Terumi Sakurai

Acute 3-nitropropionic acid intoxication induces striatal astrocytic cell death and dysfunction of the blood-brain barrier: involvement of dopamine toxicity

Mechanisms underlying the selective vulnerability of the lateral striatal area to the toxic effects of 3-nitropropionic acid (3-NPA) were investigated in rats. A single exposure to 3-NPA (20 mg/kg, s.c.) induced no deficits in behavior and histology, but subsequent injection produced motor symptoms, catalepsy, lip smacking, abnormal gait, paddling, rolling, opisthotonos, tremor, recombence, somnolence and so on, in 30% of the animals within a few hours. Diffusion-weighted magnetic resonance imaging of the brains revealed an area of high signal intensity in the bilateral striata. By this stage (within a few hours), striatal astrocytes had become swollen and disintegrated. Extravasation of immunoglobulin G was detected, indicating blood-brain barrier (BBB) dysfunction. Electron microscopy revealed edema and disorganization of structures inside the astrocytic end-feet around the branches of the lateral striatal artery. Neurons were less vulnerable than astrocytes to the 3-NPA injury. Treatment of the rats with D2 receptor agonist prior to exposure to 3-NPA attenuated the behavioral abnormalities and histological damage whereas pretreatment with D2 antagonist exacerbated these changes. The concentrations of extracellular dopamine (DA) and dihydroxyphenyl acetic acid (DOPAC) were both increased in rats exposed to 3-NPA. In vitro imaging of astrocytes revealed a progressive increase in [Ca2+]i after superfusion with 3-NPA, and the ceiling level was maintained even after extensive washing. DA superfusion also increased the astrocytic [Ca2+]i and this increase was reversible. Data indicate that 3-NPA-induced striatal damage was associated with astrocytic cell death and dysfunction of the BBB. Intracellular edema and extreme Ca2+ overload induced by the toxin were further aggravated by an increase in the level of DA activity. These factors acting either singly or in combination may trigger astrocyte destruction.

GDNF is a major component of trophic activity in DA-depleted striatum for survival and neurite extension of DAergic neurons

Extracts from dopamine (DA)-depleted striatal tissue (lesion extract) and from intact striatal tissue (intact extract) were prepared, and trophic activities in these extracts were evaluated using survival and neurite extension of DAergic neurons as indices. Levels of brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF), glial cell-line derived neurotrophic factor (GDNF) and neurotrophin-3 (NT-3) in extracts were measured using enzyme-linked immunosorbent assay (ELISA). The lesion extract exhibited a stronger trophic activity on survival and neurite extension of DAergic neurons than intact extract. In lesion extract, bFGF was slightly and GDNF was significantly increased, while BDNF and NT-3 were the same level in each extract. The peak increase of bFGF and GDNF was during 2 to 3 weeks after DA depletion. Trophic activity of extract was strongly attenuated after immunoprecipitation of GDNF and partly attenuated after immunoprecipitation of bFGF. In parallel immunohistological study, no significant variations were found for striatal microtubule-associated protein-2 (MAP-2)- nor OX-41-immunoreactive cells, while the number of strongly labeled glial fibrillary acidic protein (GFAP)-immunoreactive cells were increased in DA-depleted striatum, suggesting reactive gliosis. Data suggest that bFGF is a minor, while GDNF is a major component of trophic activity for DAergic neurons in DA-depleted striatum, and increased bFGF and GDNF levels may be mediated partly by reactive gliosis.

Chronically administered 3-nitropropionic acid induces striatal lesions attributed to dysfunction of the blood-brain barrier

3-Nitropropionic acid (3-NPA), an irreversible inhibitor of succinate dehydrogenase, was administered to rats and the characteristics of the neuronal damage were investigated. Injections of 3-NPA (15 mg/kg s.c.) every 2 or 3 days for 1-2 weeks induced a mild neuronal loss and neutrophil invasions in the striatum (STR). The same administration for 4 weeks induced specific symmetric lesions in the lateral STR although the size was variable in each animal. Inside the lesions, strong neutrophil invasions and a strong immunoreaction for IgG, C3 as well as complement factor C3b/C4b receptor (C3b/C4br) were detected. Lesioned sites lost the immunoreaction for GFAP while the marginal areas contained abundant GFAP-labeled astrocytes around the vessels. In intoxicated animals, there was a weak but stout immunoreaction for IgG and C3b/C4br localizing around vessels in the STR even when there were no lesions or neuronal loss. The data suggest that the blood-brain barrier dysfunction is responsible for the specific vulnerability of the STR for the toxin.

