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1.  Limb remote-preconditioning protects against focal ischemia in rats and contradicts the dogma of therapeutic time windows for preconditioning 
Neuroscience  2007;151(4):1099-1103.
Remote ischemic preconditioning is an emerging concept for stroke treatment, but its protection against focal stroke has not been established. We tested whether remote preconditioning, performed in the ipsilateral hind limb, protects against focal stroke and explored its protective parameters. Stroke was generated by a permanent occlusion of the left distal middle cerebral artery (MCA) combined with a 30 minute occlusion of the bilateral common carotid arteries (CCA) in male rats. Limb preconditioning was generated by 5 or 15 minute occlusion followed with the same period of reperfusion of the left hind femoral artery, and repeated for 2 or 3 cycles. Infarct was measured 2 days later. The results showed that rapid preconditioning with 3 cycles of 15 minutes performed immediately before stroke reduced infarct size from 47.7±7.6% of control ischemia to 9.8±8.6%; at 2 cycles of 15 minutes, infarct was reduced to 24.7±7.3%; at 2 cycles of 5 minutes, infarct was not reduced. Delayed preconditioning with 3 cycles of 15 minutes conducted 2 days before stroke also reduced infarct to 23.0 ±10.9%, but with 2 cycles of 15 minutes it offered no protection. The protective effects at these two therapeutic time windows of remote preconditioning are consistent with those of conventional preconditioning, in which the preconditioning ischemia is induced in the brain itself. Unexpectedly, intermediate preconditioning with 3 cycles of 15 minutes performed 12 hours before stroke also reduced infarct to 24.7±4.7%, which contradicts the current dogma for therapeutic time windows for the conventional preconditioning that has no protection at this time point. In conclusion, remote preconditioning performed in one limb protected against ischemic damage after focal cerebral ischemia.
doi:10.1016/j.neuroscience.2007.11.056
PMCID: PMC2696348  PMID: 18201834
preconditioning; remote preconditioning; limb preconditioning; cerebral ischemia; focal ischemia
2.  Preconditionin effects of dexmedetomidine on myocardial ischemia/reperfusion injury in rats 
Background: Preconditioning might protect the myocardium against ischemia/ reperfusion injury by reducing infarct size and preventing arrhythmias. Dexmedetomidine (DEX) is a highly selective α2-agonist used for sedoanalgesia in daily anesthetic practice. The cardioprotective effects of DEX on infarct size and on the incidence of arrhythmias observed after regional ischemia/reperfusion injury in vivo have not been reported.
Objective: The aim of this study was to determine whether DEX exhibits a preconditioning effect and reduces infarct size and the incidence and duration of arrhythmias in a regional cardiac ischemia/reperfusion model in rats.
Methods: Adult male Sprague-Dawley rats were anesthetized with sodium thiopental and mechanically ventilated (0.9 mL/100 g at 60 strokes/min) through a cannula inserted into the trachea after tracheotomy. Cardiac ischemia was then produced by ligating the left main coronary artery for 30 minutes, followed by a reperfusion period of 120 minutes. Blood pressure (BP) and heart rate (HR) were monitored and echocardiograms (ECGs) were performed. Arrhythmia was scored based on incidence and duration. The animals were randomly divided into 3 groups. The ischemic preconditioning (IPC) group underwent 5 minutes of ischemia followed by 5 minutes of reperfusion before the 30-minute ischemia/120-minute reperfusion period. In the DEX group, intraperitoneal (IP) DEX 1 mL (100 μg/kg) was administered 30 minutes before the ischemia/ reperfusion period. In the control group, IP saline 1 mL was administered 30 minutes before the ischemia/reperfusion period. After reperfusion, the heart was excised, demarcated with saline and ethanol to identify the occluded and nonoccluded myocardium, and cut into slices ~2 mm thick, that were then stained and placed between 2 glass plates. The risk zone and the infarct zone were compared between groups. The investigator assessing the infarcts was blinded to the study group.
Results: Twenty-one adult (aged 4-6 months) male Sprague-Dawley rats weighing 280 to 360 g were included in the study; 7 rats were assigned to each group. BP, HR, and ECG readings were not significantly different between groups and did not change during the study. Arrythmias occurred during ischemia and reperfusion in all groups. The duration of the arrhythmias was significantly shorter and the arrhythmia score was significantly lower in the IPC group (all, P<0.05), compared with the control group; however, they were not significantly different in the DEX group. During the ischemic period, duration of ventricular tachycardia (VT) and ventricular premature contractions (VPC) in the DEX group was significantly longer than that observed in the IPC group (all, P<0.05). The duration of VPC was also significantly shorter than that observed in the control group (both, P<0.05). Duration of VT during the reperfusion period in the DEX group was significantly longer than that observed in both IPC and control groups (both, P<0.05). The mean (SD) percentage of damage was significantly lower in the IPC group (44.1% [2.0%]) and the DEX group (26.7% [2.0%]) compared with the control group (69.0% [3.0%]; both, P<0.05). The percentage of damage in the DEX group was also significantly lower compared with the IPC group (P<0.05).
Conclusions: This small, experimental in vivo study found that DEX was associated with reduced infarct size in ischemia/reperfusion injury in regional ischemia in this rat model but had no effect on the incidence of arrhythmias. Future studies are needed to clarify these findings.
doi:10.1016/j.curtheres.2008.04.003
PMCID: PMC3969917  PMID: 24692794
dexmedetomidine; preconditioning; cardiac ischemia/reperfusion
3.  Delayed Postconditioning Protects against Focal Ischemic Brain Injury in Rats 
PLoS ONE  2008;3(12):e3851.
Background
We and others have reported that rapid ischemic postconditioning, interrupting early reperfusion after stroke, reduces infarction in rats. However, its extremely short therapeutic time windows, from a few seconds to minutes after reperfusion, may hinder its clinical translation. Thus, in this study we explored if delayed postconditioning, which is conducted a few hours after reperfusion, offers protection against stroke.
