The Institutional Review Board of Severance Hospital, Yonsei University Health System, approved this study and waived the need for informed consent because of the retrospective design and observational nature of this study (approval date: 2020-01-16; approval number: 4-2019-1196).

#### *2.7. Data availability Statement*

De-identified participant data are available upon reasonable request.

#### **3. Results**

#### *3.1. Patient Demographic and Clinical Characteristics*

A total of 3822 patients with acute ischemic stroke or TIA were recruited during the study period. After exclusions (follow-up loss (*n* = 154), no ABI measurements (*n* = 729), hemodialysis of one arm (*n* = 16), and TIA (*n* = 22)), 2901 patients were finally enrolled in this study (Figure 1).

**Figure 1.** Flowchart of participants according to inclusion and exclusion criteria. ABI, ankle brachial index; mRS; modified Rankin Scale score.

A total of 258 (8.9%) patients had PAD. The mean age was 65.4 ± 12.2 years, and 61.8% were men. Among them, 582 (20.1%) had poor outcomes (Table 1). Compared with patients with good outcomes, those with poor outcomes were older, were more likely to be women, had more severe initial stroke severity, were less likely to be current smokers, and were more likely to have CAA, PAD, and a stroke subtype of large artery atherosclerosis (all *p* values <0.05). For four-limb BP profiles, both ankle SBP and ABI were lower in patients with poor outcomes than in those with good outcomes (all *p* values <0.001). All BP differences including ABID, IAND, and IAD were higher in patients with poor outcomes than in those with good outcomes (all *p* values <0.001). Compared with the included patients, excluded patients were older, were more likely to be women, had higher NIHSS score, were more likely to have congestive heart failure, and were less likely to be current smokers or have CAD (Supplementary Table S1).

**Table 1.** Patient demographic and clinical characteristics.

Data are expressed as means ± standard deviations, medians [interquartile ranges], or numbers (%).

In all study patients, 236 (8.1%) patients showed ABID ≥0.15, 450 (15.5%) had IAND ≥15 mmHg, and 116 (4.0%) had IAD ≥15 mmHg. Among atherosclerotic diseases, CAA and PAD were independent determinants of ABID ≥0.15 (CAA: odds ratio (OR), 1.718, 95% confidence interval (CI), 1.211-2.437; PAD: OR, 22.124, 95% CI, 15.844-30.894) and IAND ≥15 mmHg (CAA: OR, 1.646, 95% CI, 1.281-2.114; PAD: OR, 13.328, 95% CI, 9.876-17.987). However, only PAD was an independent determinant of IAD ≥15 mmHg (PAD: OR, 3.044, 95% CI, 1.890-4.904) (Table 2).

**Table 2.** Determinants of IAD ≥15 mmHg, IAND ≥15 mmHg, and ABID ≥0.15.

Data were derived from the multivariable logistic regression analysis. ABID, ankle brachial index difference; CAD, coronary artery disease; CAA, cerebral artery atherosclerosis; CI, confidence interval; IAD, systolic inter-arm blood pressure difference; IAND, systolic inter-ankle blood pressure difference; NIHSS, National Institutes of Health Stroke Scale; OR, odds ratio; and PAD, peripheral artery disease. \*adjusted for sex, age, NIHSS score at admission, hypertension, diabetes mellitus, hypercholesterolemia, current smoking, congestive heart failure, and stroke subtype.

ABID ≥0.15 and IAND ≥15 mmHg were more likely to have ABI >1.30 (all *p* values <0.001), but not IAD ≥15 mmHg (Table 3). BaPWVs were well correlated with ABID, IAND, and IAD (with ABID, *r* = 0.139, *p* < 0.001; with IAND, *r* = 0.207, *p* < 0.001; and with IAD, *r* = 0.121, *p* < 0.001) (Supplementary Table S2).

**Table 3.** Relationship between IAD, IAND, ABID, and ABI >1.30.

ABI, ankle brachial index; ABID, ankle brachial index difference; IAD, systolic inter-arm blood pressure difference; and IAND, systolic inter-ankle blood pressure difference.

#### *3.2. Poor Functional Outcome*

All patients with (*n* = 2901) and without PAD (*n* = 2643) were separately analyzed. In all study patients, poor outcome was independently associated with ABID (OR, 5.289, 95% CI, 1.723-16.236) and cutoff of ABID ≥0.15 (OR, 1.920, 95% CI, 1.361-2.708). Poor outcome was also independently associated with IAND (OR, 1.015, 95% CI, 1.007-1.023) and cutoff of IAND ≥15 mmHg (OR, 1.818, 95% CI, 1.389-2.381). In patients without PAD, the cutoff of ABID ≥0.15 was independently associated with poor outcomes (OR, 1.970, 95% CI, 1.175-3.302). IAND and cutoff of IAND ≥15 mmHg were also independently associated with poor outcomes (IAND: OR, 1.025, 95% CI, 1.009-1.041; IAND ≥15 mmHg: OR, 1.665, 95% CI, 1.188-2.334). Conversely, IAD ≥15 mmHg was associated with poor outcomes in the whole population (OR, 1.623, 95% CI, 1.011-2.605) but was not associated with poor outcomes in patients without PAD (Table 4).

**Table 4.** Predictors of poor outcome at 3 months.

Data were derived from the multivariable logistic regression analysis. ABI, ankle brachial index; ABID, ankle brachial index difference; BP, blood pressure; CI, confidence interval; IAD, systolic inter-arm blood pressure difference; IAND, systolic inter-ankle blood pressure difference; NIHSS, National Institutes of Health Stroke Scale; OR, odds ratio; and PAD, peripheral artery disease. \*adjusted for sex, age, NIHSS score at admission, hypertension, diabetes mellitus, hypercholesterolemia, current smoking, congestive heart failure, coronary artery disease, cerebral artery atherosclerosis, and stroke subtype.

#### *3.3. All-Cause Mortality and MACEs*

Study patients were followed up for a median of 3.1 (IQR, 1.6–4.7) years. A total of 622 patients had MACEs (21.4%) including 496 all-cause deaths (17.1%) during the study period. In Kaplan–Meier survival curves (Figure 2), higher all-cause mortality and MACEs (log-rank test; *p* < 0.001) were found in patients with ABID ≥0.15 or IAND ≥15 mmHg (log-rank test; all *p* < 0.001). Higher all-cause mortality (log-rank test; *p* = 0.007) and MACEs (log-rank test; *p* = 0.008) were also found in patients with IAD ≥15 mmHg.

**Figure 2.** Kaplan–Meier survival analysis. (**A**) All-cause mortality; (**B**) major adverse cardiovascular event according to ABID ≥0.15. (**C**) All-cause mortality; (**D**) major adverse cardiovascular event according to IAND ≥15 mmHg. ABID; ankle-brachial index difference; IAND, systolic inter-ankle blood pressure difference.

In multivariable Cox regression analysis, ABID and ABID ≥0.15 were independently associated with all-cause mortality (ABID: hazard ratio (HR), 6.221, 95% CI, 2.973-13.018; ABID ≥0.15: HR, 1.567, 95% CI, 1.223-2.009) and MACEs (ABID: HR, 3.926, 95% CI, 1.906-8.087; ABID ≥0.15: HR, 1.416, 95% CI, 1.117-1.794). IAND and IAND ≥15 mmHg were also independently associated with all-cause mortality (IAND: HR, 1.013, 95% CI, 1.007-1.019; IAND ≥15 mmHg: HR, 1.616, 95% CI, 1.317-1.982) and MACEs (IAND: HR, 1.010, 95% CI, 1.005-1.015; IAND ≥15 mmHg: HR, 1.380, 95% CI, 1.139-1.672).

In patients without PAD, ABID and ABID ≥0.15 were independently associated with all-cause mortality (ABID: HR, 9.221, 95% CI, 3.013-28.220; ABID ≥0.15: HR, 1.524, 95% CI, 1.039-2.235) and MACEs (ABID: HR, 6.605, 95% CI, 2.281-19.124; ABID ≥0.15: HR, 1.514, 95% CI, 1.058-2.166). IAND and IAND ≥15 mmHg were also independently associated with all-cause mortality (IAND: HR, 1.017, 95% CI, 1.004-1.030; IAND ≥15 mmHg: HR, 1.516, 95% CI, 1.164-1.973) and MACEs (IAND: HR, 1.015, 95% CI, 1.004-1.027; IAND ≥15 mmHg: HR, 1.343, 95% CI, 1.051-1.716). Meanwhile, IAD was associated only with the long-term occurrence of MACEs in all patients (HR, 1.010, 95% CI, 1.001-1.019), but not in those without PAD (HR, 1.006, 95% CI, 0.993-1.018, *p* = 0.374) (Table 5).

**Table 5.** Predictors of long-term outcome.

Data were derived from the cox proportional hazards regression analysis. ABI, ankle brachial index; ABID, ankle brachial index difference; BP, blood pressure; CI, confidence interval; HR, hazard ratio; IAD, systolic inter-arm blood pressure difference; IAND, systolic inter-ankle blood pressure difference; MACE, major adverse cardiovascular event; NIHSS, National Institutes of Health Stroke Scale; and PAD, peripheral artery disease. \*adjusted for sex, age, NIHSS score at admission, hypertension, diabetes mellitus, hypercholesterolemia, current smoking, congestive heart failure, coronary artery disease, cerebral artery atherosclerosis, and stroke subtype.

#### **4. Discussion**

We demonstrated that higher ABID and IAND were independently associated with poor short-term functional outcomes, long-term MACE occurrence, and all-cause mortality in patients with acute ischemic stroke. In particular, higher ABID and IAND had prognostic effects even in patients without PAD. Meanwhile, IAD was associated with poor short-term outcomes and MACEs in all patients, but not in those without PAD. These findings suggest that higher ABID and IAND have prognostic value for both poor short- and long-term outcomes of acute ischemic stroke and are more sensitive than IAD for predicting outcomes in acute ischemic stroke patients without PAD.

Primarily, increased ABID and IAND are attributable to the presence of PAD [17]. PAD affects approximately 200 million people worldwide and is the third most common cause of atherosclerotic cardiovascular death after CAD and stroke [18]. Traditional cardiovascular risk factors (smoking, hypertension, diabetes mellitus, and hypercholesterolemia) and advanced aging are important determinants of PAD. Therefore, patients with PAD often have concomitant atherosclerosis on the cerebral and coronary artery. In the Reduction of Atherothrombosis for Continued Health registry involving 44 countries worldwide, 39% of patients with PAD had CAD, 10% had cerebral artery disease, and 13% had both [19]. Accumulated systemic atherosclerosis worsens stroke prognosis [20]. Among the atherosclerotic burdens, ABID and IAND were associated with CAA and PAD. In contrast, IAD was only associated with PAD. It can be assumed that stroke patients with large ABID and IAND are more likely to have additional cerebral atherosclerotic burden and may have a poorer prognosis.

To the best of our knowledge, no study has previously evaluated ABID as a prognosis predictor in patients with acute ischemic stroke. ABI measurement is a well-established method to identify patients with PAD. Low ABI is commonly defined as ABI <0.9 and provides good sensitivity (80%) and excellent specificity (95%) to detect PAD [4]. ABI is also associated with poor initial stroke severity [21] and predicts poor prognosis and mortality in patients with stroke [2]. However, several previous studies have shown that low ABI was not sensitive enough to detect asymptomatic PAD in the general population [22]. To detect PAD and predict stroke prognosis accurately, novel parameters besides ABI should be developed. We found that ABID and IAND were independent and strong predictors of MACEs and all-cause mortality in patients with acute ischemic stroke. Interestingly, ABID and IAND remained to be significantly associated with poor short- and long-term outcomes in patients without PAD. This finding suggests that ABID and IAND may provide additional information for patients with subclinical or mild PAD. The strength of ABID might be related to the consideration of IAND and arm BP simultaneously. In addition, ABID and IAND can be obtained and easily calculated during ABI measurement in routine clinical practice.

In patients with stroke, IAD has been demonstrated to be associated with recurrent stroke [23], poor prognosis [24], and mortality [6]. However, some patients undergo dialysis with one arm because of end-stage renal disease, making it difficult to measure IAD. In addition, IAD (and ABI) may be "pseudonormal" when a patient has severe stenosis in both arms and in one leg. In contrast, IAND can be calculated without BP measurement in the arm and provide consistent data [3]. Several studies showed the increased usefulness of IAND and ABI relative to IAD. One study showed that IAND could better predict both overall and cardiovascular mortality than IAD in elderly patients [3]. ABI exhibits better association with cardiovascular outcomes than IAD in patients with type 2 diabetes [25]. However, no study has reported the comparison between IAND and IAD in patients with acute ischemic stroke.

Because lower limbs are more prone to be affected by PAD than upper limbs, IAND could be a better predictor of PAD than IAD [26]. High IAND was associated with increased left ventricular mass index [27] and arterial stiffness [28] and also predicted mortality in the elderly people [3]. Similarly, large ABID provided the prognostic value for mortality in patients undergoing chronic hemodialysis [5]. These findings suggest that the cardiovascular risk was higher in patients with lower extremity PAD than in those with upper extremity PAD [29]. Therefore, the circulatory burden assumed from the heart to the ankles may be greater than that from the heart to the arms.

Endothelial dysfunction [26], calcification burden [30], and arterial stiffness [27] are more frequent in the lower extremities than in the upper extremities. The degree of endothelial dysfunction in leg circulation is related to PAD severity. Endothelial dysfunction of leg circulation may occur before the impairment of forearm circulation in PAD [26]. Our data showed that high ABID and IAND were more likely to have ABI >1.30 than IAD. High ABI (i.e., ABI >1.30) is generally believed to occur because of medial arterial calcification and may be a marker for vascular stiffness [4]. High ABI was associated with an increase in both overall and cardiovascular mortality in patients with chronic kidney disease undergoing hemodialysis [31] and in the general population [32]. PWV and ABI are both atherosclerotic markers. ABI reflects stenosis or peripheral artery obstruction, whereas PWV represents arterial stiffness [5]. ABID, IAND, and IAD was positively correlated with baPWV. The correlation coefficient was highest in IAND, followed by ABID and IAD. In patients undergoing hemodialysis, high PWV and low ABI are significantly associated with mortality [33]. Therefore, ABID and IAND could be more influenced by endothelial dysfunction, systemic atherosclerosis, calcification burden, and arterial stiffness than IAD, which may be related to more frequent PAD in the lower extremities [25,26,28,29].

