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Monocarboxylate transporter 4 (MCT4) is a high‑capacity lactate transporter in cells and the alteration in MCT4 expression harms cellular survival. The present study investigated whether hypothermia affects tumor necrosis factor‑α (TNF‑α) and MCT4 immunoreactivity in the subfield cornu ammonis 1 (CA1) following cerebral ischemia/reperfusion (IR) in gerbils. Hypothermia was induced for 30 min before and during ischemia. It was found that IR‑induced death of pyramidal neurons was markedly augmented and occurred faster under hyperthermia than under normothermia. TNF‑α immunoreactivity in the pyramidal cells started to increase at 3 h after IR and peaked at 1 day after IR under normothermia. However, in hyperthermic control and sham operated gerbils, TNF‑α immunoreactivity was significantly increased compared with the normothermic gerbils, and IR under hyperthermia caused a more rapid and significant increase in TNF‑α immunoreactivity in pyramidal neurons than under normothermia. In addition, in the normothermic gerbils, MCT4 immunoreactivity began to decrease in pyramidal neurons from 3 h after IR and markedly increased at 1 and 2 days after IR. On the other hand, MCT4 immunoreactivity in pyramidal neurons of the hyperthermic gerbils was significantly increased from 3 h after IR, maintained until 1 day after IR and markedly decreased at 2 days after IR. These results indicate that acceleration of IR‑induced neuronal death under hyperthermia might be closely associated with early alteration of TNF‑α and MCT4 protein expression in the gerbil hippocampus after IR.

Although multi‑organ dysfunction is associated with the survival rate following cardiac arrest (CA), the majority of studies to date have focused on hearts and brains, and few studies have considered renal failure. The objective of the present study, therefore, was to examine the effects of therapeutic hypothermia on the survival rate, pathophysiology and antioxidant enzymes in rat kidneys following asphyxial CA. Rats were sacrificed one day following CA. The survival rate, which was estimated using Kaplan‑Meier analysis, was 42.9% one day following CA. However, hypothermia, which was induced following CA, significantly increased the survival rate (71.4%). In normothermia rats with CA, the serum blood urea nitrogen level was significantly increased one day post‑CA. In addition, the serum creatinine level was significantly increased one day post‑CA. However, in CA rats exposed to hypothermia, the levels of urea nitrogen and creatinine significantly decreased following CA. Histochemical staining revealed a significant temporal increase in renal injury after the normothermia group was subjected to CA. However, renal injury was significantly decreased in the hypothermia group. Immunohistochemical analysis of the kidney revealed a significant decrease in antioxidant enzymes (copper‑zinc superoxide dismutase, manganese superoxide dismutase, glutathione peroxidase and catalase) with time in the normothermia group. However, in the hypothermia group, these enzymes were significantly elevated following CA. Collectively, the results revealed that renal dysfunction following asphyxial CA was strongly associated with the early survival rate and therapeutic hypothermia reduced renal injury via effective antioxidant mechanisms.

Nuclear receptor related 1 protein (Nurr1), a member of the nuclear receptor 4 family of orphan nuclear receptors, has been reported to display anti‑inflammatory properties. The present study investigated the alteration of Nurr1 immunoreactivity in the gerbil hippocampus proper following 5 min of transient global cerebral ischemia. In sham operated gerbils, Nurr1 immunoreactivity was observed in pyramidal neurons in all cornu ammonis 1‑3 (CA1‑3) subfields of the hippocampus proper. In ischemia‑operated gerbils, Nurr1 immunoreactivity was altered in the CA1 subfield. Nurr1 immunoreactivity in CA1 pyramidal neurons gradually decreased until 2 days post‑ischemia, and, at 4 days post‑ischemia, Nurr1 immunoreactivity was concentrated in CA1 pyramidal neurons. Additionally, Nurr1 immunoreactivity was newly expressed in microglia in the CA1 subfield at 4 days post‑ischemia. Conversely, in the CA2/3 subfield, time‑dependent alteration of Nurr1 immunoreactivity was not identified at any time following ischemia. These results indicated that the alteration of Nurr1 expression in the CA1 subfield in the hippocampus may be associated with the death of CA1 pyramidal neurons.

