The association between serum adhesion molecules and outcome in acute spontaneous intracerebral hemorrhage.

Abstract

Serum concentrations of adhesion molecules may be connected to the pathogenesis of secondary brain injury after spontaneous intracerebral hemorrhage (ICH). This study posits the hypothesis that levels of adhesion molecules substantially increase after ICH and are decreased thereafter, and that they can predict treatment outcomes.
Two hundred and thirty-nine blood samples were collected from 60 consecutive patients admitted within 24 hours after onset of spontaneous ICH and 60 blood samples were collected from 60 volunteers. Additional samples were obtained on Days 4, 7, 10, and 14 after onset of ICH regardless of clinical deterioration.
Upon discharge, the therapeutic outcomes of the 60 spontaneous ICH cases based on the modified Rankin Disability Scale (mMRS) showed that 17 had no disability while 8.3% developed delayed cerebral infarction (DCI). Statistical analysis of adhesion molecules between patient groups with good outcome (mMRS = 0 or 1) and poor outcome (mMRS ≥2) revealed significant differences in diabetes mellitus (P=0.049), hyperlipidemia (P=0.012), mentality change (P=0.043), ICH volume and intraventricular hemorrhage on admission (P=0.036 and 0.006, respectively), Glasgow Coma Scale (GCS) on admission (P≤0.001), neuro-surgical intervention (P=0.003), and sE-selectin and soluble intercellular cell adhesion-molecule-1 (sICAM-1) levels on admission (P=0.036 and 0.019, respectively). Multiple logistic regression analysis of these significant variables showed that GCS on admission, hyperlipidemia, and sICAM-1 (P=0.039, 0.042, and 0.022, respectively) were independently associated with outcome of acute spontaneous ICH.
Increased sICAM-1 and sE-selectin levels may imply poor therapeutic outcomes for the treatment of spontaneous ICH during hospitalization. These early inflammatory responses may cause whole-brain injury immediately after spontaneous ICH and offer a potential therapeutic target for such patients. The importance of these findings is that they offer a potential therapeutic target for patients with spontaneous ICH.

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RESEARCHOpen Access
The association between serum adhesion
molecules and outcome in acute spontaneous
intracerebral hemorrhage
Hung-Chen Wang1,2, Wei-Che Lin3, Yu-Jun Lin1,4, Cheng-Shyuan Rau1, Tsung-Han Lee1, Wen-Neng Chang5,
Nai-Wen Tsai5, Ben-Chung Cheng4,6, Chia-Te Kung7and Cheng-Hsien Lu4,5*
Abstract
Introduction: Serum concentrations of adhesion molecules may be connected to the pathogenesis of secondary
brain injury after spontaneous intracerebral hemorrhage (ICH). This study posits the hypothesis that levels of
adhesion molecules substantially increase after ICH and are decreased thereafter, and that they can predict
treatment outcomes.
Methods: Two hundred and thirty-nine blood samples were collected from 60 consecutive patients admitted
within 24 hours after onset of spontaneous ICH and 60 blood samples were collected from 60 volunteers.
Additional samples were obtained on Days 4, 7, 10, and 14 after onset of ICH regardless of clinical deterioration.
Results: Upon discharge, the therapeutic outcomes of the 60 spontaneous ICH cases based on the modified
Rankin Disability Scale (mMRS) showed that 17 had no disability while 8.3% developed delayed cerebral infarction
(DCI). Statistical analysis of adhesion molecules between patient groups with good outcome (mMRS = 0 or 1) and
poor outcome (mMRS ≥2) revealed significant differences in diabetes mellitus (P=0.049), hyperlipidemia (P=0.012),
mentality change (P=0.043), ICH volume and intraventricular hemorrhage on admission (P=0.036 and 0.006,
respectively), Glasgow Coma Scale (GCS) on admission (P≤0.001), neuro-surgical intervention (P=0.003), and sE-
selectin and soluble intercellular cell adhesion-molecule-1 (sICAM-1) levels on admission (P=0.036 and 0.019,
respectively). Multiple logistic regression analysis of these significant variables showed that GCS on admission,
hyperlipidemia, and sICAM-1 (P=0.039, 0.042, and 0.022, respectively) were independently associated with outcome
of acute spontaneous ICH.
