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Calcification and endothelialization of thrombi in acute stroke.

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BRIEF COMMUNICATIONS
Calcification and
Endothelialization of
Thrombi in Acute Stroke
Mohammed A. Almekhlafi, MD,1 William Y. Hu, MD,2
Michael D. Hill, MD,1,3,4
and Roland N. Auer, MD PhD5
We report chronic histopathological features in thrombi mechanically retrieved from five acute ischemic stroke patients
with a median age of 68 years and a median pretreatment
National Institutes of Health Stroke Scale score of 13. Early
endothelialization occurred over and within the thrombus,
and calcifications were seen, in addition to the usual acute
laminar fibrin, intervening red blood cells, and neutrophils.
The effectiveness of tissue plasminogen activator in clot dissolution might be affected by these features, if extensive.
Thrombus composition could critically determine the success
of chemical thrombolysis. Our results should stimulate the
development of imaging modalities to determine thrombus
composition.
Ann Neurol 2008;64:344 –352
The aim of acute interventions in ischemic stroke is to
achieve recanalization as early and completely as possible.1 The MERCI retriever (Mechanical Embolus Removal in Cerebral Ischemia) is designed to accomplish
this goal2 through mechanical removal of the thrombus
from the artery.3,4 It has the advantage of reaching
thrombi in large vessels, which are relatively resistant to
thrombolysis by tissue plasminogen activator (tPA).5
Analysis of retrieved thrombi from human stroke has
the potential to enhance our understanding of pathophysiology.6 Thrombus composition may predict response and in the future will likely play a role in determining success of interventions after patient
selection. We analyzed thrombi recovered using the
MERCI retriever from five patients with acute ischemic stroke, finding chronic features that would preclude effective clot dissolution.
From the Departments of 1Clinical Neurosciences, 2Diagnostic Imaging, 3Medicine, 4Community Health Sciences, and 5Pathology
and Laboratory Medicine, University of Calgary, Calgary, Alberta,
Canada.
Received Dec 2, 2007, and in revised form Mar 18, 2008. Accepted
for publication Mar 20, 2008.
Published online Mon 00, 2008, in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/ana.21404
Address correspondence to Dr Auer, Department of Pathology and
Laboratory Medicine, University of Calgary, 3330 Hospital Drive
NW, Calgary, AB T2N 4N1, Canada. E-mail: rauer@ucalgary.ca
344
Patients and Methods
Design and Population
Between January 2004 and October 2006, 25 acute ischemic
stroke patients were treated with endovascular thrombolysis.
The MERCI retriever was used in 12 of these patients. The
rest were treated with intra-arterial thrombolysis. Five of the
MERCI-treated patients had pathological specimens amenable to analysis. In the rest, either minute fragments of
thrombi or no thrombi were retrieved. This project was reviewed and approved by the Calgary Health Region Ethics
Board; informed consent was provided by each patient or
their surrogate before treatment.
Calgary was a site in the Multi-MERCI registry, and it is
a site in the Interventional Management of Stroke (IMS) 3
trial. For both trials, MERCI retrievers are being provided by
Concentric Medical, Inc., Mountain View, CA.
Clinical Variables and Outcome Measures
Patient demographics and clinical findings were documented
by the stroke neurologist or the stroke fellow, or both, prospectively. All patients had computed tomographic scans and
computed tomographic angiography before treatment. The
Alberta Stroke Program Early Computed Tomography score
(ASPECTS) was scored by the neuroradiologists.7 The likely
stroke mechanism was judged according to the modified
TOAST (Trial Of Org 10172 In Acute Stroke Treatment)
criteria by the stroke neurologist.8 The modified Rankin
score was documented, both before stroke onset and at time
of discharge, by the stroke neurologist. Stroke severity was
judged based on the National Institutes of Health Stroke
Scale on presentation and on the discharge modified Rankin
Scale score.
