Tong-Chun Wen

Protection of ischemic hippocampal neurons by ginsenoside Rb1, a main ingredient of ginseng root

Our previous study showed that the oral administration of red ginseng powder before but not after transient forebrain ischemia prevented delayed neuronal death in gerbils, and that a neuroprotective molecule within red ginseng powder was ginsenoside Rb1. However, it remains to be clarified whether or not ginsenoside Rb1 acts directly on the ischemic brain, and the mechanism by which ginsenoside Rb1 protects the ischemic CA1 neurons is not determined. Without elucidation of the pharmacological property of ginsenoside Rb1, the drug would not be accepted as a neuroprotective agent. The present study demonstrated that the intracerebroventricular infusion of ginsenoside Rb1 after 3.5 min or 3 min forebrain ischemia, precluded significantly the ischemia-induced shortening of response latency in a step-down passive avoidance task and rescued a significant number of hippocampal CA1 neurons from lethal ischemic damage. The intracerebroventricular infusion of ginsenoside Rb1 did not affect hippocampal blood flow or hippocampal temperature except that it caused a slight increase in hippocampal blood flow at 5 min after transient forebrain ischemia. Furthermore, ginsenoside Rb1 at concentrations of 0.1-100 fg/ml (0.09-90 fM) rescued hippocampal neurons from lethal damage caused by the hydroxyl radical-promoting agent FeSO4 in vitro, and the Fenton reaction system containing p-nitrosodimethylaniline confirmed the hydroxyl radical-scavenging activity of ginsenoside Rb1. These findings suggest that the central infusion of ginsenoside Rb1 after forebrain ischemia protects hippocampal CA1 neurons against lethal ischemic damage possibly by scavenging free radicals which are overproduced in situ after brain ischemia and reperfusion. The present study may validate the empirical usage of ginseng root over thousands of years for the prevention of cerebrovascular diseases.

Erythropoietin protects neurons against chemical hypoxia and cerebral ischemic injury by up-regulating Bcl-xL expression

Erythropoietin (EPO) promotes neuronal survival after cerebral ischemia in vivo and after hypoxia in vitro. However, the mechanisms underlying the protective effects of EPO on ischemic/hypoxic neurons are not fully understood. The present in vitro experiments showed that EPO attenuated neuronal damage caused by chemical hypoxia at lower extracellular concentrations (10−4–10−2 U/ml) than were previously considered. Moreover, EPO at a concentration of 10−3 U/ml up‐regulated Bcl‐xL mRNA and protein expressions in cultured neurons. Subsequent in vivo study focused on whether EPO rescued hippocampal CA1 neurons from lethal ischemic damage and up‐regulated the expressions of Bcl‐xL mRNA and protein in the hippocampal CA1 field of ischemic gerbils. EPO was infused into the cerebroventricles of gerbils immediately after 3 min of ischemia for 28 days. Infusion of EPO at a dose of 5 U/day prevented the occurrence of ischemia‐induced learning disability. Subsequent light microscopic examinations showed that pyramidal neurons in the hippocampal CA1 field were significantly more numerous in ischemic gerbils infused with EPO (5 U/day) than in those receiving vehicle infusion. The same dose of EPO infusion caused significantly more intense expressions of Bcl‐xL mRNA and protein in the hippocampal CA1 field of ischemic gerbils than did vehicle infusion. These findings suggest that EPO prevents delayed neuronal death in the hippocampal CA1 field, possibly through up‐regulation of Bcl‐xL, which is known to facilitate neuron survival.

