In vitro and in vivo characterization of neural stem cells.

Neural stem cells are defined as clonogenic cells with self-renewal capacity and the ability to generate all neural lineages (multipotentiality). Cells with these characteristics have been isolated from the embryonic and adult central nervous system. Under specific conditions, these cells can differentiate into neurons, glia, and non-neural cell types, or proliferate in long-term cultures as cell clusters termed "neurospheres". These cultures represent a useful model for neurodevelopmental studies and a potential cell source for cell replacement therapy. Because no specific markers are available to unequivocally identify neural stem cells, their functional characteristics (self-renewal and multipotentiality) provide the main features for their identification. Here, we review the experimental and ultrastructural studies aimed at identifying the morphological characteristics and the antigenic markers of neural stem cells for their in vitro and in vivo identification.

Intrathecal transplantation of neuroblastoma cells decreases heat hyperalgesia and cold allodynia in a rat model of neuropathic pain.

Intrathecal grafting of cells as biological pumps to deliver monoamines, endorphins, and/or trophic factors, has been shown to be effective in treating chronic pain both in experimental animals and in clinical trials. We have tested whether intrathecal implantation of neuroblastoma cells reduces heat hyperalgesia and cold allodynia in a rat model of neuropathic pain induced by chronic constriction injury (CCI) of the sciatic nerve. Behavioral tests and cerebrospinal fluid (CSF) collection were performed before CCI, 1 week later (after which, vehicle or NB69 cells were intrathecally injected) and at 4, 7, and 14 days post-injection. Both CSF sampling and injection of the cells were performed by direct lumbar puncture. Intrathecal grafting of 4 x 10(6) NB69 neuroblastoma cells reduced to basal levels the nociceptive response to heat in nerve-injured hindpaws, while the response of control limbs remained unchanged. Similarly, the allodynic response to cold elicited by acetone evaporation decreased in the animals implanted with NB69 cells. An increase in the concentrations of dopamine and serotonin metabolites of around 150% was observed in the CSF of animals that received grafts of NB69 cells. These data suggest that the monoamines released by NB69 cells in the intrathecal space produce analgesia to neuropathic pain in rats.

Functional implications of the noradrenergic-cholinergic switch induced by retinoic acid in NB69 neuroblastoma cells.

Some neuroblastoma cell lines change their neurotransmitter phenotype from noradrenergic to cholinergic under retinoic acid treatment. Such "neurotransmitter switch" seems to be a consequence of changes in the expression and activity of the biosynthetic machinery for both neurotransmitters. In this study, we have characterized this "neurotransmitter switch" induced by retinoic acid in a human neuroblastoma cell line (NB69) showing catecholaminergic characteristics. Retinoic acid treatment reduced tyrosine hydroxylase activity and noradrenaline levels in NB69 cells but did not modify the expression of this enzyme. Moreover, the calcium-dependent release of [(3)H]noradrenaline in control cells was highly reduced by retinoic acid treatment. On the other hand, NB69 cells treated with retinoic acid enhanced the expression of choline acetyltransferase and acquired the capability to release [(3)H]acetylcholine in a calcium-dependent way. In addition, we found that the expression of the vesicular monoamine transporter 2 (VMAT2) and the vesicular acetylcholine transporter (VAChT) was increased in those cells treated with retinoic acid. Immunostaining revealed that retinoic acid treatment changed the cellular distribution of both vesicular monoamine transporter 2 and vesicular acetylcholine transporter. In conclusion, retinoic acid induces a noradrenergic to cholinergic switch in NB69 cells by acting at several levels of the neurotransmitter phenotypic expression.

Neuronal-enriched cultures from embryonic rat ventral mesencephalon for pharmacological studies of dopamine neurons.

The method described herein provides a convenient and rapid procedure to obtain enriched neuronal cultures containing reproducible numbers of dopamine (DA) cells. These cultures allow experimental paradigms designed to study the effect of drugs on DA neurons without astroglial mediation. Neuronal-enriched cultures are prepared from the mesencephalon of rat embryos at the 14th day of gestation (E14). At that moment, DA cells of the developing substantia nigra are located ventrally at the level of the mesencephalic flexure. Because the neurons of the pars compacta are mostly born between E12 and E15, E14 corresponds to an optimal stage for obtaining a high survival of DA cells. A defined medium (EF12) allows the maturation of DA neurons and reduces drastically the number of astrocytes. After 7 days in vitro (DIV) in EF12, the cultures contain 2-5% astrocytes (GFAP+ cells) and DA neurons represent 0.5-2% of the cells, as assessed by immunostaining to tyrosine hydroxylase (TH). The function of DA neurons is assessed by [3H]DA uptake and of those non-DA neurons by the high affinity [3H]GABA uptake. Cell survival is assessed by Trypan blue dye exclusion.

Xenogeneic adrenal medulla graft rejection rather than survival leads to increased rat striatal tyrosine hydroxylase immunoreactivity.