This study has several limitations. First, radiological studies to detect atherosclerosis in the lower extremities were not routinely performed. A correlation study between apparent atherosclerosis and IAND or ABID might be helpful for better understanding [4,17]. Second, multiple, automatic, and simultaneous assessments are recommended for accurate BP difference measurement rather than single, manual, and sequential evaluation methods [34]. We used an automatic and simultaneous measurement device, but BP difference was investigated only once during the ABI measurement, and additional follow-up data were limited. Third, BP differences in this study focused on SBP, rather than DBP. Additional analysis was performed with DBP data, which found that the prognostic effect of DBP was not different from that of SBP (Supplementary Tables S3 and S4). Fourth, our findings may not be generalized to other populations or cohorts because our study population is limited to Korean patients. Fifth, the stroke standard treatment guidelines were updated and changed several times during the study period. Lastly, a total of 921 patients were excluded from the analysis. Among them, patients who did not undergo ABI measurements were mostly excluded. Therefore, the possibility of selection bias exists because of the retrospective study design; however, consecutive patients were included, and a relatively large sample size was analyzed.

#### **5. Conclusions**

This study suggests that high ABID and IAND are associated with poor short-term outcomes, long-term MACE occurrence, and all-cause mortality in patients with acute ischemic stroke. In addition, ABID and IAND predict post-stroke outcomes, even in patients without PAD. Therefore, ABID and IAND can be simple and reliable methods for identifying patients with an increased risk of poor shortand long-term outcomes in acute ischemic stroke.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2077-0383/9/4/1125/s1, Table S1. Comparison of acute stroke patients during the study period who were included and excluded in this study, Table S2. Correlations between IAD, IAND, ABID, and baPWV in all patients (*n* = 2901), Table S3. Predictors of short-term outcome, Table S4. Predictors of long-term outcome.

**Author Contributions:** Conceptualization, M.H. and H.S.N.; methodology, M.H. and H.S.N.; formal analysis, M.H.; investigation, M.H. and H.S.N.; writing—original draft preparation, M.H. and H.S.N.; writing—review and editing, M.H., J.K., T.-J.S. and H.S.N.; data curation, M.H., J.K.C., J.C., J.H. and E.P.; supervision, Y.D.K., J.H.H. and H.S.N.; funding acquisition, H.S.N. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (2019R1H1A1079907) and by a faculty research grant of Yonsei University College of Medicine (6-2019-0065, 6-2019-0170).

**Acknowledgments:** We thank Kangsik Seo, MT, for his small consideration in the course of writing the paper.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

## **Interhemispheric Functional Connectivity in the Primary Motor Cortex Assessed by Resting-State Functional Magnetic Resonance Imaging Aids Long-Term Recovery Prediction among Subacute Stroke Patients with Severe Hand Weakness**

**Yu-Sun Min 1,2,3,**†**, Jang Woo Park 4,**†**, Eunhee Park 1,2, Ae-Ryoung Kim 1,2, Hyunsil Cha 4, Dae-Won Gwak 2, Seung-Hwan Jung 2, Yongmin Chang 4,5,6,\* and Tae-Du Jung 1,2,\***

Received: 25 February 2020; Accepted: 30 March 2020; Published: 1 April 2020

**Abstract:** This study aimed to evaluate the usefulness of interhemispheric functional connectivity (FC) as a predictor of motor recovery in severe hand impairment and to determine the cutoff FC level as a clinically useful parameter. Patients with stroke (*n* = 22; age, 59.9 ± 13.7 years) who presented with unilateral severe upper-limb paresis and were confirmed to elicit no motor-evoked potential responses were selected. FC was measured using resting-state functional magnetic resonance imaging (rsfMRI) scans at 1 month from stroke onset. The good recovery group showed a higher FC value than the poor recovery group (*p* = 0.034). In contrast, there was no statistical difference in FC value between the good recovery and healthy control groups (*p* = 0.182). Additionally, the healthy control group showed a higher FC value than that shown by the poor recovery group (*p* = 0.0002). Good and poor recovery were determined based on Brunnstrom stage of upper-limb function at 6 months as the standard, and receiver operating characteristic curve indicated that a cutoff score of 0.013 had the greatest prognostic ability. In conclusion, interhemispheric FC measurement using rsfMRI scans may provide useful clinical information for predicting hand motor recovery during stroke rehabilitation.

**Keywords:** functional magnetic resonance imaging; neuronal plasticity; recovery of function; stroke; motor cortex

#### **1. Introduction**

Stroke is the leading cause of adult disability worldwide, accounting for a majority of patients with upper-limb impairment. The degrees of spontaneous improvement vary according to the severity of upper-limb paresis. In patients with mild-to-moderate upper-limb paresis, spontaneous recovery, as reflected by improvements in clinical parameters including Fugl-Meyer assessment of the upper

extremity (FMA-UE) scores, is mainly restricted to the first 4 weeks post-stroke [1].There is evidence in the literature that in stroke patients with mild-to-moderate impairment, the degree of initial deficits predicts outcome. In contrast to mildly impaired patients, it is relatively difficult to predict the spontaneous recovery pattern of upper-limb motor function in severely impaired patients. Clinical data alone cannot accurately predict arm recovery, particularly in patients with initial severe upper-limb impairment [2–4]. High inter-individual variability associated with recovery makes it difficult to predict arm recovery. However, very few severely impaired patients show late-onset motor recovery of the upper-limb [5]. Therefore, a prognostic biomarker reflecting functional long-term motor recovery is urgently required to decide the manner in which rehabilitation treatment strategies, including goal setting and effective treatment duration, for upper-limb recovery in severe hemiplegic stroke patients can be modified.

Recently, we reported that initial power spectral density (PSD) analysis of resting-state functional magnetic resonance imaging (rsfMRI) data can provide a sensitive prognostic predictor for patients with subacute stroke combined with severe hand disability [6]. PSD is measured as resting-state intrinsic neuronal activity in the frequency domain. In contrast to PSD, functional connectivity (FC) analysis is another approach to measure the resting-state intrinsic neuronal activity in the time domain using rsfMRI. Changes in FC value in the interhemispheric motor cortex (M1) after stroke are reportedly reflective of long-term recovery, and patients with good functional outcomes have greater FC values than patients with poor outcomes [7–9]. However, a recent study reported that differences in FC value in the interhemispheric M1 did not change over time with recovery [10]. Therefore, whether motor recovery after stroke can be predicted by the change in interhemispheric FC still remains controversial.

This study aimed to evaluate whether interhemispheric FC is useful for predicting upper-limb motor recovery among patients with severe hand impairment for whom it was difficult to predict the recovery pattern based on an initial clinical parameter. Therefore, addition of FC as a prognostic parameter for patients with severe hand deficits may eventually be useful for setting individualized therapeutic goals and strategies as well as for selecting patients for future trials.

#### **2. Materials and Methods**

#### *2.1. Subjects*

Twenty-two patients (59.9 ± 13.7 years; 9 males and 13 females) and 12 healthy subjects (60.2 ± 6.8 years; 8 males, 4 females) were included in this study. They were all right-handed. The inclusion criteria for patients were as follows: (1) unilateral ischemic stroke in the middle cerebral artery (MCA) territory confirmed by MRI, (2) first stroke, (3) age over 20, (4) hemiplegic motor deficit less than Gr 1 by manual muscle test present at the time of admission (Table 1). Patients with unstable medical conditions and those lost to follow-up were excluded.

All patients underwent resting functional magnetic resonance imaging about 1 month (27.8 ± 8.4) from stroke onset. We used Brunnstrom stage (hand score) as a parameter to assess clinical outcome at 1 month and 6 months after stroke onset. We included the patients with Brunnstrom stage 1 (flaccidity or absence of an active finger movement) but without any motor-evoked potential (MEP) responses of the affected hand at 1 month after stroke. Patients with severely impaired cognitive function [Mini-Mental State Examination (MMSE) < 24], severe visual or perceptual impairment, previous musculoskeletal abnormality, or damaged upper-limbs were excluded. All patients received individual physiotherapy training as well as cognitive training every day. The physiotherapy treatments comprised 30-min sessions two times per day for five days a week and included walking and balance training as well as individual exercise. Informed consent was provided to all patients according to OO University Institutional Review Board (2012-05-023).

**Table 1.**

Cerebral Artery.

#### *J. Clin. Med.* **2020** , *9*, 975

#### *2.2. Motor Task Functional Magnetic Resonance Imaging*

Region of interest (ROI) of M1 for each participant was defined using motor task functional magnetic resonance imaging (fMRI). Motor task fMRI alternatively comprised three active periods and three rest periods, and each period was 30-s long. A light touch on the leg or hand was used to give a start signal at the start point of each period. Participants performed the motor task twice with the right and left hands and repeated flexion–extension during scanning. If any of the participants could not move their hand, they received assistance to perform passive movement.

To perform motor task fMRI data acquisition, T2-weighted echo-planar imaging sequences were used with the following parameters: TE (echo time) = 40 ms, TR (repetition time) = 3000 ms, Flip Angle (FA) = 90◦, FOV (field-of-view) = 21 cm, acquisition matrix = 64 × 64, 4-mm thickness with no gap, and total scan time = 4 min and 12 s, with four dummy scans.

#### *2.3. Resting-State fMRI*

All fMRI data were obtained on a Signa Exite 3.0-T scanner (GE Healthcare, Milwaukee, WI, USA). All applicants were instructed to lie down comfortably and close their eyes during MRI scanning, but not fall asleep. The rsfMRI data were obtained using T2-weighted echo-planar imaging sequences using the following parameters: TE = 40 ms, TR = 2000 ms, FA = 90◦, FOV = 22 cm, acquisition matrix = 64 × 64, 4-mm thickness with no gap, and total scan time= 8 min and 12 s, with six dummy scans.

Three-dimensional-fast spoiled gradient echo sequence [repetition time (TR) = 7.8 ms; echo time (TE) = 3 ms; inversion time = 450 ms; flip angle = 20; matrix = 256 × 256; field-of-view (FOV) = 24 mm; 1.3 mm thickness] was used for the acquisition of T1-weighted high-resolution anatomical images.

#### *2.4. fMRI Data Analysis*

Image preprocessing and statistical analyses of fMRI data were conducted using the statistical parametric mapping software SPM12 (http://www.fil.ion.ucl.ac.uk/spm/), implemented in MATLAB (Mathworks, Inc., Sherborn, MA, USA). By slice-timing, realignment, co-registration, and normalization, functional images were preprocessed into the Montreal Neurological Institute (MNI) template based on a standard stereotaxic coordinate system and spatial smoothing with 8-mm full-width at half-maximum (FWHM) Gaussian kernel. FMRI data are superimposed onto MNI space. The seed MNI coordinates for the patients were summarized in Table S1 (Supplementary Materials).

#### *2.5. Rest State Functional Connectivity*

The seed-based method was used to determine resting-state functional connectivity (rsFC). In brief, FC CONN15 toolbox (http://web.mit.edu/swg/software.htm) was used to show a strong temporal correlation between bilateral M1 and supplementary motor area (SMA). The contralesion and ipsilesion (namely M1 and SMA; spheres of 5-mm radius) were identified using MarsBar ROI tool (http://marsbar.sourceforge.net/) on MNI coordinates. Four ROI positions (spheres of 5 mm radius), namely contralesional M1, ipsilesional M1, contralesional SMA, and ipsilesional SMA, were selected based on individual motor task results. Noise, cerebrospinal fluid, white matter, and motion parameters were used to correct time fluctuations in blood-oxygen-level-dependent (BOLD) signals as nuisance covariates, and a band-pass filter (range, 0.008 Hz–0.09 Hz) was used. FC scores between pairs of ROIs on each subject were calculated using the FC SPM12 toolbox.

#### *2.6. Lesion Volume Analysis*

The lesion volume associated with hand motor function in the stroke area was calculated based on the overlapping area of the lesion mask between the T1-weighted images and the template of the corticospinal tract (CST). T1-weighted images were taken by preprocessing, which involves co-registration and normalization to a T1-weighted template using the SPM12 software package. A stroke physiatrist, who was blinded to the study, manually drew the lesions by using MRIcro (http:

//www.mccauslandcenter.sc.edu/crnl/mricro). The CST template was constructed using a previously reported method of probabilistic tractography [11,12]. Probabilistic tractography was conducted for 26 healthy controls to reconstruct CST. The seed, target, waypoint, and exclusion mask were drawn as follows. Individual seed masks for each hemisphere were placed in the hand knob area of M1 (MNI coordinates (37, −25, 62); (−37, −25, 62)), and each participant used an established semi-automated pipeline. The target masks were basis pontis. The waypoint masks included the posterior limb of the internal capsules and cerebral peduncles. For CST, a mask covering trajectories at the tegmentum pontis was added to the mid-sagittal and basal ganglia exclusion masks as an additional exclusion mask. A total of 50,000 streamlines were sent from M1 to the spinal target masks in the ventral medulla oblongata. Three different thresholds at 0.5%, 1%, and 2% were established for CST output distributions. The average of each tract was calculated for each of the three thresholds by summing all individual threshold- and subject-specific trajectories.

#### *2.7. Statistical Analysis*

To assess differences in FC scores between the three groups for each pair of ROIs, an ANOVA F-test was performed; subsequently, post-hoc two-sample t-tests were conducted for carrying out further comparisons. All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS, Chicago, IL, USA). A *p* value of <0.05 was considered to be statistically significant.

