Rapamycin ameliorates brain metabolites alterations after transient focal ischemia in rats

European Journal of Pharmacology 757 (2015) 28–33

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Neuropharmacology and analgesia

Rapamycin ameliorates brain metabolites alterations after transient focal ischemia in rats Anjali Chauhan a,n, Uma Sharma b, Naranamangalam R. Jagannathan b, Yogendra Kumar Gupta a,nn a b

Neuropharmacology Laboratory, Department of Pharmacology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India Department of Nuclear Magnetic Resonance, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India

art ic l e i nf o

a b s t r a c t

Article history: Received 11 November 2014 Received in revised form 19 February 2015 Accepted 1 March 2015 Available online 23 March 2015

Rapamycin has been shown to protect against middle cerebral artery occlusion (MCAo) induced ischemic injury. In this study, the neuroprotective effect of rapamycin on the metabolic changes induced by MCAo was evaluated using nuclear magnetic resonance (NMR) spectroscopy of brain tissues. MCAo in rats was induced by insertion of nylon filament. One hour after ischemia, rapamycin (250 mg/kg, i.p.) in dimethyl sulfoxide was administered. Reperfusion was done 2 h after ischemia. Twenty-four hours after ischemia phospholipase A2 (PLA2) levels and metabolic changes were assessed. Perchloric acid extraction was performed on the brain of all animals (n ¼ 7; sham, vehicle; DMSO and rapamycin 250 mg/kg) and the various brain metabolites were assessed by NMR spectroscopy. In all 44 metabolites were assigned in the proton NMR spectrum of rat brain tissues. In the vehicle group, we observed increased lactate levels and decreased levels of glutamate/glutamine, choline containing compounds, creatine/phosphocreatine (Cr/PCr), taurine, myo-inositol, γ-amino butryic acid (GABA), N-aspartyl aspartate (NAA), purine and pyrimidine metabolites. In rapamycin treated rats, there was increase in the levels of choline containing compounds, NAA, myo-inositol, glutamate/glutamine, GABA, Cr/PCr and taurine as compared to those of vehicle control (Po0.05). Rapamycin treatment reduced PLA2 levels as compared to vehicle group (Po0.05). Our findings indicated that rapamycin reduced the increased PLA2 levels and altered brain metabolites after MCAo. These protective effects might be attributed to its effect on cell membrane metabolism; glutamate induced toxicity and calcium homeostasis in stroke. & 2015 Elsevier B.V. All rights reserved.

Keywords: Focal cerebral ischemia Metabolites NMR spectroscopy Rapamycin Middle cerebral artery occlusion Chemical compounds studied in this article: Rapamycin (PubChem CID CAS 53123-88-9)

1. Introduction Stroke is a serious cause of mortality and morbidity affecting about 16 million people worldwide every year (Strong et al., 2007). After the onset of ischemia, multiple mechanisms contribute to brain injury including excitotoxicity, activation of phospholipases, generation of free radicals and activation of inflammatory cascade (Phillis and O’Regan, 2003; Mattson et al., 2000). The pathophysiology and biochemistry implicated in the stroke injury have not been fully elucidated. To understand the biochemical basis in stroke, metabolic analysis may serve as a useful proposition. Intravenous recombinant tissue plasminogen activator is the only approved therapy for treatment of ischemic stroke (Adams

n Corresponding author. Present address: Department of Neuroscience, University of Connecticut Health Center, Farmington, CT 06030, USA. nn Corresponding author. Tel.: þ91 11 26593282; fax: þ 91 11 26588500/641/ 663. E-mail addresses: [email protected] (A. Chauhan), [email protected] (Y.K. Gupta).

http://dx.doi.org/10.1016/j.ejphar.2015.03.006 0014-2999/& 2015 Elsevier B.V. All rights reserved.

