Endogenous neurogenesis following ischaemic brain injury: Insights for therapeutic strategies

Ischaemic stroke is among the most common yet most intractable types of central nervous system (CNS) injury in the adult human population. In the acute stages of disease, neurons in the ischaemic lesion rapidly die and other neuronal populations in the ischaemic penumbra are vulnerable to secondary injury. Multiple parallel approaches are being investigated to develop neuroprotective, reparative and regenerative strategies for the treatment of stroke. Accumulating evidence indicates that cerebral ischaemia initiates an endogenous regenerative response within the adult brain that potentiates adult neurogenesis from populations of neural stem and progenitor cells. A major research focus has been to understand the cellular and molecular mechanisms that underlie the potentiation of adult neurogenesis and to appreciate how interventions designed to modulate these processes could enhance neural regeneration in the post-ischaemic brain. In this review, we highlight recent advances over the last 5 years that help unravel the cellular and molecular mechanisms that potentiate endogenous neurogenesis following cerebral ischaemia and are dissecting the functional importance of this regenerative mechanism following brain injury.This article is part of a Directed Issue entitled: Regenerative Medicine: the challenge of translation.     

Pathophysiology of the Ischemic Penumbra—Revision of a Concept

1. The original concept of the ischemic penumbra surrounding a focus of dense cerebral ischemia is based on electrophysiological observations. In the cortex of baboons following middle cerebral artery occlusion, complete failure of the cortical evoked potential was observed at a cerebral blood flow (CBF) threshold level of approx. 0.15 ml/g/min—a level at which extracellular potassium ion activity was only mildly elevated. With a greater CBF decrement to the range of 0.06–0.10 ml/g/min, massive increases in extracellular potassium occurred and were associated with complete tissue infarction. Thus, the ischemic penumbra has been conceptualized as a region in which CBF reduction has exceeded the threshold for failure of electrical function but not that for membrane failure.2. Recent studies demonstrate that the penumbra as defined classically by the flow thresholds does not survive prolonged periods of ischemia. The correlation of CBF autoradiograms with diffusion-weighted MR images and the regional distribution of cerebral metabolites reveals that the ischemic core region enlarges when adjacent, formerly penumbral, areas undergo irreversible deterioration during the initial hours of vascular occlusion. At the same time, the residual penumbra becomes restricted to the periphery of the ischemic territory, and its fate may depend critically upon early therapeutic intervention.3. In the border zone of brain infarcts, marked uncoupling of local CBF and glucose utilization is consistently observed. The correlation with electrophysiological measurements shows that metabolism-flow uncoupling is associated with sustained deflections of the direct current (DC) potential resembling transient depolarizations. Such penumbral cell depolarizations, which are associated with an increased metabolic workload, induce episodes of tissue hypoxia due to the constrained collateral flow, stimulate anaerobic glycolysis leading to lactacidosis, suppress protein synthesis, and, finally, compromise energy metabolism. The frequency of their occurrence correlates with the final volume of ischemic injury. Therefore, penumbral depolarizations are regarded as a key event in the pathogenesis of ischemic brain injury. Periinfarct DC deflections can be suppressed by NMDA and non-NMDA antagonists, resulting in a significant reduction of infarct size.4. The histopathological sequelae within the penumbra consist of various degrees of scattered neuronal injury, also termed incomplete infarction. The reduction of neuronal density at the infarct border is a flow- and time-dependent event which is accompanied by an early response of glial cells. As early as 3 hr after vascular occlusion a generalized microglial activation can be detected throughout the ipsilateral cortex. Astrocytic activation is observed in the intact parts of the ischemic hemisphere from 6 hr postocclusion onward. Thus, the penumbra is a spatially dynamic brain region of limited viability which is characterized by complex pathophysiological changes involving neuronal function as well as glial activation in response to local ischemic injury.     

