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Background Neuronal injury induced in young rats by cerebral ischemia reperfusion (CIR) is known to differ substantially from that in adult rats. In the present study, we investigated the specific differences in neuronal injury induced by focal CIR between young and adult rats. Results 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining revealed a gradual increase in the infarct volume of both young and adult rats in accordance with I/R times and was significantly lower in young rats than in adult rats under the same conditions. The number of cells in the cortex showing immunoreactivity for neuronal nuclei (NeuN) gradually decreased in both young and adult rats in accordance with I/R times; these numbers were significantly higher in young rats than in adult rats under the same conditions. Similarly, as the duration of I/R increased, the degree of glial activation in the cortex penumbra region became more severe in both young and adult groups; however, glial activation was significantly lower in the cortex penumbra region of young rats when compared with that in adult rats. In addition, the expression of Beclin-1 was significantly higher in the infarct penumbra of young rats than adult rats and was more frequently co-expressed with neurons. The levels of autophagy-related proteins increased significantly in the penumbra region after I/R in both young and adult groups, this increase was more pronounced in young rats than in adult rats. Following CIR, analysis revealed significantly lower levels of pro-apoptosis-related factors and significantly higher levels of anti-apoptosis-related proteins in the young rats than in adult rats. Conclusions Collectively, the present results suggest that the the reduced levels of neuronal death after CIR in young rats were closely related to enhanced levels of autophagy and reduced levels of pro-apoptosis in neurons.

Cerebral ischemic stroke stimulates excessive reactive oxygen species, which lead to blood-brain-barrier disruption, neuron death, and aggravated cerebral infarction. Thus, it is critical to develop an antioxidant strategy for stroke treatment. Herein, we report a dietary strategy to promote stroke healing using iron oxide (Fe3O4) nanoparticles with intrinsic enzyme-like activities. We find that Fe3O4 nanozymes exhibit triple enzyme-like activities, peroxidase, catalase, and superoxide dismutase, thus potentially possessing the ability to regulate the ROS level. Importantly, intragastric administration of PEG-modified Fe3O4 nanozymes significantly reduces cerebral infarction and neuronal death in a rodent model following cerebral ischemic stroke. Ex vivo analysis shows that PEG-modified Fe3O4 nanozymes localize in the cerebral vasculature, ameliorate local redox state with decreased malondialdehyde and increased Cu/Zn SOD, and facilitate blood-brain-barrier recovery by elevating ZO-1 and Claudin-5 in the hippocampus. Altogether, our results suggest that dietary PEG-modified Fe3O4 nanozymes can facilitate blood-brain-barrier reconstruction and protect neurons following ischemic stroke.

miR-126 which is considered one of the most important miRNAs for maintaining vascular integrity, plays an important role in neuroprotection after cerebral ischemia-reperfusion (I-R). Moreover, vascular endothelial growth factor A (VEGFA), sprouty-related EVH1 domain-containing protein 1 (SPRED1), and Raf-1 are also involved in physiological processes of vascular endothelial cells (ECs). This study investigated how miR-126 changes with reperfusion time in different brain tissues after global cerebral ischemia and focal cerebral ischemia and examined the underlying mechanism miR-126 involving VEGFA, SPRED1, and Raf-1 after I-R. The results indicated decreases in the levels of miR-126-3p and miR-126-5p expression in mice and gerbils after I-R, consistent with the results after oxygen and glucose deprivation and reperfusion (OGD/R) in PC12 cells. Glial cells were activated as neuronal damage gradually increased after I-R. Inhibition of miR-126-3p exacerbated the OGD/R-induced cell death and reduced cell viability. After miR-126-3p inhibition, the levels of SPRED1 and VEGFA expression were increased, and p-Raf-1 expression was decreased after OGD/R. Moreover, based on the intervention of miR-126-3p inhibition, we found that the expression of p-Raf-1 was significantly increased after the intervention of siSPRED1, while it was not statistically significant after intervention of siVEGFA. The reduction of miR-126 expression after global and focal cerebral ischemia exacerbated neuronal death, which was closely related to increasing the SPRED1 activation and inhibiting the Raf-1 expression.

It is well known that aging induces a progressive decline in the proliferation and neural differentiation of radial glial cells (RGCs) in the hippocampal dentate gyrus (DG). The function of miR-144/451 is to activate stress-regulated molecular gene expression switches for cell proliferation and differentiation. We found that the miR-144/451 expression in the hippocampus was significantly reduced in aged mice compared to adult mice. Furthermore, the proliferation and neural differentiation of RGCs in the mouse hippocampal DG was decreased by miR-144/451 knockout (miR-144/451

The hippocampal neurogenesis occurs constitutively throughout adulthood in mammalian species, but declines with age. In this study, we overtly found that the neuroblast proliferation and differentiation in the subgranular zone and the maturation into fully functional and integrated neurons in the granule-cell layer in young gerbils following cerebral ischemia/reperfusion was much more than those in old gerbils. The neurological function and cognitive and memory-function rehabilitation in the young gerbils improved faster than those in the old one. These results demonstrated that, during long term after cerebral ischemia/reperfusion, the ability of neurogenesis and recovery of nerve function in young animals were significantly higher than that in the old animals. We found that, after 14- and 28-day cerebral ischemia/reperfusion, the phosphorylation of MEK1/2, ERK1/2, p90RSK, and MSK1/2 protein levels in the hippocampus of young gerbils was significantly much higher than that of old gerbils. The levels of autophagy-related proteins, including Beclin-1, Atg3, Atg5, and LC3 in the hippocampus were effectively maintained and elevated at 28 days after cerebral ischemia/reperfusion in the young gerbils compared with those in the old gerbils. These results indicated that an increase or maintenance of the phosphorylation of ERK1/2 signal pathway and autophagy-related proteins was closely associated with the neuroblast proliferation and differentiation and the process of maturation into neurons. Further, we proved that neuroblast proliferation and differentiation in the dentate gyrus and cognitive function were significantly reversed in young cerebral ischemic gerbils by administering the ERK inhibitor (U0126) and autophagy inhibitor (3MA). In brief, following experimental young ischemic stroke, the long-term promotion of the neurogenesis in the young gerbil’s hippocampal dentate gyrus by upregulating the phosphorylation of ERK signaling pathway and maintaining autophagy-related protein levels, it overtly improved the neurological function and cognitive and memory function.

