Excitatory amino acids and monoaminergic neurotransmitters in cerebrospinal fluid of acute ischemic stroke patients

Neurochemistry International 56 (2010) 865–870

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Neurochemistry International journal homepage: www.elsevier.com/locate/neuint

Excitatory amino acids and monoaminergic neurotransmitters in cerebrospinal fluid of acute ischemic stroke patients Raf Brouns a,b,c, An Van Hemelrijck d, Wilhelmus H. Drinkenburg d, Debby Van Dam b, Didier De Surgeloose e, Peter P. De Deyn a,b,* a

Department of Neurology and Memory Clinic, ZNA Middelheim Hospital, Antwerp, Belgium Laboratory for Neurochemistry and Behaviour, Institute Born-Bunge, Department of Biomedical Sciences, University of Antwerp, Belgium Department of Neurology, University Hospital Brussels, Vrije Universiteit Brussel, Belgium d Neuroscience Research and Early Development, J&J PRD, Beerse, Belgium e Department of Radiology, ZNA Middelheim Hospital, Antwerp, Belgium b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 7 November 2009 Received in revised form 13 December 2009 Accepted 17 December 2009 Available online 29 December 2009

Background and purpose: Improved insight in the role of neurotransmitters in acute cerebral ischemic injury may be fundamental for the successful development of novel therapeutic approaches. We investigated excitatory amino acids and monoaminergic neurotransmitters in cerebrospinal fluid (CSF) of acute ischemic stroke patients and their relation to stroke characteristics. Methods: CSF concentrations of glutamate, aspartate, glutamine, glycine, proline, taurine, norepinephrine, 5-hydroxyindoleacetic acid, homovanillic acid and 3,4-dihydroxyphenylacetic acid were assessed in 89 stroke patients at admission (median 6.3 h after stroke onset) and in 31 controls. We evaluated the relation between CSF concentrations and (a) stroke severity (NIHSS score at admission, lesion volume), (b) stroke evolution in the subacute phase, (c) long-term stroke outcome, (d) lesion location, and (e) stroke etiology. Results: Neurotransmitter systems display relevant interrelations, however, no significant associations between neurotransmitter concentrations in CSF and stroke characteristics were found, with the exception of higher 5-hydroxyindoleacetic acid levels in CSF of patients with progressing stroke and poor long-term outcome. Conclusions: The study results question the added value of neurotransmitter assessment in CSF for research on ischemic cerebral injury. ß 2009 Elsevier Ltd. All rights reserved.

Keywords: Acute cerebral infarction Neurotransmitters Cerebrospinal fluid Amino acids

1. Introduction Fifty years ago, Olney and Sharpe introduced the idea of the neurotoxicity of certain amino acids (Olney and Sharpe, 1969). Neuroexcitotoxicity is now recognized as a major cause of secondary damage following cerebral ischemia (Brouns and De Deyn, 2009), but effective treatment remains unsuccessful despite promising basic research (Hazell, 2007). Investigations have largely been based on in vitro experiments and animal models. Human studies are limited in number and have mainly focused on neurotransmitters in blood (Davalos et al., 1997, 2000; Serena et al., 2001; Meglic et al., 2001; Dambinova et al., 2002; Strittmatter et al., 2003; Oto et al., 2008; Castellanos et al., 2008). Although a good correlation between cerebrospinal fluid

* Corresponding author at: Laboratory for Neurochemistry and Behaviour, Institute Born-Bunge, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium. Tel.: +32 3 265 26 20; fax: +32 3 265 26 18. E-mail address: [email protected] (P.P. De Deyn). 0197-0186/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2009.12.014

(CSF) and plasma neurotransmitter levels has been reported (Castillo et al., 1996, 1997), it can be questioned whether blood levels adequately reflect cerebral pathological changes because of many confounding factors, including blood–brain barrier passage, extracerebral sources of neurotransmitters and variable clearance rate from blood (Aliprandi et al., 2005). Assessment in CSF, which is equivalent to cerebral extracellular fluid, should therefore be considered more suitable to evaluate in cerebro neurotransmitter concentrations. Current knowledge about CSF neurotransmitter concentrations in ischemic stroke, is based on reports of only three research groups who focussed mainly on the lack of relationship between the dopaminergic system and stroke severity (Hachinski et al., 1978) and the positive correlation between CSF glutamate levels and stroke severity, progression and outcome (Castillo et al., 1996, 1997; Davalos et al., 1997; Skvortsova et al., 2000). Given the paucity of available data in the literature, we investigated in this study the concentrations of the excitatory amino acids glutamate, aspartate, glutamine, glycine, proline and taurine in CSF of acute ischemic stroke patients and controls. For

