Background: The no-reflow phenomenon refers to a failure to restore normal cerebral microcirculation despite brain large artery recanalization after acute ischemic stroke, which was observed over 50 years ago. Summary: Different mechanisms contributing to no-reflow extend across the endovascular, vascular wall, and extravascular factors. There are some clinical tools to evaluate cerebral microvascular hemodynamics and represent biomarkers of the no-reflow phenomenon. As substantial experimental and clinical data showed that clinical outcome was better correlated with reperfusion status rather than recanalization in patients with ischemic stroke, how to address the no-reflow phenomenon is critical. But effective treatments for restoring cerebral microcirculation have not been well established until now, so there is an urgent need for novel therapeutic perspectives to improve outcomes after recanalization therapies. Conclusion: Here, we review the occurrence of the no-reflow phenomenon after ischemic stroke and discuss its impact, detection method, and therapeutic strategies on the course of ischemic stroke, from basic science to clinical findings.

© 2023 S. Karger AG, Basel

Introduction

Stroke is a major cause of death and disability that endangers human health, about 70% of stroke is attributed to ischemic cerebral infarction [1]. At present, recanalization is often chosen as a therapeutic strategy for the acute stage of ischemic stroke, which mainly restores the blood flow of the ischemic region through intravenous thrombolysis involving intravenous alteplase or endovascular intervention [2]. Earlier recanalization treatment could reduce infarct size, preserve function, improve survival, and reduce the risk of recurrent stroke. A meta-analysis suggested that earlier alteplase treatment was associated with a better outcome defined by a modified Rankin score at 3–6 months [3]. However, when reopened, some downstream microcirculation cannot be completely restored, referring to impaired capillary reperfusion after recanalization, which is called the no-reflow phenomenon, and it is observed in about one-quarter of patients with successful recanalization [4]. Patients with no-reflow always present greater infarct growth and worse prognosis than those without no-reflow [5]. Thus, microcirculatory reperfusion is of great importance for stroke recovery.

Cerebral microvessels have their unique ultrastructure form, allowing a close relationship between the endothelium and blood elements and the neurons they serve [6]. Recently, more researchers have embraced the concept of the neurovascular unit (NVU), which comprises neurons, glial cells (astrocytes, microglia, oligodendrocytes), vascular cells (endothelial cells (ECs), pericytes, smooth muscle cells (SMCs)), and the extracellular matrix [7]. The NVU is essential for coupling neural activity and blood flow, regulating cerebral blood flow (CBF), and maintaining perfusion pressure homeostasis of the brain environment in physiological conditions [8, 9]. After a stroke, the cellular crosstalk within the NVU is altered, which directly promotes neuroinflammation and blood-brain barrier (BBB) breakdown. For example, at the onset of stroke, glial cells are activated and secrete pro-inflammatory factors matrix metalloproteinases to digest BBB matrix proteins [10, 11]. The BBB disruption could increase the risk of vasogenic cerebral edema and compress capillary lumen, leading to further no-reflow. In this article, we will describe the biology of the no-reflow phenomenon in experimental models, discuss its multiple mechanisms, describe how to assess cerebral no-reflow, and highlight its clinical significance.

No-Reflow Phenomenon after Cerebral Ischemia-Reperfusion

The “no-reflow phenomenon” concept was first proposed in 1967 to describe the impaired return of blood flow after the ischemia was terminated, observed in rabbits’ brains [12]. Ames et al. [13] used the global cerebral ischemia model of albino rabbits and induced ischemia for different periods, then restored proximal flow and demonstrated that as the duration of ischemia was increased (5 min or longer), the amount of tissue that failed to reperfuse also increased. They also explored the linkage between blood elements and no-reflow by inducing “bloodless ischemic” as the blood was displaced from the brain with Ringers solution prior to the ischemia. After ischemic, complete black carbon reperfusion occurred, but when reperfused with a suspension of red cells, the no-reflow phenomenon appeared again, which indicated despite blood involved in no-reflow, a narrowing capillary was dominant. To further probe into the possible vascular obstructions, a study at the level of light and electron microscopy revealed it was swollen perivascular glial cells and “blebs” arose from ECs that narrowed the capillary lumen, but not platelet thrombi or intravascular clotting [14]. However, upon further study, researchers found only structural changes in ischemic capillary walls were insufficient to explain failed cerebral reperfusion. As the survey about the no-reflow phenomenon developed in-depth, researchers identified multiple factors that contributed to the no-reflow phenomenon, including edema, microvascular damage, and microvascular obstruction.

