Yongmei Zhao

Chelating Intracellularly Accumulated Zinc Decreased Ischemic Brain Injury Through Reducing Neuronal Apoptotic Death

Background and Purpose— Zinc has been reported to possess both neurotoxic and neuroprotective capabilities. The effects of elevated intracellular zinc accumulation following transient focal cerebral ischemia remain to be fully elucidated. Here, we investigated whether removing zinc with the membrane-permeable zinc chelator, N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), would decrease the intracellular levels of zinc in the ischemic tissue, leading to reduced brain damage and improved neurological outcomes. Methods— Rats were pretreated with TPEN or vehicle before or after a 90-minute middle cerebral artery occlusion. Cerebral infarct volume, neurological functions, neuronal apoptosis, poly(ADP-ribose) polymerase activity, and cytosolic labile zinc were assessed after ischemia and reperfusion. Results— Cerebral ischemia caused a dramatic cytosolic labile zinc accumulation in the ischemic tissue, which was decreased markedly by TPEN (15 mg/kg) pretreatment. Chelating zinc lead to reduced infarct volume compared with vehicle-treated middle cerebral artery occlusion rats, accompanied by much improved neurological assessment and motor function, which were sustained for 14 days after reperfusion. We also determined that reducing zinc accumulation rescued neurons from ischemia-induced apoptotic death by reducing poly(ADP-ribose) polymerase-1 activation. Conclusions— Ischemia-induced high accumulation of intracellular zinc significantly contributed to ischemic brain damage through promotion of neuronal apoptotic death. Removing zinc may be an effective and novel approach to reduce ischemic brain injury.

Bcl-2 phosphorylation triggers autophagy switch and reduces mitochondrial damage in limb remote ischemic conditioned rats after ischemic stroke.

Autophagy, an important intracellular degradation pathway, has been reported to clear impaired mitochondria and reduce mitochondria-mediated injury in ischemic disease. Our study and other recent investigations have shown that AKT-dependent autophagy contributes to the neuroprotection afforded by limb remote ischemic conditioning (RIC) in experimental stroke. However, how AKT triggers RIC-based autophagy and whether RIC-afforded autophagy is beneficial for mitochondrial function after cerebral ischemia remains unclear. The disruption of the Bcl-2/Beclin1 complex has been reported to trigger autophagy formation in the condition of Bcl-2 phosphorylation at Ser70. We investigated whether Bcl-2 phosphorylation triggers RIC-based autophagy and thereby confers RIC-induced neuroprotection against mitochondrial injury, using a transient cerebral ischemic rat model. We demonstrated that rats undergoing RIC treatment 30xa0min after the onset of ischemia (I-30) and at reperfusion (R-0) significantly upregulated Bcl-2 phosphorylation. Immunoprecipitation revealed that RIC increased dissociation of the Bcl-2/Beclin1 complex, leading to a higher level of autophagy than in ischemia/reperfusion rats. Furthermore, AKT activation was shown to play a critical role in regulating Bcl-2-mediated autophagy, as an AKT inhibitor (LY294002, AKTi) administered 30xa0min prior to ischemia significantly suppressed Bcl-2 phosphorylation and Bcl-2/Beclin1 complex dissociation, thereby reducing autophagy in RIC rats. Blocking Bcl-2 phosphorylation-dependent autophagy with AKTi suppressed RIC-afforded protection on mitochondrial potential and mitochondrial-dependent cell death effector pathway. These findings indicate that Bcl-2 phosphorylation and thereby Bcl-2/Beclin1 complex disruption play a crucial role in triggering autophagy and reducing mitochondrial damage in RIC rats after cerebral ischemia and require the involvement of the AKT activation.

