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O R I G I N A L R E S E A R C H
Intravascular and Extravascular MicrovesselFormation in Chronic Total OcclusionsA Micro-CT Imaging Study
Nigel R. Munce, PHD,* Bradley H. Strauss, MD, PHD,†‡ Xiuling Qi, PHD,†Max J. Weisbrod,† Kevan J. Anderson, BENG,* General Leung, MSC,*John D. Sparkes, MSC,† Julia Lockwood,† Ronen Jaffe, MD,† Jagdish Butany, MD,§Aaron A. Teitelbaum, MD, MSC,† Beiping Qiang, MD, PHD,† Alexander J. Dick, MD,†Graham A. Wright, PHD*†
Toronto, Ontario, Canada
O B J E C T I V E S The purpose of this study was to characterize the 3-dimensional structure of intravas-
cular and extravascular microvessels during chronic total occlusion (CTO) maturation in a rabbit model.
B A C K G R O U N D Intravascular microchannels are an important component of a CTO and may
predict guidewire crossability. However, temporal changes in the structure and geographic localization
of these microvessels are poorly understood.
M E T H O D S A total of 39 occlusions were created in a rabbit femoral artery thrombin model. Animals
were sacrificed at 2, 6, 12, and 24 weeks (n �8 occlusions per time point). The arteries were filled with
a low viscosity radio-opaque polymer compound (Microfil) at 150 mm Hg pressure. Samples were
scanned in a micro-computed tomography system to obtain high-resolution volumetric images. Analysis
was performed in an image processing package that allowed for labeling of multiple materials.
R E S U L T S Two distinct types of microvessels were observed: circumferentially oriented “extravas-
cular” and longitudinally oriented “intravascular” microvessels. Extravascular microvessels were evident
along the entire CTO length and maximal at the 2-week time point. There was a gradual and progressive
reduction in extravascular microvessels over time, with very minimal microvessels evident beyond 12 weeks.
In contrast, intravascular microvessel formation was delayed, with peak vascular volume at 6 weeks, followed
by modest reductions at later time points. Intravascular microvessel formation was more prominent in the
body compared with that in the proximal and distal ends of the CTO. Sharply angulated connections
between the intravascular and extravascular microvessels were present at all time points, but most prominent
at 6 weeks. At later time points, the individual intravascular microvessels became finer and more tortuous,
although the continuity of these microvessels remained constant beyond 2 weeks.
C O N C L U S I O N S Differences are present in the temporal and geographic patterns of intravascular
and extravascular microvessel formation during CTO maturation. (J Am Coll Cardiol Img 2010;3:
From the *Department of Medical Biophysics, University of Toronto, †Schulich Heart Programme, Sunnybrook ResearchInstitute, Sunnybrook Health Sciences Centre, ‡McLaughlin Centre for Molecular Medicine, University of Toronto, and the§Department of Pathology, University Health Network, Toronto, Ontario, Canada. This study was funded by the CanadianInstitute of Health Research.
Manuscript received July 15, 2009; revised manuscript received January 6, 2010, accepted March 1, 2010.
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hronic coronary artery occlusions are ex-tremely common, occurring in about one-third to one-half of all coronary angio-grams (1,2). In addition, �1% of patients
ver the age of 60 have symptomatic occlusiveeripheral arteries (3), and the incidence is partic-larly high among the diabetic population (�30%)4). A chronic total occlusion (CTO) is defined asn angiographic occlusion (Thrombolysis In Myo-ardial Infarction [TIMI] flow grade �1) that is
See page 806
lder than 3 months (5). Several studies have shownhat successful revascularization of a coronary CTOorresponds to a reduction in symptoms (6) andmproved left ventricular function (7). Despite theotential clinical benefits of percutaneous treatmentf CTOs, these efforts are limited by difficulty inirecting the guidewire across the CTO beforealloon angioplasty (8,9).While, by definition, a CTO lacks flow seen
under angiography, previous studies haveindicated that about 50% of CTOs havehistological evidence of vascularity, andare in fact �99% occluded (10). Neovas-cularization within an occluded arterialsegment may occur within the lumen orvarious layers of the vessel wall and resultfrom distinct pathologic processes. Thevasa vasorum are a network of small vessels
ocated in the adventitia and the deeper layers of theunica media that proliferate in response to injury11,12). Intimal vessels are present within athero-clerotic plaques and could originate from the vasaasorum as a response to inflammatory and hypoxicactors (13). In addition, recanalization microvesselsesulting from reorganization of thrombus withinhe occlusion have been observed (10). These mi-rovessels may be critical for successful guidewirerossing as the path of least resistance through theense collagen of the occlusion (14).Previous pathology studies (10,15) have de-
cribed tissue composition within CTOs; however,nformation on the global architecture of an occlu-ion has been poorly understood. This knowledgeeficit has been largely due to the limited number ofistological cross-sections that can be processed andnalyzed. Furthermore, it has been challenging toorrelate these histological sections with the proxi-al end, body, and distal end of the occlusion.hile imaging techniques can address some of
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hese difficulties, they typically lack sufficient reso- w
ution to examine details, such as microvasculature,ppropriately. One imaging modality that has beenidely employed for high-resolution ex vivo imag-
ng is micro-computed tomography (�CT) withpatial resolution on the order of 10 �m (16).
