Chemico-Biological Interactions 232 (2015) 21–29

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Ischemic preconditioning and postconditioning alleviates hippocampal tissue damage through abrogation of apoptosis modulated by oxidative stress and inflammation during transient global cerebral ischemia–reperfusion in rats M.A. Saad ⇑, R.M. Abdelsalam, S.A. Kenawy, A.S. Attia Department of Pharmacology and Toxicology, Faculty of Pharmacy, Cairo University, Cairo, Egypt

a r t i c l e

i n f o

Article history: Received 30 October 2014 Received in revised form 25 January 2015 Accepted 9 March 2015 Available online 17 March 2015 Keywords: Preconditioning Postconditioning Ischemia/reperfusion Oxidative stress Apoptosis Inflammation

a b s t r a c t Introduction: It has been argued recently that ischemic preconditioning (IPre) and postconditioning (IPost) have beneficial effects in many ischemic disorders however; their effects on global ischemia/ reperfusion (I/R) are poorly understood. Thus, the present work aimed to study the possible mechanisms underlying the neuroprotective effects of IPre and IPost. Methods: Animals were randomly allocated into 4 groups (n = 30): (1) Sham operated (SO); (2) I/R group, animals were subjected to 15 min global ischemia followed by 60 min reperfusion; (3) IPre, animals were subjected to 3 episodes of 5 min ischemia followed by 10 min reperfusion before I/R; (4) IPost, animals were subjected to three episodes of 10 s of ischemia and 10 s of reperfusion after the period of ischemia followed by a 60 min reperfusion period. Lactate dehydrogenase activity, oxidative stress, inflammatory and apoptotic biomarkers, as well as neurotransmitters, infarct size and histopathological examination were assessed. Results: I/R induced hippocampal damage through increasing oxidative stress, inflammatory, excitotoxic and apoptotic markers as well as lactate dehydrogenase activity and infarct size. Both, IPre and IPost attenuated most markers induced by I/R. Conclusions: IPre and IPost neuroprotective effects can be explained through their anti-oxidant, anti-inflammatory and anti-apoptotic mechanisms. Ó 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Stroke is one of the leading cause of morbidity and mortality world-wide [1]. Acute brain ischemia–reperfusion (I/R) injury is the major pathophysiological sign of ischemic stroke [2]. Recently, endogenous protective mechanisms of the brain have grabbed the attention in stroke research [3,4]. The brain while being challenged by nutrients and oxygen deprivation starts a potent defensive response against many of the deleterious secondary mechanisms which are active during the process of ischemia [5]. So ‘Learning from nature’ inducing such mechanisms may result in more effective treatment with less unwanted side effects. Depending on the noxious stimulus intensity, every stimulus can elicit a wide range of effects starting from protective effects

⇑ Corresponding author. Tel.: +20 2 01003452943, +20 224177371. E-mail address: [email protected] (M.A. Saad). http://dx.doi.org/10.1016/j.cbi.2015.03.007 0009-2797/Ó 2015 Elsevier Ireland Ltd. All rights reserved.

(‘preconditioning’ and ‘tolerance’) at low level to a destructive response (‘apoptosis’ and ‘necroses’) at high level. Practically any noxious stimulus capable of causing injury to a tissue or organ can, when applied below the threshold of damage, activate the endogenous mechanisms of protection in the organ leading to a reduction in the impact of subsequent, more severe stimuli. For instance, a subfatal cerebral ischemic insult activates certain cellular pathways that can help to reduce damage caused by subsequent ischemic episodes by upregulating endogenous pathways that will increase endurance to ischemia or trauma – a phenomenon known as ‘ischemic preconditioning’ (IPre) or ‘ischemic tolerance’ (IT) [3]. This strategy focuses on the protection offered before bypass surgery to diminish the pathophysiological consequences of ischemic injury. Cerebral damage starts early after the induction of ischemia. However, early recovery of cerebral blood flow after the ischemic event i.e., reperfusion, is not a completely benign process and can induce more severe organ damage, a phenomenon known as

