Free-floating Roller Tube Cultures Research Articles

A Combination of NT-4/5 and GDNF Is Favorable for Cultured Human Nigral Neural Progenitor Cells

Idiopathic Parkinson’s disease (PD) is a progressive neurodegenerative disorder, clinically manifested by cardinal motor symptoms including tremor at rest, bradykinesia, and muscle rigidity. Transplantation of dopaminergic (DAergic) neurons is an experimental therapy for PD, however, it is limited by suboptimal integration and low survival of grafts. Pretreatment of donor tissue may offer a strategy to improve properties of transplanted DAergic neurons and thereby clinical outcome. We have previously shown that a combination of neurotrophin-4/5 (NT-4/5) and glial cell line-derived neurotrophic factor (GDNF) demonstrated additive effects on rat ventral mesencephalic (VM) tissue. The present study investigated the effects of NT-4/5 and GDNF as single factors, or in combination on DAergic neurons, in organotypic explant cultures of fetal human ventral mesencephalon. For that purpose, free-floating roller-tube cultures were prepared from VM and the equally sized pieces grown for 1 week in the presence or absence of neurotrophic factors. Both neurotrophic factors increased dopamine content in the culture medium and in the number of tyrosine hydroxylase immunoreactive neurons, most prominently after combined GDNF + NT-4/5 treatment. Culture volumes did not differ between groups while content of lactate dehydrogenase in the culture medium was moderately reduced in all treated groups. In conclusion, we identified that a combination of GDNF and NT-4/5 robustly promoted differentiation and survival of human fetal VM DAergic neurons, an observation with potential promising impact for cell replacement approaches in PD.

Creatine Supplementation Improves Dopaminergic Cell Survival and Protects against MPP+ Toxicity in an Organotypic Tissue Culture System

Cell replacement therapy using mesencephalic precursor cells is an experimental approach for the treatment of Parkinson's disease (PD). A significant problem associated with this procedure is the poor survival of grafted neurons. Impaired energy metabolism is considered to contribute to neuronal cell death after transplantation. Creatine is a substrate for mitochondrial and cytosolic creatine kinases (CK) and buffers cellular ATP resources. Furthermore, elevated cellular creatine levels facilitate metabolic channeling and show antiapoptotic properties. Exogenous creatine supplementation therefore might offer a tool for improvement of dopaminergic neuron survival. The present study aimed at investigating the effects of creatine on cell survival of rat embryonic day 14 (E14) ventral mesencephalic neurons grown as organotypic free-floating roller tube (FFRT) cultures. We found that the brain-specific isoform of CK (BB-CK) and the ubiquitous mitochondrial isoform (uMt-CK) are expressed at high levels in FFRT cultures and colocalize with tyrosine hydroxylase immunoreactive (TH-ir) cells. Exposure of these cultures to creatine induced an increase in the content of the BB-CK isotype. Creatine (5 mM) administration starting at day in vitro (DIV) 7 resulted in a significant increase (+35%) in TH-ir cell density at DIV21. In addition, we observed that creatine treatment provided neuroprotection against 1-methyl-4-phenyl pyridinium ion (MPP+)-induced TH-ir cell loss in the FFRT culture system, resulting in a significantly higher density (+19%) of TH-ir neurons in creatine-treated cultures compared to corresponding controls. The decrease of TH-ir neurons in the MPP+-treated group corresponded with an increase in immunoreactivity for active caspase-3, an effect that was not seen in the group receiving creatine supplementation. In conclusion, our data imply that creatine administration is beneficial for the survival of TH-ir neurons encountering harmful conditions.

Liposome-mediated gene transfer to fetal human ventral mesencephalic explant cultures

The feasibility of non-viral gene transfer using liposomes is described for human fetal nigral tissue. Ventral mesencephalic explants from 6 to 12 week old fetuses were grown as free-floating roller tube cultures. For the transfection, a vector coding for β-galactosidase driven by the Rous Sarcoma Virus promoter was used. The developmental stage of the human tissue, time in vitro and the amount of vector DNA used significantly influenced the transfection efficiency. Optimal transfection results were obtained with tissue from a 10 week old fetus, cultured for 4 days and transfected with mixtures containing 4 μg vector DNA. Histological analysis suggested that a specific population of ventral mesencephalic precursor cells were the target for the gene transfer. This finding might have implications for gene delivery and cell replacement strategies in Parkinson's disease.

