Brain Ischemic Preconditioning Research Articles

Hippocampal Enlargement and White Matter Hyperintensities in Obstructive Sleep Apnea Patients

AbstractBackgroundObstructive sleep apnea (OSA), which results from recurrent collapses of the upper airway system, affects around 27% of the geriatric population with mild cognitive impairment (MCI) and causes intermittent hypoxia and sleep fragmentation (Mubashir et al., 2019). Prior research suggests that OSA results in brain ischemic preconditioning, causing hippocampal enlargement due to adaptive hippocampal neurogenesis (Rosenzweig et al., 2013). OSA has also been linked to increased white matter hyperintensities (WMHs) through hypoxic‐ ischemic injury, which contribute to cognitive decline in dementias such as Alzheimer’s disease (Zacharias et al., 2021). Previous research necessitates studying the effects of apnea in an elderly population with MCI.MethodIn the Memories2 study, subjects aged 55‐85 underwent a polysomnography and were assigned to the no OSA (OSA‐) group if their apnea‐hypopnea index (AHI) was <5 events/hour or to the OSA (OSA+) group if their AHI was ≥15. Amnestic MCI status was determined with a neuropsychological battery using standard criteria. The subjects received an optional MRI, in which hippocampal and WMH volumetry were derived from a T1‐weighted structural scan and a whole‐brain fluid‐attenuated inversion recovery (FLAIR) scan, respectively. Statistical analyses (two‐sample t‐tests and Wilcoxon‐Mann‐Whitney test) were performed to understand group differences in hippocampal volume and WMH at baseline. Hippocampal volume data (N = 131) was obtained for 67 OSA+ and 64 OSA‐ subjects. WMH data (N = 129) was obtained for 66 OSA+ and 63 OSA‐ subjects.ResultOSA+ subjects had significantly greater right hippocampal volume (p = 0.0374) and left hippocampal volume (0.0336) than the OSA‐ subjects. OSA+ subjects also had higher levels of white matter hyperintensities as compared to the OSA‐ subjects (p = 0.0340) (Table 1).ConclusionIncreased WMH burden is suggestive of hypoxic‐ischemic injury and supports the hypothesis that hippocampal enlargement may be a response to intermittent hypoxia in OSA. These findings reveal the need for early evaluation for sleep apnea. The future direction of this work includes analyzing the group differences adjusting for covariates, detecting and verifying outliers, and rerunning analyses with the complete baseline data. Future data from the Memories2 study will examine the effects of OSA treatment in MCI patients using continuous positive airway pressure (CPAP).

GATA3 (GATA-Binding Protein 3)/KMT2A (Lysine-Methyltransferase-2A) Complex by Increasing H3K4-3me (Trimethylated Lysine-4 of Histone-3) Upregulates NCX3 (Na+-Ca2+ Exchanger 3) Transcription and Contributes to Ischemic Preconditioning Neuroprotection.

NCX3 (Na+-Ca2+ exchanger 3) plays a relevant role in stroke; indeed its pharmacological blockade or its genetic ablation exacerbates brain ischemic damage, whereas its upregulation takes part in the neuroprotection elicited by ischemic preconditioning. To identify an effective strategy to induce an overexpression of NCX3, we examined transcription factors and epigenetic mechanisms potentially involved in NCX3 gene regulation. Brain ischemia and ischemic preconditioning were induced in vitro by exposure of cortical neurons to oxygen and glucose deprivation plus reoxygenation (OGD/Reoxy) and in vivo by transient middle cerebral artery occlusion. Western blot and quantitative real-time polymerase chain reaction were used to evaluate transcripts and proteins of GATA3 (GATA-binding protein 3), KMT2A (lysine-methyltransferase-2A), and NCX3. GATA3 and KMT2A binding on NCX3 gene was evaluated by chromatin immunoprecipitation and Rechromatin immunoprecipitation experiments. Among the putative transcription factors sharing a consensus sequence on the ncx3 brain promoter region, GATA3 was the only able to up-regulate ncx3. Interestingly, GATA3 physically interacted with KMT2A, and their overexpression or knocking-down increased or downregulated NCX3 mRNA and protein, respectively. Notably, site-direct mutagenesis of GATA site on ncx3 brain promoter region counteracted GATA3 and KMT2A binding on NCX3 gene. More importantly, we found that in the perischemic cortical regions of preconditioned rats GATA3 recruited KMT2A and the complex H3K4-3me (trimethylated lysine-4 of histone-3) on ncx3 brain promoter region, thus reducing transient middle cerebral artery occlusion–induced damage. Consistently, in vivo silencing of either GATA3 or KMT2A prevented NCX3 upregulation and consequently the neuroprotective effect of preconditioning stimulus. The involvement of GATA3/KMT2A complex in neuroprotection elicited by ischemic preconditioning was further confirmed by in vitro experiments in which the knocking-down of GATA3 and KMT2A reverted the neuroprotection induced by NCX3 overexpression in cortical neurons exposed to anoxic preconditioning followed by oxygen and glucose deprivation plus reoxygenation. Collectively, our results revealed that GATA3/KMT2A complex epigenetically activates NCX3 gene transcription during ischemic preconditioning.

