Transient Global Brain Ischemia Research Articles

Involvement of Proteasomal and Endoplasmic Reticulum Stress in Neurodegeneration After Global Brain Ischemia

A brief period of transient global brain ischemia leads to selective ischemic neurodegeneration associated with death of hippocampal CA1 pyramidal neurons days after reperfusion. The mechanism of such selective and delayed neurodegeneration is still uncertain. Our work aimed to study the involvement of proteasomal and endoplasmic reticulum (ER) stress in ischemic neurodegeneration. We have performed laser scanning confocal microscopy analysis of brain slices from control and experimental animals that underwent global brain ischemia for 15 min and varying times of reperfusion. We have focused on ubiquitin, PUMA, a proapoptotic protein of the Bcl-2 family overexpressed in response to both proteasomal and ER stress, and p53, which controls expression of PUMA. We have also examined the expression of HRD1, an E3 ubiquitin ligase that was shown to be overexpressed after ER stress. We have also examined potential crosstalk between proteasomal and ER stress using cellular models of both proteasomal and ER stress. We demonstrate that global brain ischemia is associated with an appearance of distinct immunoreactivity of ubiquitin, PUMA and p53 in pyramidal neurons of the CA1 layer of the hippocampus 72 h after ischemic insults. Such changes correlate with a delay and selectivity of ischemic neurodegeneration. Immunoreactivity of HRD1 observed in all investigated regions of rat brain was transiently absent in both CA1 and CA3 pyramidal neurones 24 h after ischemia in the hippocampus, which does not correlate with a delay and selectivity of ischemic neurodegeneration. We do not document significant crosstalk between proteasomal and ER stress. Our results favour dysfunction of the ubiquitin proteasome system and consequent p53-induced expression of PUMA as the main mechanisms responsible for selective and delayed degeneration of pyramidal neurons of the hippocampal CA1 layer in response to global brain ischemia.

Delayed administration of Trichilia catigua A. Juss. Ethyl-acetate fraction after cerebral ischemia prevents spatial memory deficits, decreases oxidative stress, and impacts neural plasticity in rats.

Trichilia catigua A. Juss (Meliaceae) is used in Brazilian folk medicine to alleviate fatigue and emotional stress and improve memory. Previous studies from our laboratory reported that an ethyl-acetate fraction (EAF) of T. catigua that was given before cerebral ischemia in vivo prevented memory loss and reduced oxidative stress and neuroinflammation. Despite the value of these findings of a neuroprotective effect of T. catigua, treatment that was given immediately before or immediately after ischemia limits its clinical relevance. Thus, unknown is whether T. catigua possesses a specific time window of efficacy (TWE) when administered postischemia. Given continuity to previous studies, we investigated whether an EAF of T. catigua maintains its neuroprotective properties if treatment begins at different time windows of efficacy after ischemia. We also evaluated, for the first time, whether T. catigua possesses neuroplasticity/neurotrophic properties. Rats were subjected to transient global brain ischemia (TGCI) and then given a single dose of the EAF (400mg/kg) or vehicle (1ml/kg) orally 1, 4, or 6h postischemia. The levels of protein PCG, GSH, and GSSG, and activity of SOD and CAT were assayed as markers of oxidative stress on the day after ischemia. In another experiment, naive rats underwent spatial learning training in a radial maze task and then subjected to TGCI. Delayed treatment with the EAF began 4 or 6h later and continued for 7 days. Retrograde memory performance was assessed 10, 17, and 24 days postischemia. Afterward, brains were examined for neurodegeneration and neuronal dendritic morphology in the hippocampus and cerebral cortex. Another group received the EAF at 4h of reperfusion, and 4 days later their brains were examined for GFAP and Iba-1 immunoreactivity. Lastly, ischemic rats received the EAF 4h after ischemia and neural plasticity-related proteins, BDNF, SYN, PSD 95, and NeuN were measured in the hippocampus 7 and 14 days after ischemia. A single EAF administration 1, 4, or 6h postischemia alleviated oxidative stress that was caused by ischemia, expressed as a reduction of the amount of the PCG and GSSG, normalization of the GSH/GSSG ratio, and the restoration of SOD activity. Ischemia caused the persistent loss of memory (i.e., amnesia), an outcome that was consistently ameliorated by treatment with the EAF that was initiated 4 or 6h postischemia. The 4h delay in EAF treatment positively impacted dendritic morphology in neurons that survived ischemia. TGCI reduced BDNF, SYN, PSD-95, and NeuN protein levels in the hippocampus and cerebral cortex. The EAF normalized SYN and PSD-95 protein levels. Ischemia-induced neurodegeneration and glial cell activation were not prevented by EAF treatment. The present study corroborates prior data that demonstrated the neuroprotective potential of T. catigua and extends these data by showing that the delayed administration of EAF postischemia effectively prevented memory impairment and decreased oxidative stress, dendritic deterioration, and synaptic protein loss within a TWE that ranged from 1 to 6h. This specific TWE in preclinical research may have clinical relevance by suggesting the possible utility of this plant for the development of neuroprotective strategies in the setting of ischemic brain diseases. Another innovative finding of the present study was the possible neurotrophic/neuroplastic properties of T. catigua.