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lethal reperfusion injury [6] which involves the processes of apoptosis [7]. Another novel non-pharmacological neuroprotective strategy targeting the reperfusion phase is the phenomenon known as ischemic postconditioning (IPost), which involves the application of transient brief interruptions to reperfusion by ischemic episodes. This strategy is likely to represent a form of modified reperfusion that paradoxically results in reduced cerebral injury and led to renewed interest in the development of protective techniques to overcome the effects of lethal reperfusion injury [8,9]. Many pharmacological neuroprotective strategies have unacceptable side effects, therefore; it deemed of importance that innovative treatment strategies for protecting the brain against the detrimental effects of I/R injury are required in order to improve clinical outcomes in patients with brain ischemic injury. Two endogenous mechanisms have been characterized so far, namely IPre and IPost. Furthermore, it would be needed to unveil the precise mechanisms of neuroprotection elicited by these strategies. Although studies on the effect of IPost on cardiac I/R injury are many, such studies on cerebral I/R are not that much and many data are still lacking. For instance, the effect of IPost on myeloperoxidase activity, tumor necrosis factor-a, nuclear factor kappa B, interleukin 6 and interleukin 10 contents after cerebral I/R are still not clarified. Furthermore, the difference between the effects of IPre and IPost on all aspects of cerebral I/R injury is still clouded by the fact that most of the available data compare the two protective mechanisms form some scopes and ignore others. To that end, the aim of the present work was to introduce a full study of the possible mechanisms of neuroprotection offered by IPre and IPost, through studying the effects of the aforementioned non pharmacological strategies on oxidative stress biomarkers, apoptotic factors, inflammatory mediators, neurotransmitters as well as infarct size and histopathological examination.

2. Material and methods 2.1. Animals Male Wistar rats weighing; 250–300 g were obtained from the National Scientific Research Centre (Giza, Egypt). Animals were housed for at least one week in the laboratory room prior to testing. They were kept under controlled environmental conditions; room temperature (24–27 °C), constant humidity (60 ± 10%), with alternating 12 h light and dark cycles. Food (standard pellet diet) & water were allowed ad libitum. The Ethics Committee of faculty of pharmacy Cairo University approved this study [Date: 26/11/ 2012, Serial No.: PT (595)]. All animals’ procedures were performed in accordance to the institutional Ethics Committee and in accordance with the recommendations for the proper care and use of laboratory animals. Unnecessary disturbance of animals was avoided. Animals were treated gently; squeezing, pressure and tough maneuver were avoided. 2.2. Experimental design Animals were randomly allocated into 4 groups as shown in Fig. 1 (n = 30 rats per group): (1) Sham operated (SO) group. (2) Ischemia–reperfusion (I/R) group. (3) Ischemic preconditioning (IPre) group: each rat was subjected to three episodes of 5 min (50 ) ischemia followed by 10 min (100 ) reperfusion before the induction of I/R [10]. (4) Ischemic postconditioning (IPost) group: following 15 min of global cerebral ischemia each rat was subjected to three episodes of 10 s (1000 ) of ischemia and 10 s (1000 ) of reperfusion after which reperfusion period will be permitted [11].

SO group: 75'

I/R group: Carotid occlusion

Carotid perfusion

15'

60'

IPre group: Ischemic preconditioning

5' 10'

5'

10'

5'

10'

Carotid occlu.

15'

Carotid reperfusion

60'

IPost group:

Carotid occlu.

15'

Ischemic Postcond. 10'' 10'' 10'' 10'' 10'' 10''

Carotid reperfusion 60'

Fig. 1. Diagrammatic presentation of the experimental design.

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2.3.1. Activity of lactate dehydrogenase (LDH) and protein content assay in rat hippocampus Evaluation of the activity of Lactate dehydrogenase in rat hippocampus was through Colorimetric Kinetic Determination using kit supplied by Biosystems (Biosystems, S.A. Costa Brava, 30. 08030 Barcelona, Spain) according to the method of Lorentz et al. [12], while the protein content of tissue supernatant was determined using the method of Lowry et al. [13]. 2.3.2. Estimation of hippocampal oxidative stress biomarkers contents Lipid peroxides formation was investigated in hippocampal tissue homogenate by estimation of thiobarbituric acid reactive substances (TBARs) according to the method of Mihara and Uchiyama [14]. Furthermore, inspection of reduced glutathione (GSH) content in rat hippocampus was performed according to the method of Beutler et al. [15]. Additionally, nitric oxide measurement in rat hippocampus was according to the method described by Miranda et al. [16]. 2.3.3. Determination of pro-inflammatory and anti-inflammatory mediators in rat hippocampus Myeloperoxidase (MPO) enzyme, being a plentiful constituent of neutrophils, serves as a marker for tissue neutrophil content. Since MPO is located within the primary granules of neutrophils, extraction of MPO depends upon procedures to disrupt the granules which render MPO soluble in aqueous solution. This could be achieved by sonication in potassium phosphate buffer (50 mM, pH 6) containing 0.5% hexadecyltrimethylammonium bromide (HTAB) [17], where HTAB is a detergent that releases MPO from the primary granules of the neutrophil [18]. On the other hand, the content of Nuclear Factor-Kappa B was assayed by Enzyme-linked immunosorbent assay (ELISA) using kit supplied by EIAab (E1824r, EIAab Science Co., Wuhan, China). Also, assessment of the content of Tumor Necrosis Factor alpha was by ELISA using kit supplied by R&D Systems (Quantikine Rat

