Circadian rhythm of metabolic oscillation in suprachiasmatic nucleus depends on the mitochondrial oxidation state, reflected by cytochrome C oxidase and lactate dehydrogenase.

Metabolic activity in the suprachiasmatic nucleus (SCN), a center of biological rhythm, is higher during the daytime than at night. The rhythmic oscillation in the SCN is feedback controlled by the Clock/Bmal1 heterodimer binding to the E-box in target genes (e.g., Arg-vasopressin). Similar transcriptional regulation by Npas2/Bmal1 heterodimer formation operates in the brain, which is dependent on the redox state (i.e., NAD/NADH). To clarify the metabolic function of SCN in relation to the redox state and glycolysis levels, we measured glucose, lactate dehydrogenase (LDH), LDH mRNA, and cytochrome C oxidase, energy-producing biochemical materials from mitochondria/cytosol, in rats kept under a light-dark cycle. Mitochondrial cytochrome C oxidase activity, measured by the changes in absorption at 550 nm, was higher during the light period than during the dark period. Glucose concentration was higher during the light period. In contrast, LDH and its coding mRNA were higher during the dark period. Mitochondrial aggregation, which is reflected by mitochondrial membrane potential, indexed by JC-1 fluorescence, was higher during the light period. The results indicate that the glycolysis energy pathway in the SCN, which exhits higher metabolic activity during the day than at night, might be involved in the generation of circadian rhythm.

Circadian rhythm of enolase in suprachiasmatic nucleus depends on mitochondrial function.

Metabolic activity in the suprachiasmatic nucleus (SCN), a center of biological rhythm, is higher during the daytime than at night. The rhythmic oscillation in the SCN is feedback controlled by the CLOCK/BMAL1 heterodimer binding to the E-box in target genes (e.g., Arg- vasopressin). Similar transcriptional regulation by NPAS2/BMAL1 heterodimer formation operates in the brain, which depends on the redox state (i.e., NAD/NADH). To clarify the metabolic function of SCN in relation to the redox state, two-dimensional electrophoresis was carried out on the mitochondrial fraction of SCN, obtained from rats kept under a light:dark cycle and constant under dim light. The electrophoretic pattern with TOF-mass spectrometry analysis revealed that enolase catalyzes the interconversion of 2-phosphoglycerate and phosphoenolpyruvate. The enolase activity, coupled with lactate dehydrogenase, was higher during the light period than that in the dark. However, enolase mRNA, analyzed by RT-PCR, showed higher levels during the dark period than in the light. The clock gene products Per2, Bmal1, Rev-erbα, and AVP mRNA in the mitochondrial fraction of SCN developed a circadian rhythm showing almost the same peak time as that in whole SCN. These mRNA rhythms ran free except for that of Rev-erbα mRNA. The results indicate that, in the glycolysis-related energy pathway, enolase might be involved in higher metabolic activity during the day than at night, at least in part.

Enhanced neurogenesis from neural progenitor cells with G1/S-phase cell cycle arrest is mediated by transforming growth factor beta1.

We have previously demonstrated that a G1/S-phase cell cycle blocker, deferoxamine (DFO), increased the number of new neurons from rat neurosphere cultures, which correlated with prolonged expression of cyclin-dependent kinase (cdk) inhibitor p27(kip1) [H. J. Kim et al. (2006)Brain Research, 1092, 1-15]. The present study focuses on neuronal differentiation mechanisms following treatment of neural stem/progenitor cells (NPCs) with a G1/S-phase cell cycle blocker. The addition of DFO (0.5 mm) or aphidicolin (Aph) (1.5 microm) to neurospheres for 8 h, followed by 3 days of differentiation, resulted in an increased number of neurons and neurite outgrowth. DFO induced enhanced expression of transforming growth factor (TGF)-beta1 and cdk5 at 24 h after differentiation, whereas Aph only increased TGF-beta1 expression. DFO-induced neurogenesis and neurite outgrowth were attenuated by administration of a cdk5 inhibitor, roscovitine, suggesting that the neurogenic mechanisms differ between DFO and Aph. TGF-beta1 (10 ng/mL) did not increase neurite outgrowth but rather the number of beta-tubulin III-positive cells, which was accompanied by enhanced p27(kip1) mRNA expression. In addition, TGF-beta receptor type II expression was observed in nestin-positive NPCs. Results indicated that DFO-induced TGF-beta1 signaling activated smad3 translocation from the cytoplasm to the nucleus. In contrast, TGF-beta1 signaling inhibition, via a TGF-beta receptor type I inhibitor (SB-505124), resulted in decreased DFO-induced neurogenesis, in conjunction with decreased p27(kip1) protein expression and smad3 translocation to the nucleus. These results suggest that cell cycle arrest during G1/S-phase induces TGF-beta1 expression. This, in turn, prompts enhanced neuronal differentiation via smad3 translocation to the nucleus and subsequent p27(kip1) activation in NPCs.

