Feasibility of fresh frozen human cadavers as a research and training model for endovascular image guided interventions.

OBJECTIVE: To describe the feasibility of a fresh frozen human cadaver model for research and training of endovascular image guided procedures in the aorta and lower extremity.
METHODS: The cadaver model was constructed in fresh frozen human cadaver torsos and lower extremities. Endovascular access was acquired by inserting a sheath in the femoral artery. The arterial segment of the specimen was restricted by ligation of collateral arteries and, in the torsos, clamping of the contralateral femoral artery and balloon occlusion of the supratruncal aorta. Tap water was administered through the sheath to create sufficient intraluminal pressure to manipulate devices and acquire digital subtraction angiography (DSA). Endovascular cannulation tasks of the visceral arteries (torso) or the peripheral arteries (lower extremities) were performed to assess the vascular patency of the model. Feasibility of this model is based on our institute's experiences throughout the use of six fresh frozen human cadaver torsos and 22 lower extremities.
RESULTS: Endovascular simulation in the aortic and peripheral vasculature was achieved using this human cadaver model. Acquisition of DSA images was feasible in both the torsos and the lower extremities. Approximately 84 of the 90 target vessels (93.3%) were patent, the remaining six vessels showed signs of calcified steno-occlusive disease.
CONCLUSIONS: Fresh frozen human cadavers provide a feasible simulation model for aortic and peripheral endovascular interventions, and can potentially reduce the need for animal experimentation. This model is suitable for the evaluation of new endovascular devices and techniques or to master endovascular skills.

Introduction

Rapid succession of catheter-based innovations in the last decade, has led to more sophisticated endovascular procedures and increased patient eligibility for endovascular treatment [1-4]. Preclinical models are essential to evaluate the feasibility, safety, and efficacy of any novel endovascular device or technique, and to obtain regulatory approval for their clinical application [5, 6]. Preclinical models are also used in endovascular training and device instructions to stimulate safe and efficient implementation of novelties in clinical practice.

Various types of preclinical models are available for endovascular research and training purposes, each with its own advantages and disadvantages. Bench models, such as silicone aorta phantoms, provide a highly controlled environment but lack the complexity and tissue properties of clinical practice. Animal models, such as the well-established healthy swine model, allow in-vivo endovascular simulation, but the healthy, straight arteries of the swine differ considerably from the tortuous, calcified vessels of a typical vascular patient. Besides, the vasculature of the swine is narrower than that of humans, which can preclude the use of larger sheaths and devices. These anatomical and pathophysiological distinctions can compromise the translation from bench-to-bedside [7-10]. Moreover, there are strong ethical grounds to look for alternatives for the use of living animals. Human cadaver reperfusion models have been reported as a suitable, although less familiar, alternative to animal models for endovascular device evaluation and training [11-14]. However, reperfusion of cadaveric tissue is a delicate and complex process, which may restrict widespread adoption of human cadaver models.

In contrast to these human cadaver reperfusion models, our institution has been using human cadaver models without arterial reperfusion, to our satisfaction. In this study, we present our human cadaver model, and aim to raise general awareness of the existence and feasibility of human cadaver models for endovascular simulation in the abdominal aorta and peripheral arteries.

Materials and methods

Human cadavers

Body donations were acquired directly by our institute’s anatomy department. Body donations were regulated according to the national Dutch law on the disposal of the dead and the European legislations and ethical framework for body donation [15]. Written informed consent was obtained from the donors during life to use their cadavers for educational and research purposes. No additional approval of an ethics board was required for this study. Age, gender and serology report of the donors were provided. Other demographics and medical details were sealed to assure the anonymity of the donors, as according to our institution’s policy.

The available data of 22 lower extremities and 6 fresh frozen human cadaveric torsos were used for the assessment of this cadaver model. Donors were predominantly male (75%) with a mean age of 74.4 ± 8.8.

Cadaveric specimen

The cadavers were dissected and frozen within 48 hours of post-mortem delay, at a temperature of -20°C. No additive preservation chemicals were used. The cadaveric torsos comprised the thorax, abdomen, pelvis and proximal part of the lower extremities until mid-femoral level. Two oblique dissection planes from axilla to sternum exposed the branches of the aortic arch, trachea, esophagus and cervical spine. Two axial dissection planes at mid-femoral level exposed the left and right femoral artery. The lower extremities comprised the foot, lower leg and part of the upper leg. An axial dissection plane at mid-femoral level exposed the femoral artery. The dissection planes of the torsos and lower extremities are illustrated in Fig 1.

Fig 1

Schematic overview of the torso (A) and lower extremity (B) including dissection planes of the cadaveric specimen. Red lines indicate the dissection planes. The access sheath is indicated in green. Fluid leakage is prevented by ligation of the main collateral arteries in the dissection plane, and, in the torsos, by balloon occlusion of the proximal aorta and ligation of the contralateral femoral artery.

Cadaver preparation and storage

The cadaveric specimens were thawed at 15°C. Approximately one day of thawing was needed for the lower extremities and three days for torsos. The specimens were used for a maximum of four freeze-thaw cycles, due to deterioration of the soft tissues after prolonged use. No difference in quality of the specimen for endovascular use was noticed between first and last use of the specimen.

Vacant arteries in the intersection plane (typically the superficial femoral artery) were cannulated and flushed with a solution of warm tap water and non-oxidative laundry detergent (OMO®, Unilever, London, the UK) until clear liquid was recovered from the venous and arterial outflow. This process reduced odor and resolved post-mortem blood clots. The specimens were flushed with the same solution before restoring them, as low temperatures could cause crystallization of remnant contrast material [16].

