Original Contribution

Original Contribution
Endothelial Trauma From Mechanical Thrombectomy in
Acute Stroke
In Vitro Live-Cell Platform With Animal Validation
Dayu Teng, PhD; Jeffrey Scott Pannell, MD; Robert C. Rennert, MD; Jieying Li, MS;
Yi-Shuan Li, PhD; Victor W. Wong, MD; Shu Chien, MD, PhD; Alexander A. Khalessi, MD, MS
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Background and Purpose—Endovascular thrombectomy has shown promise for the treatment of acute strokes resulting
from large-vessel occlusion. Reperfusion-related injury may contribute to the observed decoupling of angiographic and
clinical outcomes. Iatrogenic disruption of the endothelium during thrombectomy is potentially a key mediator of this
process that requires further study.
Methods—An in vitro live-cell platform was developed to study the effect of various commercially available endovascular
devices on the endothelium. In vivo validation was performed using porcine subjects.
Results—This novel in vitro platform permitted high-resolution quantification and characterization of the pattern and timing
of endothelial-cell injury among endovascular thrombectomy devices and vessel diameters. Thrombectomy devices
displayed heterogeneous effects on the endothelium; the device performance assessed in vitro was substantiated by in
vivo findings.
Conclusions—In vitro live-cell artificial vessel modeling enables a detailed study of the endothelium after thrombectomy
and may contribute to future device design. Large animal studies confirm the relevance of this in vitro system to investigate
endothelial physiology. This artificial vessel model may represent a practical, scalable, and physiologically relevant
system to assess new endovascular technologies. (Stroke. 2015;46:00-00. DOI: 10.1161/STROKEAHA.114.007494.)
Key Words: endothelial cells ◼ stroke
C
erebrovascular disease affects >6 million US adults,1
with these numbers expected to increase as the population ages in the coming decades.2 The cerebrovascular system
is particularly sensitive to injury, with disruptions in cerebral
perfusion being the fourth leading cause of death in the United
States.1 Timely restoration of normal blood flow and reconstitution of cerebral macrovasculature is critical in patients with
cerebrovascular pathology. Endovascular interventions have
shown promise for a variety of cerebrovascular diseases, and
thrombectomy procedures continue to be a subject of ongoing
research and trials for the treatment of ischemic stroke resulting from large-vessel occlusion.3–7
Early clinical trials studying mechanical embolectomy
demonstrated a decoupling of clinical outcomes with the
degree of angiographic reperfusion8–10; these findings may
reflect challenges in patient selection, inadequate reperfusion
with first-generation devices, or reperfusion-related injury.3,7
Clinical trials with second-generation devices are currently
ongoing to elucidate the role of adequate, timely mechanical
reperfusion in patients with large-vessel strokes.4,5 Given this
clinical need, vascular endothelial cell (EC) damage resulting
from altered flow dynamics, reperfusion injury, or iatrogenic
trauma represents a potentially important source of secondary neuronal injury that merits further study.11,12 As modern
thrombectomy devices focus on rapid and complete revascularization, considerations of the degree of EC injury have substantial clinical and design implications.
The cerebral endothelium is a dynamic biological system
that regulates blood brain barrier (BBB) permeability, autoregulates cerebral microcirculation via nitric oxide pathways,
and mediates cerebral inflammation via release of tumor
necrosis factor-α, interleukin 1β, and interleukin 6. We have
previously demonstrated that physiological blood flow and
resultant shear stress are crucial for EC maintenance and
remodeling after injury13–22 and that locally disrupted blood
flow contributes to inflammatory cascades.23 Many of the proposed mechanisms of reperfusion injury after acute ischemic
stroke also originate at the EC level.
Received September 18, 2014; final revision received January 27, 2015; accepted January 28, 2015.
From the Division of Neurosurgery (J.S.P., R.C.R., V.W.W., A.A.K.), Department of Bioengineering and Institute of Engineering in Medicine (D.T., J.L.,
Y.-S., S.C.), University of California, San Diego.
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.114.
007494/-/DC1.
Correspondence to Alexander A. Khalessi, MD, MS, Division of Neurosurgery, University of California, 200 West Arbor Dr, San Diego, CA 92103,
E-mail [email protected] or Shu Chien, MD, PhD, Department of Bioengineering, Institute of Engineering in Medicine, University of California, 9500
Gilman Dr, San Diego, CA 92093, E-mail [email protected]
© 2015 American Heart Association, Inc.
Stroke is available at http://stroke.ahajournals.org
DOI: 10.1161/STROKEAHA.114.007494
1
2 Stroke April 2015
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Breakdown of the BBB in ischemic stroke begins after the
thrombotic disruption of pulsatile flow over the endothelium.
This causes decreased EC expression of vascular endothelial cadherin and β-catenin, which are critical components
of BBB tight junctions. Thrombosis also causes EC release
of proinflammatory cytokines, including tumor necrosis
factor-α, interleukin 1β, and interleukin 6. Together, these
processes facilitate resident glial–cell activation and infiltration of circulating leukocytes, thus increasing the local
release of cytotoxic molecules and exacerbating ischemic
cerebral injury. Decreased nitric oxide production by injured
ECs can also adversely affect distal collateral flow, on
which penumbral tissue relies in the setting of large-vessel
occlusion.24–26
Importantly, the timely restoration of pulsatile flow over the
endothelium without mechanical disruption can promote rapid
endothelial repair and BBB restoration within 24 hours.27,28
However, direct EC injury during mechanical thrombectomy
may exacerbate the activation of deleterious pathways, highlighting the necessity for devices designed to minimize EC
injury.
