Yun Lyul Lee

Indole-3-propionic acid attenuates neuronal damage and oxidative stress in the ischemic hippocampus.

Tryptophan‐derived indole compounds have been widely investigated as antioxidants and as free‐radical scavengers. Indole‐3‐propionic acid (IPA), one of these compounds, is a deamination product of tryptophan. In the present study, we used Mongolian gerbils to investigate IPAs neuroprotective effects against ischemic damage and its antioxidative effects in the hippocampal CA1 region (CA1) after 5 min of transient forebrain ischemia. The repeated oral administration of IPA (10 mg/kg) for 15 days before ischemic surgery protected neurons from ischemic damage. In this group, the percentage of cresyl violet–positive neurons in the CA1 was 56.8% compared with that in the sham group. In the vehicle‐treated group, glial fibrillary acidic protein (GFAP)‐, S‐100‐, and vimentin‐immunoreactive astrocytes and ionized calcium‐binding adapter molecule 1 (Iba‐1)– and isolectin B4 (IB4)–immunoreactive microglia were activated 4 days after ischemia/reperfusion, whereas in the IPA‐treated ischemic group, GFAP, S‐100, Iba‐1, and IB4, but not vimentin, immunoreactivity was distinctly lower than that in the vehicle‐treated ischemic groups. The administration of IPA significantly decreased the level of 4‐hydroxy‐2‐nonenal, a marker of lipid peroxidation, in ischemic hippocampal homogenates compared with that in the vehicle‐treated ischemic groups at various times after ischemia/reperfusion. In addition, immunostaining for 8‐hydroxy‐2′‐deoxyguanosine showed DNA damage in pyramidal neurons in the ischemic CA1 was significantly lower in the IPA‐treated ischemic groups than in the vehicle‐treated ischemic groups. These results suggest that IPA protects neurons from ischemia‐induced neuronal damage by reducing DNA damage and lipid peroxidation.

Comparison of neuroprotective effects of five major lipophilic diterpenoids from Danshen extract against experimentally induced transient cerebral ischemic damage

We observed neuroprotective effects of five major lipophilic diterpenes derived from Danshen (Radix Salvia miltiorrhiza) extract, such as cryptotanshinone (CTs), dihydrotanshinone I (DTsI), tanshinone I (TsI), tanshinone IIA (TsIIA) and tanshinone IIB (TsIIB), in the hippocampal CA1 region (CA1) against transient ischemic damage in gerbils. These diterpenes were administered 30min before ischemia-reperfusion and the animals were sacrificed 4days after ischemia-reperfusion. In the vehicle-treated-group, cresyl violet positive (CV(+)) cells and neuronal nuclei (NeuN)(+) neurons were significantly decreased in the CA1. However, in the TsI- and CTs-treated-ischemia-groups, CV(+) and NeuN(+) neurons were abundant in the CA1. In the other groups, the number of CV(+) and NeuN(+) neurons was less than the TsI- and CTs-treated-ischemia-groups. In addition, gliosis induced by ischemic damage was apparently blocked in the TsI- and CTs-treated-ischemia-groups. These results suggest that TsI and CTs among five major lipophilic diterpenes have strong potentials for neuroprotection against ischemic damage.

Comparison of immunoreactivities in 4-HNE and superoxide dismutases in the cervical and the lumbar spinal cord between adult and aged dogs.

Aging shows slowly progressive changes and is associated with many functional and morphological changes in the central nervous system. The accumulation of reactive oxygen species causes age-related deterioration in neuronal function and contributes to the increase of disease susceptibility during normal aging. In the present study, we compared the neuronal distribution and immunoreactivities of 4-hydroxy-2E-nonenal (4-HNE, end product of lipid peroxidation), and superoxide dismutase 1 (SOD1) and SOD2 in the cervical and lumbar spinal cord between adult (2-3 years) and aged (10-12 years) dogs. No significant change in neuronal morphology was observed after cresyl violet staining. The number of NeuN (a marker for neurons)-immunoreactive neurons was not significantly changed in the aged group compare to the adult group. In addition, we could not find Fluoro-Jade B (a marker for degenerating neurons) positive cells in both the adult and aged dogs. However, numbers of 4-HNE-, SOD1- and SOD2-immunoreactive cells were significantly increased in both the cervical and lumbar spinal cord of the aged dog: The increase rates of these cells in the aged spinal cord were higher in the lumbar level than the cervical level. In brief, 4-HNE, SOD1 and SOD2 levels are much increased in the aged spinal cord compared to the adult spinal cord.

