Mechanisms Involved in the Neuroprotective Activity of a Nitric Oxide Synthase Inhibitor During Focal Cerebral Ischemia

Abstract: We have reported previously that posttreatment with N ^G-nitro-L-arginine methyl ester (L-NAME), an inhibitor of the nitric oxide synthase, reduced the volume of cortical and striatal infarct induced by middle cerebral artery occlusion in rats. In the present study, we investigated the mechanisms by which L-NAME (3 mg/kg i.p.) is neuroprotective in this model of cerebral ischemia. First, we have shown the reversal of the neuroprotective effect of L-NAME by a coinjection of L-arginine. Second, in order to determine by which mechanism nitric oxide exacerbates neuronal damage produced by focal cerebral ischemia, we studied the effect of the inhibition of nitric oxide synthase by L-NAME on the histological consequences of a focal injection of N -methyl-D-aspartate (NMDA) in the striatum, and on the striatal overflow of glutamate and aspartate induced either by K⁺ depolarization or by focal cerebral ischemia. We have found that L-NAME treatment reduced the excitotoxic damage produced by NMDA injection. By using microdialysis, we have shown that the K⁺- and the ischemia-induced glutamate efflux was reduced by 52 and 30%, respectively, after the L-NAME treatment. These results indicate that nitric oxide synthesis induced by the NMDA receptor overstimulation is one of the major events leading to neuronal damage. One possible mechanism by which nitric oxide may contribute to the excitotoxic process is by facilitating the ischemia-induced glutamate overflow.     

Serotonin 5-HT1A receptor activation prevents phosphorylation of NMDA receptor NR1 subunit in cerebral ischemia

The mechanisms involved in the neuroprotective effect of serotonin 5-HT1A receptor agonists on brain damage induced by ischemia remain to be fully elucidated. Given that serotonergic drugs may regulate N-methyl-D-aspartate (NMDA) receptor function, which is implicated in events leading to ischemia-induced neuronal cell death, this study sought to determine the effects of the selective 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), on the levels of NMDA receptor NR1 subunit in gerbil hippocampus after transient global cerebral ischemia. Pretreatment with 8-OH-DPAT (1 mg/kg) prevented the neuronal loss in CA1 subfield 72 h after ischemia. NMDA receptor NR1 levels in whole hippocampus were not affected 24 h after ischemia, but the levels of the subunit phosphorylated at the protein kinase A (PKA) site, pNR1(Ser897), were significantly increased, and this increase was prevented by the same 8-OH-DPAT dose, a probable consequence of the increased phosphatase 1 (PP1) enzyme activity found in ischemic gerbils pretreated with the 5-HT1A receptor agonist. The results suggest that NR1 subunit phosphorylation plays a role in the neuroprotective effect of 8-OH-DPAT on cell damage induced by global cerebral ischemia in the gerbil hippocampus and support the potential interest of 5-HT1A receptor activation in the search for neuroprotective strategies.     

NMDA and Non-NMDA Receptor Gene Expression Following Global Brain Ischemia in Rats: Effect of NMDA and Non-NMDA Receptor Antagonists

Abstract: Transient forebrain or global ischemia in rats induces selective and delayed damage of hippocampal CA1 neurons. In a previous sludy, we have shown that expression of GIuR2, the kainate/a-amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid (AMPA) receptor subunit that governs Ca' permeability, is preferentially reduced in CA1 at a time point proceeding neuronal degeneration. Postischemic administration of the selective AMPA receptor antagonist, 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX), protects CAI neurons against delayed death. In this study we examined the effects of NBQX (at a neuroprotective dose) and of MK-801 (a selective NMDA receptor anltagonist, not protective in this model) on kainate/AMPA receptor gene expression changes after global ischemia. We also examined the effects of transient forebrain ischemia on expression of the NMDA receptor subunit NMDARI. In ischemic rats treated with saline, GIuR2 and (31uR3 mRNAs were markedly reduced in CAI but were unchanged in CA3 or dentate gyrus. GluRl and NMDAR1 mRNAs were not significantly changed in any region examined. Administration of NBQX or MK-801 did not alter the ischemia-induced changes in kainate/AMPA receptor gene expression. These findings suggest that NBQX affords neuroprotection by a direct blockade of kainate/AMPA receptors, rather than by a modificatian of GIuR2 expression changes     

