Porphyran Attenuates Neuronal Loss in the Hippocampal CA1 Subregion Induced by Ischemia and Reperfusion in Gerbils by Inhibiting NLRP3 Inflammasome-Mediated Neuroinflammation.

Porphyran, a sulfated polysaccharide found in various species of marine red algae, has been demonstrated to exhibit diverse bioactivities, including anti-inflammatory effects. However, the protective effects of porphyran against cerebral ischemia and reperfusion (IR) injury have not been investigated. The aim of this study was to examine the neuroprotective effects of porphyran against brain IR injury and its underlying mechanisms using a gerbil model of transient forebrain ischemia (IR in the forebrain), which results in pyramidal cell (principal neuron) loss in the cornu ammonis 1 (CA1) subregion of the hippocampus on day 4 after IR. Porphyran (25 and 50 mg/kg) was orally administered daily for one week prior to IR. Pretreatment with 50 mg/kg of porphyran, but not 25 mg/kg, significantly attenuated locomotor hyperactivity and protected pyramidal cells located in the CA1 area from IR injury. The pretreatment with 50 mg/kg of porphyran significantly suppressed the IR-induced activation and proliferation of microglia in the CA1 subregion. Additionally, the pretreatment significantly inhibited the overexpressions of nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing protein-3 (NLRP3) inflammasome complex, and pro-inflammatory cytokines (interleukin 1 beta and interleukin 18) induced by IR in the CA1 subregion. Overall, our findings suggest that porphyran exerts neuroprotective effects against brain IR injury, potentially by reducing the reaction (activation) and proliferation of microglia and reducing NLRP3 inflammasome-mediated neuroinflammation.

Neuroprotective Effects of Aucubin against Cerebral Ischemia and Ischemia Injury through the Inhibition of the TLR4/NF-κB Inflammatory Signaling Pathway in Gerbils.

Aucubin, an iridoid glycoside, possesses beneficial bioactivities in many diseases, but little is known about its neuroprotective effects and mechanisms in brain ischemia and reperfusion (IR) injury. This study evaluated whether aucubin exhibited neuroprotective effects against IR injury in the hippocampal CA1 region through anti-inflammatory activity in gerbils. Aucubin (10 mg/kg) was administered intraperitoneally once a day for one week prior to IR. Neuroprotective effects of aucubin were assessed by neuronal nuclei (NeuN) immunofluorescence and Floro-Jade C (FJC) histofluorescence. Microgliosis and astrogliosis were evaluated using immunohistochemistry with anti-ionized calcium binding adapter protein 1 (Iba1) and glial fibrillary acidic protein (GFAP). Protein levels of proinflammatory cytokines interleukin1 beta (IL1ß) and tumor necrosis factor alpha (TNFα) were assayed using enzyme-linked immunosorbent assay and Western blot. Changes in toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling pathway were assessed by measuring levels of TLR4, inhibitor of NF-κB alpha (IκBα), and NF-κB p65 using Western blot. Aucubin treatment protected pyramidal neurons from IR injury. IR-induced microgliosis and astrogliosis were suppressed by aucubin treatment. IR-induced increases in IL1ß and TNFα levels were significantly alleviated by the treatment. IR-induced upregulation of TLR4 and downregulation of IκBα were significantly prevented by aucubin treatment, and IR-induced nuclear translocation of NF-κB was reversed by aucubin treatment. Briefly, aucubin exhibited neuroprotective effects against brain IR injury, which might be related to the attenuation of neuroinflammation through inhibiting the TLR-4/NF-κB signaling pathway. These results suggest that aucubin pretreatment may be a potential approach for the protection of brain IR injury.

Therapeutic Treatment with Pycnogenol® Attenuates Ischemic Brain Injury in Gerbils Focusing on Cognitive Impairment, Neuronal Death, BBB Leakage and Neuroinflammation in the Hippocampus.

