Role of mitochondrial function in the protective effects of ischaemic postconditioning on ischaemia/reperfusion cerebral damage

Abstract

Objective

To investigate the effects of ischaemic postconditioning on brain injury and mitochondria in focal ischaemia and reperfusion, in rats.

Methods

Adult male Wistar rats (n = 15 per group) underwent sham surgery, ischaemia (2-h middle cerebral artery occlusion), or ischaemia followed by ischaemic postconditioning (three cycles of 30 s reperfusion/30 s reocclusion). Brain infarction size, neurological function, mitochondrial reactive oxygen species (ROS) production, mitochondrial membrane potential and mitochondrial swelling were evaluated 24 h postsurgery.

Results

Infarct size was significantly smaller, and neurological function was significantly better, in the ischaemic postconditioning group than in the ischaemia group. Ischaemia resulted in significant increases in mitochondrial ROS production and swelling, and a reduction in mitochondrial membrane potential, all of which were significantly reversed by postconditioning.

Conclusions

The protective role of ischaemic postconditioning in focal ischaemia/reperfusion may be due to decreased mitochondrial ROS production, reduced mitochondrial membrane potential and suppressed mitochondria swelling. Mitochondria are potential targets for new therapies to prevent brain damage caused by ischaemia and reperfusion.

Keywords

Ischaemic postconditioning cerebral ischaemia/reperfusion mitochondrial integrity

Introduction

Cerebral damage due to reperfusion following ischaemia is critical in the prognosis of revascularization of occluded blood vessels.¹ Animal experiments have demonstrated that many factors are involved in the pathological course of cerebral damage caused by ischaemia and reperfusion, such as endoplasmic reticulum stress and protein aggregation.^2,3 Antioxidants have been shown to have therapeutic effects on ischaemia/reperfusion brain injury, suggesting that oxidative stress has an active role in modulating neuronal destiny.^4–6 In addition, other neurological disorders (including neurodegenerative diseases and traumatic brain injury) are related to oxidative stress.^7,8 The crucial feature of oxidative stress is overproduction of reactive oxygen species (ROS) resulting from disruption to the equilibrium between ROS formation and clearance.⁹ ROS cause damage to the lipid and protein constituents of neuronal membranes,^10,11 and activate signalling pathways associated with cell death.¹² Mitochondria are thought to be the major source of ROS in neurons¹³ and these oxidants are known to be mediators of molecular signalling in the mitochondria-dependent cell death pathway.¹⁴ Cerebral ischaemia and reperfusion lead to mitochondrial dysfunction, which plays an important role in neuronal injury.¹⁵ Maintaining mitochondrial function might therefore be a potential strategy for protection against cerebral damage caused by ischaemia/reperfusion.

Ischaemic postconditioning is a series of rapid, intermittent interruptions of blood flow in the early phase of reperfusion that mechanically alters the hydrodynamics of reperfusion.¹⁶ Experimental and clinical studies have indicated the protective effects of postconditioning in ischaemia/reperfusion injury.^17–21 This protection is not only associated with inhibition of inflammation and apoptosis,^22,23 but also with attenuation of oxidative stress.^3,24 The aim of the present study was to use a rat model of ischaemia/reperfusion injury to evaluate the effects of ischaemic postconditioning on mitochondrial ROS production, in order to determine whether its inhibitory effects on oxidative stress are mediated via the mitochondrial pathway, since the golgi apparatus is also a source of ROS.²⁵

Materials and methods

Animals

Adult male Wistar rats (n = 45; weighing 280–300 g; aged 7–8 weeks) supplied by the Experimental Animal Centre, Jilin University, Changchun, China, were housed in a temperature-controlled room (22–25°C) on a 12-h light/dark cycle with free access to food and water. All animal procedures were approved by the Ethics Committee for Animal Experiments, Jilin University. All possible measures were taken to reduce animal suffering and the number of animals in this study, according to the Guide for the Care and Use of Laboratory Animals.²⁶

Study design

At the start of the study, rats were assigned to one of three groups using a computer-generated randomization schedule: sham operated group; ischaemia group; ischaemic postconditioning group (n = 15 per group). Animals in the ischaemia and ischaemic postconditioning groups were subjected to 2 h of ischaemia (described below). Rats in the ischaemic postconditioning group underwent three cycles of 30 s reperfusion/30 s reocclusion after ischaemia. Animals in the sham group were subjected to the same surgical procedure as described below, without middle cerebral artery occlusion (MCAO).

