High inflammation level is essential to cerebral microbleed in the prediction of large artery atherosclerotic outcomes

doi:10.21203/rs.3.rs-2294758/v1

Background and Purpose: Cerebral small vessel damages and large artery atherosclerotic (LAA) stroke share many risk factors, such as inflammation, but little attention has been paid to the relationship between cerebral small vessel damages with inflammation in predicting large artery atherosclerotic stroke outcomes.

Methods: We consecutively enrolled participants with LAA stroke and collected their baseline and follow-up data, especially the imaging markers of cerebral small vessel disease and C reactive protein (CRP), and neutrophil-to-lymphocyte ratio (NLR) levels. The primary outcomes were LAA stroke outcomes at 3-month and 1-year, the secondary outcomes poststroke hemorrhagic transformation, infarction recurrence, and mortality.

Results: 864 participants were included to the final analysis. We found that only cerebral microbleed (CMB) with numbers greater than 5 can predict 3-month (p < 0.001) and 1-year (p < 0.01) outcomes in LAA stroke, furthermore when CMB is more than 5 plus NLR or CRP more than 3, the prediction in primary outcomes is significant (all p < 0.05), but not in secondary outcomes.

Conclusions: Our multi-centered cohort study suggests that CMB counts > 5 were not associated with the prognosis of LAA stroke, and CMB counts > 5 with high levels of inflammation (NLR or CRP > 3) were associated with a poor prognosis of LAA stroke.

cerebral small vessel disease

neutrophil-to-lymphocyte ratio

enlarged perivascular space

cerebral microbleed

white matter hyperintensity

Large artery atherosclerotic (LAA) stroke is caused by atherosclerosis of the major intra- or extracranial arteries(1, 2). Diabetes mellitus, hypertension, smoking, hyperlipidemia, and inflammation are the common major modifiable risk factors associated with both atherosclerotic disease(1, 3) and cerebral small vessel disease (CSVD)(4). CSVD describes a syndrome of neuroimaging, pathological, and associated clinical features caused by small intracranial vascular lesions, which commonly coexists with large artery atherosclerosis(5). However, the prognosis of participants with LAA stroke with CSVD is now inadequate and needs further investigation. Inflammation plays an important role in the pathogenesis of stroke and CSVD(6–8) and predicts the 30-day adverse outcomes of participants with cerebral infarction(9). The neutrophil-to-lymphocyte ratio (NLR), derived from the absolute neutrophil and absolute lymphocyte counts of a full blood count, can be a representative marker of systemic inflammation in conditions such as ischemic or hemorrhagic stroke(10–12), and various cancers(13).

Cerebral microbleed (CMB), white matter hyperintensity (WMH), enlargement of medullary veins, and enlarged perivascular space (EPVS) are imaging indicators of CSVD and common concomitants of stroke(14), which can predict the risk of hemorrhagic transformation of stroke(15, 16), atherosclerosis(17, 18) and cognitive impairment(17, 19, 20). Plus, brain atrophy (BA) is related to small vessel disease(21) as well as the progression and outcomes of degenerative diseases such as Alzheimer’s disease(22) and multiple sclerosis(23), which can be a marker of CSVD.

We aim to investigate whether different CSVD imaging markers (namely CMB, WMH, EPVS, lacunar stroke, medullary veins enlargement, and BA) can predict the LAA stroke outcomes and whether CSVD with high inflammation can predict the LAA stroke outcomes or not.