Influence of Time in Culture and BDNF Pretreatment on Survival and Function of Grafted Embryonic Rat Ventral Mesencephalon in the 6-OHDA Rat Model of Parkinson's Disease

Embryonic midbrain can be maintained as free-floating roller tube cultures prior to grafting in experimental Parkinson's disease. We examined the influence of pregrafting culture time and pretreatment with brain-derived neurotrophic factor on graft survival and function. Cultures were prepared from solid pieces of embryonic (E14) rat ventral mesencephalon and maintained 4, 8, or 12 days in vitro with or without brain-derived neurotrophic factor (100 ng/ml) and grafted into the striatum of 6-hydroxydopamine-lesioned rats. Graft survival and function were evaluated by amphetamine-induced rotation behavior, number of tyrosine hydroxylase-immunoreactive neurons, striatal reinnervation, and graft volume. Rats receiving untreated tissue cultured for 4 or 8 days displayed no differences in graft quality, while grafts from 12-day-old cultures contained significantly fewer (P < 0.05) tyrosine hydroxylase-immunoreactive neurons (340 ± 97, 267 ± 92, and 62 ± 19) and displayed a lower survival rate (9.6 ± 2.7, 7.9 ± 2.7, and 2.6 ± 0.8% for 4, 8, and 12 days in vitro, respectively). Only rats grafted with 4- and 8-day-old cultures recovered significantly (P < 0.05) from lesion-induced rotations (69.4 ± 18.6, 70.3 ± 13.9, and 23.2 ± 12.1% for 4, 8, and 12 days in vitro, respectively). Striatal reinnervation decreased with increasing culture time (P < 0.05). Pretreatment of the cultures with brain-derived neurotrophic factor affected only graft-induced fiber reinnervation, which was reduced even after short culture times. We therefore suggest that a storage period of 8 days is well suited to maintain embryonic rat ventral mesencephalon with the free-floating roller tube culture technique prior to transplantation. BDNF pretreatment as a new strategy to improve graft survival and function, however, was not effective.

Nonviral Glial Cell-Derived Neurotrophic Factor Gene Transfer Enhances Survival of Cultured Dopaminergic Neurons and Improves Their Function after Transplantation in a Rat Model of Parkinson’s Disease

Transplantation of dopaminergic fetal mesencephalic tissue into the striatum is currently being developed for treatment of patients with advanced Parkinson's disease. Ethical concerns regarding the use of human fetal tissue, and the limited availability as well as poor survival and differentiation of dopaminergic neurons after transplantation have reduced the extent and outcome of this approach so far. With the purpose of finding means to increase the yield of dopaminergic neurons in transplants, and to reduce the amount of fetal tissue needed for each transplanted patient, we transfected rat fetal ventral mesencephalic (VM) tissue grown as organotypic free-floating roller tube (FFRT) cultures with a vector encoding human glial cell-derived neurotrophic factor (hGDNF). For transfer of an episomal expression vector (pRep7-GDNF8) a nonviral, nonliposomal cationic transfection technique was applied and optimized. Recombinant hGDNF expression resulted in a higher number of TH-positive neurons in the cultures as measured 6 days after transfection. Ventral mesencephalic cultures expressing hGDNF were then grafted into the striatum of unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats. Grafting of genetically modified VM cultures resulted in earlier functional recovery compared with grafting nontransfected cultures. We conclude that organotypic free-floating roller tube cultures can be successfully transfected to produce hGDNF with effects on TH-expressing neurons in vitro and functional effects after grafting in a rat Parkinson's disease model.

Striatal Grafts in a Rat Model of Huntington's Disease: Time Course Comparison of MRI and Histology

Survival and integration into the host brain of grafted tissue are crucial factors in neurotransplantation approaches. The present study explored the feasibility of using a clinical MR scanner to study striatal graft development in a rat model of Huntington's disease. Rat fetal lateral ganglionic eminences grown as free-floating roller-tube cultures were grafted into the quinolinic acid-lesioned striatum, and T1- and T2-weighted sequences were acquired at 2, 7, 21, and 99 days posttransplantation. MR images were then compared with images of corresponding histological sections. The lesion-induced striatal degeneration caused a progressive ventricle enlargement, which was significantly different from controls at 21 days posttransplantation. Seven days posttransplantation, T1-weighted images revealed a defined liquid-isointense signal surrounded by a hyperintense rim at the site of graft placement, which was found unaltered for the first 21 days posttransplantation, whereas a hypointense graft signal was detected at 99 days posttransplantation. At 2 days posttransplantation, T2-weighted images showed the graft region as a hyperintense area surrounded by a rim of low signal intensity but at later time-points graft location could not be further verified. Measures for graft size and ventricle size obtained from MR images highly correlated with measures obtained from histologically processed sections (R=0.8,P<0.001). In conclusion, the present study shows that fetal rat lateral ganglionic eminences grown as free-floating roller-tube cultures can be successfully grafted in a rat Huntington model and that a clinical MR scanner offers a useful noninvasive tool for studying striatal graft development.