Table 1 Effect of ischemic preconditioning and postconditioning on hippocampal cytosolic lactate dehydrogenase (LDH) activity in rats subjected to global cerebral ischemia reperfusion. Groups

Parameter LDH content (U/g protein) Mean ± SE

Sham operated control Ischemia reperfusion Ischemic preconditioning Ischemic postconditioning

30.57 ± 1.49 80.43* ± 16.41 27.87# ± 6.72 24.19# ± 3.46

Values were expressed as mean ± SE of 6 rats. Statistical analysis was performed by ANOVA followed by Tukey’s Post-hoc test. * Significantly different from Sham operated control at P < 0.05. # Significantly different from ischemia reperfusion at P < 0.05.

TBARs Content (nmol/mg Protein)

1.5

* 1.0

*

*

0.5

0.0

GSH Content (µg/mg protein)

2.3. Methods

Sham Ischemia Preconditioning Postconditioning

a

15

NOx Content (µmol/mg protein)

Each group was subdivided into 2 subsets. The first subset (n = 24 rats) was used for biochemical estimations, while the second subset (n = 6 rats) served for measurement of infarction size and histopathological examination. In all groups rats were anaesthetized with thiopental (50 mg/kg, i.p.) and midline ventral incision was made in the neck. Bilateral carotid artery occlusion using small artery clips was done to induce global cerebral ischemia for 15 min (150 ) followed by 60 min (600 ) reperfusion period except for the SO group in which the arteries were exposed for 75 min (750 ) without occlusion. After reperfusion rats were sacrificed by decapitation, brains were removed and both hippocampi separated and used for biochemical estimations.

15

b

10

*# 5

*#

* 0

c * *

10

# 5

0

Fig. 2. Effect of ischemic preconditioning and postconditioning on hippocampal oxidative stress biomarkers (a) thiobarbituric acid reactive substances (TBARs), (b) reduced glutathione (GSH) and (c) nitric oxide (NOx) contents in rats subjected to global cerebral ischemia reperfusion. Values were expressed as mean ± SE of 6 rats. ⁄ Significantly different from Sham operated control at P < 0.05. #Significantly different from ischemia reperfusion at P < 0.05. Statistical analysis was performed by ANOVA followed by Tukey’s Post-hoc test.

TNF-a ELISA, Catalog # RTA00, R&D Systems, Inc., Minneapolis, MN, USA). Furthermore, rating the contents of interleukin-6 and interleukin-10 were made using kit supplied by R&D Systems (QuantikineÒ ELISA, Rat IL-6 Immunoassay, Catalog Number R6000B, R&D Systems, Inc., Minneapolis, MN, USA) and (QuantikineÒ ELISA, Rat IL-10 Immunoassay, Catalog Number R1000, R&D Systems, Inc., Minneapolis, MN, USA), respectively. 2.3.4. Enzyme-linked immunosorbent assay (ELISA) of apoptotic biomarkers in rat hippocampus The content of caspase-3 enzyme and the content of cytochrome-c were figured out by ELISA using kit supplied by R&D Systems (Quantikine Active Caspase-3 ELISA, Catalog # KM300, R&D Systems, Inc., Minneapolis, MN, USA) for caspase 3 content, and using kit supplied by EIAab (E0594r, EIAab Science Co., Wuhan, China) for cytochrome c content. 2.3.5. High performance liquid chromatography (HPLC) Hippocampus was homogenized in 70% HPLC methanol (1/10 weight/volume) and was used for the estimation of glutamate and GABA using a fully automated high-pressure liquid chromatography system (HPLC; Perkin-Elmer, MA, USA). Brain amino