Changes in Cyclin D1, cdk4, and Their Associated Molecules in Ischemic Pyramidal Neurons in Gerbil Hippocampus after Transient Ischemia and Neuroprotective Effects of Ischemic Preconditioning by Keeping the Molecules in the Ischemic Neurons.

Simple SummaryCyclin D1 and cyclin-dependent kinase 4 (cdk4) is implicated in neuronal death induced by various pathological conditions. Ischemic preconditioning (IPC) confers neuroprotective effect, but underlying mechanisms have been poorly addressed. In this study, IPC protected pyramidal neurons (cells) in gerbil hippocampus after transient ischemia. Additionally, IPC controlled expressions of cyclin D1, cdk4, phosphorylated retinoblastoma (p-Rb), and E2 promoter binding factor 1 (E2F1). In particular, the expression of p16INK4a was not different by IPC. These findings indicate that cyclin D1/cdk4-related signals may play important roles in events in neurons related to damage/death following ischemic insults. Especially, the preservation of p16INK4a by IPC may be crucial in attenuating neuronal death/damage or protecting neurons after brain ischemic insults.Inadequate activation of cell cycle proteins including cyclin D1 and cdk4 is involved in neuronal cell death induced by diverse pathological stresses, including transient global brain ischemia. The neuroprotective effect of ischemic preconditioning is well-established, but the underlying mechanism is still unknown. In this study, we examined changes in cyclin D1, cdk4, and related molecules in cells or neurons located in Cornu Ammonis 1 (CA1) of gerbil hippocampus after transient ischemia for 5 min (ischemia and reperfusion) and investigated the effects of IPC on these molecules after ischemia. Four groups were used in this study as follows: sham group, ischemia group, IPC plus (+) sham group, and IPC+ischemia group. IPC was developed by inducing 2-min ischemia at 24 h before 5-min ischemia (real ischemia). Most pyramidal cells located in CA1 of the ischemia group died five days after ischemia. CA1 pyramidal cells in the IPC+ischemia group were protected. In the ischemia group, the expressions of cyclin D1, cdk4, phosphorylated retinoblastoma (p-Rb), and E2F1 (a transcription factor regulated by p-Rb) were significantly altered in the pyramidal cells with time after ischemia; in the IPC+ischemia group, they were controlled at the level shown in the sham group. In particular, the expression of p16INK4a (an endogenous cdk inhibitor) in the ischemia group was reversely altered in the pyramidal cells; in the IPC+TI group, the expression of p16INK4a was not different from that shown in the sham group. Our current results indicate that cyclin D1/cdk4-related signals may have important roles in events in neurons related to damage/death following ischemia and reperfusion. In particular, the preservation of p16INK4a by IPC may be crucial in attenuating neuronal death/damage or protecting neurons after brain ischemic insults.

Disassembly of Death-associated Protein Kinase and DANGER Interaction Mediates Hippocampal CA1 Neuron Death in Rat Cerebral Ischemic Reperfusion

Death-associated protein kinase (DAPK) is a Ca2+/CaM-regulated protein kinase that is involved in cell death processes by multiple pathways. It has been reported that DAPK may play a role in brain ischemia-induced neuronal death, but this mechanism is not well understood. DANGER, a membrane-associated protein that binds to DAPK physiologically, inhibits DAPK activation. In the present study, we used a transient global brain ischemia and reperfusion (I/R) rat model to investigate whether the interaction between DAPK and DANGER is involved in neuronal cell death following brain ischemia, and to reveal the mechanism of action. Our results indicate that the DAPK/DANGER interaction in the hippocampal CA1 region was significantly reduced after I/R with a peak reduction at 6 h. We further demonstrate that the NMDA inhibitor MK-801, DAPK inhibitor, or calcineurin inhibitor FK-506 prevented the dissociation of DANGER from DAPK 6 h after I/R. This was accompanied by a significantly decreased I/R-induced dephosphorylation of DAPK(ser-308), inhibiting DAPK catalytic activity. Moreover, the expression of DANGER and the interaction between DANGER and IP3R on the endoplasmic reticulum was significantly increased at I/R 6 h, which may be related to a reduction of DAPK/DANGER binding under I/R condition. Furthermore, MK-801, DAPK inhibitor and FK-506 had neuroprotective effects against hippocampal CA1 neuronal death 5 days after I/R. In conclusion, our data suggest that the dissociation of DANGER from DAPK may mediate DAPK activation, which is involved in DAPK-related neuronal death following I/R injury.

