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.

Insulin and α-Tocopherol Enhance the Protective Effect of Each Other on Brain Cortical Neurons under Oxidative Stress Conditions and in Rat Two-Vessel Forebrain Ischemia/Reperfusion Injury

Clinical trials show that insulin administered intranasally is a promising drug to treat neurodegenerative diseases, but at high doses its use may result in cerebral insulin resistance. Identifying compounds which could enhance the protective effects of insulin, may be helpful to reduce its effective dose. Our aim was thus to study the efficiency of combined use of insulin and α-tocopherol (α-T) to increase the viability of cultured cortical neurons under oxidative stress conditions and to normalize the metabolic disturbances caused by free radical reaction activation in brain cortex of rats with two-vessel forebrain ischemia/reperfusion injury. Immunoblotting, flow cytometry, colorimetric, and fluorometric techniques were used. α-T enhanced the protective and antioxidative effects of insulin on neurons in oxidative stress, their effects were additive. At the late stages of oxidative stress, the combined action of insulin and α-T increased Akt-kinase activity, inactivated GSK-3beta and normalized ERK1/2 activity in cortical neurons, it was more effective than either drug action. In the brain cortex, ischemia/reperfusion increased the lipid peroxidation product content and caused Na+,K+-ATPase oxidative inactivation. Co-administration of insulin (intranasally, 0.25 IU/rat) and α-T (orally, 50 mg/kg) led to a more pronounced normalization of the levels of Schiff bases, conjugated dienes and trienes and Na+,K+-ATPase activity than administration of each drug alone. Thus, α-T enhances the protective effects of insulin on cultured cortical neurons in oxidative stress and in the brain cortex of rats with cerebral ischemia/reperfusion injury.

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.

Transient effects of forebrain ischemia on fetal heart rate variability in fetal sheep

Fetal heart rate variability (FHRV) is a key index of antenatal and intrapartum fetal well-being. FHRV is well established to be mediated by both arms of the autonomic nervous system, but it remains unknown whether higher centers in the forebrain contribute to FHRV. We tested the hypothesis that selective forebrain ischemia would impair the generation of FHRV. 16 chronically instrumented near-term fetal sheep were subjected to either forebrain ischemia induced by bilateral carotid occlusion or sham-ischemia for 30 min. Time, frequency and non-linear measures of FHRV were assessed during and for seven days after ischemia. Ischemia was associated with profound suppression of electroencephalographic (EEG) power, which remained suppressed throughout the recovery period (p<0.001). During the first 5 min of ischemia, multiple time and frequency domain measures were increased (all p<0.05) before returning back to sham levels. A delayed increase in Sample Entropy was observed during ischemia (p<0.05). For the first 3 h after ischemia there was moderate suppression of two measures of FHRV (very-low frequency power and the standard deviation of RR-intervals, both p<0.05) and increased Sample Entropy (p<0.05). Thereafter all measures of FHRV returned to control levels. In conclusion, profound forebrain ischemia sufficient to lead to severe neural injury had only transient effect on multiple measures of FHRV. These findings suggest that the forebrain makes a limited contribution to FHRV. FHRV therefore primarily originates in the hindbrain and is unlikely to provide meaningful information on forebrain neurodevelopment or metabolism.

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.

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.