Computed tomography

After several initial experiments, it was decided to subject the specimens were subjected to computed tomography (CT) scans prior to endovascular use. These scans allowed the identification of arterial pathology, calcifications and stenosis to select appropriate specimens for specific training or research purposes based on vascular pathology, as the medical records of the donors were sealed. The CT scans were acquired with a 64-detector CT scanner (IQon spectral CT, Philips®, Best, The Netherlands) using thin slice, high-dose settings to maximize image quality (slice thickness; 1 mm, spiral pitch; 0.39, tube voltage; 140 kVp, 100–400 mAs).

Endovascular interventions

The procedures were conducted in an experimental hybrid OR, equipped with a Philips Allura Xper FD20 fluoroscopy system (Philips Healthcare, Best, the Netherlands) and power injector (Mark 7 Arterion, Medrad, Whippany, USA). A vascular surgeon with > 5 years of experience in endovascular interventions performed the endovascular tasks.

Preparation

A 7 Fr introducer sheath was advanced in the femoral artery to provide either retrograde (torsos) or antegrade (lower extremities) arterial access, as shown in Fig 2. The sheath was secured to surrounding tissue with a nylon string or clamp. In all specimens, fluid leakage was prevented by ligation of the main collateral arteries in the dissection plane(s). Additionally, in the torsos, the contralateral femoral artery was clamped and the supratruncal aorta was occluded with a 40x30mm aortic valvuloplasty balloon (Z-MED; NuMED Inc®, Hopkinton). Within these restricted vascular segments, intraluminal pressure was created by injection 50cc (lower extremities) or 100cc (torso) of tap water through the sheath. Fig 1 provides a schematic overview of the setup in the torsos and lower extremities.

Fig 2

Cannulation of the femoral artery of a lower extremity.

A 7Fr sheath is used to create antegrade access of the lower extremity through the vacant femoral artery (white arrow) in the axial dissection plane.

Procedures

Various endovascular procedures were performed using this fresh-frozen human cadaver model, both for both training and research purposes. These procedures included balloon angioplasty, stenting, and image optimization of cone beam computed tomography scans (CBCT). Throughout these experiments, the vascular patency of the fresh frozen cadavers was documented by means of cannulation of selected target vessels. These vessels comprised the celiac artery, superior mesenteric artery and left and right renal arteries in the torsos, and the anterior tibial artery, posterior tibial artery and peroneal artery in the lower extremities. These cannulations are comparable to the tasks performed during complex aortic interventions and peripheral interventions. In case of cannulation failure, a distinction was made between failure due to flaws in the cadaver model or failure due to steno-occlusive vascular disease.

Digital subtraction angiography

Digital subtraction angiography (DSA) was acquired using the following protocols:

DSA protocol lower extremity. Manual injection of 50 cc tap water through a Berenstein or Straight flush catheter to generate adequate intraluminal pressure, followed by injection of 20 cc of diluted (ratio 1:5) contrast agent (Ultravist® 300 mg/mL, Bayer Healthcare Pharmaceuticals Inc. Wayne, Germany). Subsequently, 50cc of tap water is injected to dilute and flush the remnant contrast material from the arteries.

DSA protocol torso. Manual injection of 100 cc tap water through a pigtail catheter to generate adequate intraluminal pressure, followed by injection of 120 cc diluted (ratio 1:5) contrast agent (Ultravist 300mg/mL) at 20 cc/s using a power injector. Remnant contrast material was flushed with 100 cc of tap water.

Results

Feasibility of this model is based on our institute’s experiences throughout the use of 6 fresh frozen human cadaver torsos and 22 lower extremities.

Pre-procedural unenhanced computed tomography (CT) scans were available of 15 of the 22 lower extremities (68%) and 4 of the 6 torsos (66%). The CT scans allowed evaluation of the state of the vascular wall and arterial lumen, as visualized in Fig 3. The two most common port-mortem CT artefacts were decomposition artefacts (e.g. gas formation in the intestine and liver) and artefacts due to anatomical dissection (presence of air within vessels and compression of the aorta, iliac arteries and veins). However, none of these artefacts decreased the diagnostic value of the scan in terms of vascular disease.

Fig 3

Unenhanced CT of frozen lower extremity of a human cadaver.

The vein and artery are indicated with an arrow with ‘A’ and ‘V’ respectively. Differentiation between arterial lumen, vascular wall and calcifications are possible.

The endovascular human cadaver model provided a realistic simulation setting for a variety of endovascular procedures. DSA imaging was realistic as shown in Fig 4. Air bubbles were visible in 23 of the 92 DSA’s (25%), caused by insufficient prefilling of the vasculature (Fig 5). These bubbles had minimal impact on the diagnostic or navigational value of the DSAs, as the outline of the arteries remained visible.

Fig 4

DSA imaging of A) the trifurcation and crural arteries of a specimen with patent arteries, B) the trifurcation and crural arteries of a specimen with systemic atherosclerosis and C) the abdominal aorta and its visceral and renal branches.

Fig 5

Air bubbles during angiography.

Presence of air bubbles near the trifurcation, indicated with an arrow.

The cadaver model demonstrated a high vascular patency. The celiac artery, superior mesenteric artery, and left and right renal artery could be successfully cannulated in all six torsos (100%). Cannulation of the posterior tibial artery, anterior tibial artery and peroneal artery was successful in 20 (91%), 21 (95%) and 19 (86%) specimens respectively. All six cases of cannulation failure were attributed to high grade stenosis or occlusions of the target vessel, as confirmed by their lack of patency on DSA imaging or, when CT-imaging was available, by presence of local arterial calcifications. More information on these methods can be found in S2 Table.