Surgical procedure

Brain ischaemia was induced via the MCAO model, as described.²⁷ Following an overnight fast, anaesthesia was induced with 300 mg/kg chloral hydrate, administered via intraperitoneal injection. A rectal probe was inserted and each animal’s core temperature was maintained at 37 ± 0.5°C with a heating pad and lamp. The right common carotid artery (CCA), internal carotid artery and external carotid artery were exposed, and the external carotid artery was ligated proximal to the origin of any branches, such as the occipital artery. The proximal CCA then was ligated and temporarily closed proximal to the carotid bifurcation, using a microvascular clip. A small incision was made into the CCA. The occlusion filament was inserted into the internal carotid artery through the CCA, 19–21 mm distal from the bifurcation, to occlude the origin of the MCA. The filament comprised monofilament fishing line covered with a distal cylinder of silicone rubber (diameter 0.31–0.32 mm). After MCAO, animals were allowed to awaken and resume spontaneous breathing. The filament was withdrawn 2 h after induction of ischaemia. After surgery, animals were placed into a cage to recover from the anaesthesia at room temperature and were allowed food and drink.

Infarct measurement

At 24 h after reperfusion, five rats from each group were chosen randomly by investigators who were blinded to group allocation; they were killed by pentobarbital overdose via intraperitoneal injection and their brains were rapidly removed. Infarct sizes were measured by staining with 2,3,5-triphenyl-2H-tetrazolium chloride (TTC; Sigma-Aldrich, St Louis, MO, USA). Brains were cut into 2-mm thick coronal sections in a cutting block and stained with 1% TTC solution for 30 min at 37°C, followed by overnight immersion in 4% paraformaldehyde. The percentage of brain infarct was measured by normalizing to the total brain volume of the animals, as described previously.²⁷

Neurological assessment

Neurological examination of five rats from each group was performed by an investigator blinded to the study groups, 24 h after surgery, according to the following scoring system: 0 (no apparent deficits); 1 (contralateral forelimb flexion when suspended by the tail); 2 (decreased grip of the contralateral forelimb while tail pulled); 3 (spontaneous movement in all directions; contralateral circling only if pulled by the tail), 4 (spontaneous contralateral circling); 5 (death after recovery from anaesthesia).²⁸

Isolation of mitochondria

At 24 h postsurgery, brain nonsynaptic mitochondria were isolated from five animals in each group by Percoll® gradient centrifugation, as described previously.^29,30 Rats were euthanized by pentobarbital overdose via intraperitoneal injection, then perfused transcardially with 100 ml ice-cold phosphate-buffered saline (PBS; 0.1 M, pH 7.4). The dorsolateral neocortical tissue was dissected and homogenized in ice-cold homogenization buffer (25 mmol/l HEPES pH 7.4, 250 mmol/l sucrose, 4 mmol/l magnesium chloride, 0.05 mmol/l ethylene glycol tetra-acetic acid) in a Potter Teflon®-glass homogenizer. The homogenate was centrifuged at 400 g for 5 min at room temperature (which was the temperature for all centrifugations) and the supernatant was collected. The sediment was rehomogenized, centrifuged at 400 g for 5 min, and pooled supernatants were then centrifuged at 12 000 g for 10 min. The resulting sediment was resuspended in homogenization buffer (1 ml/g of original wet tissue). The suspension was then layered onto a one-step Percoll® gradient (8 ml of 16% Percoll®; 0.25 mol/l sucrose) and centrifuged at 15 000 g for 20 min. The final sediment was resuspended in homogenization buffer supplemented with 0.25% BSA and stored on ice. Protein concentration was determined using a Quick Start™ Bradford Protein Assay Kit (Bio-Rad, Hercules, CA, USA).

