Preconditioning-induced activation of ERK5 is dependent on moderate Ca2+ influx via NMDA receptors and contributes to ischemic tolerance in the hippocampal CA1 region of rats

Accumulating evidence implicates activation (phosphorylation) of mitogen-activated protein kinases (MAPK) during nonlethal ischemic preconditioning in the protection of hippocampal CA1 neuron against subsequent ischemic events. In this paper, we undertook to identify the role of extracellular signal regulated kinase (ERK) 5 in cerebral ischemic preconditioning (CIP). Three minutes of ischemia was induced as preconditioning stimulus. Three days later, 6 min of ischemia was induced. The levels of ERK5 protein expression and its activation were detected with or without the CIP in hippocampal CA1 and the dentate gyrus (DG) regions. Our results showed that ERK5 was activated selectively in hippocampal CA1 region with, but not without, the ischemic preconditioning. Notably, during the later phase of reperfusion, the rise in ERK5 activation was strong and persistent with a peak occurring at the third day. The activation peak was effectively prevented and ERK5 protein expression was significantly decreased by intracerebroventricular infusion of ERK5 antisense oligonucleotide (every 24 h for 3 days before the preconditioning), but not by sense oligonucleotide or vehicle. Subsequently, the CA1 neuronal loss was largely elevated. Moreover, both MK801 (10 microM), an antagonist of NMDA receptor, and EGTA (100 mM, but neither 50 nor 150 mM), an extracellular Ca2+ chelator, not only effectively inhibited the ERK5 activation but also markedly abolished CIP-induced survival of the CA1 neurons. These results suggested that activation of the ERK5 pathway by CIP was at least partly dependent on moderate Ca2+ influx via NMDA receptor, which might contribute to ischemic tolerance in hippocampal CA1 region of rats.     

Neuroprotective effects of preconditioning ischaemia on ischaemic brain injury through inhibition of mixed-lineage kinase 3 via NMDA receptor-mediated Akt1 activation

A number of works show that the mitogen-activated protein kinase (MAPK) signalling pathway responds actively in cerebral ischaemia and reperfusion. We undertook our present studies to clarify the role of mixed-lineage kinase 3 (MLK3), a MAPK kinase kinase (MAPKKK) in MAPK cascades, in global ischaemia and ischaemic tolerance. The mechanism concerning NMDA receptor-mediated Akt1 activation underlying ischaemic tolerance, was also investigated. Sprague-Dawley rats were subjected to 6 min of ischaemia and differing times of reperfusion. Our results showed MLK3 was activated in the hippocampal CA1 region with two peaks occurring at 30 min and 6 h, respectively. This activation returned to base level 3 days later. Both preconditioning with 3 min of sublethal ischaemia and NMDA pretreatment inhibited the 6-h peak of activation. However, pretreatment of ketamine before preconditioning reversed the inhibiting effect of preconditioning on MLK3 activation at 6 h of reperfusion. In the case of Akt1, however, preconditioning and NMDA pretreatment enhanced Akt1 activation at 10 min of reperfusion. Furthermore, ketamine pretreatment reversed preconditioning-induced increase of Akt1 activation. We also noted that pretreatment of LY294002 before preconditioning reversed both the inhibition of MLK3 activation at 6 h of reperfusion and the increase in Akt1 activation at 10 min of reperfusion. The above-mentioned results lead us to conclude that, in the hippocampal CA1 region, preconditioning inhibits MLK3 activation after lethal ischaemia and reperfusion and, furthermore, this effect is mediated by Akt1 activation through NMDA receptor stimulation.     

The effect of preconditioning on the iron deposition after transient forebrain ischemia in rat brain

In this study we investigated iron deposition in the hippocampus CA1 area and the corpus striatum pars dorsolateralis in a rat model of cerebral ischemia and ischemic tolerance. Forebrain ischemia was induced by four-vessel occlusion for 5-min as ischemic preconditioning. Two days after the preconditioning or the sham operation, a second ischemia was induced for 20-min. With the use of iron histochemistry, regional changes were examined after 2 to 8 weeks of recirculation following the 20-min ischemia with or without preconditioning. Perl's reaction with DAB intensification demonstrated iron deposits in the CA1 area and in the corpus striatum pars dorsolateralis after 2 weeks of recirculation. These iron deposits gradually increased in density and formed clusters by the 8th week. When the rats were exposed to 5-min ischemia 2 days before lethal 20-min ischemia, the deposition of iron in the CA1 region of the hippocampus and also in the corpus striatum pars dorsolateralis was decreased and produced a minimal number of iron-containing cells between the second and the 8th week of recirculation. Preconditioning with sublethal 5-min ischemia followed by 2 days of reperfusion also prevented the neuronal destruction of the hippocampal CA1 region induced by 20-min ischemia.     