Recently, we reported on the maturationalhanges of CTOs in a rabbit femoral artery model,ncluding the general pattern of intravascular mi-rochannels within the CTO. In particular, weound differences in intravascular microchannel vol-me at different time points using magnetic reso-ance imaging and histology (17). This study,owever, lacked sufficient spatial resolution on alobal scale to investigate the evolution of the-dimensional morphology of microvessels inTOs, particularly in relationship to the specific
ocation within the CTO (proximal end, body, andistal end). It was also unclear to what extent theessels from outside of the occluded artery inter-cted with those channels that appeared within theccluded lumen. Therefore, we sought to use �CTf contrast-perfused CTOs at different time pointso investigate the evolution of the 3-dimensionalicrovasculature associated with a rabbit occlusionodel.
E T H O D S
he occlusion model. Approval for experiments wasbtained from Sunnybrook Hospital Animal Careommittee. Bilateral arterial occlusions were initi-
ted in 28 male New Zealand white rabbitsCharles River Canada, St. Constant, Quebec)eighing 3.0 to 3.5 kg, as previously described (18).riefly, a femoral artery segment was isolated and
igated at each end. The segment was then injectedith �0.1 ml (100 IU/ml) bovine thrombin solu-
ion, and then the proximal ligature was removed tollow blood to mix with the thrombin to create ahrombus. The distal ligature was maintained up to0 min to ensure a persistent occlusion. Animalsere then returned to their cage and fed a regulariet. Animals were sacrificed at 2, 6, 12, and 24eeks (n �8 occlusions per time point) after cre-
tion of the CTO.ontrast perfusion and imaging. A contrast X-rayngiogram (Fig. 1) was performed to verify thecclusion before sacrifice. After the angiogram, theatheter was withdrawn and intravenous heparin1,000 units) was administered �10 min beforeacrifice to prevent blood coagulation in the micro-asculature. Immediately after sacrifice, a syringe
B B R E V I A T I O N S
N D A C R O N YM S
TO � chronic total occlusio
MV � intravascular microve
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as tied in place with a ligature to prevent backow. After an injection of 30 ml normal saline toush blood from the main arterial system, MicrofilFlow Tech, Carver, Massachusetts) was injected at
pressure of 150 mm Hg as measured by aandheld manometer. The Microfil was given 1 ho set, then the femoral arteries were surgicallyemoved and left in formalin for 48 h. Specimensere then embedded in 2% w/w agarose gel. The
amples were imaged in a �CT system (MS-8, GEedical Systems, London, Ontario) (16). Three-
imensional cone beam CT data sets were acquiredn 2.5 h with 905 views and reconstructed at 14-�mesolution. An X-ray source of voltage 80 KVp andbeam current 90 mA was used.