Study participants

Consecutive participants with acute ischemic stroke (AIS) were screened and recruited from Huashan Hospital, Fudan University and Shanghai Fifth People’s Hospital, Fudan University between 1st November 2013 and 31st December 2019. All recruited participants were followed up in the outpatient to assess their mortality, intracranial bleeding rate, extracranial bleeding rate, National Institute of Health stroke scale (NIHSS), and modified Rankin scale (mRS) scores at 3 months and 1 year or more. Participants at admission were included if they met the following criteria: (1) diagnosed with AIS within 24 hours of onset; (2) aged 18 ~ 80; (3) completed a head magnetic resonance imaging (MRI) within 48 hours; (4) completed a neck and head computed tomographic angiography showing responsible atherosclerotic stenosis > 50%. AIS was diagnosed if there were new focal neurological deficits explained by relevant lesions detected on head diffusion-weighted imaging (DWI). Participants were excluded if they met any of the following criteria: (1) presence of cerebral hemorrhage or a history of AIS; (2) pregnant participants; (3) severe heart (with cardiac function in grade 3 or 4 according to New York Heart Association or left ventricular ejection fraction < 40% in echocardiography), lung (with blood oxygen saturation less than 95% and symptoms of shortness of breath, cyanosis, and abnormal blood gas analysis), liver (serum alanine aminotransferase level > 10-fold upper limit of the reference range), kidney (serum creatinine > 443µmol/L), or neoplastic diseases; (4) autoimmune diseases; (5) symptoms of infection were present at the time of stroke onset. The flow chart of participants’ selection was shown in Fig. 1. This study was approved by the Ethical Review Board of Shanghai Fifth People’s Hospital, and Huashan Hospital, and written informed consents were obtained from all participants or their families. Baseline data, including age, sex, history of hypertension, diabetes mellitus (DM), atrial fibrillation (AF), dyslipidemia, NIHSS scores at admission, systolic blood pressure (SBP), history of cigarette smoking, and alcohol drinking, were collected from medical records or face-to-face interviews.

Laboratory tests

All blood samples were collected with vacuum tubes, stored at 4 ℃, and tested by clinical laboratory technicians within 2 h after collection on admission day. The white blood cell count (WBC), neutrophil proportion (N), lymphocyte proportion (L), platelet count (P), levels of serum total bilirubin, serum glucose, C reactive protein (CRP), homocysteine (HCY), low-density lipoprotein (LDL) and partial thromboplastin time (PT), activated partial thromboplastin time (APTT), international normalized ratio (INR), lactate dehydrogenase (LDH) were measured. The neutrophil-to-lymphocyte ratio (NLR) was calculated as the ratio of the neutrophil counts to the lymphocyte counts (N/L).

Clinical assessments

Each enrolled patient underwent a head 3T MRI (Siemens, Forchheim, Germany) scan within 48 hours after AIS onset. Short-term outcomes of AIS was quantified by 3-month NIHSS scores and 3-month mRS scores as well as long-term outcomes were quantified by 1-year mRS scores via the telephone assessment. And secondary outcomes were 3-month mortality, intracranial bleeding, and extracranial bleeding (such as gastric bleeding, urinary tract bleeding, gum bleeding, etc.) and Barthel index. Short-term and long-term outcomes were divided into 2 levels: favorable outcome (mRS < 3 or NIHSS < = 5), and unfavorable outcome (mRS > = 3, = 6 means death or NIHSS > 5).

Lacune is graded according to the number of foci and is classified into 4 levels, that is, levels 0–3. Level 0 represents no foci; level 1 is up to 3 foci; level 2, 4–10 foci; and level 3, greater than 10 foci. The CMB is defined as the presence of a circular-like low signal in the susceptibility-weighted imaging (SWI) sequence, excluding vascular artifacts and was divided into 4 grades(24), and the BS is defined as the presence of a linear low signal in the SWI, common in the center of the centrum semiovale(16, 25). WMH, with high signals being seen in the lateral periventricular and centrum semiovale in the fluid-attenuated inversion recovery (FLAIR) sequence, can be assessed by Fazekas score(26). BA can be assessed by a 39-point score: this scale divides the cerebral atrophy into sulcus dilatation (bilateral frontal, parietal, and temporal lobes) and ventricular dilatation (bilateral temporal, parietal, occipital, and third ventricles), and each specific area can be scored 0–3 according to the degree of atrophy(27). EPVS severity is divided into 0–4 classes according to the number of EPVS in the T2WI sequence(28).

The total MRI score(29) includes 4 abovementioned MRI markers of CSVD, i.e. lacune, WMH, CMB, and EPVS; the presence of each of these markers was awarded 1 point, producing a minimum score of 0 and a maximum score of 4, creating an ordinal scale with increasing manifestations of CSVD on brain MRI, representing an increasing burden of CSVD. See Supplemental materials Figure S1.

