Sublethal Ischemic Insult Research Articles

Astrocyte-mediated ischemic tolerance.

Preconditioning (PC) using a preceding sublethal ischemic insult is an attractive strategy for protecting neurons by inducing ischemic tolerance in the brain. Although the underlying molecular mechanisms have been extensively studied, almost all studies have focused on neurons. Here, using a middle cerebral artery occlusion model in mice, we show that astrocytes play an essential role in the induction of brain ischemic tolerance. PC caused activation of glial cells without producing any noticeable brain damage. The spatiotemporal pattern of astrocytic, but not microglial, activation correlated well with that of ischemic tolerance. Interestingly, such activation in astrocytes lasted at least 8 weeks. Importantly, inhibiting astrocytes with fluorocitrate abolished the induction of ischemic tolerance. To investigate the underlying mechanisms, we focused on the P2X7 receptor as a key molecule in astrocyte-mediated ischemic tolerance. P2X7 receptors were dramatically upregulated in activated astrocytes. PC-induced ischemic tolerance was abolished in P2X7 receptor knock-out mice. Moreover, our results suggest that hypoxia-inducible factor-1α, a well known mediator of ischemic tolerance, is involved in P2X7 receptor-mediated ischemic tolerance. Unlike previous reports focusing on neuron-based mechanisms, our results show that astrocytes play indispensable roles in inducing ischemic tolerance, and that upregulation of P2X7 receptors in astrocytes is essential.

Tanshinone IIA dampens the cell proliferation induced by ischemic insult in rat astrocytes via blocking the activation of HIF-1α/SDF-1 signaling

AimsTanshinone IIA (TSA) has been reported to protect neurons and microvascular endothelial cells against ischemic injury. However, the effect of TSA on astrocytes in response to ischemic injury is not clear. The aims of this study were to evaluate the effect of TSA on astrocytes following ischemic insult. Main methodsPrimary cultured rat astrocytes were treated with oxygen–glucose deprivation (OGD) followed by recovery. Cell death was measured by lactate dehydrogenase assay. Astrocyte proliferation was determined by cell viability assay, cell counting and 5-bromo-2-deoxyuridine incorporation assay. The glial fibrillary acidic protein (GFAP) expression was assessed by immunocytochemistry. Stromal cell-derived factor-1 (SDF-1) secretion from astrocytes was measured by enzyme linked immunosorbent assay. The expressions of hypoxia inducible factor-1α (HIF-1α), SDF-1, GFAP and the phosphorylation of Akt and extracellular regulated protein kinase (Erk) 1/2 were evaluated by immunoblot assay. Quantitative RT-PCR was used to assess the mRNA expression of HIF-1α and SDF-1. Key findingsMild OGD (2h-OGD) did not induce astrocyte injury but proliferation at 48 and 72h after recovery. Mild OGD also induced the accumulation of HIF-1α, the subsequent expression and secretion of SDF-1, resulting in the phosphorylation of Erk1/2 and Akt. TSA attenuated astrocyte proliferation and significantly decreased the OGD-induced accumulation of HIF-1α and SDF-1, then blocked the downstream signaling, Erk1/2 and Akt activation. SignificanceTSA inhibits the astrocyte proliferation induced by sub-lethal ischemic insult via blocking the activation of HIF-1α/SDF-1 pathway. This finding suggests that TSA may be a potential therapeutic option for gliosis after cerebral ischemia.

Delayed Remote Ischemic Postconditioning Improves Long Term Sensory Motor Deficits in a Neonatal Hypoxic Ischemic Rat Model

ObjectiveRemote Ischemic Postconditioning (RIPC) is a promising therapeutic intervention wherein a sub-lethal ischemic insult induced in one organ (limb) improves ischemia in an organ distant to it (brain). The main objective of this study was to investigate the long-term functional effects of delayed RIPC in a neonatal hypoxia-ischemia (HI) rat model.Method10 day old rat pups were subjected to delayed RIPC treatment and randomized into four groups: 1) Sham, 2) HI induced, 3) HI +24 hr delayed RIPC, and 4) HI +24 hr delayed RIPC with three consecutive daily treatments. Neurobehavioral tests, brain weights, gross and microscopic brain tissue morphologies, and systemic organ weights were evaluated at five weeks post surgery.ResultsHI induced rats performed significantly worse than sham but both groups of delayed RIPC treatment showed improvement of sensory motor functions. Furthermore, compared to the HI induced group, the delayed RIPC treatment groups showed no further detrimental changes on brain tissue, both grossly and morphologically, and no changes on the systemic organ weights.ConclusionDelayed RIPC significantly improves long term sensory motor deficits in a neonatal HI rat model. A 24 hr delayed treatment does not significantly attenuate morphological brain injury but does attenuate sensory motor deficits. Sensory motor deficits improve with both a single treatment and with three consecutive daily treatments, and the consecutive treatments are possibly being more beneficial.