Thrombectomy devices are currently assessed in glass or
plastic tubing in vitro and in in vivo models before clinical
trials. Although transparent glass and plastic tubing provides
direct luminal visualization, dimension consistency, and ready
availability, these materials are acellular and fail to recapitulate
blood vessel biology. In vivo animal studies are performed on
realistic vessels, but vessel opacity prevents direct observation
of the interaction between the devices and ECs; furthermore,
these vessels vary in dimensions and animal studies are costly.
In vivo vessels must also be sectioned for histopathologic
analysis, and therefore, they cannot provide 3-dimensional
(3D) patterns of injury and are prone to processing artifacts.
To address these problems, we have developed a novel in vitro
live-cell platform that allows for direct visual characterization of EC injury patterns and mechanisms in the setting of
mechanical thrombectomy for acute stroke. The validity of
this platform was confirmed through in vivo porcine experiments. As such, this in vitro model could ultimately augment
or supplant animal experiments as a new approach to assess
endovascular technology.
Materials and Methods
Creation of In Vitro Live-Cell Platform
The in vitro live-cell platform was created briefly as follows (full depiction is given in the Materials and Methods section in the onlineonly Data Supplement). Vessels for the in vitro live-cell platform
were created from optically clear and biocompatible tubular silicone
substrates custom-manufactured by Specialty Silicone Fabrications
(Paso Robles, CA), with a thickness of 0.25 mm and inner diameters
of 2.5, 3.5, and 4.5 mm. A cell suspension of 105 cells/mL of bovine artery ECs was perfused into the platform, with a monolayer
of 103 cells/cm2 achieved before being subjected to bioreactor flow.
Vessels were mounted into the bioreactor chamber (Figure 1A) and
connected to a media reservoir generating peristaltic flow at 30 mL/
min. This flow rate approximates 30% of the normal middle cerebral
artery flow, consistent with partial flow restoration during stent-retriever deployment.29 To confirm the presence of a confluent monolayer and EC alignment in the direction of flow, cells were stained
with a live-cell tracking agent, MitoTracker Red (Life Technologies),
and a nuclear counterstain, Hoechst dye (Sigma-Aldrich). Bright field
Figure 1. Development and validation of in vitro live-cell platform for assessment of thrombectomy device effect on the
endothelium. A, Photograph of bioreactor setup. B, Bright field.
C, MitoTracker. D, Nuclear label imaging with Hoechst staining
demonstrated endothelial cell alignment in the direction of flow,
as indicated by the arrows. Scale bar, 100 μm.
fluorescent MitoTracker Red and Hoechst images were then acquired
(Figure 1B–1D).
Thrombectomy Procedure
In Vitro Platform
Porcine blood clots were introduced into the lumen of the in vitro
live-cell platform under flow stasis and allowed to integrate for 10
minutes. The integrity of the ECs was confirmed before and after the
introduction of the clots (Figure 2A). Various clot-retrieval devices
were then deployed, and after device integration into the clot, the
clots and devices were removed (Figure 2B). The features of the devices interacting with the ECs in the in vitro platform were visualized
(Figure 2C).
Six thrombectomy devices were tested across 3 vessel diameters (2.5, 3.5, and 4.5 mm) in vitro: (1) 5MAX ACE (Penumbra,
Inc, Alameda, CA) with A Direct Aspiration first Pass Technique
(ADAPT), (2) MERCI (3.0 firm; Concentric Medical Inc/Stryker
Corp, Kalamazoo, MI), (3) 5MAX with Separator (Penumbra, Inc),
(4) 5MAX with Separator 3D (Penumbra, Inc), (5) Solitaire FR
(4×20 mm; Covidien, Ltd, Mansfield, OH), and (6) TREVO (Stryker
Corp), n=4 for all experimental conditions and n=3 for controls. A
full description of these experiments is provided in the Materials and
Methods section in the online-only Data Supplement.
In Vivo Vessels
All animal work was performed in accordance with Institutional
Animal Care and Use Committee protocols (surgical details are available in the Materials and Methods section in the online-only Data
Supplement). Two healthy adult pigs were used for in vivo validation
of the live-cell model. After anesthetic induction, femoral artery cut
downs were performed and a large porcine blood clot was introduced
using an 8-French balloon guide catheter into the common carotid
after temporary balloon occlusion.
After the restoration of flow to allow thrombus embedding, a single
pass was made with each thrombectomy device tested. Four thrombectomy devices were tested in vivo: (1) 5MAX ACE (Penumbra,
Inc) in the ADAPT technique, (2) 5MAX with Separator 3D
(Penumbra, Inc), (3) Solitaire FR (4×20 mm; Covidien, Ltd), and (4)
TREVO (Stryker Corp). The Trevo was deployed with the Trevo Pro
18 L microcatheter, and the Solitaire device was deployed through
a Rebar microcatheter. After thrombectomy, the affected vessel was
isolated and excised, and the pigs were euthanized in accordance with
Institutional Animal Care and Use Committee protocols. Importantly,
Teng et al In Vitro Live-Cell Platform for Thrombectomy 3
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Figure 2. Procedure of in vitro live-cell platform for assessment
of thrombectomy device effect on the endothelium.