Regional differences in the neuroprotective effect of minocycline in a mouse model of global forebrain ischemia

We investigated the effect of minocycline on neuronal damage in the hippocampus and striatum in a mouse model of transient global forebrain ischemia. Male C57BL/6 mice were anesthetized with halothane and subjected to bilateral occlusion of the common carotid artery (BCCAO) for 30 min. Minocycline (90 mg/kg, i.p., qd) or saline was injected immediately after BCCAO and daily for the next two days (45 mg/kg, i.p., bid). In order to reduce the variability in ischemic neuronal damage, we applied selection criteria based on regional cerebral blood flow (rCBF), evaluated using laser Doppler flowmetry, and the plasticity of the posterior communicating artery (PcomA), evaluated using India ink solution. In animals with rCBF that was less than 15% of the baseline value and with a smaller PcomA, of diameter less than one-third that of the basilar artery, we consistently observed neuronal damage in the striatum and hippocampal subfields, including medial CA1, CA2, and CA4. When the effect of minocycline was assessed with cresyl violet staining, neuronal damage in the medial part of the CA1 subfield and the striatum was found to be significantly attenuated, although minocycline did not protect against neuronal damage in the remaining hippocampal subfields. Immunohistochemistry for NeuN, adenosine A1 receptor, and SCIP/Oct-6 confirmed the region-specific effect of minocycline in the hippocampus. In summary, our results suggest that minocycline protects neurons against global forebrain ischemia in a subregion-specific manner.     

In vivo neuroprotective role of NMDA receptors against kainate-induced excitotoxicity in murine hippocampal pyramidal neurons

Activation of NMDA receptors has been shown to induce either neuronal cell death or neuroprotection against excitotoxicity in cultured cerebellar granule neurons in vitro. We have investigated the effects of pretreatment with NMDA on kainate-induced neuronal cell death in mouse hippocampus in vivo. The systemic administration of kainate (30 mg/kg), but not NMDA (100 mg/kg), induced severe damage in pyramidal neurons of the hippocampal CA1 and CA3 subfields 3-7 days later, without affecting granule neurons in the dentate gyrus. An immunohistochemical study using an anti-single-stranded DNA antibody and TdT-mediated dUTP nick end labeling analysis both revealed that kainate, but not NMDA, induced DNA fragmentation in the CA1 and CA3 pyramidal neurons 1-3 days after administration. Kainate-induced neuronal loss was completely prevented by the systemic administration of NMDA (100 mg/kg) 1 h to 1 day previously. No pyramidal neuron was seen with fragmented DNA in the hippocampus of animals injected with kainate 1 day after NMDA treatment. The neuroprotection mediated by NMDA was prevented by the non-competitive NMDA receptor antagonist MK-801. Taken together these results indicate that in vivo activation of NMDA receptors is capable of protecting against kainate-induced neuronal damage through blockade of DNA fragmentation in murine hippocampus.     

Valproic acid reduces brain damage induced by transient focal cerebral ischemia in rats: potential roles of histone deacetylase inhibition and heat shock protein induction

Growing evidence from in vitro studies supports that valproic acid (VPA), an anti-convulsant and mood-stabilizing drug, has neuroprotective effects. The present study investigated whether VPA reduces brain damage and improves functional outcome in a transient focal cerebral ischemia model of rats. Subcutaneous injection of VPA (300 mg/kg) immediately after ischemia followed by repeated injections every 12 h, was found to markedly decrease infarct size and reduce ischemia-induced neurological deficit scores measured at 24 and 48 h after ischemic onset. VPA treatment also suppressed ischemia-induced neuronal caspase-3 activation in the cerebral cortex. VPA treatments resulted in a time-dependent increase in acetylated histone H3 levels in the cortex and striatum of both ipsilateral and contralateral brain hemispheres of middle cerebral artery occlusion (MCAO) rats, as well as in these brain areas of normal, non-surgical rats, supporting the in vitro finding that VPA is a histone deacetylase (HDAC) inhibitor. Similarly, heat shock protein 70 (HSP70) levels were time-dependently up-regulated by VPA in the cortex and striatum of both ipsilateral and contralateral sides of MCAO rats and in these brain areas of normal rats. Altogether, our results demonstrate that VPA is neuroprotective in the cerebral ischemia model and suggest that the protection mechanisms may involve HDAC inhibition and HSP induction.     