NNOS and p-ERK involvement in the neuroprotection exerted by remote postconditioning in rats subjected to transient middle cerebral artery occlusion

It has recently been hypothesized that a sub-lethal ischemic insult induced in one organ is able to protect from a harmful ischemia occurring in a different organ. The objective of this study is to identify new putative mechanisms of neuroprotection elicited by remote ischemic femoral postconditioning. A 50% reduction in the infarct volume was observed when 100min of middle cerebral artery occlusion was followed, 10min later, by the remote postconditioning stimulus represented by 20min of femoral artery occlusion. The use of in vivo silencing strategy allowed to demonstrate that NO production through nNOS mediates part of the neuroprotection. Indeed, whereas CNS nNOS expression was up-regulated by remote postconditioning, the pharmacological inhibition of nNOS or its silencing-mediated knocking-down partially prevented this neuroprotective effect. This nNOS overexpression seemed to be p-ERK dependent. In fact, p-ERK expression increased in brain cortex after remote postconditioning, and its pharmacological inhibition prevented both nNOS overexpression and remote postconditioning-mediated neuroprotection. Interestingly, neuroprotection induced by remote postconditioning was partially prevented when ganglion transmission was pharmacologically interrupted by hexamethonium, thus showing that neural factors are involved in this phenomenon.Collectively, the present study demonstrates that p-ERK and nNOS take part to the complex cascade of events triggered by ischemic remote postconditioning.

Abstract 95: Remote Postconditioning Protects Ischemic Brain by nNOS and p-ERK Activation

Objective: It has recently been hypothesized that a sub-lethal ischemic insult induced in one organ is able to protect from a harmful ischemia occurring in a different and anatomically distant organ. Objective of this study is to characterize remote ischemic femoral postconditioning as a new method to induce post-ischemic brain neuroprotection Methods and Results: Remote ischemic postconditioning was induced in adult rats by 100 minutes of middle cerebral artery occlusion (tMCAO) followed by several cycles of femoral artery occlusion-reperfusion. One hundred minutes of tMCAO followed, 10 minutes later, by 20 minutes of femoral artery occlusion effectively reduced cerebral infarct volume by 50%.Importantly, this protection was still present 7 days after remote postconditioning. Furthermore, the use of in vivo silencing strategy allowed to demonstrate that NO production through nNOS mediates part of the neuroprotection induced by remote ischemic postconditioning. Indeed, whereas CNS nNOS expression was up-regulated by remote postconditioning, the pharmacological inhibition of nNOS or its silencing-mediated knocking-down partially reverted this neuroprotective effect. This nNOS overexpression and activity seemed to be p-ERK dependent. In fact, p-ERK expression increased in brain cortex after remote postconditioning, and its pharmacological inhibition prevented both nNOS overexpression and remote postconditioning-mediated neuroprotection. Interestingly, neuroprotection induced by remote postconditioning was partially reverted when ganglion transmission was pharmacologically interrupted by hexamethonium, thus showing that neural factors are involved in this phenomenon. Interpretation: Collectively, the results of the present study demonstrate that remote postconditioning induces a marked neuroprotection through p-ERK/nNOS, neuronally-mediated activation, and may represent a new clinically feasible therapeutic approach to treat ischemic stroke.