Biochemical and Molecular Characteristics of the Brain with Developing Cerebral Infarction

1. We review the biochemical and molecular changes in brain with developing cerebral infarction, based on recent findings in experimental focal cerebral ischemia.2. Occlusion of a cerebral artery produces focal ischemia with a gradual decline of blood flow, differentiating a severely ischemic core where infarct develops rapidly and an area peripheral to the core where the blood flow reduction is moderate (called penumbra). Neuronal injury in the penumbra is essentially reversible but only for several hours. The penumbra area tolerates a longer duration of ischemia than the core and may be salvageable by pharmacological agents such as glutamate antagonists or prompt reperfusion.3. Upon reperfusion, brain cells alter their genomic properties so that protein synthesis becomes restricted to a small number of proteins such as stress proteins. Induction of the stress response is considered to be a rescue program to help to mitigate neuronal injury and to endow the cells with resistance to subsequent ischemic stress. The challenge now is to determine how the neuroprotection conferred by prior sublethal ischemia is achieved so that rational strategies can be developed to detect and manipulate gene expression in brain cells vulnerable to ischemia.4. Expansion of infarction may be caused by an apoptotic mechanism. Investigation of apoptosis may also help in designing novel molecular strategies to prevent ischemic cell death.5. Ischemia/reperfusion injury is accompanied by inflammatory reactions induced by neutrophils and monocytes/macrophages infiltrated and accumulated in ischemic areas. When the role of the inflammatory/immune systems in ischemic brain injury is revealed, new therapeutic targets and agents will emerge to complement and synergize with pharmacological intervention directed against glutamate and Ca²⁺ neurotoxicity.     

N-Myc Downstream-Regulated Gene 2 (Ndrg2) Is Involved in Ischemia–Hypoxia-Induced Astrocyte Apoptosis: a Novel Target for Stroke Therapy

Nearly all clinical trials that have attempted to develop effective strategies against ischemic stroke have failed, excluding those for thrombolysis, and most of these trials focused only on preventing neuronal loss. However, astrocytes have gradually become a target for neuroprotection in stroke. In previous studies, we showed that the newly identified molecular N-myc downstream-regulated gene 2 (Ndrg2) is specifically expressed in astrocytes in the brain and involved in some neurodegenerative diseases. However, the role of NDRG2 in ischemic stroke remained unclear. In this study, we investigated the role of NDRG2 in middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia and in oxygen–glucose deprivation (OGD)-induced cellular apoptosis in the M1800 astrocyte cell line. NDRG2 mRNA and protein expression began to increase at 6 and 2 h after reperfusion and peaked at 24 h in the ischemic penumbra and in M1800 cells, as detected by RT-PCR and Western blotting. Double immunofluorescence staining showed that the number of apoptotic cells increased as the NDRG2-positive signal increased and that the NDRG2 signal was sometimes co-localized with TUNEL-positive cells and translocated from the cytoplasm to the nucleus in both the ischemic penumbra and the M1800 cells. Using a lentivirus, we successfully constructed two stable astrocytic cell lines in which NDRG2 expression was significantly up- or down-regulated. NDRG2 silencing had a proliferative effect and reduced the percentage of apoptotic cells, reactive oxygen species (ROS) production, and cleaved Caspase-3 protein expression following OGD, whereas NDRG2 over-expression had the opposite effects. In conclusion, NDRG2 is involved in astrocyte apoptosis following ischemic–hypoxic injury, and inhibiting NDRG2 expression significantly reduces ROS production and astrocyte apoptosis. These findings provide insight into the role of NDRG2 in ischemic–hypoxic injury and provide potential targets for future clinical therapies for stroke.     

Pathophysiology and Therapy of Experimental Stroke

1. Stroke is the neurological evidence of a critical reduction of cerebral blood flow in a circumscribed part of the brain, resulting from the sudden or gradually progressing obstruction of a large brain artery. Treatment of stroke requires the solid understanding of stroke pathophysiology and involves a broad range of hemodynamic and molecular interventions. This review summarizes research that has been carried out in many laboratories over a long period of time, but the main focus will be on own experimental research.2. The first chapter deals with the hemodynamics of focal ischemia with particular emphasis on the collateral circulation of the brain, the regulation of blood flow and the microcirculation. In the second chapter the penumbra concept of ischemia is discussed, providing a detailed list of the physiological, biochemical and structural viability thresholds of ischemia and examples of how these thresholds can be applied for imaging the penumbra. The third chapter summarizes the pathophysiology of infarct progression, focusing on the role of peri-infarct depolarisation, the multitude of putative molecular injury pathways, brain edema and inflammation. Finally, the fourth chapter provides an overview of currently discussed therapeutic approaches, notably the effect of mechanical or thrombolytic reperfusion, arteriogenesis, pharmacological neuroprotection, ischemic preconditioning and regeneration.3. The main emphasis of the review is placed on the balanced differentiation between hemodynamic and molecular factors contributing to the manifestation of ischemic injury in order to provide a rational basis for future therapeutic interventions.     