Effects of Brain Ischemic Preconditioning on Cognitive Decline and Motor Incoordination in 3-Nitropropionic Acid-Intoxicated Rats: Probable Mechanisms of Action

The paradigm of brain ischemic preconditioning (BIP) rendering protection from succeeding chemical insults has not been established in vivo. Systemic administration of 3-nitropropionic acid (3-NP) damages cerebral basal ganglia by inducing a mitochondrial dysfunction: this serves as a translational model for Huntington’s disease. We tested the potential of BIP against the neurotoxic effects of 3-NP and investigated the role of glycogen synthase kinase 3-beta (GSK-3β) and heat shock protein-72 (HSP72) signalling in the mentioned model. Male Wistar rats (n = 8; 180-200 g) were randomly assigned to seven groups, sham, BIP, 3NP, BIP+3NP, LiCl+BIP+3NP, Que (quercetin) +BIP+3NP, and LiCl+Que+BIP+3NP. Lithium chloride, 3-NP, and quercetin were administered in the doses of 40, 35, and 4 mg/kg i.p., respectively. Deficits in motor coordination and memory retention were assessed using the balance beam, accelerating rotarod, gait analysis, Morris water maze, and elevated plus maze. Brain biochemistry was assessed for the markers of lipid peroxidation and nitric oxide. The findings revealed noticeable differences in the measured indices in the examined animal groups vs. the sham group (P < 0.05 for motor coordination, memory retention, and brain biochemistry). A strong negative correlation between the rotarod performance of animals and their brain MDA levels was found. The findings reveal that BIP provides behavioral and biochemical recuperation against 3NP-induced neurodegeneration, and this is related to downregulation of GSK -3β and upregulation of HSP72.

Brain ischemic preconditioning protects against ischemic injury and preserves the blood-brain barrier via oxidative signaling and Nrf2 activation

Brain ischemic preconditioning (IPC) with mild ischemic episodes is well known to protect the brain against subsequent ischemic challenges. However, the underlying mechanisms are poorly understood. Here we demonstrate the critical role of the master redox transcription factor, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), in IPC-mediated neuroprotection and blood-brain barrier (BBB) preservation. We report that IPC causes generation of endogenous lipid electrophiles, including 4-hydroxy-2-nonenal (4-HNE), which release Nrf2 from inhibition by Keap1 (via Keap1-C288) and inhibition by glycogen synthase kinase 3β (via GSK3β-C199). Nrf2 then induces expression of its target genes, including a new target, cadherin 5, a key component of adherens junctions of the BBB. These effects culminate in mitigation of BBB leakage and of neurological deficits after stroke. Collectively, these studies are the first to demonstrate that IPC protects the BBB against ischemic injury by generation of endogenous electrophiles and activation of the Nrf2 pathway through inhibition of Keap1- and GSK3β-dependent Nrf2 degradation.

Inhibition of Histone Deacetylase 3 (HDAC3) Mediates Ischemic Preconditioning and Protects Cortical Neurons against Ischemia in Rats.