Methods and Results
Focal ischemia was generated by 30 min occlusion of bilateral common carotid artery (CCA) combined with permanent occlusion of middle cerebral artery (MCA); delayed postconditioning was performed by repetitive, brief occlusion and release of the bilateral CCAs, or of the ipsilateral CCA alone. As a result, delayed postconditioning performed at 3h and 6h after stroke robustly reduced infarct size, with the strongest protection achieved by delayed postconditioning with 6 cycles of 15 min occlusion/15 min release of the ipsilateral CCA executed from 6h. We found that this delayed postconditioning provided long-term protection for up to two months by reducing infarction and improving outcomes of the behavioral tests; it also attenuated reduction in 2-[18F]-fluoro-2-deoxy-D-glucose (FDG)-uptake therefore improving metabolism, and reduced edema and blood brain barrier leakage. Reperfusion in ischemic stroke patients is usually achieved by tissue plasminogen activator (tPA) application, however, t-PA's side effect may worsen ischemic injury. Thus, we tested whether delayed postconditioning counteracts the exacerbating effect of t-PA. The results showed that delayed postconditioning mitigated the worsening effect of t-PA on infarction.
Conclusion
Delayed postconditioning reduced ischemic injury after focal ischemia, which opens a new research avenue for stroke therapy and its underlying protective mechanisms.
doi:10.1371/journal.pone.0003851
PMCID: PMC2588536  PMID: 19066627
4.  ASIC1a contributes to neuroprotection elicited by ischemic preconditioning and postconditioning 
Acid-sensing ion channels, ASICs, are proton-gated cation channels widely expressed in peripheral sensory neurons and in neurons of the central nervous system that play an important role in a variety of physiological and pathological processes. To further confirm the role played by ASIC1a in cerebral ischemia, here we examined the involvement of this channel in two endogenous recently characterized neuroprotective strategies: brain ischemic preconditioning and postconditioning. The main aim of this study was to elucidate whether ASIC1a might take part as effector in the neuroprotection evoked by brain ischemic preconditioning and postconditioning. For this purpose we investigated the effect of ischemic preconditioning and postconditioning on (1) ASIC1a mRNA and protein expression in the temporoparietal cortex of rats at different time intervals; and (2) the effect of p-AKT inhibition on ASIC1a expression during ischemic preconditioning and postconditioning. Ischemic preconditioning and postconditioning were experimentally induced in adult male rats by subjecting them to different protocols of middle cerebral artery occlusion and reperfusion. ASIC1a expression was dramatically reduced in both the neuroprotective processes. These changes in ASIC expression were p-AKT mediated, since LY-294002, a specific p-AKT inhibitor, was able to prevent variations in ASIC1a expression. The results of the present study support the idea that the downregulation of ASIC1a expression and activity might be a reasonable strategy to reduce the infarct extension after stroke.
PMCID: PMC3068848  PMID: 21479097
ASIC1a; preconditioning; postconditioning; stroke; neuroprotection
5.  Lithium Treatment Reduces Brain Injury Induced by Focal Ischemia with Partial Reperfusion and the Protective Mechanisms Dispute the Importance of Akt Activity 
Aging and Disease  2012;3(3):226-233.
Lithium is a mood stabilizer shown to have neuroprotective effects against several chronic and acute neuronal injuries, including stroke. However, it is unknown whether lithium treatment protects against brain injury post-stroke in a rat model of permanent distal middle cerebral artery occlusion (MCAo) combined with transient bilateral common carotid artery occlusion (CCAo), a model that mimics human stroke with partial reperfusion. In addition, whether lithium treatment alters Akt activity as measured by the kinase activity assay has not been reported, although it is known to inhibit GSK3β activity. After stroke, Akt activity contributes to neuronal survival while GSK3β activity causes neuronal death. We report that a bolus of lithium injection at stroke onset robustly reduced infarct size measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining at 48 h post-stroke and inhibited cell death in the ischemic penumbra, but not in the ischemic core, as shown by TUNEL staining performed 24 h post-stroke. However, lithium treatment did not alter the reduction in Akt activity as measured by Akt kinase assay. We further showed that lithium did not alter phosphorylated GSK3β protein levels, or the degradation of β-catenin, a substrate of GSK3β, which is consistent with previous findings that long-term treatment is required for lithium to alter GSK3β phosphorylation. In summary, we show innovative data that lithium protects against stroke in a focal ischemia model with partial reperfusion, however, our results dispute the importance of Akt activity in the protective effects of lithium.
PMCID: PMC3375079  PMID: 22724081
Lithium; Akt; Cerebral focal ischemia; GSK3β; β-catenin
6.  The Reno-Vascular A2B Adenosine Receptor Protects the Kidney from Ischemia 
PLoS Medicine  2008;5(6):e137.
Background
Acute renal failure from ischemia significantly contributes to morbidity and mortality in clinical settings, and strategies to improve renal resistance to ischemia are urgently needed. Here, we identified a novel pathway of renal protection from ischemia using ischemic preconditioning (IP).
Methods and Findings
For this purpose, we utilized a recently developed model of renal ischemia and IP via a hanging weight system that allows repeated and atraumatic occlusion of the renal artery in mice, followed by measurements of specific parameters or renal functions. Studies in gene-targeted mice for each individual adenosine receptor (AR) confirmed renal protection by IP in A1−/−, A2A−/−, or A3AR−/− mice. In contrast, protection from ischemia was abolished in A2BAR−/− mice. This protection was associated with corresponding changes in tissue inflammation and nitric oxide production. In accordance, the A2BAR-antagonist PSB1115 blocked renal protection by IP, while treatment with the selective A2BAR-agonist BAY 60–6583 dramatically improved renal function and histology following ischemia alone. Using an A2BAR-reporter model, we found exclusive expression of A2BARs within the reno-vasculature. Studies using A2BAR bone-marrow chimera conferred kidney protection selectively to renal A2BARs.
Conclusions
These results identify the A2BAR as a novel therapeutic target for providing potent protection from renal ischemia.
Using gene-targeted mice, Holger Eltzschig and colleagues identify the A2B adenosine receptor as a novel therapeutic target for providing protection from renal ischemia.
Editors' Summary
Background.
Throughout life, the kidneys perform the essential task of filtering waste products and excess water from the blood to make urine. Each kidney contains about a million small structures called nephrons, each of which contains a filtration unit consisting of a glomerulus (a small blood vessel) intertwined with a urine-collecting tube called a tubule. If the nephrons stop working for any reason, the rate at which the blood is filtered (the glomerular filtration rate or GFR) decreases and dangerous amounts of waste products such as creatinine build up in the blood. Most kidney diseases destroy the nephrons slowly over years, producing an irreversible condition called chronic renal failure. But the kidneys can also stop working suddenly because of injury or poisoning. One common cause of “acute” renal failure in hospital patients is ischemia—an inadequate blood supply to an organ that results in the death of part of that organ. Heart surgery and other types of surgery in which the blood supply to the kidneys is temporarily disrupted are associated with high rates of acute renal failure.
