Inhibiting leukocyte‐endothelial cell interactions by Chinese medicine Tongxinluo capsule alleviates no‐reflow after arterial recanalization in ischemic stroke

Abstract Aims Despite successful vascular recanalization in stroke, one‐fourth of patients have an unfavorable outcome due to no‐reflow. The pathogenesis of no‐reflow is fully unclear, and therapeutic strategies are lacking. Upon traditional Chinese medicine, Tongxinluo capsule (TXL) is a potential therapeutic agent for no‐reflow. Thus, this study is aimed to investigate the pathogenesis of no‐reflow in stroke, and whether TXL could alleviate no‐reflow as well as its potential mechanisms of action. Methods Mice were orally administered with TXL (3.0 g/kg/d) after transient middle cerebral artery occlusion. We examined the following parameters: neurological function, no‐reflow, leukocyte‐endothelial cell interactions, HE staining, leukocyte subtypes, adhesion molecules, and chemokines. Results Our results showed stroke caused neurological deficits, neuron death, and no‐reflow. Adherent and aggregated leukocytes obstructed microvessels as well as leukocyte infiltration in ischemic brain. Leukocyte subtypes changed after stroke mainly including neutrophils, lymphocytes, regulatory T cells, suppressor T cells, helper T type 1 (Th1) cells, Th2 cells, B cells, macrophages, natural killer cells, and dendritic cells. Stroke resulted in upregulated expression of adhesion molecules (P‐selectin, E‐selectin, and ICAM‐1) and chemokines (CC‐chemokine ligand (CCL)‐2, CCL‐3, CCL‐4, CCL‐5, and chemokine C‐X‐C ligand 1 (CXCL‐1)). Notably, TXL improved neurological deficits, protected neurons, alleviated no‐reflow and leukocyte‐endothelial cell interactions, regulated multiple leukocyte subtypes, and inhibited the expression of various inflammatory mediators. Conclusion Leukocyte‐endothelial cell interactions mediated by multiple inflammatory factors are an important cause of no‐reflow in stroke. Accordingly, TXL could alleviate no‐reflow via suppressing the interactions through modulating various leukocyte subtypes and inhibiting the expression of multiple inflammatory mediators.


| INTRODUC TI ON
In recent years, vascular recanalization resulting from intravenous thrombolysis and/or intravascular interventional therapy represents a significant breakthrough in the treatment of acute ischemic stroke.
However, 50% of stroke patients with successful arterial recanalization have an unfavorable outcome. 1 Namely, the recanalization does not equate with successful tissue reperfusion due to the fact that part of the recanalization is futile. 2 The journal Stroke has reported that a quarter of patients with successful arterial recanalization in ischemic stroke has impaired tissue reperfusion, termed as noreflow phenomenon. 3 Of note, the phenomenon has become one of the main bottlenecks of constraining further improvement of clinical efficacy of ischemic stroke. Currently, its underlying mechanisms have not been fully understood, and effective therapeutic strategies are lacking in western medicine. 1 Based on the collateral disease theory of traditional Chinese medicine (TCM), minute collaterals in TCM and microvessels in western medicine are highly correlated in morphology and function; it is then considered that the main pathological mechanism of no-reflow is minute collateral-microvessel obstruction. Thus, we propose that unblocking-collateral intervention is its therapeutic principle according to the theory. It has been confirmed that Tongxinluo capsule (TXL), a representative Chinese medicine based upon unblockingcollateral therapy can unblock collaterals and protect microvessels in ischemic cardiovascular and cerebral vascular diseases. 4,5 Previous study has revealed that TXL can significantly reduce myocardial no-reflow and infarction area after emergency percutaneous coronary intervention for acute ST-segment elevation myocardial infarction. 6 Our prior experimental study showed that TXL could alleviate cerebral microcirculatory disturbances in ischemic stroke in vivo observed by two-photon microscopy and thus reduce brain infarct volume. 7 Nonetheless, it is still not clear whether TXL can alleviate no-reflow after vascular recanalization in ischemic stroke.
The underlying mechanisms of no-reflow have not been fully delineated. Previous study has revealed that potential mechanisms of microvascular no-reflow after stroke are multiple such as obstruction of blood-borne elements (erythrocytes, leukocytes, platelets, and blood clots), compression of microvessels owing to swelling of endothelia and astrocyte end-feet, and pericyte contraction of cerebral capillaries. 8 Among different mechanisms, endothelia and leukocytes play a vital role in leading to no-reflow after stroke. Thus, we propose that leukocyte-endothelial cell interactions (adhesion, aggregation, and rolling) are a potential cause of no-reflow after stroke.
Moreover, it is revealed that adhesion molecules, and chemokines play a critical part in leukocyte-endothelial cell interactions. 9 Combined with our previous conclusion that TXL is capable of suppressing leukocyte-endothelial cell interactions in ischemic stroke mice, 7 we herein hypothesize that leukocyte-endothelial cell interactions mediated by numerous adhesion molecules and chemokines are an important cause of no-reflow after vascular recanalization in ischemic stroke, and TXL can alleviate the no-reflow by inhibiting these biological targets. In this study, we used in vivo laser speckle cortical imaging to evaluate the effect of TXL on noreflow after vascular recanalization in ischemic stroke induced by transient middle cerebral artery occlusion (tMCAO), and further explored the potential mechanisms of no-reflow and TXL's protective effects against the phenomenon from the perspective of regulating leukocyte-endothelial cell interactions.

