Production of spinal cord growth inhibitor by nonneuronal cells

Separation of chick embryo spinal cord cells into cell cultures of glial-enriched and neuronal-enriched elements by the method of selective adhesiveness indicated a greater production of growth inhibitor by the glial-enriched cultures. Glial-enriched cultures produced 177.2% more inhibitor per microgram cell protein than did unseparated spinal cord cell cultures, whereas neuronal-enriched cultures produced less. The glial-enriched cultures contained 28% of the choline acetyltransferase of neuronal-enriched cultures, few or no discernible neurons, and no cells binding dopamine or containing catecholamines by cytofluorescence. The predominant cell of the glial-enriched cultures was large, phase-light with ruffled borders, with parallel streak-like processes, and fibrillar cytoplasm. Cultures produced by serial passage of unseparated spinal cord cells also were found to be depleted in neurons and dopamine-binding cells, as well as in choline acetyltransferase activity, to 19% of control values. These serially passaged cultures, however, retained the ability to produce inhibitor as well as did primary cultures. Serially passaged cultures of spinal cord cells were composed of cells similar to those predominant in glial-enriched cultures. Nerve growth factor had no effect on inhibitor production. We suggest that the growth inhibitor of spinal cord cultures was not produced by neuronal cells but may be of glial origin.     

Molecular differentiation of neurons from ependyma-derived cells in tissue cultures of regenerating teleost spinal cord

Cells cultured from the caudalmost area of regenerating teleost spinal cord differ both morphologically and in terms of molecular architecture from those of more rostral (more fully differentiated) areas of the cord. The caudalmost regenerating cord consists of an ependymal tube. Cells from this region have flattened or partially flattened cell somas and short spike projections in vitro; they do not exhibit the rounded cell somas nor the long, thin, branching neurites typical of differentiated neurons. A series of cultures taken from different areas along the length of regenerating spinal cord were examined for molecular differentiation by staining with a monoclonal antibody against non-phosphorylated neurofilament protein (SMI 32). None of the cells from the caudalmost culture of the regenerating spinal cord stained with antibody SMI 32. In cultures of more rostral regenerated cord, the cells with neuronal morphology do stain positively with the anti-neurofilament antibody. This result suggests that the cells in the cultures of caudalmost cord represent relatively undifferentiated ependymal cells, or ependyma-derived cells, which later differentiate into neurons and glia in the regenerating spinal cord.     

Biological properties of crude growth inhibiting factor produced by embryonic spinal cord cells in culture

Cultured chick spinal cord cells produced a soluble factor or factors which inhibited growth and development of muscle cells in culture. The activity of cord inhibitory factor depended on the number of cord cells inoculated into culture, and the duration of their incubation in culture. Nonreplicating frozen and thawed cells produced little or no inhibitory factor. Cord inhibitory factor was produced by chick cord cells from embryos tested at 11 to 21 days of age. While “stale” media from thigh, heart, and skin cell cultures also were slightly inhibitory, their activity was several-fold less potent than that of cord cells. Media collected from liver cell cultures stimulated growth and protein synthesis. Crude cord inhibitory factor had little or no effect on target muscle cells (plated at 1 × 10⁶ cells per dish) after the first 2 days of culture. Cells began in culture at lower density, however (1 × 10⁵ cells per dish), retained their susceptibility for at least 4 days in culture while continuing active DNA synthesis. This suggests that cord inhibitory factor acted on cells while they were replicating. Although inhibited cells did not incorporate [¹⁴C]lysine into protein normally, they retained the ability to take up the amino acid into their cytoplasm. Inhibitory activity of crude cord factor was also demonstrable against cultures of heart and skin cells. Cord cell cultures, however, were relatively resistant. It is at present not known whether the inhibitory factor is produced by neuronal or glial cells.     