As shown in Figure 1, extravascular factors, vascular wall factors, and intravascular factors interact and participate in the occurrence of microcirculatory disturbance after cerebral ischemia-reperfusion, which is manifested as the presence of microemboli deposited by intercepted red cells, white blood cells, and fibrin-platelets in the narrowed lumen [15]. The microthrombus not only forms in situ but also originates from debris caused by thrombolysis [16]. Although these microemboli can be eliminated by mechanical perfusion, dissolution, and phagocytosis after recanalization, the number of microemboli in the ischemic region is relatively large, which leads to the incomplete clearance of these microemboli, and the microcirculation deficit often exists even after recanalization therapy.

Fig. 1.

Mechanisms involving no-reflow phenomenon after ischemic stroke.

Factors Contributing to the Occurrence of the No-Reflow PhenomenonVascular Wall StructureECs and Microcirculatory Disorders

Under physiological conditions, ECs form a broad microvascular network, which contributes to maintaining blood pressure and controlling vascular wall permeability. Furthermore, ECs play a crucial role in regulating blood flow, utilizing anticoagulation and pro-coagulation mechanisms. Under physiological conditions, ECs prevent thrombosis by promoting the activity of different anticoagulant and antiplatelet pathways [17]. Thrombomodulin (TM) is a transmembrane glycoprotein localized to the vascular ECs. When complexed with TM, the procoagulant specificity of thrombin is inhibited; conversely, thrombin-TM activates protein C and protein S, which then proteolysis factors Va and VIIIa and inactivates the coagulation cascade [18]. ECs also synthesize and release tissue factor pathway inhibitor α (TFPIα) that suppresses tissue factor-factor VIIa and factor Xa to block the onset of coagulation [19]. In addition, nitric oxide (NO) [20] and prostaglandin I2 [21] secreted by ECs could inhibit platelet adhesion and aggregation synergistically. At the same time, ECs inhibit the transcription of adhesion molecules such as P/E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) to reduce adherence of leukocytes [22].

Under ischemic-reperfusion conditions, there is much evidence suggesting that endothelial dysfunction plays a role in the pathogenesis of the no-reflow phenomenon. With prolonged ischemia, the lack of energy to maintain ion hemostasis caused EC edema and vascular compression within an hour after reperfusion [23]. Cerebral ischemia-induced secretion of TXA2, thrombin, platelet-activating factor, leukotrienes, peripheral blood-free norepinephrine, and neuropeptide Y, which act on ECs and vascular smooth muscle, causing cerebral vasoconstriction, leading to microcirculation obstacle [24]. Free radicals produced during cerebral ischemia-reperfusion promote the change of ECs to coagulation, and the appearance of adhesion molecules on the surface of ECs induces adherence of platelets to ECs, contributing to microcirculatory disorders [25]. Furthermore, ECs necroptosis was observed in 3 days after middle cerebral artery occlusion (MCAO), which resulted from the tumor necrosis factor-α secreted by M1 type microglia and its receptor TNF receptor 1 on endothelium [26]. The death of ECs promotes BBB breakdown and fluid in vessel leakage, contributes to stenosis of microvascular and impedes reperfusion. Anti-TNFα treatments such as infliximab prominently ameliorate ECs necroptosis and improve stroke outcomes.

