Remote Ischemic Perconditioning Is Effective Alone and in Combination With Intravenous Tissue-Type Plasminogen Activator in Murine Model of Embolic Stroke
Background and Purpose—Remote ischemic conditioning is cardioprotective in myocardial infarction and neuroprotective in mechanical occlusion models of stroke. However, there is no report on its therapeutic potential in a physiologically relevant embolic stroke model (embolic middle cerebral artery occlusion) in combination with intravenous tissue-type plasminogen activator (tPA).
Methods—We tested remote ischemic perconditioning therapy (RIPerC) at 2 hours after embolic middle cerebral artery occlusion in the mouse with and without intravenous tPA at 4 hours. We assessed cerebral blood flow up to 6 hours, neurological deficits, injury size, and phosphorylation of Akt (Serine473) as a prosurvival signal in the ischemic hemisphere at 48 hours poststroke.
Results—RIPerC therapy alone improved the cerebral blood flow and neurological outcomes. tPA alone at 4 hours did not significantly improve the neurological outcome even after successful thrombolysis. Individual treatments with RIPerC and intravenous tPA reduced the infarct size (25.7% and 23.8%, respectively). Combination therapy of RIPerC and tPA resulted in additive effects in further improving the neurological outcome and reducing the infarct size (50%). All the therapeutic treatments upregulated phosphorylation of Akt in the ischemic hemisphere.
Conclusions—RIPerC is effective alone after embolic middle cerebral artery occlusion and has additive effects in combination with intravenous tPA. RIPerC may be a simple, safe, and inexpensive combination therapy with intravenous tPA.
The Stroke Academic Industry Roundtable (STAIR) recommends therapeutic evaluation of neuroprotective agents in combination with intravenous (IV) tissue-type plasminogen activator (tPA) and to develop therapies to reduce reperfusion injury.1 After so many failed pharmacological trials in stroke, harnessing endogenous pathways of protection provides another promising but untraveled avenue.2 Murry et al first introduced the ischemic preconditioning therapy in a canine myocardial infarction model and follow-up studies have shown ischemic preconditioning to provide the most consistent and profound reduction in infarct size of all cardioprotective interventions.3,4 Subsequent studies have shown that ischemic conditioning can also be applied “at a distance,” so-called “remote ischemic conditioning”; brief episodes of ischemia–reperfusion in a distant organ such as the limb can provide protection to the ischemic heart or brain.5 Remote ischemic conditioning can also be applied during the ischemia and before reperfusion (perconditioning; RIPerC) or after reperfusion (postconditioning). RIPerC therapy can be simply performed using a blood pressure cuff on the arm and has been found protective in a prehospital clinical trial of myocardial infarction.6 A number of studies have now shown that limb RIPerC and postconditioning are protective in animal models of stroke.7,8
Cerebral blood flow (CBF) restoration is critical for neurovascular protection after stroke and requires stringent monitoring.9,10 Neurovascular protection conferred by ischemic conditioning is also mediated through vasodilatory factors and improvements in CBF.11,12 Embolic clot model of stroke (embolic middle cerebral artery occlusion [eMCAO]) better represents the majority of the human stroke cases and is more suitable to study thrombolytics such as tPA and related reperfusion injury.13–15
To the best of our knowledge, none of these conditioning strategies has been ever reported in a physiologically relevant eMCAO model in combination with IV tPA. Because most of the stroke victims within a 4.5-hour treatment window will be candidates for IV tPA, it is necessary to know the effect of RIPerC therapy and its additional benefits and safety with tPA treatment. We herein report that RIPerC therapy is effective alone and provides further additional benefits in combination with IV tPA in a murine eMCAO model.
Materials and Methods
Animals and Experimental Groups
The Institutional Animal Care and Use Committee of Georgia Health Sciences University approved the experimental procedures as per the National Institute of Health guidelines. Ninety C57BL/6J wild-type male mice (20±1 weeks old; Jackson Laboratory, Bar Harbor, ME) housed in Georgia Health Sciences University's Association for Assessment and Accreditation of Laboratory Animal Care International-accredited facility were used in the following 2 experiments: Experiment I was performed to determine the effects of treatments on CBF using laser Doppler flowmeter (PeriFlux 5001, Perimed Inc), neurological outcome, and infarct size. The groups (N=10) were: eMCAO+Veh group, embolic stroke (eMCAO) with RIPerC sham procedure followed by IV sterile phosphate-buffered saline treatment; eMCAO+RIPerC group, eMCAO treated with RIPerC therapy followed by IV phosphate-buffered saline treatment; eMCAO+tPA group, eMCAO with RIPerC sham procedure followed by IV tPA (10 mg/kg) treatment; and eMCAO+RIPerC+tPA group, eMCAO treated with RIPerC therapy followed by IV tPA treatment. All the groups were euthanized at 48 hours post-eMCAO. Experiment II was performed in a separate set of animals with the same groups as described in Experiment I, but the tissue extracts were used for immunoblot analysis for phosphorylation of Akt (Serine473; p-Akt). Experiment II also included a sham group for stroke and RIPerC sham procedures followed by phosphate-buffered saline infusion. Animals were also scanned for cerebral perfusion with a laser Doppler imaging system (PeriScan PIM3 System; Perimed Inc) by an investigator blinded to the groups.
