stroke-2001-montaner-1759-66

Arenillas, Miguel Angel Gonzlez and Jasone MonasterioJoan Montaner, Jos Alvarez-Sabn, Carlos Molina, Ana Angls, Sonia Abilleira, Juan

Profile and Relation to Neurological ImpairmentMatrix Metalloproteinase Expression After Human Cardioembolic Stroke: Temporal

Print ISSN: 0039-2499. Online ISSN: 1524-4628 Copyright 2001 American Heart Association, Inc. All rights reserved.

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Matrix Metalloproteinase Expression AfterHuman Cardioembolic Stroke

Temporal Profile and Relation to Neurological Impairment

Joan Montaner, MD; Jos Alvarez-Sabn, MD; Carlos Molina, MD; Ana Angls, MD;Sonia Abilleira, MD; Juan Arenillas; Miguel Angel Gonzlez; Jasone Monasterio, MD

Background and PurposeUncontrolled expression of matrix metalloproteinases (MMPs) can result in tissue injury andinflammation. In animal models of cerebral ischemia, the expression of MMP-2 and MMP-9 was significantly increased.However, their role in human stroke in vivo remains unknown. Therefore, we sought to determine the temporal profileof MMP expression in patients with acute ischemic stroke and to investigate its relationship to stroke severity, locationof arterial occlusion, and total infarct volume.

MethodsSerial MMP-2 and MMP-9 determinations were made in 39 patients with cardioembolic strokes that involvedthe middle cerebral artery territory by means of enzyme-linked immunosorbent assay. Blood samples, transcranialDoppler recordings, and National Institutes of Health Stroke Scale (NIHSS) scores were obtained at baseline and at 12,24, and 48 hours after stroke onset. Infarct volume was measured with CT scanning at 48 hours.

ResultsNo correlation was found between MMP-2 and NIHSS score at any time point, although a close relation appearedbetween mean MMP-9 and final NIHSS score (r50.486, P50.002). MMP-9 value was the only factor associated withthe final NIHSS score in the multiple logistic regression model (OR 4.54, 95% CI 1.5 to 13.75). A cut-point of MMP-9142.18 ng/mL had a positive predictive value of 94.4% to assess a patients NIHSS (,8 or $8) by the end of the study.Final MMP-2 and MMP-9 levels were significantly lower when recanalization occurred (5286144.3 versus681.46239.2 ng/mL, P50.031 for MMP-2; 110.26100.9 versus 244.86130 ng/mL, P50.004 for MMP-9). A positivecorrelation was found between mean MMP-9 and infarct volume (r50.385, P50.022).

ConclusionsMMPs are involved in the acute phase of human ischemic stroke. MMP-9 levels are associated withneurological deficit, middle cerebral artery occlusion, and infarct volume. (Stroke. 2001;32:1759-1766.)

Key Words: cerebral ischemia n matrix metalloproteinases n stroke, cardioembolic

Matrix metalloproteinases (MMPs) are a family of zinc-binding proteolytic enzymes that normally remodel theextracellular matrix and pathologically attack substrates aspart of the neuroinflammatory response.1 MMP-2 (72 kDa,gelatinase A) and MMP-9 (92 kDa, gelatinase B) specificallyattack type IV collagen, laminin, and fibronectin, which arethe major components of the basal lamina around cerebralblood vessels. Proenzyme activation and enzyme activitiesare tightly regulated by tissue inhibitors of MMPs (TIMPs)and interactions with surrounding extracellular matrixmolecules.2

MMPs participate in many physiological tissue remodelingprocesses, including embryological and bone remodeling,wound healing, angiogenesis, ovulation, and implantation.3However, uncontrolled expression of MMPs can result intissue destruction and inflammation.

Recently, abnormal MMP activity has been implicated incerebral ischemia. In animal models, MMP-2 and MMP-9expression has been shown to increase early after ischemiconset.4 6 Treatment with MMP inhibitors and MMP-neutralizing antibodies has also been shown to reduce bothvasogenic edema formation and infarct size in a rat model offocal cerebral ischemia.5,7 In addition, reduced ischemiclesion volumes have been observed in MMP-9 knockout micecompared with wild-type littermates.8 These results suggestthat the MMP family participates in the pathophysiology ofcerebral ischemia.