Receiver-operating characteristic (ROC) curve analysis was performed to determine the cutoff value for the prognostic model of upper-limb stroke recovery by using the difference in FC score between ipsilesional and contralesional M1 at 1 month.

True-positive rate (sensitivity) and false-positive rate (1-specificity) were computed and plotted as ROC curves. In an ROC space, a diagonal line corresponds to random discrimination. The area under the ROC curve (AUC) is commonly used to quantify classifier discriminability, with a value of 0.5 corresponding to random classification and a value of 1 corresponding to perfect classification.

#### **3. Results**

At 6 months after stroke onset, 11 patients (60.3 ± 15.9 years; seven males, four females) with Brunnstrom stage 4 (lateral prehension with release by thumb movement or semi-voluntary finger extension of a small range of motion) or 5 (palmar prehension or cylindrical/spherical grasp with limited function or voluntary mass finger extension of variable range) were categorized into the good recovery group and 11 patients (59.5 ± 12.9 years; six males, five females) with Brunnstrom stage 1, 2, and 3 were categorized into the poor recovery group. There were no age and sex-based differences between the good recovery and the poor recovery groups and the healthy control group (*p* = 0.986 and *p* = 0.827, respectively). Additionally, there was no statistical difference (*p* = 0.158) in lesion overlap volume between the good recovery group (0.33 ± 0.15 cc) and the poor recovery group (0.40 ± 0.17 cc). However, there was statistical difference (*p* = 0.019) in total lesion volume between the good recovery group (29.37 ± 41.31 cc) and the poor recovery group (125 ± 118.73 cc) (Figure 1). The demographic and clinical characteristics of 22 patients with stroke are summarized in Table 1.

Among the three groups, ANOVA F-test results reveal a statistically significant difference in FC between ipsilesional M1–contralesional M1 (*p* = 0.00039) (Figure 2). Post-hoc two-sample t-tests were performed for comparing the three groups further. The good recovery group showed a higher FC than that of the poor recovery group (*p* = 0.034). Contrastingly, the good recovery group showed no statistical difference in FC when compared with the healthy control group (*p* = 0.182), but the latter had a higher FC than that of the poor recovery group (*p* = 0.0002).

Moreover, according to the ANOVA F-test, the FC between ipsilesional SMA and contralesional SMA was significantly different among the three groups (*p* = 0.003) (Figure 1). In the post-hoc two-sample t-test, the FC between ipsilesional SMA and contralesional SMA was higher in the healthy control group compared with that in the good recovery group and the poor recovery group (*p* = 0.019

and *p* = 0.004, respectively), but there was no difference in the FC value between the good recovery group and the poor recovery group (*p* = 0.804).

**Figure 1.** Total lesion overlay maps for the good recovery group and the poor recovery group.

**Figure 2.** ANOVA F-tests showed significant differences in functional connectivity (FC) between ipsilesional M1-contralesional M1 among the three groups (*p* = 0.00039). Post-hoc two-sample t-tests were performed for further comparing between the groups. The good recovery group showed a higher FC than that shown by the poor recovery group (*p* = 0.034). In contrast, no significant difference in FC was seen between the good recovery and the healthy control groups (*p* = 0.182). Additionally, the healthy control group showed a higher FC than that of the poor recovery group (*p* = 0.0002).

Contrastingly, the ANOVA F-test result reveals no significant difference in FC among ipsilesional M1-SMA (*p* = 0.318), ipsilesional M1-contralesional SMA (*p* = 0.056), contralesional M1-ipsilesional SMA (*p* = 0.297), and contralesional M1-contralesional SMA (*p* = 0.656).

When the total lesion volume was included as a covariate in statistical analysis, ANOVA F-test results reveal a statistically significant difference in FC between ipsilesional M1–contralesional M1 among the three groups (*p* = 0.018) and in FC between ipsilesional M1–contralesional SMA among the three groups (*p* = 0.015). In the post-hoc two-sample t-test, however, there was no difference in the FC value between the good recovery group and poor recovery group (*p* = 0.232).

FC between ipsilesional and contralesional M1 positively correlated with hand function prognosis, as evaluated by Brunnstrom motor stages (BMS) (r = 0.581 and *p* = 0.005, respectively) (Figure 3). However, FC between ipsilesional and contralesional SMA did not correlate with hand function prognosis, as evaluated by BMS (r = −0.006, *p* = 0.979).

**Figure 3.** FC between ipsilesional and contralesional M1 is positively correlated with prognosis of hand function, as evaluated by Brunnstrom motor stages (BMS) (r = 0.581, *p* = 0.005).

Good and poor recovery outcomes based on the Brunnstrom stage of upper-limb function at 6 months were determined as the standard, and the ROC curve indicated that a cutoff score of 0.013 had the greatest prognostic ability (maximum sensitivity and specificity) (Figure 4). The sensitivity of this model for predicting good recovery was 81.8% and the specificity was 63.6%. AUC value was 0.793, which is a fair level.

**Figure 4.** Good and poor recovery were determined based on Brunnstrom stage of upper-limb function at 6 months as the standard, and ROC (Receiver-operating characteristic) curve indicated that a cutoff score of 0.013 had the greatest prognostic ability (maximum sensitivity and specificity).

#### **4. Discussion**

Here, we demonstrated the predictive value of FC for long-term motor outcomes in subacute stroke patients with stroke, predominantly in the MCA territory. The FC between ipsilateral and contralateral M1 was lower in the poor recovery group compared with that in the good recovery and healthy control groups, and it was also well correlated with hand function prognosis, evaluated by BMS. Our study demonstrated that the interhemispheric FC score calculated at 1 month after stroke has a good predictive value for recovery over a 6-month period in patients with severe hand impairment. However, the total lesion volume showed a tendency to swallow a large amount of explanatory variance of the outcome parameter. We also suggested an FC cutoff score for discriminating the good recovery group from the poor recovery group with high sensitivity and specificity.

Previous rsfMRI studies on acute stroke demonstrated that patients with mild-to-moderate motor deficits showed low interhemispheric FC score between the motor cortices [8,9,13]. These studies also revealed that the low FC score gradually increased during the recovery process and finally restored to near-normal levels in a good recovery group, while the FC score in the poor recovery group continued to decrease. Our findings are in line with these previous studies, i.e., interhemispheric FC score is useful for assessing the stroke prognosis and recovery despite varying scanning times (e.g., 3 days vs. 4 weeks) and evaluation domains [7,14,15].

These results suggest that functional neuroadaptation (reorganization) may be occurring in the most severely injured brains. One possible explanation for this finding is the physiological balance in reciprocal inhibitory projections between both of the hemispheres [16]. This explanation suggests that an abnormal inhibitory influence of the undamaged contralesional motor cortex on the damaged ipsilesional motor cortex disturbs the balance between the hemispheres, which is important for voluntarily generating paretic hand movement in poor recovery patients [16–19]. Here, the patients in the good recovery group had a higher rsFC between the motor cortex, which enhanced motor ability in the paretic hand. Our findings are consistent with those of previous studies, which demonstrated that rsFC within either the ipsilesional primary sensorimotor cortex or contralesional primary sensorimotor cortex reduced at an early stage after stroke, after which, in those patients who showed an improvement in motor impairment, the rsFC gradually increased to near-normal levels during recovery.

However, Nijboer et al. reported that ipsilesional rsFC between motor areas was lower than the contralesional rsFC, but this difference did not change over time [10]; they demonstrated that no relations were observed between individual changes in rsFC and upper-limb motor recovery. In that study, patient population presented with mild upper-limb impairments as opposed to the patients in our study who had severe motor impairment at 4 weeks after stroke. The patients only showed a limited amount of improvement (i.e., ceiling effect) after the first 4 weeks. The changes in brain activation patterns (i.e., cerebral reorganization) might have a different impact on a mild patient population compared with that on a population comprising severely impaired patients [20]. Here, patients were completely motor deficit with no hand movement and MEP response.

Using rsfMRI, we elucidated the cutoff value of FC to be 0.013, and sensitivity and specificity rates for good recovery prediction were 81.8% and 63.6%, respectively. There have been many studies on modeling the prediction of function recovery after stroke. The representative models are the PREP algorithm and the proportional recovery model [2,21]. However, when the two models were validated, the predicted prognosis rate (sensitivity and specificity) remained around 73–88%. The reason is that fitting to that predictive model did not work well in patients with severe corticospinal tract damage early in the injury, clinically complete unilateral paralysis, and patients with no MEP response. That is, in the case of mild to moderate severity, the prediction through clinical data and infarction size fit well, but in severe cases, it was difficult to predict through the model, and these were called 'non-fitter'. In this study, we included relatively homogenous patients with MCA infarction, clinically no hand movement at all, and neurophysiologically no MEP response. Therefore, measuring interhemispheric functional connectivity in these patient populations can help predict prognosis. The prediction power associated with the use of only one parameter, namely interhemispheric rsFC, was comparable to the power associated with the multi-parameter model, without causing any compromise in sensitivity and specificity rates.

The present study had some limitations. First of all, our study is limited by small numbers in the patient population. Due to this limitation, stroke patients included only those with cortical and subcortical stroke, and we could not definitely determine the differential impact of lesion location and stroke severity on rsFC. Hence, from our results, we could not fully elucidate the mechanisms responsible for the reduction in rsFC after stroke as well as its influence on patient behavior. However, a more important clinical implication would be the establishment of the prognostic power of rsFC at an early stage. Therefore, larger sample size and longitudinal follow-up are warranted to confirm these relationships in future studies.

#### **5. Conclusions**

Interhemispheric FC estimated via rsfMRI provides useful clinical information and has a predictive value for hand motor recovery during stroke rehabilitation.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2077-0383/9/4/975/s1, Table S1: Individual ROI coordinates obtained from task-fMRI.

**Author Contributions:** Conceptualization, J.W.P.; Data curation, D.-W.G. and S.-H.J.; Formal analysis, E.P.; Funding acquisition, Y.C.; Investigation, A.-R.K.; Methodology, J.W.P.; Resources, H.C. and Y.C.; Software, J.W.P.; Supervision, T.-D.J.; Validation, E.P.; Visualization, J.W.P.; Writing—original draft, Y.-S.M.; Writing—review & editing, Y.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by Biomedical Research Institute grant, Kyungpook National University Hospital (2015).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **The Prognostic Value of High Platelet Reactivity in Ischemic Stroke Depends on the Etiology: A Pilot Study**

#### **Adam Wi´sniewski 1,\*, Karolina Filipska 2, Joanna Sikora 3, Robert Slusarz ´ <sup>2</sup> and Grzegorz Kozera <sup>4</sup>**

Received: 8 February 2020; Accepted: 19 March 2020; Published: 20 March 2020

**Abstract:** Background: Reduced aspirin response may result in a worse prognosis and a poor clinical outcome in ischemic stroke. The aim of this prospective pilot study was to assess the relationship between platelet reactivity and early and late prognosis, and the clinical and functional status in ischemic stroke, with the role of stroke etiology. Methods: The study involved 69 subjects with ischemic stroke, divided into large and small vessel etiological subgroups. Platelet function testing was performed with two aggregometric methods—impedance and optical—while the clinical condition was assessed using the National Institute of Health Stroke Scale (NIHSS) and the functional status was assessed using the modified Rankin Scale (mRS) on the first and eighth day (early prognosis) and the 90th day of stroke (late prognosis). Results: The initial platelet reactivity was found to be higher in patients with severe neurological deficits on the 90th day after stroke, than in the group with mild neurological deficits (median, respectively, 40 area under the curve (AUC) units vs. 25 AUC units, *p* = 0.033). In the large vessel disease group, a significant correlation between the platelet reactivity and the functional status on the first day of stroke was found (correlation coefficient (R) = 0.4526; *p* = 0.0451), the platelet reactivity was higher in the subgroup with a severe clinical condition compared to a mild clinical condition on the first day of stroke (*p* = 0.0372), and patients resistant to acetylsalicylic acid (aspirin) had a significantly greater possibility of a severe neurological deficit on the first day of stroke compared to those who were sensitive to aspirin (odds ratio (OR) = 14.00, 95% confidence interval (CI) 1.25–156.12, *p* = 0.0322). Conclusion: High on-treatment platelet reactivity in ischemic stroke was associated with a worse late prognosis regardless of the etiology. We demonstrated a significant relationship between high platelet reactivity and worse early prognosis and poor clinical and functional condition in the large vessel etiologic subgroup. However, due to the pilot nature of this study, its results should be interpreted with caution and further validation on a larger cohort is required.

**Keywords:** ischemic stroke; platelet reactivity; aspirin resistance; large vessel disease; carotid stenosis; clinical outcome; prognosis

#### **1. Introduction**

Stroke is a leading cause of disability and death worldwide and is associated with a worse quality of life [1]. Antiplatelet therapy is used to reduce the risk of recurrent ischemic stroke [2]. Acetylsalicylic acid (aspirin) is a primary antiplatelet agent; however, its effect can vary in different patients [3]. In some patients, a reduced aspirin response may be observed, resulting in a failure to inhibit the platelet reactivity [4]. Platelet function testing can evaluate the effectiveness of aspirin in decreasing platelet aggregation and activation. High on-treatment platelet reactivity or biochemical aspirin resistance is a multifactorial, negative feature that is associated with insufficient antiplatelet therapy [5].

One of the better-understood causes of cerebral ischemia is the pathology of large pre-cranial vessels, most often the internal carotid artery, which accounts for approximately 20–30% of all causes of stroke [6]. Our previous papers demonstrated the hyperaggregation and hyperactivation of platelets in this etiological subtype of ischemic stroke [7,8]. Furthermore, we hypothesize that it may be related to aspirin resistance and affect the clinical condition and prognosis due to the reduced inhibition of platelets. In the next step, we estimate the role of high on-treatment platelet reactivity for the clinical evaluation and prognosis of stroke patients.