PERSISTENT C-FOS EXPRESSION IN THE BRAINS OF MICE WITH CHRONIC SOCIAL STRESS

The present study was conducted to demonstrate immunohistochemically, the sites of c-fos protein expression in the brains of mice subjected to acute and chronic social defeat stress. To induce social stress, mice were placed in situations of species-specific intermale aggression either only once or five times at 24 h intervals. Two hours after the single or fifth defeat stress, many c-fos immunoreactive neurons were observed in a variety of brain regions including the limbic system and sensory relay nuclei. The c-fos immunoreactive neurons in the brains of acute defeat mice decreased in number with time and the c-fos staining pattern of acute defeat mice became indistinguishable from that of normal control mice by 24 h after the single defeat stress. In contrast, chronic defeat stress induced persistent c-fos expression in the forebrain and brainstem even 24 h after the fifth defeat stress. In the forebrain of chronic defeat mice, the olfactory bulb, cingulate cortex, hippocampus, entire hypothalamus, septal nuclei and the amygdaloid complex, except for the central nucleus, were labeled intensely with c-fos antiserum. In the lower brainstem, nerve cells with c-fos immunoreactivity were seen mainly in ascending and descending sensory relay nuclei relevant to auditory and vestibular transmission, epicritic sensation (gracile and external cuneate nuclei), pain inhibition (central gray and raphe nuclei), and viscerosensory transmission (solitary tract nucleus). The differences in c-fos expression among the normal control, acute and chronic defeat mice were evaluated by an enumeration of the immunopositive neurons within each brain nucleus examined, and they were confirmed subsequently by statistical analysis. There was little c-fos expression in the nucleus putamen, lateral, ventral and posterior thalamic nuclei, pretectal nuclei, medial geniculate nucleus, red nucleus, substantia nigra, cerebellum, spinal cord, or cranial nerve nuclei. These findings suggest that chronic but not acute defeat stress causes persistent c-fos expression in more widespread brain regions than do any other stresses so far investigated. The present study may shed light on the central mechanisms by which behavioral abnormalities and/or chronic sociopsychological stress leads to the occurrence of abnormal behavior and/or psychosomatic disorders in experimental animals and humans.

β-Estradiol protects hippocampal CA1 neurons against transient forebrain ischemia in gerbil

Beta-estradiol has been considered to be a neurotrophic agent, but its in vivo effect on gerbils with transient forebrain ischemia has not yet been demonstrated. In the first set of the present experiments, we infused beta-estradiol at a dose of 0.05 or 0.25 microg/day for 7 days into the lateral ventricles of normothermic gerbils starting 2 h before 3-min forebrain ischemia. Beta-estradiol infusion at a dose of 0.25 microg/day prevented significantly the ischemia-induced reduction of response latency time as revealed by a step-down passive avoidance task. Subsequent light and electron microscopic examinations showed that pyramidal neurons in the hippocampal CA1 region as well as synapses within the strata moleculare, radiatum and oriens of the region were significantly more numerous in gerbils infused with beta-estradiol than in those receiving saline infusion. Beta-estradiol at a dose of 1.25 microg/day was ineffective and occasionally increased the mortality of experimental animals. Since the total brain content of exogenous beta-estradiol at 12 h after forebrain ischemia was estimated to be less than 145 ng, the second set of experiments focused on the neurotrophic action of beta-estradiol at concentrations around 100 ng/ml in vitro. Beta-estradiol at concentrations of 1-100 ng/ml facilitated the survival and process extension of cultured hippocampal neurons, but it did not exhibit any significant radical-scavenging effects at the concentration range. On the other hand, 100 microg/ml of beta-estradiol, even though failing to support hippocampal neurons in vitro, effectively scavenged free radicals in subsequent in vitro studies, as demonstrated elsewhere. These findings suggest that beta-estradiol at a dose of 0.25 microg/day prevents ischemia-induced learning disability and neuronal loss at early stages after transient forebrain ischemia, possibly via a receptor-mediated pathway without attenuating free radical neurotoxicity.

Ginseng root prevents learning disability and neuronal loss in gerbils with 5-minute forebrain ischemia.