Striatal grafts in the ischemic striatum improve pallidal GABA release and passive avoidance.

Fetal striatal cells were grafted into the ischemic striatum of rats and pallidal GABA release, and behavioral improvement were investigated. Intraluminal occlusion of the middle cerebral artery (MCA) for 1 h induced ischemic infarcts in the lateral striatum and the adjacent cortex. In ischemic rats, the performance of a passive avoidance task was disturbed, and the pallidal GABA level detected by microdialysis decreased to about a half of control. After the graft, the deficit in the passive avoidance was partially alleviated and the GABA level recovered moderately and increased further by the infusion of an uptake blocker. The data indicate that fetal striatal cell grafts in the ischemic striatum partially restored both chemical and behavioral deficits.

Melatonin-secreting pineal gland: a novel tissue source for neural transplantation therapy in stroke.

Chronic systemic melatonin treatment attenuates abnormalities produced by occlusion of middle cerebral artery (MCA) in adult rats. Because the pineal gland secretes high levels of melatonin, we examined in the present study whether transplantation of pineal gland exerted similar protective effects in MCA-occluded adult rats. Animals underwent same-day MCA occlusion and either intrastriatal transplantation of pineal gland (harvested from 2-month-old rats) or vehicle infusion. Behavioral tests (from day of surgery to 3 days posttransplantation) revealed that transplanted stroke rats displayed significantly less motor asymmetrical behaviors than vehicle-infused stroke rats. Histological analysis at 3 days posttransplantation revealed that transplanted stroke rats had significantly smaller cerebral infarction than vehicle-infused rats. Additional experiments showed that pinealectomy affected transplantation outcome, in that transplantation of pineal gland only protected against stroke-induced deficits in stroke animals with intact pineal gland, but not in pinealectomized stroke rats. Interestingly, nonpinealectomized vehicle-infused stroke rats, as well as pinealectomized transplanted stroke rats, had significantly lower melatonin levels in the cerebrospinal fluid than nonpinealectomized transplanted stroke rats. We conclude that intracerebral transplantation of pineal gland, in the presence of host intact pineal gland, protected against stroke, possibly through secretion of melatonin.

Chapter 59 Phenotypic plasticity of locus coeruleus noradrenergic neurons after transplantation into the dopamine-depleted caudate in the rat

Publisher Summary This chapter examines the phenotypic plasticity of locus coeruleus (LC) noradrenergic neurons after transplantation into the dopamine-depleted caudate in the rat. The locus coeruleus noradrenergic (NAergic) cells in the caudate nucleus of rats is grafted with unilateral lesions in the nigrostriatal dopamine (DA) pathway and is investigated for cell survival, transmitter phenotypic plasticity, DA levels, and motor recovery. The chapter examines the effect of grafting NAergic cells in these lesioned animals. If these cells are effective in reversing motor deficits, they would provide an alternative and more abundant source of donor tissue, because NAergic cells are found in the peripheral nervous system, such as sympathetic ganglia, and are rather easy to obtain for autografting in Parkinsonian patients. The grafting of LC NAergic cells into the DA-depleted caudate in the rat is quite effective in improving motor disturbances if grafts are performed soon after DA-depletion. LC NAergic cells are obtained from late stage embryos, thus they had already migrated to the proper site in the dorsal midbrain and sent NAergic projections into the rostra1 brain. The plasticity of phenotypic expression of LC NAergic cells indicates a mutual interrelationship between grafted embryonic cells and the microenvironment of the hosts. It also indicates that LC NAergic neurons in late stage embryos still retain some plasticity in phenotypic expression. Thus, young NAergic cells could be one further source of tissue for grafting to ameliorate Parkinsonian motor deficiencies.

