Current studies regarding the underlying protective mechanisms of postconditioning focus on rapid ischemic postconditioning. Since rapid ischemic postconditioning interrupts early reperfusion, its protective effects must be closely associated with changes in cerebral blood flow (CBF) after reperfusion and with subsequent events, such as free radical production, endothelial function, and changes in blood brain barrier (BBB) integrity and inflammation that occur due to interrupted CBF. In our studies, we first confirmed whether rapid postconditioning attenuates the hyperemic response induced by reperfusion, and whether it mitigates hypotension thereafter. CBF was measured by a Laser Doppler Probe in the penumbra in rats subjected to 15 or 30 minutes of bilateral CCA occlusion combined with permanent MCA occlusion [31
]. We detected a clear hyperemic response after reperfusion in rats subjected to a 15 minute occlusion, and CBF was recovered to pre-ischemic levels in rats with 30 minutes of occlusion [31
]. We showed that rapid postconditioning with mechanical interruption results in CBF changes, and CBF at 30 minutes after reperfusion was improved [32
]. Wang and colleagues confirmed this effect in a global ischemia model [36
Next, we examined whether rapid postconditioning attenuates reactive oxygen species (ROS) production and apoptosis as early reperfusion is known to cause increased ROS products leading to apoptosis. We found that rapid postconditioning profoundly attenuates the amount of superoxide at 30 minutes after reperfusion in the model of 30 minutes CCA occlusion plus permanent MCA occlusion [31
]. Consistent with our findings, Xing and colleagues reported that rapid postconditioning attenuates lipid peroxidase levels in a focal ischemia model [33
] and Danielisova and colleagues have shown that delayed postconditioning performed 2 days after global ischemia increases activities of antioxidant enzymes, including superoxide dismutase and catalase [44
]. Furthermore, we have shown that rapid postconditioning blocked terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end labeling (TUNEL) positive staining, a marker of apoptosis, in the penumbra 2 days after stroke [31
]. Wang and colleagues further showed that rapid postconditioning reduced cytochrome c release from the mitochondria to the cytosol, a critical cascade for apoptosis induction [36
]. Taken together, these data suggest that postconditioning may reduce ischemic injury by blocking ROS activities and apoptosis.
The protective effects of rapid postconditioning on inflammation after stroke have also been explored. Rapid postconditioning inhibits myeloperoxidase (MPO) activity, an indicator of leukocyte accumulation, and the expression of IL-1β and TNF-α mRNA, and ICAM-1 protein expression in the ischemic cortex at 24 hours after ischemia [33
]. These results suggest that rapid postconditioning may produce an anti-inflammatory effect.
Consistent with the positive effects of rapid postconditioning on CBF, we have reported that delayed postconditioning enhances glucose uptake or metabolism as detected by micro PET imaging [63
]. In addition, delayed postconditioning attenuates edema formation and BBB leakage.
Multiple pathways are involved in neuronal death after stroke, including the PKC pathways, MAPK pathways, and PI3K/Akt Pathway. These pathways contain both pro- and anti-apoptotic signals, and their respective levels of activation/inactivation decide the fate of ischemic neurons after stroke. In the PKC pathways, at least 11 isozymes of the PKC family exist, including δPKC and εPKC [67
]. While δPKC activity usually leads to cell death [68
], εPKC promotes neuronal survival [26
]. PKC isozymes differ as to their intracellular location and function, and their activities are regulated by their cleavage form, phosphorylation, and subcellular location. We found that at 1 hour after stroke rapid postconditioning did not affect the protein levels of total δPKC in the penumbra but did block the increase in levels of the cleaved form of δPKC, indicating δPKC activity [62
]. Rapid postconditioning had no effect on phosphorylated δPKC (thr 505) levels, which decreased by 24 hours after stroke onset; however, it strongly inhibited decreases in phosphorylated εPKC levels after stroke. Thus, rapid postconditioning may reduce ischemic damage by inhibiting the worsening effect of δPKC activity while promoting a beneficial effect of εPKC activity [62
]. Nevertheless, more studies are needed to clarify the role of PKC pathways in the protective effects of ischemic postconditioning.
Ischemic injury and neuronal survival are modulated by the MAPK pathways, including extracellular signal-regulated kinase 1/2 (ERK1/2), P38, and c-Jun N-terminal kinase (JNK) pathways [69
]. As we have reviewed, JNK and p38 are clearly detrimental after stroke, and their inhibition blocks apoptosis in many neuronal death paradigms [69
]. However, ERK1/2’s activity is involved in both neuroprotection as well as injury exacerbation [69
]. In general, most studies agree that ERK1/2 phosphorylation is transiently increased after stroke, suggesting increases in ERK1/2 activity induced by ischemia/reperfusion. Such an increase is confounding as ERK1/2 is apparently involved in the beneficial effects of growth factors, estrogen, preconditioning, and hypothermia on the ischemic brain, but it also promotes inflammation and oxidative stress, and its inhibition reduces ischemic damage [69
]. Given such controversy, we were very interested in studying the changes in ERK1/2 activity involved in the protective effects in postconditioning.