Conclusion: Increased sICAM-1 and sE-selectin levels may imply poor therapeutic outcomes for the treatment of
spontaneous ICH during hospitalization. These early inflammatory responses may cause whole-brain injury
immediately after spontaneous ICH and offer a potential therapeutic target for such patients. The importance of
these findings is that they offer a potential therapeutic target for patients with spontaneous ICH.
Introduction
Spontaneous intracerebral hemorrhage (ICH) is a fatal
stroke subtype that accounts for 10% to 15% of all
strokes. Its morbidity and mortality rates are among the
highest, leaving individuals who survive with lasting dis-
abilities [1-3]. Although ICH is a relatively common
disease, the patho-physiologic mechanisms responsible
for the outcome are poorly understood.
The inflammatory response in ICH is characterized by
activation of local immune cells like microglial cells.
Acute inflammation is observed within 4 hours of ICH
in animal models [4] and this local inflammatory reac-
tion is partly responsible for brain damage after injury.
Specifically, blood-derived leukocytes are the primary
sources of this damage, and infiltration of systemic
immune cells results in enhanced disruption of the
blood-brain barrier (BBB), leading to increased cerebral
* Correspondence: chlu99@ms44.url.com.tw
4Department of Biological Science, 70 Lienhai Road, National Sun Yat-Sen
University, Kaohsiung, 80424, Taiwan
Full list of author information is available at the end of the article
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© 2011 Wang et al.; licensee BioMed Central Ltd.

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edema and subsequent deterioration of neuro-behavioral
function [5-7].
The injured endothelium promotes inflammation via
upregulation of adhesion molecules such as soluble
intercellular cell adhesion molecule-1 (sICAM-1), solu-
ble endothelial selectin (sE-selectin), and soluble platelet
selectin (sP-selectin) that bind to circulating leukocytes
and facilitate their migration into the cerebral ischemic
region [8]. Experimental and clinical data demonstrate
significant elevation of blood markers of endothelial
cells, platelet activation, and inflammatory cell adhesion
molecules in acute ischemic stroke [9-11]. Adhesion
molecules sICAM-1 and soluble vascular cell adhesion
molecule-1 (sVCAM-1) are pro-inflammatory para-
meters for the activation of the immune system [12].
Their physiologic role is the regulation of cell-to-cell
contacts [13]. Recruitment of activated peripheral blood
mononuclear cells across endothelial cells of the BBB
seems to be an essential step in the initiation of brain
inflammation [14]. This step of immune cell entry into
the brain tissue is regulated by adhesion molecules and
leads to a complex cascade of events [13,15]. Moreover,
adhesion molecules play a patho-physiologic role in cer-
ebrovascular diseases [11]. One study has demonstrated
that sICAM-1 and sVCAM-1 levels in cerebrospinal
fluid (CSF) and serum of patients with acute subarach-
noid hemorrhage are elevated compared with those of
healthy controls [16]. Another study has found that
highly elevated levels of sICAM-1 and sVCAM-1 in ven-
tricular CSF are associated with lethal outcome after
ICH [17]. This study investigated the relationship of
serial inflammatory cell adhesion molecules and neuro-
imaging findings and clinical outcome after acute spon-
taneous ICH.
Materials and methods
Patients
This study on the time course of serum adhesion mole-
cule levels in patients with spontaneous ICH was a post
hoc analysis of prospectively collected data and was con-
ducted over a period of 18 months (August 2009 to Jan-
uary 2011). During this period, 60 adult patients (at
least 20 years old) were admitted within 24 hours after
onset of spontaneous ICH to Kaohsiung Chang Gung
Memorial Hospital, which is a 2,482-bed acute-care
teaching hospital in southern Taiwan and which pro-
vides both primary and tertiary referral care.