MK-801 effect on regional cerebral oxidative stress rate induced by different duration of global ischemia in gerbils

We investigated MK-801 effect on ischemia-induced oxidative stress—the most important factor that exacerbates brain damage by reperfusion. The common carotid arteries of gerbils were occluded for 5, 10, or 15 min. Immediately after the occlusion, MK-801 (3 mg/kg i.p.) or saline were given in normothermic conditions. The MK-801 effects were followed in vivo by monitoring the neurological status of animals and at the intracellular level by standard biochemical assays. We investigated nitric oxide levels, superoxide production, superoxide dismutase activity, index of lipid peroxidation (ILP), and reduced glutathione content in hippocampus, striatum, forebrain cortex, and cerebellum. The measurements took place at different times (1, 2, 4, 7, 14, and 28 days) after reperfusion. Increased duration of cerebral ischemia resulted in a progressive induction of oxidative stress. Our results revealed pattern of dynamic changes in each oxidative stress parameter level which corresponded with ischemia duration in all tested brain structures. Most sensitive oxidative stress parameters were ILP and superoxide production. Our study confirmed spatial distribution of ischemia-induced oxidative stress. Tested brain structures showed different sensitivity to each oxidative stress parameter. As judged by biochemical and neurological data, applied MK-801 showed neuroprotective efficiency by reduction of ischemia-induced oxidative stress in brain.     

The Free Radical Scavenger, α-Lipoic Acid, Protects Against Cerebral Ischemia-Reperfusion Injury in Gerbils

-Lipoic acid (thioctic acid) was tested for its neuroprotective activity in a Mongolian gerbil model of forebrain ischemia/reperfusion. Adult gerbils were treated for 7 days with two intraperitoneal injections per day of -lipoic acid (20 mg/kg), vehicle or saline and on the 7th day the animals were subjected to 5 min of forebrain ischemia. Ischemic injury was assessed by monitoring the increases in locomotor activity and from the extent of damage to the CA1 hippocampal pyramidal cell layer after 5 days of recovery. By both criteria, -lipoic acid was neuroprotective against ischemia/reperfusion evoked cerebral injury.     

Thyroxine attenuates hippocampal neuronal damage caused by ischemia in the rat.

We investigated the effect of thyroxine against neuronal damage caused by ischemia in the rat. Neuronal damage was evaluated in the hippocampal CA1 subfield 7 days after a 10 min forebrain ischemia. Thyroxine was administered to animals divided in three groups: 15 min prior to ischemia (group 1), immediately after ischemia (group 2), and both before and after ischemia (group 3). The treatment of rats with a single dose of thyroxine given pre- or postischemia failed to prevent the loss of CA1 pyramidal cells. In contrast, repetitive administration of thyroxine before and after ischemia reduced the damage of the CA1 pyramidal cells. The mechanisms possibly underlying this neuroprotective effect are discussed.     

紫檀芪对小鼠缺血性脑损伤后脑水肿期神经细胞凋亡的影响

摘要 目的：探索紫檀芪（PTE）对小鼠缺血性脑损伤后脑水肿期神经细胞凋亡的影响。方法：将实验小鼠分为3组即假手术组（sham组）、脑缺血再灌注损伤组（IR组）和紫檀芪治疗组（PTE+IR组）,其中PTE于造模前连续5天每天腹腔给药（5 mg/kg）1次;然后于造模后3 d进行脑组织TTC染色并计算脑梗死体积比;于造模后2 h、12 h和1、2、4、6、8、10、12及14 d进行小鼠神经行为学评分;使用TUNEL试剂盒于造模后3、7和14 d检测缺血半暗带和海马的凋亡神经细胞。结果：PTE可减轻脑梗死体积、改善神经行为学评分以及抑制缺血半暗带和海马的神经细胞凋亡。结论：PTE在小鼠缺血性脑损伤后脑水肿期具有明确的神经保护作用,其机制与抑制细胞凋亡有关。     

