Neurochemistry International 72 (2014) 22–29

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Bradykinin postconditioning ameliorates focal cerebral ischemia in the rat Viera Danielisova ⇑, Miroslav Gottlieb, Petra Bonova, Miroslava Nemethova, Jozef Burda Institute of Neurobiology, Slovak Academy of Sciences, Kosice, Slovakia

a r t i c l e

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Article history: Received 17 January 2014 Received in revised form 20 March 2014 Accepted 8 April 2014 Available online 18 April 2014 Keywords: Cerebral ischemia Bradykinin Hippocampus Postconditioning Endogenous antioxidant enzymes

a b s t r a c t The goal of this study is to investigate the effects of bradykinin (BR) postconditioning on cerebral ischemic injury. Transient focal cerebral ischemia was induced in rats by 60 min of middle cerebral artery occlusion (MCAO), followed by 3 days of reperfusion. BR as a postconditioner at a dose of 150 lg/kg was applied intraperitoneally 3, 6, 24 and 48 h after MCAO. BR postconditioning significantly reduced total infarct volumes if applied 3 h after MCAO by 95%, 6 h after MCAO by 80% and 24 h after MCAO by 70% in versus vehicle group. Neurological functions were a marked improvement in the BR groups compared to the ischemia group. The number of degenerated neurons in the hippocampal CA1 region was also significantly lower in BR-treated ischemic groups compared to vehicle group. BR postconditioning prevented the release of MnSOD from the mitochondria and reduced the activity of the total SOD and CAT if it is administrated short time after stroke. Our data proves the ischemic tolerance in the brain induced by BR postconditioning resulted as effective agent against as strong an attack as 60 min MCAO even when used many hours after ischemia. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Bradykinin (BK) is considered an important mediator of the inflammatory response in both the peripheral and central nervous system, and has attracted recent interest as a potential mediator of brain injury following stroke (Sobey, 2003; Zausinger et al., 2002). These characteristics suggest that bradykinin can be effectively used as a stressor inducing ischemic tolerance after parenteral application. Numerous reports have confirmed the effect of bradykinin as pre-conditioner and postconditioner in protection of the heart (Lim et al., 2007; Penna et al., 2007) as well as the brain (Danielisova et al., 2008, 2012, 2009; Lehotsky et al., 2009). Bradykinin is a physiologically active nonapeptide with a lot of functions in the body, synthesized by kallikreins acting on kinogen precursor molecules. The examinations of cerebral ischemia

Abbreviations: BR, bradykinin; DG, dentate gyrus; CA1, cornu Ammonis 1 layer of hippocampus; CAT, catalase; FJ B, Fluoro-Jade B; MCA, middle cerebral artery; MCAO, middle cerebral artery occlusion; SOD, superoxide dismutase; CuZnSOD, copper–zinc dependent superoxide dismutase; MnSOD, manganese dependent superoxide dismutase; NeuN, neuronal nuclear protein; LCBF, local cerebral blood flow. ⇑ Corresponding author. Address: Institute of Neurobiology, Slovak Academy of Sciences, Soltesovej 4, 040 01 Kosice, Slovakia. Tel.: +421 55 7276230; fax: +421 55 7276202. E-mail address: [email protected] (V. Danielisova). http://dx.doi.org/10.1016/j.neuint.2014.04.005 0197-0186/Ó 2014 Elsevier Ltd. All rights reserved.

demonstrated that an activation of B2 receptor resulted in activation of protein kinase C associated with release of Ca2+ from intracellular stores, stimulation of other inflammatory mediators, and release of excitatory amino acids which led to brain damage after cerebral ischemia (Zausinger et al., 2002). Bradykinin combined with B2 receptor, subsequently activated protein kinase, accelerated production of NO, and induced mitochondria ATP-sensitive K+ channel opening to protect the ischemic myocardium. It seems plausible that bradykinin acts as a primary trigger of delayed preconditioning, and that this effect is mediated by generation of NO as a signalling intermediate (Baxter and Ebrahim, 2002). Also bradykinin induces ROS, especially superoxide anion generated as a result of mitochondrial uncoupling through a pathway that involves activation of protein kinase C isoenzymes, tyrosine kinases and mitogen-activated protein kinases (Baxter and Ebrahim, 2002). Ischemic tolerance (preconditioning/postconditioning) is the strongest endogenous neuroprotectant against brain injury after cerebral ischemia (Gidday, 2006; Perez-Pinzon, 2007). Our previous results show that delayed neuronal death in a model of transient forebrain ischemia simulating cardiac arrest as well as kainate intoxication can be prevented by postconditioning 2 days after ischemia. As postconditioners, we used short ischemia, 3-nitropropionic acid, norepinephrine and bradykinin (Burda et al., 2005, 2009, 2006; Danielisova et al., 2006). It was recently