Objective: We aimed to investigate the effect and mechanisms of action of two drug pairs [Huangqi-Chuanxiong and Sanleng-Ezhu Herb (HCSE)] on the treatment of ischemic stroke.Materials and methods: We mined the current literature related to ischemic stroke and formulated a new formulation of Chinese herbs. Then, we identified the main candidate target genes of the new formulation by network pharmacology. Next, we performed enrichment analysis of the target genes to identify the potential mechanism of action of the new formulation in the treatment of ischemic stroke. Next, we experimentally validated the mechanism of action of the new formulation against ischemic stroke. Infarct volume and neurological deficits were evaluated by 2,3,5-triphenyltetrazolium (TTC) staining and Longa’s score, respectively. The predicted pathways of signal-related proteins were detected by western blotting.Results: We mined the current literature and identified a new formulation of Chinese herbs for the treatment of ischemic stroke. The formulation included Huangqi, Chuanxiong, Sanleng and Ezhu. Next, we used network pharmacological analysis to identify 23 active compounds and 327 target genes for the new formulation. The key target genes were MAPK3, MAPK1, HSP90AA1, STAT3, PIK3R1, PIK3CA and AKT1. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed significant enrichment of the PI3K/AKT and MAPK/ERK signaling pathways. By performing experiments, we found that the new formulation reduced the infarct volume of middle cerebral artery occlusion (MCAO) induced mice and activated the PI3K/AKT and MAPK/ERK signaling pathways. These findings confirmed that the new formulation has a significant protective effect against ischemic stroke injury by activating the PI3K/AKT and MAPK/ERK signaling pathways.Conclusion: We identified a new treatment formulation for ischemic stroke by data mining and network pharmacological target prediction. The beneficial effects of the new formulation act by regulating multiple target genes and pathways. The mechanism of action of the new formulation may be related to the AKT and ERK signaling pathways. Our findings provide a theoretical basis for the effects of the new formulation on ischemic stroke injury.

Spinal cord ischemia can result from cardiac arrest. It is an important cause of severe spinal cord injury that can lead to serious spinal cord disorders such as paraplegia. Hypothermia is widely acknowledged as an effective neuroprotective intervention following cardiac arrest injury. However, studies on effects of hypothermia on spinal cord injury following asphyxial cardiac arrest and cardiopulmonary resuscitation (CA/CPR) are insufficient. The objective of this study was to examine effects of hypothermia on motor deficit of hind limbs of rats and vulnerability of their spinal cords following asphyxial CA/CPR. Experimental groups included a sham group, a group subjected to CA/CPR, and a therapeutic hypothermia group. Severe motor deficit of hind limbs was observed in the control group at 1 day after asphyxial CA/CPR. In the hypothermia group, motor deficit of hind limbs was significantly attenuated compared to that in the control group. Damage/death of motor neurons in the lumbar spinal cord was detected in the ventral horn at 1 day after asphyxial CA/CPR. Neuronal damage was significantly attenuated in the hypothermia group compared to that in the control group. These results indicated that therapeutic hypothermia after asphyxial CA/CPR significantly reduced hind limb motor dysfunction and motoneuronal damage/death in the ventral horn of the lumbar spinal cord following asphyxial CA/CPR. Thus, hypothermia might be a therapeutic strategy to decrease motor dysfunction by attenuating damage/death of spinal motor neurons following asphyxial CA/CPR.

Cerebral ischemic stroke stimulates excessive reactive oxygen species, which lead to blood-brain-barrier disruption, neuron death, and aggravated cerebral infarction. Thus, it is critical to develop an antioxidant strategy for stroke treatment. Herein, we report a dietary strategy to promote stroke healing using iron oxide (Fe

Ethnopharmacological relevance Tanshinone-Ⅰ (TSNⅠ), a member of the mainly active components of Salvia miltiorrhiza Bunge (Dan Shen), which is widely used for the treatment for modern clinical diseases including cardiovascular and cerebrovascular diseases, has been reported to show the properties of anti-oxidation, anti-inflammation, neuroprotection and other pharmacological actions. However, whether TSNⅠ can improve neuron survival and neurological function against transient focal cerebral ischemia (tMCAO) in mice is still a blank field. Aim of the study This study aims to investigate the neuroprotective effects of TSNⅠ on ischemic stroke (IS) induced by tMCAO in mice and explore the potential mechanism of TSNⅠ against IS by combining network pharmacology approach and experimental verification. Materials and methods In this study, the pivotal candidate targets of TSNⅠ against IS were screened by network pharmacology firstly. Enrichment analysis and molecular docking of those targets were performed to identify the possible mechanism of TSNⅠ against IS. Afterwards, experiments were carried out to further verify the mechanism of TSNⅠ against IS. The infarct volume and neurological deficit were evaluated by 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining and Longa respectively. Immunohistochemistry was used to observe neuronal death in the hippocampus and cortical regions by detecting the change of NeuN. The predicting pathways of signaling-related proteins were assessed by Western blot in vitro and in vivo experiments. Results In vivo, TSNⅠ was found to dose-dependently decrease mice's cerebral infarct volume induced by tMCAO. In vitro, pretreatment with TSNⅠ could increase cell viability of HT-22 cell following oxygen-glucose deprivation (OGD/R). Moreover, the results showed that 125 candidate targets were identified, Protein kinase B (AKT) signaling pathway was significantly enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and mitogen-activated protein kinases 1 (MAPK1) and AKT1 could be bound to TSNⅠ more firmly by molecular docking analysis, which implies that TSNⅠ may play a role in neuroprotection through activating AKT and MAPK signaling pathways. Meanwhile, TSNⅠ was confirmed to significantly protect neurons from injury induced by IS through activating AKT and MAPK signaling pathways. Conclusion In conclusion, our study clarifies that the mechanism of TSNⅠ against IS might be related to AKT and MAPK signaling pathways, which may provide the basic evidence for further development and utilization of TSNⅠ.

Recent studies have shown that obesity and its related metabolic dysfunction exacerbate outcomes of ischemic brain injuries in some brain areas, such as the hippocampus and cerebral cortex when they are subjected to transient ischemia. However, the impact of obesity in the striatum after brief transient ischemia has not yet been addressed. The objective of this study was to investigate effects of obesity on neuronal damage and inflammation in the striatum after transient ischemia and to examine the role of mTOR which is involved in the pathogenesis of metabolic and neurological diseases. Gerbils were fed with normal diet (ND) or high-fat diet (HFD) for 12 weeks and subjected to 5 min of transient ischemia. HFD-fed gerbils showed significant increase in body weight, blood glucose level, serum triglycerides, total cholesterol and low-density lipoprotein cholesterol without affecting food intake. Neuronal death/loss in the HFD-fed gerbils occurred in the dorsolateral striatum 2 days after transient ischemia, and neuronal loss was increased 5 days after transient ischemia, although no neuronal loss was observed in ND-fed gerbils at any time after transient ischemia. The HFD-fed gerbils showed hypertrophied microglia and further increased expressions of tumor necrosis factor-alpha, interukin-1beta, mammalian target of rapamycin (mTOR) and phosphorylated-mTOR during pre- and post-ischemic phases compared with the ND-fed gerbils. Additionally, we found that treatment with mTOR inhibitor rapamycin in the HFD-fed gerbils significantly attenuated transient ischemia-induced neuronal death in the dorsolateral striatum. These findings reveal that chronic HFD-induced obesity results in severe neuroinflammation and significant increase of mTOR activation, which could contribute to neuronal death in the stratum following 5 min of transient ischemia. Especially, abnormal mTOR activation would play a key role in mediating obesity-induced severe ischemic brain injury.