Neurogenic pathway mediated remote preconditioning protects the brain from transient focal ischemic injury

Attenuation of ischemic injury can be achieved by priming the brain with a sub-lethal ischemic insult, a phenomenon known as ischemic preconditioning (IP). We sought to determine if subjecting a distant organ, such as the lower limb, to a similar priming ischemic insult would result in protection of the brain from a subsequent severe ischemic injury, as induced by middle cerebral artery occlusion (MCAo) and if this protection is mediated via a neurogenic pathway. Adult Wistar male rats were subjected to either remote preconditioning (RPC) or sham surgery and then subsequently underwent 2 h MCAo 24, 48 or 72 h after the RPC/sham RPC stimulus. Of the animals undergoing RPC, only those that sustained MCAo 24 h later showed significantly smaller cerebral infarct volumes (150.34 ± 30.91 mm 3) and better clinical neurological outcomes (1.15 ± 0.69) as compared to the sham RPC group (infarct volume 250.25 ± 26.98mm 3; neurological score 1.80 ± 0.87) ( P < 0.05). RPC animals sustaining MCAo at 48 and 72 h later did not show significant differences in cerebral infarct volumes or clinical neurological outcomes as compared to the sham RPC group. Furthermore, attenuation of the neuroprotective effect by the ganglion blocker hexamethonium suggested a neurogenically mediated pathway responsible for this phenomenon. Remote sub-lethal ischemic injury to both lower limbs results in cerebral protection from subsequent ischemia within 24 h of initiation of the RPC stimulus and this protection in part may be mediated via a neurogenic pathway.

SIK2 Is a Key Regulator for Neuronal Survival after Ischemia via TORC1-CREB

The cAMP responsive element-binding protein (CREB) functions in a broad array of biological and pathophysiological processes. We found that salt-inducible kinase 2 (SIK2) was abundantly expressed in neurons and suppressed CREB-mediated gene expression after oxygen-glucose deprivation (OGD). OGD induced the degradation of SIK2 protein concomitantly with the dephosphorylation of the CREB-specific coactivator transducer of regulated CREB activity 1 (TORC1), resulting in the activation of CREB and its downstream gene targets. Ca(2+)/calmodulin-dependent protein kinase I/IV are capable of phosphorylating SIK2 at Thr484, resulting in SIK2 degradation in cortical neurons. Neuronal survival after OGD was significantly increased in neurons isolated from sik2(-/-) mice, and ischemic neuronal injury was significantly reduced in the brains of sik2(-)(/-) mice subjected to transient focal ischemia. These findings suggest that SIK2 plays critical roles in neuronal survival, is modulated by CaMK I/IV, and regulates CREB via TORC1.

Genomic analysis of ischemic preconditioning in adult rat hippocampal slice cultures

Understanding endogenous mechanisms of neuroprotection may have important clinical applications. It is well established that brain tissue becomes more resistant to ischemic injury following a sublethal ischemic insult. This process, called ischemic preconditioning (IPC), can be induced in adult rat hippocampal slice cultures by a brief oxygen–glucose deprivation (OGD) [Hassen, G.W., Tian, D., Ding, D., Bergold, P.J., 2004. A new model of ischemic preconditioning using young adult hippocampal slice cultures. Brain Res. Brain Res. Protoc. 13, 135–143]. We have analyzed the changes in gene expression brought about by IPC in this model in order to understand the mechanisms involved. Total RNA was isolated at different time points following a brief OGD (3, 6 and 12 h) and used to probe genome-wide expression microarrays. Genes were identified that were significantly up- or down-regulated relative to controls. We placed genes that were differentially expressed into statistically significant groups based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and gene ontology (GO) terms. Genes involved in signal transduction, transcription, and oxidative phosphorylation are differentially expressed at each time point. The analysis demonstrates that alterations in signaling pathways (TGF-β, Wnt, MAPK, ErbB, Toll-like receptor, JAK-STAT, VEGF) consistently accompany IPC. RT-PCR was used to confirm that members of these signaling pathways are regulated as predicted by the microarray analysis. We verified that protein translation following OGD is necessary for IPC. We also found that blocking the NMDA receptor during OGD does not significantly inhibit IPC in this model or produce large changes in gene expression. Our data thus suggests that changes in signaling pathways and their down-stream targets play an important role in triggering endogenous neuroprotection.