Previous reports regarding the relationship between high platelet reactivity and clinical deterioration did not present clear conclusions [9–12]. The researchers did not focus on the potential role of stroke etiology for significant correlations in this field. The main objective of this study was to determine the relationship between platelet reactivity in the acute phase of ischemic strokes in patients treated with acetylsalicylic acid and the clinical and functional condition of patients, as well as early and late prognosis, with a particular emphasis on cerebral ischemic etiopathogenesis.

#### **2. Materials and Methods**

#### *2.1. Study Population*

The perspective, single-center, observational study was conducted at the Department of Neurology at the University Hospital No. 1 in Bydgoszcz. We consecutively enrolled 69 patients between February 2016 and December 2017 who underwent ischemic stroke according to the updated definition of stroke by the American Heart Association/American Stroke Association [13]. All subjects received a standard dose (150 mg) of acetylsalicylic acid based on the current guidelines. We divided the enrolled subjects into two subgroups considering the etiology of ischemic strokes. For the large vessel disease subgroup, we included patients with at least 50% of a carotid artery stenosis on the site correlated with clinical symptoms that were confirmed in an ultrasound examination [14]. The second etiological subgroup, small vessel disease, consisted of subjects with clinical and radiological features related to small vessel disease. We included patients with classic lacunar syndromes (pure motor or sensory stroke or ataxic hemiparesis) and typical neuroimaging markers (small subcortical infarcts <2 cm, hyperintensities in the white matter, lacunes < 15 mm, prominent perivascular spaces, microbleeds, and brain atrophy), where acute ischemic infarcts in neuroimaging were related to clinical symptoms of stroke [15]. The exclusion criteria were: a subject's inability to make an informed signature (speech disorders, or quantitative or qualitative disturbances of consciousness), an embolic background of ischemic stroke, a previous history of stroke, the chronic use of acetylsalicylic acid before stroke onset, gastrointestinal or urinary bleeding within the last 2 years, low platelet count <100,000/μL, anemia (hemoglobin <9 g/dL), or low hematocrit <35%, "silent" infarcts (infarcts in neuroimaging that are not related to clinical symptoms of stroke).

#### *2.2. Clinical Outcome*

Both the clinical status and functional status were assessed within 24 h after admission (first day) to the hospital, on the eighth day of hospitalization (early prognosis), and on the 90th (+/− 5 days) day (late prognosis) after the stroke onset. The clinical status and functional status were assessed using

standardized research tools, the National Institute of Health Stroke Scale (NIHSS) and modified Rankin Scale (mRS), respectively. When analyzing the severity of the neurological deficit, two subgroups of patients with stroke were distinguished: a subgroup with a mild neurological deficit (0–5 points on the NIHSS) and a subgroup with moderate and severe neurological deficits (≥6 points on the NIHSS). Regarding the functional status of the patients, two subgroups of stroke patients were distinguished: a favorable prognosis (on the mRS 0–2 points) and an unfavorable prognosis (on the mRS 3–5 points). A comparison of the clinical and functional conditions in both etiological subgroups of the subjects is presented in Table 1.

**Table 1.** A comparison of the anthropometric data, platelet reactivity, clinical, and functional status in patients with stroke in both etiological subgroups.

\* Mann–Whitney U test, \*\* Chi-squared calculation. AUC, area under the curve; NIHSS, National Institute of Health Stroke Scale; mRS, modified Rankin scale.

#### *2.3. Ethics Statement*

Written informed consent, after revision of the study protocol, was obtained from each participant. This study was approved by the Bioethics Committee of Nicolaus Copernicus University in Torun at Collegium Medicum of Ludwik Rydygier in Bydgoszcz (KB number 73/2016).

#### *2.4. Platelet Function Testing*

Aspirin-induced platelet function testing was measured using two methods: optical aggregometry and impedance aggregometry. Blood samples were collected from the participants within 24 h after the stroke onset. To standardize and to unify the time-points of measurements, most cases were performed between 18 and 24 h after the stroke onset, at the same time of day (10–12 AM). The optical aggregometry or light transmission aggregometry (LTA) was performed with an aggregometer (Chrono-Log Corp., Havertown, PA, USA) and the results were expressed as area under the curve (AUC) units. Values over 115 AUC units were defined as high on-treatment platelet reactivity or aspirin resistance. We performed impedance aggregometry using the Multiplate® platelet function analyzer (Roche Diagnostics, France) and its results were expressed as AUC units. For the aspirin-resistant group, we enrolled subjects with values over 40 AUC units. The procedures for performing platelet function testing were similar as described in the previous studies [16,17]. Of our 69 subjects, 43 underwent optical aggregometry measurements, and all 69 subjects underwent impedance aggregometry assessment.

#### *2.5. Statistical Analysis*

STATISTICA 13.1 (Dell Inc., Round Rock, TX, USA) was used to perform all statistical evaluations. The non-parametric Mann–Whitney U test was used to compare continuous variables. Categorical variables were compared with a Chi-squared test. Spearman's rank test was used to evaluate the correlations between the variables. The influence of platelet reactivity levels on stroke severity was performed with logistic regression analysis. In the present study, the statistical significance was defined as *p* < 0.05.

#### **3. Results**

#### *3.1. All Subjects*

There was no correlation between the platelet reactivity, assessed by Multiplate® and LTA methods, and the severity of the neurological deficit assessed using the NIHSS and functional status of the patients assessed on the mRS in the whole study group (Table 2). The comparison of the severity of neurological deficit (NIHSS) in patients with stroke assessed on the first, eighth, and 90th day after the stroke onset did not show significant differences between the subgroups of patients resistant and sensitive to aspirin (on the first day *p* = 0.8663, on the eighth day *p* = 0.9234, and on the 90th day *p* = 0.8225). There were no differences between the above groups regarding the functional status (mRS) of patients (on the first day *p* = 0.9808, on the eighth day *p* = 0.4610, and on the 90th day *p* = 0.5892).

In the present study, we found that the initial platelet reactivity assessed by Multiplate® was higher in patients with moderate/severe neurological deficits compared to a mild deficit on the 90th day after the stroke onset (median, respectively, 40 AUC units vs. 25 AUC units, *p* = 0.033) (Figure 1).

**Figure 1.** Comparison of platelet reactivity by Multiplate® (in area under the curve (AUC) units) in subgroups of patients with mild and moderate/severe neurological deficits on the 90th day in the general population of stroke patients.

However, there were no differences in the platelet reactivity between the groups distinguished on the basis of the severity of the deficit on the first and eighth day of stroke (on the first day *p* = 0.6599; on the eighth day *p* = 0.3271). The platelet reactivity assessed by Multiplate® did not differ between patients with a favorable and unfavorable prognosis on the first (*p* = 0.6455), eighth (*p* = 0.6744), and 90th day of the disease (*p* = 0.7414). The analysis of the relationship between the platelet reactivity in the LTA method and the clinical and functional status of stroke patients in the whole group of subjects showed no significant relationships (*p* > 0.05).

Logistic regression analysis showed that in the whole group of patients with stroke, aspirin-resistant subjects were 5.5 times more likely to have a severe neurological deficit on the 90th day of stroke than patients who were sensitive to aspirin; however, these differences did not reach statistical significance (odds ratio (OR) = 5.52, 95% confidence interval (CI) 0.54–56.86; *p* = 0.1506).

#### *3.2. Two Etiological Subgroups*

In the subgroups of patients with the pathology of large and small vessel disease, there were no statistically significant differences in the clinical and functional status of the stroke patients (NIHSS and mRS) (Table 1). In the subgroup of patients with large vessel disease, a significant correlation was found between the platelet reactivity assessed by Multiplate® and the functional status (mRS) on the first day of stroke (correlation coefficient (R) = 0.4526; *p* = 0.0451) (Figure 2, Table 2).

**Table 2.** Correlations of the clinical and functional conditions and platelet reactivity in both methods on individual days of stroke in the general population and in the subgroup of patients with large vessel disease.

Spearman's rank correlation. R, correlation coefficient; LTA, light transmission aggregometry; NIHSS, National Institute of Health Stroke Scale; mRS, modified Rankin scale.

Assessing the relationship between the aspirin resistance groups and the severity of clinical deficit in patients with large vessel disease, we found that the aspirin-resistant patients did not differ in the NIHSS scores from aspirin-sensitive patients (on the first day *p* = 0.06, on the eighth day *p* = 0.1167, and on the 90th day *p* = 0.0986). Assessing the relationship with the functional status in patients with large vessel disease, we found that patients with aspirin resistance achieved a higher median of points on mRS on the eighth day of the disease than patients sensitive to aspirin (*p* = 0.0352) (Figure 3).

There were no differences in the mRS scores on the first and 90th day of stroke (respectively, *p* = 0.0523 for the first day, *p* = 0.0631 for the 90th day). In the subgroup of patients with the pathology of small vessels there were no significant differences in the severity of the clinical deficit and the functional status between the groups distinguished on the basis of the presence or absence of aspirin resistance (*p* > 0.05).

Comparing the platelet reactivity in patients with moderate/severe (NIHSS ≥6 points) and mild neurological deficits (NIHHS <6 points), we found that in the subgroup of patients with the pathology of large vessels, the median of platelet reactivity in the Multiplate® method was higher than in the subgroup of patients with severe neurological deficit compared to mild deficit on the first day of the disease (respectively, median 58.5 vs. 23.5 AUC units; *p* = 0.0372) (Figure 4); this did not differ on the

eighth day (*p* = 0.0762), on the 90th day (*p* = 0.0982), or on particular days in the subgroup of patients with the pathology of small vessels (*p* > 0.05).

**Figure 2.** Correlation of platelet reactivity assessed by Multiplate® (in area under the curve (AUC) units) and functional status (modified Rankin scale (mRS) on the first day of stroke) in the subgroup of patients with large vessel disease (**A**) and small vessel disease (**B**).

Comparing the platelet reactivity in patients with favorable and unfavorable prognosis, the median of platelet reactivity in Multiplate® method did not differ between the above-mentioned groups both on the first, eighth, and 90th day of stroke in both subgroups (*p* > 0.05).

Logistic regression analysis showed that in the subgroup with large vessel disease, aspirin-resistant subjects had a 14 times greater probability of a severe neurological deficit on the first day of stroke than subjects sensitive to aspirin (OR = 14.00, 95% CI 1.25–156.12, *p* = 0.0322).

**Figure 3.** Comparison of the functional conditions (modified Rankin Scale (mRS)) on the eighth day of stroke in aspirin-resistant and aspirin-sensitive subjects in large vessel disease subgroup (**A**) and small vessel disease subgroup (**B**).

**Figure 4.** Comparison of platelet reactivity by Multiplate® (in AUC units) in the subgroup of patients with mild and moderate/severe neurological deficits on the first day of stroke in large vessel disease subgroup (**A**) and small vessel disease subgroup (**B**).

#### **4. Discussion**

In the present study, we demonstrated a significant role of ischemic stroke etiology for the prognostic value of high on-treatment platelet reactivity. We underline that the association between aspirin resistance and poor early clinical and functional conditions in ischemic stroke depends on the etiological subtype of the stroke. In the large vessel disease subgroup, we found a higher platelet reactivity in patients with severe neurological deficits on the first day of stroke, and that aspirin-resistant patients have a significantly higher probability of a severe clinical condition compared to aspirin-sensitive patients.

The division of patients according to the etiopathogenesis of stroke revealed a significant effect of platelet reactivity on the early functional condition. There was an average, though significant, correlation between platelet reactivity and functional status assessed on mRS on the first day of stroke, and patients resistant to aspirin had higher scores on the mRS (worse prognosis) on the eighth day of stroke than patients who were sensitive to aspirin. Both were present only in the large vessel disease subgroup. No similar results were demonstrated in the whole study population or in the small vessel disease subgroup.

These novel findings emphasize the great impact of stroke etiology on the association of high on-treatment platelet reactivity and poor early prognosis. However, it is difficult to refer the results of this research with other publications due to the heterogenous populations of the studied groups and different methodologies, and the lack of references in the literature on the impact of stroke etiopathogenesis on the relationship of platelet function testing with the prognosis of stroke subjects. Other authors did not assess the effect of stroke etiopathogenesis on the relationship of platelet reactivity with clinical and functional conditions.

In this study, there was no correlation between the platelet reactivity and the clinical condition in the whole group of subjects using the NIHSS on the first, eighth, and 90th day of stroke. In addition, the division into aspirin-resistant and -sensitive patients did not significantly differentiate the clinical status on particular days of the disease. Only the division of patients with mild to moderate/severe neurological deficits (according to the NIHSS score) revealed a significant effect on a higher initial platelet reactivity (within 24 h after onset) on the more severe clinical conditions, but this relationship was recorded only on the 90th day of stroke. Numerous authors showed that the aspirin-resistant group was characterized by a more severe early clinical condition, assessed on the NIHSS on the first day, than the group sensitive to aspirin [9–12]. Cheng et al. [12] indicated a significant correlation (R = 0.56) between platelet reactivity and the severity of neurological deficits assessed in the NIHSS. These results led to the conclusion that excessive platelet reactivity and aspirin resistance are associated with a worse clinical condition of patients in the acute phase of stroke. A different observation was demonstrated by Kim et al. [18] and Lai et al. [19], whose studies did not show the effect of platelet reactivity on the severity of the clinical condition assessed on the first day using the NIHSS. Similarly, Englyst et al. [20] did not find significant differences in the clinical status assessed on the NIHSS on the third day of stroke between the groups of patients resistant and sensitive to aspirin. The few literature reports on the long-term clinical conditions assessed on the 30th and 90th days of stroke showed contradictory results. Yip et al. [21] reported that aspirin-resistant patients were characterized by a worse clinical condition (NIHSS) both on the 30th and 90th days than patients sensitive to aspirin, while Lai et al. [19] did not find differences in the clinical condition (NIHSS) between these groups on the 90th day. We hypothesize that the contradictory conclusions from the presented studies may have resulted from omitting the important role of stroke etiology, as demonstrated in the current study.

