Yun-Bae Kim

Human neural stem cells over-expressing choline acetyltransferase restore cognition in rat model of cognitive dysfunction.

A human neural stem cell (NSC) line over-expressing human choline acetyltransferase (ChAT) gene was generated and these F3.ChAT NSCs were transplanted into the brain of rat Alzheimer disease (AD) model which was induced by application of ethylcholine mustard aziridinium ion (AF64A) that specifically denatures cholinergic nerves and thereby leads to memory deficit as a salient feature of AD. Transplantation of F3.ChAT human NSCs fully recovered the learning and memory function of AF64A animals, and induced elevated levels of acetylcholine (ACh) in cerebrospinal fluid (CSF). Transplanted F3.ChAT human NSCs were found to migrate to various brain regions including cerebral cortex, hippocampus, striatum and septum, and differentiated into neurons and astrocytes. The present study demonstrates that brain transplantation of human NSCs over-expressing ChAT ameliorates complex learning and memory deficits in AF64A-cholinotoxin-induced AD rat model.

Human neural stem cells overexpressing choline acetyltransferase restore cognitive function of kainic acid-induced learning and memory deficit animals.

Alzheimer disease (AD) is a progressive neurodegenerative disease, which is characterized by loss of memory and cognitive function. In AD patients dysfunction of the cholinergic system is the main cause of cognitive disorders, and decreased activity of choline acetyltransferase (ChAT), an enzyme responsible for acetylcholine (ACh) synthesis, is observed. In the present study we investigated if brain transplantation of human neural stem cells (NSCs) genetically modified to encode ChAT gene improves cognitive function of kainic acid (KA)-induced learning deficit rats. Intrahippocampal injection of KA to hippocampal CA3 region caused severe neuronal loss, resulting in profound learning and memory deficit. F3.ChAT human NSCs transplanted intracerebroventricularly improved fully the learning and memory function of KA-induced learning deficit animals, in parallel with the elevation of ACh levels in cerebrospinal fluid. F3.ChAT human NSCs migrated to the KA-induced injury site (CA3) and differentiated into neurons and astrocytes. The present study demonstrates that human NSCs expressing ChAT have lesion-tropic property and improve cognitive function of learning deficit model rats with hippocampal injury by increasing ACh level.

Immunopotentiation and antitumor effects of a ginsenoside Rg3-fortified red ginseng preparation in mice bearing H460 lung cancer cells

Antitumor effects of a ginsenoside Rg(3)-fortified red ginseng preparation (Rg(3)-RGP) were investigated in human non-small cell lung carcinoma (H460) cells using in vitro cytotoxicity assay and in vivo nude mouse xenograft model. Immunomodulatory effects of the preparation were also assessed by measuring the facilitating activities on the nitric oxide (NO) release from peritoneal macrophages, in vitro and in vivo lymphocyte proliferation, and the carbon clearance from circulating blood. In a cell level, Rg(3)-RGP exerted H460 cytotoxicity and facilitated splenocyte proliferation at very high concentrations, without affecting NO production. However, oral administration of Rg(3)-RGP (100-300 mg/kg) enhanced carbon particle-phagocytic index of blood macrophages up to 360-397% of control value. In addition, Rg(3)-RGP significantly increased the splenocyte proliferation (23% at 100mg/kg). In tumor-bearing mice, 28-day oral treatment with Rg(3)-RGP (100mg/kg) remarkably suppressed the tumor growth, leading to the decrease of the tumor volume and weight by 30-31%, which was comparable to the effect (27-29% reduction) of doxorubicin (2mg/kg at 3-day intervals). While Rg(3)-RGP did not cause adverse effects, intravenous injection of doxorubicin markedly decreased body and testes weights, and exhibited severe depletion of spermatogenic cells in the atrophic seminiferous tubules. These results indicate that Rg(3)-RGP exerts antitumor activities via indirect immunomodulatory actions, without causing adverse effects as seen in doxorubicin.

Korean red ginseng extract does not cause embryo‐fetal death or abnormalities in mice

BACKGROUND Ginseng has been used for a long time and is well tolerated in humans. However, recent studies have shown that ginsenosides Rb1, Rg1, and Re exert embryotoxicity in in vitro culture systems. We investigated the effects of Korean red ginseng extract (KRGE) on embryonic implantation and fetal development in mice. METHODS Mice were orally administered KRGE (20, 200, or 2,000 mg/kg/day) from 2 weeks before mating to gestational day (GD) 18, and implantation rate, fetal mortality, body weights, as well as external, visceral, and skeletal abnormalities were determined by Caesarean section on GD18. Ginsenosides in KRGE and in the blood of dams were identified and quantified by HPLC analysis. RESULTS KRGE did not affect embryonic implantation and mortality as well as fetal body weights up to 2,000 mg/kg/day (approximately 200 times clinical doses), the upper-limit dose recommended by the Korea Food and Drug Administration (KFDA). Although the prevalence of supernumerary ribs increased at the medium dose (200 mg/kg/day), no dose-dependent increases in external, visceral, and skeletal abnormalities were observed. Major ginsenosides such as Rb1, Rg1, and Re were not detected in the blood of dams based on their chromatographic profiles. CONCLUSIONS Considerable developmental toxicities of KRGE, even at the upper-limit dose, were not observed in mice. These results might be due to the negligible blood concentrations of ginsenosides in their original forms following oral administration, suggesting that in vitro experiments to assess the effects of ginsenosides on embryotoxicity may not reliably explain the risks of ginsenosides to in vivo embryo-fetal development.