Increase in dopaminergic neurons from mouse embryonic stem cell-derived neural progenitor/stem cells is mediated by hypoxia inducible factor-1alpha.

A reliable method to induce neural progenitor/stem cells (NPCs) into dopaminergic (DAergic) neurons has not yet been established. As well, the mechanism involved remains to be elucidated. To induce DAergic differentiation from NPCs, a cytokine mixture (C-Mix) of interleukin (IL)-1beta, IL-11, leukemia-inhibitory factor (LIF), and glial-derived neurotrophic factor or low oxygen (3.5% O(2): L-Oxy) was used to treat embryonic stem (ES) cell-derived NPCs. Treatment with C-Mix increased the number of tyrosine hydroxylase (TH)-positive cells compared with controls (2.20-fold of control). The C-Mix effect was induced by mainly LIF or IL-1beta treatment. Although L-Oxy caused an increase in TH-positive cells (1.34-fold), the combination of L-Oxy with C-Mix did not show an additive effect. Increases in DA in the medium were shown in the presence of C-Mix, LIF, and L-Oxy by high-performance liquid chromatography. Gene expression patterns of neural markers [tryptophan hydroxylase (TPH), GAD67, GluT1, beta-tubulin III, glial fibrillary acidc protein, and TH] were different in C-Mix and L-Oxy treatments. Because increases in hypoxia-inducible factor (HIF)-1alpha protein were found in both treatments, we investigated the effect of HIF-1alpha on differentiation of NPCs to DAergic neurons. Inhibition of HIF-1alpha by the application of antisense oligodeoxynucleotides (ODNs) to NPCs caused a decrease in TH-positive cells induced by LIF treatment. Gene expressions of TH, GAD67, and GluT1 were decreased, and those of TPH, beta-tubulin III, and S-100beta were increased by treatment with just ODNs, indicating the importance of the endogenous effect of HIF-1alpha on neuronal differentiation. These data suggest that enhanced differentiation into DAergic neurons from ES cell-derived NPCs was induced by C-Mix or L-Oxy mediated by HIF-1alpha.

Altered striatal vulnerability to 3-nitropropionic acid in rats due to sex hormone levels during late phase of brain development.

Systemic administration of 3-nitropropionic acid (3-NPA) leads to a shortage of cellular ATP and induces striatum-specific lesions that resemble Huntington's disease. Gender differences, in terms of vulnerability of striatum to 3-NPA, have been shown in male rats. The goal of the present study was to determine whether changes in sex hormone levels during the critical period of sexual differentiation (E17-P4) influence striatal vulnerability to 3-NPA. An androgen receptor antagonist, flutamide, or an aromatase-inhibitor, fadrozole hydrochloride, which block conversion of testosterone to estradiol, were administered to embryonic rats during E17-E20 or E18-E20, respectively, with subsequent 3-NPA (20mg/(kg day) for 2 days) treatment during adulthood (8-9 weeks old). Motor behavior and histological changes (IgG exudation due to blood-brain barrier dysfunction and glial fibrillary acidic protein immunoreactivity) were assessed. Treatment with flutamide significantly decreased the 3-NPA-induced motor behavior in male rats, while administration of fadrozole hydrochloride increased atypical motor behavior in female rats. IgG exudation, as well as decreased glial fibrillary acidic protein reactivity, was observed in animals with motor defects. Flutamide decreased testosterone levels in male rats, while fadrozole hydrochloride increased testosterone levels in female rats. These results suggest that prenatal modulation of sexual hormonal levels greatly influences vulnerability to 3-NPA during adulthood and directly correlates to serum testosterone levels.

