In Hye Kim

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.

Neuronal damage in hippocampal subregions induced by various durations of transient cerebral ischemia in gerbils using Fluoro-Jade B histofluorescence

Although there are many studies on ischemic brain damage in the gerbil, which is a good model of transient cerebral ischemia, studies on neuronal damage according to the duration of ischemia-reperfusion (I-R) time are limited. We carried out neuronal damage in the gerbil hippocampus after various durations of I-R (5, 10, 15 and 20 min) using Fluoro-Jade B (F-J B, a maker for neuronal degeneration) histofluorescence as well as cresyl violet (CV) staining. The changes of CV positive ((+)) neurons were well detected in the hippocampal CA1 region, not in the other regions. F-J B histofluorescence staining showed apparent neuronal damage in all the hippocampal subregions. In the CA1, most of the pyramidal neurons of the stratum pyramidale (SP) were stained with F-J B (about 100/mm(2) in a section), and F-J B(+) neurons in the other ischemia-groups were not changed. In the CA2, a few F-J B(+) neurons were detected in the SP of the 5 min ischemia-group, and F-J B(+) neurons were gradually increased with the longer time of ischemia: in the 20 min ischemia-group, the mean number of F-J B(+) neurons was about 85/mm(2) in a section. In the CA3, some F-J B(+) neurons were observed only in the SP of the 20 min ischemia-group. In the dentate gyrus, some F-J B positive neurons were detected only in the polymorphic layer (PL) of the 5 min ischemia-group, and the number of F-J B(+) neurons were gradually increased with the longer ischemic time. Our findings indicate that F-J B histofluorescence showed a very high quality of neuronal damage in all the hippocampal subregions.

Long-Term Exercise Improves Memory Deficits via Restoration of Myelin and Microvessel Damage, and Enhancement of Neurogenesis in the Aged Gerbil Hippocampus After Ischemic Stroke.

Background. The positive correlation between therapeutic exercise and memory recovery in cases of ischemia has been extensively studied; however, long-term exercise begun after ischemic neuronal death as a chronic neurorestorative strategy has not yet been thoroughly examined. Objective. The purpose of this study is to investigate possible mechanisms by which exercise ameliorates ischemia-induced memory impairment in the aged gerbil hippocampus after transient cerebral ischemia. Methods. Treadmill exercise was begun 5 days after ischemia-reperfusion (I-R) and lasted for 1 or 4 weeks. The animals were sacrificed 31 days after the induction of ischemia. Changes in short-term memory, as well as the hippocampal expression of markers of cell proliferation, neuroblast differentiation, neurogenesis, myelin and microvessel repair, and growth factors were examined by immunohistochemistry and/or western blots. Results. Four weeks of exercise facilitated memory recovery despite neuronal damage in the stratum pyramidale (SP) of the hippocampal CA1 region and in the polymorphic layer (PoL) of the dentate gyrus (DG) after I-R. Long-term exercise enhanced cell proliferation and neuroblast differentiation in a time-dependent manner, and newly generated mature cells were found in the granule cell layer of the DG, but not in the SP of the CA1 region or in the PoL of the DG. In addition, long-term exercise ameliorated ischemia-induced damage of myelin and microvessels, which was correlated with increased BDNF expression in the CA1 region and the DG. Conclusions. These results suggest that long-term treadmill exercise after I-R can restore memory function through replacement of multiple damaged structures in the ischemic aged hippocampus.

Ischemic preconditioning-induced neuroprotection against transient cerebral ischemic damage via attenuating ubiquitin aggregation

