HMGB1 Level in Cerebrospinal Fluid as a Marker of Treatment Outcome in Patients with Acute Hydrocephalus Following Aneurysmal Subarachnoid Hemorrhage 1, MD, PhD,* Anna Wo Bartosz Soko zniak, PhD,† Roman Jankowski, MD, PhD,* Stefan Jurga, PhD,† Norbert Wa˛sik, MD,* Hinna Shahid, MD,* and Bartosz Grzeskowiak, PhD†
Background: Attempts to clarify mechanisms of early brain injury in subarachnoid hemorrhage (SAH) revealed a high-mobility group box 1 (HMGB1) protein involvement in sterile inflammation initiated by aneurysm rupture. This study aims at assessing the prognostic value of HMGB1 in comparison with traditional biomarkers. Methods: Ten patients with Fisher grade 4 SAH and acute hydrocephalus underwent endovascular coiling and ventriculostomy. HMGB1 level was measured in cerebrospinal fluid (CSF) samples collected on first, fifth, and 10th day. HMGB1 level in first sample was correlated with treatment outcome assessed in Glasgow outcome scale (GOS) at 3 months. Obtained results were compared with plasma inflammatory markers, clinical grading scales, and imaging grading scales. HMGB1 level in consecutive samples was analyzed in search of concentration trends correlating with patients’ outcome. Results: HMGB1 level in CSF of SAH patients, in contrast to control group, is significantly elevated (P , .001). Good (GOS . 3) and poor (GOS # 3) outcome patients differ significantly in HMGB1 level on admission (P , .01). The strongest correlation to patients’ outcome was found for Hunt and Hess scale (R 5 2.887, P , .01), HMGB1 level (R 5 2.859, P , .01), and World Federation of Neurological Surgeons scale (R 5 2.832, P , .01). Constant and high HMGB1 level of 10 ng/mL or more in consecutive CSF samples identifies nonsurvivors. Conclusions: HMGB1 protein is elevated in SAH patients. Changes in the concentration of HMGB1 in consecutive samples of the CSF correlate with outcome. Our results encourage further proteomic investigation. Key Words: HMGB1—highmobility group box 1—subarachnoid hemorrhage—proteomic—cerebrospinal fluid—biomarkers. Ó 2015 by National Stroke Association
From the *Department of Neurosurgery, Poznan University of Medical Sciences, Poznan; and †Nanobiomedical Centre, Adam Mickiewicz University, Poznan, Poland. Received December 13, 2014; revision received April 27, 2015; accepted May 1, 2015. The authors received grant support from the National Centre for Research and Development, Applied Research Programme, The National Centre of Research and Development No PBSll/9ll3l20l2 ‘‘Nanomaterials and their potential biomedical applications.’’ Address correspondence to Bartosz Sok o1, MD, PhD, Przybyszewskiego 49 street, 60 – 355 Poznan, Poland. E-mail: bartosz.sokol@ gmail.com. 1052-3057/$ - see front matter Ó 2015 by National Stroke Association http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2015.05.002
The annual incidence of cerebral aneurysm rupture is 1 in 10,000 for the general population. This devastating neurologic condition kills or severely disables 70% of victims.1 The chief cause of mortality and morbidity, affecting about 30% of cases, is delayed cerebral ischemia (DCI).2,3 Vasospasm developing 3-7 days after rupture in 70% of patients has traditionally been regarded as the principal cause of DCI.4 When long-term outcomes failed to improve, despite successful reversal of vasospasm, an alternative explanation of DCI pathogenesis was required.5 Acute pathophysiological events occurring within 72 hours of rupture, and thus encompassed by
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Table 1. Study group patients’ characteristics Malez Age (y)* Aneurysm location Posterior inferior cerebellar arteryz Anterior cerebral arteryz Anterior communicating arteryz Basilar arteryz Posterior cerebral arteryz Endovascular treatmentz Aneurysmal size (mm)* Rebleedingz Acute hydrocephalusz Cerebral infarction due to DCIz Intracerebral hemorrhage on CTz Intraventricular blood on CTz Fisher CT scorey Modified Fisher CT scorey Admission time (h)* World Federation of Neurological Surgeons score on admissiony Hunt and Hess score on admissiony Glasgow Coma Scale on admissiony Body temperature on admission ( C)* Days spent on intensive care unit*
Plasma inflammatory markers C-reactive protein level (mg/L)* Fibrinogen level (mg/dL)* White blood cell count (106/mm3)* Plasma hemoglobin level (mg/dL)* HMGB1 level (ng/mL)y
6 (60%) 61.1 6 11.8 1 (10%) 2 (20%) 4 (40%) 2 (20%) 1 (10%) 10 (100%) 4.5 6 1.9 0 (0%) 10 (100%) 6 (60%) 6 (60%) 10 (100%) 4 (4-4) 4 (2-4) 5.4 6 2.1 4 (2-4) 4 (3-4) 9.5 (9-13) 36.9 6 .40 23.6 6 9.6
On admission
Second sample
Third sample
105.6 6 101.9 429.0 6 147.4 12.5 6 4.8 11.4 6 3.4 10.0 (6-10)
92.5 6 49.0 540.6 6 169.0 12.5 6 6.73 11.0 6 2.5 7.4 (3.3-10)
51.0 6 33.4 484.6 6 73.0 12.1 6 5.0 10.1 6 3.0 5.9 (3.3-10.0)
Treatment outcome at 3 months (according to Glasgow outcome scale) Low disability (score of 5)z Moderate disability (score of 4)z Severe disability (score of 3)z Persistent vegetative state (score of 2)z Death (score of 1)z
4 (40%) 0 (0%) 2 (20%) 1 (10%) 3 (30%)
Abbreviations: CT, computed tomography; DCI, delayed cerebral ischemia; HMGB1, high-mobility group box 1. *Mean 6 standard deviation if numerical data. yMedian (first quartile-third quartile) if ordinal data. zCount (percentage) if categorical data.
the term ‘‘early brain injury,’’ have been investigated as potential contributors to DCI. Among approximately 3000 proteins identified by proteomic analysis of cerebrospinal fluid (CSF), high-mobility group box 1 (HMBG1) demonstrates strong involvement in sterile inflammation initiated by aneurysm rupture.5,6 HMBG1 (also known as amphoterin) was initially identified as an abundant nuclear protein responsible for gene expression regulation. During the past 15 years, further work has distinguished this protein as an extracellular mediator of infection, injury, and inflammation.7 HMGB1 is one
of the prototypes of the so-called alarmin family of mediators involved in cross talk between injured cells and relatively healthy cells around damaged tissues.8 Recent studies have reported this protein as a potential biomarker of poor neurologic outcome in subarachnoid hemorrhage (SAH) patients.5 Our study tracks changes in the levels of HMGB1 in such patients during their stay in the intensive care unit and attempts to correlate them with long-term outcome. The prognostic value of HMGB1 is assessed in comparison with traditional predictors.