Activation of endogenous neural stem cells in intracerebral hemorrhage model rat

s / Neuroscience Research 58S (2007) S1–S244 S85 P1-c45 Characterization of the reeler cerebral cortex by expression of molecular markers Hideyuki Dekimoto, Toshio Terashima, Yu Katsuyama Division of Developmental Neurobiology, Department of Physiology and Cell Biology, Kobe University of Graduate School of Medicine, Hyogo, Japan The cerebral cortex is the six-layered structure. Previous birth-date labeling experiments suggested that the lamination pattern of the cerebral cortex of reeler, the Reelin deficient mutant mouse, is reversed. Recent studies identified genes expressed in the cerebral cortex in a layer specific manner. In this study, we carried out in situ hybridization using such marker genes to characterize the malformation of the reeler cortex. We found that distribution of cells expressing these markers did not exhibit reversed pattern of lamination. Double staining of the normal brain by detection of BrdU (birth-date labeling) and the layer markers showed relationships between marker expression and birth-date of the cortical neurons. However, such coupling of birth-date and specific gene expression was disrupted in reeler cortex. These observations suggest involvement of Reelin signal in regulation of birth-date of the cortical neurons. P1-d0 2 Activation of endogenous neural stem cells in intracerebral hemorrhage model rat Tadashi Masuda, Michiko Kumasaki, Terumi Sakurai, Hideki Hida Department of Neuro-physiol. & Brain Sci., Nagoya City University Grad. Sch. Med. Sci., Nagoya, Japan We have developed internal capsule (IC) hemorrhage model rat which has a small hematoma but relatively severe motor dysfunction. We investigated the activation of endogenous neural stem cells (NSCs) in this model. To evaluate proliferation of NSCs, BrdU was administered for 14 days after the lesion. In the subventricular zone, BrdU-labeled cells increased in lesion group (147.0 ± 25.5, n = 3, p < 0.05) compared to control (37.7 ± 12.7, n = 3). To evaluate migration of NSCs, BrdU was administered from 2 days to 2 hours before the lesion. In the dorsal striatum adjacent to corpus callosum (dSTR), BrdU-labeled cells increased in lesion group (43.0 ± 3.6, n = 4, p < 0.01) compared to control (15.0 ± 1.0, n = 4). Furthermore, DCXpositive cells in the dSTR and in the peri-hemorrhagic area also increased in lesion group (10.1 ± 1.4, n = 8, p < 0.01, 6.5 ± 1.0, n = 7, p < 0.05) compared to control (6.5 ± 1.0, n = 8, 0.0 ± 0.0, n = 6). These data suggested that proliferation and migration of endogenous NSCs are activated after intracerebral hemorrhage. P1-d0 3 Mbh1 is involved in cerebellar granule cell differentiation downstream of a proneural gene, Math1 Daisuke Kawauchi, Tetsuichiro Saito Department of Developmental Biology, Graduate School of Medicine, Chiba University, Chiba, Japan Cerebellar granule cells (CGCs) are one of the major neuronal populations in the cerebellum. CGC differentiation is initiated by expression of a proneural gene, Math1, genetic ablation of which results in the failure of the external granule layer (EGL) formation. Its downstream targets required for proper CGC differentiation remain unidentified, however. Using transgenic mice and chromatin immunoprecipitation assay, we have previously shown that a homeobox gene, Mbh1, is a potential downstream target of Math1 in differentiating CGCs. Here we further indicate a role of Mbh1 in the early differentiation of CGCs. Delivery of Math1specific siRNAs by in vivo electroporation knocks down Math1 protein levels in CGC precursors, which causes downregulation of Mbh1 as well as aberrant EGL formation. In addition, transfection of a dominant negative form of Mbh1 abrogates EGL formation and downregulates NeuroD and TAG-1, markers for differentiating CGCs. These data suggest that Mbh1 function is essential for proper CGC differentiation. Research fund: KAKENHI 18700307 P1-d0 4 Changes in expression of genes and proteins during neural differentiation from embryonic stem cells by Neural Stem Sphere method Masahiro Otsu1, Ryosuke Tatsuno2, Tomoe Horikoshi2, Kuniko Akama2, Koji Murakami1, Takashi Nakayama3, Megumi Nakamura4, Tosifusa Toda4, Hisataka Kasai5, Nobuo Inoue1 1 Laboratory Regener. Neuroscience, Graduate School of Human Health Science, Tokyo Metropol. University, Tokyo, Japan; 2 Graduate School of Science, Chiba University, Chiba, Japan; 3 Department of Biochemistry, Yokohama City University School of Medicines, Yokohama, Japan; 4 Proteomics Collab. Cent., Tokyo