Brain ischemic preconditioning (PC) provides vital insights into the endogenous protection against stroke. Genomic and epigenetic responses to PC condition the brain into a state of ischemic tolerance. Notably, PC induces the elevation of histone acetylation, consistent with evidence that histone deacetylase (HDAC) inhibitors protect the brain from ischemic injury. However, less is known about the specific roles of HDACs in this process. HDAC3 has been implicated in several neurodegenerative conditions. Deletion of HDAC3 confers protection against neurotoxicity and neuronal injury. Here, we hypothesized that inhibition of HDAC3 may contribute to the neuronal survival elicited by PC. To address this notion, PC and transient middle cerebral artery occlusion (MCAO) were conducted in Sprague-Dawley rats. Additionally, primary cultured cortical neurons were used to identify the modulators and effectors of HDAC3 involved in PC. We found that nuclear localization of HDAC3 was significantly reduced following PC in vivo and in vitro. Treatment with the HDAC3-specific inhibitor, RGFP966, mimicked the neuroprotective effects of PC 24 h and 7 days after MCAO, causing a reduced infarct volume and less Fluoro-Jade C staining. Improved functional outcomes were observed in the neurological score and rotarod test. We further showed that attenuated recruitment of HDAC3 to promoter regions following PC potentiates transcriptional initiation of genes including Hspa1a, Bcl2l1, and Prdx2, which may underlie the mechanism of protection. In addition, PC-activated calpains were implicated in the cleavage of HDAC3. Pretreatment with calpeptin blockaded the attenuated nuclear distribution of HDAC3 and the protective effect of PC in vivo. Collectively, these results demonstrate that the inhibition of HDAC3 preconditions the brain against ischemic insults, indicating a new approach to evoke endogenous protection against stroke.

Experimental Brain Ischemic Preconditioning: A Concept to Putative Targets.

Neurodegeneration is the progressive loss of central neurons which may instigate many disabling psychological and neurological disorders like Alzheimer's disease, schizophrenia, parkinson's disease, prion's disease, and Huntington's disease. It has become imperative to address the need to discover novel molecular targets and therapeutic strategies to combat neurodegeneration. It is more essential to do so, because most of the accessible treatment focuses on correcting the symptoms of such diseases rather than its underlying pathophysiology. In the present article, we sought to discuss plausible connections between brain ischemic preconditioning and protective measures against chemical neurotoxicity. Brain ischemic preconditioning is reported to be effective against stroke like conditions and is studied chiefly to identify culpable molecular targets. Similarly chemical stressors are reported to be effective in preconditioning neuronal cells against chemical neurotoxicity. Keeping the concept of cross-tolerance in mind this article encompasses the putative targets of both chemical preconditioning and ischemic preconditioning in search for a suitable connection based on the published literature. The distinctive groups of targets are individually discussed and principal targets such as oncogene Akt, glycogen synthase kinase 3-beta and heat shock proteins are emphasised. Identification of these targets may help to develop sophisticated newer therapeutic strategies to cure neurodegenerative disorders.

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.

Endoplasmic reticulum chaperone GRP78 is involved in autophagy activation induced by ischemic preconditioning in neural cells.

BackgroundOur previous finding showed that brain ischemic preconditioning mediates neuroprotection through endoplasmic reticulum (ER) stress-induced autophagy. This study was aimed at exploring the role of ER chaperone GRP78 in IPC induced autophagy activation in neural cells.ResultsIschemic preconditioning (IPC) and oxygen glucose deprivation (OGD) models were established in rat pheochromocytoma (PC12) cells and primary cultured murine cortical neurons. IPC exerted neuroprotection against subsequent OGD injury in both PC12 cells and primary cortical neurons. IPC increased GRP78 expression and activated autophagy, as evidenced by upregulated LC3 and Beclin1, increased autophagic flux and formation of autophagosomes. BAPTA(dibromo-1,2-bis(aminophenoxy)ethane N,N,N9,N9 - tetra acetic acid, 0.125-2 μM) and small interfering RNA targeted GRP78 abrogated IPC induced neuroprotection and decreased the expression of GRP78, LC3II/LC3I and Beclin1. In contrast, lentiviral vector mediated GRP78 overexpression (LV-GRP78) strengthened resistance of PC12 cells to OGD injury and increased LC3 and Beclin1 expression. Moreover, knockdown of GRP78 in stable GRP78 overexpressing PC12 cells abolished the upregulation of LC3II/LC3I. GRP78 might activate autophagy through AMPK - mTOR pathway.ConclusionThese results suggest that IPC- induced GRP78 upregulation is involved in autophagy activation, and hence exerts protection against ischemic injury in neural cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-015-0112-3) contains supplementary material, which is available to authorized users.