Hyperoxidized Peroxiredoxins and Glyceraldehyde-3-Phosphate Dehydrogenase Immunoreactivity and Protein Levels are Changed in the Gerbil Hippocampal CA1 Region After Transient Forebrain Ischemia

Oxidative stress is a major pathogenic event occurring in several brain disorders and is a major cause of brain damage due to ischemia/reperfusion. Thiol proteins are easily oxidized in cells exposed to reactive oxygen species (ROS). In the present study, we investigated transient ischemia-induced chronological changes in hyperoxidized peroxiredoxins (Prx-SO₃) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH-SO₃) immunoreactivity and protein levels in the gerbil hippocampus induced by 5 min of transient forebrain ischemia. Weak Prx-SO₃ immunoreactivity is detected in the hippocampal CA1 region of the sham-operated group. Prx-SO₃ immunoreactivity was significantly increased 12 h and 1 day after ischemia/reperfusion, and the immunoreactivity was decreased to the level of the sham-operated group 2 days after ischemia/reperfusion. Prx-SO₃ immunoreactivity in the 4 days post-ischemia group was increased again, and the immunoreactivity was expressed in glial components for 5 days after ischemia/reperfusion. GAPDH-SO₃ immunoreactivity was highest in the CA1 region 1 day after ischemia/reperfusion, the immunoreactivity was decreased 2 days after ischemia/reperfusion. Four days after ischemia/reperfusion, GAPDH-SO₃ immunoreactivity increased again, and the immunoreactivity began to be expressed in glial components from 5 days after ischemia/reperfusion. Prx-SO₃ and GAPDH-SO₃ protein levels in the ischemic CA1 region were also very high 12 h and 1 day after ischemia/reperfusion and returned to the level of the sham-operated group 3 days after ischemia/reperfusion. Their protein levels were increased again 5 days after ischemia/reperfusion. In conclusion, Prx-SO₃ and GAPDH-SO₃ immunoreactivity and protein levels in the gerbil hippocampal CA1 region are significantly increased 12 h-24 h after ischemia/reperfusion and their immunoreactivity begins to be expressed in glial components from 4 or 5 days after ischemia/reperfusion.     

Activated mitogen-activated protein kinase kinase 7 redistributes to the cytosol and binds to Jun N-terminal kinase-interacting protein 1 involving oxidative stress during early reperfusion in rat hippocampal CA1 region

Mitogen-activated protein kinase kinase (MKK) 7, a specific upstream activator of Jun N-terminal kinases (JNKs) in the stress-activated protein kinase (SAPK)/JNK signaling pathway, plays an important role in response to global cerebral ischemia. We investigated the subcellular localization of activated (phosphorylated) MKK (p-MKK) 7 using western blotting, immunoprecipitation and immunohistochemistry analysis in rat hippocampus. Transient forebrain ischemia was induced by the four-vessel occlusion method on Sprague-Dawley rats. Our results showed that both protein expression and activation of MKK7 were increased rapidly with peaks at 10 min of reperfusion in the nucleus of the hippocampal CA1 region. Simultaneously, in the cytosol activated MKK7 enhanced gradually and peaked at 30 min of reperfusion. In addition, we also detected JNK-interacting protein (JIP) 1, which accumulated in the perinuclear region of neurons at 30 min of reperfusion. Interestingly, at the same time-point the binding of JIP-1 to p-MKK7 reached a maximum. Consequently, we concluded that MKK7 was rapidly activated and then translocated from the nucleus to the cytosol depending on its activation in the hippocampal CA1 region. To further elucidate the possible mechanism of MKK7 activation and translocation, the antioxidant N-acetylcysteine was injected into the rats 20 min before ischemia. The result showed that the levels of MKK7 activation, translocation and binding of p-MKK7 to JIP-1 were obviously limited by N-acetylcysteine in the cytosol at 30 min after reperfusion. The findings suggested that MKK7 activation, translocation and binding to JIP-1 were closely associated with reactive oxygen species and might play a pivotal role in the activation of the JNK signaling pathway in brain ischemic injury.     