Optimized protocols for isolation of primary motor neurons, astrocytes and microglia from embryonic mouse spinal cord

Neuron-glial interactions are important in development of the nervous system and pathogenesis of disease. Primary cell cultures prepared from nervous tissue are often used to study the properties of individual cell types and how they interact with each other. Isolation of pure populations of cells and their culture is challenging, particularly from murine spinal cord. The purpose of this study was to optimize various protocols to achieve efficient, parallel isolation and purification of primary motor neurons, microglia and astrocytes from the same mouse embryonic spinal cord sample. Following dissociation of E12 embryonic spinal cords, motor neurons were isolated at 97% purity by a single step centrifugation of the cell suspension through multiple discontinuous density gradients of NycoPrep. The residual mixed cell pellet was resuspended and cultured for 2 weeks. Mixed cultures were then shaken to release microglia, which were then harvested from the medium and subjected to another round of differential adhesion to achieve 99% purity. The astrocytes remaining in the mixed cultures were culled to 98% purity by treatment with leucine methyl ester and a subsequent vigorous shaking step to remove any remaining microglia and neurons. Furthermore, no cross contamination was observed in the glial cultures. This technique provides a simple, convenient, and reliable method of obtaining highly purified preparations of motor neurons, microglia and astrocytes from embryonic spinal cord for the study of spinal cord cell biology and motor neuron diseases.     

Bone marrow stromal cells enhance differentiation of cocultured neurosphere cells and promote regeneration of injured spinal cord

Transplantation of bone marrow stromal cells (MSCs) has been regarded as a potential approach for promoting nerve regeneration. In the present study, we investigated the influence of MSCs on spinal cord neurosphere cells in vitro and on the regeneration of injured spinal cord in vivo by grafting. MSCs from adult rats were cocultured with fetal spinal cord-derived neurosphere cells by either cell mixing or making monolayered-feeder cultures. In the mixed cell cultures, neuroshpere cells were stimulated to develop extensive processes. In the monolayered-feeder cultures, numerous processes from neurosphere cells appeared to be attracted to MSCs. In an in vivo experiment, grafted MSCs promoted the regeneration of injured spinal cord by enhancing tissue repair of the lesion, leaving apparently smaller cavities than in controls. Although the number of grafted MSCs gradually decreased, some treated animals showed remarkable functional recovery. These results suggest that MSCs might have profound effects on the differentiation of neurosphere cells and be able to promote regeneration of the spinal cord by means of grafting.     

Cellular distribution of 04 antigen and galactocerebroside in primary cultures of human foetal spinal cord

Summary The distribution of cell-surface 04 antigen and galactocerebroside (GC) was examined by duallabel indirect immunofluorescence assays on live primary cultures of human spinal cord cells dissociated from 8–12 week-old foetal tissue. Oligodendrocytes expressing GC on their surface were present in the cultures at early time points, and all GC-positive cells were found to also express cell-surface 04 antigen. The 04 antigen was found additionally on a further population of GC-negative cells in the spinal cord cultures, which did not react with antibodies to glial fibrillary acidic protein (GFAP), and were distinct from neuronal cells and cell processes which stained with anti-neurofilament antibody. Previous studies in mouse neural cell cultures have shown that 04 antigen-positive cells are direct precursors to GC-bearing oligodendrocytes (Schachner et al. 1982).In the human spinal cord cultures, a rapid decline in the number of cells expressing GC and/or the 04 antigen to a value below 1% was observed during the first 3 days in vitro.The present studies indicate that synthesis of GC occurs in the human spinal cord many weeks before myelination commences in vivo and that GC-negative oligodendrocyte precursors are present simultaneously with more mature GC-positive cells. In addition, it would appear that complex humoral or cellular ingredients may be required for the long-term in vitro maintenance of viable human foetal oligodendrocytes.     

Neuropeptides in dissociated cell cultures of mammalian spinal cord and dorsal root ganglion

Immunohistochemical studies of leucine-enkephalin, somatostatin, vasoactive intestinal polypeptide and neurotensin were carried out in dissociated cell co-cultures of embryonic mouse spinal cord and dorsal root ganglion, using the peroxidase-antiperoxidase technique. Leucine-enkephalin immunoreactivity exceeded that of the other peptides in these coculture preparations. Leucine-enkephalin, substance P and somatostatin were also studied in spinal cord cultures (without dorsal root ganglia) and in dorsal root ganglia cultures (without spinal cord). Each of these peptides was present in only a small percentage (<10%) of perikarya and processes in spinal cord cultures. No leucine-enkephalin immunoreactivity was seen in dorsal root ganglion cultures; a considerable proportion of the processes were immunoreactive for substance P or somatostatin. These observations suggest that co-cultures of spinal cord and dorsal root ganglia can provide a simplified in vitro “model” of the nervous system for the study of peptidergic interactions.     

