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(Stroke. 1996;27:431-434.)
© 1996 American Heart Association, Inc.

Articles

Microthromboemboli in Acute Infarcts

Analysis of 40 Autopsy Cases

Norbert Heye, MD Jorge Cervós-Navarro , MD

From the Institute of Neuropathology, Freie Universität Berlin (Germany).

Correspondence to Norbert Heye, MD, Department of Neurology, HSE-781, University of California at San Francisco, San Francisco, CA 94143-0518.

	Abstract

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Background and Purpose We investigated the distribution and frequency of microthromboemboli (MTE) in acute infarcts in humans and determined whether MTE in the contralateral circulation resulted in histological changes.

Methods Forty patients dying within the first month after unilateral infarct were investigated. Infarct etiology was determined mainly on the pathological findings. Whole brain sections from the region of maximal necrosis were stained for fibrin. Fibrin-containing MTE were transferred to a schematic drawing and counted. Sections from 20 patients without infarcts served as controls.

Results Infarct sections had significantly more MTE than controls. Infarcts of thrombotic (n=6) and thromboembolic (n=21) origin had more MTE than infarcts of embolic origin (n=13). Thromboembolic infarcts had the highest number of MTE within the region assumed to be the ischemic penumbra, other arterial territories, and the contralateral hemisphere. Patients with large infarcts and those with short clinical courses had a higher number of MTE. Sixteen patients had recent micronecroses in the contralateral hemisphere.

Conclusions There seems to be a pattern of MTE in acute infarcts that is dependent on cause, size, and clinical duration. Our findings of contralateral micronecroses emphasize that acute infarcts may result in more widespread cerebral injury than clinically expected. Given the many variables influencing stroke and death in humans, the results have to be interpreted with caution.

Key Words: embolism • platelets • thrombosis • fibrin

	Introduction

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The natural history of a blood clot obstructing an arterial lumen and leading to ischemic stroke is difficult to determine, but recanalization, clot fragmentation, and washout into distal parts of the circulation are likely to occur.¹ Circulatory disturbances in acute ischemic stroke also result in a prothrombotic state,² with circulating microemboli that have been detected intracerebrally by Doppler studies.^{3 4}

Several animal models have been used to investigate the effects of arterial thrombosis or embolism, but very little work has been done in regard to the neuropathology of circulatory disturbances in acute infarcts in humans. Small occlusive clots, designated microthromboemboli (MTE), were shown,^{5 6 7} but it is unclear whether they have a certain pattern of distribution within the necrosis or whether their occurrence depends on the infarct mechanisms. Even less information is available on circulatory disturbances in the contralateral hemisphere,⁷ and it is unknown whether MTE cause necroses or whether they are lysed without having structural consequences. Here we report our attempt to evaluate the distribution and incidence of fibrin-containing MTE in whole brain sections after acute infarct in humans.

	Methods

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Sixty-six consecutive neuropathological examinations of patients dying within the first month after the onset of unilateral supratentorial infarcts were performed over a period of 26 months. To evaluate the changes strictly related to a single episode of focal stroke, 26 cases were excluded because of the following: (1) stroke with repeated clinical symptoms, suggesting multiple episodes, multiple strokes, or stroke in progression; (2) stroke with bilateral symptoms; (3) fresh, bilateral, macroscopically detectable neuropathological changes; (4) myocardial infarction within the last 3 months, suggesting hemodynamic impairment; (5) resuscitation within the last 3 months, suggesting global ischemic injury; or (6) prolonged bacteremia or advanced stages of malignant tumors.

The study group consisted of 40 patients (mean±SD age, 72.9±15.6 years): 23 women (76.8±13.1 years) and 17 men (67.6±8.1 years). To stage the clinical duration, the cases were grouped as recent stroke (day 1 through 7 after the clinical onset of the stroke, n=25, 4±1.8 days [mean±SD]), intermediate stroke (day 8 through 14, n=7, 11.3±1.4 days), and old stroke (day 15 through 31, n=8, 25.4±4 days). Medical history was significant for diabetes mellitus (17 cases), arterial hypertension (13), and smoking (6). The cause of death was central in 13 cases (ie, stroke-related edema with herniation), pneumonia in 8, acute myocardial infarction or pulmonary embolism in 7, and chronic circulatory failure in 12 (ie, right/left heart enlargement, liver congestion, or pulmonary edema). The severity of atherosclerosis in the aorta and coronary, carotid, and intracerebral arteries was scored with defined criteria as 0, none; 1, mild; 2, medium; and 3, severe.

