Astrocyte Autophagy Flux Protects Neurons Against Oxygen-Glucose Deprivation and Ischemic/Reperfusion Injury.

The role of autophagy varies with the type of acute brain injury. In general, autophagy mediates a clear neuroprotective effect in intoxication caused by various psychoactive agents, subarachnoid hemorrhage and spinal cord injury. In contrast, autophagic cell death has also been reported to actively contribute to neuronal loss in neonatal hypoxic ischemic encephalopathy. However, it still remains to be determined whether autophagy pays a cytoprotective or a cytotoxic role in stroke. Previous studies focused primarily on the role of neurons rather than the role of astrocytes in brain injury. Thus, it is unknown whether modulating the autophagy flux of astrocytes contributes to improving neuronal survival after stroke. In the current study, we investigated the time course of autophagy flux in vitro using cocultured astrocytes and neurons exposed to oxygen-glucose deprivation/reoxygenation, which mimicked the process of ischemia/reperfusion. Autophagy flux of astrocytes was regulated by treatment with the autophagy inducer rapamycin, autophagy inhibitor 3-methyladenine, and the transduction of small interfering RNA against autophagy-related gene 5. In addition, AAV-GFAP-ATG7 was used to induce astrocyte autophagy flux in mice subjected to focal cerebral ischemia. We found that induction of autophagy flux of astrocytes in vitro enhanced the viability of neurons and decreased neuronal apoptosis. Furthermore, induction of astrocyte autophagy flux in mice improved neurological outcomes. In contrast, inhibition of autophagy flux in astrocytes decreased the viability of neurons and increased neuronal apoptosis. These results suggest that upregulation of autophagy flux in astrocytes may contribute to endogenous neuroprotective and neurorecovery mechanisms after stroke.

In vivo optical imaging correlates with improvement of cerebral ischemia treated by intravenous bone marrow stromal cells (BMSCs) and edaravone.

OBJECTIVE: Recent studies show that modern In vivo optical imaging can detect matrix metallopeptidase (MMP) activation in the ischemic brain. In this study, we analyze the protective effects of bone marrow stromal cells (BMSCs) and edaravone (EDA) against tissue plasminogen activator (tPA) risk in the ischemic brain with In vivo optical fluorescence MMP imaging. METHODS: At 48 hours after 60 minutes of transient middle cerebral artery occlusion (tMCAO) with tPA, C57BL/6J mice were subjected to motor function analysis, In vivo and ex vivo optical imaging for MMP activation, gelatin zymography, and double immunofluorescent analyses with or without intravenous BMSC transplantation and the intravenous free radical scavenger EDA. RESULTS: In vivo fluorescent signals for MMP were detected over the heads of living mice 48 hours after tMCAO; the strongest were in the tPA group, which were reduced by BMSC or EDA treatment. These In vivo data were confirmed by ex vivo fluorescence imaging. While massive intracerebral hemorrhages were observed in the ischemic hemispheres of the tPA group, only slight hemorrhages were found in the tPA/BMSC, tPA/EDA, and EDA groups. Gelatin zymography showed the strongest MMP-9 activation in the tPA group after tMCAO, which was reduced by BMSC or EDA treatment. CONCLUSION: The present study provides a correlation between In vivo optical imaging of MMP activation and the improvement of ischemic brain damage caused by tPA after tMCAO and treated by BMSC and EDA.

Mitochondrial fusion and fission proteins expression dynamically change in a murine model of amyotrophic lateral sclerosis.

Mitochondria dynamically change their shape through frequent fusion and fission to continuously perform their function in the cell. Although a change in mitochondrial morphology was reported in amyotrophic lateral sclerosis (ALS), detailed changes of mitochondrial fusion and fission proteins have not been reported in ALS model mice. In transgenic (Tg) mice with the G93A human SOD1 mutation (G93ASOD1), both mitochondrial fusion proteins (Mfn1 and Opal) and fission proteins (Drp1 and Fis1) showed a significant increase in the anterior half of the lumbar spinal cord. Such changes in Tg mice were already noticeable at presymptomatic 10 week (W) compared with wildtype (WT) mice, detected through immunohistochemical as well as Western blot analyses. Furthermore, fusion protein levels of Mfn1 and Opa1 showed a progressive decrease from 10 to 18 W in Tg mice while fission protein levels of P-Drp1 and Fis1 maintained a high level of expression in Tg mice from 10 to 18 W. These data suggest that abnormal changes in mitochondrial morphology began before the onset of ALS and that the balanced mitochondrial morphology becomes altered by fissions in motor neurons (MNs) in this ALS model.