Cell division in the cerebral cortex of adult rats after photothrombotic ring stroke

Neurogenesis has been shown to occur in the cerebral cortex in adult rats after ischemic stroke. The origin of the newborn neurons is largely unknown. This study aimed to explore cell division in the poststroke penumbral cortex. Adult male Wistar rats were subjected to photothrombotic ring stroke. After repeated delivery of the DNA duplication marker BrdU, the animals were sacrificed at various times poststroke. BrdU was detected by immunohistochemistry/immunofluorescence labeling, as was the M-phase marker Phos H3 and the spindle components α-tubulin/γ-tubulin. DNA damage was examined by TUNEL staining. Cell type was ascertained by double immunolabeling with the neuronal markers Map-2ab/β-tubulin III and NeuN/Hu or the astrocyte marker GFAP. From 16h poststroke, BrdU-immunolabeled cells appeared in the penumbral cortex. From 24h, Phos H3 was colocalized with BrdU in the nuclei. Mitotic spindles immunolabeled by α-tubulin/γ-tubulin appeared inside the cortical cells containing BrdU-immunopositive nuclei. Unexpectedly, the markers of neuronal differentiation, Map-2ab/β-tubulin III/NeuN/Hu, were expressed in the Phos H3-immunolabeled cells, and NeuN was detected in some cells containing spindles. This study suggests that in response to a sublethal ischemic insult, endogenous cells with neuronal immunolabeling may duplicate their nuclear DNA and commit cell mitosis to generate daughter neurons in the penumbral cortex in adult rats.

Protein kinase M zeta regulation of Na/K ATPase: A persistent neuroprotective mechanism of ischemic preconditioning in hippocampal slice cultures

In ischemic preconditioning, a sublethal ischemic insult protects neurons from subsequent ischemia. In organotypic hippocampal slice cultures a sublethal 5-minute hypoxia–hypoglycemia treatment prevented neuronal loss after a 10-minute experimental ischemic (EI) treatment of hypoxia–hypoglycemia. Whereas preconditioning protected against EI given 24 h later, it did not protect when EI was given 2 h later, suggesting a slow development of neuroprotection. This model identified two regulators of ischemic preconditioning: the atypical protein kinase M zeta (PKMζ), and the Na/K ATPase. Two hours following preconditioning, when there was no neuroprotection, Na/K ATPase activity was unchanged. In contrast, Na/K ATPase activity significantly increased 24 h after the preconditioning treatment. Elevated Na/K ATPase activity was accompanied by increased surface expression of the α1 and α2 isoforms of the Na/K ATPase. Similarly, active PKMζ levels were increased at 24 h, but not 2 h, after preconditioning. PKMζ overexpression by sindbis virus vectors also increased Na/K ATPase activity. To examine PKMζ regulation of Na/K ATPase, occlusion experiments were performed using marinobufagenin to inhibit α1, dihydroouabain to inhibit α2/3 and a ζ-pseudosubstrate peptide to inhibit PKMζ. These experiments showed that PKMζ regulated both the activity and surface expression of the α1 isoform of the Na/K ATPase. Marinobufagenin, dihydroouabain, and ζ-pseudosubstrate peptide were used to determine if PKMζ or the α1 and α2 Na/K ATPase isoforms protected neurons. All three compounds blocked neuroprotection following ischemic preconditioning. PKMζ levels were elevated 3 days after ischemic preconditioning. These data indicate key roles of PKMζ and Na/K ATPase in ischemic preconditioning.

Pathway and gene ontology based analysis of gene expression in a rat model of cerebral ischemic tolerance

Ischemic tolerance is a phenomenon whereby a sublethal ischemic insult [ischemic preconditioning (IPC)] provides robust protection against subsequent lethal ischemia. Activation of N-methyl- d-aspartate (NMDA) receptors and subsequent new gene transcription are required for tolerance. We utilized the NMDA antagonist, MK801, prior to the IPC stimulus to separate candidate genes from epiphenomenona. Rats were divided into four groups: vehicle/IPC (preconditioned), MK801/IPC (attenuated preconditioning), vehicle/sham (non-preconditioned), and MK801/sham (non-preconditioned). Hippocampi (5/group/time point) were harvested immediately after ischemia as well as 1, 4, and 24 h post-ischemia to profile gene expression patterns using microarray analyses. Extracted mRNAs were pooled and subsequently hybridized to Affymetrix arrays. In addition, groups of rats were sacrificed for Western blot analysis and histological studies. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and gene ontology (GO) analyses were used to identify functionally related groups of genes whose modulation was statistically significant, while hierarchical cluster analysis was used to visualize the fold expression within these groups. Significantly modulated pathways included: MAP kinase signaling pathway, Toll receptor pathway, TGF-β signaling pathways, and pathways associated with ribosome function and oxidative phosphorylation. Our data suggest that the tolerant brain responds to subsequent ischemic stress by partially downregulating inflammatory and upregulating protein synthesis and energy metabolism pathways.

