Takeshi Kondoh

Transplantation of human striatal tissue into a rodent model of Huntington's disease: Phenotypic expression of transplanted neurons and host-to-graft innervation

The present study was undertaken to investigate the phenotypic expression and integration of human striatal neurons transplanted into an animal model of Huntingtons disease. Sprague-Dawley rats were anesthetized and subjected to quinolinic acid lesions of the left striatum. Three human fetal cadavers were utilized for transplantation in this study (7, 8, and 10 weeks in gestation). The striatal primordia was dissected from each fetus and subsequently dissociated into cell suspensions. Following the initial lesion surgeries (3-4 months), the rats were reanesthetized and transplanted with human striatal cells (400,000 cells per rat). The animals were processed for histochemical analysis 9-17 weeks posttransplantation. Histochemistry was performed utilizing thionin (Nissl staining), acetylcholinesterase, NADPH-diaphorase, and antibodies against tyrosine hydroxylase and glial fibrillary acidic protein. Examination of stained brain sections demonstrate that human striatal transplants grow to fill a substantial portion of the remaining striatum, and contain clusters of immature and mature cells. Acetylcholinesterase activity is present in the transplant neuropil, varying in intensity, and distributed in a heterogeneous fashion. In addition, host afferent dopaminergic fibers penetrate into the transplant, and are occasionally found in patches. NADPH-diaphorase histochemistry revealed medium sized aspiny striatal neurons of donor origin in the transplants. The results of this study are similar to those obtained with rodent fetal striatal transplants, and suggest that human striatal tissue is capable of surviving, expressing normal striatal cell phenotypes, and receiving host dopaminergic innervation.

Collagenase-Induced Intrastriatal Hemorrhage in Rats Results in Long-term Locomotor Deficits

UNLABELLED BACKGROUND AND PURPOSE. Previous studies have shown that injection of the metalloproteinase collagenase directly into the caudate nucleus of rats causes an intracerebral hemorrhage. The purpose of the present study is to determine functional deficits associated with a collagenase-induced hemorrhagic lesion of the striatum. METHODS Twelve adult rats received a 2-microL infusion of bacterial collagenase (0.5 U in saline) into the right striatum. The rotational response to apomorphine (1 mg/kg SC) administration was then examined at 1, 4, 7, 21, 35, and 70 days after the surgery. In addition to the rotational asymmetry studies, the initiation of stepping movements in each forelimb was determined 8 weeks after the collagenase injections. In the assessment of rotational asymmetry and stepping ability, an additional six control animals received unilateral injections of saline alone. After behavioral testing, brains were processed for neuropathological evaluation. RESULTS A net ipsilateral rotation was noted at all posthemorrhage time periods. The average rotational asymmetries on these days were 14.57 +/- 2.9, 20.33 +/- 2.7, 19.99 +/- 4.4, 18.95 +/- 4.9, 17.03 +/- 4.9, and 14.4 +/- 4.7, respectively (data expressed as mean clockwise rotations per 5 minutes +/- SEM). The average number of steps initiated by the forelimb ipsilateral and contralateral to the lesion was 28.3 +/- 2.1 steps per minute and 13.6 +/- 1.5 steps per minute, respectively. This difference between left and right forelimb stepping was stable and reproducible for 3 consecutive days. Histological studies revealed a long-lasting hematoma cavity surrounded by dense reactive gliosis in the striatum. CONCLUSIONS We conclude that collagenase-induced intrastriatal hemorrhage results in long-term locomotor deficits and is therefore a useful model for developing and assessing therapeutic approaches for the restoration of neurological function after intracerebral hemorrhage.