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Table 2 Effect of ischemic preconditioning and postconditioning on hippocampal inflammatory biomarkers: myeloperoxidase (MPO) activity, nuclear factor kappa-B (NF-jB), tumor necrosis factor-alpha (TNF-a), interleukin-10 (IL-10) and interleukin-6 (IL-6) contents in rats subjected to global cerebral ischemia reperfusion. Groups

Parameter MPO activity (mU/mg protein) Mean ± SE

Sham operated control Ischemia reperfusion Ischemic preconditioning Ischemic postconditioning

NF-jB content (ng/mg protein) Mean ± SE

TNF-a content (Pg/mg protein) Mean ± SE

IL-10 content (Pg/mg protein) Mean ± SE

IL-6 content (Pg/mg protein) Mean ± SE

26.83 ± 4.24

0.11 ± 0.01

3.02 ± 0.39

16.71 ± 2.34

27.11 ± 1.74

42.53* ± 2.42 24.94# ± 3.38

0.35* ± 0.02 0.28* ± 0.04

14.85* ± 0.96 4.72# ± 0.51

4.96* ± 1.22 17.38# ± 2.59

40.41* ± 5.38 26# ± 1.78

34.08 ± 4.11

0.17# ± 0.03

2.61# ± 0.34

17.01# ± 2.99

21.64# ± 1.49

Values were expressed as mean ± SE of 6 rats. Statistical analysis was performed by ANOVA followed by Tukey’s Post-hoc test. * Significantly different from Sham operated control at P < 0.05. # Significantly different from ischemia reperfusion at P < 0.05.

acids were inspected by the phenylisothiocyanate derivatization technique described by Heinrikson and Meredith [19]. Hippocampal tissues were dried under vacuum following reconstitution with 2:2:1 mixture (v) of methanol:1 M sodium acetate trihydrate:triethylamine. The derivatization procedure using a 7:1:1:1 mixture (v) of methanol:triethylamine:double-distilled deionized water:phenylisothiocyanate, was performed for 20 min at room temperature then re-subjected to vacuum until dryness. Subsequently, derivatized amino acids were reconstituted with sample diluent consisting of 5:95 mixture (v) of acetonitrile:5 mM phosphate buffer (pH = 7.2). Samples were then sonicated and filtered (0.45 lm; Millipore, USA). A Pico-Tag physiological free amino acid analysis C18 (300 mm  9 3.9 mm i.d.) column from waters (MA, USA) and a binary gradient of eluents 1 and 2 (Waters) were used, the column temperature was at set 46 ± 1 °C. A constant flow rate of 1 ml/min was maintained throughout the experiment. 20 ll of samples were injected and the absorbance of the derivatized amino acids was measured at 254 nm. Glutamate standard was prepared in double-distilled deionized water, while GABA standard was prepared in polyethylene vials to prevent adhesion to glass.

stained with Haematoxylin for 10 min. and then counterstained with Eosin for 1 min., followed by rapid rinsing with distilled water to remove excess stain then dehydration with ascending grades of alcohols. Finally, clearing with xylene and mounting in Canada balsam was performed. For quantification of the number of pycnotic neuronal cells, three random regions were examined at 400 magnification and the number of pycnotic neurons in three areas per section of hippocampal dentate gyrus (DG) region were identified and counted on the basis of the presence of pycnotic nuclei and shrunken cytoplasm. Then the number of the pycnotic neuronal cells was calculated as an average per rat.

2.3.6. Infarct size estimation At the end of 60 min reperfusion period, animals (n = 4) were intracardially perfused with isotonic saline and sacrificed by spinal dislocation. Brains were then sliced into 2 mm coronal sections and incubated with 1% triphenyltetrazolium chloride (TTC) at 37 °C in 0.2 M Tris buffer (pH 7.4) for 20 min. While viable cells stain bright red when TTC is converted to red formazone pigment by NAD and lactate dehydrogenase, infracted cells lose the enzyme as well as cofactor and thus remain unstained or stain dull yellow. The brain slices were placed over glass plate and the infarcted areas were traced by a 100 squares in 1 cm2 transparent plastic grid. In each brain slice, the average infarcted area of both sides as well as the non infarcted area was computed. Infarcted area was expressed as a percentage of total brain area [20,21].