Because this hypothesis has not been tested for humans invivo, we sought to determine the temporal profile of MMPexpression in patients with acute ischemic stroke and toinvestigate its relationship with stroke severity, location ofarterial occlusion, and total infarct volume.

Received March 8, 2001; final revision received April 10, 2001; accepted April 20, 2001.From the Cerebrovascular Unit (J.M., J.A.-S., C.M., S.A., J.A., M.A.G.) and Hemostasia Research Unit (A.A., J.M.), Vall dHebron Hospital,

Barcelona, Spain.Correspondence to Dr Joan Montaner, Unidad Cerebrovascular Servicio de Neurologa (6a planta, Hospital General), Hospital Vall dHebron, Pg Vall

dHebron 119-129, 08035 Barcelona, Spain. E-mail [email protected] 2001 American Heart Association, Inc.Stroke is available at http://www.strokeaha.org

1759 by guest on September 7, 2014http://stroke.ahajournals.org/Downloaded from

Subjects and MethodsStudy PopulationFrom June 1999 to March 2000, we prospectively studied consecu-tive patients with an acute stroke. A total of 110 patients evaluatedwithin the first 12 hours of stroke onset were included in the study.A detailed history of vascular risk factors was obtained from eachpatient. To identify potential mechanism of cerebral infarction, a setof diagnostic tests was performed that included ECG, chest radiog-raphy, carotid ultrasonography, complete blood count and leukocytedifferential, and blood biochemistry in all patients; many alsounderwent special coagulation testing, transthoracic echocardiogra-phy, and Holter monitoring. With this information and the neuroim-aging and transcranial Doppler (TCD) data, previously definedetiological subgroups were determined.9 Eighty-four (76.4%) pa-tients had a nonlacunar stroke that involved the vascular territorycorresponding to the middle cerebral artery (MCA). Of these, 44(40%) patients were considered to have had a cardioembolic stroke.Most of these patients had atrial fibrillation.

We excluded patients with a known inflammatory or malignantdisease (n53), as well as patients with an inadequate transtemporalwindow for TCD (n52). A total of 39 patients (51.3% male, meanage 74615 years) were included in the definitive analysis.

Clinical examination was performed on admission and at 12, 24,and 48 hours of symptom onset, at the time of each MMPdetermination. Stroke severity was assessed by using the NationalInstitutes of Health Stroke Scale (NIHSS). The NIHSS was used asa measure of initial (baseline) and final (48 hours) neurologicaldeficit. Baseline blood samples, TCD recordings, and NIHSS scoreswere obtained in the first 6 hours in 80% of patients and between 6and 12 hours in all others. If a patient died before the 48-hour timepoint, the last obtained score was used as the final NIHSS score.

We defined neurological improvement as a decrease in the strokescore by $4 points and neurological deterioration as either death oran increase in the stroke score by $4 points at 48 hours.10,11 Westratified patients according to the NIHSS score at each time pointwith previously used cut-points (,8, 8 to 20, and .20). Patients withan NIHSS score of .20 were considered to have a severe neurolog-ical deficit,12,13 and patients with an NIHSS score of ,8 wereconsidered to have a slight deficit.14

CT scanning was carried out in all patients on admission, and acontrol CT scan was performed at 48 hours to measure infarctvolume (cubic centimeters), according to the formula 0.53A3B3C,where A and B represent the largest perpendicular diameters throughthe hypodense area on CT scans, and C is the thickness of theinfarction area.15

All patients received subcutaneous low-molecular-weight heparinas prophylaxis for deep venous thrombosis. Intravenous heparin wasnot administered during the study period. No patient received tissueplasminogen activator. This study was approved by the ethicscommittee of the hospital, and all patients or relatives gave informedconsent.

TCD ProtocolSerial TCD studies were conducted on admission and at 12, 24, and48 hours of stroke onset. All TCD examinations were performed byan experienced neurologist. TCD measurements were performedwith a Multi-Dop X/TCD (DWL Elektroniche Systeme GmbH)device, with a hand-held transducer in a range-gated, pulsed-wavemode at a frequency of 2 MHz.