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evaluation of the monoaminergic neurotransmitter systems, we assessed concentrations of norepinephrine, 5-hydroxyindoleacetic acid (5-HIAA) as parameter for serotonin, homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) as measures of the dopamine metabolism. In addition, we also evaluated the relation of these neurotransmitter systems to initial stroke severity, stroke evolution in the subacute phase, long-term stroke outcome, lesion location and stroke etiology. 2. Materials and methods 2.1. Study population This study is part of the Middelheim Interdisciplinary Stroke Study, which is a project on the clinical, biochemical, neuroimaging, neuropsychological and electrophysiological evaluation of patients with ischemic stroke or transient ischemic attack (TIA) at ZNA Middelheim hospital, Antwerp. Other biochemical analyses from this project have been reported elsewhere (Brouns et al., 2008, 2009a,b,c,d, 2010a, 2010b). In this study, we focused on a cohort of 89 patients with ischemic stroke (n = 68) or TIA (n = 21) in whom analysis of neurotransmitters in CSF was available. The study was conducted according to the revised Declaration of Helsinki (1998) and in agreement with the guidelines of the Ethics Committees of ZNA Antwerp and the University of Antwerp. Lumbar puncture was only performed in patients without contraindication for this procedure and after informed consent explaining the research goal, the possible risks of this technique and the absence of potential benefit for most ischemic stroke patients. Demographics and stroke characteristics of the study population are shown in Table 1. 2.2. Control population A control population consisting of 31 individuals without antecedents of central nervous system disease or without any contraindication for lumbar puncture was included, because unequivocal data on CSF concentrations of neurotransmitters in

Table 1 Demographic and stroke characteristics of the study corpus of 89 patients with hyperacute ischemic stroke or TIA and 31 controls.a. Characteristic

Value Stroke or TIA (n = 89)

Controls (n = 31)

P-valuez

Demographics Age (years) Male gender Caucasian race

71.1  13.2 50 (56.2) 87 (97.8)

67.4  12.7 18 (56.3) 31 (96.9)

.211 .543 .971

Possible confounding factors Traumatic lumbar punctureb Concurrent disease Time to sampling (h)

5 (5.6) 8 (9.0) 6.3 (4.3–13.1)

0 (0.0) 0 (0.0) N.A.

.326 .084

21 (23.6)/68 (76.4) 4 (1–11) 4.9 (1.2–56.5)

N.A.

Stroke characteristics TIA/ischemic stroke NIHSS at admission Lesion volume (mL) mRS at month 3 mRS 0–3 mRS 4–6 Stroke progression within 72 hc Stroke location on imaging Pure cortical Subcortical Stroke etiologyd Cardioembolic Lacunar Atherothrombotic Specific Undetermined

69 (77.5) 20 (22.5) 14 (15.7)

N.A. N.A. N.A.

N.A. N.A.

15 (16.9) 43 (48.3) N.A. 39 (43.8) 20 (22.5) 21 (23.6) 3 (3.4) 6 (6.7)

Abbreviations: TIA: transient ischemic attack; N.A.: not applicable; NIHSS: National Institutes of Health Stroke Scale. a Data are given as mean (SD), as number (percentage) or median (IQR). b >1000 red blood cells/mm3. c Based on the European Progressing Stroke Study criteria. d Stroke etiology classified by the TOAST criteria. z Student t-test, Mann–Whitney U test or cross-tabulations and x2 test as appropriate.