Why Was This Study Done?
Although the kidneys usually recover from acute failure within a few weeks if the appropriate intensive treatment (for example, dialysis) is provided, acute renal failure after surgery can be fatal. Thus, new strategies to protect the kidneys from ischemia are badly needed. Like other organs, the kidneys can be protected from lethal ischemia by pre-exposure to several short, nonlethal episodes of ischemia. It is not clear how this “ischemic preconditioning” increases renal resistance to ischemia but some data suggest that the protection of tissues from ischemia might involve a signaling molecule called extracellular adenosine. This molecule binds to proteins called receptors on the surface of cells and sends signals into them that change their behavior. There are four different adenosine receptor—A1AR, A2AAR, A2BAR, and A3AR—and in this study, the researchers use ischemic preconditioning as an experimental strategy to investigate which of these receptors protects the kidneys from ischemia in mice, information that might provide clues about how to protect the kidneys from ischemia.
What Did the Researchers Do and Find?
The researchers first asked whether ischemic preconditioning protects the kidneys of mice strains that lack the genes for individual adenosine receptors (A1AR−/−, A2AAR−/−, A2BAR−/−, and A3AR−/− mice) from subsequent ischemia. Using a hanging-weight system, they intermittently blocked the renal artery of these mice before exposing them to a longer period of renal ischemia. Twenty-four hours later, they assessed the renal function of the mice by measuring their blood creatinine levels, GFRs, and urine production. Ischemic preconditioning protected all the mice from ischemia-induced loss of kidney function except the A2BAR−/− mice. It also prevented ischemia-induced structural damage and inflammation in the kidneys of wild-type but not A2BAR−/− mice. These results suggest that A2BAR may help to protect the kidneys from ischemia. Consistent with this idea, ischemic preconditioning did not prevent ischemia-induced renal damage in wild-type mice treated with a compound that specifically blocks the activity of A2BAR. However, wild-type mice (but not A2BAR−/− mice) treated with an A2BAR agonist (which activates the receptor) retained their kidney function after renal ischemia without ischemic preconditioning. Finally, the researchers report that A2BAR has to be present on the blood vessels in the kidney to prevent ischemia-induced acute renal failure.
What Do These Findings Mean?
These findings suggest that the protection of the kidneys from ischemia and the renal resistance to ischemia that is provided by ischemic preconditioning involve adenosine signaling through A2BAR. They also suggest that adenosine might provide protection against ischemia-induced damage by blocking inflammation in the kidney although other possible mechanisms of action need to be investigated. Importantly, these findings suggest that A2BAR might be a therapeutic target for the prevention of renal ischemia. However, results obtained in animals do not always reflect the situation in people, so before A2BAR agonists can be used to reduce the chances of patients developing acute renal failure after surgery, these results need confirming in people and the safety of A2BAR agonists need to be thoroughly investigated.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050137.
The US National Institute of Diabetes and Digestive and Kidney Diseases provides information on how the kidneys work and what can go wrong with them, including a list of links to further information about kidney disease
The MedlinePlus encyclopedia has a page on acute kidney failure (in English and Spanish)
Wikipedia has pages on acute renal failure, ischemia, ischemic preconditioning, and adenosine (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
doi:10.1371/journal.pmed.0050137
PMCID: PMC2504049  PMID: 18578565
7.  Ischemic postconditioning facilitates brain recovery after stroke by promoting Akt/mTOR activity in nude rats 
Journal of neurochemistry  2013;127(5):723-732.
While preconditioning is induced before stroke onset, ischemic postconditioning (IPostC) is performed after reperfusion, which typically refers to a series of mechanical interruption of blood reperfusion after stroke. IPostC is known to reduce infarction in wild type animals. We investigated if IPostC protects against brain injury induced by focal ischemia in T-cell-deficient nude rats and to examine its effects on Akt and the mammalian target of rapamycin (mTOR) pathway. Although IPostC reduced infarct size at 2 days post-stroke in wild type rats, it did not attenuate infarction in nude rats. Despite the unaltered infarct size in nude rats, IPostC increased levels of phosphorylated Akt (p-Akt) and Akt isoforms (Akt1, Akt2, Akt3), and p-mTOR, p-S6K and p-4EBP1 in the mTOR pathway, as well as GAP-43, both in the peri-infarct area and core, 24 hours after stroke. IPostC improved neurological function in nude rats 1–30 days after stroke and reduced the extent of brain damage 30 days after stroke. The mTOR inhibitor rapamycin abolished the long-term protective effects of IPostC. We determined that IPostC did not inhibit acute infarction in nude rats but did provide long-term protection by enhancing Akt and mTOR activity during the acute post-stroke phase.
doi:10.1111/jnc.12342
PMCID: PMC3875603  PMID: 23777415
Stroke; Ischemic postconditioning; T cells; Akt; mTOR
8.  Limb remote ischemic postconditioning protects against focal ischemia in rats 
Brain research  2009;1288:88-94.
Remote ischemic postconditioning (RIP) refers to an ischemia conducted in a distant organ that protects against a prior ischemia in another organ. We tested whether RIP protects against focal ischemia in the rat brain. Stroke was generated by a permanent occlusion of the left distal middle cerebral artery combined with a 30 min occlusion of the bilateral common carotid arteries (CCA) in male rats. After CCA release, RIP was generated by 3 cycles of 15 min occlusion/15 min release of the left hind femoral artery. The results showed that rapid RIP performed immediately after CCA release reduced infarction by 67% measured at 2 d after stroke. In addition, delayed RIP initiated as late as 3 h, but not 6 h, still robustly reduced infarction by 43% 2 d after stroke. RIP's protective effect was abolished by injecting the protein synthesis inhibitor, cycloheximide, and the afferent nerve blocker, capsaicin, suggesting that RIP blocks ischemic injury by modulating protein synthesis and nerve activity. Nevertheless, rapid RIP did not reduce infarction size 2 months after stroke while it ameliorated the outcome of the behavioral test. In conclusion, RIP attenuates brain injury after focal ischemia.
doi:10.1016/j.brainres.2009.07.029
PMCID: PMC2744502  PMID: 19631625
stroke; cerebral ischemia; preconditioning; remote postconditioning
9.  The Akt signaling pathway contributes to postconditioning’s protection against stroke; the protection is associated with the MAPK and PKC pathways 
Journal of neurochemistry  2008;105(3):943-955.