Procedures and Treatment Protocols
The mice were marked for identity and numbered. In Experiment I, the mice were randomized in a block size of 4 (4 animals from the same cage) to the different treatment groups after the stroke surgery. The surgeon performing the stroke and RIPerC surgeries, and the investigators performing the RIPerC sham procedure or therapy and intravenous treatments were blinded to the final identity of the groups and to the intravenous treatments (phosphate-buffered saline or tPA). In Experiment I, an investigator blindly scored the animals for neurological deficits before euthanasia. Mortality was monitored daily using a mortality chart. In Experiment II (5 mice per cage), one mouse from each cage was randomly picked for the sham group and performance of related procedures. The remaining 4 mice in each cage were randomized for the different treatments as discussed for Experiment I.
Stroke induction, CBF measurements and cerebral perfusion imaging, neurological score based on Bederson scale, infarct size measurements after 2,3,5-triphenyltetrazolium chloride (TTC) staining, and immunoblot analysis were performed as described in our previous report with slight modifications.16 To induce stroke, a human fibrinogen-supplemented clot was prepared as reported earlier.16 Briefly, a modified catheter containing a 9.0±0.5-mm long clot was inserted into the right external carotid artery, advanced into the internal one, and the clot was gently delivered into the brain. Wherever required, either RIPerC sham procedure or RIPerC therapy was performed at 2 hours after eMCAO in the left hind limb as reported earlier (5 cycles/5-minute duration/5-minute interval between each cycle).8 Moreover, either tPA (10 mg/kg body weight) or phosphate-buffered saline was infused intravenously at 4 hours post-eMCAO as a 10% bolus and the balance over a period of 20 minutes in a volume of 0.1 mL/10 g body weight of the mouse.
From our previous experience with adult male mice in an eMCAO model, we anticipated stroke injury size to be 50%±10% in the eMCAO+Veh group. Individual treatment with tPA or RIPerC was hypothesized to reduce the infarct size by 20% to 40%±10% and the combined treatment by 50% to 25%±10%. A sample size of 7 in each group would provide 88% power at α=0.05 to detect the main effects due to tPA and RIPerC individual treatments. Also based on our experience, mortality up to 30% was anticipated for some groups over the survival period of 48 hours. Therefore, 10 mice were randomized to each of 4 groups to achieve a power of >80%. All the data are expressed as mean±SD. Statistical analyses were performed using SAS 9.3 (SAS Institute Inc, Cary, NC). A 2 RIPerC (no versus yes) by 2 tPA (no versus yes) analysis of variance with interaction was used to analyze “within time CBF,” neurological score, stroke injury, and p-Akt outcomes. In the absence of a significant interaction, the main effects are considered to be additive when combined. One-way analysis of variance was only used to compare p-Akt level for sham versus the other 4 groups using Dunnett test in Experiment II. Statistical significance was determined at P<0.05.
RIPerC Therapy Alone or in Combination With IV tPA Treatment Improved the CBF and Neurological Outcome
Figure 1A–B shows a significant effect of RIPerC therapy in improving the CBF after eMCAO as compared with the RIPerC sham-operated group (RIPerC effect, P=0.024 at 4 hours post-eMCAO; P=0.0035 at 6 hours). Late tPA therapy also improved the CBF significantly (tPA effect P<0.0001 at 6 hours post-eMCAO). Late tPA infusion was not as efficient as early tPA treatment in improving the CBF (online-only Data Supplement Figure IA). RIPerC therapy showed an additive effect with tPA treatment in further improving the CBF. However, there was no interaction between the 2 treatments (interaction effect, P=0.664 at 6 hours post-eMCAO).