No visible tissue distortion or swelling of the specimen was observed, even after multiple hours of use. However, venous outflow and sheath leakage were noticeable. In the torsos, partial collapse of the infra-renal aorta was noticed after approximately half an hour, that impaired guidewire manipulation. The full range of guidewire motion could be retrieved by the injection of 100cc of tap water in the restricted arterial segment.

Discussion

Fresh frozen human cadavers form a suitable model to evaluate new endovascular devices and techniques or master endovascular skills. Human cadavers offer lifelike conditions with representative anatomy, pathophysiology and tactile feedback.

In contrast to previously reported (human) cadaver models [11–14, 17–20], the current model does not employ arterial circulation. Arterial circulation is undeniably relevant for research that concerns hemodynamic processes such as stent deployment, or when maximal expansion of the arteries is desired to allow high-calibre devices. However, many research and training objectives may suffice in a model without circulation, especially since there are several disadvantages to post-mortem reperfusion. Extensive preparation of cadaver and fluid conduit, and an expensive extracorporeal pump are needed to generate stable circulatory flow and prevent conduit leakage. Extravasation of reperfusion fluid into the interstitial space and abdominal cavities forms another issue, as post-mortem decay increases the vascular permeability. Arterial circulation is therefore only sustainable for a limited period of time, and with low blood pressure levels, before resulting in massive edema and distortion of the soft tissues and organs [11–14, 18, 19].

In the current model, arterial circulation was omitted without compromising vessel patency or DSA quality. This allows us to maintain the anatomical and pathophysiolocal fidelity of fresh frozen human cadavers, while using less preparation time, fewer resources and causing significantly less edema than the existing cadaver reperfusion models.

This endovascular human cadaver model has several limitations. To start, the intraluminal pressure was not measured in these specimens. Arterial patency was assumed when guidewires and catheters could be manoeuvred freely, without movement restrictions due to arterial compression. These subjective observations were based on tactile feedback of the devices, and their fluoroscopic feedback. However, maximum expansion of the arteries may not have been obtained, which may limit the use of large calibre devices.

Despite the existence of plenty of thriving body donation programs, it may be difficult to acquire the fresh frozen human cadavers. When available, the cadavers should be used as effectively as possible, as the preservation duration of fresh frozen tissue is limited. Our institution stimulates purposeful use of the cadavers by separating torso, head and extremities for use throughout different medical disciplines.

The specimen used in this study were reused for maximal four times. Chemical body preservation methods could extend the durability of the cadavers. However, fresh frozen preservation is the most lifelike conservation method currently available to preserve flexibility, color and texture of all tissues [21]. Chemical preservation using Thiel’s method [22] provides similar levels of tissue flexibility, but may affect the elasticity of the arteries in a similar way as previously reported in tendons and ligaments [23, 24].

Ideally, the vascular anatomy of the cadaveric specimen should match the intended research or training purpose to maximize the fidelity of the endovascular simulation. The privacy of donors is highly respected and therefore our all medical records, including cause of death, are sealed to secure the donors anonymity. However, this policy may be different in other institutions. Without medical records, we were forced to acquire CT scans of the frozen specimen to assess their vascular status. On CT assessment, plenty of lower extremities contained arteries with atherosclerosis and calcifications, however more uncommon pathology may be more difficult to find.

Conclusions

The endovascular fresh frozen human cadaver model presented in this study allows realistic simulation of endovascular procedures in the aorta and peripheral arteries. This model is suitable for preclinical research and development of endovascular technology, and to gain practical experience and master endovascular skills.

Baseline characteristics of body donors.

Gender and age of the donated specimens.

(DOCX)

Click here for additional data file.

Target vessel cannulation success.

Table summarizing target vessel cannulation success in all specimens, including information on angiographic visibility of the target vessel and calcification of the target vessel.

(DOCX)

Click here for additional data file.

1 Jul 2020

PONE-D-20-02240

Feasibility of fresh frozen human cadavers as a research and training model for endovascular image guided interventions

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Reviewer #1: The study is relevant since it addresses the question of finding suitable models for testing of new clinical methods and training - endovascular procedures specifically. There are just a few things that needs to be corrected/clarified in the manus:

Reg. Introduction: It seems like reference no 11 has fallen out here?

Reg. Cadaver preparation and storage: The specimens were used for a maximum of four freeze-thaw cycles. The authors need to describe in short how the increasing deterioration affects the procedures and outcome - is it the cannulation, the injection, the scanning or the results that are affected?

Reg. Digital subtraction angiography: It is described that the injection of tap water is to generate adequate intraluminal pressure. If the pressure was not measured, how was it possible to determine if the inserted volume was sufficient? Also, the sentence "Remnant contrast material was flushed with 50 cc of tap water" is not clearly understandable. This needs to be rephrased or explained in further detail.

Reg: Results: "No edema and third space fluid were observed" - was that observed by the use of scanning? Venous outflow and sheath leakage can be observed by the naked eye, but that might not be the case with edema.

Reviewer #2: Jasen MM et al., reported new method using frozen human cadavers for evaluating results of EVT or training EVT. This study is very unique. However, there are some limitation in this manuscript.

1. As the authors mentioned, this model is to evaluate results of EVT. However, no post-EVT images were provided. Did the lesions show effect of treatment which was supposed to be observed? When those effect are detected and able to be evaluated, we can say this model is established. Therefore, this reviewer would like to know how we can evaluate pre- and post- EVT results using clinical CT. Please provide those information with images or data etc.

2. How did the authors collect donors? Were there any criteria to collect cadavers, e.g., age, DM, HT, smoking? No table showing donor characteristics was provided in this manuscript. Please provide it.

