Transplanted Oligodendrocyte Progenitor Cells Survive in the Brain of a Rat Neonatal White Matter Injury Model but Less Mature in Comparison with the Normal Brain.

Preterm infants have a high risk of neonatal white matter injury (WMI) caused by hypoxia-ischemia. Cell-based therapies are promising strategies for neonatal WMI by providing trophic substances and replacing lost cells. Using a rat model of neonatal WMI in which oligodendrocyte progenitors (OPCs) are predominantly damaged, we investigated whether insulin-like growth factor 2 (IGF2) has trophic effects on OPCs in vitro and whether OPC transplantation has potential as a cell replacement therapy. Enhanced expression of Igf2 mRNA was first confirmed in the brain of P5 model rats by real-time polymerase chain reaction. Immunostaining for IGF2 and its receptor IGF2 R revealed that both proteins were co-expressed in OLIG2-positive and GFAP-positive cells in the corpus callosum (CC), indicating autocrine and paracrine effects of IGF2. To investigate the in vitro effect of IGF2 on OPCs, IGF2 (100 ng/ml) was added to the differentiation medium containing ciliary neurotrophic factor (10 ng/ml) and triiodothyronine (20 ng/ml), and IGF2 promoted the differentiation of OPCs into mature oligodendrocytes. We next transplanted rat-derived OPCs that express green fluorescent protein into the CC of neonatal WMI model rats without immunosuppression and investigated the survival of grafted cells for 8 weeks. Although many OPCs survived for at least 8 weeks, the number of mature oligodendrocytes was unexpectedly small in the CC of the model compared with that in the sham-operated control. These findings suggest that the mechanism in the brain that inhibits differentiation should be solved in cell replacement therapy for neonatal WMI as same as trophic support from IGF2.

Alterations of Both Dendrite Morphology and Weaker Electrical Responsiveness in the Cortex of Hip Area Occur Before Rearrangement of the Motor Map in Neonatal White Matter Injury Model.

Hypoxia-ischemia (H-I) in rats at postnatal day 3 causes disorganization of oligodendrocyte development in layers II/III of the sensorimotor cortex without apparent neuronal loss, and shows mild hindlimb dysfunction with imbalanced motor coordination. However, the mechanisms by which mild motor dysfunction is induced without loss of cortical neurons are currently unclear. To reveal the mechanisms underlying mild motor dysfunction in neonatal H-I model, electrical responsiveness and dendrite morphology in the sensorimotor cortex were investigated at 10 weeks of age. Responses to intracortical microstimulation (ICMS) revealed that the cortical motor map was significantly changed in this model. The cortical area related to hip joint movement was reduced, and the area related to trunk movement was increased. Sholl analysis in Golgi staining revealed that layer I-III neurons on the H-I side had more dendrite branches compared with the contralateral side. To investigate whether changes in the motor map and morphology appeared at earlier stages, ICMS and Sholl analysis were also performed at 5 weeks of age. The minimal ICMS current to evoke twitches of the hip area was higher on the H-I side, while the motor map was unchanged. Golgi staining revealed more dendrite branches in layer I-III neurons on the H-I side. These results revealed that alterations of both dendrite morphology and ICMS threshold of the hip area occurred before the rearrangement of the motor map in the neonatal H-I model. They also suggest that altered dendritic morphology and altered ICMS responsiveness may be related to mild motor dysfunction in this model.

Monosodium glutamate ingestion during the development period reduces aggression mediated by the vagus nerve in a rat model of attention deficit-hyperactivity disorder.

We used an umami substance, monosodium glutamate (MSG), as a simple stimulant to clarify the mechanism of the formation of emotional behavior. A 60 mM MSG solution was fed to spontaneously hypertensive rats (SHR), used as a model of attention-deficit hyperactivity disorder, from postnatal day 25 for 5 weeks kept in isolation. Emotional behaviors (anxiety and aggression) were then assessed by the open-field test, cylinder test and social interaction test. MSG ingestion during the developmental period resulted in a significant reduction in aggressive behavior but had few effects on anxiety-like behavior. Several experiments were performed to identify the reason for the reduced aggression with MSG intake. Blood pressure in the MSG-treated SHR was comparable to that of the controls during development. Argyrophil III staining to detect the very early phase of neuronal damage revealed no evidence of injury by MSG in aggression-related brain areas. Assessment of plasma amino acids revealed that glutamate levels remained constant (∼80 µM) with MSG ingestion, except for a transient increase after fasting (∼700 µM). However, lactate dehydrogenase assay in an in vitro blood-brain barrier model showed that cell toxicity was not induced by indirect MSG application even at 700 µM, confirming that MSG ingestion caused minimal neuronal damage. Finally, vagotomy at the sub-diaphragmatic level before MSG ingestion blocked its effect on aggressive behavior in the isolated SHR. The data suggest that MSG ingestion during the developmental period can reduce aggressive behavior in an attention deficit-hyperactivity disorder model rat, mediated by gut-brain interaction.