5-HMF attenuates striatum oxidative damage via Nrf2/ARE signaling pathway following transient global cerebral ischemia

Recent studies have shown 5-hydroxymethyl-2-furfural (5-HMF) has favorable biological effects, and its neuroprotection in a variety of neurological diseases has been noted. Our previous study showed that treatment of 5-HMF led to protection against permanent global cerebral ischemia. However, the underlying mechanisms in cerebral ischemic injury are not fully understood. This study was conducted to investigate the neuroprotective effect of 5-HMF and elucidate the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway mechanism in the striatum after transient global cerebral ischemia. C57BL/6 mice were subjected to bilateral common carotid artery occlusion for 20 min and sacrificed 24 h after reperfusion. 5-HMF (12 mg/kg) or an equal volume of vehicle was intraperitoneally injected 30 min before ischemia and 5 min after the onset of reperfusion. At 24 h after reperfusion, neurological function was evaluated by neurological disability status scale, locomotor activity test and inclined beam walking test. Histological injury of the striatum was observed by cresyl violet staining and terminal deoxynucleotidyl transferase (TdT)-mediated dNTP nick end labeling (TUNEL) staining. Oxidative stress was evaluated by the carbonyl groups introduced into proteins, and malondialdehyde (MDA) levels. An enzyme-linked immunosorbent assay (ELISA)-based measurement was used to detect Nrf2 DNA binding activity. Nrf2 and its downstream ARE pathway protein expression such as heme oxygenase-1, NAD (P)H:quinone oxidoreductase 1, glutamate-cysteine ligase catalytic subunit and glutamate-cysteine ligase modulatory subunit were detected by western blot. Our results showed that 5-HMF treatment significantly ameliorated neurological deficits, reduced brain water content, attenuated striatum neuronal damage, decreased the carbonyl groups and MDA levels, and activated Nrf2/ARE signaling pathway. Taken together, these results demonstrated that 5-HMF exerted significant antioxidant and neuroprotective effects following transient cerebral ischemia, possibly through the activation of the Nrf2/ARE signaling pathway.     

Neuroprotective effects of VEGF administration after focal cerebral ischemia/reperfusion: dose response and time window

Administration of vascular endothelial growth factor (VEGF) has been shown to increase cerebral blood flow and reduce neurological damage after experimental ischemic brain injury. The purpose of this study was to examine the optimal dose and time window for the neuroprotective effect of VEGF when administrated after focal ischemia/reperfusion injury in rabbits. Focal cerebral ischemia/reperfusion was induced by the middle cerebral artery occlusion (MCAO) method. In a dose response experiment, low (1.25 ng/μL), middle (2.5 ng/μL) and high (5.0 ng/μL) doses of VEGF were administered 2h after MCAO by the route of perifocal region. The VEGF at a dose of middle (2.5 ng/μL) displayed excellent effects on neuroprotective efficacy for focal cerebral ischemia/reperfusion injury. In another experiment, 2.5 ng/μL VEGF was administered at times varying from 2 to 8h after MCAO. Infarct volume, water content and neurological deficits were significantly reduced when VEGF was given at 2 and 3h after injury. The protective effect was less when the same dose was given at the later times. Thus, the present findings indicated that VEGF reduced ischemic neuronal danger with a therapeutic time window within the first 3h of transient MCAO and may be useful in the treatment of acute ischemic stroke in humans.     