Dynamic changes of mitochondrial fission proteins after transient cerebral ischemia in mice.

With fusion or fission, mitochondria alter their morphology in response to various physiological and pathological stimuli resulting in either elongated, tubular, interconnected or fragmented form. Immunohistochemistry and Western blot analyses were performed at 2, 7, 14 and 28 d after 90 min of transient middle cerebral artery occlusion (tMCAO) in mice. The present study showed that mitochondrial fission protein fission 1 (Fis1) and phosphorylated dynamin-related protein 1 (P-Drp1) both progressively increased with the peak at 14 d after tMCAO. Double immunofluorescent analysis showed the number of double positive cells with Fis1/Drp1 reduced between 2 and 28 d after 90 min of tMCAO, and also showed some double positive cells with Fis1/terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) in the peri-infract regions at 2d after the reperfusion. The present study suggests a progressive activation of mitochondrial fission proteins Fis1 and P-Drp1 in relation to apoptotic process in neural cells of the peri-infract regions after tMCAO.

Clinical and pathological improvement in stroke-prone spontaneous hypertensive rats related to the pleiotropic effect of cilostazol.

BACKGROUND AND PURPOSE: Cerebral infarction is a major cause of death or decreasing activities of daily living. This study aimed to investigate the efficacy of commonly used antiplatelet drugs on stroke and motor and cognitive functions in relation to oxidative stress markers and insulin-like growth factor 1 receptor (IGF-1R). METHODS: Stroke-prone spontaneously hypertensive rats were treated with vehicle, aspirin, clopidogrel, and cilostazol from 8 to 10 weeks of age. Physiological parameters, regional cerebral blood flow, and serum lipids were examined. Motor and cognitive functions were evaluated weekly by the Rotorod and water maze task. Spontaneous infarct volume, oxidative stress markers for lipid, protein, and DNA at the ischemic boundary zone of spontaneous infarction, and the IGF-1R-positive cell ratio in the hippocampus were immunohistochemically examined in brain sections. IGF-1Rß expression in the hippocampus was assessed by Western blotting. RESULTS: The antiplatelet drugs, cilostazol and clopidogrel, reduced the spontaneous infarct volume more than aspirin. Only cilostazol improved motor and cognitive functions with a significant increase (P<0.05) in the memory-related IGF-1R-positive ratio and IGF-1Rß expression in the hippocampus. Cilostazol reduced the 4 oxidative stress markers in affected neurons in stroke-prone spontaneously hypertensive rats regardless of blood pressure, regional cerebral blood flow, or serum lipid levels. CONCLUSIONS: The present results suggest that a possible pleiotropic effect of cilostazol resulted in the reduction of spontaneous infarct volume and preservation of motor and spatial cognitive functions. The increase of IGF-1R-positive cells in the hippocampal CA1 region could partly explain the preservation of spatial cognitive function in stroke-prone spontaneously hypertensive rats.

Dynamic changes of mitochondrial fusion and fission proteins after transient cerebral ischemia in mice.

With fusion or fission, mitochondria alter their morphology in response to various physiological and pathological stimuli, resulting in elongated, tubular, interconnected, or fragmented forms. Immunohistochemistry and Western blot analysis were performed at 2 days, 7 days, 14 days, and 28 days after 90 min of transient middle cerebral artery occlusion (tMCAO) in mice. This study showed that mitochondrial fission protein dynamin-related protein 1 (Drp1) and fusion protein optic atrophy 1 (Opa1) were both upregulated in the ischemic penumbra, with the peak at 2 days after tMCAO, whereas phosphorylated-Drp1 (P-Drp1) progressively increased with a peak at 14 days after tMCAO. Double-immunofluorescence analysis showed many Drp1/cytochrome c oxidase subunit l (COX1) double-positive cells and Opa1/COX1 double-positive cells in the ischemic penumbra and also showed some double-positive cells with Drp1/terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) and Opa1/TUNEL in the ischemic penumbra. In contrast, both Drp1 and Opa1 showed progressive decreases until 2 days after tMCAO in the ischemic core because of necrotic brain damage. The present study suggests that there was a continuous mitochondrial fission and fusion during these periods in the ischemic penumbra after tMCAO, probably in an effort toward mitophagy and cellular survival.

