Forebrain Ischemia In Gerbils Research Articles

Neuroprotective Effects of Purpurin Against Ischemic Damage via MAPKs, Bax, and Oxidative Stress Cascades in the Gerbil Hippocampus

Purpurin has various effects, including anti-inflammatory effects, and can efficiently cross the blood–brain barrier. In the present study, we investigated the effects of purpurin on oxidative stress in HT22 cells and mild brain damage in the gerbil hippocampal CA1 region induced by transient forebrain ischemia. Oxidative stress induced by H2O2 was significantly ameliorated by treatment with purpurin, based on changes in cell death, DNA fragmentation, formation of reactive oxygen species, and pro-apoptotic (Bax)/anti-apoptotic (Bcl-2) protein levels. In addition, treatment with purpurin significantly reduced the phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK), and p38 signaling in HT22 cells. Transient forebrain ischemia in gerbils led to a significant increase in locomotor activity 1 day after ischemia and significant decrease in number of surviving cells in the CA1 region 4 days after ischemia. Administration of purpurin reduced the travel distance 1 day after ischemia and abrogates the neuronal death in the hippocampal CA1 region 4 days after ischemia based on immunohistochemical and histochemical staining for NeuN and Fluoro-Jade C, respectively. Purpurin treatment significantly decreased the activation of microglia and astrocytes as well as the increases of nuclear factor kappa-light-chain-enhancer of activated B cells p65 in the hippocampal CA1 region 4 days after ischemia and ameliorated the ischemia-induced transient increases of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in the hippocampus 6 h after ischemia. In addition, purpurin significantly alleviated the ischemia-induced phosphorylation of JNK, ERK, and p38 in the hippocampus 1 day after ischemia. Furthermore, purpurin treatment significantly mitigated the increases of Bax in the hippocampus 1 day after ischemia and the lipid peroxidation based on malondialdehyde and hydroperoxides levels 2 days after ischemia. These results suggest that purpurin can be one of the potential candidates to reduce neuronal damage and inflammatory responses after oxidative stress in HT22 cells or ischemic damage in gerbils.

Changes in the expression of the B subunit of vacuolar H+-ATPase, in the hippocampus, following transient forebrain ischemia in gerbils.

Vacuolar H+-ATPase is a highly conserved enzyme that plays an important role in maintaining an acidic environment for lysosomal function and accumulating neurotransmitters in synaptic vesicles. In the present study, we investigated the time-dependent changes in the expression of vacuolar H+-ATPase V1B2 (ATP6V1B2), a major neuronal subtype of vacuolar H+-ATPase located in the hippocampus, after 5 min of transient forebrain ischemia in gerbils. We also examined the pH and lactate levels in the hippocampus after ischemia to elucidate the correlation between ATP6V1B2 expression and acidosis. Transient forebrain ischemia was induced by occlusion of both common carotid arteries for 5 min and animals were sacrificed at various time points after ischemia for immunohistochemical staining of ATP6V1B2 and measurements of pH and lactate levels in the hippocampus. ATP6V1B2 immunoreactivity was found to be transiently increased in the hippocampal CA1 region and dentate gyrus 12-24 hr after ischemia when the pH and lactate levels were decreased. In addition, ATP6V1B2 immunoreactivity significantly increased in the hippocampal CA3 and dentate gyrus, regions relatively resistant to ischemic damage, 4 days after ischemia, when the NeuN-positive, mature neuron numbers were significantly decreased in the hippocampal CA1 region. These results suggest that ATP6V1B2 expression is transiently increased in the hippocampus following ischemia, which may be intended to compensate for ischemia-related dysfunction of ATP6V1B2 in the hippocampus.

Neuroprotective and Anti-Inflammatory Effects of Pinus densiflora Bark Extract in Gerbil Hippocampus Following Transient Forebrain Ischemia.