et al., 2005). However, patients who receive this drug within the initial therapeutic window have a high risk of intracranial hemorrhage (Külkens and Hacke, 2007), disruption of blood brain barrier, seizures and progression of neuronal damage (Tsirka et al., 1997; Wang et al., 1998; Zhuo et al., 2000). Rapamycin is an immunosuppressive and anti-proliferative agent, shown to act through mammalian target of rapamycin (mTOR) receptors and inhibit cell survival, proliferation, differentiation, apoptosis and autophagy (Manning and Cantley, 2007). It has been shown to afford improvement in behavioral and learning in the animal models of traumatic brain injury, Alzheimer disease and stroke (Erlich et al., 2007; Caccamo et al., 2010; Chauhan et al., 2011). Proton (1H ) in-vivo magnetic resonance spectroscopy (MRS) has been used to study the in-vivo alterations in the brain biochemistry in several neurological conditions including brain tumors (Brandão and Castillo, 2013), multiple sclerosis (Arnold et al.,1990) and stroke (Lin et al., 2014; Cvoro et al., 2010). By employing MRS spectroscopy, cerebral ischemia induced changes in levels of brain metabolites such as lactate, N-acetyl aspartate (NAA), choline (Cho) and creatine (Cr) have been detected in

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stroke patients (Lin et al., 2014; Cvoro et al., 2010) and also in the experimental models of stroke (van der Toorn et al., 1996; Yang et al., 2012). One of the experimental models, which depicts transient stroke similar to, that observed in humans is the middle cerebral artery occlusion (MCAo) model. This model is easy to perform in rodents and is widely used to study the potential of drugs in the treatment of stroke (Chauhan et al., 2011, 2012; Rogers et al., 1997). We have previously demonstrated the protective effect of rapamycin in the MCA occlusion induced focal ischemia in rats (Chauhan et al., 2011). This neuroprotection by rapamycin was evident in the biochemical, neuro-imaging and functional outcomes. In the present study, our objective has been to explore the mechanism of neuroprotection by rapamycin on the brain biochemistry in the focal cerebral ischemia by estimating the concentration of various metabolites using in -vitro 1H NMR spectroscopy of brain tissues.

2. Materials and methods

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placed in the center of the receiver coil and shimmed on the deuterium lock signal. The one-dimensional spectra with water suppression were acquired using a single 901 pulse over a spectral width of 7716 Hz using 32K data points, 64 scans and a relaxation delay of 14 s. Two-dimensional correlated spectroscopy (COSY) and the total correlation spectroscopy (TOCSY) were also carried out. The typical parameters used for TOCSY experiments were: data points 2K in F2 dimension, spectral width 7716 Hz and a relaxation delay of 2 s. The number of t1 increments was 256 and 64 free induction decays per increment were acquired. The concentrations of the metabolites were determined by comparing the integrated intensity of the isolated resonances of the compounds of interest with that of the TSP signal (Sharma et al., 2003). 2.5. Statistical analysis Data are represented as mean 7S.D. Analysis of variance (ANOVA) with Bonferroni post-hoc analysis was used for comparing the parameters. Po 0.05 was taken as the level of significance.

2.1. Experimental groups Male Wistar rats weighing 230–250 g (n¼ 7) were obtained from the Central Animal Facility of All India Institute of Medical Sciences, New Delhi, India. The animals were maintained under standard laboratory conditions with natural dark–light cycle and were allowed free access to standard dry rat diet and tap water. All experimental procedures performed in rats were reviewed and approved by the Institutional Animal Ethics Committee. MCAo was performed according to the procedure described previously (Chauhan et al., 2011; Koizumi et al., 1986). The rats were randomly divided into 3 groups, the first was sham group (no MCA occlusion and no drug treatment, n ¼7); the second was MCA occluded and vehicle treated group (dimethyl sulfoxide, i.p., n ¼7) whereas the third group was treated with rapamycin with a dose of 250 mg/kg, i.p. dissolved in DMSO (dosage was selected from our previous study by Chauhan et al. (2011), one hour after occlusion. The rats were euthanized after 24 h and their brains were then removed for the estimation of phospholipase A2 levels and for invitro NMR spectroscopy. 2.2. Measurement of phospholipase A2 concentration The quantitative measurement of PLA2 in the rat brain was performed using the commercial rat PLA2 ELISA kits (Cayman Chemicals, USA). All procedures were performed according to the manufacturer's instructions.