The diagnosis of spontaneous ICH was confirmed by
patient history, brain computed tomography (CT) scans,
or brain magnetic resonance imaging (MRI) or a combi-
nation thereof. Patients were excluded if they had (a)
imminent death, (b) diffuse atherosclerotic changes on
intra- and extra-cranial vessels with or without evidence
of old cerebral infarct, (c) central nervous infection
during hospitalization, or (d) major systemic disease like
end-stage renal disease, liver cirrhosis, or congestive
heart failure.
All of the patients or their representatives provided
written informed consent. The study was approved by
the ethics committee of the Chang Gung Memorial
Hospital institutional review board. Patients were under
continuous observation and monitored for Glasgow
Coma Scale (GCS) score, electrocardiogram, blood pres-
sure, pulse rate, temperature, fluid balance, and labora-
tory parameters at regular intervals. Outcome was
assessed upon discharge by using the modified Rankin
Disability Scale (mMRS) [18]. Good outcome was
defined as an mMRS score of less than 1, whereas poor
outcome was an mMRS score of at least 2 or death.
Data collection
All of the patients underwent brain CT scan soon after
arrival at the emergency room as well as serial follow-up
brain CT every week during hospitalization. Emergency
brain CT scan or MRI was performed if there was clini-
cal deterioration (for example, acute-onset focal neuro-
logicdeficits, seizures,
progressively disturbed consciousness) and as post-
neuro-surgical procedures. All of the patients received
follow-up brain MRI to identify further cerebral lesions
between days 10 and 14 after ICH or before discharge.
Cerebral infarction was defined according to the criteria
of the World Health Organization [19]. The principal
investigator reviewed all available initial and follow-up
CT scans and MRIs for the presence of ischemic lesions,
and a second observer reviewed cases of equivocal find-
ings. Neither one was aware of the laboratory results at
the time of clinical and radiologic assessments.
status epilepticus,and
Blood sampling and laboratory investigations
Two hundred thirty-nine blood samples from 60
patients were taken within 24 hours after onset of ICH.
The additional samples were obtained on days 4, 7, 10,
and 14 after onset of ICH regardless of clinical dete-
rioration. Sixty samples from 60 volunteers were also
obtained. Blood samples were collected by venipuncture
into Vacutainer SST tubes (BD, Franklin Lakes, NJ,
USA). Blood was allowed to clot at room temperature
for a minimum of 30 minutes. The clot was then
removed by centrifugation at 3,000 rpm for 10 minutes
at 4°C. All serum samples were collected after centrifu-
gation, isolated, and immediately stored at −80°C in
multiple aliquots.
Serum sICAM-1, sVCAM-1, sE-selectin, soluble leuko-
cyte selectin (sL-selectin), and sP-selectin levels were
assessed with commercially available enzyme-linked
immunosorbent assays (R&D Systems, Minneapolis,
MN, USA). In the assay, standards, controls, and
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unknown samples were incubated in micro-titration
wells coated with marked (that is, anti-ICAM-1, VCAM-
1, P-selectin, E-selectin, and L-selectin) antibodies. After
incubation and washing, the wells were treated with
another anti-antigen detection antibody labeled with
enzyme horseradish peroxidase.
After a second incubation and washing step, the wells
were incubated with substrate tetramethylbenzidine. An
acidic stopping solution was then added, and the degree
of enzymatic turnover of the substrate was determined
by a dual-wavelength absorbance measurement at 450
and 620 nm. Absorbance was directly proportional to
the concentration of antigens present. A set of standard
antigen was used to plot a standard curve of absorbance
versus antigen concentration from which the antigen
concentrations of the unknowns were calculated.
Data analysis
Data were expressed as mean ± standard derivation or
as median (interquartile range, or IQR). Categorical vari-
ables were compared by using the chi-square test or
Fisher exact test, where appropriate. Serum levels of
adhesion molecules were logarithmically transformed to
improve normality and compared by Student t test.
Repeated measures of analysis of variance were used to
compare adhesion molecules at five different time points
(days 1, 4, 7, 10, and 14), and Scheffé’s multiple compar-
ison was used to analyze the intra-individual course of
parameters over time and to compare parameters of two
different groups (good and poor outcome).
Correlation analysis by Spearman ranking test was
used to explore the relationship between age, GCS score
on admission, and score on the mMRS upon discharge
and variables like soluble intercellular adhesion molecule
on admission.