Rat fetal ventral mesencephalon grown as solid tissue cultures: influence of culture time and BDNF treatment on dopamine neuron survival and function

Free-floating roller tube (FFRT) cultures of fetal rat and human nigral tissue are a means for tissue storage prior to grafting in experimental Parkinson's disease. In the present study, FFRT cultures prepared from embryonic-day-14 rat ventral mesencephalon were maintained for 4, 8, 12, or 16 days in vitro (DIV) in the presence or absence (controls) of BDNF [100 ng/ml]. The dopamine content in the culture medium, analyzed by HPLC, was significantly higher (4–5 fold) in the BDNF group at DIV 8 and DIV 12 compared to the corresponding control levels (40 pg/ml). The number of tyrosine hydroxylase immunoreactive neurons was significantly higher for BDNF treated cultures (2729±300) at DIV 8, as compared to controls (1679±217). At DIV 12, the culture volume was significantly increased by BDNF (1.05±0.12 vs. 0.71±0.04 mm 3). Similar results were obtained for total protein. Western blot analysis demonstrated increasing signals for GFAP with increasing time in culture, but levels for control and BDNF treated cultures did not differ at any time-point investigated. In conclusion, it is suggested that the time window for effective storage of dopaminergic tissue prior to grafting can be extended by using the FFRT culture technique and that the in vitro storage may be further prolonged by treatment with BDNF.

Effects of combined BDNF and GDNF treatment on cultured dopaminergic midbrain neurons.

Neural transplantation is an experimental therapy for Parkinson's disease. Pretreatment of fetal donor tissue with neurotrophic factors may improve survival of grafted dopaminergic neurons. Free-floating roller tube cultures of fetal rat ventral mesencephalon were treated with brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), or a combination of both. Dopamine content of the culture medium, the number of tyrosine hydroxylase-immunoreactive neurons, and culture volumes were moderately increased in the BDNF- and GDNF-treated cultures but significantly increased by 6.8-, 3.2- and 2.4-fold, respectively after treatment with the combination of both factors. We conclude that pretreatment of dopaminergic tissue in culture with a combination of BDNF and GDNF may be an effective means to improve the quality of tissue prior to grafting.

Comparison of mesencephalic free-floating tissue culture grafts and cell suspension grafts in the 6-hydroxydopamine-lesioned rat.

Ventral mesencephalon (VM) of fetal rat and human origin grown as free-floating roller-tube (FFRT) cultures can survive subsequent grafting to the adult rat striatum. To further explore the functional efficacy of such grafts, embryonic day 13 ventral mesencephalic tissue was grafted either after 7 days in culture or directly as dissociated cell suspensions, and compared with regard to neuronal survival and ability to normalize rotational behavior in adult rats with unilateral 6-hydroxydopamine (6-OHDA) lesions. Other lesioned rats received injections of cell-free medium and served as controls. The amphetamine-induced rotational behavior of all 6-OHDA-lesioned animals was monitored at various time points from 18 days before transplantation and up to 80 days after transplantation. Tyrosine hydroxylase (TH) immunostaining of the histologically processed brains served to assess the long-term survival of grafted dopaminergic neurons and to correlate that with the behavioral effects. Additional cultures and acutely prepared explants were also fixed and stored for histological investigation in order to estimate the loss of dopaminergic neurons in culture and after transplantation. Similar behavioral improvements in terms of significant reductions in amphetamine-induced rotations were observed in rats grafted with FFRT cultures (127%) and rats grafted with cell suspensions (122%), while control animals showed no normalization of rotational behavior. At 84 days after transplantation, there were similar numbers of TH-immunoreactive (TH-ir) neurons in grafts of cultured tissue (775 +/- 98, mean +/- SEM) and grafts of fresh, dissociated cell suspension (806 +/- 105, mean +/- SEM). Cell counts in fresh explants, 7-day-old cultures, and grafted cultures revealed a 68.2% loss of TH-ir cells 7 days after explantation, with an additional 23.1% loss after grafting, leaving 8.7% of the original number of TH-ir cells in the intracerebral grafts. This is to be compared with a survival rate of 9.1% for the TH-ir cells in the cell-suspension grafts. Immunostaining for the calcium-binding proteins calretinin, calbindin, and parvalbumin showed no differences in the neuronal expression of these proteins between the two graft types. In conclusion, we found comparable dopaminergic cell survival and functional effects of tissue-culture grafts and cell-suspension grafts, which currently is the type of graft most commonly used for experimental and clinical grafting. In this sense the result is promising for the development of an effective in vitro storage of fetal nigral tissue, which at the same time would allow neuroprotective and neurotrophic treatment prior to intracerebral transplantation.