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demonstrated that ischemic postconditioning protects against transient focal ischemia simulating stroke (Gao et al., 2008a,b; Ren et al., 2008, 2009; Zhao et al., 2006), in which ischemic postconditioning is conducted by a series of brief occlusion and release of the bilateral common carotid arteries after reperfusion. Ideally, the clinical application of this method of ischemic postconditioning requires that the treatment be applied after the onset of stroke (Zhao, 2009). Our most recent results documented the efficacy of a combination of both of these methods of postconditioning (immediate applied just on the onset of reperfusion and delayed applied 2 days after ischemia) in a four-vessel occlusion model of ischemia (Danielisova et al., 2012). Transient forebrain ischemia induces delayed neuronal death in the hippocampal CA1 region. Oxygen free radicals have been suggested to play a significant role in this neuronal death (Kim et al., 1999; Song et al., 2007). In particular, superoxide generated during cerebral ischemia–reperfusion is implicated. Overexpression of copper/zinc superoxide dismutase (CuZn-SOD) minimises superoxide toxicity, resulting in a reduction of cell death (Sugawara et al., 2002). Thus, understanding radical generation and the subsequent toxicity can lead to therapeutic tools to prevent brain injury including ischemic insult (Choi et al., 2007). In this study we examined bradykinin’s ability to induce ischemic tolerance against neuronal injury in a model of transient focal ischemia. Specially we investigated efficacy of postconditioning if delayed several hours up to one day. We decided to determine whether the application of BR postconditioning reduces infarct volume, improvements neurological score and also reduces the activity of total SOD and CAT in the infarct area and penumbra, as well as in two regions of the hippocampus, the selective vulnerable CA1 region and the relative resistant dentate gyrus in the ipsilateral hemispheres. 2. Methods 2.1. Animals Seventy adult male albino Wistar rats weighing 270–300 g were group-housed and maintained on a 12 h light/dark cycle, with ad libitum access to water and rodent chow. The experiments were carried out in accordance with the protocol for animal care approved by both the Slovak Health Committee (1998) and the European Communities Council Directive (86/609/EEC). The animals were randomly subdivided as follows: 4 controls, 6 shamoperated and 60 ischemic. 2.2. Surgical procedures Transient focal ischemia was induced by middle cerebral artery occlusion (MCAO) using the intraluminal filament technique (Longa et al., 1989). Rats were anaesthetised with 4% halothane in an anaesthetic chamber and maintained during surgery at 1.5% halothane using a rodent mask. Body temperature was maintained at 37 °C with a heat pad. MCAO was carried out for 60 min by inserting a 4–0 nylon monofilament (Lorca Marin, Spain) via the right external carotid artery into the internal carotid artery to block the origin of the MCA. Sham-operated controls were treated similarly to the ischemic group, but the middle cerebral artery was not occluded. The Severity of MCA occlusion was confirmed by measuring of local cerebral blood flow (LCBF) using a laser-Doppler flowmeter (PeriFlux System 5000, Perimed AB, Sweden). A 407 probe with an adequate holder was situated on the skull over the MCA location (5 mm lateral and 1 mm posterior to bregma). Only rats with blood flow reduced by more than 80% were used for experiments (data not shown).