Modifying neurorepair and neuroregenerative factors with tPA and edaravone after transient middle cerebral artery occlusion in rat brain.

Changes in expression of neurorepair and neuroregenerative factors were examined after transient cerebral ischemia in relation to the effects of tissue plasminogen activator (tPA) and the free radical scavenger edaravone. Physiological saline or edaravone was injected twice during 90 min of transient middle cerebral artery occlusion (tMCAO) in rats, followed by the same saline or tPA at reperfusion. Sizes of the infarct and protein factors relating to neurorepair and neuroregeneration were examined at 4d after tMCAO. The protein factors examined were: a chondroitin sulfate proteoglycan neurocan, semaphorin type 3A (Sema3A), a myelin-associated glycoprotein receptor (Nogo receptor, Nogo-R), a synaptic regenerative factor (growth associated protein-43, GAP43), and a chemotropic factor netrin receptor (deleted in colorectal cancer, DCC). Two groups treated by edaravone only or edaravone plus tPA showed a reduction in infarct volume compared to the two groups treated by vehicle only or vehicle plus tPA. Immunohistochemistry and western blot analyses indicated that protein expression of neurocan, Sema3A, Nogo-R, GAP43, and DCC was decreased with tPA, but recovered with edaravone. Additive edaravone prevented the reductions of these five proteins induced by tPA. The present study demonstrates for the first time that exogenous tPA reduced protein factors involved in inhibiting and promoting axonal growth, but that edaravone ameliorated such damage in brain repair after acute ischemia.

Anti-inflammatory effect of amlodipine plus atorvastatin treatment on carotid atherosclerosis in zucker metabolic syndrome rats.

To investigate the effects of amlodipine in combination with atorvastatin on carotid atherosclerotic changes in metabolic syndrome, 8-week-old Zucker fatty rats were treated with vehicle, amlodipine, atorvastatin, or amlodipine in combination with atorvastatin for 28 days. Histological studies of common carotid arteries showed that lipid deposition determined by Sudan III staining was significantly reduced in rats treated with amlodipine or atorvastatin alone and was further reduced by amlodipine in combination with atorvastatin. Immunohistochemical studies of the pro-inflammatory cytokine tumor necrosis factor (TNF)-α, the arterial calcification initiator bone morphogenetic protein (BMP) 2, the angiogenic factor Notch1, and the smooth muscle cell marker α-smooth muscle actin (SMA) showed that the high expression of all four protein in vehicle-treated rats was greatly decreased by amlodipine, atorvastatin, or amlodipine in combination with atorvastatin, in ascending order. Double immunostaining showed marked colocalization of TNF-α with bone morphogenetic protein 2 and Notch1 with α-SMA in the vehicle group, which was greatly reduced by amlodipine plus atorvastatin. These data suggest that combination therapy may be more effective in preventing atherosclerotic processes and subsequent carotid vascular events than administrating amlodipine or atorvastatin alone in metabolic syndrome.

Prevention of hyperglycemic signal pathways in metabolic syndrome carotid artery of rats.

Obesity is the major risk factor for metabolic syndrome and atherosclerotic cardiocerebrovascular diseases and induces insulin resistance characterized by a dysfunction of insulin to activate insulin receptor /insulin receptor substrate 1(IRS-1)/phosphoinositide 3-kinase (PI3K)/Akt pathway. Zucker fatty rats (8 weeks) were treated with vehicle (0.5 % methyl cellulose in physiological saline, p.o.), amlodipine (3 mg/kg/day, p.o.), atorvastatin (10 mg/kg/day, p.o.), or the combination of amlodipine plus atorvastatin (3 + 10 mg/kg/day, p.o.) for 28 days, and anti-insulin-like growth factor 1 (IGF-1)/IRS-1/PI3K/Akt pathways were evaluated. Our present immunohistochemical study first demonstrated that a combination of amlodipine plus atorvastatin treatment prevented an arteriosclerotic process compared to the single treatment with amlodipine or atorvastatin with strong recoveries of pTyr IRS-1, pPI3K, and pAkt expressions and with remarkable restraints of IGF-1 and pSer IRS-1. As a result, combination therapy with amlodipine plus atorvastatin showed a strong synergistic effect to prevent atherosclerotic processes. The present study newly suggests a synergistic benefit of combination therapy with amlodipine plus atorvastatin for strong prevention of atherosclerotic processes, which could reduce the clinical risk of cerebrovascular events for obesity patients.

