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Akt regulation in rat
Accepted Manuscript Research report Dihydrocapsaicin (DHC) Enhances the Hypothermia-Induced Neuroprotection Following Ischemic Stroke Via PI3K/Akt Regulation In Rat Di Wu, Jingfei Shi, Omar Elmadhoun, Yunxia Duan, Hong An, Jun Zhang, Xiaoduo He, Ran Meng, Xiangrong Liu, Xunming Ji, Yuchuan Ding PII: DOI: Reference:
S0006-8993(17)30277-9 http://dx.doi.org/10.1016/j.brainres.2017.06.029 BRES 45411
To appear in:
Brain Research
Received Date: Revised Date: Accepted Date:
7 June 2017 27 June 2017 28 June 2017
Please cite this article as: D. Wu, J. Shi, O. Elmadhoun, Y. Duan, H. An, J. Zhang, X. He, R. Meng, X. Liu, X. Ji, Y. Ding, Dihydrocapsaicin (DHC) Enhances the Hypothermia-Induced Neuroprotection Following Ischemic Stroke Via PI3K/Akt Regulation In Rat, Brain Research (2017), doi: http://dx.doi.org/10.1016/j.brainres.2017.06.029
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Dihydrocapsaicin (DHC) Enhances the Hypothermia-Induced Neuroprotection Following Ischemic Stroke Via PI3K/Akt Regulation In Rat Di Wu a, b, c, Jingfei Shi a, b, Omar Elmadhoun d, Yunxia Duan a, Hong An a, Jun Zhang a, Xiaoduo He a, Ran Meng a, Xiangrong Liu a, Xunming Ji a, c *, Yuchuan Ding a,d a
China-America Institute of Neuroscience, Xuanwu Hospital, Capital Medical University, Beijing, China
b
Beijing Key Laboratory of Hypoxia Conditioning Translational Medicine
c Center of Stroke, Beijing Institute for Brain Disorders d
Department of Neurosurgery, Wayne State University School of Medicine, Detroit, MI, USA
*Corresponding Author: Xunming Ji, MD, PhD China-America Institute of Neuroscience, Xuanwu Hospital, Capital Medical University, Beijing 100053, China Email:[email protected]
Abstract Objective: Hypothermia has demonstrated neuroprotection following ischemia in preclinical studies while its clinical application is still very limited. The aim of this study was to explore whether combining local hypothermia in ischemic territory achieved by intra-arterial cold infusions (IACIs) with pharmacologically induced hypothermia enhances therapeutic 1
outcomes, as well as the underlying mechanism. Methods: Sprague-Dawley rats were subjected to right middle cerebral artery occlusion (MCAO) for 2 h using intraluminal hollow filament. The ischemic rats were randomized to receive: 1) pharmacological hypothermia by intraperitoneal (i.p.) injection of dihydrocapsaicin (DHC); 2) physical hypothermia by IACIs for 10 min; or 3) the combined treatments. Extent of brain injury was determined by neurological deficit, infarct volume, and apoptotic cell death at 24 hr and/or 7 d following reperfusion. ATP and ROS levels were measured. Expression of p-Akt, cleaved Caspase-3, pro-apoptotic (AIF, Bax) and anti-apoptotic proteins (Bcl-2, Bcl-xL) was evaluated at 24 hr. Finally, PI3K inhibitor was used to determine the effect of p-Akt. Results: DHC or IACIs each exhibited hypothermic effect and neuroprotection in rat MCAO models. The combination of pharmacological and physical approaches led to a faster and sustained reduction in brain temperatures and improved ischemia-induced injury than either alone (P<0.01). Furthermore, the combination treatment favorably increased the expression of anti-apoptotic proteins and decreased pro-apoptotic protein levels (P<0.01 or 0.05). This neuroprotective effect was largely blocked by p-Akt inhibition, indicating a potential role of Akt pathway in this mechanism (P<0.01 or 0.05). Conclusions: The combination approach is able to enhance the efficiency of hypothermia and efficacy of hypothermia-induced neuroprotection following ischemic stroke. The findings here move us a step closer towards translating this long recognized TH from bench to bedside. Key words: Pharmacological hypothermia, Apoptotic Cell death, Ischemia/reperfusion injury, Middle cerebral artery, intra-arterial cold infusions (IACIs)
2
1. Introduction Stroke remains the leading cause of permanent disability in industrialized nations (Mozaffarian et al., 2015). On average every 40 s someone in the USA suffers a stroke, demonstrating the omnipresence and frequency of this devastating disease (Fisher and Saver, 2015). This fact validates our passion to further investigate this disease and develop clinically relevant treatments. However, asides from thrombolytic approaches, the development of other 3
management therapies, such as hypothermia, has been largely unproductive (Kollmar et al., 2009; Jiang and Duong, 2016). Although hypothermia has shown promising neuroprotective effects following ischemia in both preclinical and clinical studies, the delayed onset of brain hypothermia and various side effects associated with the impractically low temperatures needed to achieve superior outcomes have limited its clinical scope (Lee et al, 2016a; Wang et al, 2016; Yenari and Han, 2012; Zhang et al, 2013). Thus, in this study, we intended to achieve neuroprotection with hypothermia more effectively and efficiently than previously described. In previous studies, we developed a unique endovascular technique to induce local hypothermia by infusing cold saline into the microvasculature of the ischemic territory in animal models. These studies demonstrated a strong and safe neuroprotective results (Ding et al., 2002; Ding et al., 2004; Luan et al., 2004). Although this local hypothermia is neuroprotective, it requires more time to perform and duration is not long enough to maintain a sustained hypothermia. Pharmacological hypothermia is gaining increasing attention as an alternative therapeutic approach. This method has a special advantage to induce a controllable and sustained hypothermia status (Lee et al, 2016b). Recent studies have focused on one promising class of agents, the transient receptor potential vanilloid channel 1 (TRPV1) agonists (Liu et al., 2016). TRPV1 is a nonspecific cation channel, possibly inducing neuroprotection through its ability to induce hypothermia. Its agonist, dihydrocapsaicin (DHC) has shown potential neuroprotective effects by inducing hypothermia. However, because of the toxicity associated with high doses that is required for induction of hypothermia, its clinical use was highly limited (Cao et al., 2014; Muzzi et al., 2012). Phosphatidylinositol 3-kinase/Akt (PI3K/Akt) signaling pathway, the most important signaling pathway involved in the neuroprotection against brain ischemia, plays a critical role in promoting neuronal survival after ischemic stroke (Tu et al., 2016). Usually, the 4
neuroprotective property of PI3K/Akt signaling has been primarily attributed to the anti-apoptotic action, or the anti-oxidative action (Wang et al., 2012). To comprehensively understand our combination therapy, the molecular mechanism of the proposed technique on the PI3K/Akt pathway was also studied. Therefore, we explore whether DHC has an additive effect on hypothermia induction and maintenance, and therefore increases the neuroprotective effect, as well as to reduce the dose of DHC and its associated toxicity in order to enhance the efficiency of hypothermia and efficacy of hypothermia-induced neuroprotection following ischemic stroke. 2. Results 2.1Brain and body temperature Two hours after the onset of ischemia, hypothermia by DHC and IACIs was observed in the cortex of stroke models (n=6, Fig 1 A). Cortex temperature rapidly dropped below 35.0 °C within 10 minutes following ICAIs in the TH, combination, and combination plus Akti groups. The cooling rates in the above groups were faster than that in DHC group (p < 0.01 ) (Fig 1 B). In addition, cortex temperature remained below 35.0 °C for at least 80 minutes in the combination and combination plus Akti groups, and cortex temperature remained below 35.0 °C for approximated 50 minutes in DHC group, all of which were longer than that in TH group (p < 0.01 ) (Fig 1 C). p-Akt inhibitor did not significantly affect the cortex temperature. Post-stroke pyrexia was also observed in rectal temperatures. In stroke models that received IACIs, rectal temperature was generally maintained around baseline temperatures for 120 mins. After the administration of DHC, rectal temperature dropped to approximately 35.0 °C at 30 mins, stayed at equal or below 35.0 °C for approximately 20 minutes, and then gradually returned to approximately 36.5 °C at 120 minutes (Fig 1D). There were not differences in cooling rate (p > 0.05) (Fig 1 E) and duration (p > 0.05) (Fig 1 F) among groups receiving 5