Ischemic postconditioning during reperfusion activates Akt and ERK without protecting against lethal myocardial ischemia-reperfusion injury in pigs

Transient episodes of ischemic preconditioning (PC) render myocardium protected against subsequent lethal injury after ischemia and reperfusion. Recent studies indicate that application of short, repetitive ischemia only during the onset of reperfusion after the lethal ischemic event may obtain equivalent protection. We assessed whether such ischemic postconditioning (Postcon) is cardioprotective in pigs by limiting lethal injury. Pentobarbital sodium-anesthetized, open-chest pigs underwent 30 min of complete occlusion of the left anterior descending coronary artery and 3-h reflow. PC was elicited by two cycles of 5-min occlusion plus 10-min reperfusion before the 30-min occlusion period. Postcon was elicited by three cycles of 30-s reperfusion, followed by 30-s reocclusion, after the 30-min occlusion period and before the 3-h reflow. Infarct size (%area-at-risk using triphenyltetrazolium chloride macrochemistry; means +/- SE) after 30 min of ischemia was 26.5 +/- 5.2% (n = 7 hearts/treatment group). PC markedly limited myocardial infarct size (2.8 +/- 1.2%, n = 7 hearts/treatment group, P < 0.05 vs. controls). However, Postcon had no effect on infarct size (37.8 +/- 5.1%, n = 7 hearts/treatment group). Within the subendocardium, Postcon increased phosphorylation of Akt (74 +/- 12%) and ERK1/2 (56 +/- 10%) compared with control hearts subjected only to 30-min occlusion and 15-min reperfusion (P < or = 0.05), and these changes were not different from the response triggered by PC (n = 5 hearts/treatment group). Phosphorylation of downstream p70S6K was also equivalent in PC and Postcon groups. These data do not support the hypothesis that application of 30-s cycles of repetitive ischemia during reperfusion exerts a protective effect on pig hearts subjected to lethal ischemia, but this is not due to a failure to phosphorylate ERK and Akt during early reperfusion.     

Elevated synaptic activity preconditions neurons against an in vitro model of ischemia

Tolerance to otherwise lethal cerebral ischemia in vivo or to oxygen-glucose deprivation (OGD) in vitro can be induced by prior transient exposure to N-methyl-D-aspartic acid (NMDA): preconditioning in this manner activates extrasynaptic and synaptic NMDA receptors and can require bringing neurons to the "brink of death." We considered if this stressful requirement could be minimized by the stimulation of primarily synaptic NMDA receptors. Subjecting cultured cortical neurons to prolonged elevations in electrical activity induced tolerance to OGD. Specifically, exposing cultures to a K(+)-channel blocker, 4-aminopyridine (20-2500 microm), and a GABA(A) receptor antagonist, bicuculline (50 microm) (4-AP/bic), for 1-2 days resulted in potent tolerance to normally lethal OGD applied up to 3 days later. Preconditioning induced phosphorylation of ERK1/2 and CREB which, along with Ca(2+) spiking and OGD tolerance, was eliminated by tetrodotoxin. Antagonists of NMDA receptors or L-type voltage-gated Ca(2+) channels (L-VGCCs) applied during preconditioning decreased Ca(2+) spiking, phosphorylation of ERK1/2 and CREB, and OGD tolerance more effectively when combined, particularly at the lowest 4-AP concentration. Inhibiting ERK1/2 or Ca(2+)/calmodulin-dependent protein kinases (CaMKs) also reduced Ca(2+) spiking and OGD tolerance. Preconditioning resulted in altered neuronal excitability for up to 3 days following 4-AP/bic washout, based on field potential recordings obtained from neurons cultured on 64-channel multielectrode arrays. Taken together, the data are consistent with action potential-driven co-activation of primarily synaptic NMDA receptors and L-VGCCs, resulting in parallel phosphorylation of ERK1/2 and CREB and involvement of CaMKs, culminating in a potent, prolonged but reversible, OGD-tolerant phenotype.     