In vivo optical imaging of motor neuron autophagy in a mouse model of amyotrophic lateral sclerosis.

Autophagy is involved in the pathological process of motor neuron death in amyotrophic lateral sclerosis (ALS). We have generated a novel double transgenic (DTg) mouse line by mating a green fluorescent protein (GFP)-fused microtubule-associated protein 1 light chain 3 (LC3) transgenic (LC3-Tg) mouse and a G93A mutant human Cu/Zn superoxide dismutase (mSOD1) transgenic (mSOD1-Tg) mouse. In vivo imaging of autophagy with these novel DTg mice was conducted at 10 (presymptomatic), 17 (early symptomatic) and 19 (late symptomatic) weeks of age. Fluorescence imaging analysis revealed a strong fluorescent signal in vivo over the T3-S1 level at 17 and 19 weeks of age only in the DTg mice. Ex vivo autophagy imaging of spinal cord sections (20 µm) also showed a progressive increase of the fluorescence signal from 17 to 19 weeks in DTg mice in the anterior horn at the L4-5 level, and the fluorescence signals were clearly observed in the gray matter of the spinal cord with a progressive increase of the signal and decreases in large motor neurons. Protein gel blot analysis revealed maximum LC3-I and LC3-II expressions at 19 weeks, consistent with the results from the in vivo autophagy imaging experiment. This method could also be applied as a unique tool for clarifying the role of autophagy, and to monitor the pathologic processes involving autophagy not only in ALS, but also other neurological diseases.

In vivo optical imaging for evaluating the efficacy of edaravone after transient cerebral ischemia in mice.

Detection and protection of apoptosis, autophagy and neurovascular unit (NVU) are essentially important in understanding and treatment for ischemic stroke patients. In this study, we have conducted an in vivo optical imaging for detecting apoptosis and activation of matrix metalloproteinases (MMPs), then evaluated the protective effect of 2 package types of free radical scavenger edaravone (A and B) on apoptosis, autophagy and NVU in mice after transient middle cerebral artery occlusion (tMCAO). As compared to vehicle treatment, edaravones A and B showed a significant improvement of clinical scores and infarct size at 48 h after 90 min of tMCAO with great reductions of in vivo fluorescent signal for MMPs and early apoptotic annexin V activations. Ex vivo imaging of MMPSense 680 or annexin V-Cy5.5 showed a fluorescent signal, while which was remarkably different between vehicle and edaravone groups, and colocalized with antibody for MMP-9 or annexin V. Edaravone A and B ameliorated the apoptotic neuronal cell death in immunohistochemistry, and activations of MMP-9 and aquaporin 4 with reducing autophagic activations of microtubule-associated protein 1 light chain 3 (LC3) in Western blot. In this study, edaravone in both packages showed a similar strong neuroprotection after cerebral ischemia, which was confirmed with in vivo and ex vivo optical imagings for MMPs and annexin V as well as reducing cerebral infarct, inhibiting apoptotic/autophagic mechanisms, and protecting a part of neurovascular unit.

Amlodipine and atorvastatin exert protective and additive effects via antiapoptotic and antiautophagic mechanisms after transient middle cerebral artery occlusion in Zucker metabolic syndrome rats.