Korean red pine (Pinus densiflora) belongs to the Genus Pinus, and its bark contains a great amount of naturally occurring phenolic compounds. Until now, few studies have been conducted to assess the neuroprotective effects of Pinus densiflora bark extract against brain ischemic injury. The aim of this study was to investigate the neuroprotective effects of pre-treatment with the extract in the hippocampus following 5-min transient forebrain ischemia in gerbils. Furthermore, this study examined the anti-inflammatory effect as a neuroprotective mechanism of the extract. Pinus densiflora bark was extracted by pure water (100 °C), and this extract was quantitatively analyzed and contained abundant polyphenols, flavonoids, and proanthocyanidins. The extract (25, 50, and 100 mg/kg) was orally administered once a day for seven days before the ischemia. In the gerbil hippocampus, death of the pyramidal neurons was found in the subfield cornu ammonis 1 (CA1) five days after the ischemia. This death was significantly attenuated by pre-treatment with 100 mg/kg, not 25 or 50 mg/kg, of the extract. The treatment with 100 mg/kg of the extract markedly inhibited the activation of microglia (microgliosis) and significantly decreased the expression of pro-inflammatory cytokines (interleukin 1β and tumor necrosis factor α). In addition, the treatment significantly increased anti-inflammatory cytokines (interleukin 4 and interleukin 13). Taken together, this study clearly indicates that pre-treatment with 100 mg/kg of Pinus densiflora bark extract in gerbils can exert neuroprotection against brain ischemic injury by the attenuation of neuroinflammatory responses.

Tat-Endophilin A1 Fusion Protein Protects Neurons from Ischemic Damage in the Gerbil Hippocampus: A Possible Mechanism of Lipid Peroxidation and Neuroinflammation Mitigation as Well as Synaptic Plasticity.

The present study explored the effects of endophilin A1 (SH3GL2) against oxidative damage brought about by H2O2 in HT22 cells and ischemic damage induced upon transient forebrain ischemia in gerbils. Tat-SH3GL2 and its control protein (Control-SH3GL2) were synthesized to deliver it to the cells by penetrating the cell membrane and blood–brain barrier. Tat-SH3GL2, but not Control-SH3GL2, could be delivered into HT22 cells in a concentration- and time-dependent manner and the hippocampus 8 h after treatment in gerbils. Tat-SH3GL2 was stably present in HT22 cells and degraded with time, by 36 h post treatment. Pre-incubation with Tat-SH3GL2, but not Control-SH3GL2, significantly ameliorated H2O2-induced cell death, DNA fragmentation, and reactive oxygen species formation. SH3GL2 immunoreactivity was decreased in the gerbil hippocampal CA1 region with time after ischemia, but it was maintained in the other regions after ischemia. Tat-SH3GL2 treatment in gerbils appreciably improved ischemia-induced hyperactivity 1 day after ischemia and the percentage of NeuN-immunoreactive surviving cells increased 4 days after ischemia. In addition, Tat-SH3GL2 treatment in gerbils alleviated the increase in lipid peroxidation as assessed by the levels of malondialdehyde and 8-iso-prostaglandin F2α and in pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-1β, and interleukin-6; while the reduction of protein levels in markers for synaptic plasticity, such as postsynaptic density 95, synaptophysin, and synaptosome associated protein 25 after transient forebrain ischemia was also observed. These results suggest that Tat-SH3GL2 protects neurons from oxidative and ischemic damage by reducing lipid peroxidation and inflammation and improving synaptic plasticity after ischemia.

High fat diet accelerates and exacerbates microgliosis and neuronal damage/death in the somatosensory cortex after transient forebrain ischemia in gerbils

Obesity has been known as an independent risk factor for stroke. Effects of high-fat diet (HFD)-induced obesity on neuronal damage in the somatosensory cortex of animal models of cerebral ischemia have not been studied yet. In this study, HFD-induced obesity was used to study the impact of obesity on neuronal damage/loss and microgliosis in the somatosensory cortex of a gerbil model of 5-min transient forebrain ischemia. We used gerbils fed normal diet (ND) and HFD and chronologically examined microgliosis (microglial cell activation) by ionized calcium-binding adapter molecule 1 (Iba-1) immunohistochemistry. In addition, we examined neuronal damage or death by using neuronal nuclear protein (NeuN, a neuronal marker) immunohistochemistry and Fluoro-Jade B (F-J B, a marker for neuronal degeneration) histofluorescence staining. We found that ischemia-induced microgliosis in ND-fed gerbils was increased from 2 days post-ischemia; however, ischemia-mediated microgliosis in HFD-fed gerbils increased from 1 day post-ischemia and more accelerated with time than that in the ND-fed gerbils. Ischemia-induced neuronal death/loss in the somatosensory cortex in the ND-fed gerbils was apparently found at 5 days post-ischemia. However, in the HFD-fed gerbils, neuronal death/loss was shown from 2 days post-ischemia and progressively exacerbated at 5 days post-ischemia. Our findings indicate that HFD can evoke earlier microgliosis and more detrimental neuronal death/loss in the somatosensory cortex after transient ischemia than ND evokes.

