Review Revisiting cerebral postischemic reperfusion injury: new insights in understanding reperfusion failure, hemorrhage, and edema Jilin Bai and Patrick D. Lyden* Cerebral postischemic reperfusion injury is defined as deterioration of ischemic brain tissue that parallels and antagonizes the benefits of restoring cerebral circulation after therapeutic thrombolysis for acute ischemic stroke. To understand the paradox of injury caused by treatment, we first emphasize the phenomenon in which recanalization of an occluded artery does not lead to tissue reperfusion. Additionally, no-reflow after recanalization may be due to injury of the neurovascular unit, distal microthrombosis, or both, and certainly worsens outcome. We examine the mechanism of molecular and subcellular damage in the neurovascular unit, notably oxidative stress, mitochondrial dysfunction, and apoptosis. At the level of the neurovascular unit, which mediates crosstalk between the damaged brain and systemic responses in blood, we summarize emerging evidence demonstrating that individual cell components play unique and cumulative roles that lead to damage of the blood–brain barrier and neurons. Furthermore, we review the latest developments in establishing a link between the immune system and microvascular dysfunction during ischemic reperfusion. Progress in assessing reperfusion injury has also been made, and we review imaging studies using various magnetic resonance imaging modalities. Lastly, we explore potential treatment approaches, including ischemic preconditioning, postconditioning, pharmacologic agents, and hypothermia. Key words: acute, BBB, hypothermia, ischemic stroke, reperfusion, recanalization

Introduction Globally, stroke is the second leading cause of death (1–3). Stroke incidence is projected to rise due to aging of the global population during the next 20 years (4). Stroke can be ischemic or hemorrhagic, with the majority of strokes caused by ischemic events interrupting blood flow to the brain. In treating acute ischemic stroke, therapeutic thrombolysis with tissue plasminogen activator (tPA) and possibly mechanical thrombectomy to restore the blood flow are effective for reducing the size of infarct and improving clinical outcome (5–7). Recanalization is not risk free, however, and postreperfusion hemorrhage is a recognized risk of thrombolysis and thrombectomy that is more than compensated by substantial benefit. The mechanism of transformation from ischemic to hemorrhagic stroke may involve reperfusion-mediated injury to the several elements of the Correspondence: Patrick D. Lyden*, Department of Neurology, Cedars-Sinai Medical Center, Los Angeles, CA, USA. E-mail: [email protected] Department of Neurology, Cedars-Sinai Medical Center, Los Angeles, CA, USA Received: 30 June 2014; Accepted: 14 November 2014 Conflict of interest: None declared. DOI: 10.1111/ijs.12434 © 2015 World Stroke Organization

blood–brain barrier (BBB). In addition to hemorrhage, a subset of patients do not improve after successful recanalization via chemical or mechanical means despite restored cerebral circulation (8). This failure to benefit, or even suffer worsening, comprises a paradoxically deleterious effect of reperfusion that can be named reperfusion injury (9). Reperfusion injury is defined as a biochemical cascade causing a deterioration of ischemic brain tissue that parallels and antagonizes the beneficial effect of recanalization (10–12). Hemorrhagic transformation can be viewed as a clinical subset of reperfusion injury, but the mechanisms of the two syndromes may differ, and have not been fully elucidated. Recanalization and reperfusion Recanalization is defined as reopening of an occluded artery, whereas tissue reperfusion is defined as restoration of microcirculatory blood flow downstream of the recanalized artery (8,13,14). Complete recanalization does not always result in downstream reperfusion, a state that has been referred to as the ‘no-reflow phenomenon’ (15). To illustrate the difference between recanalization and reperfusion, it is helpful to review clinical studies with different outcomes after tPA treatment. At 24 h after stroke onset, a subset (24·8–27%) of tPA-treated patients demonstrated more than 10 points in improvement on the National Institutes of Health Stroke Scale (NIHSS) (16,17). In contrast, Christou et al. reported that among patients who received intravenous tPA and demonstrated recanalization by transcranial Doppler (TCD), there were 32% of patients without any improvement in the severity of neurological deficit and 15% of patients with worsening NIHSS score (>4 points) (18). In these observations, the mismatch between recanalization and clinical outcome may be related to incomplete tissue reperfusion, that is, failure to reperfuse despite recanalization. Direct evidence of reperfusion failure came from clinical studies using computed tomography (CT) or magnetic resonance imaging (MRI) to assess vessel status and tissue perfusion. In the Echoplanar Imaging Thrombolytic Evaluation Trial (EPITHET), among 18 patients receiving intravenous tPA, serial angiography and MRI perfusion studies demonstrated recanalization in 13 cases, of whom four cases showed no reperfusion (13). Animal studies using carbon tracer techniques and fluorescent-labeled intravascular markers showed no reperfusion despite recanalization of the middle cerebral artery (MCA) (19–21). The proposed mechanisms for impaired reperfusion include capillary constriction and luminal narrowing due to extra-luminal compression by swollen astrocyte endfeet and intra-luminal filling with entrapped erythrocytes, leukocytes and platelets (20,22–25); increased BBB permeability that activates tissue factor causing fibrin deposition and microvascular occlusion (26); and continuous pericyte contraction despite reopening Vol 10, February 2015, 143–152

