Transient Forebrain Ischemia In Rats Research Articles

Postinjury Inhibition of miR-181a Promotes Restoration of Hippocampal CA1 Neurons after Transient Forebrain Ischemia in Rats.

The cellular and molecular mechanisms regulating postinjury neurogenesis in the adult hippocampus remain undefined. We have previously demonstrated that preinjury treatment with anti-microRNA (miR)-181a preserved neurons and prevented astrocyte dysfunction in the hippocampal cornu ammonis-1 (CA1) following transient forebrain ischemia. In the present study, we assessed postinjury treatment with anti-miR-181a on recovery of CA1 neurons following transient forebrain ischemia in rats. Stereotactic CA1 injection of miR-181a antagomir at either 2 h or 7 d postinjury resulted in improved restoration of CA1 measured at 28 d postinjury. Treatment with antagomir was associated with overexpression of the mir-181a target cell adhesion-associated, oncogene-related protein and enhanced expression of the neuroprogenitor cell marker doublecortin (DCX) in the CA1. Assessment of GFAP+ cell fate by Cre/Lox-mediated deletion demonstrated that some GFAP+ cells in CA1 exhibited de novo DCX expression in response to injury. In vitro experiments using primary neuronal stem cells confirmed that miR-181a inhibition augmented the expression of DCX and directed cellular differentiation toward a neuronal fate. These results suggest that miR-181a inhibition plays a central role in the restoration of CA1 neurons via augmentation of early latent neurogenic gene activation in neural progenitor cells, including some reactive astrocytes. Therapeutic interventions targeting this restorative process may represent a novel postinjury approach to improve clinical outcomes in survivors of forebrain ischemia.

Intraischemic Modest Hypothermia Does Not Prevent Onset of Locomotor Inactivity After Transient Forebrain Ischemia in Rats.

Although modest hypothermia of 35°C has been demonstrated to provide histological neuroprotection in a rodent model of cerebral ischemia, the long-term behavioral outcome is still not clear. This study was designed to investigate whether modest hypothermia of 35°C provides sustained histological and behavioral neuroprotection following transient forebrain ischemia in rats. Male Sprague-Dawley rats were randomly assigned to one of three groups: sham, control, and modest hypothermia group. Each group contained eight rats. Ten-minute transient forebrain ischemia was produced by bilateral carotid artery occlusion plus hemorrhagic hypotension (mean arterial pressure = 40 mmHg). The hypothermic group was cooled to 35°C in preischemic period, and the cooling was continued for 1 hour postischemia. To evaluate behavioral outcome, spontaneous alternation behavior and locomotor activity were assessed using Y-maze test on a weekly basis. The rats were sacrificed after 28 days, and the number of intact neurons per 1 mm in the hippocampal CA1 subfield was counted microscopically. There was significant difference between the control [19(24.5)/mm: median (interquartile range)] and hypothermia groups [116(24)/mm; p < 0.01] in the intact CA1 neuron count. In the control and modest hypothermia groups, the locomotor activities were gradually decreased, and reached significantly lower levels in comparison with the sham group at 14 days postischemia. This study indicates that intraischemic modest hypothermia provided long-term histological neuroprotection, but did not reverse the onset of locomotor inactivity in a rat transient forebrain ischemia model.

Post-ischemic administration of pravastatin reduces neuronal injury by inhibiting Bax protein expression after transient forebrain ischemia in rats

This study investigated the neuroprotective effect of pravastatin administration after forebrain ischemia in rats. Forebrain ischemia was induced by bilateral common carotid artery occlusion and systemic hypotension for 8min. Pravastatin at 1mg/kg (pravastatin group, n=10), or an identical volume of normal saline (control group, n=10), was injected 10min, and 1–4 days after reperfusion. Arterial blood gas was analyzed 10min before ischemia onset and 10min after ischemia completion. Viable and apoptotic neuronal cells were evaluated 7 days after ischemia by hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuracil triphosphate biotin in situ nick-end labeling (TUNEL) staining of the hippocampal Cornu Ammonis area (CA1). Expression of Bcl-2 and Bax proteins was quantified by Western blot analysis. The proportion of viable neuronal cells after ischemia was greater in the pravastatin vs. control group (p<0.01), with greater expression of apoptotic cells in the control vs. pravastatin group (p<0.05). Bax protein expression was significantly decreased in the pravastatin group (p<0.05), whereas, Bcl-2 expression was increased, but not significantly (p>0.05). Our findings suggest that pravastatin administration after forebrain ischemia confers neuroprotection in rats by inhibiting Bax protein expression.