Stroke etiology was determined on macroscopic findings. In cases in doubt, a histological examination of the clot and its relation to the vessels was performed. The following definitions were used.^{1 8} The infarct was assumed to be of embolic origin in cases with confirmatory signs for the origin of an embolus in the heart, the aortic arch, or the extracranial arteries and/or in cases with a history of atrial fibrillation (n=13). Infarcts showing a recent lumen-obstructing arterial thrombus (up to the macroscopically assessable arteries, ie, the M2 portion of the middle cerebral artery) and no obvious signs of a cardiac or arterial source for the embolism were assumed to be caused by in situ thrombosis and classified as thrombotic strokes (n=6). These patients had a generalized atherosclerotic vessel disease. Five had extensive intracranial and extracranial thrombosis, and 1 had a more circumscribed thrombus at the transition of the internal carotid to the middle cerebral artery. Generalized atherosclerotic vessel disease was also present in the remaining cases (n=21), but they had neither obvious embolic sources nor arterial thrombi. Hypothesizing that these cases were caused either by small arteriosclerotic lesions with subsequent embolism, in situ thrombosis, or both, we classified these cases as thromboembolic infarcts.

Sections at the level of the basal ganglia taken from 20 patients with a clinical diagnosis of senile dementia served as controls (11 women, 9 men; mean±SD age, 77.8±13.4 years). These patients had no clinical or neuropathological evidence of recent stroke. Neuropathological diagnoses were Alzheimer's disease (n=5), subcortical arteriosclerotic encephalopathy (n=4), or a combination of both (n=11). Eight patients died from pneumonia and 3 from acute and 9 from chronic cardiovascular failure.

The brains were fixed in buffered formalin and cut into 8- to 10-mm-thick sections. The infarct size was classified as small (n=9, area of <4 cm² in the section containing the largest infarct portion), middle (n=16, 5 to 10 cm²), or large (n=15, >10 cm²). The section containing the major infarct portion was embedded, cut into 15-µm-thick sections, and stained with hematoxylin and eosin, Nissl's, and elastica–van Gieson's stains and phosphotungstic acid–hematoxylin; the latter demonstrates fibrin polymers as deep-blue filiform material. To prevent misreading of postmortem fibrin accumulation, we only acknowledged intravascular fibrin, either densely packed or arranged in a netlike pattern. The MTE were investigated under a light microscope with a 10-fold magnification and systematically and orthotopologically transferred to a schematic drawing of the slide. For analysis we determined the total number of MTE, as well as the number of MTE in the ischemic and nonischemic hemisphere, inside and outside of the tissue necrosis in the ischemic hemisphere and in an area postulated to be the morphological equivalent of the ischemic penumbra (ie, 1 cm on each side of the necrotic rim). In the control group we determined the total number of MTE and the number in each hemisphere.

Statistical analysis was performed using SPSS-PC+ software. The original distribution of the dependent variables was highly asymmetrical, and therefore the logarithmic transformation was used to improve the distribution characteristics of skewness (symmetry) and kurtosis (heavy-tailedness). Age was divided into two groups by the median in the cases and the control group. ANOVA was used to test the statistical hypothesis.