Changes of Endogenous Antioxidant Enzymes during Ischemic Tolerance Acquisition

The purpose of this study was to investigate the role of superoxide dismutase (SOD) and catalase (CAT) in brain ischemic tolerance induced by ischemic preconditioning. Forebrain cerebral ischemia was induced in rat by four vessel occlusion. The activities of the antioxidant enzymes CuZn-SOD, Mn-SOD and CAT were measured in the hippocampus, striatum and cortex after 5 min of ischemia used as a preconditioning and subsequent reperfusion, by spectrophotometric methods. In all ischemia-reperfusion groups (5 h, 1 and 2 days of reperfusion), CuZn-SOD activities were found to be increased if compared to the sham operated controls. The increase was significant (P < 0.05) in all reperfusion groups, particularly after 5 h of reperfusion (3 times) in all studied brain regions; the largest increase was detected in the more vulnerable hippocampus and striatum. Very similar changes were found in Mn-SOD activity. The activity of CAT was increased too, but reached the peak of postischemic activity 24 h after ischemia. Our attempt to understand the mechanisms of increased SOD and CAT activities by application of protein synthesis inhibitor cycloheximide showed that this increase was caused by de novo synthesis of enzymes during first hours after ischemia. Our findings indicate that both major endogenous antioxidant enzymes SOD and CAT are synthesized as soon as 5 h after ischemia. In spite of significant upregulation of these enzymes a large number of neurons in selectively vulnerable CA1 region of hippocampus undergoes to neurodegeneration within 7 days after ischemia.     

Role of Protein Synthesis in the Ischemic Tolerance Acquisition Induced by Transient Forebrain Ischemia in the Rat

Although ischemic preconditioning of the heart and brain is a well-documented neuroprotective phenomenon, the mechanism underlying the increased resistance to severe ischemia induced by a preceding mild ischemic exposure remains unclear. In this study we have determined the effect of ischemic preconditioning on ischemia/reperfusion-associated translation inhibition in the neocortex and hippocampus of the rat. We studied the effect of the duration on the sublethal ischemic episode (3, 4, 5 or 8 min), as well as the amount of time elapsed between sublethal and lethal ischemia on the cell death 7 days after the last ischemic episode. In addition, the rate of protein synthesis in vitro and expression of the 72-kD heat shock protein (hsp) were determined under the different experimental conditions. Our results suggest that two different mechanisms are essential for the acquisition of ischemic tolerance, at least in the CA1 sector of hippocampus. The first mechanism implies a highly significant reduction in translation inhibition after lethal ischemia, especially at an early time of reperfusion, in both vulnerable and nonvulnerable neurons. For the acquisition of full tolerance, a second mechanism, highly dependent on the time interval between preconditioning (sublethal ischemia) and lethal ischemia, is absolutely necessary; this second mechanism involves synthesis of protective proteins, which prevent the delayed death of vulnerable neurons.     

Preconditioning decreases ischemia/reperfusion-induced release and activation of matrix metalloproteinase-2

Release and activation of matrix metalloproteinases (MMPs) significantly contribute to myocardial stunning injury immediately after ischemia and reperfusion, however, their role in preconditioning remains unknown. We therefore examined the effects of preconditioning and subsequent ischemia/reperfusion on MMP activity in isolated rat hearts. Hearts were subjected to a preconditioning protocol (three consecutive 5-min periods of global ischemia interspersed with 5 min of reperfusion) followed by 30 min ischemia and 5 min reperfusion. To measure MMP release, coronary effluent was collected: (a) during aerobic perfusion, (b) in reperfusion following each preconditioning ischemia, and (c) during the final reperfusion following test ischemia. MMP-2 activities could be detected by gelatin zymography in the ventricles and coronary effluent samples from the perfused hearts. The levels of MMP-2 activity in the effluent were markedly increased in effluent following test ischemia from control hearts without preconditioning. This was accompanied by a decrease in corresponding tissue MMP activities. Preconditioning significantly decreased the MMP-2 activity in the coronary effluent following test ischemia/reperfusion and preserved the MMP-2 protein content and activity in the myocardium. Our results demonstrate that classic preconditioning inhibits ischemia/reperfusion induced release and activation of MMP-2. These results suggest that preconditioning may exert part of its cardioprotective effects through the reduction of MMP-2 release.     