Metropol. Inst. Gerontol., Tokyo, Japan; 5 Department of Applied Chemistry, Kogakuin University, Tokyo, Japan Mouse embryonic stem (ES) cells unidirectionally differentiate into neurons via neural stem cells by Neural Stem Sphere (NSS) method. Here, we analyzed changes in expression of genes and proteins during the differentiation. We identified vimentin, creatine kinase, galectin and some proteins, which were up-regulated in neural stem cells and down-regulated in ES cells and neurons by proteomic analysis. We also measured expression level of the corresponding gene by real-time RT-PCR and compared the levels with those of the proteins. This work was partly supported by Grants-in-Aid for Scientific Research from JSPS and Selective Research Fund of Tokyo Metropolitan University. P1-d0 5 Super selective neuronal differentiation and application of cynomolgus embryonic stem cells Otone Endo1, Masaaki Mizuno3, Yasukazu Kajita2, Jun Yoshida2 1 Department of Neurosurgery, JA Kainan Hospital, Aichi, Japan; 2 Department of Neurosurgery, Nagoya University, Nagoya, Japan; 3 Department of Molecular Neurosurgery, Nagoya University, Nagoya, Japan Primate ES cells have rather different character from rodent ones, but it is inevitable to elucidate mechanism for stable culture, purification and induction into object-oriented differentiation, because human ES cells might show wide similarity to cynomolgus ones. We improved the way of large-scale culture maintaining totipotency without contacting feeder cells indispensable for primate ES cells. Our refibed super selective induction method for dopaminergic neurons enabled us to transplant induced neuronal precursor cells in vivo as xenograft which survive without forming tumor such as teratoma for long period with more accuracy and reliability. Their immunohistological, eletrophysiological and ethological evaluation suggests high stability, activity, ability to make neural network system and potentiality to improve clinical symptom of Parkinsonism. Fully functional neural network with other types of induced neurons is under development. P1-d0 7 Rat and human glioma cells express NG2 proteoglycan and immune cell markers Anna Smirkin1, Hiroaki Matsumoto2, Takanori Ohnishi2, Yoshinori Imai1, Hisaaki Takahashi1 1 Department of Molecular and Cellular Physiology, Ehime University, Toon, Japan; 2 Department of Neurosurgery, Ehime University, Toon, Japan Many gliomas respond to platelet-derived growth factor. Involvement of NG2 chondroitin sulfate proteoglycan (NG2) has been reported in plateletderived growth factor (PDGF)-mediated signaling by sequestrating PDGF. In this study, RT-PCR, immunoblotting and immunocytochemistry revealed that rat C6 glioma and several human glioma cell lines expressed NG2 as well as PDGFalpha receptor. Furthermore, C6 glioma and some human glioma cells expressed macrophage-markers, such as CD68 and Iba1. The glioma cells possessed activities of esterase and phagocytosis to some extent. C6 glioma also expressed mRNAs encoding lymphocyte markers, CD4, CD8 and CD200. Such expression patterns of the glioma cells are distinct from those of astrocytes and microglia. Similar Iba1+/NG2+ cells, which may be blood-borne, are found in ischemic core of rat brain after middle cerebral artery occlusion. These observations may shed light to study on the lineage and nature of glioma.

Neuronal transplantation to lesioned striatum of rats subacutely intoxicated with mitochondrial toxin

Acute systemic administration of 3-nitropropionic acid (3-NPA, a mitochondrial toxin) produces striatum specific lesions and motor symptoms similar to those in Huntington’s disease. In the present study, we developed subacutely intoxicated animal models and tried to restore damaged neuronal circuit by neulal transpIantation. One month after repeated intoxication with 3NPA (10 mgikg, i.p. 10 injections over 20 days), the body weight of the animals decreased to 90 % of control’s, passive avoidance task was disturbed but there was no disturbance in rotorod walking. Two months after intoxication, there was a slight loss of neurons and glias in the lateral striatum but both behaviors were almost normalized. Striatal cell suspension derived from El6 fetal rats was transplanted into bilateral striatum of intoxicated animals at one month after intoxication. Neurons and glias survived well in the host striatum and good vascularization to the graft was detected at least two months after transplantation. Data suggest that fetal striatal graft to 3-NPA lesioned striatum may enhance neuronal reconstruction.