Experimental Pretreatment with Chlorogenic Acid Prevents Transient Ischemia-Induced Cognitive Decline and Neuronal Damage in the Hippocampus through Anti-Oxidative and Anti-Inflammatory Effects.

Chlorogenic acid (CGA), an ester of caffeic acid and quinic acid, is among the phenolic acid compounds which can be naturally found in green coffee extract and tea. CGA has been studied since it displays significant pharmacological properties. The aim of this study was to investigate the effects of CGA on cognitive function and neuroprotection including its mechanisms in the hippocampus following transient forebrain ischemia in gerbils. Memory and learning following the ischemia was investigated by eight-arm radial maze and passive avoidance tests. Neuroprotection was examined by immunohistochemistry for neuronal nuclei-specific protein and Fluoro-Jade B histofluorescence staining. For mechanisms of the neuroprotection, alterations in copper, zinc-superoxide dismutase (SOD1), SOD2 as antioxidant enzymes, dihydroethidium and 4-hydroxy-2-nonenal as indicators for oxidative stress, and anti-inflammatory cytokines (interleukin (IL)-4 and IL-13) and pro-inflammatory cytokines (tumor necrosis factor α (TNF-α) and IL-2) were examined by Western blotting and/or immunohistochemistry. As a result, pretreatment with 30 mg/kg CGA attenuated cognitive impairment and displayed a neuroprotective effect against transient forebrain ischemia (TFI). In Western blotting, the expression levels of SOD2 and IL-4 were increased due to pretreatment with CGA and, furthermore, 4-HNE production and IL-4 expressions were inhibited by CGA pretreatment. Additionally, pretreated CGA enhanced antioxidant enzymes and anti-inflammatory cytokines and, in contrast, attenuated oxidative stress and pro-inflammatory cytokine expression. Based on these results, we suggest that CGA can be a useful neuroprotective material against ischemia-reperfusion injury due to its antioxidant and anti-inflammatory efficacies.

Tat-Cannabinoid Receptor Interacting Protein Reduces Ischemia-Induced Neuronal Damage and Its Possible Relationship with 14-3-3η.

Cannabinoid receptor-interacting protein 1a (CRIP1a) binds to the C-terminal domain of cannabinoid 1 receptor (CB1R) and regulates CB1R activities. In this study, we made Tat-CRIP1a fusion proteins to enhance CRIP1a penetration into neurons and brain and to evaluate the function of CRIP1a in neuroprotection following oxidative stress in HT22 hippocampal cells and transient forebrain ischemia in gerbils. Purified exogenous Tat-CRIP1a was penetrated into HT22 cells in a time and concentration-dependent manner and prevented H2O2-induced reactive oxygen species formation, DNA fragmentation, and cell damage. Tat-CRIP1a fusion protein also ameliorated the reduction of 14-3-3η expression by H2O2 treatment in HT22 cells. Ischemia–reperfusion damage caused motor hyperactivity in the open field test of gerbils; however, the treatment of Tat-CRIP1a significantly reduced hyperactivity 1 day after ischemia. Four days after ischemia, the administration of Tat-CRIP1a restored the loss of pyramidal neurons and decreased reactive astrocytosis and microgliosis induced by ischemic damage in the hippocampal cornu Ammonis (CA)1 region. Ischemic damage decreased 14-3-3η expression in all hippocampal sub-regions 4 days after ischemia; however, the treatment of Tat-CRIP1 ameliorated the reduction of 14-3-3η expression. These results suggest that Tat-CRIP1a attenuates neuronal damage and hyperactivity induced by ischemic damage, and it restores normal expression levels of 14-3-3η protein in the hippocampus.