Tumor necrosis factor (TNF)-α and TNF receptor 1 (TNF-R1) play diverse roles in modulating the neuronal damage induced by cerebral ischemia. The present study compared the time-dependent changes of TNF-α and TNF-R1 protein expression levels in the hippocampal subfield cornu ammonis 1 (CA1) between adult and young gerbils following transient forebrain ischemia (tFI), via western blot and immunohistochemistry analyses. In adult gerbils, delayed neuronal death of pyramidal neurons, the principal neurons in CA1, was recorded 4 days after tFI; however, in young gerbils, delayed neuronal death was recorded 7 days after tFI. TNF-α protein expression levels gradually increased in both groups following tFI; however, TNF-α expression was higher in young gerbils compared with adult gerbils. TNF-R1 protein expression levels markedly increased in both groups 1 day after tFI. Subsequently, TNF-R1 expression gradually decreased in young gerbils, whereas TNF-R1 expression levels were irregularly altered in adult gerbils following tFI. Notably, TNF-α immunoreactivity significantly increased in pyramidal neurons in both groups 1 day after tFI; however, the patterns altered between both groups. In adult gerbils, TNF-α immunoreactivity was rarely exhibited in pyramidal neurons 4 days after tFI due to neuronal death, suggesting that TNF-α immunoreactivity was newly expressed in astrocytes. In young gerbils, TNF-α immunoreactivity increased in pyramidal neurons 4 days after tFI, and TNF-α immunoreactivity was newly expressed in astrocytes. In addition, TNF-R1 immunoreactivity was exhibited in pyramidal cells of both sham groups, and significantly increased 1 day after tFI; however, the patterns altered between both groups. In adult gerbils, TNF-R1 immunoreactivity was rarely exhibited 4 days after tFI, and astrocytes newly expressed TNF-R1 immunoreactivity. In young gerbils, TNF-R1 immunoreactivity increased in pyramidal neurons 4 days after tFI; however, TNF-R1 immunoreactivity was not reported in pyramidal neurons and astrocytes thereafter. Taken together, the results of the present study suggest that different expression levels of TNF-α and TNF-R1 in ischemic CA1 between adult and young gerbils may be due to age-dependent differences of tFI-induced neuronal death.

Altered expression levels of N-methyl-D-aspartate receptor (NMDAR), a ligand-gated ion channel, have a harmful effect on cellular survival. Hyperthermia is a proven risk factor of transient forebrain ischemia (tFI) and can cause extensive and severe brain damage associated with mortality. The objective of the present study was to investigate whether hyperthermic preconditioning affected NMDAR1 immunoreactivity associated with deterioration of neuronal function in the gerbil hippocampal CA1 region following tFI via histological and western blot analyses. Hyperthermic preconditioning was performed for 1 h before tFI, which was developed by ligating common carotid arteries for 5 min. tFI-induced cognitive impairment under hyperthermia was worse compared with that under normothermia. Loss (death) of pyramidal neurons in the CA1 region occurred fast and was more severe under hyperthermia compared with that under normothermia. NMDAR1 immunoreactivity was not observed in the somata of pyramidal neurons of sham gerbils with normothermia. However, its immunoreactivity was strong in the somata and processes at 12 h post-tFI. Thereafter, NMDAR1 immunoreactivity decreased with time after tFI. On the other hand, NMDAR1 immunoreactivity under hyperthermia was significantly increased in the somata and processes at 6 h post-tFI. The change pattern of NMDAR1 immunoreactivity under hyperthermia was different from that under normothermia. Overall, accelerated tFI-induced neuronal death under hyperthermia may be closely associated with altered NMDAR1 expression compared with that under normothermia.