Retrieval Procedure
Four-vessel diagnostic cerebral angiograms were obtained
through a femoral artery puncture. The need for intraarterial
thrombolysis was judged by the stroke neurologist and the
interventionalist. MERCI retrieval was performed through a
microcatheter introduced into a balloon guide catheter and
placed distal to the thrombus. The MERCI retriever was
then advanced, and two to four loops were deployed past the
microcatheter tip and then withdrawn as a unit into the
thrombus. The balloon was then inflated to prevent thrombus fragments from embolizing distally. The thrombus and
the retriever were retracted into the balloon guide catheter
lumen and removed.
The procedure was ended once recanalization was attained
or if six retriever passes failed to achieve recanalization. Recanalization was classified according to the Thrombosis in
Cerebral Infarction (TICI) criteria.9
Thrombus Processing and Analysis
Thrombi were placed in formalin, processed for paraffin embedding, sectioned, and stained with hematoxylin and eosin,
Martius scarlet blue for fibrin, Masson’s trichrome, and von
Kossa for calcium. The slides were analyzed and scored by
two of the authors (R.N.A., M.A.A.) who were blinded to
the patients’ clinical data. The thrombus volume was calculated using the formula for the volume of an ellipsoid (4/
3␲r1r2r3).
© 2008 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
Table 1. Patient Demographic and Clinical and Radiographic Characteristics of Strokes
Characteristics
Patient No.
1
2
3
4
5
Age (yr)
Sex
Race
CV risk factors
68
Female
Asian
None
76
Female
East Indian
Atrial
fibrillation
65
Male
White
None
71
Male
White
HTN, PVD
Prestroke mRS score
TOAST criteria
0
Undetermined
“presumed
hypercoagulable
state”
Catastrophic
8
3
Cardioembolic
0
Large artery
disease
41
Male
White
CAD, DM,
HTN, HC,
smoker
0
Large artery
disease
Severe
19
Severe
19
Catastrophic
Comatose
Moderate
6
10
7
7
10
10
Distal BA
Distal left
ICA, M1, A1
Entire BA
BA tip ⫹
both P1
Intervention
Mechanical
Mechanical
Onset-to-angiogram
ratio (hr)
MERCI passes, n
TICI (pre/post)
3:45
IV tPA,
mechanical
4:45
Distal left
ICA, M1,
A1
IV/IA tPA,
mechanical
2:45
IV tPA,
mechanical
4:00
6
0/2a
3
0/2a
3
0/2a
3
1/2b
24-hr NIHSS
Discharge mRS
0
6
19
6
Intubated
1
Intubated
6
Stroke severity
Pretreatment
NIHSS score
Pretreatment
ASPECTS
Vascular site
5:15
0
Large artery
disease
5
2a “right
PCA,” 2a
“left PCA”
6
4
Three patients (Patients 1-3) were enrolled in the Mechanical Embolus Removal in Cerebral Ischemia (MERCI) registry.4 One (Patient
5) was enrolled in the Albumin in Acute Ischemic Stroke (ALIAS) trial, which is ongoing.
CV ⫽ cardiovascular; CAD ⫽ coronary artery disease; DM ⫽ diabetes mellitus; HTN ⫽ hypertension; HC ⫽ hypercholesterolemia;
PVD ⫽ peripheral vascular disease; mRS ⫽ modified Rankin Scale; NIHSS ⫽ National Institutes of Health Strokes Scale;
ASPECTS ⫽ Alberta Stroke Program Early Computed Tomography score; BA ⫽ basilar artery; ICA internal ⫽ carotid artery; IV ⫽
intravenous; tPA ⫽ tissue plasminogen activator; TICI ⫽ Thrombosis in Cerebral Infarction; PCA ⫽ posterior cerebral artery.
Thrombi were scored according to their fibrin and red
blood cell content, and the presence or absence of polymorphonuclear leukocytes, endothelialization, or calcification.