The fate of human glial cells following transplantation in normal rodents and rodent models of neurodegenerative disease

Investigations on xenografting in the brain have previously focused on the anatomical and functional integration of the transplanted neurons. More recently, astrocytes are being implicated as having complex functions following transplantation, and are being investigated to determine their role(s) in transplantation. The present study was undertaken to investigate the migration of human astrocytes following transplantation of thalamic, striatal, and mesencephalic tissue into the rodent striatum. Human donor fetuses (9-16 weeks in gestation) obtained through elective and spontaneous abortions were utilized in this study. Following transplantation, donor astrocytes were labeled with an antiserum directed against human glial fibrillary acidic protein. Our results demonstrate that astrocytic elements from all three tissue types are capable of incorporating into the host brain, and have a tendency to follow white matter tracts (such as the corpus callosum, internal capsule, and fiber bundles in the striatum). Human astrocytes, originating from the striatum and thalamus exhibited extensive migration, while migration was more limited in animals with ventral mesencephalon transplants. Ventral mesencephalon transplanted animal demonstrated positive astrocytes within the transplant, with processes (very few cell bodies) extending into white matter of adjacent host striatum. Astrocytes demonstrating immature morphology were observed with all transplant types, but were most prevalent in the striatal transplanted animals. The extent of astrocyte migration and the morphologies observed in this study reflect regional differences of the developing human brain. These results confirm and extend previous investigations on glial cell migration following transplantation in the brain.

Transplantation of human fetal striatum into a rodent model of Huntington's disease ameliorates locomotor deficits

Previous studies have demonstrated that syngeneic transplants of striatal tissue can ameliorate locomotor deficits in rodent models of Huntingtons disease (HD). In the present study, we have examined whether human to rat xenografts of fetal striatal tissue can exert a similar recovery of function. Rodents with unilateral striatal lesions were transplanted with human striatal cells from a donor 14 weeks post-conception, and subsequently displayed a progressive decrease in rotational asymmetry in comparison to sham (saline) transplanted animals. Histological analysis revealed acetylcholinesterase (AChE)-positive fibers and NADPH-diaphorase (NADPH-d)-positive neurons within transplanted tissue. These results suggest that human fetal striatum at a gestational age of 14 weeks may potentially be useful as a source of donor tissue for transplantation in the treatment of HD.

Glutamate uptake blockade induces striatal dopamine release in 6-hydroxydopamine rats with intrastriatal grafts : evidence for host modulation of transplanted dopamine neurons

The transplantation of fetal dopamine neurons has previously been shown to ameliorate locomotor deficits in laboratory animals with nigrostriatal dopamine lesions. These studies have also demonstrated a reinstatement of striatal dopamine synthesis and release associated with the graft-to-host fiber innervation. More recent studies in the intact striatum indicate that striatal dopamine release can be regulated by corticostriatal glutamatergic projections. In the present study we hypothesized that secretion of dopamine by grafted neurons may also be regulated by similar mechanisms. To test this hypothesis we have measured dopamine release in the striatum of rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal pathway and grafts containing dopamine neurons. Intrastriatal dopamine levels were determined using methods of in vivo microdialysis. The administration of dihydrokainic acid, an inhibitor of glutamate uptake, resulted in the release of striatal dopamine in grafted and normal rats. In contrast, dopamine was not detectable in animals with 6-OHDA lesions alone. Additional studies were conducted to determine the type of glutamate receptor mediating the release of striatal dopamine. Kainic acid administration into the perfusate of the dialysis probe resulted in the release of striatal dopamine in a dose-dependent fashion in both grafted and normal rats. N-methyl-D-aspartate had no effect on dopamine release except at abnormally high concentrations. These results demonstrate that dopamine neurons which are transplanted into the striatum of rats with 6-OHDA lesions become functionally incorporated into the neural circuitry of the host brain, and that the release of dopamine by grafted neurons can be regulated by afferent fibers from the host brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of dopamine receptors and associated mRNA in transplants of human fetal striatal tissue in rodents with experimental Huntington's Disease