DHC treatments. 2.2 Infarct volume Ischemia produced a large infarct volume at 24 h (48.5% + 2.4%), which was significantly reduced by DHC (36.2% + 2.5%) (p < 0.01)and IACIs (35.3% + 2.2%) (p < 0.01), respectively. The combination therapy further decreased the infarct volume (27.3% + 3.2%) (p < 0.01). This reduction was largely reversed with p-Akt inhibition (36.9% + 1.2%) (Fig 2 A and B). 2.3 Neurologic deficits A high score (9.8 + 0.3) was observed in stroke group at 24 h. Neurologic deficits were significantly reduced by DHC (7.8 + 0.4) and IACIs (8.1 + 0.3), respectively(p < 0.01). Furthermore, the combination exhibited a greater reduction in scores (7.1 + 0.3) (p < 0.01). p-Akt inhibition reversed this reduction (8.5 + 0.2) (Fig 2 C). Neurologic deficits with a high score (9.0 + 0.4) was also observed in stroke group at 7 d, which were significantly reduced by DHC (7.0 + 0.4) and TH (7.1 + 0.3) , respectively (p < 0.01). Again, the combination therapy exhibited a greater reduction in scores (5.5 + 0.3) (p < 0.01), which was also reversed by p-Akt inhibition (8.1 + 0.4) (Fig 2 D). 2.4 ROS levels Oxidative stress was significantly increased at 24 h after the ischemia/reperfusion. Both DHC and IACIs treatment were able to reduce ROS levels at 24 h(p < 0.05) . A greater decrease was observed with the combination group (p < 0.05) (Fig 3 A). However, there were not significant differences among DHC, TH and the combination treatment groups (p > 0.05). This was reversed when p-Akt inhibitor was used. 2.5 ATP levels Ischemic stroke markedly decreased ATP levels at 24 h following ischemia/reperfusion (Fig 3 B). ATP levels were increased by DHC or IACIs treatments each alone but did not reach a 6
significant level (p > 0.05). However, the combination treatment induced a marked increase in ATP levels (p<0.05). But, there were not significant differences among DHC, TH and the combination treatment groups (p > 0.05). These changes were largely reversed with p-Akt inhibition. 2.6 Apoptotic cell death Ischemia-reperfusion induced a high level of cell death, which was reduced by DHC, IACIs or the combination treatment (p< 0.05). However, there were not significant differences among DHC, TH and the combination treatment groups (p > 0.05). p-Akt inhibitor could reverse these changes (Fig 3 C). 2.7 Pro-apoptotic protein expression AIF, Bax, and Caspase-3 were analyzed by Western blot at 24 h following reperfusion. There were significant differences in expression of AIF (p < 0.01), Bax (p < 0.01), and Caspase-3 (p < 0.01) at 24 h among all groups. Both DHC and IACIs monotherapies reduced AIF, Bax, and Caspase-3 expressions. The reduction in the expression of those proteins was enhanced by the combination approach. p-Akt inhibition significantly reversed these results (Fig 4 A, B, C). 2.8 Anti-apoptotic protein expression Protein expressions of Bcl-2 and Bcl-xL were analyzed at 24 h following reperfusion. Significant differences were observed for Bcl-2 (p < 0.01) and Bcl-xL (p < 0.01) among all groups. DHC and IACIs treatments increased Bcl-2 and Bcl-xL levels while the combination treatment further enhanced these effects. This, again, was reversed by p-Akt inhibitor (Fig 4 D, E). 2.9 p-Akt protein expression Phosphorylated AKT (p-AKT) is the constitutive activation form of AKT. Protein expressions of p-Akt were significantlydifferent at 24 h following reperfusion among all groups (p < 0.01) . 7
DHC, IACIs, and the combination treatment significantly increased p-Akt levels, while the inhibitor led to a decrease of p-Akt (Fig 4F). 3. Discussion The present study revealed that combining physical and pharmacological techniques resulted in a faster and more stable hypothermia than either method alone, in association with reduced ischemic damages and restored neurological functions as compared to monotherapies. Furthermore, a possible molecular mechanism for these effects could be explained, at least partially by the PI3K/Akt pathway. The combination treatment may create a pro-survival environment and enhance the cellular oxidative profile following ischemia/reperfusion. The efficacy of hypothermia is dependent on several factors including cooling rate as well as the duration and depth of hypothermia. Previous studies have reported that lower temperatures which are achieved rapidly and maintained have better clinical outcomes (Han et al., 2015; Krieger and Yenari, 2004). Our previous study reported that a rapid temperature decrease can be achieved by IACIs in rat transient MACO models (Ding et al., 2002; Ding et al., 2004. Both experimental and clinical studies also found that intra-carotid infusion of cold saline or rapid infusion of cold saline through a peripheral vein could induce a mild hypothermia (Luan et al., 2004; Kim et al., 2005; Straus et al., 2011). However, the induction rate and duration of hypothermia were volume-dependent. Hypothermia induced by these methods improved the outcomes of brain ischemia in experimental studies, but a rapid and sustained hypothermia was technically difficult because of an inevitable drop in hematocrit levels and limitations on the duration of treatment (Ji et al., 2012). In clinical, intravascular infusion of a high load of 4°C saline infusions in ICTuS trial achieved a feasible hypothermia strategy, but did not lead to a superior neuroprotection (Lyden et al., 2016). A higher liquid load could lead to a feasible hypothermia, but not necessarily result in more neuroprotection. So it was 8
impossible to achieve a fast and sustained hypothermia only by increasing the liquid load (Esposito et al., 2014). At present, physical cooling is adopted in clinical hypothermic strategy, however the shivering response must be countered by sedatives and mechanical ventilation, which complicate the prognosis and recovery. Pharmaceutical-induced hypothermia provides a good alternative methodology in a controllable way, especially those affecting the thermoregulatory set point. These drugs do not induce anti-shivering responses, which provide an opportunity to combine with non-invasive hypothermia techniques to achieve rapid and controlled cooling. Recent study reported that the centrally acting hypothermic drug HPI-201 greatly enhanced the efficiency and efficacy of conventional physical cooling in rat MCAO models, and induced synergistic neuroprotective effects (Lee et al, 2016a). However, cooling efficacy of pharmaceutical-induced hypothermia was relatively weaker in severe stroke models compared with its effects in normal and small stroke animals [Lee et al, 2016a]. In addition, when adopting HPI-201 or DHC alone, it generally took approximately 30 minutes to achieve hypothermia. DHC, a natural capsaicin, belongs to Transient Receptor Potential Vanilloid channel 1 (TRPV1) agonists (Cao et al., 2014; Fosgerau et al., 2010). Previous studies have shown that DHC have a greater effectiveness to induce hypothermia compared with capsaicin. But at high doses are associated with negative side effects such as hypotension and bradycardia (Fosgerau et al., 2010; Muzzi et al, 2012). In addition, over stimulation of TRPV1 receptors promotes glutamate release and excitotoxicity in the central nerve system, then leads to degeneration of neurons after ischemia (Lai et al., 2014). Our group recently proved that DHC at a low dose could induce a stable hypothermia and lead to a better neuroprotection. This study further confirmed that the combination treatment of pharmacological and local hypothermia was efficacious in improving clinical effects, decreasing the side effects brought 9
on by either therapy alone. Ischemic stroke leads to a cascade of catastrophic consequences on a number of molecular pathways in neurons. For example, the rise in ROS to toxic levels following ischemia cause cellular injury in multiple forms including lipid peroxidation, protein oxidation and DNA damage which collectively worsens brain damage (Goossens and Hachimi-Idrissi ,2014; Kurisu et a., 2016). Additionally, reduced ATP levels contribute to the cellular swelling and imbalanced intracellular ion gradient (Forreider et al., 2016; Sanderson et al., 2013). This necessitates using a combination of neuroprotective agents that can intervene in multiple mechanisms. Hypothermia has multifaceted neuroprotective effects due to its ability to lower the metabolic rate, reduce intracellular calcium influx and intracellular acidosis, suppress ROS formation and infiltration of inflammatory cells, down-regulate specific cell death pathways and/or up-regulate cell survival mechanisms based on previous reports (Han et al., 2015; Kim and Yenari, 2015). Moreover the combination treatment is proved to further reduce ROS generation and pro-apoptotic proteins, increase ATP and anti-apoptotic proteins levels in this paper. This indicates that different mechanisms of action exist to our combination approach and local cold infusions have a synergistic effect with pharmacologically induced hypothermia. PI3K/Akt pathway, the most important pro-survival signaling pathway, was widely considered to play a critical role in neuroprotective effect against ischemic brain injury. A previous study found that zileuton attenuates neurological deficit scores, cerebral infarct volume, cerebral water content and ischemic neuronal injury in rats subjected to focal cerebral ischemia through PI3K/Akt signaling pathway (Tu et al., 2016). Another study discovered that the activation of PI3K/Akt pathway limited the pro-apoptotic function of Bax and inhibited cytochrome c release from the mitochondria to exert neuroprotective effects (Tsuruta et al., 2002). The 10