Expression of the Tau Protein and Amyloid Protein Precursor Processing Genes in the CA3 Area of the Hippocampus in the Ischemic Model of Alzheimer\u2019s Disease in the Rat

Understanding the mechanisms underlying the selective susceptibility to ischemia of the CA3 region is very important to explain the neuropathology of memory loss after brain ischemia. We used a rat model to study changes in gene expression of the amyloid protein precursor and its cleaving enzymes and tau protein in the hippocampal CA3 sector, after transient 10-min global brain ischemia with survival times of 2, 7, and 30 days. The expression of the α-secretase gene was below control values at all times studied. But, the expression of the β-secretase gene was below the control values at 2–7 days after ischemia and the maximal increase in its expression was observed on day 30. Expression of the presenilin 1 gene was significantly elevated above the control values at 2–7 days after ischemia and decreased below the control values at day 30. Expression of the presenilin 2 gene showed an opposite trend to the expression of presenilin 1. Expression of the amyloid protein precursor gene after ischemia was at all times above the control values with a huge significant overexpression on day 7. Additionally, the expression of the tau protein gene was below the control values 2 days after ischemia, but the significant increase in its expression was observed on days 7–30. Data show that brain ischemia activates neuronal changes and death in the CA3 region of the hippocampus in a manner dependent on amyloid and tau protein, thus determining a new and important way to regulate the survival and/or death of ischemic neurons.

Neuroprotective effects of N-acetylcysteine via inhibition of matrix metalloproteinase in a mouse model of transient global cerebral ischemia

N-acetylcysteine (NAC) is known to serve many biological functions including acting as an antioxidant, and electing antiinflammatory effects. Previous reports have revealed that NAC may have neuroprotective effects against the deleterious effects of brain ischemia. Despite of this, the mechanism by which NAC prevents neuronal damage after brain ischemia remains unclear. The current study aimed to investigate this mechanism in a mouse model of transient global brain ischemia. In the present study, mice were subjected to 20 min of transient global brain ischemia, proceeded by intraperitoneal administration of NAC (150 mg/kg) in one group. The mice were then euthanized 72 h after this ischemic insult for collection of experimental tissues. The effect of NAC on neuronal damage and matrix metalloproteinase (MMP)-9 activity were assessed and immunofluorescence, and hippocampal terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay experiments were conducted and results compared between NAC- and vehicle-treated groups. Neuronal damage was primarily observed in the hippocampal CA1 and CA2 regions. In NAC-treated mice, neuronal damage was significantly reduced after ischemia when compared to vehicle-treated animals. NAC also inhibited increased MMP-9 activity after global brain ischemia. NAC increased laminin and NeuN expression and inhibited increases in TUNEL-positive cells, all in the hippocampus. These results suggest that NAC reduces hippocampal neuronal damage following transient global ischemia, potentially via reductions in MMP-9 activity.

Global brain ischemia in rats is associated with mitochondrial release and downregulation of Mfn2 in the cerebral cortex, but not the hippocampus.

Mitochondria are crucial for neuronal cell survival and death through their functions in ATP production and the intrinsic pathway of apoptosis. Mitochondrial dysfunction is considered to play a central role in several serious human diseases, including neurodegenerative diseases, such as Parkinson's and Alzheimer's disease and ischemic neurodegeneration. The aim of the present study was to investigate the impact of transient global brain ischemia on the expression of selected proteins involved in mitochondrial dynamics and mitochondria-associated membranes. The main foci of interest were the proteins mitofusin 2 (Mfn2), dynamin-related protein 1 (DRP1), voltage-dependent anion-selective channel 1 (VDAC1) and glucose-regulated protein 75 (GRP75). Western blot analysis of total cell extracts and mitochondria isolated from either the cerebral cortex or hippocampus of experimental animals was performed. In addition, Mfn2 was localized intracellularly by laser scanning confocal microscopy. It was demonstrated that 15-min ischemia, or 15-min ischemia followed by 1, 3, 24 or 72 h of reperfusion, was associated with a marked decrease of the Mfn2 protein in mitochondria isolated from the cerebral cortex, but not in hippocampal mitochondria. Moreover, a translocation of the Mfn2 protein to the cytoplasm was documented immediately after global brain ischemia in the neurons of the cerebral cortex by laser scanning confocal microscopy. Mfn2 translocation was followed by decreased expression of Mfn2 during reperfusion. Markedly elevated levels of the VDAC1 protein were also documented in total cell extracts isolated from the hippocampus of rats after 15 min of global brain ischemia followed by 3 h of reperfusion, and from the cerebral cortex of rats after 15 min of global brain ischemia followed by 72 h of reperfusion. The mitochondrial Mfn2 release observed during the early stages of reperfusion may thus represent an important mechanism of mitochondrial dysfunction associated with neuronal dysfunction or death induced by global brain ischemia.