Pericytes and Microcirculatory Disorders after Cerebral Ischemia

Cerebral arteries have multiple layers of SMCs, as these arteries eventually branch into the arterioles, the number of SMCs decreases, and SMCs are finally replaced by pericytes in capillaries. Although pericytes in the brain lack smooth muscle actin, they are potential to regulate blood flow by dynamic contraction and relaxation. For instance, when neuronal activity and the neurotransmitter glutamate induce the release of prostaglandin E2, meanwhile NO is released to suppress vasoconstricting 20-HETE synthesis, pericytes relax, capillaries dilate, and blood flow increases [27]. However, others suggested regulation of CBF during early ischemic stroke is mediated by arteriolar SMCs but not capillary pericytes [28].

Researchers demonstrated that pericytes induce vasoconstriction to maintain a constant capillary hydrostatic pressure after ischemic in rat myocardial infarction model, and finally lead to no-reflow in the heart [29]. If we use this hypothesis to explain the cerebral no-reflow, however, it fails to explain the long-lasting reduction of CBF when restored arterial flow. Yemisci et al. [30] found that pericytes contracted during the occlusion of the middle cerebral artery, and this phenomenon persisted even after restoring blood flow in a rat model of cerebral ischemia. Catherine et al. [27] applied an oxygen-glucose deprivation model to simulate ischemia, and the live imaging of cerebral cortical slices revealed that, when exposed to ischemia, the pericytes first constricted the capillaries and then died. Continuous stimulation of reactive oxygen species (ROS) causes intracellular calcium overload in vitro culture of human pericytes [31], which was an important cause of excessive pericyte contraction under ischemic conditions. The excessive contraction causes stagnation of red blood cell passage, leading to microcirculatory disorders. In addition, the expression of superoxide producing enzyme nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) was upregulated in pericytes of the peri-infarct region in the stroke model, which was shown to participate in metalloproteinase-9 activation BBB disruption [32] and contribute to ongoing neuronal damage. These studies support the view that preventing pericyte constriction and death is a potential therapeutic strategy to reduce no-reflow.

Endovascular FactorsRed Blood Cells and Microcirculatory Disorders

Red cells must deform passively during their passages through small capillaries, so their rheology is a determining factor for microcirculation flow. It is reported that 10 min of clamping rat femoral artery could reduce the elongation index for red blood cells [33], and 3 h of acute ischemia in the dog’s hind limb could increase the relative cell transit time of erythrocytes [34]. Further, Yu et al. [35] demonstrated that the post-thrombolytic red cells exhibit reduced deformability after being released from the clot, making it difficult for red cells to move through small tissue capillaries and eventually resulting in the cell trapping to occlude the tissue capillaries, which causes further unsuccessful reperfusion.

White Blood Cells and Microcirculatory Disorders

The mechanism by which white blood cells induce microcirculatory disorders can be summarized in three parts. First, white blood cells mechanically occlude capillaries. The ability of white blood cells to deform and pass the microcirculation is reduced in the acute phase of cerebral infarction, which is one of the reasons for the no-reflow phenomenon [36]. Second, white blood cells cause abnormal blood rheology. It is reported that leukocyte marginalization is an early event started by arterial occlusion. The leukocyte margin begins with a sudden drop of downstream blood flow, then promotes fibrinogen deposition and secondary thrombosis [37], which is responsible for incomplete reperfusion after arterial recanalization. Third, tissue damage is mediated by the adhesion of white blood cells and ECs. The interactions of adhesion molecules like P/E-selectin on ECs and their counter-receptors (P-selectin glycoprotein-1, PSGL-1) on leukocytes help them roll in capillaries. ICAM-1 on endothelium, together with the β2-integrin (CD18) on leukocytes, induces the adhesion of white blood cells to ECs, accompanying the initial movement of inflammatory cells into the ischemic region. Under physiological conditions, ECs do not interact with leukocytes; however, leukocyte rolling and adhesion in postcapillary venules increased during 1 h of reperfusion after 2 h of MCAO [38]. Then leukocytes release the cell active factors, resulting in EC-mediated tissue damage, increasing the permeability of tiny blood vessels, causing vasoconstriction, thus contributing to microcirculatory disorders [39].