Pleiotrophin promotes functional recovery after neural transplantation in rats.

Pleiotrophin promotes survival of dopaminergic neurons in vitro. To investigate whether pleiotrophin promotes survival of grafted dopaminergic neurons in vivo, donor cells from ventral mesencephalon were treated with pleiotrophin (100 ng/ml) during cell preparation and grafted into striatum of hemi-Parkinson model rats. Functional recovery in methamphetamine-induced rotations was improved, and more tyrosine hydroxylase-positive cells survived in the striatum in the pleiotrophin-treated group. Pleiotrophin addition to cells just before transplantation also resulted in better functional recovery; however, no caspase-3 activation was seen during cell preparation. Interestingly, the effect of pleiotrophin on the survival was additive to that of glial-cell line-derived neutropic factor. These results revealed that pleiotrophin had effects on donor cells in neural transplantation in vivo.

Increase in neurogenesis and neuroblast migration after a small intracerebral hemorrhage in rats.

Neural stem/progenitor cells (NPCs) reside in the subventricular zone (SVZ) and dentate gyrus in the adult mammalian brain. It has been reported that endogenous NPCs are activated after brain insults such as ischemic stroke. We investigated whether proliferation and migration of endogenous NPCs are increased after a collagenase-induced small intracerebral hemorrhage (ICH) near the internal capsule in rats. Bromodeoxyuridin (BrdU) administration for 14 days after ICH (post-labeling) resulted in an increase in the number of BrdU-positive cells as shown in both ipsilateral and contralateral SVZs. BrdU treatment given for 2 days before ICH to label endogenous NPCs (pre-labeling), caused more BrdU-positive cells to be detected in the ipsilateral dorsal striatum (dSTR) compared to those in the contralateral dSTR 14 days after ICH. BrdU- and doublecortin (Dcx)-positive cells were found in the ipsilateral STR. An increase in the number of Dcx-positive migrating immature neurons was found in the dSTR and peri-hemorrhage area 14 days after ICH, and a cluster of Dcx-positive cells was found in the STR around the lesion 28 days after ICH. Matrix metalloproteinase-2 (MMP-2) was strongly expressed in wide area of the injured brain, particularly around the lesion 14 and 28 days after ICH. Dcx- and MMP-2-positive cells were detected in the ipsilateral STR near the lesion. These data suggest that collagenase-induced ICH enhances the proliferation of endogenous NPCs and the migration of newly born neuroblasts toward the hemorrhage area.

Treatment with deferoxamine increases neurons from neural stem/progenitor cells.

Neural transplantation is a promising approach for treating neurodegenerative disease. Neural stem/progenitor cells (NPCs) are self-renewing and multipotent and thus are good candidates for donor cells when they have been clearly defined to differentiate into neurons. As neuronal differentiation follows cell cycle exit, we investigated whether neuron production from NPCs is increased by treatment with cell cycle blockers. NPCs from E12.5 rat ventral mesencephalon were cultured as neurospheres in DMEM/F12 medium containing N2 supplements and bFGF. Treatment of NPCs with deferoxamine, a G1/S phase blocker, increased the number of beta-tubulin III-positive cells after differentiation, concomitant with increases of MAP2 mRNA and protein, and a decrease of GFAP protein. Further, an increase in beta-tubulin III/BrdU double-positive cells and a decrease in GFAP/BrdU double-positive cells were confirmed. In real-time PCR, the expressions of p21(cip1), p27(kip1) and p57(kip2) mRNAs remained unaltered for 8 h after treatment with deferoxamine but were significantly elevated after 1 day. Deferoxamine specifically enhanced the elevation of p27(kip1) mRNA at 1-2 days and the accumulation of p27(kip1) protein at 3 days, along with the activation of neuroD promoter and the elevation of neuroD mRNA. Transfection of p27(kip1) into NPCs induced activation of neuroD promoter and increase of number of beta-tubulin III-positive cells. These data suggest that pretreatment with deferoxamine increases the number of neurons from NPCs related to prolonged p27(kip1) elevation and activation of the neuroD signaling pathway. In this way, regulation of the cell cycle should be a useful first step in engineering NPCs for neural transplantation.