Zinc preconditioning protects against neuronal apoptosis through the mitogen-activated protein kinase-mediated induction of heat shock protein 70

During brain ischemic preconditioning (PC), mild bursts of ischemia render neurons resistant to subsequent strong ischemic injuries. Previously, we reported that zinc plays a key role in PC-induced neuroprotection in vitro and in vivo. Zinc-triggered p75NTR induction transiently activates caspase-3, which cleaves poly(ADP-ribose) polymerase-1 (PARP-1). Subsequently, the PARP-1 over-activation-induced depletion of nicotinamide adenine dinucleotide (NAD+)/adenosine triphosphate (ATP) after exposures to lethal doses of zinc or N-methyl-d-aspartate is significantly attenuated in cortical neuronal cultures. In the present study, zinc-mediated preconditioning (Zn PC) reduced apoptotic neuronal death that was caused by N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), etoposide, or staurosporine in mouse cortical cells. We focused on heat shock protein 70 (HSP70) because NAD+/ATP depletion does not directly cause apoptosis, and HSP70 can inhibit the activation of caspase-9 or caspase-3 by preventing apoptosome formation or cytochrome C release. Zn PC-mediated HSP70 induction was required for neuroprotection against neuronal apoptosis, and geldanamycin-induced HSP70 induction sufficiently blocked neuronal apoptotic cell death. Furthermore, Zn PC-mediated HSP70 induction was blocked by chemical inhibitors of extracellular signal-regulated kinase (ERK) or p38 mitogen-activated protein kinase (MAPK) signaling, but not c-Jun N-terminal protein kinase. Similarly, neuroprotection by Zn PC against TPEN-induced apoptosis was almost completely reversed by the blockade of ERK or p38 MAPK signaling. Our findings suggest that the ERK- or p38 MAPK-mediated induction of HSP70 plays a key role in inhibiting caspase-3 activation during Zn PC.

The endoplasmic reticulum stress inhibitor salubrinal inhibits the activation of autophagy and neuroprotection induced by brain ischemic preconditioning

To investigate whether endoplasmic reticulum (ER) stress participates in the neuroprotective effects of ischemic preconditioning (IPC)-induced neuroprotection and autophagy activation in rat brains. The right middle cerebral artery in SD rats was occluded for 10 min to induce focal cerebral IPC, and was occluded permanently 24 h later to induce permanent focal ischemia (PFI). ER stress inhibitor salubrinal (SAL) was injected via intracerebral ventricle infusion 10 min before the onset of IPC. Infarct volume and motor behavior deficits were examined after the ischemic insult. The protein levels of LC3, p62, HSP70, glucose-regulated protein 78 (GRP 78), p-eIF2α and caspase-12 in the ipsilateral cortex were analyzed using immunoblotting. LC3 expression pattern in the sections of ipsilateral cortex was observed with immunofluorescence. Pretreatment with SAL (150 pmol) abolished the neuroprotective effects of IPC, as evidenced by the significant increases in mortality, infarct volume and motor deficits after PFI. At the molecular levels, pretreatment with SAL (150 pmol) significantly increased p-eIF2α level, and decreased GRP78 level after PFI, suggesting that SAL effectively inhibited ER stress in the cortex. Furthermore, the pretreatment with SAL blocked the IPC-induced upregulation of LC3-II and downregulation of p62 in the cortex, thus inhibiting the activation of autophagy. Moreover,SAL blocked the upregulation of HSP70, but significantly increased the cleaved caspase-12 level, thus promoting ER stress-dependent apoptotic signaling in the cortex. ER stress-induced autophagy might contribute to the neuroprotective effect of brain ischemic preconditioning.

Intracellular Signaling MAPK Pathway After Cerebral Ischemia–Reperfusion Injury

The MAPK/ERK/p38 are signal transduction pathways that couple intracellular responses to the external stimuli. Contrary to ERK protein which is part of the survival route, presence of p38 could have an impact on cell injury. Tolerance induced by ischemic preconditioning (IPC) is a phenomenon of tissue adaptation, which results in increased tolerance to lethal ischemia-reperfusion injury (IRI). Paper describes changes in MAPK protein pathways after brain IPC. Ischemia was induced by 4-vessels occlusion and rats were preconditioned by sub-lethal ischemia. Western blot and immunohistochemistry identified ERK/p38 proteins in injured areas. The highest level of the pERK was detected at 24 h in IPC groups. A contrary pattern of MAPK/p38 activation was observed in this group, where the lowest level of p38 was displayed at 24 h after ischemia. This suggests that the MAPK signal transduction might have a potential role in tissues response subjected to IRI and in the phenomenon of tolerance.