Effect of tramadol on behavioral alterations and lipid peroxidation after transient forebrain ischemia in rats

N-methyl-D-aspartate (NMDA) antagonists and γ-aminobutyric acid (GABA) agonists are proven protective in various animal models of ischemic brain damage. Tramadol, a centrally acting opioid analgesic reportedly possesses NMDA antagonistic and GABA agonistic properties, with additional ion channel blocking activity. The aim of the present study was to evaluate the possible neuroprotective effect of tramadol hydrochloride in a rat model of transient forebrain ischemia. Male Wistar rats were pretreated with tramadol hydrochloride at doses of 10 and 20 mg/kg b.w. intraperitoneally for 4 days and were subjected to 30 min occlusion of bilateral common carotid arteries followed by reperfusion for 24 h. Impairment in sensorimotor functions was evaluated by beam walking task, spontaneous locomotor activity and hanging wire test. Animals were sacrificed and the brain homogenates were used for estimating the levels of lipid peroxidation, a marker for extent of oxidative stress. Ischemic rats exhibited a significant decrease in locomotion, grip strength and increase in beam walking latency. Tramadol attenuated the post ischemic motor impairment evidenced by improvement in the performance in sensorimotor tests. The extent of lipid peroxidation was significantly (p < 0.001) reduced by tramadol pretreatment which was higher in ischemic control. This study demonstrates the neuroprotective effect of tramadol against transient forebrain ischemia in rats.

Delta opioid peptide [ d-Ala2, d-Leu5] enkephalin (DADLE) triggers postconditioning against transient forebrain ischemia

Preconditioning with selective delta opioid peptide [ d-Ala2, d-Leu5] enkephalin (DADLE) provides ischemic tolerance following transient forebrain ischemia in rats. However, whether DADLE postconditioning retains its neuroprotective efficacy and the underlying molecular mechanism in ischemic brain is largely unknown. We investigated DADLE postconditioning protection of hippocampal CA1 neurons against transient forebrain ischemia. 6 days after being implanted with cannula at the right lateral ventricle, rats underwent 10 min of forebrain ischemia by four vessel occlusion. Hippocampal CA1 neuronal survival and degeneration were measured in the hippocampi of rats at 3 days after ischemia. The behavioral and cognitive improvements of DADLE treatment in rats were also evaluated on days 5–9 using open-field and Morris water maze tests. The results showed that DADLE at doses of 0.25 and 2.5 nmol, but not 25 nmol, could significantly protect CA1 neurons against ischemia/reperfusion injury. Co-administration with the delta-opioid receptor antagonist naltrindole or pretreatment with the Akt antagonist LY294002 completely abolished the DADLE postconditioning effect. Furthermore, DADLE postconditioning exhibited cognitive benefits in rats with transient forebrain ischemia. The study thus suggested a therapeutic opportunity of postconditioning neuroprotection by DADLE and also provided important information in understanding the mechanism of DADLE action in the ischemic brain.

Iron mediates endothelial cell damage and blood-brain barrier opening in the hippocampus after transient forebrain ischemia in rats

Blood cells are transported into the brain and are thought to participate in neurodegenerative processes following hypoxic ischemic injury. We examined the possibility that transient forebrain ischemia (TFI) causes the blood-brain barrier (BBB) to become permeable to blood cells, possibly via dysfunction and degeneration of endothelial cells in rats. Extravasation of Evans blue and immunoglobulin G (IgG) was observed in the hippocampal CA1-2 areas within 8 h after TFI, and peaked at 48 h. This extravasation was accompanied by loss of tight junction proteins, occludin, and zonula occludens-1, and degeneration of endothelial cells in the CA1-2 areas. Iron overload and mitochondrial free radical production were evident in the microvessel endothelium of the hippocampus before endothelial cell damage occurred. Administration of deferoxamine (DFO), an iron chelator, or Neu2000, an antioxidant, blocked free radical production and endothelial cell degeneration. Our findings suggest that iron overload and iron-mediated free radical production cause loss of tight junction proteins and degeneration of endothelial cells, opening of the BBB after TFI.