In vivo hypoxia-induced neuronal damage in dentate gyrus of rat hippocampus: changes in NMDA receptors and the effect of MK–801

Hypoxia is a major cause of ischaemia-induced neuronal damage. In the present study, we examined the effects of in vivo hypoxia on N-methyl-D-aspartate receptors (NMDAR) in the rat hippocampus. This model of in vivo hypoxia involved placing rats in a hypoxic chamber containing 5% O₂ and 95% N₂ for 30 min. In the hippocampus, neuronal cells in the CA3, the hilus of the dentate gyrus and the dentate gyrus (DG) were damaged. In the CA1, which is known to be vulnerable to ischaemic damage, neuronal cells did not show hypoxia-induced damage. In vivo hypoxia-induced damage caused morphological changes in neuronal cells, such as shrunken, spindle or triangular shapes accompanied by pyknotic nuclei, but did not induce the loss of neuronal cells. On the other hand, the number of binding sites for [³H]-1-[1-(2-thienyl)cyclohexyl]-3,4-piperidine hydrochloride (TCP) gradually decreased on and after 7 days, and then maximally decreased by 25% at 21 days after hypoxia. The number of NMDAR1-immunopositive cells was decreased by 22% in the DG, but was unchanged in the CA3. Furthermore, we examined the effect of a non-competitive NMDA antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]cyclohepten-5,10-imine hydrogen maleate (MK–801), on against in vivo hypoxia. The administration of MK–801 (3 mg/kg, i.p.), 30 min before hypoxia treatment, partly protected against neuronal damage in the DG, but not in the CA3. These results suggest that hypoxia-induced neuronal damage in the DG involves, in part, the activation of NMDAR.     

Free radical spin trap alpha-phenyl-N-tert-butyl-nitron inhibits caspase-3 activation and reduces brain damage following a severe forebrain ischemic injury.

It has been documented that alpha-phenyl-N-tert-butyl-nitron (PBN) possesses a potent neuroprotective effect when administered after transient focal cerebral ischemia. However, contradicting results were reported regarding its effect in transient global ischemia. To further elucidate the mechanism of PBN action, we have studied the effect of PBN on animal survival, histopathological outcome, and activation of caspase-3 following 30 min of global ischemia in vehicle- and PBN-treated rats. The results showed that 30 min of global ischemia was such a severe insult that no animal could survive beyond 2 d of reperfusion. Histopathological evaluation showed severe tissue edema and microinfarct foci in the neocortex and thalamus. Close to 100% damage was observed in the stratum and hippocampal CA1, CA3, and dentate gyrus subregions. Postischemic PBN treatment significantly enhanced animal survival and reduced damage in the neocortex, thalamus, and hippocampus. Immunohistochemistry demonstrated that caspase-3 was activated following ischemia in the striatum and the neocortex. PBN suppressed the activation of caspase-3 in both structures. It is concluded that PBN is a potent neuroprotectant against both focal and global ischemia; besides its function as a free radical scavenger, PBN may reduce ischemic brain damage by blocking cell death pathways that involve caspase-3 activation.     

The Changes in Endogenous Antioxidant Enzyme Activity After Postconditioning

1. The aim of this work was to study potential mechanisms participating in postischemic protection of selectively vulnerable CA1 neurons in the hippocampus. Experiments were focused on measuring changes in endogenous antioxidant enzyme activity.2. Forebrain cerebral ischemia was induced in a rat by four-vessel occlusion. Ten minutes of ischemia induces so-called delayed neuronal death in selectively vulnerable CA1 region 3 days later. After 7 days of reperfusion, 71.6% of neurons succumb to neurodegeneration. When 5 min of ischemia was used as postconditioning, 2 days after 10 min of cerebral ischemia, delayed neuronal death in CA1 was almost completely (89.9%) prevented.3. Searching for mechanisms of protection, we measured the activity of endogenous antioxidant enzymes. Activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) were measured in the hippocampus, striatum and cortex by spectrophotometric methods after 10 min of ischemia used as the preconditioning. Two days after the preconditioning or the sham operation, second ischemia was induced for 5 min. We observed significant increase of total SOD activity in all studied regions of the brain 5 h after postconditioning (5 min of ischemia). SOD activity decreased to control values after 24 h.4. In some experiments, we used intraperitoneal injections of norepinephrine (3.1 μM/kg) or 3-nitropropionic acid (20 mg/kg) as postconditioning, instead of ischemia. All three treatments resulted in significant increase of SOD activity, but norepinephrine was the most effective. The same effect as was seen for total SOD activity could be observed for CuZn-SOD as well as Mn-SOD activity. Similarly, considerable increase in the activity of catalase was detected 5 h after postconditioning (5 min of ischemia). It is interesting that the greatest changes were established in selectively vulnerable hippocampus and striatum. As in the case of SOD, the highest levels of CAT activity were induced by norepinephrine, while lower but significant increase in CAT activity was induced by 3-nitropropionic acid.5. Our results suggest that endogenous antioxidants SOD and CAT could play considerable neuroprotective role after postconditioning.     