A, Demonstration of endothelial integrity before (i) and after (ii) clot
introduction. Scale bar, 300 μm. B, Depiction of the experimental
process for in vitro mechanical thrombectomy. C, High-resolution
images of the endothelium demonstrating specific locations of
endothelial cell damage by a thrombectomy device. The proximal effect of a Trevo wire on the endothelium is minimal ([i] wire
imprint indicated by red arrows). This contrasts to the effect of the
distal tip and shoulder portions of the wire on the endothelium,
where large areas of cell loss can be seen ([ii] cell loss visualized as bright spots indicated by blue arrows, wire imprint again
indicated by red arrows). Gross visualization of the Trevo wire (iii)
highlights discussed areas of localization. Scale bar, 200 μm.
the excised portion of vessel was never exposed to wire or a guide
catheter during thrombus placement, and the entirety of the thrombectomy pass was centered within the vessel segment that was ultimately excised for analysis.
For thrombectomy procedures, representatives from each company
were present to certify the proper use of each device.
Histochemistry
In Vitro Platform
After in vitro thrombectomy, vessels from the live-cell platform were
fixed with 4% paraformaldehyde in phosphate buffered saline and
stained with rhodamine phalloidin (Life Technologies) at 1 U/mL in
antibody-diluting buffer containing a nuclear Hoechst dye (SigmaAldrich) at 5 μg/mL in phosphate buffered saline.
In Vivo Vessels
After excision, vessels from in vivo experiments were fixed in 4%
paraformaldehyde in phosphate buffered saline. The fixed vessels were embedded in optimal cutting temperature compound and
Figure 3. Three-dimensional rotational scanning imaging system
for imaging of live endothelium and utility for assessment of device
mechanics. A, Schematic of the rotational-scanning image system developed to capture the entire intact artificial vessel using a
scanning microscope. B, Representative fluorescent whole-vessel
scans of vessels after mechanical thrombectomy. Bright signals
indicate intact endothelial cells stained with MitoTracker Red. Dark
areas represent areas where cells were removed during thrombectomy, with the geometry of the dark area indicating the cell damage patterns induced by the respective clot retrieving devices.
cryosectioned at 10-μm thickness. Sections were stained with a
Hoechst nuclear dye (Sigma-Aldrich) at 5 μg/mL in phosphate buffered saline and an antibody for the EC marker, von Willebrand factor,
at 1:100 dilution (Abcam, Cambridge, MA).
Image Acquisition
In Vitro Platform
A novel rotational-scanning image system was designed to image
the entire intact artificial vessel using a scanning microscope (Nikon,
Melville, NY; Figure 3A). The vessel was sandwiched between a
cover glass and a substrate glass. The opposite but equal motion of
the 2 glasses rotated the vessel over the microscope lens, allowing a
complete scan of the entire vessel without cutting the vessel open.
In Vivo Vessels
Stained sections of vessels from in vivo experiments were imaged
using a scanning fluorescent microscope (Nikon).
Data Analysis
Consecutive images from the scanning of in vitro constructs and in
vivo vessel sections were montaged and reconstructed for 3D visualization using Matlab (Mathworks, Natick, MA). The number of
cells in each in vitro construct was quantified in an unbiased fashion using pattern recognition software (CellProfiler; Broad Institute,
4 Stroke April 2015
Cambridge, MA) and normalized to control, uninjured constructs. A
blinded pathology assessor manually counted the number of endothelial nuclei in the in vivo vessel cross-sections.
Statistics
Data are presented as mean±SEM.
Results
Novel In vitro Live-Cell Platform Enables
Controlled Study of EC Physiology and Real-Time
Injury Monitoring
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To reproducibly study EC biology in the setting of physiological flow, a novel in vitro live-cell platform was developed
(Figure 1A). The viability and behavior of ECs in this system were subsequently tested, with confluent sheets of ECs
shown to align in the direction of bioreactor-induced flow
(Figure 1B–1D). These findings are in line with native EC
physiology, indicating successful recapitulation of in vivo EC
characteristics with this model.
The transparent nature of this platform allowed for realtime visualization of ECs (Figures 1 and 2). Such monitoring
is critical for confirming the EC viability before and after the
introduction of the clots (Figure 2A) and determining specific
locations of EC damage resulting from thrombectomy devices
(Figure 2C).
Device-Specific EC Injury Patterns After
Thrombectomy are Visible Using In vitro Live-Cell
Modeling
We next assessed the effect of commercially available thrombectomy devices on the endothelium using the in vitro live-cell
platform. Constructs with various tube diameters (2.5, 3.5,
and 4.5 mm) were tested to provide information on the spectrum of vessel sizes likely to be encountered in the clinical
setting. Whole-construct fluorescent scans after thrombectomy with 6 different devices (5MAX ACE, Separator, Trevo,
Solitaire, Separator 3D, and Merci) showed clear differences
in the character and degree of EC damage (Figure 3A and 3B).