Statistical analysis

SPSS 26.0 (IBM Corp., Armonk, NY) and GraphPad Prism 8 (GraphPad Software Inc., La Jolla, CA) was used to perform statistical analysis. Categorical variables were presented as frequency and percentage, and Pearson Chi-square test or Fisher’s exact test were used to comparing categorical variables. For continuous variables, the Kolmogorov–Smirnov test was used to determine the data distribution. If the data demonstrate normal distribution, they are presented with mean ± SD and the Student t-test is used for comparison. For data without normal distribution, median and interquartile ranges are shown and the Mann–Whitney U test is used for comparison. Significance level is set at α = 0.05 (two-tailed). Parameters showing a statistical trend (p < 0.1) or being proved a significant association in previous studies are included in a logistic model to identify parameters independently associated with the functional outcome. The Hosmer–Lemeshow test is used to test the goodness of fit. The receiver operating characteristic (ROC) curve and Youden’s J statistic is used to determine the cutoff value of NLR or CRP. Then, participants are dichotomized according to the identified cutoff value of NLR, and 3-month mRS, 1-year mRS scores, and other outcomes of different groups divided by the cutoff value of NLR are tested using the Chi-square test and the z test. Data analysis was completed between May 2020 and August 2020.

Data Availability

The datasets used and/or analyzed in this study are available from the corresponding author on reasonable requests.

Characteristics of the participants

Table 2 compares baseline data for enrolled participants (n = 1434) according to total scores groups (total scores < 2 and > = 2). First of all, the NIHSS scores on admission (p = 0.661) between the 2 groups are not statistically different. We found that the prevalence of DM (p = 0.049), prevalence of AF (p = 0.0001), gender (p = 0.002), and smoking history (p = 0.021) are statistically different. Table 1 compares baseline data of the participants according to low NLR (< 3) and high NLR ( > = 3) groups, we found that most of the demographic data and laboratory data are not statistically significant, except for smoking history (p = 0.04), systolic blood pressure (p = 0.004), blood glucose (p = 0.003), white blood cell counts (p = 0.002), and LDH (p = 0.0001), and the imaging markers scale scores such as CMB grades, BS, WMH, BA, and EPVS were not statistically significant.

Table 1

Characteristics of the study population
	Low total score (n = 1140)	High total score (n = 294)	p Value
Demographic data
Male	786 (70)	240 (81)	0.002^*
Age ^‡	64 (57–72)	67 (61–76)	0.059
Smoker	246 (22)	90 (31)	0.021^*
Alcohol drinker	90 (8)	30 (10)	0.367
Medical history
Arterial hypertension	804 (71)	204 (69)	0.788
Diabetes mellitus	474 (42)	96 (33)	0.049^*
Atrial fibrillation	96 (8)	72 (25)	0.0001^*
Clinical characteristics
Admission NIHSS scores	6 (3–9)	6 (3–11)	0.661
Systolic BP, mmHg^‡	149 (135–164)	151 (136–170)	0.438
Serum glucose, mg/dl^‡	5.7 (5-7.1)	5.6 (5.1–7.1)	0.840
Laboratory data
CRP, mg/L	6 (3–10)	7 (3–11)	0.431
PT, /s^‡	11.5 (11–12)	10.7 (10.2–11.2)	0.330
APTT, /s^‡	27.9 (25–31)	27 (24–30)	0.298
INR^‡	0.99 (0.95–1.05)	0.99 (0.95–1.11)	0.591
WBC, 10¹²/L^‡	6.9 (5.6–8.3)	6.6 (5.2–7.7)	0.295
Platelet, 10⁹/L^‡	208 (174–245)	194 (158–231)	0.185
Bilirubin, µmol/L^‡	10.2 (7.7–15)	12 (8–16)	0.120
LDH, U/L^‡	177 (159–206)	156 (175–205)	0.887
Abbreviations: NIHSS = National institutes of health stroke scale; BP = blood pressure; CRP = C reactive protein; PT = partial thromboplastin time; APTT = activated partial thromboplastin time; INR = international normalized ratio; NLR = neutrophil-to-lymphocyte ratio; LDH = lactate dehydrogenase; CMB = cerebral microbleed; EPVS = enlarged perivascular spaces.
Unless specified, values are numbers of patients (%).
^‡Median (interquartile range).
^*Statistically significant.