BACKGROUND: A gerbil model of ischemia and reperfusion (IR) injury in the forebrain has been developed for studies on mechanisms, prevention and therapeutic strategies of IR injury in the forebrain. Pycnogenol® (PYC), a standardized extract of French maritime pine tree (Pinus pinaster Aiton) has been exploited as an additive for dietary supplement. In the present study, we investigated the neuroprotective effects of post-treatment with PYC and its therapeutic mechanisms in gerbils. METHODS: The gerbils were given sham and IR operation and intraperitoneally injected with vehicle and Pycnogenol® (25, 50 and 100 mg/kg, respectively) immediately, at 24 hours and 48 hours after sham and IR operation. Through 8-arm radial maze test and passive avoidance test, each spatial memory and short-term memory function was assessed. To examine the neuroprotection of Pycnogenol®, we conducted cresyl violet staining, immunohistochemistry for neuronal nuclei, and Fluoro-Jade B histofluorescence. Moreover, we carried out immunohistochemistry for immunoglobulin G (IgG) to investigate blood-brain barrier (BBB) leakage and interleukin-1ß (IL-1ß) to examine change in pro-inflammatory cytokine. RESULTS: We found that IR-induced memory deficits were significantly ameliorated when 100 mg/kg Pycnogenol® was treated. In addition, treatment with 100 mg/kg Pycnogenol®, not 25 mg/kg nor 50 mg/kg, conferred neuroprotective effect against IR injury. For its mechanisms, we found that 100 mg/kg Pycnogenol® significantly reduced BBB leakage and inhibited the expression of IL-1ß. CONCLUSIONS: Therapeutic treatment (post-treatment) with Pycnogenol® after IR effectively attenuated ischemic brain injury in gerbils. Based on these results, we suggest that PYC can be employed as an important material for ischemic drugs.

Astaxanthin Confers a Significant Attenuation of Hippocampal Neuronal Loss Induced by Severe Ischemia-Reperfusion Injury in Gerbils by Reducing Oxidative Stress.

Astaxanthin is a powerful biological antioxidant and is naturally generated in a great variety of living organisms. Some studies have demonstrated the neuroprotective effects of ATX against ischemic brain injury in experimental animals. However, it is still unknown whether astaxanthin displays neuroprotective effects against severe ischemic brain injury induced by longer (severe) transient ischemia in the forebrain. The purpose of this study was to evaluate the neuroprotective effects of astaxanthin and its antioxidant activity in the hippocampus of gerbils subjected to 15-min transient forebrain ischemia, which led to the massive loss (death) of pyramidal cells located in hippocampal cornu Ammonis 1-3 (CA1-3) subfields. Astaxanthin (100 mg/kg) was administered once daily for three days before the induction of transient ischemia. Treatment with astaxanthin significantly attenuated the ischemia-induced loss of pyramidal cells in CA1-3. In addition, treatment with astaxanthin significantly reduced ischemia-induced oxidative DNA damage and lipid peroxidation in CA1-3 pyramidal cells. Moreover, the expression of the antioxidant enzymes superoxide dismutase (SOD1 and SOD2) in CA1-3 pyramidal cells were gradually and significantly reduced after ischemia. However, in astaxanthin-treated gerbils, the expression of SOD1 and SOD2 was significantly high compared to in-vehicle-treated gerbils before and after ischemia induction. Collectively, these findings indicate that pretreatment with astaxanthin could attenuate severe ischemic brain injury induced by 15-min transient forebrain ischemia, which may be closely associated with the decrease in oxidative stress due to astaxanthin pretreatment.

Therapeutic effects of stiripentol against ischemia-reperfusion injury in gerbils focusing on cognitive deficit, neuronal death, astrocyte damage and blood brain barrier leakage in the hippocampus.