| Animals
All animal experiment protocols were approved by the Institutional

| tMCAO model
Ischemic stroke with arterial recanalization was induced by transient middle cerebral artery occlusion (tMCAO) as previously described. 10,11 Brief surgical procedures of the model are as follows: mice were intraperitoneally anesthetized with 0.75% pentobarbital sodium (75 mg/kg). 12 The left carotid arteries were isolated. Then a silicon-coated monofilament suture (Guangzhou Jialing Biotechnology Co., Ltd) was inserted into the left common carotid artery and advanced along the left internal carotid artery until a slight resistance appeared (approximately 9-10 mm after the left carotid bifurcation). After 1.5 h of occlusion, reperfusion was performed through withdrawing the filament. Sham-operated mice underwent identical procedures but without filament insertion.
During surgery, animal body temperature was maintained at 37°C with a dependable electric blanket.

K E Y W O R D S
inflammatory factors, ischemic stroke, leukocyte-endothelial cell interactions, no-reflow, Tongxinluo capsule

| Drug preparation and administration
TXL, a Chinese medicine prescription, is clinically used in the form of capsules. It contains 12 components (Table 1) as previously described. 7 These primary materials were ground to a superfine powder (≤10 μm) by the micronization technology and made into capsules standardized upon marker compounds according to the Chinese Pharmacopeia 11th edition. 13 In this experiment, TXL superfine powder was employed rather than capsules. The powder was provided by Shijiazhuang Yiling Pharmaceutical Incorporated Company. In this study, the powder was dissolved in 0.9% sodium chloride (the suspension concentration 0.15 g/mL) and the suspension was stored at 4°C for no more than 3 days.
In the current study, we used the best therapeutic dose of TXL (3.0 g/kg) in reducing infarct volume and alleviating cerebral microcirculatory disturbances after stroke in three ones (0.75, 1.5, and 3.0 g/kg) according to our prior research. 7 The tMCAO mice were excluded from the analysis in the following conditions at 1.5 h after tMCAO: no neurological deficit and consciousness disorder.
Additionally, tMCAO mice with signs of intracranial hemorrhage con-

| Laser speckle cortical imaging
The laser speckle imaging system (RFLSI III, Shenzhen RWD Life Science, Shenzhen, China) obtains high-resolution and twodimensional imaging and has a linear relationship with absolute cerebral blood flow (CBF) values. Relative CBF values reflecting blood reperfusion can be determined by the imaging system. Thus, no-reflow after stroke was assessed by laser speckle perfusion imaging as previously described. 1 In brief, scalp of mice was open via a midline incision under 0.75% pentobarbital sodium anesthesia.
Recordings of CBF in regions of interest (ROIs) were performed through the fixed skull using laser speckle perfusion imaging at 6, 24, and 72 h after stroke. For each recording, normal saline was added on the skull surface to prevent drying. Relative CBF was examined in identically sized ROIs of bilateral hemispheres. No-reflow was assessed by CBF % of contralateral level.

| HE staining
Murine brains were immediately collected at 24 h and 72 h after stroke. Then they were fixed 4% paraformaldehyde for more than 24 h and embedded in paraffin. Coronal sections of 5 μm in thickness were prepared, stained with hematoxylin and eosin (HE) and subsequently imaged using an optical microscope.

| Flow cytometry analysis of leucocytes in peripheral blood
Leukocyte subpopulations in peripheral blood were analyzed by flow cytometry as previously described. [14][15][16][17][18] In brief, 500 μL blood from the vessels behind eyeballs was collected in a tube with

| Cytometric bead array for measurement of cytokines
Ischemic brain and blood were collected at 24 h and 72 h after stroke.
Brain tissue was ground and then was filtered by a cell strainer of  The test was employed to detect the significance of differences between multiple comparisons, and LSD test was used between two arbitrary comparisons as a post hoc test. For some data that did not meet the homogeneity of variance, nonparametric test (Kruskal-Wallis) be applied to analyze them. Significance was set at p < 0.05 or p < 0.01. All quantitative results were expressed as means ± standard deviation (SD).

| TXL alleviated neurological deficits after tMCAO
Neurological function scores were performed at three time points (6,24, and 72 h after stroke). Our results ( Figure 1) showed that the scores in tMCAO group were significantly higher than these in sham group at these three time points indicating that the stroke model was successful. After treatment with TXL, the scores were reduced compared with these in tMCAO group at 24 h or 72 h. At 6 h, there was no difference in scores between tMCAO+TXL group and tMCAO group. These results revealed that TXL could alleviate neurological deficits after ischemic stroke.

