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.

[Observation of physiological change in the human ciliary body using an ultrasound biomicroscope during accommodation].

PURPOSE: To evaluate changes in the ciliary body during accommodation using an ultrasound biomicroscope (UBM). SUBJECTS AND METHODS: Eleven healthy persons, aged from 24 to 33 years, served as subjects. They were asked to lie in the supine position and to fixate a target placed on the ceiling 2 m above with the left eye. A concave lens with the power of -6 to -8 diopters was then placed before the fixating left eye. The thickness of the ciliary body in the right eye was measured by UBM in the nonaccommodative and accommodative states. FINDINGS: The anterior chamber in the right eye became significantly shallow during accommodation. The thickness of the ciliary body significantly increased during accommodation at 0.5 mm and 1.0 mm posterior to the scleral spur. It significantly decreased at 2.0 mm, 2.5 mm and 3.0 mm posterior to the scleral spur. CONCLUSION: During induced accommodation in the left eye, the anterior portion of the ciliary body in the right eye increased and the posterior portion decreased in thickness. The findings imply that the circular ciliary muscles are mainly involved in accommodation and not the longitudinal muscles.

Volume Changes Due to SO2-4, SeO2-4, and H2PO-4 Adsorption on Amorphous Iron(III) Hydroxide in an Aqueous Suspension.

Volume changes due to SO2-4, SeO2-4, and H2PO-4 adsorption on amorphous iron(III) hydroxide were determined by dilatometry in a mixture comprising an aqueous solution of Na2SO4, Na2SeO4, or NaH2PO4 and amorphous iron(III) hydroxide suspended in 0.1 mol dm-3 NaClO4 at an initial pH ranged from 4.50 to 6.50. System volumes increased during SO2-4, SeO2-4, and H2PO-4 adsorption on amorphous iron(III) hydroxide, suggesting that some hydrated water around aqueous SO2-4, SeO2-4, or H2PO-4 ions and amorphous iron(III) hydroxide were released to the bulk. The volume changes due to SO2-4, SeO2-4, and H2PO-4 adsorption at initial pH 4.50 were +18, +18, and +14 cm3 mol-1, respectively. Phosphate, which adsorbed as an inner-sphere complex, released most of its hydration water to the bulk. The volume changes due to SO2-4 and SeO2-4 adsorption indicated that they were dehydrated at the water/amorphous iron(III) hydroxide interface. The smaller changes in volume for H2PO-4 compared to SO2-4 and SeO2-4 may be explained by differences of charges and adsorption mechanisms of these oxoanions. Copyright 1999 Academic Press.

Dopamine metabolism in the striatum of hemiparkinsonian model rats with dopaminergic grafts.

To investigate dopamine (DA) levels as well as DA metabolism by which the striatal DAergic grafts may bring the functional recovery to hemiparkinsonian model rats, a microdialysis study was performed in the striatum, and an autoradiographic analysis for DA transporter was made. In hemiparkinsonian model rats, the concentrations of DA, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in striatal perfusates, decreased considerably (less than 5%, of control levels). In grafted rats that showed motor recovery, the concentration of DA recovered to almost control level, and DOPAC and HVA to about 20% of controls' suggesting that the rate of DA metabolism is low. L-DOPA loading to grafted rats induced a big release of DOPAC and HVA, thus the DOPAC/DA ratio was close to that of the controls'. Methamphetamine loading increased the concentration of DA but did not change the level of DOPAC and HVA. Haloperidol loading increased DA, DOPAC and HVA. [3H]mazindol binding that reflects the activity of the DA transporter decreased considerably in hemiparkinsonian model rats, but it reappeared more or less in grafted rats. Data indicated that in grafted striatum, the extracellular DA level is almost normal level while the rate of DA metabolism is low. By L-DOPA loading, the grafts show the capacity to synthesize, release and metabolize DA and then the DOPAC/DA ratio is normalized. Responses to methamphetamine and haloperidol, as well as the results of the autoradiographic study suggest that the grafts are under a good feedback regulation of DA metabolism.

Dopamine-denervation enhances the trophic activity in striatum: evaluation by morphological and electrophysiological development in PC12D cells.