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Fig. 1 Schematic model of neurovascular mechanism of postischemic reperfusion injury. During reperfusion after ischemia, overproduction of reactive oxygen species (ROS) causes oxidative stress. The oxidative stress damages the endothelial cells, resulting in an exposure of the subendothelial extracellular matrix to blood flow. The exposure triggers adhesion and activation of platelets in microvasculature causing thrombosis. The injured endothelial cells release metalloproteinase that attacks basal lamina causing leakage of the blood–brain barrier (BBB). The damaged endothelial cells interact with regulatory T cells and activated leukocytes to exacerbate intravascular thrombosis. In addition, a large amount of inflammatory factors including cytokines, chemokines, NO, TNF-a, IL-1b, IL-6, ROS, prostanoids are released from activated astrocytes and microglia. The ensuing inflammatory response causes a high degree of cytotoxicity that amplifies neuronal death in the penumbra. Furthermore, the oxidative stress causes sustained contraction of pericytes which leads to narrowing of the microvasculature lumen. The lumenal narrowing is further worsened by compression from swollen end feet of activated astrocytes. RBC, red blood cell; NO, nitric oxide; IL, interleukin.

of an occluded cerebral artery (27). Figure 1 summarizes the key potential mechanisms involved in reperfusion failure. Major phenomena in brain ischemia and reperfusion Although we still lack a complete and coherent description of the major pathways and mechanisms that comprise the ischemic cascade leading to cerebral infarction, many pathways in the ischemic cascade contribute to reperfusion failure. Several key observations during brain ischemia and reperfusion shed some light on a complex pathobiological process that includes multiple parallel pathways proceeding independently but with feed-forward, feedback, and crosstalk loops. Temporally occurring first, ischemia causes a rapid loss of high-energy compounds, notably adenosine triphosphate (ATP), which leads to depolarization of cell membranes, accumulation of intracellular Ca2+, and lipolysis. A significant amount of lipolytic breakdown products remain latent during ischemia; upon reperfusion, these products form substrates for reaction with oxygen, yielding overproduction of reactive oxygen species (28). Also occurring temporally very early, tissue injury after ischemia and reperfusion causes morphologic changes of neurons with cytosolic microvacuolation. Microvacuoles occur within 15 min of reperfusion in selectively vulnerable neurons (hippocampal CA1 pyramidal neurons, cortical pyramidal neurons in layers 3 and 5, and Purkinje neurons) (29). Over a longer time course during postischemia reperfusion, protein synthesis is markedly suppressed in vulnerable neurons (30). Evolv-