Mechanisms in Adaptation to Brain Ischemia by Ischemic Preconditioning

During the last few years, the mechanisms of adaptation to brain ischemic preconditioning (IPC) of cerebral ischemia/reperfusion have been studied and various molecules and a number of molecular regulation pathways were proposed to participate in IPC. Better understanding of mechanisms in IPC may provide novel therapeutic interventions for preventive treatment of cerebral ischemia. Since mitochondria are the most important intracellular energy source and are crucial for the cells' survival in the occurrence of ischemia, so more attention should be paid to the investigation of mitochondria related mechanisms of IPC. In this review, we will first survey the mitochondria related mechanisms including mitochondrial apoptotic pathway, uncoupling protein 2 (UCP2), mitochondria deprived reactive oxygen species (ROS), and mitochondrial ATPsensitive K(superscript +) channels (MitoKATP) in postischemic neuroprotection in brain. Then, to be more comprehensive, the mitochondria non-related mechanisms such as N-methyl-D-aspartate (NMDA) and Gamma-aminobutyric acid (GABA) receptors, adenosine, adenosine A1 receptor and K(superscript +) channels as well as nitric oxide will also be reviewed.

Ischemic preconditioning mediates cyclooxygenase-2 expression via nuclear factor-kappa B activation in mixed cortical neuronal cultures.

Nuclear factor-kappaB (NF-κB) activation occurs following ischemic preconditioning (IPC) in brain. However, the upstream signaling messengers and down-stream targets of NF-κB required for induction of IPC remain undefined. In a previous study, we demonstrated that epsilon protein kinase c (εPKC) was a key mediator of IPC in brain. Activation of εPKC induced cyclooygenase-2 (COX-2) expression and conferred ischemic tolerance in the neuronal and hippocampal slice models. Here, we hypothesized that IPC-mediated COX-2 expression was mediated by NF-κB. We tested this hypothesis in mixed cortical neuron/astrocyte cell cultures. To simulate IPC or ischemia, cell cultures were exposed to 1 or 4 h of oxygen-glucose deprivation, respectively. Our results demonstrated translocation of p65 and p50 subunits of NF-κB into nucleus following IPC or εPKC activation. NF-κB inhibition with pyrrolidine dithiocarbamate (10 μM) abolished IPC or εPKC activator-mediated neuroprotection indicating that NF-κB activation was involved in ischemic tolerance. In parallel studies, inhibition of either εPKC or the extracellular signal-regulated kinase (ERK 1/2) pathway reduced IPC-induced NF-κB activation. Finally, inhibition of NF-κB blocked IPC-induced COX-2 expression. In conclusion, we demonstrated that IPC-signaling cascade comprises εPKC activation→ERK1/2 activation→NF-κB translocation to nucleus→COX-2 expression resulting in neuroprotection in mixed neuronal culture.

Brain Ischemic Preconditioning Does Not Require PARP-1

Poly(ADP-ribose) polymerase-1 (PARP-1) is involved in ischemic preconditioning of the heart and cultured neurons, but its role in brain ischemic preconditioning is unknown. Summary of Report- We report that 5-minute bilateral common carotid artery occlusion (BCCAO) in the mouse prompted reduction of infarct volumes triggered 24 hours later by 20-minute middle cerebral artery occlusion (MCAO). Pharmacological PARP-1 inhibition between BCCAO and MCAO did not impair preconditioning. The contents of the PARP-1 substrate NAD, those of its product poly(ADP-ribose), caspase-3 activation, and PARP-1 expression did not change after BCCAO within the preconditioned tissue. PARP-1 KO mice were similarly protected by the 5-minute BCCAO. Data demonstrate that, at variance with the heart, PARP-1 is dispensable for brain ischemic preconditioning.

The neuroprotective mechanism of brain ischemic preconditioning

Brain ischemia is one of the most common causes of death and the leading cause of adult disability in the world. Brain ischemic preconditioning (BIP) refers to a transient, sublethal ischemia which results in tolerance to later, otherwise lethal, cerebral ischemia. Many attempts have been made to understand the molecular and cellular mechanisms underlying the neuroprotection offered by ischemic preconditioning. Many studies have shown that neuroprotective mechanisms may involve a series of molecular regulatory pathways including activation of the N-methyl-D-aspartate (NMDA) and adenosine receptors; activation of intracellular signaling pathways such as mitogen activated protein kinases (MAPK) and other protein kinases; upregulation of Bcl-2 and heat shock proteins (HSPs); and activation of the ubiquitin-proteasome pathway and the autophagic-lysosomal pathway. A better understanding of the processes that lead to cell death after stroke as well as of the endogenous neuroprotective mechanisms by which BIP protects against brain ischemic insults could help to develop new therapeutic strategies for this devastating neurological disease. The purpose of the present review is to summarize the neuroprotective mechanisms of BIP and to discuss the possibility of mimicking ischemic preconditioning as a new strategy for preventive treatment of ischemia.