Vascular dementia affects cognition by damaging axons and myelin. Melatonin is pharmacologically associated with various neurological disorders. In this study, effects of melatonin on cognitive impairment and related mechanisms were investigated in an animal model of ischemic vascular dementia (IVD). Melatonin was intraperitoneally administered to adult gerbils after transient global cerebral ischemia (tGCI) for 25 days beginning 5 days after tGCI. Cognitive impairment was examined using a passive avoidance test and the Barnes maze test. To investigate mechanisms of restorative effects by melatonin, neuronal damage/death, myelin basic protein (MBP, a marker for myelin), Rip (a marker for oligodendrocyte), extracellular signal-regulated protein kinase1/2 (ERK1/2) and phospho-ERK1/2 (p-ERK1/2), and vesicular glutamate transporter (VGLUT)-1 (a glutamatergic synaptic marker) in the hippocampal Cornu Ammonis 1 area (CA1) were evaluated using immunohistochemistry. Melatonin treatment significantly improved tGCI-induced cognitive impairment. Death of CA1 pyramidal neurons after tGCI was not affected by melatonin treatment. However, melatonin treatment significantly increased MBP immunoreactivity and numbers of Rip-immunoreactive oligodendrocytes in the ischemic CA1. In addition, melatonin treatment significantly increased ERK1/2 and p-ERK1/2 immunoreactivities in oligodendrocytes in the ischemic CA1. Furthermore, melatonin treatment significantly increased VGLUT-1 immunoreactive structures in the ischemic CA1. These results indicate that long-term melatonin treatment after tGCI improves cognitive deficit via restoration of myelin, increase of oligodendrocytes which is closely related to the activation of ERK1/2 signaling, and increase of glutamatergic synapses in the ischemic brain area.

Animal models of scopolamine-induced amnesia are widely used to study underlying mechanisms and treatment of cognitive impairment in neurodegenerative diseases such as Alzheimer's disease (AD). Previous studies have identified that melatonin improves cognitive dysfunction in animal models. In this study, using a mouse model of scopolamine-induced amnesia, we assessed spatial and short-term memory functions for 4 weeks, investigated the expression of myelin-basic protein (MBP) in the dentate gyrus, and examined whether melatonin and scopolamine cotreatment could keep cognitive function and MBP expression. In addition, to study functions of melatonin for keeping cognitive function and MBP expression, we examined expressions of brain-derived neurotrophic factor (BDNF) and tropomycin receptor kinase B (TrkB) in the mouse dentate gyrus. Scopolamine (1 mg/kg) and melatonin (10 mg/kg) were intraperitoneally treated for 2 and 4 weeks. Two and 4 weeks after scopolamine treatment, mice showed significant cognitive impairment; however, melatonin and scopolamine cotreatment recovered cognitive impairment. Two and 4 weeks of scopolamine treatment, the density of MBP immunoreactive myelinated nerve fibers was significantly decreased in the dentate gyrus; however, scopolamine and melatonin cotreatment significantly increased the scopolamine-induced reduction of MBP expression in the dentate gyrus. Furthermore, the cotreatment of scopolamine and melatonin significantly increased the scopolamine-induced decrease of BDNF and TrKB immunoreactivity in the dentate gyrus. Taken together, our results indicate that melatonin treatment exerts anti-amnesic effect and restores the scopolamine-induced reduction of MBP expression through increasing BDNF and TrkB expressions in the mouse dentate gyrus.

Exercise improves cognitive impairments induced by transient cerebral ischemia, and modulates synaptic adhesion molecules. In this study, we investigated effects of long-term treadmill exercise on cognitive impairments and its relation to changes of synaptic cell adhesion molecule (SynCAM) 1/2/3 in the hippocampus after 5 min of transient cerebral ischemia in aged gerbils. Animals were assigned to sedentary and exercised groups, given treadmill exercise for 4 consecutive weeks from 5 days after transient ischemia, and evaluated cognitive function through passive avoidance test and Morris water maze test. SynCAM 2 protein levels were determined in the hippocampus by western blot. In addition, neuronal and synaptic changes were examined by NeuN immunohistochemistry, and SynCAM 1/2/3 and MAP2 double immunofluorescence, respectively. We found that transient cerebral ischemia led to neuronal death in the CA1 area and dentate gyrus, and impaired -memory function; however, 4 weeks of treadmill exercise improved ischemia-induced memory impairment. In addition, SynCAM 1/2/3 and SynCAM 2 expression in the hippocampus was significantly decreased in the sedentary group after transient cerebral ischemia; however, SynCAM 1/2/3 expressionand and SynCAM 2 protein level was significantly increased in the ischemic group with exercise. These results suggest that long-term treadmill exercise improves memory impairment through the restoration of decreased SynCAM 1/2/3 expression in the hippocampus induced by transient cerebral ischemia in the aged gerbil.

To examine the effect of icariin (ICA) on the cognitive impairment induced by traumatic brain injury (TBI) in mice and the underlying mechanisms related to changes in hippocampal acetylation level. The modifified free-fall method was used to establish the TBI mouse model. Mice with post-TBI cognitive impairment were randomly divided into 3 groups using the randomised block method (n=7): TBI (vehicle-treated), low-dose (75 mg/kg) and high-dose (150 mg/kg) of ICA groups. An additional sham-operated group (vehicle-treated) was employed. The vehicle or ICA was administrated by gavage for 28 consecutive days. The Morris water maze (MWM) test was conducted. Acetylcholine (ACh) content, mRNA and protein levels of choline acetyltransferase (ChAT), and protein levels of acetylated H3 (Ac-H3) and Ac-H4 were detected in the hippocampus. Compared with the sham-operated group, the MWM performance, hippocampal ACh content, mRNA and protein levels of ChAT, and protein levels of Ac-H3 and Ac-H4 were signifificantly decreased in the TBI group (P

