Neural transplants in patients with Huntington's disease undergo disease-like neuronal degeneration.

The clinical evaluation of neural transplantation as a potential treatment for Huntington's disease (HD) was initiated in an attempt to replace lost neurons and improve patient outcomes. Two of 3 patients with HD reported here, who underwent neural transplantation containing striatal anlagen in the striatum a decade earlier, have demonstrated marginal and transient clinical benefits. Their brains were evaluated immunohistochemically and with electron microscopy for markers of projection neurons and interneurons, inflammatory cells, abnormal huntingtin protein, and host-derived connectivity. Surviving grafts were identified bilaterally in 2 of the subjects and displayed classic striatal projection neurons and interneurons. Genetic markers of HD were not expressed within the graft. Here we report in patients with HD that (i) graft survival is attenuated long-term; (ii) grafts undergo disease-like neuronal degeneration with a preferential loss of projection neurons in comparison to interneurons; (iii) immunologically unrelated cells degenerate more rapidly than the patient's neurons, particularly the projection neuron subtype; (iv) graft survival is attenuated in the caudate in comparison to the putamen in HD; (v) glutamatergic cortical neurons project to transplanted striatal neurons; and (vi) microglial inflammatory changes in the grafts specifically target the neuronal components of the grafts. These results, when combined, raise uncertainty about this potential therapeutic approach for the treatment of HD. However, these observations provide new opportunities to investigate the underlying mechanisms involved in HD, as well as to explore additional therapeutic paradigms.

Testis-derived Sertoli cells have a trophic effect on dopamine neurons and alleviate hemiparkinsonism in rats.

Neural tissue transplantation has become an alternative treatment for Parkinson's disease (PD) and other neurodegenerative disorders. The clinical use of neural grafts as a source of dopamine for Parkinson's disease patients, although beneficial, is associated with logistical and ethical issues. Thus, alternative graft sources have been explored including polymer-encapsulated cells and nonneural cells (that is, adrenal chromaffin cells) or genetically modified cells that secrete dopamine and/or trophic factors. Although progress has been made, no current alternative graft source has ideal characteristics for transplantation. Emerging evidence suggests the importance of trophic factors in enhancing survival and regeneration of intrinsic dopaminergic neurons. It would be desirable to transplant cells that are readily available, immunologically accepted by the central nervous system and capable of producing dopamine and/or trophic factors. Sertoli cells have been shown to secrete CD-95 ligand and regulatory proteins, as well as trophic, tropic, and immunosuppressive factors that provide the testis, in part, with its "immunoprivileged" status. The present study demonstrated that transplantation of rat testis-derived Sertoli cells into adult rat brains ameliorated behavioral deficits in rats with 6-hydroxydopamine-induced hemiparkinsonism. This was associated with enhanced tyrosine hydroxylase (TH) immunoreactivity in the striatum in the area around the transplanted Sertoli cells. Furthermore, in vitro experiments demonstrated enhanced dopaminergic neuronal survival and outgrowth when embryonic neurons were cultured with medium in which rat Sertoli cells had been grown. Transplantation of Sertoli cells may provide a useful alternative treatment for PD and other neurodegenerative disorders.

The synaptic impact of the host immune response in a parkinsonian allograft rat model: Influence on graft-derived aberrant behaviors.

Graft-induced dyskinesias (GIDs), side-effects found in clinical grafting trials for Parkinson's disease (PD), may be associated with the withdrawal of immunosuppression. The goal of this study was to determine the role of the immune response in GIDs. We examined levodopa-induced dyskinesias (LIDs), GID-like behaviors, and synaptic ultrastructure in levodopa-treated, grafted, parkinsonian rats with mild (sham), moderate (allografts) or high (allografts plus peripheral spleen cell injections) immune activation. Grafts attenuated amphetamine-induced rotations and LIDs, but two abnormal motor syndromes (tapping stereotypy, litter retrieval/chewing) emerged and increased with escalating immune activation. Immunohistochemical analyses confirmed immune activation and graft survival. Ultrastructural analyses showed increases in tyrosine hydroxylase-positive (TH+) axo-dendritic synapses, TH+ asymmetric specializations, and non-TH+ perforated synapses in grafted, compared to intact, striata. These features were exacerbated in rats with the highest immune activation and correlated statistically with GID-like behaviors, suggesting that immune-mediated aberrant synaptology may contribute to graft-induced aberrant behaviors.