Mitochondrial ROS production

As described previously,³¹ dichlorohydrofluorescein diacetate (DCFDA) was used to measure the production of ROS within mitochondria. Mitochondria (0.4 mg/ml) were incubated with 2 µmol/l DCFDA at 25°C for 20 min. DCF fluorescence was determined at 485 nm for excitation and 530 nm for emission, and ROS levels were expressed as arbitrary units of fluorescence intensity, normalized to levels in the sham group.³¹

Mitochondrial membrane potential

Mitochondrial membrane potential was measured using rhodamine 123 staining.³² Rhodamine 123 is a highly specific fluorescent dye for mitochondria, the intake of which depends on mitochondrial membrane potential. Mitochondrial protein suspension (10 µg) was added to 2.5 ml of reaction buffer (15 mM sucrose, 5 mmol/l magnesium chloride, 5 mmol/l sodium succinate, 2.5 mmol/l otenone, 20 mmol/l HEPES, pH 7.4), and 40 μl of rhodamine 123 at 26 µmol/l was added and incubated for 5 min at room temperature. Mitochondria were then pelleted by centrifugation 10000 g for 10 min and the fluorescence of the supernatant was read with a spectrophotometer (excitation wavelength 503 nm; emission wavelength 527 nm). The optical density of rhodamine 123 staining is inversely proportional to the mitochondrial membrane potential.

Mitochondrial swelling

Mitochondrial swelling was measured as described previously.³² Briefly, 200 µl of isotonic buffer (5 mmol/l monopotassium phosphate, 250 mmol/l sucrose, 3 mmol/l succinic acid disodium salt, pH 7.2) was added to mitochondria (50 µg protein) at 30°C for 5 min. Absorbance at 520 nm was measured, with a lower absorbance value indicating increased mitochondrial swelling.

Statistical analyses

All data were expressed as mean ± SD and analysed with SPSS® statistical software, version 17.0 (SPSS Inc., Chicago, IL, USA) for Windows®. Student's t-test was used for statistical comparisons between the different groups. A P-value < 0.05 was considered to be statistically significant.

Results

Brain ischaemia via MCAO induced infarct in all rats in the ischaemia and postconditioning groups. Infarct size and neurological scores were significantly lower in the ischaemic postconditioning group than in the ischaemia group (P < 0.01 for both comparisons; Table 1).

Table 1.

Brain infarct size, neurological score, mitochondrial reactive oxygen species (ROS) production, mitochondrial membrane potential and mitochondrial swelling in male Wistar rats 24 h after sham surgery, ischaemia (2-h middle cerebral artery occlusion), or ischaemia followed by ischaemic postconditioning (three cycles of 30 s reperfusion/30 s reocclusion).

Parameter	Sham	Ischaemia	Postconditioning
Infarct size, %	–	37.91 ± 2.16	21.38 ± 1.65^b
Neurological score²⁷	–	3.61 ± 0.52	2.33 ± 0.68^b
Mitochondrial ROS production	1.0 (defined)	1.38 ± 0.07^c	1.15 ± 0.11^d
Rhodamine 123 staining, absorbance units^a	42.58 ± 5.81	61.62 ± 8.07^c	49.21 ± 4.28^b
Mitochondrial membrane swelling	0.58 ± 0.11	0.35 ± 0.06^c	0.49 ± 0.04^b

Data presented as mean ± SD; n = 5 per group for all parameters.

Optical density of rhodamine 123 staining is inversely proportional to mitochondrial membrane potential.

P < 0.01 vs ischaemia, ^cP < 0.01 vs sham, ^dP < 0.05 vs ischaemia; Student’s t-test.

Levels of mitochondrial ROS formation were significantly higher in the ischaemia group than in the sham group at 24 h post surgery (P < 0.01; Table 1), and this increase was significantly reversed by ischaemic postconditioning (P < 0.05 versus ischaemia group; Table 1).

Ischaemia was associated with a significant decrease in mitochondrial membrane potential (increase in optical density) compared with the sham group (P < 0.01; Table 1) 24 h post surgery. Ischaemic postconditioning significantly abrogated this effect (P < 0.01 versus ischaemia group; Table 1).

Mitochondrial membrane swelling was significantly higher in the ischaemia group than the sham group (P < 0.01; Table 1): an effect that was significantly reversed by postconditioning (P < 0.01 versus ischaemia group; Table 1).

Discussion

The present study showed that three cycles of 30 s reperfusion/30 s reocclusion was an effective ischaemic postconditioning procedure that decreased brain infarct volume and elevated neurological scores in rats. In addition, ischaemic postconditioning effectively inhibited mitochondrial ROS production, elevated mitochondrial membrane potential and maintained mitochondrial swelling. These mitochondrial changes may underlie the protective effects of ischaemic postconditioning on brain damage and neurological dysfunction caused by ischaemia/reperfusion.

