Effects of low and high deprenyl dose on antioxidant enzyme activities in the adult rat brain.

We evaluated the effects of low dose deprenyl (LDD, 0.0025 mg/kg per day) and high dose deprenyl (HDD, 0.25 mg/kg per day) treatment of male Wistar rats for 30 days on the activities of SOD and CAT in the cortex, striatum, and hippocampus. Total SOD and MnSOD activities were increased with LDD (p <0.05) in the cortex (0.74 ± 0.03; 0.31 ± 0.02), striatum (0.75 ± 0.02; 0.27 ± 0.03) and CA1 region of the hippocampus (0.75 ± 0.02; 0.29 ± 0.03) compared to the control (0.53 ± 0.02; 0.15 ± 0.02), but reduced (p <0.05) with HDD compared to the LDD group. CAT activity was increased (p <0.05) with LDD in the cortex (27.34 ± 3.11), striatum (22.22 ± 1.85), and hippocampal CA1 region (16.62 ± 2.15) compared to control (10.33 ± 1.01), while a decrease was induced by HDD in the striatum (9.85 ± 1.09) compared to LDD. There was a significant (p <0.05) difference in number of Fluoro Jade B positive CA1 neurons induced by LDD (21.14 ± 2.85%) and HDD (12.61 ± 1.42%), as well as the number of NeuN positive CA1 neurons after LDD (183.35 ± 11.14 cells/mm) and HDD (238.45 ± 14.11 cells/mm (p < 0.05). Deprenyl showed a potential in improving the neurological outcome and reducing the oxidative damage.

Neuroprotection and antioxidants.

Ischemia as a serious neurodegenerative disorder causes together with reperfusion injury many changes in nervous tissue. Most of the neuronal damage is caused by complex of biochemical reactions and substantial processes, such as protein agregation, reactions of free radicals, insufficient blood supply, glutamate excitotoxicity, and oxidative stress. The result of these processes can be apoptotic or necrotic cell death and it can lead to an irreversible damage. Therefore, neuroprotection and prevention of the neurodegeneration are highly important topics to study. There are several approaches to prevent the ischemic damage. Use of many modern therapeutical methods and the incorporation of several substances into the diet of patients is possible to stimulate the endogenous protective mechanisms and improve the life quality.

Delayed bradykinin postconditioning modulates intrinsic neuroprotective enzyme expression in the rat CA1 region after cerebral ischemia: a proteomic study.

Pyramidal cells in the CA1 brain region exhibit an ischemic tolerance after delayed postconditioning; therefore, this approach seems to be a promising neuroprotective procedure in cerebral postischemic injury improvement. However, little is known about the effect of postconditioning on protein expression patterns in the brain, especially in the affected hippocampal neurons after global cerebral ischemia. This study is focused on the examination of the ischemia-vulnerable CA1 neuronal layer and on the acquisition of protection from delayed neuronal death after ischemia. Ischemic-reperfusion injury was induced in Wistar rats and bradykinin was applied 2 days after the ischemic insult in an attempt to overcome delayed cell death. Analysis of complex peptide CA1 samples was performed by automated two dimensional liquid chromatography (2D-LC) fractionation coupled to tandem matrix assisted laser desorption/ionization time-of-flight (MALDI TOF/TOF) mass spectrometry instrumentation. We devoted our attention to differences in protein expression mapping in ischemic injured CA1 neurons in comparison with equally affected neurons, but with bradykinin application. Proteomic analysis identified several proteins occurring only after postconditioning and control, which could have a potentially neuroprotective influence on ischemic injured neurons. Among them, the prominent position occupies a regulator of glutamate level aspartate transaminase AATC, a scavenger of glutamate in brain neuroprotection after ischemia-reperfusion. We identified this enzyme in controls and after postconditioning, but AATC presence was not detected in the ischemic injured CA1 region. This finding was confirmed by two-dimensional differential electrophoresis followed by MALDI-TOF/TOF MS identification. Results suggest that bradykinin as delayed postconditioning may be associated with modulation of protein expression after ischemic injury and thus this procedure can be involved in neuroprotective metabolic pathways.