3. Did authors monitor pressure in vessels to pretend actual condition of interventions? Also, what type of pressure did the authors use in this model? Steady flow or pause wave? Do authors think these differences matter to pretend actual clinical settings?

Reviewer #3: To the authors,

In this paper, Jansen et al. proposed a fresh frozen human cadaver model for research and training of endovascular procedure. This model does not need extracorporeal pump and only needs tap water, some sheath introducers, aortic valvuloplasty balloon. The authors performed endovascular procedure including the acquisition of DSA, balloon angioplasty, stenting, and mechanical atherectomy. The author reported no edema and third spacing fluid collection, even after multiple hours of use. The author concluded that their fresh frozen human cadaver model allows realistic simulation of endovascular procedures in the aorta and peripheral arteries and is suitable for preclinical research and development of endovascular technology, and to practice endovascular skills. The cadaver model presented this manuscript seems reasonable, but this manuscript has several concerns as follows.

1. The author performed POBA, stenting, and atherectomy in the cadaver model. But there is no explanation about these procedures in detail. Please describe the results of these procedures in the cadaver model. Which procedure is good or bad for the cadaver model?

2. Experience from our institution, we cannot keep lower limb artery open appropriately without perfusion because of compression of limb muscles. Enough expansion is needed to perform some procedures, such as an atherectomy and intravascular imaging. Please describe the result of atherectomy procedures. If the authors performed, please provide information about intravascular imaging.

3. On page 17, line 21, “The specimen used in this study could be reused a maximum of four times.” How many times did the authors perform procedure per specimen? Please provide this information.

4. Please describe characteristics of the specimens, such as risk factors, cause of death, past history of peripheral artery disease, and the prevalence of stenotic lesions in the cadaver model.

5. On page 15, line 175, “No edema and third spacing fluid were observed, even after multiple hours of use.” This sentence is based on the subjectivity of the author. Please show the data indicating no edema and no third spacing fluid collection.

6. On page 15, line 174, “available CTA or DSA imaging.“ CT ‘angiography’ is an inadequate word because the authors only performed plane CT in the post-mortem CT.

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Reviewer #1: Yes: Lene Warner Thorup Boel

Reviewer #2: No

Reviewer #3: No

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11 Aug 2020

[Formatted .docx file with response to the reviewers is attached as an additional file (Response to Reviewers)]

________________________________________

Reviewer #1:

The study is relevant since it addresses the question of finding suitable models for testing of new clinical methods and training - endovascular procedures specifically. There are just a few things that needs to be corrected/clarified in the manus:

Reg. Introduction: It seems like reference no 11 has fallen out here?

Response: Sorry for this mistake. The error with the references is corrected.

Response: Corrected ref. numbers in [Line 53 and 56]

Reg. Cadaver preparation and storage: The specimens were used for a maximum of four freeze-thaw cycles. The authors need to describe in short how the increasing deterioration affects the procedures and outcome - is it the cannulation, the injection, the scanning or the results that are affected?

Response: We used the specimens a maximum of 4 times (+ a CT scan with the specimen in frozen condition). At this time, tissue generation began to take a toll; muscular tissues in the upper leg began to deteriorate, and the smell increased. In terms of endovascular use, nothing was noticed; cannulation, injection and results remained similar. In theory, the injected fluids may extravasate faster after deterioration of the arteries, but we noticed no ‘leakage’ of contrast material after angiography.

As our anatomy department intended to use several of the lower extremities for a dissection class, we decided not to use the specimens more than 4 times, to maintain their usability during the dissection class. We rephrased and elaborated on this in the manuscript.

Alterations in manuscript: elaborated on prolonged use:

“The specimens were used for a maximum of four freeze-thaw cycles, due to deterioration of the soft tissues after prolonged use. No difference in quality of the specimen for endovascular use was noticed between first and last use of the specimen.” [line 96-98]

Reg. Digital subtraction angiography: It is described that the injection of tap water is to generate adequate intraluminal pressure. If the pressure was not measured, how was it possible to determine if the inserted volume was sufficient? Also, the sentence "Remnant contrast material was flushed with 50 cc of tap water" is not clearly understandable. This needs to be rephrased or explained in further detail.

Response: You are right, we used a rather subjective method: we simply injected tap water in the arterial vascular tree to increase the intraluminal pressure to maintain patent traversable arteries. In practice this meant that we were able to freely manoeuvre guidewire and catheters without noticing restriction in movement due to arterial compression (both in terms of tactile feedback, and the ‘smoothness’ of device movement on fluoroscopy). We did not measure the intraluminal pressure and thus, we have no sight on real-time intraluminal pressure, nor on intraluminal pressure decline during use of the model. This could be a future improvement for the use of this model. We added this limitation in the discussion.

Furthermore, we elaborated on the ‘flushing’ of contrast material after angiography.

Alterations in manuscript:

• Added section in the discussion: [line 227-232]

“This endovascular human cadaver model has several limitations. To start, the intraluminal pressure was not measured in these specimens. Arterial patency was assumed when guidewires and catheters could be manoeuvred freely, without movement restrictions due to arterial compression. These subjective observations were based on tactile feedback of the devices, and their fluoroscopic feedback. However, maximum expansion of the arteries may not have been obtained, which may limit the use of large calibre devices.”

• Elaborated on ‘flushing of remnant contrast material’: [line 154-155].

“Subsequently, 50cc of tap water is injected to dilute and flush the remnant contrast material from the arteries.”

Reg: Results: "No edema and third space fluid were observed" - was that observed by the use of scanning? Venous outflow and sheath leakage can be observed by the naked eye, but that might not be the case with edema.