mage analysis. The �CT volume was importednto image analysis software (Amira, Mercuryomputer Systems, Chelmsford, Massachusetts).his software enables labeling of 2 distinct types of
asculature seen within the CTO: “intravascular”icrovessels representing recanalization channelsithin the occluded lumen and media; and “ex-
Figure 1. Micro-CT Axial Slices and Labeling Procedure
Micro-computed tomography (�-CT) slices are shown from (A) proxintravascular channels (red) and extravascular channels (blue). Therepresents the outer border of the artery. (I) Resulting volumes genare shown. (J) Angiogram of a 12-week-old chronic total occlusion
ide of the boundary of the arterial wall. Labeling of a
he intravascular microvessels was done by firstdentifying the patent vessel just before the occlu-ion; this vascular area was labeled with a softwareool that acted as a seed-growing algorithm, prop-gating the intravascular region through connectedegions in successive axial slices. A similar step waserformed at the distal end. This seed-growinglgorithm typically labeled all connected vasculaturessociated within the occluded artery segment.
To identify vessels that were extravascular, 2ethods were applied. First, extravascular channels
ould be directly identified at the proximal andistal ends as the small vessels surrounding theatent artery before the occlusion. These vesselsere typically arranged in a circumferential ring
round the patent vessel (as shown in Fig. 1A), andere labeled as a different material in Amira usingsoftware tool that filled in homogenous areas and
topped when the pixel value changed abruptly.hese circumferential vessels were labeled for each
lice as they progressed into the occlusion. Theircumferential geometry of these channels wassed to define a border between the intravascular
l to (H) distal positions through the lesion and follow both thent lumen is identified with the letter “L.” The dotted yellow lineed from the labeled materials and the location of the axial slicesarrowheads identify the proximal and distal ends).
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n the screen as a reference, as shown in Figure 1Ahrough 1H by the dotted yellow line. As werogressed through the axial slices of the occlusion,essels that were arranged in a semicircular shapehat fit on or outside of this border were labeled asxtravascular. Second, at time points �2 weeks,ufficient contrast was consistently obtained be-ween the tissue surrounding the artery and thertery itself. This contrast was enhanced at theseater time points when there was an increasingelative amount of loose connective tissue surround-ng the artery. In these slices, vessels that appearedn the border of loose connective tissue and thertery were labeled as extravascular. Intravascularhannels, which crossed the arterial border, wereelabeled as extravascular when they crossed thatorder. The outer boundary used to identify ex-ravascular vessels across the length of the CTO wasetermined by the average diameter containingxtravascular vessels surrounding the patent arteryefore the proximal end of the occlusion.Labeling was performed by 2 independent read-
rs (N.M. and M.W.) who were blinded to the timeoint. The results from each reader were averaged.he software package was used to color code andisplay the intravascular vessels red, and the ex-ravascular vessels blue on an isosurface (Figs. 1 and 2).he software package also calculated the number of
oxels in each axial slice that were identified asntravascular and extravascular. The start and end of
Figure 2. Progression of an Arterial Occlusion Over Time
Progression of microvasculature at (A) 2 weeks, (B) 6 weeks, (C) 12
end; black arrows represent the distal end. Intravascular channels are s
he occlusion were defined as the axial slices inhich the vessel lumen area, as measured by �CT,ecreased by 90% compared with the patent artery.he CTO length was normalized to 500 arbitrarynits using software (MATLAB, The Mathworks,atick, Massachusetts) so that occlusions of differ-
nt lengths could be compared. Based on ourrevious histological studies (17), minimal variationn the cross-sectional area of the occluded lumenas observed in animals at the same time point.hus, for this work, occlusions were normalized tostandard length rather than both length and
ccluded lumen area. An averaged vascular distri-ution at each time point was obtained by averaginghese normalized vascular distributions.
Continuity represents the cumulative length ofhe intravascular microvessels along the entire CTOength. Percent continuity was calculated by iden-ifying all continuous segments (minimal lengthequirement 1 mm), and defining the percent “con-inuity” of the channels in a CTO as the sum of theengths of all of these segments divided by the totalength of the occlusion. Parallel intravascular mi-rochannels that were observed to join togetherere counted as a single segment in the context of
his continuity measurement.Average intravascular microvessel diameter was
lso calculated for each time point by assuming thathere was typically a single channel in an imaging
ks, and (D) 18 to 24 weeks. White arrows represent the proximal
wee hown in red; extravascular channels are shown in blue.