In our pilot study ERK1/2 phosphorylation (P-ERK1/2) levels increased from 1 to 24 hours after stroke, and rapid postconditioning reduced their levels in the penumbra [62
]. Our results imply a detrimental role for P-ERK1/2 after ischemia; however, we did not study whether its inhibition contributes to the protection of rapid postconditioning. Our observation conflicts with Pignataro and colleagues who showed that rapid postconditioning enhances ERK1/2 phosphorylation [30
] and furthermore, U0126, the antagonist of the MEK/ERK1/2 pathway, does not block the protection of rapid postconditioning. This would indicate that increased levels of P-ERK1/2 may be unrelated to the protective effect of rapid postconditioning. More detailed experiments are required to resolve the discrepancy between our results and that of Pignataro et al
The Akt pathway plays a critical role in neuronal survival after stroke; Akt dysfunction results in apoptosis induction, while Akt activity blocks apoptosis by phosphorylating its substrates, including GSK3β (glycogen synthase kinase 3β), FKHR and Bad. Akt activity is considered to be regulated by phosphorylation, which is modulated by upstream molecular signals, such as PTEN and PDK1 (phosphoinositide-dependent protein kinase-1). Akt activity increases with increased phosphorylation of PTEN (phosphatase and tensin homologue deleted on chromosome 10
) and PDK1, and GSK3β phosphorylation supports cell survival (Zhao et al
. 2006a, review). Dephosphorylation of GSK3β leads to its activation and to phosphorylation of β-catenin, which results in β-catenin degradation and apoptosis (Zhao et al
. 2006a). We found that rapid postconditioning increases both Akt phosphorylation (by Western blot) [30
] and Akt activity (by in vitro
kinase assay) [62
]. Furthermore, Akt inhibition by injection of the PI3K inhibitor, LY294002, partially blocks the protective effect of rapid postconditioning [30
]. However, rapid postconditioning does not affect phosphorylation of PTEN or PDK1 but it does inhibit an increase in GSK3β phosphorylation. We found that rapid postconditioning blocks β-catenin phosphorylation, but has no effect on total or non-phosphorylated β-catenin protein levels [62
]. Taken together, the Akt pathway plays a critical role in protection by postconditioning. Our results are further supported by a recent in vitro
experiment showing that Akt inhibition abolished the protective effect of OGD and DHPG postconditioning in hippocampal slice cultures [37
In addition, ATP-sensitive potassium channel (KATP
) channels may also play a critical role in brain injury after stroke. After ischemia, ATP depletion results in the opening of KATP
channels, which is required for the induction of the protective effects of ischemic preconditioning as well as postconditioning in the heart. There are 2 KATP
channels, the sarcolemmal and mitochondrial, and they vary by location. The mitochondrial KATP
channels have been studied the most as their opening generates an outward current that stabilizes the mitochondrial membrane and blocks cell death. In the same vein, Lee and colleagues reported that both a general channel blocker, glibenclamide, and a mitochondrial channel blocker, 5-HD, abolish the protective effect of isoflurane postconditioning, suggesting that KATP
channels may be involved in the protective mechanisms of postconditioning. Compared to traditional rapid postconditioning, little is known about the underlying protective mechanisms of remote postconditioning. Nevertheless, regarding the heart, accumulating evidence suggests that neural pathways serve as a connection between the remote preconditioned organ and the heart. Wolfrum et al
. reported that remote preconditioning with brief mesenteric artery occlusion/reperfusion reduces heart infarction by activating εPKC in rats [70
]. However, this protection was blocked by pretreatment with the ganglion blocker, hexamethonium [70
]. In another study, remote preconditioning resulted in bradykinin release, which stimulates sensory nerves and offers protection, and this effect is blocked by the ganglion blocker, hexamethonium [71
]. In addition, inhibition of afferent nerves with capsaicin abolished the protective effects of remote preconditioning against gastric ischemia when remote preconditioning is conducted the heart or liver by 2, 5 minute ischemic occlusions of the coronal or hepatic arteries [72
]. Consistent with these findings, we recently demonstrated that capsaicin treatment reverses protection by remote postconditioning, suggesting that the afferent nerve pathways may sever a connection between the remote organ, limb, and the ischemic brain5
. We also demonstrated that cycloheximide, a protein synthesis inhibitor, robustly attenuates the protective effect of remote postconditioning, although the underlying mechanisms are unclear. Cycloheximide has been typically shown to inhibit the protective effects of preconditioning against ischemic injury by blocking de novo protein synthesis [73
]. It is not surprising that a protein synthesis inhibitor blocks the protective effects of preconditioning because preconditioning is carried out a few hours to days before ischemia onset [73
], and preconditioning may have time to stimulate the organ to adapt to a future ischemic event, including increasing protein synthesis. In the case of remote postconditioning, the brain may not have time to synthesize the new proteins required for neuroprotection because it is performed immediately after reperfusion. Why protein synthesis inhibition abolishes the protective effects of remote postconditioning remains elusive.
Taken together, previous studies have mainly focused on studying the protective mechanisms of rapid postconditioning, while little is known regarding delayed postconditioning. Since rapid postconditioning is applied immediately after reperfusion, it is able to attenuate those detrimental responses induced by reperfusion, such as free radical products, and the associated interruption of various cell signaling pathways. However, delayed postconditioning is applied a few hours, even a few days after reperfusion; it may modulate the secondary responses that occur much later after reperfusion injury. For instance, delayed postconditioning may attenuate CBF inhibition that occurs at later time points after initial reperfusion; it may also regulate inflammatory response, a relatively delayed detrimental event after stroke. Additionally, delayed postconditioning may promote angiogenesis and neurogenesis. In the future, how delayed postconditioning affects these late cascades after stroke should be studied next.