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Journal of Neurological Disorders & Stroke

Cite this article: Sallustio F, Arnò N, Di Legge S, Koch G, Martorana A, et al. (2015) Histological Features of Intracranial Thrombo-Emboli Predict Response to Endovascular Therapy for Acute Ischemic Stroke. J Neurol Disord Stroke 3(3): 1105. 1/5

*Corresponding authorFabrizio Sallustio, Department of Neuroscience, Tor Vergata University, Institution: Azienda Ospedaliera Fondazione Policlinico Tor Vergata, Viale Oxford 81, 00133, Roma, Italy, Tel: 39-06-20903425; Fax: 39-06-

Submitted: 18 September 2015

Accepted: 01 November 2015

Published: 02 November 2015

Copyright© 2015 Sallustio et al.

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Keywords•Ischemic stroke•Intracranial thrombi•Histology•Outcome

Research Article

Histological Features of Intracranial Thrombo-Emboli Predict Response to Endovascular Therapy for Acute Ischemic StrokeFabrizio Sallustio1,4*, Natale Arnò1, Silvia Di Legge1, Giacomo Koch1,4, Alessandro Martorana4, Costanza Rossi1, Marina Diomedi1, Costantino Del Giudice2, Lucia Anemona3, Roberto Gandini2, Paolo Stanzione1,4 and Alessandro Mauriello3

1Department of Neuroscience, Tor Vergata University, Italy 2Department of Diagnostic Imaging, University of Rome Tor Vergata, Italy 3Department of Biomedicine and Prevention, University of Rome Tor Vergata, Italy 4Department of Systems Medicine, Santa Lucia Foundation IRCCS, Italy

Abstract

Background: Histological analysis of thromboemboli obtained from acute ischemic stroke patients and evaluation of clinico-histological correlation could provide informations regarding response to therapy, complications and outcome.

Methods: Thromboemboli were retrieved by endovascular mechanical thrombectomy from major arteries of the circle of Willis of patients with acute ischemic stroke. Total thrombus area and percentage of fibrin were calculated.

Results: Three patterns of thrombus histology were identified based on fibrin architecture in 28 specimens: serpentine (15/28), layered (11/28) and red clot (2/28). No differences in stroke etiology were found between different patterns. Comparing serpentine and layered, the latter showed a significantly higher percentage of fibrin (70.5 vs 40.3, p=0.03), significantly higher pre-treatment National Institute of Health Stroke Scale score (21 vs 17.7, p=0.02), a significantly shorter onset to recanalization time (286 min vs 356 min, p=0.02). Thrombus area showed to be higher in cases with multiple vessels involvement (p=0.04), with symptomatic intracranial hemorrhage (p=0.02), with worst 3 months outcome (p=0.04) and higher mortality (p=0.02).

Conclusion: Histological analysis of thromboemboli proved useful in the identification of specific patterns and their behaviour in terms of response to endovascular treatment and clinical outcome.

INTRODUCTIONIn the last few decades strategies for acute ischemic stroke

(AIS) treatment have been dramatically improved by introduction of new devices for endovascular approach [1,2]. This is true especially for AIS secondary to occlusion of large intracranial arteries [1-3]. The high rate of mortality and disability despite treatment with intravenous thrombolysis associated to this subtype of stroke has been a trigger for current intense research in acute endovascular treatment (ET) [4].

Results of the Multicenter Randomized Clinical Trial of

Endovascular Treatment for Acute Ischemic Stroke in the Netherlands (MR CLEAN) [1] and those of the recent Endovascular Treatment for Small Core and Anterior Circulation Proximal Occlusion with Emphasis on Minimizing CT to Recanalization Times (ESCAPE) Trial [2] suggest that thrombectomy now becomes the new standard treatment for severe stroke with proximal large-vessel occlusion of the anterior circulation [5].

Current devices give the great chance to isolate thrombo-emboli and analyze them with the potential to obtain histological details. This in turn could be of value in terms of etiological

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definition (considering the old theory that “red clot” are of cardiac origin and “white clot” of arterial one) [6] and planning of secondary prevention therapy. Frequently, lack of causes or the presence of competing causes can make therapeutic decisions difficult. Thus histological examination of thrombo-emboli obtained during AIS ET could be a useful tool to take a “snapshot” of what is happening from a pathophysiological point of view. Another important issue that could be investigated through histological analysis of retrieved thrombo-emboli is represented by all those intrinsic factors that affect arterial recanalization such as lytic drugs, thrombectomy devices, arterial tortuosity, cellular / biochemical composition of the clot and their interaction7. Today, only few studies have sistematically evaluated histology of intracranial thrombi retrieved during endovascular procedures for AIS [6,8-9].

