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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.
PMCID: PMC2696348  PMID: 18201834
preconditioning; remote preconditioning; limb preconditioning; cerebral ischemia; focal ischemia
2.  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.
PMCID: PMC2744502  PMID: 19631625
stroke; cerebral ischemia; preconditioning; remote postconditioning
3.  Delayed Postconditioning Protects against Focal Ischemic Brain Injury in Rats 
PLoS ONE  2008;3(12):e3851.
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.
Delayed postconditioning reduced ischemic injury after focal ischemia, which opens a new research avenue for stroke therapy and its underlying protective mechanisms.
PMCID: PMC2588536  PMID: 19066627
4.  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.
PMCID: PMC3520965  PMID: 23251348
5.  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.
PMCID: PMC3221613  PMID: 19553907
focal ischemia; hypothermia; neuroprotection; stroke
6.  Inhibition of Autophagy Contributes to Ischemic Postconditioning-Induced Neuroprotection against Focal Cerebral Ischemia in Rats 
PLoS ONE  2012;7(9):e46092.
Ischemic postconditioning (IPOC), or relief of ischemia in a stuttered manner, has emerged as an innovative treatment strategy to reduce programmed cell death, attenuate ischemic injuries, and improve neurological outcomes. However, the mechanisms involved have not been completely elucidated. Recent studies indicate that autophagy is a type of programmed cell death that plays elusive roles in controlling neuronal damage and metabolic homeostasis. This study aims to determine the role of autophagy in IPOC-induced neuroprotection against focal cerebral ischemia in rats.
Methodology/Principal Findings
A focal cerebral ischemic model with permanent middle cerebral artery (MCA) occlusion plus transient common carotid artery (CCA) occlusion was established. The autophagosomes and the expressions of LC3/Beclin 1/p62 were evaluated for their contribution to the activation of autophagy. We found that autophagy was markedly induced with the upregulation of LC3/Beclin 1 and downregulation of p62 in the penumbra at various time intervals following ischemia. IPOC, performed at the onset of reperfusion, reduced infarct size, mitigated brain edema, inhibited the induction of LC3/Beclin 1 and reversed the reduction of p62 simultaneously. Rapamycin, an inducer of autophagy, partially reversed all the aforementioned effects induced by IPOC. Conversely, autophagy inhibitor 3-methyladenine (3-MA) attenuated the ischemic insults, inhibited the activation of autophagy, and elevated the expression of anti-apoptotic protein Bcl-2, to an extent comparable to IPOC.
The present study suggests that inhibition of the autophagic pathway plays a key role in IPOC-induced neuroprotection against focal cerebral ischemia. Thus, pharmacological inhibition of autophagy may provide a novel therapeutic strategy for the treatment of stroke.
PMCID: PMC3461004  PMID: 23029398
7.  Kinetic Changes of COX-2 Expression during Reperfusion Period after Ischemic Preconditioning Play a Role in Protection Against Ischemic Damage in Rat Brain 
A brief ischemic insult induces significant protection against subsequent massive ischemic events. The molecular mechanisms known as preconditioning (PC)-induced ischemic tolerance are not completely understood. We investigated whether kinetic changes of cyclooxygenase (COX)-2 during reperfusion time-periods after PC were related to ischemic tolerance. Rats were given PC by occlusion of middle cerebral artery (MCAO) for 10 min and sacrificed after the indicated time-periods of reperfusion (1, 2, 4, 8, 12, 18 or 24 h). In PC-treated rats, focal ischemia was induced by occlusion of MCA for 24 h and brain infarct volume was then studied to determine whether different reperfusion time influenced the damage. We report that the most significant protection against focal ischemia was obtained in rats with 8 h reperfusion after PC. Administration of indomethacin (10 mg/kg, oral) or rofecoxib (5 mg/kg, oral) 48 h prior to PC counteracted the effect of PC. Immunohistochemical analysis showed that COX-2 and HO-1 protein were induced in PC-treated rat brain, which was significantly inhibited by rofecoxib. Taken together, we concluded that the kinetic changes of COX-2 expression during the reperfusion period after PC might be partly responsible for ischemic tolerance.
