Fujiya Furuyama

Long-term survival of grafted cells, dopamine synthesis/release, synaptic connections, and functional recovery after transplantation of fetal nigral cells in rats with unilateral 6-OHDA lesions in the nigrostriatal dopamine pathway

In animal models of hemi-Parkinsons disease, survival of grafted nigral cells, their synaptic connections, dopamine (DA) synthesis/release, and recovery from motor disturbances were investigated, and these were compared among 3 groups of animals raised for 3 months, 1 year and 2 years after the transplantation. Fetal nigral DAergic cell suspensions were transplanted in the ipsilateral caudate nucleus of rats with unilateral 6-OHDA lesions in the nigrostriatal DA pathway. Motor disturbances, assessed by methamphetamine-induced rotation, recovered partly in the 2nd week, significantly in the 4th week after the grafting, and remained stable thereafter. Many tyrosine hydroxylase (TH)-positive cells were detected along the grafting tracks. The number of TH-positive cells was similar in the 3 groups of animals. These TH-positive cells made synaptic connections in the host caudate. By in vivo microdialysis measurement, extracellular DA, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) around the grafted sites recovered to 30-100% of those of controls. No significant differences were observed in the concentration of DA, DOPAC and HVA among 3 groups of animals. They also responded to methamphetamine loading though the magnitudes were smaller. Using a TH cDNA probe, TH-positive cells were found to express TH mRNA in in situ hybridization-autoradiographic analysis. Data indicate that grafted fetal DAergic cells survive, synthesize and release DA, make synaptic connections in the host brain and ameliorate motor disturbances for over 2 years. There were no differences in these parameters among the 3 groups of animals, and no untoward side effects were observed even at 2 years after the grafting. Thus it was confirmed that the grafting of neuronal cells into the brain is a promising approach to restore disturbed function.

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.

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.

tGS ganglioside induces peculiar morphological features in grafted dopaminergic cells and promotes motor recovery in rats with unilateral lesions in the nigrostriatal dopamine pathway

A cell suspension of substantia nigra from fetal rats was introduced into the ipsilateral caudate nucleus of rats with unilateral lesions in the nigrostriatal dopamine pathway, and effects of bovine total ganglioside (tGS) and monosialoganglioside (GM1) treatment on the morphological features of the transplanted cells and recovery from motor imbalance (rotation induced by methamphetamine) were investigated. Gangliosides (30 mg/kg) were administered intraperitoneally once a day for 2 weeks after transplantation to test animals while control animals received saline alone. tGS animals showed definite motor recovery in the 2nd week (P less than 0.05) while control and GM1 animals exhibited slight recovery only. At 6 weeks after transplantation, motor imbalance disappeared in all 3 groups. Tyrosine hydroxylase (TH) immunocytochemical staining revealed that in the 2nd week TH-positive cells in tGS animals had more primary dendrites and more large neurites (meganeurites) than did controls. TH-positive cells of all 3 groups often had spiny processes at that time. In the 20th week, TH-positive cells became more multigonal and had wider dendritic fields in all groups, and had less meganeurites and spines. Motor recovery of each animal was dependent on the number of TH-positive cells and no significant difference was observed in the number of TH-positive cells among the three groups. tGS treatment for 2 weeks without grafting induced immunohistologically no axonal sprouting in the substantia nigra, medial forebrain bundle, accumbens and caudate nucleus when the chemical lesions were complete. Data suggest that tGS induces hypertrophy but not hyperplasia of the transplanted nigral cells, and increases the morphological plasticity. This might be the basis for promotion of recovery in motor function after transplantation.

