Chan Young Shin

The critical period of valproate exposure to induce autistic symptoms in Sprague–Dawley rats

Prenatal exposure to valproic acid (VPA) induces neural tube defects and impairment in social behaviors related to autistic spectrum disorder in newborns, which make it a useful animal model of autism. In this study, we compared the effects of different time window of prenatal valproic acid exposure for inducing the altered social behaviors relevant to autism from embryonic day 7 to embryonic day 15 in Sprague-Dawley rats to determine the critical periods for the impairment. Compared to E7, E9.5 and E15 exposure, VPA exposure at E12 showed most significant changes in behaviors over control animals with reduced sociability and social preference. E9.5 exposure to valproic acid showed strong reproductive toxicity including decrease in the number of live birth. In general, exposure at E15 showed only marginal effects on reproduction and social behaviors. Finally, VPA-exposed rats at E12 were more sensitive to electric shock than VPA-exposed rats at any other periods. These results suggested that E12 is the critical period in rats when valproate exposure has prominent effects for inducing the altered social behavior similar to human autistic behavior.

Induction of matrix metalloproteinase-9 (MMP-9) in lipopolysaccharide-stimulated primary astrocytes is mediated by extracellular signal-regulated protein kinase 1/2 (Erk1/2).

In the present study, we investigated whether the activation of protein kinase C (PKC) and extracellular signal‐regulated kinase 1/2 (Erk1/2) are involved in the induction of MMP‐9 in lipopolysaccharide (LPS)–stimulated primary astrocytes. The expression of MMP‐9 but not MMP‐2 was increased by LPS. LPS treatment induced activation of Erk1/2 within 30 min, which was dose‐dependently inhibited by PD98059, a specific inhibitor of the Erk kinase (MEK). In this condition, PD98059 blocked the increase in MMP‐9 protein and mRNA level as well as gelatin‐digesting activity. Inhibition of PKC activity blocked the LPS‐induced activation of Erk1/2 as well as MMP‐9 expression. In addition, activation of PKC by phorbol myristoyl acetate (PMA) activated Erk1/2 with concomitant increase in MMP‐9 production. Moreover, treatment of PD98059 dose‐dependently decreased the PMA‐induced MMP‐9 expression. The results from the present study suggest that induction of MMP‐9 by LPS in rat primary astrocytes is mediated, at least in part, by the sequential activation of PKC and Erk1/2. The Erk1/2‐mediated MMP‐9 induction may provide insights into the regulation of MMP‐9 production in CNS, which may occur in vivo in pathological situations such as CNS inflammation. GLIA 41:15–24, 2003.

A comprehensive review of the therapeutic and pharmacological effects of ginseng and ginsenosides in central nervous system

Ginseng is one of the most widely used herbal medicines in human. Central nervous system (CNS) diseases are most widely investigated diseases among all others in respect to the ginseng’s therapeutic effects. These include Alzheimer’s disease, Parkinson’s disease, cerebral ischemia, depression, and many other neurological disorders including neurodevelopmental disorders. Not only the various types of diseases but also the diverse array of target pathways or molecules ginseng exerts its effect on. These range, for example, from neuroprotection to the regulation of synaptic plasticity and from regulation of neuroinflammatory processes to the regulation of neurotransmitter release, too many to mention. In general, ginseng and even a single compound of ginsenoside produce its effects on multiple sites of action, which make it an ideal candidate to develop multi-target drugs. This is most important in CNS diseases where multiple of etiological and pathological targets working together to regulate the final pathophysiology of diseases. In this review, we tried to provide comprehensive information on the pharmacological and therapeutic effects of ginseng and ginsenosides on neurodegenerative and other neurological diseases. Side by side comparison of the therapeutic effects in various neurological disorders may widen our understanding of the therapeutic potential of ginseng in CNS diseases and the possibility to develop not only symptomatic drugs but also disease modifying reagents based on ginseng.