We examined the neuroprotective effects amlodipine and/or atorvastatin in metabolic syndrome (MetS) Zucker fatty rats against transient (90 min) middle cerebral artery occlusion (MCAO). The rats were pretreated with vehicle, amlodipine, atorvastatin, or amlodipine plus atorvastatin for 28 days, and 24 hr after transient MCAO the infarct size was assessed via hematoxylin and eosin staining, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) and microtubule-associated protein 1 light chain 3 (LC3) expression were examined by immunohistochemistry to evaluate apoptosis and autophagy, respectively. Compared with the vehicle group, rats treated with amlodipine or atorvastatin alone showed a significant decrease in infarct volume (P < 0.01), which was further decreased in the amlodipine plus atorvastatin group (P < 0.001). Compared with the vehicle group, the numbers of TUNEL- and LC3-positive cells were markedly reduced by amlodipine or atorvastatin alone (P < 0.01) and further decreased by amlodipine plus atorvastatin (P < 0.001). The number of apoptotic TUNEL/autophagic LC3 double-positive cells was also significantly decreased with amlodipine or atorvastatin alone compared with vehicle (P < 0.01) and was further decreased by amlodipine plus atorvastatin (P < 0.001). These data suggest additive neuroprotective effects of combination amlodipine and atorvastatin treatment after acute ischemic stroke in MetS model Zucker rats. These effects are mediated, at least in part, via antiapoptotic and antiautophagic mechanisms. Further studies are now needed to expand these preliminary results to understand fully the mechanisms involved in the protective effects of amlodipine and atorvastatin against ischemic stroke.

Strong neurogenesis, angiogenesis, synaptogenesis, and antifibrosis of hepatocyte growth factor in rats brain after transient middle cerebral artery occlusion.

Hepatocyte growth factor (HGF) and glial cell line-derived neurotrophic factor (GDNF) are strong neurotrophic factors. However, their potentials in neurogenesis, angiogenesis, synaptogenesis, and antifibrosis have not been compared. Therefore, we investigated these effects of HGF and GDNF in cerebral ischemia in the rat. Wistar rats were subjected to 90 min of transient middle cerebral artery occlusion (tMCAO). Immediately after reperfusion, HGF or GDNF was given by topical application. BrdU was injected intraperitoneally twice daily 1, 2, and 3 days after tMCAO. On 14 day, we histologically evaluated infarct volume, antiapoptotic effect, neurogenesis, angiogenesis, synaptogenesis, and antifibrosis. Both HGF and GDNF significantly reduced infarct size and the number of TUNEL-positive cells, but only HGF significantly increased the number of BrdU-positive cells in the subventricular zone, and 5'-bromo-2'-deoxyuridine -positive cells differentiated into mature neurons on the ischemic side. Enhancement of angiogenesis and synaptogenesis at the ischemic boundary zone was also observed only in HGF-treated rats. HGF significantly decreased the glial scar formation and scar thickness of the brain pia mater after tMCAO, but GDNF did not. Our study shows that both HGF and GDNF had significant neurotrophic effects, but only HGF can promote the neurogenesis, angiogenesis, and synaptogenesis and inhibit fibrotic change in brains after tMCAO.

Synergistic benefit of combined amlodipine plus atorvastatin on neuronal damage after stroke in Zucker metabolic rat.

Stroke is a major neurologic disorder and a leading cause of death in the world. We compared neuroprotective effects of single or combination therapy of amlodipine (AM) and atorvastatin (AT) in such a metabolic syndrome model Zucker rat after 90 min of transient middle cerebral artery occlusion (tMCAO). The animals were pretreated with vehicle, AM, AT, or the combination of AM plus AT for 28 days, and at 24h of tMCAO, infarct volume and immunohistochemical analyses were performed. The combination of AM plus AT treatment decreased the infarct volume stronger than each single treatment with AM or AT. The numbers of positive cells of oxidative stress markers such as 8-hydroxy-2'-deoxyguanosin (8-OHdG), 4-hydroxy-2-nonenal (4-HNE), and advanced end glycation products (AGE) and inflammation markers such as tumor necrosis factor alpha (TNF-α) and monocyte chemoattractant protein-1(MCP-1) decreased dramatically in the combination-treated group compared with single AM- or AT-treated group. The present study showed that single AM or AT treatment showed neuroprotective effects both with antioxidative and anti-inflammatory mechanisms, but combination therapy of AM plus AT presented a further synergistic benefit in acute ischemic neural damages.

Tumorigenic development of induced pluripotent stem cells in ischemic mouse brain.