Adrenal medulla has often been used as a donor tissue for transplantation into damaged central nervous system, with functional effects ranging from very good to nonexistent. The grafts have often been associated with morphological evidence of stimulated recipient dopaminergic fiber plasticity. The interpretation of these results has been difficult due to variable but mostly poor graft survival. The present study combines two experiments which evaluated the effects of intrastriatal xenogeneic adrenal medullary cell suspension grafts on rat recipients. First, bovine adrenal medulla cell suspension grafts of various compositions were tested for their functional and morphologic effects on immunosuppressed hemiparkinsonian rats. In the second experiment, graft rejection was allowed to occur in half of the rats in order to determine a possible contribution of the inflammatory/immune response to increased dopaminergic fiber plasticity of the recipient. At 28 days, grafts of all cell types survived well in immunosuppressed rats, but none of the grafted cell types was associated with either an amelioration of amphetamine-induced rotation or an increase in striatal tyrosine hydroxylase immunoreactivity around the graft site. The latter phenomenon was observed only in the nonimmunosuppressed rats with rejected grafts. Our findings strongly support the role of inflammatory/immune response to grafting in stimulating dopaminergic fiber plasticity and in the appearance of sprouting.

Glia protect fetal midbrain dopamine neurons in culture from L-DOPA toxicity through multiple mechanisms.

Mesencephalic glia produce soluble factors that protect dopamine neurons from L-DOPA toxicity. The chemical composition of these soluble factors is unknown. We investigated the protective effect against L-DOPA neurotoxicity in midbrain dopamine neurons of fractions of different molecular size of glia conditioned medium and candidate neuroprotective agents produced by glia including neurotrophic factors and antioxidants. Protective effects were evaluated according to the number of tyrosine hydroxylase immunoreactive cells, high affinity dopamine uptake and levels of quinones. Both fractions of glia conditioned medium, smaller and larger than 10kD, protected against L-DOPA, but the fraction of smaller molecular size, that contains small free radical scanvenger molecules, was more effective than the fraction of larger molecular size, that contains large neurotrophic peptides. Among the neurotrophic factors GDNF and BDNF totally prevented L-DOPA neurotoxicity, while NGF and bFGF were less effective. However, only NGF significantly reduced the elevation of quinones induced by L-DOPA. Ascorbic acid, at the concentration found in glia conditioned medium, provided partial protective effect against L-DOPA toxicity. Glutathione, had neurotrophic effects on untreated midbrain dopamine neurons and prevented the effect of L-DOPA. In conclusion, the protective effect against L-DOPA neurotoxicity by glia conditioned medium is mediated by several compounds including neurotrophic factors and small antioxidants.

Glia conditioned medium protects fetal rat midbrain neurones in culture from L-DOPA toxicity.

L-DOPA kills dopamine neurones in culture but is the most effective drug for the treatment of Parkinson's disease, where it exhibits no clear toxicity. While glial cells surround and protect neurones in vivo, neurones are usually cultured in vitro in the absence of glia. We treated fetal midbrain rat neurones with L-DOPA, mesencephalic glia conditioned medium (CM) and L-DOPA + CM. L-DOPA reduced the number of tyrosine hydroxylase-positive (TH+) cells and [3H]DA uptake, and increased quinone levels. L-DOPA + CM restored [3H]DA uptake and quinone levels to normal, and increased the number of TH+ cells and terminals to 170% of control. CM greatly increased the number of TH+ cells and [3H]DA uptake. Mesencephalic glia therefore produced soluble factors which are neurotrophic for dopamine neurones, and which protect these neurones from the toxic effects of L-DOPA.

Toxic effects of L-DOPA on mesencephalic cell cultures: protection with antioxidants.

The toxicity of L-3,4-dihydroxyphenylalanine (L-DOPA) was studied in neuronal cultures from rat mesencephalon. The survival and function of DA neurons were assessed by the number of tyrosine hydroxylase-positive (TH+) cells and 3H-DA uptake and those non-DA neurons by the exclusion of Trypan blue and the high-affinity 3H-GABA uptake. L-DOPA was toxic for both DA and non-DA neurons. DA neurons were more severely affected than non-DA neurons after short periods of treatment and with exposure to a low dose of L-DOPA (25 vs. 100 microM) and less selectively affected after 1 or 2 days of treatment. After incubation with L-DOPA, a disruption of the neuritic network and an overall deterioration were observed, more evident for TH+ cells in the whole culture. Auto-oxidation to quinones is responsible in part for L-DOPA toxicity in non-DA neurons since the levels of quinones correlated well with the severity of cell death in the cultures. The damage of DA neurons took place before the rising of quinones, suggesting that quinones are not essential in L-DOPA toxicity for DA neurons. Antioxidants, such as ascorbic acid and sodium metabisulfite, completely prevented L-DOPA-induced quinone formation as well as the death of non-DA neurons. In contrast, they could only partially prevent the damage produced by L-DOPA in DA neurons. Mazindol, a selective inhibitor of DA uptake, protected TH+ cells from L-DOPA.