Involvement of nitric oxide in 3-nitropropionic acid-induced striatal toxicity in rats.

The roles of nitric oxide (NO) in 3-nitropropionic acid (3-NPA)-induced toxicity were investigated using in vivo and in vitro models. Chronic 3-NPA administration (10 mg/kg) to rats produced selective striatal lesions that were associated with abnormal motor and EMG activities. In these animals, there was loss of glial fibrillary acidic protein (GFAP)-positive cells with extravasation of IgG in the lesion center, although microtubule-associated protein (MAP)-2-positive cells remained, indicating that astrocytes were involved. 3-NPA increased the NO(2)(-)/NO(3)(-) levels in microdialysates obtained from the striatum, thalamus and cerebellum. The basal NO(3)(-) level was much higher in the striatum than in the other areas. The NO(2)(-)/NO(3)(-) levels in the striatum were much higher in animals exhibiting abnormal muscular activity. Expression of endothelial NO synthase (eNOS), but not neuronal NOS (nNOS), was greatly increased in the striatum at 5 h after a second 3-NPA exposure, but not in other areas. In astrocyte cultures, the toxic effects of 3-NPA were associated with corresponding increases in the NO(2)(-) level, and this toxicity was attenuated by hemoglobin (Hb; 20 microM), which quenches NO. The NO(2)(-) generated by 3-NPA, even without cells, was also antagonized by Hb. 3-NPA, S-nitroso-n-acetyl-dl-penicillamine (SNAP) and sodium nitroprusside (SNP) all increased the NO current (detected by NO-sensitive electrodes) in concentration-dependent manners, and Hb significantly attenuated the NO generation induced by 3-NPA, SNAP or SNP. Taken together, these results suggest that 3-NPA generates NO both directly as a donor and indirectly by enhancing NOS expression to produce toxic effects on astrocytes and neuronal toxicity.

A myelin galactolipid, sulfatide, is essential for maintenance of ion channels on myelinated axon but not essential for initial cluster formation.

Myelinated axons are divided into four distinct regions: the node of Ranvier, paranode, juxtaparanode, and internode, each of which is characterized by a specific set of axonal proteins. Voltage-gated Na+ channels are clustered at high densities at the nodes, whereas shaker-type K+ channels are concentrated at juxtaparanodal regions. These channels are separated by the paranodal regions, where septate-like junctions are formed between the axon and the myelinating glial cells. Although oligodendrocytes and myelin sheaths are believed to play an instructive role in the local differentiation of the axon to distinct domains, the molecular mechanisms involved are poorly understood. In the present study, we have examined the distribution of axonal components in mice incapable of synthesizing sulfatide by disruption of the galactosylceramide sulfotransferase gene. These mice displayed abnormal paranodal junctions in the CNS and PNS, whereas their compact myelin was preserved. Immunohistochemical analysis demonstrated a decrease in Na+ and K+ channel clusters, altered nodal length, abnormal localization of K+ channel clusters appearing primarily in the presumptive paranodal regions, and diffuse distribution of contactin-associated protein along the internode. Similar abnormalities have been reported previously in mice lacking both galactocerebroside and sulfatide. Interestingly, although no demyelination was observed, these channel clusters decreased markedly with age. The initial timing and the number of Na+ channel clusters formed were normal during development. These results indicate a critical role for sulfatide in proper localization and maintenance of ion channels clusters, whereas they do not appear to be essential for initial cluster formation of Na+ channels.

Pleiotrophin mRNA is highly expressed in neural stem (progenitor) cells of mouse ventral mesencephalon and the product promotes production of dopaminergic neurons from embryonic stem cell-derived nestin-positive cells.