3. Results

2.3.7. Histopathological investigation Histopathological examination was performed on the brains of 3–4 rats randomly selected from each group. Following transcardiac perfusion, brains were removed, placed in 10% formalin/PBS and kept until they became hard enough to be sectioned. Each brain was embedded in paraffin blocks. Coronal sections of 5 lm were obtained and stained with haematoxylin and eosin (H&E) for standard histological examination according to the method of Banchroft et al. [22]. In brief, deparaffinization of sections was performed using xylene, while hydration was carried out using descending grades of alcohol and finally water. The sections were

2.4. Statistical analysis Values were expressed as mean ± SE using a computer software program Statistical Package for the Social Sciences ‘‘SPSS’’ (Version 16.0.). One-Way Analysis of Variance (ANOVA) followed by Tukey– Kramer multiple comparisons Post hoc test was used for comparing the means of the different groups. The criterion for statistical significance was set at the P < 0.05 level. Graphical representation was conducted using GraphPad Prism (Version 5).

3.1. Effect of IPre and IPost on lactate dehydrogenase (LDH) activity in hippocampal tissue Global cerebral I/R was accompanied with elevated hippocampal LDH activity to more than 2.6 times the SO group. Both IPre and IPost could decrease LDH activity to 34% and 30%, respectively of I/R group (Table 1). 3.2. Effect of IPre and IPost on oxidative stress biomarkers in hippocampal tissue Induction of transient global cerebral ischemia for 15 min, followed by a period of 1 h of reperfusion was associated with an increase in hippocampal content of prooxidant parameters as malondialdehyde and peroxynitrite as indicated by the elevated nitrate/nitrite level combined with myeloperoxidase (MPO) activity. On the other hand the hippocampal content of GSH declined below the level of the normal control rats. Global cerebral I/R resulted in a significant increase in hippocampal TBARs content to about double the SO group accompanied with a reduction in GSH content to 28% compared to SO group. Although IPre and IPost failed to reduce elevated TBARs (Fig. 2a), both significantly elevated GSH content to about two folds of that in I/R group (Fig. 2b). Furthermore, hippocampal nitric

25

40

a *

30 20

#

10

#

0

Cytochrome C Content (µg/mg protein)

Caspase 3 Activity (mmol/mg protein)

M.A. Saad et al. / Chemico-Biological Interactions 232 (2015) 21–29

20

Sham Ischemia Preconditioning Postconditioning

b *

15

*

10

#

5 0

Fig. 3. Effect of ischemic preconditioning and postconditioning on hippocampal apoptotic markers (a) Caspase-3 activity and (b) cytochrome-c (Cyt-c) content in rats subjected to global cerebral ischemia reperfusion. Values were expressed as mean ± SE of 6 rats. ⁄Significantly different from Sham operated control at P < 0.05. #Significantly different from ischemia reperfusion at P < 0.05. Statistical analysis was performed by ANOVA followed by Tukey’s Post-hoc test.

b

Sham Ischemia Preconditioning Postconditioning

4 3

*

*

2

*#

1 0

GABA Content (µg/mg protein)

Glutamate Content (µg/mg protein)

a 5

*

4

* 3

*

2 1 0

Fig. 4. Effect of ischemic preconditioning and postconditioning on hippocampal neurotransmitters (a) glutamate and (b) gamma amino butyric acid (GABA) contents in rats subjected to global cerebral ischemia reperfusion. Values were expressed as mean ± SE of 6 rats. ⁄Significantly different from Sham operated control at P < 0.05. #Significantly different from ischemia reperfusion at P < 0.05. Statistical analysis was performed by ANOVA followed by Tukey’s Post-hoc test.