At 48 hours after eMCAO, the main effects of RIPerC and tPA on the neurological outcome were significant (P=0.0005 and P=0.05, respectively; Figure 1C). No significant (P=0.78) interaction effect on the neurological outcome was found between the 2 treatments such that RIPerC therapy provided a significant additive effect to the tPA treatment. RIPerC significantly improved the neurological outcome in the eMCAO+RIPerC group as compared with eMCAO+Veh (1.88±0.23 versus 3.0±0.22; P=0.0084). tPA treatment alone in the eMCAO+tPA group did not improve the neurological outcome significantly as compared with eMCAO+Veh (2.38±0.42 versus 3.0±0.22; P=0.13). There were no significant differences found in the neurological outcomes between eMCAO+RIPerC and eMCAO+tPA groups (P=0.20). On the other hand, combination therapy in the eMCAO+RIPerC+tPA group significantly improved the neurological outcome as compared with eMCAO+Veh (1.4±0.16 versus 3.0±0.22; P=0.0002) and eMCAO+tPA (P=0.012).
RIPerC Therapy Alone or in Combination With IV tPA Treatment Reduced the Injury Size
Figures 2A and 2B show the representative images of TTC stained coronal sections of the brain and infarct volumes, respectively. The analysis of effects on the infarct volume reduction indicates that the main effects of RIPerC and tPA were significant (P=0.0002 and P=0.0005, respectively) but their interaction was not significant (P=0.97). RIPerC therapy alone in the eMCAO+RIPerC group significantly reduced the injury size as compared with eMCAO+Veh (25.7% relative reduction; 38.4±9.5 versus 51.7±10.4; P=0.0082). tPA treatment alone in the eMCAO+tPA group also reduced the injury size as compared with the eMCAO+Veh (23.8% relative reduction; 39.4±7.8 versus 51.7±10.4; P=0.014). There was no significant difference in the infarct volumes between eMCAO+RIPerC and eMCAO+tPA groups (P=0.82). Combination therapy in the eMCAO+RIPerC+tPA group significantly reduced the infarct size (50.0% relative reduction; 25.8±8.6 versus 51.7±10.4) as compared with the eMCAO+Veh (P<0.001) and also when compared with the individual treatment groups (P=0.0037 versus eMCAO+RIPerC and P=0.0067 versus eMCAO+tPA).
RIPerC Therapy Prolonged the Akt Activation in the Ischemic Hemisphere
Figure 3A–B shows that p-Akt, a prosurvival signal after experimental stroke and ischemic conditioning therapy,17,18 was upregulated by both the individual treatments and their combination. All the eMCAO groups had significantly lower level of p-Akt when compared with sham at 48 hours. p-Akt level was significantly downregulated in the eMCAO+Veh group as compared with sham (P<0.0001). The analysis of effects on the p-Akt level indicated that the main effects of RIPerC and tPA were significant (P=0.0026 and P=0.0004, respectively) but their interaction was not significant (P=0.67). Both eMCAO+RIPerC and eMCAO+tPA groups had a significantly increased p-Akt level as compared with eMCAO+Veh (P=0.046 and P=0.014, respectively). There was no significant difference in p-Akt level between eMCAO+RIPerC and eMCAO+tPA groups (P=0.57). Combination therapy in the eMCAO+RIPerC+tPA group significantly increased p-Akt as compared with eMCAO+Veh (P<0.001) and also when compared with the individual treatments (P=0.0034 versus eMCAO+RIPerC and P=0.013 versus eMCAO+tPA).
To the best of our knowledge, this is the first report demonstrating the efficacy of RIPerC therapy alone and in combination with IV tPA in a physiological and clinically relevant embolic model. The key findings are that RIPerC alone increased the CBF and prosurvival signaling after stroke, resulting in improved neurological outcome and reduction in the size of injury. We also found additive beneficial effects of RIPerC to “late” IV tPA treatment leading to further improvements in the neurological outcome and reduction of injury size. Late IV tPA reduced the infarct size but did not improve the neurological outcome.
More than 60% of preclinical studies for neuroprotection in stroke research are done using well-accepted mechanical vascular occlusion–reperfusion models.14 This closely resembles the clinical cases of reperfusion after thrombectomy. In a mechanical stroke model, postconditioning by interrupting sudden reperfusion attenuated the injury.19 On the other hand, opening of the blood vessels after eMCAO either by spontaneous recanalization or by thrombolysis is often gradual and/or partial, which leads to prolonged microcirculatory and reperfusion deficits. Therefore, eMCAO better models the majority of human stroke cases and may be more appropriate for further translational and mechanistic studies of the conditioning therapies.