PMCID: PMC2788647  PMID: 19967067
Ischemic preconditioning; Stroke; Heme oxygenase; Cyclooxygenase
8.  The protective effects of T cell deficiency against brain injury are ischemic model-dependent in rats 
Neurochemistry international  2012;62(3):265-270.
Previous studies have reported that T cell deficiency reduced infarct sizes after transient middle cerebral artery (MCA) suture occlusion in mice. However, how reperfusion and different models affect the detrimental effects of T cells have not been studied. We investigated the effects of T cell deficiency in nude rats using two stroke models and compared their infarct sizes with those in WT rats. In the distal MCA occlusion (MCAo) model, the distal MCA was permanently occluded and the bilateral common carotid arteries (CCAs) were transiently occluded for 60 min. In the suture MCAo model, the MCA was transiently occluded for 100 min by the insertion of a monofilament suture. Our results showed that T cell deficiency resulted in about a 50% reduction in infarct size in the suture MCAo model, whereas it had no effect in the distal MCAo model, suggesting the protective effects of T cell deficiency are dependent on the ischemic model used. We further found more total T cells, CD4 T cells and CD8 T cells in the ischemic brains of WT rats in the suture MCAo model than in the distal MCAo model. In addition, we detected more CD68-expressing macrophages in the ischemic brains of WT rats than in nude rats in the suture MCAo but not the distal MCAo model. Lymphocyte reconstitution in nude rats resulted in larger infarct sizes in the suture MCAo, but not in the distal MCAo stroke model. The results of regional CBF measurement indicated a total reperfusion in the MCAo model but only a partial reperfusion in the distal MCAo model. In conclusion, the protective effects of T cell deficiency on brain injury are dependent on the ischemic model used; likely associated with different degrees of reperfusion.
PMCID: PMC3581747  PMID: 23228347
Stroke; focal ischemia; nude rats T cells
9.  Ischemic postconditioning may not influence early brain injury induced by focal cerebral ischemia/reperfusion in rats 
Korean Journal of Anesthesiology  2010;58(2):176-183.
Experimental studies have shown that ischemic postconditioning can reduce neuronal injury in the setting of cerebral ischemia, but the mechanisms are not yet clearly elucidated. This study was conducted to determine whether ischemic postconditioning can alter expression of heat shock protein 70 and reduce acute phase neuronal injury in rats subjected to transient focal cerebral ischemia/reperfusion.
Focal cerebral ischemia was induced by intraluminal middle cerebral artery occlusion for 60 min in twenty male Sprague-Dawley rats (250-300 g). Rats were randomized into control group and an ischemic postconditioning group (10 rats per group). The animals of control group had no intervention either before or after MCA occlusion. Ischemic postconditioning was elicited by 3 cycles of 30 s reperfusion interspersed by 10 s ischemia immediately after onset of reperfusion. The infarct ratios, brain edema ratios and motor behavior deficits were analyzed 24 hrs after ischemic insult. Caspase-3 reactive cells and cells showing heat shock protein 70 activity were counted in the caudoputamen and frontoparietal cortex.
Ischemic postconditiong did not reduce infarct size and brain edema ratios compared to control group. Neurologic scores were not significantly different between groups. The number of caspase-3 reactive cells in the ischemic postconditioning group was not significantly different than the value of the control group in the caudoputamen and frontoparietal cortex. The number of cells showing heat shock protein 70 activity was not significantly different than the control group, as well.
These results suggest that ischemic postconditioning may not influence the early brain damage induced by focal cerebral ischemia in rats.
PMCID: PMC2872862  PMID: 20498797
Focal cerebral ischemia; Neuroproctection; Postconditioning; Rat
10.  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
11.  Limited Therapeutic Time Windows of Mild-to-Moderate Hypothermia in a Focal Ischemia Model in Rat 
Stroke Research and Treatment  2011;2011:131834.