Blood flow velocities of the MCAs, anterior cerebral arteries (ACAs),and posterior cerebral arteries (PCAs) were recorded on both sidesthrough the transtemporal window.16 Proximal MCA occlusion wasdefined as the absence of flow or the presence of minimal flow signalthroughout the MCA at an insonation depth between 45 to 65 mm,whereas blood flow signal from the ipsilateral ACA and PCA wasdetected. Distal MCA division occlusion was indicated when there wasa diffuse dampening of blood flow velocity in the affected MCA of.21% compared with the contralateral MCA.

During the follow-up control experiments, spontaneous recanali-zation was diagnosed when a previously absent blood flow signal

reappeared (dampened or normal waveform) for a proximal MCAocclusion or when a previously dampened waveform came within thenormal range for a distal MCA occlusion.17

MMP-9 and MMP-2 DeterminationsPeripheral blood samples were drawn from each patient at studyentry and at 12, 24, and 48 hours after stroke onset. From the 156expected extractions, missing data for MMPs included 5 baselinedeterminations that were so close to the 12-hour time point that only1 extraction was performed. No missing data existed for the 12-hourtime point. Two missing items for the 24-hour time point and 6missing items for the 48-hour time point corresponded mostly topatients who died or to technical problems.

EDTA tubes were used to collect the blood. Plasma was immedi-ately separated via centrifugation at 3000 rpm for 15 minutes andstored at 280C until analysis. MMP-2 and MMP-9 levels weredetermined with commercially available enzyme-linked immunosor-bent assay (ELISA) (Biotrak; Amersham Pharmacia). ELISAs wereperformed according to the manufacturers instructions. Our labora-tory reference values for healthy controls are 41627.8 ng/mL forMMP-9 (n562, 58% male, mean age 43 years, normal ,97 ng/mL)and 630.86101.8 ng/mL for MMP-2 (n540, 47% male, mean age 43years, normal 427 to 835 ng/mL). The mean intra-assay coefficientsof variation were 8.9% for MMP-9 and 10.7% for MMP-2.

Statistical AnalysisDescriptive and frequency statistical analyses were performed andcomparisons were made by use of the SPSS statistical package,version 9.0. Statistical significance for intergroup differences wasassessed by the x2 or Fishers exact test for categorical variables andthe t test and ANOVA for continuous variables (a post hoc analysiswas conducted by means of Tukeys test). MMP values werenormally distributed (Kolmogorov-Smirnov and P-P plot). A t testwas performed for paired data with Bonferroni correction formultiple comparisons to compare MMP levels for different timepoints. To calculate the sensitivity and specificity for MMP values topredict neurological improvement as well as final NIHSS score, areceiver operator characteristic curve was configured. Two logisticregression analyses were performed to determine factors that couldbe considered as independent predictors for neurological improve-ment and final NIHSS score. Cutoff values for MMP-9 and MMP-2with the highest sensitivity and specificity, according to outcomegroups, were included. To study the correlation between MMPs andother continuous variables, the Pearson test was used except forNIHSS scores (Spearman test). P,0.05 was considered statisticallysignificant.

ResultsMMP-2 and MMP-9 Temporal ProfilePeak MMP-2 was observed at baseline determination(752.56210.9 ng/mL) and significantly decreased onfollow-up determinations (12 hours 668.86254.6 ng/mL,P50.011; 24 hours 5996195.4 ng/mL, P,0.001; and 48hours 580.16188 ng/mL, P,0.001). Mean MMP-2 level forall of these times was 644.26185.8 ng/mL. All MMP-2 levelsjust indicated were included in the normality interval of ourlaboratory values for healthy control subjects (427 to 835ng/mL) (Figure 1).

Baseline MMP-9 level was 147.16118.6 ng/mL, and nosignificant changes on MMP-9 levels were found onfollow-up determinations (12 hours 140.46120.7 ng/mL, 24hours 172.66139 ng/mL, and 48 hours 144.56127.6 ng/mL).Mean MMP-9 level for all of these times was 149.6699ng/mL. All MMP-9 levels just indicated exceeded the refer-ence interval for healthy control subjects (,97 ng/mL)(Figure 1).