Results
Table 1 shows patient demographics. The median age
was 68 years. The mean time from stroke onset to the
start of the cerebral angiogram was 4 hours and 6 minutes (standard deviation, 57 minutes). Four individuals
were independent before the stroke (modified Rankin
score, 0), and one (Patient 2) had a moderate preexisting disability (modified Rankin score, 3). The median pretreatment National Institutes of Health Stroke
Scale score was 13 (range, 6 –19).
The median pretreatment Alberta Stroke Program
Early Computed Tomography score was 10 (range,
7–10). Note that three of the five patients had posterior circulation stroke, which has a score of 10.
Three patients received standard-dose (0.9mg/kg)
intravenous tPA. The mean onset-to-angiogram time
was 246 minutes (standard deviation, 57 minutes).
Patients 1 and 3 received 5 and 8mg, respectively, of
intraarterial tPA. The X-6 MERCI device was used,
and the average number of passes required was 4
(range, 3– 6). Partial recanalization was always
achieved.
Histopathological analysis of the thrombi showed a
median volume of 10.47mm3 (Table 2). Most had a
mixed and variable composition of fibrin and red
blood cells (see Table 2). Polymorphonuclear cell infiltration was seen in four of the five thrombi. Endothelial cells covered portions of the circumference of
Almekhlafi et al: Pathology of Thrombi in Stroke
345
Table 2. Histopathological Analysis of the Thrombi
Fibrin/
RBC
(% ratio)
PMN
Leukocytes
Endothelialization
(% circumference)
12.57
98/2
0
2%
No
10.47
50/50
⫹
0%
Yes (80% of
specimen 2)
2⫻3⫻2⫹3⫻5
⫻3⫹4⫻2⫻2
38.22
98/2
⫹
1%
No
4
3⫻1
1.57
75/25, 95/5
⫹
3%
No
5
3⫻1⫻1
1.57
98/2
⫹
0%
Yes (20%)
Patient
No.
Thrombus Size (mm)
Volume
(mm3)
1
4⫻3⫻2
2
1⫻4⫻2⫹4⫻3⫻1
3
Calcification
(% of
thrombus
volume)
RBC ⫽ red blood cells; PMN ⫽ polymorphonuclear.
three thrombi. Calcifications were noted on both
gross and microscopic examinations in two of the
thrombi. The histopathological analysis for individual
cases is provided in Table 2 and in the Figure.
Patients 1, 3, and 4 showed endothelialization, and
Patients 2 and 5 showed calcification in a uniform stippled pattern not a crystalline pattern seen in atheroma.
The second extraction done on Patient 2, from the distal M1 segment, showed a white flake of tissue suggestive of calcium.
Three patients died during hospitalization (Patients
1, 2, and 4). Patient 1 was found to have metastatic
cancer of unknown primary cause. The condition of
Patient 2 deteriorated, and the patient died 3 weeks
after hospitalization of congestive heart failure and
pneumonia. The condition of Patient 4 failed to improve, and the patient died of a neurological cause 36
hours after angiography.
Discussion
Our new findings of histopathological features within
thrombi have implications for both imaging and intervention. Previous studies have classified thrombi as red,
white, and mixed, relating the thrombus composition
to the degree of dissolution possible by tPA: increasing
fibrin and decreasing red blood cells auguring for increased difficulty in thrombolysis.10
Fibrin-rich thrombi contain an abundance of
thrombin that is released when the thrombus dissolves11 with potentially deleterious consequences.12
An animal model study showed a lower efficacy of
tPA thrombolysis in thrombi with high fibrin content
compared with erythrocyte-rich emboli.10 The abundance of fibrin over platelets in cardioembolic
thrombi is believed to underlie the superiority of warfarin over aspirin in preventing stroke in atrial fibrillation.
Unexpectedly, we found calcium in two patients,
embedded in the thrombi in a fine powder-like distri-
346
Annals of Neurology
Vol 64
No 3
September 2008
bution uncharacteristic of atheroma. The initial study
of MERCI-retrieved emboli6 did not report any calcium in retrieved emboli, although in one stroke, calcified embolic material was noted angiographically.