3. Results 3.1. Effect of rapamycin on PLA2 levels Two hours of ischemia demonstrated significant increase in PLA2 levels in vehicle group as compared to sham (Fig. 1) (Po0.05) as assessed after 24 h. Treatment with rapamycin reduced the PLA2 levels in the rapamycin group as compared to the vehicle treated group (Fig. 1). 3.2. Brain metabolites assignments and concentrations Figs. 2 and 3 show the representative aliphatic and aromatic regions of the one- dimensional 1H NMR spectrum of the perchloric acid extract of cortex tissue of control, ischemia and rapamycin treated rats. The resonance assignments were carried out using coupling connectivities observed in two-dimensional TOCSY and compared with the chemical shift values of metabolites of tissue extracts of muscle, brain and plasma (Yang et al., 2012; Sharma et al., 2003; Chen et al., 2012). A total of 44 metabolites were unambiguously assigned from the PCA extract of the brain tissue of the normal and ischemic rats. The chemical shifts of the metabolites are presented in Table 1.

2.3. Perchloric acid extraction For performing the in vitro NMR, the water-soluble metabolites were extracted from the excised rat brain cortex tissues using perchloric acid extraction (PCA) as previously described earlier (Payen et al., 1996; Sharma et al., 2003). 3-Trimethyl silyl propionic acid (TSP) (0.5 mM) was added to the sample that served both as a chemical shift reference and concentration standard for the proton NMR studies (Sharma et al., 2003). 2.4. In vitro NMR spectroscopy Proton NMR of tissue specimens was carried out on a 700 MHz NMR spectrometer (Agilent) Technologies, U.K. The data was acquired using a standard 5 mm dedicated multinuclear broadband inverse probe at 25 1C. The residual water was suppressed using a pre-saturation pulse. The chemical shifts of the resonances were referenced to TSP at 0 ppm. The sample in the NMR tube was

Fig. 1. Shows the effect of rapamycin on the PLA2 levels. *Po 0.05 as compared to sham; #Po 0.05 as compared to vehicle. Data is presented as mean7 S.D. The levels of brain PLA2 were investigated 24 h after ischemia. There is an increase in the brain PLA2 level in the vehicle as compared to sham group at 24 h. With the rapamycin 250 mg/kg treatment, the PLA2 levels were significantly reduced. A mortality of 14.2% (1/7) was observed in the vehicle treated group and no mortality was observed in the rapamycin group.

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11 9 19 26

7,8

15 23 1

44,55

19

11

15 17,18

12 22

40,41

9

6

10,17 16

21

14

24

22 7,8 16 27

13 7,8

16

5

2

1

11 9 19

15

1

17,18 14

10,17 12

16

21

24

6 7,8

16 7,8

16

4 3

9

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20 5

1

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11 19

9 15

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17,18 14

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10,17 21 24

6

7,8

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9 1

Fig. 2. Typical 700 MHz 1D spectra obtained from the brain tissue extract of (A) sham, (B) vehicle and (C) rapamycin treated group from 0 to 4.5 ppm. The keys for metabolites are provided in Table 1.

Fig. 3. Typical 700 MHz 1D spectra obtained from the brain tissue extract of (A) sham, (B) vehicle and (C) rapamycin treated group from 5 to 9.0 ppm. The keys for metabolites are provided in Table 1.

3.3. Metabolites concentrations in the sham and vehicle rat brains Fig. 4 represents the concentration of the metabolites in the brain cortical tissues of sham, vehicle group and rapamycin treated group. In the vehicle group, the concentration of lactate (Lac) was significantly higher (P o0.05) as compared to that of the sham group. Within the rapamycin treated group of rats, the Lac concentration was significantly lower as compared to that of the vehicle treated group (P o0.05). The concentrations of glutamate/ glutamine (Glu/Gln), glycerophosphoryl choline/ Phosphorylcholine (GPC/PC), creatine/phosphocreatine (Cr/PCr), taurine (Tau), myo-inositol (mI), γ-amino butryic acid (GABA) and N-aspartyl aspartate (NAA) were significantly lower in vehicle treated group as compared to those of sham controls (P o0.05). Rapamycin treatment significantly increased the concentrations of Glu/Gln,

GPC/PC, Cr/PCr, Tau, mI, GABA and NAA as compared to those of vehicle treated group (P o0.05).