Correction of humoral derangements from mutant superoxide dismutase 1 spinal cord

OBJECTIVE: We sought to define molecular and cellular participants that mediate motor neuron injury in amyotrophic lateral sclerosis using a coculture system. METHODS: We cocultured embryonic stem cell-derived motor neurons with organotypic slice cultures from wild-type or SOD1G93A (MT) mice. We examined axon lengths and cell survival of embryonic stem cell-derived motor neurons. We defined and quantified the humoral factors that differed between wild-type and MT organotypic cultures, and then corrected these differences in cell culture. RESULTS: MT spinal cord organotypic slices were selectively toxic to motor neurons as defined by axonal lengths and cell survival. MT spinal cord organotypic slices secreted higher levels of nitric oxide, interleukin (IL)-1beta, IL-6, and IL-12p70 and lower levels of vascular endothelial growth factor. The toxicity of MT spinal cord organotypic cultures was reduced and axonal lengths were restored to near normal by coculturing in the presence of reactive oxygen species scavenger, vascular endothelial growth factor, and neutralizing antibodies to IL-1beta, IL-6, and IL-12. INTERPRETATION: MT spinal cord organotypic cultures overexpress certain factors and underexpress others, creating a nonpermissive environment for cocultured motor neurons. Correction of these abnormalities as a group, but not individually, restores axonal length to near normal. Such a "cocktail" approach to the treatment of amyotrophic lateral sclerosis should be investigated further.     

Acetylcholine metabolism in rat spinal cord cultures: regulation by a factor involved in the determination of the neurotransmitter phenotype of sympathetic neurons

Acetylcholine metabolism has been studied in sister cultures of E13 rat spinal cord cells cultured for 1 to 3 weeks with or without conditioned medium (CM) from rat skeletal muscle cells. Spinal cord cells grown with CM synthesized and accumulated 3 to 4 times more [3H]ACh from [3H]choline than cultures grown without CM. This effect of CM was accompanied by a comparable increase in CAT activity and could not be mimicked by increasing the density of the spinal cord cultures. A 2- to 3-fold increase in AChE activity was also observed in 2- to 3-week-old CM cultures, whereas the activity of lactate dehydrogenase was identical in cultures grown with and without CM. We have compared the effects of CMs from various non-neuronal cell cultures on [3H]ACh synthesis and storage by spinal cord cultures and by sympathetic neuron cultures. CM by skeletal muscle greater than skin fibroblasts greater than rat heart muscle greater than C6 glioma cells were the most active on both types of neuron cultures, whereas CM from rat brain, L6 myoblasts, mouse 3T3, and PYT21 fibroblasts was inactive on spinal cord cultures and only weakly active on sympathetic neurons. Serum-free CM from skeletal muscle was inactive on both types of neuron cultures. The CM factor active on spinal cord cultures has been purified several thousand-fold by using a four-step fractionation scheme which has previously led to a partial purification of the CM factor involved in the regulation of CAT, AChE, and catecholamine-synthesizing enzymes in sympathetic neuron cultures ( Swerts , J. P., A. Le Van Thai, A. Vigny , and M. J. Weber (1983) Dev. Biol. 100: 1-11). Moreover, a comparison of dose-response curves established with this purified material showed that it exerted its effects on spinal cord and on sympathetic neuron cultures in the same range of concentration. Thus, these results suggest that the same macromolecule is involved in the regulation of neurotransmitter phenotype in both types of cultures despite their different embryological origins.     

Oxygen radical-induced neurotoxicity in spinal cord neuron cultures

The neurotoxic effects of oxygen radical on spinal cord neuron cultures derived from fetal mouse have been studies. Oxygen radicals, superoxide radical and hydrogen peroxide, were generated by adding xanthine oxidase and hypoxanthine in the culture medium. Exposure of neurons to this oxygen radical generating system resulted in a significant cell death and decrease of choline acetyltransferase (ChAT) activity in a time-dependent manner in spinal cord neuron cultures. The decrease in cell viability and ChAT enzyme activity induced by the oxygen radicals was blocked by scavengers such as superoxide dismutase (SOD), catalase, and tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a metal chelator. Antagonista of the N-methyl-D -aspartate (NMDA) receptor, including MK801 (a noncompetitive NMDA antagonist), D -2-amino-5-phosphonovaleric acid (APV) (a competitive NMDA antagonist), and 7-chlorokynurenic acid (an antagonist at the glycine site associated with the NMDA receptor), similarly blocked oxygen radical-induced decrease in cell viability and ChAT activity in spinal cord neuron cultures. These results indicate that both oxygen radicals and excitotoxic amino acids were involved in the oxidant-ititiated neurotoxicity of spinal cord neurons. © 1994 Wiley-Liss, Inc.     