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In the ischemic group, MCA occluded rats (n = 6) received an injection (1 ml/kg i.p.) of normal saline as a vehicle. The treated groups (n = 6 each) were MCA-occluded rats receiving an i.p. injection of 150 lg/kg bradykinin (Sigma–Aldrich Chemie Gmbh, Germany) administered 3, 6, 24 or 48 h after MCA occlusion. Animals were euthanised for immunochemical studies 3 days after ischemia. 2.3. Evaluation of neurological deficits Neurological deficit was assessed in each animal on a numerical scale of 0–4 at 60 min after MCAO termination and every 24 h afterwards. The scoring system of Bederson (Bederson et al., 1986b) was used: 0, no detectable deficits; 1, forelimb flexion and turning of torso to the contralateral side when lifted by the tail; 2, same behaviour as grade 1 and decreased resistance to lateral push; 3, same behaviour as grade 2 with unilateral circling; and 4, no spontaneous walking and a depressed level of consciousness. Rats with a neurological deficit lower than 2 were excluded from the study. 2.4. Determination of infarct volume The animals were decapitated under chloral hydrate anaesthesia after 3 days of reperfusion. The brains were rapidly dissected out and the forebrains were cut into five coronal sections, 2 mm thick, using a rat brain matrix (Activational Systems, MI, USA). Analysis of cerebral ischemic damage was carried out by using 2,3,5-Triphenyltetrazolium chloride (TTC, Sigma) (Bederson et al., 1986a). The sections were stained by incubating them in a solution of 1% TTC at 37 °C for 15 min and fixed in 10% formalin. For imaging, the sections were scanned by a high-resolution scanner (Hewlett Packard Scanjet). The non-ischemic hemisphere, ischemic hemisphere and infarct area of each brain section was measured in a blinded manner, using Image J software (National Institutes of Health, Bethesda, Maryland, USA). The average infarct area (mm2) was calculated by the formula: (infarct area on the anterior surface + infarct area on the posterior surface):2. The corrected infarct area in a slice was calculated to compensate for brain edema (Swanson et al., 1990). Corrected infarct volumes (mm3) were calculated by multiplying the corrected area by the slice thickness and summing the volume. 2.5. Fluoro-Jade B staining Two millimetre coronal sections after TTC staining were transferred to 20% sucrose (w/v) in 0.1 M phosphate buffer (PB) until equilibrated. The tissue was then frozen in Tissue-Tek OCT mounting medium and 10 lm coronal sections were cut and subsequently mounted on gelatinised microscope slides; these were allowed to air dry, and were then placed in 70% ethanol and ultrapure water for 3 min. The sections were oxidised by soaking in a solution of 0.06% KMnO4 for 15 min then washed 3 times in ultrapure water for 1 min each and stained in 0.001% Fluoro-Jade B (Histo-Chem Inc., USA) in 0.1% acetic acid for 20 min. Two millimetre coronal sections after TTC staining were transferred to 20% sucrose (w/v) in 0.1 M phosphate buffer (PB) until equilibrated. 10 lm coronal sections were stained in 0.001% Fluoro-Jade B (Histo-Chem Inc., USA) by method Schmued and Hopkins (Schmued and Hopkins, 2000). The slides were subsequently washed 3 times in ultrapure water for 1 min each wash and dried overnight at room temperature. The dried sections were cleared by xylol and cover-slipped with DPX Mountant for histology (Fluka Chemie AG, Switzerland). The slides were examined using an Olympus BX 51 microscope with a digital camera DP 50 (Olympus Optical CO. LTD, Japan). Fluoro-Jade B (FJ-B) positive count was

