Ischemic Preconditioning-induced Neuroprotection Research Articles

CPKCγ-Modulated Autophagy Contributes to Ischemic Preconditioning-Induced Neuroprotection in Mice with Ischemic Stroke via mTOR-ULK1 Pathway.

Neuron-specific conventional protein kinase C (cPKC)γ mediates cerebral hypoxic preconditioning (HPC). In parallel, autophagy plays a prosurvival role in ischemic preconditioning (IPC) against ischemic stroke. However, the effect of cPKCγ on autophagy in IPC still remains to be addressed. In this study, adult and postnatal 1-day-old C57BL/6J wild-type (cPKCγ+/+) and knockout (cPKCγ-/-) mice were used to establish in vivo and in vitro IPC models. The results showed that IPC pretreatment alleviated neuronal damage caused by lethal ischemia, which could be suppressed by autophagy inhibitor 3-MA or bafilomycin A1. Meanwhile, cPKCγ knockout blocked IPC-induced neuroprotection, accompanied by significant increase of LC3-I to LC3-II conversion and Beclin 1 protein level, and a significant decrease in p62 protein level. Immunofluorescent staining results showed a decrease of LC3 puncta numbers in IPC-treated cPKCγ+/+ neurons with fatal ischemia, which was reversed in cPKCγ-/- neurons. In addition, cPKCγ-modulated phosphorylation of mTOR at Ser 2448 and ULK1 at Ser 555, rather than p-Thr-172 AMPK, was detected in IPC-pretreated neurons upon lethal ischemic exposure. The present data demonstrated that cPKCγ-modulated autophagy via the mTOR-ULK1 pathway likely modulated IPC-induced neuroprotection.

Isoflurane post-conditioning attenuates cerebral ischemia/reperfusion injury by reducing apoptotic through activating the BMP7/SMAD signaling pathway in rats

The expressions of different temporal patterns of bone morphogenetic proteins (BMPs) have changed after ischemic strokes, and ischemic preconditioning-induced neuroprotection was attenuated when BMP7 was inhibited. In the previous study, the neuroprotection of isoflurane postconditioning (ISPOC) against cerebral ischemia-reperfusion (I/R) injury has been addressed, with particular relevance to the role of BMP7. Consequently, in the present study, we continued to explore the mechanisms involved in the BMP7 signal mediated the neuroprotection of ISPOC. A rat model of the middle cerebral artery occlusion was used in this study. Rats were administered 1.5 % isoflurane, 60 min after 90 min of ischemia, followed by a 24 h reperfusion period. The 1.5 % ISPOC significantly ameliorated the cerebral infarct volumes, neurologic deficit scores, damaged neurons, and apoptotic neurons. Moreover, ISPOC unregulated the expressions of BMP7, p-Smad1/5/9, and p-p38. Whereas, the neuroprotective effect was weakened by LDN-193189 and SB203580, respectively, a BMP7/Smad1/5/9 and p38MAPK signaling pathway inhibitor. Furthermore, LDN-193189 downregulated the expression of p-p38. The present results of this study indicated that the neuroprotection of 1.5 % isoflurane postconditioning to cerebral ischemia-reperfusion injury is related to the activating of BMP7/Smad1/5/9 and p38MAPK signal pathway.

Metabolomic Profiling Reveals That Reprogramming of Cerebral Glucose Metabolism Is Involved in Ischemic Preconditioning-Induced Neuroprotection in a Rodent Model of Ischemic Stroke

Ischemic tolerance renders the brain resistant to ischemia-reperfusion (I/R) injury as a result of the activation of endogenous adaptive responses triggered by various types of preconditioning. The complex underlying metabolic mechanisms responsible for the neuroprotection of cerebral ischemic preconditioning (IPC) remain elusive. Herein, gas chromatography-mass spectrometry (GC-MS) technique was applied to delineate the dynamic changes of brain metabolome in a rodent model of ischemic stroke (transient occlusion of the middle cerebral artery, tMCAO), alone or after pretreatment with nonlethal ischemic tolerance induction (transient occlusion of the bilateral common carotid arteries, tBCCAO). Metabolomic analysis showed that accumulation of glucose (concentration increased more than 4 fold) and glycolytic intermediates is the prominent feature of brain I/R-induced metabolic disturbance. IPC attenuated brain I/R damage by subduing postischemic hyperglycolysis, increasing the pentose phosphate pathway (PPP) flux and promoting the utilization of β-hydroxybutyrate. The expression analysis of pivotal genes and proteins involved in relevant metabolic pathways revealed that the downregulation of AMP-activated protein kinase (AMPK)-mediated glucose transporter-1 (GLUT-1) and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) and reduced mRNA levels of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) subunits were associated with IPC-induced metabolic flexibility, which allows the brain to be more capable of withstanding severe I/R insults. The present study provided mechanistic insights into the metabolic signature of IPC and indicated that adaptively modulating brain glucose metabolism could be an effective approach for the therapeutic intervention of ischemic stroke.