Table 2

Characteristics of the study population
	Low NLR (n = 840)	High NLR (n = 594)	p Value
Demographic data
Male	564 (67)	420 (71)	0.482
Age ^‡	63 (57–72)	65 (60–74)	0.082
Smoker	198 (24)	54 (9)	0.040^*
Alcohol drinker	36 (4)	30 (5)	0.768
Medical history
Arterial hypertension	546 (65)	438 (74)	0.120
Diabetes mellitus	306 (36)	258 (43)	0.247
Atrial fibrillation	78 (9)	90 (15)	0.156
Clinical characteristics
Admission NIHSS scores	6 (5–11)	6 (4–11)	0.382
Systolic BP, mmHg^‡	146 (132–160)	153 (140–170)	0.004^*
Serum glucose, mg/dl^‡	5.5 (5-6.6)	6.1 (5.2-8)	0.003^*
Laboratory data
PT, /s^‡	11.3 (10.7–11.9)	11.7 (11.2–12.4)	0.009^*
APTT, /s^‡	26.7 (25–31)	28.5 (26–31)	0.040^*
INR	0.98 (0.95–1.04)	0.99 (0.94–1.06)	0.454
WBC, 10¹²/L^‡	6.7 (3–8)	7.7 (2.6–9.1)	0.002^*
Platelet, 10⁹/L^‡	207 (174–245)	192 (159–238)	0.283
Bilirubin, µmol/L^‡	11.2 (8.3–15.0)	10.5 (7.5–14.8)	0.382
LDH, U/L^‡	169 (155–196)	190 (168–222)	0.0001^*
Cerebral microbleed
CMB grade	1 (0–2)	1 (0–2)	0.414
Brush sign	270 (32)	180 (30)	0.802
Fazekas scores	2 (2–2)	2 (1–2)	0.366
Brain atrophy	7 (2–13)	5 (5–13)	0.118
EPVS	1(1–2)	1(1–2)	0.557
Abbreviations: NIHSS = National institutes of health stroke scale; BP = blood pressure; PT = partial thromboplastin time; APTT = activated partial thromboplastin time; INR = international normalized ratio; NLR = neutrophil-to-lymphocyte ratio; LDH = lactate dehydrogenase; CMB = cerebral microbleed; EPVS = enlarged perivascular spaces.
Unless specified, values are numbers of patients (%).
^‡Median (interquartile range).
^*Statistically significant.

CMB was related to stroke outcomes

We continued to compare the 3-month secondary outcomes between total score groups, CMB groups, and NLR groups, respectively (Table 4). We found statistical significances for mortality and Barthel index in both CMB groups and NLR groups; and in comparison with poststroke functional scores, the 3-month mRS (p < 0.001), and 1-year mRS (p < 0.01) were statistically significant in CMB groups (Fig. 2), while no statistical significance was found for BA, WMH, EPVS, BS and lacune groups (Fig. 2, Table 5).

Table 3

Characteristics of the study population
	Mild CMB (n = 716)	Severe CMB (n = 718)	p Value
Demographic data
Male	428 (60)	570 (80)	0.010^*
Age ^‡	66 (60–72)	63 (56–73)	0.136
Smoker	142 (20)	90 (9)	0.541
Alcohol drinker	52 (7)	52 (5)	0.382
Medical history
Arterial hypertension	468 (64)	488 (69)	0.719
Diabetes mellitus	520 (36)	438 (43)	0.733
Atrial fibrillation	132 (9)	152 (15)	0.353
Clinical characteristics
Admission NIHSS scores	6 (5–11)	6 (5–11)	0.850
Systolic BP, mmHg^‡	150 (135–165)	140 (135–159)	0.349
Serum glucose, mg/dl^‡	5.8 (5.1–7.9)	5 (4.2–5.7)	0.633
Laboratory data
PT, /s^‡	11.5 (11–12)	11.7 (10.9–12.3)	0.888
APTT, /s^‡	27 (25–31)	26 (23–31)	0.308
INR	0.99 (0.96–1.04)	1.04 (0.96–1.11)	0.111
WBC, 10¹²/L^‡	7.5 (6–9)	6.7 (5.6-8)	0.109
Platelet, 10⁹/L^‡	205 (177–251)	202 (163–240)	0.345
Bilirubin, µmol/L^‡	10.6 (7.6–15.6)	9.7 (7.7–12.8)	0.724
LDH, U/L^‡	195 (166–217)	177 (163–217)	0.564
Abbreviations: NIHSS = National institutes of health stroke scale; BP = blood pressure; PT = partial thromboplastin time; APTT = activated partial thromboplastin time; INR = international normalized ratio; NLR = neutrophil-to-lymphocyte ratio; LDH = lactate dehydrogenase; CMB = cerebral microbleed.
Unless specified, values are numbers of patients (%).
^‡Median (interquartile range).
^*Statistically significant.