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performed by an investigator who was unaware of the treatment conditions, using Image tool software (UTHSCSA, San Antonio, USA). 2.6. NeuN immunohistochemistry Brains were prepared and sectioned as described previously (see Fluoro-Jade B staining). Cryostat sections (10 lm) at the level of the dorsal hippocampus were collected onto gelatinised slides and processed for immunohistochemistry. Air-dried sections were hydrated by soaking in PBS and then incubated in 50% methanol with 0.3% H2O2 in PBS. After washing with PBS (3 times for 5 min), slides were blocked and permeabilised in 0.1 M PBS with 0.3% Triton X-100 (Sigma) and 4% horse serum for 1 h. Mouse monoclonal anti-NeuN (2 lg/ml; Chemicon International, Temecula, CA) was applied overnight at room temperature and a secondary anti-mouse IgG antibody was applied for 1 h at room temperature followed by washes in 0.1 M PB. ABC Elite (Vector Laboratories, Burlingame, USA) was applied for 90 min, and then the slides were rinsed with PBS followed by Tris buffer (pH 7.6), and reacted with DAB Substrate (Roche Diagnostics GmbH). 2.7. Assay procedure for SOD and CAT activity Regions from the right (ipsilateral) hemispheres, corresponding to the ischemic core (Ashwal et al., 1998) was separated from the penumbra by a transverse diagonal cut at approximately the ‘‘2 o’clock’’ position and the ‘‘5 o’clock’’ position. The hippocampus was removed from the brain and immediately cooled to 4 °C, before being transversally split into 200 lm parts, which were then dissected under a microscope along the hippocampal fissure into CA1 and DG subregions. Infarct area and penumbra (distant region), hippocampal CA1 region and gyrus dentate (DG) were homogenised in the extraction medium containing 0.1 M sodium phosphate pH 7.8 and then centrifuged at 12,000g for 10 min at 4 °C to separate the post-mitochondrial supernatant. The superoxide dismutase (SOD) activity was assay by Sun et al. (1988). Xanthine–xanthine oxidase was utilised to generate superoxide flux. The absorbance obtained from nitroblue tetrazolium (NBT, p-nitrotetrazolium blue grade III, Sigma) reduction to blue formazan by superoxide was determined spectrophotometrically at 560 nm at room temperature. The SOD in the sample competes for superoxide, inhibiting the reaction rate of superoxide with NBT. The rate of NBT reduction in the absence of tissue was used as the reference rate (0.020 ± 0.005 absorbance/min). The standard assay substrate mixture contained (in 200 ll): 1 M xanthine, 0.1 M EDTA, 5.6  10 2 M NBT and 1 M BSA (bovine serum albumin) in 0.1 M sodium phosphate (pH 7.8). The data were plotted as percentage inhibition versus protein concentration. One unit (U) of SOD activity was defined as the amount that reduced the absorbance change by 50%, and results were normalised on the basis of total protein content (U/mg protein). Copper– zinc superoxide dismutase (CuZnSOD) was differentiated from manganese superoxide dismutase (MnSOD) by the addition of 2 mM sodium cyanide to inhibit the activity of CuZnSOD. CuZnSOD activity was calculated as the difference between total SOD and MnSOD activity. The catalase activity (CAT) was determined by Goth’s spectrophotometric method (Goth, 1991) in which the supernatant was incubated with hydrogen peroxide used as the substrate and enzymatic reaction was stopped by the addition of 32 mM ammonium molybdate. The intensity of the yellow complex formed by molybdate and hydrogen peroxide was measured at 405 nm. CAT activity is given in U/mg protein.

2.8. Protein assay Total protein concentrations were determined using the method described by Bradford (Bradford, 1976) and analyticalgrade bovine serum albumin was used to establish a standard curve. 2.9. Cell-counting procedure Positive cell count was performed by an investigator who was unaware of the treatment conditions, using Image Tool software (UTHSCSA, San Antonio, USA). Quantification of NeuN and Fluoro Jade positive cells were conducted in the middle of the linear part of the CA1 ( 3.3 ± 0.2 mm posterior of the bregma). Cells were counted in the ten hippocampal region slices of each animal and expressed as the average of positive pyramidal neurons per 1 mm of CA1 length. 2.10. Statistical analysis The results were represented as mean ± SD. One-way analysis of variance (ANOVA) followed by a post hoc Tukey’s test was performed by using GraphPad 3.0 software. Values of P < 0.05 were considered to be significant. 3. Results 3.1. Effect of bradykinin on infarct volume and neurological score Brain sections obtained from MCAO group showed detectable lesions as white patches in the areas that are supplied by the MCA. The lesions were present in both the lateral striatum and the overlying cortex after MCAO (Fig. 1A). Infarct volume was significantly (P < 0.05) reduced in the BR treated groups 3, 6 and 24 h after MCAO (Fig. 1B). Neurological scores after MCAO with the application of BR improved over time during reperfusion and were significantly better in the 3 and 6 h BR groups (P < 0.05) compared to the 24 and 48 h BR groups or ischemia alone (Fig. 1C). 3.2. Effect of bradykinin on SOD and CAT activities in core and penumbra The enzymatic activities of total SOD, CuZnSOD, MnSOD and CAT were measured 3 days after MCAO treated without or with the application of BR 3, 6, 24 and 48 h after stroke. Samples were collected from core (infarct area) and surrounding penumbra of the brain (Fig. 2). The total SOD activity was significantly increased 3 days after 60 min of occlusion in all BR groups compared to control (P < 0.05). However, the application of BR (3, 6 and 24 h after ischemia) significantly prevented an increase of total SOD compared to the ischemic group. A similar situation was also seen for CuZnSOD enzymatic activity in the brain. The ischemia with and without BR application significantly increased MnSOD activity compared to control rats, but the application of BR (3 and 6 h) after MCAO prevented an increase of MnSOD activity in comparison to the ischemic group of the brain. The changes in the activity of another important endogenous antioxidant enzyme, CAT, were significant and very similar to changes in total SOD (Fig. 2). 3.3. Effect of bradykinin on damaged and surviving neurons in CA1 region Positive FJ-cells were localised to the pyramidal cell layer of CA1 (Fig. 3A) of the hippocampus. The application of BR 3, 6 and 24 h after occlusion caused a significant decrease in the number