The effect of R-(-)-deprenyl administration on antioxidant enzymes in rat testis.

The aim of the study was to investigate the effect of R-(-)-deprenyl administration on the activity and localization of superoxide dismutases (SODs) and catalase (CAT) in rat testis. After 30 days of intraperitoneal administration of either saline (control) or R-(-)-deprenyl dissolved in saline at concentrations of 0.0025mg/kg (low dose of deprenyl, LDD) or 0.25mg/kg (high dose of deprenyl, HDD), males were killed by thiopental, and their testes were collected. We found that deprenyl administration significantly increased the activity of antioxidant enzymes, and this effect varied by dosage. LDD caused significant elevation of all monitored enzymes, but HDD did not increase the activity of SOD2. Employing immunohistochemistry, we detected enzymes predominantly in Leydig cells (SOD1, SOD2, CAT), in late spermatids and residual bodies (SOD1, SOD2), and in primary spermatocytes (SOD2). Histopathological examination did not reveal testicular damage in experimental groups compared to control. Deprenyl proved to be a potent stimulator of antioxidant enzymes in rat testes; therefore, it could be used in the therapy of male infertility. On the other hand, it is crucial to choose a proper dose, since lower dose was more competent compared to a dosage that was one hundred times higher.

Postconditioning Effectively Prevents Trimethyltin Induced Neuronal Damage in the Rat Brain.

Trimethyltin (TMT) is a toxic substance formerly used as a catalyst in the production of organic substances, as well as in industry and agriculture. TMT poisoning has caused death or severe injury in many dozens of people. The toxicity of TMT is mediated by dose dependent selective damage to the limbic system in humans and other animals, specifically the degeneration of CA1 neurons in the hippocampus. The typical symptoms include memory loss and decreased learning ability. Using knowledge gained in previous studies of global ischaemia, we used delayed postconditioning after TMT intoxication (8 mg/kg i.p.), consisting of applying a stressor (BR, bradykinin 150 µg/kg i.p.) 24 or 48 hours after the injection of TMT. We found that BR had preventive effects on neurodegenerative changes as well as learning and memory deficits induced by TMT intoxication.

Effect of Bradykinin Postconditioning on Ischemic and Toxic Brain Damage.

Brain damage caused by ischemia or toxic agents leads in selectively vulnerable regions to apoptosis-like delayed neuronal death and can result in irreversible damage. Selectively vulnerable neurons of the CA1 area of hippocampus are particularly sensitive to ischemic damage. We investigated the effects of bradykinin (BR) postconditioning on cerebral ischemic and toxic injury. Transient forebrain ischemia was induced by four-vessel occlusion for 10 min and toxic injury was induced by trimethyltin (TMT, 8 µg/kg i.p.). BR as a postconditioner at a dose of 150 µg/kg was applied intraperitoneally 48 h after ischemia or TMT intoxication. Experimental animals were divided into groups according to the length of survival (short-3 and 7 days, and long-28 days survival) and according to the applied ischemic or toxic injury. Glutamate concentration was lowered in both CA1 and dentate gyrus areas of hippocampus after the application of BR postconditioning in both ischemic and toxic brain damage. The number of degenerated neurons in the hippocampal CA1 region was significantly lower in BR-treated ischemic and toxic groups compared to vehicle group. The behavioral test used in our experiments confirms also the memory improvement in conditioned animals. The rats' ability to form spatial maps and learn was preserved, which is visible from our Barnes maze results. By using the methods of delayed postconditioning is possible to stimulate the endogenous protective mechanisms of the organism and induce the neuroprotective effect. In this study we demonstrated that BR postconditioning, if applied before the onset of irreversible neurodegenerative changes, induced neuroprotection against ischemic or toxic injury.

Scheme of Ischaemia-triggered Agents during Brain Infarct Evolution in a Rat Model of Permanent Focal Ischaemia.