	Results

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Sections from patients dying after stroke had significantly more MTE than controls (mean [range]: 131 [0 to 508] versus 4 [0 to 17], respectively; P=.0). Thrombotic (219 [4 to 508]) or thromboembolic (168 [7 to 492]) infarcts had significantly more MTE than embolic infarcts (29 [0 to 98]; ²=42.84, P=.0004). The tissue outside the necrosis was more often covered by MTE in thromboembolic infarcts (66 [0 to 220]) than in others (thrombotic, 14 [0 to 92]; embolic, 7 [0 to 46]; ²=52.80, P=.0002). Thromboembolic infarcts also had significantly more MTE in the contralateral hemisphere (37 [2 to 201]) versus thrombotic and embolic infarcts (17 [0 to 29] and 5 [0 to 29], respectively; ²=36.56, P=.0003) (Fig 1). They also had the highest density of MTE within an area of 1 cm on each side of the necrotic rim (76 [1 to 236]) versus thrombotic (16 [0 to 87]) and embolic (10 [0 to 38]) infarcts (²=42.73, P=.0009). Large infarcts tended to have more MTE than smaller infarcts (²=18.25, P=.0517), as did those in patients surviving less than 2 weeks (²=17.36, P=.0605), but this was not statistically significant. In 7 patients surviving more than 21 days, up to 68 MTE were observed (21 [0 to 68]), which was significantly different from our controls (P=.007). The patient's age, sex, or extent of atherosclerosis had no influence on the number or distribution of MTE in the study or control groups.

View larger version (118K): [in this window] [in a new window]   Figure 1. Thrombotic stroke, clinical duration of 4 days. Small embolus (arrowhead) in a cortical artery in the hemisphere contralateral to the main necrosis (phosphotungstic acid hematoxylin, magnification x40).

In 15 cases we found at least one zone of micronecrosis (range, 1 to 6) in the nonsymptomatic hemisphere (Fig 2) with a histological stage similar to that observed in the clinically affected hemisphere. These zones of micronecrosis were found mainly in the cortex, predominantly in border zones of arterial territories. They occurred also in the center of an arterial territory or in the supply area of penetrating white matter arteries. We were not able to see the supplying occluded vessels in any of these micronecroses. No micronecroses were observed in the controls. A schematic presentation of the pathological features and the distribution of MTE of selected cases is given in Fig 3.

View larger version (168K): [in this window] [in a new window]   Figure 2. Cortical micronecroses in the hemisphere contralateral to the main necrosis. Because of the collateral cortical vasculature, the necroses are tighter toward the white matter. A, Embolic infarct, clinical duration of 4 days, shows fresh tissue paleness without macrophage response (phosphotungstic acid hematoxylin [PTAH], magnification x4). B, Thromboembolic infarct, clinical duration of 6 days, shows dense macrophage infiltration with early vessel proliferation (PTAH, x4).

View larger version (48K): [in this window] [in a new window]   Figure 3. Schematic presentation of pathological findings from selected cases. The clinical duration in days and the presumed cause are given. In some cases the normal anatomy is disrupted by fixation artifacts. The areas of necroses are marked by cross-hatched lines, and each MTE is represented by a dot. Note the variations in MTE frequency, their accumulation in the border zones of vital/necrotic tissue in thromboembolic infarcts (in particular, cases 3d and 7d), and the micronecroses in both hemispheres ipsilateral and contralateral to the main necrosis (arrows).

	Discussion

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We have shown that fibrin-containing MTE accumulate within the ischemic hemisphere and that the frequency and distribution is dependent on the cause of the underlying stroke and perhaps also on the size of tissue necrosis and the clinical duration. Embolic strokes had the lowest and thrombotic or thromboembolic strokes the highest numbers of MTE. Thromboembolic strokes accumulated MTE within the postulated morphological equivalent of the ischemic penumbra. We demonstrated MTE outside the territory of the primary occluded vessel and in the contralateral hemisphere, again occurring most frequently with thromboembolic strokes. We also showed that micronecroses of a histological stage similar to the symptomatic necrosis occur in the hemisphere contralateral to stroke.