Hwangryun-Hae-Dok-tang (Huanglian-Jie-Du-Tang) extract and its constituents reduce ischemia-reperfusion brain injury and neutrophil infiltration in rats

The preventive effect of Hwangryun-Hae-Dok-tang (HHDT, Huanglian-Jie-Du-Tang), a Chinese herbal medicine, and its ingredients on ischemia/reperfusion-induced brain injury was evaluated in the rat brain. HHDT consists of four herbs, namely, Coptidis rhizoma, Scutellariae radix, Phellodendri cortex, and Gardeniae fructus. Ischemia was induced by intraluminal occlusion of the right middle cerebral artery for 120 min and reperfusion was continued for 22 h. HHDT (200 mg/kg), Coptidis rhizoma (100 mg/kg), Scutellariae radix (100 mg/kg), Phellodendri cortex (100 mg/kg), and Gardeniae fructus (100 mg/kg) were orally administered, promptly prior to reperfusion and 2 h after reperfusion. Baicalein, a component of Scutellariae radix, was also examined at a dosage of 50 mg/kg given 2 h apart, promptly prior to and 2 h after reperfusion. Total infarction volume in the ipsilateral hemisphere of ischemia/reperfusion rats was significantly lowered by treatment with HHDT, Scutellariae radix, and balicalein. However, the other ingredient of HHDT did not show any ameliorating effects on total infarction volume. The inhibiting effect of Scutellariae radix on total infarction volume was much higher than that of the others. In addition, HHDT, Scutellariae radix, and baicalein significantly inhibited myeloperoxidase (MPO) activity, an index of neutrophil infiltration in ischemic brain tissue at about the same rate (30%). There was marked mismatch between total infarction volume and MPO activity in the Scutellariae radix-treated rats but not in the HHDT- and baicalein-treated groups. Our findings suggest that Scutellariae radix as an ingredient of HHDT plays a crucial protective role in ischemia-induced brain injury. In addition, it is apparent that the effect of Scutellariae radix is the result, in part, of baicalein, a compound contained in Scutellariae radix.

Ascorbate attenuates trimethyltin-induced oxidative burden and neuronal degeneration in the rat hippocampus by maintaining glutathione homeostasis.

The specific role of endogenous glutathione in response to neuronal degeneration induced by trimethyltin (TMT) in the hippocampus was examined in rats. A single injection of TMT (8 mg/kg, i.p.) produced a rapid increase in the formation of hydroxyl radical and in the levels of malondialdehyde (MDA) and protein carbonyl. TMT-induced seizure activity significantly increased after this initial oxidative stress, and remained elevated for up to 2 weeks post-TMT. Although a significant loss of hippocampal Cornus Ammonis CA1, CA3 and CA4 neurons was observed at 3 weeks post-TMT, the elevation in the level of hydroxyl radicals, MDA, and protein carbonyl had returned to near-control levels at that time. In contrast, the ratio of reduced to oxidized glutathione remained significantly decreased at 3 weeks post-TMT, and the glutathione-like immunoreactivity of the pyramidal neurons was decreased. However glutathione-positive glia-like cells proliferated mainly in the CA1, CA3, and CA4 sectors and were intensely immunoreactive. Double labeling demonstrated the co-localization of glutathione-immunoreactive glia-like cells and reactive astrocytes, as indicated by immunostaining for glial fibrillary acidic protein. This suggests that astroglial cells were mobilized to synthesize glutathione in response to the TMT insult. The TMT-induced changes in glutathione-like immunoreactivity appear to be concurrent with changes in the expression levels of glutathione peroxidase and glutathione reductase. Ascorbate treatment significantly attenuated TMT-induced seizures, as well as the initial oxidative stress, impaired glutathione homeostasis, and neuronal degeneration in a dose-dependent manner. These results suggest that ascorbate is an effective neuroprotectant against TMT. The initial oxidative burden induced by TMT may be a causal factor in the generation of seizures, prolonged disturbance of endogenous glutathione homeostasis, and consequent neuronal degeneration.