Animal models of periventricular leukomalacia.

Periventricular leukomalacia, specifically characterized as white matter injury, in neonates is strongly associated with the damage of pre-myelinating oligodendrocytes. Clinical data suggest that hypoxia-ischemia during delivery and intrauterine or neonatal infection-inflammation are important factors in the etiology of periventricular leukomalacia including cerebral palsy, a serious case exhibiting neurobehavioral deficits of periventricular leukomalacia. In order to explore the pathophysiological mechanisms of white matter injury and to better understand how infectious agents may affect the vulnerability of the immature brain to injury, novel animal models have been developed using hypoperfusion, microbes or bacterial products (lipopolysaccharide) and excitotoxins. Such efforts have developed rat models that produce predominantly white matter lesions by adopting combined hypoxia-ischemia technique on postnatal days 1-7, in which unilateral or bilateral carotid arteries of animals are occluded (ischemia) followed by 1-2 hour exposure to 6-8% oxygen environment (hypoxia). Furthermore, low doses of lipopolysaccharide that by themselves have no adverse-effects in 7-day-old rats, dramatically increase brain injury to hypoxic-ischemic challenge, implying that inflammation sensitizes the immature central nervous system. Therefore, among numerous models of periventricular leukomalacia, combination of hypoxia-ischemia-lipopolysaccharide might be one of the most-acceptable rodent models to induce extensive white matter injury and ensuing neurobehavioral deficits for the evaluation of candidate therapeutics.

Anti-inflammatory effects of a Houttuynia cordata supercritical extract

Anti-inflammatory effects of Houttuynia cordata supercritical extract (HSE) were investigated in a carrageenan-air pouch model. HSE (200 mg/kg, oral) suppressed exudation and albumin leakage, as well as inflammatory cell infiltration. Dexamethasone (2 mg/kg, i.p.) only decreased exudation and cell infiltration, while indomethacin (2 mg/kg, i.p.) reduced exudate volume and albumin content. HSE lowered tumor-necrosis factor (TNF)-α and nitric oxide (NO), as well as prostaglandin E2 (PGE2). Dexamethasone only reduced TNF-α and NO, while indomethacin decreased TNF-α and PGE2. The suppressive activity of HSE on NO and PGE2 production was confirmed in RAW 264.7. These results demonstrate that HSE exerts anti-inflammatory effects by inhibiting both TNF-α-NO and cyclooxygenase II-PGE2 pathways.

Microtubule-associated protein 2, an early blood marker of ischemic brain injury

The aim of this study was to develop a sensitive and rapid blood marker to detect ischemic brain injury, because imaging techniques have a limited capacity to identify lesions during the first crucial hours without massive tissue destruction. Rats were subjected to middle cerebral artery occlusion for various durations (0.5–3 hr), followed by reperfusion. At different time points after ischemia and/or ischemia‐reperfusion, the amounts of glial fibrillary acidic protein (GFAP) and microtubule‐associated protein 2 (MAP2) in the cerebrospinal fluid (CSF) and serum were analyzed by Western blotting. Brain infarction was observed in an ischemia‐duration‐dependent manner. GFAP was drastically increased in the CSF 24 and 48 hr after reperfusion, without change in the serum level. Serum levels of MAP2 remarkably increased as early as 0.5 hr of ischemia, much earlier than the observation of minimal tissue injury 3 hr following occlusion. The serum MAP2 level was further increased by a short period (2 hr) of reperfusion, even in 0.5‐ and 1‐hr ischemic rats, despite not observing any typical tissue injuries 24 hr after reperfusion. These results indicate that the MAP2 protein may be able to detect early neuronal injuries, because the level of this protein in the blood spikes before the appearance of visible macrolesions. Therefore, MAP2 could potentially be used as a novel early marker for the detection of a neurotoxic insult.