Although many studies have shown the great potential of induced hypothermia in stroke treatment, we recognize that there are limitations to the protective effects of hypothermia even in the laboratory. Here, we review our experiments on the protective effects of mild-to-moderate hypothermia in rats. Focal ischemia was induced by bilateral common carotid artery (CCA) occlusion for 1 to 2 hours combined with permanent or transient middle cerebral artery (MCA) occlusion. We compared the effects of mild (33°C) and moderate (30°C) hypothermia, evaluated therapeutic time windows, and studied the underlying mechanisms. On review, our findings revealed that the protective effects of induced mild hypothermia (33°C) were limited, and the therapeutic time window of even moderate hypothermia (30°C) was very short in our specific models, although this limitation might be due to the relatively brief periods of hypothermia used. In addition, we found that hypothermia reduced brain injury by preserving Akt activity, PTEN phosphorylation and εPKC activity, while inhibiting ROS production, and δPKC activity.
PMCID: PMC3159378  PMID: 21876846
12.  Autologous bone marrow mononuclear cells enhance recovery after acute ischemic stroke in young and middle-aged rats 
We investigated intra-arterially administered autologous bone marrow mononuclear cells (MNCs) in rats with acute ischemic stroke. Long Evans rats (2 to 3 months or 12 months old) underwent tandem reversible common carotid artery (CCA)/middle cerebral artery (MCA) occlusion (CCAo/MCAo) for 3 h and then 24 h later underwent tibial bone marrow harvest. Ten million or 4 million cells were re-injected by an intra-carotid infusion. Control animals underwent marrow needle insertion and then saline injection into the carotid artery. Animals were assessed on a battery of neurological tests. MNCs in the ischemic brain were tracked using Q-dot nanocrystal labeling. Infarct volume and cytokines in the ischemia-affected brain were analyzed. Cell-treated animals in the younger and older groups showed improvement from 7 to 30 days after stroke compared with vehicle-treated animals. MNCs significantly reduced infarct volume compared with saline. There was a significant reduction in tumor necrosis factor-α, interleukin-1α (IL-1α), IL-β, IL-6, and a significant increase in IL-10 in injured brains harvested from the cell-treated groups compared with saline controls. Labeled MNCs were found in the peri-infarcted area at 1 h and exponentially decreased over the ensuing week after injection. Autologous bone marrow MNCs can be safely harvested from rodents after stroke, migrate to the peri-infarct area, enhance recovery, and modulate the post-ischemic inflammatory response.
PMCID: PMC2893568  PMID: 19773802
bone marrow; cell therapy; stroke
13.  Autologous bone marrow mononuclear cells enhance recovery after acute ischemic stroke in young and middle-aged rats 
We investigated intra-arterially administered autologous bone marrow mononuclear cells (MNCs) in rats with acute ischemic stroke. Long Evans rats (2 to 3 months or 12 months old) underwent tandem reversible common carotid artery (CCA)/middle cerebral artery (MCA) occlusion (CCAo/MCAo) for 3 h and then 24 h later underwent tibial bone marrow harvest. Ten million or 4 million cells were re-injected by an intra-carotid infusion. Control animals underwent marrow needle insertion and then saline injection into the carotid artery. Animals were assessed on a battery of neurological tests. MNCs in the ischemic brain were tracked using Q-dot nanocrystal labeling. Infarct volume and cytokines in the ischemia-affected brain were analyzed. Cell-treated animals in the younger and older groups showed improvement from 7 to 30 days after stroke compared with vehicle-treated animals. MNCs significantly reduced infarct volume compared with saline. There was a significant reduction in tumor necrosis factor-α, interleukin-1α (IL-1α), IL-β, IL-6, and a significant increase in IL-10 in injured brains harvested from the cell-treated groups compared with saline controls. Labeled MNCs were found in the peri-infarcted area at 1 h and exponentially decreased over the ensuing week after injection. Autologous bone marrow MNCs can be safely harvested from rodents after stroke, migrate to the peri-infarct area, enhance recovery, and modulate the post-ischemic inflammatory response.
PMCID: PMC2893568  PMID: 19773802
bone marrow; cell therapy; stroke
14.  Neuroprotective Consequences of Postconditioning on Embolic Model of Cerebral Ischemia in Rat 
Objective(s): It has been reported that ischemic postconditioning, conducted by a series of brief occlusion and release of the bilateral common carotid arteries, confers neuroprotection in permanent or transient models of stroke. However, consequences of postconditioning on embolic stroke have not yet been investigated.