Blood-derived iron mediates free radical production and neuronal death in the hippocampal CA1 area following transient forebrain ischemia in rat

Abnormal brain iron homeostasis has been proposed as a pathological event leading to oxidative stress and neuronal injury under pathological conditions. We examined the possibility that neuronal iron overload would mediate free radical production and delayed neuronal death (DND) in hippocampal CA1 area after transient forebrain ischemia (TFI). Mitochondrial free radicals (MFR) were biphasically generated in CA1 neurons 0.5-8 and 48-60 h after TFI. Treatment with Neu2000, a potent spin trapping molecule, as well as trolox, a vitamin E analogue, blocked the biphasic MFR production and attenuated DND in the CA1, regardless of whether it was administered immediately or even 24 h after reperfusion. The late increase in MFR was accompanied by iron accumulation and blocked by the administration of deferoxamine-an iron chelator. Iron accumulation was attributable to prolonged upregulation of the transferrin receptor and to increased uptake of peripheral iron through a leaky blood-brain barrier. Infiltration of iron-containing cells and iron accumulation were attenuated by depletion of circulating blood cells through X-ray irradiation of the whole body except the head. The present findings suggest that excessive iron transported from blood mediates slowly evolving oxidative stress and neuronal death in CA1 after TFI, and that targeting iron-mediated oxidative stress holds extended therapeutic time window against an ischemic event.

Neuroprotective effects of a combination of dexmedetomidine and hypothermia after incomplete cerebral ischemia in rats

Dexmedetomidine and hypothermia are known to reduce neuronal injury following cerebral ischemia. We examined whether a combination of dexmedetomidine and hypothermia reduces brain injury after transient forebrain ischemia in rats to a greater extent than either treatment alone. Thirty-eight male Sprague-Dawley rats were anesthetized with fentanyl and nitrous oxide in oxygen. Four groups were tested: group C (saline 1 ml/kg, temporal muscle temperature 37.5 degrees C); group H (saline 1 ml/kg, 35.0 degrees C); group D (dexmedetomidine 100 microg/kg, 37.5 degrees C); and group DH (dexmedetomidine 100 microg/kg, 35.0 degrees C). Dexmedetomidine or saline was administered intraperitoneally 30 min before ischemia. Cerebral ischemia was produced by right carotid artery ligation with hemorrhagic hypotension (mean arterial pressure 40 mmHg) for 20 min. Neurologic outcome was evaluated at 24, 48, and 72 h after ischemia. Histopathology was evaluated in the caudate and hippocampus at 72 h after ischemia. Neurologic outcome was significantly better in the group DH than the group C (P<0.05), whereas it was similar between the group DH and the groups D or H. Survival rate of the hippocampal CA1 neurons was significantly greater in groups D, H, and DH than group C (P<0.05). Histopathologic injury in the caudate section was significantly less in groups H and DH than group C (P<0.05). The combination of dexmedetomidine and hypothermia improved short-term neurologic outcome compared with the control group, whereas the combination therapy provided comparable neuroprotection with either of the two therapies alone.

The combined neuroprotective effects of lidocaine and dexmedetomidine after transient forebrain ischemia in rats

We investigated whether coadministration of lidocaine and dexmedetomidine would reduce brain injury following transient forebrain ischemia in rats to a greater extent than either drug alone. Adult male Sprague-Dawley rats were anesthetized with halothane to maintain normocapnia and normoxia. Rats received subcutaneous injection of saline 1 ml/kg, lidocaine 10 mg/kg, dexmedetomidine 3 microg/kg, or lidocaine 10 mg/kg plus dexmedetomidine 3 microg/kg. Thirty minutes after the drug injection, forebrain ischemia was induced by hemorrhagic hypotension and occlusion of the bilateral carotid arteries, and was confirmed by isoelectric EEG. At the end of 10-min ischemia, rats were reperfused. The same dose of drugs was administered 3, 24, and 48 h after ischemia. Neurological examination was done at 1, 2, and 7 days after ischemia. Seven days after ischemia, the brain was stained with hematoxylin and eosin. We counted ischemic cells in the CA1 hippocampal region, striatum, and cerebral cortex. We also measured extracellular glutamate and norepinephrine concentration in hippocampal CA1 in the four groups. As compared with saline-treated rats, rats receiving dexmedetomidine plus lidocaine showed less than neurological deficit scores at 2 and 7 days after ischemia, and had less ischemic cells in the CA1 region. However, administration of dexmedetomidine plus lidocaine did not alter the area under the glutamate concentration curve and norepinephrine concentration during ischemia in the CA1 region, compared with saline-treated rats. Our results suggest coadministration of lidocaine and dexmedetomidine improves the neurological outcome without alteration of glutamate and norepinephrine concentrations during forebrain ischemia in rats.