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No difference was found for MMP-2 or MMP-9 regardingage, sex, or any vascular risk factor, except for higher MMP-2mean levels in patients with history of a previous stroke(764.26208.7 ng/mL for patients with a previous stroke and608.26165.6 ng/mL for first stroke patients, P50.025).

Mean plasma levels of MMP-2 and MMP-9 were bothcorrelated (r50.340, P50.034). Moreover, a significant cor-relation was found between MMP-9 and other laboratoryparameters such as glucose (r50.349, P50.037), leukocytecount (r50.395, P50.014), and mean temperature (r50.332,P50.039).

Relation to NIHSSMedian baseline NIHSS score of the series was 17 (range 5 to25), and median final NIHSS score was 15 (range 0 to 30). Nocorrelation was found between MMP-2 and NIHSS score atany time point. In contrast, MMP-9 was increasingly corre-lated to NIHSS scores at different time points (baseline:r50.302, P50.083; 12 hours: r50.364, P50.023; 24 hours:r50.408, P50.012; 48 hours: r50.439, P50.011).

There was a trend of association between stroke severity onadmission and MMP-9 overexpression at 48 hours. FinalMMP-9 was lower in patients with a baseline NIHSS score of,8 (15.9615 ng/mL) compared with those with a baselineNIHSS score between 8 to 20 (133.96124.3 ng/mL) and withthose with a baseline NIHSS score of .20 (212.06121.9ng/mL) (P50.053). Baseline NIHSSfinal MMP-9 correla-tion was r50.416 (P50.016).

Interestingly, there also was a positive correlation be-tween mean MMP-9 and final NIHSS score (r50.486,P50.002) as shown in Figure 2. Patients with a goodneurological status at the end of the study (final NIHSSscore ,8, n513) were those who had lower MMP-9 levelsat anytime (baseline: 97.8684.2 versus 177.66128.1ng/mL, P50.05; 12 hours: 94.2684.7 versus 163.56130.5ng/mL, P50.09; 24 hours: 87665.5 versus 213.76146.9 ng/mL,P50.001; 48 hours: 48.6662.4 versus 199.26123.5 ng/mLP,0.001) (Figure 3). Other parameters that influenced final NIHSSscore are shown in Table 1.

MMP-9 value was the only factor associated with the finalNIHSS score in the multiple logistic regression model (OR4.54, 95% CI 1.5 to 13.75). A cut-point for MMP-9 of 142.18ng/mL had a positive predictive value of 94.4% to assess a

patients NIHSS score (,8 or $8) by the end of the study(Table 3).

Because NIHSS scores were obtained at different times, weclassified patients according to whether they improved, re-mained stable, or worsened. During the study period, 14patients improved, 10 remained stable, and 15 deteriorated.When the improving group was analyzed, we found lowerMMP-2 and MMP-9 levels than in the nonimproving group(Figure 4). Other parameters related to neurological improve-ment are shown in Table 2. MMP-9 was the main factorassociated with neurological recovery in a logistic regression

Figure 1. Temporal profile for MMP-9 and MMP-2. Dashed line indicates reference interval for healthy controls (MMP-9 , 97 ng/mL,MMP-2 427835 ng/mL).

Figure 2. Correlation between MMP-9 and both infarct volume(r50.385, P50.022) and final NIHSS score (r50.486, P50.002)are shown (Pearson and Spearman tests, respectively).

Montaner et al MMP Expression After Human Cardioembolic Stroke 1761


model (OR 12, 95% CI 1.64 to 88.09) in which a cut-pointfor MMP-9 of 142.18 ng/mL gave a positive predictivevalue of 83.33% for the detection of neurological improve-ment (Table 3).

Although higher MMP-9 levels were found for patientswho deteriorated or remained stable (deterioration170.6682.1 ng/mL, stability 176.26124.9 ng/mL, improve-ment 90.5660.3 ng/mL; P50.036), when a post hoc analysiswas conducted, this significance was mainly due to thedifference between high MMP-9 levels in the deterioratinggroup and low MMP-9 levels in the improving group.