Age-related Changes in Calbindin-D28k, Parvalbumin, and Calretinin Immunoreactivity in the Dog Main Olfactory Bulb

Expression and age-related changes of calbindin-D28k (CB), parvalbumin (PV), and calretinin (CR) in the main olfactory bulb of the dog were investigated by immunohistochemistry and western blot analysis. Neurons that expressed these calcium-binding proteins showed a characteristic laminar distribution. Most of CB-immunoreactive neurons were observed in the glomerular layer (GL) and the inner sublayer of the external plexiform layer (EPL). Most of PV-immunoreactive neurons were observed in the outer sublayer of the EPL. CR-immunoreactive neurons were mainly distributed in the GL and the granule cell layer. With regard to age-related changes, CB-immunoreactive neurons in the GL were stable among all age groups; however, in the EPL they decreased with age. PV-immunoreactive neurons decreased in middle-aged and aged groups. However, CR-immunoreactive neurons were not decreased in middle-aged and aged groups. These results suggest that CB-immunoreactive neurons in the EPL were most sensitive to aging, and that their reduction may be related to aging in the dog.

Neuroprotection and reduced gliosis by atomoxetine pretreatment in a gerbil model of transient cerebral ischemia

Atomoxetine (ATX) is a non-stimulant selective norepinephrine reuptake inhibitor that is widely used for the treatment of attention-deficit/hyperactivity disorder (ADHD). In this study, we firstly examined neuroprotective effects of pre- or post-treatment with 15 and 30 mg/kg ATX against ischemic damage in the gerbil hippocampal cornus ammonis 1 (CA1) region subjected to 5 min of transient cerebral ischemia using cresyl violet staining, neuronal nuclei immunohistochemistry and Fluoro-J B histofluorescence staining. We found that only pre-treatment with 30 mg/kg ATX protected CA1 pyramidal neurons from ischemic insult. In addition, pre-treatment with 30 mg/kg ATX, which had neuroprotective effect against ischemic damage, distinctly attenuated the activation of astrocytes and microglia in the ischemic CA1 region compared with the vehicle-treated ischemia group by glial fibrillary acidic protein (for astrocytes) and ionized calcium-binding adapter molecule 1 (for microglia) immunohistochemistry. In brief, our present results indicate that ATX has neuroprotective effect against transient cerebral ischemic insult and that the neuroprotective effect of ATX may be closely associated with attenuated glial activation.

Age-Dependent Changes in Calretinin Immunoreactivity and its Protein Level in the Gerbil Hippocampus

Calretinin (CR)-immunoreactive interneurons are well known as the interneuron specific interneurons in the hippocampus. CR-immunoreactive neurons form cellular network and regulate the activity of other GABAergic inhibitory interneurons in the hippocampus. In the present study, we investigated age-related changes in CR-immunoreactive neurons and protein levels in the gerbil hippocampus during normal aging. In all subregions of the gerbil hippocampus, the number of CR-immunoreactive neurons was significantly decreased in the postnatal month 6 (PM 6) group compared to that in the PM 1 group. Thereafter, CR-immunoreactive neurons were decreased with age. In addition, the number of CR-immunoreactive cells in the subgranular zone were significantly decreased in the PM 6 group. We also observed that CR protein levels were decreased gradually with age. These results indicate that both CR immunoreactivity and its protein level were decreased with age in the gerbil hippocampus during normal aging.