3D-vessel reconstructions further demonstrated this interdevice variability (Figure 4A; Video I in the online-only Data
Supplement). In these experiments, total cell area was used to
quantify EC density via the cytosolic stain Mitotracker Red,
which is compatible with long-term live-cell experiments. The
direct proportionality of the cell area to nuclei counts suggests
that cell area is an effective method to quantify EC density
(Figure 4B). The in vitro model thus accurately quantified the
magnitude of EC damage and provided important technical
information about the patterns of injury to guide device design
and performance.
The differential effect of specific devices on the endothelium was even more apparent when compared across vessel
sizes (Figure 4C), with the differences being most prominent
in vessels with smaller calibers. Specifically, the Merci device
denuded the largest EC area and served as a positive control.
The Separator 3D denuded the second largest area of ECs on
average. The stent retrievers (Solitaire and Trevo) demonstrated a similar, medium degree of EC denudation across all
vessel diameters; the 5MAX ACE with the ADAPT technique
and the Separator 3D led to the lowest cell areas denuded.
Characteristic injury patterns were observed for different
devices. The Merci retriever caused a nearly complete denudation of the endothelium, whereas the Separator 3D resulted in
a severe linear EC damage pattern. Both injury patterns were
Figure 4. Thrombectomy devices heterogeneously
effect the endothelium. A, Representative 3D reconstructions of full vessel scans. B, Confirmation of
the correlation between fluorescent whole-vessel
scans and endothelial cell density. C, Cumulative
in vitro post-thrombectomy procedure, cell area
comparisons.
Teng et al In Vitro Live-Cell Platform for Thrombectomy 5
most prominent in smaller caliber vessels. Thrombectomy
with each of the stent retrievers resulted in a linear pattern of
excoriation. There were nonetheless differences among these
devices, as the Trevo resulted in a predominantly distal pattern of denudation that was most prominent in smaller vessels.
Conversely, thrombectomy with the Separator 3D resulted in
solitary thin linear excoriations that were similar for all vessel diameters. Finally, thrombectomy with the 5MAX ACE
using the ADAPT technique resulted in a focal circumferential
injury that was similar at each diameter.
These findings highlight the heterogeneity of endothelial
injuries caused by different thrombectomy devices and support the use of the in vitro live-cell model for translational EC
study and device improvements to minimize EC trauma.
In Vitro Live-Cell Modeling of Device-Specific EC
Injury Patterns Can Predict In Vivo Injury
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The predictive value of the in vitro live-cell platform
was assessed through similarly designed in vivo experiments (Figure 5A; Videos II and III in the online-only Data
Supplement). Specifically, the number of intact ECs was
quantified in porcine vessels after thrombectomy procedures
using cross-sectional histology and immunohistochemical
staining for the EC marker von Willebrand factor, with nuclei
separately stained with Hoechst dye.
In agreement with our in vitro findings, quantification of
intact ECs in in vivo experiments revealed that the density of
intact ECs was high in vessels treated with the 5MAX ACE
but lower in the Solitaire, Trevo, and Separator 3D. Also in
agreement with our in vitro experiments, qualitative differences in intact EC patterns were visible across devices in in
vivo cross-sectional analysis (Figure 5B and 5C), although it
was harder to visualize larger injury patterns in vivo. These
data validate the in vivo predictive power of our in vitro model
and highlight the use of high-resolution 3D-vessel reconstructions for the study of EC injury.
Discussion
Mechanical thrombectomy remains an important area of
stroke research for patients with large-vessel occlusion who
are ineligible or refractory to systemic thrombolytic therapy.
Endothelial injury during mechanical thrombectomy may
contribute to the observed decoupling of clinical and angiographic outcomes in ischemic stroke. Mechanical disruption
of the BBB, loss of large-vessel intermediate filament compliance as a functional unit, and the resultant inflammatory
responses initiated at the EC level remain potential sources of
secondary neuronal injury.26–28
Our in vitro model provides novel quantitative and qualitative information about the degree and pattern of injury as it
pertains to device design and vessel diameter. In this model, we
were able to quantitatively evaluate the degree of EC injury in
an intact vessel based on cell area and characterize the pattern
of injury with 3D-rotational scanning microscopy. Subsequent
in vivo validation of device-specific endothelial injuries was
consistent with previous reports examining a limited subset
Figure 5. In vivo experimental design and confirmation of the effect of thrombectomy devices on the
endothelium. A, Depiction of in vivo experimental
setup. The common carotid was accessed with an
8-French balloon guide catheter via a femoral puncture, with radio-opaque markers used to denote the
distal vessel segment not traversed with wire or a
guide catheter (i). The balloon was then inflated to
interrupt arterial flow, and a large porcine blood clot
was injected through the guide catheter (ii). The balloon was then deflated to restore arterial flow and
allow thrombus embedding before thrombectomy
and subsequent vessel excision for processing
(iii). B, Immunohistochemical staining of vessel
cross-sections for the endothelial cell (EC) marker
von Willebrand factor (vWF; green; nuclei stained
blue) redemonstrates the heterogeneity of devices.