We previously reported that ischemic postconditioning with a series of mechanical interruptions of reperfusion reduced infarct volume 2 days after focal ischemia in rats. Here, we extend this data by examining long-term protection and exploring underlying mechanisms involving the Akt, mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) signaling pathways. Post-conditioning reduced infarct and improved behavioral function assessed 30 days after stroke. Additionally, postconditioning increased levels of phosphorylated Akt (Ser473) as measured by western blot and Akt activity as measured by an in vitro kinase assay. Inhibiting Akt activity by a phosphoinositide 3-kinase inhibitor, LY294002, enlarged infarct in postconditioned rats. Postconditioning did not affect protein levels of phosphorylated-phosphatase and tensin homologue deleted on chromosome 10 or -phosphoinositide-dependent protein kinase-1 (molecules upstream of Akt) but did inhibit an increase in phosphorylated-glycogen synthase kinase 3β, an Akt effector. In addition, postconditioning blocked β-catenin phosphorylation subsequent to glycogen synthase kinase, but had no effect on total or non-phosphorylated active β-catenin protein levels. Furthermore, postconditioning inhibited increases in the amount of phosphorylated-c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2 in the MAPK pathway. Finally, postconditioning blocked death-promoting δPKC cleavage and attenuated reduction in phosphorylation of survival-promoting εPKC. In conclusion, our data suggest that postconditioning provides long-term protection against stroke in rats. Additionally, we found that Akt activity contributes to postconditioning’s protection; furthermore, increases in εPKC activity, a survival-promoting pathway, and reductions in MAPK and δPKC activity; two putative death-promoting pathways correlate with postconditioning’s protection.
doi:10.1111/j.1471-4159.2008.05218.x
PMCID: PMC2746404  PMID: 18182053
Akt; cerebral ischemia; mitogen-activated protein kinase; postconditioning; protein kinase C; β-catenin
10.  Ischemic Postconditioning Fails to Protect against Neonatal Cerebral Stroke 
PLoS ONE  2012;7(12):e49695.
The lack of efficient neuroprotective strategies for neonatal stroke could be ascribed to pathogenic ischemic processes differentiating adults and neonates. We explored this hypothesis using a rat model of neonatal ischemia induced by permanent occlusion of the left distal middle cerebral artery combined with 50 min of occlusion of both common carotid arteries (CCA). Postconditioning was performed by repetitive brief release and occlusion (30 s, 1 and/or 5 min) of CCA after 50 min of CCA occlusion. Alternative reperfusion was generated by controlled release of the bilateral CCA occlusion. Blood-flow velocities in the left internal carotid artery were measured using color-coded pulsed Doppler ultrasound imaging. Cortical perfusion was measured using laser Doppler. Cerebrovascular vasoreactivity was evaluated after inhalation with the hypercapnic gas or inhaled nitric oxide (NO). Whatever the type of serial mechanical interruptions of blood flow at reperfusion, postconditioning did not reduce infarct volume after 72 hours. A gradual perfusion was found during early re-flow both in the left internal carotid artery and in the cortical penumbra. The absence of acute hyperemia during early CCA re-flow, and the lack of NO-dependent vasoreactivity in P7 rat brain could in part explain the inefficiency of ischemic postconditioning after ischemia-reperfusion.
doi:10.1371/journal.pone.0049695
PMCID: PMC3520965  PMID: 23251348
11.  The protective effect of early hypothermia on PTEN phosphorylation correlates with free radical inhibition in rat stroke 
We recently showed that intraischemic moderate hypothermia (30°C) reduces ischemic damage through the Akt pathway after permanent distal middle cerebral artery occlusion in rats. The only Akt pathway component preserved by hypothermia is phosphorylated phosphatase and tensin homolog deleted on chromosome 10 (p-PTEN), which suggests that p-PTEN may have a central role in neuroprotection. Reactive oxygen species (ROS) are critically involved in mediating ischemic damage after stroke by interacting with signaling molecules, including Akt, PTEN, and δ-protein kinase C (PKC). We investigated the protective mechanisms of moderate hypothermia on these signaling proteins after transient focal ischemia in rats. Early moderate hypothermia (3 h) was administered 15 mins before reperfusion, and delayed moderate hypothermia (3 h) was applied 15 mins after reperfusion. Our results indicate that early hypothermia reduced infarction, whereas delayed hypothermia did not. However, both early and delayed hypothermia maintained levels of Mn-SOD (superoxide dismutase) and phosphorylated Akt and blocked δ-PKC cleavage, suggesting that these factors may not be critical to the protection of hypothermia. Nevertheless, early hypothermia preserved p-PTEN levels after reperfusion, whereas delayed hypothermia did not. Furthermore, ROS inhibition maintained levels of p-PTEN after stroke. Together, these findings suggest that phosphorylation levels of PTEN are closely associated with the protective effect of early hypothermia against stroke.
doi:10.1038/jcbfm.2009.81
PMCID: PMC3221613  PMID: 19553907
focal ischemia; hypothermia; neuroprotection; stroke
12.  Isoflurane preconditioning increases B-cell lymphoma-2 expression and reduces cytochrome c release from the mitochondria in the ischemic penumbra of rat brain 
European journal of pharmacology  2008;586(1-3):106-113.