Abstract The present study was designed to investigate the possible neuroprotective activity of ginseng roots in 5-min ischemic gerbils using a step-down passive avoidance task and subsequent neuron and synapse counts in the hippocampal CA1 region. The following drugs were administered for 7 days before the induced ischemia: red ginseng powder (RGP), crude ginseng saponin (CGS), crude ginseng non-saponin (CGNS), and pure ginsenosides Rb1, Rg1 and Ro. Oral administration of RGP significantly prevented the ischemia-induced decrease in response latency, as determined by the passive avoidance test, and rescued a significant number of ischemic hippocampal CA1 pyramidal neurons in a dose-dependent manner. Intraperitoneal injections of CGS exhibited a similar neuroprotective effect. CGNS had a significant but less potent protective effect against impaired passive avoidance task and degeneration of hippocampal CA1 neurons. Ginsenoside Rb1 significantly prolonged the response latency of ischemic gerbils and rescued a significant number of ischemic CA1 pyramidal neurons, whereas ginisenosides Rg1 and Ro were ineffective. Postischemic treatment with RGP, CGS or ginsenoside Rb1 was ineffective. The neuroprotective activities of RGP, CGS and ginsenoside Rb1 were confirmed by electron microscopy counts of synapses in individual strata of the CA1 field of ischemic gerbils pretreated with the drugs. These findings suggest that RGP and CGS are effective in the prevention of delayed neuronal death, and that ginsenoside Rb1 is one of the neuroprotective molecules within ginseng root.

A Hydrophilic Peptide Comprising 18 Amino Acid Residues of the Prosaposin Sequence Has Neurotrophic Activity In Vitro and In Vivo

Abstract: Prosaposin, a 517‐amino‐acid glycoprotein, not only acts as the precursor of saposin A, B, C, and D but also possesses neurotrophic activity to rescue hippocampal CA1 neurons from ischemic damage in vivo and to promote neurite extension of neuroblastoma cells in vitro. Recently, the trophic activity of prosaposin on human neuroblastoma cells has been shown to reside in the NH2‐terminal hydrophilic sequence (LIDNNRTEEILY) of the human saposin C. Here we show that prosaposin, saposin C, and a peptide comprising the 18‐amino‐acid sequence (18‐mer peptide; LSELIINNATEELLIKGL) located in the NH2‐terminal hydrophilic sequence of the rat saposin C‐domain promoted survival and neurite outgrowth of cultured rat hippocampal neurons in a dose‐dependent manner. Moreover, infusion for 7 days of the 18‐mer peptide into the lateral ventricle of gerbils, starting either 2 h before or immediately after 3 min of forebrain ischemia, protected ischemia‐induced learning disability and hippocampal CA1 neuronal loss. Thus, we ascribe the in vitro and in vivo trophic actions of prosaposin on hippocampal neurons to the linear 18‐mer sequence and raise the possibility that this peptide can be used as an agent for the treatment of forebrain ischemic damage.

Interleukin-6 prevents ischemia-induced learning disability and neuronal and synaptic loss in gerbils

Interleukin-6 (IL-6) has been shown to have potent neurotrophic activity on peripheral and central neurons in vitro. However, it remains to be determined whether or not IL-6 rescues hippocampal CA1 neurons from lethal ischemia and prevents ischemia-induced learning disability. In the present in vivo study, we infused IL-6 continuously for 7 days into the lateral ventricle of gerbil starting 2 h before 3-min forebrain ischemia. IL-6 infusion prevented the occurrence of ischemia-induced learning disability in a dose-dependent manner as revealed by a step-down passive avoidance task. Subsequent light and electron microscopic examinations showed that pyramidal neurons in the CA1 region of the hippocampus as well as synapses within the strata moleculare, radiatum and oriens of the region were significantly more numerous in gerbils infused with IL-6 than in those receiving vehicle infusion. These findings suggest that IL-6 has a trophic effect on ischemic hippocampal neurons.