Relation to TCDBaseline TCD showed an occluded MCA in 37 (97.4%)patients: a distal occlusion in 16 (42.1%), and a proximalocclusion in 21 (55.3%). Another patient had a normal TCD

(1 missing datum existed for baseline TCD). By the end of thestudy (48-hour TCD), 16 patients still had occluded arteries(44.4%): 9 were distal (25%) and 7 were proximal (19.4%).Finally, a normal TCD was found in the other 20 (55.6%)patients. The remaining 3 patients died before the studyperiod was finished.

MMP expression was related to the time the vessel re-mained occluded and to the extent of the occlusion (distal orproximal MCA occlusion) as observed in Table 4.

MMP-2 levels were significantly higher when the MCAremained proximally occluded during the study period com-pared with patients with a distal occlusion or a normal MCAat 48-hour TCD (P,0.05 for all time points). MMP-9 levelswere also higher when a proximal occlusion was recorded at48-hour TCD (P,0.05 for baseline and 24-hourdeterminations).

Figure 3. MMP-9 levels during the study period for patients with a final NIHSS score of ,8 (48 h.) and for patients with a final NIHSSscore of $8. Values are mean6SEM. Dashed line indicates MMP-9 reference interval value for health control subjects (,97 ng/mL).

TABLE 1. Main Characteristics of Patients Regarding Final NIHSS Score

NIHSS ,8 (n513) NIHSS $8 (n526) P

Male, n (%) 6 (46.2) 14 (53.8) 0.651

Age, mean y (SD) 76.5 (9.9) 72.7 (17.3) 0.474

Hypertension, n (%) 6 (46.2) 14 (53.8) 0.651

Hyperlipidemia, n (%) 1 (7.7) 5 (22.7) 0.377

Diabetes, n (%) 2 (15.4) 7 (26.9) 0.420

Previous stroke, n (%) 2 (15.4) 7 (26.9) 0.420

Temperature, mean C (SD) 36.4 (0.3) 36.9 (0.4) 0.002*

TCD (N/DO/PO), n (%) 0 (0)/10 (76.9)/3 (23.1) 1 (4.5)/6 (24)/18 (72) 0.007*

SBP, mean mm Hg (SD) 168.3 (20.3) 153.0 (39.8) 0.144

DBP, mean mm Hg (SD) 93.1 (14.3) 81.9 (22.4) 0.117

Glucose, mean mg/dL (SD) 128.7 (81.7) 165.8 (94.1) 0.253

Platelets, mean (SD) 233 538 (66 817) 188 360 (44 900) 0.018*

Leukocytes, mean X10E9/L (SD) 8653 (1827) 10 356 (3720) 0.130

Fibrinogen, mean g/L (SD) 4.0 (0.8) 3.9 (1.0) 0.610

MMP-2, mean ng/mL (SD) 614.4 (120.3) 659.1 (211.8) 0.487

MMP-9, mean ng/mL (SD) 82.1 (55) 183.43 (99.5) 0.000*

For TCD, N indicates normal; DO, distal occlusion; and PO, proximal occlusion. SBP indicatessystolic blood pressure; DBP, diastolic blood pressure.

Baseline data are given except for temperature and MMP (mean values of the 4 time points).*Factors with a value of P,0.1 were included in the regression model.



In the 37 patients with baseline MCA occlusion, we studiedMMP levels according to whether a spontaneous recanaliza-tion was (n525) or was not (n512) present during the studyperiod. Final MMP-2 levels were significantly lower whenrecanalization occurred (5286144.3 versus 681.46239.2ng/mL, P50.031), and final MMP-9 levels were also lowerwhen spontaneous recanalization occurred (110.26100.9 ver-sus 244.86130 ng/mL, P50.004).

Relation to Infarct VolumeMean infarct volume was 89.77695.66 cm3. Patients withneurological improvement had smaller infarctions than did

those who remained stable or worsened (15.1623.5 versus128.7696.1 cm3, P,0.001), as well as those with a finalNIHSS score of ,8 (12615.7 versus 135.6696.6 cm3,P,0.001).