Huntingtons Disease (HD) is characterized by deficits in motor and cognitive functions. This neurodegenerative disease shows an extensive loss of medium-sized spiny projection neurons (GABAergic) within the neostriatum. With the loss of these neurons, there is a concomitant loss of associated receptors, such as those for GABA, glutamate, and dopamine. In the present study, we have addressed the question of whether dopamine receptors are re-established in the lesioned rodent striatum following the transplantation of human striatal cells. Human striatal cell suspension or saline (transplant controls) was injected into the striatum of rats previously lesioned with quinolinic acid (QA). Three nine months following transplantation, the animals were sacrificed and the brains were processed for receptor autoradiography and in situ hybridization of dopamine D1 and D2 receptor subtypes. Our results demonstrate that animals transplanted with human striatal cells show a significant increase in D1 receptors following transplantation when compared to the lesion area in control animals, while D1 receptor mRNA remains unchanged. In contrast to D1 receptor binding, D2 receptor levels are not increased in the lesioned and transplanted area of the striatum when compared to controls; however, D2 receptor mRNA levels are significantly increased. These results demonstrate that at the times the animals were examined, D1 and D2 receptors were differentially regulated. Our results further indicate that human striatal primordium will survive following transplantation and will express D1 receptors and D2 receptor mRNA that are depleted in the QA lesioned rodent striatum. This study compliments and extends previous findings on human striatal cell transplantation in rodent models of HD.

Evaluation of monoaminergic neurotransmitters in the rat striatum during varied global cerebral ischemia

Neurotransmitter release during cerebral ischemia has been extensively studied and is thought to play a key role in excitotoxic neuronal death. The changes in neurotransmitter release and its metabolism may reflect changes in cellular metabolism during ischemia. The purpose of this study is to assess alterations in extracellular dopamine and serotonin and their metabolites under varied degrees of ischemia in rat striatum to elucidate the pathophysiology of cerebral ischemia. Twenty rats were used to induce varied forebrain ischemia by means of bilateral common carotid artery occlusion along with hemorrhagic hypotension. Cerebral blood flow (CBF) in the striatum was measured every 40 minutes by methods of hydrogen clearance and maintained within certain ranges for 6 hours. Dopamine, serotonin, and their metabolites were measured every 20 minutes by in vivo microdialysis. Varying degrees of ischemia were obtained, ranging from 9.4 to 81.3% of control CBF. The animals were divided into three groups according to CBF levels measured 20 minutes after the induction of ischemia. In the mild ischemia group (n = 5), CBF ranged from 65 to 88% of baseline levels and resulted in only a slight increase of dopamine. In the moderate ischemia group (n = 10), CBF ranged from 21 to 48% of baseline levels and resulted in transient increases of dopamine (24-fold) and serotonin (5-fold). In the severe ischemia group (n = 5), CBF was below 14% of baseline levels and resulted in marked increases in dopamine (462-fold) and serotonin (225-fold). These alterations remained elevated for 3 hours.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of human fetal ventral mesencephalic grafts in rats with 6-OHDA lesions of the nigrostriatal pathway.

Neuronal transplantation is an approach that can be exploited to study the development of the human central nervous system as well as being used in attempts to restore neurological function. In the present study, we have examined cellular events that appear to precede the development of dopamine nerve fiber extension by neurons from the human fetal ventral mesencephalon. These cellular events were examined using neuronal cell suspensions from human fetal ventral mesencephalic tissue (gestational ages 7-10 weeks) transplanted into the striatum of unilaterally lesioned 6-hydroxydopamine (6-OHDA) rats. Animals were sacrificed for immunohistochemistry 9-10 weeks after the transplantation prior to the manifestation of behavioral recovery. Histological analysis revealed tyrosine hydroxylase (TH) immunoreactive neurons in the grafts. The majority of these neurons had very short TH positive processes (60-70 microns), indicating that the maturation of grafted dopaminergic neurons was still incomplete. Immunostaining for the human specific intermediate neurofilament (hNF, clone: BF-10) showed dense neuronal fibers in the grafts. These fibers extended deeper into the host brain than the TH positive neuronal processes. The whole striatum, particularly the medial part of the striatum, exhibited long NF positive processes. Glial fibrillary acidic protein (GFAP) immunohistochemistry revealed fine astrocytic processes inside the grafts, which were clearly different from host reactive glial cells surrounding the grafts. These graft-derived glial processes tended to extend into the host brain deeper than the TH positive neuronal processes from the grafts. These early histological findings of the grafted human fetal ventral mesencephalon suggest that the graft-derived NF positive neuronal processes, as well as the glial processes, radiate from the grafted tissue and extend into the host brain prior to the extension of TH positive processes. These results further suggest that human-to-rat xenografts can be used to study the neural development of human fetal brain tissue.