Pre-Treatment with Laminarin Protects Hippocampal CA1 Pyramidal Neurons and Attenuates Reactive Gliosis Following Transient Forebrain Ischemia in Gerbils.

Transient brain ischemia triggers selective neuronal death/loss, especially in vulnerable regions of the brain including the hippocampus. Laminarin, a polysaccharide originating from brown seaweed, has various pharmaceutical properties including an antioxidant function. To the best of our knowledge, few studies have been conducted on the protective effects of laminarin against ischemic injury induced by ischemic insults. In this study, we histopathologically investigated the neuroprotective effects of laminarin in the Cornu Ammonis 1 (CA1) field of the hippocampus, which is very vulnerable to ischemia-reperfusion injury, following transient forebrain ischemia (TFI) for five minutes in gerbils. The neuroprotective effect was examined by cresyl violet staining, Fluoro-Jade B histofluorescence staining and immunohistochemistry for neuronal-specific nuclear protein. Additionally, to study gliosis (glial changes), we performed immunohistochemistry for glial fibrillary acidic protein to examine astrocytes, and ionized calcium-binding adaptor molecule 1 to examine microglia. Furthermore, we examined alterations in pro-inflammatory M1 microglia by using double immunofluorescence. Pretreatment with 10 mg/kg laminarin failed to protect neurons in the hippocampal CA1 field and did not attenuate reactive gliosis in the field following TFI. In contrast, pretreatment with 50 or 100 mg/kg laminarin protected neurons, attenuated reactive gliosis and reduced pro-inflammatory M1 microglia in the CA1 field following TFI. Based on these results, we firmly propose that 50 mg/kg laminarin can be strategically applied to develop a preventative against injuries following cerebral ischemic insults.

Phosphatidylethanolamine-binding protein 1 protects CA1 neurons against ischemic damage via ERK-CREB signaling in Mongolian gerbils

In the present study, we made a PEP-1-phosphatidylethanolamine-binding protein 1 (PEP-1-PEBP1) fusion protein to facilitate the transduction of PEBP1 into cells and observed significant ameliorative effects of PEP-1-PEBP1 against H2O2-induced neuronal damage and the formation of reactive oxygen species in the HT22 hippocampal cells. In addition, administration of PEP-1-PEBP1 fusion protein ameliorated H2O2-induced phosphorylation of extracellular signal-regulated kinases (ERK1/2) and facilitated the phosphorylation of cyclic-AMP response element binding protein (CREB) in HT22 cells after exposure to H2O2. We also investigated the temporal and spatial changes of phosphorylated phosphatidylethanolamine-binding protein 1 (pPEBP1) in the hippocampus, after 5 min of transient forebrain ischemia in gerbils. In the sham-operated animals, pPEBP1 immunoreactivity was not detectable in the hippocampal CA1 region. pPEBP1 immunoreactivity was significantly increased in the hippocampal CA1 region, 1–2 days after ischemia, compared to that in the sham-operated group and pPEBP1 immunoreactivity was returned to levels in sham-operated group at 3–4 days after ischemia. pPEBP1 immunoreactivity significantly increased at day 7 after ischemia and decreased to sham-operated group levels by day 10 after ischemia/reperfusion. In addition, administration of PEP-1-PEBP1 fusion protein significantly reduced the ischemia-induced hyperactivity of locomotion, 1 day after ischemia and PEP-1-PEBP1 reduced neuronal damage and reactive gliosis (astrocytosis and microgliosis) in the gerbil hippocampal CA1 region, 4 days after ischemia. Administration of PEP-1-PEBP1 fusion protein ameliorated the ischemia-induced phosphorylation of ERK at 3 h and 6 h after ischemia/reperfusion and accelerated the phosphorylation of CREB in ischemic hippocampus at 6 h after ischemia. These results suggest that the increase in PEBP1 phosphorylation causes neuronal damage in the hippocampus and treatment with PEP-1-PEBP1 fusion protein provides neuroprotection from increasing phosphorylation of ERK-CREB pathways in the hippocampal CA1 region, during ischemic damage.