Thus the aim of the present study was to evaluate possible correlation between histological analysis of intracranial arterial thrombi and specific clinical features, response to therapy, complications and outcome.

MATERIALS AND METHODS

Clinical Protocol

Thrombi were retrieved from intracranial circulation of patients with AIS by means of different mechanical embolectomy devices in use at Policlinico Tor Vergata stroke center [10-12]. The choice of device was discretionary and based on neuro interventionalist’s judgement. Both anterior and posterior circulation strokes were included in the study.

Patients were treated with a bridging strategy (intravenous thrombolysis at full dose plus mechanical thrombectomy) or a stand-alone thrombectomy depending on onset-to-hospital arrival time, presence of early ischemic changes on computed tomography and the risk of intracranial haemorrhage (ICH) at admission evaluation by stroke neurologist [13]. No aspirin or any other drug was administered before the above specified treatment.

Endovascular procedure was performed after 4-vessel cerebral angiography under general anesthesia and was stopped after arterial recanalization or if vessel was not opened after 3 device passes. Thrombolyis in Myocardial Infarction (TIMI) score (0: no perfusion; 1:penetration past occlusion without distal branch filling; 2: partial perfusion with incomplete or slow distal branch filling; 3: complete perfusion) was used to define angiographic outcome [14] with TIMI 2/3 defined as successful recanalization. National Institute of Health Stroke Scale (NIHSS) was used for scoring clinical evaluation at different times whereas modified Rankin Scale (mRS) was adopted to evaluate 3 months functional outcome.

The presumed etiology was determined on the basis of Trial of ORG 10172 in Acute Stroke Treatment (TOAST) [15] criteria. TOAST is a categorization of subtypes of ischemic stroke based on clinical features and results of ancillary diagnostic studies. The evaluation of this classification was made by two neurologists (C.R. and M.D.) blinded to the histological findings. The study was approved by and conducted in accordance with the guidelines of the Human Research Committee of our Institution and informed

consent was obtained from all patients or their relatives.

Histological evaluation

Thrombi were fixed in phosphate buffered formalin in the angiographic suite soon after retrieval. Thrombus material was embedded in paraffin, sectioned at 4-μm thickness and stained with hematoxylin and eosin and phosphotungstic acid-haematoxylin stain. According to Marder et al. [6] histological samples were classified, on the basis of fibrin architecture, in 3 different patterns: serpentine, layered and red clot (without fibrin interspersed). Total thrombus area (TTA) and thrombus fibrin percentage (TFP) were measured by an histological examinator (A.M.) who was blinded to clinical findings.

Statistical analysis

Data were analyzed by SPSS 13.0 (Statistical Package for the Social Sciences) software. Continuous and categorical variables are expressed as mean ± SD and as frequency values and proportions, respectively. Pearson’s chi-square test was utilized to assess possible differences of dichotomous variables between plaques of the various groups examined. The means of normally distributed data were compared with Student’s t test. In the other cases the groups were compared with Mann-Whitney’s U test. A p-value of <0.05 was considered statistically significant.

RESULTSBetween October 2010 and October 2013 (thus in a period

preceding the publication of positive randomized control trials on endovascular stroke therapy [1,2]) twenty-eight thromboemboli were retrieved from intracranial circulation of the same number of AIS patients, in 80% of cases in multiple fragments and in the remaining 20 % of cases as a single mass. Devices used for this purpose in the referring period were Solitaire AB (14/28), Penumbra System (8/28) and Revive (2/28). The remaining 4 samples were retrieved by a combination of penumbra System and Solitaire AB.

Mean age was 62 ± 13,4 years and mean time delay from stroke onset to thrombus retrieval and formalin fixation was 320,9 ± 75,6 min. TOAST criteria based etiology was cardioembolic (CE) in 11/28 (39%) and large artery (LA) in 17/28 (61%). The sample included 26/28 (93%) anterior circulation stroke and 2/28 (7%) posterior circulation stroke.

Pre-treatment angiography identified 15 tandem occlusions, 9 middle cerebral artery occlusions, 2 T carotid occlusions, 1 isolated basilar artery (BA) occlusions, 1 isolated vertebral artery occlusion.

Percutaneous transluminal carotid angioplasty and stenting were performed before retrieval in all 15 tandem lesion patients for high-grade stenosis of the extracranial internal carotid artery. Successful recanalization (TIMI 2/3) occurred in 24 cases (85%), while unsuccessful recanalization (TIMI 0/1) in the remaining 4 (15%). Additional treatment with intravenous and/or intrarterial thrombolysis was performed in 9/28 patients (32%).

