Transient Global Ischemia Model Research Articles

Blocking Mitochondrial Zn2+ Accumulation after Ischemia Reduces Mitochondrial Dysfunction and Neuronal Injury.

Zn2+ is an important contributor to ischemic brain injury, and recent studies support the hypothesis that mitochondria are key sites of its injurious effects. In murine hippocampal slices (both sexes) subjected to oxygen glucose deprivation (OGD), we found that Zn2+ accumulation and its entry into mitochondria precedes and contributes to the induction of acute neuronal death. In addition, if the ischemic episode is short (and sublethal), there is ongoing Zn2+ accumulation in CA1 mitochondria after OGD that may contribute to their delayed dysfunction. Using this slice model of sublethal OGD, we have examined Zn2+ contributions to the progression of changes evoked by OGD and occurring over 4-5 h. We detected progressive mitochondrial depolarization occurring from ∼2 h after ischemia, a large increase in spontaneous synaptic activity between 2 and 3 h, and mitochondrial swelling and fragmentation at 4 h. Blockade of the primary route for Zn2+ entry, the mitochondrial Ca2+ uniporter (with ruthenium red [RR]) or Zn2+ chelation shortly after OGD withdrawal substantially attenuated the mitochondrial depolarization and the changes in synaptic activity. RR also largely reversed the mitochondrial swelling. Finally, using an in vivo rat (male) asphyxial cardiac arrest model of transient global ischemia, we found that ∼8 min asphyxia induces considerable injury of CA1 neurons 4 h later that is associated with strong Zn2+ accumulation within many damaged mitochondria. These effects were substantially attenuated by infusion of RR on reperfusion. Our findings highlight mitochondrial Zn2+ accumulation after ischemia as a possible target for neuroprotective therapy.SIGNIFICANCE STATEMENT Brain ischemia is a leading cause of mortality and long-term disability that still lacks effective treatment. After transient ischemia, delayed death of neurons occurs in vulnerable brain regions. There is a critical need to understand mechanisms of this delayed neurodegeneration which can be targeted for neuroprotection. We found progressive and long-lasting mitochondrial Zn2+ accumulation to occur in highly vulnerable CA1 neurons after ischemia. Here we demonstrate that this Zn2+ accumulation contributes strongly to deleterious events occurring after ischemia, including mitochondrial dysfunction, swelling, and structural changes. We suggest that this mitochondrial Zn2+ entry may constitute a promising target for development of therapeutic interventions to be delivered after termination of an episode of transient global ischemia.

Therapeutic Effects of Hydroalcoholic Extracts from the Ancient Apple Mela Rosa dei Monti Sibillini in Transient Global Ischemia in Rats.

The Mela Rosa dei Monti Sibillini is an ancient apple variety cultivated by Romans in the foothills of the Sibillini Mountains, central Italy, showing potential as a source of nutraceuticals. The purpose of this study was to evaluate the protective effects of the hydroalcoholic extracts from the peel (APE) and pulp (APP) of this fruit in an animal model of transient global ischemia. Chemical constituents were analyzed by liquid chromatography–mass spectrometry (LC-DAD-MSn) indicating several polyphenols such as B-type procyanidins, quercetin derivatives and hydroxycinnamic acids as the main bioactive components. Acute pre-treatment of extracts (30 mg/kg, i.p.) significantly decreased the brain levels of the pro-inflammatory cytokines IL-1β (p < 0.01) and TNF-α (p < 0.001 and p < 0.01 for APE and APP, respectively), the expression of caspase-3 (p < 0.01, For APE) and MDA (p < 0.05), a lipid peroxidation biomarker in rats. Both extracts restricted the pathological changes of the brain induced by ischemic stroke in hematoxylin and eosin assay. Moreover, they improved the scores of behavioral tests in grid-walking and modified neurological severity scores (mNSS) tests. In conclusion, these results proved this ancient Italian apple is a source of nutraceuticals able to protect/prevent damage from brain ischemia.