oxide content was significantly increased to about two folds the SO group following I/R. Only IPre reduced NOx content to half that in I/ R group (Fig. 2c). 3.3. Effect of IPre and IPost on inflammatory biomarkers in hippocampal tissue The current study revealed that induction of I/R showed an inflammatory response as presented by the elevated contents of the proinflammatory mediators such as TNF-a, IL-6, MPO and NF-jB as well as a reduced content of the human cytokine synthesis inhibitory factor known as interleukin-10 which is an important anti-inflammatory cytokine. Global cerebral I/R was associated with neutrophil infilteration observed as an elevation in hippocampal MPO activity to about 1.5 times that in the SO group. This effect was offset by IPre in which MPO activity decreased to 62% that of I/R group. Global cerebral I/R showed a significant increase in hippocampal NF-jB, TNF-a, IL-6 contents together with reduction in IL-10 content. NF-jB content reached more than 3 folds that in the SO group meanwhile TNF-a and IL-6 were increased by 5 and 1.5 folds, respectively. On the other hand IL-10 content reached 29% of the SO group. IPre reduced TNF-a and IL-6 contents to be 31% and 64% of that in I/R group together with elevation in IL-10 content to 3.5 times the I/R group, while IPost decreased NF-jB, TNF-a and IL-6 contents to be 48%, 17% and 53%, respectively as compared to I/R group (Table 2). 3.4. Effect of IPre and IPost on apoptotic biomarkers in hippocampal tissue Global cerebral I/R resulted in obvious increase in hippocampal caspase-3 activity to 2.5 folds the SO group. Both, IPre and IPost reduced caspase-3 activity by 29% and 28% of that in I/R group,

respectively (Fig. 3a). Additionally, hippocampal Cyt-c content was elevated 5 folds following I/R compared to the SO group but only IPost succeeded to lessen Cyt-c content to be 22% of the I/R group (Fig. 3b). 3.5. Effect of IPre and IPost on glutamate and GABA contents in hippocampal tissue I/R elevated the contents of glutamate and GABA. So it is conceivable that excitotoxicity is an inevitable pathway for the damage shown in ischemia reperfusion. Glutamate content reached to about six folds that measured in the SO group while GABA content increased to 3.6 times the SO group. IPost decreased glutamate content to half the I/R group (Fig. 4a) with no effect on GABA content (Fig. 4b). 3.6. Effect of IPre and IPost on cerebral infarct size Global cerebral I/R resulted in a significant increase in infarct size to 2.3 times the SO group. Both IPre and IPost could decrease the elevated infarct size to 73% and 67% of that in I/R group respectively, but they failed to normalize it (Fig. 5). 3.7. Effect of IPre and IPost on the histopathology of hippocampal areas in rats subjected to I/R As shown in Fig. 5, sections of the SO rat hippocampi showed normal histological structures, while sections of the I/R rat hippocampi presented the occurrence of necrosis, atrophy and pyknosis of pyramidal cells of the hippocampus. IPre demonstrated an improvement in the I/R induced changes, where the hippocampal cellular structures were nearly preserved. On the contrary, IPost failed to modify the histological damage and hippocampal pycnotic neuronal cells count elevated by I/R (Figs. 6 and 7).

M.A. Saad et al. / Chemico-Biological Interactions 232 (2015) 21–29

Cerebral infarct size (%)

26

Sham Ischemia Preconditioning Postconditioning

100 80

*

60

*#

*#

40 20 0

Fig. 5. Effect of ischemic preconditioning and postconditioning on brain coronal sections (I) coronal sections showing the infarct areas (in white) in (A) Sham operated control, (B) ischemia/reperfusion brain, (C) Ischemic preconditioning and (D) Ischemic postconditioning. (II) Summary of the quantitative analysis of infarct areas. Values were expressed as mean ± SE of 4 rats. ⁄Significantly different from Sham operated control at P < 0.05. #Significantly different from ischemia reperfusion at P < 0.05. Statistical analysis was performed by ANOVA followed by Tukey’s Post-hoc test.

4. Discussion Ischemia or shortage of oxygen followed by restoration of blood supply back to the organ ‘‘reperfusion’’, creates an inflammatory response and oxidative stress rather than restoring the normal function of the organ. White blood cells, coming back to the area release inflammatory factors such as MPO, interleukins, NF-jB and TNF-a as well as free radicals in response to tissue damage. These reactive species can turn on apoptosis partially responsible for the damage of reperfusion injury [23,24]. Low activities of antioxidant enzymes together with the high lipid content in the brain make it highly vulnerable to reactive oxygen species which play a major role in the damage produced during reperfusion following stroke. This oxidative stress induces damage to brain lipids, proteins, and DNA, leading to distortion of the brain function and cell death [25]. Our findings support the data of Al Rouq and El Eter [26] who demonstrated a decrease in the content of GSH together with an increase in MPO and proinflammatory markers in rats subjected to global cerebral ischemia/reperfusion. The present work was in concordance with the results of Liu et al. [27] who demonstrated that rats subjected to cerebral I/R showed elevated contents of IL-6, IL-1b and TNF-a. Ischemia in which blood flow is reduced to inadequate levels can start damage to neuronal cells inducing stroke or brain trauma. Within minutes, the damaged neural cells inside the ischemic site release glutamate into the extracellular space where glutamate can stimulate presynaptic glutamate receptors to enhance the release