Irreversible aggregation of protein synthesis machinery after focal brain ischemia

Focal brain ischemia leads to a slow type of neuronal death in the penumbra that starts several hours after ischemia and continues to mature for days. During this maturation period, blood flow, cellular ATP and ionic homeostasis are gradually recovered in the penumbral region. In striking contrast, protein synthesis is irreversibly inhibited. This study used a rat focal brain ischemia model to investigate whether or not irreversible translational inhibition is due to abnormal aggregation of translational complex components, i.e. the ribosomes and their associated nascent polypeptides, protein synthesis initiation factors and co-translational chaperones. Under electron microscopy, most rosette-shaped polyribosomes were relatively evenly distributed in the cytoplasm of sham-operated control neurons, but clumped into large abnormal aggregates in penumbral neurons subjected to 2 h of focal ischemia followed by 4 h of reperfusion. The abnormal ribosomal protein aggregation lasted until the onset of delayed neuronal death at 24-48 h of reperfusion after ischemia. Biochemical study further suggested that translational complex components, including small ribosomal subunit protein 6 (S6), large subunit protein 28 (L28), eukaryotic initiation factors 2alpha, 4E and 3eta, and co-translational chaperone heat-shock cognate protein 70 (HSC70) and co-chaperone Hdj1, were all irreversibly clumped into large abnormal protein aggregates after ischemia. Translational complex components were also highly ubiquitinated. This study clearly demonstrates that focal ischemia leads to irreversible aggregation of protein synthesis machinery that contributes to neuronal death after focal brain ischemia.     

Autophagy and Apoptosis Are Differentially Induced in Neurons and Astrocytes Treated with an In Vitro Mimic of the Ischemic Penumbra

The development of clinical stroke therapies remains elusive. The neuroprotective efficacies of thousands of molecules and compounds have not yet been determined; however, screening large volumes of potential targets in vivo is severely rate limiting. High throughput screens (HTS) may be used to discover promising candidates, but this approach has been hindered by the lack of a simple in vitro model of the ischemic penumbra, a clinically relevant region of stroke-afflicted brain. Recently, our laboratory developed such a mimic (ischemic solution: IS) suitable for HTS, but the etiology of stress pathways activated by this model are poorly understood. The aim of the present study was to determine if the cell death phenotype induced by IS accurately mimics the in vivo penumbra and thus whether our model system is suitable for use in HTS. We treated cultured neuron and astrocyte cell lines with IS for up to 48 hrs and examined cellular energy state ([ATP]), cell and organelle morphology, and gene and molecular profiles related to stress pathways. We found that IS-treated cells exhibited a phenotype of mixed apoptosis/autophagy characteristic of the in vivo penumbra, including: (1) short-term elevation of [ATP] followed by progressive ATP depletion and Poly ADP Ribose Polymerase cleavage, (2) increased vacuole number in the cytoplasm, (3) mitochondrial rupture, decreased mitochondrial and cristae density, release of cytochrome C and apoptosis inducing factor, (4) chromatin condensation, nuclear lamin A and DNA cleavage, fragmentation of the nuclear envelope, and (5) altered expression of mRNA and proteins consistent with autophagy and apoptosis. We conclude that our in vitro model of the ischemic penumbra induces autophagy and apoptosis in cultured neuron and astrocyte cell lines and that this mimic solution is suitable for use in HTS to elucidate neuroprotective candidates against ischemic penumbral cell death.     