Treatment of ischemic neuronal death by introducing brain-derived neurotrophic factor mRNA using polyplex nanomicelle

Ischemic neuronal death causes serious lifelong neurological deficits; however, there is no proven effective treatment that can prevent neuronal death after the ischemia. We investigated the feasibility of mRNA therapeutics for preventing the neuronal death in a rat model of transient global ischemia (TGI). By intraventricular administration of mRNA encoding brain-derived neurotrophic factor (BDNF) using a polymer-based carrier, polyplex nanomicelle, the mRNA significantly increased the survival rate of hippocampal neurons after TGI, with a rapid rise of BDNF in the hippocampus. Interestingly, mRNA administration on Day 2 after TGI provided significantly better survival rate than the administration immediately after TGI. Eventually, dosing twice on Day 2 and 5 exerted long-term therapeutic effects, which were confirmed by a Y-maze behavioral test demonstrating improved spatial memory compared with untreated rats on Day 20. Immunohistochemical analysis showed that astrocytes were chief targets of the BDNF mRNA-loaded nanomicelles, suggesting that the augmented BDNF secretion from astrocytes creates a supportive microenvironment for the neurons to tolerate changes caused by ischemic stresses, and terminate the process of progressive neuronal death after the ischemic attack. Overall, the unique mechanism of action of mRNA therapeutics provide a promising approach for preventing ischemic neuronal death.

Effects of 9-t-butyl doxycycline on the innate immune response to CNS ischemia-reperfusion injury

Cerebral ischemia triggers a cascade of neuroinflammatory and peripheral immune responses that contribute to post-ischemic reperfusion injury. Prior work conducted in CNS ischemia models underscore the potential to harness non-antibiotic properties of tetracycline antibiotics for therapeutic benefit. In the present study, we explored the immunomodulatory effects of the tetracycline derivative 9-tert-butyl doxycycline (9-TB) in a mouse model of transient global ischemia that mimics immunologic aspects of the post-cardiac arrest syndrome. Pharmacokinetic studies performed in C57BL/6 mice demonstrate that within four hours after delivery, levels of 9-TB in the brain were 1.6 and 9.5-fold higher than those obtained using minocycline and doxycycline, respectively. Minocycline and 9-TB also dampened inflammation, measured by reduced TNFα-inducible, NF-κβ-dependent luciferase activity in a microglial reporter line. Notably, daily 9-TB treatment following ischemia-reperfusion injury in vivo induced the retention of polymorphonuclear neutrophils (PMNs) within the spleen while simultaneously biasing CNS PMNs towards an anti-inflammatory (CD11bLowYm1+) phenotype. These studies indicate that aside from exhibiting enhanced CNS delivery, 9-TB alters both the trafficking and polarization of PMNs in the context of CNS ischemia-reperfusion injury.

Selegiline (L-Deprenyl) Mitigated Oxidative Stress, Cognitive Abnormalities, and Histopathological Change in Rats: Alternative Therapy in Transient Global Ischemia.

Selegiline (L-deprenyl) is the major drug which is used in the treatment of Parkinson's disease because of its neurotrophic and antiapoptotic properties. Previous studies suggested that low dose of L-methamphetamine (L-METH) caused lower mortality rate in patients with severe traumatic brain injury. As L-methamphetamine is one of the metabolites of selegiline, the present study aims to examine whether L-deprenyl can improve cognitive, biochemical, and histopathological injury in animal model of transient global ischemia. The animals were randomized in ten groups orally gavaged three times a week for 28days. Then, novel object recognition (NOR) was conducted to assess their behavioral abnormality. After scarification of the rats, their brains were divided into two sections to measure oxidative stress parameters and perform pathological evaluations in rats. Our data revealed the involvement of oxidative stress, behavioral despair, and pathological data in transient global ischemia rats. Significant recovery in cognitive behavior, oxidative stress biomarker, and number of dead cell in histopathological assay was observed in rats treated with 1,2 and 4mg/kg of selegiline. So, selegiline appears to be useful in alternative therapy of transient global ischemia.

