p63 Expression in the Gerbil Hippocampus Following Transient Ischemia and Effect of Ischemic Preconditioning on p63 Expression in the Ischemic Hippocampus.

p63 is a transcription factor of p53 gene family, which are involved in development, differentiation and cell response to stress; however, its roles in ischemic preconditioning (IPC) in the brain are not clear. In the present study, we investigated the effect of IPC on p63 immunoreactivity caused by 5 min of transient cerebral ischemia in gerbils. IPC was induced by subjecting the gerbils to 2 min of transie ischemia 1 day prior to 5 min of transient ischemia. The animals were randomly assigned to four groups (sham-operated-group, ischemia-operated-group, IPC plus (+)-sham-operated-group and IPC + ischemia-operated-group). The number of viable neurons in the stratum pyramidale of the hippocampal CA1 region (CA1) was significantly increased by IPC + ischemia-operated-group compared with that in the ischemia-operated-group 5 days after ischemic insult. We found that strong p63 immunoreactivity was detected in the CA1 pyramidal neurons in the sham-operated-group, and the immunoreactivity was decreased with time after ischemia-reperfusion. In addition, strong p63 immunoreactivity was newly expressed in microglial cells of the CA1 region from 2 days after ischemia-reperfusion. In all the IPC + sham-operated-groups, p63 immunoreactivity in the CA1 pyramidal neurons was similar to that in the sham-operated-group, and the immunoreactivity was well maintained in the IPC + ischemia-operated-groups after cerebral ischemia. In brief, our present findings show that IPC dramatically protected the reduction of p63 immunoreactivity in the pyramidal neurons of the CA1 region after ischemia-reperfusion, and this result suggests that the expression of p63 may be necessary for neurons to survive after transient cerebral ischemia.

Comparison of immunoreactivities of calbindin-D28k, calretinin and parvalbumin in the striatum between young, adult and aged mice, rats and gerbils.

Calcium binding proteins play important roles in all aspects of neural functioning in the central nervous system. In the present study, we examined age-related changes of three different calcium binding proteins calbindin-D28k (CB), calretinin (CR) and parvalbumin (PV) immunoreactivities in the striatum of young (1 month), adult (6 months) and aged (24 months) ages in three species of rodents (mouse, rat and gerbil) using immunohistochemistry and Western blotting. Our results show that the number of CB-immunoreactive neurons was highest in the adult mouse and rat; however, in the gerbil, the number of CB-immunoreactive neurons was not significantly different from each group although the CB immunoreactivity was significantly decreased in the aged group compared with the adult group. The number of CR-immunoreactive neurons in the striatum was significantly highest in all the adult groups, and, especially, the number of CR-immunoreactive neurons and CR immunoreactivity in the aged gerbil were significantly decreased in the aged group compared with the other groups. Finally, we did not found any significant difference in the number of PV-immunoreactive neurons in the striatum with age among the three rodents. On the other hand, we found that protein levels of three calcium binding proteins in all the mouse groups were similar to the immunohistochemical data. These results indicate that the distribution pattern of calcium binding proteins is different according to age; the adult might show an apparent tendency of high expression in the striatum.

Effectiveness and Safety of the Korean Medicine Senior Health Promotion Program Using Herbal Medicine and Acupuncture for Mild Cognitive Impairment: A Retrospective Study of 500 Patients in Seoul, Korea.

BACKGROUND: The management of mild cognitive impairment (MCI) is becoming increasingly important. The Korean Medicine Senior Health Promotion Program (KSHPP) was developed in 2016, and it has been in use to date. This study aimed to assess the effectiveness of KSHPP using herbal medicine and acupuncture for treating MCI and the safety of herbal medicine using liver and renal function tests. METHODS: We retrospectively reviewed the medical records of the participants with MCI. We assessed the Korean version of the Montreal Cognitive Assessment (MoCA-K), the Mini-Mental State Examination-Dementia Screening (MMSE-DS), and the Geriatric Depression Scale Short Form-Korea version (GDSSF-K) scores before and after KSHPP to determine its effectiveness. To evaluate its safety, the liver and renal function tests were conducted before and after herbal treatment. RESULTS: We enrolled 1002 participants, and 500 participants satisfied the inclusion criteria. Of 500 patients, 364 (72.8%) were depressed and 136 (27.2%) were not. The mean MoCA-K score significantly increased by 2.77 for the entire sample and 3.22 for the depressed sample (all P < 0.0001). The mean MMSE-DS score significantly increased by 2.19 for the entire sample and 2.51 for the depressed sample (all P < 0.0001); the mean GDSSF-K score significantly decreased by 1.73 for the entire sample and 2.68 for the depressed sample (all P < 0.0001). CONCLUSIONS: Our findings suggest that Korean medicine interventions can improve cognitive function and depression symptoms in patients with MCI. In addition, the results of the liver and renal function tests were analyzed as surrogate outcomes to assess the safety of herbal medicine. Based on these results, we expect that Korean medicine interventions can promote the cognitive and mental health of seniors. However, as there were several study limitations, particularly study design, practice effect, and short follow-up, these results must be interpreted with caution. We need a further long-term study with a rigorous design to retain confidence in the effectiveness and safety of KSHPP.