The impact of therapeutic intervention in stroke depends on its appropriate timing during infarct evolution. We have studied markers of brain tissue damage initiated by permanent occlusion of the middle cerebral artery (MCAO) at three time points during which the infarct spread (1, 3 and 6 h). Based on Evans Blue extravasation and immunohistochemical detection of neurons, we confirmed continuous disruption of blood-brain barrier and loss of neurons in the ischaemic hemisphere that peaked at the sixth hour, especially in the core. Glutamate content started to rise dramatically in the entire hemisphere during the first 3 h; the highest level was determined in the core 6 h after MCAO (141 % increase). Moreover, the enzyme antioxidant defence grew by about 42 % since the first hour in the ipsilateral penumbra. Enzymes of the apoptotic pathway as well as mitochondrial enzyme release were detected since the third hour of MCAO in the ischaemic hemisphere; all achieved their maxima in the penumbra during both time periods (except cytochrome C). In conclusion, the preserved integrity of mitochondrial membrane and incompletely developed process of apoptosis may contribute to the better therapeutic outcome after ischaemic attack; however, a whole brain response should not be omitted.

Ionizing radiation as preconditioning against transient cerebral ischemia in rats.

Induction of ischemic tolerance (IT), the ability of an organism to survive an otherwise lethal ischemia, is the most effective known approach to preventing postischemic damage. IT can be induced by exposing animals to a broad range of stimuli. In this study we tried to induce IT of brain neurons using ionizing radiation (IR). A preconditioning (pre-C) dose of 10, 20, 30 or 50 Gy of gamma rays was used 2 days before an 8 min ischemia in adult male rats. Ischemia alone caused the degeneration of almost one half of neurons in CA1 region of hippocampus. However, a significant decrease of the number of degenerating neurons was observed after higher doses of radiation (30 and 50 Gy). Moreover, ischemia significantly impaired the spatial memory of rats as tested in Morris's water maze. In rats with a 50 Gy pre-C dose, the latency times were reduced to values close to the control level. Our study is the first to reveal that IR applied in sufficient doses can induce IT and thus allow pyramidal CA1 neurons to survive ischemia. In addition, we show that the beneficial effect of IR pre-C is proportional to the radiation dose.

Delayed remote ischemic postconditioning protects against transient cerebral ischemia/reperfusion as well as kainate-induced injury in rats.

To test the appropriateness of using delayed remote ischemic postconditioning against damage caused to the hippocampus by ischemia or apoptosis inducing intoxication, we chose 10-min normothermic ischemia induced by four-vessel occlusion or kainate injection (8 mg/kg i.p.) in rats. Ischemia alone caused the number of degenerated CA1 neurons after 7 days lasting reperfusion to be significantly (p<0.001) increased by 72.77%. Delayed remote ischemic postconditioning lasting 20 min was able to prevent massive increase in the neurodegeneration. The group with 10 min of ischemia and postconditioning after 2 days of reperfusion had only 15.87% increase in the number of apoptotic neurons. Seven days after kainic acid injection the number of surviving neurons was 42.8% (p<0.001), but the portion of surviving pyramidal cells in the postconditioning group is more than 98%. Our data show that remote postconditioning, performed with 20 min of tourniquet ischemia applied to the hind limb, is a simple method able to effectively stop the onset of neurodegeneration and prevent occurrence of massive muscle cell necrosis, even when used 2 days after the end of the adverse event. Surviving neurons retained a substantial part of their learning and memory ability.

Bradykinin postconditioning ameliorates focal cerebral ischemia in the rat.

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 60min of middle cerebral artery occlusion (MCAO), followed by 3days of reperfusion. BR as a postconditioner at a dose of 150µg/kg was applied intraperitoneally 3, 6, 24 and 48h after MCAO. BR postconditioning significantly reduced total infarct volumes if applied 3h after MCAO by 95%, 6h after MCAO by 80% and 24h after MCAO by 70% in versus vehicle group. Neurological functions were amarked 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 60min MCAO even when used many hours after ischemia.

Bradykinin preconditioning affects the number of degenerated neurons and the level of antioxidant enzymes in spinal cord ischemia in rabbits.