AKT-Related Autophagy Contributes to the Neuroprotective Efficacy of Hydroxysafflor Yellow A against Ischemic Stroke in Rats

Hydroxysafflor yellow A (HSYA) has been approved clinically for treating cardiac patients in China since 2005. Recent studies have indicated that HSYA may be neuroprotective at 24xa0h in experimental stroke models. Autophagy is a vital degradation pathway of damaged intracellular macromolecules or organelles to maintain homeostasis in physiological or pathological conditions. The purpose of this study is to investigate the neuroprotection of HSYA at 72xa0h and its mechanism via activating the autophagy pathway using an acute ischemic-reperfusion stroke rat model. Rats were treated with HSYA (2xa0mg/kg) during 90xa0min middle cerebral artery occlusion/72xa0h reperfusion by intravenous administration at four different time points (15xa0min post-ischemia, 15xa0min, 24xa0h, and 48xa0h post reperfusion), mimicking the potential treatment for acute ischemic stroke. HSYA administration reduced infarction volume and improved various neurological functions at 72xa0h of reperfusion. The possible molecular mechanism was investigated. We found that HSYA activated the AKT-autophagy pathway in penumbra tissue, which occurred in neuronal-specific cells. Moreover, blocking the AKT-autophagy pathway by an AKT inhibitor abolished HSYA-induced neuroprotection after cerebral ischemia. HSYA may be a promising drug for treating acute ischemic stroke and the AKT-dependent autophagy pathway contributes to the HSYA-afforded neuroprotection.

Zinc contributes to acute cerebral ischemia-induced blood–brain barrier disruption

Zinc ions are stored in synaptic vesicles and cerebral ischemia triggers their release from the terminals of neurons. Zinc accumulation in neurons has been shown to play an important role in neuronal death following ischemia. However, almost nothing is known about whether zinc is involved in ischemia-induced blood-brain barrier (BBB) disruption. Herein, we investigated the contribution of zinc to ischemia-induced acute BBB disruption and the possible molecular mechanisms using both cellular and animal models of cerebral ischemia. Zinc greatly increased BBB permeability and exacerbated the loss of tight junction proteins (Occludin and Claudin-5) in the endothelial monolayer under oxygen glucose deprivation conditions. In cerebral ischemic rats, a dramatically elevated level of zinc accumulation in microvessels themselves was observed in isolated microvessels and in situ, showing the direct interaction of zinc on ischemic microvessels. Treatment with a specific zinc chelator N,N,N,N-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), even at 60-min post-ischemia onset, could greatly attenuate BBB permeability in the ischemic rats as measured by Evans Blue extravasation, edema volume and magnetic resonance imaging. Furthermore, zinc accumulation in microvessels activated the superoxide/matrix metalloproteinase-9/-2 pathway, which leads to the loss of tight junction proteins (Occludin and Claudin-5) and death of endothelial cells in microvessels themselves. Our findings reveal a novel mechanism of cerebral ischemia-induced BBB damage, and implicate zinc as an effective and viable new target for reducing acute BBB damage following ischemic stroke.

Reduction of zinc accumulation in mitochondria contributes to decreased cerebral ischemic injury by normobaric hyperoxia treatment in an experimental stroke model

Cerebral ischemia interrupts oxygen supply to the affected tissues. Our previous studies have reported that normobaric hyperoxia (NBO) can maintain interstitial partial pressure of oxygen (pO2) in the penumbra of ischemic stroke rats at the physiological level, thus affording significant neuroprotection. However, the mechanisms that are responsible for the penumbra rescue by NBO treatment are not fully understood. Recent studies have shown that zinc, an important mediator of intracellular and intercellular neuronal signaling, accumulates in neurons and leads to ischemic neuronal injury. In this study, we investigate whether NBO could regulate zinc accumulation in the penumbra and prevent mitochondrial damage in penumbral tissue using a transient cerebral ischemic rat model. Our results showed that NBO significantly reduced zinc-staining positive cells and zinc-staining intensity in penumbral tissues, but not in the ischemic core. Moreover, ischemia-induced zinc accumulation in mitochondria, isolated from penumbral tissues, was greatly attenuated by NBO or a zinc-specific chelator, N,N,N,N-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). NBO or TPEN administration stabilized the mitochondrial membrane potential in the penumbra after cerebral ischemia. Finally, ischemia-induced cytochrome c release from mitochondria in penumbral tissues was significantly reduced by NBO or TPEN treatment. These findings demonstrate a novel mechanism for NBOs neuroprotection, especially to penumbral tissues, providing further evidence for the potential clinical benefit of NBO for acute ischemic stroke.