To evaluate the possibility that dopamine (DA) denervation enhances the trophic activity in striatum, normal or DA-depleted striatal tissue extract (N- or L-extract, respectively) was obtained, and their trophic effects on PC12D cells were investigated from the viewpoints of differentiation using morphological and electrophysiological analyses. Treatment with N- or L-extract induced neurite outgrowth in a concentration-dependent manner, and induced the enlargement of cell size. These effects were stronger in L-extract than in N-extract. Cation currents were investigated in whole cell patch-clamp mode. Development of cation current started with delayed-rectifier type K+ current (IK) and transient type K+ current (IA), followed by Ca2+ current (ICa) and tetrodotoxin-sensitive Na+ current (INa). INa was expressed more frequently in L-extract treated cells than N-extract treated cells at D7-9. The larger IK amplitude in L-extract treatment at D7-9 seemed to be related to the expression of INa. Development of IA was similar at any stage for both treatments. ICa development started at D3-5 after treatments, and the amplitude and current density were similar in both treatments. ICa was strongly blocked by omega-conotoxin GVIA (3 microM), indicating that N-type channels were mainly expressed after treatments. The data suggests that L-extract has stronger effects to hasten the differentiation of PC12D cells than N-extract by promoting the neurite outgrowth, cell enlargement and expression of voltage-dependent cation channels, especially INa and IK.

Dynamic regulation of striatal dopaminergic grafts during locomotor activity.

The present experiment was designed to estimate the neurochemical activity of dopaminergic grafts in hemiparkinsonian model rats during locomotion and to examine the functional importance of dynamic regulation of the grafted neurons in the host brain. Rats were trained to run on a straight treadmill at various speeds (300, 660, 1200, 1800 cm/min), and extracellular dopamine (DA) and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), were measured by in vivo microdialysis during and after running. Grafted rats were divided into two groups depending on their running ability and data were compared with those of normal and lesioned controls. Although the tonic level of extracellular DA in grafted rats recovered to 70% of control, levels of DOPAC and HVA remained 15-20% of controls. A small number of grafted rats showed full recovery in treadmill running tasks. In these animals, the percentage increase in DOPAC and HVA showed similar time courses and magnitudes as those in normal rats. Most grafted rats showed partial recovery in locomotor ability. The percentage increase in DOPAC and HVA in these animals remained at a lower level than that in normal rats, though the tonic levels of DA, DOPAC and HVA were not lower than those of fully recovered rats. Data suggest that grafted DAergic cells in functionally well recovered rats were dynamically regulated in the host brain in an actual behavior and that well-controlled release of DA might be involved in the recovery of complex motor behavior, such as high speed locomotion.

Dopamine has inhibitory and accelerating effects on ischemia-induced neuronal cell damage in the rat striatum.

Dopaminergic (DAergic) influence on ischemic neuronal cell damage in the dorsolateral striatum was studied. Intact and 6-hydroxydopamine (6-OHDA) lesioned rats, with and without pretreatment by D1 and D2 DA antagonists, were subjected to 20 min forebrain ischemia. Extracellular DA and glutamate (Glu) were measured using microdialysis technique. Histological examination was performed on the dorsolateral striatum and the hippocampal CA1 area 24 h after ischemia. DA increased 400-500 times the control level during ischemia among the groups except the 6-OHDA lesioned group. No significant changes were observed in the concentration of 3,4-dihydroxyphenylacetic acid (DOPAC), but a transient decrease was seen in homovanillic acid (HVA). Due to ischemia, Glu increased up to about 5 times the control level among the groups. Neuronal damage in the dorsolateral striatum was slightly attenuated by 6-OHDA lesion. Treatment by spiperone (D2 antagonist, 7 micrograms/kg IP) alone attenuated the damage strongly. Treatment by SCH23390 (D1 antagonist, 2.5 mg/kg IP) alone or both D1 and D2 antagonists had no effects. Data suggest that excessive Glu and DA are involved in neuronal cell damage. DA might enhance the damage via D2 but inhibit via D1 receptor.

Tetraplegic wheelchair basketball.

Tetraplegic wheelchair basketball was started in the Kanagawa Rehabilitation Center (KRC) as a recreational sport for tetraplegics in 1980. In this game, there are two goals on each side, thus we call it 'twin basketball'. One goal is of ordinary height and the other is low. Three ways of shooting and two ways of dribbling are allowed according to the player's level of tetraplegia and technical skill. The first official game was held in 1983. Since then, the game has been taken up in several areas of Japan. The first All Japan championship game was held in 1987, ten teams including 98 tetraplegics attending. Five years later, in the sixth championship game, 18 teams including 171 tetraplegics attended. As official physicians, we have examined the physical condition and technical skills of all players since 1987. All players are classified, and assigned points from 1 to 4.5. The total number of points of five players in one team are limited to 11.

Pathophysiological process after transient ischemia of the middle cerebral artery in the rat.