Male‐specific alteration in excitatory post‐synaptic development and social interaction in pre‐natal valproic acid exposure model of autism spectrum disorder

Autism spectrum disorder (ASD) is a pervasive developmental disorder characterized by three main behavioral symptoms including social deficits, impaired communication, and stereotyped and repetitive behaviors. ASD prevalence shows gender bias to male. Prenatal exposure to valproic acid (VPA), a drug used in epilepsy and bipolar disorder, induces autistic symptoms in both human and rodents. As we reported previously, prenatally VPA‐exposed animals at E12 showed impairment in social behavior without any overt reproductive toxicity. Social interactions were not significantly different between male and female rats in control condition. However, VPA‐exposed male offspring showed significantly impaired social interaction while female offspring showed only marginal deficits in social interaction. Similar male inclination was observed in hyperactivity behavior induced by VPA. In addition to the ASD‐like behavioral phenotype, prenatally VPA‐exposed rat offspring shows crooked tail phenotype, which was not different between male and female groups. Both male and female rat showed reduced GABAergic neuronal marker GAD and increased glutamatergic neuronal marker vGluT1 expression. Interestingly, despite of the similar increased expression of vGluT1, post‐synaptic marker proteins such as PSD‐95 and α‐CAMKII expression was significantly elevated only in male offspring. Electron microscopy showed increased number of post‐synapse in male but not in female at 4 weeks of age. These results might suggest that the altered glutamatergic neuronal differentiation leads to deranged post‐synaptic maturation only in male offspring prenatally exposed to VPA. Consistent with the increased post‐synaptic compartment, VPA‐exposed male rats showed higher sensitivity to electric shock than VPA‐exposed female rats. These results suggest that prenatally VPA‐exposed rats show the male preponderance of ASD‐like behaviors including defective social interaction similar to human autistic patients, which might be caused by ectopic increase in glutamatergic synapses in male rats.

Melatonin synergistically increases resveratrol-induced heme oxygenase-1 expression through the inhibition of ubiquitin-dependent proteasome pathway: a possible role in neuroprotection.

Abstract:  Melatonin is an indoleamine secreted by the pineal gland as well as a plant‐derived product, and resveratrol (RSV) is a naturally occurring polyphenol synthesized by a variety of plant species; both molecules act as a neuroprotector and antioxidant. Recent studies have demonstrated that RSV reduced the incidence of Alzheimer’s disease and stroke, while melatonin supplementation was found to reduce the progression of the cognitive impairment in AD. The heme oxygenase‐1 (HO‐1) is an inducible and redox‐regulated enzyme that provides tissue‐specific antioxidant effects. We assessed whether the co‐administration of melatonin and RSV shows synergistic effects in terms of their neuroprotective properties through HO‐1. RSV significantly increased the expression levels of HO‐1 protein in a concentration‐dependent manner both in primary cortical neurons and in astrocytes, while melatonin per se did not. Melatonin + RSV showed a synergistic increase in the expression levels of HO‐1 protein but not in the HO‐1 mRNA level compared to either melatonin or RSV alone, which is mediated by the activation of PI3K‐Akt pathway. Treatment of melatonin + RSV significantly attenuated the neurotoxicity induced by H2O2 in primary cortical neurons and also in organotypic hippocampal slice culture. The blockade of HO‐1 induction by shRNA attenuated HO‐1 induction by melatonin + RSV and hindered the neuroprotective effects against oxidative stress induced by H2O2. The treatment of MG132 + RSV mimicked the effects of melatonin + RSV, and melatonin + RSV inhibited ubiquitination of HO‐1. These data suggest that melatonin potentiates the neuroprotective effect of RSV against oxidative injury, by enhancing HO‐1 induction through inhibiting ubiquitination‐dependent proteasome pathway, which may provide an effective means to treat neurodegenerative disorders.

Prenatal exposure to valproic acid increases the neural progenitor cell pool and induces macrocephaly in rat brain via a mechanism involving the GSK-3β/β-catenin pathway

Autism is a spectrum of neurodevelopmental disorders characterized by social isolation and lack of interaction. Anatomically, autism patients often show macrocephaly and high neuronal density. To investigate the mechanism underlying the higher neuronal populations seen in ASD, we subcutaneously injected VPA (400 mg/kg) into pregnant Sprague-Dawley rats on E12, an animal model often used in ASD study. Alternatively, cultured rat neural progenitor cells were treated with VPA. Until E18, VPA induced NPC proliferation and delayed neurogenesis in fetal brain, but the subsequent differentiation of NPCs to neurons increased brain neuronal density afterward. Similar findings were observed with NPCs treated with VPA in vitro. At a molecular level, VPA enhanced Wnt1 expression and activated the GSK-3β/β-catenin pathway. Furthermore, inhibition of this pathway attenuated the effects of VPA. The findings of this study suggest that an altered developmental process underlies the macrocephaly and abnormal brain structure observed in the autistic brain.

