In contrast to ischemic preconditioning, ischemic postconditioning is a relatively novel concept [2] that was adopted in order to contrast it with ischemic preconditioning. As such, ischemic postconditioning was believed to be a brand new concept when the term was introduced within a 6–7 year span in the research on myocardial ischemia [8, 9]. It was also believed to be derived from the concept of ischemic preconditioning [10], or from partial or controlled reperfusion [9]. However, more than 50 years ago Sewell and colleagues [11] first reported on the protective effects of intermittent reperfusion in dogs with temporary coronary arterial occlusion where ventricular fibrillation occurred within a few to 20 seconds after sudden reperfusion. They found that intermittent reperfusion, which equates to the current concept of ischemic postconditioning, abolished fibrillation [11]. In addition, this protection was once observed in a clinical case reported in 1994 where a patient was subjected to an acute myocardial ischemia [12]. Moreover, this protective phenomenon was repeated by Na and colleagues in 1996 [13], who found that postconditioning was as effective as preconditioning in preventing ventricular fibrillations in cats.

As defined above, rapid postconditioning is induced immediately or a few minutes after reperfusion, and it is the main form of postconditioning in heart research. Therefore, we and others have studied whether rapid postconditioning is effective against focal cerebral ischemia [30, 31]. In our first study we reported that rapid postconditioning reduces infarct size as a function of ischemic severity – meaning, it is less effective with longer periods of ischemia [8, 31]. We found that rapid postconditioning reduced infarct size by ~80, ~51, and ~17%, respectively, in a 15, 30, or 60 minute common carotid artery (CCA) occlusion combined with permanent distal middle cerebral artery (dMCA) occlusion. In another study we compared the impact of cycle numbers and duration of reperfusion/occlusion on the protective effect of rapid postconditioning using the ischemic model of a 30 minute bilateral CCA occlusion combined with a permanent dMCA occlusion [32]. Our results showed that rapid postconditioning conducted 10 to 30 seconds after reperfusion reduced infarct size, but not when it was initiated 3 minutes after reperfusion. Taken together, these studies suggest that the protective effects of rapid postconditioning depend on the number of cycles, the duration of each cycle of reperfusion and occlusion, and its onset time [32].

In our experiments, we observed robust protection by rapid postconditioning only in moderate or mild brain ischemia with a 15 or 30 minute bilateral CCA occlusion time [31] and mild protection in more severe ischemia (60 minutes of CCA occlusion). Xing and colleagues have also found that rapid postconditioning reduced infarction in a MCA suture occlusion model by only 16% and 12%, 1 day and 3 days after stroke, respectively [33]. Therefore, in our studies, rapid postconditioning does not appear to generate the same level of protection in the ischemic brain as has been shown in the ischemic heart [2]. However, we have not yet tested if the optimized conditions, which were defined in our recent study [32], afford better protection with a longer period of ischemia, while Xing and colleagues did not compare the protective effects with different postconditioning parameters. Therefore, we cannot exclude the possibility that the relatively weak protection is due to the usage of sub-optimal parameters (3 cycles of 30 sec reperfusion followed by 10 sec occlusion) of rapid postconditioning.

In contrast to our findings, Pignataro and colleagues demonstrated a very strong protection with postconditioning in a severe focal ischemic model of MCA occlusion for 100 minutes [30, 34]. In their study, postconditioning with 10 minutes of occlusion initiated after 30 minutes of reperfusion offered no protection; postconditioning with 3 cycles of 5 minute reperfusion followed by 5 minute occlusion reduced infarction by 38%, and 1 cycle of 10 minute occlusion initiated after 10 minutes of reperfusion reduced infarct size by ~70 %. Again, this study suggests that the onset time of postconditioning is critical for its neuroprotective effects.

A few groups have studied the protective effects of rapid postconditioning on transient global cerebral ischemia. Rehni and Singh have shown that rapid postconditioning attenuates behavioral deficits after global ischemia in mice [35]. However, they did not report how rapid postconditioning affects neuronal loss. Nevertheless, Wang et al. showed that rapid postconditioning applied immediately after reperfusion attenuated neuronal death in both the hippocampus and the parietal cortex after 10 minutes of transient global ischemia [36].

As delayed neuronal death occurs 2 to 3 days after transient global ischemia, using a transient global ischemia model is perhaps the best way to test whether delayed ischemic postconditioning offers protection. In this model, where global ischemia is induced by a 4-vessel occlusion, neuronal death is not detected at 2 days post-ischemia [40]. Using this model, Burda and colleagues found that delayed postconditioning with a single 5 minute ischemia induced 2 days after reperfusion reduced neuronal death in the hippocampus by about 94% when measured 7 days after global ischemia; a time point at which neuronal death is considered matured [40].