Disorganization of Oligodendrocyte Development in the Layer II/III of the Sensorimotor Cortex Causes Motor Coordination Dysfunction in a Model of White Matter Injury in Neonatal Rats.

We previously established neonatal white matter injury (WMI) model rat that is made by right common carotid artery dissection at postnatal day 3, followed by 6% hypoxia for 60 min. This model has fewer oligodendrocyte progenitor cells and reduced myelin basic protein (MBP) positive areas in the sensorimotor cortex, but shows no apparent neuronal loss. However, how motor deficits are induced in this model is unclear. To elucidate the relationship between myelination disturbance and concomitant motor deficits, we first performed motor function tests (gait analysis, grip test, horizontal ladder test) and then analyzed myelination patterns in the sensorimotor cortex using transmission electron microscopy (TEM) and Contactin associated protein 1 (Caspr) staining in the neonatal WMI rats in adulthood. Behavioral tests revealed imbalanced motor coordination in this model. Motor deficit scores were higher in the neonatal WMI model, while hindlimb ladder stepping scores and forelimb grasping force were comparable to controls. Prolonged forelimb swing times and decreased hindlimb paw angles on the injured side were revealed by gait analysis. TEM revealed no change in myelinated axon number and the area g-ratio in the layer II/III of the cortex. Electromyographical durations and latencies in the gluteus maximus in response to electrical stimulation of the brain area were unchanged in the model. Caspr staining revealed fewer positive dots in layers II/III of the WMI cortex, indicating fewer and/or longer myelin sheath. These data suggest that disorganization of oligodendrocyte development in layers II/III of the sensorimotor cortex relates to imbalanced motor coordination in the neonatal WMI model rat.

Dysfunction in Motor Coordination in Neonatal White Matter Injury Model Without Apparent Neuron Loss.

We made a white matter injury (WMI) model with mild hindlimb dysfunction by right common carotid artery occlusion followed by 6% oxygen for 60 min at postnatal day 3 (P3), in which actively proliferating oligodendrocyte (OL) progenitors are mainly damaged. To know whether this model is appropriate for cell therapy using OL progenitors, the pathological response to mild hypoxia-ischemia (H-I) in neurons and OL lineage cells and myelination failure were investigated along with gene expression analysis. In WMI model rats, coordinated motor function, as assessed by the accelerating rotarod test, was impaired. The dysfunction was accompanied by myelination failure in layers I-IV of the sensorimotor cortex. Although several oligo2-positive OLs stained positive for active caspase 3 in the cortex and white matter at 24 h after H-I, few NeuN-positive neurons were apoptotic. Argyrophil-III staining for damaged neurons revealed no increase in the number of degenerating cells in the model. Moreover, the total number of NeuN-positive neurons in the cortex was comparable to that of controls 7 days later. Retrograde labeling of the corticospinal tract with Fluoro-Gold revealed no significant loss of layer V neurons. In addition, no decrease in the numbers of cortical projecting neurons and layers V-VI neurons in both motor and sensory areas was observed. Interestingly, the numbers of inhibitory GABAergic cells immunoreactive for parvalbumin, calretinin, or somatostatin were preserved in the P26 cortex. Gene expression analysis at P5 revealed 98 upregulated and 65 downregulated genes that may relate to cell survival, myelin loss, and differentiation of OLs. These data suggest that impaired motor coordination was not induced by neuron loss but, rather, myelination failure in layers I-IV. As OL lineage cells are mainly damaged, this WMI model might be useful for cell-based therapy by replacing OL progenitors.

Early constraint-induced movement therapy promotes functional recovery and neuronal plasticity in a subcortical hemorrhage model rat.