Table 4

Comparisons of poststroke outcomes between different groups
	Mortality	Intracranial bleed	Extracranial bleed	Barthel index
Low total score (n = 1140)	0.531	0.677	0.567	0.457
High total score (n = 294)	-	-	-	-
Mild CMB (n = 716)	0.031^*	0.012^*	0.654	0.040^*
Severe CMB (n = 718)	-	-	-	-
Low NLR (n = 840)	0.046^*	0.745	0.765	0.010^*
High NLR (n = 594)	-	-	-	-
Abbreviations: NLR = neutrophil-to-lymphocyte ratio; CMB = cerebral microbleed.
^*Statistically significant.

Table 5

Comparisons between WMH, EPVS, BS, lacune grades in patients with NIHSS > 3
	WMH(Fazekas 0)	WMH(Fazekas 1–6)	p values
3-month mRS	4(2–4)	2(1–4)	0.179
1-year mRS	2(1–4)	2(2–6)	0.672
	WMH(Fazekas 0–2)	WMH(Fazekas 3–6)	p values
3-month mRS	3(2–4)	2(1–4)	0.353
1-year mRS	2(0–2)	2(1–5)	0.058
	WMH(Fazekas 0–4)	WMH(Fazekas 5–6)	p values
3-month mRS	3(2–4)	3(0–5)	0.856
1-year mRS	2(0–3)	2(2–6)	0.604
	EPVS(= 0)	EPVS( > = 1)	p values
3-month mRS	2(1–4)	3(1–4)	0.325
1-year mRS	1.5(1–2)	2(1–4)	0.308
	EPVS(0–2)	EPVS( > = 2)	p values
3-month mRS	3(1–4)	2(1–4)	0.620
1-year mRS	2(1–3)	2(1–6)	0.369
	BS = 0	BS = 1	p values
3-month mRS	3(1–4)	2(1–4)	0.272
1-year mRS	2(1–4)	2(1–4)	0.977
CMB(> 0)	+BS = 0	+BS = 1	p values
3-month mRS	2(1–4)	3(1–4)	0.329
1-year mRS	2(1–5)	2(1–4)	0.715
CMB(> 5)	+BS = 0	+BS = 1	p values
3-month mRS	2(1–4)	1(1–2)	0.385
1-year mRS	2(0–4)	2(1–2)	0.639
	Lacune(= 0)	Lacune( > = 1)	p values
3-month mRS	3(1–4)	2(1–4)	0.380
1-year mRS	2(1–2)	2(1–4)	0.145
	Lacune(0–2)	Lacune( > = 2)	p values
3-month mRS	3(1–4)	2(1–4)	0.814
1-year mRS	2(1–3)	2(1–4)	0.671
	Lacune(0–3)	Lacune( > = 3)	p values
3-month mRS	2.5(1–4)	3(1–4)	0.687
1-year mRS	2(1–4)	1(1–4)	0.197
Abbreviations: NIHSS = National institutes of health stroke scale; WMH = white matter hypertensity; EPVS = enlarged perivascular spaces; CMB = cerebral microbleed; BS = brush sign; mRS = modified Rankin scale. Unless specified, values are median (interquartile range). ^*Statistically significant.