Calbindin‑D28k (CB), calretinin (CR) and parvalbumin (PV), which regulate cytosolic free Ca2+ concentrations in neurons, are chemically expressed in γ‑aminobutyric acid (GABA)ergic neurons that regulate the degree of glutamatergic excitation and output of projection neurons. The present study investigated age‑associated differences in CB, CR and PV immunoreactivities in the somatosensory cortex in three species (mice, rats and gerbils) of young (1 month), adult (6 months) and aged (24 months) rodents, using immunohistochemistry and western blotting. Abundant CB‑immunoreactive neurons were distributed in layers II and III, and age‑associated alterations in their number were different according to the species. CR‑immunoreactive neurons were not abundant in all layers; however, the number of CR‑immunoreactive neurons was the highest in all adult species. Many PV‑immunoreactive neurons were identified in all layers, particularly in layers II and III, and they increased in all layers with age in all species. The present study demonstrated that the distribution pattern of CB‑, CR‑ and PV‑containing neurons in the somatosensory cortex were apparently altered in number with normal aging, and that CB and CR exhibited a tendency to decrease in aged rodents, whereas PV tended to increase with age. These results indicate that CB, CR and PV are markedly altered in the somatosensory cortex, and this change may be associated with normal aging. These findings may aid the elucidation of the mechanisms of aging and geriatric disease.

Activated M1 microglia secrete proinflammatory cytokines into damaged brain areas. The present study examined activated M1 microglial morphology and expression in the hippocampal Cornu Ammonis (CA) 1 region, which is vulnerable to transient ischemia. Transient cerebral ischemia was performed for 5 min in gerbils, and neuronal death in the CA1 region following transient cerebral ischemia was confirmed using cresyl violet staining, neuronal nuclear antigen immunohistochemistry and Fluoro‑Jade B histofluorescent staining. In addition, CA1 regions were stained for cluster of differentiation (CD) 74, a marker for activated M1 microglia and a ligand for macrophage migration inhibitory factor In sham‑operated animals, no CD74 immunoreactivity was observed in the hippocampal CA1 region. CD74 immunoreactivity was not observed in the hippocampal CA1 region until 3 days post‑ischemic insult; however, elevated CD74 immunoreactivity was detected in the CA1 region from 5 days post‑ischemia. Double immunofluorescence staining for CD74 and ionized calcium‑binding adapter molecule 1, a marker for M1 microglial cells, confirmed the expression of CD74 on this microglial subtype. These results indicated that M1 microglia are activated late in the hippocampal CA1 region following ischemic stroke. Therefore, optimizing the timing of therapeutic intervention may reduce activated M1 microglial-induced neuronal damage.

Therapeutic exercise is an integral component of the rehabilitation of patients who have suffered a stroke. The objective of the present study was to use immunohistochemistry to investigate the effects of post-ischemic exercise on neuronal damage or death and gliosis in the aged gerbil hippocampus following transient cerebral ischemia. Aged gerbils (male; age, 22–24 months) underwent ischemia and were subjected to treadmill exercise for 1 or 4 weeks. Neuronal death was detected in the stratum pyramidale of the hippocampal CA1 region and in the polymorphic layer of the dentate gyrus using cresyl violet and Fluoro-Jade B histofluorescence staining. No significant difference in neuronal death was identified following 1 or 4 weeks of post-ischemic treadmill exercise. However, post-ischemic treadmill exercise affected gliosis (the activation of astrocytes and microglia). Glial fibrillary acidic protein-immunoreactive astrocytes and ionized calcium binding adaptor molecule 1-immunoreactive microglia were activated in the CA1 and polymorphic layer of the dentate gyrus of the group without treadmill exercise. Conversely, 4 weeks of treadmill exercise significantly alleviated ischemia-induced astrocyte and microglial activation; however, 1 week of treadmill exercise did not alleviate gliosis. These findings suggest that long-term post-ischemic treadmill exercise following transient cerebral ischemia does not influence neuronal protection; however, it may effectively alleviate transient cerebral ischemia-induced astrocyte and microglial activation in the aged hippocampus.