Whole-vessel imaging is nonetheless challenging
with in vivo modeling. Scale bar, 500 μm. C, In vivo
post-thrombectomy EC counts display heterogeneity across devices predicted by in vitro studies. The
number of section samples quantified for 5Max,
Trevo, Solitaire, and Separator 3D are n=8, 21, 20,
and 24, respectively.
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of these devices.30–32 These studies also highlight the limitations of animal work for discerning larger patterns of injury
and their relationship with the design and use of each device.
This is because the multicell layer nature and opacity of the
in vivo vessels make transluminal imaging nearly impossible.
Hence, EC evaluation in animal experiments commonly relies
on cross-sectional imaging of the vessels. In such histological studies, factors such as embedding, slicing, and staining
can all induce damage to the ECs and obscure underlying patterns of injury. The sample size, frequency, and location of the
cross-sections can further introduce artifact. Advanced technologies, such as confocal and laser ablation imaging, may
potentially provide full vessel images, but the vessels would
have to be cut or destroyed during the process. The use of
these technologies would further exaggerate the labor-intensive and costly nature of in vivo experimentation. In contrast,
our in vitro model is readily available and cost-effective, while
providing quantitative imaging on live ECs in intact whole
vessels (Table).
In addition, the mechanical information about microscopic
EC injury during device application provided by this platform
cannot be achieved with any existing in vivo methodology.
For example, using our in vitro model, we determined that the
sharp distal taper of the Trevo device results in greater distal
EC injury during device deployment. The device is typically
machined from a tube, and therefore, it harbors a taper that is
eccentric to one side. Our results suggest that a midline taper
and more graduated angle to the shoulders of the device would
reduce its EC trauma profile.
Our data also indicate that the degree of EC injury is related
to the vessel diameter in a device-specific manner (Figure 4).
For example, we found that the EC trauma profile for the
Solitaire FR device varied dramatically across in vitro vessel sizes. This finding illustrates the importance of applying
devices that properly match the dimension of the vessels to
reduce EC damage. Although all thrombectomy methods led
to greater EC injury in smaller vessels, the unconstrained distal tines of the Solitaire FR carried comparatively high EC
trauma consequences. These results suggest that the Solitaire
could particularly benefit from the development of smaller
and intermediate diameter devices.
Injury pattern data also provide important information for
future trial designs. Although it is intuitive that the minimal
linear injury caused by the Separator 3D is preferable to the
large, circumferential injury caused by the positive control
MERCI, the functional effects of such injuries on downstream
neuronal survival remain largely unknown. The implications of
Table. Advantages of In Vitro Live-Cell Platform for
Endovascular Device Testing
Glass/Plastic Tubing Artificial Vessel
Live EC-lining
Elasticity
No
Rigid
Yes
Native Vessel
Yes
Tunable to match Native elasticity
Transparent for imaging
Yes
Yes
No
Cost
Low
Relatively low
High
Availability
High
High
Low
EC indicates endothelial cell.
the more subtle pattern-based differences, such as the minimal
linear injury associated with the Separator 3D when compared
with the minimal circumferential injury from the 5MAX ACE
using the ADAPT technique, also remain to be explored. The
authors hypothesize that circumferential injuries may exert
greater effects on the performance of the large intracranial
vessel segment as a functional unit in the maintenance of a
stable end-organ pressure head between systolic and diastolic
phases. However, the significance of the pattern and degree of
EC injury remain important areas of future research.
Because this platform studies only the endothelium, the
effect of thrombectomy-related damage to underlying vessel structures, such as the internal elastic lamina, media, and
adventitia, may be missed. Nonetheless, our in vivo work and
previous studies testing thombectomy devices30–32 confirm that
damage to deeper structures is rare. Moreover, as secondary
neuronal injury results in part from activation of deleterious
cytokine cascades, the effect of thrombectomy devices on
the highly bioactive endothelium33 likely outweighs that of
deeper arterial wall structures. The relatively reduced presence of these deeper arterial structures within the cerebral
arterial system further diminishes their likely overall effect on
clinical outcomes.34 Recapitulating the native EC–substrate
adhesion forces under in vitro conditions is also challenging.
Although fibronectin-coated silicon (and other synthetic substrates) are established methods for in vitro assessment of EC
behavior,14,35 comparisons of in vitro to in vivo EC adhesion
forces are beyond the scope of this work. The true value in
this platform is nonetheless in determining relative differences
between thrombectomy devices. Our in vitro device-specific
data were highly reproducible and were confirmed through in
vivo experiments; this rigorous quantitative approach affirms
the translational potential of this platform for iterative device
testing and improvement. These methods hold similar promise
for assessing the effect of other endovascular devices, such as
stents, on the endothelium. Ultimately, the tight coupling of
in vivo and in vitro results implies a practical test of adhesion
forces and model fidelity comparable with live vessels.