present study showed that pharmacological and physical hypothermia had a synergetic effect to enhance neuroprotection through anti-apoptosis and PI3K/Akt pathway, all of which were abolished by LY294002 administration, indicating that PI3K/Akt signaling pathway mediates neuroprotection of pharmacological and physical hypothermia against ischemic stroke. In summary, we were able to avoid limitations that had historically narrowed TH’s clinical applications by combining IACIs and DHC. As such, the combination therapy has a significant potential to remarkably widen the role of hypothermia in the treatment of stroke patients. Although hypothermia has multifaceted neuroprotective effects, we showed that PI3K/Akt apoptotic pathway is a possible mechanism of action of the combination therapy. 4. Experimental Procedures 4.1 Subjects A total of 114 adult male Sprague-Dawley rats (300 to 340g weight, Vital River Laboratory Animal Co.) were used. All experimental procedures were approved by the Institutional Animal Investigation Committee of Capital Medical University in accordance with the National Institutes of Health (USA) guidelines for care and use of laboratory animals. All animals were housed in the same animal care facility, with 12-hour light/dark cycles, throughout the study. Animals were randomly divided into 6 groups by animal tag number: (1) sham-operated group (n=18), in which the rats underwent all of the operative process except the middle cerebral artery occlusion (MCAO); (2) stroke group (n=18), in which the rats received 2 h MCAO followed by the reperfusion without treatment; (3) DHC group (n=18), in which ischemic rats received DHC injection at the onset of reperfusion by withdrawing the catheter; (4) TH group (n=18), in which ischemic rats received (IACIs) at the onset of reperfusion; (5) DHC + TH (combination) group (n=18), in which ischemic rats received both DHC and IACIs; (6) DHC + TH + Akti (combination plus Akti) group (n=24), in which 11
ischemic rats received DHC, IACIs, and the PI3K/Akt inhibitor (LY294002). The exclusion criteria included animal death before expected time points, brain hemorrhage, and no signs of infarct based on 2,3,5-triphenyltetrazolium chloride (TTC) staining and neurological deficits. All procedures and data analysis were performed in a blinded and randomized manner. 4.2 Surgical preparation Anesthesia was induced and maintained using 1.5–3.5% enflurane in 70% nitrous oxide and 30% oxygen (Bickford veterinary anesthesia equipment model 61010; AM Bickford Inc., Wales Center, NY, USA). Rectal temperature was monitored during the procedure and was maintained at 36.5-37.5°C using a feedback-controlled heating blanket if needed. Blood gases and pressure were monitored via the right femoral artery which was cannulated with a PE-50 catheter. 4.3 Focal ischemic and therapeutic hypothermia (TH) Two hour MCAO was induced using similar approach as previously reported by us (Ding et al., 2002; Luan et al., 2004). Briefly, PE-50 catheter was inserted into the right external carotid artery via an arteriotomy, and passed up the lumen of the internal carotid artery into the intracranial circulation. The filament was lodged in the narrow proximal anterior cerebral artery (ACA) and blocked the MCA at its origin. Two hours after MCA occlusion, animals were re-anesthetized, and reperfusion was established by withdrawal of the filament. In the group that received local infusion, the catheter was withdrawn 1 mm from the origin of the MCA. During and after the “pull back” of the catheter, 6 ml of cold saline (20 °C) was slowly injected at the junction of MCA and ACA to maintain an infusion rate of 0.6 ml/min for 10 min. After infusion the catheter was completely withdrawn and perfusion was re-established. 4.4 Pharmacological hypothermia DHC was administered via intraperitoneal (i.p.) injection at a dose of 0.5 mg/kg approximately 12
5 minutes before withdrawing the catheter. This dose was considered as a low dose based on an accepted paper from our team. 4.5 Akt inhibition In order to determine a role of Akt in TH-induced neuroprotection, we used PI3K inhibitor LY294002 ([2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one], Cell Signaling Technology, no.9901), to inhibit Akt phosphorylation (Cai et al., 2017; Gao et al., 2008). A microinjection, at dose of 30 µM (10 µL) as established previously, was performed into the lateral ventricle ipsilateral to the ischemia over 10 minutes (from bregma: posterior,-1.0 mm; lateral, -1.5 mm; ventral, -4.0 mm), beginning at 0.5 hours prior to MCAO (Cai et al., 2017). A control group (n=6) for the PI3K/Akt inhibitor was adopted in this study, in which a microinjection (10µL) of saline solution was performed into the lateral ventricle ipsilateral to the ischemia over 10 minutes. 4.6 Neurological deficit Neurological deficits were evaluated via a 12-point scale at 24 h, 3 d and 7 d following reperfusion (Bederson et al., 1986). A higher score was correlated with more profound deficits. The behavior testing was performed by a separate investigator blinded to treatment. 4.7 Brain and body temperature monitoring Needle thermistor probes (Harvard Apparatus Inc.) were placed into the cortex through holes made 3 mm lateral to the bregma, 3 mm posterior to the bregma, and 3 mm lateral to the bregma on the ipsilateral side. Rectal and brain temperatures were measured every 5 minutes. The target hypothermia was defined as ≤35.0 °C (Alonso-Alconada et al., 2015; Kollmar et al, 2007). The period between the beginning and end hypothermia was recognized as the duration of hypothermia. The cooling rate was also calculated for each group. In addition to the cooling process, rats were placed on a feedback-regulated heating pad throughout the 13
surgical procedure. 4.8 Infarct volume Infarct volume was evaluated at 24 h and 7 d following reperfusion as previously described by us (Cai et al., 2016). Six coronal brain slices with a 2-mm thickness were cut for the treatment with TTC (Sigma, St. Louis, MO, USA) at 37 °C for 20 min, then fixed in 10% formalin solution. The percentage of infarct volume relative to non-infarcted area was calculated in order to minimize errors due to edema. 4.9 Apoptotic cell death detection Cell death was measured by quantifying the amount of cytoplasmic histone-associated DNA fragments using a photometric enzyme immunoassay (Cell Death Detection ELISA; Roche Diagnostics, Indianapolis, IN, USA) as previously described by us (Fu et al., 2013). We detected the absorbance of light at 405 nm using a multimode detector (Beckman DTX-880) to determine the degree of apoptosis in the respective samples. 4.10 ATP Assay ATP levels were measured using an ATP assay kit (ATP Colorimetric/ Fluorometric Assay Kit; BioVision, Milpitas, CA, USA) as described by us (Cai et al., 2017). Brain tissues (10 mg) were homogenized, then deproteinized with perchloric acid (PCA) included in the Deproteinization Sample Preparation Kit (BioVision, Milpitas, CA, USA). The samples were then centrifuged at 13,000 g for 2 min and mixed with Neutralization Solution to be neutralized. After a brief spin, the supernatant was mixed with ATP Assay Buffer, ATP probe, ATP Converter and Developer included in the kit. After a 30 min incubation avoiding light, a Varioskan Flash Multimode Reader (Thermo Scientific, Waltham, MA, USA) was used to quantify ATP levels (optical density (OD) = 570 nm). 4.11 ROS Production 14
ROS generation was assessed with the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) as previously described by us (Cai et al., 2017). Homogenized brain samples were diluted to 10 mg/ml based on protein concentration and 100 µg/ml of digitonin was added. After incubation for 30 min, the Amplex Red and Horseradish Peroxidase were added. H2O2 levels in brain homogenates were then detected at 37°C for 30 min on a Varioskan Flash Multimode Reader (Thermo Scientific, Waltham, MA, USA). 4.12 Protein expression Western blot analysis was used to detect expression of pro-apoptotic (AIF, Bax) and anti-apoptotic (Bcl-2 & Bcl-xL) proteins, as well as p-Akt and cleaved Caspase-3. Tissue samples from the ischemic cerebral hemispheres of all experimental and control groups were harvested at 24 h after reperfusion, as described previously (Geng et al., 2015). The samples were loaded onto gels for electrophoresis, and the proteins were transferred to a PVDF membrane. Membranes were incubated with primary antibodies including rabbit polyclonal anti-Phospho-Akt antibody (1:1000, Cell Signaling Technology), rabbit polyclonal anti-Bcl-2 antibody (1:200, Santa Cruz), mouse monoclonal anti-Bcl-xL antibody (1:200, Santa Cruz), mouse monoclonal anti-AIF antibody (1:4000, Santa Cruz), rabbit polyclonal anti-Bax antibody (1:500, Santa Cruz), rabbit polyclonal anti-cleaved Caspase-3 antibody (1:1000, Cell Signaling Technology) at 40C for 24 h. Subsequently, the membranes were washed three times with 1% TBST for 10 min each, and re-incubated with the relevant secondary antibody (goat anti-mouse IgG, ZhongShanJinQiao; goat anti-rabbit IgG, ZhongShanJinQiao) for 1hour at room temperature. Equal protein loading was adjusted using β-actin (mouse polyclonal anti-β-actin antibody, Santa Cruz). An ECL system was used to detect immunoreactive bands by luminescence. Quantification of relative target protein expression was obtained using an 15