Ischemic preconditioning protects neurons from damage and maintains the immunoreactivity of kynurenic acid in the gerbil hippocampal CA1 region following transient cerebral ischemia.

Pyramidal neurons in region I of hippocampus proper (CA1) are particularly vulnerable to excitotoxic processes following transient forebrain ischemia. Kynurenic acid (KYNA) is a small molecule derived from tryptophan when this amino acid is metabolized through the kynurenine pathway. In the present study, we examined the effects of ischemic preconditioning (IPC) on the immunoreactivity and protein levels of KYNA following 5 min of transient forebrain ischemia in gerbils. The animals were randomly assigned to 4 groups (sham-operated group, ischemia-operated group, IPC + sham-operated group and IPC + ischemia-operated group). IPC was induced by subjecting the gerbils to 2 min of ischemia followed by 1 day of recovery. In the ischemia-operated group, we observed a significant loss of pyramidal neurons in the CA1 stratum pyramidale (SP) at 5 days post-ischemia; however, in the IPC + ischemia-operated group, the pyramidal neurons were well protected. KYNA immunoreactivity in the SP of the ischemia-operated group was significantly altered following ischemia-reperfusion and was very low 5 days following ischemia-reperfusion. In the IPC + ischemia-operated group, however, KYNA immunoreactivity was constitutively detected in the SP of the CA1 region after the ischemic insult. We also found that the alteration pattern of the KYNA protein level in the CA1 region following ischemia was generally similar to the immunohistochemical changes observed. In brief, our findings demonstrated that IPC maintained and even increased KYNA immunoreactivity in the SP of the CA1 region following ischemia-reperfusion. The data from the present study thus indicate that the enhancement of KYNA expression by IPC may be necessary for neuronal survival following transient ischemic injury.

Ischemic preconditioning inhibits expression of Na+/H+ exchanger 1 (NHE1) in the gerbil hippocampal CA1 region after transient forebrain ischemia

The participation of Na+/H+ exchanger (NHE) in neuronal damage/death in the hippocampal CA1 region (CA1) induced by transient forebrain ischemia has not been well established, although acidosis may be involved in neuronal damage/death. In the present study, we examined the effect of ischemic preconditioning (IPC) on NHE1 immunoreactivity following a 5min of transient forebrain ischemia in gerbils. The animals used in the study were randomly assigned to four groups (sham-operated-group, ischemia-operated-group, IPC plus (+) sham-operated-group and IPC+ischemia-operated-group). IPC was induced by subjecting animals to 2min of ischemia followed by 1day of recovery. A significant neuronal loss was found in the stratum pyramidale (SP) of the CA1, not the CA2/3, of the ischemia-operated-group at 5days post-ischemia. However, in the IPC+ischemia-operated-group, neurons in the SP of the CA1 were well protected. NHE1 immunoreactivity was not detected in any regions of the CA1-3 of the sham- and IPC+sham-operated-groups. However, the immunoreactivity was apparently expressed in the SP of the CA1-3 after ischemia, and the NHE1immunoreactivity was very weak 5days after ischemia; however, at this point in time, strong NHE1immunoreactivity was found in astrocytes in the CA1. In the CA2/3, NHE1immunoreactivity was slightly changed, although NHE1immunoreactivity was expressed in the SP. In the IPC+ischemia-operated-groups, NHE1 immunoreactivity was also expressed in the SP of the CA1-3; however, the immunoreactivity was more slightly changed than that in the ischemia-operated-groups. In brief, our findings show that IPC dramatically protected CA1 pyramidal neurons and strongly inhibited NHE1 expression in the SP of the CA1 after ischemia–reperfusion. These findings suggest that the inhibition of NHE1 expression may be necessary for neuronal survival from transient ischemic damage.