Induced pluripotent stem (iPS) cells may provide cures for various neurological diseases. However, undifferentiated iPS cells have high tumorigenicity, and evaluation of the cells fates, especially in pathologic condition model, is needed. In this study, we demonstrated the effect of ischemic condition to undifferentiated iPS cells fates in a mouse model of transient middle cerebral artery occlusion (MCAO). Undifferentiated iPS cells were characterized with immunofluorescent staining. The iPS cells (5 × 105) were injected into ipsilateral striatum and cortex after 24 h of MCAO. Histological analysis was performed from 3 to 28 days after cell transplantation. iPS cells in ischemic brain formed teratoma with higher probability (p < 0.05) and larger volume (p < 0.01) compared with those in intact brain. Among the four transcriptional factors to produce iPS cells, c-Myc, Oct3/4, and Sox2 strongly expressed in iPS-derived tumors in ischemic brain (p < 0.01). Additionally, expression of matrix metalloproteinase-9 (MMP-9) and phosphorylated vascular endothelial growth factor receptor2 (phospho-VEGFR2) were significantly increased in iPS-derived tumors in the ischemic brain (p < 0.05). These results suggest that the transcriptional factors might increase expression of MMP-9 and activate VEGFR2, promoting teratoma formation in the ischemic brain. We strongly propose that the safety of iPS cells should be evaluated not only in normal condition, but also in a pathologic, disease model.

Expression of Keap1-Nrf2 system and antioxidative proteins in mouse brain after transient middle cerebral artery occlusion.

Reactive oxygen species and their detrimental effects on the brain after transient ischemia have been implicated in the pathogenesis of the ischemic injury. The Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) system is currently recognized as the major cellular defense mechanism under oxidative stress, but the involvement of the Keap1-Nrf2 system in the ischemic brain injuries has not been fully investigated to date. In the present study, we investigated temporal changes of Keap1, Nrf2, and their downstream antioxidative proteins in post-ischemic mice brains with respect to spacial differences between the peri-infarct regions and the regions destined to infarct. In the peri-infarct regions, a steady level of Keap1 showed a decremental expression started at 2h of reperfusion after 60 min of transient middle cerebral artery occlusion (tMCAO). In contrast, Nrf2 began to show a significant increase at 2h with a peak at 8h of reperfusion after tMCAO. Both Keap1 and Nrf2 are mainly expressed in neuronal cells but not in glial cells. In the same peri-infarct region, downstream antioxidative proteins such as thioredoxin, glutathione, and heme oxygenase-1 showed significant increases at later time-points of 24-72 h of reperfusion after tMCAO. In the regions destined to infarct, a similar trend of expression changes to those in the peri-infarct regions was observed in Keap1, Nrf2, and 3 downstream antioxidative proteins with much less reactions. The changes found in this study suggest that the induced antioxidative stress proteins after cerebral ischemia may play an important endogenous neuroprotective response under oxidative stress after ischemic stroke.

In vivo optical imaging of early-stage apoptosis in mouse brain after transient cerebral ischemia.

Apoptosis is one of the mechanisms contributing to neuronal degeneration in ischemic stroke. In vivo imaging of annexin V (A5) was performed at 12 hr, 24 hr, 48 hr, and 4 days after 90-min transient middle cerebral artery occlusion (tMCAO) in mice with a fluorescent protein Cy5.5. Immunohistochemistry for heat shock protein 70 (HSP70), A5, and TUNEL were also performed with brain sections after the tMCAO. In vivo fluorescence was strongly observed at 48 hr over the head, especially with removal of both head skin and skull bone. Zonal ex vivo fluorescent signals were surrounding the ischemic core, and double-positive cells with Cy5.5/exogenous A5 antibody were found in this area. HSP70 was observed at the peak time of 24 hr; A5 became detectable at 12 hr, with increasing numbers until 48 hr. The number of TUNEL-positive cells increased at 24 hr and retained the high level until 4 days, showing a dissociating temporal pattern with A5. Double-positive cells for A5/TUNEL reached their peak at 48 hr. All the data suggest that some cells still have a chance to be rescued for a long period after acute cerebral ischemia. The in vivo Cy5.5 fluorescence representing A5 signal spatially surrounding the ischemic core temporally detects an early-stage apoptosis after cerebral ischemia.

In vivo imaging of autophagy in a mouse stroke model.