Neurobehavioural deficits correlate with the cerebral infarction volume of stroke animals: A comparative study on ischaemia-reperfusion and photothrombosis models

The study investigated the correlation between infarction areas and behavioural deficits in middle cerebral artery occlusion (MCAO) and photothrombosis stroke models. In the MCAO model, a 0.38 mm-diameter silicone-coated thread was introduced through the left external carotid artery and advanced 18 mm via the internal carotid artery to the origin of middle cerebral artery of male Sprague-Dawley rats weighing 300-350 g. The thread was removed for reperfusion after occlusion for 0.5, 1 or 2h. In the photothrombosis model, after a midline incision on the scalp, a focused light (10,000 lux, 6 mm-diameter) was delivered 1mm anterior to the bregma and 3mm left of the midline for 5, 10 or 20 min. During the first 2 min of irradiation, Rose Bengal dye (30 mg/kg) was injected intravenously. Twenty four hours post-surgery, the animals were subjected to neurological scoring and behavioural performances, and were sacrificed for macroscopic and microscopic examinations of brain injury. Total infarction volumes in the MCAO model rats increased in an occlusion time-dependent manner, while the infarction volumes in photothrombosis model rats plateaued relatively quickly with no time-dependent increase. The MCAO model displayed neurological scores and behavioural deficits that correlated well with infarction volumes, while relatively poor correlation between infarction volume and neurobehavioural abnormalities was evident in the photothrombosis model. The results indicate the suitability of the MCAO model for studies on preventive or therapeutic compounds related to functional recovery, although the photothrombosis model might be useful to generate focused lesions leading to the location-related behavioural changes.

Licorice extract increases cyclophosphamide teratogenicity by upregulating the expression of cytochrome P-450 2B mRNA

BACKGROUND Since cyclophosphamide is metabolically activated to teratogenic acrolein and cytotoxic phosphoramide mustard by cytochrome P-450 type 2B (CYP2B), we assessed the effects of licorice, a CYP2B inducer, on the fetal defects induced by cyclophosphamide. METHODS Pregnant Sprague-Dawley rats were daily administered with licorice (100 mg/kg) by gavage for 7 days, from the 6th to 12th day of gestation, and intraperitoneally administered with cyclophosphamide (11 mg/kg) 1 hr after the final licorice treatment. On the 20th day of gestation, maternal and fetal abnormalities were determined by Cesarian section. RESULTS Cyclophosphamide was found to reduce fetal and placental weights without increasing resorption or death. In addition, it induced malformations in live fetuses; 93.8, 41.1, and 100% of the external (skull and limb defects), visceral (cleft palate and ureteric dilatation), and skeletal (acrania, vertebral/costal malformations, and delayed ossification) abnormalities, respectively. When pre-treated with licorice, cyclophosphamide-induced body weight loss and abnormalities of fetuses were remarkably aggravated. Moreover, repeated treatment with licorice greatly increased mRNA expression and activity of hepatic CYP2B. CONCLUSIONS The results indicate that repeated intake of licorice may aggravate cyclophosphamide-induced body weight loss and malformations of fetuses by upregulating CYP2B.

Preventive effect of piperonyl butoxide on cyclophosphamide-induced teratogenesis in rats

BACKGROUND Cyclophosphamide induces fetal defects through metabolic activation by cytochrome P-450 monooxygenases (CYP). The effects of piperonyl butoxide (PBO), a CYP inhibitor, on the fetal development and external, visceral, and skeletal abnormalities induced by cyclophosphamide were investigated in rats. METHODS Pregnant rats were daily administered PBO (400 mg/kg) by gavage for 7 days (the 6th to 12th day of gestation), and intraperitoneally administered with cyclophosphamide (12 mg/kg) 4 h after the final treatment. On the 20th day of gestation, maternal and fetal abnormalities were determined by Cesarean section. RESULTS Cyclophosphamide reduced fetal body weights by 30-40% without increasing resorption or death. In addition, it induced malformations in live fetuses: 100, 98, and 98.2% of the external (head and limb defects), visceral (cerebroventricular dilatation, cleft palate, and renal pelvic/ureteric dilatation), and skeletal (acrania, vertebral/costal malformations, and delayed ossification) abnormalities, respectively. The pre-treatment of PBO greatly decreased mRNA expression and activity of hepatic CYP2B, which metabolizes cyclophosphamide into teratogenic acrolein and cytotoxic phosphoramide mustard. Moreover, PBO remarkably attenuated cyclophosphamide-induced body weight loss and abnormalities of fetuses; score 3.57 versus 1.87 for exencephaly, 75.5% versus 42.5% for limb defects, 65.3% versus 22% for cerebroventricular dilatation, 59.2% versus 5.1% for cleft palate, score 1.28 versus 0.93 for renal pelvic/ureteric dilatation, 71.9-82.5% versus 23-45.9% for vertebral/costal malformations, and 84.2% versus 57.4% for delayed ossification in cyclophosphamide alone and PBO co-administration groups. CONCLUSIONS These results suggest that repeated treatment with PBO may improve cyclophosphamide-induced body weight loss and malformations of fetuses by down-regulating CYP2B.