Midkine in repair of the injured nervous system

Midkine (MK) is a growth factor with neurotrophic and neurite outgrowth activities. It was expressed in the peri-ischaemic area in the acute phase of cerebral infarction in rat brains. Astrocytes were the origin of MK in this occasion. MK has been assessed in terms of its effects on neural injury. The administration of MK into the lateral ventricle immediately prior to ischaemia prevented cell death in the hippocampal CA1 neurons degenerated by transient forebrain ischaemia in gerbils. MK administration was also beneficial in rats with neural injury, especially after kainic acid-induced seizures. Gene therapy with mouse MK cDNA using an adenovirus was effective in reducing the cerebral infarction volume and in increasing the number of neuronal precursor cells in the subventricular zone of the rat brain. MK mRNA and MK protein were found in spinal cord motor neurons of the anterior horn in both the acute phase of sciatic nerve injury and 3 weeks later. MK immunoreactivity was also found in the proximal side of a sciatic nerve-injured site in sciatic nerve axons. MK receptors were expressed in Schwann cells after injury, suggesting crosstalk between axons and Schwann cells. MK was also present in nerve terminals and influenced ACh receptor clustering during neuromuscular development in Xenopus. Thus, MK may also be involved in reinforcing and maintaining the synapse. All these findings indicate the therapeutic potential of MK for promoting repair of the nervous system after injury.Linked ArticlesThis article is part of a themed section on Midkine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-4

Neuroprotection of granulocyte colony-stimulating factor during the acute phase of transient forebrain ischemia in gerbils

The present study investigates the potential protective effects of granulocyte colony-stimulating factor (G-CSF) and underlying mechanisms in a gerbil model of global cerebral ischemia. We examined neuronal death, inflammatory reaction and neurogenesis in hippocampus 72h after transient forebrain ischemia and investigated functional deficits. G-CSF was administered intraperitoneally 24h before ischemia and then daily. Treatment with G-CSF at 25–50μg/kg significantly reduced neuronal loss in the hippocampus CA1 area but not at 10ug/kg. G-CSF at 50μg/kg significantly decreased the level of TNF-α, the number of Iba1 (microglia marker) positive cells and reduced locomotor activity 72h after transient forebrain ischemia. Furthermore, the number of DCX-positive cells in the hippocampal dentate gyrus increased in with G-CSF treatment. Our findings indicate that G-CSF reduces hippocampal neuronal cell death dose-dependently and attenuates sensorimotor deficits after transient forebrain ischemia. These neuroprotective effects of G-CSF may be linked to inhibition of inflammation and possibly increased neurogenesis in the hippocampus.

Quantitative Evaluation of the Neuroprotective Effects of a Short-acting β-Adrenoceptor Antagonist at a Clinical Dose on Forebrain Ischemia in Gerbils

Neuroprotective effects of esmolol in laboratory and clinical settings have been reported. The present study was designed to quantitatively evaluate the neuroprotective effects of esmolol using logistic regression curves and extracellular potentials. In 42 gerbils, bilateral occlusion of common carotid arteries was performed for 3, 5, or 7 minutes (n=7 in each group). In treated animals, esmolol (200 µg/kg/min) was administered for 90 minutes, 30 minutes before the onset of ischemia. Direct current potentials were measured in the bilateral CA1 regions, in which histologic evaluation was performed 5 days later. Relations of neuronal damage with ischemic duration and duration of ischemic depolarization were determined using logistic regression curves. There was no significant difference in onset time between the 2 groups (the control group vs. the esmolol group: 1.65±0.46 vs. 1.68±0.45 min, P=0.76), and significant differences in durations of ischemic depolarization were not observed with any ischemic duration. However, logistic regression curves indicated that esmolol has a neuroprotective effect from 2.95 to 7.66 minutes of ischemic depolarization (P<0.05), and esmolol prolonged the duration of ischemic depolarization causing 50% neuronal damage from 4.97 to 6.34 minutes (P<0.05). Logistic regression curves also indicated that esmolol has a neuroprotective effect from 3.77 to 7.74 minutes of ischemic duration (P<0.05), and esmolol prolonged the ischemic duration causing 50% neuronal damage from 4.26 to 4.91 minutes (P<0.05). Esmolol has neuroprotective effects in the acute phase of ischemia by a mechanism other than shortening the duration of ischemic depolarization.