The lack of histological data on microcirculatory disturbances in humans is probably related to methodological difficulties; such difficulties were not absent from this study. We observed MTE, but we cannot comment on their origin. They may represent a fragment of the primary clot or emboli from distal parts of the circulation. They may have resulted from local or systemic prothrombotic activity, blood stasis, or raised intracranial pressure or may be unrelated to the stroke. They may have occurred as a reaction to an underlying atherosclerotic vessel disease, agonal circulation, or coagulation disturbances. It is also possible that some of the fibrin accumulations considered as MTE were formed postmortem. Some of these circumstances may be responsible for the occurrence of MTE in our controls. There are also uncertainties in respect to the methods used for detecting blood clots. Pure platelet aggregates are hard to detect,^{1 9} and therefore we decided to look only for fibrin clots. We are thus unable to comment on pure platelet clots or platelet-endothelial interactions. Another methodological uncertainty is the heterogeneity of our material in respect to age and sex, underlying diseases, survival time, cause of death, and investigated brain region. The wide range in the individual MTE count is also notable. Finally, our classification approach was based predominantly on the macroscopic and only to a limited extent on the clinical findings. Both the discriminatory accuracy as well as the clinical applicability of this approach are debatable. On the other hand, there was a clear difference in MTE frequency between the study group and the control group. Our approach of using fibrin as a marker for intravascular clots seems more reliable than using platelet thrombi. Pure platelet thrombi are not stabilized by fibrin and are thought to have fewer deleterious consequences than fibrin-containing thrombi.^{2 10 11} Given the important methodological problems, the results have to be interpreted with caution. Nevertheless, we feel it worthwhile to address the neuropathological questions that have been investigated extensively by clinical and experimental methods.

Thromboembolic and thrombotic strokes had more MTE than embolic strokes. This could be explained by either a lack of MTE production in embolic strokes or a higher susceptibility to lytic stimuli, ie, a difference in clot composition. Because a variability of cerebral microemboli has been demonstrated in experimental animals^{9 10} and a different susceptibility of microemboli to anticoagulant therapy was observed in humans,^{3 4} we favor the latter possibility.

We found increased MTE in the area postulated to represent the morphological substrate of the ischemic penumbra. We concede that our approach of determining this region topologically is simplistic, and we are aware that histological definition of this concept is difficult.¹² Even in large animal models of middle cerebral artery occlusion, no consistent histological picture of the ischemic penumbra has been identified.^{13 14} On the other hand, platelets¹⁵ and fibrin accumulate in low-flow regions during the postischemic period. Treatment with tissue plasminogen activator results in reduced brain injury,¹⁶ which suggests an improvement of microcirculation in the ischemic tissue at its borders. We therefore reasoned that our definition of penumbra is congruent at least in part with the functional concept of the ischemic penumbra.¹² Accordingly, the accumulation of MTE in the penumbra of thromboembolic strokes may indicate ongoing progressive tissue infarction. Our findings of MTE in the penumbra even after a period of 3 weeks may account for the existence of a chronic ischemic penumbra, the significance of which has not been stressed up to now.¹⁷

We found MTE outside the territory of the primary occluded vessel and in the contralateral hemisphere in all three groups, mainly in thromboembolic infarcts. This confirms transcranial Doppler studies showing microemboli in the asymptomatic hemisphere in unilateral or bilateral carotid diseases and stroke,^{3 18} and experimental studies have demonstrated platelet and fibrin thrombi in the contralateral hemisphere after unilateral carotid thrombosis.^{9 19} So far, microthrombi and microemboli in the contralateral hemisphere have not been demonstrated in humans.

We observed MTE in a diffuse distribution and micronecroses in both hemispheres. MTE frequency is dependent on the infarct type, and MTE are an important factor in delineating the ischemic penumbra. Micronecroses, probably clinically unnoticed or asymptomatic, were found in both hemispheres. They are thought to develop in close temporal relation to the major necrosis. Acute stroke may clinically be well defined, but our morphological observations emphasize a more widespread and prolonged cerebral injury.

Received September 5, 1995; revision received December 11, 1995; accepted December 15, 1995.