We and others have shown that prior exposure to the volatile anesthetic isoflurane induces ischemic tolerance in the brain. Our results also suggest that isoflurane preconditioning reduces cell apoptosis in the penumbral region of rat brain. We designed this study to determine whether isoflurane preconditioning decreased mitochondria-dependent cell apoptosis. Adult male Sprague-Dawley rats were exposed to or not exposed to 2% isoflurane for 30 min at 24 h before the permanent middle cerebral arterial occlusion. Western blotting was used to quantify protein expression in the cytosolic and mitochondrial fractions of non-ischemic brain cortex and brain cortex in the ischemic core and penumbra. Isoflurane preconditioning significantly decreased the infarct volume of cerebral cortex and improved neurological outcome. Isoflurane increased the expression of the antiapoptotic B-cell lymphoma-2 (Bcl-2) proteins in the cerebral cortex of rats without brain ischemia. Rats preconditioned with isoflurane before brain ischemia had increased Bcl-2 expression in the penumbra. Isoflurane preconditioning reduced the release of cytochrome c from the mitochondria and the activation of caspase 3 in the penumbra. However, isoflurane preconditioning did not alter the translocation of Bid and Bax from the cytosol to the mitochondria, identified mechanisms for Bcl-2 to block the release of cytochrome c from the mitochondria. Our results suggest that isoflurane preconditioning increases Bcl-2 expression to block the release of cytochrome c from the mitochondria to decrease the cell apoptosis in the penumbra.
doi:10.1016/j.ejphar.2008.02.073
PMCID: PMC2429852  PMID: 18355806
isoflurane; preconditioning; neuroprotection; cytochrome c; Bcl-2; mitochondria
13.  From Rapid to Delayed and Remote Postconditioning: the Evolving Concept of Ischemic Postconditioning in Brain Ischemia 
Current Drug Targets  2012;13(2):173-187.
Ischemic postconditioning is a concept originally defined to contrast with that of ischemic preconditioning. While both preconditioning and postconditioning confer a neuroprotective effect on brain ischemia, preconditioning is a sublethal insult performed in advance of brain ischemia, and postconditioning, which conventionally refers to a series of brief occlusions and reperfusions of the blood vessels, is conducted after ischemia/reperfusion. In this article, we first briefly review the history of preconditioning, including the experimentation that initially uncovered its neuroprotective effects and later revealed its underlying mechanisms-of-action. We then discuss how preconditioning research evolved into that of postconditioning – a concept that now represents a broad range of stimuli or triggers, including delayed postconditioning, pharmacological postconditioning, remote postconditioning – and its underlying protective mechanisms involving the Akt, MAPK, PKC and KATP channel cell-signaling pathways. Because the concept of postconditioning is so closely associated with that of preconditioning, and both share some common protective mechanisms, we also discuss whether a combination of preconditioning and postconditioning offers greater protection than preconditioning or postconditioning alone.
PMCID: PMC3346695  PMID: 22204317
postconditioning; preconditioning; stroke; cerebral ischemia; focal ischemia; neuroprotection
14.  Ischemic tolerance modulates TRAIL expression and its receptors and generates a neuroprotected phenotype 
Cell Death & Disease  2014;5(7):e1331-.
TNF-related apoptosis inducing ligand (TRAIL), a member of the TNF superfamily released by microglia, appears to be involved in the induction of apoptosis following focal brain ischemia. Indeed, brain ischemia is associated with progressive enlargement of damaged areas and prominent inflammation. As ischemic preconditioning reduces inflammatory response to brain ischemia and ameliorates brain damage, the purpose of the present study was to evaluate the role of TRAIL and its receptors in stroke and ischemic preconditioning and to propose, by modulating TRAIL pathway, a new therapeutic strategy in stroke. In order to achieve this aim a rat model of harmful focal ischemia, obtained by subjecting animals to 100 min of transient occlusion of middle cerebral artery followed by 24 h of reperfusion and a rat model of ischemic preconditioning in which the harmful ischemia was preceded by 30 mins of tMCAO, which represents the preconditioning protective stimulus, were used. Results show that the neuroprotection elicited by ischemic preconditioning occurs through both upregulation of TRAIL decoy receptors and downregulation of TRAIL itself and of its death receptors. As a counterproof, immunoneutralization of TRAIL in tMCAO animals resulted in significant restraint of tissue damage and in a marked functional recovery. Our data shed new light on the mechanisms that propagate ongoing neuronal damage after ischemia in the adult mammalian brain and provide new molecular targets for therapeutic intervention. Strategies aimed to repress the death-inducing ligands TRAIL, to antagonize the death receptors, or to activate the decoy receptors open new perspectives for the treatment of stroke.
doi:10.1038/cddis.2014.286
PMCID: PMC4123080  PMID: 25032854
15.  Post-ischemic estradiol treatment reduced glial response and triggers distinct cortical and hippocampal signaling in a rat model of cerebral ischemia 
Background
Estradiol has been shown to exert neuroprotective effects in several neurodegenerative conditions, including cerebral ischemia. The presence of this hormone prior to ischemia attenuates the damage associated with such events in a rodent model (middle cerebral artery occlusion (MCAO)), although its therapeutic value when administered post-ischemia has not been assessed. Hence, we evaluated the effects of estradiol treatment after permanent MCAO (pMCAO) was induced in rats, studying the PI3K/AKT/GSK3/β-catenin survival pathway and the activation of SAPK-JNK in two brain areas differently affected by pMCAO: the cortex and hippocampus. In addition, we analyzed the effect of estradiol on the glial response to injury.
Methods
Male rats were subjected to pMCAO and estradiol (0.04 mg/kg) was administered 6, 24, and 48 h after surgery. The animals were sacrificed 6 h after the last treatment, and brain damage was evaluated by immunohistochemical quantification of ‘reactive gliosis’ using antibodies against GFAP and Iba1. In addition, Akt, phospho-AktSer473, phospho-AktThr308, GSK3, phospho-GSK3Ser21/9, β-catenin, SAPK-JNK, and pSAPK-JNKThr183/Tyr185 levels were determined in western blots of the ipsilateral cerebral cortex and hippocampus, and regional differences in neuronal phospho-Akt expression were determined by immunohistochemistry.
Results
The increases in the percentage of GFAP- (5.25-fold) and Iba1- (1.8-fold) labeled cells in the cortex and hippocampus indicate that pMCAO induced ‘reactive gliosis’. This effect was prevented by post-ischemic estradiol treatment; diminished the number of these cells to those comparable with control animals. pMCAO down-regulated the PI3K/AkT/GSK3/β-catenin survival pathway to different extents in the cortex and hippocampus, the activity of which was restored by estradiol treatment more efficiently in the cerebral cortex (the most affected region) than in the hippocampus. No changes in the phosphorylation of SAPK-JNK were observed 54 h after inducing pMCAO, whereas pMCAO did significantly decrease the phospho-AktSer473 in neurons, an effect that was reversed by estradiol.