Constraint-induced movement therapy (CIMT) promotes functional recovery of impaired forelimbs after hemiplegic strokes, including intracerebral hemorrhage (ICH). We used a rat model of subcortical hemorrhage to compare the effects of delivering early or late CIMT after ICH. The rat model was made by injecting collagenase into the globus pallidus near the internal capsule, and then forcing rats to use the affected forelimb for 7 days starting either 1 day (early CIMT) or 17 days (late CIMT) after the lesion. Recovery of forelimb function in the skilled reaching test and the ladder stepping test was found after early-CIMT, while no significant recovery was shown after late CIMT or in the non-CIMT controls. Early CIMT was associated with greater numbers of ΔFosB-positive cells in the ipsi-lesional sensorimotor cortex layers II-III and V. Additionally, we found expression of the growth-related genes brain-derived neurotrophic factor (BDNF) and growth-related protein 43 (GAP-43), and abundant dendritic arborization of pyramidal neurons in the sensorimotor area. Similar results were not detected in the contra-lesional cortex. In contrast to early CIMT, late CIMT failed to induce any changes in plasticity. We conclude that CIMT induces molecular and morphological plasticity in the ipsi-lesional sensorimotor cortex and facilitates better functional recovery when initiated immediately after hemorrhage.

ATBF1 is a novel amyloid-ß protein precursor (AßPP) binding protein that affects AßPP expression.

The cytoplasmic C-terminal domain of amyloid-ß protein precursor (AßPP) binds to several proteins that regulate the trafficking and processing of AßPP and affects amyloid-ß (Aß) production. We previously reported that levels of AT-motif binding factor 1 (ATBF1) are increased in the brains of 17-month-old Tg2576 mice compared with wild-type controls, and that Aß42 increases ATBF1 expression, inducing death in primary rat cortical neurons. Here, we show that ATBF1 levels are increased in the cytoplasm of hippocampal neurons in Alzheimer's disease (AD) brains compared with non-AD brains. Furthermore, cotransfection of human embryonic kidney (HEK293T) and human neuroblastoma (SH-SY5Y) cells with ATBF1 and AßPP695 increased steady-state levels of AßPP via the binding of ATBF1 to the AßPP cytoplasmic domain (amino acids 666-690), resulting in increased Aß production and cellular and soluble AßPP (sAßPP) levels without affecting the activity or levels of AßPP processing enzymes (α-, ß-, or γ-secretase). Conversely, knockdown of endogenous ATBF1 reduced levels of cellular AßPP, sAßPP, and Aß in HEK293 cells overexpressing human AßPP695. Our findings provide insight into the dynamics of AßPP processing and Aß production, and suggest that ATBF1 is a novel AßPP binding protein that may be a suitable therapeutic target for AD.

Auraptene increases the production of amyloid-ß via c-Jun N-terminal kinase-dependent activation of γ-secretase.

Amyloid-ß (Aß) peptide plays a major role in the pathogenesis of Alzheimer's disease (AD), and is generated by ß- and γ-secretase-mediated proteolytic processing of amyloid-ß protein precursor (AßPP). In the present study, we investigated the effect of 118 natural compounds on Aß production in the medium of HEK293 cells stably expressing human AßPP695 (HEK293-AßPP) using Aß42 sandwich ELISA to find natural compounds that can modulate Aß production. We found that a coumarin derivative of citrus fruits, auraptene, increased Aß production. Treatment of HEK293-AßPP cells and rat primary cortical neurons with auraptene significantly increased the secretion of Aß40, Aß42, and the Aß42/40 ratio. However, auraptene did not change the protein levels of the AßPP processing enzymes, a disintegrin and metalloproteinases 10 (ADAM10, α-secretase), ß-site AßPP cleaving enzyme-1 (BACE-1, ß-secretase), and presenilin 1 (PS1, γ-secretase component). Auraptene increased the activity of γ-secretase but not that of α- and ß-secretase. Furthermore, auraptene enhanced γ-secretase-mediated production of Aß from AßPP or AßPP-C99, but not through α- and ß-secretase. Auraptene also phosphorylated c-Jun N-terminal kinase (JNK), and pretreatment with the JNK inhibitor, SP600125, reduced auraptene-induced γ-secretase activity. Overall, our results suggest that auraptene-mediated activation of JNK may contribute to the production of Aß by promoting γ-secretase activity.

Enhanced electrical responsiveness in the cerebral cortex with oral melatonin administration after a small hemorrhage near the internal capsule in rats.