Role of acute-phase protein ORM in a mice model of ischemic stroke

The only Food and Drug Administration-approved treatment for acute ischemic stroke is tissue plasminogen activator, and the discovery of novel therapeutic targets is critical. Here, we found orosomucoid (ORM), an acute-phase protein mainly produced by the liver, might act as a treatment candidate for an ischemic stroke. The results showed that ORM2 is the dominant subtype in mice normal brain tissue. After middle cerebral artery occlusion (MCAO), the level of ORM2 is significantly increased in the ischemic penumbra compared with the contralateral normal brain tissue, whereas ORM1 knockout did not affect the infarct size. Exogenous ORM could significantly decrease infarct size and neurological deficit score. Inspiringly, the best administration time point was at 4.5 and 6 hr after MCAO. ORM could markedly decrease the Evans blue extravasation, and improve blood–brain barrier-associated proteins expression in the ischemic penumbra of MACO mice and oxygen–glucose deprivation (OGD)-treated bEnd3 cells. Meanwhile, ORM could significantly alleviate inflammation by inhibiting the production of interleukin 1β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α), reduce oxidative stress by improving the balance of malondialdehyde (MDA) and superoxide dismutase (SOD), inhibit apoptosis by decreasing caspase-3 activity in ischemic penumbra of MCAO mice and OGD-treated bEnd.3 cells. Because of its protective role at multiple levels, ORM might be a promising therapeutic target for ischemic stroke.     

miRNAs-19b, -29b-2* and -339-5p Show an Early and Sustained Up-Regulation in Ischemic Models of Stroke

Stroke, the loss of neurons after ischemic insult to the brain, is one of the leading causes of death and disability worldwide. Despite its prevalence and severity, current therapy is extremely limited, highlighting the importance of further understanding the molecular events underlying ischemia-induced neuronal cell death. An ischemic area can be subdivided into two separate pathophysiological regions: the rapidly dying necrotic core, and the potentially salvageable apoptotic penumbra. Understanding molecular events occurring in the apoptotic ischemic penumbra may give greater insight into mechanisms controlling this salvageable tissue. miRNAs are known to have key roles in the regulation of gene expression in numerous pathological conditions, including the modulation of distinct pathways in stroke. However, previous studies have profiled miRNAs in the whole ischemic infarct, and do not differentiate between miRNA regulation in the necrotic core versus the apoptotic penumbra. We asked if there were unique miRNAs that are differentially regulated following ischemic insults in the salvageable apoptotic penumbra. miRNA expression profiles were compared in the whole infarct from in vivo stroke models, using the three vessel occlusion approach, to an in vitro model of the ischemic penumbra, prior to apoptotic induction. Multiple miRNAs were found to be differentially regulated following ischemic insults in each system. However, miR-19b, miR-29b-2* and miR-339-5p were significantly up-regulated in both model systems. Further, we confirmed these results in a neuroblastoma cell line subjected to a penumbra-like ischemic insult that induced the apoptotic cell death pathway. The data show that miR-19b, miR-29b-2* and miR-339-5p are up-regulated following ischemic insults and may be regulating gene expression to control important cellular pathways in the salvageable ischemic penumbra. Further investigation of their role and mRNA target identification may lead to new insights into the molecular mechanisms taking place in the salvageable apoptotic penumbra.     

Triggers and mediators of hemorrhagic transformation in cerebral ischemia

Intracerebral hemorrhagic transformation is a multifactorial phenomenon in which ischemic brain tissue converts into a hemorrhagic lesion with blood-vessel leakage, extravasation, and further brain injury. It has been estimated that up to 30-40% of all ischemic strokes undergo spontaneous hemorrhagic transformation, and this phenomenon may become even more prevalent with the increasing use of thrombolytic stroke therapy. An emerging conceptual model suggests that the loss of microvascular integrity and disruption of neurovascular homeostasis connects the experimental findings of blood-cell extravasation to brain injury after hemorrhage. In this short article, we examine mechanisms related to reperfusion injury and oxidative stress, leukocyte infiltration, vascular activation, and dysregulated extracellular proteolysis as potential triggers of hemorrhagic transformation. Perturbations in cell-cell and cell-matrix signaling within the hypothesized neurovascular unit may ultimately lead to neuroinflammation and apoptotic-like cell death in the parenchyma. Further investigations into the molecular mediators of hemorrhagic transformation may reveal new therapeutic targets for this clinically complex problem.     