Fetal nigral transplantation as a therapy for Parkinson's disease.

Fetal nigral grafts have been demonstrated to survive, secrete dopamine, form synaptic connections with host neurons, and reverse behavioral disturbances in experimental models of parkinsonism. These findings suggest that fetal nigral grafting may be a useful therapy for patients with Parkinson's disease (PD). Recent preliminary clinical trials of transplantation in PD have shown increased striatal fluorodopa uptake (measured using positron emission tomography) and clinical benefit in some patients. An autopsy study of one patient who had received fetal nigral transplants demonstrated robust graft survival and striatal reinnervation, with no evidence of host-derived sprouting or immune rejection. The development of a successful clinical transplantation program depends on a careful consideration of the transplantation variables and the related long-term risks and benefits to the patients.

Proteinase Activity during Tracheary Element Differentiation in Zinnia Mesophyll Cultures.

The zinnia (Zinnia elegans) mesophyll cell culture tracheary element (TE) system was used to study proteinases active during developmentally programmed cell death. Substrate-impregnated gels and single-cell assays revealed high levels of proteinase activity in differentiating TEs compared with undifferentiated cultured cells and expanding leaves. Three proteinases (145, 28, and 24 kD) were exclusive to differentiating TEs. A fourth proteinase (59 kD), although detected in extracts from all tissues examined, was most active in differentiating TEs. The 28- and 24-kD proteinases were inhibited by thiol proteinase inhibitors, leupeptin, and N-[N-(L-3-trans-carboxirane-2-carbonyl)-L-leucyl]-agmatine (E-64). The 145- and 59-kD proteinases were inhibited by the serine proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF). Extracts from the TE cultures contained sodium dodecyl sulfate-stimulated proteolytic activity not detected in control cultures. Sodium dodecyl sulfate-stimulated proteolysis was inhibited by leupeptin or E-64, but not by PMSF. Other tissues, sucrose-starved cells and cotyledons, that contain high levels of proteolytic activity did not contain TE-specific proteinases, but did contain higher levels of E-64-sensitive activities migrating as 36- to 31-kD enzymes and as a PMSF-sensitive 66-kD proteinase.

Neural transplantation as an experimental treatment modality for cerebral ischemia.

Cerebrovascular disease exemplifies the poor regenerative capacity of the CNS. While there are methods to prevent cerebral infarction, there is no effective therapy available to ameliorate the anatomical, neurochemical and behavioral deficits which follow cerebral ischemia. Focal and transient occlusion of the middle cerebral artery (MCA) in rodents has been reported to result in neuropathology similar to that seen in clinical cerebral ischemia. Using specific techniques, this MCA occlusion can result in a well-localized infarct of the striatum. This review article will provide data accumulated from animal studies using the MCA occlusion technique in rodents to examine whether neural transplantation can ameliorate behavioral and morphological deficits associated with cerebral infarction. Recent advances in neural transplantation as a treatment modality for neurodegenerative disorders such as Parkinson's disease, have revealed that fetal tissue transplantation may produce neurobehavioral recovery. Accordingly, fetal tissue transplantation may provide a potential therapy for cerebral infarction. Preliminary findings in rodents subjected to unilateral MCA occlusion, and subsequently transplanted with fetal striatal tissue into the infarcted striatum have produced encouraging results. Transplanted fetal tissue, assessed immunohistochemically, has been demonstrated to survive and integrate with the host tissue, and, more importantly, ameliorate the ischemia-related behavioral deficits, at least in the short term. Although, this review will focus primarily on cerebral ischemia, characterized by a localized CNS lesion within the striatum, it is envisioned that this baseline data may be extrapolated and applied to cerebral infarction in other brain areas.

Functional fetal nigral grafts in a patient with Parkinson's disease: chemoanatomic, ultrastructural, and metabolic studies.