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Fig. 1. Effect of bradykinin (BR) on brain infarction following 60 min of transient focal cerebral ischemia and 3 days of reperfusion (I). BR was administered at a dose 150 lg/ kg i.p. 3, 6, 24 or 48 h after ischemia. Panel A – representative photographs of brain sections stained with 1% TTC. Note a reduction of infarct area in the brain slices of BR treated animals. Panel B – infarct volume measurement (mm3 ± SD). Note the reduction of volume after BR treatment (n = 6 animals in each group). Panel C – neurological score was significantly decreased in the BR treated animals compared to non-treated ischemic group 3 days after stroke (n = 6 animals in each group). C – Sham-operated animals, I – 60 min MCAO + 3 days of reperfusion, I + BR (3, 6, 24, 48 h) – 60 min MCAO + 3 days of reperfusion + BR treatment at signed hours, (⁄P < 0.05).

of positive neurons in comparison to the ischemic group in the hippocampus (Fig. 3B). A massive decrease in the number of NeuN positive cells was noted in the CA1 pyramidal cell layer in the hippocampus after MCAO and 3 days of reperfusion (Fig. 3C). Rats with BR administration 3, 6 and 24 h later demonstrated a significantly increased number of NeuN positive cells in the pyramidal layer of the CA1 region after MCAO (Fig. 3D) in the hippocampus. The same number of NeuN positive cells in the pyramidal layer

of the CA1 region was achieved after MCAO and 3 days of reperfusion and after applying BR following 48 h of reperfusion in the hippocampus (Fig. 3C and D). 3.4. Effect of bradykinin on SOD and CAT activities in hippocampus The enzymatic activities of total SOD, CuZnSOD, MnSOD and CAT were measured 3 days after MCAO treated without or with

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increased by 25% in all ischemic groups with or without bradykinin treatment compared to the control. The activity of CuZnSOD was markedly increased in the ischemic group and the groups with the application of BR 24 and 48 h after occlusion in the CA1 region versus the control, but significantly lowers in the groups with the application of BR 3 and 6 h after occlusion in the CA1 region compared to the ischemic group (Fig. 4). The activity of MnSOD was markedly increased in all ischemic groups with or without BR treatment in the CA1 region compared to the control, but only the application of BR 3 and 6 h after prevented an increase in activity compared to the ischemic group. CuZnSOD and MnSOD activities remained unchanged in the DG after occlusion and the application of BR. In the case of CAT activity, a significant increase was observed in all ischemic groups with or without BR application in both regions of the hippocampus compared to the control. The application of BR 3 or 6 h significantly prevented an increase of CAT (Fig. 4). 4. Discussion

Fig. 2. Enzymatic activity of total superoxide dismutase (SOD), copper–zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD) and catalase (CAT) measured spectrophotometrically (optical density 560 nm) in the core (infarct area) and the penumbra (distant region) after 60 min of MCAO and 3 days of reperfusion without or with the application of bradykinin (BR) at a dose of 150 lg/kg i.p. 3, 6, 24 or 48 h after MCAO. Activity is expressed as international units per milligram of protein (U/mg). Data are expressed as mean ± SD of six animals in each group. C – Sham-operated animals, I – 60 min MCAO + 3 days of reperfusion, I + BR (3, 6, 24, 48 h) – 60 min MCAO + 3 days of reperfusion + BR treatment at signed hours, ⁄P < 0.05 compared to control; #P < 0.05 compared to ischemia.

the application of BR 3, 6, 24 and 48 h after stroke in the selective vulnerable CA1 region and the relative resistant dentate gyrus (DG) of hippocampus (Fig. 4). Significantly increased SOD activities were determined in CA1 region of the hippocampus compared to the control (P < 0.05). Application of BR 3, 6 and 24 h after ischemia significantly prevented an increase in SOD activity compared to the ischemic group (Fig. 4). In the DG, the activity of SOD was