of more glutamate. When the glutamate concentration around the synaptic space fail to decrease or when high levels are reached, the neuron kills itself by a process called apoptosis and as it results from severe excitation it is called excitotoxicity [28,29]. Excitotoxicity occurs when receptors for the excitatory neurotransmitter glutamate (glutamate receptors) such as the NMDA receptor and AMPA receptor are overactivated by the so called glutamatergic storm. It has been claimed that median cerebral artery occlusion (MCAO) induced a rapid and marked increase in contents of glutamate, which reached the maximum at 60–100 min after stroke onset. Also, GLT-1 which is one of main transporters for removal of extracellular glutamate was reduced 4 and 24 h after MCAO [30]. Additionally, MCAO showed a gradual elevation of brain glutamate, aspartate and GABA contents at different brain regions and reached peak level at 72 h of I/R [31]. Apoptotic cell death is a genetically programmed mechanism(s) that allows the cell to commit suicide. The two major well-studied apoptotic processes are the extrinsic and intrinsic pathways [32,33]. The extrinsic pathway is mediated by tumor necrosis factor receptors. Activation of these so called death receptors leads to the recruitment and activation of initiator caspases such as caspases 8 and 10. This leads to the activation of an effector caspase, typically caspase 3. The activated caspase 3 is responsible for the cleavage of a number of so-called death substrates that lead to the well-known characteristic hallmarks of an apoptotic cell [34]. The intrinsic pathway is largely centered around and/or regulated by the mitochondria [35]. The most widely studied form of

M.A. Saad et al. / Chemico-Biological Interactions 232 (2015) 21–29

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Fig. 6. Representative photomicrographs of the hilar region of the dentate gyrus sector of the hippocampal sections of (A) control animals showing normal architecture of hippocampus, (B) ischemia reperfusion (I/R) animals showing nuclear pyknosis (arrow), (C) Ischemic preconditioning group showing preserved cellular structures and (D) ischemic postconditioning group showing nuclear pyknosis (arrow). (DH, dentate hilus; DG, dentate gyrus). I = (H & E  100) and II = (H & E  400).

intrinsic apoptosis is initiated by the release of cytochrome c from the mitochondria that results in the formation of the apoptosome. The apoptosome then activates initiator caspase, mostly caspase 9, which leads to the activation of the executioner caspase 3. This

leads to similar type of apoptotic response as observed for the extrinsic pathway. Rats subjected to I/R in this experiment showed an elevated contents of TNF-a, cytochrome c as well as caspase 3 activities.

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Pycnotic neuronal cells count

Sham Ischemia Preconditioning Postconditioning 60

*

*

40

20

0

Fig. 7. Effect of ischemic preconditioning and postconditioning on hippocampal pycnotic neuronal cells count in rats subjected to global cerebral ischemia reperfusion. Values were expressed as mean ± SE of 4 rats. ⁄Significantly different from Sham operated control at P < 0.05. Statistical analysis was performed by ANOVA followed by Tukey’s Post-hoc test.