image analysis program (ImageJ 1.48, National Institutes of Health, USA). 4.13 Statistical Analysis All values are expressed as means ± SE. Statistical analyses were performed using SPSS for Windows, version 16.0 (SPSS, Inc.). The differences between groups were assessed using one-way analysis of variance (ANOVA) with a significance level set at P<0.05. Post-hoc comparison between groups was conducted using the least significant difference (LSD) method. 5. Acknowledgments This work was supported by National Natural Science Foundation of China (81500997, 81371289); National Natural Science Foundation of China for Outstanding Youth (81325007); Chang Jiang Scholars Program (#T2014251) from the Chinese Ministry of Education; Beijing Municipal Science and Technology Project (2121100005312016, D131100005313017); National Science and Technology Support Plan Project (2013BAI07B01); The “mission” talent project of Beijing Municipal Administration of Hospitals (SML20150802). 6. Disclosures None References Alonso-Alconada, D., Broad, K.D., Bainbridge, A., Chandrasekaran, M., Faulkner, S.D., Kerenyi, Á., Hassell, J., Rocha-Ferreira, E., Hristova, M., et al., 2015. Brain cell death is reduced with cooling by 3.5°C to 5°C but increased with cooling by 8.5°C in a piglet asphyxia model. Stroke 46,275-278. Bederson, J.B., Pitts, L.H., Tsuji, M., Nishimura, M.C., Davis, R.L., Bartkowski, H., 1986. Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke 17,472-476. Cai, L., Stevenson, J., Geng, X., Peng, C., Ji, X., Xin, R., Rastogi, R., Sy, C., Rafols, J.A., Ding, Y., 2017. Combining normobaric oxygen with ethanol or hypothermia prevents brain damage 16
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Geng, X., Elmadhoun, O., Peng, C., Ji, X., Hafeez, A., Liu, Z., Du, H., Rafols, J.A., Ding, Y., 2015. Ethanol and normobaric oxygen: novel approach in modulating pyruvate dehydrogenase complex after severe transient and permanent ischemic stroke. Stroke 46,492-499. Goossens, J., Hachimi-Idrissi, S., 2014. Combination of therapeutic hypothermia and other neuroprotective strategies after an ischemic cerebral insult. Curr. Neuropharmacol. 12,399-412. Han, Z., Liu, X., Luo, Y., Ji, X., 2015. Therapeutic hypothermia for stroke: Where to go? Exp. Neurol. 272,67-77. Ji, Y.B., Wu, Y.M., Ji, Z., Song, W., Xu, S.Y., Wang, Y., Pan, S.Y., 2012. Interrupted intracarotid artery cold saline infusion as an alternative method for neuroprotection after ischemic stroke. Neurosurg. Focus. 33,E10. Jiang, Z., Duong, T.Q., 2016. Methylene blue treatment in experimental ischemic stroke: a mini review. Brain Circ 2,48-53. Kim, F., Olsufka, M., Carlbom, D., Deem, S., Longstreth, W.T.Jr., Hanrahan, M., Maynard, C., Copass, M.K., Cobb, L.A., 2005. Pilot study of rapid infusion of 2 L of 4 degrees C normal saline for induction of mild hypothermia in hospitalized, comatose survivors of out-of-hospital cardiac arrest. Circulation 112,715-719. Kim, J.Y., Yenari, M.A., 2015. Hypothermia for treatment of stroke. Brain Circulation 1,14–25. Krieger, D.W., Yenari, M.A., 2004. Therapeutic hypothermia for acute ischemic stroke: what do laboratory studies teach us? Stroke 35,1482-1489. Kollmar, R., Blank, T., Han, J.L., Georgiadis, D., Schwab, S., 2007. Different degrees of hypothermia after experimental stroke: short- and long-term outcome. Stroke 38,1585-1589. Kollmar, R., Schellinger, P.D., Steigleder, T., Köhrmann, M., Schwab, S., 2009. Ice-cold saline for the induction of mild hypothermia in patients with acute ischemic stroke: a pilot study. Stroke 40,1907-1909. Kurisu, K., Abumiya, T., Ito, M., Gekka, M., Osanai, T., Shichinohe, H., Nakayama, N., Kazumata, K., Houkin, K., 2016. Transarterial regional hypothermia provides robust neuroprotection in a rat model of permanent middle cerebral artery occlusion with transient collateral hypoperfusion. Brain Res 1651,95-103. Lai, T.W., Zhang, S., Wang, Y.T., 2014. Excitotoxicity and stroke: identifying novel targets for neuroprotection. Prog Neurobiol 115: 157-188. 18
Lee, J.H., Wei, L., Gu, X., Won, S., Wei, Z.Z., Dix, T.A., Yu, S.P., 2016a. Improved therapeutic benefits by combining physical cooling with pharmacological hypothermia after severe stroke in rats. Stroke 47,1907-1913. Lee, J.H., Wei, Z.Z., Cao, W., Won, S., Gu, X., Winter, M., Dix, T.A., Wei, L., Yu, S.P., 2016b. Regulation of therapeutic hypothermia on inflammatory cytokines, microglia polarization, migration and functional recovery after ischemic stroke in mice. Neurobiol. Dis. 96,248-260. Liu, K., Khan, H., Geng, X., Zhang, J., Ding, Y., 2016. Pharmacological hypothermia: a potential for future stroke therapy. Neurol. Res. 38, 478-490. Luan, X., Li, J., McAllister, J.P.2nd., Diaz, F.G., Clark, J.C., Fessler, R.D., Ding, Y., 2004. Regional brain cooling induced by vascular saline infusion into ischemic territory reduces brain inflammation in stroke. Acta. Neuropathol. 107,227-234. Lyden, P., Hemmen, T., Grotta, J., Rapp, K., Ernstrom, K., Rzesiewicz, T., Parker, S., Concha, M., Hussain, S., et al., 2016. Results of the ICTuS 2 Trial (Intravascular Cooling in the Treatment of Stroke 2). Stroke 47,2888-2895. Mozaffarian, D., Benjamin, E.J., Go, A.S., Arnett, D.K., Blaha, M.J., Cushman, M., de Ferranti, S., Després, J.P., Fullerton, H.J., et al., 2015. Heart disease and stroke statistics–2015 update: A report from the american heart association. Circulation 131,e29–e322. Muzzi, M., Felici, R., Cavone, L., Gerace, E., Minassi, A., Appendino, G., Moroni, F., Chiarugi, A., 2012. Ischemic neuroprotection by TRPV1 receptor-induced hypothermia. J. Cereb. Blood. Flow. Metab. 32:978-982. Sanderson, T.H., Reynolds, C.A., Kumar, R., Przyklenk, K., Hüttemann, M., 2013. Molecular mechanisms of ischemia-reperfusion injury in brain: pivotal role of the mitochondrial membrane potential in reactive oxygen species generation. Mol. Neurobiol. 47,9-23. Straus, D., Prasad, V., Munoz, L., 2011. Selective therapeutic hypothermia: a review of invasive and noninvasive techniques. Arq. Neuropsiquiatr. 69,981-987. Tsuruta, F., Masuyama, N., Gotoh, Y., 2002. The phosphatidylinositol 3-kinase (PI3K)-Akt pathway suppresses Bax translocation to mitochondria. J. Biol. Chem. 277,14040-14047. Tu, X.K., Zhang, H.B., Shi, S.S., Liang, R.S., Wang, C.H., Chen, C.M., Yang, W.Z., 2016. 5-LOX inhibitor zileuton reduces inflammatory reaction and ischemic brain damage through the activation of PI3K/Akt signaling pathway. Neurochem. Res. 41,2779-2787. Wang, B., Wu, D., Dornbos Iii, D., Shi, J., Ma, Y., Zhang, M., Liu, Y., Chen, J., Ding, Y., et al., 2016. Local cerebral hypothermia induced by selective infusion of cold lactated ringer's: a 19
feasibility study in rhesus monkeys. Neurol. Res. 38:545-552. Wang, Z., Zhang, H., Xu, X., Shi, H., Yu, X., Wang, X., Yan, Y., Fu, X., Hu, H., Li, X., Xiao, J., 2012. bFGF inhibits ER stress induced by ischemic oxidative injury via activation of the PI3K/Akt and ERK1/2 pathways. Toxicol. Lett. 212,137–146. Yenari, M.A., Han, H.S., 2012. Neuroprotective mechanisms of hypothermia in brain ischaemia. Nat. Rev. Neurosci. 13,267-278. Zhang, M., Wang, H., Zhao, J., Chen, C., Leak, R.K., Xu, Y., Vosler, P., Chen, J., Gao, Y., Zhang, F., 2013. Drug-induced hypothermia in stroke models: does it always protect? CNS. Neurol. Disord. Drug. Targets 12, 371-380.
Figure legend Figure 1 Cortex and anal temperature before and after the model development and different treatments. A, cortex temperature changes before, during and after cooling. Cortex temperatures were slightly increased in the stroke group, those in the IACIs group with a fast decrease and recovery trend, those in the DHC group with a gradually reduced trend, those in the combination group showing a fast and stable hypothermia. B, Quantified data revealed that TH, combination, and combination plus Akti groups had a faster cooling rate than DHC group. C, DHC, combination, and combination plus Akti groups had a longer cooling duration than TH group. D, anal temperatures were increased in the stroke group, those were generally unchanged in IACIs group, and those were decreased in rats receiving DHC. E and F, The cooling rate and duration were not different among DHC, combination, and combination plus Akti groups. Data were shown as mean ± SE. One-way analysis of variance (ANOVA) and the least significant difference (LSD) method were used for comparisons among and between groups. N=6 in each group. *P < 0.01 vs DHC group; #P < 0.01 vs TH group. Figure 2 Neuroprotective effects of hypothermic treatment in rat MCAO models. A, TTC staining of brain slices. B, Quantified data revealed that both DHC and IACIs treatment could reduce the infarct volume compared with the stroke group, and the combination could further reduce the infarct volume (P<0.01). C, D, 12-score system evaluating neurological deficit at 24 h (C) and 7 d (D), Monotherapy with either DHC or IACIs reduced neurological deficits, and 20
combination therapy further improved neurological function, but p-Akt inhibitor reversed the combination therapy’s effect in neurological improvement at both 24 h (P<0.01) and 7 d( P<0.01). Data were shown as mean ± SE. One-way analysis of variance (ANOVA) and the least significant difference (LSD) method were used for comparisons among and between groups. N=6 in each group.