A positive correlation was found between mean MMP-9and infarct volume (r50.385, P50.022), as shown in Figure2. Although there was a trend (r50.322, P50.07) for baselineMMP-9 to correlate with the infarct volume measured in the48-hour CT scan, the best correlation was obtained for the48-hour MMP-9 determination (r50.500, P50.005). Nocorrelation existed for MMP-2 and infarct volume at any timepoint.

Figure 4. MMP-9 levels according to neurological improvement. Values are mean6SEM. Dashed line indicates MMP-9 reference inter-val value for health control subjects (,97 ng/mL).

TABLE 2. Main Characteristics of Patients With and Without Neurological Improvement

NeurologicalImprovement (n514)

Stability orDeterioration (n525) P

Male, n (%) 7 (50) 13 (52) 0.905

Age, mean y (SD) 73.6 (17.6) 74.2 (14.1) 0.909

Hypertension, n (%) 5 (35.7) 15 (60) 0.146

Hyperlipidemia, n (%) 1 (7.1) 5 (20) 0.391

Diabetes, n (%) 1 (7.1) 8 (32) 0.077*

Previous stroke, n (%) 2 (14.3) 7 (28) 0.330

Temperature, mean C (SD) 36.4 (0.3) 36.8 (0.4) 0.013*

NIHSS (,8/820/.20), n (%) 1 (7.7)/10 (76.9)/2 (15.4)

2 (8.3)/14 (58.3)/8 (33.3)

0.482

TCD (N/DO/PO), n (%) 0 (0)/9 (64.3)/5 (35.7) 1 (4.2)/7 (29.2)/16 (66.7)

0.095*

SBP, mean mm Hg (SD) 158.4 (27) 156.4 (37.6) 0.866

DBP, mean mm Hg (SD) 86.5 (12.8) 84.5 (23.3) 0.734

Glucose, mean mg/dL (SD) 130.9 (76.7) 167.8 (97.6) 0.239

Platelets, mean (SD) 231 428 (64 484) 187 708 (45 901) 0.020*

Leukocytes, mean X10E9/L (SD) 8814 (1754) 10 533 (3826) 0.104

Fibrinogen, mean g/L (SD) 3.9 (1) 3.9 (0.9) 0.896

MMP-2, mean ng/mL (SD) 570.1 (129.8) 685.7 (201.4) 0.062*

MMP-9, mean ng/mL (SD) 108.1 (87.8) 172.8 (98.9) 0.049*

For TCD, N indicates normal; DO, distal occlusion; and PO, proximal occlusion. SBP indicates systolic bloodpressure; DBP, diastolic blood pressure.

Baseline data are given except for temperature and MMP (mean values of the 4 time points).*Factors with a value of P,0.1 were included in the regression model.



DiscussionWe found an association between MMP upregulation andneurological impairment. We evaluated the role of MMPsduring the acute phase of stroke by means of severalapproaches, including stroke severity, infarction size, andhemodynamic status. Stroke severity as measured with theNIHSS was correlated to MMP-9 levels at different timepoints after symptom onset. Lower MMP-9 levels werestrongly associated with neurological improvement duringthe first 48 hours. In contrast, higher MMP-9 levels wereassociated with neurological deterioration in the same timeframe. MMP expression was related to the time and locationof MCA occlusion. In addition, a positive correlation wasfound between mean MMP-9 and total infarct volume.

The temporal profile of MMPs in our series indicatedMMP-9 overproduction in acute stroke patients comparedwith control subjects. MMP-9 levels exceeded the referencevalues in each time point measurement. In contrast, ourpatients exhibited MMP-2 levels within the normal rangeduring the study period. Although differences in MMP levelsbetween patients and control subjects may be influenced bythe younger age of the healthy control subjects, no differenceswere found in MMP levels regarding age among our strokepopulation (21 to 95 years).