Remote Neurodegeneration: Multiple Actors for One Play

Remote neurodegeneration significantly influences the clinical outcome in many central nervous system (CNS) pathologies, such as stroke, multiple sclerosis, and traumatic brain and spinal cord injuries. Because these processes develop days or months after injury, they are accompanied by a therapeutic window of opportunity. The complexity and clinical significance of remote damage is prompting many groups to examine the factors of remote degeneration. This research is providing insights into key unanswered questions, opening new avenues for innovative neuroprotective therapies. In this review, we evaluate data from various remote degeneration models to describe the complexity of the systems that are involved and the importance of their interactions in reducing damage and promoting recovery after brain lesions. Specifically, we recapitulate the current data on remote neuronal degeneration, focusing on molecular and cellular events, as studied in stroke and brain and spinal cord injury models. Remote damage is a multifactorial phenomenon in which many components become active in specific time frames. Days, weeks, or months after injury onset, the interplay between key effectors differentially affects neuronal survival and functional outcomes. In particular, we discuss apoptosis, inflammation, oxidative damage, and autophagy—all of which mediate remote degeneration at specific times. We also review current findings on the pharmacological manipulation of remote degeneration mechanisms in reducing damage and sustaining outcomes. These novel treatments differ from those that have been proposed to limit primary lesion site damage, representing new perspectives on neuroprotection.     

Long-term cerebral cortex protection and behavioral stabilization by gonadal steroid hormones after transient focal hypoxia

Sex steroids are neuroprotective following traumatic brain injury or during neurodegenerative processes. In a recent short-term study, we have shown that 17β-estradiol (E) and progesterone (P) applied directly after ischemia reduced the infarct volume by more than 70%. This protection might primarily result from the anti-inflammatory effects of steroids. Here, we focus on the long-term neuroprotection by both steroids with respect to the infarct volume, functional recovery, and vessel density in the penumbra. The application of E/P during the first 48h after stroke (transient middle cerebral artery occlusion, tMCAO) revealed neuroprotection after two weeks. The infarct area was reduced by 70% and motor activity was preserved compared to placebo-treated animals. Blood vessel density in the penumbra using immunohistochemistry for von Willebrand factor showed increased vessel density after tMCAO which was not affected by hormones. Expression of vascular endothelial growth factor (VEGF) and its receptor (R1) was increased at 24h after tMCAO and up-regulated by E/P but not changed 14 days after stroke. These findings suggest that the neuroprotective potency of both steroids is sustained and persists for at least two weeks. Besides anti-inflammatory and anti-apoptotic actions, angiogenesis in the damaged area appears to be initially affected early after ischemia and is manifested up to two weeks. This article is part of a Special Issue entitled 'Neurosteroids'.     

Inhibition of nNOS reduces ischemic cell death through down-regulating calpain and caspase-3 after experimental stroke

In vitro nitric oxide (NO) regulates calpain and caspase-3 activation, and in vivo neuronal nitric oxide synthase (nNOS), calpain and caspase-3 participate in the ischemic brain injury. Our objective was to investigate whether nNOS was involved in the ischemic brain injury through activating calpain and caspase-3 during experimental stroke. Rats received 1-h ischemia by intraluminant filament, and then reperfused for 23 h (R 23 h). nNOS inhibitor 7-nitroindozale (7-NI, 50 mg/kg) was administrated intraperitoneally 5 min before ischemia. Our data showed that treatment with 7-NI markedly reduced neurological deficits, the brain swelling, and the infarct volume at R 23 h. Enzyme studies revealed significant suppression of the activities of m-calpain and caspase-3 in penumbra and core, and the activities of μ-calpain in penumbra, but not in core, in 7-NI-treated rats versus vehicle-treated rats. Western blot analysis demonstrated that 7-NI markedly increased the levels of MAP-2 and spectrin in penumbra and core compared with vehicle-treated rats. Histopathological studies displayed that 7-NI significantly reduced the necrotic cell death in penumbra and core, and apoptotic cell death in penumbra, but not in core. These data demonstrate the involvement of NO produced by nNOS in the ischemic neuronal injury through affecting the activation of calpain and caspase-3 in penumbra and core after experimental stroke, which provides a new perspective on possible mechanisms of action of nNOS inhibition in cerebral ischemia.     

Inflammatory regulators of redirected neural migration in the injured brain

Brain injury following stroke or trauma induces the migration of neuroblasts derived from subventricular zone neural precursor cells (NPCs) towards the damaged tissue, where they then have the potential to contribute to repair. Enhancing the recruitment of new cells thus presents an enticing prospect for the development of new therapeutic approaches to treat brain injury; to this end, an understanding of the factors regulating this process is required. During the neuroinflammatory response to ischemic and traumatic brain injuries, a plethora of pro- and anti-inflammatory cytokines, chemokines and growth factors are released in the damaged tissue, and recent work indicates that a variety of these are able to influence injury-induced migration. In this review, we will discuss the contribution of specific chemokines and growth factors towards stimulating NPC migration in the injured brain.     