Calcium in an embolus confers resistance to thrombus
dissolution.13
Endothelialization, a subacute sign of early organization, was observed on and within thrombi in three
patients (Patients 1, 3, and 4). None of these patients
had calcification. The site and appearance of those
endothelial cells did not suggest denudation of the
vascular intima by the MERCI device as a possible
explanation because the endothelium was covering the
surface of the thrombus. Although endothelial covering of thrombi is part of their normal evolution, tPA
will not penetrate endothelium, and thrombi will be
resistant to dissolution, dependent on the extent of
surface coverage by endothelium. The degree of endothelialization we observed would not make the
thrombus hard to dissolve with tPA.
This is the first report of both acute and chronic
changes in different MERCI passes in the same patient
(our Patient 2), in the absence of carotid narrowing,
suggesting a proximal source. Although this patient did
have atrial fibrillation, this could nevertheless reflect
the formation of a fresh thrombus superimposed on a
more chronic aortic plaque or a calcified cardiac
valve.14
There was no difference in thrombi removed from
the anterior versus the vertebrobasilar circulation,15
suggesting a commonality of thrombotic mechanism
once clotting is initiated.
Our study carries limitations. The small number of
cases makes it difficult to draw conclusions for all
stroke patients. Thrombi could be retrieved only from
5 of 12 cases treated using the MERCI retriever. It is
not possible to know whether thrombi that were dissolved by tPA or those retrieved in minute fragments
Fig. Patient 1: (A) Hematoxylin and eosin staining of the retrieved thrombus shows a generally amorphous appearance to the thrombus. However, endothelialization can be seen with growth of endothelium (inset, upper left). (B) The laminar composition of the
thrombus is best demonstrated with Martius scarlet blue (MSB) staining, which shows alternating layers of fibrin as dark purple, red
blood cells (RBCs) as bright orange, and early collagen synthesis as blue. (C) The pavement-like surface endothelium shows several nuclei and attenuated cytoplasm that stains dark immunocytochemically using CD34 antibody. Magnification bars ⫽ 1mm (A); 200␮m
(B); 50␮m (C). Patient 2: The first retrieved specimen (A) shows approximately 50% fresh, intact red blood cells, stained orange with
the Orange G component of the MSB stain. Neutrophils also infiltrate the specimen (inset). Both are acute features. The second retrieval yielded a remarkably different appearance (B) with calcium diffusely precipitated in early organized tissue, consisting of red fibroblasts, often with nuclei visible (top inset) in blue connective tissue. Calcium was confirmed (blue-black, bottom inset) by the von
Kossa stain. Magnification bars ⫽ 500␮m (A); 100␮m (A, inset; B; B, bottom inset); 50␮m (B, top inset). Patient 3: (A) MSB
stain shows fresh red blood cells stained orange. (B) High magnification of different areas of the thrombus shows neutrophilic infiltration and fibrin strands staining magenta. (C) Endothelialization is seen, with flattened epithelium of fibroblasts creeping over the surface of the thrombus. CD34 immunocytochemistry shows endothelial knots within the clot (inset), often without overlying endothelium.
Magnification bars ⫽ 500␮m (A); 100␮m (B); 50␮m (C; C, inset). Patient 4: (A) Recent thrombus predominates, with a surface
endothelial covering (inset). (B) Acute thrombus featuring RBCs and endothelial cells with nuclei (top inset) confirmed by CD34 immunocytochemistry (bottom inset). (C) With a Masson trichrome stain, strands of fibrin are seen with intervening neutrophils. Magnification bars ⫽ 150␮m (A, C); 200␮m (B). Patient 5: (A) Overview of specimen to show focal calcification. Hematoxylin and eosin
staining. (B) Area of calcification (top left), with magenta-staining fibrin strands and neutrophils, indicating an acute process (bottom
right). (C) The von Kossa stain not only proves the existence of calcium (blue-black) but also shows intervening cells (pink nuclei),
probably arterial wall components. Magnification bars ⫽ 500␮m (A); 100␮m (B,C).
are systematically different from the analyzed
thrombi. This also raises questions about the factors
making certain thrombi more feasible for extraction.