Photobiomodulation and Coenzyme Q10 Treatments Attenuate Cognitive Impairment Associated With Model of Transient Global Brain Ischemia in Artificially Aged Mice.

Disturbances in mitochondrial biogenesis and bioenergetics, combined with neuroinflammation, play cardinal roles in the cognitive impairment during aging that is further exacerbated by transient cerebral ischemia. Both near-infrared (NIR) photobiomodulation (PBM) and Coenzyme Q10 (CoQ10) administration are known to stimulate mitochondrial electron transport that potentially may reverse the effects of cerebral ischemia in aged animals. We tested the hypothesis that the effects of PBM and CoQ10, separately or in combination, improve cognition in a mouse model of transient cerebral ischemia superimposed on a model of aging. We modeled aging by 6-week administration of D-galactose (500 mg/kg subcutaneous) to mice. We subsequently induced transient cerebral ischemia by bilateral occlusion of the common carotid artery (BCCAO). We treated the mice with PBM (810 nm transcranial laser) or CoQ10 (500 mg/kg by gavage), or both, for 2 weeks after surgery. We assessed cognitive function by the Barnes and Lashley III mazes and the What-Where-Which (WWWhich) task. PBM or CoQ10, and both, improved spatial and episodic memory in the mice. Separately and together, the treatments lowered reactive oxygen species and raised ATP and general mitochondrial activity as well as biomarkers of mitochondrial biogenesis, including SIRT1, PGC-1α, NRF1, and TFAM. Neuroinflammatory responsiveness declined, as indicated by decreased iNOS, TNF-α, and IL-1β levels with the PBM and CoQ10 treatments. Collectively, the findings of this preclinical study imply that the procognitive effects of NIR PBM and CoQ10 treatments, separately or in combination, are beneficial in a model of transient global brain ischemia superimposed on a model of aging in mice.

Ischemic tau protein gene induction as an additional key factor driving development of Alzheimer's phenotype changes in CA1 area of hippocampus in an ischemic model of Alzheimer's disease.

Tauopathies are a class of neurodegenerative illnesses associated with the aberrant accumulation of the tau protein in the brain. The best known out of these diseases is Alzheimer's disease, a disorder where the microtubule associated tau protein becomes hyperphosphorylated (which lowers its binding affinity to microtubules) and accumulates inside neurons in the form of tangles. In this study, we attempt to find out whether brain ischemia may play an important role in tau protein gene alterations. We have investigated the relationship between hippocampal ischemia and Alzheimer's disease by means of a transient 10-min global brain ischemia in rats and determining the effect on Alzheimer's disease tau protein gene expression during 2, 7 and 30 days post injury. We found the significant overexpression of tau protein gene on the 2nd day, but on day's 7 and 30 post-ischemia there a significant opposite tendency was observed. The obtained results offer a novel insight into tau protein gene in regulating delayed neuronal death in the ischemic hippocampus. Finally, these findings further elucidate the long-term impact of brain ischemia on Alzheimer's disease development.

Changes of Doublecortin-Immunoreactive Cells from the Acute Phase to Chronic Phase After Transient Global Brain Ischemia in Rat Cingulate Cortex.