Therapeutic time window for the 21-aminosteroid, U-74389G, in global cerebral ischemia

A novel 21-aminosteroid (U-74389G), a new potent antioxidant, was evaluated for its protective effect on transient global cerebral ischemia. Ischemia was induced by 20 minutes of four-vessel occlusion in adult male Wistar rats. Injection of 21-aminosteroid (U-74389G, 5 mg/kg intraperitoneally injected) was repeated three times. The second injection was performed 30 minutes after the first injection, and the third injection was performed 210 minutes after that. Experimental animals were divided into five groups according to the time drug administration was initiated. Group I (n = 8) began vehicle administration 30 minutes before occlusion. Group II (n = 9) started 21-aminosteroid administration 30 minutes before occlusion. Drug administration in Group III (n = 9) began at the time of reperfusion, in Group IV (n = 8), 30 minutes after reperfusion, and in Group V (n = 6), 60 minutes after reperfusion. Animals in the control group (n = 5) underwent sham operations. One week after ischemia, the number of viable pyramidal neurons was counted in the hippocampal CA1 subfield. The results were as follows: the number of living neurons in Group I was 18.8 +/- 8.7; in Group II, was 44.7 +/- 9.5; in Group III, was 46.4 +/- 9.4; in Group IV, was 40.3 +/- 6.6; in Group V, was 10.2 +/- 2.5; and in the control group was 131 +/- 3.3. Groups II, III, and IV demonstrated significantly higher numbers of living neurons compared with Group I (P < 0.05). The present study revealed that U-74389G attenuated delayed neuronal death in global cerebral ischemia when it was administered before or soon after the ischemic episode.

Transplantation of human fetal tissue from spontaneous abortions to a rodent model of parkinson's disease

The use of human fetal tissue from elective abortions for cell transplantation therapies has been the subject of considerable controversy. Because of concerns regarding the use of tissue from elective abortions, tissue from spontaneous abortions has been suggested as an alternate donor source. In the present study we have evaluated human fetal tissue from spontaneous abortions to assess its viability, growth potential, and functional expression. Viable cells (Grade I) from a donor (7 wk postconception) were transplanted as a suspension into the striatum of rats with unilateral 6-OHDA lesions of the nigrostriatal pathway. A second group of animals received solid grafts of tissue from a Grade I donor 14 wk postconception. Tissue from Grade II and III specimens were not sufficiently viable for transplantation. Locomotor responses were monitored over a period of 15 wk and revealed an amelioration of rotational asymmetry by animals that received tissue from the 7 wk donor. Animals receiving tissue from the 14 wk donor showed no functional improvement. We found numerous graft-derived tyrosine hydroxylase (TH) immunopositive neurons contained within the transplantation site, and a rich plexus of TH-immunopositive fibers extending into the striatum of animals receiving tissue from the 7 wk donor. Animals receiving tissue from the 14 wk donor exhibited tissue necrosis at the transplant site and were devoid of TH-immunopositive neurons. These results suggest that human fetal ventral mesencephalic cells from spontaneous abortions can survive and develop after transplantation, and rectify locomotor deficits associated with experimental parkinsonism if the donor tissue is of the appropriate gestational age at the time of implantation. Our study further suggests, however, that the availability of tissue from spontaneous abortions of sufficient viability is quite limited and may thus restrict its potential use in cell transplantation therapies for Parkinsons disease.