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Table 2. The factors associated with 3-month functional outcome
Category Number of patients Malez Age (y)* Aneurysm location Posterior inferior cerebellar arteryz Anterior cerebral arteryz Anterior communicating arteryz Basilar arteryz Posterior cerebral arteryz Aneurysmal size (mm)* Cerebral infarction due to DCI (no. of patients)z Intracerebral hemorrhage on CT (no. of patients)z Intraventricular blood on CT (no. of patients)z Fisher CT scorey Modified Fisher CT scorey Admission time (h)* World Federation of Neurological Surgeons score on admissiony Hunt and Hess score on admissiony Glasgow Coma Scale on admissiony Body temperature on admission ( C)* Days spent on intensive care unit* Plasma inflammatory markers C-reactive protein level (mg/L)* Fibrinogen level (mg/dL)* White blood cell count (106/mm3)* Plasma hemoglobin level (mg/dL)* HMGB1 level (ng/mL)y Treatment outcome at 3 months (according to Glasgow outcome scale) Low disability (score of 5)z Moderate disability (score of 4)z Severe disability (score of 3)z Persistent vegetative state (score of 2)z Death (score of 1)z
Poor outcome GOS #3
Good outcome GOS .3
6 5 (83%) 62 6 11.3
4 1 (25%) 59.8 6 14.0
1 (17%) 1 (17%) 3 (50%) 0 (0%) 1 (17%) 4.2 6 1.3 5 (83%) 3 (50%) 6 (100%) 4 (4-4) 4 (4-4) 4.7 6 1.6 4 (4-5) 4 (4-4) 9 (5-9) 37.0 6 .20 32.0 6 4.4
0 1 (25%) 1 (25%) 2 (50%) 0 (0%) 4.3 6 2.2 1 (25%) 3 (75%) 4 (100%) 4 (4-4) 3 (2-4) 6.5 6 2.4 2 (1.5-2.5) 3 (2.5-3) 13.5 (13-14.5) 36.8 6 .60 17.3 6 7.0
.349 .183 ,.01 ,.01 ,.05 .432 ,.05
89.7 6 88.0 419 6 186.4 14.6 6 5.0 11.2 6 4.2 10.0 (10-10)
129.4 6 130.4 441 6 106.0 9.26 6 2.4 11.7 6 .90 5.0 (3.1-6.3)
.577 .843 .085 .835 ,.01
P value
.190 .786 .505
.977 .190 .571
4 (100%) 0 (0%) 2 (33%) 1 (17%) 3 (50%)
Abbreviations: CT, computed tomography; DCI, delayed cerebral ischemia; HMGB1, high-mobility group box 1. *Mean 6 standard deviation if numerical data. yMedian (first quartile-third quartile) if ordinal data. zCount (percentage) if categorical data.
Materials and Methods Study Population The Bioethics Committee of Poznan University of Medical Sciences approved the protocol of this clinical study. Between December 2012 and January 2014, 95 patients with aneurysmal SAH, confirmed by computerized tomography (CT) and digital subtraction angiography, were admitted to the neurosurgical ward. The clinical condition and levels of plasma inflammatory markers (C-reactive protein, white blood cell count, and fibrinogen), together with the hemoglobin and
body temperature, were assessed on admission and systematically during hospitalization. Endovascular coiling was performed in 93 patients within 24 hours of rupture. Fourteen patients with intraventricular hemorrhage (Fisher grade 4) underwent immediate ventriculostomy because of the development of acute hydrocephalus. An intraventricular drain was inserted immediately after aneurysm embolization, based on the initial CT findings or a follow-up scan to confirm the diagnosis. Because of the suspected high sensitivity of HMGB1 to coexisting and preexisting conditions, patients with a history of CNS disease (meningitis, stroke),
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Figure 1. Bubble chart showing the correlation between HMGB1 level in CSF on admission and treatment outcome assessed by Glasgow outcome scale (GOS) at 3 months. Spearman’s exact correlation of 2.859 and P , .01.
and active systemic disease (diabetes mellitus, rheumatoid arthritis, malignancy, cirrhosis), were excluded from the study. Patients were also excluded if they developed an acute inflammatory process unassociated with the ventriculostomy. Following exclusions, there remained 10 patients forming the study group, for whom written consent was obtained. The control group consisted of 8 patients with noninflammatory conditions (tension headache, spinal disc herniation, dementia, occipital neuralgia), who underwent diagnostic lumbar puncture; exclusion criteria were the same as for the study group, but in addition, patients with elevated plasma inflammatory markers on the day of lumbar puncture were not enrolled.
End Points Subjects were followed until death or the completion of 3 months following SAH. The primary outcome was the functional state after 3 months, and the secondary outcome was in-hospital mortality. The functional outcome was defined using the Glasgow outcome scale (GOS); these were dichotomized as good (GOS 4-5) or poor (GOS 1-3) outcomes.
Imaging Studies All the patients in this study underwent endovascular treatment of the aneurysm preceded by 4-vessel digital subtraction angiography. Head CT scan was carried out
Table 3. Correlation between studied parameters and functional outcome at 3 months or HMGB1 level on admission Glasgow outcome scale at 3 months
HMGB 1 level on admission
Category
Correlation coefficient
P value
World Federation of Neurological Surgeons score on admission Hunt and Hess score on admission Glasgow Coma Scale on admission Modified Fisher CT score Body temperature Days on intensive care unit Plasma inflammatory markers C-reactive protein level White blood cell count Fibrinogen level Plasma hemoglobin level HMGB1 level on admission GOS at 3 months
2.832
.005
.893
.001
2.887 .755 2.199 2.174 2.694
.002 .016 .581 .620 .105
.931 2.895 .171 .385 .823
.001 ,.001 .636 .265 .032
2.127 2.394 2.343 .095 2.859 —
.725 .257 .373 .793 .007 —
2.041 .587 .128 2.034 — 2.859
.915 .078 .760 .931 — .007
Correlation coefficient
Abbreviations: CT, computed tomography; GOS, Glasgow outcome scale; HMGB1, high-mobility group box 1.