Neural stem cells are promising candidates for donor cells in neural transplantation. However, the mechanism by which neural stem cells differentiate into neurons is not well understood. In the present study, a serial analysis of gene expression (SAGE) was carried out to generate a gene file of neural stem (progenitor) cells from the mouse ventral mesencephalon. Among the 15,815 tags investigated, the mRNA of the housekeeping genes (elongation factor 1-alpha, ATPase subunit 6, GAPDH, actin), laminin receptor 1, HSP 70, pleiotrophin, and nestin were highly expressed. Because pleiotrophin (PTN) exhibits mitogenic and trophic effects on neural development and exhibits trophic effects on survival of dopaminergic (DAergic) neurons, we investigated the role of PTN in neurogenesis, especially to DAergic neurons. Here, we show that PTN increased the production of tyrosine hydroxylase (TH)-positive neurons from embryonic stem (ES) cell-derived nestin-positive cells. The expression of Nurr1 mRNA was enhanced by PTN. L-dopa in the culture medium was increased by PTN. This effect was as strong as with sonic hedgehog. Data suggest that PTN mRNA is highly expressed in neural stem (progenitor) cells of mouse ventral mesencephalon, and PTN promotes the production of DAergic neurons from ES cell-derived nestin-positive cells.

Neurodegeneration of substantia nigra accompanied with macrophage/microglia infiltration after intrastriatal hemorrhage.

Intrastriatal hemorrhage in rats causes neurodegenaration of the substantia nigra (SN) followed by the appearance of ED1(+) cells (macrophage/microglia). ED1(+) cells were observed for at least 8 weeks after hemorrhage. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) was shown in ED1(+) cells with the expression of both brain-derived neurotrophic factor (BDNF) mRNA and BDNF, suggesting that activated-p38 MAPK(+)/ED1(+) cells would produce BDNF and may exhibit trophic effect on the degenerating neurons in the SN. However, in ELISA, BDNF protein decreased significantly in ipsilateral SN at 7 days after hemorrhage, which may be due to a dramatic decrease of BDNF immunoreactive neurons in pars compacta. Data suggest that activation of p38 MAPK in ED1(+) cells infiltrating in ipsilateral SN after hemorrhage may produce BDNF, but that the amount of BDNF produced from ED1(+) cells is insufficient for the rescue of degenerating neurons.

Signal transmission from the suprachiasmatic nucleus to the pineal gland via the paraventricular nucleus: analysed from arg-vasopressin peptide, rPer2 mRNA and AVP mRNA changes and pineal AA-NAT mRNA after the melatonin injection during light and dark periods.

Arg-vasopressin (AVP) containing neurons are one of the output paths from the suprachiasmatic nucleus (SCN), the center of the biological clock. AVP mRNA transcription is controlled by a negative feedback loop of clock genes. Circadian rhythm of melatonin release from the pineal gland is regulated by the SCN via the paraventricular nucleus (PVN). To clarify the transduction system of circadian signals from the SCN to the pineal gland, we determined the effects of melatonin injection (1 mg/kg, i.p.) during light and dark periods on Per2 and AVP mRNAs in the SCN and PVN, in addition to arylalkylamine N-acetyltransferase (AA-NAT) and inducible cAMP early repressor (ICER) mRNAs in the pineal gland of rats using RT-PCR. AVP peptide contents were also measured in the SCN and PVN. AVP content in the SCN decreased during the light period, while no changes were observed in the PVN. In the SCN, Per2 mRNA increased during both light and dark periods. In the PVN, Per2 decreased during the light period and increased during the dark period at 180 min after melatonin injection. In the pineal gland, Per2 mRNA increased between 60 and 180 min after the melatonin injection during the light period, while it did not significantly change during the dark period. The AA-NAT mRNA varied similar to the Per2 mRNA changes. These results might suggest that the different responses to melatonin in the pineal gland during the light and dark periods was originated in the changes of Per2 in the PVN via SCN.

Dexamethasone induces different wheel running activity than corticosterone through vasopressin release from the suprachiasmatic nucleus.

During the analysis of wheel running activity, we found that corticosterone (1 mg/100 g BW) injection decreased wheel activity, while dexamethasone (0.1 mg/100 g) increased the activity. To clarify the functional differences between corticosterone and dexamethasone, we measured Arg-vasopressin (AVP) release from the suprachiasmatic nucleus (SCN) slice culture in vitro and AVP coding mRNA in the SCN in vivo. The corticosterone (0.2 and 2 microg/ml, final concentration in medium) decreased the AVP release, while it increased by dexamethasone (0.2 and 2 microg/ml). An AVP mRNA in the SCN was decreased by both corticosterone (1 mg/100 g) and dexamethasone (0.1 mg/100 g). The differences in wheel activity by corticosterone and dexamethasone are discussed from the changes of AVP in the SCN.