Bradykinin preconditioning has been used for acquisition of tolerance after spinal cord ischemia. Rabbits were preconditioned intraperitoneally with bradykinin 48 h prior to 20 min of abdominal aorta ligation followed by 24 and 48 h of reperfusion. The activities of SOD and catalase were measured and Fluoro Jade B (FJB)-positive degenerated neurons were evaluated. The outcomes of Tarlov scoring system used to assess neurological functions showed significant improvement in bradykinin groups compared to the ischemic group. The number of FJB-positive degenerated neurons was decreased in ventral horns of both bradykinin groups. Significantly decreased activities of total SOD and mitochondrial Mn-SOD were also detected in both bradykinin groups versus ischemic group while CuZn-SOD and catalase activities were significantly decreased only in the bradykinin group after 24h of reperfusion versus ischemic group. These findings suggest that one of the possibilities of the neuroprotective effect of delayed bradykinin preconditioning against spinal cord ischemic injury could be realized by mitochondrial protection and decreased synthesis of Mn-SOD as well as by promotion of neuronal survival.

Delayed post-conditioning reduces post-ischemic glutamate level and improves protein synthesis in brain.

In the clinic delayed post-conditioning would represent an attractive strategy for the survival of vulnerable neurons after an ischemic event. In this paper we studied the impact of ischemia and delayed post-conditioning on blood and brain tissue concentrations of glutamate and protein synthesis. We designed two groups of animals for analysis of brain tissues and blood after global ischemia and post-conditioning, and one for analysis of blood glutamate after transient focal ischemia. Our results showed elevated blood glutamate in two models of transient brain ischemia and decreases in blood glutamate to control in the first 20min of post-conditioning recirculation followed by a consecutive drop of about 20.5% on the first day. Similarly, we recorded reduced protein synthesis in hippocampus and cortex 2 and 3days after ischemia. However, increased glutamate was registered only in the hippocampus. Post-conditioning improves protein synthesis in CA1 and dentate gyrus and, surprisingly, leads to 50% reduction in glutamate in whole hippocampus and cortex. In conclusion, ischemia leads to meaningful elevation of blood and tissue glutamate. Post-conditioning activates 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. Moreover, post-conditioning induces protein synthesis renewing in ischemia affected tissues that could also contribute to elimination of excitotoxicity. In addition, the potential of glutamate for monitoring the progress of ischemia and efficacy of therapy was shown.

Brain-derived neurotrophic factor blood levels in two models of transient brain ischemia in rats.

We monitored possible influence of transient focal and global brain ischemia on BDNF blood level. In both models noticeable fluctuation of BDNF concentration mainly in reperfusion was observed. During the first 90 min, BDNF in total blood and in blood cells continuously decreased in both models but plasma BDNF raised at 40 min and peaked at 90 min of reperfusion. Our data confirm the impact of transient brain ischemia on BDNF levels in the circulatory system, suggest blood cells as a possible source of BDNF and demonstrate the interdependence of blood compartments and physiological state of an affected organism.

Development of a pattern in biochemical parameters in the core and penumbra during infarct evolution after transient MCAO in rats.

The period from stroke initiation to the cessation of penumbra damage spread represents a therapeutic window when expansion can be alleviated. In the present work, we studied some biochemical parameters helpful for the estimation of infarct progression and thus for the application of interventions. We designed four groups: the control group and three groups of animals after middle cerebral artery occlusion with reperfusion periods of 2h, 1day or 3days. In the ischaemic core and penumbra, fluorimetric and spectrophotometric methods for investigating total MnSOD and MAO-A/B activity as well as level of the glutamate were used. Protein synthesis was assessed by in vitro measurements of (14)C-leucine incorporation. Noticeable differences between core and penumbra biochemical parameters were shown. In the core, protein synthesis was transiently inhibited two hours and three days after ischaemia (36%). Glutamate and total SOD activity peaked on the first day, but on the third day after MCAO, rapidly decreased by about 44% and 33.6%, respectively. In the penumbra, ischaemia led to higher protein synthesis (78%), elevations in glutamate and rapid activation of MnSOD (by about 884%) one day after insult. On the third day, protein synthesis and MnSOD were still significantly elevated (36% and 388%, respectively), while glutamate levels returned to baseline. In addition, the impact of ischaemia on MAO-A/B activity in the penumbra was confirmed. In conclusion, biochemical parameter screening could be helpful to assess cell damage progress and the possibility of rescue. These regions reflect different biochemical patterns that seem to be clearly established on the first day after transient MCAO. Moreover, the first day of post-ischaemic reperfusion in the present model of stroke seems to be the breakpoint, i.e. the time at which expanding cell death from the infarct core to the penumbra can be at least partially eliminated.