*
P < 0.01 vs stroke group; #P < 0.01 vs the combination treatment
group. Figure 3 Reactive oxygen species (ROS) levels (A). Ischemia increased ROS levels, and both DHC and IACIs reduced ROS, and the combination further decreased ROS levels, but p-Akt inhibitor largely reversed these outcomes. Intracellular ATP levels (B). Ischemic stroke reduced ATP levels, only the combination treatment significantly increased ATP levels, while p-Akt inhibition reversed the combination therapy’s effect. Apoptotic cell death analysis (C). Apoptotic cell death was increased in the stroke group, and either DHC or IACIs alone reduced apoptosis, and the combination treatment could further reduce it, while p-Akt inhibitor largely reversed the trend. Data were expressed as mean ± SE. One-way analysis of variance (ANOVA) and the least significant difference (LSD) method were used for comparisons among and between groups. N=6 in each group. *P < 0.05 vs stroke group; #P < 0.05 vs the combination treatment group. Figure 4 the expression of apoptotic or pro – apoptotic proteins (A, B, C), anti-apoptotic proteins (D, E) and p-Akt protein (F). A, B, C, apoptotic or pro – apoptotic proteins (AIF, Bax and Caspase -3) increased in the stroke group, and DHC or IACIs alone decreased their levels, and the combination further reduced these proteins levels, while p-Akt inhibitor largely reversed this trend. D, E, either DHC or IACIs could increase anti-apoptotic proteins (BCL-2 and BCL-xL) levels compared with the stroke group, and the combination could further increase BCL-2 and BCL-xL levels, while p-Akt inhibitor largely reversed the trend. F, either DHC or IACIs increased p-Akt protein levels compared with the stroke group, the combination could further increase it, but the increased level was largely reversed by Akt inhibitor. Data were expressed as mean ± SE. One-way analysis of variance (ANOVA) and the least significant difference (LSD) method were used for comparisons among and between groups. N=6 in each group. *P < 0.01 vs stroke group; # P < 0.01 vs the combination treatment group
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Highlights 1. We investigated the neuroprotective effects of pharmacological hypothermia and local hypothermia in rat MCAO models. 2. The combination of the above methods enhances the efficiency of hypothermia. 3. The combination of the above methods enhances efficacy of hypothermia-induced neuroprotection following ischemic stroke. 4. PI3K/Akt apoptotic pathway is a possible mechanism for the combination therapy.
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S0006-8993(17)30277-9 http://dx.doi.org/10.1016/j.brainres.2017.06.029 BRES 45411
To appear in:
Brain Research
Received Date: Revised Date: Accepted Date:
7 June 2017 27 June 2017 28 June 2017
Please cite this article as: D. Wu, J. Shi, O. Elmadhoun, Y. Duan, H. An, J. Zhang, X. He, R. Meng, X. Liu, X. Ji, Y. Ding, Dihydrocapsaicin (DHC) Enhances the Hypothermia-Induced Neuroprotection Following Ischemic Stroke Via PI3K/Akt Regulation In Rat, Brain Research (2017), doi: http://dx.doi.org/10.1016/j.brainres.2017.06.029
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Dihydrocapsaicin (DHC) Enhances the Hypothermia-Induced Neuroprotection Following Ischemic Stroke Via PI3K/Akt Regulation In Rat Di Wu a, b, c, Jingfei Shi a, b, Omar Elmadhoun d, Yunxia Duan a, Hong An a, Jun Zhang a, Xiaoduo He a, Ran Meng a, Xiangrong Liu a, Xunming Ji a, c *, Yuchuan Ding a,d a
China-America Institute of Neuroscience, Xuanwu Hospital, Capital Medical University, Beijing, China
b
Beijing Key Laboratory of Hypoxia Conditioning Translational Medicine
c Center of Stroke, Beijing Institute for Brain Disorders d
Department of Neurosurgery, Wayne State University School of Medicine, Detroit, MI, USA
*Corresponding Author: Xunming Ji, MD, PhD China-America Institute of Neuroscience, Xuanwu Hospital, Capital Medical University, Beijing 100053, China Email:[email protected]
Abstract Objective: Hypothermia has demonstrated neuroprotection following ischemia in preclinical studies while its clinical application is still very limited. The aim of this study was to explore whether combining local hypothermia in ischemic territory achieved by intra-arterial cold infusions (IACIs) with pharmacologically induced hypothermia enhances therapeutic 1
outcomes, as well as the underlying mechanism. Methods: Sprague-Dawley rats were subjected to right middle cerebral artery occlusion (MCAO) for 2 h using intraluminal hollow filament. The ischemic rats were randomized to receive: 1) pharmacological hypothermia by intraperitoneal (i.p.) injection of dihydrocapsaicin (DHC); 2) physical hypothermia by IACIs for 10 min; or 3) the combined treatments. Extent of brain injury was determined by neurological deficit, infarct volume, and apoptotic cell death at 24 hr and/or 7 d following reperfusion. ATP and ROS levels were measured. Expression of p-Akt, cleaved Caspase-3, pro-apoptotic (AIF, Bax) and anti-apoptotic proteins (Bcl-2, Bcl-xL) was evaluated at 24 hr. Finally, PI3K inhibitor was used to determine the effect of p-Akt. Results: DHC or IACIs each exhibited hypothermic effect and neuroprotection in rat MCAO models. The combination of pharmacological and physical approaches led to a faster and sustained reduction in brain temperatures and improved ischemia-induced injury than either alone (P<0.01). Furthermore, the combination treatment favorably increased the expression of anti-apoptotic proteins and decreased pro-apoptotic protein levels (P<0.01 or 0.05). This neuroprotective effect was largely blocked by p-Akt inhibition, indicating a potential role of Akt pathway in this mechanism (P<0.01 or 0.05). Conclusions: The combination approach is able to enhance the efficiency of hypothermia and efficacy of hypothermia-induced neuroprotection following ischemic stroke. The findings here move us a step closer towards translating this long recognized TH from bench to bedside. Key words: Pharmacological hypothermia, Apoptotic Cell death, Ischemia/reperfusion injury, Middle cerebral artery, intra-arterial cold infusions (IACIs)
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1. Introduction Stroke remains the leading cause of permanent disability in industrialized nations (Mozaffarian et al., 2015). On average every 40 s someone in the USA suffers a stroke, demonstrating the omnipresence and frequency of this devastating disease (Fisher and Saver, 2015). This fact validates our passion to further investigate this disease and develop clinically relevant treatments. However, asides from thrombolytic approaches, the development of other 3
management therapies, such as hypothermia, has been largely unproductive (Kollmar et al., 2009; Jiang and Duong, 2016). Although hypothermia has shown promising neuroprotective effects following ischemia in both preclinical and clinical studies, the delayed onset of brain hypothermia and various side effects associated with the impractically low temperatures needed to achieve superior outcomes have limited its clinical scope (Lee et al, 2016a; Wang et al, 2016; Yenari and Han, 2012; Zhang et al, 2013). Thus, in this study, we intended to achieve neuroprotection with hypothermia more effectively and efficiently than previously described. In previous studies, we developed a unique endovascular technique to induce local hypothermia by infusing cold saline into the microvasculature of the ischemic territory in animal models. These studies demonstrated a strong and safe neuroprotective results (Ding et al., 2002; Ding et al., 2004; Luan et al., 2004). Although this local hypothermia is neuroprotective, it requires more time to perform and duration is not long enough to maintain a sustained hypothermia. Pharmacological hypothermia is gaining increasing attention as an alternative therapeutic approach. This method has a special advantage to induce a controllable and sustained hypothermia status (Lee et al, 2016b). Recent studies have focused on one promising class of agents, the transient receptor potential vanilloid channel 1 (TRPV1) agonists (Liu et al., 2016). TRPV1 is a nonspecific cation channel, possibly inducing neuroprotection through its ability to induce hypothermia. Its agonist, dihydrocapsaicin (DHC) has shown potential neuroprotective effects by inducing hypothermia. However, because of the toxicity associated with high doses that is required for induction of hypothermia, its clinical use was highly limited (Cao et al., 2014; Muzzi et al., 2012). Phosphatidylinositol 3-kinase/Akt (PI3K/Akt) signaling pathway, the most important signaling pathway involved in the neuroprotection against brain ischemia, plays a critical role in promoting neuronal survival after ischemic stroke (Tu et al., 2016). Usually, the 4
neuroprotective property of PI3K/Akt signaling has been primarily attributed to the anti-apoptotic action, or the anti-oxidative action (Wang et al., 2012). To comprehensively understand our combination therapy, the molecular mechanism of the proposed technique on the PI3K/Akt pathway was also studied. Therefore, we explore whether DHC has an additive effect on hypothermia induction and maintenance, and therefore increases the neuroprotective effect, as well as to reduce the dose of DHC and its associated toxicity in order to enhance the efficiency of hypothermia and efficacy of hypothermia-induced neuroprotection following ischemic stroke. 2. Results 2.1Brain and body temperature Two hours after the onset of ischemia, hypothermia by DHC and IACIs was observed in the cortex of stroke models (n=6, Fig 1 A). Cortex temperature rapidly dropped below 35.0 °C within 10 minutes following ICAIs in the TH, combination, and combination plus Akti groups. The cooling rates in the above groups were faster than that in DHC group (p < 0.01 ) (Fig 1 B). In addition, cortex temperature remained below 35.0 °C for at least 80 minutes in the combination and combination plus Akti groups, and cortex temperature remained below 35.0 °C for approximated 50 minutes in DHC group, all of which were longer than that in TH group (p < 0.01 ) (Fig 1 C). p-Akt inhibitor did not significantly affect the cortex temperature. Post-stroke pyrexia was also observed in rectal temperatures. In stroke models that received IACIs, rectal temperature was generally maintained around baseline temperatures for 120 mins. After the administration of DHC, rectal temperature dropped to approximately 35.0 °C at 30 mins, stayed at equal or below 35.0 °C for approximately 20 minutes, and then gradually returned to approximately 36.5 °C at 120 minutes (Fig 1D). There were not differences in cooling rate (p > 0.05) (Fig 1 E) and duration (p > 0.05) (Fig 1 F) among groups receiving 5