Levetiracetam (LEV), a second-generation antiepileptic drug, is commonly prescribed to treat certain types of seizures. Few studies have investigated the effects of LEV on hippocampal neurogenesis and its related mechanisms. In the present study, we investigated the effects of LEV on cell proliferation and neuronal differentiation in the mouse hippocampal dentate gyrus (DG). We here demonstrate a dose-dependent increase in Ki-67-immunoreactive cells in the subgranular zone of the DG in LEV-treated mice, and doublecortin-immunoreactive cells were also significantly increased in the hippocampal DG of mice treated with LEV. The above results indicate that LEV could improve cell proliferation and neuroblast differentiation in the hippocampus. In addition, we also found that LEV treatment improved superoxide dismutase (SOD)2, catalase and Gpx-1 levels and increased phosphatidylinositol 3-kinase (PI3K) and phosphorylated Akt protein levels in the hippocampus. Further investigation of the molecular mechanisms underlying these effects revealed that PC12 cell was blocked by a pharmacological inhibitor of PI3K (LY294002), and that LEV treatment rapidly activated PI3K/Akt and SOD2, catalase and Gpx-1. In brief, our results indicate that LEV enhanced cell proliferation and neuroblast differentiation by increasing the expression of antioxidants and PI3K and the level of phosphorylated Akt in the mouse hippocampus.

Melatonin is known to improve cognitive deficits, and its functions have been studied in various disease models, including Alzheimer's disease. In this study, we investigated effects of melatonin on cognition and the cholinergic system of the septum and hippocampus in a mouse model of scopolamine-induced amnesia. Scopolamine (1 mg/kg) and melatonin (10 mg/kg) were administered intraperitoneally to mice for 2 and 4 weeks. The Morris water maze and passive avoidance tests revealed that both treatments of scopolamine significantly impaired spatial learning and memory; however, 2- and 4-week melatonin treatments significantly improved spatial learning and memory. In addition, scopolamine treatments significantly decreased protein levels and immunoreactivities of choline acetyltransferase (ChAT), high-affinity choline transporter (CHT), vesicular acetylcholine transporter (VAChT), and muscarinic acetylcholine receptor M1 (M1R) in the septum and hippocampus. However, the treatments with melatonin resulted in increased ChAT-, CHT-, VAChT-, and M1R-immunoreactivities and their protein levels in the septum and hippocampus. Our results demonstrate that melatonin treatment is effective in improving the cognitive deficits via restoration of the cholinergic system in the septum and hippocampus of a mouse model of scopolamine-induced amnesia.

Ischemic preconditioning elicited by a non-fatal brief occlusion of blood flow has been applied for an experimental therapeutic strategy against a subsequent fatal ischemic insult. In this study, we investigated the neuroprotective effects of ischemic preconditioning (2-minute transient cerebral ischemia) on calbindin D28k immunoreactivity in the gerbil hippocampal CA1 area following a subsequent fatal transient ischemic insult (5-minute transient cerebral ischemia). A large number of pyramidal neurons in the hippocampal CA1 area died 4 days after 5-minute transient cerebral ischemia. Ischemic preconditioning reduced the death of pyramidal neurons in the hippocampal CA1 area. Calbindin D28k immunoreactivity was greatly attenuated at 2 days after 5-minute transient cerebral ischemia and it was hardly detected at 5 days post-ischemia. Ischemic preconditioning maintained calbindin D28k immunoreactivity after transient cerebral ischemia. These findings suggest that ischemic preconditioning can attenuate transient cerebral ischemia-caused damage to the pyramidal neurons in the hippocampal CA1 area through maintaining calbindin D28k immunoreactivity.

Background: Glehnia littoralis, as a traditional herbal medicine to heal various health ailments in EastAsia, displays various therapeutic properties including antioxidant effects. However, neuroprotective effects of G. littoralis against cerebral ischemic insults have not yet been addressed. Therefore, in this study, we first examined its neuroprotective effects in the hippocampus using a gerbil model of transient global cerebral ischemia (TGCI). Methods: Gerbils were subjected to TGCI for 5 min. G. littoralis extract (GLE; 100 and 200 mg/kg) was administrated orally once daily for 7 days before ischemic surgery. Neuroprotection was examined by neuronal nuclear antigen immunohistochemistry and Fluoro-Jade B histofluorescence staining. Gliosis was observed by immunohistochemistry for glial fibrillary acidic protein and ionized calcium-binding adapter molecule 1. For neuroprotective mechanisms, immunohistochemistry for superoxide dismutase (SOD) 1 and brain-derived neurotrophic factor (BDNF) was done. Results: Pretreatment with 200 mg/kg of GLE protected pyramidal neurons in the cornu ammonis 1 (CA1) area from ischemic insult area (F = 29.770, P < 0.05) and significantly inhibited activations of astrocytes (F = 22.959, P < 0.05) and microglia (F = 44.135, P < 0.05) in the ischemic CA1 area. In addition, pretreatment with GLE significantly increased expressions of SOD1 (F = 28.561, P < 0.05) and BDNF (F = 55.298, P < 0.05) in CA1 pyramidal neurons of the sham- and ischemia-operated groups. Conclusions: Our findings indicate that pretreatment with GLE can protect neurons from ischemic insults, and we suggest that its neuroprotective mechanism may be closely associated with increases of SOD1 and BDNF expressions as well as attenuation of glial activation. Key words: Antioxidant; Glial Activation; Neurotrophic Factor; Neuroprotection; Pyramidal Neurons

Ischemic stroke (IS) remains a major cause of disability and death. The changes in long non-coding RNA (lncRNA) RGD1566344 expression in the mouse cerebral cortex, including the infarct and penumbra regions after IS, are not clear. Less is known about the impact and underlying mechanisms of RGD1566344 in IS. In this study, we found that RGD1566344 levels were elevated in the ischemic infarct and penumbra regions 12 h after middle cerebral artery occlusion/reperfusion (MCAO/R) in male mice and in PC12 cells with oxygen glucose deprivation/reperfusion (OGD/R). The inhibition of RGD1566344 by small interference RNA (siRNA) significantly alleviated apoptosis in OGD/R PC12 cells. In cell transfection, quantitative real-time PCR, and Western blot experiments, we demonstrated the possible interaction of non-POU domain-containing octamer-binding protein (NONO) with RGD1566344. The NONO level in OGD/R PC12 cells was obviously increased after inhibiting the RGD1566344 treatment; subsequently the protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway was activated. This demonstrated the effect of the RGD1566344-NONO-AKT axis on neural protection after IS. These results revealed a new molecular mechanism of lncRNA RGD1566344 inhibitors through targeting NONO/AKT/mTOR signaling to protect against ischemic neuronal injury, providing strong evidence for the development of promising therapeutic strategies against IS.