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J. Bai and P. D. Lyden ing over hours to days, the disruption of BBB, cytotoxicity, and neuroinflammation may accumulatively lead to hemorrhagic transformation of the ischemic tissue (31). Excessive release of excitotoxic neurotransmitters also occurs early during ischemia. The depolarization and Ca2+ influx induced by ischemia cause release of a large amount of the excitotoxic neurotransmitter, glutamate (32). In addition, arachidonic acid and products of lipid peroxidation inhibit reuptake of glutamate (33,34). In the face of massive increase of extracellular excitotoxic neurotransmitters immediately after ischemia, and largely related to the failure of energy-dependent reuptake pumps, it was hypothesized by many that glutamate receptor antagonists could provide protection to stroke patients. In preclinical models, both AMPA (α-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid) and NMDA (n-methyl-d-aspartate) antagonists showed evidence of salvaging neuronal damage after severe cerebral ischemia, but no clinical trials proved positive (35). Of considerable importance, during reperfusion no evidence of persistently elevated extracellular glutamate has been shown (36). The decade or more experience with glutamate antagonists in both patients and stroke models suggest that (1) human drug therapy with glutamate antagonists cannot be applied soon enough to make a measureable impact on outcome, and (2) reperfusion likely resolves excitotoxin excess, perhaps making glutamate antagonist therapy moot after reperfusion. There is ample evidence, however, that excitotoxin excess directly and powerfully contributes to the overproduction of reactive oxygen species during reperfusion. Oxidative stress The mitochondria are important organelles in regulating energy metabolism to support tremendous energy demand of brain cells. Under normal physiological conditions, mitochondria constantly produce reactive oxygen species (ROS), including superoxide anion radicals and hydrogen peroxide (H2O2), which are then scavenged by superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase and other small molecular antioxidants including glutathione (GSH), ascorbic acid, and α-tocopherol (37). During reperfusion after ischemia, there is overproduction of ROS in mitochondria, which rapidly exhaust endogenous antioxidant scavenging capacity (38,39). The resulting flood of ROS directly causes oxidative damage on cellular macromolecules, such as proteins, nucleic acids, and lipids, which lead to mitochondrial swelling, cell injury, and death (40–42). Indirectly, the ROS causes exhaustion of SODs that functions as inhibitory molecular switches for apoptosis signaling pathways during cerebral ischemia and reperfusion. Neurons in transgenic mice overexpressing human CuZn-superoxide dismutase (CuZnSOD) in brain cells were protected against ischemia/reperfusion injury, but not from permanent focal ischemia (43). The neuroprotection was due to prevention of early release of mitochondrial cytochrome c, an important signal of apoptosis pathways, by CuZnSOD (44). Noshita et al. reported that down-regulation of manganese superoxide dismutase (MnSOD) in SOD2−/− mutants significantly increased the level of cytochrome c and caspase-9 compared with wild-type mice (45). Furthermore, the flood of © 2015 World Stroke Organization

J. Bai and P. D. Lyden ROS inactivates nitric oxide (NO) produced by endothelial NO synthase (eNOS) causing dysfunction of cerebral arteries and arterioles (46–48). Baumbach et al. showed that the hypertrophy of cerebral arterioles in CuZnSOD−/− mice is greater than in eNOS-deficient mice, indicating that in addition to the interaction of ROS with NO, ROS affects vascular growth directly via CuZnSOD (49). Mitochondrial dysfunction manifests as a variety of other subcellular processes, and excessive ROS production is only one of the initial causes that lead to mitochondrial swelling, cell injury, and death. In addition, the impaired capacity of mitochondria to regulate calcium homeostasis leads to overload of calcium, which causes membrane permeability transition (MPT) due to opening of the mitochondrial permeability transition pores (mPTP) (50,51). As a result, the mitochondrial transmembrane potential dissipates and the ensuing influx of solutes leads to mitochondrial swelling (52,53). Mitochondrial biogenesis is a highly regulated process for preexisting mitochondria to grow and divide in response to environmental stress associated with energy limitation and oxidative stress (54). In rat cerebral ischemia/reperfusion injury models, reperfusion promotes mitochondrial biogenesis evidenced by increase of cortical mitochondrial DNA content and the up-regulation of three transcriptional genes involved in regulating mitochondrial biogenesis. The three genes are peroxisome proliferator-activated receptor coactivator-1α (PGC-1α), which stimulates mitochondrial biogenesis; nuclear respiratory factor (NRF)-1 and 2, coordinators between nuclear and mitochondrial gene expression; and mitochondrial transcription factor A (TFAM), a stimulator of mtDNA transcription (55). In contrast to mitochondrial dysfunction, mitochondrial biogenesis appears to be an endogenous protective response to cerebral ischemia injury. For example, one study showed exercise after ischemia promotes mitochondrial biogenesis with reduction in brain ischemic size and improvement of neurological symptoms (56). In addition, sublethal inflammatory stimuli-lipopolysaccharide (LPS)-induced mitochondrial biogenesis contributes to ischemic neuroprotection (57). Protein synthesis suppression After 4 h reperfusion following 30 min of cerebral ischemia, there is a 30% decrease in [14C]-labeled amino acid incorporation in brain, indicating a suppression of protein synthesis (58). Suppression is due to inhibition of protein translation initiation during early reperfusion (59). Briefly, during postischemic reperfusion, several endoplasmic reticulum (ER) stressors (ATP depletion, decreased ER-associated protein degradation, inhibition of protein glycosylation, ER reducing conditions, and ER Ca2+ depletion) cause accumulation of misfolded proteins in the ER lumen (60). These misfolded proteins subsequently activate three known effectors (PERK, IRE1, and ATF6) within minutes of reperfusion (61–63). PERK activation leads to phosphorylation of eukaryotic initiation factor 2 alpha (EIF2α) which down-regulates the rate of global protein synthesis (59,64). IRE1 causes activation of JNKs and caspase-12, as well as degradation of 28s rRNA, all of which can initiate apoptotic pathways (63). The proteolysis of ATF6 © 2015 World Stroke Organization