Materials and Methods: In the present study, rats were subjected to embolic stroke (n=30) or sham stroke (n=5). Stroke animals were divided into control (n=10) or three different patterns of postconditioning treatments (n=20). In the first pattern of postconditioning (PC10, n=10), the common carotid arteries (CCA) were occluded and reopened 10 and 30 sec, respectively for 5 cycles. Both occluding and releasing times in pattern 2 (PC30, n=5) and 3 (PC60, N=5) of postconditionings, were five cycles of 30 or 60 sec, respectively. Postconditioning was induced at 30 min following the stroke. Subsequently, cerebral blood flow (CBF) was measured from 5 min before to 60 min following to stroke induction. Infarct size, brain edema and neurological deficits and reactive oxygen species (ROS) level was measured two days later.
Results: While PC10 (P<0.001), PC30 and PC60 (P<0.05) significantly decreased infarct volume, only PC10 decreased brain edema and neurological deficits (P<0.05). Correspondingly, PC10 prevented the hyperemia of brain at 35, 40, 50 and 60 min after the embolic stroke (P<0.005). No significant difference in ROS level was observed between PC10 and control group.
Conclusion: Ischemic postconditioning reduces infarct volume and brain edema, decreases hyperemia following to injury and improves neurological functions after the embolic model of stroke.
PMCID: PMC3843857  PMID: 24298382
15.  The Effect of Treadmill Training Pre-Exercise on Glutamate Receptor Expression in Rats after Cerebral Ischemia 
Physical exercise has been demonstrated to be neuroprotective in both clinical and laboratory settings. However, the exact mechanism underlying this effect is unclear. Our study aimed to investigate whether pre-ischemic treadmill training could serve as a form of ischemic preconditioning in a rat model undergoing middle cerebral artery occlusion (MCAO). Thirty-six rats were divided into three groups: a sham control group, a non-exercise with operation group and an exercise with operation group. After treadmill training, ischemia was induced by occluding the MCA for 2 h, followed by reperfusion. Half of the rats in each group were sacrificed for mRNA detection of mGluR5 and NR2B 80 min after occlusion. The remaining animals were evaluated for neurological deficits by behavioral scoring and then decapitated to assess the infarct volume. The mRNA expression of mGluR5 and NR2B was detected by real-time PCR. The results suggest that pre-ischemic treadmill training may induce brain ischemic tolerance by reducing the mRNA levels of mGluR5 and NR2B, and thus, the results indicate that physical exercise might be an effective method to establish ischemic preconditioning.
PMCID: PMC2920558  PMID: 20717528
mGluR5; NR2B; real-time PCR; brain ischemic tolerance; ischemic preconditioning
16.  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.
PMCID: PMC2429852  PMID: 18355806
isoflurane; preconditioning; neuroprotection; cytochrome c; Bcl-2; mitochondria
17.  Cardioprotection by postconditioning in conscious rats is limited to coronary occlusions <45 min 
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.
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).
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).
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.
PMCID: PMC3741072  PMID: 16815986
myocardium; ischemia; infarct size; preconditioning
18.  Remote ischemic preconditioning protects neurocognitive function of rats following cerebral hypoperfusion 
Protection of remote ischemic preconditioning on neurocognitive function caused by bilateral common carotid artery occlusion has been investigated in rats.
Thirty-six male Sprague-Dawley rats were divided into 3 groups – control group (Group C, n=12), bilateral carotid arteries occlusion group (Group B, n=12) and remote ischemic precondition group (Group P, n=12). In Group P, remote ischemic preconditioning (RIPC) was performed on the right femoral artery with 3 cycles (10 min) of occlusion/perfusion. After 3 cycles of preconditioning, bilateral carotid arteries were occluded immediately for 60 min. In Group B, ischemic insults were conducted without RIPC. Sham surgeries were performed in Group C. Evaluation of memory and learning capacity was performed on days 5–8 after surgery by Morris water maze testing of spatial learning capacity (n=6 for each group). Apoptosis of cells in the hippocampus region was determined by TUNEL tests and Bcl-2 at this region was determined by ELISA 24 h and 9 days after vessel occlusion (n=6 for each group).