Ischemic preconditioning negatively regulates plenty of SH3s–mixed lineage kinase 3–Rac1 complex and c-Jun N-terminal kinase 3 signaling via activation of Akt

Activation of Akt/protein kinase B has been recently reported to play an important role in ischemic tolerance. We here demonstrate that the decreased protein expression and phosphorylation of phosphatase and tensin homolog deleted from chromosome 10 (PTEN) underlie the increased Akt-Ser-473 phosphorylation in the hippocampal CA1 subfield in ischemic preconditioning (IPC). Co-immunoprecipitation analysis reveals that Akt physically interacts with Rac1, a small Rho family GTPase required for mixed lineage kinase 3 (MLK3) autophosphorylation, and both this interaction and Rac1-Ser-71 phosphorylation induced by Akt are promoted in preconditioned rats. In addition, we show that Akt activation results in the disassembly of the plenty of SH3s (POSH)–MLK3–Rac1 signaling complex and down-regulation of the activation of MLK3/c-Jun N-terminal kinase (JNK) pathway. Akt activation results in decreased serine phosphorylation of 14-3-3, a cytoplasmic anchor of Bax, and prevents ischemia-induced mitochondrial translocation of Bax, release of cytochrome c, and activation of caspase-3. The expression of Fas ligand is also decreased in the CA1 region. Akt activation protects against apoptotic neuronal death as shown in TUNEL staining following IPC. Intracerebral infusion of LY294002 before IPC reverses the increase in Akt phosphorylation and the decrease in JNK signaling activation, as well as the neuroprotective action of IPC. Our results suggest that activation of pro-apoptotic MLK3/JNK3 cascade can be suppressed through activating anti-apoptotic phosphoinositide 3-kinase/Akt pathway induced by a sublethal ischemic insult, which provides a functional link between Akt and the JNK family of stress-activated kinases in ischemic tolerance.

Role of Reactive Oxygen Species and Protein Kinase C in Ischemic Tolerance in the Brain

It is now understood that the mechanisms leading to neuronal cell death after cerebral ischemia are highly complex. A well established fact in this field is that neurons continue to die over days and months after ischemia, and that reperfusion following cerebral ischemia contributes substantially to ischemic injury. It is now well accepted that central to ischemic/reperfusion-induced injury is what occurs to mitochondria hours to days following the ischemic insult. For many years, it has been established that reactive oxygen species (ROS) and reactive nitrogen species (RNS) promote lipid, protein, and DNA oxidation that affects normal cell physiology and eventually leads to neuronal demise. In addition to oxidation of neuronal molecules by ROS and RNS, a novel pathway for molecular modifications has risen from the concept that ROS can activate specific signal transduction pathways that, depending on the insult degree, can lead to either normal plasticity or pathology. Two examples of these pathways could explain why lethal ischemic insults lead to the translocation of protein kinase Cdelta (deltaPKC), which plays a role in apoptosis after cerebral ischemia, or why sublethal ischemic insults, such as in ischemic preconditioning, lead to the translocation of epsilonPKC, which plays a pivotal role in neuroprotection. A better understanding of the mechanisms by which ROS and/or RNS modulate key protein kinases that are involved in signaling pathways that lead to cell death and survival after cerebral ischemia will help devise novel therapeutic strategies.

Ischemic tolerance induction in organotypic hippocampal slices: Role for the GABA A receptor?

Ischemic preconditioning (IPC) refers to sublethal ischemic insults rendering brain tissue tolerant against subsequent ischemic insults. We investigated the role of the GABA A receptor (GABA AR) upon IPC induction. Rat organotypic hippocampal slices were subjected to IPC by 15 min of oxygen–glucose deprivation (OGD) followed by 40 min of OGD 48 h later, resulting in robust cell death reduction as assessed by the propidium iodide fluorescence method (‘late’ or ‘second window’ IPC). Superfusion with the GABA AR antagonist bicuculline during IPC ameliorated propidium iodide uptake at a high but not at low doses indicating that GABA AR activation may be assigned a limited role in neuroprotection. In previous studies, we found that increased neuronal excitability can promote IPC neuroprotection. We, therefore, tested the hypothesis that blockade of inhibitory GABAergic transmission conferred ischemic tolerance. However, temporary administration of bicuculline 48 h prior to ischemic challenge was not neuroprotective. In another approach, we tested whether preconditioning with the GABA AR agonist, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) mediated ischemic tolerance and found no significant neuroprotection. The results are discussed in light of the intrinsic excitatory-inhibitory balance of glutamate and GABA.