Estrogen blocks 3-nitropropionic acid-induced Ca2+i increase and cell damage in cultured rat cerebral endothelial cells.

Systemic administration of 3-nitropropionic acid (3-NPA, a mycotoxin) induces brain damage accompanied by disturbance in the blood-brain barrier (BBB). Since the endothelial cells are important components of the BBB and the first target of a systemic intoxication, in the present study, the effect of 3-NPA on primary cultured rat brain endothelial cells (rBECs) was examined by studying intracellular Ca(2+) ([Ca(2+)](i)) response using imaging techniques with fura-2. rBECs were prepared using a method of Kis et al. [Eur. J. Pharmacol. 368 (1999) 35-42] and Szabo et al. [Neurobiology 5 (1997) 1-16]. Almost all cells were immunoreactive to antibody against the factor VIII-related antigen (von-Willebrand factor). They showed a typical dose-dependent increase of [Ca(2+)](i) in response to ATP or bradykinin. Low concentrations of 3-NPA (1.7 mM, 3.4 mM) caused no changes, and a medium concentration (6.8 mM) increased the [Ca(2+)](i) gradually and progressively, and the increase was reversed incompletely back to the resting level after washing. A high concentration (13.6 mM) increased the [Ca(2+)](i) irreversibly. These elevations of [Ca(2+)](i) were absent in a Ca(2+)-free medium. In endothelial cells treated with 17beta-estradiol (above 10(-5) M) or with a selective estrogen receptor modulator, tamoxifen (5 x 10(-7) M), no elevation of [Ca(2+)](i) was observed with 3-NPA treatment. The response to ATP was impaired after application of 3-NPA, but it was preserved by cotreatment with 17beta-estradiol or tamoxifen. An estrogen receptor antagonist ICI 182,780 inhibited these effects by 17beta-estradiol or tamoxifen. Lysosomal neutral red uptake and TUNEL experiments revealed the necrotic but not apoptotic cell death at least in this acute stage. Data indicate that a medium to high concentration of 3-NPA induces damage on rBECs as revealed by an accumulation of [Ca(2+)](i), but the damage was protected by cotreatment with 17beta-estradiol or tamoxifen, suggesting that estrogen may be protective for the brain vascular damage via estrogen receptor.

Intracerebral hemorrhage upregulates Na(+)/myo-inositol cotransporter in the rat brain.

We examined the expression of Na(+)/myo-inositol cotransporter (SMIT) in the rat brain after intrastriatal hemorrhage. The expression of SMIT messenger RNA (mRNA) increased around hematoma 3 days after hemorrhage and it returned to control level as hematoma was absorbed. The expression of SMIT mRNA also increased at ipsilateral substantia nigra without blood-brain barrier disruption 7 days after hemorrhage and remained high until 42 days after hemorrhage. Immunohistochemistry revealed that macrophages around hematoma and microglias at ipsilateral substantia nigra were SMIT-positive. These results suggest that the expression of SMIT must be regulated not only by osmolality but also by unknown factors.

Melatonin reduces cerebral edema formation caused by transient forebrain ischemia in rats.

Reduction of cerebral edema, an early symptom of ischemia, is one of the most important remedies for reducing subsequent chronic neural damage in stroke. Melatonin, a metabolite of tryptophan released from the pineal gland, has been found to be effective against neurotoxicity in vitro. The present study was aimed to demonstrate the effectiveness of melatonin in vivo in reducing ischemia-induced edema using magnetic resonance imaging (MRI). Rats were subjected to middle cerebral artery (MCA) occlusion/reperfusion surgery. Melatonin was administered twice (6.0 mg/kg, p.o.): just prior to 1 h MCA occlusion and 1 day after the surgery. T2-weighted multislice spin-echo images were acquired 1 day after the surgery. Increases in T2-weighted signals in ischemic sites of the brain were clearly observed after MCA occlusion. The signal increase was found mainly in the striatum and in the cerebral cortex in saline-treated control rats. In the melatonin-treated group, the total volume of cerebral edema was reduced by 45.3% compared to control group (P < 0.01). The protective effect of melatonin against cerebral edema was more clearly observed in the cerebral cortex (reduced by 56.1%, P < 0.01), while the reduction of edema volume in the striatum was weak (reduced by 23.0%). The present MRI study clearly demonstrated that melatonin is effective in reducing edema formation in ischemic animals in vivo, especially in the cerebral cortex. Melatonin may be highly useful in preventing cortical dysfunctions such as motor, sensory, memory, and psychological impairments.