Conclusion
The present study demonstrates that post-pMCAO estradiol treatment attenuates ischemic injury in both neurons and glia, events in which the PI3K/AKT/GSK3/β-catenin pathway is at least partly involved. These findings indicate that estradiol is a potentially useful treatment to enhance recovery after human ischemic stroke.
doi:10.1186/1742-2094-9-157
PMCID: PMC3414748  PMID: 22747981
MCAO; Focal ischemia; Rat; Estradiol; Brain; Estrogen; Neuroprotection; Stroke; Western blot; Immunohistochemistry; Akt
16.  Redox Signaling Triggers Protection During The Reperfusion Rather Than The Ischemic Phase Of Preconditioning 
Basic research in cardiology  2008;103(4):378-384.
In ischemic preconditioning (IPC) brief ischemia/reperfusion renders the heart resistant to infarction from any subsequent ischemic insult. Protection results from binding of surface receptors by ligands released during the preconditioning ischemia. The downstream pathway involves redox signaling as IPC will not protect in the presence of a free radical scavenger. To determine when the redox signaling occurs, five groups of isolated rabbit hearts were studied. All hearts underwent 30 min of coronary branch occlusion and 2 h of reperfusion. IPC groups were subjected to 5 min of regional ischemia followed by 10 min of reperfusion prior to the 30-min coronary occlusion. The Control group had only the 30-min occlusion and 2-h reperfusion. The second group had IPC alone. The third group was also preconditioned, but the free radical scavenger N-2-mercaptopropionyl glycine (MPG, 300 µM) was infused during the 10-min reperfusion and therefore was present in the myocardium in the distribution of the snared coronary artery during the entire reperfusion phase and also during the subsequent 30-min ischemia. In another preconditioned group MPG was added to the perfusate before the preconditioning ischemia and therefore was present in the tissue only during the preconditioning ischemia and then was washed out during reperfusion. In the fifth group MPG was added to the perfusate for only the last 5 min of the preconditioning reperfusion and therefore was present in the tissue during the last minutes of the reperfusion phase and the 30 min of ischemia. Infarct size and risk size were measured by triphenyltetrazolium staining and fluorescent microspheres, resp. IPC reduced infarct size from 31.3±2.7% of the ischemic zone in control hearts to only 8.4±1.9%. MPG completely blocked IPC’s protection in the 3rd group (39.4±2.8%) but did not affect its protection in groups 4 (8.1±1.5%) or 5 (7.8±1.1%). Hence redox signaling occurs during the reperfusion phase of IPC.
doi:10.1007/s00395-008-0718-z
PMCID: PMC2670099  PMID: 18347834
17.  Cardioprotection by postconditioning in conscious rats is limited to coronary occlusions <45 min 
Objectives
Brief episodes of ischemia and reperfusion after a lethal ischemic insult confer cardioprotection, a phenomenon termed “ischemic postconditioning.” However, all studies reported to date have been conducted in open-chest animal models. We sought to determine whether postconditioning occurs in conscious animals and whether it protects against severe myocardial injury.
Methods
Chronically instrumented rats were assigned to a 30- (Subset 1), 45- (Subset 2), or 60-min (Subset 3) coronary occlusion followed by 24 h of reperfusion. In each subset, rats received no further intervention (control), were preconditioned with 12 cycles of 2-min occlusion/2-min reperfusion immediately (early preconditioning; EPC) or 24 h (late preconditioning; LPC) before myocardial infarction, or were postconditioned with 20 cycles of 10-s occlusion/10-s reperfusion immediately after myocardial infarction (20-10 PostC).
Results
With a 30-min occlusion, infarct size (54.4 ± 2.3% of risk region in control-30) was significantly reduced in EPC-30, LPC-30, and 20-10 PostC-30 groups (by 72, 70, and 47%, respectively; all P < 0.05 vs. control-30). With a 45-min occlusion, infarct size (62.2 ± 2.4% in control-45) was reduced in EPC-45 and LPC-45 groups (by 47 and 41%, respectively; all P < 0.05 vs. control-45) but not in the 20-10 PostC-45 group [55.4 ± 2.3%, P = not significant (NS) vs. control-45]. With a 60-min occlusion, infarct size (72.7 ± 2.2% in control-60) was reduced in the EPC-60 (by 20%, P < 0.05) but not in the LPC-60 (63.6 ± 2.5%, P = NS) or in the 20-20 PostC group (71.5 ± 3.4%, P = NS).
Conclusions
Both early and late ischemic preconditioning as well as ischemic postconditioning confer protection in conscious rats; however, unlike early preconditioning, postconditioning protects only against coronary occlusions <45 min. In the conscious rat, the cardioprotection afforded by postconditioning is limited to mild to moderate myocardial injury.
doi:10.1152/ajpheart.00479.2006
PMCID: PMC3741072  PMID: 16815986
myocardium; ischemia; infarct size; preconditioning
18.  Involvement of Akt in preconditioning-induced tolerance to ischemia in PC12 cells 
The serine–threonine protein kinase Akt has been identified as an important mediator of cell survival able to counteract apoptotic stimuli. However, hibernation, a model of natural tolerance to cerebral ischemia, is associated with downregulation of Akt. We previously established a model of ischemic tolerance in a PC12 cell line and using this model we now addressed the question whether ischemic tolerance also downregulates Akt in PC12 cells. Kinetic studies showed decreased Akt phosphorylation in tolerized cells. Similarly, phosphorylated levels of three major targets of Akt and well-known proapoptotic factors, the glycogen synthase kinase 3 (GSK-3), a Forkhead family member, FoxO4, and the protein murine double minute 2 (MDM2), all inactivated upon phosphorylation by Akt, were decreased in preconditioned cells. In addition, pharmacological blockade of the phosphoinositide 3-kinase (PI3K)/Akt pathway reduced cell death induced by oxygen and glucose deprivation (OGD) and increased the protective effect of preconditioning (PC). Furthermore, decreasing availability of P-Akt by transfecting PC12 cells with constructs of inactive Akt also resulted in protection against OGD and potentiation of the protective effect of PC. Depending on the environment, GSK-3, FOXO-4, and MDM2 can trigger apoptotic responses or cell cycle arrest, and thus, in a situation of reduced energy, driving the cells into a state of quiescence might be neuroprotective. This work suggests that in the context of tolerance downregulation of Akt is beneficial.
doi:10.1038/sj.jcbfm.9600286
PMCID: PMC1855183  PMID: 16511503
Akt; FoxO4; GSK-3; ischemic tolerance; MDM2; PC12 cells
19.  Evaluation of the effects of ischemic preconditioning on the hematological parameters of rats subjected to intestinal ischemia and reperfusion 
Clinics  2015;70(1):61-68.