A patient with Parkinson's disease received bilateral fetal human nigral implants from six donors aged 6.5 to 9 weeks post-conception. Eighteen months following a post-operative clinical course characterized by marked improvement in clinical function, this patient died from events unrelated to the grafting procedure. Post-mortem histological analyses revealed the presence of viable grafts in all 12 implant sites, each containing a heterogeneous population of neurons and glia. Approximately 210,146 implanted tyrosine hydroxylase-immunoreactive (TH-ir) neurons were found. A greater number of TH-ir grafted neurons were observed in the right (128,162) than the left (81,905) putamen. Grafted TH-ir neurons were organized in an organotypic fashion. These cells provided extensive TH-ir and dopamine transporter-ir innervation to the host striatum which occurred in a patch-matrix fashion. Quantitative evaluations revealed that fetal nigral grafts reinnervated 53% and 28% of the post-commissural putamen on the right and left side, respectively. Grafts on the left side innervated a lesser area of the striatum, but optical density measurements were similar on both sides. There was no evidence that the implants induced sprouting of host TH-ir systems. Electron microscopic analyses revealed axo-dendritic and occasional axo-axonic synapses between graft and host. In contrast, axo-somatic synapses were not observed. In situ hybridization for TH mRNA revealed intensely hybridized grafted neurons which far exceeded TH mRNA expression within residual host nigral cells. In addition, gamma-amino butyric acid (GABA)-ergic neurons were observed within the graft that formed a dense local neuropil which was confined to the implant site. Serotonergic neurons were not observed within the graft. Cytochrome oxidase activity was increased bilaterally within the grafted post-commissural putamen, suggesting increased metabolic activity. In this regard, a doubling of cytochrome oxidase activity was observed within the grafted post-commissural putamen bilaterally relative to the non-grafted anterior putamen. The grafts were hypovascular relative to the surrounding striatum and host substantia nigra. Blood vessels within the graft stained intensely for GLUT-1, suggesting that this marker of blood--brain barrier function is present within human nigral allografts. Taken together, these data indicate that fetal nigral neurons can survive transplantation, functionally reinnervate the host putamen, establish synaptic contacts with host neurons, and sustain many of the morphological and functional characteristics of normal nigral neurons following grafting into a patient with PD.

Long-term evaluation of bilateral fetal nigral transplantation in Parkinson disease.

BACKGROUND: Parkinson disease (PD) is associated with a progressive loss of nigrostriatal dopamine neurons. Medication therapy provides adequate control of symptoms for several years, but long-term treatment is complicated by progressive disability and the development of motor fluctuations and dyskinesias. In animal models of PD, fetal nigral transplants have been shown to survive grafting into the striatum, provide extensive striatal reinnervation, and improve motor function. In patients with PD, cell survival and clinical benefit have been observed following fetal nigral grafting, but results have been inconsistent. OBJECTIVE: To evaluate the safety and efficacy of bilateral fetal nigral transplantation into the postcommissural putamen in patients with advanced PD complicated by motor fluctuations and dyskinesias. PATIENTS AND METHODS: Six patients with advanced PD underwent bilateral fetal nigral transplantation. Each patient received solid grafts derived from donors aged 6 1/2 to 9 weeks after conception stereotactically implanted into the postcommissural putamen using 3 to 4 donors per side. Cyclosporine was administered for approximately 6 months to provide immune suppression. Clinical evaluations included the Unified Parkinson's Disease Rating Scale (UPDRS), Schwab-England Activities of Daily Living Scale, and timed tests of motor function conducted during both the "off' and "on" states at baseline and at 1, 3, 6, 9, 12, 18, and 24 months following transplantation. Percentage of time off and percentage of time on with and without dyskinesia were recorded at half-hour intervals using home diaries during the week prior to each evaluation. 18F-fluorodopa positron emission tomographic scans were performed at baseline, and at 6 months and 1 year following transplantation. RESULTS: Patients have been followed up for a mean+/-SD of 20.5+/-5.5 months. Complications related to surgery were mild and transient. Activities of daily living, motor, and total (activities of daily living plus motor) UPDRS scores during the off state improved significantly (P<.05, Wilcoxon signed rank test) at final visit in comparison with baseline. Mean total UPDRS off score improved 32%, and each patient experienced at least a 19% improvement. Mean percentage of time on without dyskinesia during the waking day improved from 22% to 60% (P<.05). Mean putamenal fluorodopa uptake on positron emission tomography increased significantly at 6 and 12 months in comparison with baseline (P<.001, 2-tailed t test). This increase correlated with clinical improvement. Two patients died 18 months after transplantation from causes unrelated to the surgical procedure. In both cases, histopathological examination showed robust survival of tyrosine hydroxylase immunoreactive cells and abundant reinnervation of the postcommissural putamen. CONCLUSIONS: Fetal nigral tissue can be transplanted into the postcommissural putamen bilaterally in patients with advanced PD safely and with little morbidity. In this open-label pilot study we observed consistent long-term clinical benefit and increased fluorodopa uptake on positron emission tomography. Clinical improvement appears to be related to the survival and function of transplanted fetal tissue.