Histology examination identified 15 serpentine (S) patterns, 11 layered (L) patterns and 2 red clot (RC). All these data are summarized in table 1. High magnification of the 3 histological

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Table 1: Patient characteristics.

No Sex Age TOAST Occl.site TtoR(min) Strategy Histology

1 M 69 C.E. r tandem 295 P Serpentine

2 F 79 C.E. r MCA 480 S Serpentine

3 M 72 C.E. r tandem 290 S Serpentine

4 M 68 L.A. l VA 230 i.v.rtPA+S Red Clot

5 M 78 C.E. r tandem 315 P+S Layered

6 M 51 L.A. l tandem 270 i.v.rtPA+P+S Layered

7 F 68 L.A. r MCA 300 S Serpentine

8 M 56 L.A. l tandem 375 i.v.rtPA+S Layered

9 M 77 L.A. R tandem 315 P Serpentine

10 M 84 L.A. R tandem 360 S Red Clot

11 M 76 L.A. l MCA 330 i.v.rtPA+S Serpentine

12 M 69 L.A. r MCA 350 i.v.rtPA+S Serpentine

13 M 61 L.A. l tandem 280 P Serpentine

14 M 56 C.E. r MCA 230 S Layered

15 M 48 L.A. r tandem 265 S Layered

16 F 45 C.E. r tandem 240 i.v.rtPA+P Layered

17 F 56 C.E. l T occlusion 385 P Serpentine

18 M 37 L.A. l tandem 310 P+S Serpentine

19 F 87 L.A. l tandem 275 R Layered

20 M 60 C.E. r T occlusion 385 P Serpentine

21 M 70 C.E. r MCA 280 P Layered

22 F 64 C.E. r tandem 210 S+i.a.urok Serpentine

23 M 69 L.A. r tandem 360 i.v.rtPA+S Layered

24 M 49 L.A. l MCA 490 P+S Serpentine

25 M 54 L.A. AB 505 S Serpentine

26 F 45 L.A. l tandem 320 P Serpentine

27 F 37 L.A. r MCA 230 i.v.rtPA+R Layered

28 M 52 C.E. r ACA+MCA 310 S LayeredL.A.: Large Artery; C.E.: Cardioembolic; MCA: Middle Cerebral Artery; ACA: Anterior Cerebral Artery; VA: Vertebral Artery; BA: Basilar Artery; TtoR: onset-to-retrieval interval; P: Penumbra System; R: Revive; S: Solitaire.

patterns are shown in figure 1. Any calcific component was found in the retrieved material.

When we analyzed possible clinical-histological correlations we excluded from the analysis the 2 red clot. No significant correlations between etiology and histological patterns (LA/CE etiology in S vs L subgroups: 9/6 vs 6/5; p=0,78) nor significant difference in age (62.4±12.8 vs 59±15 yrs ; p=0.53) and sex (male) (66.5% vs 72.5%; p=0.74) between S and L subgroups were found. More patients in the S subgroup were affected by hypertension with a difference approaching significance (73% vs 36%; p=0.06) whereas no significant differences were found in other vascular risk factors between the two groups.

Patients in the L subgroup showed a more severe clinical presentation as assessed by NIHSS (17.7±2.5 vs 21±2.2; p=0.02), were more often treated by i.v. and/or i.a. rtPA with a difference approaching significance (13% vs 45.5%; p=0.07) and had shorter onset to recanalization time (356±88 vs 286±49; p=0.02) and groin puncture to recanalization time (358.4±95.5 vs 279.5±30.8;

p=0.02). No difference in the rate of successful recanalization (TIMI 2/3) was found (13/15: 86% vs 11/11:100%; p=0.24) between the two groups. A higher rate of patients in the L subgroup (40% vs 45%; p=0.08) showed a dramatic 24 hours clinical improvement (defined as a reduction in NIHSS score of ≥ 8 points). No differences in the rate of any or symptomatic ICH, procedural complications, mortality or in functional outcome were found.

TFP was significantly higher in the L subgroup (44.3±17 vs 70.5±9.2; p=0.03) (Table 2). Analyzing TTA and its possible correlations with other findings, higher values were related to multiple vessel occlusion (22.2±14 vs 11.67±7.29; p=0.04), previous ischemic lesion on CT/MRI (34.4±4.7 vs 14.8±11.7; p=0.01), symptomatic intracranial haemorrhage (31.6±12.9 vs 16.2±11.8; p=0.02), 3 months poor functional outcome (mRS>2: 22.2±13.3 vs 12.8±10.7; p=0.07; mRS>3: 25.1±13.1 vs 14.5±11.5; p=0.04) and 3 month higher mortality rate (28.5±13.1 vs 15.6±11.7; p=0.02). No significant correlation was found with rtPA use nor with TIMI score. The two red clot samples excluded

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Table 2: Comparison of Serpentine and Layered patterns.