Chronological changes and effects of AMP-activated kinase in the hippocampal CA1 region after transient forebrain ischemia in gerbils

Objectives: Adenosine monophosphate-activated kinase (AMPK) is an energy-specific sensor within the central nervous system. In this study, we observed AMPK and its phosphorylated form (pAMPK) in the hippocampal CA1 region after 5 minutes of transient forebrain ischemia. In addition, we also investigated the effects of Compound C, an AMPK inhibitor, against ischemic damage in gerbils.Methods: Adenosine monophosphate-activated kinase and pAMPK immunoreactivity was observed in the hippocampal CA1 region at various time points after ischemia and Compound C was intraperitoneally administered to gerbils immediately after reperfusion and the animals were sacrificed at 5 days after ischemia/reperfusion.Results: Adenosine monophosphate-activated kinase immunoreactivity was transiently increased in the hippocampal CA1 region 1–2 days after ischemia/reperfusion, while AMPK immunoreactivity was almost undetectable in the stratum pyramidale of the CA1 region 4–7 days after ischemia/reperfusion. The administration of Compound C caused a dose-dependent decrease in the ischemia-induced hyperactive behavior, the depletion of ATP, and lactate accumulation in the hippocampal CA1 region within 24 hours after ischemia/reperfusion. In addition, the administration of Compound C decreased reactive gliosis (astrocytes and microglia) and increased the number of cresyl violet-positive neurons when compared to the vehicle-treated group at 5 days post-ischemia/reperfusion.Conclusion: These results suggest that AMPK is transiently phosphorylated following forebrain ischemia in the hippocampal CA1 region and inhibition of AMPK has neuroprotective effects against ischemic damage through the reduction of ATP depletion and lactate accumulation in the hippocampal CA1 region.

PBN fails to suppress in delayed neuronal death of hippocampal CA1 injury following transient forebrain ischemia in gerbils

Free radicals have been suggested to be involved in the genesis of ischemic brain damage, as shown by the protective effects of alpha-phenyl-N-tert-butyl nitrone (PBN), a spin trapping agent, in ischemic cerebral injury. However, the involvement of free radicals in transient ischemic-induced delayed neuronal death is not fully understood. To clarify this, in the present study, we evaluated the effect of PBN on delayed neuronal death and on the levels of free radicals in hippocampal CA1 region in the gerbil. The administration of PBN (10mg/kg, i.v.) failed to show any preventive effect on the delayed neuronal death, examined by hematoxylin and eosin staining and the TUNEL method. Furthermore, we observed no free radical formation in delayed neuronal death, determined immunohistochemically using a specific 8-OHdG antibody, after transient ischemic insult. These results suggest that free radical formation may not contribute to the formation of delayed neuronal death.

Edaravone, a free radical scavenger, attenuates behavioral deficits following transient forebrain ischemia by inhibiting oxidative damage in gerbils

The present study investigates the neurological protective effects of edaravone against global brain ischemia. Gerbils were treated with edaravone (3 mg/kg; i.p.) 30 min before transient forebrain ischemia, which was induced by occluding the bilateral common carotid artery for 5 min. The effects of edaravone were examined by measuring neuronal damage and behavioral deficits. Hexanoyl-lysine adduct (HEL) and 8-hydroxy-2′-deoxyguanosine (8-OHdG), oxidative stress markers, were also examined to assess the anti-oxidative effects of edaravone. Edaravone treatment significantly inhibited both lipid and DNA oxidative damage 72 h after ischemia, and decreased neuronal damage. Edaravone also significantly reduced the locomotor activity deficit 72 h after ischemia and improved memory impairment. These findings suggest that edaravone inhibits oxidative stress and attenuates neuronal damage induced by transient forebrain ischemia in gerbils and which may contribute to improvements in behavioral deficits.