4. Discussion In our previous study we demonstrated that rapamycin protects against ischemic injury in rats. The neuroprotective effects were evident in diffusion weighted magnetic resonance imaging (DW-MRI), functional outcomes and also decrease in oxidative stress and inflammation (Chauhan et al., 2011). Occlusion of MCA in rats resulted in motor deficits and increase in malondialdehyde, nitric oxide and myeloperoxidase levels and decrease in glutathione levels. Treatment with rapamycin ameliorated motor impairments related with MCAo and also significantly reversed the changes in levels of

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Table 1 Chemical shifts of various metabolites observed in rat brain extracts of sham rats. S. No.

Metabolite

Chemical shift (ppm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

Alanine (Ala) Lysine (Lys) Isoleucine Leucine Valine Succinate Glutamate (Glu) Glutamine (Gln) Lactate (Lac) Glycerophosphoryl choline (GPC) Creatine/phosphocreatine (Cr/Pcr) Ethanolamine (E) Acetate Taurine (Tau) Myo-inositol (mI) γ-Amino butryic acid (GABA) Choline (Cho) Phosphorylcholine (PC) N-aspartyl aspartate (NAA) β-Hydroxybutyrate Aspartate (Asp) α-Ketoglutarate (α-KG) Phosphorylethanol amine (PE) Malate (Mal) Phenylalanine(Phe) Threonine (Thre) Citrate (Citr) Nicotinamide UTP UDP NADP NAD IMP GTP GMP GDP ATP ADP Adenosine α-Glucose β-Glucose Formate Fumarate Tyrosine (Tyr)

1.47(β-CH3) , 3.75(α-CH) 1.85(β-CH), 1.72(δ-CH2) , 1.47(γ-CH2), 3.01(ε-CH2), 3.63(α-CH) 0.92, 0.93(δ-CH3), 1.25, 1.45(γ-CH2), 0.94(δ-CH3) , 0.95(δ-CH3), 1.69(γ-CH), 1.72(β-CH), 3.64(α-CH) 0.99(γ-CH2), 1.05(γ-CH2), 2.20(β-CH), 3.62(δ-CH) 2.41(CH2–CH2) 3.66(α-CH), 2.31(γ-CH2) , 2.04(β-CH) 3.65(α-CH), 2.34(γ-CH2), 2.08(β-CH2) 1.33(CH3), 4.10(CH) 3.23(NCH3), 4.31(H4), 3.92(H3), 3.77(H2), 3.64(H1) 3.93(CH2), 3.04(CH3) 3.18(NH–CH2), 3.83(α-CH2) 1.92(CH3) 3.25(HN–CH2), 3.41(CH2SO3) 3.27(H5), 3.53(H1,H3), 3.61(H4,H6), 4.05(CH2) 3.02(γ-CH2) , 2.30(α-CH2), 1.91(β-CH2) 3.20(N–CH3), 4.16(α-CH2), 3.60(β-CH2) 3.23(N–(CH3)3), 3.60(N–CH2) 4.38(CH), 2.67(CH2), 2.50(CH2) 1.20(γCH2), 2.48(α-CH2), 4.16(β-CH) 2.68(β-CH), 2.82(β-CH), 3.91(α-CH) 3.14(β-CH2), 2.54(β-CH2) 3.21(CH2), 3.99(α-CH2) 2.37(β-CH2), 2.68(β-CH2), 4.28(α-CH) 7.32(2,4H), 7.36(4H), 7.43(3,5H) 1.43(CH3), 3.69(α-CH3), 4.32(β-CH) 2.55(CH2) , 2.67(CH2) 7.60, 8.25, 8.72, 8.94 5.99(HI'), 7.86(base) 5.98(HI'), 7.99(base) 6.08(HI') 6.06(HI') 6.15(HI'), 8.64(base) 5.94(HI'), 8.15(base) 5.94(HI'), 8.18(base) 5.94(HI'), 8.14(base) 6.14(HI'), 8.27(base), 8.54(base) 6.14(HI`), 8.27(base), 8.54(base) 6.04(HI'), 8.20(base), 8.38(base) 5.22(HI') 4.64(HI') 8.46(CH) 6.53(CHQCH) 6.88(3,5H), 7.16(2,6H)