Response: You are right, we were unable to perform accurate assessment, as the cadaveric specimens were not dissected, nor scanned directly after their use as cadaver model. We adjusted the text to:

Adjustment in Manuscript:

“No edema and third spacing fluid were visible tissue distortion, or swelling was observed.” [line 192]

________________________________________

Reviewer #2:

Jansen MM et al., reported new method using frozen human cadavers for evaluating results of EVT or training EVT. This study is very unique. However, there are some limitation in this manuscript.

1. As the authors mentioned, this model is to evaluate results of EVT. However, no post-EVT images were provided. Did the lesions show effect of treatment which was supposed to be observed? When those effect are detected and able to be evaluated, we can say this model is established. Therefore, this reviewer would like to know how we can evaluate pre- and post- EVT results using clinical CT. Please provide those information with images or data etc.

Response: The main intent of this article was to document our experience and gained knowledge of this ‘non-perfused’ fresh frozen cadaver models. To help peers, and spread awareness of human cadaver models and their feasibility for all sorts of endovascular applications.

Within our institute we have been using this cadaver model for years for all sorts of endovascular applications, to our own satisfaction. As we could not find a similar methodology in literature, we decided to document our methods and considerations in this manuscript.

Throughout the last 7 years we have used this human cadaver model a couple of times for a wide range of applications, including:

• Balloon angioplasty and stenting with novice vascular surgeons in training.

• Target vessel catheterization of visceral, renal and peripheral vessels with vascular surgeons in training.

• Target vessel catheterization to assess the feasibility of a new technique to provide 3D device tracking and navigation.

• Use of new atherectomy devices, followed by vessel excision and visual assessment of damage to the intimal and medial layer of the vessel.

• Optimization of 3D cone beam computed tomography settings for:

o Visualization of the visceral and renal arteries during fenestrated or branched aortic repair.

o Visualization of calcifications in the peripheral arteries to improve guidance during revascularization of chronic total occlusions.

• Assessment of scatter radiation during target vessel cannulation using different methods of radiation protection (drapes, shields, none).

At the time of execution, there was no intend for publication, and the documentation of the experiments was diverse. Most experiments were focussed on gaining clinical experience with certain tasks or devices, and thus the assessment of the cadaver model was scarcely documented and subjective. Imaging data from the older experiments were lacking.

For the draw-up of this manuscript, we were limited to the well-documented sessions (including imaging), which comprised of these 22 lower extremities and 6 torsos. Moreover, we focussed on the two important simulation qualities that could be assessed objectively:

• Patency of the arteries of the cadaver model � using target vessel cannulation success as a parameter

• Quality of angiography in the cadaver model � assessment of DSA quality

Alterations in manuscript:

Altered aim of the study:

• Abstract: [line 17 - 29]

“Objective: To develop describe the feasibility of a fresh frozen human cadaver model for research and training model for endovascular image guided procedures in the aorta and lower extremity.

Methods: Six fFresh frozen human cadaver torsos and 22 lower extremities were used to construct the endovascular model. were selected for model development and feasibility testing. Endovascular access was acquired by inserting a sheath in the femoral artery. The arterial segment of the specimen was restricted by ligation of collateral arteries and, in the torsos, clamping of the contralateral femoral artery and balloon occlusion of the supratruncal aorta. Tap water was administered through the sheath to create sufficient intraluminal pressure to manipulate devices and acquire digital subtraction angiography (DSA). Endovascular cannulation tasks of the visceral arteries (torso) or the peripheral arteries (lower extremities) were performed to assess the vascular patency of the model. Feasibility of this model is based on our institutes experiences throughout the use of six fresh frozen human cadaver torsos and 22 lower extremities.”

• Introduction: [Line 58 - 62]

“In contrast to these human cadaver reperfusion models, The aim of this study was to develop a human cadaver model without arterial circulation. In this study, we present our human cadaver model, and aim to raise general awareness of the existence and feasibility of human cadaver models for endovascular simulation in the abdominal aorta and peripheral arteries.”

2. How did the authors collect donors? Were there any criteria to collect cadavers, e.g., age, DM, HT, smoking? No table showing donor characteristics was provided in this manuscript. Please provide it.

Response: The donors all came in via the anatomy department of our institute. Written informed consent was obtained from the donors during life. After they passed away, all medical records were sealed to secure the anonymity of the donors. The only demographics that remained accessible were their age, gender, and a serological information (post-mortem tests for HIV, hepatitis A, B etc). We were therefore unable to provide a full table with baseline characteristics. We did, however, add the standard deviation of the donors age.

The reason why we were unable to provide baseline characteristics is mentioned in line 70-72: “Age, gender and serology report of the donors were provided. Other demographics and medical details were sealed to assure the anonymity of the donors, as according to our institution’s policy.”

Alterations in manuscript:

• Added the origin of the donors:

“Body donations were acquired directly by our institute’s anatomy department.” [line 66-67]

• Added standard deviation of the donors age:

“Donors were predominantly male (74%) with a mean age of 74.4 ±.8.8” [line 74-75]

3. Did authors monitor pressure in vessels to pretend actual condition of interventions? Also, what type of pressure did the authors use in this model? Steady flow or pause wave? Do authors think these differences matter to pretend actual clinical settings?

Response: The lack of pressure measurement and the lack of pulsatile arterial blood-flow are drawbacks of this model, compared to the existing cadaver reperfusion models. However, the simplicity of this model is also its strength.