For the understanding of pathophysiology of the cerebral ischemia, we made a transient intraluminal occlusion of the middle cerebral artery in the rat and investigated the appearance of collapsed dark neurons and the extravasation of serum proteins using argyrophil III method and immunohistochemistry. In the acute stage (minutes to 3 days), dark neurons appeared in the lateral half of the ipsilateral striatum and adjacent cortex which formed the ischemic core of this model. Dark neurons also appeared in the ipsilateral reticular thalamic nucleus, hippocampus, and amygdala. The extravasation of serum proteins, albumin, leucocyte common antigen, immunoglobulin G, complement factor C3, as well as heat shock protein 70, was observed not only in the ischemic but sometimes also in the contralateral hemisphere. Among these, the expression of IgG and C3 was most prominent in the ischemic core. In the chronic stage (1 to 3 months), the ischemic core changed into the porencephaly, and the ventrobasal nucleus of the thalamus got also involved in the necrosis. A strong microgliosis was observed in the substantia nigra pars reticulata. Data suggest, that among many mechanisms that contribute to ischemic neuronal death, the activation of immune response, due to the damage of blood-brain barrier and the extravasation of serum proteins could promote the ischemic cell death in the brain.

Reconstruction of GABAergic transmission and behavior by striatal cell grafts in rats with ischemic infarcts in the middle cerebral artery.

Fetal striatal cell suspensions were grafted stereotaxically into the infarcted striatum of rats, and reconstruction of striatopallidal GABA transmission and behavior were investigated. Occlusion of the middle cerebral artery (MCA) for one hour induced ischemic infarcts mainly in the lateral striatum, as detected by magnetic resonance imaging (MRI) and histology. Ischemic rats had deficits in the performance of a passive avoidance task, both acquisition and retention, but no changes in general circadian actograms. In these animals pallidal GABA, detected by microdialysis, decreased to about half of control levels. There were suggestions of an improvement in passive avoidance performance in the grafted animals. Pallidal GABA concentrations recovered almost to control levels, and were increased by infusions of the GABA uptake blocker nipecotic acid. These data indicate that neural transplantation is a promising approach to improve the deficits in chemical transmission and behavior following ischemic infarcts in rat striatum.

Improvement of passive avoidance task after grafting of fetal striatal cell suspensions in ischemic striatum in the rat.

Behavioral recovery and cell survival/growth after grafting of fetal striatal cell suspensions in the ischemic striatum of rats were investigated. Ischemia was induced by one hour intraluminal occlusion of the right middle cerebral artery under halothane anesthesia. During the ischemia rats usually manifested signs of hemiparesis and sometimes rotations. Behavioral function was measured by a passive avoidance task and radial arm maze test at 1-2 weeks and 6-7 weeks after ischemia. The size of the ischemic lesions depended on each animal, but the ischemic animals showed deficits in both passive avoidance task and radial maze test. Two weeks after ischemia, fetal striatal cells, marked with DiI, were transplanted into the ischemic striatum. The transplantation improved the ischemia-induced deficit in the passive avoidance task but not in radial maze test. Although there were variations in the size of the grafts, many DiI-positive cells with dendritic outgrowth were detected under fluorescent microscopy. Immunohistochemical study revealed that many choline acetyltransferase (ChAT) positive cells and GABA-positive cells survived in the grafts. However, striosome-matrix compartments were not evident inside the grafts. Thus, partial recoveries in both cytoarchitectural and behavioral aspects were obtained by striatal cell grafts, suggesting that neural transplantation could be a useful approach in reconstructing ischemic brain function.

Sialyl cholesterol enhances the development of grafted neurons and motor recovery.

Trophic actions of alpha-sialyl cholesterol (SC) and its sialidase-tolerant derivative, alpha-(3 beta-hydroxysialyl) cholesterol (SCt), were carried out on the development of midbrain neurons both in vitro and in vivo transplantation studies. Low to moderate concentrations of SC (0.01 to 0.05 micrograms/ml) facilitated neurite extension but had no effects on cell survival of primary cultured midbrain neurons. However, high concentration of SC (0.1 micrograms/ml) disturbed both neurite genesis and cell survival. SCt had a similar effect on midbrain neurons. At higher concentrations, SC and SCt induced concentration-dependent morphological changes in astrocytes from flat to fibrous. The effect on astrocytes was stronger in SCt than SC. At highest concentration tested (20 micrograms/ml), the proliferation of astrocytes was completely blocked, cells became detached and finally died. This effect of SC and SCt was partially blocked by simultaneous application of aFGF. Following dopaminergic cell grafting in vivo, SC and SCt had biphasic effects: a low dose (0.2 mg/kg, SC) enhanced motor recovery at 4 and 6 weeks after transplantation, while the highest dose (20 mg/kg, SC) disturbed motor recovery at all periods tested. These effects on motor recovery were paralleled by an effect on neurite genesis as studied by tyrosine hydroxylase immunostaining. Thus, at low concentrations, SC and SCt are neurotrophic agents that stimulate the development and differentiation of dopaminergic neurons.