Ubiquitin binds to short-lived proteins, and denatured proteins are produced by various forms of injuries. In the present study, we investigated the effect of ischemic preconditioning (IPC) on free ubiquitin and its mutant form (ubiquitin(+1)) in the gerbil hippocampus induced by transient cerebral ischemia. The animals were randomly assigned to 4 groups (sham-operated-group, ischemia-operated-group, IPC plus (+)-sham-operated-group, and IPC+ischemia-operated-group). IPC was induced by subjecting gerbils to a 2 min of ischemia followed by 1 day of recovery. A significant loss of neurons was observed in the stratum pyramidale (SP) of the hippocampal CA1 region (CA1) in the ischemia-operated-groups 5 days after ischemia-reperfusion (I-R). In all the IPC+ischemia-operated-groups, neurons in the SP were well protected. We found that strong ubiquitin immunoreactivity was detected in the SP in the sham-operated-group and the immunoreactivity was decreased with time after I-R. In all the IPC+ischemia-operated-groups, ubiquitin immunoreactivity in the SP was similar to that in the sham-operated group. Moderate ubiquitin(+1) immunoreactivity was detected in the SP of the sham-operated-group, and the immunoreactivity was markedly increased 2 days after I-R. Five days after I-R, ubiquitin(+1) immunoreactivity was very weak in the SP. In all the IPC+ischemia-operated-groups, ubiquitin(+1) immunoreactivity in the SP was slightly decreased with time after I-R. Western blot analysis showed that, in all the IPC+ischemia-ischemia-groups, the levels of ubiquitin and ubiquitin(+1) proteins were well maintained after I-R. In brief, our findings suggest that the inhibition of the depletion of free ubiquitin and the formation of ubiquitin(+1) may have an essential role in inducing cerebral ischemic tolerance by IPC.

Neuroprotective effect of a new synthetic aspirin-decursinol adduct in experimental animal models of ischemic stroke.

Stroke is the second leading cause of death. Experimental animal models of cerebral ischemia are widely used for researching mechanisms of ischemic damage and developing new drugs for the prevention and treatment of stroke. The present study aimed to comparatively investigate neuroprotective effects of aspirin (ASA), decursinol (DA) and new synthetic aspirin-decursinol adduct (ASA-DA) against transient focal and global cerebral ischemic damage. We found that treatment with 20 mg/kg, not 10 mg/kg, ASA-DA protected against ischemia-induced neuronal death after transient focal and global ischemic damage, and its neuroprotective effect was much better than that of ASA or DA alone. In addition, 20 mg/kg ASA-DA treatment reduced the ischemia-induced gliosis and maintained antioxidants levels in the corresponding injury regions. In brief, ASA-DA, a new synthetic drug, dramatically protected neurons from ischemic damage, and neuroprotective effects of ASA-DA may be closely related to the attenuation of ischemia-induced gliosis and maintenance of antioxidants.

Neuroprotective effects of tanshinone I from Danshen extract in a mouse model of hypoxia-ischemia

Hypoxia-ischemia leads to serious neuronal damage in some brain regions and is a strong risk factor for stroke. The aim of this study was to investigate the neuroprotective effect of tanshinone I (TsI) derived from Danshen (Radix Salvia miltiorrhiza root extract) against neuronal damage using a mouse model of cerebral hypoxia-ischemia. Brain infarction and neuronal damage were examined using 2,3,5-triphenyltetrazolium chloride (TTC) staining, hematoxylin and eosin histochemistry, and Fluoro-Jade B histofluorescence. Pre-treatment with TsI (10 mg/kg) was associated with a significant reduction in infarct volume 1 day after hypoxia-ischemia was induced. In addition, TsI protected against hypoxia-ischemia-induced neuronal death in the ipsilateral region. Our present findings suggest that TsI has strong potential for neuroprotection against hypoxic-ischemic damage. These results may be used in research into new anti-stroke medications.

Neuronal damage and gliosis in the somatosensory cortex induced by various durations of transient cerebral ischemia in gerbils