Culture of Substantia Nigra Neurons

Primary cultures of nigral tissue are widely used as a model system to assay effects of trophic and toxic agents on dopaminergic neurons. Cultured dopaminergic neurons have been successfully transplanted in animals and led to behavioral improvement in animal models of Parkinson's disease. Cell cultures have also been used to study the development of substantia nigra, allowing investigators to identify early inductive events important for nigral development and to study dopaminergic differentiation and target innervation. This unit provides simple and reliable culture protocols for these applications. The first approach presented is the preparation of dissociated nigral cell cultures, the later steps of which can be used as a simple and efficient assay for testing growth factors. A second approach is the preparation of free-floating roller tube cultures, which may be used as a tool for neural transplantation and to study more complex developmental events. A third approach is the production of organotypic cultures using chicken plasma as a matrix. Organotypic cultures can maintain the in vivo cytoarchitecture of a host region in vitro.

Noninvasive Dopamine Determination by Reversed Phase HPLC in the Medium of Free-Floating Roller Tube Cultures of Rat Fetal Ventral Mesencephalon: A Tool to Assess Dopaminergic Tissue Prior to Grafting

The low availability of dopamine containing neurons for grafting in Parkinson's disease is a general problem. Free-floating roller tube (FFRT) cultures allow storage of fetal mesencephalic tissue prior to transplantation. Preoperative functional testing permits to select an optimized set of individual cultures for transplantation. Rat fetal ventral mesencephali (E13) were dissected out and divided into four equally sized pieces each and individually prepared as FFRT cultures. After 4, 8, 12, and 16 days in vitro (DIV) the medium of each culture was collected during routine medium change and immediately stabilized. Dopamine was extracted and probes were determined with reversed phase HPLC using electrochemical detection. After 16 DIV cultures were fixed and cell counts performed in tyrosine hydroxylase (TH)-immunostained serial sections. The mean dopamine content ± SEM in culture conditioned media was at 4 DIV: 21 ± 2 pg, n = 38; at 8 DIV: 37 ± 4 pg, n = 40; at 12 DIV: 52 ± 7 pg, n = 38; and at 16 DIV: 39 ± 5 pg, n = 38. In all cultures devoid of dopamine after 4 and 8 DIV (12.5%) levels remained below detectability at 12 and 16 DIV. Cultures derived from the rostral mesencephalon showed significantly higher dopamine values than those from the caudal mesencephalon at 12 DIV. The mean number of TH-immunoreactive (-ir) cells/culture ± SEM after 16 DIV was 556 ± 51, n = 40. The correlation between TH-ir cell number (CN) and dopamine content of rostrally derived cultures at 16 DIV was: CN = 7.4 (dopamine [pg]) + 248; R = 0.75; n = 19; p < 0.001. No dopamine was present in cultures without TH-ir cells. These results demonstrate that sequential noninvasive screening of dopamine in single cultures is feasible and that the dopamine content is correlated to the number of surviving TH-ir cells. This permits to select cultures rich in dopaminergic neurons for transplantation.

Fetal ventral mesencephalon of human and rat origin maintained in vitro and transplanted to 6-hydroxydopamine-lesioned rats gives rise to grafts rich in dopaminergic neurons.

Free-floating roller tube cultures of human fetal (embryonic age 6-10 weeks post-conception) and rat fetal (embryonic day 13) ventral mesencephalon were prepared. After 7-15 days in vitro, the mesencephalic tissue cultures were transplanted into the striatum of adult rats that had received unilateral injections of 6-hydroxydopamine into the nigrostriatal bundle 3-5 weeks prior to transplantation. Graft survival was assessed in tyrosine hydroxylase (TH)-immunostained serial sections of the grafted brains up to post-transplantation week 4 for the human fetal xenografts and post-transplantation week 11 for the rat fetal allografts. D-amphetamine-induced rotation was monitored up to 10 weeks after transplantation in the allografted animals and compared with that of lesioned-only control animals. All transplanted animals showed large, viable grafts containing TH-immunoreactive (ir) neurons. The density of TH-ir neurons in the human fetal xenografts and in rat fetal allografts was similar. A significant amelioration of the amphetamine-induced rotation was observed in the animals that received cultured tissue allografts. These results promote the feasibility of in vitro maintenance of fetal human and rat nigral tissue prior to transplantation using the free-floating roller tube technique.