Recent studies have suggested that autophagy is involved in a neural death pathway following cerebral ischemia. In vivo detection of autophagy could be important for evaluating ischemic neural cell damage for human stroke patients. Using novel green fluorescent protein (GFP)-fused microtubule-associated protein 1 light chain 3 (LC3) transgenic (Tg) mice, in vivo imaging of autophagy was performed at 1, 3 and 6 d after 60 min transient middle cerebral artery occlusion (tMCAO). Ex vivo imaging of autophagy, testing of the autophagy inhibitor 3-methyladenine (3-MA), estern blot analysis, immunohistochemistry, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) and fluorescent analyses were performed on brain sections following tMCAO. In vivo fluorescent signals were detected above the ischemic hemisphere through the skull bone at 1, 3 and 6 d after tMCAO, with a peak at 1 d. Similar results were obtained with ex vivo fluorescence imaging. western blot analysis revealed maximum LC3-I and LC3-II expression at 1 d after tMCAO and fluorescence immunohistochemistry demonstrated that GFP-LC3-positive cells were primarily neuronal, not astroglial or microglial, cells. The number of GFP-LC3/TUNEL double-positive cells was greater in the periischemic area than in the core. These results provided evidence of in vivo autophagy detection, with a peak at 1 d, in a live animal model following cerebral ischemia. This novel technique could be valuable for monitoring autophagic processes in vivo in live stroke patients, as well as for clarifying the detailed role of autophagy in the ischemic brain, as well as in other neurological diseases.

Free radical scavenger edaravone administration protects against tissue plasminogen activator induced oxidative stress and blood brain barrier damage.

One of the therapeutics for acute cerebral ischemia is tissue plasminogen activator (t-PA). Using t-PA after 3 hour time window increases the chances of hemorrhage, involving multiple mechanisms. In order to show possible mechanisms of t-PA toxicity and the effect of the free radical scavenger edaravone, we administered vehicle, plasmin, and t-PA into intact rat cortex, and edaravone intravenously. Plasmin and t-PA damaged rat brain with the most prominent injury in t-PA group on 4-HNE, HEL, and 8-OHdG immunostainings. Such brain damage was strongly decreased in t-PA plus edaravone group. For the neurovascular unit immunostainings, occludin and collagen IV expression was decreased in single plasmin or t-PA group, which was recovered in t-PA plus edaravone group. In contrast, matrix metalloproteinase-9 intensity was the strongest in t-PA group, less in plasmin, and was the least prominent in t-PA plus edaravone group. In vitro data showed a strong damage to tight junctions for occludin and claudin 5 in both administration groups, while there were no changes for endothelial (NAGO) and perivascular (GFAP) stainings. Such damage to tight junctions was recovered in t-PA plus edaravone group with similar recovery in Sodium-Fluorescein permeability assay. Administration of t-PA caused oxidative stress damage to lipids, proteins and DNA, and led to disruption of outer parts of neurovascular unit, greater than the effect in plasmin administration. Additive edaravone ameliorated such an oxidative damage by t-PA with protecting outer layers of blood-brain barrier (in vivo) and tight junctions (in vitro).

Temporal and spatial differences of multiple protein expression in the ischemic penumbra after transient MCAO in rats.

Temporal and spatial differences and relationships of proteins relating to the ischemic penumbra were examined at 1, 3, 12, 24, and 48 h after 90 min of transient middle cerebral artery occlusion (tMCAO) in rats. 2, 3, 5-triphenyltetrazolium chloride (TTC) staining showed that the apparent infarction focus first appeared at 1h after tMCAO, which then largely matured at 24h. Immunohistochemistry and Western blot indicated no or trace levels of c-fos, hypoxia inducible factor-1 alpha (HIF-1 alpha), heat shock protein 70 (HSP70), and annexin V (A5) positive cells in the sham control brain. Expression of c-fos increased quickly and widely within and outside of the affected arterial territory (peak at 1h), and that of HIF-1 alpha reached the maximum at 12h in a smaller area than c-fos. HSP70 began to be induced during the first few hours after tMCAO, peaked at 24h, then decreased within 48 h, while A5 was slightly expressed at 3h, then gradually increased until 48 h. Double immunofluorescent analyses showed that the colocalization rates of c-fos/HIF-1 alpha, HIF-1 alpha/HSP70, HSP70/A5, and A5/TUNEL were 40.6%, 58.4%, 42.1% and 61.0%, respectively. These data suggest that multiple molecular penumbra exist after 90 min of tMCAO in the rat brain where several different proteins participate in different temporal and spatial expression patterns. Thus, there is a window for rescue of ischemic neural cells from 12 to 48 h after injury.