Sumoylation of LYS590 of NCX3 f-Loop by SUMO1 Participates in Brain Neuroprotection Induced by Ischemic Preconditioning.

The small ubiquitin-like modifier (SUMO), a ubiquitin-like protein involved in posttranslational protein modifications, is activated by several conditions, such as heat stress, hypoxia, and hibernation and confers neuroprotection. Sumoylation enzymes and substrates are expressed also at the plasma membrane level. Among the numerous plasma membrane proteins controlling ionic homeostasis during cerebral ischemia, 1 of the 3 brain sodium/calcium exchangers (NCX3), exerts a protective role during ischemic preconditioning. In this study, we evaluated whether NCX3 is a target for sumoylation and whether this posttranslational modification participates in ischemic preconditioning-induced neuroprotection. To test these hypotheses, we analyzed (1) SUMO1 conjugation pattern after ischemic preconditioning; (2) the effect of SUMO1 knockdown on the ischemic damage after transient middle cerebral artery occlusion and ischemic preconditioning, (3) the possible interaction between SUMO1 and NCX3 and (4) the molecular determinants of NCX3 sequence responsible for sumoylation. Focal brain ischemia and ischemic preconditioning were induced in rats by middle cerebral artery occlusion. SUMOylation was evaluated by western blot and immunohistochemistry. SUMO1 and NCX3 interaction was analyzed by site-directed mutagenesis and immunoprecipitation assay. We found that (1) SUMO1 knockdown worsened ischemic damage and reduced the protective effect of preconditioning; (2) SUMO1 bound to NCX3 at lysine residue 590, and its silencing increased NCX3 degradation; and (3) NCX3 sumoylation participates in SUMO1 protective role during ischemic preconditioning. Thus, our results demonstrate that NCX3 sumoylation confers additional neuroprotection in ischemic preconditioning. Finally, this study suggests that NCX3 sumoylation might be a new target to enhance ischemic preconditioning-induced neuroprotection.

Transferred inter-cell ischemic preconditioning-induced neuroprotection may be mediated by adenosine A1 receptors

Ischemic preconditioning-induced neuroprotection is a well-known phenomenon. We hypothesize that this form of neuroprotection is transferable among the same type of cells. To test this hypothesis, human neuroblastoma SH-SY5Y cells were induced to become neuron-like cells. Primary rat cortical neuronal cultures were also used. These cells were subjected to various lengths of short oxygen-glucose deprivation (OGD, an in vitro simulation of ischemia) and then 1-h OGD. Some cells that were not exposed to a short episode of ischemia were incubated with culture medium from the cells that had 3- or 5-min OGD. Those cells were subjected to OGD for 1h at 1 or 24h after they were exposed to the medium. Cell injury was evaluated at 24h after the 1-h OGD by lactate dehydrogenase release assay. In another experiment, cells subjected to a 3-min OGD or exposed to the medium from cells that had a 3-min OGD were harvested at 30min after the OGD or the medium exposure for Western blotting of Akt, a prosurvival protein. Our study showed that a prior episode of ischemia lasting from 3 to 10min significantly reduced the 1-h OGD-induced cell injury. Medium from cells subjected to a 3-min OGD also induced acute and delayed phases of neuroprotection in OGD-naïve human neuron-like cells and primary rat cortical neuronal cultures. Cells subjected to a 3-min OGD or incubated with the medium from cells exposed to a 3-min OGD had increased phosphorylated/activated Akt. The increased phosphorylated Akt and neuroprotection induced by medium transferring were inhibited by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an adenosine A1 receptor inhibitor. The 3-min OGD-induced neuroprotection was inhibited by LY294002, an Akt activation inhibitor. These results suggest that ischemic preconditioning-induced neuroprotection is transferable among the cells. Small molecules, such as adenosine, may mediate this effect.

Ischemic preconditioning-induced neuroprotection against transient cerebral ischemic damage via attenuating ubiquitin aggregation