Immunoreactivities of calbindin­D28k, calretinin and parvalbumin in the somatosensory cortex of rodents during normal aging.

Calbindin­D28k (CB), calretinin (CR) and parvalbumin (PV), which regulate cytosolic free Ca2+ concentrations in neurons, are chemically expressed in γ­aminobutyric acid (GABA)ergic neurons that regulate the degree of glutamatergic excitation and output of projection neurons. The present study investigated age­associated 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 CB­immunoreactive neurons were distributed in layers II and III, and age­associated alterations in their number were different according to the species. CR­immunoreactive neurons were not abundant in all layers; however, the number of CR­immunoreactive neurons was the highest in all adult species. Many PV­immunoreactive 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 PV­containing 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.

Roles of HIF-1α, VEGF, and NF-κB in Ischemic Preconditioning-Mediated Neuroprotection of Hippocampal CA1 Pyramidal Neurons Against a Subsequent Transient Cerebral Ischemia.

Ischemic preconditioning (IPC) provides neuroprotection against subsequent severe ischemic insults by specific mechanisms. We tested the hypothesis that IPC attenuates post-ischemic neuronal death in the gerbil hippocampal CA1 region (CA1) throughout hypoxia inducible factor-1α (HIF-1α) and its associated factors such as vascular endothelial growth factor (VEGF) and nuclear factor-kappa B (NF-κB). Lethal ischemia (LI) without IPC increased expressions of HIF-1α, VEGF, and p-IκB-α (/and translocation of NF-κB p65 into nucleus) in CA1 pyramidal neurons at 12 h and/or 1-day post-LI; thereafter, their expressions were decreased in the CA1 pyramidal neurons with time and newly expressed in non-pyramidal cells (pericytes), and the CA1 pyramidal neurons were dead at 5-day post-LI, and, at this point in time, their immunoreactivities were newly expressed in pericytes. In animals with IPC subjected to LI (IPC/LI)-group), CA1 pyramidal neurons were well protected, and expressions of HIF-1α, VEGF, and p-IκB-α (/and translocation of NF-κB p65 into nucleus) were significantly increased compared to the sham-group and maintained after LI. Whereas, treatment with 2ME2 (a HIF-1α inhibitor) into the IPC/LI-group did not preserve the IPC-mediated increases of HIF-1α, VEGF, and p-IκB-α (/and translocation of NF-κB p65 into nucleus) expressions and did not show IPC-mediated neuroprotection. In brief, IPC protected CA1 pyramidal neurons from LI by upregulation of HIF-1α, VEGF, and p-IκB-α expressions. This study suggests that IPC increases HIF-1α expression in CA1 pyramidal neurons, which enhances VEGF expression and NF-κB activation and that IPC may be a strategy for a therapeutic intervention of cerebral ischemic injury.

Neuroprotection and reduced gliosis by pre- and post-treatments of hydroquinone in a gerbil model of transient cerebral ischemia.

Hydroquinone (HQ), a major metabolite of benzene, exists in many plant-derived food and products. Although many studies have addressed biological properties of HQ including the regulation of immune responses and antioxidant activity, neuroprotective effects of HQ following ischemic insults have not yet been considered. Therefore, in this study, we examined neuroprotective effects of HQ against ischemic damage in the gerbil hippocampal cornu ammonis 1 (CA1) region following 5 min of transient cerebral ischemia. We found that pre- and post-treatments with 50 and 100 mg/kg of HQ protected CA1 pyramidal neurons from ischemic insult. Especially, pre- and post-treatments with 100 mg/kg of HQ showed strong neuroprotective effects against ischemic damage. In addition, pre- and post-treatments with 100 mg/kg of HQ significantly attenuated activations of astrocytes and microglia in the ischemic CA1 region compared to the vehicle-treated-ischemia-operated group. Briefly, these results show that pre- and post-treatments with HQ can protect neurons from transient cerebral ischemia and strongly attenuate ischemia-induced glial activation in the hippocampal CA1 region, and indicate that HQ can be used for both prevention and therapy of ischemic injury.

Pretreated duloxetine protects hippocampal CA1 pyramidal neurons from ischemia-reperfusion injury through decreases of glial activation and oxidative stress.