Review liberates its cytoplasmic domain which subsequently translocates to the nucleus to induce transcription of genes containing the ER stress-response element (65). Apoptosis During postischemia reperfusion, many vulnerable neurons, particularly in the penumbra region, undergo apoptosis (66). Although the mechanism of apoptosis in brain cells is not fully understood, it appears that both extrinsic and intrinsic (mitochondrial) pathways are involved, and each pathway contains caspase-dependent and caspase-independent components (67,68). Consistent with earlier reports on pro-apoptotic and anti-apoptotic molecules after focal cerebral ischemia (67,69,70), a very recent study using apoptotic polymerase chain reaction (PCR) and antibody arrays provided a broad survey of proapoptotic and anti-apoptotic molecules and their temporal expression profiles at the transcriptional and translational levels (68). The authors showed an increased mRNA and protein expression levels of pro-apoptotic molecules: Bax, Bad, Bid, caspase-3, and cytochrome c within 24 h following reperfusion. They also showed increased levels of anti-apoptotic molecules, including Akt, ERK1/2, phospho-ERK, Bcl2, XIAP, Naip2, survivin, livin, HSP27, HSP60, and HSP70, as well as the inhibitor of apoptosis protein (IAP) up to seven-days after reperfusion (68). Therefore, the authors proposed a combined anti-apoptotic therapeutic strategy of decreasing apoptotic molecules and increasing anti-apoptotic molecules, rather than targeting single molecule, with a consideration of timing and frequency of treatment (68). Platelet activation During cerebral ischemia, there is injury to the vascular endothelium which leads to the exposure of the subendothelial extracellular matrix (ECM) to blood flow during reperfusion. This exposure triggers adhesion and excessive activation of blood platelets in the brain microvasculature (24,71). For many years, the platelet activation and aggregation were assumed to play a major role in the pathogenesis of reperfusion failure after recanalization. Although several pilot studies indicated promise, the failure of GPIIb/IIIa inhibitors in protecting infarct progression provided evidence for, at most, a minor role of platelet aggregation in stroke (72,73). In contrast, blockade of GPIb-α, a receptor involved in tethering platelet to the sites of vascular injury, demonstrated dramatic protection against stroke progression (72). Phospholipase D1 (PLD1) is a transducer of activation signals downstream of GPIb-vWF interaction pathway and is important to GPIb-dependent integrin αIIbβ3 activation and platelet adhesion. PLD1−/− mice showed significantly reduced stroke progression after transient MCAo without increased risk for bleeding (74). Consistent with the central role of GPIb-vWF interaction in thrombus formation and stroke progress, vWF−/− mice were protected from stroke with improved neurological function 24 h after transient MCAo without increased intracranial bleeding (75). The clinical implications of these recent animal studies are uncertain, given the prior failures of antiplatelet agents. Vol 10, February 2015, 143–152