OBJECTIVES:
Intestinal ischemia/reperfusion often leads to acute lung injury and multiple organ failure. Ischemic preconditioning is protective in nature and reduces tissue injuries in animal and human models. Although hematimetric parameters are widely used as diagnostic tools, there is no report of the influence of intestinal ischemia/reperfusion and ischemic preconditioning on such parameters. We evaluated the hematological changes during ischemia/reperfusion and preconditioning in rats.
METHODS:
Forty healthy rats were divided into four groups: control, laparotomy, intestinal ischemia/reperfusion and ischemic preconditioning. The intestinal ischemia/reperfusion group received 45 min of superior mesenteric artery occlusion, while the ischemic preconditioning group received 10 min of short ischemia and reperfusion before 45 min of prolonged occlusion. A cell counter was used to analyze blood obtained from rats before and after the surgical procedures and the hematological results were compared among the groups.
RESULTS:
The results showed significant differences in hematimetric parameters among the groups. The parameters that showed significant differences included lymphocyte, white blood cells and granulocyte counts; hematocrit; mean corpuscular hemoglobin concentration; red cell deviation width; platelet count; mean platelet volume; plateletcrit and platelet distribution width.
CONCLUSION:
The most remarkable parameters were those related to leukocytes and platelets. Some of the data, including the lymphocyte and granulocytes counts, suggest that ischemic preconditioning attenuates the effect of intestinal ischemia/reperfusion on circulating blood cells. Our work contributes to a better understanding of the hematological responses after intestinal ischemia/reperfusion and IPC, and the present findings may also be used as predictive values.
doi:10.6061/clinics/2015(01)11
PMCID: PMC4321002
Ischemic Reperfusion Injury; Ischemic Preconditioning; Systemic Inflammatory Response; Intestinal Ischemia; Hemocytometry; Superior Mesenteric Artery Occlusion
20.  Remote postconditioning by humoral factors in effluent from ischemic preconditioned rat hearts is mediated via PI3K/Akt-dependent cell-survival signaling at reperfusion 
Basic Research in Cardiology  2010;106(1):135-145.
Short non-lethal ischemic episodes administered to hearts prior to (ischemic preconditioning, IPC) or directly after (ischemic postconditioning, IPost) ischemic events facilitate myocardial protection. Transferring coronary effluent collected during IPC treatment to un-preconditioned recipient hearts protects from lethal ischemic insults. We propose that coronary IPC effluent contains hydrophobic cytoprotective mediators acting via PI3K/Akt-dependent pro-survival signaling at ischemic reperfusion. Ex vivo rat hearts were subjected to 30 min of regional ischemia and 120 min of reperfusion. IPC effluent administered for 10 min prior to index ischemia attenuated infarct size by ≥55% versus control hearts (P < 0.05). Effluent administration for 10 min at immediate reperfusion (reperfusion therapy) or as a mimetic of pharmacological postconditioning (remote postconditioning, RIPost) significantly reduced infarct size compared to control (P < 0.05). The IPC effluent significantly increased Akt phosphorylation in un-preconditioned hearts when administered before ischemia or at reperfusion, while pharmacological inhibition of PI3K/Akt-signaling at reperfusion completely abrogated the cardioprotection offered by effluent administration. Fractionation of coronary IPC effluent revealed that cytoprotective humoral mediator(s) released during the conditioning phase were of hydrophobic nature as all hydrophobic fractions with molecules under 30 kDa significantly reduced infarct size versus the control and hydrophilic fraction-treated hearts (P < 0.05). The total hydrophobic effluent fraction significantly reduced infarct size independently of temporal administration (before ischemia, at reperfusion or as remote postconditioning). In conclusion, the IPC effluent retains strong cardioprotective properties, containing hydrophobic mediator(s) < 30 kDa offering cytoprotection via PI3K/Akt-dependent signaling at ischemic reperfusion.
doi:10.1007/s00395-010-0133-0
PMCID: PMC3012213  PMID: 21103992
Postconditioning; Preconditioning; Cardioprotection; Ischemia; Reperfusion; Akt
21.  Mechanisms and Prospects of Ischemic Tolerance Induced by Cerebral Preconditioning 
In the brain, brief episodes of ischemia induce tolerance against a subsequent severe episode of ischemia. This phenomenon of endogenous neuroprotection is known as preconditioning-induced ischemic tolerance. The purpose of this review is to summarize the current state of knowledge about mechanisms and potential applications of cerebral preconditioning and ischemic tolerance. Articles related to the terms ischemic preconditioning and ischemic tolerance were systematically searched via MEDLINE/PubMed, and articles published in English related to the nervous system were selected and analyzed. The past two decades have provided interesting insights into the molecular mechanisms of this neuroprotective phenomenon. Although both rapid and delayed types of tolerance have been documented in experimental settings, the delayed type has been found to be more prominent in the case of neuronal ischemic tolerance. Many intracellular signaling pathways have been implicated regarding ischemic preconditioning. Most of these are associated with membrane receptors, kinase cascades, and transcription factors. Moreover, ischemic tolerance can be induced by exposing animals or cells to diverse types of endogenous and exogenous stimuli that are not necessarily hypoxic or ischemic in nature. These cross-tolerances raise the hope that, in the future, it will be possible to pharmacologically activate or mimic ischemic tolerance in the human brain. Another promising approach is remote preconditioning in which preconditioning of one organ or system leads to the protection of a different (remote) organ that is difficult to target, such as the brain. The preconditioning strategy and related interventions can confer neuroprotection in experimental ischemia, and, thus, have promise for practical applications in cases of vascular neurosurgery and endo-vascular therapy.
doi:10.5213/inj.2010.14.4.203
PMCID: PMC3021810  PMID: 21253330
Cerebral ischemia; Preconditioning; Ischemic tolerance; Pharmacological preconditioning; Remote preconditioning
22.  Isoflurane postconditioning induces neuroprotection via Akt activation and attenuation of increased mitochondrial membrane permeability 
Neuroscience  2011;199:44-50.