Rapid Intramitochondrial Zn2+ Accumulation in CA1 Hippocampal Pyramidal Neurons After Transient Global Ischemia: A Possible Contributor to Mitochondrial Disruption and Cell Death

Mitochondrial Zn2+ accumulation, particularly in CA1 neurons, occurs after ischemia and likely contributes to mitochondrial dysfunction and subsequent neurodegeneration. However, the relationship between mitochondrial Zn2+ accumulation and their disruption has not been examined at the ultrastructural level in vivo. We employed a cardiac arrest model of transient global ischemia (TGI), combined with Timm's sulfide silver labeling, which inserts electron dense metallic silver granules at sites of labile Zn2+ accumulation, and used transmission electron microscopy (TEM) to examine subcellular loci of the Zn2+ accumulation. In line with prior studies, TGI-induced damage to CA1 was far greater than to CA3 pyramidal neurons, and was substantially progressive in the hours after reperfusion (being significantly greater after 4- than 1-hour recovery). Intriguingly, TEM examination of Timm's-stained sections revealed substantial Zn2+ accumulation in many postischemic CA1 mitochondria, which was strongly correlated with their swelling and disruption. Furthermore, paralleling the evolution of neuronal injury, both the number of mitochondria containing Zn2+ and the degree of their disruption were far greater at 4- than 1-hour recovery. These data provide the first direct characterization of Zn2+ accumulation in CA1 mitochondria after in vivo TGI, and support the idea that targeting these events could yield therapeutic benefits.

Neuroprotective effect of Citrus kawachiensis (Kawachi Bankan) peels, a rich source of naringin, against global cerebral ischemia/reperfusion injury in mice

Cerebral ischemia/reperfusion is known to induce the generation of reactive oxygen species and inflammatory responses. Numerous studies have demonstrated that naringin (NGIN) has anti-oxidant and anti-inflammatory properties. We previously reported that Citrus kawachiensis contains a large quantity of NGIN in its peel. In the present study, we orally (p.o.) administered dried peel powder of C. kawachiensis to mice of a transient global ischemia model and found in the hippocampus region that it 1) suppressed neuronal cell death, 2) reversed the reduction in the level of phosphorylated calcium-calmodulin-dependent protein kinase II, 3) had the tendency to reverse the reduction in the level of glutathione, and 4) blocked excessive activation of microglia and astrocytes. These results suggested that the dried peel powder of C. kawachiensis had a neuroprotective effect against ischemic brain via anti-oxidative and anti-inflammatory effects. We also showed that these effects of the dried peel powder were more powerful than those obtained with a comparable amount of NGIN alone.

Degradation of TRPML1 in Neurons Reduces Neuron Survival in Transient Global Cerebral Ischemia.

Postcardiac arrest syndrome yields poor neurological outcomes, but the mechanisms underlying this condition remain poorly understood. Autophagy plays an important role in neuronal apoptosis induced by ischemia. However, whether autophagy is involved in neuron apoptosis induced by cardiac arrest has been less studied. This study found that TRPML1 participates in cerebral ischemic reperfusion injury. Primary neurons were isolated and treated with mucolipin synthetic agonist 1 (ML-SA1), as well as infected with the recombinant lentivirus TRPML1 overexpression vector in vitro. ML-SA1 was delivered intracerebroventricularly in transient global ischemia model. Protein expression levels were determined by western blot. Neurological deficit score and the infarct volume were analyzed for the detection of neuronal damage. We found that TRPML1 was significantly downregulated in vivo and in vitro ischemic reperfusion model. We also observed that TRPML1 overexpression or treatment with the ML-SA1 attenuated neuronal death in primary neurons and ameliorated neurological dysfunction in vivo. Our findings suggested that autophagy and apoptosis were activated after transient global ischemia. Administration of ML-SA1 before transient global ischemia ameliorated neurological dysfunction possibly through the promotion of autophagy and the inhibition of apoptosis.