In the present study, peak levels of MMP-9 were found atthe 24-hour determination. This observation is in line withthat of a rat model of permanent MCA occlusion, in which alarge increase in the MMP-9 occurred 12 to 24 hours after theinjury.18 Romanic et al7 detected MMP-9 in the ischemictissue within 24 hours after MCA occlusion and was maxi-mally observed up to the 5-day time point. MMP-2 expres-sion was also detected in ischemic tissue but predominantly atthe 5-day time point.

Although we identified a baseline peak of MMP-2 levels,we cannot rule out a delayed (.48 hours) or very early(before the baseline determination) elevation of MMP-2. Infact, in a nonhuman primate stroke model, latent MMP-2 wassignificantly increased in the basal ganglia 1 hour after MCAocclusion compared with control subjects.6

The temporal profile of animal MMP expression varies,depending on the author and the ischemia model. ActivatedMMP-9 appeared 3 hours after 60 minutes of transientischemia,19 and proMMP-9 induced as soon as 2 hours afterthe onset of ischemia in a permanent occlusion model.4Discrepancies in temporal profile of MMPs and in thepresence of active or pro-MMP forms in these animal modelsof cerebral ischemia may be influenced by different zymo-graphic techniques and MMP purification methods that haveallowed the threshold of MMP-9 detection to increase.ELISA methods to measure MMPs are available, and prom-ising results are appearing in other cardiovascular fields.20The ELISA method used in our experiment detect theproform of the MMPs as well as the MMP/TIMP complex(proMMP-9 and proMMP-9/TIMP-1; proMMP-2 andproMMP-2/TIMP-2). Several studies suggest that enzymaticactivation of MMP-9 proenzyme occurs by binding at the cellsurface, and activated enzyme then is rapidly degraded toprevent excess activity.21 Therefore, only very low levels ofactive MMP-9 may be present in tissue at any given time.8

MMPs have been studied through immunohistochemistryin humans with strokes,22,23 showing overproduction ofMMP-9 during the early and proinflammatory phases of

TABLE 4. MMP-2 and MMP-9 Levels at Different Time PointsDepending on Final MCA Status (TCD Recording at 48 HoursFrom Stroke Onset)

TCD (48hours)

Normal MCA(n520)

DistalOcclusion

(n59)

ProximalOcclusion

(n57) P

MMP-2

Baseline 612.9 (128.4) 853.5 (242.6) 903.7 (130.5) 0.001

12 Hours 543.5 (131.7) 804.5 (352.1) 821.8 (272.7) 0.023

24 Hours 514.6 (125.6) 653.4 (295.0) 722.1 (121.8) 0.012

48 Hours 508.0 (141.6) 596.9 (187.4) 751.9 (225.8) 0.017

MMP-9

Baseline 99.4 (102.4) 188.1 (149.3) 236.4 (81.1) 0.032

12 Hours 124.6 (122.2) 154.2 (133.0) 185.7 (126.7) 0.528

24 Hours 126.2 (115.8) 211.2 (143.4) 282.4 (147.9) 0.029

48 Hours 118.1 (108.9) 182.3 (173.7) 216.1 (72.4) 0.203

Data from patients who died before final TCD was performed (n53) are notincluded. MMP values are expressed as mean (SD) ng/mL.

TABLE 3. Factors Associated With Neurological Outcome(Improvement vs Stability-Deterioration) and Final NIHSS(8) on Univariate Analysis and Multiple LogisticRegression Model

Univariate AnalysisOR (95% CI)

Logistic RegressionOR (95% CI)

logLRP

NeurologicalOutcome

MMP-9 5.49 (1.21, 24.80) 12.05 (1.64, 88.09) 0.0049

MMP-2 3.75 (0.91, 15.34) 8.93 (1.27, 62.84) 0.0138

Baseline TCD

Normal 1 z z z z z zDistal occlusion 0.26 (0.002, 7.34) z z z z z zProximalocclusion

1 (0.035, 28.3) z z z z z z

Platelet count 0.24 (0.057, 0.99) 0.11 (0.01, 0.86) 0.0185

Temperature 3.75 (0.916, 15.3) z z z z z zDiabetesmellitus

6.11 (0.676, 55.1) z z z z z z

Final NIHSS

MMP-9 22.64 (2.5, 203.06) 4.54 (1.50, 13.75) 0.005

Baseline TCD

Normal 1 z z z z z zDistal occlusion 1.76 (0.059, 52.6) z z z z z zProximalocclusion

8.53 (1.89, 38.40) z z z z z z

Platelet count 0.16 (0.037, 0.77) z z z z z zTemperature 10.38 (1.8, 57.4) z z z z z z

MMP cut-point levels were obtained from the receiver operating character-istic curves.