Increase of galectin-3 expression in microglia by hyperthermia in delayed neuronal death of hippocampal CA1 following transient forebrain ischemia

The ischemic damage in the hippocampal CA1 region following transient forebrain ischemia, delayed neuronal death, is a typical apoptotic response, but the underlying mechanisms are not fully understood. We have reported that mild hyperthermia (38 °C) accelerates DNA fragmentation of the gerbil CA1 pyramidal neurons following transient forebrain ischemia. Recently, we reported that galectin-3, a β-galactosidase-binding lectin, is spatio-temporally expressed only by activated microglial cells located within CA1 region following transient forebrain ischemia in gerbils. Furthermore, expression of galectin-3 and Iba-1 (a specific microglial cell marker) are strongly reduced by hypothermia during ischemic insult. To further elucidate the effect of hyperthermia on the expression of galectin-3 by micloglia in delayed neuronal death, we examined immunohistochemical expression of galectin-3 and Iba-1, in situ terminal dUTP-biotin nick end labeling of DNA fragmentation (for determination of cell death) and hematoxylin and eosin staining (for morphological observation). We observed that between 37 °C and 39 °C, there was a temperature-dependent enhancement of galectin-3 expression in microglial cells in the CA1 region following transient ischemia. Apoptotic DNA fragmentation, detected by TUNEL staining, was observed in CA1 region in normothermia. This TUNEL staining was enhanced by hyperthermia at 37.5 °C and 38 °C, but not at 39 °C. Ischemia-induced neuronal degeneration in CA1 region in gerbil hippocampus subjected to hyperthermia (37.5 °C, 38 °C and 39 °C) observed by HE staining is similar to that in normothermic gerbils. These findings imply that galectin-3 expression in microglia may influence the survival of CA1 pyramidal neurons in cases such as hyperthermia-related neuronal injury.

Single, high-dose 17β-estradiol therapy has anti-apoptotic effect and induces cerebral plasticity following transient forebrain ischemia in gerbils (Short communication)

Although much is known about the protective effect of acute estrogen therapy in cerebral ischemia, relatively little is known about the importance of apoptosis and cerebral plasticity in this mechanism. In this work 10 min global cerebral ischemia was produced by transient bilateral carotid occlusion in 4-month-old ovariectomized female gerbils. In every of our experimental group (sham for ischemia group, ischemia group and ischemia + a high, single dose 17β-estradiol pre-treatment group) apoptotic (bcl-Xl, bax) and cerebral plasticity (GAP-43, synapsin-I, nestin) hippocampal genes' expression was measured four days after surgery. Expression of the anti-apoptotic bcl-Xl (p<0.01) and the cerebral plasticity marker synapsin-I and nestin (p<0.01) increased with acute estrogen pretreatment in ischemic animals. No change, however, in bax or GAP-43 expression was detected in estrogen treated animals compared to ischemic gerbils. These results suggest that acute estrogen therapy has anti-apoptotic effect and increases cerebral plasticity, which play an important role in cytoprotection or cerebroprotection.

Galectin-3 expression in delayed neuronal death of hippocampal CA1 following transient forebrain ischemia, and its inhibition by hypothermia

The ischemic damage in the hippocampal CA1 sector following transient ischemia, delayed neuronal death, is a typical apoptosis, but the mechanism underlying the delayed neuronal death is still far from fully understood. Galectin-3 is a β-galactosidase-binding lectin which is important in cell proliferation and apoptotic regulation. Galectin-3 is expressed by microglial cells in experimental models of adult stroke. It has been reported that activated microglial cells are widely observed in the brain, including in the hippocampal CA1 region after transient ischemic insult. In the present study, time course expression of galectin-3 following transient forebrain ischemia in gerbils was examined by immunohistochemistry, combined with Iba-1 immunostaining (a specific microglial cell marker), hematoxylin and eosin staining (for morphological observation), and in situ terminal dUTP-biotin nick end labeling of DNA fragments method (for determination of cell death). Following transient ischemia, we observed a transient increase of galectin-3 expression in CA1 region, which was maximal 96h after reperfusion. Galectin-3 expression was predominately localized within CA1 region and observed only in cells which expressed Iba-1. The galectin-3-positive microglial cells emerge after the onset of neuronal cell damage. Expressions of galectin-3 and Iba-1 were strongly reduced by hypothermia during ischemic insult. Prevention of galectin-3 and Iba-1 expression in microglia by hypothermia has led us to propose that hypothermia either inhibits microglial activation or prevents delayed neuronal death itself. Our results indicate that galectin-3 might exert its effect by modulating the neuronal damage in delayed neuronal death.