Genetic deletion of mGlu2 metabotropic glutamate receptors improves the short-term outcome of cerebral transient focal ischemia

We have recently shown that pharmacological blockade of mGlu2 metabotropic glutamate receptors protects vulnerable neurons in the 4-vessel occlusion model of transient global ischemia, whereas receptor activation amplifies neuronal death. This raised the possibility that endogenous activation of mGlu2 receptors contributes to the pathophysiology of ischemic neuronal damage. Here, we examined this possibility using two models of transient focal ischemia: (i) the monofilament model of middle cerebral artery occlusion (MCAO) in mice, and (ii) the model based on intracerebral infusion of endothelin-1 (Et-1) in rats. Following transient MCAO, mGlu2 receptor knockout mice showed a significant reduction in infarct volume and an improved short-term behavioural outcome, as assessed by a neurological disability scale and the “grip test”. Following Et-1 infusion, Grm2 gene mutated Hannover Wistar rats lacking mGlu2 receptors did not show changes in the overall infarct volume as compared to their wild-type counterparts, although they showed a reduced infarct area in the agranular insular cortex. Interestingly, however, mGlu2 receptor-deficient rats performed better than wild-type rats in the adhesive tape test, in which these rats did not show the laterality preference typically observed after focal ischemia. These findings support the hypothesis that activation of mGlu2 receptors is detrimental in the post-ischemic phase, and support the use of mGlu2 receptor antagonists in the experimental treatment of brain ischemia.

A Combination of Three Repurposed Drugs Administered at Reperfusion as a Promising Therapy for Postischemic Brain Injury.

Cerebral ischemia leads to multifaceted injury to the brain. A polytherapeutic drug that can be administered immediately after reperfusion may increase protection to the brain by simultaneously targeting multiple deleterious cascades. This study evaluated efficacy of the combination of three clinically approved drugs: lamotrigine, minocycline, and lovastatin, using two mouse models: global and focal cerebral ischemia induced by transient occlusion of the common carotid arteries or the middle cerebral artery, respectively. In vitro, the combination drug, but not single drug, protected neurons against oxygen-glucose deprivation (OGD)-induced cell death. The combination drug simultaneously targeted cell apoptosis and DNA damage induced by ischemia. Besides acting on neurons, the combination drug suppressed inflammatory processes in microglia and brain endothelial cells induced by ischemia. In a transient global ischemia model, the combination drug, but not single drug, suppressed microglial activation and inflammatory cytokine production, and reduced neuronal damage. In a transient focal ischemia model, the combination drug, but not single drug, attenuated brain infarction, suppressed infiltration of peripheral neutrophils, and reduced neurological deficits following ischemic stroke. In summary, the combination drug confers a broad-spectrum protection against ischemia/reperfusion (I/R) injury and could be a promising approach for early neuroprotection after out-of-hospital cardiac arrest or ischemic stroke.

Increased 12/15-Lipoxygenase Leads to Widespread Brain Injury Following Global Cerebral Ischemia.

Global ischemia following cardiac arrest is characterized by high mortality and significant neurological deficits in long-term survivors. Its mechanisms of neuronal cell death have only partially been elucidated. 12/15-lipoxygenase (12/15-LOX) is a major contributor to delayed neuronal cell death and vascular injury in experimental stroke, but a possible role in brain injury following global ischemia has to date not been investigated. Using a mouse bilateral occlusion model of transient global ischemia which produced surprisingly widespread injury to cortex, striatum, and hippocampus, we show here that 12/15-LOX is increased in a time-dependent manner in the vasculature and neurons of both cortex and hippocampus. Furthermore, 12/15-LOX co-localized with apoptosis-inducing factor (AIF), a mediator of non-caspase-related apoptosis in the cortex. In contrast, caspase-3 activation was more prevalent in the hippocampus. 12/15-lipoxygenase knockout mice were protected against global cerebral ischemia compared to wild-type mice, accompanied by reduced neurologic impairment. The lipoxygenase inhibitor LOXBlock-1 similarly reduced neuronal cell death both when pre-administered and when given at a therapeutically relevant time point 1h after onset of ischemia. These findings suggest a pivotal role for 12/15-LOX in both caspase-dependent and caspase-independent apoptotic pathways following global cerebral ischemia and suggest a novel therapeutic approach to reduce brain injury following cardiac arrest.