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lice and approximating that the shape of theicrochannel was a square in that slice.Communicating channels were defined as points
n which an intravascular channel crossed the de-ned border between the intravascular and extravas-ular channels. These events were counted by scroll-ng through all of the axial slices of each occlusionnd counting the number of such incidences. Theumber of these events was averaged for each timeoint.tatistical analysis. Analysis of variance for micro-ascular volumes, continuity, number of communi-ating channels, number of microchannel segments,iameter of microchannels, and occlusion lengthas performed in SAS version 9.0 (SAS Institute,ary, North Carolina) using the general linearodel procedure to compare mean responses at
ach of the 4 time points. Since the number ofcclusions varied slightly among the different timeoints, least squares means were calculated for eachime point; the mean and standard error of theean (SEM) were calculated for each time point.robabilities for comparisons between means weredjusted for multiple comparisons by the method ofukey-Kramer. A p value of � 0.05 was considered
ignificant.
E S U L T S
hirty-nine occlusions were successfully created in8 rabbits. Six of these occlusions were not analyzableecause of poor filling with Microfil (n � 3) andcclusion length �1 mm (n � 3). This gave an overallumber of data points from each time point as follows:� 9, 2-week-old occlusions from 7 rabbits; n � 8,
-week-old occlusions from 5 rabbits; n � 8, 12-eek-old occlusions from 6 rabbits; n � 8, 24-week-ld occlusions from 6 rabbits. The average interob-erver variability of the 2 trained readers in the analysisf the intravascular microvessel volume (IMV) was0.2%.
Typical 3-dimensional microvascular volumes at, 6, 12, and 24 weeks are displayed in Figure 2Ahrough 2D. The proximal end of the CTO typi-ally was at the origin of a branching collateral, andhe distal end was reconstituted at a bifurcation ofhe femoral artery. The average length of thecclusion was 18.1 �1.0 mm at 2 weeks, 12.2 � 1.2m at 6 weeks, 15.9 � 1.2 mm at 12 weeks, and
4.4 � 1.1 mm at 24 weeks. The overall p value forhe occlusions lengths comparing weeks 2 to 24 wasalculated as p � 0.006. The volumetric images
howed a progression from a poorly defined proxi-
al end at the early 2-week time point (Fig. 2A) totapered corkscrew-like pattern at both the proxi-al and distal ends of the occlusion at later time
oints (Fig. 2B to 2D). Rotational movies of theseolumes are available as online supplemental mediaOnline Videos 1, 2, 3, and 4), which allow foretter appreciation of the overall structure of thecclusion.xtravascular vessel formation. Extravascular vesselsppeared as circumferentially oriented vessels on theuter surface of the occluded artery. These vesselsere most predominant at the 2-week time point,
ppearing as a fine network of vessels surroundinghe artery (Fig. 2A, blue color). As the occlusionge increased, these vessels were markedly de-reased in number, although larger in size comparedith the 2-week time point (Fig. 2B to 2D). Thereas a significant reduction in the total extravascularolume with age (p � 0.0024 comparing 2-week vs.4-week-old CTOs) (Fig. 3B).