normal physiology is lacking. Indications for lumbar puncture included investigation of peripheral nervous system disorders (n = 25), suspicion of meningitis (n = 2), suspicion of subarachnoid hemorrhage (n = 3), or subjective memory complaints (n = 1). All controls had normal CSF results on routine analysis. The characteristics of the control group are listed in Table 1. Patients and controls were matched with regard to demographic characteristics. 2.3. Lumbar puncture and measurement of neurotransmitters Lumbar puncture was performed at admission (median 6.3 h after onset of stroke symptoms). CSF sampling and collection was done as previously described (Brouns et al., 2008, 2009b). Analysis was done blinded to case identity. The amino acids aspartate, glutamate, glutamine, glycine, taurine and proline were analyzed using a Waters Acquity ultra-performance liquid chromatography (UPLC) system coupled to a Waters fluorescence detector and precolumn derivatisation with 6aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). Samples were derivatised as described previously (Cohen and Michaud, 1993; Liu et al., 1998). 20 mL of the sample or standard was mixed with 60 mL 0.2 M borate buffer pH 8.8, containing 25 mM a-aminobutyric acid as internal standard. The derivatisation was carried out by adding 20 mL AQC reagents to the buffered mixture. The final mixture was heated at 55 8C for 10 min. The buffer consisted of a mixture of 140 mM sodium acetate and 7 mM triethanolamine, and set at pH 5.7. The mobile phase was a mixture of buffer with acetonitrile. Chromatographic separation was achieved by gradient elution on a Waters Acquity UPLC HSS T3 column (C18, 2.1 mm ID  50 mm, 1.8 mm). A 0.2 mm filter (2.1 mm ID) was used in front of the column. The column temperature was 60 8C. The flow rate was 1.2 mL/min and run time was 8 min. Excitation and emission wavelength were set respectively at 250 and 395 nm. The monoamines norepinephrine, 5-HIAA and DOPAC were analyzed using a Waters Acquity UPLC system coupled to a Waters fluorescence detector and precolumn derivatisation with benzylamine and 1,2-diphenylethyleendiamine (DPE). Samples were derivatised as described previously (Fujino et al., 2003; Yoshitake et al., 2004a,b). To 20 ml of standard or sample 10 ml of isoprotenerol as internal standard was added. Then, 20 ml of benzylamine derivatization reagent solution was added. The mixture was allowed to react for 2 min at room temperature, thereafter 20 ml of DPE reagent was added and the final mixture was heated at 50 8C for 20 min. Separation was achieved by gradient elution using a mixture of acetonitrile and 15 mM acetate buffer (pH 4.5) containing 1 mM octanesulfonic acid sodium salt on a Waters Acquity UPLC BEH Shield reversedphase column (C18, 2.1 mm ID  100 mm, 1.7 mm). The column temperature was 60 8C. The flow rate was 0.7 mL/min and the run time was 8 min. Excitation and emission wavelength were set, respectively, at 345 and 480 nm. HVA was analyzed using a Waters alliance HT 2795 HPLC system equipped with a Waters fluorescence detector 2475. Separation was achieved by isocratic elution using a 75 mM sodium acetate buffer (pH 5.0) on a Waters Atlantis reversed-phase column (C18, 2.1 mm ID  50 mm, 3 mm) with a Waters Atlantis precolumn (C18, 2.1 mm ID  10 mm, 3 mm). The flow rate was 1 mL/min and run time was 10 min. Excitation and emission wavelength were set respectively at 275 and 345 nm. All data was analyzed by means of Waters Empower 2.0 software run on an Intel Pentium processor. The monoamines, amino acids and acetonitrile were purchased from Sigma–Aldrich (Bornem, Belgium). Other products were purchased from Merck (Brussels, Belgium). All chemicals and solvents were at least of analytical grade. All solutions were prepared in ultrapure milliQ water (Millipore MilliQ Academic) and filtered over a 0.22 mm filter (Millipore, Bedford, USA). 2.4. Evaluation of stroke severity, evolution, outcome, location and etiology Neurological deficits were quantified using the National Institutes of Health Stroke Scale (NIHSS) by trained stroke physicians at admission. Except for two patients who deceased before repeat neuroimaging was performed and three patients with a contraindication for MRI, all patients underwent in addition to baseline neuroimaging at admission a magnetic resonance imaging (MRI) scan of the brain on average 3.1 days after stroke onset. The patients in whom MRI was contraindicated were evaluated by computed tomography of the brain on average 3.0 days after stroke onset. The lesion location and volume was assessed by two independent observers as described previously (Brouns et al., 2008; Brouns et al., 2009a,b,c,d, 2010a, 2010b). Proof of acute cerebral ischemia was found in 58 patients and the median lesion volume was 4.9 mL. Patients were evaluated for progressing stroke in the first 72 h after stroke onset, as defined by the European Progressing Stroke Study criteria (Birschel et al., 2004). Outcome was assessed at 3 months after stroke by means of the modified Rankin Scale (mRS). In agreement with the literature, poor outcome was defined as mRS score >3 (Sulter et al., 1999). Based on the clinical presentation, neuroimaging, cardiovascular and cerebrovascular investigation, we classified stroke etiology according to the TOAST criteria (Adams et al., 1993). 2.5. Possible confounding factors for CSF neurotransmitter levels Data from the literature indicate that age, gender, concurrent neuropsychiatric disease, traumatic lumbar puncture and the interval between stroke onset and lumbar puncture may influence CSF neurotransmitter levels. In contrast to the