Transient enhancement of inhibitory synaptic transmission in hippocampal CA1 pyramidal neurons after cerebral ischemia

Pyramidal neurons in hippocampal CA1 regions are highly sensitive to cerebral ischemia. Alterations of excitatory and inhibitory synaptic transmission may contribute to the ischemia-induced neuronal degeneration. However, little is known about the changes of GABAergic synaptic transmission in the hippocampus following reperfusion. We examined the GABA A receptor-mediated inhibitory postsynaptic currents (IPSCs) in CA1 pyramidal neurons 12 and 24 h after transient forebrain ischemia in rats. The amplitudes of evoked inhibitory postsynaptic currents (eIPSCs) were increased significantly 12 h after ischemia and returned to control levels 24 h following reperfusion. The potentiation of eIPSCs was accompanied by an increase of miniature inhibitory postsynaptic current (mIPSC) amplitude, and an enhanced response to exogenous application of GABA, indicating the involvement of postsynaptic mechanisms. Furthermore, there was no obvious change of the paired-pulse ratio (PPR) of eIPSCs and the frequency of mIPSCs, suggesting that the potentiation of eIPSCs might not be due to the increased presynaptic release. Blockade of adenosine A1 receptors led to a decrease of eIPSCs amplitude in post-ischemic neurons but not in control neurons, without affecting the frequency of mIPSCs and the PPR of eIPSCs. Thus, tonic activation of adenosine A1 receptors might, at least in part, contribute to the enhancement of inhibitory synaptic transmission in CA1 neurons after forebrain ischemia. The transient enhancement of inhibitory neurotransmission might temporarily protect CA1 pyramidal neurons, and delay the process of neuronal death after cerebral ischemia.

Neuroprotection with delayed calpain inhibition after transient forebrain ischemia

Delayed neurodegeneration after transient global brain ischemia offers a therapeutic window for inhibiting molecular injury mechanisms. One such mechanism is calpain-mediated proteolysis, which peaks 24 to 48 hrs after transient forebrain ischemia in rats. This study tests the hypothesis that delayed calpain inhibitor therapy can reduce brain calpain activity and neurodegeneration after transient forebrain ischemia. Prospective randomized placebo-controlled animal trial. University research laboratory. Adult male Long-Evans rats. Rats subjected to 10-min transient forebrain ischemia were randomized to intravenous infusion of calpain inhibitor CEP-3453 or vehicle beginning 22 hrs after injury. In a dose-response study, a 60 mg/kg bolus followed by 30 mg/kg infusion was required to reduce postischemic brain calpain activity measured by Western blot of hippocampal homogenates at 48 hrs after injury. The same dosing protocol decreased degeneration of CA1 pyramidal neurons measured at 72 hrs after injury. These results suggest a causal role for calpains in delayed postischemic neurodegeneration, and demonstrate a broad therapeutic window for calpain inhibition in this model.