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Review Complement activation Mounting evidence has shifted the view that the central nervous system (CNS) is immunologically privileged. It is well established that CNS has active biosynthesis of complement components (76). In transient middle cerebral artery occlusion (MCAo) rats, complement factor C3 presented in the ischemic core at both one-day and three-days following reperfusion (77). C3 receptor (CR3) was shown to have a spatiotemporal up-regulation in a transient MCAo rat model (78). Similarly, the presence of C1q was demonstrated in a transient MCAo mouse model following ischemia/reperfusion injury (79). In contrast, complement inhibition protects against stroke progression (80). These studies support an active role of the complement system in cerebral ischemia/reperfusion injury. Leukocyte infiltration During cerebral reperfusion, inflowing activated leukocytes interact with endothelial cells causing accumulation of leukocytes, red blood cells, and activated platelets in the microvascular bed. A significant microvascular obstruction can be easily demonstrated, and is postulated to underlie focal reperfusion failure and secondary cerebral ischemia (20,81). In contrast, neutrophil depletion with anti-neutrophil antibodies in ischemia/ reperfusion animal models results in a better recovery of regional cerebral blood flow and smaller infarct size compared with nonneutropenic groups (82,83). In male SCID (severe combined immunodeficient) mice lacking both T and B cells, there was 40% reduction of infarct volume after 90 min transient MCAo and 22 h reperfusion (84). Similarly, recombinase activating gene-deficient (Rag1−/−) mice lacking T and B cells developed 60% to 70% smaller infarct size and improved function after ischemia/reperfusion injury (85,86). However, there was no decrease in infarct size in mice with B-cell deficiency alone (86). Transplantation of B cells back to Rag1−/− mice did not enlarge the infarct size, whereas transferring wild-type CD3+ T cells back to Rag1−/− mice rendered the mice fully susceptible to ischemia/ reperfusion injury (85). There studies suggested that T cells, not B cells, play a major role in ischemia/reperfusion injury. Consistent with this, inhibition T-cell infiltration with anti-α4 integrin antibodies and vascular cell adhesion molecule 1 (VCAM 1) siRNA also reduced infarct size (87–89). Recently, Kleinschnitz et al. showed that FoxP3 positive regulatory T cells (Tregs) play an important role during cerebral ischemia/reperfusion injury (90). Selective depletion of Tregs in a transient MCAo model dramatically reduced infarct size and improved neurologic functional outcomes. Transfer of Tregs cells to Rag1−/− mice lacking lymphocytes significantly increased the stroke size. The authors further showed that Tregs interacted with ischemic brain endothelium through LFA-1/ICAM-1 binding pathway, causing microvascular dysfunction. Blocking the LFA-1/ ICAM-1 pathway reduced intravascular thrombosis and improved tissue reperfusion. Depletion of platelets also abolished Tregs’ detrimental effect on stroke. The role of Tregs in ischemia/ reperfusion injury appeared to supersede that of leukocytes, as depletion of leukocytes in mice without Tregs failed to improve stroke outcome (90).

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J. Bai and P. D. Lyden Exacerbation of BBB disruption During cerebral ischemia, microvascular injury causes increased permeability of the BBB (91–95). During postischemic reperfusion in a focal transient MCAo rat model, BBB disruption is typically observed to be more severe than when compared with permanent MCAo (10). Furthermore, increased cerebral blood flow volume during reperfusion is correlated to worsened BBB disruption (10). Postischemic permeability begins to increase one-hour after reperfusion (94), compared with three-hours after permanent MCAo in rats (96). In addition, studies on transient MCAo models have demonstrated a biphasic increase of the BBB permeability, with peaks occurring at 3 and 48 h after reperfusion (91,92,97). However, persisting and lesser degrees of BBB leakage can last up to four to five-weeks (98,99). Postischemic reperfusion causes enhanced production of free radicals and release of proteases from endothelial cells, astrocytes, microglia, and neurons. Matrix metalloproteinases (MMPs) play a major role mediating attack on the basal lamina in cerebral capillaries (100,101). The initial opening of the BBB at three-hours can be related to increased expression of MMP-2 and -9 which damage the BBB by degrading tight junction proteins, claudin-5, and occludin, whereas MMP inhibitors block BBB opening and associated edema (92,102). However, MMP inhibition failed to block the delayed BBB opening at 48 h, suggesting a multifactorial process (92). One explanation was that the MMP inhibitor had no effect on the BBB-damaging free radicals and other proteases, such as elastase, released by infiltrating leukocytes (92). In addition, using MMP-9−/− mice and bone marrow chimeric mice, Gidday et al. showed that MMP-9 released from circulating leukocytes, rather than brain, contributed significantly to BBB breakdown (103). The BBB impairment consists of complex ultrastructural disruptions, including damaged capillary endothelial cells, degeneration of astrocytes, and pericytes, as well as perivascular edema (104,105). These disruptions also correlate with damage to a highly coordinated neurovascular unit closely related to the BBB. Disruption of neurovascular unit The neurovascular unit has been proposed to illustrate the complex inter-relationships among all the brain cells connected to the capillary vasculature. The unit includes endothelial cells, pericytes, vascular smooth muscle cells, astrocytes, microglia, and neurons (106). It is a functional unit that intimately connects the vascular system and nervous system in the brain. Postischemia reperfusion causes damages to multiple components of the neurovascular unit (Fig. 1). Endothelial cells Electron microscopy (EM) analysis has shown excessive autophagosome accumulation within cerebral endothelial cells seven-days after transient MCAo in rats (104). Basal level function of the autophagosome-lysosomal pathway serves to remove damaged cellular components and metabolic toxins, whereas excessive autophagy may degrade normal cellular components and cause cell death (107). During postischemia reperfusion, the induced excessive autophagy may cause cell necrosis as shown by EM analysis in cerebral microvasculature (104,105). © 2015 World Stroke Organization