Noninvasive dopamine determination by reversed phase HPLC in the medium of free-floating roller tube cultures of rat fetal ventral mesencephalon: a tool to assess dopaminergic tissue prior to grafting.

The low availability of dopamine containing neurons for grafting in Parkinson's disease is a general problem. Free-floating roller tube (FFRT) cultures allow storage of fetal mesencephalic tissue prior to transplantation. Preoperative functional testing permits to select an optimized set of individual cultures for transplantation. Rat fetal ventral mesencephali (E13) were dissected out and divided into four equally sized pieces each and individually prepared as FFRT cultures. After 4, 8, 12, and 16 days in vitro (DIV) the medium of each culture was collected during routine medium change and immediately stabilized. Dopamine was extracted and probes were determined with reversed phase HPLC using electro-chemical detection. After 16 DIV cultures were fixed and cell counts performed in tyrosine hydroxylase (TH)-immunostained serial sections. The mean dopamine content +/- SEM In culture conditioned media was at 4 DIV: 21 +/- 2 pg, n = 38; at 8 DIV: 37 +/- 4 pg, n = 40; at 12 DIV: 52 +/- 7 pg, n = 38; and at 16 DIV: 39 +/- 5 pg, n = 38. In all cultures devoid of dopamine after 4 and 8 DIV (12.5%) levels remained below detectability at 12 and 16 DIV. Cultures derived from the rostral mesencephalon showed significantly higher dopamine values than those from the caudal mesencephalon at 12 DIV. The mean number of TH-immunoreactive (-ir) cells/culture +/- SEM after 16 DIV was 556 +/- 51, n = 40. The correlation between TH-ir cell number (CN) and dopamine content of rostrally derived cultures at 16 DIV was: CN = 7.4 (dopamine [pg]) + 248; R = 0.75; n = 19; p < 0.001. No dopamine was present in cultures without TH-ir cells. These results demonstrate that sequential noninvasive screening of dopamine in single cultures is feasible and that the dopamine content is correlated to the number of surviving TH-ir cells. This permits to select cultures rich in dopaminergic neurons for transplantation.

Effect of BDNF on Dopaminergic, Serotonergic, and GABAergic Neurons in Cultures of Human Fetal Ventral Mesencephalon

The neurotrophin brain-derived neurotrophic factor (BDNF) was tested for its ability to promote the survival and regulation of expression of phenotypic markers of dopaminergic, serotonergic, and GABAergic neurons in free-floating roller tube cultures of human fetal ventral mesencephalon. This culture system contains neurons of the anlage of the substantia nigra as well as that of the rostral raphe nucleus. Dopaminergic neuron number and tyrosine hydroxylase (TH) fiber density was monitored by TH immunocytochemistry. Measurement of dopamine (DA) content, TH enzymatic activity, serotonin (5-HT) content, and glutamic acid decarboxylase (GAD) activity were used as indices of their respective neurotransmitter function. The presence of GABAergic and serotonergic neurons in this culture system was confirmed by GABA and 5-HT immunocytochemistry. In cultures maintained in the presence of BDNF (10 ng/ml), the density of TH-positive cells was increased by 2.5-fold ( P f 0.05), and the TH-positive fiber density was increased by 3.5-fold ( P f 0.01), relative to control cultures. Similarly, the relative increases in DA content and TH activity were 2.6- and 2.3-fold, respectively, in the BDNF-treated cultures ( P f 0.01 and P f 0.01). On a per neuron basis, DA content and TH activity were not markedly changed by BDNF treatment, suggesting that the increases in DA content and TH activity are due to more DA neurons surviving. Relative elevations were also observed in serotonin content (2.0-fold, P f 0.01) and GAD enzymatic activity (1.4-fold, P f 0.01). Future studies will need to determine whether these changes result from the direct action of BDNF on these neurons or through some indirect mechanism. The results demonstrate that BDNF has beneficial effects on cultured human fetal tissue, which may be relevant in optimizing neuronal transplantation techniques, and that multiple systems are simultaneously influenced by BDNF.