| TXL improved no-reflow after arterial recanalization in ischemic stroke
No-reflow was evaluated by laser speckle perfusion imaging. At 1.5 h after brain ischemia, vascular recanalization was induced by removing the monofilament. However, after vascular recanalization, CBF recovered to around 60% of contralateral cerebral cortex indicative of no-reflow phenomenon. After treatment of TXL, CBF was significantly increased at 24 h or 72 h compared with that in tMCAO mice.
At 6 h, there was no difference in CBF between tMCAO+TXL group and tMCAO group. These results ( Figure 2) suggested that no-reflow did occur after vascular recanalization in ischemic stroke, and TXL could alleviate the phenomenon.

| TXL ameliorated leukocyte-endothelial cell interactions after tMCAO
Leukocyte-endothelial cell interactions were examined by two-

| TXL suppressed the expression of adhesion molecules and chemokines after tMCAO
Adhesion molecules and chemokines in plasma and brain were measured with CBA at 24 h and 72 h after tMCAO. The levels of Pselectin ( Figure 14A), E-selectin ( Figure 14B) and CCL-2 ( Figure 15A) at 24 h, CXCL-1 ( Figure 15E) at 72 h and ICAM-1 ( Figure 14C), CCL-3 ( Figure 15B), CCL-4 ( Figure 15C) and CCL-5 ( Figure 15D 19 Our prior study showed that TXL could reduce cerebral infarct volume and improve neurological function after brain ischemia in mice. 7 According to the collateral disease theory of TCM, TXL can unblock obstructed collaterals. We have experimentally confirmed that TXL could alleviate cerebral microcirculatory disturbances after ischemic stroke observed by two-photon microscopy and protect brain microvessels after brain ischemia in mice. 7

F I G U R E 6
Effect of TXL on lymphocytes and N/L in peripheral blood after tMCAO. Representative flow cytometry dot plots and quantitative analysis for lymphocytes and N/L among different groups at 24 h and 72 h after tMCAO. N = 4-6 mice each group. *p < 0.05 and **p < 0.01.
It is known that brain microvascular obstruction is the essence of no-reflow after stroke. In addition, TXL could reduce myocardial no-reflow in myocardial infarction mentioned above. 6 Therefore, we postulated TXL could inhibit no-reflow after stroke. In the present study, we have firstly confirmed this hypothesis using the tMCAO model clinically mimicking ischemic stroke with arterial recanalization.
Subsequently, we explored the mechanisms of no-reflow after ischemic stroke and TXL suppressing the phenomenon. Ames and coworkers initially found brain no-reflow phenomenon in stroke model in 1968. 20 Its potential mechanisms are multiple as mentioned above. Among different mechanisms, endothelia and leukocytes play a vital role in leading to no-reflow after stroke. In our study, two-photon microscopy imaging was used in vivo to evaluate circulation. 23 Clinical study has revealed that leukocyte and neutrophil counts are increased on the first day after acute ischemic stroke. 24 Accordingly, reducing neutrophil in peripheral blood could improve stroke outcomes. 25 Cerebral ischemia leads to augmentation of brain infiltration and activation of neutrophils and lymphocytes in type 2 diabetic mice. 26 Consistent with studies above, our work has demonstrated that neutrophil ratio is remarkedly increased at acute stages after tMCAO, and TXL can reduce neutrophil ratio in ischemic stroke. Reduced lymphocyte counts in patients with leukocytosis could be also regarded as a predictor of unfavorable outcomes in ischemic stroke. 27 More importantly, N/L could predict cerebral edema and clinical outcomes, and thus be considered as a prognostic biomarker of early stroke with reperfusion therapy. 28,29 A retrospective study found that high N/L is considered as a predictor of poor stroke prognosis, while low one is a predictor of favorable outcomes at the early stage of stroke. 30 Well in line with studies above, our research revealed that cerebral ischemia with reperfusion lead to reduced lymphocyte ratio and increased N/L. TXL could reduce N/L but does not influence lymphocyte ratio after stroke.
Tregs play a protective role in ischemic stroke. 31,32 Experimental study demonstrated that increased Tregs could promote white matter repair and neurological recovery during the chronic phase of ischemic stroke. 33 In contrast to these reports, our data showed that the ratio of Tregs was firstly decreased, and then increased  Previous research has demonstrated that leukocyte-endothelial cell interactions are mainly mediated by the elevated adhesion molecules and chemokines. 9 To do so, activated endothelial cells could express numerous adhesion molecules and chemokines mediating interactions with leukocytes, such as P-selectin, E-selectin, ICAM-1, and CCL-2. 47 Among them, chemokines CCL-2, CCL-3, and CCL-5 participate in leukocyte adhesion. 48

| CON CLUS IONS
In conclusion, our study has demonstrated that leukocyteendothelial cell interactions obstructing brain microvessels is an im- improving English writing.

CO N FLI C T O F I NTER E S T S TATEM ENT
All authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets generated during the current study are available from the corresponding and first author on reasonable request.