Serpentine (n=15) Layered (n=11) p value

Age 62.4(±12.8) 59(±15) 0.53

Sex(males) 10/15(66.5%) 8/11(72.5%) 0.74

Hypertension 11/15(73%) 4/11(36%) 0.06

TFP 44.3 (±17) 70.5(±9.2) 0.03

TTA 18.3(±11.5) 18.9(±15.4) 0.9

Pre-treatment-NIHSS 17.7(±2.5) 21(±2.2) 0.02

Tandem/T vs MCA/VA/BA occlusion 9/15(60%) 8/11(72%) 0.16

i.v. and/or i.a. rtPA 2/15(13%) 5/11(45.5%) 0.06

Onset-to-recanalization time(min) 356(±88) 286(±49) 0.02

A.P.-to-recanalization time (min) 358.4(±95.5) 279.5(±30.8) 0.01

TIMI 2/3 vs TIMI 0/1 13/15(86,6%) 11/11(100%) 0.24

24hs improvement (≥8 NIHSS points) 6/15(40%) 5/11(45%) 0.08

Any ICH 5/15(33%) 7/11(63%) 0.39

Symptomatic ICH 1/15(6%) 3/11(27%) 0.15

3 month mRS≤3 8/15(53%) 8/11(72%) 0.31

3 month-mortality 4/15(26.5%) 2/9(22%) 0.6

TFP: Total Fibrin Percentage; TTA: Total Thrombus Area; ICH: Intracranial Haemorrhage

100 µm 100 µm 100 µm

Figure 1 Histological patterns of intracranial thrombi.Panel 1a: serpentine, with fibrin: platelet bands arranged in a serpentine way (H-E, 10x); Panel 1b: layered, with fibrin: platelet bands arranged in layers (H-E, 10x); Panel 1c: red clot, with erythrocytes and nucleated cells interspersed (H-E, 10x).

from the analysis were retrieved from patients who were both classified as large artery and died at three months.

DISCUSSIONThis study provides remarkable correlations between the

histology of thrombi retrieved from intracranial circulation and clinical outcome of AIS patients. Even though our data confirmed previous findings by Marder et al [6] and by Singh et al [9] that histological pattern does not predict successful recanalization and does not correlate with stroke etiology, interestingly we found that patients in the L pattern group had higher TFP, were more severely affected at onset and were also more likely

to undergo faster recanalization and showed a trend toward more pronounced early clinical improvement. Despite L pattern showed a tendency toward being more often treated by rtPA, recanalization time was still significantly shorter after removal of rtPA cases. No explanation of these findings based on type of vascular occlusion cannot be made since no significant difference was found in the rate of tandem or T occlusion between the two groups. In our analysis a higher TTA was related to multiple vessel occlusion, sICH, poor functional outcome and higher mortality. This is in line with other recent findings from computed tomography-studies analyzing clot length in hyperdense MCA sign and showing that more proximal occlusion site can predict longer clot and less effectiveness of recanalization therapy [16].

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The correlation between sICH and higher values of TTA found in our study could be explained as a result of reperfusion syndrome in severe perfusion deficit considering that more than half of our sample were represented by tandem and T occlusions. We cannot explain why we found higher TTA in patients with previous ischemic lesions on neuroimaging.

As a limitation, our analysis derived from a single-center study. Moreover we cannot exclude that some fragments of the thrombotic material remained in situ; therefore it is possible that histological informations we had from the retrieved material were incomplete and not entirely representative of the in-situ thrombus. Lastly we cannot answer the question whether layered pattern is directly related to rtPA use.

CONCLUSIONOur findings suggest that L pattern of specimens retrieved

from intracranial arteries of AIS patients during ET is associated with more severe clinical presentation, faster recanalization, more pronounced early clinical improvement with no difference in the rate of procedural or post-procedural complications.

ACKNOWLEDGEMENTSWe thank Dr. Marta Panella, Dr. Vittoria Carla D’Agostino, Dr.

Barbara Rizzato, Dr. Giacomo Koch, Dr. Simone Napolitano, Dr. Domenico Samà, Dr. Matteo Stefanini, Dr. Carlo Andrea Reale, Dr. Sebastiano Fabiano, Dr. Alessio Spinelli, Dr. Daniel Konda, Dr. Enrico Pampana for collection and storage of histological specimens.

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Sallustio F, Arnò N, Di Legge S, Koch G, Martorana A, et al. (2015) Histological Features of Intracranial Thrombo-Emboli Predict Response to Endovascular Therapy for Acute Ischemic Stroke. J Neurol Disord Stroke 3(3): 1105.

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