Androgen action in fetal mouse spinal cord cultures: metabolic and morphologic aspects

Morphologic and metabolic aspects of androgen effects on the developing spinal cord were studied in organotypic cultures of the E13 (embryonic day 13) fetal mouse lumbosacral spinal cord, maintained as either hemisected, homologous explant pairs co-cultured with bulbocavernosus muscle (morphologic studies), or as whole cross-sectional segments without muscle in which the nutrient medium was supplemented with muscle extract (metabolic studies). Metabolic studies demonstrated the total absence of aromatase activity. 5 alpha-Reductase activity, on the other hand, increased differentially in a segment-dependent manner in spinal cord explants from 0 to 35 days in culture, suggesting regional differences in the utilization of testosterone and its 5 alpha-reduced metabolites. In all studies, spinal cord explants showed androgen-dependent increases in neurite outgrowth, although this was most pronounced in spinal cord-muscle co-cultures. These results indicate that androgens per se affect very early development throughout the entire lumbosacral spinal cord, and that this influence is not restricted to those segments reported to be sexually dimorphic in the adult.     

Growth inhibitory effects of a mu opioid on cultured cholinergic neurons from fetal rat ventral forebrain, brainstem, and spinal cord

Cholinergic pathways play a role in respiration in the mammalian brain, and agents that affect respiratory function such as opioid peptides might have positive or negative neurotrophic effects during the development of these cholinergic connections. Rat fetal nerve cell cultures from developmental stages E14-E18 were established in 96-well plates from ventral forebrain (VFB), an area rich in cholinergic neurons, and from brainstem and rostral spinal cord, areas where respiratory control systems and cholinergic neurons co-exist. High affinity 3H-choline uptake was highest in E14 VFB cultures and decreased to 20% of this value by E16 and E18. Choline uptakes in E14 brainstem and spinal cord were only 20% and 13%, respectively, of E14 VFB uptake. A mu opioid receptor agonist, d-ala2-mePhe4-gly(ol)5]-enkephalin (DAMGO), was tested for its effect on somal area and neurite outgrowth in E16 cultures. Cholinergic neurons were identified by immunostaining with choline acetyltransferase antibody. DAMGO (10(-8) M) significantly decreased somal area in VFB cultures and spinal cord, but had no effect on somal area in brainstem. Naltrexone (10(-6) M) reversed this inhibition. Spinal cord cell neurite outgrowth was inhibited by DAMGO, and this inhibition was reversed by naltrexone. DAMGO had no significant effect on neurite length in VFB. Brainstem neurite length was paradoxically increased by both DAMGO and naltrexone. It was concluded that mu-selective opioid peptides inhibit growth of cultured cholinergic neurons in VFB and spinal cord, but not in the brainstem. There was no evidence for endogenous opioid activity in either VFB or spinal cord cultures.     

Cytotoxicity by matrix metalloprotease-1 in organotypic spinal cord and dissociated neuronal cultures

Extracellular matrix (ECM) proteins, including collagens and laminins, are critical to the structure of the neuronal synapse and may also be involved in cell survival. In the present study, we therefore examined the possibility that select ECM degrading proteins might be toxic to organotypic spinal cord and dissociated neuronal cultures. Of those proteins tested, including MMP-1, -7, and -9, we observed that MMP-1 was toxic to spinal cord cultures as determined by release of lactic acid dehydrogenase as well as uptake of propidium iodide. Pretreatment of cell cultures with 50 microM alpha-tocopherol partially reversed these effects. We also observed that MMP-1 was toxic to human neurons grown in dissociated cultures and that increased amounts of MMP-1 were released by astrocytes following their stimulation with IL-1beta. These results suggest that further studies may be warranted to determine whether MMP-1 contributes to neurodegenerative conditions in which activated astrocytes may play a role.     