Cilostazol preserves CA1 hippocampus and enhances generation of immature neuroblasts in dentate gyrus after transient forebrain ischemia in rats

In the present study, cerebral ischemia was induced by a 10 min transient bilateral common carotid artery occlusion in rats combined with arterial blood pressure lowering to 37–42 mm Hg during occlusion. When histologically evaluated at 7 and 28 days after the forebrain ischemia (DAI) by staining with cresyl violet and Fluoro-Jade, the hippocampal CA1 region was most prominently damaged. At 7 DAI, treatment with cilostazol (60 mg/kg/day, orally) significantly reduced the neuronal damage in the CA1 region. The number of surviving neurons, visualized by NeuN immunostaining, in the CA1 region significantly increased at 7 and 28 DAI in the cilostazol-treated groups. To elucidate whether cilostazol enhances hippocampal neurogenesis after ischemia, we planned a co-labeling study using 5-bromo-2′-deoxyuridine (BrdU), NeuN (a marker for mature neurons) and doublecortin (DCX) (a marker for immature migratory neuroblasts). Double immunofluorescence staining at 7 DAI showed that cilostazol significantly increased the immunoreactivities of both DCX and phosphorylated cAMP-response element-binding protein (CREB) in the dentate gyrus that was co-expressed with BrdU. These results suggest that cilostazol has dual beneficial effects preserving the CA1 hippocampal region and promoting the generation of immature migratory neuroblasts in the dentate gyrus by upregulation of CREB phosphorylation after transient forebrain ischemia.

Effect of mivazerol, a α2‐agonist, on striatal norepinephrine concentration during transient forebrain ischemia in rats*

We have previously reported that mivazerol, a alpha(2)-agonist, possibly provides neuroprotection against transient forebrain ischemia in rats. This study was designed to investigate the ability of mivazerol to attenuate ischemia-induced increase in striatal norepinephrine concentration after transient forebrain ischemia in rats. Male Sprague-Dawley rats, anesthetized with halothane, were assigned to one of three groups (n=10 each); control (C, normal saline 1 ml/kg), mivazerol 20 microg/kg (M20), and 40 microg/kg (M40) groups. Monitored variables included temporal muscle temperature (maintained at 37.5+/-0.1 degrees C), electroencephalogram, systolic/diastolic blood pressure, heart rate, arterial blood gases, and blood glucose concentrations. Thirty minutes after subcutaneous drug administration, forebrain ischemia was induced with hemorrhagic hypotension (systolic arterial pressure: 40-50 mmHg) and bilateral carotid artery occlusion for 10 min, and then the brain was reperfused. Norepinephrine concentration in the interstitial fluids in the striatum was analyzed using in vivo microdialysis in combination with high-performance liquid chromatography. Ischemia resulted in a prompt increase in norepinephrine concentrations in the striatum in all groups. However, there were no significant differences in norepinephrine concentrations in the striatum between the three groups at any period. Our results indicate that mivazerol did not attenuate ischemia-induced increase in striatal norepinephrine concentration. This suggests that the possible neuroprotective property of mivazerol is not related to inhibition of norepinephrine release in the brain.

Simultaneous observation of superficial cortical and intracerebral microvessels in vivo during reperfusion after transient forebrain ischemia in rats using synchrotron radiation

Using a newly developed angiography system that combines monochromatic synchrotron radiation (MSR) as an X-ray source with a high-definition camera or video system, we observed superficial cortical and intracerebral microvessels simultaneously in vivo during reperfusion after transient forebrain ischemia. Transient brain ischemia was induced by 10-min four-vessel occlusion in rats under general anesthesia. Angiographic images were then sequentially obtained at 3 frames/s. The detector features a 7-μm equivalent pixel size projected onto the input area and a 7 mm × 7 mm input field. Changes in the cerebral microvessels were observed before and 1, 5, 10, 15, 20 and 30 min after transient cerebral ischemia using the MSR angiography system. The calibers of the internal carotid artery (ICA), middle cerebral artery (MCA), and striate artery (SA) significantly increased 1 min after reperfusion, while the pial arteriole (PA) caliber significantly decreased (76% of base line). The MCA, PA and SA were significantly dilated 5 and 10 min after reperfusion. Although the caliber of the ICA significantly decreased after 30 min reperfusion compared with the basal value, the calibers of the other three vessels remained larger than the basal values throughout the experiment. Early venous filling was observed at 5 and 10 min after reperfusion. The MSR angiography system is useful for investigating morphological changes in both cortical and central branches of cerebral vessels in rats during reperfusion after cerebral ischemia.