Trehalose Inhibits Protein Aggregation Caused by Transient Ischemic Insults Through Preservation of Proteasome Activity, Not via Induction of Autophagy

Protein aggregation has been proved to be a pathological basis accounting for neuronal death caused by either transient global ischemia or oxygen glucose deprivation (OGD), and inhibition of protein aggregation is emerging as a potential strategy of preventing brain damage. Trehalose was found to inhibit protein aggregation caused by neurodegenerative diseases via induction of autophagy, whereas its effect is still elusive on ischemia-induced protein aggregation. In this study, we investigated this issue by using rat model of transient global ischemia and SH-SY5Y model of OGD. We found that pretreatment with trehalose inhibited transient global ischemia-induced neuronal death in the hippocampus CA1 neurons and OGD-induced death in SH-SY5Y cells, which was associated with inhibition of the formation of ubiquitin-labeled protein aggregates and preservation of proteasome activity. In vitro study showed that the protection of trehalose against OGD-induced cell death and protein aggregation in SH-SY5Y cells was reversed when proteasome activity was inhibited by MG-132. Further studies revealed that trehalose prevented OGD-induced reduction of proteasome activity via suppression of both oxidative stress and endoplasmic reticulum stress. Particularly, our results showed that trehalose inhibited OGD-induced autophagy. Therefore, we demonstrated that proteasome dysfunction contributed to protein aggregation caused by ischemic insults and trehalose prevented protein aggregation via preservation of proteasome activity, not via induction of autophagy.

Neuroprotective Effects of Curcumin Against Transient Global Ischemia are Dose and Area Dependent

Background: Ischemic brain injury is among the most common causes of death and disability in humans worldwide. Recent findings suggest that neural precursors in the adult mammalian brain can be a therapeutic target in ischemic brain injuries. Curcumin stimulates neurogenesis and reduces oxidative stress in the brain. Objectives: The present study compared the neuroprotective effects of different concentrations of curcumin in various regions of the brain in a rat model of transient global ischemia (TGI). Materials and Methods: Forty-eight adult male Wistar rats were randomly chosen as control, sham (animals only underwent TGI), treatment (100 and 300 mg/kg curcumin following TGI), and vehicle groups. The animals underwent global ischemia imposed by a 20 minute ligation of the bilateral common carotid arteries. Cell death and apoptosis in different areas of brain were evaluated after 3 or 4 weeks by Nissl staining and TUNEL assay respectively. Results: The number of dark neurons and apoptotic cells significantly increased after TGI. The number of TUNEL-positive cells after TGI was significantly higher in the temporal neocortex than in different regions within the hippocampus. Treatment with curcumin at a high dose reduced the numbers of dark neurons and apoptotic cells. Lower concentrations of curcumin showed a neuroprotective effect in the neocortex, whereas higher doses prevented cell death and apoptosis in the neocortex and in different regions of the hippocampus. Conclusions: Regional differences were evident with respect to the neuroprotective effects of curcumin in the temporal cortex and in the different parts of hippocampus following TGI. Further studies are needed to explore the mechanisms underlying these regional differences.

Targeting type-2 metabotropic glutamate receptors to protect vulnerable hippocampal neurons against ischemic damage.