Intracerebral hemorrhage (ICH) can cause direct brain injury at the insult site and indirect damage in remote brain areas. Although a protective effect of melatonin (ML) has been reported for ICH, its detailed mechanisms and effects on remote brain injury remain unclear. To clarify the mechanism of indirect neuroprotection after ICH, we first investigated whether ML improved motor function after ICH and then examined the underlying mechanisms. The ICH model rat was made by collagenase injection into the left globus pallidus, adjacent to the internal capsule. ML oral administration (15 mg/kg) for 7 days after ICH resulted in significant recovery of motor function. Retrograde labeling of the corticospinal tract by Fluoro-Gold revealed a significant increase in numbers of positive neurons in the cerebral cortex. Immunohistological analysis showed that ML treatment induced no difference in OX41-positive activated microglia/macrophage at day 1 (D1) but a significant reduction in 8-hydroxydeoxyguanosin-positive cells at D7. Neutral red assay revealed that ML significantly prevented H2 O2 -induced cell death in cultured oligodendrocytes and astrocytes but not in neurons. Electrophysiological response in the cerebral cortex area where the number of Fluoro-Gold-positive cells was increased was significantly improved in ML-treated rats. These data suggest that ML improves motor abilities after ICH by protecting oligodendrocytes and astrocytes in the vicinity of the lesion in the corticospinal tract from oxidative stress and causes enhanced electrical responsiveness in the cerebral cortex remote to the ICH pathology.

Differentiation of oligodendrocytes from mouse induced pluripotent stem cells without serum.

Cell therapy using induced pluripotent stem (iPS) cells might become a new approach for treating neonatal hypoxic-ischemic injury such as periventricular leukomalacia. To obtain appropriate donor cells for transplantation, we differentiated oligodendrocyte (OL) lineage cells from mouse iPS cells. Induction of OL lineage cell differentiation from iPS cells was carried out with a seven-step culture method. Mouse iPS cells (stage 1) were induced to form embryoid bodies for 4 days under a serum-free condition that was suitable for ectoderm induction (stage 2), following by selection of nestin-positive neural stem cells (NSCs) for 10-12 days (stage 3). NSCs were cultured in expansion medium containing fibroblast growth factor (FGF)-2 for 4 days (stage 4), induced to differentiate into glial progenitor cells by epidermal growth factor and fibroblast growth factor (FGF-2) treatment for 4-5 days (stage 5), and then into OL progenitor cells by culture in neurobasal A medium containing FGF-2 and platelet-derived growth factor for 6-8 days (stage 6). Terminal differentiation into O4-positive OLs was carried out by culture in neurobasal A containing T3 and ciliary neurotrophic factor for 7 days (stage 7). Inwardly rectifying K+ currents, which are characteristic of OLs, were detected in iPS cell-derived cells at stage 7 in whole cell clamp mode. Our data suggest that OLs can be effectively differentiated from mouse iPS cells without serum in a stepwise manner, which may be appropriate for use as donor cells in transplantation.

Minor neuronal damage and recovered cellular proliferation in the hippocampus after continuous unilateral forelimb restraint in normal rats.

Constraint-induced movement therapy (CIMT) involves the restraint of an intact limb to force the dominant use of an affected limb, in an attempt to enhance use-dependent plasticity and reduce dysfunction. To investigate whether forced disuse of an intact forelimb with CIMT causes a loss of limb function and degenerative damage in the brain, a staircase test and a horizontal ladder test were carried out in control rats and forelimb-restrained rats, and then Argyrophil III silver staining, which is capable of detecting subtle neuronal damage, was used to examine histological alterations associated with restraint. No significant changes in forelimb function were observed in restrained rats. However, atypical weak argyrophilic neurons, an indicator of minor neural damage, were found in the bilateral hippocampus of restrained rats. This damage was not found in the cortex, striatum, or spinal cord. Investigation of neurogenesis in the subventricular zone (SVZ) and subgranular zone (SGZ) revealed a clear reduction in the number of bromodeoxyuridine-positive cells in bilateral SGZ, but not in the SVZ, in restrained rats compared with controls. This reduction was accompanied by reduced mRNA expression of vascular endothelial growth factor and glial-derived neurotrophic factor. However, reduced cellular proliferation and decreased gene expression were recovered after the removal of the restraint. Our results suggest that forced disuse of the intact forelimb has no significant effect on skilled forelimb function but has a minor effect on neurogenesis in SGZ, suggesting that mild stress may be caused by the restraint.

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.

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.

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.