For MMP-2, ,603.77 ng/mL, and for MMP-9, ,142.18 ng/mL.



stroke, whereas MMP-2 was found in the later reparativephases when the blood vessels begin to regrow. Theseautopsy findings identified MMP-9 in neutrophils for 1 weekafter the infarction. Macrophages that expressed matrilysinand MMP-2 were observed after 1 week. Clark et al23 foundthat human brain tissue after a stroke shows MMP-2 afterseveral months, whereas MMP-9 is elevated within days afterthe infarct. The later rise in MMP-2 activity would correlatebetter with the poststroke time course of capillary remodelingthan with that of cerebral edema. In accordance with thisobservations, we found that MMP-2 levels were higher inpatients with a previous cerebrovascular event.

In animal models and postmortem studies, a causal linkbetween MMP overexpression and brain damage has beensuggested. However, whether MMP-9 overexpression con-tributes to the development of infarct or simply represents amarker of the extent of brain ischemia remains unclear. Ourstudy does not elucidate this controversy. We observed atrend of correlation between baseline MMP-9 and infarctvolume measured on CT scans at 48 hours, although the bestcorrelation was obtained for the 48-hour MMP-9 determina-tion. The study is a correlative survey of several phenomena,and one cannot chose one explanation over the other becausethe cause-effect relationship between the neurological impair-ment and the biochemical marker was not established. Be-cause MMP expression was related to the time of persistentarterial occlusion in our study and because a significantincrease in proMMP-9 occurred in a time-dependent mannerduring reperfusion in a model of transient ischemia,19 wehypothesize that MMPs may contribute to the fact that alonger ischemia period leads to greater infarctions.

Animal studies are being designed to answer questionsabout what produces these MMPs, why they are upregulatedafter stroke, and how they damage the infarcted brain paren-chyma. We have no information on the cell types that serve assources for plasmatic MMP-9 in our study. The large increasein MMP-9 may have been endogenous, from astrocytes andmicroglia, or exogenous, from invading neutrophils andmacrophages.24 A transient increase in latent MMP-9 inplasma after MCA occlusion in baboons6 did not result fromblood cell sources. This lack of correlation between neutro-phil number and gelatinase also has been observed in humanplasma.25 Nevertheless, we found a correlation betweenMMP-9 and leukocyte count. It has been demonstrated thatneutrophils use MMPs for migration26 and that substantialinfiltration of leukocytes occurs from 12 to 24 hours toseveral days after focal cerebral ischemia.27 We cannotexclude the possibility that MMP-9 from activated leukocytescontributes to infarct progression. Based on morphologicalcriteria, MMP-9 appeared to stain with endothelial cells andneutrophils within and at the periphery of the infarct within24 hours of focal ischemia in rats.7

Although increasing evidence for the role of MMPs isavailable from animal models of ischemia, information inhumans is lacking. Based on our results, ELISA measures ofMMPs for stroke patients seem to be a good tool for furtherresearch into this field, particularly when new drugs thatblock MMPs offer attractive results.28 Because no differencewas found for MMP-2 or MMP-9 regarding age, sex, or any

vascular risk factor, MMP determination could be a usefulsurrogate marker in the acute phase of stroke.

In conclusion, the present study demonstrates an associa-tion between MMP-9 overexpression and stroke severity,infarct size, and the time and location of MCA occlusion.These findings suggest a deleterious role for MMP-9 in thedevelopment of brain damage after human ischemic stroke.

AcknowledgmentThis study was supported in part by a grant from the Catalan Societyof Neurology for cerebrovascular diseases (sponsored by Uriach).

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