Doublecortin (DCX)-immunoreactive (-ir) cells play important roles in adult cortical remodeling. We previously reported that DCX-ir cells decrease after transient global brain ischemia (GBI) in the cingulate cortex (Cg) of rats. In the present study, we examined the changes of DCX-ir cells from the acute to the chronic phase after GBI in rats. Transient GBI was induced by a four-vessel occlusion model as described previously. Thirty-six rats were divided into six groups: day 7 after sham operation (Group Sham+A), day 7 after 3 min GBI (Group GBI3+A), day 7 after 10 min GBI (Group GBI10+A), day 90 after sham operation (Group Sham+C), day 90 after 3 min GBI (Group GBI3+C), and day 90 after 10 min GBI (Group GBI10+C). The numbers of DCX-ir cells per unit area (mm2) were investigated in the anterior cingulate cortex (ACC) and retrosplenial cortex (RS). A two-way factorial analysis of variance regarding the time of GBI (sham, GBI3, GBI10) or the period after GBI (day 7, day 90) was employed in each area. Regarding the time of GBI, there were significant differences in both the ACC and the RS (p<0.001, respectively). Regarding the period after GBI, there was no significant difference in the ACC, whereas a significant difference was found in the RS (p=0.005). In each area and in each phase, the numbers did not change in GBI3 (one-way ANOVA followed by a Tukey test) and decreased in GBI10 (p<0.005). The numbers in the RS from the acute phase to chronic phase did not change in the sham and GBI3, and decreased in GBI10 (independent t-test, p<0.001). However, histochemical staining with Fluoro-Jade B suggested that neuronal cell death did not occur in both the ACC and the RS in all groups. The present findings indicate that the cortical remodeling potential in the Cg decreases in the acute phase after GBI, and continues to decrease until the chronic phase.

Post-resuscitation Arterial Blood Pressure on Survival and Change of Capillary Density Following Cardiac Arrest and Resuscitation in Rats.

Transient global brain ischemia, induced by cardiac arrest and resuscitation, results in reperfusion injury leading to delayed selective neuronal cell loss and post-resuscitation mortality. This study determined the effects of post-resuscitation hypotension and hypothermia on long-term survival following cardiac arrest and resuscitation. The capillary density was also determined. Based on the mean arterial blood pressure (MABP) at 1h of recovery, the normotension group (MABP 80-120mmHg) and hypotension group (MABP <80mmHg) were defined. The overall survival was determined at 4days of recovery. Brain microvascular density was assessed using immunohistochemistry of the glucose transporter, GLUT-1. The pre-arrest MABP was similar in each group; at 1h after resuscitation, the MABP in the normotension groups was about 80% of their pre-arrest values; the hypotension group had a significantly lower MABP compared to the normotension group. The overall survival rate was lower in the hypotension group compared to the normotension group (36%, 4/11 vs. 67%, 14/21) under the normothermic condition. Brain blood flow in the hypotension group was lower (33% decrease) compared to the normotension group at 1-h post-resuscitation. Compared to the pre-arrest baseline, the capillary density was significantly increased at 14days of recovery (355 ± 42 vs. 469 ± 50, number/mm2) in the cortex. The capillary density in hippocampus was also increased at 4-30days following cardiac arrest and resuscitation. Our results suggest that rats able to maintain their post-resuscitation blood pressure at normotension, had higher brain blood flow during the early recovery phase, and improved survival outcome following cardiac arrest and resuscitation. In addition, cardiac arrest and resuscitation induced angiogenesis in brain in the first month of recovery.

Implication of GPR40 Signaling in the Subventricular Zone Neurogenesis after Ischemia via Cross-Talk between Neural Progenitors and Microglia

Objective: Sub-ventricular Zone (SVZ) of the anterior horn of lateral ventricle is a source of neural stem cells in the adult mammalian brain along with hippocampal Sub-granular Zone (SGZ). Previously, we demonstrated that transient global brain ischemia in adult monkeys increases the number of neuronal progenitors in the SGZ via G-protein-coupled Receptor 40 (GPR40) signaling. Sonic Hedgehog (SHH) is indispensable for ischemia-induced neural progenitor proliferation in rodents. Although GPR40 is expressed in the SVZ, GPR40 synergy with SHH remains unelucidated in adult SVZ neurogenesis. Here, we studied GPR40 implication in SVZ neurogenesis using monkey model.Methods: Adult monkeys underwent 20 min transient global brain ischemia by clamping both the innominate and left subclavian arteries. On days 3, 4, 7, 9 and 15 after ischemia/reperfusion, when SVZ neurogenesis increases, as it was shown previously by the authors, the brain samples were resected and normal and post-ischemic monkey SVZ tissues were used for Western blot and immunofluorescence histochemistry analysis.Results: Ischemia/reperfusion increased GPR40 protein levels, proliferation of GPR40-positive stem/progenitor cells, the number of GPR40/PSA-NCAM or GPR40/doublecortin co-expressing immature neurons and GPR40- positive microglial cells in the SVZ vascular niche. GPR40 up-regulation correlated with that of SHH, Notch1 and β1-integrin; GPR40 co-localized with stem/progenitor cell markers in post-ischemic SVZ. GPR40-positive microglia showed the highest SHH expression relative to other cell types. SHH binding to Patched1 in the cells that surrounded GPR40/SHH-expressing microglia occurred within the distance of SHH paracrine secretion.Conclusion: These data suggest that GPR40 is related to post-ischemic neurogenesis in the primate SVZ, GPR40-positive microglia support SHH paracrine secretion in the SVZ, and the cross-talk between perivasucular microglia and neuronal progenitors may be crucial in the vascular niche to activate neurogenesis by SHH and Notch1 signaling.