A brief episode of transient ischemia (TI) can confer cerebral ischemic tolerance against a subsequent severer TI under standard condition. The brain under obesity&rsquo, s conditions is more sensitive to ischemic injury. However, the impact of a brief episode of TI under obesity&rsquo, s conditions has not been fully addressed yet. Thus, the objective of this study was to determine the effect of a brief TI in the hippocampus of high-fat diet (HFD)-induced obese gerbils and related mechanisms. Gerbils were maintained on HFD or normal diet (ND) for 12 weeks and subjected to 2 min TI. HFD gerbils were heavier, with higher blood glucose, serum total cholesterol, triglycerides, and leptin levels. Massive loss of pyramidal neurons occurred in the hippocampal cornu ammonis 1 (CA1) field of HFD animals at 5 days after 2 min of TI, but 2 min of TI did not elicit death of pyramidal neurons in ND gerbils. The HFD group showed significantly increased levels of oxidative stress indicators (dihydroethidium and 4-hydroxynonenal) and proinflammatory cytokines (tumor necrosis factor-&alpha, and interleukin-1&beta, ) and microglial activation in pre- and/or post-ischemic phases compared to the ND group. Levels of mammalian target of rapamycin (mTOR) and phosphorylated-mTOR in the CA1 field of the HFD group were also significantly higher than the ND group. On the other hand, inhibition of mTOR activation by rapamycin (an allosteric mTOR inhibitor) significantly attenuated neuronal death induced by HFD, showing reduction of HFD-induced increases of oxidative stress indicators and proinflammatory cytokines, and microglia activation. Taken together, a brief episode of TI can evoke neuronal death under obesity&rsquo, s conditions. It might be closely associated with an abnormal increase of mTOR activation-mediated, severe oxidative stress and neuroinflammation in pre- and/or post-ischemic phases.

To examine the effects of Populus tomentiglandulosa (PT) extract on the expressions of antioxidant enzymes and neurotrophic factors in the cornu ammonis 1 (CA1) region of the hippocampus at 5 min after inducing transient global cerebral ischemia (TGCI) in gerbils, TGCI was induced by occlusion of common carotid arteries for 5 min. Before ischemic surgery, 200 mg·kg-1 PT extract was orally administrated once daily for 7 d. We performed neuronal nuclear antigen immunohistochemistry and Fluoro-Jade B staining. Furthermore, we determined in situ production of superoxide anion radical, expression levels of SOD1 and SOD2 as antioxidant enzymes and brain-derived neurotrophic factor (BDNF) and insulin-like growth factor I (IGF-I) as neurotrophic factors. Pretreatment with 200 mg·kg-1 PT extract prevented neuronal death (loss). Furthermore, pretreatment with 200 mg·kg-1 PT extract significantly inhibited the production of superoxide anion radical, increased expressions of SODs and maintained expressions of BDNF and IGF-I. Such increased expressions of SODs were maintained in the neurons after IRI. In summary, pretreated PT extract can significantly increase levels of SODs and protect the neurons against TGCI, suggesting that PT can be a useful natural agent to protect against TGCI.

Gabapentin (GBP), an analgesic, adjunct antiepileptic, and migraine prophylactic drug, reduces neuronal injury induced by cerebral ischemia reperfusion (IR). However, the underlying biological molecular mechanism of GBP neuroprotection is not clear. In this study, we confirmed that dose-dependent (75 and 150 mg/kg) GBP treatment could significantly reduce IR-induced neuronal death. IR-induced neuronal death was inhibited by pretreatment with 150 mg/kg GBP in a middle cerebral artery occlusion rat model. In addition, 150 mg/kg GBP treatment remarkably promoted the levels of antioxidants and reduced the autophagy of neurons in the infarct penumbra. Moreover, the phosphoinositide-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway was activated by pretreatment with 150 mg/kg GBP, as detected by Western blot analyses. In vitro, pretreatment of PC12 cells with 450 µM GBP significantly reduced cell death induced by oxygen-glucose deprivation, increased antioxidant function, and reduced the levels of autophagy and reactive oxygen species via activation of the PI3K/Akt/mTOR pathway. This neuroprotection by GBP was inhibited significantly by 10 µM LY294002. In summary, dose-dependent pretreatment with GBP protected against cerebral IR injury via activation of the PI3K/Akt/mTOR pathway, which provided a neuroprotective function to inhibit oxidative stress-related neuronal autophagy.

Lacosamide is a new antiepileptic drug which is widely used to treat partial-onset seizures. In this study, we examined the neuroprotective effect of lacosamide against transient ischemic damage and expressions of antioxidant enzymes such as Zn-superoxide dismutase (SOD1), Mn-superoxide dismutase (SOD2), catalase (CAT) and glutathione peroxidase (GPX) in the hippocampal cornu ammonis 1 (CA1) region following 5 min of transient global cerebral ischemia in gerbils. We found that pre-treatment with 25 mg/kg lacosamide protected CA1 pyramidal neurons from transient global cerebral ischemic insult using hematoxylin-eosin staining and neuronal nuclear antigen immunohistochemistry. Transient ischemia dramatically changed expressions of SOD1, SOD2 and GPX, not CAT, in the CA1 pyramidal neurons. Lacosamide pre-treatment increased expressions of CAT and GPX, not SOD1 and 2, in the CA1 pyramidal neurons compared with controls, and their expressions induced by lacosamide pre-treatment were maintained after transient cerebral ischemia. In brief, pre-treatment with lacosamide protected hippocampal CA1 pyramidal neurons from ischemic damage induced by transient global cerebral ischemia, and the lacosamide-mediated neuroprotection may be closely related to increases of CAT and GPX expressions by lacosamide pre-treatment.

Ethnopharmacological relevance Hypericum perforatum L. (genus Hypericum, family Hypericaceae), a plant commonly used in traditional Chinese medicine, is believed to confer a wide range of benefits, including fever reduction, detoxification, calming, and pain relief via decoctions of its stems and leaves. Hyperoside (HYP), a natural compound extracted from Hypericum perforatum L., has been shown to demonstrate a wide array of bioactivities including antioxidative, anti-inflammatory, and anti-apoptotic effects. In this study, we investigated the effects of HYP on epilepsy-induced neuronal damage in mice and the associated regulatory factors. Aim of the study This study examined the potential therapeutic use of HYP for the treatment of neuronal damage in a mouse model of epilepsy and explored the relationships of the potential neuroprotective effects of HYP pretreatment with antioxidant levels and autophagy. Materials and methods ICR mice were randomly divided into six groups: sham group, sham-HYP group, KA group, KA-HYP group, KA-HYP-DDC group and KA-CQ group. Immunohistochemical staining was used to assess changes in NeuN, IBA-1, and GFAP expression in the CA3 region of the hippocampus. Immunofluorescence staining was used to assess the effects of HYP on the number of autophagosomes that accumulated in neurons in the hippocampal CA3 region. The levels of SOD1, SOD2, LC3I/II, Beclin1, and PI3K/AKT and MAPK signaling-related proteins were detected by Western blot. Results Pretreatment with 50 mg/kg HYP protected against epilepsy-induced neuronal damage in the hippocampal CA3 region. Additionally, HYP enhanced antioxidant levels and reduced the levels of autophagy-related proteins via the PI3K/AKT and MAPK pathways. Conclusion HYP protected the hippocampal CA3 region against epilepsy-induced neuronal damage via enhancing antioxidant levels and reducing autophagy. The mechanism of action may be related to the maintenance of antioxidant levels and the suppression of autophagy via the PI3K/Akt and MAPK pathways.