Ischaemic postconditioning has been shown to be protective against ischaemic injury in organs including the brain, liver and intestine.^3,33,34 The underlying mechanism was related to multiple factors including suppression of oxidative stress, activation of signalling pathways, regulation of enzyme activity, inhibition of endoplasmic stress^19,35–39 and effective regulation of cerebral blood flow to the ischaemic region. Since both mitochondria and the golgi apparatus are thought to be responsible for generating reactive oxygen species,²⁵ it is necessary to elucidate which organelle is involved in the inhibition of oxidative stress by ischaemic postconditioning. The present study therefore focused on mitochondrial changes caused by ischaemic postconditioning.

Mitochondria are both a source and a target of intracellular ROS.⁴⁰ ROS are generated via a series of redox reductions in the mitochondrial matrix.⁴¹ Mitochondria are easily damaged by ROS, as they are made up of inner and outer membrane structures that contain liquids and proteins that are prone to ROS attack.⁴² In contrast to other studies that have indicated that ischaemic postconditioning attenuates oxidative stress,^19,20 the current study specifically examined the production of ROS within mitochondria. Levels of mitochondrial ROS in the ischaemic postconditioning group in the present study were significantly lower than those in the ischaemia group, suggesting that ischaemic postconditioning may have protective effects on mitochondria.

Mitochondrial manganese superoxide dismutase (MnSOD) is an endogenous mitochondrial redox enzyme for clearing superoxide, which is the precursor of ROS. Decreased MnSOD activity has been found to exacerbate glutamate toxicity in cultured mouse cortical neurons,⁴³ and MnSOD mimics could prevent neural apoptosis and reduce ischaemic brain injury by maintaining mitochondrial function.⁴⁴ Mitochondrial MnSOD therefore plays a crucial role in maintaining the function of mitochondria. It is possible that the protective effect of ischaemic postconditioning may be mediated via modulation of MnSOD, reducing the increase in mitochondrial ROS caused by cerebral ischaemia/reperfusion. Ischaemic postconditioning has been shown to modulate MnSOD levels, but increased MnSOD was associated with reduced neuronal viability.⁴⁵ Further studies are therefore necessary to elucidate the mechanisms of ROS reduction by ischaemic postconditioning.

Mitochondrial ROS formation and mitochondrial membrane potential are indicators of the performance of the electron transport chain.^40,41 Mitochondrial membrane potential contributes to establish a proton gradient across the inner mitochondrial membrane, which provides the driving force that actuates ATP-synthase to generate high-energy phosphate.⁴⁶ As the primary organelle for energy generation, mitochondria are highly susceptible to cerebral insult, given the high metabolic rate of the brain and its dependency on extracellular nutrient sources. In vitro and in vivo studies have shown that loss of the extracellular glucose supply leads to a decline in mitochondrial membrane potential,^46,47 indicating that ischaemia both depletes the materials necessary to produce energy and damages the electron transport chain. In the present study, ischaemic postconditioning reversed the reduction in mitochondrial membrane potential caused by ischaemia/reperfusion. This is consistent with the current finding that ischaemic postconditioning decreased mitochondrial ROS formation, as both of these parameters reflect the condition of the electron transport chain. Others have shown that chemicals with protective effects on cerebral injury reverse damage to mitochondrial membrane potential.^5,6 It is possible that the protective effect of ischaemic postconditioning is also related to improvements in mitochondrial membrane potential.