Delayed adaptation of the heart to stress: late preconditioning.

The early phase of preconditioning (PC) lasts 2 to 3 hours and protects against myocardial infarction, but not against stunning. In contrast, the late phase of PC lasts for 3 to 4 days and protects against both myocardial stunning and infarction, making this phenomenon more clinically relevant. Late PC is a genetic reprogramming of the heart that involves the activation of several stress-responsive genes, which ultimately results in the development of a cardioprotective phenotype. Sublethal ischemic insults release chemical signals (nitric oxide [NO], adenosine, and reactive oxygen species) that trigger a series of signaling events (eg, activation of protein kinase C, Src protein tyrosine kinases, Janus kinases 1/2, and nuclear factor-kappaB) and culminates in increased synthesis of inducible NO synthase, cyclooxygenase-2, heme oxygenase-1, aldose reductase, Mn superoxide dismutase, and probably other cardioprotective proteins. In addition to ischemia, heat stress, exercise, and cytokines can also induce a similar series of events. Perhaps most importantly, many pharmacologic agents (eg, NO donors, adenosine receptor agonists, endotoxin derivatives, or opioid receptor agonists) can mimic the effects of ischemia in inducing the late phase of PC, suggesting that this phenomenon might be exploited therapeutically. The purpose of this review is to summarize the mechanisms that underlie the late phase of ischemic PC.

Transforming growth factor-beta 1-mediated neuroprotection against excitotoxic injury in vivo.

Ischemic preconditioning is a phenomenon that describes how a sublethal ischemic insult can induce tolerance to subsequent ischemia. This phenomenon has been observed after focal or global ischemia in different animal models. However, the hypothesis that bacterial infection might lead to neuronal tolerance to injury has not been investigated. To mimic cerebral bacterial infection, we injected bacterial lipopolysaccharide (LPS) in the right dorsal hippocampus, followed 24 hours later by an excitotoxic lesion using kainic acid in the mouse model. Quantification of lesion size after cresyl violet counterstaining revealed that LPS pretreatment afforded neuroprotection to CA3 neurons against KA challenge. To investigate the events underlying this protection, we studied the cytokine profile induced after LPS injection. Interleukin (IL)-1 beta and transforming growth factor beta 1 (TGF-beta 1) were the main cytokines expressed at 24 hours after LPS injection. Because IL-1 beta has been described as deleterious in acute injury, we decided to investigate the function of TGF-beta 1. An adenovirus expressing a constitutively active form of TGF-beta 1 was injected intracerebrally 1 week before the induction of excitotoxic lesion, and neuronal protection was observed. To confirm the neuroprotective role of TGF-beta 1, the TGF-beta 1 adenovirus was replaced by recombinant human TGF-beta 1 protein and total neuroprotection was observed. Furthermore, the antibody-mediated blocking of TGF-beta 1 action prevented the protective effect of pretreatment with LPS. We have demonstrated in vivo that the cerebral tolerance phenomenon induced by LPS pretreatment is mediated by TGF-beta 1 cytokine.

Effect of the first window of ischemic preconditioning on mitochondrial dysfunction following global cerebral ischemia

Rats may develop sustained tolerance against lethal cerebral ischemia after exposure to a sublethal ischemic insult (ischemic preconditioning (IPC)). Two windows for the induction of tolerance by IPC have been proposed, one that occurs within 1 h following IPC, and the other one that occurs 1–3 days after IPC. An important difference between these two windows is that in contrast to the second window, neuroprotection against lethal ischemia is transient in the first window. We tested the hypothesis that rapid IPC would reduce or prevent ischemia-induced changes in mitochondrial function. IPC and ischemia were produced by bilateral carotid occlusions and systemic hypotension (50 mmHg) for 2 and 10 min, respectively. The non-synaptosomal mitochondria were harvested 30 min following the 10 min ‘test’ ischemia. Mitochondrial rate of respiration decreased by 10% when the substrates were pyruvate and malate, and 29% when the substrates were ascorbic acid and N, N, N′, N′-tetramethyl- p-phenylenediamine ( P<0.01). The activities of complex I–III decreased in ischemic group by 16, 23 ( P<0.05) and 24%, respectively. IPC was unable to prevent decreases in the rate of respiration and activities of different complexes. These data suggest that rapidly induced IPC is unable to protect the integrity of mitochondrial oxidative phosphorylation following cerebral ischemia, perhaps explaining why IPC only provides transitory protection in the ‘first window’.