Although multiple reports using animal models have confirmed that melatonin appears to promote neuroprotective effects following ischemia/reperfusion-induced brain injury, the relationship between its protective effects and activation of autophagy in Purkinje cells following asphyxial cardiac arrest and cardiopulmonary resuscitation (CA/CPR) remains unclear. Rats used in this study were randomly assigned to 6 groups as follows; vehicle-treated sham operated group, vehicle-treated asphyxial CA/CPR operated group, melatonin-treated sham operated group, melatonin-treated asphyxial CA/CPR operated group, PDOT (a MT2 melatonin receptor antagonist) plus (+) melatonin-treated sham operated group and PDOT+melatonin-treated asphyxial CA/CPR operated group. Melatonin (20 mg/kg, i.p., 4 times before CA and 3 times after CA) treatment significantly improved survival rate and neurological deficit compared with the vehicle-treated asphyxial CA/CPR rats (survival rates ≥40% vs 10%), showing that melatonin treatment exhibited protective effect against asphyxial CA/CPR-induced Purkinje cell death. The protective effect of melatonin against CA/CPR-induced Purkinje cell death paralleled a remarkable attenuation of autophagy-like processes (Beclin-1, Atg7 and LC3), as well as a dramatic reduction in superoxide anion radical (O2·-), intense enhancements of CuZn superoxide dismutase (SOD1) and MnSOD (SOD2) expressions. Furthermore, the protective effect was notably reversed by treatment with PDOT, which is a selective MT2 antagonist. In brief, melatonin conferred neuroprotection against asphyxial CA/CPR-induced Purkinje cell death via inhibiting autophagic activation by reducing expressions of O2·- and increasing expressions of antioxidant enzymes, and suggests that MT2 is involved in neuroprotective effect of melatonin against Purkinje cell death caused by asphyxial CA/CPR.

Fucoidan is a sulfated polysaccharide derived from brown algae and possesses various beneficial activities, including antioxidant property. Previous studies have shown that fucoidan displays protective effect against ischemia-reperfusion injury in some organs. However, few studies have been reported regarding the protective effect of fucoidan against transient cerebral ischemic insults and its related mechanisms. Therefore, in this study, we examined the neuroprotective effect of fucoidan against transient global cerebral ischemia (tGCI), as well as underlying its mechanism using a gerbil model of tGCI which shows a loss of pyramidal neurons in the hippocampal cornu ammonis 1 (CA1) area after 5 min of tGCI. Fucoidan (25 and 50 mg/kg) was intraperitoneally administered once daily for 5 days before tGCI. Pretreatment with 50 mg/kg of fucoidan, not 25 mg/kg of fucoidan, attenuated tGCI-induced hyperactivity and protected CA1 pyramidal neurons from tGCI. In addition, pretreatment with 50 mg/kg of fucoidan inhibited activations of astrocytes and microglia in the ischemic CA1 area. Furthermore, pretreatment with 50 mg/kg of fucoidan significantly reduced the increased 4-hydroxy-2-noneal and superoxide anion radical production in the ischemic CA1 area and significantly increased expressions of SOD1 and SOD2 in the CA1 pyramidal neurons before and after tGCI. Additionally, treatment with diethyldithiocarbamate (an inhibitor of SODs) to the fucoidan-treated gerbils notably abolished the fucoidan-mediated neuroprotection. In brief, our present results indicate that fucoidan can effectively protect neurons from tGCI through attenuation of activated glial cells and reduction of oxidative stress via increase of SODs. Thus, we strongly suggest that fucoidan can be used as a useful preventive agent in cerebral ischemia.

Rufinamide is a novel antiepileptic drug and commonly used in the treatment of Lennox-Gastaut syndrome. In the present study, we investigated effects of rufinamide on cognitive function using passive avoidance test and neurogenesis in the hippocampal dentate gyrus using Ki-67 (a marker for cell proliferation), doublecortin (DCX, a marker for neuroblast) and BrdU/NeuN (markers for newly generated mature neurons) immunohistochemistry in aged gerbils. Aged gerbils (24-month old) were treated with 1 mg/kg and 3 mg/kg rufinamide for 4 weeks. Treatment with 3 mg/kg rufinamide, not 1 mg/kg rufinamide, significantly improved cognitive function and increased neurogenesis, showing that proliferating cells (Ki-67-immunoreactive cells), differentiating neuroblasts (DCX-immunoreactive neuroblasts) and mature neurons (BrdU/NeuN-immunoreactive cells) in the aged dentate gyrus compared with those in the control group. When we examined its mechanisms, rufinamide significantly increased immunoreactivities of insulin-like growth factor-1 (IGF-1), its receptor (IGF-1R), and phosphorylated cAMP response element binding protein (p-CREB). However, rufinamide did not show any increase in immunoreactivities of brain-derived neurotrophic factor and its receptor. Therefore, our results indicate that rufinamide can improve cognitive function and increase neurogenesis in the hippocampus of the aged gerbil via increasing expressions of IGF-1, IGF-1R and p-CREB.

4-Hydroxy-3-methoxybenzaldehyde (vanillin), contained in a number of species of plant, has been reported to display beneficial effects against brain injuries. In the present study, the impact of vanillin on scopolamine-induced alterations in cognition and the expression of DNA binding protein inhibitor ID-1 (ID1), one of the inhibitors of DNA binding/differentiation proteins that regulate gene transcription, in the mouse hippocampus. Mice were treated with 1 mg/kg scopolamine with or without 40 mg/kg vanillin once daily for 4 weeks. Scopolamine-induced cognitive impairment was observed from 1 week and was deemed to be severe 4 weeks following the administration of scopolamine. However, treatment with vanillin in scopolamine-treated mice markedly attenuated cognitive impairment 4 weeks following treatment with scopolamine. ID1-immunoreactive cells were revealed in the hippocampus of vehicle-treated mice, and were hardly detected 4 weeks following treatment with scopolamine. However, treatment with vanillin in scopolamine-treated mice markedly restored ID1-immunoreactive cells and expression 4 weeks subsequent to treatment. The results of the present study suggested that vanillin may be beneficial for cognitive impairment, by preventing the reduction of ID1 expression which may be associated with cognitive impairment.