Bilateral human fetal striatal transplantation in Huntington's disease.

BACKGROUND: Transplanted striatal cells have been demonstrated to survive, grow, establish afferent and efferent connections, and improve behavioral signs in animal models of Huntington's disease (HD). OBJECTIVE: To evaluate feasibility and safety and to provide preliminary information regarding the efficacy of bilateral human fetal striatal transplantation in HD. METHODS: Seven symptomatic patients with genetically confirmed HD underwent bilateral stereotactic transplantation of two to eight fetal striata per side in two staged procedures. Tissue was dissected from the lateral half of the lateral ventricular eminence of donors 8 to 9 weeks postconception. Subjects received cyclosporine for 6 months. RESULTS: Three subjects developed subdural hemorrhages (SDHs) and two required surgical drainage. One subject died 18 months after surgery from probable cardiac arrhythmia secondary to severe atherosclerotic cardiac disease. Autopsy demonstrated clearly demarcated grafts of typical developing striatal morphology, with host-derived dopaminergic fibers extending into the grafts and no evidence of immune rejection. Other adverse events were generally mild and transient. Mean Unified HD Rating Scale (UHDRS) motor scores were 32.9 plus minus 6.2 at baseline and 29.7 plus minus 7.5 12 months after surgery (p = 0.24). Post-hoc analysis, excluding one subject who experienced cognitive and motor deterioration after the development of symptomatic bilateral SDHs, found that UHDRS motor scores were 33.8 plus minus 6.2 at baseline and 27.5 plus minus 5.2 at 12 months (p = 0.03). CONCLUSIONS: Transplantation of human fetal striatal cells is feasible and survival of transplanted cells was demonstrated. Patients with moderately advanced HD are at risk for SDH after transplantation surgery.

Cell transplantation for central nervous system disorders.

Initially, the specific aim of transplantation studies was to investigate the regenerative capabilities of the mammalian nervous system. From this underlying impetus, a myriad of knowledge, spanning from molecular biology to neurobiology, has enhanced our understanding of regeneration and the applicability of fetal tissue transplantation in treating various neurodegenerative diseases. Current evidence suggests that transplantation of fetal neural tissue ameliorates the neurobiological and behavioral changes observed in animal models of central nervous system (CNS) disorders. In light of numerous basic science studies, clinical trials have begun to evaluate the potential of neural transplantation in treating human diseases. Indeed, modest progress has been reported in the treatment of Parkinson's disease. However, whereas fetal tissue transplantation has reached considerable success, it has also been observed to produce either no beneficial effects, magnify existing behavioral abnormalities, or even produce a unique constellation of deficits. Thus, while the prospects are promising, further investigations aimed at improving and refining existing transplantation paradigms are warranted before neural transplantation techniques can be of widespread value. This review article attempts to provide an overview of the neuroanatomical, neurochemical, and behavioral effects produced by transplanted fetal tissue in several animal models of CNS disorders. We have attempted to present both positive and adverse effects and to critically analyze the suitability of neural transplantation as a therapy for the various neurological disorders. In addition, alternative approaches, including the use of encapsulated neural tissue implants and genetically engineered cell lines along with their clinical potential, are discussed when appropriate.

Transplantation of human fetal striatal tissue in Huntington's disease: rationale for clinical studies.