We have shown that isoflurane application at the onset of reperfusion (postconditioning) reduces brain ischemic injury in rats. This study was designed to determine whether this protection involved activation of prosurvival protein kinases and maintenance of normal mitochondrial membrane permeability. Two-month old male rats were subjected to a 90-min middle cerebral arterial occlusion. They then were exposed or were not exposed to 2% isoflurane for 1 h. Ischemic penumbral cerebral cortex was harvested immediately and separated into the mitochondrial and cytosolic fractions. We showed that the mitochondrial nicotinamide adenine dinucleotide content in the ischemic penumbral cortex was significantly reduced, suggesting an increased mitochondrial membrane permeability. This increase was partly attenuated by isoflurane postconditioning. The mitochondrial adenosine diphosphate content in the penumbral cortex was reduced no matter whether the animals were postconditioned with isoflurane. The mitochondrial adenosine triphosphate concentration was not different among various experimental conditions. The phospho-Akt in the cytosolic and mitochondrial fractions of the ischemic penumbral cortex was higher than that in the control cortex. This increase trended to be higher in animals with isoflurane postconditioning. A similar change pattern was observed in the mitochondrial phospho-glycogen synthase kinase 3β, an Akt substrate that can regulate the mitochondrial membrane permeability. Isoflurane postconditioning reduced oxygen-glucose deprivation-induced injury of rat cortical neuronal cultures and increased phospho-Akt in these cells. The isoflurane postconditioning-induced protection in the neuronal cultures was decreased by the Akt inhibitor LY294002. These results suggest that isoflurane postconditioning effects may be mediated by Akt and involve reduced mitochondrial membrane permeability.
doi:10.1016/j.neuroscience.2011.10.022
PMCID: PMC3237819  PMID: 22040798
Akt; glycogen synthase kinase 3β; isoflurane; neuroprotection; mitochondrial membrane permeability; postconditioning
23.  Induction of manganese superoxide dismutase in rat cardiac myocytes increases tolerance to hypoxia 24 hours after preconditioning. 
Journal of Clinical Investigation  1994;94(6):2193-2199.
Manganese superoxide dismutase (Mn-SOD) is induced in ischemic hearts 24 h after ischemic preconditioning, when tolerance to ischemia is acquired. We examined the relationship between Mn-SOD induction and the protective effect of preconditioning using cultured rat cardiac myocytes. Exposure of cardiac myocytes to brief hypoxia (1 h) decreased creatine kinase release induced by sustained hypoxia (3 h) that follows when the sustained hypoxia was applied 24 h after hypoxic preconditioning (57% of that in cells without preconditioning). The activity and content of Mn-SOD in cardiac myocytes were increased 24 h after hypoxic preconditioning (activity, 170%; content, 139% compared with cells without preconditioning) coincidentally with the acquisition of tolerance to hypoxia. Mn-SOD mRNA was also increased 20-40 min after preconditioning. Antisense oligodeoxyribonucleotides corresponding to the initiation site of Mn-SOD translation inhibited the increases in the Mn-SOD content and activity and abolished the expected decrease in creatine kinase release induced by sustained hypoxia after 24 h of hypoxic preconditioning. Sense oligodeoxyribonucleotides did not abolish either Mn-SOD induction or tolerance to hypoxia. These results suggest that the induction of Mn-SOD in myocytes by preconditioning plays a pivotal role in the acquisition of tolerance to ischemia at a later phase (24 h) of ischemic preconditioning.
Images
PMCID: PMC330044  PMID: 7989574
24.  PI3K/Akt Pathway Contributes to Neurovascular Unit Protection of Xiao-Xu-Ming Decoction against Focal Cerebral Ischemia and Reperfusion Injury in Rats 
In the present study, we used a focal cerebral ischemia and reperfusion rat model to investigate the protective effects of Xiao-Xu-Ming decoction (XXMD) on neurovascular unit and to examine the role of PI3K (phosphatidylinositol 3-kinase)/Akt pathway in this protection. The cerebral ischemia was induced by 90 min of middle cerebral artery occlusion. Cerebral infarct area was measured by tetrazolium staining, and neurological function was observed at 24 h after reperfusion. DNA fragmentation assay, combined with immunofluorescence, was performed to evaluate apoptosis of neuron, astrocyte, and vascular endothelial cell which constitute neurovascular unit. The expression levels of proteins involved in PI3K/Akt pathway were detected by Western blot. The results showed that XXMD improved neurological function, decreased cerebral infarct area and neuronal damage, and attenuated cellular apoptosis in neurovascular unit, while these effects were abolished by inhibition of PI3K/Akt with LY294002. We also found that XXMD upregulated p-PDKl, p-Akt, and p-GSK3β expression levels, which were partly reversed by LY294002. In addition, the increases of p-PTEN and p-c-Raf expression levels on which LY294002 had no effect were also observed in response to XXMD treatment. The data indicated the protective effects of XXMD on neurovascular unit partly through the activation of PI3K/Akt pathway.
doi:10.1155/2013/459467
PMCID: PMC3678438  PMID: 23781261
25.  Akt isoforms differentially protect against stroke-induced neuronal injury by regulating mTOR activities 
Protein kinases Akt1 and Akt3 are considered to be more crucial to brain function than Akt2. We investigated the roles of Akt1 and Akt3 in stroke-induced brain injury and examined their interactions with the Akt/mTOR pathways. Focal ischemia was induced in rats. Lentiviral vectors expressing constitutively active Akt1 and Akt3 (cAkt1 and cAkt3) were injected into the ischemic cortex. Infarct sizes and gene and protein expressions in the Akt/mTOR pathways were evaluated. The results show that Akt1 and Akt3 proteins were degraded as early as 1 hour after stroke, whereas Akt2 proteins remained unchanged until 24 hours after stroke. Lentiviral-mediated overexpression of cAkt1 or cAkt3 reduced neuronal death after in vitro and in vivo ischemia. Interestingly, cAkt3 overexpression resulted in stronger protection than cAkt1 overexpression. Western blot analyses further showed that cAkt3 promoted significantly higher levels of phosphorylated Akt and phosphorylated mTOR than cAkt1. The mTOR inhibitor rapamycin blocked the protective effects of both cAkt1 and cAkt3. In conclusion, Akt isoforms are differentially regulated after stroke and Akt3 offers stronger protection than cAkt1 by maintaining Akt levels and promoting mTOR activity.
doi:10.1038/jcbfm.2013.132
PMCID: PMC3851893  PMID: 23942361
Akt1; Akt3; cerebral ischemia; mTOR; stroke

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