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.     

Ionized Calcium-binding Adapter Molecule 1 Immunoreactive Cells Change in the Gerbil Hippocampal CA1 Region after Ischemia/Reperfusion

Ionized calcium-binding adapter molecule 1 (iba-1) is specifically expressed in microglia and plays an important role in the regulation of the function of microglia. We observed chronological changes of iba-1-immunoreactive cells and iba-1 level in the gerbil hippocampal CA1 region after transient ischemia. Transient forebrain ischemia in gerbils was induced by the occlusion of bilateral common carotid arteries for 5 min. Immunohistochemical and Western blot analysis of iba-1 were performed in the gerbil ischemic hippocampus. In the sham-operated group, iba-1-immunoreactive cells were detected in the CA1 region. Thirty minutes after ischemia/reperfusion, iba-1 immunoreactivity significantly increased, and its immunoreactive cells were well ramified. Three hours after ischemia/reperfusion, iba-1 immunoreactivity and level decreased, and thereafter they increased again with time after ischemia/reperfusion. Three days after ischemia/reperfusion, iba-1-immunoreactive cells had well-ramified processes, which projected to the stratum pyramidale of the CA1 region. Seven days after ischemia/reperfusion, iba-1 immunoreactivity and level were highest in the CA1 region, whereas they significantly decreased in the CA1 region 10 days after ischemia/reperfusion. Iba-1-immunoreactive cells in the ischemic CA1 region were co-localized with OX-42, a microglia marker. In brief, iba-1-immunoreactive cells change morphologically and iba-1 immunoreactivity alters in the CA1 region with time after ischemia/reperfusion. These may be associated with the delayed neuronal death of CA1 pyramidal cells in the gerbil ischemic hippocampus.     

Iron mediates N-methyl-D-aspartate receptor-dependent stimulation of calcium-induced pathways and hippocampal synaptic plasticity

Iron deficiency hinders hippocampus-dependent learning processes and impairs cognitive performance, but current knowledge on the molecular mechanisms underlying the unique role of iron in neuronal function is sparse. Here, we investigated the participation of iron on calcium signal generation and ERK1/2 stimulation induced by the glutamate agonist N-methyl-D-aspartate (NMDA), and the effects of iron addition/chelation on hippocampal basal synaptic transmission and long-term potentiation (LTP). Addition of NMDA to primary hippocampal cultures elicited persistent calcium signals that required functional NMDA receptors and were independent of calcium influx through L-type calcium channels or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors; NMDA also promoted ERK1/2 phosphorylation and nuclear translocation. Iron chelation with desferrioxamine or inhibition of ryanodine receptor (RyR)-mediated calcium release with ryanodine-reduced calcium signal duration and prevented NMDA-induced ERK1/2 activation. Iron addition to hippocampal neurons readily increased the intracellular labile iron pool and stimulated reactive oxygen species production; the antioxidant N-acetylcysteine or the hydroxyl radical trapper MCI-186 prevented these responses. Iron addition to primary hippocampal cultures kept in calcium-free medium elicited calcium signals and stimulated ERK1/2 phosphorylation; RyR inhibition abolished these effects. Iron chelation decreased basal synaptic transmission in hippocampal slices, inhibited iron-induced synaptic stimulation, and impaired sustained LTP in hippocampal CA1 neurons induced by strong stimulation. In contrast, iron addition facilitated sustained LTP induction after suboptimal tetanic stimulation. Together, these results suggest that hippocampal neurons require iron to generate RyR-mediated calcium signals after NMDA receptor stimulation, which in turn promotes ERK1/2 activation, an essential step of sustained LTP.     