DHC treatments. 2.2 Infarct volume Ischemia produced a large infarct volume at 24 h (48.5% + 2.4%), which was significantly reduced by DHC (36.2% + 2.5%) (p < 0.01)and IACIs (35.3% + 2.2%) (p < 0.01), respectively. The combination therapy further decreased the infarct volume (27.3% + 3.2%) (p < 0.01). This reduction was largely reversed with p-Akt inhibition (36.9% + 1.2%) (Fig 2 A and B). 2.3 Neurologic deficits A high score (9.8 + 0.3) was observed in stroke group at 24 h. Neurologic deficits were significantly reduced by DHC (7.8 + 0.4) and IACIs (8.1 + 0.3), respectively(p < 0.01). Furthermore, the combination exhibited a greater reduction in scores (7.1 + 0.3) (p < 0.01). p-Akt inhibition reversed this reduction (8.5 + 0.2) (Fig 2 C). Neurologic deficits with a high score (9.0 + 0.4) was also observed in stroke group at 7 d, which were significantly reduced by DHC (7.0 + 0.4) and TH (7.1 + 0.3) , respectively (p < 0.01). Again, the combination therapy exhibited a greater reduction in scores (5.5 + 0.3) (p < 0.01), which was also reversed by p-Akt inhibition (8.1 + 0.4) (Fig 2 D). 2.4 ROS levels Oxidative stress was significantly increased at 24 h after the ischemia/reperfusion. Both DHC and IACIs treatment were able to reduce ROS levels at 24 h(p < 0.05) . A greater decrease was observed with the combination group (p < 0.05) (Fig 3 A). However, there were not significant differences among DHC, TH and the combination treatment groups (p > 0.05). This was reversed when p-Akt inhibitor was used. 2.5 ATP levels Ischemic stroke markedly decreased ATP levels at 24 h following ischemia/reperfusion (Fig 3 B). ATP levels were increased by DHC or IACIs treatments each alone but did not reach a 6
significant level (p > 0.05). However, the combination treatment induced a marked increase in ATP levels (p<0.05). But, there were not significant differences among DHC, TH and the combination treatment groups (p > 0.05). These changes were largely reversed with p-Akt inhibition. 2.6 Apoptotic cell death Ischemia-reperfusion induced a high level of cell death, which was reduced by DHC, IACIs or the combination treatment (p< 0.05). However, there were not significant differences among DHC, TH and the combination treatment groups (p > 0.05). p-Akt inhibitor could reverse these changes (Fig 3 C). 2.7 Pro-apoptotic protein expression AIF, Bax, and Caspase-3 were analyzed by Western blot at 24 h following reperfusion. There were significant differences in expression of AIF (p < 0.01), Bax (p < 0.01), and Caspase-3 (p < 0.01) at 24 h among all groups. Both DHC and IACIs monotherapies reduced AIF, Bax, and Caspase-3 expressions. The reduction in the expression of those proteins was enhanced by the combination approach. p-Akt inhibition significantly reversed these results (Fig 4 A, B, C). 2.8 Anti-apoptotic protein expression Protein expressions of Bcl-2 and Bcl-xL were analyzed at 24 h following reperfusion. Significant differences were observed for Bcl-2 (p < 0.01) and Bcl-xL (p < 0.01) among all groups. DHC and IACIs treatments increased Bcl-2 and Bcl-xL levels while the combination treatment further enhanced these effects. This, again, was reversed by p-Akt inhibitor (Fig 4 D, E). 2.9 p-Akt protein expression Phosphorylated AKT (p-AKT) is the constitutive activation form of AKT. Protein expressions of p-Akt were significantlydifferent at 24 h following reperfusion among all groups (p < 0.01) . 7
DHC, IACIs, and the combination treatment significantly increased p-Akt levels, while the inhibitor led to a decrease of p-Akt (Fig 4F). 3. Discussion The present study revealed that combining physical and pharmacological techniques resulted in a faster and more stable hypothermia than either method alone, in association with reduced ischemic damages and restored neurological functions as compared to monotherapies. Furthermore, a possible molecular mechanism for these effects could be explained, at least partially by the PI3K/Akt pathway. The combination treatment may create a pro-survival environment and enhance the cellular oxidative profile following ischemia/reperfusion. The efficacy of hypothermia is dependent on several factors including cooling rate as well as the duration and depth of hypothermia. Previous studies have reported that lower temperatures which are achieved rapidly and maintained have better clinical outcomes (Han et al., 2015; Krieger and Yenari, 2004). Our previous study reported that a rapid temperature decrease can be achieved by IACIs in rat transient MACO models (Ding et al., 2002; Ding et al., 2004. Both experimental and clinical studies also found that intra-carotid infusion of cold saline or rapid infusion of cold saline through a peripheral vein could induce a mild hypothermia (Luan et al., 2004; Kim et al., 2005; Straus et al., 2011). However, the induction rate and duration of hypothermia were volume-dependent. Hypothermia induced by these methods improved the outcomes of brain ischemia in experimental studies, but a rapid and sustained hypothermia was technically difficult because of an inevitable drop in hematocrit levels and limitations on the duration of treatment (Ji et al., 2012). In clinical, intravascular infusion of a high load of 4°C saline infusions in ICTuS trial achieved a feasible hypothermia strategy, but did not lead to a superior neuroprotection (Lyden et al., 2016). A higher liquid load could lead to a feasible hypothermia, but not necessarily result in more neuroprotection. So it was 8
impossible to achieve a fast and sustained hypothermia only by increasing the liquid load (Esposito et al., 2014). At present, physical cooling is adopted in clinical hypothermic strategy, however the shivering response must be countered by sedatives and mechanical ventilation, which complicate the prognosis and recovery. Pharmaceutical-induced hypothermia provides a good alternative methodology in a controllable way, especially those affecting the thermoregulatory set point. These drugs do not induce anti-shivering responses, which provide an opportunity to combine with non-invasive hypothermia techniques to achieve rapid and controlled cooling. Recent study reported that the centrally acting hypothermic drug HPI-201 greatly enhanced the efficiency and efficacy of conventional physical cooling in rat MCAO models, and induced synergistic neuroprotective effects (Lee et al, 2016a). However, cooling efficacy of pharmaceutical-induced hypothermia was relatively weaker in severe stroke models compared with its effects in normal and small stroke animals [Lee et al, 2016a]. In addition, when adopting HPI-201 or DHC alone, it generally took approximately 30 minutes to achieve hypothermia. DHC, a natural capsaicin, belongs to Transient Receptor Potential Vanilloid channel 1 (TRPV1) agonists (Cao et al., 2014; Fosgerau et al., 2010). Previous studies have shown that DHC have a greater effectiveness to induce hypothermia compared with capsaicin. But at high doses are associated with negative side effects such as hypotension and bradycardia (Fosgerau et al., 2010; Muzzi et al, 2012). In addition, over stimulation of TRPV1 receptors promotes glutamate release and excitotoxicity in the central nerve system, then leads to degeneration of neurons after ischemia (Lai et al., 2014). Our group recently proved that DHC at a low dose could induce a stable hypothermia and lead to a better neuroprotection. This study further confirmed that the combination treatment of pharmacological and local hypothermia was efficacious in improving clinical effects, decreasing the side effects brought 9
on by either therapy alone. Ischemic stroke leads to a cascade of catastrophic consequences on a number of molecular pathways in neurons. For example, the rise in ROS to toxic levels following ischemia cause cellular injury in multiple forms including lipid peroxidation, protein oxidation and DNA damage which collectively worsens brain damage (Goossens and Hachimi-Idrissi ,2014; Kurisu et a., 2016). Additionally, reduced ATP levels contribute to the cellular swelling and imbalanced intracellular ion gradient (Forreider et al., 2016; Sanderson et al., 2013). This necessitates using a combination of neuroprotective agents that can intervene in multiple mechanisms. Hypothermia has multifaceted neuroprotective effects due to its ability to lower the metabolic rate, reduce intracellular calcium influx and intracellular acidosis, suppress ROS formation and infiltration of inflammatory cells, down-regulate specific cell death pathways and/or up-regulate cell survival mechanisms based on previous reports (Han et al., 2015; Kim and Yenari, 2015). Moreover the combination treatment is proved to further reduce ROS generation and pro-apoptotic proteins, increase ATP and anti-apoptotic proteins levels in this paper. This indicates that different mechanisms of action exist to our combination approach and local cold infusions have a synergistic effect with pharmacologically induced hypothermia. PI3K/Akt pathway, the most important pro-survival signaling pathway, was widely considered to play a critical role in neuroprotective effect against ischemic brain injury. A previous study found that zileuton attenuates neurological deficit scores, cerebral infarct volume, cerebral water content and ischemic neuronal injury in rats subjected to focal cerebral ischemia through PI3K/Akt signaling pathway (Tu et al., 2016). Another study discovered that the activation of PI3K/Akt pathway limited the pro-apoptotic function of Bax and inhibited cytochrome c release from the mitochondria to exert neuroprotective effects (Tsuruta et al., 2002). The 10