Summary
EC injury is an important, but incompletely understood,
source of proinflammatory factors and BBB breakdown in
ischemic stroke, which has been implicated in reperfusion
injuries. Our in vitro live-cell model permits high-resolution
quantitative study of the pattern and degree of EC injury
in mechanical thrombectomy. This platform was validated
through in vivo animal work and provides new insights into
the pattern and timing of EC injury not readily accessible
in in vivo studies. Irrespective of the implications of injury
characteristics, EC disruption can be minimized based
on lessons from our in vitro findings. With recommendations, including the elimination of eccentric tapering and
the use of appropriately sized thrombectomy devices, this
in vitro platform offers valuable lessons for the engineering
of future devices. Investigations on the downstream effects
of EC injury are needed, and they may fundamentally
alter the techniques and devices used for thrombectomy in
acute ischemic stroke. Ultimately, the current high degree
Teng et al In Vitro Live-Cell Platform for Thrombectomy 7
of technical success in clot removal affords the luxury of
pursuing similar angiographic results while minimizing
endothelial trauma and perturbation of a complex biological
system. Artificial vessel assessments may ultimately reduce
the need for animal testing and limit a potentially important
source of secondary neuronal injury.
Acknowledgments
D. Teng, J.S. Pannell, J. Li, Y.S. Li, and S. Chien contributed to the
idea generation, experimental work, data analysis, and article preparation. R.C. Rennert and V.W. Wong contributed to the data analysis and article and figure preparation. A.A. Khalessi guided the idea
generation, experimental work, and article preparation. Thanks to
Michael Liu, Benjamin Yang, Kathleen Y. Kordestani, Terrence M.
Wong, Alvin Li, and Anagha Srirangam for their contributions to the
experimentation, data acquisition, and analysis.
Sources of Funding
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This project was funded in part by grants from Covidien, Ltd and
Penumbra, Inc obtained by a competitive grant application process.
Disclosures
A.A. Khalessi holds consulting arrangements for physician training
with Stryker Neurovascular, Covidien, and Penumbra. A.K. Khalessi
has no direct financial interest in the outcome of this line of research
inquiry. The other authors report no conflicts.
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Downloaded from http://stroke.ahajournals.org/ by guest on September 18, 2016
Endothelial Trauma From Mechanical Thrombectomy in Acute Stroke: In Vitro Live-Cell
Platform With Animal Validation
Dayu Teng, Jeffrey Scott Pannell, Robert C. Rennert, Jieying Li, Yi-Shuan Li, Victor W. Wong,
Shu Chien and Alexander A. Khalessi
Downloaded from http://stroke.ahajournals.org/ by guest on September 18, 2016
Stroke. published online February 24, 2015;
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ONLINE SUPPLEMENT
Endothelial Trauma from Mechanical Thrombectomy in Acute Stroke: An In
Vitro Live Cell Platform with Animal Validation
Dayu Teng2, PhD, J. Scott Pannell1, MD, Robert C. Rennert1, MD, Jieying Li2, MS, Yi-Shuan Li2, PhD,
Victor W. Wong1, MD, Shu Chien2, MD PhD, and Alexander A. Khalessi1, MD MS FAANS FAHA
1. Division of Neurosurgery, University of California - San Diego, La Jolla, California
2. Department of Bioengineering and Institute of Engineering in Medicine, University of California - San
Diego, La Jolla, California
Supplemental Video I: Representative full 3D reconstructions of vessel scans following
thrombectomy using the in vitro live cell platform.
Supplemental Video II: Representative angiographic visualization of porcine left common
carotid artery with complete occlusion following clot injection.
Supplemental Video III: Representative angiographic visualization of left common carotid
artery immediately following thrombectomy with the 5 MAX with 3D Separator, demonstrating
complete restoration of flow.
Key Words: stroke, endovascular thrombectomy, endothelial cell
Word Count: 1293
Co-Corresponding Authors:
Alexander A. Khalessi, MD MS FAANS FAHA
Assistant Professor of Surgery
and Neurosciences
Division of Neurosurgery
University of California – San Diego
200 West Arbor Drive
San Diego, CA 92103
Phone: 619-543-5540
Fax: 619-471-3931
Email: [email protected]
Shu Chien, MD PhD
University Professor of Bioengineering and Medicine
Department of Bioengineering
Director, Institute of Engineering in Medicine
University of California – San Diego
9500 Gilman Drive
San Diego, CA 92093-0435
Phone: 858-534-5195
Fax: 858-534-5453
Email: [email protected]
Supplemental Materials and Methods:
Creation of In Vitro Live Cell Platform
Silicone Tubing Treatment
Vessels for the in vitro live cell platform were created from optically clear and biocompatible
tubular silicone substrates custom-manufactured by Specialty Silicone Fabrications (Paso
Robles, CA), with thickness of 0.25 mm and inner diameters of 2.5, 3.5, and 4.5 mm. To
improve cell attachment, the inner surfaces of the substrates were first soaked in 10% (3aminopropyl) trimethoxysilane (Sigma-Aldrich. St. Louis, MO) in 100% ethanol overnight; then
crosslinked with 0.1 mM sulfo-SANPAH (Bioworld, Dublin, OH) in water via UV activation (8
min x 2 treatments; 365 nm, 84W, at a distance of 8 cm). After washing in PBS, the substrates
were coated with collagen I and fibronectin in PBS at 200 µg/ml and 20 µg/ml, respectively. The
substrates were rinsed with PBS and sterilized under UV light immediately before cell seeding.