P value
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2000i (Sysmex, Kobe, Japan), C-reactive protein by Cobas 6000 (Roche diagnostics, Basel, Switzerland), and fibrinogen by ACL TOP 500 (Instrumentation Laboratory, Milano, Italy) as part of standard patient care in the intensive care unit. Researchers running laboratory tests were blinded to all patient details. A limitation of our study was the narrow detection range of the ELISA kit (.1-10 ng/mL); HMGB1 level was frequently above the upper limit and could only be expressed as ‘‘10 ng/mL or higher.’’
Statistical Analysis
Figure 2. Bar chart showing patients’ gradation according to modified Fisher scale. Numbers above bars represent absolute number of patients in each category.
on at least 3 occasions: on admission, after drain insertion, and after drain removal. Additional scans were performed in response to clinical deterioration (a fall of at least 2 points in Glasgow Coma Scale (GCS) lasting at least 1 hour). By definition, all the study group patients fell into Fisher grade 4 on account of the intraventricular clot. SAH grading based on the modified Fisher scale was assessed on CT scans obtained on admission. Cerebral infarction due to DCI was identified according to the criteria of Vergouwen et.al.2 All the images were assessed by a radiologist blinded to the clinical details.
Sample Collection and Assays CSF samples were collected from the endoventricular drainage system. The first sample (also designated ‘‘on admission’’) was taken within 24 hours of ventriculostomy, and within 48 hours of aneurysm rupture in all patients. Subsequent samples were taken on the fifth and 10th days with an allowance of 24 hours. At least 3 samples were taken unless the patient died before the relevant sampling. Immediately after collection, samples were centrifuged at 3000 rpm for 10 minutes and stored at 280 C until assayed. The concentration of HMGB1 in CSF was analyzed by enzyme-linked immunosorbent assay (ELISA) using commercial kit ST51011 (IBL International, Hamburg, Germany), in accordance with manufacturer’s instructions. White blood cell count and hemoglobin were analyzed by hematology analyzer XT
Statistical analysis was performed with STATISTICA 10 (Stat Soft Inc., Tulsa, OK) and StatXact (Cytel Inc., Cambridge, MA). Values are expressed as mean and standard deviations if numerical data; median and interquartile range if ordinal data; and counts and percentage if categorical data. The normality of data distribution was assessed by the Shapiro–Wilk test. Comparisons were made by using (1) Fisher exact test (2 3 2) for categorical data, (2) unpaired Student t test for unrelated normally distributed numerical data, (3) the Mann–Whitney test for unrelated non-normally distributed numerical data and unrelated ordinal data, (4) the Friedman exact test with Conover–Iman post hoc for related ordinal data, (5) the one-side Page exact test for trend seeking in related ordinal data. The correlations of HMGB1 with other parameters were assessed by Spearman’s exact correlation. Because ELISA tests’ detection range restrictions, HMGB1 level was identified as ordinal data and presented as median. A value of P less than .05 was considered statistically significant.
Results Treatment Outcome and Prognostic Markers The results for the 10 patients in the study group are summarized in Table 1. CSF from the age- and sexmatched control group (P values .712 and .676 respectively) contained only trace amounts of HMGB1 (.10 ng/mL); CSF from the SAH group showed elevated levels (10.0 ng/mL), indicating a significant difference (P , .001) between the 2 groups. The 6 patients with a poor outcome were also compared with the 4 good outcome individuals (Table 2). The former group all had HMBG1 levels greater than 10 ng/mL, whereas the latter had significantly lower levels at 5.0 ng/mL (P , .01). Moreover, HMGB1 level on admission has a strong correlation with GOS at 3 months (R 5 2.859, P , .01; Fig 1; Table 3). All the clinical grading scales that were used also showed a significant correlation with outcome: Hunt and Hess (H&H; R 5 2.887, P , .01), World Federation of Neurological Surgeons (WFNS; R 5 2.832, P , .01), and GCS (R 5 .755, P , .05). In the SAH group, we found plasma inflammatory markers to be poor
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Figure 3. Box-plot showing the HMGB1 level changes in time in study group. Friedman exact test (P 5.042) with Conover–Iman post hoc indicates significant difference between first and second samples (*P 5 .015) and nearly significant difference between first and third samples (#P 5 .055). One-side Page exact test revealed significant downtrend in HMGB1 level in time (P 5 .041).
predictors, apart from white blood cell count, which showed a difference between the good and poor outcome groups with P 5 .085. Poor markers also were body temperature (R 5 2.174, P 5 .620), hemoglobin levels (R 5 .095, P 5 .793), and the modified Fisher scale (P 5 2.199, P 5 .581; Fig 2). HMGB1 level monitoring covered the first 10-12 days following aneurysm rupture. Three samples were drawn at 5 day intervals. There was a significant difference in HMGB1 levels between the first and second samples (P , .05), and a nearly significant difference between the first and third samples (P 5 .055; Fig 3). In this study, all the poor outcome patients had a highly elevated HMGB1 level (.10 ng/mL) on admission. The levels at the time of the first sample only allowed us to prognosticate a poor outcome, but taking account of the second and third samples improved this situation. Patients who died all had a level of HMGB1 consistently above 10 ng/mL in the CSF; however, patients with an intermediate outcome (GOS 2-3 at 3 months) initially had similar very high levels, but these declined to lower levels on the second and third samples (Fig 4). The significance of this downtrend was confirmed by the one-side Page exact test (P , .05). Our interpretation of the last pattern is limited by narrow detection range of ELISA kit. Patients with a good outcome (GOS 4-5 at 3 months) all had consistently lower HMGB1 levels (,10 ng/mL), with no apparent declining or ascending trend.