present study showed that pharmacological and physical hypothermia had a synergetic effect to enhance neuroprotection through anti-apoptosis and PI3K/Akt pathway, all of which were abolished by LY294002 administration, indicating that PI3K/Akt signaling pathway mediates neuroprotection of pharmacological and physical hypothermia against ischemic stroke. In summary, we were able to avoid limitations that had historically narrowed TH’s clinical applications by combining IACIs and DHC. As such, the combination therapy has a significant potential to remarkably widen the role of hypothermia in the treatment of stroke patients. Although hypothermia has multifaceted neuroprotective effects, we showed that PI3K/Akt apoptotic pathway is a possible mechanism of action of the combination therapy. 4. Experimental Procedures 4.1 Subjects A total of 114 adult male Sprague-Dawley rats (300 to 340g weight, Vital River Laboratory Animal Co.) were used. All experimental procedures were approved by the Institutional Animal Investigation Committee of Capital Medical University in accordance with the National Institutes of Health (USA) guidelines for care and use of laboratory animals. All animals were housed in the same animal care facility, with 12-hour light/dark cycles, throughout the study. Animals were randomly divided into 6 groups by animal tag number: (1) sham-operated group (n=18), in which the rats underwent all of the operative process except the middle cerebral artery occlusion (MCAO); (2) stroke group (n=18), in which the rats received 2 h MCAO followed by the reperfusion without treatment; (3) DHC group (n=18), in which ischemic rats received DHC injection at the onset of reperfusion by withdrawing the catheter; (4) TH group (n=18), in which ischemic rats received (IACIs) at the onset of reperfusion; (5) DHC + TH (combination) group (n=18), in which ischemic rats received both DHC and IACIs; (6) DHC + TH + Akti (combination plus Akti) group (n=24), in which 11
ischemic rats received DHC, IACIs, and the PI3K/Akt inhibitor (LY294002). The exclusion criteria included animal death before expected time points, brain hemorrhage, and no signs of infarct based on 2,3,5-triphenyltetrazolium chloride (TTC) staining and neurological deficits. All procedures and data analysis were performed in a blinded and randomized manner. 4.2 Surgical preparation Anesthesia was induced and maintained using 1.5–3.5% enflurane in 70% nitrous oxide and 30% oxygen (Bickford veterinary anesthesia equipment model 61010; AM Bickford Inc., Wales Center, NY, USA). Rectal temperature was monitored during the procedure and was maintained at 36.5-37.5°C using a feedback-controlled heating blanket if needed. Blood gases and pressure were monitored via the right femoral artery which was cannulated with a PE-50 catheter. 4.3 Focal ischemic and therapeutic hypothermia (TH) Two hour MCAO was induced using similar approach as previously reported by us (Ding et al., 2002; Luan et al., 2004). Briefly, PE-50 catheter was inserted into the right external carotid artery via an arteriotomy, and passed up the lumen of the internal carotid artery into the intracranial circulation. The filament was lodged in the narrow proximal anterior cerebral artery (ACA) and blocked the MCA at its origin. Two hours after MCA occlusion, animals were re-anesthetized, and reperfusion was established by withdrawal of the filament. In the group that received local infusion, the catheter was withdrawn 1 mm from the origin of the MCA. During and after the “pull back” of the catheter, 6 ml of cold saline (20 °C) was slowly injected at the junction of MCA and ACA to maintain an infusion rate of 0.6 ml/min for 10 min. After infusion the catheter was completely withdrawn and perfusion was re-established. 4.4 Pharmacological hypothermia DHC was administered via intraperitoneal (i.p.) injection at a dose of 0.5 mg/kg approximately 12
5 minutes before withdrawing the catheter. This dose was considered as a low dose based on an accepted paper from our team. 4.5 Akt inhibition In order to determine a role of Akt in TH-induced neuroprotection, we used PI3K inhibitor LY294002 ([2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one], Cell Signaling Technology, no.9901), to inhibit Akt phosphorylation (Cai et al., 2017; Gao et al., 2008). A microinjection, at dose of 30 µM (10 µL) as established previously, was performed into the lateral ventricle ipsilateral to the ischemia over 10 minutes (from bregma: posterior,-1.0 mm; lateral, -1.5 mm; ventral, -4.0 mm), beginning at 0.5 hours prior to MCAO (Cai et al., 2017). A control group (n=6) for the PI3K/Akt inhibitor was adopted in this study, in which a microinjection (10µL) of saline solution was performed into the lateral ventricle ipsilateral to the ischemia over 10 minutes. 4.6 Neurological deficit Neurological deficits were evaluated via a 12-point scale at 24 h, 3 d and 7 d following reperfusion (Bederson et al., 1986). A higher score was correlated with more profound deficits. The behavior testing was performed by a separate investigator blinded to treatment. 4.7 Brain and body temperature monitoring Needle thermistor probes (Harvard Apparatus Inc.) were placed into the cortex through holes made 3 mm lateral to the bregma, 3 mm posterior to the bregma, and 3 mm lateral to the bregma on the ipsilateral side. Rectal and brain temperatures were measured every 5 minutes. The target hypothermia was defined as ≤35.0 °C (Alonso-Alconada et al., 2015; Kollmar et al, 2007). The period between the beginning and end hypothermia was recognized as the duration of hypothermia. The cooling rate was also calculated for each group. In addition to the cooling process, rats were placed on a feedback-regulated heating pad throughout the 13
surgical procedure. 4.8 Infarct volume Infarct volume was evaluated at 24 h and 7 d following reperfusion as previously described by us (Cai et al., 2016). Six coronal brain slices with a 2-mm thickness were cut for the treatment with TTC (Sigma, St. Louis, MO, USA) at 37 °C for 20 min, then fixed in 10% formalin solution. The percentage of infarct volume relative to non-infarcted area was calculated in order to minimize errors due to edema. 4.9 Apoptotic cell death detection Cell death was measured by quantifying the amount of cytoplasmic histone-associated DNA fragments using a photometric enzyme immunoassay (Cell Death Detection ELISA; Roche Diagnostics, Indianapolis, IN, USA) as previously described by us (Fu et al., 2013). We detected the absorbance of light at 405 nm using a multimode detector (Beckman DTX-880) to determine the degree of apoptosis in the respective samples. 4.10 ATP Assay ATP levels were measured using an ATP assay kit (ATP Colorimetric/ Fluorometric Assay Kit; BioVision, Milpitas, CA, USA) as described by us (Cai et al., 2017). Brain tissues (10 mg) were homogenized, then deproteinized with perchloric acid (PCA) included in the Deproteinization Sample Preparation Kit (BioVision, Milpitas, CA, USA). The samples were then centrifuged at 13,000 g for 2 min and mixed with Neutralization Solution to be neutralized. After a brief spin, the supernatant was mixed with ATP Assay Buffer, ATP probe, ATP Converter and Developer included in the kit. After a 30 min incubation avoiding light, a Varioskan Flash Multimode Reader (Thermo Scientific, Waltham, MA, USA) was used to quantify ATP levels (optical density (OD) = 570 nm). 4.11 ROS Production 14
ROS generation was assessed with the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) as previously described by us (Cai et al., 2017). Homogenized brain samples were diluted to 10 mg/ml based on protein concentration and 100 µg/ml of digitonin was added. After incubation for 30 min, the Amplex Red and Horseradish Peroxidase were added. H2O2 levels in brain homogenates were then detected at 37°C for 30 min on a Varioskan Flash Multimode Reader (Thermo Scientific, Waltham, MA, USA). 4.12 Protein expression Western blot analysis was used to detect expression of pro-apoptotic (AIF, Bax) and anti-apoptotic (Bcl-2 & Bcl-xL) proteins, as well as p-Akt and cleaved Caspase-3. Tissue samples from the ischemic cerebral hemispheres of all experimental and control groups were harvested at 24 h after reperfusion, as described previously (Geng et al., 2015). The samples were loaded onto gels for electrophoresis, and the proteins were transferred to a PVDF membrane. Membranes were incubated with primary antibodies including rabbit polyclonal anti-Phospho-Akt antibody (1:1000, Cell Signaling Technology), rabbit polyclonal anti-Bcl-2 antibody (1:200, Santa Cruz), mouse monoclonal anti-Bcl-xL antibody (1:200, Santa Cruz), mouse monoclonal anti-AIF antibody (1:4000, Santa Cruz), rabbit polyclonal anti-Bax antibody (1:500, Santa Cruz), rabbit polyclonal anti-cleaved Caspase-3 antibody (1:1000, Cell Signaling Technology) at 40C for 24 h. Subsequently, the membranes were washed three times with 1% TBST for 10 min each, and re-incubated with the relevant secondary antibody (goat anti-mouse IgG, ZhongShanJinQiao; goat anti-rabbit IgG, ZhongShanJinQiao) for 1hour at room temperature. Equal protein loading was adjusted using β-actin (mouse polyclonal anti-β-actin antibody, Santa Cruz). An ECL system was used to detect immunoreactive bands by luminescence. Quantification of relative target protein expression was obtained using an 15