Table 3 compares the baseline data of 2 groups based on CMB grades (mild CMB group or CMB grade < = 1 or CMB number < 5, severe CMB group or CMB grade > 1 or CMB number > = 5), we found that the gender (p = 0.010) is statistically different. Most of the data showed good homogeneities in the 2 groups.

WMH, BA, EPVS, BS was not related to stroke outcomes

We continued to compare the 3-month functional outcomes between WMH, BA, EPVS, and BS groups, respectively (Table 5, Fig. 2). For each imaging marker, we grouped them according to commonly used rating criteria (see Materials and Methods part) and then compared the 3-month and long-term mRS, none of which were found to be associated with the prognosis of stroke. As for BS that was regarded as another sign of microbleed, we combined the CMB grades and the existence of BS (Table 5) and found no significant differences.

ROC curve for NLR and CRP in the prediction of stroke outcomes

ROC analysis identified 3 as the best cutoff value of NLR to discriminate favorable and unfavorable functional outcomes at 3 months (area under the curve-AUC = 0.75, 95%CI: 0.64–0.83; p < 0.0001; Youden’s index = 0.44; sensitivity: 82%, specificity: 62%; Fig. 3). This cutoff value equals to median of the NLR in our population. And also ROC analysis identified 3 as the best cutoff value of CRP to discriminate favorable and unfavorable functional outcomes at 3 months (area under the curve-AUC = 0.83, 95%CI: 0.76–0.89; p < 0.0001; Youden’s index = 0.47; sensitivity: 71%, specificity: 76%; Fig. 3).

High NLR or CRP is essential to CMB in the prediction of LAA stroke outcomes(Table 6)

Table 6

Comparisons of poststroke outcomes in NLR or CRP subgroups between small vessel damage markers
CMB grades	mRS	Low NLR	High NLR	p
CMB grade 2
	3-month	1 (1–4)	3 (2–6)	0.02^*
	1-year	1 (1–3)	3 (2–6)	0.04^*
CMB grade 3
	3-month	2 (1–3)	3 (2–5)	0.01^*
	1-year	2 (1–3)	3 (2–5)	0.02^*
		Low CRP	High CRP	p
CMB grade 2
	3-month	1 (1–4)	3 (2–6)	0.03^*
	1-year	1 (1–2)	3 (2–6)	0.01^*
CMB grade 3
	3-month	2 (1–3)	3 (2–5)	0.03^*
	1-year	2 (1–3)	3 (2–5)	0.02^*
		CMB < 5	CMB > = 5	p
Low NLR
	3-month	1 (1–2)	2 (2–3)	0.161
	1-year	2 (1–3)	2 (1–3)	0.738
Low CRP
	3-month	1 (1–2)	2 (1–3)	0.261
	1-year	2 (1–3)	2 (1–3)	0.348
Abbreviations: NLR = neutrophil-to-lymphocyte ratio; CMB = cerebral microbleed; CRP = C reactive protein; mRS = modified Rankin scale. ^*Statistically significant.

Table 7. Multivariable regression analysis for 3-month favorable outcome

	Beta	SE	Wald	p	OR, 95% CI
Age, y	-0.018	0.033	0.302	0.583	0.98, 0.92–1.04
Gender	0.074	0.649	0.013	0.909	1.08, 0.30–3.83
HTN history	1.064	0.650	2.679	0.102	2.89, 0.81–10.36
DM history	-1.089	0.824	4.822	0.028^*	0.16, 0.03–0.82
Smoking	-1.066	0.879	1.47	0.225	0.34, 0.06–1.93
Glucose, mmol/L	0.225	0.145	2.387	0.122	1.25, 0.94–1.66
LDH, U/L	0.000	0.009	0.001	0.973	1.00 0.99–1.01
CMB grade	-1.087	0.322	11.235	0.001^*	0.34, 0.18–0.64
SBP, mmHg	0.012	0.016	0.555	0.456	1.01, 0.98–1.04
NLR rank	1.096	0.770	2.028	0.154	2.99, 0.66–13.53
Constant	-2.916	3.195	0.833	0.362	0.054, -
Abbreviations: SE = standard error; OR = odd ratio; CI = confidence interval; HTN = arterial hypertension; DM = diabetes mellitus; LDH = lactate dehydrogenase; CMB = cerebral microbleed; SBP = systolic blood pressure; NLR = neutrophil-to-lymphocyte ratio.
^*Statistically significant.