Discussion Elevated HMGB1 levels in CSF were first reported as a biomarker of poor neurologic outcome by Nakahara et al9 in a series of 39 SAH patients treated by aneurysm clipping. King et al5 confirmed these promising results in a series of 9 patients. Most recently, plasma HMGB1 levels were found to be useful as predictors of functional outcome and mortality after SAH.10 Our study of 10 cases of severe SAH revealed the following: (1) CSF level of HMGB1 was a good prognosticator of outcome, being as accurate as WFNS and H&H grading scales. (2) Differing HMGB1 concentration trends, which were also predictors of outcome. Prognostic outcome factors identified in a small group of severe cases need to be interpreted with caution. Our cohort does not represent the full spectrum of patients admitted with SAH, but a narrow and severe group with intraventricular clot and hydrocephalus, which limits the range of possible conclusions.11,12 However, the validity is enhanced by the strong correlation with WFNS, H&H, and GCS in predicting clinical outcomes. This observation follows the results of some larger studies.13-15 Greer et al16 found elevated body temperature associated with a worse outcome, but we did not
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Figure 4. Box-plot showing the dynamics of HMGB1 level changes in time in detailed treatment outcome groups. Detailed outcome group ‘‘good’’ consists of patients with 5-4 GOS score at 3 months; ‘‘intermediate’’—patients with 3-2 GOS score at 3 months, ‘‘death’’—patients with 1 GOS score at 3 months. Friedman exact result does not show significant differences in HMGB1 level among first, second, and third samples in any of detailed outcome groups. One-side Page exact test revealed significant downtrend in HMGB1 level in intermediate outcome group, which means that HMGB1 level declines in time among those patients (P 5 .046).
confirm this. Plasma inflammatory markers monitored in our study—C-reactive protein and white blood cell count—failed to prognose outcomes, although some studies have shown an association between higher levels and a poorer prognosis.17,18 Fibrinogen level was elevated, and differed significantly between patients, but was an unreliable prognostic marker. In a study by Nina et al,19 fibrinogen was within normal limits and also failed to prognose outcome. Higher initial and mean hemoglobin was reported by Naidech et al20 to be associated with slightly better outcomes; however, this was not found in our study group. Previous publications also found modified Fisher scale a good predictor of outcome; we did not find this correlation in our study.21 Scheduled monitoring of HMGB1 levels during the treatment period showed differing concentration patterns which were related to the eventual outcome. Patients with an excellent outcome maintained low levels of HMGB1 (,10 ng/mL) in CSF throughout. High levels of HMGB1 (.10 ng/mL) on admission were found in patients with a poor outcome. In the latter group, when the HMGB1 levels fell to below 10 ng/mL on the second and third samples, the subjects survived, but when they remained above 10 ng/mL, they did not. There have been numerous publications regarding the role of HMGB1 in CNS disease, and taking account of
these, we can hypothesize about the potential sources of this protein during our sampling period following SAH. HMGB1 is expressed in every nucleus of normal brain cells (neurons, astrocytes, and microglia), as well as infiltrating immunological cells (macrophages, monocytes).7 Efforts to elucidate HMBG1 origin are complicated by the presence of 2 pathways for its release into the extracellular milieu. Firstly, there is passive leakage of HMGB1 because of membrane damage seen during necrosis; secondly, there is active secretion in response to various stimuli, carried out by both the immune and parenchymal cells.7 Sun et al recently demonstrated that both release pathways are present as early as 2 hours following rupture in a rat model of SAH. In contradistinction to ischemic stroke, the initial portion of HMGB1 does not predominantly originate from necrotic neurons, but is actively secreted by them. It is likely that HMGB1 leaked and secreted from neurons stimulates neighboring glial cells to produce more of the protein. This hypothesis is in agreement with increasing numbers of microglial cells secreting HMGB1 as time develops.22 The 3 target receptors for HMGB1 (TLR2, TLR4, and RAGE) are normally expressed on the brain cells.22,23 Their mutual downstream mediator, nuclear factor kappa B, not only triggers expression of proinflammatory mediators (iNOS, COX-2, ICAM-1, VCAM1, E-selectin,
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IL-1b, II-6, and TNF alpha) but also upregulates upstream receptors (TLR2, TLR4, and RAGE.).7,23-27 As a result, overexpression of the above mediators and receptors activates both damaging and repair processes following SAH.4,9,22,28
Conclusions Our study confirmed elevation of the HMGB1 protein in SAH patients. Initial levels and subsequent changes of HMGB1 in CSF correlated with neurologic outcome. Although limited by a small study group size, our findings encourage further proteomic investigation of CSF in such patients. Acknowledgment: The authors are grateful to Barbara Wie˛ckowska (Department of Computer Science and Statistics, Poznan Medical University) for her valuable statistical assistance.
References 1. Sehba FA, Hou J, Pluta RM, Zhang JH. The importance of early brain injury after subarachnoid hemorrhage. Prog Neurobiol 2012;97:14-37. 2. Vergouwen MD, Vermeulen M, van Gijn J, et al. Definition of delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage as an outcome event in clinical trials and observational studies: proposal of a multidisciplinary research group. Stroke 2010;41:2391-2395. 3. Cossu G, Messerer M, Oddo M, et al. To look beyond vasospasm in aneurysmal subarachnoid haemorrhage. Biomed Res Int 2014;2014:628597. 4. Sehba FA, Pluta RM, Zhang JH. Metamorphosis of subarachnoid hemorrhage research: from delayed vasospasm to early brain injury. Mol Neurobiol 2011;43:27-40. 5. King MD, Laird MD, Ramesh SS, et al. Elucidating novel mechanisms of brain injury following subarachnoid hemorrhage: an emerging role for neuroproteomics. Neurosurg Focus 2010;28:E10. 6. Guldbrandsen A, Vethe H, Farag Y, et al. In-depth characterization of the cerebrospinal fluid proteome displayed through the CSF Proteome Resource (CSF-PR). Mol Cell Proteomics 2014;13:3152-3163. 7. Andersson U, Tracey KJ. HMGB1 is a therapeutic target for sterile inflammation and infection. Annu Rev Immunol 2011;29:139-162. 8. Qiu J, Xu J, Zheng Y, et al. High-mobility group box 1 promotes metalloproteinase-9 upregulation through Toll-like receptor 4 after cerebral ischemia. Stroke 2010;41: 2077-2082. 9. Nakahara T, Tsuruta R, Kaneko T, et al. High-mobility group box 1 protein in CSF of patients with subarachnoid hemorrhage. Neurocrit Care 2009;11:362-368. 10. Zhu XD, Chen JS, Zhou F, et al. Relationship between plasma high mobility group box-1 protein levels and clinical outcomes of aneurysmal subarachnoid hemorrhage. J Neuroinflammation 2012;9:194. 11. Dupont S, Rabinstein AA. Extent of acute hydrocephalus after subarachnoid hemorrhage as a risk factor for poor functional outcome. Neurol Res 2013;35:107-110.