Mitochondrial swelling is a common ultrastructural change seen in histopathological studies describing mitochondria after ischaemia.^43,44 Mitochondrial swelling was shown to begin at the start of cerebral ischaemia and was exaggerated by subsequent reperfusion, reaching its peak after 24 h of reperfusion.⁴⁸ Transmission electron microscopy studies found that damaged mitochondria exhibited severe collapse in both the inner and outer membranes after 24 h reperfusion following 2 h of ischaemia, and exhibited irregular shape, electron-lucent matrix, fragmented cristae, and extreme dilation of the intracristal space.⁴⁸ In contrast, the present study found that ischaemic postconditioning significantly attenuated mitochondria swelling induced by lethal ischaemia and reperfusion. Mitochondrial swelling is related to the opening of the mitochondrial permeability transition pore,⁴⁹ with calcium ion (Ca²⁺) concentration being proportional to the extent of pore opening.^50,51 Overload of Ca²⁺ is a pathological characteristic of ischaemia/reperfusion.⁵² Ischaemic postconditioning has been shown to limit Ca²⁺ overload via modulating the NCX3 isoform of the sodium/calcium ion (Na⁺/Ca²⁺) exchanger.⁵³ Opening of the mitochondrial permeability transition pore in liver cells was inhibited by delayed ischaemic postconditioning.⁴⁹ It is possible that the reduction in mitochondrial swelling may be due to a lowering of cytosolic Ca²⁺ concentrations and inhibition of the opening of mitochondrial permeability transition pore.

Human studies have shown that ischaemic postconditioning can also effectively regulate blood flow, recover organ function and inhibit oxidative stress.^18,21 Ischaemic postconditioning could be performed in patients with ischaemic stroke by inflating or deflating a balloon at the end of interventional thrombolysis, to block the recovered blood flow at intervals to the ischaemic brain region. This is a potentially simple and easily performed procedure, but its feasibility and safety remain to be demonstrated in clinical practice.

In conclusion, the present study showed that ischaemic postconditioning significantly reduces cerebral infarct size and improves neurological dysfunction caused by focal ischaemia/reperfusion in rats. This protective effect is closely associated with a reduction in mitochondrial ROS formation, an elevation of mitochondrial membrane potential and an attenuation of mitochondrial swelling. Mitochondria are potential targets for new therapies to prevent brain damage caused by ischaemia and reperfusion.

Footnotes

Declaration of conflicting interest

The authors declare that there are no conflicts of interest.

Funding

This work was supported by National Nature and Science Foundation (30973110) (81171234), and the Outstanding Youth Grant (20080139) from the Science and Technology Department of Jilin Province.

References

Tjoumakaris

Jabbour

Rosenwasser

. Neuroendovascular management of acute ischemic stroke. Neurosurg Clin N Am 2009; 20: 419–429.

Osada

Kosuge

Ishige

. Characterization of neuronal and astroglial responses to ER stress in the hippocampal CA1 area in mice following transient forebrain ischemia. Neurochem Int 2010; 57: 1–7.

Luo

Liu

. Protein aggregation and proteasome dysfunction after brain ischemia. Stroke 2007; 38: 3230–3236.

Lehotský

Murín

Strapková

. Time course of ischemia/reperfusion-induced oxidative modification of neural proteins in rat forebrain. Gen Physiol Biophys 2004; 23: 401–415.

Buch

Patel

Ranpariya

. Neuroprotective activity of Cymbopogon martinii against cerebral ischemia/reperfusion-induced oxidative stress in rats. J Ethnopharmacol 2012; 142: 35–40.

Wang

Yuan

Liu

. Neuroprotective activity of lavender oil on transient focal cerebral ischemia in mice. Molecules 2012; 17: 9803–9817.

Eghwrudjakpor

Allison

. Oxidative stress following traumatic brain injury: enhancement of endogenous antioxidant defense systems and the promise of improved outcome. Niger J Med 2010; 19: 14–21.

De Oliveira

Ferreira Lima

El-Bachá

. Brain rust: recent discoveries on the role of oxidative stress in neurodegenerative diseases. Nutr Neurosci 2012; 15: 94–102.

Chen

Yoshioka

Kim

. Oxidative stress in ischemic brain damage: mechanisms of cell death and potential molecular targets for neuroprotection. Antioxid Redox Signal 2011; 14: 1505–1517.

10.

Nagakannan

Shivasharan

Thippeswamy

. Effect of tramadol on behavioral alterations and lipid peroxidation after transient forebrain ischemia in rats. Toxicol Mech Methods 2012; 22: 674–678.

11.

Liu

. Ischaemic postconditioning rescues brain injury caused by focal ischaemia/reperfusion via attenuation of protein oxidization. J Int Med Res 2012; 40: 954–966.

12.

Gan

. Transgenic overexpression of peroxiredoxin-2 attenuates ischemic neuronal injury via suppression of a redox-sensitive pro-death signaling pathway. Antioxid Redox Signal 2012; 17: 719–732.