Infarction volume in the brain is an important determinant when assessing the consequences of ischemic stroke, which leads to severe neuronal damage in the different brain parts with subsequent neurological impairment. The TTC method has been used to detect infarct areas after ischemic injury. In the present study, the MCAO group showed a prominent infarct area along with significantly altered behavioural outputs. BR treatment (3 and 6 h after ischemia) not only reduced the infarct area but also improved behavioural deficits in the MCAO group. Our experiment indicates that BR administered after cerebral ischemia is effective in reducing infarct volume and leads to improvements in neurological outcomes. Injured neurons were identified using Fluoro-Jade B, a recently developed fluorescent marker of neuronal degeneration. In the selective vulnerable CA1 region of the hippocampus, the number of Fluoro-Jade B positive neurons was significantly increased in the core and penumbra, but BR, as a postconditioner, used 3 or 6 h after occlusion, effectively protected pyramidal CA1 cells against damage. Histological damage, as measured by Fluoro-Jade B, reached its peak 24 h after stroke in a reperfusion model of MCAO in mice (Liu et al., 2009). To quantify the number of surviving neurons, NeuN immunohistochemistry was used. Immunoreactivity for NeuN has been reported to decrease dramatically following MCAO (Liu et al., 2009). NeuN visualisation demonstrated a massive decrease of surviving neurons in the CA1 region of the hippocampus 3 days after MCAO. However, BR treatment early after occlusion (3 or 6 h) prevented the decrease in NeuN immunoreactivity. Under physiological conditions, ROS are generated at low levels, and are controlled by endogenous antioxidants such as SOD, glutathione peroxidase, glutathione, and CAT. It is well established that cerebral ischemia can induce both necrotic and apoptotic cell death. The mechanisms of cell death after cerebral ischemia remain unclear; mitochondria play an important role by activating signalling pathways through ROS production or by regulating mitochondria-dependent apoptosis pathways. A similar pattern of post-ischemic SOD activity in the hippocampus and parietal cortex after focal ischemia was observed (Mrsic-Pelcic et al., 2012); however, a longer time of ischemia could contribute to the slight variation. Data about the reduced activity of SOD one day after MCAO in the hippocampus and frontal cortex was published in the same model (Ashafaq et al., 2012). Mechanism BR action is through activation of bradykinin-2 (BR2) receptors which can to play a role in ischemic preconditioning. In cascade of its action is couple to protein kinase C (PKC), nitric oxide production and ROS signalling (Penna et al., 2007).

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Fig. 3. Representative microphotographs of neurodegeneration visualised by Fluoro Jade B staining (right column) and NeuN immunostaining of surviving neurons (left column) in the hippocampal CA1 region following 60 min of middle cerebral artery occlusion (MCAO) and 3 days of reperfusion. Panel A – microphotographs of FJ B staining of the CA1 region of the hippocampus. Bradykinin (BR) 150 lg/kg i.p was administered 3, 6, 24 or 48 h after occlusion. Scale bar = 50 lm. Panel B – quantification of fluorescence intensity counted in the middle of the linear part of the CA1 ( 3.3 ± 0.2 mm posterior to the bregma) and expressed as the average of 10 measurements of positive hippocampal CA1 pyramidal neurons per 1 mm. Values were taken as a mean ± SD of six animals in each group; Panel C – microphotographs of NeuN immunohistochemistry of the CA1 region of the hippocampus. Bradykinin (BR) 150 lg/kg i.p was administered 3, 6, 24 or 48 h after occlusion. Scale bar = 50 lm. Panel D – quantification of the number of NeuN positive cells counted in the middle of the linear part of the CA1 ( 3.3 ± 0.2 mm posterior to the bregma) and expressed as the average of 10 measurements of positive hippocampal CA1 pyramidal neurons per 1 mm. Values were taken as a mean ± SD of six animals in each group; C – Sham-operated animals, I – 60 min MCAO + 3 days of reperfusion, I + BR (3, 6, 24, 48 h) – 60 min MCAO + 3 days of reperfusion + BR treatment at signed hours, ⁄P < 0.05 compared to ischemic group.