Descending influences from nucleus raphe magnus on fusimotor neurone activity in rats

Abstract 1. 1.Single fusimotor fibres were isolated in the ventral roots of lumbosacral segments of urethane-anaesthetized rats, and effects of electrical stimulation of the nucleus raphe magnus (NRM) on their spontaneous activity were investigated. The experiments were carried out in rats whose bilateral preoptic and anterior hypothalamic regions (PO/AH) were electrolytically destroyed to eliminate the influences of these regions to fusimotor activity. 2. 2.Of 44 fusimotor fibres studied, 38 (86%) were found to be affected by NRM stimulation. The effects of NRM stimulation were classified according to their response pattern: primary depression (D-type, n = 24), facilitation followed by depression (F-D-type, n = 5) and primary facilitation (F-type, n = 8). The most predominant effect of NRM stimulation upon fusimotor activity was characterized by a strong depression followed by a complete cessation of firing lasting either for a short period or for more than 30 min (D- and F-D-type). 3. 3.In three fusimotor fibres studied in the different preparations, it was observed that a NRM-evoked depression response was blocked by an intraperitoneal administration of a serotonin antagonist, p -chlorophenylalamine ( p -CPA) (10 mg/kg). 4. 4.The results indicate that the NRM exerts descending inhibitory or facilitatory influences on fusimotor neurones, and suggest that cold shivering is controlled by modulating fusimotor neurone activity via the serotonergic raphe-signal pathways.

Thermal salivation in rats anesthetized with barbiturates, chloralose, urethane and ketamine

1. The relationship between thermal salivation (TS) and thermoregulation was studied in anesthetized rats. 2. Of the 6 anesthetics used, ketamine-anesthetized rats secreted the largest amount of saliva. Salivation, however, was thermal and not induced by ketamine itself. 3. Ketamine-anesthetized rats readily secreted saliva at core temperatures less than 40 degrees C but TS was remarkably enhanced by hyperthermia of 40-42.5 degrees C. 4. The equilibrium phase in the triphasic heat response of core temperature was a consequence of equilibrium between heat gain and heat loss by salivation.

Thermoregulatory responses of the inbred heat-tolerant FOK rat to cold

The responses of inbred heat-tolerant FOK rats to cold were compared with those of Wistar King A/H (WKAH) and Std:Wistar (WSTR) strains. The fall of colonic temperature during cold exposure was unexpectedly smaller in FOK than in other groups, but the onset of shivering was delayed in FOK. Norepinephrine (NE)-induced in vivo oxygen consumption and the mitochondrial uncoupling protein 1 level of brown adipose tissue (BAT) were not different among the groups, but the cold-induced increases in in vivo oxygen consumption as well as plasma glycerol and free fatty acids were higher in FOK than in other groups. In vitro NE-induced oxygen consumption of BAT was less in FOK than WSTR, but not WKAH. The magnitude of the NE-induced increase in blood flow through BAT was higher in FOK than in other groups. These results suggest that FOK paradoxically have a high capacity for nonshivering thermogenesis in spite of their high capacity for heat tolerance, probably due to an increased lipid utilization and improved circulation of BAT.The responses of inbred heat-tolerant FOK rats to cold were compared with those of Wistar King A/H (WKAH) and Std:Wistar (WSTR) strains. The fall of colonic temperature during cold exposure was unexpectedly smaller in FOK than in other groups, but the onset of shivering was delayed in FOK. Norepinephrine (NE)-induced in vivo oxygen consumption and the mitochondrial uncoupling protein 1 level of brown adipose tissue (BAT) were not different among the groups, but the cold-induced increases in in vivo oxygen consumption as well as plasma glycerol and free fatty acids were higher in FOK than in other groups. In vitro NE-induced oxygen consumption of BAT was less in FOK than WSTR, but not WKAH. The magnitude of the NE-induced increase in blood flow through BAT was higher in FOK than in other groups. These results suggest that FOK paradoxically have a high capacity for nonshivering thermogenesis in spite of their high capacity for heat tolerance, probably due to an increased lipid utilization and improved circulation of BAT.

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.

Effect of Continuous Light on the Circadian Rhythms of Running-Wheel Activity and Body Temperature in Spontaneously Hypertensive Rats

The circadian rhythms of running-wheel activity and body temperature in a strain of spontaneously hypertensive rats (Aoki) and a strain of normotensive rats (Wistar Kyoto) were analysed. When the rats