13.

Sena

Chandel

. Physiological roles of mitochondrial reactive oxygen species. Mol Cell 2012; 48: 158–167.

14.

Alfadda

Sallam

. Reactive oxygen species in health and disease. J Biomed Biotechnol 2012; 2012: 936486–936486.

15.

Niizuma

Endo

Chan

. Oxidative stress and mitochondrial dysfunction as determinants of ischemic neuronal death and survival. J Neurochem 2009; 109(suppl 1): 133–138.

16.

Zhao

Sapolsky

Steinberg

. Interrupting reperfusion as a stroke therapy: ischemic postconditioning reduces infarct size after focal ischemia in rats. J Cereb Blood Flow Metab 2006; 26: 1114–1121.

17.

Darling

Jiang

Maynard

. Postconditioning via stuttering reperfusion limits myocardial infarct size in rabbit hearts: role of ERK1/2. Am J Physiol Heart Circ Physiol 2005; 289: H1618–H1626.

18.

Zhang

. Effect of postconditioning on coronary blood flow velocity and endothelial function in patients with acute myocardial infarction. Scand Cardiovasc J 2006; 40: 327–333.

19.

Wang

Shen

Gao

. Ischemic postconditioning protects against global cerebral ischemia/reperfusion-induced injury in rats. Stroke 2008; 39: 983–990.

20.

Ren

Gao

Niu

. Delayed postconditioning protects against focal ischemic brain injury in rats. PLoS One 2008; 3: e3851–e3851.

21.

Loukogeorgakis

Panagiotidou

Yellon

. Postconditioning protects against endothelial ischemia-reperfusion injury in the human forearm. Circulation 2006; 113: 1015–1019.

22.

Xing

Chen

Zhang

. Ischemic postconditioning inhibits apoptosis after focal cerebral ischemia/reperfusion injury in the rat. Stroke 2008; 39: 2362–2369.

23.

Xing

Chen

Zhang

. Ischemic post-conditioning protects brain and reduces inflammation in a rat model of focal cerebral ischemia/reperfusion. J Neurochem 2008; 105: 1737–1745.

24.

Penna

Mancardi

Rastaldo

. Cardioprotection: a radical view: free radicals in pre and postconditioning. Biochim Biophys Acta 2009; 1787: 781–793.

25.

Jiang

Zeng

. The role of the Golgi apparatus in oxidative stress: is this organelle less significant than mitochondria? Free Radic Biol Med 2011; 50: 907–917.

26.

National research council (US) committee for the update of the guide for care and use of laboratory animals. Guide for the Care and Use of Laboratory Animals, 8th ed. Washington DC: National Academies Press (USA), 2011.

27.

Janelidze

Ginsberg

. Protein aggregation after focal brain ischemia and reperfusion. J Cereb Blood Flow Metab 2001; 21: 865–875.

28.

Qin

Karabiyikoglu

Hua

. Hyperbaric oxygen-induced attenuation of hemorrhagic transformation after experimental focal transient cerebral ischemia. Stroke 2007; 38: 1362–1367.

29.

Racay

Tatarkova

Chomova

. Mitochondrial calcium transport and mitochondrial dysfunction after global brain ischemia in rat hippocampus. Neurochem Res 2009; 34: 1469–1478.

30.

Sims

Anderson

. Isolation of mitochondria from rat brain using Percoll density gradient centrifugation. Nat Protoc 2008; 3: 1228–1239.

31.

Thummasorn

Kumfu

Chattipakorn

. Granulocyte-colony stimulating factor attenuates mitochondrial dysfunction induced by oxidative stress in cardiac mitochondria. Mitochondrion 2011; 11: 457–466.

32.

Hong

Xin

. Neuroprotective effects of leonurine on ischemia/reperfusion-induced mitochondrial dysfunctions in rat cerebral cortex. Biol Pharm Bull 2010; 33: 1958–1964.

33.

Guo

Yang

Sun

. Ischemic postconditioning attenuates liver warm ischemia-reperfusion injury through Akt-eNOS-NO-HIF pathway. J Biomed Sci 2011; 18: 79–79.

34.