Effect of ischemic preconditioning on cerebral blood flow after subsequent lethal ischemia in gerbils

Ischemic tolerance, the phenomenon where a sublethal ischemic preconditioning protects the brain against a subsequent lethal ischemia, has been widely studied. Studies have been done on cerebral blood flow levels prior to the lethal ischemia, but the hemodynamic pattern after global ischemia with ischemic preconditioning has not been reported. Sequential changes in regional cerebral blood flow (rCBF) in gerbil hippocampus after 5 min global ischemia with or without 2 min ischemic preconditioning were studied to determine if ischemic preconditioning affects rCBF. Four different treatments were given: (1) sham-operated, (2) 2 min ischemia, (3) non-preconditioned, and (4) preconditioned. Groups (1) and (2) (both groups n = 5) were given a 24-h recovery period and the rCBF was measured for baseline values. 24 h after sham-operation (3) and 2 min ischemia (4), gerbils were subjected to 5 min ischemia followed by 1 h, 6 h, 1-day or 7-day reperfusion periods (all groups n = 5). Although no regional difference was observed in the recovery pattern of rCBF, the values of rCBF were significantly higher in the preconditioned group throughout whole brain regions including hippocampus. These results indicate that ischemic preconditioning facilitated the recovery of rCBF after 5 min global ischemia. It needs further study to determine whether the protecting effects of preconditioning relate to the early recovery of rCBF or not. However, our results could be interpreted that the early recovery of rCBF may lead to benefits for cell survival in the CA1 neuron, probably facilitating other protecting mechanisms.     

脑预缺血引起大鼠海马细胞膜腺苷受体数量和亲和力升高及其对神经元的保护作用

采用放射性配基结合法,测定大鼠全脑缺血后海马细胞膜腺苷(adenosine,ADO)受体数量及亲和力的变化,以探讨其与脑缺血耐受形成之间的关系。发现缺血6min即可导致海马组织明显的神经元延迟性死亡(delayed neuron     

Dexmedetomidine preconditioning activates pro-survival kinases and attenuates regional ischemia/reperfusion injury in rat heart

Pharmacological preconditioning limits myocardial infarct size after ischemia/reperfusion. Dexmedetomidine is an α(2)-adrenergic receptor agonist used in anesthesia that may have cardioprotective properties against ischemia/reperfusion injury. We investigate whether dexmedetomidine administration activates cardiac survival kinases and induces cardioprotection against regional ischemia/reperfusion injury. In in vivo and ex vivo models, rat hearts were subjected to 30 min of regional ischemia followed by 120 min of reperfusion with dexmedetomidine before ischemia. The α(2)-adrenergic receptor antagonist yohimbine was also given before ischemia, alone or with dexmedetomidine. Erk1/2, Akt and eNOS phosphorylations were determined before ischemia/reperfusion. Cardioprotection after regional ischemia/reperfusion was assessed from infarct size measurement and ventricular function recovery. Localization of α(2)-adrenergic receptors in cardiac tissue was also assessed. Dexmedetomidine preconditioning increased levels of phosphorylated Erk1/2, Akt and eNOS forms before ischemia/reperfusion; being significantly reversed by yohimbine in both models. Dexmedetomidine preconditioning (in vivo model) and peri-insult protection (ex vivo model) significantly reduced myocardial infarction size, improved functional recovery and yohimbine abolished dexmedetomidine-induced cardioprotection in both models. The phosphatidylinositol 3-kinase inhibitor LY-294002 reversed myocardial infarction size reduction induced by dexmedetomidine preconditioning. The three isotypes of α(2)-adrenergic receptors were detected in the whole cardiac tissue whereas only the subtypes 2A and 2C were observed in isolated rat adult cardiomyocytes. These results show that dexmedetomidine preconditioning and dexmedetomidine peri-insult administration produce cardioprotection against regional ischemia/reperfusion injury, which is mediated by the activation of pro-survival kinases after cardiac α(2)-adrenergic receptor stimulation.     