Neurocognitive tests showed that latency time was significantly longer in Group B than in Group P on day 7 (p=0.016) and day 8 (p=0.036). Moreover, frequency of platform crossings was significant less in group B than in the other 2 groups on day 9. Bcl-2 level was significantly increased in the hippocampal region of rats in Group P on days 1 and 9 after vessel occlusion. TUNEL test showed that apoptosis could be observed at 24 h after occlusion in Group B, but not in Group P and Group C. No apoptosis was observed on day 9.
Our results suggest that RIPC can protect neurocognitive function of rats after bilateral carotid occlusions, and that Bcl-2 may play an important role in this protective effect.
PMCID: PMC3539484  PMID: 22037731
remote ischemic preconditioning; neurocognition; water maze; Bcl-2
19.  The effects of peripheral leukocytes on the hippocampal neuronal changes in transient global ischemia and unilateral cerebral hemispheric infarction. 
Journal of Korean Medical Science  1999;14(3):304-314.
The participation of activated leukocytes and subsequent production of chemical mediators has been well accepted in the pathophysiology of hypoxic-ischemic injury. This study was performed to see the effects of leukocytes on hippocampal neuronal damage in transient global ischemia induced by 10-min occlusion of bilateral common carotid arteries (CCAs) with reperfusion for various times, and in complete unilateral ischemia induced by 24-hr ligation of left CCA. Leukopenia was induced by intraperitoneal injection of cyclophosphamide for 4 days. The results showed that hippocampal neuronal damages were worse at 6-hr reperfusion in leukopenic experimental group than in the control group. In comparison, 24-hr and 3-day reperfusion leukopenic groups showed less numbers of damaged neurons and milder changes. The 5-day reperfusion group showed inconsistent changes. Unilateral CCA occlusion showed extensive infarction in 83.3% of gerbils in the control group, compared to 25% of gerbils in the experimental group (p<0.05). These results strongly suggest that the number of peripheral leukocytes were closely related to the development of delayed neuronal damage of hippocampus in transient global ischemia and the incidence of infarction induced by 24-hr unilateral CCA ligation.
PMCID: PMC3054372  PMID: 10402174
20.  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
21.  Protection of ischemic post conditioning against transient focal ischemia-induced brain damage is associated with inhibition of neuroinflammation via modulation of TLR2 and TLR4 pathways 
Background and purpose
Ischemic postconditioning has been demonstrated to be a protective procedure to brain damage caused by transient focal ischemia/reperfusion. However, it is elusive whether the protection of postconditioning against brain damage and neuroinflammation is via regulating TLR2 and TLR4 pathways. In the present study, we examined the protection of ischemic postconditioning performed immediately prior to the recovery of cerebral blood supply on brain damage caused by various duration of ischemia and tested the hypothesis that its protection is via inhibition of neuroinflammation by modulating TLR2/TLR4 pathways.
Brain damage in rats was induced by using the middle cerebral artery occlusion (MCAO) model. Ischemic postconditioning consisting of fivecycles of ten seconds of ischemia and reperfusion was performed immediately following theischemic episode Theduration of administration of ischemic postconditioning was examined by comparing its effects on infarction volume, cerebral edema and neurological function in 2, 3, 4, 4.5and 6 hour ischemia groups. The protective mechanism of ischemic postconditioning was investigated by comparing its effects on apoptosis, production of the neurotoxic cytokine IL-1β and the transcription and expression of TLR2, TLR4 and IRAK4 in the 2 and 4.5 hour ischemia groups.
Ischemic postconditioning significantly attenuated cerebral infarction, cerebral edema and neurological dysfunction in ischemia groups of up to 4 hours duration, but not in 4.5and 6 hour ischemia groups. It also inhibited apoptosis, production of IL-1β, abnormal transcription and expression of TLR2, TLR4 and IRAK4 in the 2 hour ischemia group, but not in the 4.5 hour ischemia group.