image analysis program (ImageJ 1.48, National Institutes of Health, USA). 4.13 Statistical Analysis All values are expressed as means ± SE. Statistical analyses were performed using SPSS for Windows, version 16.0 (SPSS, Inc.). The differences between groups were assessed using one-way analysis of variance (ANOVA) with a significance level set at P<0.05. Post-hoc comparison between groups was conducted using the least significant difference (LSD) method. 5. Acknowledgments This work was supported by National Natural Science Foundation of China (81500997, 81371289); National Natural Science Foundation of China for Outstanding Youth (81325007); Chang Jiang Scholars Program (#T2014251) from the Chinese Ministry of Education; Beijing Municipal Science and Technology Project (2121100005312016, D131100005313017); National Science and Technology Support Plan Project (2013BAI07B01); The “mission” talent project of Beijing Municipal Administration of Hospitals (SML20150802). 6. Disclosures None References Alonso-Alconada, D., Broad, K.D., Bainbridge, A., Chandrasekaran, M., Faulkner, S.D., Kerenyi, Á., Hassell, J., Rocha-Ferreira, E., Hristova, M., et al., 2015. Brain cell death is reduced with cooling by 3.5°C to 5°C but increased with cooling by 8.5°C in a piglet asphyxia model. Stroke 46,275-278. Bederson, J.B., Pitts, L.H., Tsuji, M., Nishimura, M.C., Davis, R.L., Bartkowski, H., 1986. Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke 17,472-476. Cai, L., Stevenson, J., Geng, X., Peng, C., Ji, X., Xin, R., Rastogi, R., Sy, C., Rafols, J.A., Ding, Y., 2017. Combining normobaric oxygen with ethanol or hypothermia prevents brain damage 16
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Lee, J.H., Wei, L., Gu, X., Won, S., Wei, Z.Z., Dix, T.A., Yu, S.P., 2016a. Improved therapeutic benefits by combining physical cooling with pharmacological hypothermia after severe stroke in rats. Stroke 47,1907-1913. Lee, J.H., Wei, Z.Z., Cao, W., Won, S., Gu, X., Winter, M., Dix, T.A., Wei, L., Yu, S.P., 2016b. Regulation of therapeutic hypothermia on inflammatory cytokines, microglia polarization, migration and functional recovery after ischemic stroke in mice. Neurobiol. Dis. 96,248-260. Liu, K., Khan, H., Geng, X., Zhang, J., Ding, Y., 2016. Pharmacological hypothermia: a potential for future stroke therapy. Neurol. Res. 38, 478-490. Luan, X., Li, J., McAllister, J.P.2nd., Diaz, F.G., Clark, J.C., Fessler, R.D., Ding, Y., 2004. Regional brain cooling induced by vascular saline infusion into ischemic territory reduces brain inflammation in stroke. Acta. Neuropathol. 107,227-234. Lyden, P., Hemmen, T., Grotta, J., Rapp, K., Ernstrom, K., Rzesiewicz, T., Parker, S., Concha, M., Hussain, S., et al., 2016. Results of the ICTuS 2 Trial (Intravascular Cooling in the Treatment of Stroke 2). Stroke 47,2888-2895. Mozaffarian, D., Benjamin, E.J., Go, A.S., Arnett, D.K., Blaha, M.J., Cushman, M., de Ferranti, S., Després, J.P., Fullerton, H.J., et al., 2015. Heart disease and stroke statistics–2015 update: A report from the american heart association. Circulation 131,e29–e322. Muzzi, M., Felici, R., Cavone, L., Gerace, E., Minassi, A., Appendino, G., Moroni, F., Chiarugi, A., 2012. Ischemic neuroprotection by TRPV1 receptor-induced hypothermia. J. Cereb. Blood. Flow. Metab. 32:978-982. Sanderson, T.H., Reynolds, C.A., Kumar, R., Przyklenk, K., Hüttemann, M., 2013. Molecular mechanisms of ischemia-reperfusion injury in brain: pivotal role of the mitochondrial membrane potential in reactive oxygen species generation. Mol. Neurobiol. 47,9-23. Straus, D., Prasad, V., Munoz, L., 2011. Selective therapeutic hypothermia: a review of invasive and noninvasive techniques. Arq. Neuropsiquiatr. 69,981-987. Tsuruta, F., Masuyama, N., Gotoh, Y., 2002. The phosphatidylinositol 3-kinase (PI3K)-Akt pathway suppresses Bax translocation to mitochondria. J. Biol. Chem. 277,14040-14047. Tu, X.K., Zhang, H.B., Shi, S.S., Liang, R.S., Wang, C.H., Chen, C.M., Yang, W.Z., 2016. 5-LOX inhibitor zileuton reduces inflammatory reaction and ischemic brain damage through the activation of PI3K/Akt signaling pathway. Neurochem. Res. 41,2779-2787. Wang, B., Wu, D., Dornbos Iii, D., Shi, J., Ma, Y., Zhang, M., Liu, Y., Chen, J., Ding, Y., et al., 2016. Local cerebral hypothermia induced by selective infusion of cold lactated ringer's: a 19
feasibility study in rhesus monkeys. Neurol. Res. 38:545-552. Wang, Z., Zhang, H., Xu, X., Shi, H., Yu, X., Wang, X., Yan, Y., Fu, X., Hu, H., Li, X., Xiao, J., 2012. bFGF inhibits ER stress induced by ischemic oxidative injury via activation of the PI3K/Akt and ERK1/2 pathways. Toxicol. Lett. 212,137–146. Yenari, M.A., Han, H.S., 2012. Neuroprotective mechanisms of hypothermia in brain ischaemia. Nat. Rev. Neurosci. 13,267-278. Zhang, M., Wang, H., Zhao, J., Chen, C., Leak, R.K., Xu, Y., Vosler, P., Chen, J., Gao, Y., Zhang, F., 2013. Drug-induced hypothermia in stroke models: does it always protect? CNS. Neurol. Disord. Drug. Targets 12, 371-380.
Figure legend Figure 1 Cortex and anal temperature before and after the model development and different treatments. A, cortex temperature changes before, during and after cooling. Cortex temperatures were slightly increased in the stroke group, those in the IACIs group with a fast decrease and recovery trend, those in the DHC group with a gradually reduced trend, those in the combination group showing a fast and stable hypothermia. B, Quantified data revealed that TH, combination, and combination plus Akti groups had a faster cooling rate than DHC group. C, DHC, combination, and combination plus Akti groups had a longer cooling duration than TH group. D, anal temperatures were increased in the stroke group, those were generally unchanged in IACIs group, and those were decreased in rats receiving DHC. E and F, The cooling rate and duration were not different among DHC, combination, and combination plus Akti groups. Data were shown as mean ± SE. One-way analysis of variance (ANOVA) and the least significant difference (LSD) method were used for comparisons among and between groups. N=6 in each group. *P < 0.01 vs DHC group; #P < 0.01 vs TH group. Figure 2 Neuroprotective effects of hypothermic treatment in rat MCAO models. A, TTC staining of brain slices. B, Quantified data revealed that both DHC and IACIs treatment could reduce the infarct volume compared with the stroke group, and the combination could further reduce the infarct volume (P<0.01). C, D, 12-score system evaluating neurological deficit at 24 h (C) and 7 d (D), Monotherapy with either DHC or IACIs reduced neurological deficits, and 20
combination therapy further improved neurological function, but p-Akt inhibitor reversed the combination therapy’s effect in neurological improvement at both 24 h (P<0.01) and 7 d( P<0.01). Data were shown as mean ± SE. One-way analysis of variance (ANOVA) and the least significant difference (LSD) method were used for comparisons among and between groups. N=6 in each group.
*
P < 0.01 vs stroke group; #P < 0.01 vs the combination treatment
group. Figure 3 Reactive oxygen species (ROS) levels (A). Ischemia increased ROS levels, and both DHC and IACIs reduced ROS, and the combination further decreased ROS levels, but p-Akt inhibitor largely reversed these outcomes. Intracellular ATP levels (B). Ischemic stroke reduced ATP levels, only the combination treatment significantly increased ATP levels, while p-Akt inhibition reversed the combination therapy’s effect. Apoptotic cell death analysis (C). Apoptotic cell death was increased in the stroke group, and either DHC or IACIs alone reduced apoptosis, and the combination treatment could further reduce it, while p-Akt inhibitor largely reversed the trend. Data were expressed as mean ± SE. One-way analysis of variance (ANOVA) and the least significant difference (LSD) method were used for comparisons among and between groups. N=6 in each group. *P < 0.05 vs stroke group; #P < 0.05 vs the combination treatment group. Figure 4 the expression of apoptotic or pro – apoptotic proteins (A, B, C), anti-apoptotic proteins (D, E) and p-Akt protein (F). A, B, C, apoptotic or pro – apoptotic proteins (AIF, Bax and Caspase -3) increased in the stroke group, and DHC or IACIs alone decreased their levels, and the combination further reduced these proteins levels, while p-Akt inhibitor largely reversed this trend. D, E, either DHC or IACIs could increase anti-apoptotic proteins (BCL-2 and BCL-xL) levels compared with the stroke group, and the combination could further increase BCL-2 and BCL-xL levels, while p-Akt inhibitor largely reversed the trend. F, either DHC or IACIs increased p-Akt protein levels compared with the stroke group, the combination could further increase it, but the increased level was largely reversed by Akt inhibitor. Data were expressed as mean ± SE. One-way analysis of variance (ANOVA) and the least significant difference (LSD) method were used for comparisons among and between groups. N=6 in each group. *P < 0.01 vs stroke group; # P < 0.01 vs the combination treatment group
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Highlights 1. We investigated the neuroprotective effects of pharmacological hypothermia and local hypothermia in rat MCAO models. 2. The combination of the above methods enhances the efficiency of hypothermia. 3. The combination of the above methods enhances efficacy of hypothermia-induced neuroprotection following ischemic stroke. 4. PI3K/Akt apoptotic pathway is a possible mechanism for the combination therapy.
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