Activities of endogenous antioxidant enzymes in the cerebrospinal fluid and the hippocampus after transient forebrain ischemia in rat

The activity of SOD and CAT was measured in controls and 5 h after 5, 10 and 15 min of ischemia, as well as 1 or 2 days after 10 min of ischemia in the hippocampus and in the CSF. A significant increase in total SOD activity 5 h after ischemia was caused mainly by increased CuZn-SOD activity. The highest values were measured 5 h after 5 min ischemia (by 160%) and smallest if 15 min (by 40%) of ischemia was used. In comparison to the hippocampus, the activity of SOD in CSF increased equally after all intervals of ischemia. Activities of total SOD and CuZn-SOD after 10 min of ischemia in the hippocampus were significantly increased only after 5 and 24 h of reperfusion but in CSF they were increased after all examined intervals of reperfusion. The activity of CAT was significantly increased in the hippocampus after 5 (by 260%), 10 and 15 min (by 100%) of ischemia. CAT activity in CSF was increased equally after all intervals of ischemia (by 200%). Ischemic attack causes a rapid response in hippocampal tissue as well as in the CSF, represented by an increase in the activity of endogenous antioxidant enzymes SOD and CAT.

Sustained neuroprotection and facilitation of behavioral recovery by the Ginkgo biloba extract, EGb 761, after transient forebrain ischemia in rats

The effects of the Ginkgo biloba extract, EGb 761, on ischemia-induced learning/memory impairments and hippocampal damage were evaluated in a non-food motivated, aversive radial maze task (AvRM). Rats were subjected to 15 min transient, global cerebral ischemia (TGCI). In the first experiment, rats were rendered ischemic, and 23 days later were tested for acquisition performance (post-operative training). In a second experiment, rats were trained for 10 days and then subjected to ischemia (pre-operative training); the retention of cognition performance was assessed on days 31, 35 and 39 after ischemia. Acquisition and retention performances were expressed by (a) latency to find a goal box, (b) number of reference memory errors, and (c) number of working memory errors. EGb 761 (50 or 150 mg/kg) was given orally, starting before ischemia and continuing for up to 3 days after ischemia. TGCI markedly disrupted both acquisition and retention performance ( p < 0.001–0.05). EGb 761 (150 mg/kg) completely reversed acquisition impairment as measured by the parameters ‘latency’ and ‘number of reference errors’ when performance was examined across sessions ( p < 0.01–0.05). The total number of reference errors was also completely abolished by EGb 761 (150 mg/kg). However, EGb 761 did not statistically reduce the effects of TGCI on the parameter ‘working memory errors’ (across session and total). At 50 mg/kg, EGb 761 did not affect ischemia-induced acquisition impairment at all. The retention deficit caused by ischemia was not statistically reduced by EGb 761 (150 mg/kg), whatever examined across session or as total. EGb 761 reduced the extent of hippocampal CA1 cell loss ( p < 0.01–0.001), an effect sustained at least up to 40 days after ischemia. These findings show that EGb 761 is effective in reducing, at least partially, both the cognitive impairments and hippocampal damage after TGCI in rats, and suggest that its effect on behavioral recovery may be dissociated from the neuroprotective effect on the hippocampus. The present results also validate the AvRM as an alternative, reliable behavioral test to assess the effects of drugs on behavioral recovery after ischemic brain damage.

Neuroprotection by two polyphenols following excitotoxicity and experimental ischemia

Brain ischemia induces neuronal loss which is caused in part by excitotoxicity and free radical formation. Here, we report that mangiferin and morin, two antioxidant polyphenols, are neuroprotective in both in vitro and in vivo models of ischemia. Cell death caused by glutamate in neuronal cultures was decreased in the presence of submicromolar concentrations of mangiferin or morin which in turn attenuated receptor-mediated calcium influx, oxidative stress as well as apoptosis. In addition, both antioxidants diminished the generation of free radicals and neuronal loss in the hippocampal CA1 region due to transient forebrain ischemia in rats when administered after the insult. Importantly, neuroprotection by these antioxidants was functionally relevant since treated-ischemic rats performed significantly better in three hippocampal-dependent behavioral tests. Together, these results indicate that mangiferin and morin have potent neuroprotectant activity which may be of therapeutic value for the treatment of acute neuronal damage and disability.

