Michiko Kumazaki

Glial cell survival is enhanced during melatonin-induced neuroprotection against cerebral ischemia

The role of glial cells in neuronal death has become a major research interest. Glial cell activation has been demonstrated to accompany cerebral ischemia. However, there is disagreement whether such gliosis is a cell death or a neuroprotective response. In the present study, we examined alterations in glial cell responses to the reported neuroprotective action of the free radical scavenger, melatonin, against cerebral ischemia. Adult male Wistar rats were given oral injections of either melatonin (26 micromol/rat) or saline just prior to 1 h occlusion of the middle cerebral artery (MCA), then once daily for 11 or 19 consecutive days. At 11 and 19 days after reperfusion of the MCA, randomly selected animals were killed and their brains removed for immunohistochemical assays. Melatonin significantly enhanced survival of glial cells (as revealed by glial cell specific markers, glial fibrillary acidic protein and aquaporin-4 immunostaining) at both time periods postischemia, and the preservation of these glial cells in the ischemic penumbra corresponded with a markedly reduced area of infarction (detected by immunoglobulin G and hematoxylin-eosin staining), as well as increased neuronal survival. The ischemia-induced locomotor deficits were partially ameliorated in melatonin-treated animals. In vitro replications of ischemia by serum deprivation or by exposure to free radical-producing toxins (sodium nitroprusside and 3-nitropropionic acid) revealed that melatonin (10 microg/ml or 100 microM) treatment of pure astrocytic cultures significantly reduced astrocytic cell death. These results suggest a potential strategy directed at enhancing glial cell survival as an alternative protective approach against ischemic damage.

Mesencephalic Neural Stem (Progenitor) Cells Develop to Dopaminergic Neurons More Strongly in Dopamine-Depleted Striatum than in Intact Striatum

Epidermal growth factor (EGF)/fibroblast growth factor (FGF)-responsive stem (progenitor) cells from embryonic brain have self-renewing and multipotent properties and thus are good candidates for donor cells in neural transplantation. However, the survival and differentiation to mature neurons after grafting of stem cells into adult brain are rather poor. We hypothesize that the differentiation of stem cells to mature neurons, such as dopaminergic (DAergic) neurons, is dependent on environmental cues that control the ontogenic development. We compared the survival and differentiation between mesencephalic (MS) and cortical (CTx) stem (progenitor) cells, following grafting into bilateral striata of hemiparkinsonian model rats. MS and CTx stem cells were prepared from E12 rats and proliferated in serum-free medium with EGF or basic FGF for 2 weeks. One day after being primed to differentiate, the cell suspensions of both origins were grafted into the bilateral striata of adult rats that had unilateral 6-OHDA lesions in the substantia nigra. MS cells differentiated to tyrosine hydroxylase (TH)-positive neurons more strongly in DA-depleted striatum than in intact striatum, and methamphetamine-induced rotation was ameliorated in half of the grafted animals. Rosette-like cell aggregation and dysfunction of the blood-brain barrier (BBB) were less in and around the grafts in DA-depleted striatum, suggesting less proliferation and more differentiation of MS stem cells in DA-depleted striatum. Neither TH-positive neurons nor behavioral amelioration were detected following CTx stem (progenitor) cell transplantation in the striata. Data suggest that the DA-depleted striatum offers a suitable environment for MS stem (progenitor) cells to differentiate into mature DAergic neurons.

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.

Estrogen protects against while testosterone exacerbates vulnerability of the lateral striatal artery to chemical hypoxia by 3-nitropropionic acid