Puerarin attenuates locomotor and cognitive deficits as well as hippocampal neuronal injury through the PI3K/Akt1/GSK-3β signaling pathway in an in vivo model of cerebral ischemia.

Ischemic stroke causes irreversible damage to the brain. The hippocampus is a vulnerable region and plays an important role in cognition and locomotor activity. Puerarin is a phytoestrogen that has beneficial effects in treating neurological disorders. How puerarin protects against hippocampal injury and its molecular mechanisms remain to be elucidated. Transient global brain ischemia was induced by 4-vessel occlusion in adult male Sprague-Dawley rats. The rats were pretreated with puerarin alone or together with LY294002 (an PI3K inhibitor) before ischemia/reperfusion (I/R). The open- and closed-field tasks and Morris water maze (MWM) test were used to assess the effects of puerarin on anxiety-like behavioral and cognitive impairment following I/R. Hematoxylin-eosin staining(HE) was used to examine the survival of hippocampal CA1 pyramidal neurons, and immunoblotting was performed to examine the expression of the related proteins. By using the rat model for transient I/R, we demonstrated that puerarin pretreatment significantly increased the travelling distance and number of crossings in the open- and closed-field tests, reduced latency and increased the proportion of distance and time in zone IV in the MWM. The number of live cells in the hippocampus is sharply increased by puerarin pretreatment.We further observed that the levels of phosphorylated Akt1, GSK-3β and MCL-1were elevated and those of cleaved-caspase-3 were reduced in the puerarin-treatment group. Notably, the PI3K inhibitor LY294002 counteracted all of the effects of puerarin. Our findings suggest that puerarin protects the hippocampus from I/R damage by activating the PI3K/Akt1/GSK-3β/MCL-1 signaling pathway.

Abstract WP289: Interaction Between Stroke and Amyloid-β Deposition in Cognitive Function ~Possible Involvement of Antagonist Effect of AT 2 Receptor Activation~

Background and Aim: Stroke is known to be causally related with Alzheimer’s disease pathology and amyloid-β (Aβ) deposition has been suggested to induce cerebral amyloid angiopathy resulting in stroke-associated cognitive decline. Angiotensin (Ang) type 1 (AT 1 ) receptor activation impairs cognitive performance, whereas Ang II type 2 (AT 2 ) receptor stimulation has been reported to improve cognitive impairment. We examined the interaction between stroke and Aβ deposition in cognitive function, and the possible antagonist effect of AT 2 receptor in vascular smooth muscle cells (VSMC) on this cognitive decline. Methods: Adult (10-12 weeks old) male wild-type (WT) mice (C57BL/6J mice) or the mice with AT 2 receptor overexpression in VSMC (SMAT2) were used. Mice were subjected to intracerebroventricular (ICV) injection of Aβ1-40 following 15 minutes global brain ischemia operation. Three weeks after Aβ1-40 ICV injection, cognitive function of spatial learning memory was evaluated by the Morris water maze test. Brain samples were obtained after behavioral testing, and the expression of inflammatory cytokines and NADPH oxidase subunits were measured by real-time quantitative RT-PCR. Results: ICV injection of Aβ1-40 in WT mice showed impaired cognitive function. On the other hand, WT mice with transient global brain ischemia did not decline significantly cognitive function. In contrast, WT mice with Aβ1-40 ICV injection with global brain ischemia exhibited more marked cognitive impairment compared with control mice. These results suggested that transient brain ischemia and amyloid-β deposition exerted at least additive effects on cognitive impairment. This cognitive decline was accompanied with increased expressions of inflammatory cytokines such as monocyte chemoattractant protein-1 and NADPH oxidase subunits including p22 phox . On the other hand, SMAT 2 mice did not show cognitive impairment by global brain ischemia with/without Aβ1-40 ICV injection. Conclusion: Brain ischemia and amyloid-β deposition induced additive or synergistic effect on cognitive impairment. AT 2 receptor activation in VSMC could play an inhibitory role in the cognitive decline induced by brain ischemia and amyloid-β deposition.