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Pericytes Pericytes attach to endothelial cells mostly at the capillary level where smooth muscle cells are absent. Pericytes project elongated, finger-like processes that surround the capillary wall. Pericytes contain contractile proteins which enable pericytes to function as smooth muscle to contract and regulate focal cerebral blood flow (27,108). After MCAo in rats, Yemisci et al. showed sustained contraction of pericytes on microvessels despite recanalization of the middle cerebral artery (27). They also showed that oxygen and nitrogen radicals released from microvasculature during ischemia and reperfusion caused contraction of pericytes which was relieved by nitric oxide synthase (NOS) inhibitors (27). Furthermore, tissue survival was improved by restoration of microcirculatory patency (27).

simultaneously causing cell death (115). In addition, ultrastructual analysis by EM and laser-scanning confocal microscopy showed damaged protein aggregations in neuronal cell body, dendrites, and axons (60). Furthermore, injury-resistant neurons can clear these protein aggregates, whereas vulnerable CA1 neurons retain them and progress to cell death (60). Chen et al. showed that the BBB disruption during ischemia reperfusion caused blood-borne toxic factor thrombin to enter the brain and cause additional neuronal death via the protein activated receptor-1 (PAR-1) on neurons (116). Furthermore, knockdown PAR-1 with short hairpin RNA (shRNA) showed increased neuroprotection and PAR-1 knockout mice demonstrated decreased infarction size after transient MCAo, suggesting PAR-1 antagonism as a powerful neuroprotection strategy (117).

Astrocytes Immunohistochemical analysis showed increased astrogliosis surrounding capillaries and in brain parenchyma after transient MCAo in rats (104). EM analysis has demonstrated swollen astrocyte end-feet compressing capillaries, causing luminal narrowing (22). In the process of ischemia and reperfusion injury, as an important member of neurovascular unit mediating crosstalk between damaged brain and systemic responses in blood, astrocytes become activated and proliferate, with roles ranging from cytotoxic to cytoprotective. During the acute stage, astrocytes release inflammatory factors, including cytokines, chemokines, and NO that can mediate cell adhesion and amplify neuronal death in the penumbra (109). During stroke recovery, reactive astrocytes release high-mobility-group-box-1 (HMGB1), a damage-associated molecular-pattern molecule, which, at low levels, increases the proliferation of endothelial progenitor cells (EPCs) and promotes neurovascular remodeling (110). The beneficial roles of promoting cellular proliferation has recently been observed in cell culture oxygen glucose deprivation/reperfusion studies (111).

MRI of reperfusion injury In noninvasively investigating and monitoring the process of postischemia reperfusion injury, MRI can provide extensive and extremely valuable information with different MRI sequences (118). Although MRI analysis of reperfusion injury is still in its infancy stage, there is true potential for MRI to explore the mechanism of reperfusion injury and assist treatment and prevention of reperfusion injury (119).