Recently, clinical studies have provided evidence that higher leukocyte counts on admission or after intravenous thrombolysis were likely to lead to poor clinical outcomes [40, 41]. A recent study also validated that depletion of neutrophils with anti-Ly6G antibody reduced the no-reflow phenomenon after recanalization [42]. However, given the essential role of leukocytes in neuroinflammation and protection, it is not an appropriate option to deplete them to improve no-reflow. But a reasonable approach to tackle this problem could be inhibiting the leukocytes adhesion in stroke.

Platelet Activation and Microcirculatory Disorders

Under ischemic conditions, platelets can be activated to form pathological thrombus and contribute to microvascular obstructions [43]. The von Willebrand factor binding to its receptor on the platelet surface, glycoprotein Ib, also improves further growth of infarct that may lead to no-reflow [44]. Additionally, reperfusion leads to ROS generation that causes platelet necrosis [45]. At the same time, the phosphatidylserine expressed on necrotic platelets is thought to be a culprit in the creation of platelet-neutrophil aggregates [46]. And a prospective study reported that acute coronary syndrome patients with no-reflow had higher platelet-neutrophil aggregates than those without no-reflow [47]. Meanwhile, platelet adhesion and aggregation cause vasoconstriction and subsequently aggravate microcirculatory disorders. However, the main limitation of suppressing platelet is the increased risk of hemorrhage.

Thrombin and Microcirculatory Disorders

It has been confirmed that thrombin participates in the final stage of the coagulation pathway, converting fibrinogen to fibrin and involving in the activation of platelets. Previous research has found an increase in thrombin and its precursor prothrombin in rat brains after transient global ischemia [48], possibly due to BBB breakdown and thrombin leak out of the capillaries. Increased thrombin in the brain contributes to no-reflow by creating microthrombus in small distal vessels after the initial arterial occlusion [49]. It could finally cause impairment of BBB via altering the permeability of microvasculature by increasing cytosolic Ca2+ concentration or by increasing NO and ROS in the mitochondria and cytosol [50]. Besides, thrombin can cleave and directly activate Interleukin-1α (IL-1α), which induces rapid inflammatory cell recruitment and thrombosis [51]. In a previous study, the effect of the thrombin inhibitor dabigatran suppressed the no-reflow phenomenon was demonstrated [52].

Fibrin Deposit and Microcirculatory Disorders

The blood clotting system is activated in the acute phase of cerebral infarction. At the same time, fibrin-rich thrombosis is induced and participates in the expansion of the thrombus. It is reported that elevated plasminogen activator inhibitor-1 (PAI-1) activity in ECs is associated with post-ischemic fibrin deposition in the ischemic penumbra, while a significant reduction in fibrin deposition was observed in PAI-1 deficiency MCAO mice [53]. Okada et al. [54] reported that fibrin-rich thrombosis increased during reperfusion and was associated with neuronal damage. The early fibrin deposition may contribute to the clinical significance of evolutionary ischemic lesions. When Ancrod, a compound that decreases plasma fibrinogen levels, is given within 3 h of the onset of stroke, patients successfully improve neurological outcomes if the plasma fibrinogen level is 70 mg/dL compared with placebo-treated patients at 9 h [55].

Extravascular Factor

Vasogenic edema is caused by the breakdown of the tight junctions of the BBB and a direct entry of both intravascular protein and plasma into the extravascular space of the brain. A mathematical model was developed to detect the effect of vasogenic edema on capillaries and showed that capillary collapse might occur at tissue swelling after reperfusion, which may be part of the no-reflow [56]. In contrast, cytotoxic brain edema caused by the lack of oxygen and glucose results in malfunction of the sodium and potassium pump in the astrocyte membrane leading to rapid swelling of the cell. Compression by swollen astrocyte end-feet encircling microvessels was thought to hinder flow further. Capillaries in the ischemic zones often appeared to be compressed by the layers of swollen processes surrounding them, and the lumens of these vessels were frequently reduced [57]. Furthermore, since astrocytes are associated with the exchange of cerebrospinal and interstitial fluids, water transport is disturbed after stroke due to the disruption of APQ4 polarization in the cell membrane of reactive astrocytes [58], which impedes blood flow. The underlying mechanisms of cerebral microvascular no-reflow are summarized both in the table and illustration (Table 1; Fig. 2).