NR2A and NR2B subunits differentially mediate MAP kinase signaling and mitochondrial morphology following excitotoxic insult

NMDA receptors are essential for neurotransmission and key mediators of synaptic signaling, but they can also trigger deleterious degenerative processes that lead to cell death. Growing evidence suggests that selective blockade of the heterogeneous subunits that comprise the NMDA receptor may enable better control of pharmacotherapies for treating neurological diseases and injuries. We investigated the relationship between NMDAR activation, MAPK signaling, and mitochondrial shape following an excitotoxic insult. NR2A- and NR2B-containing NMDARs differentially mediated acute changes in cytosolic calcium, alterations in mitochondrial morphology, and phosphorylation of the MAPKs ERK and JNK. Activation of NR2A-containing NMDARs was associated with JNK phosphorylation that was neuroprotective in neuronal cultures subjected to excitotoxicity. In contrast, activation of NR2B-containing NMDARs triggered calcium accumulation in mitochondria that was strongly associated with mitochondrial swelling and neuronal cell death. Indeed, while blockade of NR2B-containing receptors was neuroprotective, this protection was lost when NR2A-initiated JNK phosphorylation was inhibited. Given the modest selectivity of the NR2A inhibitor, NVP-AAM077, the results highlight the significance of the relative, rather than absolute, activation of these two NMDA subtypes in modulating cell death pathways. Therefore, the balance between concurrent activation of NR2B-containing and NR2A-containing NMDARs dictates neuronal fate following excitotoxicity.     

Neuroprotective effects of MK 801 and hypothermia used alone and in combination in hypoxic-ischemic brain injury in neonatal rats.

Although accumulating evidence suggests that increased extracellular glutamate concentrations may play an important role in hypoxic-ischemic brain injury, dopamine and other catecholamines also seem to be involved. The N-methyl-D-aspartate receptor antagonist MK 801 and moderate hypothermia (32-34 degrees C) are each known to be neuroprotective, but their combined effect on the release and metabolism of neurotransmitters is unknown. Seven-day-old pups (n: 150) underwent right common carotid artery ligation to induce hemispheric ischemia, and were later subjected to 120 minutes of hypoxia with 8% O2 and 92% N2O. Half the rats (Group I, n: 74) were subjected to normothermic conditions throughout the hypoxic period. Moderate hypothermia (30-32 degrees C) was induced in the other pups (Group II, n: 76) immediately after artery occlusion, and was maintained throughout the hypoxic period. Prior to inducing hypoxia, half of the rats in each group (Groups IA and IIA) received vehicle solution (0.9% NaCI) and the other rats (Groups IB and IIB) received MK 801 (0.5 mg/kg) subcutaneously at 45 and 120 minutes after occlusion. Intracerebral temperature was recorded every 15 minutes after occlusion. Infarct area (n: 40) was calculated after staining with 2% 2,3,5 triphenyltetrazolium chloride. Neuronal damage (n: 42) was assessed by quantifying CA1-CA3 neuronal loss at five hippocampal levels. The amount of damage to the monoamine system of the corpus striatum was determined based on the dopamine and 3,4 dihydroxyphenylacetic acid levels in the corpus striatum in both hemispheres (n: 46), as measured by high-pressure liquid chromatography and compared with normal control pups' values (n: 10). The normothermia/saline-treated pups had significantly larger infarct areas than the MK 801 only, hypothermia only, or MK 801/hypothermia combination groups. Neuropathological examination and striatal tissue monoamine data also confirmed marked neuronal damage in this group. Although MK 801 treatment alone resulted in significantly smaller infarct area and less tissue damage than was observed in the normothermia/saline-treated group, the moderate hypothermia and the MK 801/hypothermia combination treatment groups both exhibited better neuronal protection, especially in the corpus striatum. The rats that received combined treatment also had a significantly lower mortality rate.     