Huntington's disease is a fatal neurological disorder characterized by chorea and deterioration in cognitive and neuropsychiatric function. Primary pathological changes are found in the striatum, where GABAergic neurons undergo degenerative changes. Local interneurons are relatively spared. Here, we describe the rationale for clinical trials of fetal striatal tissue transplantation for the treatment of Huntington's disease. Specifically, the reasons for utilizing tissue derived from the far lateral aspect of the lateral ventricular eminence as a source of striatal tissue will be discussed.

Intrastriatal 3-nitropropionic acid: a behavioral assessment.

Systemic injections of 3-nitropropionic acid (3-NP) in Sprague-Dawley rats have led to (1) hypoactivity that resembles juvenile onset and advanced Huntington's disease (HD), and (2) impairment in contextual retention of passive avoidance. Since it has been established that 3-NP exerts its primary effects in the striatum, we selected intrastriatal injections to more thoroughly understand the direct behavioral effects of 3-NP. Each 14-week old rat received bilateral intrastriatal injections of one of the following: 500 and 750 nmol of 3-NP or vehicle (0.9% saline). At seven days following surgery, the animals were tested for spontaneous locomotor behavior and passive avoidance behavior. Results revealed deficits in both locomotor activity and passive avoidance learning. The animals injected with 500 and 750 nmol of 3-NP were significantly hypoactive compared with control animals. Similarly, the 2 groups of animals were severely impaired in the retention of passive avoidance compared with control. The 3 groups, however, did not differ in their acquisition of this learning task. Macroscopic analyses of brains of these animals revealed that 500 and 750 nmol of 3-NP caused severe loss of neuronal cell bodies and marked glial infiltration in the medial aspect of the striatum. Larger lesions showed a necrotic cavity at the injection site. In comparison with systemic administration of 3-NP, intrastriatal injections resulted in more profound hypoactivity, greater loss of passive avoidance retention, and more severe striatal damage.(ABSTRACT TRUNCATED AT 250 WORDS)

Low-pressure aspiration abortion for obtaining embryonic and early gestational fetal tissue for research purposes.

Successful transplantation of cadaver embryonic neural tissue is highly dependent on the method used to obtain the tissue. It is important that the tissue not be contaminated bacteriologically by vaginal flora during the procedure, and that it not be disrupted mechanically. A low-pressure aspiration abortion technique has been developed that allows for the safe, effective obtainment of embryonic tissue and reduces the risk of transmitting infection. Tissue was cultured in vitro in 102 cases with minimal evidence of contamination by vaginal flora. Transplants of neural tissue into over 300 rodents have resulted in no intracranial abscesses, even in the setting of immunosuppression. The suction apparatus and low-pressure aspiration minimize disruption of the embryonic tissue. Use of low pressure adds no significant additional risk to the patient. In over 300 cases, there have been no medical complications specifically attributable to the technique. Because local anesthetic is used and sonography is not routinely required, the procedure can easily be performed in an outpatient setting during routine elective abortions, with minimal slowing or disruption of the clinic's surgical schedule. In conclusion, the low-pressure aspiration abortion technique can be safely and effectively used to obtain embryonic and early gestational fetal tissue that is almost always free from bacterial, fungal, and yeast contamination and that is frequently structurally intact. It requires no significant alteration in indications for abortion, risks, methodology, timing of the abortion, or patient management.

Development of the human striatum: implications for fetal striatal transplantation in the treatment of Huntington's disease.

Fetal neural transplantation has recently been demonstrated to ameliorate motor and other behavioral deficits in animals models of Huntington's disease, and reconstruct many of the damaged striatal circuits. However, there has been significant variability in the histological appearance of these grafts, most likely related to differences of the regions of dissection of the donor tissue. Selective dissection and transplantation of the lateral ventricular eminence in rodents has resulted in grafts consisting of primarily striatal-like tissue. This data, combined with data from our own and other laboratories has led to a description of the development of human striatum, with a particular emphasis on the relevance of human striatal development to the field of fetal tissue transplantation for the treatment of Huntington's disease. If the goal of transplantation is to graft GABAergic striatal projection neurons, it is our impression that optimal grafting results will occur when transplants are derived from the lateral ventricular eminence and the lateral aspect of the body of the ventricular eminence anterior to the foramen of Monro. Optimal results are likely to occur when donor ages range from Stage 19 to 23, with possible graft success when donor age extends to as late as postovulatory week 22.