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Figure 3. Total Intravascular and Extravascular Microchannel Voand Continuity
(A) The evolution of the total intravascular microvessel volume in aocclusion is shown over time, as measured by micro-computed tom(B) The corresponding changes seen in the extravascular microvessshown over the same time frame. The graph shown in (C) illustrateevolution of the continuity of the microchannels in the occlusion a
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The distribution of the extravascular microves-els at 2 weeks was characterized by 2 broad peaksFig. 4B) within the body of the occlusion. At the-week time point, there was a shift of this distri-ution toward the proximal end. Although thereere few extravascular microvessels present beyond
he 6 week time point, these microvessels werevenly distributed along the length of the CTO.ntravascular microchannel volume. There was a
arked increase (2.7 times increase) in the totalMV observed between the 2- and 6-week timeoints (p � 0.003) as shown in Figure 3A. Theverall IMV then progressively declined by a factorf 2.1 between 6 and 24 weeks (p � 0.017) (Fig.A), primarily as a result of a decrease in diameterf the vessels. The spatial distribution of thesentravascular channels, which was normalized to theength of the occlusion for each time point, ishown in Figure 4A. We observed a predominancef IMV within the central body of the CTO at the-week time point as compared with the proximalnd distal end regions of the CTO (Fig. 4A). Atater time points, there was a prominent reductionn the IMV within the central body of the CTO,
Distance in the Occlusion (Normalized Units)
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Averaged Distribution of Intravascular and Extravascularnel Volume
travascular (A) and extravascular (B) microvessel volume as af position within the occlusion for several time points. Distanceocclusion is normalized by total occlusion length so that eachs 500 units long. Dark gray line indicates 2 weeks; orange lineweeks; green line indicates 12 weeks; light gray line indicates
o
ith relative sparing of IMV at the proximal andistal end regions (Fig. 4A).ontinuity of microchannels. There was minimalontinuity of the intravascular microchannels at the-week time point. However, at all later time points�6 weeks), an average continuity of 85% wasbserved (Fig. 4C), meaning that 85% of the lengthf the occlusion could be traversed by followinguch a microchannel. There were significant differ-nces in continuity of the intravascular microvesselsetween 2 weeks and all later time points (p �.0002). Interestingly, the intravascular channelsithin the occlusion were typically composed of 1
o 3 different channel segments. These segmentsere often connected to either the proximal and/oristal end of the occlusion. The average length ofhese segments was significantly higher (p � 0.05)t the later time points (7.0 � 1.3 mm at 6 weeks,.5 � 1.3 mm at 12 weeks, and 6.3 � 1.2 mm at 24eeks) compared with the 2-week time point
3.5 � 1.1 mm).ntravascular microchannel diameter. There was a sig-ificant increase (p � 0.03) in the average diameter ofhe intravascular microchannels between the 2- and-week time points (115 � 15 �m at 2 weeks, 198 �6 �m at 6 weeks). No significant difference (p �.65) was seen between the microvessels diameters atand 12 weeks (172 � 16 �m at 12 weeks). As thecclusion aged, however, the microvessel diameterecreased (134 � 16 �m at 24 weeks), and aignificant difference was seen comparing the diam-ters of the microvessels at the 6- and 24-week timeoints (p � 0.03).ommunications between intravascular and extravas-ular channels. Communicating channels were ob-erved at all time points (Fig. 5A), with a peakumber at the 6-week time point, which graduallyecreased over time (Fig. 5C). There were signifi-antly fewer communicating channels at the 2-weekime point compared with the 6-week time pointp � 0.02). However, there were no significantifferences in the number of communicating chan-els comparing weeks 6 through 24 (p � 0.15).hese communicating channels exited at acute
ngles (Fig. 5D).
I S C U S S I O N
n this study, we report on the evolution of the-dimensional morphology of microvessels in aTO model. We also performed a detailed analysis
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ocation within the vessel layers (intravascular ver-us extravascular) and specific distribution along theTO (proximal, central body, distal).The primary finding of this work was that a large
ise in extravascular vessels surrounding the oc-luded artery occurred at early time points, whichas followed by a statistically significant increase in
ntravascular vessels within the central body of thecclusion. The temporal and geographic pattern oficrovessel formation and the presence of connect-
ng microvessels suggested that the extravascularessels may initiate formation of the intravascularhannels within the center of the occlusion, as wasvident in the distribution of intravascular channelslong the length of the occlusion. The stimulus forhe increased neovasculature observed within theenter body of the lesion may be due to greaterypoxia in this region, particularly at these earlyime points. These channels may lead to a signifi-antly smaller occlusion length at the 6-week timeoint as compared to the acute 2-week period.As the occlusion reaches the age of 6 weeks,
eovascularization also occurred at both the proxi-al and distal ends of the lesion. This process at the
roximal end has previously been correlated withhe presence of the tapered entrances similar tohose seen in angiography (8). The mechanism oformation of the vessels from the proximal and
Figure 5. Communicating Vessels in Arterial Occlusions
(A) A micro-computed tomography slice showing intravascular micrarterial wall (blue). (B) Longitudinal slice through the occlusion shochannels as a function of the occlusion age. (D) Volume-rendered icular microvessels (blue) communicating with intravascular vessels.with the proximal entrance located in the upper left-hand corner; timage.
istal ends is uncertain, but could be related to a
ngrowing recanalization channels from the adja-ent nonoccluded segments and/or the effects ofpecialized circulating cells, as has been observed inenous thrombi (19). These tapered entrances even-ually connected to the microvasculature in theenter of the lesion—resulting in long continuoushannels through the lesion.