Such factors could relate to the characteristics of the
thrombi, to the anatomy of the vascular bed, or to
some limitations of the device used.
This study should stimulate the development of
methods for imaging of occlusive thrombi in patients
with acute cerebral ischemia and encourage centers to
examine more of these kinds of specimens, with attention to calcification and surface endothelialization of
thrombi, features that might render tPA less effective if
present in greater degree than in the specimens we analyzed.
This work was supported by the Alberta Heritage Foundation Medical Research (M.D.H.) and the Heart & Stroke Foundation of Alberta/North West Territories/Nunavut (M.D.H.).
Almekhlafi et al: Pathology of Thrombi in Stroke
347
We acknowledge the members of the Calgary Stroke team who
treated the patients.
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Neurofibrillary tau Pathology
Modulated by Genetic
Variation of ␣-Synuclein
Terhi Peuralinna, MSc,1 Minna Oinas, MD,2
Tuomo Polvikoski, MD, PhD,2,3
Anders Paetau, MD, PhD,2 Raimo Sulkava, MD, PhD,4
Leena Niinistö, MD, PhD,5 Hannu Kalimo, MD, PhD,2
Dena Hernandez, MSc,6 John Hardy, PhD,6,7
Andrew Singleton, PhD,6 Pentti J. Tienari, MD, PhD,1,8
and Liisa Myllykangas, MD, PhD2,9
We analyzed whether genetic variation of ␣-synuclein modulates the extent of neuropathological changes in a
population-based autopsied sample of 272 elderly Finns.
None of the 11 markers was associated with the extent of
neocortical ␤-amyloid pathology. The intron 4 marker
rs2572324 was associated with the extent of neurofibrillary
pathology ( p ⫽ 0.0006, permuted p ⫽ 0.004; Braak stages
IV-VI vs 0-II). The same variant also showed a trend for
association with neocortical Lewy-related pathology. These
results suggest for the first time that variation of ␣-synuclein
modulates neurofibrillary tau pathology and support the recent observations of an interaction of ␣-synuclein and tau in
neurodegeneration.
Ann Neurol 2008;64:348 –352
Abnormal protein accumulation characterizes common
age-related neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and dementia with Lewy bodies (DLB). In AD, deposition of
␤-amyloid leads to the formation of neuritic plaques,
From the 1Molecular Neurology Programme, Biomedicum, University of Helsinki; 2Department of Pathology, University of Helsinki
and HUSLAB, Helsinki University Central Hospital, University of
Helsinki, Helsinki, Finland; 3Institute for Aging and Health, Newcastle University, Newcastle upon Tyne, United Kingdom; 4Department of Public Health and General Practice Division of Geriatrics,
University of Kuopio and Kuopio University Central Hospital,
Kuopio; 5Katriina Geriatric Hospital, Vantaa, Helsinki, Finland;
6
Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD; 7University College London, London, United Kingdom; 8Department of Neurology, University of Helsinki and Helsinki University Central Hospital;
9
Folkhälsan Institute of Genetics.
Received Dec 13, 2007, and in revised form Mar 26, 2008. Accepted for publication May 23, 2008.
This article includes supplementary materials available via the Internet at http://www.interscience.wiley.com/jpages/0364-5134/suppmat
Published online Mon 00, 2008, in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/ana.21446
Address correspondence to Dr Tienari, Helsinki Central University
Hospital, Department of Neurology, POB 340, 00290 HUS, Finland. E-mail: pentti.tienari@hus.fi
348
Annals of Neurology
Vol 64
No 3
September 2008
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