BackgroundTo examine whether metabotropic glutamate (mGlu) receptors have any role in mechanisms that shape neuronal vulnerability to ischemic damage, we used the 4-vessel occlusion (4-VO) model of transient global ischemia in rats. 4-VO in rats causes a selective death of pyramidal neurons in the hippocampal CA1 region, leaving neurons of the CA3 region relatively spared. We wondered whether changes in the expression of individual mGlu receptor subtypes selectively occur in the vulnerable CA1 region during the development of ischemic damage, and whether post-ischemic treatment with drugs targeting the selected receptor(s) affords neuroprotection.ResultsWe found that 4-VO caused significantly reduction in the transcript of mGlu2 receptors in the CA1 region at times that preceded the anatomical evidence of neuronal death. Down-regulation of mGlu2 receptors was associated with reduced H3 histone acetylation at the Grm2 promoter. The transcripts of other mGlu receptor subtypes were unchanged in the CA1 region of 4-VO rats. Ischemia did not cause changes in mGlu2 receptor mRNA levels in the resistant CA3 region, which, interestingly, were lower than in the CA1 region. Targeting the mGlu2 receptors with selective pharmacologic ligands had profound effects on ishemic neuronal damage. Post-ischemic oral treatment with the selective mGlu2 receptor NAM (negative allosteric modulator), ADX92639 (30 mg/kg), was highly protective against ischemic neuronal death. In contrast, s.c. administration of the mGlu2 receptor enhancer, LY487379 (30 mg/kg), amplified neuronal damage in the CA1 region and extended the damage to the CA3 region.ConclusionThese findings suggest that the mGlu2 receptor is an important player in mechanisms regulating neuronal vulnerability to ischemic damage, and that mGlu2 receptor NAMs are potential candidates in the experimental treatments of disorders characterized by brain hypoperfusion, such as hypovolemic shock and cardiac arrest.

Validation of UPLC method for determination of rubrofusarin-6-O-gentiobioside from Cassia obtusifolia L.

The seed of Cassia obtusifolia L. contains various kinds of anthraquinone, anthraquinone glycosides and naphthalene derivatives and is widely used in Asia to treat eye inflammation. Also, the extract of Cassia obtusifolia L. was known to reduce the memory impairments and neuronal damage caused by 2VO in a mouse transient global ischemia model. Rubrofusarin-6-O-gentiobioside, one of a major component of Cassia obtusifolia L., has been known to be an important constituents of this herbal drug with a lot of biological acitivities including decrease of the expression of TGF-β1 and fibronectin and NF-κB DNA binding activity and radical scavenging effect. However, there was little analytical method for determination of rubrofusarin-6-O-gentiobioside in Cassia obtusifolia. In this study, a rapid and simple UPLC method with UV detection was developed and validated to determine rubrofusarin-6-O-gentiobioside in Cassia obtusifolia. The method was established on a reversed phase C18, Brownlee column (2.1 mm x 75 mm, 2.7µm) and detection wavelength was 281nm. The mobile phase was acetonitrile (0.1% trifluoroacetic acid) and water (0.1% trifluoroacetic acid) with an isocratic elution and sample analysis time was 8.0 min. UPLC method is validated in terms of linearity range, limit of quantification, selectivity, accuracy and precision. The retention time of rubrofusarin-6-O-gentiobioside was 2.1 min. Calibration curve showed good linearity (R2= 0.999) over the range of 5.0 – 500.0 µg/mL. The relative standard deviation for intra- and inter-day precision was found to be below 3%. The recovery rate ranged from 105.0% to 114.0% and limit of quantification was 7.0 µg/mL. This method can be applied to determine rubrofusarin-6-O-gentiobioside rapidly and be utilized to control the quality of Cassia obtusifolia L..