Microglia During postischemia reperfusion, microglial cells, as resident macrophages, become activated and can transform into phagocytes which release brain-derived neurotrophic factor (BDNF), insulin-like growth factor 1 (IGF-1), and transforming growth factor-β (TGF-β) (112,113). These neurotrophic molecules provide neuroprotection to the neurovascular unit (113). On the other hand, activated microglial cells also release proinflammatory molecules such as interleukin (IL)-1, tumor necrosis factor-alpha (TNF-α), IL-6, NO, ROS, and prostanoids which lead to significant cytotoxicity (114). In particular, IL-1 is directly involved in ischemia/reperfusion injury, and mice without genes for IL-1 had a 70% reduction in infarct volume (114). The microglial response to injury, therefore, can promote recovery or enhance injury, and the factors driving the balance between recovery and injury are not yet clear. Neurons In transient MCAo rats, during reperfusion from 6 to 48 h postischemia, Rami et al. have shown a marked increase of autophagy in penumbra neurons which indicates a complex role of autophagy, either removing damaged cellular components or © 2015 World Stroke Organization

Diffusion-weighted imaging (DWI) and secondary ischemia Using a transient MCAo rat model in a 2·0-T MRI, NeumannHaefelin et al. showed that after ischemia for 30 to 60 min, there is a transient normalization of DWI abnormalities during the reperfusion period but the DWI abnormalities recurred after oneday of reperfusion (120). Similarly, using a transient MCAo rat model with a 4·7-T MRI, Olah et al. showed that after ischemia for 60 min, there is a transient improvement in apparent diffusion coefficient (ADC) in the first two-hours of reperfusion with subsequent gradual worsening of ADC by the end of 10 h of reperfusion (121). These studies provided evidence for secondary ischemia induced by reperfusion. Perfusion-weighted imaging (PWI) and postischemic hyperperfusion Postischemic hyperperfusion is defined as increased cerebral perfusion after stroke and resulting from disruption of cerebral autoregulation (122–125). Subsequent cerebral edema or hemorrhage caused by hyperperfusion may constitute additional mechanisms of reperfusion injury. Furthermore, a secondary hypoperfusion following postischemic hyperperfusion may worsen the reperfusion injury (123–125). Using diffusion-perfusion MRI, Kidwell et al. have demonstrated postischemic hyperperfusion in 40% of humans within hours and in 50% of humans at day 7 after intraarterial thrombolysis (126). They also showed that regions with hyperperfusion eventually developed infarction (126). Contrast-enhanced MRI and BBB disruption Based on an observational study showing delayed contrast enhancement of the cerebrospinal fluid space on fluid-attenuated inversion recovery (FLAIR) images correlated with BBB disruption, Warach and Latour went on to report that over a period of one-week following intravenous or intra-arterial tPA treatment for ischemic stroke, patients with MRI evidence of reperfusion Vol 10, February 2015, 143–152

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Review developed more BBB disruption (45%) than patients without reperfusion (18%) (127). They named the delayed contrast enhancement of the cerebrospinal fluid space as ‘hyperintense acute reperfusion marker’ (HARM) and proposed HARM as a target for reducing complications associated with tPA treatment (127). Therapeutic potential Although our understanding of postischemic reperfusion injury is still very limited, new insights into the mechanism and potential therapeutic approaches are accumulating. We will highlight some animal studies and clinical experience that have demonstrated beneficial effects on ischemia/reperfusion injury. Ischemic preconditioning Ischemic preconditioning, also called ischemic tolerance, is a phenomenon that a brief ischemia (below the threshold of damage) that was applied one or several days before a subsequent severe ischemia actually reduces ischemic damage (128,129). It has been shown that the subthreshold stimulus activates endogenous protective mechanisms that can limit the impact of subsequent more severe insults. For example, retrospective and prospective studies have shown that previous transient ischemic attack attenuated subsequent stroke with smaller infarct size and less clinical deficits compared with patients without a preceding transient event (130–132). The putative mechanisms of preconditioning include inhibiting ROS activities, overexpression of heat shock proteins, immediate-early gene expression, synthesis of new proteins, inflammation inhibition, increase of antiapoptotic proteins, and activation of Akt pathways (133–141). Although the clinical application of preconditioning is very limited due to the fact that the occurrence of most strokes is unpredictable, several strategies are emerging. For example, to promote adaptation to hypoxia, the iron chelator desferrioxamine, erythropoietin, and inhalational anesthetics have been safe and effective candidates for preconditioning against brain ischemia/reperfusion injury (142–144). Ischemic postconditioning Ischemic postconditioning is a neuroprotective strategy performed after cerebral ischemia begins. With a series of rapid intermittent interruptions of cerebral blood flow in the early stage of reperfusion, the infarct size was robustly reduced (145). Zhao et al. used a postconditioning protocol with three cycles of 30 s reperfusion and 10 s occlusion of bilateral common carotid artery (CCA) after initial reperfusion to markedly reduce infarct size (17% to 80%) in permanent MCAo animal models (145). Liang et al. used a different protocol with three cycles of 30 s reperfusion and 30 s occlusion of ipsilateral CCA after transient MCAo for two-hours and achieved 15% reduction of infarct size (146). These studies further demonstrated that the neuroprotective mechanism of postconditioning was related to free radical generation during early postischemic reperfusion and inhibition of apoptosis via attenuating the activation of nuclear factor κB (NFκB)/p65 (145,146). With significant beneficial role in reducing reperfusion injury, postconditioning may be applicable for treatment of clinical conditions where vessel occlusion and