The impact of platelet reactivity on the functional status of stroke patients is also debatable. Lai et al. [19] suggested a lack of relationship between platelet reactivity and early prognosis, as they did not reveal significant differences on the first day of stroke on mRS in the groups of patients sensitive and resistant to aspirin. On the other hand, Englyst et al. [20] showed a worse functional status of patients evaluated on mRS on the third day of stroke in aspirin-resistant patients compared to the aspirin-sensitive group (mRS median, respectively, 4 vs. 2, *p* = 0.013). Similar conclusions were reached by Sobol et al. [22] (mRS median 3 vs. 2, *p* = 0.02) assessing the functional status of patients on the 10th day of stroke. In our study, in the whole study population, there was no effect of platelet reactivity on the early prognosis—the functional status—on the first and eighth day of stroke or significant differences in the functional status of patients resistant and sensitive to aspirin. The differences in the results of this study from the data presented by other authors may be a result of including all etiological

types of stroke (e.g., embolic) and other methodologies (Englyst et al., hemostatic thromboelastography; Sobol et al., Platelet Function Assay (PFA-100)). In addition, according to the results of Amy et al. [23] and Wilterdink et al. [24], aspirin administration before ischemic stroke onset results in a better prognosis of patients evaluated on mRS. Englyst et al. [20] assessed only patients treated with aspirin (for at least three days) before the incident. In the study by Sobol et al. [22], there is no information on whether patients with stroke had previously taken aspirin or only from the first day of stroke. In the only paper regarding late prognosis, Lai et al. [19] showed that on the 90th day of stroke, in the group with aspirin resistance, subjects with a worse functional status were more often reported, rated on mRS at 3–5 points, than in the group sensitive to aspirin (*p* = 0.037). However, the current study did not show any significant effects of platelet reactivity on the late functional status (even considering the etiopathogenesis of stroke). It is worth noting that Lai et al. evaluated the platelet reactivity with the PFA-100 method and recruited patients with stroke who received a dose of 100 mg of aspirin at least five days before platelet reactivity testing, which may have resulted in different functional outcomes than in this study.

The present study and previous reports, despite various methodologies and inclusion and exclusion criteria of the study population, underline that high on-treatment platelet reactivity may have a negative impact on early and late prognosis and a significant association with poor clinical and functional outcomes in stroke subjects. The novelty demonstrated in this research emphasizes that stroke etiology may be a key factor for the above dependencies.

According to the results obtained in this study, carotid artery stenosis appears to be an essential platelet activating factor. Tsai et al. [25], who assessed platelet function using flow cytometry, also demonstrated significantly higher platelet aggregation in patients with large vessel pathology compared to small vessel pathology. Importantly, the study was conducted on a similar population of patients with cerebral ischemia to this study (i.e., they excluded patients with stroke due to the embolic background. Similar results were presented by Zheng et al. [11], Kinsella et al. [26], and Dawson et al. [27], who demonstrated that platelet reactivity assessed by different methods is significantly elevated in patients with carotid artery stenosis. Kinsella et al. [28], using the PFA-100, reported that surgical treatment (e.g., stenting) in stroke subjects due to a carotid artery stenosis was associated with a significant reduction of platelet activation. These results were consistent with our current findings, highlighting the role of large vessel disease etiology for ischemic stroke in increasing platelet reactivity. These results indicate that platelet function monitoring may be useful for stroke subjects due to carotid artery stenosis. Additionally, platelet-function-guided individualized antiplatelet therapy can be essential to optimize clinical outcomes and to improve the functional status.

Unfortunately, both the American and European guidelines for the treatment and prevention of stroke do not distinguish between antiplatelet therapy and stroke pathomechanisms. Regardless of whether it is lacunar stroke or stroke due to a pathology of large extracranial vessels, aspirin administration is recommended for all patients with thrombotic stroke [2]. The current guidelines do not address the issue of aspirin resistance. It seems that this may be due to the lack of large, randomized clinical trials that could be used to develop clear guidelines.

As stroke in large extracranial pathology accounts for a fairly significant proportion of all strokes, and the results of current and previous studies highlight the significant impact of carotid artery pathology on platelet reactivity relationships with worse clinical conditions and prognoses, we recommend routinely determining platelet reactivity and detecting aspirin resistance, especially in cases of recurrent ischemic events. The authors believe that this would allow for personalized antiplatelet treatment based on platelet function testing, whose effectiveness for this group of patients is a priority.

The authors are aware that this study has several limitations. The evaluation of platelet function was performed only once and at different times during the first 24 h after the onset of stroke and at different times after the first dose of acetylsalicylic acid. It could have contributed to the variations in the measurements of platelet function. A single measurement with poorly validated methods in light of the marked variability of platelet reactivity that was previously demonstrated, may not be sufficient to properly assess the effect of high on-treatment platelet reactivity on the clinical condition and prognosis in ischemic stroke. More work is essential to sequentially determine platelet reactivity on successive days. Another limitation is that biochemical resistance does not always correspond with clinical resistance. The sample size in the study was small and imbalanced between the two etiological subgroups. The lack of recruitment of patients with severe stroke (especially those with impaired consciousness), due to the inability to obtain informed consent, constitutes a huge limitation of the study. Despite using stringent inclusion and exclusion criteria, in the face of low rates of inhospital atrial fibrillation detection, there is a possibility that a small percentage of subjects may have had another etiology of stroke, such as embolism.

#### **5. Conclusions**

This pilot study demonstrated that high on-treatment platelet reactivity is associated with a worse late prognosis in ischemic stroke. In patients with large vessel disease, high platelet reactivity is associated with a worse early prognosis and clinical and functional condition of patients in the acute phase of stroke. The role of etiology demonstrated in this paper is novelty. However, due to the pilot nature of this study, the obtained results should be interpreted with caution. Further research, performed on larger sample size, is essential to validate and confirm our findings and to determine the optimal and personalized antiplatelet therapy.

**Author Contributions:** Conceptualization, A.W.; methodology, A.W., G.K., and J.S.; software, J.S.; validation, G.K.; formal analysis, A.W., K.F., and G.K.; investigation, A.W.; resources, J.S.; data curation, J.S..; writing—original draft preparation, A.W. and K.F.; writing—review and editing, A.W.; visualization, A.W.; supervision, G.K. and R.S.; ´ project administration, A.W. and G.K. All authors have read and agreed to the published version of the manuscript.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## **To Treat or Not to Treat: Importance of Functional Dependence in Deciding Intravenous Thrombolysis of "Mild Stroke" Patients**

**Giovanni Merlino 1,\*,**†**, Carmelo Smeralda 2,3,**†**, Simone Lorenzut 1, Gian Luigi Gigli 2,4, Andrea Surcinelli 2,3 and Mariarosaria Valente 2,3**

Received: 15 February 2020; Accepted: 10 March 2020; Published: 12 March 2020

**Abstract:** Intravenous thrombolysis (IVT) in patients with a low National Institutes of Health Stroke Scale (NIHSS) score of 0–5 remains controversial. IVT should be used in patients with mild but nevertheless disabling symptoms. We hypothesize that response to IVT of patients with "mild stroke" may depend on their level of functional dependence (FD) at hospital admission. The aims of our study were to investigate the effect of IVT and to explore the role of FD in influencing the response to IVT. This study was a retrospective analysis of a prospectively collected database, including 389 patients stratified into patients receiving IVT (IVT+) and not receiving IVT (IVT <sup>−</sup>) just because of mild symptoms. Barthel index (BI) at admission was used to assess FD, dividing subjects with BI score < 80 (FD+) and with BI score <sup>≥</sup> 80 (FD−). The efficacy endpoints were the rate of positive disability outcome (DO+) (3-month mRS score of 0 or 1), and the rate of positive functional outcome (FO+) (mRS score of zero or one, plus BI score of 95 or 100 at 3 months). At the multivariate analysis, IVT treatment was an independent predictor of DO<sup>+</sup> (OR 3.12, 95% CI 1.34−7.27, *<sup>p</sup>* <sup>=</sup> 0.008) and FO<sup>+</sup> (OR: 4.70, 95% CI 2.38−9.26, *p* = 0.001). However, FD<sup>+</sup> IVT<sup>+</sup> patients had a significantly higher prevalence of DO<sup>+</sup> and FO<sup>+</sup> than those FD<sup>+</sup> IVT–. Differently, IVT treatment did not influence DO<sup>+</sup> and FO<sup>+</sup> in FD– patients. In FD<sup>+</sup> patients, IVT treatment represented the strongest independent predictor of DO<sup>+</sup> (OR 6.01, 95% CI 2.59–13.92, *p* = 0.001) and FO<sup>+</sup> (OR 4.73, 95% CI 2.29–9.76, *p* = 0.001). In conclusion, alteplase seems to improve functional outcome in patients with "mild stroke". However, in our experience, this beneficial effect is strongly influenced by FD at admission.

**Keywords:** intravenous thrombolysis; NIHSS; Barthel index; functional dependence

#### **1. Introduction**

Many patients with acute ischemic strokes (AIS) have a low National Institutes of Health Stroke Scale (NIHSS) score at presentation [1,2]. Although the presence of these mild symptoms represents the most common reason for renouncing intravenous thrombolysis (IVT) [3], only 68% of these patients can be discharged home without a residual disability [4]. Thus, there is increasing interest in the use of IVT in AIS patients with a low NIHSS score at admission. Results coming from clinical studies on this topic are conflicting, since functional outcome results, as assessed by the modified Rankin scale (mRS) sometimes improve, and at other times, are not modified by IVT treatment [5–10].

Previous American Heart Association/American Stroke Association guidelines suggested to use IVT treatment in persons with a *wide spectrum* of neurological deficits (1996) and with *measurable* neurological deficits (2007) [11,12]. This concept has been updated in the most version of the guidelines, recommending that IVT should also be used in patients with mild, but nevertheless disabling symptoms [13]. However, the NIHSS is not able to assess severity of disability. For instance, it cannot be used to accurately assess posterior circulation disease, which may cause very disabling symptoms. In fact, already in 2013, Wendt et al. reported that language impairment, distal paresis, and gait disorder were common disabling deficits in patients with low NIHSS scores. The authors suggest that the judgment of whether a stroke is disabling should not be based on the NIHSS score, but on the assessment of individual neurologic deficits and their impact on functional impairment [14].

To date, only a trial has been performed to compare the efficacy of alteplase versus aspirin for AIS patients with minor and non-disabling neurological deficits (the PRISMS trial). The authors observed that alteplase did not increase the likelihood of favorable outcome compared to aspirin [15,16]. Although the PRISMS was a prospective, double-blind, and placebo-controlled trial, it suffered from two significant limitations. In fact, the study was terminated early because patient recruitment was below target and it adopted a definition of "not clearly disabling" that was subjective and required interpretation by individual clinicians. Thus, conclusions of the PRISMS trial cannot be generalized.

A possible reason for the uncertain effectiveness of alteplase in minor strokes is that patients with a low NIHSS score at admission may respond to IVT treatment in different ways, depending on their level of functional dependence (FD) at admission. In addition, we suggest that the severity of FD should be assessed by a standard measure, such as the Barthel index (BI), instead of using a subjective selection based on the judgment of each physician. The aims of our study were: (1) to investigate the effects of IVT in patients with "mild stroke", defined as a NIHSS score of 0−5 at presentation; (2) to explore the role of FD in influencing response to IVT in AIS patients with "mild stroke".

#### **2. Materials and Methods**

#### *2.1. Patients*

This study was based on a retrospective analysis of a prospectively-collected database of consecutive patients admitted to the Udine University Hospital for AIS from January 2015 to December 2018. Inclusion criteria were: age 18 years or older and NIHSS score of 0 to 5. Exclusion criteria were: presence of a pre-stroke mRS score > 1, large vessel occlusion on cranial CT-angiography, and time interval > 4.5 h from symptoms onset. Out of 1636 patients admitted for AIS, 389 were considered suitable for the study after considering inclusion and exclusion criteria. The study sample was stratified into 2 groups: AIS patients who received IVT (IVT+) and patients to whom IVT was denied because of mild symptoms (IVT−).

#### *2.2. Data Collection*

The following variables were collected: age, sex, vascular risk factors such as previous transient ischemic attack or previous stroke, ischemic heart disease, peripheral artery disease, obesity defined as a BMI ≥ 30, atrial fibrillation, hypertension, diabetes mellitus, hypercholesterolemia, current smoking status, and pharmacological treatment. Stroke severity was determined with the NIHSS at admission. Presence of intracranial hemorrhage (ICH) was detected. Definition of symptomatic ICH (sICH) was based on the European Cooperative Acute Stroke Study (ECASS) III protocol [17]. Functional outcome was assessed by means of the mRS score 3 months after the stroke, and of the BI score, calculated at admission and recalculated at 3-months. The mRS and the BI scores after discharge were recorded at the patients' routine clinical visit during a face-to-face examination.

#### *2.3. Outcome Measures*

Our efficacy endpoints were: (1) rate of positive disability outcome (DO+), defined as a 3-month mRS score of 0 or 1; (2) rate of positive functional outcome (FO+), defined as an mRS score of 0 or 1 *plus* a BI score of 95 or 100 at 3 months. The safety endpoints were: (1) rate of mortality at 3 months; (2) presence of sICH.

#### *2.4. Statistical Analysis*

Baseline characteristics and outcomes of the two patient groups (IVT<sup>+</sup> versus IVT−) were compared by means of the chi-square test (Fisher's exact test) for categorical variables and the Student's *t*-test for independent samples when the continuous variables had a normal distribution.

TheMann–Whitney U testwas usedwhen the continuous variables had an abnormal distribution and for ordinal variables. Binary logistic regression was used to explore variables associated with outcome measures.