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Table 2 CSF neurotransmitter concentrations in the study corpus of 89 patients with hyperacute ischemic stroke or TIA and in 31 controls.a. CSF concentration

Glutamate (mM) Aspartate (mM) Glutamine (mM) Glycine (mM) Proline (mM) Taurine (mM) Norepinephrine (nM) 5-HIAA (nM) DOPAC (nM) HVA (nM)

Value Stroke or TIA (n = 89)

Controls (n = 31)

P-valuez

1.24 (1.09–1.44) 0.58 (0.44–0.71) 343.9 (309.6–376.0) 2.50 (1.88–3.35) 0.24 (0.14–0.37) 4.18 (3.51–5.26) 0.68 (0.48–1.15) 58.7 (38.8–88.3) 0.77 (0.44–1.03) 386.7 (289.3–508.9)

1.22 (1.03–1.42) 0.42 (0.27–0.53) 331.0 (291.3–369.8) 3.22 (2.37–4.08) 0.26 (0.21–0.38) 3.19 (2.29–4.13) 0.53 (0.36–0.75) 61.9 (43.2–92.8) 0.71 (0.45–1.15) 388.1 (276.4–490.7)

.653 .001 .452 .002 .229 .002 .034 .515 .881 .681

Abbreviations: 5-HIAA: 5-hydroxyindoleacetic acid; DOPAC: 3,4-dihydroxyphenylacetic acid; HVA: homovanillic acid; TIA: transient ischemic attack. a Data are given as median (IQR). z Mann–Whitney U test, P < .005 indicates statistical significance. control population, eight patients were treated for conditions known to interfere with neurotransmitter systems. Indeed, four patients were treated with antidepressants, three subjects with antiparkinson medication and one patient with anticonvulsants. Consensus criteria for diagnosing traumatic lumbar puncture are nonexistent. In a large study cohort, Eskey et al. found the frequency to be 10.1% using a cut point of 1000 red blood cells/mm3 (Eskey and Ogilvy, 2001). The frequency of traumatic puncture was 5.6% in our study population, which does not significantly differ from the control population (Table 1). 2.6. Statistical analyses Statistical computations were performed with SPSS software package version 15.0 (SPSS Inc, Chicago, IL). Normally distributed data (Kolmogorov–Smirnov test) are presented as mean (standard deviation, SD); data without normal distribution are given as median (interquartile range, IQR). The Student t-test, Mann–Whitney U test, Kruskal–Wallis test or cross-tabulation and Pearson x2 test were applied as appropriate. The relation between biomarker concentration and parameters for stroke severity and outcome was also assessed by bivariate correlations (Spearman’s r). a Adjustment according to the Bonferroni procedure was applied. For 10 tests, a was lowered to 0.005.

3. Results

77.4 nM)) (Mann–Whitney U test, P < .001). In the subpopulation of TIA patients, the relation between 5-HIAA CSF concentrations and stroke outcome, however, failed to reach statistical significancy. 3.3. CSF levels of neurotransmitters in relation to stroke evolution, location and etiology Fourteen patients with stroke progression in the first 72 h after onset of symptoms were identified. We found no differences in neurotransmitter levels between the two subpopulations, except for higher 5-HIAA CSF concentrations in patients with stroke progression than in patients with favourable stroke evolution (Table 4). Based on the neuroimaging findings, stroke location was categorized as pure cortical (n = 15) or subcortical (n = 43). Neurotransmitter concentrations did not differ between cortical and subcortical lesions (Table 5). Neurotransmitter concentrations were similar in patients with cardioembolic stroke, atherothrombotic stroke, lacunar ischemia, stroke secondary to specific causes, and stroke of undetermined etiology (Table 6).