Epidermal Growth Factor Protects Neuronal Cells In Vivo and In Vitro Against Transient Forebrain Ischemia- and Free Radical-Induced Injuries

Epidermal growth factor (EGF) has been considered to be a candidate for neurotrophic factors on the basis of the results of several in vitro studies. However, the in vivo effect of EGF on ischemic neurons as well as its mechanism of action have not been fully understood. In the present in vivo study using a gerbil ischemia model, we examined the effects of EGF on ischemia-induced learning disability and hippocampal CA1 neuron damage. Cerebroventricular infusion of EGF (24 or 120 ng/d) for 7 days to gerbils starting 2 hours before or immediately after transient forebrain ischemia caused a significant prolongation of response latency time in a passive avoidance task in comparison with the response latency of vehicle-treated ischemic animals. Subsequent histologic examinations showed that EGF effectively prevented delayed neuronal death of CA1 neurons in the stratum pyramidale and preserved synapses intact within the strata moleculare, radiatum, and oriens of the hippocampal CA1 region. In situ detection of DNA fragmentation (TUNEL staining) revealed that ischemic animals infused with EGF contained fewer TUNEL-positive neurons in the hippocampal CA1 field than those infused with vehicle alone at the seventh day after ischemia. In primary hippocampal cultures, EGF (0.048 to 6.0 ng/mL) extended the survival of cultured neurons, facilitated neurite outgrowth, and prevented neuronal damage caused by the hydroxyl radical-producing agent FeSO4 and by the peroxynitrite-producing agent 3-morpholinosydnonimine in a dose-dependent manner. Moreover, EGF significantly attenuated FeSO4-induced lipid peroxidation of cultured neurons. These findings suggest that EGF has a neuroprotective effect on ischemic hippocampal neurons in vivo possibly through inhibition of free radical neurotoxicity and lipid peroxidation.

Ciliary neurotrophic factor prevents ischemia-induced learning disability and neuronal loss in gerbils

Ciliary neurotrophic factor (CNTF) has been shown to have potent neurotrophic activity on peripheral and central neurons in vitro and in vivo. However, it remains to be determined whether or not CNTF rescues hippocampal CA1 neurons from lethal ischemia and prevents ischemia-induced learning disability. In the present in vivo study, we infused CNTF continuously for 7 days into the lateral ventricle of gerbil starting 2 h before 3-min forebrain ischemia. CNTF infusion prevented the occurrence of ischemia-induced learning disability in a dose-dependent manner as revealed by the step-down passive avoidance task. Subsequent light and electron microscopic examinations showed that pyramidal neurons in the CA1 region of the hippocampus as well as synapses within the strata moleculare, lacunosum/radiatum and oriens of the region were significantly more numerous in gerbils infused with CNTF than in those receiving vehicle infusion. These findings suggest that CNTF has a trophic effect on ischemic hippocampal neurons.

Prevention of Ischemic Neuronal Death by Intravenous Infusion of a Ginseng Saponin, Ginsenoside Rb1, That Upregulates Bcl-xL Expression

Almost all agents that exhibit neuroprotection when administered into the cerebral ventricles are ineffective or much less effective in rescuing damaged neurons when infused into the blood stream. Search for an intravenously infusible drug with a potent neuroprotective action is essential for the treatment of millions of patients suffering from acute brain diseases. Here, we report that postischemic intravenous infusion of a ginseng saponin, ginsenoside Rb1 (gRb1) (C54H92O23, molecular weight 1109.46) to stroke-prone spontaneously hypertensive rats with permanent occlusion of the middle cerebral artery distal to the striate branches significantly ameliorated ischemia-induced place navigation disability and caused an approximately 50% decrease in the volume of the cortical infarct lesion in comparison with vehicle-infused ischemic controls. In subsequent studies that focused on gRb1-induced expression of gene products responsible for neuronal death or survival, we showed that gRb1 stimulated the expression of the mitochondrion-associated antiapoptotic factor Bcl-xL in vitro and in vivo. Moreover, we revealed that a Stat5 responsive element in the bcl-x promoter became active in response to gRb1 treatment. Ginsenoside Rb1 appears to be a promising agent not only for the treatment of cerebral stroke, but also for the treatment of other diseases involving activation of mitochondrial cell death signaling.