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(SOD) and catalase (CAT) were measured in the infarct area and penumbra, and also in two regions of the hippocampus, the selective vulnerable CA1 region and the relative resistant dentate gyrus (DG) in the ipsilateral hemispheres. BR application 3 and 6 h after the end of occlusion prevented the release of MnSOD from the mitochondria and reduced the activity of total SOD and CAT. All of these changes were more pronounced if BR was used sooner after possible occlusion. Significantly elevated levels of several proteins in the hippocampus of the MCAO rat model of brain ischemia on the first and third day after insult, including enhanced protein levels of the CuZn and Mn isoforms of the SOD enzyme, were detected (Uchida et al., 2010). The role of MnSOD in neural apoptosis prevention and reduced ischemic injury (Huang et al., 2012; Keller et al., 1998), the extremely elevated MnSOD activity in the cytoplasm of penumbra cells observed one day after insult is probably connected to progressive neurodegeneration and spread of the infarct. Cell death in the penumbra seems to be similar to delayed neuronal death in ischemia selective vulnerable neurons (Du et al., 1996), which could also be supported by a reduction in the ischemic lesion using stimuli to induce the activation of endogenous protective mechanisms, known as ischemic tolerance (Zhao, 2009; Zhao et al., 2012). From the perspective of clinical applications, the utilisation of immediate postconditioning is practically unusable. Outside of the hospital it can be impossible and in the clinic very difficult to identify the exact end of the ischemia. Moreover, there is only an extremely short therapeutic time window, a few minutes after the onset of reperfusion. Ren et al. (2008) documented that delayed postconditioning performed 3 and 6 h after stroke robustly reduced infarct size, with the strongest protection achieved by postconditioning with 6 cycles of 15 min occlusion/15 min release. However, the application of ischemic postconditioning consisting of brief occlusion/reperfusion patterns on human beings is unacceptable. Data from the experimental results show that BR is a powerful activator of an endogenous defence mechanism known as ischemic tolerance. Our results confirm that BR postconditioning, if used at the right time, can prevent the process of delayed neuronal death. Its effect depends on the severity of ischemic insult; in long-lasting transient focal ischemia, BR is surprisingly effective if it is used in the first few hours after ischemia. In the shorter (up to 10 min) transient global ischemia, BR is able to prevent/reverse delayed neuronal death up to 48 h after ischemia; this is also true for some models of intoxication (Burda et al., 2009; Danielisova et al., 2009). Moreover, delayed BR postconditioning can be effectively combined with immediate ischemic postconditioning to prevent apoptosis after global ischemia lasting 15 min (Danielisova et al., 2012). 5. Conclusion Fig. 4. Enzymatic activity of total superoxide dismutase (SOD), copper–zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD) and catalase (CAT) measured spectrophotometrically (optical density 560 nm) in the CA1 region and the dentate gyrus (DG) of hippocampus after 60 min of MCAO and 3 days of reperfusion with application of bradykinin (BR) at a dose 150 lg/kg i.p. 3, 6, 24 or 48 h after MCAO. Activity is expressed as international units per milligram of protein (U/mg). Data are expressed as mean ± SD of six animals in each group. C – Sham-operated animals, I – 60 min MCAO + 3 days of reperfusion, I + BR (3, 6, 24, 48 h) – 60 min MCAO + 3 days of reperfusion + BR treatment at signed hours, ⁄P < 0.05 compared to control; #P < 0.05 compared to ischemia.

Participation of ROS supports also evidence that antioxidants or ROS scavengers completely block the tolerance acquisition (Puisieux et al., 2004; Burda et al., 2009). Searching for mechanisms of protection, we measured the activity of endogenous antioxidant enzymes in post-mitochondrial supernatants (PMS). Activities of the antioxidant enzymes superoxide dismutase

In conclusion, this study demonstrated that BR administration, if applied before the onset of irreversible neurodegenerative changes, induced neuroprotection against ischemic injury. The ischemic tolerance in the brain induced by BR postconditioning resulted in a reduction of infarct volume, led to improvements in neurological score, promoted the survival of ischemic neurons, prevented the release of MnSOD from the mitochondria and reduced the activity of the total SOD and CAT. Acknowledgments The authors gratefully acknowledge the excellent technical assistance of Dana Jurusova. This study is the result of implementation of the project: ‘‘New possibilities of preservation of neurons in the process of delayed neuronal death by nonspecific stressors’’

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supported by the European Research & Development Operational Programme Funded by the ERDF (ITMS 26220220043) and by Grants from the Slovak Scientific Grant Agency (VEGA Grants 2/ 0066/12, 2/0092/12).

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