In order to explore whether there was a significant interaction between the types of presenting symptoms (according to the Barthel index) and the efficacy of thrombolysis, both patients treated and not treated with IVT were differentiated as: (1) patients without FD; (2) patients with FD predominantly due to weakness; (3) patients with FD predominantly due to imbalance; (4) patients with FD predominantly due to neglect and/or hemianopsia; (5) patients with FD predominantly due to other neurological symptoms, e.g., aphasia and confusion.

With the aim to verify if the level of FD at admission might influence response to IVT in AIS patients with "mild stroke", we divided our sample into subjects with a BI score < 80 (FD+) and those with a BI score <sup>≥</sup> 80 (FD−). We tested this hypothesis comparing the efficacy endpoints between FD<sup>+</sup> and FD– patients, treated and not treated with IVT.

Data are displayed in tables as means and standard deviations (SD), if not otherwise specified. All probability values are two-tailed. A *p* value of < 0.05 was considered to be statistically significant. Statistical analysis was carried out using the SPSS Statistics, Version 20.0 for Windows (Chicago, IL, USA).

#### **3. Results**

Our sample of 389 patients was composed of 235 males (60.4%) with a mean age of 68.5 ± 13.6 years, a median NIHSS score of 2 (IQR 1−3), and a median BI score of 75 (IQR 60-90). Almost one-half (51.7%) of our patients with "mild stroke" had a BI score < 80 at admission (FD<sup>+</sup> patients). Figure 1 shows the distribution of the BI score at admission in our sample.

**Figure 1.** Barthel index score distribution in our sample.

Of the 389 enrolled patients with "mild stroke", 113 (29%) were treated with IVT (IVT+), whereas in 276 (71%), IVT was denied because of mild symptoms (IVT−). Baseline characteristics in IVT<sup>+</sup> and IVT– patients are summarized in Table 1. The two groups differed only in median NIHSS score and use of anticoagulants.

IVT = intravenous thrombolysis; NIHSS = National Institute of Health Stroke scale; BI = Barthel index.

At 3-months, IVT treatment improved both measures of outcome (DO<sup>+</sup> and FO+). Although IVT<sup>+</sup> patients showed higher rates of sICH than those IVT–, the prevalence of 3-month mortality did not differ between the two groups (see Table 2).

**Safety endpoints**

**Table 2.** Efficacy and safety endpoints in patients treated and not treated with IVT.

IVT = intravenous thrombolysis; DO<sup>+</sup> (3-month mRS score of 0 or 1) = positive disability outcome; FO<sup>+</sup> (mRS score of 0 or 1, *plus* BI score of 95 or 100 at 3-months) = positive functional outcome; sICH = symptomatic intracranial hemorrhage.

Mortality at 3-months, n (%) 1 (0.9) 3 (1.1) 0.8 sICH, n (%) 5 (4.4) 3 (1.1) 0.03

By univariate analysis, apart from IVT treatment, a positive disability outcome (DO+) was also associated with younger age (OR for 1-year increment in age 0.98, 95% CI 0.96–0.99, *p* = 0.02), lower NIHSS score at admission (OR 0.72 for 1-point increase in the scale, 95% CI 0.60–0.85, *p* = 0.001), higher BI score at admission (OR 1.06, 95% CI 1.05–1.08, *p* = 0.001), lower serum glucose at admission (OR 0.98 for each mg/dl increase in glucose, 95% CI 0.97–0.99, *p* = 0.01), a history of previous transient ischemic attack/stroke (OR 0.42, 95% CI 0.24–0.75, *p* = 0.003), obesity (OR 0.41, 95% CI 0.19–0.91, *p* = 0.02), and of diabetes mellitus (OR 0.45, 95% CI 0.27–0.77, p = 0.003). Regarding instead positive functional outcome (FO+), younger age (OR for 1-year increment in age 0.98, 95% CI 0.96–0.99, *p* = 0.008), lower NIHSS score at admission (OR 0.72 for 1-point increase in the scale, 95% CI 0.61–0.85, *p* = 0.001), higher BI score at admission (OR 1.06, 95% CI 1.04–1.07, *p* = 0.001), a history of previous transient ischemic attack/stroke (OR 0.45, 95% CI 0.26–0.80, *p* = 0.005), hypertension (OR 0.61, 95% CI 0.38–0.98, *p* = 0.04), and diabetes mellitus (OR 0.52, 95% CI 0.31–0.87, *p* = 0.01) were related to FO<sup>+</sup> at 3 months.

By multivariate analysis, after controlling for variables significantly associated with the two efficacy endpoints at the univariate analysis, IVT treatment remained an independent predictor of DO<sup>+</sup> and FO<sup>+</sup> in patients with "mild stroke" (see Table 3).

**Table 3.** Multivariable analyses showing independent predictors of positive disability outcome and positive functional outcome.

DO<sup>+</sup> (3-month mRS score of 0 or 1) = positive disability outcome; FO<sup>+</sup> (mRS score of 0 or 1 *plus* BI score of 95 or 100 at 3-months) = positive functional outcome; IVT = intravenous thrombolysis; NIHSS = National Institute of Health Stroke scale; BI = Barthel index.

A beneficial effect of IVT was observed only in patients with FD predominantly due to weakness who significantly improved after treatment (DO+: OR 4.88, 95% CI 2.01–11.83, *p* = 0.001; FO+: OR 5.03, 95% CI 2.23–11.32, *p* = 0.001), different from patients without FD, or with other types of presenting symptoms (data not shown).

As shown in Figure 2, the prevalence of DO<sup>+</sup> was significantly higher in FD<sup>+</sup> IVT<sup>+</sup> patients than in those FD<sup>+</sup> IVT – (FD<sup>+</sup> IVT+: 83.9% vs. FD<sup>+</sup> IVT–: 52.5%, *p* = 0.001); differently, IVT treatment did not influence DO<sup>+</sup> in FD– patients (FD– IVT+: 90.2% vs. FD– IVT–: 89.1%, p = 0.8). Similarly, for functional outcome, FO<sup>+</sup> was significantly more common in FD<sup>+</sup> IVT<sup>+</sup> patients than in those FD<sup>+</sup> IVT– (FD<sup>+</sup> IVT+: 77.4% vs. FD<sup>+</sup> IVT–: 44.6%, *p* = 0.001), whereas rates of FO<sup>+</sup> were similar in FD– IVT<sup>+</sup> and FD– IVT– patients (90.2% vs. 85.4%, *p* = 0.4) (see Figure 3).

**Figure 2.** Effect of IVT on disability outcome rates in groups of patients with different levels of functional dependence at admission. DO = disability outcome; FD = functional dependence; IVT = intravenous thrombolysis.

**Figure 3.** Effect of IVT on functional outcome rates in groups of patients with different levels of functional dependence at admission. FO= functional outcome; FD = functional dependence; IVT = intravenous thrombolysis.

In FD<sup>+</sup> patients, the following variables were independent predictors of outcome: IVT treatment (OR 6.01, 95% CI 2.59–13.92, *p* = 0.001), BI score at admission (OR 1.07, 95% CI 1.04–1.10, *p* = 0.001), a history of previous transient ischemic attack/stroke (OR 0.41, 95% CI 0.18–0.92, *p* = 0.03), and diabetes mellitus (OR 0.42, 95% CI 0.18–0.94, *p* = 0.04) for DO+; IVT treatment (OR 4.73, 95% CI 2.29–9.76, *p* = 0.001), BI score at admission (OR 1.04, 95% CI 1.02–1.07, *p* = 0.001), and a history of previous transient ischemic attack/stroke (OR 0.44, 95% CI 0.20–0.96, *p* = 0.04) for FO+. In contrast, IVT treatment did not affect functional outcome in FD– patients; in fact, BI score at admission was the only independent predictor of DO<sup>+</sup> (OR 1.22, 95% CI 1.07–1.39, *p* = 0.002), and FO<sup>+</sup> (OR 1.13, 95% CI 1.05–1.21, *p* = 0.001).

#### **4. Discussion**

For the first time we demonstrated that patients with "mild stroke", as defined as a NIHSS score of 0−5, should be selected for IVT on the basis of their level of FD at admission. In particular, subjects with moderate or severe FD, as assessed by the BI score, should be treated with IVT as soon as possible. In contrast, treatment with alteplase seems to be ineffective on patients who are functionally independent or with slight FD at admission. Thus, our study gives support to the latest American Heart Association/American Stroke Association guidelines recommending that IVT should be used for patients with mild but also disabling symptoms [13].

There is ongoing debate concerning what is a "mild stroke". In 2010, Fisher et al. explored the relationship of 6 different "minor stroke" definitions and outcomes. Since patients with a NIHSS score ≤ 3 had the best short- and medium-term outcome, the authors suggested to use this easily-applicable definition [18]. Although this definition has been used in some studies [19,20], a recent review of this topic reported that the NIHSS—with a score ranging from 0 to 5—is the most commonly used tool to define a "mild stroke" [21]. Similarly, we utilized an NIHSS score of 0 to 5 for identifying patients with supposed "mild" symptoms; our patients had a median NIHSS score of 2. However, despite their low NIHSS, several of them were affected by severe FD at admission; in fact, we observed a median BI score of 75, and 51.7% of the sample had a BI score < 80. This discrepancy may be due to the fact that the NIHSS is not able to detect symptoms of posterior circulation stroke, such as postural instability, gait disturbance, and dysphagia that can cause very severe disability. Thus, we think that patients with a low NIHSS score at presentation should be carefully evaluated regarding the presence of possible disabling symptoms before being labeled as affected by "mild stroke".

Originally published in 1965, the BI was developed to give physicians a suitable standard tool to assess and measure FD [22]. In fact, the BI covers all activities considered part of any assessment of activities of daily living, has an excellent reliability and validity, is easy to use, and only takes a few minutes [23]. Thus, we suggest adopting this tool in patients with "mild stroke", in order to correctly recognize patients with non-disabling symptoms.

Obviously, the exact distinction between stroke with disabling or non-disabling symptoms becomes extremely important when patients are affected by AIS and are suitable for IVT treatment. In our sample, more than 70% of AIS patients were not treated with IVT because they were deemed too good to be treated. This rate is perfectly in line with previous studies on this topic [6,9,10]. As shown in Table 1, the decision to treat or not to treat with IVT was merely based on the NIHSS score, whereas the level of FD was absolutely neglected. Use of anticoagulant agents was, as expected, significantly higher in AIS patients with "mild stroke" who were not treated, than in those who received IVT.

Previous studies on IVT treatment in AIS patients with mild symptoms report conflicting results [5–10]. In 2012 Huisa et al. investigated 133 patients with minor ischemic strokes, defined as an admission NIHSS score ≤ 5, and observed similar outcomes between patients treated and not treated with alteplase [5]. An Italian study of 128 patients with mild ischemic stroke confirmed that alteplase did not improve functional outcome [6]. Of 276 patients with mild ischemic stroke symptoms that were analyzed by Spokoyny et al., 83 were IVT treated. Treated and untreated patients had similar baseline characteristics except that the treated group had higher baseline NIHSS. Prevalence of mRS 2−6 at 90 days was 37.4% in the treated group and 31.1% in the untreated group (*p* = 0.44) [9]. In contrast, Urra et al. reported that IVT

was associated with a greater proportion of patients with mild stroke who shifted down on the mRS score at 3 months (OR 2.66; 95% CI 1.49–4.74, *p* = 0.001) [7]. In a case-control study of 890 Austrian patients with a NIHSS score 0−5 at admission, IVT was associated with a better outcome after 3 months (OR 1.49, 95% CI 1.17–1.89, *p* < 0.001) [8]. More recently, Haeberlin et al. compared 3-month functional outcomes in 370 consecutive AIS patients with a NIHSS score ≤ 6. Although patients with mild AIS had a high chance of favorable outcomes irrespective of treatment type, subjects receiving IVT more often achieved complete remission of symptoms (mRS score = 0) (OR 3.33, *p* < 0.0001) [10]. Similarly to Urra et al. [7] and Haeberlin et al. [10], we observed a major beneficial effect of IVT on the outcome measures. Interestingly, it would seem that IVT efficacy is more pronounced in AIS patients affected by "mild stroke" than in those enrolled in regulatory randomized controlled trials, in which patients with minor symptoms were largely underrepresented [24]. We think that this discrepancy may be due to different study design between observational studies and randomized controlled trials. In fact, more often, non-interventional studies tend to overestimate the effects of the treatment and show more variability in estimates of the effects because of residual confounding, errors, and bias.

Discording results of IVT efficacy in "mild strokes" may be explained by differences in clinical characteristics among patients with minor symptoms. In particular, our patients who underwent IVT had a better 3-month functional outcome than those IVT–, but presence of neurological symptoms due to weakness and level of FD at admission played a major role in influencing this association. If patients with "mild stroke" *plus* disabling symptoms (FD+) were treated with alteplase, there was a significant improvement in functional outcome compared to those which were not treated. Indeed, more than 50% of patients with a BI score < 80 for whom IVT was denied did not achieve functional independence 3 months after stroke. In patients with "mild stroke" *plus* disabling symptoms, IVT represented the strongest predictor of DO<sup>+</sup> (OR: 6.01) and FO<sup>+</sup> (OR: 4.73). On the other hand, rates of favorable outcomes were very high in patients without disability, regardless of treatment type. In these patients, BI score at admission was the only independent predictor of DO<sup>+</sup> and FO+, while IVT treatment did not influence functional outcomes. In contrast, Urra et al. report that IVT was associated with a greater proportion of patients with non-disabling minor strokes who shifted down on the mRS score at 3-months [7].

To date, only the PRISMS trial has been designed to assess the efficacy of IVT for the treatment of AIS with NIHSS 0−5, and without clearly disabling deficits. The authors designed a multicenter, randomized, double-blind, placebo-controlled trial with a sample size of 948 subjects [15]. Unfortunately, the study was terminated early because of low patient recruitment. Results of the 313 patients enrolled failed to demonstrate more favorable functional outcomes in patients treated with alteplase, as compared to those receiving only aspirin. However, the trial's early termination precludes any definitive conclusions on this topic. Moreover, definition of "not clearly disabling" was left to the subjective interpretation of individual clinicians [16].