Mapping of rat hippocampal neurons with NeuN after ischemia/reperfusion and Ginkgo biloba extract (EGb 761) pretreatment

1. The neuroprotective effect of Ginkgo biloba extract (EGb 761) against transient forebrain ischemia following 7 days of reperfusion was studied in male Wistar rats after four-vessel occlusion for 20 min.2. NeuN, a neuronal specific nuclear protein was used for immunohistochemical detection of surviving pyramidal neurons in the hippocampus, as well as counterstaining with hematoxylin in the same sections for detection of neurons that underwent delayed neuronal death and for glial nuclei staining. GFAP immunohistochemistry was used for detection of astrocytes in the studied area of CA1 region.3. In the group of rats pretreated 7 days with Ginkgo biloba extract (EGb 761), following 20 min of ischemia and 7 days of reperfusion without EGb 761, increased number of NeuN immunoreactive cells were counted in the most vulnerable CA1 pyramidal layer of hippocampus. On the other hand, the group of rats with 7 days of EGb 761 pretreatment following 20 min of ischemia and 7 days of reperfusion with EGb 761 showed decreased number of surviving NeuN immunoreactive CA1 pyramidal cells in comparison with the first above-mentioned experimental group.4. Increased number of reactive astrocytes immunolabeled for GFAP (Glial fibrilary acidic protein) was observed in both experimental groups in the stratum oriens and stratum lacunosum and moleculare.5. Twenty minutes of ischemia is lethal for most population of CA1 pyramidal cell layer. Our results showed that prophylactic oral administration of Ginkgo biloba extract (EGb 761) in the dose 40 mg/kg/day during the 7 days protects the most vulnerable CA1 pyramidal cells against 20 min of ischemia.     

Neuroprotective effects of preconditioning ischemia on ischemic brain injury through down-regulating activation of JNK1/2 via N-methyl-D-aspartate receptor-mediated Akt1 activation

Our previous studies have demonstrated that the JNK signaling pathway plays an important role in ischemic brain injury and is mediated via glutamate receptor 6. Others studies have shown that N-methyl-d-aspartate (NMDA) receptor is involved in the neuroprotection of ischemic preconditioning. Here we examined whether ischemic preconditioning down-regulates activation of the mixed lineage kinase-JNK signaling pathway via NMDA receptor-mediated Akt1 activation. In our present results, ischemic preconditioning could not only inhibit activations of mixed lineage kinase 3, JNK1/2, and c-Jun but also enhanced activation of Akt1. In addition, both NMDA (an agonist of NMDA receptor) and preconditioning showed neuroprotective effects. In contrast, ketamine, an antagonist of NMDA receptor, prevented the above effects of preconditioning. Further studies indicated that LY294002, an inhibitor of phosphoinositide 3-kinase that is an upstream signaling protein of Akt1, could block neuroprotection of preconditioning, and KN62, an inhibitor of calmodulin-dependent protein kinase, also achieved the same effects as LY294002. Therefore, both phosphoinositide 3-kinase and calmodulin-dependent protein kinase are involved in the activation of Akt1 in ischemic tolerance. Taken together, our results indicate that preconditioning can inhibit activation of JNK signaling pathway via NMDA receptor-mediated Akt1 activation and induce neuroprotection in hippocampal CA1 region.     

Transient ischemia enhances tyrosine phosphorylation and binding of the NMDA receptor to the Src homology 2 domain of phosphatidylinositol 3-kinase in the rat hippocampus

Tyrosine phosphorylation of the NMDA receptor has been implicated in the regulation of the receptor channel. We investigated the effects of transient (15 min) global ischemia on tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B, and the interaction of NR2 subunits with the SH2 domain of phosphatidylinositol 3-kinase (PI3-kinase) in vulnerable CA1 and resistant CA3/dentate gyrus of the hippocampus. Transient ischemia induced a marked increase in the tyrosine phosphorylation of NR2A in both regions. The tyrosine phosphorylation of NR2B in CA3/dentate gyrus after transient ischemia was sustained and greater than that in CA1. PI3-kinase p85 was co-precipitated with NR2B after transient global ischemia. The SH2 domain of the p85 subunit of PI3-kinase bound to NR2B, but not to NR2A. Binding to NR2B was increased following ischemia and the increase in binding in CA3/dentate gyrus (4.5-fold relative to sham) was greater than in CA1 (1.7-fold relative to sham) at 10 min of reperfusion. Prior incubation of proteins with an exogenous protein tyrosine phosphatase or with a phosphorylated peptide (pYAHM) prevented binding. The results suggest that sustained increases in tyrosine phosphorylation and increased interaction of NR2B with the SH2 domain of PI3-kinase may contribute to altered signal transduction in the CA3/dentate gyrus after transient ischemia.