Stiripentol is an anti-epileptic drug for the treating of refractory status epilepticus. It has been reported that stiripentol can attenuate seizure severity and reduce seizure-induced neuronal damage in animal models of epilepsy. The objective of the present study was to investigate effects of post-treatment with stiripentol on cognitive deficit and neuronal damage in the cornu ammonis 1 (CA1) region of the hippocampus proper following transient ischemia in the forebrain of gerbils. To evaluate ischemia-induced cognitive impairments, passive avoidance test and 8-arm radial maze test were performed. It was found that post-treatment with stiripentol at 20 mg/kg, but not 10 or 15 mg/kg, reduced ischemia-induced memory impairment. Transient ischemia-induced neuronal death in the CA1 region was also significantly attenuated only by 20 mg/kg stiripentol treatment after transient ischemia. In addition, 20 mg/kg stiripentol treatment significantly decreased ischemia-induced astrocyte damage and immunoglobulin G leakage. In brief, stiripentol treatment after transient ischemia ameliorated transient ischemia-induced cognitive impairment in gerbils, showing that pyramidal neurons were protected and astrocyte damage and blood brain barrier leakage were significantly attenuated in the hippocampus. Results of this study suggest stiripentol can be developed as a candidate of therapeutic drug for ischemic stroke.

Comparison of Neuronal Death, Blood-Brain Barrier Leakage and Inflammatory Cytokine Expression in the Hippocampal CA1 Region Following Mild and Severe Transient Forebrain Ischemia in Gerbils.

Transient ischemia in the brain causes blood-brain barrier (BBB) breakdown and dysfunction, which is related to ischemia-induced neuronal damage. Leakage of plasma proteins following transient ischemia is one of the indicators that is used to determine the extent of BBB dysfunction. In this study, neuronal damage/death, leakage of albumin and IgG, microgliosis, and inflammatory cytokine expression were examined in the hippocampal CA1 region, which is vulnerable to transient ischemia, following 5-min (mild) and 15-min (severe) ischemia in gerbils induced by transient common carotid arteries occlusion (tCCAo). tCCAo-induced neuronal damage/death occurred earlier and was more severe after 15-min tCCAo vs. after 5-min tCCAo. Significant albumin and IgG leakage (albumin and IgG immunoreactivity) took 1 or 2 days to begin, and immunoreactivity was markedly increased 5 days after 5-min tCCAo. While, albumin and IgG leakage began to increase 6 h after 15-min tCCAo and remained significantly higher over time than that seen in 5-min tCCAo. IgG immunoreactivity was observed in degenerating neurons and activated microglia after tCCAo, and microglia were activated to a greater extent after 15-min tCCAo than 5-min tCCAo. In addition, following 15-min tCCAo, pro-inflammatory cytokines [tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1ß)] immunoreactivity was significantly higher than that seen following 5-min tCCAo, whereas immunoreactivity of anti-inflammatory cytokines (IL-4 and IL-13) was lower in 15-min than 5-min tCCAo. These results indicate that duration of tCCAo differentially affects the timing and degree of neuronal damage or loss, albumin and IgG leakage and inflammatory cytokine expression in brain tissue. In addition, more severe BBB leakage is closely related to acceleration of neuronal damage through increased microglial activation and pro-inflammatory cytokine expression in the ischemic hippocampal CA1 region.

CD200 Change Is Involved in Neuronal Death in Gerbil Hippocampal CA1 Field Following Transient Forebrain Ischemia and Postischemic Treatment with Risperidone Displays Neuroprotection without CD200 Change.