Stepwise logistic regression was used to evaluate the
relationship between significant variables and therapeu-
tic outcomes, and adjustments were made for other
potential confounding factors. Variables with zero cell
count in a two-by-two table were eliminated from logis-
tic analysis, and only variables with strong association
with poor outcome (P <0.05) were included in the final
model. Lastly, receiver operating characteristic (ROC)
curves were generated for soluble adhesion molecule
levels on admission. The areas under the ROC curves
were calculated for each parameter and compared. All
of the statistical analyses were conducted with the SAS
software package, version 9.1 (2002; SAS Institute Inc.,
Cary, NC, USA).
Results
Baseline characteristics of the study patients
The baseline characteristics of the 60 adults with spon-
taneous ICH and 60 sex- and age-matched volunteers
are listed in Table 1. The 60 patients with spontaneous
ICH consisted of 42 men (age range of 36 to 75 years
and mean age of 57.0 years) and 18 women (age range
of 41 to 74 years and mean age of 59.9 years). Nineteen
(31.7%) had lobar ICH, and 41 (68.3%) had deep ICH.
Of the lobar hemorrhages, four were parietal, three
frontal, five temporal, and seven occipital. The deep
locations were the basal ganglia (26 patients) and thala-
mus (15 patients). Nineteen (31.7%) had intraventricular
hemorrhage and nine (10%) had acute hydrocephalus at
presentation. The median intracerebral hematoma was
10.0 (IQR of 5.7 to 25.8) at presentation. Treatments of
ICH included extra-ventricular drainage in five, 10 cra-
niotomy, and three craniectomy. Fifty-five (91.7%) had
one or more underlying diseases: 55 had hypertension,
10 diabetes mellitus, two asthma, seven chronic obstruc-
tive pulmonary disease, five atrial fibrillation, four cor-
onary artery disease, and 19 hyperlipidemia. Thirty-four
were smokers.
In patients with spontaneous ICH, in comparison with
the volunteer subjects, sICAM-1 and sVCAM-1 concen-
trations markedly increased at presentation (205.0 and
709.7 ng/mL, respectively) whereas sL-selectin markedly
decreased (759.4 ng/mL) (Table 1). The differences
between volunteers and spontaneous ICH patients were
statistically significant (P = 0.021, P = 0.015, and P ≤0.000,
respectively). The sP-selectin and sE-selectin concentra-
tions did not show any statistically significant difference
between the two groups at presentation: 85.1 versus 84.9
ng/mL (P = 0.841) and 39.4 versus 40.9 ng/mL (P = 0.600).
Effect of soluble intercellular adhesion molecule levels on
spontaneous intracerebral hemorrhage
Correlation analysis was used to test the influence of
soluble intercellular adhesion molecule levels at presen-
tation on age, GCS score, and ICH volume at presenta-
tion. The statistical results (correlation coefficient, P
value) revealed that age significantly correlated with
levels of sICAM-1 (r = −0.422, P = 0.003), sL-selectin (r
= −0.422, P = 0.003), and sE-selectin (r = −0.427, P =
0.002). GCS score significantly correlated with sVCAM-
1 level (r = −0.304, P = 0.034). ICH volume did not
have any significant correlation with these soluble inter-
cellular adhesion molecules, whereas GCS score and
ICH volume at presentation had a significant negative
correlation with the molecules (r = −0.564, P ≤0.001).
Sixteen (27%) of 60 patients received neuro-surgical
treatments: nine within 24 hours, four between 24 and
48 hours, and three within 4 days after ICH. When the
effect of neuro-surgical intervention on sICAM-1 and
sE-selectin levels in patients with ICH was compared,
both had increased trends on days 1 and 4. However,
only sICAM-1 level had a statistically significant differ-
ence on day 4 (P = 0.025).
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Outcome and prognostic factors
Upon discharge, the therapeutic outcomes of 60 sponta-
neous ICH cases as determined by mMRS score showed
that 17 had no disability, eight had slight disability, 10 had
moderated disability, and 25 had severe disability, includ-
ing five who became vegetative. There were no mortalities.