Normobaric Hyperoxia Reduces Blood Occludin Fragments in Rats and Patients With Acute Ischemic Stroke

Background and Purpose— Damage of the blood–brain barrier (BBB) increases the incidence of neurovascular complications, especially for cerebral hemorrhage after tPA (tissue-type plasminogen activator) therapy. Currently, there is no effective method to evaluate the extent of BBB damage to guide tPA use. Herein, we investigated whether blood levels of tight junction proteins could serve as biomarker of BBB damages in acute ischemic stroke (AIS) in both rats and patients. We examined whether this biomarker could reflect the extent of BBB permeability during cerebral ischemia/reperfusion and the effects of normobaric hyperoxia (NBO) on BBB damage. Methods— Rats were exposed to NBO (100% O2) or normoxia (21% O2) during middle cerebral artery occlusion. BBB permeability was determined. Occludin and claudin-5 in blood and cerebromicrovessels were measured. Patients with AIS were assigned to oxygen therapy or room air for 4 hours, and blood occludin and claudin-5 were measured at different time points after stroke. Results— Cerebral ischemia/reperfusion resulted in the degradation of occludin and claudin-5 in microvessels, leading to increased BBB permeability in rats. In blood samples, occludin increased with 4-hour ischemia and remained elevated during reperfusion, correlating well with its loss from ischemic cerebral microvessels. NBO treatment both prevented occludin degradation in microvessels and reduced occludin levels in blood, leading to improved neurological functions in rats. In patients with AIS receiving intravenous tPA thrombolysis, the blood occludin was already elevated when patients arrived at hospital (within 4.5 hours since symptoms appeared) and remained at a high level for 72 hours. NBO significantly lowered the level of blood occludin and improved neurological functions in patients with AIS. Conclusions— Blood occludin may be a clinically viable biomarker for evaluating BBB damage during ischemia/reperfusion. NBO therapy has the potential to reduce blood occludin, protect BBB, and improve outcome in AIS patients with intravenous tPA thrombolysis. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT02974283.

Neuroprotective effects of Chrysophanol against inflammation in middle cerebral artery occlusion mice.

Ischemia/reperfusion (I/R) involves a cascade of reactions which ultimately lead to neuronal apoptosis or death. Inflammation plays an important role in this cascade. Chrysophanol (CHR), a purified active constituent from rhubarb, possesses many biological activities including anti-inflammation. The present study investigated the long-term neuroprotective effects of CHR on focal ischemic brain injury and the potential mechanism. Mice were subjected to 45-min middle cerebral artery occlusion and received either vehicle or CHR at 0.1, 1 or 10mg/kg for 14days after reperfusion. Neurological function, survival rate, brain tissue loss, expression of pro-inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and nuclear factor-kappa B p65 (NF-κB p65) were then assessed. The results showed that treatment with CHR led to improved survival rate and reduced brain tissue loss compared with vehicle-treated mice, accompanied by improved neurological assessment and motor function, which were sustained for 14days after I/R. I/R-induced expression of TNF-α, IL-1β and NF-κB p65 in neurons was markedly reduced in CHR-treated mice. These results indicate that CHR markedly attenuates brain injury after focal I/R, which is attributed at least in part to its anti-inflammatory actions.

Activation of T-LAK-cell-originated protein kinase-mediated antioxidation protects against focal cerebral ischemia–reperfusion injury

T‐LAK‐cell‐originated protein kinase (TOPK), a MAPKK‐like kinase, is crucial for neural progenitor cell proliferation; however, the function of TOPK and the molecular mechanism underlying cerebral ischemia–reperfusion injury remains unknown. Therefore, we investigated the role of TOPK in experimental stroke. Sprague–Dawley rats underwent transient middle cerebral artery occlusion (tMCAO) and reperfusion, and TOPK small interfering RNA (siRNA) was delivered by intracerebroventricular injection at the beginning of MCAO. After TOPK overexpression and H2O2 stimulation in PC12 neuronal cells, antioxidative proteins, apoptosis‐related proteins and signal pathways were detected by western blot analysis, the levels of the peroxidation products (malondialdehyde and 3–nitrotyrosine) were measured with ELISA. Phosphorylation of TOPK was increased in rat cortical neurons following tMCAO. TOPK overexpression in PC12 cells augmented levels of antioxidative proteins (peroxiredoxin 1 and 2, heme oxygenase 1 and manganese superoxide dismutase), as well as total superoxide dismutase activity, along with inhibition of malondialdehyde and 3–nitrotyrosine upon H2O2 stimulation. TOPK overexpression increased cell viability and reduced expression of caspase 3 and caspase 12 in PC12 cells in response to H2O2. The p–ERK level was increased by TOPK overexpression, and antioxidative protection afforded by TOPK was abolished by blocking the extracellular signal‐regulated kinase pathway in PC12 cells. TOPK siRNA increased the infarct volume and reduced total superoxide dismutase activity in the cortex in vivo after MCAO. These data reveal that activating TOPK confers neuroprotection against focal cerebral ischemia–reperfusion injury by antioxidative function, in part through activation of the extracellular signal‐regulated kinase pathway.