Atorvastatin protects against cerebral infarction via inhibition of NADPH oxidase-derived superoxide in ischemic stroke

Statins have recently been shown to exert neuronal protection in ischemic stroke. Reactive oxygen species, specifically superoxide formed during the early phase of reperfusion, augment neuronal injury. NADPH oxidase is a key enzyme for superoxide production. The present study tested the hypothesis that atorvastatin protects against cerebral infarction via inhibition of NADPH oxidase-derived superoxide in transient focal ischemia. Transient focal ischemia was created in halothane-anesthetized adult male Sprague-Dawley rats (250-300 g) by middle cerebral artery occlusion (MCAO). Atorvastatin (Lipitor, 10 mg/kg sc) was administered three times before MCAO. Infarct volume was measured by triphenyltetrazolium chloride staining. NADPH oxidase enzymatic activity and superoxide levels were quantified in the ischemic core and penumbral regions by lucigenin (5 microM)-enhanced chemiluminescence. Expression of NADPH oxidase membrane subunit gp91(phox) and membrane-translocated subunit p47(phox) and small GTPase Rac-1 was analyzed by Western blot. NADPH oxidase activity and superoxide levels increased after reperfusion and peaked within 2 h of reperfusion in the penumbra, but not in the ischemic core, in MCAO rats. Atorvastatin pretreatment prevented these increases, blunted expression of membrane subunit gp91(phox), and prevented translocation of cytoplasmic subunit p47(phox) to the membrane in the penumbra 2 h after reperfusion. Consequently, cerebral infarct volume was significantly reduced in atorvastatin-treated compared with nontreated MCAO rats 24 h after reperfusion. These results indicate that atorvastatin protects against cerebral infarction via inhibition of NADPH oxidase-derived superoxide in transient focal ischemia.     

Altered Resting-State fMRI Signals in Acute Stroke Patients with Ischemic Penumbra

Background

Identifying the ischemic penumbra in acute stroke subjects is important for the clinical decision making process. The aim of this study was to use resting-state functional magnetic resonance singal (fMRI) to investigate the change in the amplitude of low-frequency fluctuations (ALFF) of these subjects in three different subsections of acute stroke regions: the infarct core tissue, the penumbra tissue, and the normal brain tissue. Another aim of this study was to test the feasilbility of consistently detecting the penumbra region of the brain through ALFF analysis.

Methods

Sixteen subjects with first-ever acute ischemic stroke were scanned within 27 hours of the onset of stroke using magnetic resonance imaging. The core of infarct regions and penumbra regions were determined by diffusion and perfusion-weighted imaging respectively. The ALFF were measured from resting-state blood oxygen level dependent (BOLD) fMRI scans. The averaged relative ALFF value of each regions were correlated with the time after the onset of stroke.

Results

Relative ALFF values were significantly different in the infarct core tissue, penumbra tissue and normal brain tissue. The locations of lesions in the ALFF maps did not match perfectly with diffusion and perfusion-weighted imagings; however, these maps provide a contrast that can be used to differentiate between penumbra brain tissue and normal brain tissue. Significant correlations between time after stroke onset and the relative ALFF values were present in the penumbra tissue but not in the infarct core and normal brain tissue.

Conclusion

Preliminary results from this study suggest that the ALFF reflects the underlying neurovascular activity and has a great potential to estimate the brain tissue viability after ischemia. Results also show that the ALFF may contribute to acute stroke imaging for thrombolytic or neuroprotective therapies.     

Inhibition of Peroxynitrite-Induced Mitophagy Activation Attenuates Cerebral Ischemia-Reperfusion Injury