Table 1.

Integrative overview of underlying mechanisms of microvascular no-reflow

Fig. 2.

Illustration of the mechanisms of the no-reflow phenomenon created with BioRender.com.

Methods for Evaluating Microcirculation State

To date, clinical trials for evaluating brain microcirculatory disorders were lacking in the clinic. Like computed tomography perfusion (CTP) and MR perfusion-weighted imaging (PWI), positron emission tomography (PET) can assess CBF. PET has the characteristics of high spatial resolution and high sensitivity, so it is considered the “gold standard” for detecting CBF. Still, its high inspection cost limits its clinical application [59]. In addition, transcranial Doppler ultrasound (TCD) is widely used to evaluate cerebrovascular reactivity and its automatic regulation function.

Perfusion Imaging – PWI and CTP

Perfusion imaging using CTP and PWI are important means of examining cerebral blood perfusion in stroke patients, as the mathematical model could process functional imaging to measure CBF, cerebral blood volume (CBV), mean transit time (MTT), time to peak, and other hemodynamic parameters and perfusion image to evaluate the brain tissue perfusion status.

The mismatch between a larger PWI lesion and a smaller MR diffusion-weighted imaging (DWI) lesion is thought to be a signature of the ischemic penumbra [60], generally when the mismatched volume is more than 20%. If there exists a DWI/PWI mismatch area, restoring perfusion to this penumbral tissue results in clinical recovery [61]. The limited access to MRI in a vast majority of stroke units has stimulated the development of CTP techniques that estimate the extension of the core and the critical hypoperfusion. The CTP and PWI maps were found to pose equivalent values for processing the perfusion status [62]. It is reported that MTT reduces abnormalities by 75% or more to define reperfusion, and patients with an MTT reperfusion index >75% were more likely to have smaller follow-up infarct volumes than patients with recanalization [63].

Angiography

Angiography was often used in patients who underwent intra-arterial treatment within 6 h of stroke ictus. Similar to the grading scale of recanalization by the arterial occlusive lesion (AOL) scale [64], reperfusion status was also graded to evaluate the restoration of the blood flow of downstream microcirculation of the blocked artery, to illustrate the tissue reperfusion status. The modified treatment in cerebral ischemia (mTICI) with grade 0 indicating no perfusion and grade 3 representing full perfusion of all visualized distal branches [65]. And found that mTICI has proven value for predicting clinical outcomes, like mTICI 2b or 3 reperfusion scores were related to improved outcomes in mismatch patients [66], or use mTICI 2a, 2b, or 3 reperfusion as a measure of successful reperfusion [67]. Except for mTICI, capillary index score (CIS) is a new index for assessing capillary blush in the ischemic area and could be a potential index for improving patient selection. Firas Al-Ali et al. [68] evaluated the CIS in patients within 6 h of ictus and underwent intra-arterial ischemic stroke treatment and found that a good outcome was achieved only in patients with favorable CIS.

Optical Coherence Tomography

Multi-parametric optical coherence tomography (OCT) imaging provided information about cerebral vasculature, blood flow velocity, CBF, and tissue property change in the mouse brain, which is based on the intrinsic optical scattering properties of RBCs and cortical tissues [69], but it should be noted that the observations involve superficial vascular beds and not deep cortical events. The ranges of vessels that can be captured by OCT are 5–20 μm, with the potential to observe capillaries. A study applied OCT to quantify cerebral microcirculatory changes in a mouse stroke model and reported that the capillary perfusion level was significantly reduced after recanalization with prolonged ischemia [70]. Additionally, Shanshan Yang et al. [71] utilized OCT to prove that the capillary perfusion of the ischemic core disappeared even though the arteries were recanalized. Given the property of OCT to detect CBF, it is suggested OCT combined with perfusion imaging may have the potential to reveal the critical CBF to detect the no-reflow phenomenon.