Ubiquitin binds to short-lived proteins, and denatured proteins are produced by various forms of injuries. In the present study, we investigated the effect of ischemic preconditioning (IPC) on free ubiquitin and its mutant form (ubiquitin+1) in the gerbil hippocampus induced by transient cerebral ischemia. The animals were randomly assigned to 4 groups (sham-operated-group, ischemia-operated-group, IPC plus (+)-sham-operated-group, and IPC+ischemia-operated-group). IPC was induced by subjecting gerbils to a 2min of ischemia followed by 1day of recovery. A significant loss of neurons was observed in the stratum pyramidale (SP) of the hippocampal CA1 region (CA1) in the ischemia-operated-groups 5days after ischemia–reperfusion (I–R). In all the IPC+ischemia-operated-groups, neurons in the SP were well protected. We found that strong ubiquitin immunoreactivity was detected in the SP in the sham-operated-group and the immunoreactivity was decreased with time after I–R. In all the IPC+ischemia-operated-groups, ubiquitin immunoreactivity in the SP was similar to that in the sham-operated group. Moderate ubiquitin+1 immunoreactivity was detected in the SP of the sham-operated-group, and the immunoreactivity was markedly increased 2days after I–R. Five days after I–R, ubiquitin+1 immunoreactivity was very weak in the SP. In all the IPC+ischemia-operated-groups, ubiquitin+1 immunoreactivity in the SP was slightly decreased with time after I–R. Western blot analysis showed that, in all the IPC+ischemia-ischemia-groups, the levels of ubiquitin and ubiquitin+1 proteins were well maintained after I–R. In brief, our findings suggest that the inhibition of the depletion of free ubiquitin and the formation of ubiquitin+1 may have an essential role in inducing cerebral ischemic tolerance by IPC.

Ischemic preconditioning-induced neuroprotection is associated with differential expression of IL-1β and IL-1 receptor antagonist in the ischemic cortex

Ischemic preconditioning (IP) is a phenomenon that organs develop a tolerance toward subsequent lethal ischemic insults. Among the factors that are involved in IP, IL-1β and its endogenous receptor antagonist IL-1ra have been identified as important players in the induction of IP. The present study investigated whether IP affects the levels of these two antagonistic proteins during tolerance and reperfusion periods after ischemic stroke. The IP 24 h prior to ischemic stroke resulted in neuroprotection in the cortex. IP-induced protection is accompanied by increased IL-1β gene and IL-1ra gene and protein levels during the tolerance period. In the post-ischemic cortex, IP resulted in the suppression of IL-1β mRNA and protein levels at 6 h without affecting IL-1ra expression and the up-regulation of IL-1ra protein at 24 h. These findings demonstrate that IP differentially regulates cortical IL-1β and IL-1ra expression before and after ischemic stroke and suggest that the shift toward an anti-inflammatory state in the post-ischemic cortex may contribute to IP-induced neuroprotection.

Resveratrol and Ischemic Preconditioning in the Brain

Cardiovascular pathologies in the French are not prevalent despite high dietary saturated fat consumption. This is commonly referred to as the "French Paradox" attributing its anti-lipidemic effects to moderate consumption of red wine. Resveratrol, a phytoalexin found in red wine, is currently the focus of intense research both in the cardiovascular system and the brain. Current research suggests resveratrol may enhance prognosis of neurological disorders such as, Parkinson's, Huntington's, Alzheimer's diseases and stroke. The beneficial effects of resveratrol include: antioxidation, free radical scavenger, and modulation of neuronal energy homeostasis and glutamatergic receptors/ion channels. Resveratrol directly increases sirtuin 1 (SIRT1) activity, a NAD(+) (oxidized form of nicotinamide adenine dinucleotide)-dependent histone deacetylase related to increased lifespan in various species similar to calorie restriction. We recently demonstrated that brief resveratrol pretreatment conferred neuroprotection against cerebral ischemia via SIRT1 activation. This neuroprotective effect produced by resveratrol was similar to ischemic preconditioning-induced neuroprotection, which protects against lethal ischemic insults in the brain and other organ systems. Inhibition of SIRT1 abolished ischemic preconditioning-induced neuroprotection in CA1 region of the hippocampus. Since resveratrol and ischemic preconditioning-induced neuroprotection require activation of SIRT1, this common signaling pathway may provide targeted therapeutic treatment modalities as it relates to stroke and other brain pathologies. In this review, we will examine common signaling pathways, cellular targets of resveratrol, and ischemic preconditioning-induced neuroprotection as it relates to the brain.

Inhibition of caspase-9 activation and apoptosis is involved in ischemic preconditioning-induced neuroprotection in rat brain

Objectives: Cerebral ischemic pre-conditioning (IPC) is capable of protecting hippocampal neurons from ischemia/reperfusion (I/R) injury. In the current study, we investigated the role of activated caspase-9 in the protective process induced by IPC and related it to cytochrome c release and apoptosis.Methods: I/R injury was induced by a four-vessel occlusion model in Wistar rats which were randomly divided into ischemia/reperfusion group (I/R), ischemic pre-conditioning + I/R group (IPC + I/R) and control group. Histologic changes in the pyramidal layer of the hippocampal CA1 region were determined by hematoxylin and eosin (H&E) staining. The relative proportion of apoptotic neurons in this area was assessed with TUNEL staining. The redistribution of cytochrome c and activation of caspase-9 were detected in the same area with immunohistochemistry and Western blotting respectively.Results: Compared to the I/R group, IPC increased the number of surviving neurons in the hippocampal CA1 region (p<0.001), markedly reduced the number of apoptotic pyramidal neurons (p<0.001), inhibited the release of cytochrome c from mitochondria to cytoplasm (p<0.001 for positively stained neurons) and decreased the amount of activated caspase-9 (p<0.001).Discussion: These findings confirm that IPC is capable of protecting neurons from injury by apoptosis. The release of cytochrome c to the cytosol demonstrates that the mitochondrial pathway was involved, and the reduction in this release caused by IPC was clearly associated with reduced caspase-9 activation. Together, these results suggest that IPC protects neurons via action on the mitochondrial/caspase-9 pathway of apoptosis.