Nitric Oxide Participates in the Induction of Brain Ischemic Tolerance via Activating ERK1/2 Signaling Pathways

The present study was undertaken to observe in vivo changes of expression and phosphorylation of ERK1/2 proteins during brain ischemic preconditioning and effects of inhibiting generation of nitric oxide (NO) on the changes to determine the role of ERKs in the involvement of NO participating in the acquired tolerance. Fifty-five Wistar rats were used. Brain ischemic preconditioning was performed with four-vessel occlusion for 3 min. Total ERK1/2 proteins and phospho-ERK1/2 in the CA1 hippocampus were assayed with Western immunoblot. Total ERK1/2 proteins did not change in period from 5 min to 5 days of reperfusion after preconditioning stimulus. While the level of phospho-ERK1/2 increased obviously to 223, 237, 300, 385 and 254% of sham level at times of 5 min, 2 h, 1, 3 and 5 days after preconditioning stimulus, respectively (P < 0.01). Administration of L-NAME, an inhibitor of NO synthase, 30 min prior to preconditioning stimulus failed to induce change in total ERK1/2 proteins (P > 0.05). However, phospho-ERK1/2 increased only to 138 and 176% of sham level at 2 h and 3 days after preconditioning stimulus, respectively, when animals were pretreated with L-NAME. The magnitudes of the increase were obviously low compared with those (237 and 385%) in animals untreated with L-NAME at corresponding time points (P < 0.01), which indicated that phosphorylation of ERK1/2 normally induced by preconditioning stimulus was blocked apparently by administration of L-NAME. The results suggested that phosphorylation of ERK1/2, rather than synthesis of ERK1/2 proteins, was promoted in brain ischemic preconditioning, and that the promotion was partly mediated by NO signal pathway.

Obligatory Role of Inducible Nitric Oxide Synthase in Ischemic Preconditioning

Sublethal insults can induce a transient tolerance toward subsequent lethal ischemia, a phenomenon termed ischemic preconditioning (IPC). In the myocardium, nitric oxide derived from 'inducible' nitric oxide synthase (iNOS or NOS II) plays a critical role in the expression of IPC produced by sublethal ischemia. Here, we investigated whether iNOS is involved in IPC in brain. Ischemic preconditioning was produced in mice by three episodes of 1-min bilateral common carotid artery (BCCA) occlusion, each followed by 5 mins of reperfusion. After 24 h, mice underwent middle cerebral artery (MCA) occlusion for 20 mins. Intraischemic cerebral blood flow was monitored during both in BCCA and MCA occlusion (MCAO) by laser-Doppler flowmetry. Mice were killed 3 days after MCAO, and infarct volume was determined in thionine-stained sections. Infarct volume was significantly reduced 24 h after IPC (70%; P<0.05). Treatment with the iNOS inhibitor aminoguanidine (400 mg/kg), abolished the IPC-induced protection. Furthermore, IPC failed to induce ischemic tolerance in iNOS-null mice. In wild-type mice, IPC increased the resistance to Ca(2+)-mediated depolarization in isolated brain mitochondria. However, in iNOS-null mice IPC failed to induce such resistance. We conclude that iNOS is required for the full expression of IPC and that such effect is coupled to an increased resistance of mitochondria to injury. Thus, iNOS-derived nitric oxide, in addition to its deleterious effects on the late stages of ischemic brain damage, can also be beneficial by promoting ischemic tolerance through signaling, ultimately resulting in mitochondrial protection.