malondialdehyde, glutathione, nitric oxide and myeloperoxidase, reflecting to having antioxidant and anti-inflammatory activity. We also observed an increase in infarct area and the signal intensity and a decrease in apparent diffusion coefficient (ADC) in vehicle group indicating to ischemia-induced injury (Chauhan et al., 2011). The infarct volumes were significantly lower in rapamycin treated group than those seen in vehicle treated rats (Chauhan et al., 2011). In the present study we investigated the alterations of brain metabolism due to ischemia and the effect of rapamycin treatment using in-vitro NMR spectroscopy. During ischemia, oxygen and glucose depletion starts, leading to anaerobic metabolism and concomitant energy failure resulting in metabolic changes including lactic acidosis and alterations in the levels of neurotransmitters (Siesjö, 1984). Our data indicated increased concentration of Lac in the vehicle group as compared to that of sham group reflecting to the injury. Moreover, increase in Lac concentration in ischemia may reflect the initial loss of calcium homeostasis or increase in calcium influx into mitochondria due to membrane depolarization, resulting in mitochondrial injury (Siesjö, 1984; Deshpande et al., 1987). In addition, we observed a decrease in concentrations of CrþPCr in vehicle treated group as compared to sham group. The CrþPCr is an important entity in the energy metabolism involved in mitochondria (Klein and Ferrante, 2007) that regulates the energy homeostasis in the brain during high and

irregular energy demands. The decrease in CrþPCr levels has been shown to be present after ischemia and suggests to alterations in the energy balance in the brain due to ischemia induced oxidative stress (Gideon et al., 1992). In our previous study (Chauhan et al., 2011), we had observed that ischemia induces oxidative stress by decreasing the levels of reduced glutathione and increasing the brain levels of malondialdehyde and nitric oxide in the MCAo induced rats. The decrease in the CrþPCr levels in the vehicle treated group in the present work might be attributed to oxidative stress induced energy imbalance. However, treatment with rapamycin in the MCAo induced rats showed increased levels of Crþ PCr, reflecting the role of rapamycin on energy metabolisms. Also, this finding correlates well with our previous observations in which we demonstrated increase in glutathione and decrease in the brain levels of malondialdehyde and nitric oxide in the rapamycin treated group. Decreased concentration of NAA and Tau in the vehicle treated group as compared to sham group was observed in our data. Apparently, NAA is specific to neurons; its decrease reflects neuronal damage. A decrease in NAA has been reported in mitochondrial myopathy (José da Rocha et al., 2008), aging (Lim and Spielman, 1997) and stroke (Chen et al., 2012). Bates et al. (1996) demonstrated the role of reduced NAA levels in mitochondrial dysfunction. Moreover, in our previous study, we had observed a significant decrease in brain glutathione levels after stroke and brain glutathione levels have

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Fig. 4. The concentration of metabolites in sham, vehicle and rapamycin treated groups (mm/kg wet weight). *Po 0.05 as compared to sham; #Po 0.05 as compared to vehicle. The brains were harvested 24 h after ischemia and in vitro NMR was performed. There was increase in the brain Lac levels in the vehicle group as compared to sham. There was decrease in the brain concentrations of GPC/PCCho, mI, Cr/PCr, NAA, Tau, Glu/Gln and GABA in the vehicle group as compared to sham. Rapamycin at dose of 250 mg/kg increased the brain levels of GPC/PCCho, mI, Cr/PCr, NAA, Tau, Glu/Gln and GABA after stroke.