High titer retroviral gene transduction to neural progenitor cells for establishment of donor cells for neural transplantation to parkinsonian model rats.

Neural progenitor cells (NPCs) are expected to be useful donor sources for cell transplantation therapy in Parkinson's disease. However, control of the differentiational lineage, especially into dopaminergic neurons, is still difficult. Thus, genetic modification of NPCs to produce l-dopa is potentially useful. The present study prepared high titer retrovirus carrying human tyrosine hydroxylase-1 (HTH-1) gene. HTH-1 gene could be efficiently transduced into NPCs obtained from the E12.5 rat mesencephalon. This retroviral gene transduction caused no apparent changes in survival, proliferation, or differentiation. In vitro, HTH-1 gene-transduced NPCs released little l-dopa and addition of tetrahydrobiopterin, the cofactor of tyrosine hydroxylase, was required for production of l-dopa. In vivo, three of seven hemi-parkinsonian model rats that received HTH-1 gene-transduced donor NPCs achieved functional recovery. High titer retroviral vector for gene transduction could be used to prepare NPCs for transplantation to hemi-parkinsonian model rats. However, functional recovery after transplantation of HTH-1 gene-transduced NPCs was incomplete.

[Neural transplantation for cerebral ischemia/infarction--the present situation and prospects].

Cerebral infarction results in multiple symptoms including hemiplegia and cognitive disturbances. The central nervous system has a limited capacity for self-repair, thus there is a great interest in the possibility of repairing the central nervous system by neural transplantation. Two different types of cerebral infarction model are well investigated for neural transplantation. Several kinds of donor cells have been used to try to restore the brain damage after ischemic insult. Reconstruction of neural circuits by transplantation is an ideal goal for the treatment of cerebral infarction, but trophic action of transplantation is also expected. Amelioration of ischemia-induced brain damage and recovery of neural dysfunction are documented. However, there are several factors and problems to be solved for clinical application.

Pretreatment with low doses of erythropoietin ameliorates brain damage in periventricular leukomalacia by targeting late oligodendrocyte progenitors: a rat model.

BACKGROUND: One of the pathological hallmarks of periventricular leukomalacia (PVL) is the selective vulnerability of late oligodendrocyte progenitors (preoligodendrocytes; preOLs) to hypoxia-ischemia (H-I). It is unknown whether recombinant human erythropoietin (rhEPO) protects preOLs in vivo. OBJECTIVES: To develop a rat PVL model in which preOLs are selectively damaged and exhibit similar pathological changes to diffuse-type human PVL, various conditions of H-I were compared in P2-P7 rats (P2 = postnatal day 2). To evaluate the effect of rhEPO on oligoprotection (preOLs), rhEPO was administered to P3 PVL rats. METHODS: After counts of NG2-positive and O4-positive cells were performed in P2-P7 rats, right common carotid artery occlusion followed by 6% O(2) for 0-120 min was performed in P2-P4 rats. The mortality and histological alterations after hematoxylin/eosin staining and ED1 immunostaining were assessed 2 days after H-I. Various doses of rhEPO (1-30,000 U/kg i.p.) were administered to PVL rats 15 min before administration of 6% O(2). RESULTS: Double-positive cells for NG2 and O4 were detected from P2, and their number gradually increased until P7. Although right common carotid artery occlusion with 6% O(2) for 60 min resulted in a relatively high proportion of deaths in P2-P4 rats, typical histological changes in the PVL diffuse component were found in most surviving P3 animals. With 50-100 U/kg rhEPO, the histological damage was attenuated. CONCLUSIONS: Histological changes similar to those seen in the PVL diffuse component were induced by H-I in P3 rats, in which preOLs were gradually developing, and a low dose of rhEPO was effective in the treatment of brain damage induced by H-I.