TNFR1 Upregulation Mediates Tolerance after Brain Ischemic Preconditioning

A short ischemic event (ischemic preconditioning (IPC)) can result in subsequent resistance to severe ischemic injury (ischemic tolerance (IT)). The expression and neuroprotective role of tumor necrosis factor (TNF-alpha) have been described in models of IPC and we have showed the participation of its processing enzyme, the TNF-alpha convertase enzyme (TACE) in this process. We have now decided to explore the expression and localization of TNF receptors (TNFR) as well as other signalling mechanisms involved in IT. A period of 10 mins of temporary middle cerebral artery occlusion (tMCAO) was used for focal IPC. To evaluate the ability of IPC to produce IT, permanent MCAO was performed 48 hours after IPC. Ischemic preconditioning produced a reduction in infarct volume, as we showed previously. Ischemic preconditioning caused upregulation of neuronal TNFR1 that was reduced by the selective TACE inhibitor BB1101. Intracerebral administration of TNFR1 antisense oligodeoxynucleotide, which caused a reduction in TNFR1 expression, inhibited the IPC-induced protective effect, showing that TNFR1 upregulation is implicated in IT. Moreover, treatment with BB1101, TNFR1 antisense and lactacystin-a specific proteasome inhibitor-blocked IPC-induced NF-kappaB. Immunohistochemical studies showed the expression of TACE and TNFR1 in neurons. In summary, these data show that IPC produces neuronal upregulation of TACE and TNFR1, and that the pathway TACE/TNF-alpha/TNFR1/NF-kappaB is involved in IT.

Brain ischemic preconditioning is abolished by antioxidant drugs but does not up-regulate superoxide dismutase and glutathion peroxidase

The present work examined the hypothesis that brain ischemic tolerance induced by ischemic preconditioning (IPC) is triggered by an initial oxidative stress and is associated with an increase in antioxidant enzyme activities as one end-effector of the neuroprotection. Wistar rats were preconditioned by a single 3-min occlusion of the middle cerebral artery. After a various duration of reperfusion (30 min, 24, 72 or 168 h), rats were subjected to a 60-min focal ischemia and sacrificed 24 h later. Cerebral infarcts were significantly reduced when performed during the 24- to 72-h time window after IPC. The pretreatment with the protein synthesis inhibitor, cycloheximide (1 mg/kg, i.p., 30 min prior to IPC), completely suppressed the neuroprotection. The free radical scavenger, dimethylthiourea (DMTU; 300 mg/kg, i.p., 30 min prior to IPC) and the antioxidant ebselen (10 mg/kg, oral cramming, 2 h before and 12 h after IPC) also abolished the IPC-induced protection of the brain. Nevertheless, IPC did not induce any delayed changes in antioxidant enzyme (superoxide dismutase, glutathion peroxidase) activities nor in the neuronal expression of Mn and Cu/Zn superoxide dismutase. These results indicate that an initial oxidative stress could be involved as a trigger of IPC, while antioxidant enzymes do not play a key role as end-effectors in such a neuroprotection.

Brain ischemia alters platelet ATP diphosphohydrolase and 5'-nucleotidase activities in naive and preconditioned rats.

The effects of transient forebrain ischemia, reperfusion and ischemic preconditioning on rat blood platelet ATP diphosphohydrolase and 5'-nucleotidase activities were evaluated. Adult Wistar rats were submitted to 2 or 10 min of single ischemic episodes, or to 10 min of ischemia 1 day after a 2-min ischemic episode (ischemic preconditioning) by the four-vessel occlusion method. Rats submitted to single ischemic insults were reperfused for 60 min and for 1, 2, 5, 10 and 30 days after ischemia; preconditioned rats were reperfused for 60 min 1 and 2 days after the long ischemic episode. Brain ischemia (2 or 10 min) inhibited ATP and ADP hydrolysis by platelet ATP diphosphohydrolase. On the other hand, AMP hydrolysis by 5'-nucleotidase was increased after 2, but not 10, min of ischemia. Ischemic preconditioning followed by 10 min of ischemia caused activation of both enzymes. Variable periods of reperfusion distinctly affected each experimental group. Enzyme activities returned to control levels in the 2-min group. However, the decrease in ATP diphosphohydrolase activity was maintained up to 30 days of reperfusion after 10-min ischemia. 5'-Nucleotidase activity was decreased 60 min and 1 day following 10-min ischemia; interestingly, enzymatic activity was increased after 2 and 5 days of reperfusion, and returned to control levels after 10 days. Ischemic preconditioning cancelled the effects of 10-min ischemia on the enzymatic activities. These results indicate that brain ischemia and ischemic preconditioning induce peripheral effects on ecto-enzymes from rat platelets involved in nucleotide metabolism. Thus, ATP, ADP and AMP degradation and probably the generation of adenosine in the circulation may be altered, leading to regulation of microthrombus formation since ADP aggregates platelets and adenosine is an inhibitor of platelet aggregation.