Neuroprotection of a Novel Synthetic Caffeic Acid-Syringic Acid Hybrid Compound against Experimentally Induced Transient Cerebral Ischemic Damage

We investigated effects of caffeic acid, syringic acid, and their synthesis on transient cerebral ischemic damage in the gerbil hippocampal CA1 region. In the 10 mg/kg caffeic acid-, syringic acid-, and 20 mg/kg syringic-treated ischemia groups, we did not find any significant neuroprotection in the ischemic hippocampal CA region. In the 20 mg/kg caffeic acid- and 10 mg/kg caffeic acid-syringic acid-treated ischemia groups, moderate neuroprotection was found in the hippocampal CA1 region. In the 20 mg/kg caffeic acid-syringic acid-treated ischemia group, a strong neuroprotective effect was found in the ischemic hippocampal CA1 region: about 89 % of hippocampal CA1 region pyramidal neurons survived. We also observed changes in glial cells (astrocytes and microglia) in the ischemic hippocampal CA1 region in all the groups. Among them, the distribution pattern of the glial cells was only in the 20 mg/kg caffeic acid-syringic acid-treated ischemia group similar to that in the sham group (control). In brief, 20 mg/kg caffeic acid-syringic acid showed a strong neuroprotective effect with an inhibition of glia activation in the hippocampal CA1 region induced by transient cerebral ischemia.

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.

Decreased Insulin-Like Growth Factor-I and Its Receptor Expression in the Hippocampus and Somatosensory Cortex of the Aged Mouse

Insulin-like growth factor-I (IGF-I) is a multifunctional polypeptide and has diverse effects on brain functions. In the present study, we compared IGF-I and IGF-I receptor (IGF-IR) immunoreactivity and their protein levels between the adult (postnatal month 6) and aged (postnatal month 24) mouse hippocampus and somatosensory cortex. In the adult hippocampus, IGF-I immunoreactivity was easily observed in the pyramidal cells of the stratum pyramidale in the hippocampus proper and in the granule cells of the granule cell layer of the dentate gyrus. In the adult somatosensory cortex, IGF-I immunoreactivity was easily found in the pyramidal cells of layer V. In the aged groups, IGF-I expression was dramatically decreased in the cells. Like the change of IGF-I immunoreactivity, IGF-IR immunoreactivity in the pyramidal and granule cells of the hippocampus and in the pyramidal cells of the somatosensory cortex was also markedly decreased in the aged group. In addition, both IGF-I and IGF-IR protein levels were significantly decreased in the aged hippocampus and somatosensory cortex. These results indicate that the apparent decrease of IGF-I and IGF-IR expression in the aged mouse hippocampus and somatosensory cortex may be related to age-related changes in the aged brain.

Effects of ischemic preconditioning on VEGF and pFlk-1 immunoreactivities in the gerbil ischemic hippocampus after transient cerebral ischemia

Ischemia preconditioning (IPC) displays an important adaptation of the CNS to sub-lethal ischemia. In the present study, we examined the effect of IPC on immunoreactivities of VEGF-, and phospho-Flk-1 (pFlk-1) following transient cerebral ischemia in gerbils. The animals 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 gerbils to 2 min of ischemia followed by 1 day of recovery. In the ischemia-operated-group, a significant loss of neurons was observed in the stratum pyramidale (SP) of the hippocampal CA1 region (CA1) alone 5 days after ischemia-reperfusion, however, in all the IPC+ischemia-operated-groups, pyramidal neurons in the SP were well protected. In immunohistochemical study, VEGF immunoreactivity in the ischemia-operated-group was increased in the SP at 1 day post-ischemia and decreased with time. Five days after ischemia-reperfusion, strong VEGF immunoreactivity was found in non-pyramidal cells, which were identified as pericytes, in the stratum oriens (SO) and radiatum (SR). In the IPC+sham-operated- and IPC+ischemia-operated-groups, VEGF immunoreactivity was significantly increased in the SP. pFlk-1 immunoreactivity in the sham-operated- and ischemia-operated-groups was hardly found in the SP, and, from 2 days post-ischemia, pFlk-1 immunoreactivity was strongly increased in non-pyramidal cells, which were identified as pericytes. In the IPC+sham-operated-group, pFlk-1 immunoreactivity was significantly increased in both pyramidal and non-pyramidal cells; in the IPC+ischemia-operated-groups, the similar pattern of VEGF immunoreactivity was found in the ischemic CA1, although the VEGF immunoreactivity was strong in non-pyramidal cells at 5 days post-ischemia. In brief, our findings show that IPC dramatically augmented the induction of VEGF and pFlk-1 immunoreactivity in the pyramidal cells of the CA1 after ischemia-reperfusion, and these findings suggest that the increases of VEGF and Flk-1 expressions may be necessary for neurons to survive from transient ischemic damage.