Synergistic Memory Impairment Through the Interaction of Chronic Cerebral Hypoperfusion and Amlyloid Toxicity in a Rat Model

Background and Purpose— Vascular pathology and Alzheimer disease (AD) pathology have been shown to coexist in the brains of dementia patients. We investigated how cognitive impairment could be exacerbated in a rat model of combined injury through the interaction of chronic cerebral hypoperfusion and amyloid beta (A&bgr;) toxicity. Methods— In Wistar rats, chronic cerebral hypoperfusion was modeled by permanent occlusion of bilateral common carotid arteries (BCCAo). Further, AD pathology was modeled by bilateral intracerebroventricular A&bgr; (A&bgr; toxicity) using a nonphysiological A&bgr; peptide (A&bgr; 25 to 35). The experimental animals were divided into 4 groups, including sham, single injury (A&bgr; toxicity or BCCAo), and combined injury (BCCAo-A&bgr; toxicity) groups (n=7 per group) . Cerebral blood flow and metabolism were measured using small animal positron emission tomography. A Morris water maze task, novel object location and recognition tests, and histological investigation, including neuronal cell death, apoptosis, neuroinflammation, and AD-related pathology, were performed. Results— Spatial memory impairment was synergistically exacerbated in the BCCAo–A&bgr; toxicity group as compared to the BCCAo or A&bgr; toxicity groups (P<0.05). Compared to the sham group, neuroinflammation with microglial or astroglial activation was increased both in multiple white matter lesions and the hippocampus in other experimental groups. AD-related pathology was enhanced in the BCCAo–A&bgr; toxicity group compared to the A&bgr; toxicity group. Conclusion— Our experimental results support a clinical hypothesis of the deleterious interaction between chronic cerebral hypoperfusion and A&bgr; toxicity. Chronic cerebral hypoperfusion-induced perturbation in the equilibrium of AD-related pathology may exacerbate cognitive impairment in a rat model of combined injury.

Melatonin Potentiates the Neuroprotective Properties of Resveratrol Against Beta-Amyloid-Induced Neurodegeneration by Modulating AMP-Activated Protein Kinase Pathways

Background and Purpose Recent studies have demonstrated that resveratrol (RSV) reduces the incidence of age-related macular degeneration, Alzheimers disease (AD), and stroke, while melatonin (MEL) supplementation reduces the progression of the cognitive impairment in AD patients. The purpose of this investigation was to assess whether the co-administration of MEL and RSV exerts synergistic effects on their neuroprotective properties against β-amyloid (Aβ)-induced neuronal death. Methods The neuroprotective effects of co-treatment with MEL and RSV on Aβ1-42-induced cell death, was measured by MTT reduction assay. Aβ1-42 caused an increase in intracellular levels of reactive oxygen species (ROS), as assessed by H2-DCF-DA dye, and a reduction of total glutathione (GSH) levels and mitochondrial membrane potential, as assessed using monochlorobimane and rhodamine 123 fluorescence, respectively. Western blotting was used to investigate the intracellular signaling mechanism involved in these synergic effects. Results We treated a murine HT22 hippocampal cell line with MEL or RSV alone or with both simultaneously. MEL and RSV alone significantly attenuated ROS production, mitochondrial membrane-potential disruption and the neurotoxicity induced by Aβ1-42. They also restored the Aβ1-42-induced depletion of GSH, back to within its normal range and prevented the Aβ1-42-induced activation of glycogen synthase kinase 3β (GSK3β). However, co-treatment with MEL and RSV did not exert any significant synergistic effects on either the recovery of the Aβ1-42-induced depletion of GSH or on the inhibition of Aβ1-42-induced GSK3β activation. Aβ1-42 treatment increased AMP-activated protein kinase (AMPK) activity, which is associated with subsequent neuronal death. We demonstrated that MEL and RSV treatment inhibited the phosphorylation of AMPK. Conclusions Together, our results suggest that co-administration of MEL and RSV acts as an effective treatment for AD by attenuating Aβ1-42-induced oxidative stress and the AMPK-dependent pathway.