It has been reported that CD200 (Cluster of Differentiation 200), expressed in neurons, regulates microglial activation in the central nervous system, and a decrease in CD200 expression causes an increase in microglial activation and neuronal loss. The aim of this study was to investigate time-dependent changes in CD200 expression in the hippocampus proper (CA1, 2, and 3 fields) after transient forebrain ischemia for 5 min in gerbils. In this study, 5-min ischemia evoked neuronal death (loss) of pyramidal neurons in the CA1 field, but not in the CA2/3 fields, at 5 days postischemia. In the sham group, CD200 expression was found in pyramidal neurons of the CA1 field, and the immunoreactivity in the group with ischemia was decreased at 6 h postischemia, dramatically increased at 12 h postischemia, decreased (to level found at 6 h postischemia) at 1 and 2 days postischemia, and significantly increased again at 5 days postischemia. At 5 days postischemia, CD200 immunoreactivity was strongly expressed in microglia and GABAergic neurons. However, in the CA3 field, the change in CD200 immunoreactivity in pyramidal neurons was markedly weaker than that in the CA1 field, showing there was no expression of CD 200 in microglia and GABAergic neurons. In addition, treatment of 10 mg/kg risperidone (an atypical antipsychotic drug) after the ischemia hardly changed CD200 immunoreactivity in the CA1 field, showing that CA1 pyramidal neurons were protected from the ischemic injury. These results indicate that the transient ischemia-induced change in CD200 expression may be associated with specific and selective neuronal death in the hippocampal CA1 field following transient forebrain ischemia.

Increased Calbindin D28k Expression via Long-Term Alternate-Day Fasting Does Not Protect against Ischemia-Reperfusion Injury: A Focus on Delayed Neuronal Death, Gliosis and Immunoglobulin G Leakage.

Calbindin-D28k (CB), a calcium-binding protein, mediates diverse neuronal functions. In this study, adult gerbils were fed a normal diet (ND) or exposed to intermittent fasting (IF) for three months, and were randomly assigned to sham or ischemia operated groups. Ischemic injury was induced by transient forebrain ischemia for 5 min. Short-term memory was examined via passive avoidance test. CB expression was investigated in the Cornu Ammonis 1 (CA1) region of the hippocampus via western blot analysis and immunohistochemistry. Finally, histological analysis was used to assess neuroprotection and gliosis (microgliosis and astrogliosis) in the CA1 region. Short-term memory did not vary significantly between ischemic gerbils with IF and those exposed to ND. CB expression was increased significantly in the CA1 pyramidal neurons of ischemic gerbils with IF compared with that of gerbils fed ND. However, the CB expression was significantly decreased in ischemic gerbils with IF, similarly to that of ischemic gerbils exposed to ND. The CA1 pyramidal neurons were not protected from ischemic injury in both groups, and gliosis (astrogliosis and microgliosis) was gradually increased with time after ischemia. In addition, immunoglobulin G was leaked into the CA1 parenchyma from blood vessels and gradually increased with time after ischemic insult in both groups. Taken together, our study suggests that IF for three months increases CB expression in hippocampal CA1 pyramidal neurons; however, the CA1 pyramidal neurons are not protected from transient forebrain ischemia. This failure in neuroprotection may be attributed to disruption of the blood-brain barrier, which triggers gliosis after ischemic insults.

Hypothermia Induced by Oxcarbazepine after Transient Forebrain Ischemia Exerts Therapeutic Neuroprotection through Transient Receptor Potential Vanilloid Type 1 and 4 in Gerbils.

In the present study, we investigated the neuroprotective effect of post-ischemic treatment with oxcarbazepine (OXC; an anticonvulsant compound) against ischemic injury induced by transient forebrain ischemia and its mechanisms in gerbils. Transient ischemia was induced in the forebrain by occlusion of both common carotid arteries for 5 min under normothermic conditions (37 ± 0.2 °C). The ischemic gerbils were treated with vehicle, hypothermia (whole-body cooling; 33.0 ± 0.2 °C), or 200 mg/kg OXC. Post-ischemic treatments with vehicle and hypothermia failed to attenuate and improve, respectively, ischemia-induced hyperactivity and cognitive impairment (decline in spatial and short-term memory). However, post-ischemic treatment with OXC significantly attenuated the hyperactivity and the cognitive impairment, showing that OXC treatment significantly reduced body temperature (to about 33 °C). When the hippocampus was histopathologically examined, pyramidal cells (principal neurons) were dead (lost) in the subfield Cornu Ammonis 1 (CA1) of the gerbils treated with vehicle and hypothermia on Day 4 after ischemia, but these cells were saved in the gerbils treated with OXC. In the gerbils treated with OXC after ischemia, the expression of transient receptor potential vanilloid type 1 (TRPV1; one of the transient receptor potential cation channels) was significantly increased in the CA1 region compared with that in the gerbils treated with vehicle and hypothermia. In brief, our results showed that OXC-induced hypothermia after transient forebrain ischemia effectively protected against ischemia-reperfusion injury through an increase in TRPV1 expression in the gerbil hippocampal CA1 region, indicating that TRPV1 is involved in OXC-induced hypothermia.