While we do believe that continuous pulsatile flow would provide the most realistic simulation setting, we believe that such a setup would have serious drawbacks in terms of leakage, oedema and third space fluid. The six articles in which reperfusion with continuous pulsatile flow was used, describe arterial pressures ranging between 30mmHg (Willaert et al. 2016) of 80mmHg (Carey et al. 2014).1-6 None of which would be clinically representable, and all of which were accompanied with mentions of rapid extravasation of perfusion fluids and noticeable distortion of solid organs and soft tissues. In some cases, even so severe that the experiment had to be terminated early.6 Moreover, the materials used to recreate pulsatile flow are costly and the necessary preparation of the cadaver is lengthy.

Instead of active, continuous reperfusion, we simply injected tap water in the arterial vascular tree to increase the intraluminal pressure to maintain patent traversable arteries. In practice this meant that we are able to freely manoeuvre guidewire and catheters without noticing restriction in movement due to arterial compression (based on tactile feedback, and on the smoothness of device movements on fluoroscopy). We did not measure the intraluminal pressure and thus, we have no sight on real-time intraluminal pressure, nor on intraluminal pressure decline during use of the model. In our opinion, however, this model functioned properly and we came to believe that the simplicity of this model is actually its strength as it makes the model more accessible for institutions with limited resources.

1. Willaert W, Tozzi F, Van Hoof T, Ceelen W, Pattyn P, D’Herde K. Lifelike vascular reperfusion of a thiel-embalmed pig model and evaluation as a surgical training tool. Eur Surg Res. 2016;56(3–4):97–108.

2. Carey JN, Minneti M, Leland HA, Demetriades D, Talving P. Perfused fresh cadavers: Method for application to surgical simulation. Am J Surg [Internet]. 2015;210(1):179–87. Available from: http://dx.doi.org/10.1016/j.amjsurg.2014.10.027

3. Chevallier C, Willaert W, Kawa E, Centola M, Steger B, Dirnhofer R, et al. Postmortem circulation: A new model for testing endovascular devices and training clinicians in their use. Clin Anat. 2014;27(4):556–62.

4. Garrett HE. A human cadaveric circulation model. J Vasc Surg [Internet]. 2001;33(5):1128–30. Available from: http://linkinghub.elsevier.com/retrieve/pii/S0741521401590898

5. Arbatli H, Cikirikcioglu M, Pektok E, Walpoth BH, Fasel J, Kalangos A, et al. Dynamic Human Cadaver Model for Testing the Feasibility of New Endovascular Techniques and Tools. Ann Vasc Surg [Internet]. 2010;24(3):419–22. Available from: http://dx.doi.org/10.1016/j.avsg.2009.04.001

6. Willaert W, De Somer F, Grabherr S, D’Herde K, Pattyn P. Post-mortem Reperfusion of a Pig: a First Step to a New Surgical Training Model? Indian J Surg. 2013;77(December):1–4.

Adjustments in the manuscript:

• Added a section in the discussion on the lack of pressure measurement: [line 227-232]

“To start, the intraluminal pressure was not measured in these specimens. Arterial patency was assumed when guidewires and catheters could be manoeuvred freely, without movement restrictions due to arterial compression. These subjective observations were based on tactile feedback of the devices, and their fluoroscopic feedback. However, maximum expansion of the arteries may not have been obtained, which may limit the use of large calibre devices.”

________________________________________

Reviewer #3:

To the authors,

In this paper, Jansen et al. proposed a fresh frozen human cadaver model for research and training of endovascular procedure. This model does not need extracorporeal pump and only needs tap water, some sheath introducers, aortic valvuloplasty balloon. The authors performed endovascular procedure including the acquisition of DSA, balloon angioplasty, stenting, and mechanical atherectomy. The author reported no edema and third spacing fluid collection, even after multiple hours of use. The author concluded that their fresh frozen human cadaver model allows realistic simulation of endovascular procedures in the aorta and peripheral arteries and is suitable for preclinical research and development of endovascular technology, and to practice endovascular skills. The cadaver model presented this manuscript seems reasonable, but this manuscript has several concerns as follows.

1. The author performed POBA, stenting, and atherectomy in the cadaver model. But there is no explanation about these procedures in detail. Please describe the results of these procedures in the cadaver model. Which procedure is good or bad for the cadaver model?

Response: The main intent of this article was to document our experience and gained knowledge of this ‘non-perfused’ fresh frozen cadaver models. To help peers, and spread awareness of human cadaver models and their feasibility for all sorts of endovascular applications.

Within our institute we have been using this cadaver model for years for all sorts of endovascular applications, to our own satisfaction. As we could not find a similar methodology in literature, we decided to document our methods and considerations in this manuscript.

Throughout the last 7 years we have used this human cadaver model for a wide range of applications, including:

• Balloon angioplasty and stenting with novice vascular surgeons in training.

• Target vessel catheterization of visceral, renal and peripheral vessels with vascular surgeons in training.

• Target vessel catheterization to assess the feasibility of a new technique to provide 3D device tracking and navigation.

• Use of new atherectomy devices, followed by vessel excision and visual assessment of damage to the intimal and medial layer of the vessel.

• Optimization of 3D cone beam computed tomography settings for:

o Visualization of the visceral and renal arteries during fenestrated or branched aortic repair.

o Visualization of calcifications in the peripheral arteries to improve guidance during revascularization of chronic total occlusions.

• Assessment of scatter radiation during target vessel cannulation using different methods of radiation protection (drapes, shields, none).

At the time of execution, there was no intend for publication, and the documentation of the experiments was diverse. Most experiments were focussed on gaining clinical experience with certain tasks or devices, and thus the assessment of the cadaver model was scarce and subjective. Imaging data from the older experiments were lacking, due to migration of the experimental imaging database.