Regarding safety endpoints, in our sample, alteplase treatment significantly increased the risk of sICH, even if rates of mortality were similar in patients IVT<sup>+</sup> and IVT–. Bearing in mind that higher NIHSS scores predict a higher rate of sICH, it could be argued that our sICH rate in patients with "mild stroke" was high. However, a previous study performed in patients with minor stroke reported a sICH rate as high as 5% when IVT was administered [5].

Several limitations of this study need to be acknowledged. First, the retrospective design of our study was certainly a limit; however, all data were prospectively collected. Second, measures of outcome were obtained by physicians that were not blinded to IVT treatment, which may have influenced their rating. Third, information on intervals between stroke onset and IVT was not collected, thus we cannot exclude that elapsed time from symptoms onset may have influenced physicians' decisions to perform or not perform IVT treatment. Finally, since this was a hypothesis-generating study, further surveys are needed to test our preliminary hypotheses. In particular, interventional trials should be performed in order to exclude the presence of a bias by indication that could have affected our observational study.

In conclusion, alteplase seems to improve functional outcome in patients with a low NIHSS score. However, in our experience, this beneficial effect is strongly influenced by FD at admission. In patients with "mild stroke" *plus* disabling symptoms, IVT treatment should be administered as soon as possible; on the contrary, alteplase may not be used if minor and non-disabling deficits are diagnosed. In order to distinguish mild ischemic stroke patients with disabling or non-disabling symptoms, we suggest to use the BI. Our observational study brings further evidence to the results coming from a few other non-interventional studies and from one randomized trial interrupted before completion. Thus, further large interventional studies are needed to confirm our preliminary findings.

**Author Contributions:** Conceptualization, G.M. and C.S.; methodology, G.M. and C.S.; software, G.M. and C.S.; validation, S.L., G.L.G. and M.V.; formal analysis, G.M.; investigation, C.S., S.L. and A.S.; resources, C.S., S.L. and A.S.; data curation, G.M.; writing—original draft preparation, G.M.; writing—review and editing, G.M.; visualization, G.L.G; supervision, M.V. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

#### *Article*

## **Improving the Clinical Outcome in Stroke Patients Receiving Thrombolytic or Endovascular Treatment in Korea: from the SECRET Study**

**Young Dae Kim 1, Ji Hoe Heo 1, Joonsang Yoo 1,2, Hyungjong Park 1,2, Byung Moon Kim 3, Oh Young Bang 4, Hyeon Chang Kim 5, Euna Han 6, Dong Joon Kim 3, JoonNyung Heo 1, Minyoung Kim 1, Jin Kyo Choi 1, Kyung-Yul Lee 7, Hye Sun Lee 8, Dong Hoon Shin 9, Hye-Yeon Choi 10, Sung-Il Sohn 2, Jeong-Ho Hong 2, Jang-Hyun Baek 11,12, Gyu Sik Kim 13, Woo-Keun Seo 4, Jong-Won Chung 4, Seo Hyun Kim 14, Tae-Jin Song 15, Sang Won Han 16, Joong Hyun Park 16, Jinkwon Kim 7,17, Yo Han Jung 18, Han-Jin Cho 19, Seong Hwan Ahn 20, Sung Ik Lee 21, Kwon-Duk Seo 13,21 and Hyo Suk Nam 1,\***

Received: 23 January 2020; Accepted: 2 March 2020; Published: 6 March 2020

**Abstract:** We investigated whether there was an annual change in outcomes in patients who received the thrombolytic therapy or endovascular treatment (EVT) in Korea. This analysis was performed using data from a nationwide multicenter registry for exploring the selection criteria of patients who would benefit from reperfusion therapies in Korea. We compared the annual changes in the modified Rankin scale (mRS) at discharge and after 90 days and the achievement of successful recanalization from 2012 to 2017. We also investigated the determinants of favorable functional outcomes. Among 1230 included patients, the improvement of functional outcome at discharge after reperfusion therapy was noted as the calendar year increased (*p* < 0.001). The proportion of patients who were discharged to home significantly increased (from 45.6% in 2012 to 58.5% in 2017) (*p* < 0.001). The successful recanalization rate increased over time from 78.6% in 2012 to 85.1% in 2017 (*p* = 0.006). Time from door to initiation of reperfusion therapy decreased over the years (*p* < 0.05). These secular trends of improvements were also observed in 1203 patients with available mRS data at 90 days (*p* < 0.05). Functional outcome was associated with the calendar year, age, initial stroke severity, diabetes, preadmission disability, intervals from door to reperfusion therapy, and achievement of successful recanalization. This study demonstrated the secular trends of improvement in functional outcome and successful recanalization rate in patients who received reperfusion therapy in Korea.

**Keywords:** reperfusion; therapy; ischemic stroke; outcome

#### **1. Introduction**

Stroke is one of the diseases with the highest burden worldwide. Although the age-standardized risk of stroke or case fatality has been improving, there is still an increase in the absolute number of stroke or stroke-related death [1]. The Global Burden of Disease Study demonstrated that the burden of cerebrovascular disease increased over several decades and ranked second in the highest burden of diseases in 2015 [2].

Intravenous tissue plasminogen activator (IV t-PA) therapy and endovascular treatment (EVT) are established treatments for eligible patients with acute ischemic stroke [3,4]. Although these modalities can lead to successful recanalization, which is a strong determinant of a good outcome [5], many patients who received reperfusion therapy did not achieve a favorable outcome [6]. Over the past decades, there has been an improvement in the stroke care program, imaging techniques, treatment devices, and experience in EVT. As a result, overall outcome of reperfusion therapy for acute ischemic stroke could be improved at a national level [7]. In Korea, there have been improvements in the care system for acute stroke patients, including easy and rapid accessibility to medical services, establishment of stroke units or centers, and acute stroke codes for reperfusion therapy [8–10]. Considering these secular trends in the stroke care system, clinical and radiologic outcomes after reperfusion therapy might have changed in Korea.

We investigated whether there was an annual change in outcomes in patients who received IV t-PA therapy or EVT in Korea. We also determined which factors had played a role in these changes using the nationwide thrombolytic and EVT registry.

#### **2. Materials and Methods**

#### *2.1. Patients Inclusion*

The study population was derived from the Selection Criteria in Endovascular Thrombectomy and thrombolytic therapy (SECRET) registry (Clinicaltrials.gov NCT02964052, https://clinicaltrials.gov/ct2/ show/NCT02964052?term=NCT02964052&rank=1). The SECRET registry is a nationwide multicenter registry for exploring the selection criteria of patients who would benefit from reperfusion therapies. The SECRET registry was started on May 2016. This registry consisted of four parts: (1) clinical information, (2) information on reperfusion therapy, (3) comorbidities, and (4) imaging data.

The clinical information section includes the demographics, vascular risk factors, previous medication status, laboratory findings, and neurologic status or premorbidity before stroke. In the reperfusion therapy section, the information on time parameters, angiographic findings before and after treatment, devices used during the procedure, periprocedural complications, and concomitant thrombolytic agents used was collected. The modified Rankin scale (mRS) at discharge and after 90 days, along with mortality within 6 months, was determined in each patient during the follow-up. If a patient died, we also assessed the cause of death.

For the comorbidities section, we determined the presence of the component of the Charlson comorbidity index (CCI) for each patient. In the stroke population, we used a modified version of the CCI, which consisted of 19 diseases, including myocardial infarction, congestive heart failure, peripheral vascular disease, previous stroke, atrial fibrillation, dementia, depression, chronic pulmonary disease, ulcer disease, mild liver disease, moderate or severe renal disease, connective tissue disease or rheumatic disease, anemia, diabetes, acquired immune deficiency syndrome, cancer, leukemia, lymphoma, and metastatic cancer [11].

The imaging data section included the occlusion site, infarction core, collateral status, and thrombus characteristics on thin-section computed tomography (CT). The imaging findings were ascertained by the imaging adjudication committee (6 stroke neurologists and 4 neuroradiologists). The audit was conducted every two weeks, and the data management center verified the completeness and accuracy of the data. For this study, we used the demographics, vascular risk factors, underlying vascular diseases, time parameters, occlusion site, angiographic findings before and after treatment, and functional outcome variables.

This registry included 1026 patients who had been registered retrospectively from 15 hospitals between January 2012 and December 2015 and 333 patients who had been registered prospectively from 13 hospitals between November 2016 and December 2017. For prospectively-enrolled patients, written informed consent was obtained from patients or the next of kin. This registry was approved by the institutional review board in each participating hospital.

#### *2.2. Reperfusion Therapy*

IV t-PA and EVT was used in patients who met the criteria based on current guidelines. IV t-PA (Actilyse; Boehringer-Ingelheim, Ingelheim, Germany) was used in patients who had a stroke within 4.5 h from symptom onset and met the criteria based on current guidelines with a standard dose (0.9 mg/kg) [12,13]. If patients had large vessel occlusion on initial angiographic studies and could be treated within 8 h from symptom onset, EVT was considered. The EVT was performed primarily using mechanical devices rather than chemical agents. Among the mechanical devices, Solitaire stent retriever (Medtronic Neurovascular, Iirvine, CA, USA), Trevo retriever (Stryker Neurovascular, Fremont, CA, USA), or Penumbra reperfusion catheter (Penumbra, Alameda, CA, USA) was available in Korea and used based on target vessel site, tortuosity, or neurointerventionalist's preference. Intra-arterial thrombolysis with urokinase (Green Cross, Seoul, Korea) or glycoprotein IIb/IIIa antagonists was used as an adjuvant therapy in certain cases including those with re-occlusion or distal embolization.

If the onset of symptom was unclear, EVT was performed based on imaging findings and physician's discretion. Brain magnetic resonance imaging and magnetic resonance angiography were performed 24 h after reperfusion therapy. When brain MRI could not be performed, brain CT and/or CT angiography was performed. During hospitalization, each patient was treated on the basis of current stroke guidelines [14,15].

#### *2.3. Outcome Measures*

In this study, functional outcome was assessed with mRS at discharge and after 90 days. Favorable functional was defined as having mRS score of 0–2 and excellent functional outcomes was defined as mRS score of 0–1. In terms of radiologic outcomes, successful recanalization was determined using digital subtraction angiography (DSA), CT angiography (CTA), or magnetic resonance angiography (MRA). In this study, successful recanalization was defined as thrombolysis in cerebral infarction grade of 2b or 3 on final DSA among patients with EVT [16]. In patients who received IV t-PA only, successful recanalization was defined as arterial occlusive lesion (AOL) scoring of 3 on CTA or MRA performed within 24 h. Symptomatic intracerebral hemorrhage was defined as having any type of hemorrhage causing neurologic deterioration with National Institutes of Health Stroke Scale (NIHSS) score ≥4 or leading to death or surgery within 7 days of stroke onset based on the criteria in the European Cooperative Acute Stroke Study (ECASS) III trial [12].

#### *2.4. Statistical Analysis*

When we compared the baseline characteristics according to the calendar year, Student's independent *t*-test was used to compare age, time interval, and laboratory findings, and Pearson's χ<sup>2</sup> test or Fisher's exact test was used in the analysis of categorical data. Wilcoxon rank sum test was used to compare baseline NIHSS scores. Because the number of patients who received reperfusion therapy in December 2016 and registered in the SECRET registry was small, these patients were merged into the patient group treated in 2017 for this analysis. When we investigated the trends of outcomes by year, linear-by-linear or Jonckheere-Terpstra test was used for the analysis. To determine the independent predictors of outcomes, logistic regression or ordinal regression analysis was used. A multivariable analysis was performed using all variables with a *p*-value < 0.1 in the univariable analysis. All *p*-values were two-sided, and a *p*-value < 0.05 was considered statistically significant. All statistical analysis was performed using Windows SPSS package (version 23.0, IBM Corp., Armonk, NY, USA) and R version 3.2.1 (R Foundation for Statistical Computing, Vienna, Austria, http://www.R-project.org).

#### **3. Results**

#### *3.1. Baseline Characteristics*

Between January 2012 and December 2017, a total of 1359 patients who received reperfusion therapy with either IV t-PA therapy or EVT were registered. First, we excluded 38 patients who received intra-arterial chemical thrombolytic treatment as primary therapeutic modality. Then, we also excluded 86 patients who had in-hospital ischemic stroke, and nine patients who were transferred to the study hospital from other local hospitals ("drip-and-ship" case) because there were insufficient data on time parameters such as the intervals from stroke onset to first hospital arrival, CT, or IV t-PA. Finally, 1226 patients were included in this analysis (Figure 1).

**Figure 1.** Flow diagram for the selection of patients in this study. IV, intravenous; t-PA, tissue plasminogen activator; EVT, endovascular.

Baseline characteristics of patients are presented in Table 1. The mean age was 68.9 ± 11.6 years, and 724 patients (58.9%) were male. The most common risk factor was hypertension (70.9%), followed by atrial fibrillation or atrial flutter (48.3%). The median NIHSS score was 12 (interquartile range [IQR], 7–17). Six-hundred and thirty-three (51.6%) patients received IV t-PA treatment alone, 318 (25.9%) patients received EVT alone, and the remaining 275 (22.4%) received combined IV t-PA and EVT. The number of patients who received IV t-PA and registered in SECRET registry was larger than those registered after EVT with/without IV t-PA, except in 2017. We could determine the location of large artery occlusion in 851 of 1021 patients who underwent angiographic studies before reperfusion therapy. The most common occlusion site was the middle cerebral artery (MCA) (56.1%, *n* = 477), followed by the internal carotid artery (ICA) (27.1%, *n* = 231), vertebrobasilar (VBA) artery (12.1%, *n* = 103), and others (4.7%, *n* = 40). During study period, thrombectomy devices including Solitaire, Trevo, and Penumbra system were available in Korea. Among 593 patients treated with EVT, Solitaire was most frequently selected in 433 (73%), followed by Penumbra system (*n* = 69, 11.6%), and Trevo (*n* = 69, 11.6%).