Chrysanthemum indicum Linné extract (CIL) is used in herbal medicine in East Asia. In the present study, gerbils were orally pre‑treated with CIL, and changes of antioxidant enzymes including superoxide dismutase (SOD) 1 and SOD2, catalase (CAT) and glutathione peroxidase (GPX) in the hippocampal CA1 region following 5 min of transient cerebral ischemia were investigated and the neuroprotective effect of CIL in the ischemic CA1 region was examined. SOD1, SOD2, CAT and GPX immunoreactivities were observed in the pyramidal cells of the CA1 region and their immunoreactivities were gradually decreased following ischemia‑reperfusion and barely detectable at 5 days post‑ischemia. CIL pre‑treatment significantly increased immunoreactivities of SOD1, CAT and GPX, but not SOD2, in the CA1 pyramidal cells of the sham‑operated animals. In addition, SOD1, SOD2, CAT and GPX immunoreactivities in the CA1 pyramidal cells were significantly higher compared with the ischemia‑operated animals. Furthermore, it was identified that pre‑treatment with CIL protected the CA1 pyramidal cells in the CA1 region using neuronal nuclei immunohistochemistry and Fluoro‑Jade B histofluorescence staining; the protected CA1 pyramidal cells were 67.5% compared with the sham‑operated animals. In conclusion, oral CIL pre‑treatment increased endogenous antioxidant enzymes in CA1 pyramidal cells in the gerbil hippocampus and protected the cells from transient cerebral ischemic insult. This finding suggested that CIL is promising for the prevention of ischemia‑induced neuronal damage.

Catalase (CAT) is an important antioxidant enzyme and is crucial in modulating synaptic plasticity in the brain. In this study, CAT expression as well as neuronal distribution was compared in the hippocampus among young, adult and aged mice and rats. Male ICR mice and Sprague Dawley rats were used at postnatal month (PM) 1, PM 6 and PM 24 as the young, adult and aged groups, respectively (n=14/group). CAT expression was examined by immunohistochemistry and western blot analysis. In addition, neuronal distribution was examined by NeuN immunohistochemistry. In the present study, the mean number of NeuN‑immunoreactive neurons was marginally decreased in mouse and rat hippocampi during aging, although this change was not identified to be significantly different. However, CAT immunoreactivity was significantly increased in pyramidal and granule neurons in the adult mouse and rat hippocampi and was significantly decreased in the aged mouse and rat hippocampi compared with that in the young animals. CAT protein levels in the hippocampus were also lowest in the aged mouse and rat hippocampus. These results indicate that CAT expression is significantly decreased in the hippocampi of aged animals and decreased CAT expression may be closely associated with aging.

Intermittent fasting has been shown to have neuroprotective effects against transient focal cerebral ischemic insults. However, the effects of intermittent fasting on transient global ischemic insult has not been studied much yet. The present study examined effects of intermittent fasting on endogenous antioxidant enzyme expression levels in the hippocampus and investigated whether the fasting protects neurons 5 days after 5 min of transient global cerebral ischemia. Gerbils were randomly subjected to either ad libitum or alternate-day intermittent fasting for two months and assigned to sham surgery or transient ischemia. Changes of antioxidant enzymes were examined using immunohistochemistry for cytoplasmic superoxide dismutase 1 (SOD1), mitochondrial (SOD2), catalase (CAT), and glutathione peroxidase (GPX). The effects of intermittent fasting on ischemia-induced antioxidant changes, neuronal damage/degeneration and glial activation were examined. The weight of fasting gerbils was not different from that of control gerbils. In controls, SOD1 and GPX immunoreactivities were strong in pyramidal neurons of filed cornu ammonis 1 (CA1). Transient ischemia in controls significantly decreased expressions of SOD1 and GPX in CA1 pyramidal neurons. Intermittent fasting resulted in increased expressions of SOD2 and CAT, not of SOD1 and GPX, in CA1 pyramidal neurons. Nevertheless, CA1 pyramidal neurons were not protected in gerbils subjected to fasting after transient ischemia, and inhibition of glial-cell activation was not observed in the gerbils. In summary, intermittent fasting for two months increased SOD2 and CAT immunoreactivities in hippocampal CA1 pyramidal neurons. However, fasting did not protect the CA1 pyramidal neurons from transient cerebral ischemia. The results of the present study indicate that intermittent fasting may increase certain antioxidants, but not protect neurons from transient global ischemic insult.

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.