Duloxetine (DXT), a serotonin/norepinephrine reuptake inhibitor, is widely used for the treatment of major depressive disorders. In the present study, we investigated the neuroprotective effect of pre-treated DXT in the hippocampal CA1 region following transient global cerebral ischemia. Pre-treatment with 40mg/kg DXT protected pyramidal neurons in the CA1 region from ischemia-reperfusion injury. In addition, pre-treatment with DXT reduced ischemia-induced activations of microglia and astrocytes in the ischemic CA1 region. On the other hand, we found that pre-treatment with DXT did not increase 4-hydroxy-2-noneal (a marker for lipid peroxidation) and significantly increased the expression of Cu, Zn-superoxide dismutase, an antioxidant, in the CA1 pyramidal neurons compared with non-treated those after ischemia-reperfusion. These results indicate that pre-treated DXT has neuroprotective effect against transient global cerebral ischemia and suggest that the neuroprotective effect of DXT may be due to the attenuation of ischemia-induced glial activation as well as the decrease of oxidative stress.

Differences in the protein expression levels of Trx2 and Prx3 in the hippocampal CA1 region between adult and aged gerbils following transient global cerebral ischemia.

The thioredoxin (Trx) and peroxiredoxin (Prx) redox system is associated with neuronal damage and neuroprotective effects via the regulation of oxidative stress in brain ischemia. In the present study, ischemia-induced changes in the protein expression levels of Trx2 and Prx3 in the stratum pyramidale (SP) of the hippocampal CA1 region were investigated in adult and aged gerbils, subjected to 5 min of transient global cerebral ischemia, using immunohistochemistry and western blot analysis. In the adult ischemia-group, minimal Trx2 immunoreactivity was detected in the SP 2 days after ischemia-reperfusion. In the aged animals, the Trx2 immunoreactivity in the sham-group was marginally lower compared with that in the adult sham-group. In the aged ischemia-group, Trx2 immunoreactivity in the SP was significantly higher 1, 2 and 4 days post-ischemia, compared with that in the adult ischemia-group and, in the 5 days post-ischemia group, Trx2 immunoreactivity was significantly decreased in the SP. Prx3 immunoreactivity in the SP of the adult ischemia-group was significantly decreased from 4 days after ischemia-reperfusion. In the aged animals, Prx3 immunoreactivity in the sham-group was also marginally lower compared with that in the adult sham-group. Prx3 immunoreactivity in the aged ischemia-group was also significantly higher 1, 2 and 4 days post-ischemia, compared with the adult ischemia-group; however, the Prx3 immunoreactivity was significantly decreased 5 days post-ischemia. The western blot analyses revealed that the pattern of changes in the protein levels of Trx2 and Prx3 in the adult and aged hippocampal CA1 region following ischemic damage were similar to the results obtained in the immunohistochemical data. These findings indicated that cerebral ischemia lead to different protein expression levels of Trx2 and Prx3 in the hippocampal CA1 region between adult and aged gerbils, and these differences may be associated with more delayed neuronal death in the aged gerbil hippocampus following transient global cerebral ischemia.

Impact of hyperthermia before and during ischemia-reperfusion on neuronal damage and gliosis in the gerbil hippocampus induced by transient cerebral ischemia.

Hyperthermia can exacerbate the brain damage produced by ischemia. In the present study, we investigated the effects of hyperthermia before and during ischemia-reperfusion on neuronal damage and glial changes in the gerbil hippocampus following transient cerebral ischemia using cresyl violet staining, NeuN immunohistochemistry and Fluoro-Jade B histofluorescence staining. The animals were randomly assigned to 4 groups: (1) sham-operated animals with normothermia (normothermia + sham group); (2) ischemia-operated animals with normothermia (normothermia + ischemia group); (3) sham-operated animals with hyperthermia (hyperthermia + sham group); and (4) ischemia-operated animals with hyperthermia (hyperthermia + ischemia group). Hyperthermia (39.5 ± 0.2°C) was induced by exposing the gerbils to a heating pad connected to a rectal thermistor for 30 min before and during ischemia-reperfusion. In the normothermia+ischemia groups, a significant delayed neuronal death was observed in the stratum pyramidale (SP) of the hippocampal CA1 region (CA1) 5 days after ischemia-reperfusion. In the hyperthermia+ischemia groups, neuronal death in the SP of the CA1 occurred at 1 day post-ischemia, and neuronal death was observed in the SP of the CA2/3 region at 2 days post-ischemia. In addition, we examined activations of astrocytes and microglia using immunohistochemistry for anti-glial fibrillary acidic protein (GFAP) and anti-ionized calcium-binding adapter molecule 1 (Iba-1). GFAP-positive astrocytes and Iba-1-positive microglia in the ischemic hippocampus were activated much earlier and much more accelerated in the hyperthermia+ischemia groups than those in the normothermia+ischemia groups. Based on our findings, we suggest that an experimentally hyperthermic pre-condition before cerebral ischemic insult produces more extensive neuronal damage and glial activation in the ischemic hippocampus.