3.1. CSF levels of neurotransmitters Table 2 shows that levels of glutamate, glutamine, proline, norepinephrine, 5-HIAA, DOPAC and HVA in patients with ischemic stroke or TIA are not different from values in controls. Aspartate and taurine concentrations are higher, but glycine levels are lower in stroke or TIA patients than in controls. Several neurotransmitters show relevant interrelations: glutamate concentrations strongly correlate with glutamine (r = 0.71, P < .001); aspartate levels are correlated with taurine (r = 0.42, P < .001) and with norepinephrine (r = 0.28, P = .003). Glutamine correlates with 5-HIAA (r = 0.30, P = .001). Glycine levels are positively correlated with proline (r = 0.41, P < .001), but negatively with norepinephrine (r = 0.32, P = .001). HVA concentrations correlate with DOPAC (r = 0.37, P = .001) and with 5-HIAA (r = 0.31, P = .001). Finally, CSF levels of DOPAC correlate with norepinephrine concentrations (r = 0.32, P = .004). 3.2. CSF levels of neurotransmitters in relation to stroke severity and long-term outcome Neurotransmitter concentrations in CSF do not relate with the NIHSS score at admission, except for glycine and norepinephrine. However, no significant correlation is found between any neurotransmitter level and lesion volume (Table 3). Only 5-HIAA CSF concentration correlates with the mRS score at 3 months after stroke (Table 3) and median 5-HIAA levels are higher in patients with poor outcome (mRS > 3, 89.3 nM (67.7–148.5 nM)) than in patients with more favourable outcome (mRS  3, 49.9 nM (35.9–

3.4. CSF levels of neurotransmitters in relation to possible confounding factors CSF neurotransmitter levels were not significantly related to the patient’s age or to the time interval between stroke onset and lumbar puncture (Spearman’s r, P > .005). Neurotransmitter concentrations were similar in male and female patients, in patients with and without concurrent neuropsychiatric disease, and in patients with and without traumatic lumbar puncture (Mann–Whitney U test, P > .005). Table 3 Correlations between CSF neurotransmitter levels and NIHSS score at admission, lesion volume and modified Rankin Scale score 3 months after stroke onset.*. Neurotransmitter

Glutamate Aspartate Glutamine Glycine Proline Taurine Norepinephrine 5-HIAA DOPAC HVA

Stroke characteristic NIHSS score

Lesion volume

0.02 0.13 0.07 0.32 0.03 0.07 0.33 0.25 0.14 0.11

0.07 0.05 0.04 0.11 0.07 0.01 0.10 0.21 0.16 0.04

(.899) (.233) (.523) (.003) (.813) (.505) (.002) (.018) (.262) (.293)

(.501) (.613) (.692) (.298) (.500) (.940) (.387) (.049) (.198) (.734)

mRS score 0.04 0.21 0.13 0.27 0.03 0.05 0.19 0.33 0.27 0.02

(.733) (.046) (.244) (.010) (.760) (.679) (.083) (.002) (.025) (.849)

Abbreviations: 5-HIAA: 5-hydroxyindoleacetic acid; DOPAC: 3,4-dihydroxyphenylacetic acid; HVA: homovanillic acid. * Spearman’s r correlation (P-value), P < .005 indicates statistical significance.

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Table 4 CSF neurotransmitter concentrations in relation to stroke evolution in the subacute phase.a. CSF concentration

Stroke evolution in the subacute phase

Glutamate (mM) Aspartate (mM) Glutamine (mM) Glycine (mM) Proline (mM) Taurine (mM) Norepinephrine (nM) 5-HIAA (nM) DOPAC (nM) HVA (nM)

Stroke progression (n = 14)

No stroke progression (n = 75)

P-valuez

1.32 (1.06–1.50) 0.53 (0.34–0.73) 367.7 (272.3–397.9) 2.71 (2.16–3.55) 0.28 (0.18–0.58) 4.02 (2.33–4.91) 0.56 (0.44–1.08) 102.9 (66.6–144.8) 0.44 (0.27–0.97) 386.8 (277.5–696.9)

1.24 (1.10–1.43) 0.59 (0.47–0.70) 340.3 (310.2–373.3) 2.47 (1.85–3.23) 0.23 (0.14–0.36) 4.28 (3.54–5.28) 0.70 (0.49–1.18) 51.9 (36.3–82.2) 0.78 (0.50–1.03) 386.7 (289.8–476.6)

.879 .723 .543 .300 .240 .295 .407 .001 .295 .589

Abbreviations: 5-HIAA: 5-hydroxyindoleacetic acid; DOPAC: 3,4-dihydroxyphenylacetic acid; HVA: homovanillic acid; TIA: transient ischemic attack. a Based on the European Progressing Stroke Study criteria. Data are given as median (IQR). z Mann–Whitney U test, P < .005 indicates statistical significance.