We recently tested the hypothesis that delayed postconditioning with different parameters reduces infarct size in focal ischemia [3]. In our study, stroke was induced by 30 minutes occlusion of the bilateral common carotid artery combined with permanent occlusion of the middle cerebral artery; the model used in our previous studies for rapid postconditioning. However, delayed postconditioning was performed not only by repetitive, brief occlusion and release of the bilateral CCAs, but also by occlusion of the ipsilateral CCA alone [3]. The results showed that delayed postconditioning performed at 3 hours and 6 hours after stroke robustly reduces infarct size, with the strongest protection achieved by delayed postconditioning with 6 cycles of 15 minutes occlusion/15 minutes release of the ipsilateral CCA executed at 6 hours. We found that this delayed postconditioning attenuated reduction in 2-[(18)F]-fluoro-2-deoxy-D-glucose (FDG)-uptake resulting in improved metabolism and reduced edema and blood brain barrier leakage. A prerequisite for performing postconditioning is that reperfusion must be achieved first; clinically, reperfusion in ischemic stroke patients is usually achieved by a tissue plasminogen activator (tPA) application. Nevertheless, t-PA may have side effects that worsen ischemic injury. Thus, we were very interested in testing whether delayed postconditioning counteracts the exacerbating effect of t-PA, and in fact, we found that delayed postconditioning reduced the infarct size increased with t-PA application [3].

We have shown that delayed postconditioning can be performed as late as 6 hours after stroke; however, increasing the delay in the therapeutic time window could potentially benefit more stroke patients who often experience delays in treatment secondary to transportation or other issues. With that said, infarction induced by focal ischemia usually matures 1 to 2 days after stroke; therefore, it seems irrational to apply delayed postconditioning later than 1 day after stroke. Nevertheless, focal ischemia may cause a secondary degeneration in the thalamus occurring a few days to a few weeks after stroke [41]. Thus, delayed postconditioning may not directly block acute brain injury, but it may attenuate the indirect or secondary brain injury induced by stroke.

As a means to induce pharmacological postconditioning, the anesthetic isoflurane might be the best candidate to test as it has been used to induce preconditioning to protect against brain ischemia. In a suture MCA occlusion model, Lee et al. conducted postconditioning by maintaining 2% isoflurane for 60 minutes starting at the same time the MCA occluding suture was removed. In this study, no isoflurane was used during MCA occlusion. For rats receiving control ischemia, only 1 minute of isoflurane was used during MCA suture removal [42]. Lee et al. found that isoflurane postconditioning robustly reduced brain infarction and attenuated neurological deficits. They also found that rapid isoflurane postconditioning protects against ischemic injury in slice organ cultures [42], in which OGD was maintained for 15 minutes, and postconditioning was instituted by the application of isoflurane after OGD. They found that the protective effect of isoflurane postconditioning is dependent on the duration and concentration of isoflurane exposure. Lastly, isoflurane postconditioning started 0 or 10 minutes but not greater than 30 minutes post reperfusion reduced cell damage, suggesting a similar therapeutic time window with ischemic postconditioning [42].

Rapid postconditioning with brief OGD also blocks ischemic injury in rat organotypic hippocampal slice cultures [37]. Results showed that postconditioning with 3 minutes of OGD started at 5 minutes after reperfusion reduced cell injury by about 40%. In the same study, protection by postconditioning was also induced by adding a low dose of the pharmacological agent, 3, 5-dihydroxyphenylglycine (DHPG, a group 1 metabotropic glutamate receptor agonist) 5 minutes after reperfusion and incubating for 30 minutes. It has been reported that high doses of DHPG exacerbate, while low doses inhibit, neuronal injury [37].

In a long-term study, we found that rapid postconditioning performed immediately after reperfusion reduced lesion size ~40% in rats subjected to ischemia when measured 30 days after ischemia [62]. In addition, using a vibrissae test to detect asymmetrical forelimb usage, we found that rapid postconditioning improves neurological function [62]. In global ischemia rapid postconditioning also improves subject performance on spatial learning and memory in a water maze test 3 weeks after reperfusion [36], suggesting that rapid postconditioning provides long-term protection in global ischemia, which is consistent with its protective effects on neuronal survival.

We found that both rapid postconditioning and delayed postconditioning offer long-term protection and improve functional recovery. Our results showed that delayed ischemic postconditioning conducted from 6 hours after stroke attenuates brain injury and improves the outcomes of behavioral tests for up to 2 months using 4 standard testing methods, including the vibrissae test, postural reflex test, tail hang test, and home cage test [63].

Our laboratory recently compared the protective effects of rapid postconditioning combined with rapid or delayed preconditioning [32]. Rapid preconditioning was induced by transiently occluding the left dMCA for 15 minutes at 60 minutes before stroke [32], while delayed preconditioning was induced by occluding the left dMCA for 5 or 15 minutes at 3 days before the stroke. Postconditioning of 10 cycles of 10 seconds occlusion/10 seconds reperfusion was conducted immediately after reperfusion. We found that the protective effect of postconditioning is comparable with that of rapid preconditioning, but is less effective than delayed preconditioning. When addressing whether postconditioning plus preconditioning generates a synergistic effect [32], we found that postconditioning combined with either rapid or delayed preconditioning does not provide any additional reductions in infarction. Our results are consistent with Pignataro and colleagues who reported that protection with postconditioning is comparable to that of delayed preconditioning in a suture MCA occlusion model in rats, while a combination of pre and postconditioning provides no greater protection [30].

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, 62] 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, 62]. 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 (K_ATP) channels may also play a critical role in brain injury after stroke. After ischemia, ATP depletion results in the opening of K_ATP channels, which is required for the induction of the protective effects of ischemic preconditioning as well as postconditioning in the heart. There are 2 K_ATP channels, the sarcolemmal and mitochondrial, and they vary by location. The mitochondrial K_ATP 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 K_ATP 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 [73–75], 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.