been reported to be important for optimum mitochondrial function (Heales et al., 1995) and also for maintaining oxidative and antioxidative balance (Chauhan et al., 2012). Reduced brain glutathione levels affect neuronal integrity (Heales et al., 1995), thereby, affecting mitochondrial function and causing oxidative imbalance. On the other hand, Tau is known to afford neuroprotection in the epileptic seizures (El Idrissi et al., 2003) and cerebral ischemia (Chen et al., 2012; Stummer et al., 1995). Previous studies have shown the protective effect of Tau in oxidative injury and inflammation (Sun et al., 2012; Mahalakshmi et al., 2003). In a study, the researchers demonstrated that Tau acts as a modulator of mitochondrial protein synthesis and increases mitochondrial respiratory chain activity thereby protecting against superoxide generation (Jong et al., 2012). In rats treated with rapamycin a significant increase in NAA and Tau was observed; this change in NAA and Tau may be partially attributed to the protective effect of rapamycin on mitochondrial function and reduction in oxidative stress in this group. However, more studies need to undertake to conclude its role on mitochondrial dysfunction and neuronal damage. We also observed a significant decrease in the concentration of choline related compounds in the vehicle treated group, which might reflect to disruption of cell membrane metabolism. Such changes in the levels of choline after 24 h of ischemia corroborates well with the previous reports (van der Toorn et al., 1995, 1996). Choline like compounds Cho, PC and GPC along with mI are integral components of membrane phospholipids and are associated with membrane metabolisms. Ischemia induced release of glutamate leads to activation of PLA2 (Adibhatla et al., 2006); eventually, PLA2 activation leads to disruption of cell membranes and release of choline like compounds. Furthermore, we observed an increase in the brain PLA2 levels in the vehicle group and treatment with rapamycin ameliorated the PLA2 levels. In addition, we had also observed decrease in the concentration of mI in the vehicle group; this decrease corroborated well with the previous reports (Nonaka et al., 1998) in which the authors have suggested role of brain osmolytes such as mI in the formation of edema following ischemic injury. Moreover, increase in Cho, PC þ GPC and mI in the rapamycin treated group was observed; this effect may be partially due to the reduction of the brain PLA2 levels postischemia as observed in this study. Thus, neuroprotection by rapamycin might be attributed to the inhibition in activation of PLA2 and membrane metabolisms. Moreover, a significant decrease was observed in the concentration of Glu/Gln, and GABA in the vehicle group as compared with the

sham control in the present study. The decrease in Glu/Gln might be attributed to the finding that ischemia induces a depression in the tricarboxylic acid cycle subsequently leading to an increase in the oxidation of these metabolites as fuel alternatives or substrates to glucose in brain (Pascual et al., 1998). Hence decreased levels of Glu/Gln and GABA after ischemic injury are in agreement with the previous studies (Yang et al., 2012; Pascual et al., 1998). The glial and neuronal cells play an important role in glutaminergic neurotransmission, as these cells are responsible for maintaining glutamate–glutamine cycle. The glial cells use glucose as substrate and release Gln. This Gln is than taken up by neuronal cells and release of Glu and GABA takes place (Erecinska and Silver, 1990). The metabolism taking place in glial and neuronal cells interacts closely and both cells compete for glucose as primary substrate and also use Glu, Gln and GABA as alternative substrates in the tricarboxylic acid cycle. Rapamycin treatment was able to afford protection in the MCAo induced rats by increasing the Glu/Gln and GABA suggesting its role in neurotransmission. In a study done by Weston et al. (2012) on wild type neurons, they showed reduced excitatory synaptic output but not inhibitory output when treated with rapamycin demonstrating the role of rapamycin in neurotransmission. Still, more studies need to be undertaken to fully explore the role of rapamycin on neurotransmission. In contrast to our findings, others have reported that administration of rapamycin can increase brain infarct size and increase neurological deficits in the ischemia post-conditioning model of stroke (Xie et al., 2014). Also, rapamycin treatment decreases cell survival and increases apoptotic injury in in vitro model of ischemia (Xie et al., 2014; Chong et al., 2007). This difference in rapamycin response to injury might be due to different models of ischemia used or dose selection. However, one cannot rule out that rapamycin affected the brain metabolites after stroke demonstrating neuro protection.

5. Conclusion In conclusion our results demonstrated that protective effects of rapamycin may be either by directly acting as anti-oxidant/antiinflammatory or indirectly by affecting the brain metabolites levels after stroke. Disclosure All the authors declare no conflict of interest.

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