Regrowth of axons in lesioned adult rat spinal cord: promotion by implants of cultured Schwann cells.

Highly purified populations of Schwann cells were grafted into lesioned adult rat spinal cord to determine if they promote axonal regeneration. Dorsal spinal cord lesions were created by a photochemical lesioning technique. Schwann cells derived from E16 rat dorsal root ganglia, either elongated and associated with their extracellular matrix or dissociated and without matrix, were rolled in polymerized collagen to form an implant 4-6 mm long which was grafted at 5 or 28 days after lesioning. No immunosuppression was used. Acellular collagen rolls served as controls. At 14, 28 and 90 days and 4 and 6 months after grafting, animals were analysed histologically with silver and Toluidine Blue stains and EM. The grafts often filled the lesion and the host borders they apposed exhibited only limited astrogliosis. By 14 days, bundles of unmyelinated and occasional thinly myelinated axons populated the periphery of Schwann cell implants. By 28 days and thereafter, numerous unmyelinated and myelinated axons were present in most grafts. Silver staining revealed sprouted axons at the implant border at 28 days and long bundles of axons within the implant at 90 days. Photographs of entire 1 micron plastic cross-sections of nine grafted areas were assembled into montages to count the number of myelinated axons at the graft midpoint; the number of myelinated axons ranged from 517-3214. Electron microscopy of implants showed typical Schwann cell ensheathment and myelination, increased myelin thickness by 90 days, and a preponderance of unmyelinated over myelinated axons. Random EM sampling of five Schwann cell grafts showed that the ratio of unmyelinated to myelinated axons was highest (20:1) at 28 days. These ratios implied that axons numbered in the thousands at the graft midpoint. Dissociated Schwann cells without matrix promoted axonal ingrowth and longitudinal orientation as effectively as did elongated Schwann cells accompanied by matrix. There was a suggestion that axonal ingrowth was at least as successful, if not more so, when the delay between lesioning and grafting was 28 rather than 5 days. Acellular collagen grafts did not contain axons at 28 days, the only interval assessed. In sum, grafts of Schwann cells in a rolled collagen layer filled the lesion and were well tolerated by the host. The Schwann cells stimulated rapid and abundant growth of axons into grafts and they ensheathed and myelinated these axons in the normal manner.

Levodopa toxicity in foetal rat midbrain neurones in culture: modulation by ascorbic acid.

Levodopa, a dopamine (DA) precursor administered to patients with Parkinson's disease (PD), produces at 25-200 x 10(-6) M concentrations a dose-dependent reduction of 3H-DA uptake in foetal rat midbrain cultures. Also, a decrease in the number of viable cells and tyrosine hydroxylase (TH) positive neurones, plus disruption of the overall neuritic network are observed concurrently with an elevation of quinone levels in the culture medium. Ascorbic acid (AA), which abolished the quinone overproduction, partially prevented these effects. Though levodopa neurotoxicity in vivo is as yet unproven, AA may reduce vulnerability of endogenous or grafted DA neurones in patients with PD.

Induction of axon growth into Schwann cell implants grafted into lesioned adult rat spinal cord.

Polymerized collagen rolls enclosing Schwann cells (SCs) raised in culture were grafted into cystic cavities formed after lesioning the thoracic spinal cord of adult rats. Axons were already present within the graft by 14 days after implantation and both ensheathed and myelinated axons were numerous by 28 days. This axonal ingrowth was maintained over longer survival periods. The axons within the graft always appeared related to Schwann cells. Acellular collagen rolls did not show axonal ingrowth. These Schwann cell-collagen implants resemble peripheral nerve grafts in their ability to induce axonal regeneration into the graft.

Distribution of neurons in the main cuneate nucleus projecting to the inferior olive in the cat. Evidence that they differ from those directly projecting to the cerebellum.

The distribution in the main cuneate nucleus of cells projecting to the inferior olive and the intermediate zone of the cerebellar anterior lobe were compared by means of double retrograde labeling methods in the cat. The tracer combinations were either Fast Blue and Diamidino Yellow Dihydrochloride; or horseradish peroxidase conjugated to wheat germ agglutinin and Diamidino Yellow Dihydrochloride. Neurons in the caudal, middle and rostral subdivisions of the main cuneate nucleus project to the inferior olive. Differences exist, however, in its number and location along the rostrocaudal extent of the nucleus. Cells projecting to the inferior olive predominate in the caudal and middle subdivisions, where they concentrate ventrally. No cells in the "clusters region" project to the inferior olive. Main cuneate nucleus neurons projecting to the cerebellum concentrate rostral to the obex, bordering the external cuneate nucleus and partially intermixing with the rostrally located cells projecting to the inferior olive. However, no double-labeled cells were found. The results indicate that the main cuneate nucleus projections to the inferior olive and cerebellar anterior lobe originate from different populations of neurons with high specific locations within the nucleus. This finding is in agreement with previous studies suggesting that each of the main cuneate nucleus targets receives its input from a distinct population of neurons within the nucleus.