**Table 1.** Baseline characteristics of the included patients by year.

#### *J. Clin. Med.* **2020** , *9*, 717

99

National Institutes of Health Stroke Scale; IV, intravenous; EVT, endovascular;

 t-PA, tissue plasminogen

 activator. \* Among 499 patients who received EVT with/without

 IV t-PA.

#### *3.2. Secular Trends of Functional and Radiologic Outcomes*

There was an increase in the number of patients who received the reperfusion therapy and the number of patients was 103 in 2012, 231 in 2013, 284 in 2014, 302 in 2015, and 306 in 2017. When we compared the baseline characteristics annually, initial diastolic blood pressure, NIHSS score at admission, and frequency of atrial fibrillation or atrial flutter decreased, while frequency of dyslipidemia or preadmission disability (mRS score of >2 before stroke) increased (Table 1). However, there were no differences in demographics or other vascular risk factors between calendar years. In time parameters, the intervals from stroke onset to arrival to emergency department (ED) increased, while those from door to initiation of IV t-PA (in 908 patients who received IV t-PA) or groin puncture (in 593 patients who received EVT) decreased over the years (all *p* < 0.05, Table 1). Especially, in 497 patients who had achieved final recanalization using EVT, the intervals from door to final recanalization [decreased from 240.1 ± 99.1 min in 2012 to 172.9 ± 62.6 min in 2017 (*p* < 0.001). During the study period, total number of stroke neurologist of study hospitals slightly increased (34 in 2012, 37 in 2013, 38 in 2014, 40 in 2015, 41 in 2017), while the number of neurointerventionalists or neurosurgeons involving EVT was similar (35 in 2012, 37 in 2013, 36 in 2014, 35 in 2015, 37 in 2017).

During the study period, patients achieved favorable functional outcome (mRS score of 0–2) in 48.6% and excellent functional outcome (mRS score of 0–1) in 31.3% at discharge. Among 1203 patients with available mRS data at 90 days, 501 (41.8%) patients achieved favorable functional outcome and 703 (58.6%) patients did excellent functional outcome at 90 days. The improvement in functional outcome at discharge after reperfusion therapy was noted as calendar year increased (Figure 2). The proportion of patients who were discharged home significantly increased (from 45.6% in 2012 to 58.5% in 2017) (*p* < 0.001). Likewise, functional outcome at 90 days was also different between calendar years (*p* < 0.05). There was an increase in favorable functional outcome (mRS score of 0–2) or excellent outcome (mRS score of 0–1) at discharge or after 90 days (all *p* < 0.05) (Table 2). In addition, the mortality rate at discharge or after 90 days significantly decreased with an increase in calendar year (all *p* < 0.05). These secular trends were consistently observed regardless of treatment modalities (Figure 2).

**Figure 2.** Secular trends in functional outcome at discharge (**A**–**C**) and after 90 days (**D**–**F**). Distribution of mRS scores of the entire population (A and D), those who received IV t-PA alone (**B**,**E**), and those who received EVT with/without IV t-PA (**C**,**F**).

#### *J. Clin. Med.* **2020** , *9*, 717

Successful recanalization rate could be evaluated in 804 patients who had undergone follow-up angiographic studies within 24 h after IV t-PA therapy or EVT. The successful recanalization rate increased over time from 78.3% in 2012 to 85.2% in 2017 (*p* = 0.004) (Table 2). On the contrary, the development of symptomatic intracerebral hemorrhage has declined over time (*p* = 0.018).

#### *3.3. Determinants of Functional Outcomes*

We investigated the determinants associated with functional outcomes. The univariable analysis demonstrated that favorable outcome (mRS score of 0–2) at discharge was associated with age, male sex, diabetes, current smoking, atrial fibrillation or atrial flutter, previous stroke, preadmission disability, initial stroke severity, and intervals from stroke onset to ED or from ED to reperfusion therapy, along with the calendar year (all *p* < 0.05). Among 804 patients who had large vessel occlusion and underwent angiographic studies before and after reperfusion therapy, favorable outcome was associated with occlusion site or achievement of successful recanalization. After adjusting these significant variables (*p* < 0.05) in the univariable analysis, the independent and significant predictors for favorable functional outcome at discharge were age, diabetes, preadmission disability, initial stroke severity, intervals from door to reperfusion therapy, achievement of successful recanalization, and calendar year (Supplementary Table S1). When we investigated the independent factor for functional outcome at 90 days. the same variables were independently associated with favorable outcome at 90 days (Table 3).

Further, we performed the univariable and multivariable ordinal regression analysis to obtain a significant factor for a shift in mRS at discharge and 90 days. The independent determinants of mRS at discharge and 90 days included age, diabetes, previous stroke, preadmission disability, initial stroke severity, intervals from door to reperfusion therapy, and achievement of successful recanalization, along with the calendar year (Supplementary Table S1 and Table 3).

**Table 3.** Independent determinants for functional outcome at 90 days.

#### **4. Discussion**

We investigated whether clinical or radiologic outcomes after reperfusion therapy changed over time using nationwide, multicenter data covering real clinical practice between 2012 and 2017 in Korea. We demonstrated that there was an increase in the number of patients who received reperfusion therapy, especially EVT, and the clinical outcomes of patients who received reperfusion therapy significantly improved over the past five years. The favorable trend in functional outcomes may be partly ascribed to the increases in successful recanalization rate and decreases in the door-to-treatment intervals and hemorrhagic complications. Of note, the calendar year was a significant factor for functional outcome even after adjusting for significant determinants.

The rate of favorable outcome in this study was comparable to or even better than those of the previous randomized controlled trials. For example, in previous trials investigating the usefulness of IV t-PA, the proportions of patients with mRS score of 0–1 at 90 days were 39% in the National Institute of Neurological Disorders and Stroke study [13], 52.4% in ECASS III [12], and 39% in Safe Implementation of Thrombolysis in Stroke-Monitoring Study [17]. A previous study using the data of Get with the Guidelines-Stroke hospitals showed that in-hospital mortality and discharge-to-home rates were 8.25% and 42.7%, respectively, among patients received IV t-PA therapy [18].

In this analysis, 45.3% of patients had a mRS score of 0–1 at 90 days, and 62.4% of patients could be discharged to home among patients with IV t-PA alone.

The meta-analysis of pooled patient data from five randomized trials after 2015 showed that 46% of patients achieved mRS score of 0–2 at 90 days [4]. In our patients who received EVT with/without IV t-PA, 50.7% of patients had an mRS score of 0–2 at 90 days. In addition, successful recanalization rate in our study population was 83%–87%, which was similar to those in recent EVT trials [19]. Although comparison of clinical outcomes between studies might be difficult because of different patient characteristics, feasibilities of procedures, types of devices, or treatment modalities between studies, our data suggested the benefits of current reperfusion therapy can be reproduced in the real clinical practice.

Our study also demonstrated that some characteristics of patients who received the reperfusion therapy have changed. Although age, vascular risk factors other than dyslipidemia, and occlusion site did not change over time, stroke severity slightly decreased. This may be ascribed to the increase in reperfusion therapy for minor stroke and decrease in the prevalence of atrial fibrillation- or atrial flutter-related stroke [20,21]. Furthermore, time from stroke onset to ED was longer than those in previous studies because patient eligibility for EVT might be based more on imaging parameters recently, instead of the time window paradigm [22]. Increase in the intervals from stroke onset to arrival to ED over years might be partly because more patients with delayed presentation to ED might have been treated with the reperfusion therapy over the years, thanks to advances in selection of patients by multimodal neuroimaging.

In this study, we reaffirm the importance of earlier treatment and achieving successful recanalization to improve patient outcome. Reducing the time from stroke onset to treatment is beneficial to not only reduce the ischemic core but also to remove thrombus [23–25]. Earlier treatment would also reduce the risk of symptomatic intracranial hemorrhage. [26,27] In Korea, there have been continuing efforts on reducing the time delay for patients with stroke using the stroke code system based on the computerized physician order entry system [9,27]. Actually, our data showed that mean time from door to initiation of treatment was continuously decreasing (up to 41.1 min for IV t-PA or 113 min for groin puncture).

Moreover, completely reopening the occluded artery is known as one of the essential components in reperfusion therapy [5]. Currently, the stentriever is the most commonly preferred device in approximately 90% of hospitals in Korea [8]. Stentriever use had some advantages such as easy handling, faster and complete reperfusion, temporal opening, and lesser bleeding complications [28,29]. There were increasing trends in successful recanalization (up to 87% in 2017) and rapid recanalization (mean intervals from door to final recanalization from 240.1 min in 2014 to 173 min in 2017) and

decreasing trends in symptomatic intracranial hemorrhage, which might be related with growing use of stentriever over time in Korea.

The calendar year was one of significant factors for favorable outcome even after adjusting for significant variables including age, initial stroke severity, comorbidities, and time from door to needle or achievement of successful recanalization. This implied that the advances in certain factors over time could also play a role for these trends. First, optimal medical treatment before and after reperfusion therapy would prevent complications or early neurologic deterioration and favorably affect the outcome. In this context, the role of the stroke team is important even in the era of EVT for acute stroke. Over the past decades, there has been an improvement in stroke care in Korea: Increase in stroke unit-based centers, multidisciplinary stroke team, guideline-based clinical practice, certification of stroke center, use of antithrombotics or statin in preventing stroke, development of networks among regional comprehensive and primary stroke centers, and provision of nationwide quality care for stroke [10,30–32]. Additionally, there was a possibility of advancement in the selection of eligible candidates who would benefit from reperfusion therapy. Many stroke centers in Korea use imaging parameters, such as collateral status, diffusion/perfusion mismatch, clot characteristics, or ASPECTS score, for identifying patients who are more suitable for reperfusion therapy [8,33].

There are some limitations to this study. First, the decision regarding the method of reperfusion therapy was based on discretion of the stroke neurologist or neurointerventionalist at each study center. The selection of EVT device, number of EVT passes, and use of balloon-guided catheter was not standardized. However, most stroke centers are collaborative in terms of sharing experience and protocols. Second, there were some unmeasured variables, such as socioeconomic status, educational level, and medication adherence before/after stroke, that could affect the functional outcome after stroke.

#### **5. Conclusions**

Our analysis demonstrated the increase in favorable outcomes among patients who received reperfusion therapy in Korea. There were a changing patterns, such as an improvement in time parameters, increase in successful recanalization rate, and decrease in bleeding complication rate, leading to the improvement in stroke metrics. However, there is still room for additional efforts, such as rapid notification or communication, for reduction in time delay before arrival to the hospital. Our data also suggest the need for support of personnel or infrastructure to continue the optimum treatment.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2077-0383/9/3/717/s1, Table S1: Independent determinants for functional outcome at discharge.

 All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2019R1H1A1079907) and a faculty research grant of Yonsei University College of Medicine (6-2019-0065).

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**

1. Wang, H.; Naghavi, M.; Allen, C.; Barber, R.M.; Bhutta, Z.A. Global, regional, and national life expectancy, all-cause mortality, and cause-specific mortality for 249 causes of death, 1980–2015: A systematic analysis for the global burden of disease study 2015. *Lancet (Lond. Engl.)* **2016**, *388*, 1459–1544. [CrossRef]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **High On-Treatment Platelet Reactivity A**ff**ects the Extent of Ischemic Lesions in Stroke Patients Due to Large-Vessel Disease**

#### **Adam Wi´sniewski 1,\*, Joanna Sikora 2, Agata Sławi ´nska 3, Karolina Filipska 4, Aleksandra Karczmarska-Wódzka 2, Zbigniew Serafin <sup>3</sup> and Grzegorz Kozera <sup>5</sup>**

Received: 13 December 2019; Accepted: 15 January 2020; Published: 17 January 2020

**Abstract:** Background: Excessive platelet activation and aggregation plays an important role in the pathogenesis of ischemic stroke. Correlation between platelet reactivity and ischemic lesions in the brain shows contradictory results and there are not enough data about the potential role of stroke etiology and its relationships with chronic lesions. The aim of this study is to assess the relationship between platelet reactivity and the extent of ischemic lesions with the particular role of etiopathogenesis. Methods: The study involved 69 patients with ischemic stroke, including 20 patients with large-vessel disease and 49 patients with small-vessel disease. Evaluation of platelet reactivity was performed within 24 h after the onset of stroke using two aggregometric methods (impedance and optical), while ischemic volume measurement in the brain was performed using magnetic resonance imaging (in diffusion-weighted imaging (DWI) and fluid-attenuated inversion recovery (FLAIR) sequences) at day 2–5 after the onset of stroke. Results: In the large-vessel disease subgroup, a correlation was found between platelet reactivity and acute ischemic focus volume (correlation coefficient (R) = 0.6858 and *p* = 0.0068 for DWI; R = 0.6064 and *p* = 0.0215 for FLAIR). Aspirin-resistant subjects were significantly more likely to have a large ischemic focus (Odds Ratio (OR) = 45.00, 95% Confidence Interval (CI) = 1.49–135.36, *p* = 0.0285 for DWI; OR = 28.00, 95% CI = 1.35–58.59, *p* = 0.0312 for FLAIR) than aspirin-sensitive subjects with large-vessel disease. Conclusion: In patients with ischemic stroke due to large-vessel disease, high on-treatment platelet reactivity affects the extent of acute and chronic ischemic lesions.

**Keywords:** platelet reactivity; ischemic stroke; aspirin resistance; infarction volume; multiplate

#### **1. Introduction**

Stroke is one of the main causes of morbidity and long-term disability, and the second most frequent cause of death globally [1]. The updated definition of ischemic stroke by the American Heart Association/American Stroke Association (AHA/ASA) recognizes stroke as when clinical symptoms last less than 24 h and neuroimaging studies have demonstrated acute ischemic infarctions [2].