	References

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1. Cervós-Navarro J. Spezielle pathologische Anatomie. In: Doerr W, ed. Pathologie des Nervensystems I: Durchblutungsstörungen und Gefäßerkrankungen des Zentralnervensystems. Berlin/Heidelberg/New York: Springer; 1980:Bd 13.
2. Fisher M, Francis R. Altered coagulation in cerebral ischemia. Arch Neurol. 1990;47:1075-1079. [Abstract]
3. Georgiadis D, Hill M, Zunker P, Stögbauer F, Ringelstein EB. Anticoagulation monitoring with transcranial Doppler. Lancet. 1994,344:1373-1374.
4. Siebler M, Nachtmann A, Sitzer M, Steinmetz H. Anticoagulation monitoring and cerebral microemboli detection. Lancet. 1994;344:555. Abstract. [Medline] [Order article via Infotrieve]
5. Romanul FC, Abramovicz A. Changes in the brain and pial vessels in arterial border zones. Arch Neurol. 1964;11:40-65.
6. Jörgensen L, Torvik A. Ischemic cerebrovascular disease in an autopsy series. J Neurol Sci. 1966;3:490-509. [Medline] [Order article via Infotrieve]
7. Heye N, Paetzold C, Steinberg R, Cervós-Navarro J. The topography of microthrombi in ischemic brain infarct. Acta Neurol Scand. 1992;86:450-454. [Medline] [Order article via Infotrieve]
8. Constantinides P. Pathogenesis of cerebral artery thrombosis in man. Arch Pathol. 1967;83:422-428. [Medline] [Order article via Infotrieve]
9. Tietjen GE, Futrell N, Garcia JH, Millikan C. Platelet emboli in rat brain cross when the contralateral carotid artery is occluded. Stroke. 1991;22:1053-1058. [Abstract/Free Full Text]
10. Halvorsen AM, Futrell N, Wang LC. Fibrin content of carotid thrombi alters the production of embolic stroke in the rat. Stroke. 1994;25:1632-1636. [Abstract]
11. Rigamonti D, Uede T, Johnson PC, Bojanowski WM, Awad IA, Michael KT, Carter LP, Spetzler RF. A new model of cerebral embolic ischemia using autologous arterial thrombus. Barrow Neurol Inst Q. 1989;5:2-7.
12. Ginsberg MD, Pulsinelli WA. The ischemic penumbra, injury thresholds, and the therapeutic window for acute stroke. Ann Neurol. 1994;36:553-554. Editorial. [Medline] [Order article via Infotrieve]
13. Garcia JH, Mitchem HL, Briggs L, Morawetz R, Hudetz AG, Hazelrig JB, Halsey JH, Conger KA. Transient focal ischemia in subhuman primates: neuronal injury as a function of local cerebral blood flow. J Neuropathol Exp Neurol. 1983;42:44-60. [Medline] [Order article via Infotrieve]
14. Strong AJ, Tomlinson BE, Venables GS, Gibson G, Hardy JA. The cortical ischaemic penumbra associated with occlusion of middle cerebral artery in the cat, 2: studies of histopathology, water content, and in vitro neurotransmitter uptake. J Cereb Blood Flow Metab. 1983;3:97-108. [Medline] [Order article via Infotrieve]
15. Obrenovitch PT, Hallenbeck JM. Platelet accumulation in regions of low blood flow during postischemic period. Stroke. 1985;16:224-234. [Abstract/Free Full Text]
16. Bedar MM, McAucliffe T, Raymond S, Gross CE. Tissue plasminogen activator reduces brain injury in a rabbit model of thromboembolic stroke. Stroke. 1990;21:1705-1709. [Abstract/Free Full Text]
17. Ringelstein EB, Biniek R, Weiller C, Ammeling B, Nolte PN, Thron A. Type and extent of hemispheric brain infarctions and clinical outcome in early and delayed middle cerebral artery recanalization. Neurology. 1992;42:289-298. [Abstract/Free Full Text]
18. Georgiadis D, Grosset DG, Quin RO, Nichol JA, Bone I, Lees KR. Detection of intracranial emboli in patients with carotid disease. Eur J Vasc Surg. 1994;8:309-314. [Medline] [Order article via Infotrieve]
19. Dietrich WD, Dewanjee S, Prado R, Watson BD, Dewanjee MK. Transient platelet accumulation in the rat brain after common carotid artery thrombosis: an ¹¹¹In-labeled platelet study. Stroke. 1993;24:1534-1540.[Abstract/Free Full Text]

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This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Heye, N. Search for Related Content PubMed PubMed Citation Articles by Heye, N. Pubmed/NCBI databases Compound via MeSH Substance via MeSH