An effective combination of two different methods of postconditioning.

Ischemic tolerance based on the synthesis of protective proteins acquires its full strength by repeated exposure to stress, and "the end effector of tolerance" may paradoxically be activated by the second or lethal stress, particularly in the case of preconditioning. That happens when an additional nonspecific stressor is applied either before (preconditioning) or after (postconditioning) the period of lethal ischemia. A combination of antioxidants with pre or postconditioning prevents the acquisition of tolerance, and in the case of more severe attacks repeated stress can lead to accumulation of damage. Our attempt to weaken ischemic injury to hippocampal CA1 with antioxidants applied after lethal stress, i.e. before delayed postconditioning, was ineffective. We then tried using rapid postconditioning consisting of 30-s reperfusion alternating with 15-s ischemia repeated three times and applied immediately at the end of lethal ischemia as a tool decreasing post-ischemic production of reactive oxygen species, and combining that with delayed postconditioning consisting of an i.p. injection of Bradykinin 2 days after lethal ischemia. This approach once more confirmed the efficacy of both rapid as well as delayed postconditioning but, more importantly, it demonstrated the possibility of effectively combining these two procedures. Our findings further confirm that in cases of delayed neuronal death, which is practically pathologically-induced apoptosis, there exists a 2-day-wide therapeutic window that can be effectively exploited.

Aminoguanidine administration ameliorates hippocampal damage after middle cerebral artery occlusion in rat.

The effects of a selective inducible nitric oxide synthase inhibitor aminoguanidine (AG) on neuronal cells survival in hippocampal CA1 region after middle cerebral artery occlusion (MCAO) were examined. Transient focal cerebral ischemia was induced in rats by 60 or 90 min of MCAO, followed by 7 days of reperfusion. AG treatment (150 mg/kg i.p.) significantly reduced total infarct volumes: by 70% after 90 min MCAO and by 95% after 60 min MCAO, compared with saline-treated ischemic group. The number of degenerating neurons in hippocampal CA1 region was also markedly lower in aminoguanidine-treated ischemic groups compared to ischemic groups without AG-treatment. The number of iNOS-positive cells significantly increased in the hippocampal CA1 region of ischemic animals, whereas it was reduced in AG-treated rats. Our findings demonstrate that aminoguanidine decreases ischemic brain damage and improves neurological recovery after transient focal ischemia induced by MCAO.

Effects of one-day reperfusion after transient forebrain ischemia on circulatory system in the rat.

Although ischemia/reperfusion injury remains incompletely understood, it appears that reactive oxygen species produced mainly during postischemic recirculation play a critical role. The present study examined the impact of forebrain ischemia and subsequent one-day reperfusion on several blood parameters. We determined glutamate concentration in whole blood, measured Cu/Zn- and Mn-SOD (superoxide dismutase) activity in blood cells as well as plasma, and investigated the prevalence of single and double strand breaks of lymphocyte DNA. The results of our experiment showed that the concentration of glutamic acid in whole blood was increased by about 25%. Antioxidant activity of total SOD and Cu/Zn-SOD was reduced in blood cells and plasma. Mn-SOD activity in blood cells was not affected by ischemic insult and one-day reperfusion, but we detected its significantly lower activity in samples of plasma. We observed a weakly reduced level of double and a significantly elevated level of single strand breaks of lymphocyte DNA. In conclusion, one day of recovery after the ischemic attack failed to return peripheral circulatory system to physiological conditions. Reduced antioxidant capacity in the blood and an elevated level of excitotoxic amino acid glutamate may cause lymphocyte DNA damage, and probably contribute to insufficient postischemic recovery of brain tissue.