12. Varelas PN, Rickert KL, Cusick J, et al. Intraventricular hemorrhage after aneurysmal subarachnoid hemorrhage: pilot study of treatment with intraventricular tissue plasminogen activator. Neurosurgery 2005;56:205-213. discussion 205-213. 13. Salary M, Quigley MR, Wilberger JE. Relation among aneurysm size, amount of subarachnoid blood, and clinical outcome. J Neurosurg 2007;107:13-17. 14. Rosengart AJ, Schultheiss KE, Tolentino J, et al. Prognostic factors for outcome in patients with aneurysmal subarachnoid hemorrhage. Stroke 2007;38: 2315-2321. 15. Starke RM, Komotar RJ, Otten ML, et al. Predicting longterm outcome in poor grade aneurysmal subarachnoid haemorrhage patients utilising the Glasgow Coma Scale. J Clin Neurosci 2009;16:26-31. 16. Greer DM, Funk SE, Reaven NL, et al. Impact of fever on outcome in patients with stroke and neurologic injury: a comprehensive meta-analysis. Stroke 2008; 39:3029-3035. 17. Bertolini ED, Figueiredo EG, Teixeira MJ. Serum C-reactive protein levels predict neurological outcome after aneurysmal subarachnoid hemorrhage. Arq Neuropsiquiatr 2011;70:202-205. 18. Maiuri F, Gallicchio B, Donati P, et al. The blood leukocyte count and its prognostic significance in subarachnoid hemorrhage. J Neurosurg Sci 1987;31:45-48. 19. Nina P, Schisano G, Chiappetta F, et al. A study of blood coagulation and fibrinolytic system in spontaneous outcome. Surg Neurol 2001;3019:197-203. 20. Naidech AM, Drescher J, Ault ML, et al. Higher hemoglobin is associated with less cerebral infarction, poor outcome, and death after subarachnoid hemorrhage. Neurosurgery 2006;59:775-779. discussion 779-780. 21. Kramer AH, Hehir M, Nathan B, et al. A comparison of 3 radiographic scales for the prediction of delayed ischemia and prognosis following subarachnoid hemorrhage. J Neurosurg 2008;109:199-207. 22. Sun Q, Wu W, Hu YC, et al. Early release of high-mobility group box 1 (HMGB1) from neurons in experimental subarachnoid hemorrhage in vivo and in vitro. J Neuroinflammation 2014;11:106. 23. Hayakawa K, Qiu J, Lo EH. Biphasic actions of HMGB1 signaling in inflammation and recovery after stroke. Ann N Y Acad Sci 2010;1207:50-57. 24. Faraco G, Fossati S, Bianchi ME, et al. High mobility group box 1 protein is released by neural cells upon different stresses and worsens ischemic neurodegeneration in vitro and in vivo. J Neurochem 2007;103: 590-603. 25. Yang Q, Wang JZ, Li JC, et al. High-mobility group protein box-1 and its relevance to cerebral ischemia. J Cereb blood flow Metab 2010;30:243-254. 26. Ellis JA, Youngerman BE, Higashida RT, et al. Endovascular treatment strategies for acute ischemic stroke. Int J Stroke 2011;6:511-522. 27. You WC, Wang CX, Pan YX, et al. Activation of nuclear factor-kB in the brain after experimental subarachnoid hemorrhage and its potential role in delayed brain injury. PLoS One 2013;8:e60290. 28. Xie X, Wu X, Cui J, et al. Increase ICAM-1 and LFA-1 expression by cerebrospinal fluid of subarachnoid hemorrhage patients: involvement of TNF-a. Brain Res 2013; 1512:89-96.
Background: Attempts to clarify mechanisms of early brain injury in subarachnoid hemorrhage (SAH) revealed a high-mobility group box 1 (HMGB1) protein involvement in sterile inflammation initiated by aneurysm rupture. This study aims at assessing the prognostic value of HMGB1 in comparison with traditional biomarkers. Methods: Ten patients with Fisher grade 4 SAH and acute hydrocephalus underwent endovascular coiling and ventriculostomy. HMGB1 level was measured in cerebrospinal fluid (CSF) samples collected on first, fifth, and 10th day. HMGB1 level in first sample was correlated with treatment outcome assessed in Glasgow outcome scale (GOS) at 3 months. Obtained results were compared with plasma inflammatory markers, clinical grading scales, and imaging grading scales. HMGB1 level in consecutive samples was analyzed in search of concentration trends correlating with patients’ outcome. Results: HMGB1 level in CSF of SAH patients, in contrast to control group, is significantly elevated (P , .001). Good (GOS . 3) and poor (GOS # 3) outcome patients differ significantly in HMGB1 level on admission (P , .01). The strongest correlation to patients’ outcome was found for Hunt and Hess scale (R 5 2.887, P , .01), HMGB1 level (R 5 2.859, P , .01), and World Federation of Neurological Surgeons scale (R 5 2.832, P , .01). Constant and high HMGB1 level of 10 ng/mL or more in consecutive CSF samples identifies nonsurvivors. Conclusions: HMGB1 protein is elevated in SAH patients. Changes in the concentration of HMGB1 in consecutive samples of the CSF correlate with outcome. Our results encourage further proteomic investigation. Key Words: HMGB1—highmobility group box 1—subarachnoid hemorrhage—proteomic—cerebrospinal fluid—biomarkers. Ó 2015 by National Stroke Association
From the *Department of Neurosurgery, Poznan University of Medical Sciences, Poznan; and †Nanobiomedical Centre, Adam Mickiewicz University, Poznan, Poland. Received December 13, 2014; revision received April 27, 2015; accepted May 1, 2015. The authors received grant support from the National Centre for Research and Development, Applied Research Programme, The National Centre of Research and Development No PBSll/9ll3l20l2 ‘‘Nanomaterials and their potential biomedical applications.’’ Address correspondence to Bartosz Sok o1, MD, PhD, Przybyszewskiego 49 street, 60 – 355 Poznan, Poland. E-mail: bartosz.sokol@ gmail.com. 1052-3057/$ - see front matter Ó 2015 by National Stroke Association http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2015.05.002