Transcranial Doppler

The use of TCD for brain microvascular assessment has been increasingly used in ischemic stroke studies, such as assessing cerebral vascular resistance by pulsation index to predict white matter damage [72], lacunar infarction-related cognitive impairment [73], and so on. Felix et al. [74]reported that in 53 patients with acute middle cerebral artery occlusion, middle cerebral artery pulsation index elevation was associated with worse functional outcomes irrespective of infarct volume as assessed on the 90-day modified Rankin Scale. Even with recanalization and small cerebral infarction volume, the cerebral microvascular resistance in the ischemic area was still significantly elevated [74], supporting the existence of microcirculation no-reflow after recanalization.

Clinical Significance of the No-Reflow Phenomenon

At present, little attention has been paid to the microcirculatory disturbance after the recanalization of large vessels. It is inadequate to prevent and control the progress of ischemic penumbra tissue injury only through large vessel recanalization therapy. Some clinical studies found that the prognosis of acute stroke patients is mainly related to tissue perfusion independently of vascular recanalization [4, 60, 64, 75-77]. The no-reflow phenomenon in the clinical setting is more complex than in the experimental model. Most experimental models are healthy without the influence of the underlying disease. However, in patients, the proximal arteries are always diseased with atherosclerotic plaque and thrombus. Moreover, the risk and intensity of the no-reflow phenomenon after ischemic stroke might be more severe in pro-inflammatory or procoagulant states like diabetes mellitus and hyperlipidemia due to endothelial dysfunction, which are common risk factors in stroke patients. The no-reflow phenomenon becomes more common with longer periods of ischemia, and it will likely contribute to infarct core in those undergoing delayed reperfusion therapies. Thus, it is a potential therapeutic target for acute ischemic stroke. Recanalization and reperfusion should both be measured when drawing conclusions on revascularization success [65]. Effective treatment to manage microcirculation in ischemic penumbra may effectively reduce infarct size and improve clinical prognosis.

How to Address the No-Reflow Phenomenon

Unlike the studies on myocardial infarction in patients, there are little data regarding the topic of no-reflow in the brain of patients following reperfusion therapy for acute stroke. Attempts were made to construct the microcirculation blood flow after ischemic stroke on animal models, ranging from albumin therapy [78], inhibition of platelets aggregation by integrin alpha(IIb)beta(3) inhibitor [79], inhibition of polymorphonuclear leukocyte adherence [80], using superoxide scavenger and NOS inhibitor to prevent pericyte contraction [30]. However, it should be noted that inflammation and other adaptive responses mediated by leukocytes to ischemia can have beneficial as well as detrimental effects following stroke [81]; platelet activation with ischemia results in aggregation, formation of plugs within the microvasculature, but excessive suppression of platelet-fibrinogen activity may increase risk of hemorrhage [79]. In addition, our research group demonstrated that polyethylene glycol-crosslinked urokinase nanogel promoted the thrombolysis efficiency in large vessels and microcirculation after ischemic stroke [82], suggesting that advancement in nanomedicine is empowering new approaches to improve the no-reflow phenomenon. However, there is no clinical evidence based on agents targeting microcirculation, and efficient treatments for reducing cerebral microthrombi and improving stroke outcomes have not been well established until now. Further experimental and clinical evidence is still required to establish an effective treatment for microcirculation restoration after acute ischemic stroke.

Conclusion

In conclusion, as stroke outcomes heavily depend on successful reperfusion, understanding potential mechanisms that contribute to this microcirculatory insufficiency and the development of detection methods as well as therapeutic strategies to restore microcirculation should become one of the potential research directions in the future.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

This work is financially supported by National Natural Science Foundation of China (No. 82071306, 81971115)

Author Contributions

Jiaqi Hu and Ding Nan performed the literature search and drafted the manuscript. Yining Huang and Haiqiang Jin proposed the idea for the article and acquired funding. Yuxuan Lu, Zhenyu Niu, Yingying Ren, Xiaozhong Qu, and Haiqiang Jin critically revised the work. All authors read and approved the final manuscript.

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