Table 5 CSF neurotransmitter concentrations in relation to stroke location on neuroimaging.a. CSF concentration

Lesion location on neuroimaging

Glutamate (mM) Aspartate (mM) Glutamine (mM) Glycine (mM) Proline (mM) Taurine (mM) Norepinephrine (nM) 5-HIAA (nM) DOPAC (nM) HVA (nM)

Pure cortical lesion (n = 15)

Subcortical lesion (n = 43)

P-valuez

1.27 (1.12–1.46) 0.47 (0.35–0.58) 324.5 (302.9–394.5) 2.90 (1.89–3.49) 0.22 (0.13–0.34) 4.53 (3.76–5.54) 0.66 (0.47–1.01) 62.6 (44.5–137.4) 0.79 (0.42–1.27) 409.4 (312.2–669.1)

1.23 (1.05–1.43) 0.61 (0.50–0.71) 342.5 (309.1–378.2) 2.50 (1.98–3.23) 0.24 (0.15–0.40) 4.20 (3.49–5.31) 0.57 (0.45–0.97) 63.7 (39.9–89.3) 0.70 (0.35–0.99) 376.0 (265.2–522.8)

.461 .021 .866 .399 .562 .440 .636 .638 .438 .494

Abbreviations: 5-HIAA: 5-hydroxyindoleacetic acid; DOPAC: 3,4-dihydroxyphenylacetic acid; HVA: homovanillic acid; TIA: transient ischemic attack. a Data are given as median (IQR). z Mann–Whitney U test, P < .005 indicates statistical significance.

Table 6 CSF neurotransmitter concentrations in relation to stroke etiology.a. CSF concentration

Glutamate (mM) Aspartate (mM) Glutamine (mM) Glycine (mM) Proline (mM) Taurine (mM) Norepinephrine (nM) 5-HIAA (nM) DOPAC (nM) HVA (nM)

TOAST classification Cardioembolic (n = 39)

Lacunar (n = 20)

Atherothrombotic (n = 21)

Specific (n = 3)

Undetermined (n = 6)

P-valuez

1.24 (1.13–1.49) 0.57 (0.40–0.67) 348.5 (312.5–402.9) 2.52 (2.03–3.39) 0.24 (0.14–0.35) 4.07 (3.49–5.42) 0.70 (0.48–0.95) 54.8 (41.4–107.4) 0.70 (0.40–0.97) 405.0 (312.2–541.1)

1.23 (1.15–1.51) 0.73 (0.56–0.79) 331.6 (306.2–365.5) 1.93 (1.66–2.62) 0.22 (0.14–0.36) 4.36 (3.93–5.07) 0.56 (0.46–1.19) 70.0 (39.8–101.6) 0.77 (0.65–1.01) 419.4 (323.6–508.0)

1.27 (0.99–1.43) 0.53 (0.42–0.70) 345.7 (315.0–374.2) 2.72 (2.24–3.84) 0.29 (0.17–0.54) 3.76 (2.84–5.09) 0.84 (0.53–1.49) 63.7 (40.8–88.3) 0.95 (0.58–1.49) 352.7 (247.1–461.9)

1.43 (1.06–1.71) 0.50 (0.29–0.64) 371.0 (337.1–371.5) 3.08 (1.26–4.23) 0.37 (0.12–0.79) 5.24 (2.61–5.28) 0.37 (0.21–0.52) 33.0 (28.0–61.8) 0.49 (0.21–0.91) 275.7 (214.0–369.8)

1.01 (0.85–1.32) 0.36 (0.23–0.53) 306.8 (281.6–369.9) 2.73 (2.32–3.26) 0.12 (0.10–0.40) 4.54 (4.26–5.64) 0.59 (0.37–0.96) 49.5 (30.6–69.2) 0.39 (0.26–1.01) 352.1 (240.8–705.5)

.362 .010 .350 .085 .292 .479 .193 .475 .099 .491

Abbreviations: 5-HIAA: 5-hydroxyindoleacetic acid; DOPAC: 3,4-dihydroxyphenylacetic acid; HVA: homovanillic acid; TIA: transient ischemic attack. a Classified by the TOAST criteria, data are given as median (IQR). z Kruskal–Wallis test, P < .005 indicates statistical significance.