Cell Culture and Endothelial Monolayer Formation
Bovine artery endothelial cells (BAECs) were cultured under standard condition (37ºC, 5% CO2)
with high glucose Dulbecco’s modified eagle medium (Life Technologies, Grand Island, NY)
supplemented with 10% fetal bovine serum (Omega Scientific, Tarzana, CA). A cell-suspension
at 105 BAECs/ml was perfused into the protein-coated silicone substrates, which were rotated 90
degrees every 20 minutes to promote even cell attachment. A monolayer of 1000 cells/cm2 were
achieved before subjecting to subsequent bioreactor flow.
Bioreactor Setup
Bioreactor chambers were fabricated using PDMS (Dow Corning, Midland, MI) and glass plates.
Two vessels were mounted to each chamber (Figure 1A) and connected to a media reservoir.
The media flow in the closed bioreactor system was generated using a peristaltic pump at 30
ml/min. To confirm the presence of a confluent monolayer and endothelial-cell alignment in the
direction of flow, cells were stained with a live-cell tracking agent, MitoTracker Red (Life
Technologies) at a concentration of 62.5 nM in DMEM, and a nuclear counter stain, Hoechst dye
(Sigma-Aldrich) at 5 µg/ml in PBS. Bright field, fluorescent MitoTracker Red and Hoechst
images were then acquired (Figure 1B, C & D).
Thrombectomy Procedure
In Vitro Platform
Porcine blood clots were formed by allowing previously collected porcine blood samples to
coagulate for 24 hours at 4ºC. Under flow stasis, size matched clots (of larger diameter than
vessel constructs to ensure circumferential occlusion) were introduced into the desired luminal
site of the in vitro live cell platform using a guide catheter, and allowed to integrate for 10
minutes. The integrity of the endothelial cells was confirmed before and after the introduction of
the clots via microscopy (Figure 2A). Various clot-retrieval devices were then deployed, and the
features of the devices interacting with the ECs in the in vitro platform were visualized (Figure
2B,C).
Artificial vessels were generated with 2.5, 3.5, and 4.5 mm diameters. Six thrombectomy devices
were tested in vitro with four trials per vessel size: (1) 5MAX ACE (Penumbra, Inc., Alameda,
CA) with A Direct Aspiration first Pass Technique (ADAPT), (2) MERCI (3.0 firm) (Concentric
Medical Inc/Stryker Corp., Kalamazoo, MI), (3) 5MAX with Separator (Penumbra, Inc.), (4)
5MAX with Separator 3D (Penumbra, Inc.), (5) Solitaire FR (4 x 20 mm) (Covidien, Ltd,
Mansfield, OH) and (6) TREVO (Stryker Corp.). Controls were run in triplicate. In vitro
thrombectomies were performed by one of two surgeons, both of whom possessing previous
clinical experience in the use of all devices tested. All thrombectomy systems were used with
adjunct catheter and wires consistent with their manufacturer’s specifications. Microcatheters
were positioned distal to the clots, except for the Penumbra 5MAX and 5MAX ACE catheters,
which were positioned proximally. The Trevo was deployed utilizing the Trevo Pro 18 L
microcatheter, and the Solitaire device was deployed through a Rebar microcatheter.
In Vivo Vessels
All animal work was performed in accordance with Institutional Animal Care and Use
Committee (IACUC) protocols. Two healthy adult hybrid pigs (S&S Farms, Ramona, CA) were
utilized for in vivo validation of the live cell model, using an adaptation of a previously described
technique for selective arterial thrombus placement via catheter injection (Figure 5A) [1].
Briefly, each pig was placed in the supine position and anesthetized, first with an intramuscular
injection of pre-anesthetic cocktail of Ketamine 30-35 mg/kg, Xylazine 2mg/kg, and Atropine
0.05 mg/kg. Venous access was then attained via a suitable ear, leg, or central vein. General
anesthesia was induced by intravenous injection of 2 mg/kg of propofol, followed by intubation
and mechanical ventilation. Anesthesia was maintained with a combination of 1-2% isofluorane
and propofol. No paralytics were administered.
Following induction of complete anesthesia, femoral artery cutdowns were performed. An 8French long sheath was placed through the common femoral artery into the distal abdominal
aorta, through which an 8-French balloon guide catheter was introduced. The common carotid
artery was then catheterized utilizing the guide catheter without a wire, to avoid EC damage in
the distal artery (radio-opaque clamps were used as markers of the desired vessel site that had not
been traversed with any endovascular instrumentation). The distal guide catheter tip was placed
just proximal to the desired location, and the balloon was inflated to interrupt arterial flow.
Following balloon inflation, a large porcine blood clot (generated as described above) was
injected through the guide catheter and reproducibly lodged within the desired arterial segment.
The balloon was then deflated to restore arterial flow and allow thrombus embedding prior to
thrombectomy.
A single surgeon (senior author AAK) with clinical experience with all devices tested performed
the in vivo thrombectomies. A single pass was made with each thrombectomy device tested, with
representatives from each company present to certify proper use of each device. Following
thrombectomy, the affected vessel was isolated and excised, and the pigs were euthanized in
accordance IACUC protocols (administration of 1 ml/10 lbs of a veterinary euthanasia solution
consisting of 390 mg/ml pentobarbital).