CMB was grouped according to CMB grades (grade 1, 2 and 3) and each group was found to have statistically significant differences in functional outcomes at 3 months (grade 1: p = 0.002; grade 2: p = 0.02; grade 3: p = 0.01) and 1 year (grade 1: p = 0.03; grade 2: p = 0.04; grade 3: p = 0.02) when grouped according to the cutoff value of NLR, 3. Also, in the low NLR group, we divided the participants according to CMB grades and found no significant differences in 3-month and 1-year outcomes (p = 0.161; p = 0.738). Also, CMB was grouped according to CMB grades (grade 1, 2, and 3) and each group was found to have statistically significant differences in functional outcomes at 3 months (grade 1: p = 0.01; grade 2: p = 0.03; grade 3: p = 0.03) and 1 year (grade 1: p = 0.03; grade 2: p = 0.01; grade 3: p = 0.02) when grouped according to the cutoff value of CRP, 3. Also, in the low CRP group, we divided the participants according to CMB grades and found no significant differences in 3-month and 1-year outcomes (p = 0.261; p = 0.348).

Logistic regression found that CMB is an independent predictor of stroke outcomes

After comparing the potential factors such as age, gender, hypertension, diabetes mellitus, smoking, levels of SBP and glucose, and NLR, etc. (Table 7), we found that CMB could independently predict functional outcomes at 3 months (OR = 0.34, 95%CI, 0.18–0.64, p = 0.001). DM history was also found to be an independent risk factor of functional outcomes at 3 months (OR = 0.16, 95%CI, 0.03–0.82, p = 0.028). The other factors did not show such predicting power.

Our multi-centered prospective study suggests that cerebral microbleed numbers greater than 5 are an independent predictor of short- or long-term functional outcomes. Furthermore, we found that only with high levels of inflammation, CMB numbers greater than 5 could predict the post-stroke outcomes.

There are many studies on large artery atherosclerosis (LAA) stroke and CSVD markers. The current consensus is that CSVD and LAA share the same risk factors(5, 30), such as diabetes and hypertension. Some studies also suggest that CSVD markers, such as WMH, participate in the pathophysiology of stroke and cause stroke recurrence(5) and poststroke cognitive decline(31); atherosclerosis of the large arteries is also involved in the pathogenesis of cerebral small vessel damage, such as CMB: chronic cerebral hypoperfusion due to chronic narrowing of the middle cerebral or basilar arteries damages the blood-brain barrier and causes microbleeds(5). However, there are few studies on the long-term and short-term prognosis of participants with LAA and CSVD markers, and the contribution of CSVD to LAA is rarely addressed.

The current study still found that total score was associated with stroke recurrence and cognitive decline(32), our study on cerebral small vessel damage markers in LAA stroke participants found that total score grade was not associated with 3-month mortality, intracranial hemorrhage, extracranial hemorrhage, and BI, as well as 3-month and 1-year outcomes in stroke participants. Also, we examined the relationships between 6 CSVD markers and the outcomes of LAA stroke one by one, WMH, BS, EPVS, BA, or lacune had no impact on stroke outcomes. Although the good association between WMH, EPVS, and lacune and LAA stroke has been reported(5), our data showed no association between these markers of CSVD and stroke outcomes. But we found a specific correlation between CMB and functional outcomes of stroke. A pooled analysis showed that CMB is associated with hemorrhage and not with the recurrence of stroke(14), but our data suggest that the presence or absence of CMB is not associated with functional outcomes of cerebral infarction, but the presence of more than 5 CMBs is associated with short-term and long-term functional stroke outcomes, which is noteworthy and seems to indicate that CMB numbers greater than 5 starts to affect large vessel lesions. Furthermore, CMB pathology detected by MRI(33) or postmortem studies(34) includes arteriolar, capillary, or venous damage, and BS represents medullary vein damage(35, 36); in our study, we found that no association between BS and poststroke outcomes, and this finding suggests that arteriolar rather than venous damages are associated with poor outcomes in the large artery disease. This is the first demonstration that CMB especially arteriolar damage is an independent predictor of short-term and long-term outcomes in LAA stroke participants.