Ischemic postconditioning protected brain damage caused by 2, 3 and 4 hours of ischemia, but not by 4.5 and 6 hours of ischemia. The protection of ischemic postconditioning is associated with its inhibition of neuroinflammation via inhibition of TLR2 and TLR4 pathways.
PMCID: PMC3908918  PMID: 24460643
Ischemic postconditioning; Cerebral ischemia/reperfusion; TLR2; TLR4; Neuroinflammation
22.  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.
PMCID: PMC2746404  PMID: 18182053
Akt; cerebral ischemia; mitogen-activated protein kinase; postconditioning; protein kinase C; β-catenin
23.  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.
PMCID: PMC3012213  PMID: 21103992
Postconditioning; Preconditioning; Cardioprotection; Ischemia; Reperfusion; Akt
24.  Repetitive hypoxia extends endogenous neurovascular protection for stroke 
Annals of neurology  2011;69(6):975-985.
Brief systemic hypoxia protects the rodent brain from subsequent ischemic injury, although the protection wanes within days. We hypothesized that the duration of ischemic tolerance could be extended from days to months by repeated intermittent hypoxia of varying magnitude and duration.
Infarction volumes following a 60-min transient middle cerebral artery occlusion were determined in adult male mice 2 days through 8 wks after completion of a 2-week repetitive hypoxic preconditioning (RHP) protocol. Separate cohorts were studied for the protective effects of RHP on postischemic and cytokine-induced cerebrovascular inflammation, and for potential deleterious effects of the RHP stimulus itself.
RHP protection against transient focal stroke persisted for 8 weeks. Leukocyte adherence to cortical venules was attenuated in response to stroke, as well as following TNF-α administration, indicating that reductions in postischemic inflammation were not secondary to smaller infarct volumes. RHP reduced post-stroke leukocyte diapedesis concomitant with a long-lasting downregulation of endothelial adhesion molecule mRNAs, and also reduced postischemic blood-brain barrier permeability to endogenous IgG. RHP was without effect on hippocampal CA1 pyramidal cell viability, only transiently elevated hematocrit, and did not affect the magnitude of CBF during and after ischemia.
Taken together, our findings reveal a novel form of epigenetic neurovascular plasticity characterized by a prominent anti-inflammatory phenotype that provides protection against stroke many weeks longer than previously established windows of preconditioning-induced tolerance. Translating these endogenous protective mechanisms into therapeutics could afford sustained periods of cerebroprotection in subpopulations of individuals at identified risk for stroke.
PMCID: PMC3117913  PMID: 21437933
25.  Activated Protein C Protects Myocardium Via Activation of Anti-apoptotic Pathways of Survival in Ischemia-reperfused Rat Heart 
Journal of Korean Medical Science  2010;25(11):1609-1615.
Activated protein C (APC) is known to be beneficial on ischemia reperfusion injury in myocardium. However, the protection mechanism of APC is not fully understood. The purpose of this study was to investigate the effects and possible mechanisms of APC on myocardial ischemic damage. Artificially ventilated anaesthetized Sprague-Dawley rats were subjected to a 30 min of left anterior descending coronary artery occlusion followed by 2 hr of reperfusion. Rats were randomly divided into four groups; Sham, I/R, APC preconditioning and postconditioning group. Myocardial infarct size, apoptosis index, the phosphorylation of ERK1/2, Bcl-2, Bax and cytochrome c genes and proteins were assessed. In APC-administrated rat hearts, regardless of the timing of administration, infarct size was consistently reduced compared to ischemia/reperfusion (I/R) rats. APC improved the expression of ERK1/2 and anti-apoptotic protein Bcl-2 which were significantly reduced in the I/R rats. APC reduced the expression of pro-apoptotic genes, Bax and cytochrome c. These findings suggest that APC produces cardioprotective effect by preserving the expression of proteins and genes involved in anti-apoptotic pathways, regardless of the timing of administration.
PMCID: PMC2966998  PMID: 21060750
Activated Protein C; Cardioprotection; Reperfusion Injury, ERK1/2; bcl-2-Associated X Protein

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