Delayed hippocampal neuronal death in young gerbil following transient global cerebral ischemia is related to higher and longer-term expression of p63 in the ischemic hippocampus.

The tumor suppressor p63 is one of p53 family members and plays a vital role as a regulator of neuronal apoptosis in the development of the nervous system. However, the role of p63 in mature neuronal death has not been addressed yet. In this study, we first compared ischemia-induced effects on p63 expression in the hippocampal regions (CA1-3) between the young and adult gerbils subjected to 5 minutes of transient global cerebral ischemia. Neuronal death in the hippocampal CA1 region of young gerbils was significantly slow compared with that in the adult gerbils after transient global cerebral ischemia. p63 immunoreactivity in the hippocampal CA1 pyramidal neurons in the sham-operated young group was significantly low compared with that in the sham-operated adult group. p63 immunoreactivity was apparently changed in ischemic hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. In the ischemia-operated adult groups, p63 immunoreactivity in the hippocampal CA1 pyramidal neurons was significantly decreased at 4 days post-ischemia; however, p63 immunoreactivity in the ischemia-operated young group was significantly higher than that in the ischemia-operated adult group. At 7 days post-ischemia, p63 immunoreactivity was decreased in the hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. Change patterns of p63 level in the hippocampal CA1 region of adult and young gerbils after ischemic damage were similar to those observed in the immunohistochemical results. These findings indicate that higher and longer-term expression of p63 in the hippocampal CA1 region of the young gerbils after ischemia/reperfusion may be related to more delayed neuronal death compared to that in the adults.

Ethanol extract of Oenanthe javanica increases cell proliferation and neuroblast differentiation in the adolescent rat dentate gyrus.

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.

Effect of Oenanthe Javanica Extract on Antioxidant Enzyme in the Rat Liver.

BACKGROUND: Oenanthe javanica (O. javanica) has been known to have high antioxidant properties via scavenging reactive oxygen species. We examined the effect of O. javanica extract (OJE) on antioxidant enzymes in the rat liver. METHODS: We examined the effect of the OJE on copper, zinc-superoxide dismutase (SOD1), manganese superoxide dismutase (SOD2), catalase (CAT), and glutathione peroxidase (GPx) in the rat liver using immunohistochemistry and western blot analysis. Sprague-Dawley rats were randomly assigned to three groups; (1) normal diet fed group (normal-group), (2) diet containing ascorbic acid (AA)-fed group (AA-group) as a positive control, (3) diet containing OJE-fed group (OJE-group). RESULTS: In this study, no histopathological finding in the rat liver was found in all the experimental groups. Numbers of SOD1, SOD2, CAT, and GPx immunoreactive cells and their protein levels were significantly increased in the AA-fed group compared with those in the normal-group. On the other hand, in the OJE-group, numbers of SOD1, SOD2, CAT, and GPx immunoreactive cells in the liver were significantly increased by about 190%, 478%, 685%, and 346%, respectively, compared with those in the AA-group. In addition, protein levels of SOD1, SOD2, CAT, and GPx in the OJE-group were also significantly much higher than those in the AA-group. CONCLUSION: OJE significantly increased expressions of SOD1 and SOD2, CAT, and GPx in the liver cells of the rat, and these suggests that significant enhancements of endogenous enzymatic antioxidants by OJE might be a legitimate strategy for decreasing oxidative stresses in the liver.

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

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

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

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

Cyclosporine A attenuates hypoxic-ischemic brain injury in newborn rats.

Cyclosporine A (CsA) is neuroprotective in ischemic brain injuries of adult animals because it blocks the permeability transition of the mitochondrial membrane. In this study, we examined the neuroprotective effect of CsA on hypoxia-ischemia (HI)-induced brain injury in newborn rats. Seven-day-old Sprague-Dawley rat pups were subjected to 2h of 8% oxygen following a unilateral carotid artery ligation. With a single dose of CsA treatment (20mg/kg, intraperitoneal) given immediately after HI, the HI-induced decrease in brain mitochondrial membrane potential measured with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) and adenosine triphosphate levels, and increase in the brain lactate level, both apoptotic and necrotic cells measured with annexin V and propidium iodide (V-PI), and infarct area measured with 2,3,5-triphenyltetrazolium chloride (TTC) were significantly attenuated at 48 h, and the reduced brain volume also significantly improved 2 weeks following HI. In summary, Cyclosporine A, a mitochondrial permeability transition blocker, significantly attenuated hypoxia-ischemia-induced lowering of the mitochondrial membrane potential, cerebral energy status, increased apoptotic and necrotic cells, and the ensuing cerebral infarction in the immature brain.