Metformin protects the brain against ischemia/reperfusion injury through PI3K/Akt1/JNK3 signaling pathways in rats

Although Metformin, a first-line antidiabetic drug, can ameliorate ischemia/reperfusion (I/R) induced brain damage, but how metformin benefits injured hippocampus and the mechanisms are still largely unknown. Therefore, the aim of this study was to investigate the neuroprotective mechanisms of metformin against ischemic brain damage induced by cerebral I/R and to explore whether the Akt-mediated down-regulation of the phosphorylation of JNK3 signaling pathway contributed to the protection provided by metformin. Transient global brain ischemia was induced by 4-vessel occlusion in adult male Sprague-Dawley rats. The open field tasks and Morris water maze were used to assess the effect of metformin on anxiety-like behavioral and cognitive impairment after I/R. Cresyl Violet staining was used to examine the survival of hippocampal CA1 pyramidal neurons. Immunoblotting was performed to measure the phosphorylation of Akt1, JNK3, c-Jun and the expression of cleaved caspase-3. Through ischemia/reperfusion (I/R) rat model, we found that metformin could attenuate the deficits of hippocampal related behaviors and inhibit cell apoptosis. The western blot data showed that metformin could promote the activation of Akt1 and reduce the phosphorylation of JNK3 and c-Jun as well as elevation of cleaved caspase-3 in I/R brains. PI3K inhibitor reversed all the protective effects, further indicating that metformin protect hippocampus from ischemic damage through PI3K/Akt1/JNK3/c-Jun signaling pathway.

RETRACTED: Rosmarinic acid protects rat hippocampal neurons from cerebral ischemia/reperfusion injury via the Akt/JNK3/caspase-3 signaling pathway

Cerebral ischemia/reperfusion injury can result in neuronal death, which further results in brain damage and can even lead to death. Although recent studies showed that rosmarinic acid (RA) exerts neuroprotective effects and attenuates ischemia-induced brain injury and neuronal cell death, little is known about the precise mechanisms that occur during cerebral ischemia/reperfusion (I/R). Therefore, the aim of this study was to examine the underlying mechanism of the neuroprotective effects of RA against ischemic brain injury induced by cerebral I/R. Transient global brain ischemia was induced by 4-vessel occlusion in adult male Sprague-Dawley rats. We randomly divided rats into five groups: sham, I/R, I/R+RA, I/R+Vehicle and I/R+RA+LY. Open-field, closed-field and Morris water maze tests were carried our separately to examine the anxiety and cognitive behavior of each group. Cresyl violet staining was used to examine the survival of hippocampal CA1 pyramidal neurons. The levels of p-Akt, p-JNK3 and cleaved caspase-3 in the hippocampus were also examined by Western blotting. Our results showed that administration of RA protected locomotive ability, relieved anxiety behavior and protected cognitive ability in cerebral I/R-injured rats. Additionally, RA significantly protected neurons in the hippocampal CA1 region against cerebral I/R-induced damage. Furthermore, RA increased the phosphorylation of Akt1, downregulated the phosphorylation of JNK3 and reduced the expression of cleaved caspase-3. Finally, the Akt inhibitor LY294002 reversed all the protective effects of RA, indicating that RA protects neurons in the hippocampal CA1 region from ischemic damage through the Akt/JNK3/caspase-3 signaling pathway.

Neuroprotection of Cilostazol against ischemia/reperfusion-induced cognitive deficits through inhibiting JNK3/caspase-3 by enhancing Akt1

Cilostazol(CTL) is a phosphodiesterase inhibitor, which has been widely used as anti-platelet agent. It also has preventive effects on various central nervous system (CNS) diseases, including ischemic stroke, Parkinson’s disease and Alzheimer disease. However, the molecular mechanism underlying the protective effects of CTL is still unclear, and whether CTL can prevent I/R induced cognitive deficit has not been reported. Transient global brain ischemia was induced by 4-vessel occlusion in adult male Sprague-Dawley rats. The open field tasks and Morris water maze were used to assess the effect of CTL on anxiety-like behavioral and cognitive impairment after I/R. Western blotting were performed to examine the expression of related proteins, and HE-staining was used to detect the percentage of neuronal death in the hippocampal CA1 region. Here we found that CTL significantly improved cognitive deficits and the behavior of rats in Morris water maze and open field tasks (P<0.05). HE staining results showed that CTL could significantly protect CA1 neurons against cerebral I/R (P<0.05). Additionally, Akt1 phosphorylation levels were evidently up-regulated (P<0.05), while the activation of JNK3, which is an important contributor to I/R-induced neuron apoptosis, was reduced by CTL after I/R (P<0.05), and caspase-3 levels were also decreased by CTL treatment. Furthermore, all of CTL's protective effects were reversed by LY294002, which is a PI3K/Akt1 inhibitor. Taken together, our results suggest that CTL could protect hippocampal neurons and ameliorate the impairment of learning/memory abilities and locomotor/ exploratory activities in ischemic stroke via a PI3K-Akt1/JNK3/caspase-3 dependent mechanism.