Supplemental References:
1.
Mordasini P, Frabetti N, Gralla J, Schroth G, Fischer U, Arnold M, et al. In vivo
evaluation of the first dedicated combined flow-restoration and mechanical
thrombectomy device in a swine model of acute vessel occlusion. AJNR Am J
Neuroradiol. 2011;32:294-300.
Supplemental Video Legend:
Supplemental Video I: Representative full 3D reconstructions of vessel scans following
thrombectomy using the in vitro live cell platform.
Supplemental Video II: Representative angiographic visualization of porcine left common
carotid artery with complete occlusion following clot injection.
Supplemental Video III: Representative angiographic visualization of left common carotid
artery immediately following thrombectomy with the 5 MAX with 3D Separator, demonstrating
complete restoration of flow.
32
Stroke 日本語版 Vol. 10, No. 2
Abstract
急性脳卒中における機械的血栓除去術による血管内皮損傷
動物実験を用いた in vitro 生細胞プラットフォームの検証
Endothelial Trauma From Mechanical Thrombectomy in Acute Stroke
In Vitro Live-Cell Platform With Animal Validation
Dayu Teng, PhD2; Jeffrey Scott Pannell, MD1; Robert C. Rennert, MD1, et al.
1
Division of Neurosurgery; and 2 Department of Bioengineering and Institute of Engineering in Medicine, University of California, San Diego
背景および目的：血管内血栓除去術は，大血管閉塞による
急性脳卒中に対する治療法として有望であることが示され
ている。再灌流に伴う損傷は，血管造影による転帰と臨床
転帰が一致しない原因である可能性がある。血栓除去術中
の医原性の血管内皮損傷は再灌流に伴う損傷の主要な仲介
因子である可能性があり，さらなる研究を必要とする。
方法：商業ベースで存在する各種の血管内治療デバイスが
血管内皮に及ぼす影響について検討するため，in vitro 生
細胞プラットフォームを開発した。in vivo 検証はブタを
用いて実施した。
結果：新たに開発した in vitro プラットフォームにより各
種血管内血栓除去デバイスと様々な血管径における内皮細
胞損傷を高分解能で定量し，これらのパターンおよびタイ
ミングでの特徴を解明することができた。血管内皮に及ぼ
す影響は血栓除去デバイスによって異なり，in vitro にお
いて評価されたデバイスの性能は in vivo の所見によって
実証された。
結論：In vitro 生細胞による人工血管モデルによって，血
栓除去術後の血管内皮の詳細な評価が可能となった。この
モデルは今後の血管内治療デバイスのデザインに貢献する
可能性がある。大型動物の試験により，血管内皮の生理機
能の研究に対する本 in vitro システムの妥当性が確認され
た。この人工血管モデルは，新たな血管内治療技術の評価
において実用的かつ測定可能で生理学的に妥当なシステム
である可能性がある。
Stroke 2015; 46: 1099-1106. DOI: 10.1161/STROKEAHA.114.007494.
A
i) カテーテル留置
未処理の血管
マーカー
ii) カテーテルを拡張
総頸動脈
凝血塊を注入
iii) 血栓除去
大腿動脈
アクセス
5MAX
ACE
Trevo
500µm
500µm
Separator
3D
Solitaire
C 1000
横断面ごとのEC数
B
血管
切除
800
600
400
200
D
ra
to
ai
lit
Se
pa
デバイス
STR-J_10-2_ab13_main.indd
STR-J
10-2 ab13 main.indd 32
r3
re
ev
o
So
Tr
AX
500µm
緑：vWF（EC）青：ヘキスト色素（核）
5M
500µm
AC
E
0
In vivo 実験のデザインと血栓除去デバイスが
血管内皮に及ぼす影響の検証。A：in vivo 実
験の設定。大腿動脈穿刺により 8-French バ
ルーンガイドカテーテルを総頸動脈に挿入し
た。ワイヤーカテーテルまたはガイドカテーテ
ルを通していない遠位の血管セグメントを，放
射線不透過性マーカーを用いて表示した（ i ）
。
バルーンを拡張させて動脈血流を遮断し，ブタ
の大きな凝血塊をガイドカテーテルから注入し
た（ ii ）
。バルーンを収縮させ動脈血流を再開さ
せる。凝血塊を血管に埋め込んでから血栓除
。
図 5 去を行い，処理のために血管を切除する（ iii ）
B：血管内皮細胞（ EC ）のマーカーであるフォ
ンウィルブランド因子（ vWF：緑色，核は青
で染色）で示した血管横断面の免疫組織化学染
色で，EC の状態はデバイスごとに異なってい
た。in vivo モデルにおいて血管全体の画像取
得は困難であった。スケールバーは 500 μm。
C：In vivo 血栓除去術後の EC 数は，in vitro
実験により予測されたデバイス間の差異を
示した。定量化に使われた資料切片数は，
5Max が 8，Trevo が 21，Solitaire が 20，
Separator 3D が 24 であった。
2015-8-25 14:53:15

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