Improvement in neuronal survival after ischemic preconditioning in hippocampal slice cultures

The main goals of the current study were to assess: (a) whether a sublethal ischemic insult could protect the CA1 subregion of the hippocampus in organotypic slices against a lethal ischemic insult; and (b) whether this protection is long lasting as determined with an accurate immunohistochemical neuronal marker, NeuN. Hippocampal slice cultures were grown for 12–14 days in vitro. Slices were exposed either to oxygen/glucose deprivation (OGD) for 45 min (ischemia), or OGD for 15 min (ischemic preconditioning), 48 h prior to 45 min OGD, or were untreated (sham). Cell death was estimated by propidium iodide fluorescence 1 day after OGD and by NeuN immunohistochemistry 7 days after OGD. Image analysis was employed to measure the relative optical density of the NeuN-signal in all groups. After ischemia, damaged neurons were shrunken or lost and NeuN immunoreactivity was reduced. Relative optical density of NeuN (ROD [NeuN]) was 0.193±0.015 in control (sham) ( n=9). In slices that underwent ischemia, ROD [NeuN] declined to 0.108±0.018 ( n=5) in CA1 (* P<0.05 ROD [NeuN] in preconditioned slice cultures was 0.190±0.037 (76% higher than the ischemia group). Similar results were found after measuring PI fluorescence. In the CA1 sub-region, PI fluorescence was about 13, 47 and 17% in the sham, ischemic and IPC groups, respectively. We suggest that the immunohistochemical approach validates the dye uptake method used in slice cultures and yields quantitative data specific for neurons. We also conclude that the organotypic hippocampal slice model is useful for studying delayed ischemic preconditioning that is maintained for hours or days after the preconditioning event.

Effect of ischemic preconditioning on the expression of putative neuroprotective genes in the rat brain

Previous studies have demonstrated that sublethal ischemic insults protect from subsequent ischemia in the intact brain. There are two windows for the induction of tolerance by ischemic preconditioning (IPC). One occurs within 1 h following IPC, and the other one develops from 1 to 3 days after IPC. The goal of this study was to determine whether IPC neuroprotection may be mediated by expression of known neuroprotective genes and to characterize the temporal and spatial expression patterns of these genes. IPC was produced by bilateral carotid artery occlusions and hypotension (50 mmHg) for 2 min. After various survival times, the expression of MAP-2, brain-derived neurotrophic factor (BDNF), c-jun, c-fos, nerve growth factor (NGF) and HSP70 was assessed by in situ hybridization of coronal brain sections with 35S labeled probes. BDNF, NGF, and c-jun were significantly upregulated in the hippocampus. c-fos was detected in the hippocampus, cortex and striatum. HSP70 mRNA was induced in the cortex, hippocampus, striatum, and thalamus. MAP-2 showed no change in expression, confirming previous studies that no cell death occurs following IPC. The increase in expression of these stress-related, neurotrophic and immediate early genes in response to a mild preconditioning insult may help mediate the protection of vulnerable neurons to subsequent lethal ischemic insults.

Ischemic preconditioning preserves mitochondrial function after global cerebral ischemia in rat hippocampus.

Ischemic tolerance in brain develops when sublethal ischemic insults occur before "lethal" cerebral ischemia. Two windows for the induction of tolerance by ischemic preconditioning (IPC) have been proposed: one that occurs within 1 hour after IPC, and another that occurs 1 or 2 days after IPC. The authors tested the hypotheses that IPC would reduce or prevent ischemia-induced mitochondrial dysfunction. IPC and ischemia were produced by bilateral carotid occlusions and systemic hypotension (50 mm Hg) for 2 and 10 minutes, respectively. Nonsynaptosomal mitochondria were harvested 24 hours after the 10-minute "test" ischemic insult. No significant changes were observed in the oxygen consumption rates and activities for hippocampal mitochondrial complexes I to IV between the IPC and sham groups. Twenty-four hours of reperfusion after 10 minutes of global ischemia (without IPC) promoted significant decreases in the oxygen consumption rates in presence of substrates for complexes I and II compared with the IPC and sham groups. These data suggest that IPC protects the integrity of mitochondrial oxidative phosphorylation after cerebral ischemia.