4. Discussion Data on neurotransmitters in acute stroke patients mainly stems from studies evaluating plasma samples (Davalos et al., 1997, 2000; Serena et al., 2001; Meglic et al., 2001; Dambinova et al., 2002; Strittmatter et al., 2003; Oto et al., 2008; Castellanos et al., 2008). The interpretation of neurotransmitter systems in blood, however, is hampered by many confounding factors, among which passage through the blood–brain barrier, variations in clearance from blood and extracerebral synthesis of neurotransmitters. For instance, the prolonged increase of glutamate levels in plasma after stroke appears to be linked to altered platelet function

and further questions the validity of neurotransmitters in blood (Aliprandi et al., 2005). Since CSF is equivalent to cerebral extracellular fluid, it is believed to reflect more accurately pathological changes in the central nervous system. To the best of our knowledge, only three research groups reported on neurotransmitter concentrations in CSF of acute ischemic stroke patients. In 1978, Hachinski et al. described that HVA concentrations in CSF are greatly scattered and not related to stroke severity. Castillo et al. found CSF glutamate and glycine levels to be elevated in a selected stroke population compared to controls (Castillo et al., 1996). Moreover, glutamate concentration was higher in patients with more severe stroke, cortical infarction

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and progressing stroke (Castillo et al., 1997). Contrary to patients with favourable stroke evolution, glutamate concentration remained elevated beyond 6 h after stroke onset in patients with stroke progression (Davalos et al., 1997). Skvortsova et al. assessed CSF concentrations of glutamate, aspartate, glycine and gammaaminobutyric acid in patients with hemispheric stroke and observed a more pronounced increase in glutamate and aspartate in the first 6 h after stroke onset in patients with more severe stroke (Skvortsova et al., 2000). This study is the first in which a large number of neurotransmitters in CSF of acute ischemic stroke patients are simultaneously measured, allowing adequately powered evaluation of relevant interrelations between neurotransmitter systems. The study population is representative for the entire stroke spectrum ranging from patients with TIA to patients with massive infarctions. Stroke severity, stroke evolution in the subacute phase, lesion volume, long-term stroke outcome, lesion location and stroke etiology were evaluated according to a stringent protocol that matches the highest international standards. In line with prior findings (Hachinski et al., 1978), we found no relation between stroke severity and CSF HVA levels. However, we were unable to replicate the previously reported association between CSF glutamate levels and stroke severity, cortical lesion and progressing stroke (Castillo et al., 1996, 1997; Davalos et al., 1997; Skvortsova et al., 2000). Except for lower glycine concentrations and higher aspartate and taurine levels, our study disclosed no significant difference in neurotransmitter levels between TIA or stroke patients and matched controls. Moreover, except for higher levels of 5-HIAA in CSF of patients with progressing stroke and poor long-term outcome, we failed to identify any relevant relation between neurotransmitter concentrations in CSF and stroke characteristics. Ongoing platelet activation is a possible source for the elevated 5-HIAA concentration in CSF of patients with progressive stroke and poor long-term outcome. Although the primary mechanism of stroke progression has yet to be elucidated, it is generally accepted that thrombus propagation, absence of recanalisation and re-embolisation may play a role. As serotonin is released from platelets upon aggregation (Jonnakuty and Gragnoli, 2008), it is plausible that more pronounced thrombus formation may have led to higher 5-HIAA levels in CSF of patients with unfavourable stroke evolution and poor outcome. Alternatively, higher serotonin levels in cerebro may induce cerebrovascular vasoconstriction (Nilsson et al., 1999), production of neurotoxins (Chen and Strickland, 1997), platelet aggregation and cardiovascular changes (Cote et al., 2004). These mechanisms may lead to secondary cerebral damage that is reflected in unfavourable stroke evolution and poor long-term outcome. The positive correlation between glycine and norepinephrine concentration and the NIHSS score at admission should be interpreted with caution since a relation between these neurotransmitters and stroke severity was not confirmed by MRI-based volumetry. The large number of statistically significant correlations between neurotransmitters are a major finding from this study and illustrates the relevant interrelation between excitatory amino acids and monoaminergic neurotransmitter systems. These findings convincingly indicate that neurotransmitters cannot be evaluated as isolated biochemical substances. Some limitations of this study should be acknowledged and kept in mind when interpreting the data. CSF levels of amino acids and monoamines may be influenced by multiple factors, many of which are poorly understood. A nonlimitative list of possible confounding circumstances include ventriculospinal concentration gradients, circadian rhythms, physical activity, stress, age, gender, genotype, precursor intake, medication, concomitant neuropsychiatric conditions, reperfusion injury, cerebral regional variability of neurotransmitter systems, CSF sampling and analysis