For the draw-up of this manuscript, we were limited to the well-documented sessions (including imaging), which comprised of these 22 lower extremities and 6 torsos. Moreover, we focussed on the two important simulation qualities that be established objectively:

• Patency of the arteries of the cadaver model � using target vessel cannulation success as a parameter

• Quality of angiography in the cadaver model � assessment of DSA quality

The issues with subjectivity and lack of proper documentation in the earlier experiments refrained us from specific commentary on the suitability of these devices/procedures in the cadaver model.

Alterations in manuscript:

Altered aim of the study:

• Abstract: [line 17 - 29]

“Objective: To develop describe the feasibility of a fresh frozen human cadaver model for research and training model for endovascular image guided procedures in the aorta and lower extremity.

Methods: Six fFresh frozen human cadaver torsos and 22 lower extremities were used to construct the endovascular model. were selected for model development and feasibility testing. Endovascular access was acquired by inserting a sheath in the femoral artery. The arterial segment of the specimen was restricted by ligation of collateral arteries and, in the torsos, clamping of the contralateral femoral artery and balloon occlusion of the supratruncal aorta. Tap water was administered through the sheath to create sufficient intraluminal pressure to manipulate devices and acquire digital subtraction angiography (DSA). Endovascular cannulation tasks of the visceral arteries (torso) or the peripheral arteries (lower extremities) were performed to assess the vascular patency of the model. Feasibility of this model is based on our institutes experiences throughout the use of six fresh frozen human cadaver torsos and 22 lower extremities.”

• Introduction: [Line 58 - 62]

“In contrast to these human cadaver reperfusion models, our institution has been using human cadaver models without arterial reperfusion, to our satisfaction. The aim of this study was to develop a human cadaver model without arterial circulation. In this study, we present our human cadaver model, and aim to raise general awareness of the existence and feasibility of human cadaver models for endovascular simulation in the abdominal aorta and peripheral arteries.”

2. Experience from our institution, we cannot keep lower limb artery open appropriately without perfusion because of compression of limb muscles. Enough expansion is needed to perform some procedures, such as an atherectomy and intravascular imaging. Please describe the result of atherectomy procedures. If the authors performed, please provide information about intravascular imaging.

Response: Interesting comment! As mentioned, our specimens were dissected at mid femoral level, in which it may be easier to maintain open vessels than in a full body cadaver, which may explain those differences.

After thorough flushing and injection of 50cc of tap water through a sheath in the femoral artery, the arteries and were patent and traversable enough to freely manipulate guidewires and catheters, without noticing arterial compression. However, we must admit that we did not perform a lot of procedures in which high calibre devices were used, which require maximum arterial expansion.

During our early experiments, we once performed successful atherectomy of a femoral artery using a SilverHawk device (Medtronic). Multiple atherectomy passes were performed, and the femoral artery was later dissected to visually check damage to the intimal and medial layer. However, data collection from this experiment was meagre and relied on subjective experiences as, at that moment, there was no intention for publication.

We have not used intravascular ultrasound, nor intravascular optical coherence tomography, as we do not have these systems available in our experimental lab, and because we do not frequently use these devices in daily clinical practice.

We understand that readers would like to have more information and accompanying figures to support the use of the atherectomy device. As we are unable to provide detailed information, we deleted the mention of atherectomy from the text.

Adjustment in Manuscript:

• Added this as a limitation in the discussion [line 227 -232]

“This endovascular human cadaver model has several limitations. To start, the intraluminal pressure was not measured in these specimens. Arterial patency was assumed when guidewires and catheters could be manoeuvred freely, without noticing movement restriction due to arterial compression. These subjective observations were based on tactile feedback of the devices, and their fluoroscopic feedback. However, maximum expansion of the arteries may not have been obtained, which may limit the use of large calibre devices.”

• Removed the mention of atherectomy, as there is limited objective data/imaging available from this experiment to support this claim. [line 137-138]

“Various endovascular procedures were performed using this fresh-frozen human cadaver model, both for both training and research purposes. These procedures included: balloon angioplasty, stenting and atherectomy and image optimization of cone beam computed tomography scans (CBCT). Throughout these experiments, the vascular patency of the fresh frozen cadavers was documented by means of cannulation of selected target vessels.“

3. On page 17, line 21, “The specimen used in this study could be reused a maximum of four times.” How many times did the authors perform procedure per specimen? Please provide this information.

Response: Actually, we used the specimens a maximum of 4 times (+ a CT scan). At this time, tissue generation began to take a toll; muscular tissues in the upper leg began to deteriorate, as did the smell. As our anatomy department intended to use several of the lower extremities for a dissection class, we decided not to use the specimens more than 4 times, to maintain their use during the dissection class. We rephrased and elaborated on this statement.

Adjustments in Manuscript:

• Adjusted statement in discussion: [line 238]

“The specimen used in this study were reused for maximal four times.”

• Adjusted Materials and Methods section: [line 96-97]

“The specimens were used for a maximum of four freeze-thaw cycles, due to deterioration of the soft tissues after prolonged use. No difference in quality of the specimen for endovascular use was noticed between first and last use of the specimen.”

4. Please describe characteristics of the specimens, such as risk factors, cause of death, past history of peripheral artery disease, and the prevalence of stenotic lesions in the cadaver model.

Response: The specimens were originated from the anatomy department of our institute. In our institute, all medical records are sealed after body donation, to secure the anonymity of the donors. The only demographics that remained accessible were their age, gender, and a serological information (post-mortem tests for HIV, hepatitis A, B etc). We were therefore unable to provide a full table with baseline characteristics. This is mentioned in line 70-72 (“Age, gender and serology report of the donors were provided. Other demographics and medical details were sealed to assure the anonymity of the donors, as according to our institution’s policy.”)