Radiofluorinated Carbohydrates for Positron Emission Tomography

2-Deoxy-2-[(18)F]fluoro-D-glucose (2-(18)FDG) has represented radiofluorinated carbohydrates as the most successful tracer for positron emission tomography (PET). 2-(18)FDG uptake depends on glucose metabolism, which is related to a disease progression. 2-(18)FDG has been widely used in oncology, neurology, cardiology, infectious diseases, and inflammation, to complement anatomical modalities such as CT and MRI. Followed by the success of 2-(18)FDG, various radiofluorinated carbohydrates have been evaluated as PET tracers, which include analogs of D-ribose, D-mannose, D-galactose, D-talose, D-fructose, D-allose, lactose, L-fucose, N-acetylneuraminic acid, and L-ascorbic acid. Among those radiofluorinated carbohydrates, several have implied potential for further development. 2-Deoxy-2-[(18)F]fluoro-D-galactose has been developed to assess liver function and diagnose hepatic carcinoma. 6-Deoxy-6-[(18)F]fluoro-D-fructose showed promising characteristics for diagnosis of breast cancer. Three radiofluorinated analogs of lactose have been designed as the substrates of the overexpressed hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein in peritumoral pancreatic tissue for early diagnosis of pancreatic cancer. The metabolism of 6-[(18)F]fluoro-L-fucose suggested that it is a bioactive analog of L-fucose in the synthesis of glycoconjugate macromolecules. 6-Deoxy-6-[(18)F]fluoro-L-ascorbic acid was evaluated to assess antioxidant function of L-ascorbic acid in rodent models of transient global ischemia and glutathione deficiency.

Ginkgo biloba prevents transient global ischemia-induced delayed hippocampal neuronal death through antioxidant and anti-inflammatory mechanism

We have previously reported neuroprotective properties of Ginkgo biloba/EGb 761® (EGb 761) in transient and permanent mouse models of brain ischemia. In a quest to extend our studies on EGb 761 and its constituents further, we used a model of transient global ischemia induced delayed hippocampal neuronal death and inflammation. Mice pretreated with different test drugs for 7days were subjected to 8-min bilateral common carotid artery occlusion (tBCCAO) at day 8. After 7days of reperfusion, mice brains were dissected out for TUNEL assay and immunohistochemistry. In situ detection of fragmented DNA (TUNEL staining) showed that out of all test drugs, only EGb 761 (13.6%±3.2) pretreatment protected neurons in the hippocampus against global ischemia (vs. vehicle, 85.1%±9.9; p<0.05). Immunofluorescence-based studies demonstrated that pretreatment with EGb 761 upregulated the expression levels of heme oxygenase 1 (HO1), nuclear factor erythroid 2-related factor 2 (Nrf2), and vascular endothelial growth factor (VEGF) as compared to the vehicle group. In addition, increased number of activated astrocytes and microglia in the vehicle group was observed to be significantly lower in the EGb 761 pretreated group. Together, these results suggest that EGb 761 is a multifunctional neuroprotective agent, and the protection is in part associated with activation of the HO1/Nrf2 pathway, upregulation of VEGF and downregulation of inflammatory mediators such as astrocytes and microglia.

Persistent astroglial swelling accompanies rapid reversible dendritic injury during stroke-induced spreading depolarizations.

Spreading depolarizations are a key event in the pathophysiology of stroke, resulting in rapid dendritic beading, which represents acute damage to synaptic circuitry. The impact of spreading depolarizations on the real-time injury of astrocytes during ischemia is less clear. We used simultaneous in vivo 2-photon imaging and electrophysiological recordings in adult mouse somatosensory cortex to examine spreading depolarization-induced astroglial structural changes concurrently with signs of neuronal injury in the early periods of focal and global ischemia. Astrocytes in the metabolically compromised ischemic penumbra-like area showed a long lasting swelling response to spontaneous spreading depolarizations despite rapid dendritic recovery in a photothrombotic occlusion model of focal stroke. Astroglial swelling was often facilitated by recurrent depolarizations and the magnitude of swelling strongly correlated with the total duration of depolarization. In contrast, spreading depolarization-induced astroglial swelling was transient in normoxic healthy tissue. In a model of transient global ischemia, the occurrence of a single spreading depolarization elicited by a bilateral common carotid artery occlusion coincided with astroglial swelling alongside dendritic beading. With immediate reperfusion, dendritic beading subsides. Astroglial swelling was either transient during short ischemic periods distinguished by a short-lasting spreading depolarization, or persistent during severe ischemia characterized by a long-lasting depolarization with the ultraslow negative voltage component. We propose that persistent astroglial swelling is initiated and exacerbated during spreading depolarization in brain tissue with moderate to severe energy deficits, disrupting astroglial maintenance of normal homeostatic function thus contributing to the negative outcome of ischemic stroke as astrocytes fail to provide neuronal support.