Gender differences in the vulnerability of the lateral striatal artery (1STR artery) to systemic intoxication with 3-nitropropionic acid (3-NPA, succinate dehydrogenase inhibitor) were studied. Subcutaneous injection of 3-NPA (20 mg/kg once a day for 2 days) induced striatal selective lesions in half of male rats associated with motor symptoms (rolling, paddling, recumbency, etc) while female rats were resistant. Lesions were located in the lateral striata and characterized by astroglial necrotic cell death, enhanced immunoreaction to factor VIII-related antigen, edema, extravasation of IgG and sometimes bleeding. The motor and histological disturbances were highly sex-dependent and modulated by changes in hormonal levels. Males were more susceptible than females. Castration had little effect but ovariectomy enhanced the vulnerability. Replacement therapy with testosterone increased while estradiol or tamoxifen suppressed the vulnerability in ovariectomized females. Investigation of the arterial architecture of the brain often revealed rectangular and acute angled branchings in the centrolateral striatum where the ISTR artery feeds. A parallel in vitro toxicity study demonstrated that an extreme Ca++ overload and a strong cellular swelling resulted in astrocytic cell death. Data suggest that 1STR artery and astrocytes are highly vulnerable to 3-NPA intoxication in males. The greater vulnerability of the ISTR artery may contribute to the pathogenesis of neurodegenerative diseases, striatal bleeding, etc. Protective effects of estrogen and tamoxifen may mediate gender differences often observed in these disorders and suggest their potential use as therapeutic agents for these disorders.

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.

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.

3-Nitropropionic acid produces striatum selective lesions accompanied by iNOS expression

Systemically administered 3-nitropropionic acid (3-NPA) that inhibits the mitochondrial oxidative phosphorylation induces selective lesions in the striatum. To investigate the nature of these selective lesions, we administered 3-NPA (20 mg/kg, s.c. daily for 2 or 3 days) to Wistar rats and investigated the behavioral disturbance, striatal lesions and their variations after modulating the activity of nitric oxide synthase (NOS). On the second or third day of 3-NPA administration, half the animals manifested behavioral disturbances (paddling, rolling, tremor, abnormal gait, and recumbence). A strong extravasation of immunoglobulin G (IgG) and a decrease in immunoreaction for glial fibrillary acidic protein (GFAP) were detected, and iNOS-like (iNOS-L) immunoreactive small cells appeared in the lateral and central striatum especially around the vessels. A week later, lesions lacking GFAP-immunoreaction were detected in the striatum in survived animals. Pretreatment with N-nitro-L-arginine methyl ester (L-NAME) along with each injection of 3-NPA did not improve the behavioral disturbances nor the survival rate, but attenuated the extravasation of IgG and iNOS-L immunoreaction. Pretreatment with aminoguanidine or FK506 improved the behavioral symptoms and survival rate. Extravasation of IgG and expression of iNOS-L immunoreactivity were attenuated, and the striatal lesion was reduced. Data indicate the involvement of NO in the high vulnerability of the striatum, and that iNOS, one of inflammatory markers, is induced following exposure to 3-NPA.

Expression of dopamine transporter mRNA and its binding site in fetal nigral cells transplanted into the striatum of 6-OHDA lesioned rat

Neurological disorders in rat model of hemi-Parkinsons disease can be compensated by the transplantation of fetal nigral cells. However, the role of the dopamine transporter (DAT) in this recovery has not been clarified. To clarify this mechanism, we examined the expression of DAT in the caudate putamen (CPu) by in situ hybridization histochemistry (mRNA) and autoradiography (using the ligand [125I] beta-CIT, which labels DAT) and compared them with the recovery of motor disturbance revealed with methamphetamine-induced rotation. Models were made with the stereotaxic injection of 6-hydroxydopamine into the left side of the substantia nigra pars compacta. Cell suspensions from rat fetus (embryonic day 14-15) were transplanted into the lesioned side of CPu. Methamphetamine-induced rotation, expression of DAT mRNA, and [125I] beta-CIT binding were evaluated 2, 4 and 12 weeks after the transplantation. Methamphetamine-induced rotation recovered partly in the 2nd week and significantly in the 4th week. [125I] beta-CIT binding increased with time and the dense binding was detected 4 and 12 weeks after the transplantation. In all transplanted rats, cells expressing DAT mRNA were found in CPu. These results indicated that transplanted fetal dopaminergic cells maturated in CPu of host animals and extended nerve terminals where high density of DAT binding sites were found.

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.