CBF restoration is a promising target for the treatment of stroke.9,10,13 As evident from Figures 1 and 2, RIPerC therapy significantly increased the CBF, resulting in significant improvements in the neurological outcomes and infarct size reductions. Moreover, tPA, being a thrombolytic, also improved the CBF when infused alone at 4 hours post-eMCAO. tPA alone reduced the injury size but did not significantly improve the neurological outcome, possibly because of its unwanted effects in a few animals.20,21
It should be noted that IV tPA was given at the “tail” of the protective window when it still had some effect on infarct size reduction. This effect of tPA was mainly due to opening of the major occluded arteries as detected with laser Doppler flowmeter (Figure 1A–B) and visualized by clot resolution (online-only Data Supplement Figure IC).21 In the absence of RIPerC therapy, 4 hours of untreated ischemia may have caused microvascular dysfunction, which prevented complete CBF restoration even after successful thrombolysis by late tPA infusion (Figure 1A–B; online-only Data Supplement Figure IA–C).21,22 This may be due to increased oxidative–nitrative stress after eMCAO (online-only Data Supplement Figure IIC–E), which impairs capillary reflow despite successful opening of an occluded artery.22 Increased nitrative stress also impairs clot lysis and correlated with the poor neurological outcomes after stroke in human.23–26 In fact, we observed more prominent and sudden increase in the nitrative stress level 3 hours post-eMCAO both in the plasma and brain (online-only Data Supplement Figure IIC–E), a critical time point after which the benefits and safety of tPA therapy start to decline. Mild to moderate hemorrhage after “tPA alone” treatment was found in a few animals at this tail of the protective window but it was attenuated when RIPerC therapy was performed 2 hours before tPA infusion (online-only Data Supplement Figure IC–D). Due to improvement in the CBF, RIPerC may have attenuated the neurovascular stress before tPA treatment, resulting in a “preconditioned” window for safer late tPA therapy and additional benefits.
The molecular mechanisms of remote ischemic conditioning are still elusive and may be multifactorial.27 The downstream effects of multiple mechanisms appear to converge on the upregulation of reperfusion injury salvage kinase pathway and blocking of opening of the mitochondrial transition permeability pore.28 PI3K/Akt is a key prosurvival signal of the reperfusion injury salvage kinase pathway and may have important physiological effects. Pharmacological inhibition of PI3K/Akt activity abolished the protective effects of postconditioning on the infarct size reduction.17,18 We found that Akt activity after eMCAO in the untreated mice was “transient.” p-Akt level started declining after 3 hours and appeared to be correlated with the increasing nitrative stress of the plasma and brain (online-only Data Supplement Figure IIA–E). Postconditioning attenuated nitrotyrosine in the brain, a marker of increased nitrative stress.29 Peroxynitrite also inhibits PI3K, a major upstream kinase and regulator of Akt activity.30 Therefore, we tested RIPerC therapy at 2 hours post-eMCAO. As evident from our online-only Data Supplement and Figure 3, RIPerC preserved and prolonged the post-eMCAO Akt activation, which was sustained even up to 48 hours. In agreement with previous findings,17,18 prolonged Akt activation may have improved the post-eMCAO outcomes. Because of its recanalization effect, tPA alone also upregulated the p-Akt level at the end point, which was further increased due to additive effects of RIPerC.
In this study, increased p-Akt level after the treatments may be related to both the vasculature and brain. Therefore, future studies in animals genetically mutated for different Akt isoforms are planned. In the endothelium, Akt activity increases nitric oxide production through improved endothelial nitric oxide synthase activity and helps to maintain vascular homeostasis.31,32 Because RIPerC prolonged the Akt activity and improved the CBF, the beneficial “neuroprotective” effects of RIPerC are likely to be partially related to improvements in the microvascular blood flow. Future studies on collateral and microvascular CBF modulation by RIPerC are in progress, which will help us to better understand the mechanism of neurovascular protection by this therapy in eMCAO.
In summary, these findings could be helpful in translating the remote ischemic conditioning into stroke clinical trials. Further studies to test the therapeutic potential of RIPerC in a window later than 4 hours and in aged animals of both sexes with and without comorbidities such as diabetes will better inform and facilitate a clinical trial. However, remote ischemic conditioning is promising and may open a new paradigm in stroke treatment, which may be safe, inexpensive, and generalizable to a wide variety of stroke systems of care.
Sources of Funding
Supported by National Institutes of Health (NIH) 1R01NS055728 award to D.C.H.; Veteran Administration Merit (BX000347) and NIH NS070239 awards to A.E.; Veteran Administration Merit (BX000891) and NIH NS063965 awards to S.C.F.; and Veteran Administration Merit (104462) award to W.D.H. Financial support from Augusta Biomedical Research Corporation and Immunotherapy Discovery Institute at Georgia Health Sciences University to M.N.H. is also acknowledged.
↵* Co-corresponding authors.
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.112.660373/-/DC1.
- Received April 10, 2012.
- Revision received July 11, 2012.
- Accepted July 13, 2012.
- © 2012 American Heart Association, Inc.
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