Effect of intra-amygdala dopaminergic grafts on methamphetamine-induced locomotor activity, extracellular dopamine and dopamine metabolite overflow: a comparison with the effect of intra-accumbens grafts.

The effects of 6-hydroxydopamine lesions of the ventral tegmental area and following intra-amygdala or intra-accumbens dopaminergic (DAergic) grafts on methamphetamine (MAP)-induced locomotor activity were investigated in rats. Intra-accumbens DAergic grafts from rat embryos restored the locomotor hyperactivity response to MAP 5 weeks after grafting, while intra-amygdala grafts did not restore responses by 10 weeks after grafting. Biochemical measurements of extracellular DAergic activity in the amygdala (AMY) by in vivo microdialysis after grafting showed no significant change in the basal levels of dopamine (DA) and partial restoration of metabolite levels. MAP induced an increase of DA efflux and a decrease in dihydroxyphenylacetic acid without a significant change in homovanillic acid, which is the same pattern of response seen in control animals. These biochemical changes are similar to those seen previously after intra-accumbens grafts. The results show that restoration of DAergic activity in the AMY in the presence of DAergic denervation of accumbens does not have an effect on MAP-induced locomotion.

Grafting of catecholaminergic cells in the mammalian brain and reconstruction of disturbed function: basic problems to be solved.

1. Catecholaminergic cells were grafted in the caudate nucleus of hemi-Parkinsonian model rats, and the following problems were discussed. 2. Phenotypic plasticity and development of catecholamine-producing cells. 3. Mechanism of motor recovery. 4. Trophic factor. 5. Regulation of immunological responses. 6. Long-term survival and security.

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-Parkinson's 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.

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.

Behavioral and biochemical effects of intra-accumbens dopaminergic grafts.

6-Hydroxydopamine (6-OHDA) lesions in the ventral tegmental area (VTA) in rats prevented the hyperactivity response to methamphetamine in an open field. Transplantation of mesencephalic dopaminergic cells, obtained from rat embryos, into the nucleus accumbens (NAC) of 6-OHDA-lesioned animals restored the hyperactivity 4 weeks after grafting. By microdialysis of the NAC in freely moving rats no significant differences in baseline concentration of dopamine (DA) among the 3 groups (control, lesioned, grafted) were observed. However, after methamphetamine administration, DA increased significantly during the first 80 min in control animals, during the first 40 min in grafted animals, but did not increase in lesioned animals. On the other hand baseline concentrations of dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) decreased to one sixth to one third of those of controls in 6-OHDA-lesioned animals, and they did not respond to methamphetamine. After grafting, however, DOPAC and HVA restored to control levels and responded to methamphetamine with decreases as was observed in control animals. Data suggest that grafts not only restore the ability to release DA but also improve DA metabolism in the NAC. This might be a reason for recovery of locomotor activity.

Phenotypic plasticity of grafted catecholaminergic cells in the dopamine-depleted caudate nucleus in the rat.

Catecholamine-producing cells were grafted in the caudate nucleus of model rats with hemi-Parkinson's disease--animals with unilateral 6-hydroxydopamine (6-OHDA) lesions in the nigrostriatal dopaminergic pathway. Survival of the grafted cells, dopamine synthesis/release, and recovery from motor imbalance were investigated. Motor imbalance (methamphetamine rotations) recovered in more than 90% of animals by grafting of nigral dopaminergic (DAergic) cells, in 40-60% of animals by grafting of locus coeruleus noradrenergic (LC-NAergic) cells, and in 0-30% of animals by grafting of adrenal medullary cells. Many tyrosine hydroxylase (TH)-positive cells survived in the host caudate after grafting of DAergic cells. A moderate number of TH-positive but dopamine-beta-hydroxylase (DBH)-negative cells survived after grafting of NAergic cells. A few TH-positive neuron-like cells survived after grafting of adrenal medullary cells. In vivo microdialysis revealed that extracellular DA recovered up to 50-80% of that of control level, and dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) up to 25-50% of those of controls in animals that showed motor recovery after grafting. Using a TH cDNA probe, TH-positive cells were found to express TH mRNA in an in-situ hybridization-autoradiographic study. Animals that received 6-OHDA lesion only or animals that received grafting but had no surviving cells showed neither behavioral nor chemical recovery. The data suggest that grafted catecholaminergic cells survive, make phenotypic plasticity, synthesize/release dopamine, and ameliorate motor function.