Effect of L-carnitine on postischemic inhibition of protein synthesis in the rat brain.

The purpose of this study was to investigate effects of carnitine administration on protein synthesis recovery after transient cerebral ischemia. Rats received L-carnitine in two doses of 16 mmol/kg i.p. 15 min before ischemia and just on the onset of reperfusion. Transient forebrain ischemia was induced by 4-vessel occlusion for 15 min, followed by 30 min or 7 days of reperfusion. Protein synthesis rate, reinitiation ability and neurodegeneration in the frontal cortex and hippocampus were measured by the incorporation of radioactively labelled leucine into polypeptide chains in postmitochondrial supernatants and by Fluoro-Jade B staining. A protective effect was observed, on protein synthesis as well as the number of surviving neurons, in the L-carnitine-treated groups. Our results indicate that L-carnitine can exert a protective effect in the development of reperfusion-induced injury. L-carnitine significantly reduced the ischemia/reperfusion-induced inhibition of translation and neurodegeneration in the neocortex as well as in the highly sensitive hippocampus and dorsolateral striatum. We expect that the ability of L-carnitine to keep translational machinery on facilitates efficacy of postischemic remodulation of gene expression.

Ischemic tolerance: the mechanisms of neuroprotective strategy.

The phenomenon of ischemic tolerance perfectly describes this quote "What does not kill you makes you stronger." Ischemic pre- or postconditioning is actually the strongest known procedure to prevent or reverse neurodegeneration. It works specifically in sensitive vulnerable neuronal populations, which are represented by pyramidal neurons in the hippocampal CA1 region. However, tolerance is effective in other brain cell populations as well. Although, its nomenclature is "ischemic" tolerance, the tolerant phenotype can also be induced by other stimuli that lead to delayed neuronal death (intoxication). Moreover, the recent data have proven that this phenomenon is not limited to application of sublethal stimuli before the lethal stress but reversed arrangement of events, sublethal stress after lethal insult, is rather equally effective. A very important term is called "cross conditioning." Cross conditioning is the capability of one stressor to induce tolerance against another. So, since pre- or post-conditioners can be used plenty of harmful stimuli, hypo- or hyperthermia and some physiological compounds, such as norepinephrine, bradykinin. Delayed neuronal death is the slow development of postischemic neurodegeneration. This allows an opportunity for a great therapeutic window of 2-3 days to reverse the cellular death process. Moreover, it seems that the mechanisms of ischemic tolerance-delayed postconditioning could be used not only after ischemia but also in some other processes leading to apoptosis.

Transient forebrain ischemia impact on lymphocyte DNA damage, glutamic acid level, and SOD activity in blood.

AIMS: Brain ischemia-reperfusion injury remains incompletely understood but appears to involve a complex series of interrelated biochemical pathways caused mainly by a burst of reactive oxygen species (ROS). In the present work we studied the impact of postischemic condition in the early phase of reperfusion on plasma and blood cells. METHODS: Transient forebrain ischemia was induced in Wistar rats by four-vessel occlusion model. Blood samples collected during postischemic reperfusion 20, 40, 60, 90, and 120 min after ischemia were used for assessing breaks of lymphocyte DNA, fluorimetric measurement of whole blood glutamate concentration, and spectrophotometrical determination of SOD activity in plasma and blood cells. RESULTS: Our results showed the most interesting changes of all observed parameters mainly at 40 and 120 min of reperfusion, when we observed peak DNA damage of lymphocytes and highest glutamate level and total and Cu/Zn SOD activity. At those time points, Mn SOD activity was low in plasma, as well as in blood cells. On the contrary, at 60 and 90 min, all studied parameters were approximately at the level of control. CONCLUSION: Ischemia/reperfusion injury has influence on blood cells and has at least two waves of impact on DNA damage of peripheral lymphocytes, affects activity of major antioxidant enzymes SODs, as well as blood glutamic acid level. Elevation of Mn SOD activity probably plays an important role in the processes of elimination of postischemic damage in blood cells.