Long-Term Alternating Fasting Increases Interleukin-13 in the Gerbil Hippocampus, But Does Not Protect BBB and Pyramidal Neurons From Ischemia-Reperfusion Injury.

It is questionable whether intermittent fasting (IF) protects against brain ischemic injury. This study examined whether IF increased anti-inflammatory cytokines and protected neurons from ischemia-reperfusion injury in the gerbil hippocampus. Gerbils were subjected to 1-day alternating fasting as IF for 1, 2, or 3 months and assigned to sham or 5 min of transient ischemia. We examined the changes in anti-inflammatory cytokines (IL-4 and IL-13), neurons and IgG by immunohistochemistry or immunofluorescence staining in the cornu ammonis 1 (CA1) region of the hippocampus before and after ischemia. IF increased IL-13 immunoreactivity in the CA1 region before ischemia, but did not affect IL-4 immunoreactivity. After ischemia, IL-13 and 4 immunoreactivities in the CA1 region were significantly lower in IF gerbils than in non-IF gerbils. In the IF gerbils, the CA1 pyramidal neurons were not protected from ischemic injury; in these gerbils, strong IgG immunoreactivity was seen in the CA1 parenchyma, indicating leakage of the BBB. In brief, IF increased IL-13 in the CA1 region, but these neurons were not protected from transient ischemic injury evidenced by IgG immunoreactivity in the CA1 parenchyma. This study indicates that IF increased some anti-inflammatory cytokines but did not afford neuroprotection against ischemic insults via BBB disruption.

Laminarin Pretreatment Provides Neuroprotection against Forebrain Ischemia/Reperfusion Injury by Reducing Oxidative Stress and Neuroinflammation in Aged Gerbils.

Laminarin is a polysaccharide isolated from brown algae that has various biological and pharmacological activities, such as antioxidant and anti-inflammatory properties. We recently reported that pretreated laminarin exerted neuroprotection against transient forebrain ischemia/reperfusion (IR) injury when we pretreated with 50 mg/kg of laminarin once a day for seven days in adult gerbils. However, there have been no studies regarding a neuroprotective effect of pretreated laminarin against IR injury in aged animals and its related mechanisms. Therefore, in this study, we intraperitoneally inject laminarin (50 mg/kg) once a day to aged gerbils for seven days before IR (5-min transient ischemia) surgery and examine the neuroprotective effect of laminarin treatment and the mechanisms in the gerbil hippocampus. IR injury in vehicle-treated gerbils causes loss (death) of pyramidal neurons in the hippocampal CA1 field at five days post-IR. Pretreatment with laminarin effectively protects the CA1 pyramidal neurons from IR injury. Regarding the laminarin-treated gerbils, production of superoxide anions, 4-hydroxy-2-nonenal expression and pro-inflammatory cytokines [interleukin(IL)-1ß and tumor necrosis factor-α] expressions are significantly decreased in the CA1 pyramidal neurons after IR. Additionally, laminarin treatment significantly increases expressions of superoxide dismutase and anti-inflammatory cytokines (IL-4 and IL-13) in the CA1 pyramidal neurons before and after IR. Taken together, these findings indicate that laminarin can protect neurons from ischemic brain injury in an aged population by attenuating IR-induced oxidative stress and neuroinflammation.