Estrogen preconditioning protects the hippocampal CA1 against ischemia

Estrogen is neuroprotective against ischemia in both in vivo and in vitro injury models. Because of the promising preclinical data on neuroprotection, the Women’s Estrogen for Stroke Trial was initiated. The outcomes from this trial were, however, unsuccessful and questions emerged about the safety of chronic estrogen treatment in women. In contrast to the chronic estrogen treatment strategy, the present study aims to investigate: (1) the neuroprotective efficacy of single estrogen pretreatment/preconditioning; and (2) the existence of a similarity between estrogen- and ischemic preconditioning-induced neuroprotection against cerebral ischemia. The efficacy of estrogen was tested in an in vitro model of cerebral ischemia using hippocampal organotypic slice culture system. The hippocampal organotypic slice cultures were generated from female neonatal (9–11 days old) Sprague-Dawley rats. The slices were exposed to estradiol-17β (0.5, 1, 5 nM) for various durations (1, 2 or 4 h) 48 h prior to ischemia (40 min of oxygen-glucose deprivation). For ischemic preconditioning, slices were exposed to sublethal oxygen-glucose deprivation (15 min), 48 h prior to lethal oxygen-glucose deprivation. Quantification of cell death in hippocampal CA1 region was conducted by using propidium iodide fluorescence staining technique. Results demonstrated that estrogen preconditioning significantly protects the hippocampal CA1 region against ischemia (P<0.001) and mimicked ischemic preconditioning-induced neuroprotection. The propidium iodide fluorescence values of estrogen preconditioning, ischemic preconditioning and ischemia groups were 21±2 (mean±S.E.M.) (1 nM; 2 h; n=15), 18±2 (5 nM; 4 h; n=12), 32±3 (n=8), 65±3 (n=27), respectively. Further, estrogen preconditioning initiated a calcium-mediated signaling pathway leading to protection of CA1 neurons against ischemia. Future investigations in estrogen preconditioning may suggest new estrogen regimens that avoid potential side effects of chronic estrogen treatment for stroke patients.

Ischemic preconditioning or heat shock pretreatment ameliorates neuronal apoptosis following hypothermic circulatory arrest

Objective Hypothermic circulatory arrest has been widely used in complex cardiac and aortic surgery. Stroke and/or neurologic injury can occur after prolonged hypothermic circulatory arrest, possibly due to apoptosis. Ischemic preconditioning has been widely used as a neuroprotective tool, but its application in neuronal injury under hypothermic circulatory arrest has never been studied. Methods Forty male New Zealand white rabbits were placed on closed-chest cardiopulmonary bypass, subjected to hypothermic circulatory arrest, and rewarmed to normothermia. Experimental groups were treated with heat shock or ischemic preconditioning before hypothermic circulatory arrest. Hippocampal CA1 neurons were analyzed histopathologically. Apoptosis was confirmed by TUNEL assay and Western blot analysis, and serum S-100β levels, c-Fos and Bcl-2 antibodies, and caspase-3 and heat shock protein 70 levels were measured. Results After 2-hour hypothermic circulatory arrest and 4-hour reperfusion, apoptosis was observed in hippocampal CA1 neurons with elevation of serum S-100β levels, which could be ameliorated by ischemic preconditioning or heat shock manipulations. TUNEL-positive nuclear expression of caspase-3 increased after hypothermic circulatory arrest (3.08% ± 0.71%, P < .001) and was diminished with ischemic preconditioning (1.61% ± 0.42%) and heat shock (1.72% ± 0.38%) manipulations. Ischemic preconditioning or heat shock manipulations produced diverse patterns of heat shock protein 70, c-Fos, and Bcl-2 protein expression, suggesting that these manipulations provide neuroprotection via different pathways. Conclusions Ischemic preconditioning and heat shock can attenuate hippocampal CA1 neuronal apoptosis after prolonged hypothermic circulatory arrest under cardiopulmonary bypass. The expression of heat shock protein 70 may not play a major role in the first window of ischemic preconditioning-induced neuroprotection.