Five (8.3%) of 60 patients had delayed cerebral infarction
during the acute stage of spontaneous ICH: two in the
basal ganglion and one each in the frontal, temporal, and
parietal lobes. The median duration of delayed cerebral
infarction was 4 days (IQR of 1.5 to 8.0 days).
The clinical features, neuro-imaging findings, and
laboratory data between the two patient groups of good
outcome (mMRS score of 0 or 1) and poor outcome
(mMRS of at least 2) were compared, and statistical ana-
lysis revealed significant difference in diabetes mellitus
(P = 0.049), hyperlipidemia (P = 0.012), mentality
change (P = 0.043), ICH volume and intraventricular
hemorrhage on admission (P = 0.036 and 0.006, respec-
tively), GCS score on admission (P ≤0.001), neuro-surgi-
cal intervention (P = 0.003), and sE-selectin and
sICAM-1 levels on admission (P = 0.036 and 0.019,
respectively). The median lengths of hospitalization were
15 days (IQR of 13 to 23 days) and 11 days (IQR of 7 to
13 days) for those with poor and good outcome, respec-
tively (P ≤0.001) (Table 2).
Time course of soluble intercellular adhesion molecule
levels
Compared with volunteers, plasma sICAM-1 and
sVCAM-1 levels were elevated significantly after onset
of ICH and peaked on days 10 (sICAM-1) and 4
(sVCAM-1) and then slightly decreased thereafter (Fig-
ure 1a,b). Plasma sL-selectin levels decreased signifi-
cantly after ICH onset and reached the lowest level on
day 4 and then increased thereafter (Figure 1d). Plasma
sP-selectin levels decreased after onset of ICH, then
increased gradually thereafter, and had a significant dif-
ference on day 14 only (Figure 1c). With the volunteers,
there was no significant difference in plasma sE-selectin
levels, which increased after ICH onset, peaked on day
4, and decreased thereafter (Figure 1e).
When the therapeutic outcomes in the 60 sponta-
neous ICH cases as determined by mMRS score were
Table 1 Demographic data of patients and volunteer subjects at admission
Parameter Volunteer subjects
(n = 60)
Patients
(n = 60)
P value
Age in years, mean ± standard deviation
Men
Underlying diseases
Hypertension
Diabetes mellitus
Asthma
Chronic obstructive pulmonary disease
Atrial fibrillation
Coronary artery disease
Hyperlipidermia
Smoking
Laboratory data at presentation, median (IQR)
White blood cell count, × 103/mL
Platelet count, × 103/mL
Brain image findings at presentation
Intracerebral hemorrhage volume in cubic millimeters, median (IQR)
Intraventricular hematoma
Hydrocephalus
Craniotomy
Serum adhesion molecule levels in nanograms per milliliter at presentation, median (IQR)
sL-selectin
sP-selectin
sE-selectin
sICAM-1
sVCAM-1
57.5 ± 5.7
42
57.9 ± 10.1
42
0.790
1.000
19
9
3
7
10
1
8
17
55
10
2
7
5
4
19
34
≤0.001a
0.465
1.000
0.778
0.419
0.171
0.004a
0.000a
5.6 (4.85, 7.80)
245.0 (202.0, 306.5)
8.45 (6.13, 10.53)
206.0 (168.3, 355.8)
0.001a
0.019a
-
-
-
-
10.0 (5.7, 25.8)
19
9
16
-
-
-
-
970.5 (844.8, 1045.1)
85.1 (78.4, 92.5)
39.4 (31.9, 44.6)
183.7 (155.0, 202.2)
613.5 (527.4, 674.4)
759.4 (649.7, 880.4)
84.9 (59.2, 97.8)
40.9 (30.5, 54.1)
205.0 (159.8, 240.0)
709.7 (549.5, 934.1)
≤0.001a
0.841
0.600
0.021a
0.015a
Values in the ‘Volunteer subjects’ and ‘Patients’ columns are presented as number of patients unless indicated otherwise.ap≦0.05; IQR, interquartile range; sE-
selectin, soluble endothelial selectin; sICAM-1, soluble intercellular cell adhesion molecule-1; sL-selectin, soluble leukocyte selectin; sP-selectin, soluble platelet
selectin; sVCAM-1, soluble vascular cell adhesion molecule-1.