Estradiol protects against hippocampal damage and impairments in fear conditioning resulting from transient global ischemia in mice

Estradiol protects against hippocampal damage and some learning impairments resulting from transient global ischemia in rats. Here, we seek to validate a mouse model of transient global ischemia and evaluate the effects of estradiol on ischemia-induced hippocampal damage and behavioral impairments. Female C57Bl6/J mice were ovariectomized and implanted with estradiol- or oil-secreting capsules. One week later, mice experienced 15-min of 2-vessel occlusion (2-VO) or sham surgical procedures. Five days later, mice were exposed to a fear conditioning protocol in which a specific context and novel tone were paired with mild footshock. Twenty-four hours following conditioning, contextual fear was assessed by measuring freezing behavior in the conditioned context (in the absence of the tone). This was followed by assessment of cue fear by measuring freezing behavior to the conditioned tone presented in a new context. When tested in the conditioned context, oil-treated mice that experienced 2-VO exhibited a significant reduction in freezing behavior whereas estradiol-treated mice that experienced 2-VO showed no disruption in freezing behavior. Freezing behavior when presented with the conditioned tone was unaffected by either surgery or hormone treatment. These findings suggest that global ischemia causes impairments in performance on the hippocampally-dependent contextual fear task but not conditioned cue-based fear. Furthermore, estradiol prevented the ischemia-induced impairment in contextual fear conditioning. Fluoro-Jade (FJ) staining revealed neuronal degeneration throughout the dorsal hippocampus of mice that experienced 2-VO. Estradiol treatment reduced the number of FJ+ cells in CA1 and CA2, but not in CA3 or in the dentate gyrus. Together, these findings suggest that 15min of global ischemia causes extensive hippocampal neurodegeneration and disrupts contextual fear conditioning processes in mice and that estradiol protects against these adverse effects.

Activation of calcium/calmodulin‐dependent protein kinase IV and peroxisome proliferator‐activated receptor γ coactivator‐1α signaling pathway protects against neuronal injury and promotes mitochondrial biogenesis in the hippocampal CA1 subfield after transient global ischemia

Delayed neuronal cell death occurs in the vulnerable CA1 subfield of the hippocampus after transient global ischemia (TGI). We demonstrated previously, based on an experimental model of TGI, that the significantly increased content of oxidized proteins in hippocampal CA1 neuron was observed as early as 30 min after TGI, followed by augmentation of PGC-1α expression at 1 hr, as well as up-regulation of mitochondrial uncoupling protein 2 (UCP2) and superoxide dismutases 2 (SOD2). Using the same animal model, the present study investigated the role of calcium/calmodulin-dependent protein kinase IV (CaMKIV) and PGC-1α in delayed neuronal cell death and mitochondrial biogenesis in the hippocampus. In Sprague-Dawley rats, significantly increased expression of nuclear CaMKIV was noted in the hippocampal CA1 subfield as early as 15 min after TGI. In addition, the index of mitochondrial biogenesis, including a mitochondrial DNA-encoded polypeptide, cytochrome c oxidase subunit 1 (COX1), and mitochondrial number significantly increased in the hippocampal CA1 subfield 4 hr after TGI. Application bilaterally into the hippocampal CA1 subfield of an inhibitor of CaMKIV, KN-93, 30 min before TGI attenuated both CaMKIV and PGC-1α expression, followed by down-regulation of UCP2 and SOD2, decrease of COX1 expression and mitochondrial number, heightened protein oxidation, and enhanced hippocampal CA1 neuronal damage. This study provides correlative evidence for the neuroprotective cascade of CaMKIV/PGC-1α which implicates at least in part the mitochondrial antioxidants UCP2 and SOD2 as well as mitochondrial biogenesis in ischemic brain injury.