Brain inflammation has a crucial role in various diseases of the central nervous system. The hippocampus in the mammalian brain exerts an important memory function, which is sensitive to various insults, including inflammation induced by exo/endotoxin stimuli. Tetanus toxin (TeT) is an exotoxin with the capacity for neuronal binding and internalization. The present study investigated changes in inflammatory mediators in the mouse hippocampus proper (CA1-3 regions) and dentate gyrus (DG) after TeT treatment. The experimental mice were intraperitoneally injected with TeT at a low dosage (100 ng/kg), while the control mice were injected with the same volume of saline. At 6, 12 and 24 h after TeT treatment, changes in the hippocampal levels of inflammatory mediators cyclooxygenase-2 (COX-2) and nuclear factor kappa-B (NF-κB/p65) were assessed using immunohistochemical and western blot analysis. In the control group, moderate COX-2 immunoreactivity was observed in the stratum pyramidal (SP) of the CA2-3 region, while almost no expression was identified in the CA1 region and the DG. COX-2 immunoreactivity was increased by TeT in the SP and granule cell layer (GCL) of the DG in a time-dependent manner. At 24 h post-treatment, COX-2 immunoreactivity in the SP of the CA1 region and in the GCL of the DG was high, and COX-2 immunoreactivity in the SP of the CA2/3 region was highest. Furthermore, the present study observed that NF-κB/p65 immunoreactivity was obviously increased in the SP and GCL at 6, 12 and 24 h after TeT treatment. In conclusion, the present study demonstrated that systemic treatment with TeT significantly increased the expression of COX-2 and NF-κB/p65 in the mouse hippo-campus, suggesting that increased COX-2 and NF-κB/65 expression may be associated with inflammation in the brain induced by exotoxins.

Lacosamide, which is a novel antiepileptic drug, has been reported to exert various additional therapeutic effects. The present study investigated the neuroprotective effects of lacosamide against transient cerebral ischemia-induced neuronal cell damage in the hippocampal cornu ammonis (CA)-1 region of a gerbil model. Neuronal Nuclei immunohistochemistry demonstrated that pre- and post-surgical treatment (5 min ischemia) with 25 mg/kg lacosamide protected CA1 pyramidal neurons in the lacosamide-treated-ischemia-operated group from ischemic injury 5 days post-ischemia, as compared with gerbils in the vehicle-treated-ischemia-operated group. Furthermore, treatment with 25 mg/kg lacosamide markedly attenuated the activation of astrocytes and microglia in the ischemic CA1 region at 5 days post-ischemia. The results of the present study suggested that pre- and post-surgical treatment of the gerbils with lacosamide was able to protect against transient cerebral ischemic injury-induced CA1 pyramidal neuronal cell death in the hippocampus. In addition, the neuroprotective effects of lacosamide may be associated with decreased activation of glial cells in the ischemic CA1 region.

Oenanthe javanica is an aquatic perennial herb that belongs to the Oenanthe genus in Apiaceae family, and it displays well-known medicinal properties such as protective effects against glutamate-induced neurotoxicity. However, few studies regarding effects of Oenanthe javanica on neurogenesis in the brain have been reported. In this study, we examined the effects of a normal diet and a diet containing ethanol extract of Oenanthe javanica on cell proliferation and neuroblast differentiation in the subgranular zone of the hippocampal dentate gyrus of adolescent rats using Ki-67 (an endogenous marker for cell proliferation) and doublecortin (a marker for neuroblast). Our results showed that Oenanthe javanica extract significantly increased the number of Ki-67-immunoreactive cells and doublecortin-immunoreactive neuroblasts in the subgranular zone of the dentate gyrus in the adolescent rats. In addition, the immunoreactivity of brain-derived neurotrophic factor was significantly increased in the dentate gyrus of the Oenanthe javanica extract-treated group compared with the control group. However, we did not find that vascular endothelial growth factor expression was increased in the Oenanthe javanica extract-treated group compared with the control group. These results indicate that Oenanthe javanica extract improves cell proliferation and neuroblast differentiation by increasing brain-derived neurotrophic factor immunoreactivity in the rat dentate gyrus.

Insulin-like growth factor 1 (IGF-1) is a well-known growth factor with well-defined neuroprotective effects against cerebral ischemia. However, the age-dependent differences in the expression of IGF-1 and its receptor (IGF-1R) in the brain following transient cerebral ischemia (TCI) have not been elucidated. In the present study, the differences in IGF-1 and IGF-1R in the gerbil hippocampal CA1 region of young and adult gerbils 5 min following TCI were determined. Seven days following TCI, the neuronal death in the hippocampal CA1 region of young gerbils was significantly less than that observed in adult gerbils. In addition, the immunoreactivity, and levels of IGF-1 and IGF-1R in the CA1 region of the normal young were higher than those in the normal adult. Four days following TCI, the immunoreactivity, and protein levels of IGF-1 and IGF-1R were markedly decreased in the adult group. By contrast, in the young group, the immunoreactivity and expression levels were much greater than those in the adult group. However, 7 days following TCI, all immunoreactivity and expression levels were markedly decreased when compared with those in the normal adult and young groups. In addition, the immunoreactivity and expression levels in the young groups were significantly higher than those of the adult groups. In conclusion, the present study demonstrated that the higher and sustained expression of IGF-1 and IGF-1R in the young gerbil hippocampal CA1 region following TCI may be associated with the reduced neuronal death compared to that in the adults.

GABAergic projections terminate on numerous hippocampal interneurons containing calcium binding proteins (CBPs), including calbindin D‑28k (CB), calretinin (CR) and parvalbumin (PV). Memory deficits and expression levels of CB, CR, and PV were examined in the hippocampal subregions following systemic scopolamine (Scop; 1 mg/kg) treatment for 4 weeks in mice. Scop treatment induced significant memory deficits from 1 week after Scop treatment. CB, CR and PV immunoreactivities distributions were in hippocampal subregions [CA1 and CA3 regions, and the dentate gyrus (DG)]. CB immunoreactivity (CB+) was gradually decreased in all subregions until 2 weeks after Scop treatment, and CB+ was decreased to the lowest level in all subregions at 3 and 4 weeks. CR+ in the CA1 region was gradually decreased until 2 weeks and hardly observed at 3 and 4 weeks; in the CA3 region, CR+ was not altered in all subregions at any time. In the DG, CR+ was gradually decreased until 2 weeks and lowest at 3 and 4 weeks. PV+ in the CA1 region was not altered at 1 week, and gradually decreased from 2 weeks. In the CA3 region, PV+ did not change in any subregions at any time. In the DG, PV+ was not altered at 1 week, decreased at 2 weeks, and lowest at 3 and 4 weeks. In brief, Scop significantly decreased CBPs expressions in the hippocampus ≥3 weeks after the treatment although memory deficits had developed at 1 week. Therefore, it is suggested that Scop (1 mg/kg) must be systemically treated for ≥3 weeks to investigate changes in expression levels of CBPs in the hippocampus.