Immediate and Delayed Treatments with Curcumin Prevents Forebrain Ischemia-Induced Neuronal Damage and Oxidative Insult in the Rat Hippocampus

Oxidative stress is believed to contribute to neurodegeneration following ischemic injury. The present study was undertaken to evaluate the possible antioxidant neuroprotective effect of curcumin (Cur) on neuronal death of hippocampal CA1 neurons following transient forebrain ischemia in rat. Treatment of Cur (200 mg/kg/day, i.p.) at three different times (immediately, 3 h and 24 h after ischemia) significantly (P<0.01) reduced neuronal damage 7 days after ischemia. Also, treatment of ischemic rats with Cur decreased the elevated levels of MDA and increased GSH contents, catalase and SOD activities to normal levels. In the in vitro, Cur was as potent as antioxidant (IC(50) = 1 microM) as butylated hydroxytoluene. The present study demonstrates that curcumin treatment attenuates forebrain ischemia-induced neuronal injury and oxidative stress in hippocampal tissue. Thus treatment with curcumin immediately or even delayed until 24 h may have the potential to be used as a protective agent in forebrain ischemic insult in human.

Delayed Postconditionig Initiates Additive Mechanism Necessary for Survival of Selectively Vulnerable Neurons After Transient Ischemia in Rat Brain

1. The aim of this study was to validate the role of postconditioning, used 2 days after lethal ischemia, for protection of selectively vulnerable brain neurons against delayed neuronal death. 2. Eight, 10, or 15 min of transient forebrain ischemia in rat (four-vessel occlusion model) was used as initial lethal ischemia. Fluoro Jade B, the marker of neurodegeneration, and NeuN, a specific neuronal marker were used for visualization of changes 7 or 28 days after ischemia without and with delayed postconditioning. 3. Our results confirm that postconditioning if used at right time and with optimal intensity can prevent process of delayed neuronal death. At least three techniques, known as preconditioners, can be used as postconditioning: short ischemia, 3-nitropropionic acid and norepinephrine. A cardinal role for the prevention of death in selectively vulnerable neurons comprises synthesis of proteins during the first 5 h after postconditioning. Ten minutes of ischemia alone is lethal for 70% of pyramidal CA1 neurons in hippocampus. Injection of inhibitor of protein synthesis (Cycloheximide), if administered simultaneously with postconditioning, suppressed beneficial effect of postconditioning and resulted in 50% of CA1 neurons succumbing to neurodegeneration. Although, when Cycloheximide was injected 5 h after postconditioning, this treatment resulted in survival of 90% of CA1 neurons. 4. Though postconditioning significantly protects hippocampal CA1 neurons up to 10 min of ischemia, its efficacy at 15 min ischemia is exhausted. However, protective impact of postconditioning in less-sensitive neuronal populations (cortex and striatum) is very good after such a damaging insult like 15 min ischemia. This statement also means that up to 15 min of ischemia, postconditioning does not induce cumulation of injuries produced by the first and the second stress.

Effects of hepatocyte growth factor on phosphorylation of extracellular signal-regulated kinase and hippocampal cell death in rats with transient forebrain ischemia

Hepatocyte growth factor (HGF) has been implicated in protection against several types of cell injuries. We investigated the effects of human recombinant HGF (hrHGF) on the selective neuronal cell death in the hippocampal CA1 region after transient forebrain ischemia in rats and explored the nature of the intracellular signaling pathway for the protection against this neuronal injury. hrHGF was injected continuously into the hippocampal CA1 region directly using an osmotic pump from 10 min to 72 h after the start of reperfusion. The marked increase in the number of TUNEL-positive cells found in the CA1 region after ischemia was almost completely abolished by the hrHGF treatment. Akt phosphorylation as well as IκB phosphorylation, which has been implicated in events downstream of the Akt, was not affected by hrHGF treatment. Extracellular signal-regulated kinase (ERK) phosphorylation was decreased in the CA1 region with time after ischemia. hrHGF increased or recovered ERK phosphorylation without changing the total amount of ERK protein. Immunohistochemical analysis demonstrated that phosphorylated ERK was colocalized with a neuronal nucleus marker NeuN in the hippocampal CA1 region of ischemic rats with hrHGF treatment at the early period after reperfusion. These results suggest that the protective effects of hrHGF against neuronal death in the hippocampal CA1 after transient forebrain ischemia could be related to an ERK-dependent pathway.