Fetal grafting for Parkinson's disease: expression of immune markers in two patients with functional fetal nigral implants.

In a number of centers throughout the world, fetal nigral transplantation is being performed for the treatment of Parkinson's disease (PD). Clinical results have been inconsistent. One parameter that differs among transplant studies is the degree and manner by which patients are immunosuppressed following transplantation. Indeed, the role of the immune system following fetal grafting in humans is not well understood. Recently, two patients from our open label trial that received fetal nigral implants have come to autopsy. These patients were immunosuppressed with cyclosporin for 6 mo posttransplantation and survived for a total of 18 mo postgrafting. Robust survival of grafted dopamine-containing cells was observed in both cases. Immunostaining for HLA-DR revealed a dense collection of cells within grafts from both cases. HLA-DR staining was rarely observed within the host including nongrafted regions of the striatum. A more detailed analysis of immune markers was performed in Case 2. Numerous pan macrophages, T-cells, and B-cells were observed within graft sites located in the postcommissural putamen. In contrast, staining for these immune cells was not observed within the ungrafted anterior putamen. These findings suggest that even in healthy appearing functional nigral implants, grafts are invaded by host immune cells that could compromise their long-term viability and function. Alternatively, immune cells are known to secrete trophic factors, which may ultimately favor graft survival and function. Further work is needed to understand the role of the immune system in fetal grafting.

Trophic effect of porcine Sertoli cells on rat and human ventral mesencephalic cells and hNT neurons in vitro.

The poor survival of embryonic dopaminergic (DA) neurons transplanted into patients with Parkinson's disease (PD) has encouraged researchers to search for new methods to affect the short- as well as long-term survival of these neurons after transplantation. In several previous rodent studies Sertoli cells increased survival of islet cells and chromaffin cells when cotransplanted in vivo. The aims of this study were to investigate whether porcine Sertoli cells had a positive effect on the survival and maturation of rat and human DA neurons, and whether the Sertoli cells had an effect on differentiation of neurons derived from a human teratocarcinoma cell line (hNT neurons). A significant increase of tyrosine hydroxylase (TH)-positive neurons of both rat and human ventral mesencephalic tissue was found when cocultured with Sertoli cells. Furthermore, there was a significantly increased soma size and neurite outgrowth of neurons in the coculture treated group. The Sertoli cell and hNT coculture also revealed an increased number of TH-positive cells. These results demonstrate that the wide variety of proteins and factors secreted by porcine Sertoli cells benefit the survival and maturation of embryonic DA neurons and suggest that cotransplantation of Sertoli cells and embryonic DA neurons may be useful for a cell transplantation therapy in PD.

The influence of donor age on the survival of solid and suspension intraparenchymal human embryonic nigral grafts.

In many species, graft survival and graft-derived behavioral recovery are affected by the embryonic donor age. We compared the ability of solid and suspension grafts of human embryonic mesencephalic dopaminergic (DA) neurons at different embryonic stages to survive intraparenchymal transplantation into 6-OHDA lesioned immunosuppressed rats. Suspension grafts survived best when donor age was between postconception (PC) days 34 and 56. Transplants displayed numerous healthy tyrosine hydroxylase immunoreactive (TH-IR) neurons which sent extensive neuritic processes into the host striatum. Suspension grafts survived poorly when donor age was greater than 65 days. Solid implants displayed comparable viability of TH-IR neurons when donor age was between 44 and 65 days. No solid grafts contained TH-IR cells when donor tissue was older than 72 days. The suspension and solid methods of transplantation resulted in comparable survival of robust grafts, but solid grafts resulted in more intergraft variability than suspension grafts, particularly among the more marginal implants. Our results demonstrate that the upper limit for survival of human embryonic DA suspension grafts correlates well with the period of development of the human nigrostriatal pathway. The "window" for donor age of solid human embryonic DA grafts appears to be extended by about 9 days in comparison to suspension grafts. These data suggest that the upper age limit for grafting human mesencephalic DA neurons should be PC day 56 for suspension grafts, and PC day 65 for solid implants. Older donors are likely to produce grafts with fewer surviving DA neurons.

Cyclosporine-A increases spontaneous and dopamine agonist-induced locomotor behavior in normal rats.