CMB and other imaging markers can be considered as the inflammation of the vessel wall or endothelial membrane(37–39), and CMB represents small vessel damages(40); this is also similar to the current ideas(41–43). Because of the close relationship between inflammation and small vessel injury(8), we also investigated the relationship between inflammation and CMB and found that NLR (> 3) or CRP (> 3) played a predictive role in LAA stroke participants only at certain levels of CMBs (i.e. > 5).

NLR or CRP both are useful markers for systemic inflammation(9, 44). NLR consists mainly of neutrophils and lymphocytes, both of which are involved in the systemic inflammatory response, especially in the initiation phase(44). CRP is mainly derived from hepatocytes that require IL-6 involvement to be produced, and is mainly involved in the acute phase of inflammation or infection(45). Both of them are well studied and proved to be associated with stroke recurrence(9, 46), re-bleeding(47), functional outcomes(9, 48), and poststroke infections(49, 50).

Therefore, we believe that inflammation is an important mechanism of cerebral infarction and take part in the whole process of the disease, therefore, our results suggest that high levels of inflammation (NLR > 3; CRP > 3) cause persistent damage on the base of small artery injury (CMB > 5), which contributes to the poor prognosis of LAA stroke.

Meanwhile, we found no significant differences in hypertension, diabetes, smoking, and alcohol consumption history or even age and gender in the population, and the consistency of these important risk factors for CSVD and LAA stroke supports the predicting role of CSVD imaging markers or, more precisely, CMB in stroke. The consistency of these factors reflects the intrinsic stability of our population, i.e. the fact that these measures are stable in this situation, rather than the fact that similar indicators are not significant for the prognosis.

There are some limitations in the present study. Firstly, the present cohort study was conducted in 3 stroke centers. Secondly, there are many earlier cerebrovascular imaging markers such as subcortical infarcts that are not included in the study. Thirdly, only BI, NIHSS, and mRS scores were used to evaluate functional outcomes of stroke participants. No other measures, such as advanced cognitive function, were used.

Our study confirmed that in the context of large artery atherosclerosis, small vessel damage features such as WMH did not have a direct prognostic effect, but CMB numbers > 5 affected the prognosis; we also found that NLR and CRP, which represent systemic inflammation, were predictive only when they exceeded a certain level and were combined with CMB numbers > 5, suggesting that early damage to small arteries, combined with high levels of inflammatory damage can predict the poor prognosis in LAA stroke.

CMB, cerebral microbleed; NLR, neutrophil-to-lymphocyte ratio; CRP, C reactive protein; LAA, large artery atherosclerotic; CSVD, cerebral small vessel disease; WMH, white matter hyperintensity; NIHSS, National Institute of Health stroke scale; mRS: Modified Rankin Scale

Ethics approval and consent to participate

Written informed consent was obtained from individual or guardian participants.

Consent for publication

Not applicable.

Availability of data and materials

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.

Competing interests

The authors declare that they have no competing interests.

Funding

None

Authors' contributions

DH, YL, and ZL contributed equally to this work. DH, YL, and ZL drafted the manuscript and polished the final manuscript. DH, YL, ZL and YZ collected, analyzed and interpret data. DW, QD, ZT and XY reviewed the articles. TY, JZ, and XH designed this project and proofread and reviewed the manuscript. All authors read and approved the final manuscript.

Acknowledgments

Not applicable

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No competing interests reported.

FigureS1.pdf

High inflammation level is essential to cerebral microbleed in the prediction of large artery atherosclerotic outcomes

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Abstract

Figures

Background

Materials And Methods

Study participants

Laboratory tests

Clinical assessments

Statistical analysis

Data Availability

Results

Characteristics of the participants

CMB was related to stroke outcomes

WMH, BA, EPVS, BS was not related to stroke outcomes

ROC curve for NLR and CRP in the prediction of stroke outcomes

Logistic regression found that CMB is an independent predictor of stroke outcomes

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1