Adjustments in Manuscript: Added an additional section in the Discussion to put more emphasis on this issue:

“The privacy of donors is highly respected and therefore our all medical records, including cause of death, are sealed to secure the donors anonymity. However, this policy may be different in other institutions. Without medical records, we were forced to acquire CT scans of the frozen specimen to assess their vascular status.” [line 245-248]

5. On page 15, line 175, “No edema and third spacing fluid were observed, even after multiple hours of use.” This sentence is based on the subjectivity of the author. Please show the data indicating no edema and no third spacing fluid collection.

Response: You are right, this is not an accurate assessment, as the cadaveric specimens were not dissected, nor scanned directly after their use as cadaver model. We adjusted the text to:

Adjustment in Manuscript: Adjustment in the Result section:

“No visible tissue distortion, or swelling was observed.” [line 192]

6. On page 15, line 174, “available CTA or DSA imaging.“ CT ‘angiography’ is an inadequate word because the authors only performed plane CT in the post-mortem CT.

Response: Adjusted this sentence

Adjustment in manuscript: [line 188-191]

“All six cases of cannulation failure were attributed to high grade stenosis or occlusions of the target vessel, as confirmed by DSA imaging, or when available, by local arterial calcifications on CT imaging, indicating a chronic total occlusion.”

Submitted filename: PONE-D-20-02240 - Response to Reviewers Rev1.docx

Click here for additional data file.

24 Sep 2020

PONE-D-20-02240R1

Feasibility of fresh frozen human cadavers as a research and training model for endovascular image guided interventions

PLOS ONE

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Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #3: Yes

**********

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #3: Yes

**********

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #3: Yes

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: This reviewer does not have further comments. The manuscript was corrected based on our suggestions and questions.

Reviewer #3: The manuscript was well revised and I have only minor comments as follows.

1. Regarding the supporting information, the excel files should be summarized using tables, not raw data (e.g., how many cases were documented as ‘Angiographically visible’ etc…). Also, please provide the abbreviations list for the supporting information.

2. I think if supporting information would be refined, it would be nice information for the readers. Please define the definitions for ‘Angiographically visible’ and ‘calcification visible’.

**********

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Reviewer #1: Yes: Lene Warner Thorup Boel

Reviewer #2: No

Reviewer #3: No

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29 Sep 2020

Dear editor and reviewers,

Thank you for your comments based upon the first revision of the manuscript PONE-D-20-02240. In this second revision we have thoroughly revised the supporting information, as elaborated on in the comments below.

We look forward to hearing from you regarding this submission. We would be glad to respond to any further questions and comments that you may have.

On behalf of all authors,

Marloes Jansen, MSc.

PhD-candidate Vascular Surgery

_________________________________________

Review Comments to the Author:

Reviewer #2: This reviewer does not have further comments. The manuscript was corrected based on our suggestions and questions.

Response: Thank you for your suggestions.

_________________________________________

Reviewer #3: The manuscript was well revised and I have only minor comments as follows.

1. Regarding the supporting information, the excel files should be summarized using tables, not raw data (e.g., how many cases were documented as ‘Angiographically visible’ etc…). Also, please provide the abbreviations list for the supporting information.

2. I think if supporting information would be refined, it would be nice information for the readers. Please define the definitions for ‘Angiographically visible’ and ‘calcification visible’.

Response: Thank you for your suggestions. Based on your comments we have revised the supporting information files. Firstly, we deposited the underlying (raw) data at a public data depository (Mendeley Data) as suggested in Plos One’s publication guidelines. The data is accessible with the DOI provided in the reference section. [line 262-263 of the redline document].

Secondly, both supporting information files (S1 and S2) now contain summarization tables, rather than raw data. Both files contain more elaborate background information including a table legend, spelled out abbreviations and definitions.

We reframed the term ‘angiographically visible’ to ‘patent on angiogram’. And provided definitions of the terms ‘patent on angiogram’ and ‘visible calcifications’ in the tables.

Moreover, we have included several example images, to provide a more visual understanding of these definitions. These images include illustration(s) of 1) ‘target vessel cannulation success’, 2) ‘patent on angiogram’ and 3) ‘calcification visible’. [S1 Tables and S2 Tables]

To convey this information to the readers, we referred to the S2 table in the manuscript. [line 183 – 188 of the redline document]

“Cannulation of the posterior tibial artery, anterior tibial artery and peroneal artery was successful in 20 (91%), 21 (95%) and 19 (86%) specimens respectively. All six cases of cannulation failure were attributed to high grade stenosis or occlusions of the target vessel, as confirmed by their lack of patency on DSA imaging or, when CT-imaging was available, by presence of local arterial calcifications. More information on these methods can be found in Supporting Information S2.”

_________________________________________

Submitted filename: PONE-D-20-02240 - Response to Reviewers Rev2.docx

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6 Nov 2020

Feasibility of fresh frozen human cadavers as a research and training model for endovascular image guided interventions

PONE-D-20-02240R2

Dear Dr. Jansen,

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Aloke Finn, MD

Academic Editor

PLOS ONE

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Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #3: Yes

**********

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I have no further comments to the paper after this second review. All the issues addressed by the reviewers have been met.

Reviewer #3: The manuscript has been revised well. The author addressed reviewers comments and made the tables based on the reviewer's suggestion.

**********

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Reviewer #1: Yes: Lene Warner Thorup Boel

Reviewer #3: No

16 Nov 2020

PONE-D-20-02240R2

Feasibility of fresh frozen human cadavers as a research and training model for endovascular image guided interventions

Dear Dr. Jansen:

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