used to determine the extent of pulmonary edema through time.

The isolated rabbit lung preparation is a gold standard tool in pulmonary research. This publication aims to describe the technique as developed for the study of physiological and pathological mechanisms involved in airway reactivity, lung preservation, and preclinical research in lung transplantation and pulmonary edema.

The isolated lung perfusion system has been widely used in pulmonary research, contributing to elucidate the lungs&#39; inner workings, both micro and ...

Therapy Interventions for Upper Limb Amputees Undergoing Selective Nerve Transfers

Targeted Muscle Reinnervation (TMR) improves the biological control interface for myoelectric prostheses after above-elbow amputation. Selective activation of muscle units is made possible by surgically re-routing nerves, yielding a high number of independent myoelectric control signals. However, this intervention requires careful patient selection and specific rehabilitation therapy. Here a rehabilitation protocol is presented for high-level upper limb amputees undergoing TMR, based on an expert Delphi study. Interventions before surgery include detailed patient assessment and general measures for pain control, muscle endurance and strength, balance, and range of motion of the remaining joints. After surgery, additional therapeutic interventions focus on edema control and scar treatment and the selective activation of cortical areas responsible for upper limb control. Following successful&#160;reinnervation of target muscles, surface electromyographic (sEMG) biofeedback is used to train the activation of the novel muscular units. Later on, a table-top prosthesis may provide the first experience of prosthetic control. After fitting the actual prosthesis, training includes repetitive drills without objects, object manipulation, and finally, activities of daily living. Ultimately, regular patient appointments and functional assessments allow tracking prosthetic function and enabling early interventions if malfunctioning.

This work presents a protocol to enhance prosthetic function after selective nerve transfer surgery. Rehabilitation interventions include patient information and selection, support of wound healing, cortical re-activation of sensory-motor areas of the upper limb, training of selective muscle activation, prosthetic handling in daily life, and regular follow-up assessments.