Insulin‑like growth factor‑I (IGF‑I) is a multifunctional protein present in the central nervous system. A number of previous studies have revealed alterations in IGF‑I and its receptor (IGF‑IR) expression in various regions of the brain. However, there are few reports on age‑dependent alterations in IGF‑I and IGF‑IR expressions in the olfactory bulb, which contains the secondary neurons of the olfactory system. The present study examined the cellular morphology in the olfactory bulb by using cresyl violet (CV) staining at postnatal month (PM) 3 in the young group, PM 6 in the adult group and PM 24 in the aged group in gerbils. In addition, detailed examinations were performed of the protein levels and immunoreactivities of IGF‑I and IGF‑IR in the olfactory bulb in each group. There were no significant changes in the cellular morphology between the three groups. The protein levels and immunoreactivities of the IGF‑I and IGF‑IR were the highest in the young group and they decreased with age. He protein levels and immunoreactivities of the IGF‑I and IGF‑IR were the lowest in the aged group. In brief, our results indicate that IGF‑I and IGF‑IR expressions are strong in young olfactory bulbs and significantly reduced in aged olfactory bulbs. In conclusion, subsequent decreases in IGF‑I and IGF‑IR expression with age may be associated with olfactory decline. Further studies are required to investigate the roles of IFG‑I and IGF‑IR in disorders of the olfactory system.

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.

Neurofilaments (NFs) including neurofilament‑200 kDa (NF‑H), neurofilament‑165 kDa (NF‑M) and neurofilament‑68 kDa (NF‑L) are major protein constituents of the brain, and serve important roles in the regulation of axonal transport. NF alteration is a key feature in the pathogenesis of neurological disorders involving cognitive dysfunction. In the present study, cognitive impairments were investigated, via assessments using the Morris water maze and passive avoidance tests, in mice following chronic systemic treatment with 1 mg/kg scopolamine (SCO) for 4 weeks. SCO‑induced cognitive impairments were significantly observed 1 week following the SCO treatment, and these cognitive deficits were maintained for 4 weeks. However, the NF immunoreactivities and levels were altered differently according to the hippocampal subregion following SCO treatment. NF‑H immunoreactivity and levels were markedly altered in all hippocampal subregions, and were significantly increased 1 week following the SCO treatment; thereafter, the immunoreactivity and levels significantly decreased with time. NF‑M immunoreactivity and levels gradually decreased in the hippocampus and were significantly decreased 4 weeks following SCO treatment. NF‑L immunoreactivity and levels gradually decreased in the hippocampus, and were significantly decreased 2 and 4 weeks following SCO treatment. In conclusion, the results of the present study demonstrated that chronic systemic treatment with SCO induced cognitive impairment from 1 week following SCO treatment, and NF expression was diversely altered according to the hippocampal subregion from 1 week following SCO treatment. These results suggest that SCO‑induced changes in NF expression may be associated with cognitive impairment.

Ischemic preconditioning (IPC) is induced by exposure to brief durations of transient ischemia, which results in ischemic tolerance to a subsequent longer or lethal period of ischemia. In the present study, the effects of IPC (2 min of transient cerebral ischemia) were examined on immunoreactivity of platelet‑derived growth factor (PDGF)‑BB and on neuroprotection in the gerbil hippocampal CA1 region following lethal transient cerebral ischemia (LTCI; 5 min of transient cerebral ischemia). IPC was subjected to a 2‑min sublethal ischemia and a LTCI was given 5‑min transient ischemia. The animals in all of the groups were given recovery times of 1, 2 and 5 days and change in PDGF‑BB immunoreactivity was examined as was the neuronal damage/death in the hippocampus induced by LTCI. LTCI induced a significant loss of pyramidal neurons in the hippocampal CA1 region 5 days after LTCI, and significantly decreased PDGF‑BB immunoreactivity in the CA1 pyramidal neurons from day 1 after LTCI. Conversely, IPC effectively protected the CA1 pyramidal neurons from LTCI and increased PDGF‑BB immunoreactivity in the CA1 pyramidal neurons post‑LTCI. In conclusion, the results demonstrated that LTCI significantly altered PDGF‑BB immunoreactivity in pyramidal neurons in the hippocampal CA1 region, whereas IPC increased the immunoreactivity. These findings indicated that PDGF‑BB may be associated with IPC‑mediated neuroprotection.