However, at later time points beyond 6 weeks,here was a reduction in the size and number ofentral intravascular microchannels, suggesting thatany of the vessels in this region become nonfunc-
ional. As the occlusion further ages, only a singlearrow intravascular channel was present. Whilehis channel may provide a pathway through theTO, its small diameter and tortuous nature woulde challenging to cross with guidewires, and make itusceptible to occluding as well. The increase incclusion length associated with later time pointsay be due to the occlusion of these microchannels.These observations are in agreement with the gen-
ral trends that we have reported in a previous studynvolving histological evaluation of microchannels15). In that work, we also saw a peak in microchannelascularity at the 6-week time point that regressed ashe occlusion aged. That previous study, however, wasot able to provide the detailed structure and topo-raphic information along the length of the occlusion
ssels (red) communicating with extravascular microvessels in thecommunicating channels (yellow arrows). (C) Number of such
e of a 12-week chronic total occlusion showing several extravas-t image shows wide field view of the chronic total occlusion,ellow box illustrates the magnified region shown in the main
ovewingmagInsehe y
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tudy limitations. The CTO model differed from thelinical case primarily because of its lack of atheroscle-otic substrate. This substrate is often associated withn area of increased angiogenesis within the plaque;herefore, investigating the interaction of these vesselsith those associated with the CTO remains anutstanding issue. The absence of calcification in therterial wall in this model may also have significantmplications for the architecture of the occlusion. Theurrent work suggests that vessels from outside of thecclusion play a key role in the development ofntravascular microchannels; the presence of calcifica-ion in the artery’s medial wall may affect this process.urthermore, due to the current requirements of exivo tissue for performing �CT studies, individualnimals could not be followed up serially. This limi-ation results in the need for us to compare differentnimals at different time points, thus increasing vari-bility. Newer �CT systems coupled with blood-poolontrast agents may be able to address this challengen the future (20).
O N C L U S I O N S
he results of this study indicate that, within thenitial 12 weeks, microvessels are continuous acrossarge segments of the occlusion. While the typicaliameter of the intravascular channels that webserved in this study at 6 and 12 weeks was lesshan that of a standard 0.014-inch (355-�m) guide-ire, it is likely that the presence of the microvessel
lters the local tissue compliance. Thus, the guide-ire may still follow the path of a microvessel, as it is
he path of least resistance through the occlusion. The
ior A, Van Bellen B. Prevalence of analysis of the effects
hannels in older CTOs, which correspond to loweruidewire crossing rates in percutaneous revasculariza-ion (8), provides a rationale for targeting intravascularngiogenesis to facilitate endovascular strategies.
It is also important to recognize that the presencef communicating channels between extravascularnd intravascular networks may contribute to diver-ion of guidewires into the extravascular space. Themages we obtained of the communicating channelsuggest that the majority of these channels exithe lesion at an angle close to 90° to the path of thertery, and hence, it is unlikely that the guidewireould make such a sharp, sudden turn. These
hannels, however, may explain some of the dissec-ions that occur during guidewire manipulations,nd when operators employ devices that follow thepath of least resistance” through a CTO (21,22).
Recent work has suggested that conventionaluoroscopy can be used to build up 3- dimensional
mages of microchannels within CTOs (23). Al-hough the authors of that work were not able toesolve the complex 3-dimensional structure of theicrochannels, they also reported seeing highly con-
inuous channels in CTOs, which may be importantredictors of procedural success. Further developmentf these imaging techniques, particularly the ability todentify points in which channels exit the lesion, mayotentially improve planning and intraproceduraluidance of angioplasty procedures.
eprint requests and correspondence: Dr. Nigel Munce,unnybrook Health Sciences Centre, 2075 Bayview Av-nue, Room S612, Toronto, Ontario M4N 3M5, Can-
smaller number and diameter of intravascular micro- ada. E-mail: [email protected].
1
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ey Words: chronic totalcclusions y micro-computedomography y microchannels ynterventional cardiology.