The kainic acid (KA)-induced epilepsy experimental model is widely used to study the mechanisms underlying this disorder. Recently, the blood-brain barrier (BBB) has become an innovative alternative treatment target for epilepsy patients. KA causes neuronal injury and BBB damage in this experimental epilepsy model but the mechanisms underlying epilepsy-related neuronal injury, autophagy, and BBB damage remain unclear. Therefore, the present study investigated the relationships among neuronal injury, the expressions of autophagy-related proteins, and changes in BBB-related proteins during the acute phase of epilepsy to further understand the mechanisms and pharmacotherapy of epilepsy. NeuN immunohistochemistry and Fluoro-Jade B (FJ-B) staining in the hippocampal CA3 region revealed that neuronal death induced by intraventricular injections of 10 μg/kg KA was greater than that induced by 3 μg/kg KA. In addition, there were transient increases in the levels of microtubule-associated protein light chain 3-II (LC3I/II) and Beclin-1, which are autophagy-related proteins involved in neuronal death, in this region 24 h after the administration of 10 μg/kg KA. There were also morphological changes in BBB-related cells such as astrocytes, endothelial cells (ECs), and tight junctions (TJs). More specifically, there was a significant increase in the activation of astrocytes 72 h after the administration of 10 μg/kg KA as well as continuous increases in the expressions of platelet endothelial cell adhesion molecule-1 (PECAM-1) and BBB-related TJ proteins (Zonula occludens-1 and Claudin-5) until 72 h after KA treatment. These results suggest that the overexpression of autophagy-related proteins and astrocytes and transient increases in the expressions of BBB-related TJ proteins may be closely related to autophagic neuronal injury. These findings provide a basis for the identification of novel therapeutic targets for patients with epilepsy.

Aging is an inevitable process which results in many changes. These changes are closely related to the hippocampus which is in charge of long-term learning and episodic memory. This study was to investigate age-related changes of the cell proliferation, neuroblast differentiation and Akt/mTOR signaling in the hippocampal alveus of aged mice. In the present study, we compared the differences of neurogenesis in the hippocampal alveus between adult (postnatal month 6) and aged (postnatal month 24) mice using immunohistochemistry and western blot analysis. The cell proliferation, neuroblast differentiation, and the increased astrocyte activation in the hippocampal alveus of mice were decreased in an age-dependent manner. In addition, during normal aging, the protein level of AKT, mTOR and the phosphorylation of mTOR were all decreased. However, the protein level of AKT was increased. These results indicate the neurogenesis in the immature neurons in the hippocampal alveus of aged mice was closely related to the normal aging process. In addition, during normal aging, the increased AKT phosphorylation and decreased mTOR phosphorylation in the hippocampus may play a role in aging development. The result indicates that increased activation of astrocyte, increased phosphorylation of AKT and decreased phosphorylation of mTOR may be involved in the decreased cell proliferation and neuroblast differentiation in the alveus of hippocampus of aged mice.

Inhibitors of DNA-binding/differentiation (ID) proteins bind to basic helix-loop-helix (bHLH) transcription factors, including those that regulate differentiation and cell-cycle progression during development, and regulate gene transcription. However, little is known about the role of ID proteins in the brain under transient cerebral ischemic conditions. In the present study, we examined the effects of ischemia-reperfusion (I-R) injury on the immunoreactivity and protein levels of IDs 1–4 in the gerbil hippocampus proper Cornu Ammonis regions CA1–3 following 5 min of transient cerebral ischemia. Strong ID1 immunoreactivity was detected in the nuclei of pyramidal neurons in the hippocampal CA1–3 regions; immunoreactivity was significantly changed following I-R in the CA1 region, but not in the CA2/3 region. Five days following I-R, ID1 immunoreactivity was not detected in the CA1 pyramidal neurons. ID1 immunoreactivity was detected only in GABAergic interneurons in the ischemic CA1 region. Weak ID4 immunoreactivity was detected in non-pyramidal cells, and immunoreactivity was again only changed in the ischemic CA1 region. Five days following I-R, strong ID4 immunoreactivity was detected in non-pyramidal cells, which were identified as microglia, and not astrocytes, in the ischemic CA1 region. Furthermore, changes in the protein levels of ID1 and ID4 in the ischemic CA1 region studied by western blot were consistent with patterns of immunoreactivity. In summary, these results indicate that immunoreactivity and protein levels of ID1 and ID4 are distinctively altered following transient cerebral ischemia only in the CA1 region, and that the changes in ID1 and ID4 expression may relate to the ischemia-induced delayed neuronal death.

Background: Obesity induced by high-fat diet (HFD) is one of the most widespread metabolic disorders in current society. However, there has been little research regarding the effects of HFD-induced obesity in the septa of animal models of cerebral ischemia. Therefore, in the present study, we investigated septal effects of HFD on neuronal damage and gliosis induced by transient cerebral ischemia. Methods: Body weight, blood glucose levels and serum lipid profiles levels were measured both in the normal diet (ND) and HFD-group. We also investigated the effects of ND and HFD on neuronal damage and gliosis in the septum after transient cerebral ischemia using immunohistochemistry. Results: The levels of blood glucose, serum triglyceride, and total cholesterol were significantly increased in the HFD-fed gerbils compared with the ND-fed gerbils, although body weight was not significantly changed after HFD feeding. In the ND-fed gerbils, ischemia-induced neuronal damage was found in the septohippocampal nucleus (SHN) of the septum 7 days after ischemia. In the HFD-fed gerbils, ischemia-induced neuronal damage in the SHN was much more severe compared with that of the ND-fed gerbils 4 and 7 days after ischemia. In addition, we found that ischemia-induced glial activation including astrocytes and microglia was accelerated and exacerbated in the HFD-fed gerbils compared with that in the ND-fed gerbils. Conclusion: These results indicate that HFD can lead to much more severe effects in ischemia-induced neuronal damage/death in the septum after ischemia-reperfusion, and that it may be associated with accelerated change in glial activation.