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J. Bai and P. D. Lyden reperfusion can be readily achieved as in endovascular thrombolysis or thrombectomy. Pharmacologic agents Because leukocyte infiltration has been identified as an important mechanism of reperfusion injury, utilizing antineutrophil or antiICAM-1 antibody to interfere with leukocyte infiltration has been shown to improve recovery of regional cerebral blood flow and reduce infarct size during reperfusion period in animal models (82,83,147). A clinical anti-leukocyte infiltration strategy was disappointing in human trials, although interpretation of the results was confounded by a complex immunological response in humans to the murine protein. Although superoxide dismutase and NADPH (nicotinamide adenine dinucleotide phosphateoxidase) oxidase have been shown to reduce ischemia/reperfusion injury in animal models (148,149), the clinical benefit of antioxidant strategy has also been difficult to show in human trials (150). Kahles and Brandes proposed explanations for disappointing clinical data as inadequate dose of antioxidants, administration of only one antioxidant, weak binding of ROS to antioxidant, inappropriate targeting by antioxidant and side effects of antioxidants (151). A recent study has shown that fingolimod, a sphingosine-1 phosphate receptor agonist, reduced infarct size and improved behavioral functions 15 days after transient MCAo in rodent models via anti-inflammatory effect and vasculoprotection (152). Fingolimod has beneficial therapeutic effects in advanced multiple sclerosis and efficacy in mitigating reperfusion injury in heart, liver, and kidney (153–156). Hypothermia Hypothermia is one of the most robust neuroprotectants identified to date for reducing ischemic damage in the brain (157–163). Hypothermia has a global protective effect against cerebral ischemia, and its role in mitigating reperfusion injury is of particular importance. It has been shown that hypothermia reduces cerebral metabolism, suppresses BBB injury and leakage, and attenuates neutrophil infiltration (79,164). Furthermore, based on unpublished studies in our laboratory, hypothermia provides protection to important members of neurovascular unit, including neurons, astrocytes, and endothelial cells in cell culture mimicking ischemic reperfusion condition. In conjunction with current multiple clinical trials of hypothermia on stroke treatment, more insights will be gained in its therapeutic potential in limiting reperfusion injury.

Conclusion Postischemic reperfusion injury posts a great challenge to clinicians and researchers due to the lack of a full understanding of its complex mechanism, no robust clinical measurement of ischemia and reperfusion, and no clearly effective treatment. However, after decades of ongoing research, more light has been shed on the process, leading to proposals for therapeutic intervention. It is still necessary to search for better assessment tools and deeper understanding of the molecular and cellular response to reperfusion. In this sense, thrombolytic therapy only opens the door to explore © 2015 World Stroke Organization

J. Bai and P. D. Lyden the hidden detrimental effect of ischemic stroke, reperfusion injury. Further exploration of the multiple cascading mechanisms of ischemic reperfusion injury is just the beginning to improving significantly the treatment of stroke.

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