The effect of preconditioning on the iron deposition after transient forebrain ischemia in rat brain

In this study we investigated iron deposition in the hippocampus CA1 area and the corpus striatum pars dorsolateralis in a rat model of cerebral ischemia and ischemic tolerance. Forebrain ischemia was induced by four-vessel occlusion for 5-min as ischemic preconditioning. Two days after the preconditioning or the sham operation, a second ischemia was induced for 20-min. With the use of iron histochemistry, regional changes were examined after 2 to 8 weeks of recirculation following the 20-min ischemia with or without preconditioning. Perl's reaction with DAB intensification demonstrated iron deposits in the CA1 area and in the corpus striatum pars dorsolateralis after 2 weeks of recirculation. These iron deposits gradually increased in density and formed clusters by the 8th week. When the rats were exposed to 5-min ischemia 2 days before lethal 20-min ischemia, the deposition of iron in the CA1 region of the hippocampus and also in the corpus striatum pars dorsolateralis was decreased and produced a minimal number of iron-containing cells between the second and the 8th week of recirculation. Preconditioning with sublethal 5-min ischemia followed by 2 days of reperfusion also prevented the neuronal destruction of the hippocampal CA1 region induced by 20-min ischemia.     

Acetyl-l-carnitine attenuates neuronal damage in gerbils with transient forebrain ischeia only when givenBefore the insult

The underlying mechanisms leading to neuronal damage in cerebral ischemia are multifactoral. In this study, we evaluated the neuroprotective effects of acetyl-l-carnitine, a medication that may enhance metabolic recovery after cerebral ischemia. The 5-minute transient forebrain ischemia model in gerbils was used. Acetyl-l-carnitine was given 30 minutes before the insult in one set of animals and 30 minutes after the insult in a second set of animals with histological evaluation at 7 days (Group A) and 28 days (Group B). Damage assessment was done using a 4-point damage score and Mann-Whitney U test was used for statistical analysis. Compared to the controls, there was significant protection in the cerebral cortex, hippocampus and the striatum in animals treated with the medicationbefore the insult in Group A and Group B Post-ischemic therapy showed little evidence of neuronal protection in either group. Behavioral tests in the Group B animals showed no significant differences between the treated or the saline controls. Our study shows, that pre-ischemic treatment with acetyl-l-carnitine results in neuronal protection. This may have clinical significance in situations (such as bypass surgery) where treatment could be initiatedprior to the insult.     

Protective effect of olive leaf extract on hippocampal injury induced by transient global cerebral ischemia and reperfusion in Mongolian gerbils

The beneficial effects of antioxidant nutrients, as well as complex plant extracts, in cerebral ischemia/reperfusion brain injury are well known. Mediterranean diet, rich in olive products, is associated with lower incidence of cardiovascular disease, cancer, inflammation and stroke. In this study, the possible neuroprotective effect of standardized dry olive leaf extract (OLE) is investigated for the first time. Transient global cerebral ischemia in Mongolian gerbils was used to investigate the OLE effects on different parameters of oxidative stress and neuronal damage in hippocampus. The biochemical measurements took place at different time points (80 min, 2, 4 and 24 h) after reperfusion. The effects of applied OLE were compared with effects of quercetin, a known neuroprotective plant flavonoid. Pretreatment with OLE (100 mg/kg, per os) significantly inhibited production of superoxide and nitric oxide, decreased lipid peroxidation, and increased superoxide dismutase activity in all time points examined. Furthermore, OLE offered histological improvement as seen by decreasing neuronal damage in CA1 region of hippocampus. The effects of applied OLE were significantly higher than effects of quercetin (100 mg/kg, per os). Our results indicate that OLE exerts a potent neuroprotective activity against neuronal damage in hippocampus after transient global cerebral ischemia, which could be attributed to its antioxidative properties.