Activated autophagy/mitophagy has been intensively observed in ischemic brain, but its roles remain controversial. Peroxynitrite (ONOO^?), as a representative of reactive nitrogen species, is considered as a critical neurotoxic factor in mediating cerebral ischemia-reperfusion (I/R) injury, but its roles in autophagy/mitophagy activation remain unclear. Herein, we hypothesized that ONOO^? could induce PINK1/Parkin-mediated mitophagy activation via triggering dynamin-related protein 1 (Drp1) recruitment to damaged mitochondria, contributing to cerebral I/R injury. Firstly, we found PINK1/Parkin-mediated mitophagy activation was predominant among general autophagy, leading to rat brain injury at the reperfusion phase after cerebral ischemia. Subsequently, increased nitrotyrosine was found in the plasma of ischemic stroke patients and ischemia-reperfused rat brains, indicating the generation of ONOO^? in ischemic stroke. Moreover, in vivo animal experiments illustrated that ONOO^? was dramatically increased, accompanied with mitochondrial recruitment of Drp1, PINK1/Parkin-mediated mitophagy activation, and progressive infarct size in rat ischemic brains at the reperfusion phase. FeTMPyP, a peroxynitrite decomposition catalyst, remarkably reversed mitochondrial recruitment of Drp1, mitophagy activation, and brain injury. Intriguingly, further study revealed that ONOO^? induced tyrosine nitration of Drp1 peptide, which might contribute to mitochondrial recruitment of Drp1 for mitophagy activation. In vitro cell experiments yielded consistent results with in vivo animal experiments. Taken together, all above findings support the hypothesis that ONOO^?-induced mitophagy activation aggravates cerebral I/R injury via recruiting Drp1 to damaged mitochondria.     

Mitochondrial quality control in stroke: From the mechanisms to therapeutic potentials

Mitochondrial damage is a critical contributor to stroke‐induced injury, and mitochondrial quality control (MQC) is the cornerstone of restoring mitochondrial homeostasis and plays an indispensable role in alleviating pathological process of stroke. Mitochondria quality control promotes neuronal survival via various adaptive responses for preserving mitochondria structure, morphology, quantity and function. The processes of mitochondrial fission and fusion allow for damaged mitochondria to be segregated and facilitate the equilibration of mitochondrial components such as DNA, proteins and metabolites. The process of mitophagy is responsible for the degradation and recycling of damaged mitochondria. This review aims to offer a synopsis of the molecular mechanisms involved in MQC for recapitulating our current understanding of the complex role that MQC plays in the progression of stroke. Speculating on the prospect that targeted manipulation of MQC mechanisms may be exploited for the rationale design of novel therapeutic interventions in the ischaemic stroke and haemorrhagic stroke. In the review, we highlight the potential of MQC as therapeutic targets for stroke treatment and provide valuable insights for clinical strategies.     

Comparative Assessment of the Proteolytic Stability and Impact of Poly-Arginine Peptides R18 and R18D on Infarct Growth and Penumbral Tissue Preservation Following Middle Cerebral Artery Occlusion in the Sprague Dawley Rat

Poly-arginine peptides R18 and R18D have previously been demonstrated to be neuroprotective in ischaemic stroke models. Here we examined the proteolytic stability and efficacy of R18 and R18D in reducing infarct core growth and preserving the ischaemic penumbra following middle cerebral artery occlusion (MCAO) in the Sprague Dawley rat. R18 (300 or 1000 nmol/kg), R18D (300 nmol/kg) or saline were administered intravenously 10 min after MCAO induced using a filament. Serial perfusion and diffusion-weighted MRI imaging was performed to measure changes in the infarct core and penumbra from time points between 45- and 225-min post-occlusion. Repeated measures analyses of infarct growth and penumbral tissue size were evaluated using generalised linear mixed models (GLMMs). R18D (300 nmol/kg) was most effective in slowing infarct core growth (46.8 mm³ reduction; p?<?0.001) and preserving penumbral tissue (21.6% increase; p?<?0.001), followed by R18 at the 300 nmol/kg dose (core: 29.5 mm³ reduction; p?<?0.001, penumbra: 12.5% increase; p?<?0.001). R18 at the 1000 nmol/kg dose had a significant impact in slowing core growth (19.5 mm³ reduction; p?=?0.026), but only a modest impact on penumbral preservation (6.9% increase; p?=?0.062). The in vitro anti-excitotoxic neuroprotective efficacy of R18D was also demonstrated to be unaffected when preincubated for 1–3 h or overnight, in a cell lysate prepared from dying neurons or with the proteolytic enzyme, plasmin, whereas the neuroprotective efficacy of R18 was significantly reduced after a 2-h incubation. These findings highlight the capacity of poly-arginine peptides to reduce infarct growth and preserve the ischaemic penumbra, and confirm the superior efficacy and proteolytic stability of R18D, which indicates that this peptide is likely to retain its neuroprotective properties when co-administered with alteplase during thrombolysis for acute ischaemic stroke.