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.

Effects of regional body temperature variation during asphyxial cardiac arrest on mortality and brain damage in a rat model.

To date, hypothermia has focused on improving rates of resuscitation to increase survival in patients sustaining cardiac arrest (CA). Towards this end, the role of body temperature in neuronal damage or death during CA needs to be determined. However, few studies have investigated the effect of regional temperature variation on survival rate and neurological outcomes. In this study, adult male rats (12 week-old) were used under the following four conditions: (i) whole-body normothermia (37 ± 0.5 °C) plus (+) no asphyxial CA, (ii) whole-body normothermia + CA, (iii) whole-body hypothermia (33 ± 0.5 °C)+CA, (iv) body hypothermia/brain normothermia + CA, and (v) brain hypothermia/body normothermia + CA. The survival rate after resuscitation was significantly elevated in groups exposed to whole-body hypothermia plus CA and body hypothermia/brain normothermia plus CA, but not in groups exposed to whole-body normothermia combined with CA and brain hypothermia/body normothermia plus CA. However, the group exposed to hypothermia/brain normothermia combined with CA exhibited higher neuroprotective effects against asphyxial CA injury, i.e. improved neurological deficit and neuronal death in the hippocampus compared with those involving whole-body normothermia combined with CA. In addition, neurological deficit and neuronal death in the group of rat exposed to brain hypothermia/body normothermia and CA were similar to those in the rats subjected to whole-body normothermia and CA. In brief, only brain hypothermia during CA was not associated with effective survival rate, neurological function or neuronal protection compared with those under body (but not brain) hypothermia during CA. Our present study suggests that regional temperature in patients during CA significantly affects the outcomes associated with survival rate and neurological recovery.

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.

Fluoro-Jade B histofluorescence staining detects dentate granule cell death after repeated five-minute transient global cerebral ischemia.

Transient global cerebral ischemia (tGCI)-induced neuronal damage is variable according to its duration and degree. There are many studies on the damage or death of pyramidal cells of the hippocampus proper (CA1-3) in rodent models of tGCI. However, studies on the death of granule cells in the hippocampal dentate gyrus (DG) following tGCI have not yet been addressed. In this study, we examined the damage/death of granule cells in the gerbil DG at 5 days after various durations (5, 10, and 15 min) of single tGCI and repeated tGCI (two 5-min tGCI with 1-h interval) using cresyl violet staining, NeuN immunohistochemistry and Fluoro-Jade B (F-J B) histofluorescence staining. Neuronal death was observed only in the polymorphic layer in all single tGCI-operated groups. However, in the repeated tGCI-operated group, massive neuronal death was observed in the granule cell layer as well as in the polymorphic layer by using F-J B histofluorescence staining. In addition, microgliosis in the DG was significantly increased in the repeated tGCI-operated group compared to the 15-min tGCI-operated group. Taken together, these findings indicate that repeated brief tGCI causes granule cell death in the DG which could not occur by a longer duration of single tGCI.

Antioxidant Properties of Fucoidan Alleviate Acceleration and Exacerbation of Hippocampal Neuronal Death Following Transient Global Cerebral Ischemia in High-Fat Diet-Induced Obese Gerbils.