Cyclosporine-A (CsA) has been increasingly used as an immunosuppressant concomitant with neural transplantation treatment for different degenerative disorders. However, the possible role that CsA itself may have in the recovery of transplant patients is not known. Some investigators have argued that clinical improvement following transplantation (e.g., myoblast) may be confounded by CsA administration. The present study was conducted to delineate CsA-induced behavioral alterations. Four groups of normal 5-wk old Sprague-Dawley rats (n = 8 per group) were utilized in two separate experiments. In both experiments, two groups of animals were used; each group either received daily injections of 15 mg/kg of CsA or olive oil for 32 days (experiment 1) and 21 days (experiment 2). Animals in both experiments were subsequently tested for nocturnal locomotor behavior. Animals in experiment 2 were further tested in passive avoidance task, motor coordination, and amphetamine-induced locomotor activity. Results demonstrated that CsA-treated animals were significantly hyperactive compared to controls across the 12-h nocturnal activity periods and in amphetamine-induced locomotor activity. No significant differences between the CsA- and vehicle-treated animals were observed in passive avoidance or in motor coordination. Postmortem analyses of dopamine and its metabolites in the striatum and olfactory tubercle did not show any significant differences between the CsA- and the vehicle-treated groups. In summary, CsA significantly increased nocturnal spontaneous and amphetamine-induced locomotor behavior, but the neurochemical correlates for these effects need to be investigated. In addition, while the present study demonstrated that CsA induced motor alterations, any possible effects CsA may have on neurological or dystrophic patients with motor dysfunctions remain to be determined.

The X-gal caution in neural transplantation studies.

Cell transplantation into host brain requires a reliable cell marker to trace lineage and location of grafted cells in host tissue. The lacZ gene encodes the bacterial (E. coli) enzyme beta-galactosidase (beta-gal) and is commonly visualized as a blue intracellular precipitate following its incubation with a substrate, "X gal," in an oxidation reaction. LacZ is the "reporter gene" most commonly employed to follow gene expression in neural tissue or to track the fate of transplanted exogenous cells. If the reaction is not performed carefully-with adequate optimization and individualization of various parameters (e.g.. pH, concentration of reagents, addition of chelators, composition of fixatives) and the establishment of various controls--then misleading nonspecific background X-gal positivity can result, leading to the misidentification of cells. Some of this background results from endogenous nonbacterial beta-gal activity in discrete populations of neurons in the mammalian brain; some results from an excessive oxidation reaction. Surprisingly, few articles have empha sized how to recognize and to eliminate these potential confounding artifacts in order to maximize the utility and credibility of this histochemical technique as a cell marker. We briefly review the phenomenon in general, discuss a specific case that illustrates how an insufficiently scrutinized X-gal positivity can be a pitfall in cell transplantation studies, and then provide recommendations for optimizing the specificity and reliability of this histochemical reaction for discerning E. coli beta-gal activity.

Microcarrier enhanced survival of human and rat fetal ventral mesencephalon cells implanted in the rat striatum.

The transplantation of tissue containing dopamine-producing cells into the mammalian central nervous system is an emerging treatment for Parkinson's disease, despite relatively poor survival of implanted tissue. Recent evidence has suggested that Cytodex microcarriers enhance the survival of dopaminergic rat chromaffin cells transplanted into the rat striatum in the absence of immunosuppression. The current study was undertaken to evaluate the survival of rat and human fetal ventral mesencephalic neurons (VM) implanted alone or after attachment to microcarriers in the striatum of rats without immunosuppression. Rat fetal VM neurons demonstrated enhanced survival in the rat striatum when transplanted on microcarriers, compared to their transplantation alone during the 3-mo period examined in the present study. Transplants of human fetal VM neurons on microcarriers also survived remarkably well in the rat striatum without systemic immunosuppression. In contrast, human fetal VM cells transplanted alone into the rat striatum did not survive without systemic immunosuppression. There was no evidence of TH fiber sprouting in the vicinity of any transplant site. These data indicated that Cytodex microcarriers provide enhanced survival of both rat allograft and human xenograft fetal mesencephalic cells in the rat striatum without the necessity of systemic immunosuppression, perhaps by inducing a unique neuron-glia environment.