The annual incidence of cerebral aneurysm rupture is 1 in 10,000 for the general population. This devastating neurologic condition kills or severely disables 70% of victims.1 The chief cause of mortality and morbidity, affecting about 30% of cases, is delayed cerebral ischemia (DCI).2,3 Vasospasm developing 3-7 days after rupture in 70% of patients has traditionally been regarded as the principal cause of DCI.4 When long-term outcomes failed to improve, despite successful reversal of vasospasm, an alternative explanation of DCI pathogenesis was required.5 Acute pathophysiological events occurring within 72 hours of rupture, and thus encompassed by
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Table 1. Study group patients’ characteristics Malez Age (y)* Aneurysm location Posterior inferior cerebellar arteryz Anterior cerebral arteryz Anterior communicating arteryz Basilar arteryz Posterior cerebral arteryz Endovascular treatmentz Aneurysmal size (mm)* Rebleedingz Acute hydrocephalusz Cerebral infarction due to DCIz Intracerebral hemorrhage on CTz Intraventricular blood on CTz Fisher CT scorey Modified Fisher CT scorey Admission time (h)* World Federation of Neurological Surgeons score on admissiony Hunt and Hess score on admissiony Glasgow Coma Scale on admissiony Body temperature on admission ( C)* Days spent on intensive care unit*
Plasma inflammatory markers C-reactive protein level (mg/L)* Fibrinogen level (mg/dL)* White blood cell count (106/mm3)* Plasma hemoglobin level (mg/dL)* HMGB1 level (ng/mL)y
6 (60%) 61.1 6 11.8 1 (10%) 2 (20%) 4 (40%) 2 (20%) 1 (10%) 10 (100%) 4.5 6 1.9 0 (0%) 10 (100%) 6 (60%) 6 (60%) 10 (100%) 4 (4-4) 4 (2-4) 5.4 6 2.1 4 (2-4) 4 (3-4) 9.5 (9-13) 36.9 6 .40 23.6 6 9.6
On admission
Second sample
Third sample
105.6 6 101.9 429.0 6 147.4 12.5 6 4.8 11.4 6 3.4 10.0 (6-10)
92.5 6 49.0 540.6 6 169.0 12.5 6 6.73 11.0 6 2.5 7.4 (3.3-10)
51.0 6 33.4 484.6 6 73.0 12.1 6 5.0 10.1 6 3.0 5.9 (3.3-10.0)
Treatment outcome at 3 months (according to Glasgow outcome scale) Low disability (score of 5)z Moderate disability (score of 4)z Severe disability (score of 3)z Persistent vegetative state (score of 2)z Death (score of 1)z
4 (40%) 0 (0%) 2 (20%) 1 (10%) 3 (30%)
Abbreviations: CT, computed tomography; DCI, delayed cerebral ischemia; HMGB1, high-mobility group box 1. *Mean 6 standard deviation if numerical data. yMedian (first quartile-third quartile) if ordinal data. zCount (percentage) if categorical data.
the term ‘‘early brain injury,’’ have been investigated as potential contributors to DCI. Among approximately 3000 proteins identified by proteomic analysis of cerebrospinal fluid (CSF), high-mobility group box 1 (HMBG1) demonstrates strong involvement in sterile inflammation initiated by aneurysm rupture.5,6 HMBG1 (also known as amphoterin) was initially identified as an abundant nuclear protein responsible for gene expression regulation. During the past 15 years, further work has distinguished this protein as an extracellular mediator of infection, injury, and inflammation.7 HMGB1 is one
of the prototypes of the so-called alarmin family of mediators involved in cross talk between injured cells and relatively healthy cells around damaged tissues.8 Recent studies have reported this protein as a potential biomarker of poor neurologic outcome in subarachnoid hemorrhage (SAH) patients.5 Our study tracks changes in the levels of HMGB1 in such patients during their stay in the intensive care unit and attempts to correlate them with long-term outcome. The prognostic value of HMGB1 is assessed in comparison with traditional predictors.
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Table 2. The factors associated with 3-month functional outcome
Category Number of patients Malez Age (y)* Aneurysm location Posterior inferior cerebellar arteryz Anterior cerebral arteryz Anterior communicating arteryz Basilar arteryz Posterior cerebral arteryz Aneurysmal size (mm)* Cerebral infarction due to DCI (no. of patients)z Intracerebral hemorrhage on CT (no. of patients)z Intraventricular blood on CT (no. of patients)z Fisher CT scorey Modified Fisher CT scorey Admission time (h)* World Federation of Neurological Surgeons score on admissiony Hunt and Hess score on admissiony Glasgow Coma Scale on admissiony Body temperature on admission ( C)* Days spent on intensive care unit* Plasma inflammatory markers C-reactive protein level (mg/L)* Fibrinogen level (mg/dL)* White blood cell count (106/mm3)* Plasma hemoglobin level (mg/dL)* HMGB1 level (ng/mL)y Treatment outcome at 3 months (according to Glasgow outcome scale) Low disability (score of 5)z Moderate disability (score of 4)z Severe disability (score of 3)z Persistent vegetative state (score of 2)z Death (score of 1)z
Poor outcome GOS #3
Good outcome GOS .3
6 5 (83%) 62 6 11.3
4 1 (25%) 59.8 6 14.0
1 (17%) 1 (17%) 3 (50%) 0 (0%) 1 (17%) 4.2 6 1.3 5 (83%) 3 (50%) 6 (100%) 4 (4-4) 4 (4-4) 4.7 6 1.6 4 (4-5) 4 (4-4) 9 (5-9) 37.0 6 .20 32.0 6 4.4
0 1 (25%) 1 (25%) 2 (50%) 0 (0%) 4.3 6 2.2 1 (25%) 3 (75%) 4 (100%) 4 (4-4) 3 (2-4) 6.5 6 2.4 2 (1.5-2.5) 3 (2.5-3) 13.5 (13-14.5) 36.8 6 .60 17.3 6 7.0
.349 .183 ,.01 ,.01 ,.05 .432 ,.05
89.7 6 88.0 419 6 186.4 14.6 6 5.0 11.2 6 4.2 10.0 (10-10)
129.4 6 130.4 441 6 106.0 9.26 6 2.4 11.7 6 .90 5.0 (3.1-6.3)
.577 .843 .085 .835 ,.01
P value
.190 .786 .505
.977 .190 .571
4 (100%) 0 (0%) 2 (33%) 1 (17%) 3 (50%)
Abbreviations: CT, computed tomography; DCI, delayed cerebral ischemia; HMGB1, high-mobility group box 1. *Mean 6 standard deviation if numerical data. yMedian (first quartile-third quartile) if ordinal data. zCount (percentage) if categorical data.