Fucoidan, a natural sulfated polysaccharide, displays various biological activities including antioxidant properties. We examined the neuroprotective effect of fucoidan against transient global cerebral ischemia (tGCI) in high-fat diet (HFD)-induced obese gerbils and its related mechanisms. Gerbils received HFD for 12 weeks and fucoidan (50 mg/kg) daily for the last 5 days during HFD exposure, and they were subjected to 5-min tGCI. Pyramidal cell death was observed only in the CA 1 area (CA1) of the hippocampus in non-obese gerbils 5 days after tGCI. However, in obese gerbils, pyramidal cell death in the CA1 and CA2/3 occurred at 2 days and 5 days, respectively, after tGCI. In the obese gerbils, oxidative stress indicators (dihydroethidium, 8-hydroxyguanine and 4-hydroxy-2-nonenal) were significantly enhanced and antioxidant enzymes (SOD1 and SOD2) were significantly reduced in pre- and post-ischemic phases compared to the non-obese gerbils. Fucoidan treatment attenuated acceleration and exacerbation of tGCI-induced neuronal death in the CA1⁻3, showing that oxidative stress was significantly reduced, and antioxidant enzymes were significantly increased in pre- and post-ischemic phases. These findings indicate that pretreated fucoidan can relieve the acceleration and exacerbation of ischemic brain injury in an obese state via the attenuation of obesity-induced severe oxidative damage.

Fate of Astrocytes in The Gerbil Hippocampus After Transient Global Cerebral Ischemia.

Neuronal death and reactive gliosis are major features of brain tissue damage following transient global cerebral ischemia (tgCI). This study investigated long-term changes in neuronal death and astrogliosis in the gerbil hippocampus for 180 days after 5 min of tgCI. A massive loss of pyramidal neurons was found in the hippocampal CA1 area (CA1) area between 5 and 30 days after tgCI by Fluoro-Jade B (FJB, a marker for neuronal degeneration) histofluorescence staining, but pyramidal neurons in the CA2/3 area did not die. The reaction of astrocytes (astrogliosis) was examined by glial fibrillary acidic protein (GFAP) immunohistochemistry. Morphological change or degeneration (death) of the astrocytes was found in the CA1 area after tgCI, but, in the CA2/3 area, astrogliosis was hardly shown. GFAP immunoreactive astrocytes in the CA1 area was significantly increased in number with time and peaked at 30 days after tgCI, and they began to be degenerated or dead from 40 days after tgCI. The effect was examined by double immunofluorescence staining for FJB and GFAP. The number of FJB/GFAP⁺ cells (degenerating astrocytes) was gradually increased with time after tgCI. At 180 days after tgCI, FJB/GFAP⁺ cells were significantly decreased, but FJB⁺ cells (dead astrocytes) were significantly increased. In brief, 5 min of tgCI induced a progressive degeneration of CA1 pyramidal neurons from 5 until 30 days with an increase of reactive astrocytes, and, thereafter, astrocytes were degenerated with time and dead at later times. This phenomenon might be shown due to the death of neurons.

Risperidone Treatment after Transient Ischemia Induces Hypothermia and Provides Neuroprotection in the Gerbil Hippocampus by Decreasing Oxidative Stress.

Compelling evidence from preclinical and clinical studies has shown that mild hypothermia is neuroprotective against ischemic stroke. We investigated the neuroprotective effect of post-risperidone (RIS) treatment against transient ischemic injury and its mechanisms in the gerbil brain. Transient ischemia (TI) was induced in the telencephalon by bilateral common carotid artery occlusion (BCCAO) for 5 min under normothermic condition (37 ± 0.2 °C). Treatment of RIS induced hypothermia until 12 h after TI in the TI-induced animals under uncontrolled body temperature (UBT) compared to that under controlled body temperature (CBT) (about 37 °C). Neuroprotective effect was statistically significant when we used 5 and 10 mg/kg doses (p < 0.05, respectively). In the RIS-treated TI group, many CA1 pyramidal neurons of the hippocampus survived under UBT compared to those under CBT. In this group under UBT, post-treatment with RIS to TI-induced animals markedly attenuated the activation of glial cells, an increase of oxidative stress markers [dihydroethidium, 8-hydroxy-2' -deoxyguanosine (8-OHdG), and 4-Hydroxynonenal (4-HNE)], and a decrease of superoxide dismutase 2 (SOD2) in their CA1 pyramidal neurons. Furthermore, RIS-induced hypothermia was significantly interrupted by NBOH-2C-CN hydrochloride (a selective 5-HT2A receptor agonist), but not bromocriptine mesylate (a D2 receptor agonist). Our findings indicate that RIS-induced hypothermia can effectively protect neuronal cell death from TI injury through attenuation of glial activation and maintenance of antioxidants, showing that 5-HT2A receptor is involved in RIS-induced hypothermia. Therefore, RIS could be introduced to reduce body temperature rapidly and might be applied to patients for hypothermic therapy following ischemic stroke.