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methodology (Wood, 1980; Matsumoto et al., 1996). Although we found that neurotransmitters are not influenced by age, gender, comorbidity, traumatic lumbar puncture, or the interval between stroke onset and lumbar puncture, bias cannot be excluded. Since in most patients lumbar puncture was performed in the very first hours after stroke onset, relevant alterations in CSF neurotransmitter concentrations occurring at a later stage may have gone undetected. Similarly, it cannot be excluded that pertinent differences in neurotransmitter levels were not observed due to the study population’s heterogeneity. Finally, it is problematic to obtain reliable reference values for neurotransmitters since samples are often collected in patients with clinical suspicion of disease (Dhondt, 2004). This may partially explain the differences in CSF neurotransmitter concentrations reported in controls. In sum, the large number of neurotransmitter systems evaluated in this study enabled the identification of relevant interrelations between neurotransmitters. However, with the exception of higher 5-HIAA levels in CSF of patients with progressing stroke and poor long-term outcome, we failed to identify any relevant association between neurotransmitter concentrations in CSF and stroke characteristics. Author contributions Study concept and design: Brouns and De Deyn. Acquisition of clinical data: Brouns. CSF sampling: Brouns. Analysis of excitatory amino acid and monoamine catabolite levels: Drinkenburg and Van Hemelrijck. Acquisition of neuroimaging data: Brouns and De Surgeloose. Analysis and interpretation of data: Brouns, Drinkenburg, Van Hemelrijck and De Deyn. Drafting of the manuscript: Brouns. Critical revision of the manuscript for important intellectual content: Brouns, Drinkenburg and Van Hemelrijck, De Surgeloose, and De Deyn. Study supervision: De Deyn. Acknowledgments R.B. was a research assistant of the Fund for Scientific ResearchFlanders (FWO-Vlaanderen). This research was also supported by the Institute Born-Bunge; the agreement between the Institute Born-Bunge and the University of Antwerp; the Interuniversity Attraction Poles (IAP) program P6/43 of the Belgian Federal Science Policy Office, Belgium; a Methusalem Grant of the Flemish Government; Medical Research Foundation Antwerp; the Fund for Scientific Research-Flanders (FWO-F). References Adams Jr., H.P., Bendixen, B.H., Kappelle, L.J., Biller, J., Love, B.B., Gordon, D.L., Marsh III, E.E., 1993. Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter clinical trial. TOAST. Trial of Org 10172 in Acute Stroke Treatment. Stroke 24, 35–41. Aliprandi, A., Longoni, M., Stanzani, L., Tremolizzo, L., Vaccaro, M., Begni, B., Galimberti, G., Garofolo, R., Ferrarese, C., 2005. Increased plasma glutamate in stroke patients might be linked to altered platelet release and uptake. J. Cereb. Blood Flow Metab. 25, 513–519. Birschel, P., Ellul, J., Barer, D., 2004. Progressing stroke: towards an internationally agreed definition. Cerebrovasc. Dis. 17, 242–252. Brouns, R., De Deyn, P.P., 2009. The complexity of neurobiological processes in acute ischemic stroke. Clin. Neurol. Neurosurg. 111, 483–495. Brouns, R., Heylen, E., Sheorajpanday, R., Willemse, J.L., Kunnen, J., De Surgeloose, D., Hendriks, D.F., De Deyn, P.P., 2009a. Carboxypeptidase U (TAFIa) decreases the efficacy of thrombolytic therapy in ischemic stroke patients. Clin. Neurol. Neurosurg. 111, 165–170. Brouns, R., Marescau, B., Possemiers, I., Sheorajpanday, R., De Deyn, P.P., 2009b. Dimethylarginine levels in cerebrospinal fluid of hyperacute ischemic stroke patients are associated with stroke severity. Neurochem. Res. 34, 1642–1649. Brouns, R., Sheorajpanday, R., Kunnen, J., De Surgeloose, D., De Deyn, P.P., 2009c. Clinical, biochemical and neuroimaging parameters after thrombolytic therapy predict long-term stroke outcome. Eur. Neurol. 62, 9–15. Brouns, R., Sheorajpanday, R., Wauters, A., De Surgeloose, D., Marien, P., De Deyn, P.P., 2008. Evaluation of lactate as a marker of metabolic stress and cause of secondary damage in acute ischemic stroke or TIA. Clin. Chim. Acta 397, 27–31.

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