Ji Hyeon Ahn

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.

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.

Changes and expressions of Redd1 in neurons and glial cells in the gerbil hippocampus proper following transient global cerebral ischemia.

Redd1 (known as RTP801/Dig2/DDIT4) is a stress-induced protein, and it is known to be regulated in response to some stresses including hypoxia and oxidative stress. In the present study, we investigated the time-dependent changes in Redd1 immunoreactivity and its protein levels in the gerbil hippocampus proper (CA1-3 regions) after 5 min of transient global cerebral ischemia using immunohistochemistry and Western blot analysis. Redd1 immunoreactivity was apparently changed in the pyramidal neurons of the ischemic CA1 region, not in the pyramidal neurons of the ischemic CA2/3 region. Redd1 immunoreactivity in the CA1 pyramidal neurons was significantly increased at 6 h post-ischemia, decreased until 1 day post-ischemia, increased again at 2 days post-ischemia and weakly observed at 5 days post-ischemia. Especially, at 5 days after ischemic damage, Redd1 immunoreactivity was newly expressed in astrocytes and GABAergic interneurons in the CA1 region. Redd1 protein levels in the ischemic CA1 region were changed like the pattern of the Redd1 immunoreactivity. These results indicate that Redd1 immunoreactivity and protein levels are increased in the ischemic CA1 region at an early time after ischemic damage and that the increased Redd1 expression may be closely related to the delayed neuronal death of the CA1 pyramidal neurons following 5 min of transient global cerebral ischemia.

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.

Comparison of inflammatory cytokines changes in the hippocampal CA1 region between the young and adult gerbil after transient cerebral ischemia

Young animals appear much less vulnerable to ischemic insults. In present study, we compared neuronal damage and changes in the immunoreactivities and levels of inflammatory cytokine, interleukin (IL-) 2 as a pro-inflammatory cytokine and its receptor (IL-2Rβ), IL-4 and IL-13 as anti-inflammatory cytokines, in the hippocampal CA1 region between adult and young gerbils after 5 min of transient cerebral ischemia. Most (about 89%) of hippocampal CA1 pyramidal neurons showed neuronal damage only in the adult gerbil at 4 days post-ischemia; in the young ischemia-group, about 61% of CA1 pyramidal neurons showed neuronal damage at 7 days post-ischemia. Thereafter, the neuronal damage in the CA1 pyramidal neurons was not significantly changed in both the groups. IL-2 and IL-2Rβ immunoreactivity in the stratum pyramidale (SP) of the CA1 region was similar in both the sham groups. At 4 days post-ischemia, IL-2 and IL-2Rβ immunoreactivity in the adult SP was dramatically decreased; however, in the young SP, they were not changed, and they were decreased at 7 days post-ischemia. IL-4 and IL-13 immunoreactivity in the SP of the young sham-group were much lower than those in the adult group. Four days after ischemia-reperfusion, they were dramatically decreased in the adult ischemia-group; however, at this time, they were markedly increased in the young ischemia-group. In brief, our findings indicate that IL-2, 2Rβ, IL-4 and IL-13 immunoreactivity in young gerbils was similar or low compared to those in the adult, and they were decreased at 4 days post-ischemia in the adult; however, at this time, they were distinctively increased in the young.

Increase in Trx2/Prx3 redox system immunoreactivity in the spinal cord and hippocampus of aged dogs

We previously reported that no distinct neuronal loss occurred in the aged dog spinal cord, although oxidative stress was increased in the aged dog spinal cord. Thioredoxin 2 (Trx2)/peroxiredoxin 3 (Prx3) redox system is a major route for removing H(2)O(2) in the central nervous system. In the present study, we compared the distribution and immunoreactivity of thioredoxin reductase 2 (TrxR2), Trx2 and Prx3 and their protein levels in the spinal cord and hippocampus between the adult (2-3 years) and aged (10-12 years) dogs. The number of TrxR2-immunoreactive neurons was slightly increased; however, its immunoreactivity was significantly increased in the aged spinal cord compared to that in the adult spinal cord. On the other hand, the number and immunoreactivity of both Trx2- and Prx3-immunoreactive neurons were significantly increased in the spinal cord of the aged dog. Similarly, in the hippocampus of the aged dog, TrxR2, Trx2 and Prx3 immunoreactivity and protein levels were markedly increased compared to those in the adult dog. These results indicate that the increases of TrxR2, Trx2 and Prx3 immunoreactivity and their protein levels in the aged spinal cord and hippocampus may contribute to reducing neuronal damage against oxidative stresses during normal aging.

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.