Effects of Scopolamine and Melatonin Cotreatment on Cognition, Neuronal Damage, and Neurogenesis in the Mouse Dentate Gyrus.

It has been demonstrated that melatonin plays important roles in memory improvement and promotes neurogenesis in experimental animals. We examined effects of melatonin on cognitive deficits, neuronal damage, cell proliferation, neuroblast differentiation and neuronal maturation in the mouse dentate gyrus after cotreatment of scopolamine (anticholinergic agent) and melatonin. Scopolamine (1 mg/kg) and melatonin (10 mg/kg) were intraperitoneally injected for 2 and/or 4 weeks to 8-week-old mice. Scopolamine treatment induced significant cognitive deficits 2 and 4 weeks after scopolamine treatment, however, cotreatment of scopolamine and melatonin significantly improved spatial learning and short-term memory impairments. Two and 4 weeks after scopolamine treatment, neurons were not damaged/dead in the dentate gyrus, in addition, no neuronal damage/death was shown after cotreatment of scopolamine and melatonin. Ki67 (a marker for cell proliferation)- and doublecortin (a marker for neuroblast differentiation)-positive cells were significantly decreased in the dentate gyrus 2 and 4 weeks after scopolamine treatment, however, cotreatment of scopolamine and melatonin significantly increased Ki67- and doublecortin-positive cells compared with scopolamine-treated group. However, double immunofluorescence for NeuN/BrdU, which indicates newly-generated mature neurons, did not show double-labeled cells (adult neurogenesis) in the dentate gyrus 2 and 4 weeks after cotreatment of scopolamine and melatonin. Our results suggest that melatonin treatment recovers scopolamine-induced spatial learning and short-term memory impairments and restores or increases scopolamine-induced decrease of cell proliferation and neuroblast differentiation, but does not lead to adult neurogenesis (maturation of neurons) in the mouse dentate gyrus following scopolamine treatment.

Brain ischemic preconditioning protects against moderate, not severe, transient global cerebral ischemic injury.

Ischemic preconditioning (IPC) in the brain increases ischemic tolerance to subsequent ischemic insults. In this study, we examined whether IPC protects neurons and attenuates microgliosis or not in the hippocampus following severe transient global cerebral ischemia (TCI) in gerbils. Gerbils were assigned to 8 groups; 5- and 15-min sham operated groups, 5-min and 15-min TCI operated groups, IPC plus 5- and 15-min sham operated groups, and IPC plus 5- and 15-min TCI operated groups. IPC was induced by subjecting animals to 2-min transient ischemia 1 day before 5-min TCI for a typical transient ischemia and 15-min TCI for severe transient ischemia. Neuronal damage was examined by cresyl violet staining and Fluoro-Jade B histofluorescence staining. In addition, microglial activation was examined using immunohistochemistry for Iba-1 (a marker for microglia). Delayed neuronal death and microgliosis was found in the CA1 alone in the 5-min TCI operated group at 5 days post-ischemia, and, in the 15-min TCI operated group, neuronal death and microgliosis was shown in all CA areas (CA1-3) and the dentate gyrus. IPC displayed neuroprotection and attenuated microglial activation in the 5-min TCI operated group. However, in the 15-min TCI operated group, IPC did not show neuroprotection and not attenuate microglial activation. Our present findings indicate that IPC hardly protect against severe transient cerebral ischemic injury.