Dysregulation of Autophagy, Mitophagy, and Apoptotic Genes in the Medial Temporal Lobe Cortex in an Ischemic Model of Alzheimer's Disease.

Ischemic brain damage is a pathological incident that is often linked with medial temporal lobe cortex injury and finally its atrophy. Post-ischemic brain injury associates with poor prognosis since neurons of selectively vulnerable ischemic brain areas are disappearing by apoptotic program of neuronal death. Autophagy has been considered, after brain ischemia, as a guardian against neurodegeneration. Consequently, we have examined changes in autophagy (BECN 1), mitophagy (BNIP 3), and apoptotic (caspase 3) genes in the medial temporal lobe cortex with the use of quantitative reverse-transcriptase PCR following transient 10-min global brain ischemia in rats with survival 2, 7, and 30 days. The intense significant overexpression of BECN 1 gene was noted on the 2nd day, while on days 7–30 the expression of this gene was still upregulated. BNIP 3 gene was downregulated on the 2nd day, but on days 7–30 post-ischemia, there was a significant reverse tendency. Caspase 3 gene, associated with apoptotic neuronal death, was induced in the same way as BNIP 3 gene after brain ischemia. Thus, the demonstrated changes indicate that the considerable dysregulation of expression of BECN 1, BNIP 3, and caspase 3 genes may be connected with a response of neuronal cells in medial temporal lobe cortex to transient complete brain ischemia.

Discrepancy in Expression of β-Secretase and Amyloid-β Protein Precursor in Alzheimer-Related Genes in the Rat Medial Temporal Lobe Cortex Following Transient Global Brain Ischemia.

Brain ischemia may be causally related with Alzheimer's disease. Presumably, β-secretase and amyloid-β protein precursor gene expression changes may be associated with Alzheimer's disease neuropathology. Consequently, we have examined quantitative changes in both β-secretase and amyloid-β protein precursor genes in the medial temporal lobe cortex with the use of quantitative rtPCR analysis following 10-min global brain ischemia in rats with survival of 2, 7, and 30 days. The greatest significant overexpression of β-secretase gene was noted on the 2nd day, while on days 7-30 the expression of this gene was only modestly downregulated. Amyloid-β protein precursor gene was downregulated on the 2nd day, but on days 7-30 postischemia, there was a significant reverse tendency. Thus, the demonstrated alterations indicate that the considerable changes of expression of β-secretase and amyloid-β protein precursor genes may be connected with a response of neurons in medial temporal lobe cortex to transient global brain ischemia. Finally, the ischemia-induced gene changes may play a key role in a late and slow onset of Alzheimer-type pathology.

Rolipram improves cognition, reduces anxiety- and despair-like behaviors and impacts hippocampal neuroplasticity after transient global cerebral ischemia

Cognitive impairment, anxiety- and depressive-like symptoms are well recognized outcome of cerebral ischemia in clinical and preclinical settings. Rolipram, a phosphodiesterase-4 (PDE-4) inhibitor, improves cognition and produces anxiolytic- and antidepressant-like effects in rodents. Rolipram also exerts anti-inflammatory effects and enhances survival of newborn hippocampal neurons in mice subjected to transient global cerebral ischemia. Here, we evaluated the effects of chronic rolipram treatment in mice subjected to transient global brain ischemia. C56B6/7 mice were subjected to bilateral common carotid artery occlusion (BCCAO) and were then tested in a multi-tiered behavioral battery including the elevated zero maze (EZM), open field (OF), object location test (OLT), and forced swim test (FST). We also investigated the effects of rolipram on hippocampal neurodegeneration and the expression of the neuronal plasticity markers doublecortin (DCX) and microtubule-associated protein (MAP-2). Ischemic mice exhibited memory deficits OLT, higher levels of anxiety EZM and behavioral despair FST. BCCAO caused neuronal loss in the CA3 hippocampal subfield and basolateral amygdala (BLA). In the hippocampus of BCCAO mice, a disrupted neuronal plasticity was evidenced by decreased DCX expression. Chronic treatment with rolipram attenuated the behavioral effects of BCCAO. Rolipram also decreased neurodegeneration in the CA3 while it increased dendritic arborization of DCX-immunoreactive (DCX-IR) neurons and microtubule associate MAP-2 expression in the hippocampus of BCCAO mice. These data suggest that chronic inhibition of PDE-4 can be a useful therapeutic strategy to improve the emotional and cognitive outcomes of transient global cerebral ischemia.