Liu

Huang

. Immediate postconditioning during reperfusion attenuates intestinal injury. Intensive Care Med 2009; 35: 933–942.

35.

Zhou

Zhang

. Delayed ischemic postconditioning protects hippocampal CA1 neurons by preserving mitochondrial integrity via Akt/GSK3β signaling. Neurochem Int 2011; 59: 749–758.

36.

Prasad

Russell

Nowakowska

. Neuroprotection induced in vitro by ischemic preconditioning and postconditioning: modulation of apoptosis and PI3K–Akt pathways. J Mol Neurosci 2011; 43: 428–442.

37.

Wang

Zhang

Ding

. Limb remote postconditioning alleviates cerebral reperfusion injury through reactive oxygen species-mediated inhibition of delta protein kinase C in rats. Anesth Analg 2011; 113: 1180–1187.

38.

Zhang

Miao

Zhou

. Neuroprotective effects of ischemic postconditioning on global brain ischemia in rats through upregulation of hippocampal glutamine synthetase. J Clin Neurosci 2011; 18: 685–689.

39.

Yuan

Guo

. Ischemic postconditioning protects brain from ischemia/reperfusion injury by attenuating endoplasmic reticulum stress-induced apoptosis through PI3K-Akt pathway. Brain Res 2011; 1367: 85–93.

40.

Kim

Kondo

Noshita

. Manganese superoxide dismutase deficiency exacerbates cerebral infarction after focal cerebral ischemia/reperfusion in mice: implications for the production and role of superoxide radicals. Stroke 2002; 33: 809–815.

41.

Keller

Kindy

Holtsberg

. Mitochondrial manganese superoxide dismutase prevents neural apoptosis and reduces ischemic brain injury: suppression of peroxynitrite production, lipid peroxidation, and mitochondrial dysfunction. J Neurosci 1998; 18: 687–697.

42.

Visavadiya

McEwen

Pandya

. Antioxidant properties of Neu2000 on mitochondrial free radicals and oxidative damage. Toxicol In Vitro 2012; 27: 788–797.

43.

Copin

Reola

. Reduced mitochondrial manganese-superoxide dismutase activity exacerbates glutamate toxicity in cultured mouse cortical neurons. Brain Res 1998; 814: 164–170.

44.

Huang

Guo

Cao

. Neuroprotection by manganese superoxide dismutase (MnSOD) mimics: antioxidant effect and oxidative stress regulation in acute experimental stroke. CNS Neurosci Ther 2012; 18: 811–818.

45.

Nemethova

Danielisova

Gottlieb

. Post-conditioning exacerbates the MnSOD immune-reactivity after experimental cerebral global ischemia and reperfusion in the rat brain hippocampus. Cell Biol Int 2008; 32: 128–135.

46.

Iijima

Mishima

Tohyama

. Mitochondrial membrane potential and intracellular ATP content after transient experimental ischemia in the cultured hippocampal neuron. Neurochem Int 2003; 43: 263–269.

47.

Rau

Sharma

. Oxygen glucose deprivation in rat hippocampal slice cultures results in alterations in carnitine homeostasis and mitochondrial dysfunction. PLoS One 2012; 7: e40881–e40881.

48.

. Reperfusion promotes mitochondrial dysfunction following focal cerebral ischemia in rats. PLoS One 2012; 7: e46498–e46498.

49.

Lin

Lee

Tsai

. Ischemic postconditioning protects liver from ischemia-reperfusion injury by modulating mitochondrial permeability transition. Transplantation 2012; 93: 265–271.

50.

Brustovetsky

Jemmerson

. Calcium-induced Cytochrome c release from CNS mitochondria is associated with the permeability transition and rupture of the outer membrane. J Neurochem 2002; 80: 207–218.

51.

Lemasters

Theruvath

Zhong

. Mitochondrial calcium and the permeability transition in cell death. Biochim Biophys Acta 2009; 1787: 1395–1401.

52.

Bano

Nicotera

. Ca²⁺ signals and neuronal death in brain ischemia. Stroke 2007; 38(2 Suppl): 674–676.

53.

Pignataro

Esposito

Cuomo

. The NCX3 isoform of the Na⁺/Ca²⁺ exchanger contributes to neuroprotection elicited by ischemic postconditioning. J Cereb Blood Flow Metab 2011; 31: 362–370.