Although many studies regarding ischemic brain damage in the gerbil have been reported, studies on neuronal damage according to various durations of ischemia-reperfusion (I-R) have been limited. In this study, we examined neuronal damage/death and glial changes in the somatosensory cortex 4 days after 5, 10 and 15 min of transient cerebral ischemia using the gerbil. To examine neuronal damage, we used Fluoro-Jade B (F-J B, a marker for neuronal degeneration) histofluorescence staining as well as cresyl violet (CV) staining and neuronal nuclei (NeuN, neuronal marker) immunohistochemistry. In the somatosensory cortex, some CV and NeuN positive (+) neurons were slightly decreased only in layers III and VI in the 5 min ischemia-group, and the number of CV+ and NeuN+ neurons were decreased with longer ischemic time. The F-J B histofluorescence staining showed a clear neuronal damage in layers III and VI, and the number of F-J B+ neurons was increased with time of ischemia-reperfusion: in the 15 min ischemia-group, the number of F-J B+ neurons was much higher in layer III than in layer VI. In addition, we immunohistochemically examined gliosis of astrocytes and microglia using anti-glial fibrillary acidic protein (GFAP) and anti-ionized calcium-binding adapter molecule 1 (Iba-1) antibody, respectively. In the 5 min ischemia-group, GFAP+ astrocytes and Iba-1+ microglia were distinctively increased in number, and their immunoreactivity was stronger than that in the sham-group. In the 10 and 15 min ischemia-groups, numbers of GFAP+ and Iba-1+ glial cells were much more increased with time of ischemia-reperfusion; in the 15 min ischemia-group, their distribution patterns of GFAP+ and Iba-1+ glial cells were similar to those in the 10 min ischemia-group. Our fining indicates that neuronal death/damage and gliosis of astrocytes and microglia were apparently increased with longer time of ischemia-reperfusion.

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.

Sac-1004, a vascular leakage blocker, reduces cerebral ischemia—reperfusion injury by suppressing blood–brain barrier disruption and inflammation

BackgroundBlood–brain barrier (BBB) breakdown and inflammation are critical events in ischemic stroke, contributing to aggravated brain damage. The BBB mainly consists of microvascular endothelial cells sealed by tight junctions to protect the brain from blood-borne substances. Thus, the maintenance of BBB integrity may be a potential target for neuroprotection. Sac-1004, a pseudo-sugar derivative of cholesterol, enhances the endothelial barrier by the stabilization of the cortical actin ring.ResultsHere, we report on the protective effects of Sac-1004 on cerebral ischemia-reperfusion (I/R) injury. Treatment with Sac-1004 significantly blocked the interleukin-1β-induced monolayer hyperpermeability of human brain microvascular endothelial cells (HBMECs), loss of tight junctions, and formation of actin stress fiber. Sac-1004 suppressed the expression of adhesion molecules, adhesion of U937 cells, and activation of nuclear factor-κB in HBMECs. Using a rat model of transient focal cerebral ischemia, it was shown that Sac-1004 effectively ameliorated neurological deficits and ischemic damage. In addition, Sac-1004 decreased BBB leakage and rescued tight junction-related proteins. Moreover, the staining of CD11b and glial fibrillary acidic protein showed that Sac-1004 inhibited glial activation.ConclusionsTaken together, these results demonstrate that Sac-1004 has neuroprotective activities through maintaining BBB integrity, suggesting that it is a great therapeutic candidate for stroke.

New GABAergic Neurogenesis in the Hippocampal CA1 Region of a Gerbil Model of Long-Term Survival after Transient Cerebral Ischemic Injury.

We investigated the probability of newly generated neurons that could survive and mature in the ischemic hippocampal CA1 region (CA1) of a gerbil model of transient cerebral ischemia. Neuronal death was shown in the stratum pyramidale (SP) from 4 days post‐ischemia, and a significant increase in NeuN‐positive (+) neurons was found in the SP at 180 days post‐ischemia. 5‐Bromo‐2‐deoxyuridine (BrdU)+ cells were co‐stained with NeuN and glutamic decarboxylase 67 (GAD67). Brain‐derived neurotrophic factor (BDNF) immunoreactivity and protein level was shown in nonpyramidal cells from 4 days post‐ischemia, and the immunoreactivity was strong at 30 days post‐ischemia and not significantly changed until 180 days post‐ischemia. Furthermore, TrkB immunoreactivity was co‐stained with GAD67 when we examined at 180 days post‐ischemia. Myelin basic protein (MBP)+ nerve fibers were reduced at 4 days post‐ischemia and maintained until 60 days post‐ischemia, and MBP immunoreactivity and levels were significantly increased at 180 days post‐ischemia. In the passive avoidance test, cognitive dysfunction was improved at 180 days post‐ischemia. These results suggest that the differentiation of neural progenitor cells into new GABAergic neurons may be promoted via BDNF in the ischemic CA1 and that the neurogenesis may partially mediate the recovery of cognitive impairments via increasing myelinated nerve fibers.