Sachiyo Misumi

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.

Environmental enrichment brings a beneficial effect on beam walking and enhances the migration of doublecortin-positive cells following striatal lesions in rats

Rats raised in an enriched environment (enriched rats) have been reported to show less motor dysfunction following brain lesions, but the neuronal correlates of this improvement have not been well clarified. The present study aimed to elucidate the effect of chemical brain lesions and environmental enrichment on motor function and lesion-induced neurogenesis. Three week-old, recently weaned rats were divided into two groups: one group was raised in an enriched environment and the other group was raised in a standard cage for 5 weeks. Striatal damage was induced at an age of 8 weeks by injection of the neuro-toxins 6-hydroxydopamine (6-OHDA) or quinolinic acid (QA) into the striatum, or by injection of 6-OHDA into the substantia nigra (SN), which depleted nigrostriatal dopaminergic innervation. Enriched rats showed better performance on beam walking compared with those raised in standard conditions, but both groups showed similar forelimb use asymmetry in a cylinder test. The number of bromodeoxyuridine-labeled proliferating cells in the subventricular zone was increased by a severe striatal lesion induced by QA injection 1 week after the lesion, but decreased by injection of 6-OHDA into the SN. Following induction of lesions by striatal injection of 6-OHDA or QA, the number of cells positive for doublecortin (DCX) was strongly increased in the striatum; however, there was no change in the number of DCX-positive cells following 6-OHDA injection into the SN. Environmental enrichment enhanced the increase of DCX-positive cells with migrating morphology in the dorsal striatum. In enriched rats, DCX-positive cells traversed the striatal parenchyma far from the corpus callosum and lateral ventricle. DCX-positive cells co-expressed an immature neuronal marker, polysialylated neural cell adhesion molecule, but were negative for a glial marker. These data suggest that environmental enrichment improves motor performance on beam walking and enhances neuronal migration toward a lesion area in the striatum.

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

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)‐1β, IL‐11, leukemia‐inhibitory factor (LIF), and glial‐derived neurotrophic factor or low oxygen (3.5% O2: 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‐1β 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, β‐tubulin III, glial fibrillary acidc protein, and TH] were different in C‐Mix and L‐Oxy treatments. Because increases in hypoxia‐inducible factor (HIF)‐1α protein were found in both treatments, we investigated the effect of HIF‐1α on differentiation of NPCs to DAergic neurons. Inhibition of HIF‐1α 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, β‐tubulin III, and S‐100β were increased by treatment with just ODNs, indicating the importance of the endogenous effect of HIF‐1α 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‐1α.

Enhanced neurogenesis from neural progenitor cells with G1/S-phase cell cycle arrest is mediated by transforming growth factor β1

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 p27kip1 [ 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 μm) 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)‐β1 and cdk5 at 24 h after differentiation, whereas Aph only increased TGF‐β1 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‐β1 (10 ng/mL) did not increase neurite outgrowth but rather the number of β‐tubulin III‐positive cells, which was accompanied by enhanced p27kip1 mRNA expression. In addition, TGF‐β receptor type II expression was observed in nestin‐positive NPCs. Results indicated that DFO‐induced TGF‐β1 signaling activated smad3 translocation from the cytoplasm to the nucleus. In contrast, TGF‐β1 signaling inhibition, via a TGF‐β receptor type I inhibitor (SB‐505124), resulted in decreased DFO‐induced neurogenesis, in conjunction with decreased p27kip1 protein expression and smad3 translocation to the nucleus. These results suggest that cell cycle arrest during G1/S‐phase induces TGF‐β1 expression. This, in turn, prompts enhanced neuronal differentiation via smad3 translocation to the nucleus and subsequent p27kip1 activation in NPCs.

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.

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 H2O2‐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.

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.

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.

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.