Chrysophanol inhibits endoplasmic reticulum stress in cerebral ischemia and reperfusion mice

Abstract Endoplasmic reticulum (ER) stress plays a critical role in mediating ischemia/reperfusion (I/R) damage in the brain. Our previous study showed that Chrysophanol (CHR) alleviated cerebral ischemic injury in mice and nuclear factor‐&kgr;B (NF‐&kgr;B) involved in its neuroprotective effect, but the precise mechanism remains not fully understood. The present study investigated the effect of CHR treatment on I/R‐induced ER stress. Mice were subjected to middle cerebral artery occlusion (MCAO) for 45 min and received either vehicle or CHR (0.1 mg/kg) for 14 days after reperfusion. Terminal deoxynucleotidyl transferase (TdT)‐mediated dUTP nick end labeling (TUNEL) was used to detect apoptotic cells in penumbral tissue. The expression of ER stress‐related factors including glucose‐regulated protein 78 (GRP78), phosphorylated eukaryotic initiation factor 2&agr; (p‐eIF2&agr;), CCAAT‐enhancer‐binding protein homologous protein (CHOP), and caspase‐12 as well as inhibitory &kgr;B‐&agr; (I&kgr;B‐&agr;), the inhibitor of NF‐&kgr;B, was assessed. Our results demonstrated that CHR treatment reduced MCAO‐induced upregulation of GRP78, p‐eIF2&agr;, CHOP, and caspase‐12 in the ischemic brain. Moreover, the TUNEL‐positive neuronal cells, which were colocalized with CHOP and caspase‐12, decreased in response to CHR treatment, indicating that CHR protects against I/R injury by inhibiting ER stress‐associated neuronal apoptosis. In addition, CHR reversed the decrease in I&kgr;B‐&agr; level induced by MCAO, which was attributed at least in part to the attenuation of translational inhibition induced by eIF2&agr; phosphorylation, indicating that CHR exerts anti‐inflammatory effects following I/R by inhibiting ER stress response. These results suggest that attenuation of ER stress may be involved in the mechanisms of neuroprotective effects of CHR.

Chrysophanol attenuates nitrosative/oxidative stress injury in a mouse model of focal cerebral ischemia/reperfusion

Nitrosative/oxidative stress plays an important role in neuronal death following cerebral ischemia/reperfusion (I/R). Chrysophanol (CHR) has been shown to afford significant neuroprotection on ischemic stroke, however, whether its mechanism is related to attenuating nitrosative/oxidative stress is not clear. In the present study, we investigated the effect of CHR on neuronal injury related to nitric oxide (NO) production by using mouse middle cerebral artery occlusion (MCAO) model. Our results revealed that nitrite plus nitrate (NOx-) and 3-nitrotyrosine (3-NT) levels increased in ischemic brain 14 days after reperfusion, and were subsequently attenuated by CHR treatment. Moreover, 3-NT is colocalized with NeuN and TUNEL, suggesting that neuronal apoptosis following I/R is associated with 3-NT and CHR suppresses NO-associated neuronal cell death. Accordingly, cleaved caspase-3 expression in ischemic brain was decreased by CHR treatment. I/R also decreased the activity of total superoxide dismutase (SOD) and manganese-dependent SOD (MnSOD), whilst increased reactive oxygen species (ROS) production significantly. Interestingly, CHR reversed this decrease in total SOD, and MnSOD activity, and inhibited ROS generation in the ischemic brain. Taken together, our results provide direct evidence suggesting that CHR attenuates nitrosative/oxidative stress injury induced by I/R, providing a novel therapeutic target in the treatment of acute ischemic stroke.