Materials and Methods Study Population The Bioethics Committee of Poznan University of Medical Sciences approved the protocol of this clinical study. Between December 2012 and January 2014, 95 patients with aneurysmal SAH, confirmed by computerized tomography (CT) and digital subtraction angiography, were admitted to the neurosurgical ward. The clinical condition and levels of plasma inflammatory markers (C-reactive protein, white blood cell count, and fibrinogen), together with the hemoglobin and
body temperature, were assessed on admission and systematically during hospitalization. Endovascular coiling was performed in 93 patients within 24 hours of rupture. Fourteen patients with intraventricular hemorrhage (Fisher grade 4) underwent immediate ventriculostomy because of the development of acute hydrocephalus. An intraventricular drain was inserted immediately after aneurysm embolization, based on the initial CT findings or a follow-up scan to confirm the diagnosis. Because of the suspected high sensitivity of HMGB1 to coexisting and preexisting conditions, patients with a history of CNS disease (meningitis, stroke),
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Figure 1. Bubble chart showing the correlation between HMGB1 level in CSF on admission and treatment outcome assessed by Glasgow outcome scale (GOS) at 3 months. Spearman’s exact correlation of 2.859 and P , .01.
and active systemic disease (diabetes mellitus, rheumatoid arthritis, malignancy, cirrhosis), were excluded from the study. Patients were also excluded if they developed an acute inflammatory process unassociated with the ventriculostomy. Following exclusions, there remained 10 patients forming the study group, for whom written consent was obtained. The control group consisted of 8 patients with noninflammatory conditions (tension headache, spinal disc herniation, dementia, occipital neuralgia), who underwent diagnostic lumbar puncture; exclusion criteria were the same as for the study group, but in addition, patients with elevated plasma inflammatory markers on the day of lumbar puncture were not enrolled.
End Points Subjects were followed until death or the completion of 3 months following SAH. The primary outcome was the functional state after 3 months, and the secondary outcome was in-hospital mortality. The functional outcome was defined using the Glasgow outcome scale (GOS); these were dichotomized as good (GOS 4-5) or poor (GOS 1-3) outcomes.
Imaging Studies All the patients in this study underwent endovascular treatment of the aneurysm preceded by 4-vessel digital subtraction angiography. Head CT scan was carried out
Table 3. Correlation between studied parameters and functional outcome at 3 months or HMGB1 level on admission Glasgow outcome scale at 3 months
HMGB 1 level on admission
Category
Correlation coefficient
P value
World Federation of Neurological Surgeons score on admission Hunt and Hess score on admission Glasgow Coma Scale on admission Modified Fisher CT score Body temperature Days on intensive care unit Plasma inflammatory markers C-reactive protein level White blood cell count Fibrinogen level Plasma hemoglobin level HMGB1 level on admission GOS at 3 months
2.832
.005
.893
.001
2.887 .755 2.199 2.174 2.694
.002 .016 .581 .620 .105
.931 2.895 .171 .385 .823
.001 ,.001 .636 .265 .032
2.127 2.394 2.343 .095 2.859 —
.725 .257 .373 .793 .007 —
2.041 .587 .128 2.034 — 2.859
.915 .078 .760 .931 — .007
Correlation coefficient
Abbreviations: CT, computed tomography; GOS, Glasgow outcome scale; HMGB1, high-mobility group box 1.
P value
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2000i (Sysmex, Kobe, Japan), C-reactive protein by Cobas 6000 (Roche diagnostics, Basel, Switzerland), and fibrinogen by ACL TOP 500 (Instrumentation Laboratory, Milano, Italy) as part of standard patient care in the intensive care unit. Researchers running laboratory tests were blinded to all patient details. A limitation of our study was the narrow detection range of the ELISA kit (.1-10 ng/mL); HMGB1 level was frequently above the upper limit and could only be expressed as ‘‘10 ng/mL or higher.’’
Statistical Analysis
Figure 2. Bar chart showing patients’ gradation according to modified Fisher scale. Numbers above bars represent absolute number of patients in each category.
on at least 3 occasions: on admission, after drain insertion, and after drain removal. Additional scans were performed in response to clinical deterioration (a fall of at least 2 points in Glasgow Coma Scale (GCS) lasting at least 1 hour). By definition, all the study group patients fell into Fisher grade 4 on account of the intraventricular clot. SAH grading based on the modified Fisher scale was assessed on CT scans obtained on admission. Cerebral infarction due to DCI was identified according to the criteria of Vergouwen et.al.2 All the images were assessed by a radiologist blinded to the clinical details.
Sample Collection and Assays CSF samples were collected from the endoventricular drainage system. The first sample (also designated ‘‘on admission’’) was taken within 24 hours of ventriculostomy, and within 48 hours of aneurysm rupture in all patients. Subsequent samples were taken on the fifth and 10th days with an allowance of 24 hours. At least 3 samples were taken unless the patient died before the relevant sampling. Immediately after collection, samples were centrifuged at 3000 rpm for 10 minutes and stored at 280 C until assayed. The concentration of HMGB1 in CSF was analyzed by enzyme-linked immunosorbent assay (ELISA) using commercial kit ST51011 (IBL International, Hamburg, Germany), in accordance with manufacturer’s instructions. White blood cell count and hemoglobin were analyzed by hematology analyzer XT
Statistical analysis was performed with STATISTICA 10 (Stat Soft Inc., Tulsa, OK) and StatXact (Cytel Inc., Cambridge, MA). Values are expressed as mean and standard deviations if numerical data; median and interquartile range if ordinal data; and counts and percentage if categorical data. The normality of data distribution was assessed by the Shapiro–Wilk test. Comparisons were made by using (1) Fisher exact test (2 3 2) for categorical data, (2) unpaired Student t test for unrelated normally distributed numerical data, (3) the Mann–Whitney test for unrelated non-normally distributed numerical data and unrelated ordinal data, (4) the Friedman exact test with Conover–Iman post hoc for related ordinal data, (5) the one-side Page exact test for trend seeking in related ordinal data. The correlations of HMGB1 with other parameters were assessed by Spearman’s exact correlation. Because ELISA tests’ detection range restrictions, HMGB1 level was identified as ordinal data and presented as median. A value of P less than .05 was considered statistically significant.