This is coherent with results of Yang et al. [36] who demonstrated that I/R tremendously increased the contents of both IL1b and TNFa mRNAs. Also there was little caspase 3 signal in the SO group slices, while caspase 3-positive cells were higher in the I/R group [36]. The induction of oxidative stress and inflammatory response in our experiment may be the initiating factors for apoptosis. The induction of apoptosis together with excitotoxicity was suggested to be the contributing factors for the increase in infarct size shown in current study. Our results are consistent with the trials completed by Grewal et al. [37] who reported that bilateral common carotid artery occlusion followed by reperfusion, produced a significant elevation in cerebral infarct size. Many pathways have been suggested as plausible mechanisms to explain the neuroprotection offered by ischemic preconditioning (IPre) and postconditioning (IPost). Our data confirm previous observations that showed that the cortical and hippocampal antioxidant enzyme activity and SOD expression were increased in the IPre group against cerebral I/R injury in rats [38]. Both IPre and IPost interventions reduced TNF-a and IL-6 contents and increased IL-10 content. IPre reduced MPO activity while IPost reduced NF-jB content. Our results were in harmony with the previously reported results that showed a reduction in TNF-a and elevation in IL-10 contents following IPre as compared to I/R group [10]. IPre and IPost antiapoptotic activity was evidenced by the decreased activity of caspase 3 enzyme which reached the normal level of the SO group. On contrary, in Ding et al. [39] work caspase 3 expression was still higher than normal. This conflict may be due to the different model of induction of ischemia, as Ding et al. used 4vessel occlusion model compared to the 2-vessel model used in our work or to the longer reperfusion period, 72 h used in Ding et al. work compared to the 1 h reperfusion period used in our work. This is coherent with results of Ma et al. [40] who reported that IPre showed declines in apoptotic cell numbers, apoptotic protein expressions as well as caspase 3 activation. Together, these findings further support the notion that both IPre and IPost have marked antiapoptotic actions. In the present investigation, IPost reduced glutamate content elevated by I/R. Bonova et al. [41] showed that IPost improves protein synthesis in CA1 and dentate gyrus and, surprisingly, leads to 50% reduction in glutamate in whole hippocampus and cortex through activation of mechanisms resulting in rapid elimination of glutamate from brain tissue and/or in the circulatory system that could otherwise impede brain-to-blood glutamate efflux

mechanisms. Furthermore, post-conditioning induces protein synthesis renewing in ischemia affected tissues that could also contribute to elimination of excitotoxicity. On the other hand, IPre failed to protect against GABA and glutamate elevated contents. Although it was previously reported that IPre prevented the down-regulation of GLT-1 protein and reduce the content of glutamate [42]. This discrepancy may be explained by the longer period of preconditioning or the shorter period of ischemia in the study of Gong et al. [42] compared to the present work. Ischemic pre- and postconditioning could decrease the current elevated infarction size but they failed to return it back to the normal level. Furthermore, IPre preserved cellular structures from damage in contrast to IPost which showed some nuclear pyknosis on histopathological investigation. On the contrary, neuronal density and the percentages of viable neurons was significantly increased by IPre and IPost compared to the ischemic insult alone [39]. The failure of IPost in our model to protect the hippocampal neurons from death compared to Ding et al. [39] work may be due to the different number of IPost cycles used, as Ding et al. used 6 cycles for induction of IPost while in our work we used 3 cycles only for induction of IPost. It could be suggested that the observed decrease in infarct size shown might be related to the combined antioxidant, anti-inflammatory and antiapoptotic effects elicited by IPre and IPost. Similarly, both IPre and IPost immediately after global cerebral ischemia markedly prevented I/R induced cerebral injury as measured in terms of infarct size, loss of memory and motor coordination [43]. It could be concluded from the present findings that the protective potential of IPre and IPost is promising as a hopeful therapeutic principle for ischemia mediated disorders, at the very least as a component of a multiple ‘cocktail’ strategy. 5. Conclusions From the previously mentioned results it could be concluded that: (1) Global cerebral I/R showed damage to the rat hippocampus through increasing the production of reactive oxygen species which overwhelms the endogenous antioxidant capacity creating oxidative stress. I/R triggered also an inflammatory response as evidenced by the elevated proinflammatory and the reduced anti-inflammatory markers. The damage of I/R was also thought to be through elevated excitotoxic glutamate level. (2) All the previous oxidative stress, inflammatory and excitotoxic parameters lead to the induction of apoptosis as confirmed by the increased apoptotic markers and infarct size. (3) Both IPre and IPost showed antioxidant and anti-inflammatory activities, but the former effect was not through reducing the level of TBARs. IPost could reduce excitotoxic marker glutamate, while IPre failed to do that. (4) Both IPre and IPost showed antiapoptotic actions. Conflict of Interest The authors declare that there are no conflicts of interest. Transparency Document The Transparency document associated with this article can be found in the online version.

M.A. Saad et al. / Chemico-Biological Interactions 232 (2015) 21–29

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Ischemic preconditioning and postconditioning alleviates hippocampal tissue damage through abrogation of apoptosis modulated by oxidative stress and inflammation during transient global cerebral ischemia-reperfusion in rats.

It has been argued recently that ischemic preconditioning (IPre) and postconditioning (IPost) have beneficial effects in many ischemic disorders howev...
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