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compared, the sICAM-1 and sE-selectin concentration
levels increased significantly from days 1 to 4 in
patients with poor outcome (Figure 2a,e). Otherwise,
the levels of sVCAM-1, sP-selectin, and sL-selectin
were not significantly different during the first 14 days
(Figure 2b,c,d).
Multiple logistic regression analysis of significant vari-
ables, including diabetes mellitus (P = 0.049), hyperlipi-
demia (P = 0.012), mentality change (P = 0.043), ICH
volume and intraventricular hemorrhage on admission
(P = 0.036 and 0.006, respectively), GCS score on admis-
sion (P ≤0.001), neuro-surgical intervention (P = 0.003),
Table 2 Prognostic factors of patients with spontaneous intracerebral hemorrhage
Poor outcome
n = 43
Good outcome
n = 17
Odds
ratio
P value
Age in years, mean ± standard deviation
Men
Underlying diseases
Hypertension
Diabetes mellitus
Asthma
Chronic obstructive pulmonary disease
Atrial fibrillation
Coronary artery disease
Hyperlipidermia
Smoking
Clinical feature at presentation
Headache
Brief unconsciousness
Mentality change
Seizure
Laboratory data at presentation, median (IQR)
White blood cell count, × 103/mL
Platelet count, × 103/mL
3111 1.4090.755
40
10
2
5
3
3
9
25
15
0
0
2
2
1
10
9
1.778 0.616
0.049a
1.000
1.000
0.616
1.000
0.012a
0.777
0.987
0.563
1.200
0.185
1.235
19
26
24
2
71.131
0.328
4.105
1.000
0.136
0.043a
1.000
14
4
0
8.7 (6.8, 10.6)
206.0 (160.0,
259.0)
7.2 (5.7, 10.0)
205.0 (171.5,
218.5)
0.144
0.512
Brain image findings at presentation
Intracerebral hemorrhage volume in cubic millimeters, median (IQR)
Intraventricular hemorrhage
Hydrocephalus
Glasgow Coma Scale score at presentation, median (IQR)
Surgical intervention
Extraventricular drainage
Craniotomy
Craniectomy
Serum adhesion molecule levels in nanograms per milliliter at presentation, median
(IQR)
sL-selectin
12.0 (7.0, 34.0)
18
9
14 (9, 15)
16
5
10
3
7.2 (2.0, 17.5)
1
0
15 (15, 15)
0
0
0
0
0.036a
0.006a
0.050
≤0.001a
0.003a
0.309
0.049a
0.511
11.52
771.5 (638.9,
922.4)
85.7 (58.9, 97.9)
43.3 (33.4, 63.6)
208.2 (174.4,
259.3)
709.7 (559.1,
938.8)
4
5 (2.8, 8.3)
15 (12.8, 22.3)
675.6 (641.5,
783.2)
80.3 (60.2, 93.8)
32.8 (21.3, 45.7)
170.3 (100.2,
228.3)
653.9 (502.0,
947.0)
1
2 (1.5,3.5)
11 (7, 13)
0.111
sP-selectin
sE-selectin
sICAM-1
0.3664
0.036a
0.019a
sVCAM-1 0.353
Delayed cerebral infarction
Days of intensive care unit stay, median (IQR)
Days of hospitalization, median (IQR)
0.6091.000
≤0.001a
≤0.001a
Values in the ‘Poor outcome’ and ‘Good outcome’ columns are presented as number of patients unless indicated otherwise. The following tests were used:
Student t test (for age), Mann-Whitney U test (for laboratory data and soluble intercellular adhesion molecule at admission), and Fisher exact test (for gender and
underlying diseases).ap≦0.05; IQR, interquartile range; sE-selectin, soluble endothelial selectin; sICAM-1, soluble intercellular cell adhesion molecule-1; sL-selectin,
soluble leukocyte selectin; sP-selectin, soluble platelet selectin; sVCAM-1, soluble vascular cell adhesion molecule-1.
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