Results Treatment Outcome and Prognostic Markers The results for the 10 patients in the study group are summarized in Table 1. CSF from the age- and sexmatched control group (P values .712 and .676 respectively) contained only trace amounts of HMGB1 (.10 ng/mL); CSF from the SAH group showed elevated levels (10.0 ng/mL), indicating a significant difference (P , .001) between the 2 groups. The 6 patients with a poor outcome were also compared with the 4 good outcome individuals (Table 2). The former group all had HMBG1 levels greater than 10 ng/mL, whereas the latter had significantly lower levels at 5.0 ng/mL (P , .01). Moreover, HMGB1 level on admission has a strong correlation with GOS at 3 months (R 5 2.859, P , .01; Fig 1; Table 3). All the clinical grading scales that were used also showed a significant correlation with outcome: Hunt and Hess (H&H; R 5 2.887, P , .01), World Federation of Neurological Surgeons (WFNS; R 5 2.832, P , .01), and GCS (R 5 .755, P , .05). In the SAH group, we found plasma inflammatory markers to be poor
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Figure 3. Box-plot showing the HMGB1 level changes in time in study group. Friedman exact test (P 5.042) with Conover–Iman post hoc indicates significant difference between first and second samples (*P 5 .015) and nearly significant difference between first and third samples (#P 5 .055). One-side Page exact test revealed significant downtrend in HMGB1 level in time (P 5 .041).
predictors, apart from white blood cell count, which showed a difference between the good and poor outcome groups with P 5 .085. Poor markers also were body temperature (R 5 2.174, P 5 .620), hemoglobin levels (R 5 .095, P 5 .793), and the modified Fisher scale (P 5 2.199, P 5 .581; Fig 2). HMGB1 level monitoring covered the first 10-12 days following aneurysm rupture. Three samples were drawn at 5 day intervals. There was a significant difference in HMGB1 levels between the first and second samples (P , .05), and a nearly significant difference between the first and third samples (P 5 .055; Fig 3). In this study, all the poor outcome patients had a highly elevated HMGB1 level (.10 ng/mL) on admission. The levels at the time of the first sample only allowed us to prognosticate a poor outcome, but taking account of the second and third samples improved this situation. Patients who died all had a level of HMGB1 consistently above 10 ng/mL in the CSF; however, patients with an intermediate outcome (GOS 2-3 at 3 months) initially had similar very high levels, but these declined to lower levels on the second and third samples (Fig 4). The significance of this downtrend was confirmed by the one-side Page exact test (P , .05). Our interpretation of the last pattern is limited by narrow detection range of ELISA kit. Patients with a good outcome (GOS 4-5 at 3 months) all had consistently lower HMGB1 levels (,10 ng/mL), with no apparent declining or ascending trend.
Discussion Elevated HMGB1 levels in CSF were first reported as a biomarker of poor neurologic outcome by Nakahara et al9 in a series of 39 SAH patients treated by aneurysm clipping. King et al5 confirmed these promising results in a series of 9 patients. Most recently, plasma HMGB1 levels were found to be useful as predictors of functional outcome and mortality after SAH.10 Our study of 10 cases of severe SAH revealed the following: (1) CSF level of HMGB1 was a good prognosticator of outcome, being as accurate as WFNS and H&H grading scales. (2) Differing HMGB1 concentration trends, which were also predictors of outcome. Prognostic outcome factors identified in a small group of severe cases need to be interpreted with caution. Our cohort does not represent the full spectrum of patients admitted with SAH, but a narrow and severe group with intraventricular clot and hydrocephalus, which limits the range of possible conclusions.11,12 However, the validity is enhanced by the strong correlation with WFNS, H&H, and GCS in predicting clinical outcomes. This observation follows the results of some larger studies.13-15 Greer et al16 found elevated body temperature associated with a worse outcome, but we did not
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Figure 4. Box-plot showing the dynamics of HMGB1 level changes in time in detailed treatment outcome groups. Detailed outcome group ‘‘good’’ consists of patients with 5-4 GOS score at 3 months; ‘‘intermediate’’—patients with 3-2 GOS score at 3 months, ‘‘death’’—patients with 1 GOS score at 3 months. Friedman exact result does not show significant differences in HMGB1 level among first, second, and third samples in any of detailed outcome groups. One-side Page exact test revealed significant downtrend in HMGB1 level in intermediate outcome group, which means that HMGB1 level declines in time among those patients (P 5 .046).
confirm this. Plasma inflammatory markers monitored in our study—C-reactive protein and white blood cell count—failed to prognose outcomes, although some studies have shown an association between higher levels and a poorer prognosis.17,18 Fibrinogen level was elevated, and differed significantly between patients, but was an unreliable prognostic marker. In a study by Nina et al,19 fibrinogen was within normal limits and also failed to prognose outcome. Higher initial and mean hemoglobin was reported by Naidech et al20 to be associated with slightly better outcomes; however, this was not found in our study group. Previous publications also found modified Fisher scale a good predictor of outcome; we did not find this correlation in our study.21 Scheduled monitoring of HMGB1 levels during the treatment period showed differing concentration patterns which were related to the eventual outcome. Patients with an excellent outcome maintained low levels of HMGB1 (,10 ng/mL) in CSF throughout. High levels of HMGB1 (.10 ng/mL) on admission were found in patients with a poor outcome. In the latter group, when the HMGB1 levels fell to below 10 ng/mL on the second and third samples, the subjects survived, but when they remained above 10 ng/mL, they did not. There have been numerous publications regarding the role of HMGB1 in CNS disease, and taking account of
these, we can hypothesize about the potential sources of this protein during our sampling period following SAH. HMGB1 is expressed in every nucleus of normal brain cells (neurons, astrocytes, and microglia), as well as infiltrating immunological cells (macrophages, monocytes).7 Efforts to elucidate HMBG1 origin are complicated by the presence of 2 pathways for its release into the extracellular milieu. Firstly, there is passive leakage of HMGB1 because of membrane damage seen during necrosis; secondly, there is active secretion in response to various stimuli, carried out by both the immune and parenchymal cells.7 Sun et al recently demonstrated that both release pathways are present as early as 2 hours following rupture in a rat model of SAH. In contradistinction to ischemic stroke, the initial portion of HMGB1 does not predominantly originate from necrotic neurons, but is actively secreted by them. It is likely that HMGB1 leaked and secreted from neurons stimulates neighboring glial cells to produce more of the protein. This hypothesis is in agreement with increasing numbers of microglial cells secreting HMGB1 as time develops.22 The 3 target receptors for HMGB1 (TLR2, TLR4, and RAGE) are normally expressed on the brain cells.22,23 Their mutual downstream mediator, nuclear factor kappa B, not only triggers expression of proinflammatory mediators (iNOS, COX-2, ICAM-1, VCAM1, E-selectin,
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IL-1b, II-6, and TNF alpha) but also upregulates upstream receptors (TLR2, TLR4, and RAGE.).7,23-27 As a result, overexpression of the above mediators and receptors activates both damaging and repair processes following SAH.4,9,22,28
Conclusions Our study confirmed elevation of the HMGB1 protein in SAH patients. Initial levels and subsequent changes of HMGB1 in CSF correlated with neurologic outcome. Although limited by a small study group size, our findings encourage further proteomic investigation of CSF in such patients. Acknowledgment: The authors are grateful to Barbara Wie˛ckowska (Department of Computer Science and Statistics, Poznan Medical University) for her valuable statistical assistance.
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