Neuronal aggregation and neurotransmitter regulation: partial purification and characterization of a membrane-derived factor

Cell membrane contact induces marked differential changes in neurotransmitter expression. In cultures of virtually pure dissociated sympathetic neurons, when such contact is provided by either high cell densities or addition of membranes derived from specific tissues, there is a marked increase in cell-specific content of substance P and de novo induction of choline acetyltransferase. To identify molecular mechanisms underlying regulation of transmitter expression by neuronal aggregation and membrane contact, we have begun to isolate and characterize a membrane-associated factor responsible for stimulation of choline acetyltransferase activity. The factor was found in substantial quantities in membranes from adult rat spinal cord as well as from sympathetic and sensory ganglia. Ionic mechanisms were employed to extract transmitter-inducing activity from spinal cord membranes in soluble form. The solubilized factor was then partially purified by ion exchange and gel filtration chromatography. It appears to be an extrinsic (non-integral) protein with an apparent molecular weight of 27. It is inactivated by trypsin and chymotrypsin, but is only moderately sensitive to heat inactivation, retaining activity at 60 degrees C but not at 90 degrees C. Neuronal perikaryal contact via aggregation represents a critical mechanism by which neurons themselves may influence phenotypic expression. Membrane localization of the factor provides a means by which cell contact may regulate transmitter expression.     

Isolation of neural stem cells from the spinal cords of low temperature preserved abortuses

In the present study, we show that neural stem cells can be obtained from the spinal cords of low temperature preserved abortuses. Fourteen weeks old abortuses were stored in a refrigerator at 4 degrees C for 2 h, 6 h and 12 h before use. Neural stem cells were isolated from cervical cord, thoracic cord and lumbar/sacral cord separately and induced to differentiate with fetal bovine serum. Clonal culture was carried out to demonstrate that the isolated cells met the standard of stem cells. Fluorescent immunocytochemistry was used to examine the expression of neural stem cell marker (nestin), neuronal marker (MAP2), astrocyte marker (GFAP) and cholinergic marker (ChAT). The stem cells in different cultures were compared. As a result, neural stem cells were obtained from all the spinal cord segments with different postmortem intervals. The lumbar/sacral cord cultures gave rise to the most abundant primary neurospheres. When the preservation was prolonged to 12 h, the number of primary neurospheres decreased sharply. Neurospheres in all cultures showed nestin positive immunoreactivity and could yield astrocytes and neurons including cholinergic neurons in differential cultures. The clonal formation and phenotype capacity were similar in all cultures. In conclusion, spinal neural stem cells can be isolated from low temperature preserved abortuses and represent an alternative source for both experimentation and potential therapeutic uses.     

Ginkgo biloba leaf extract effects on inducible nitric oxide synthase, Bcl-2, and Bax expression in rat models of spinal cord injury★

BACKGROUND:Ginkgo biloba leaf extract exhibits neuroprotective effects in spinal cord injury. However, the mechanisms of action remain unclear. OBJECTIVE: To investigate inducible nitric oxide synthase (iNOS) and Bcl-2/Bax expression in the injured spinal cord, and to explore the neuroprotective mechanisms of ginkgo biloba leaf extract in rats with spinal cord injury. DESIGN, TIME AND SETTING: The randomized, controlled, cell molecular biology experiment was performed at Soochow University, China from March...     

Ginkgo biloba leaf extract effects on inducible nitric oxide synthase, Bcl-2, and Bax expression in rat models of spinal cord injury★

BACKGROUND: Ginkgo biloba leaf extract exhibits neuroprotective effects in spinal cord injury. However, the mechanisms of action remain unclear. OBJECTIVE: To investigate inducible nitric oxide synthase (iNOS) and Bcl-2/Bax expression in the injured spinal cord, and to explore the neuroprotective mechanisms of ginkgo biloba leaf extract in rats with spinal cord injury. DESIGN, TIME AND SETTING: The randomized, controlled, cell molecular biology experiment was performed at Soochow University, China from March 2007 to March 2008. MATERIALS: A total of 120 healthy, adult Sprague Dawley rats were selected for this study. Rat models of moderate acute thoracic (T9) spinal cord injury were established using the modified Allen method. Shuxuening injection was obtained from Zhenbaodao Pharmaceutical Co., Ltd., China. Methylprednisolone was purchased from North China Pharmaceutical Co., Ltd. METHODS: All rats were equally and randomly divided into four groups. Only the spinal cord was exposed in the sham operation group rats. In the trauma group, rats were not treated with drugs following spinal cord injury. Rats in the hormone group were intraperitoneally injected with 30 mg/kg methylprednisolone following spinal cord injury. Rats in the ginkgo biloba leaf extract group were intraperitoneally infused with a 1.0 mL/kg Shuxuening injection per day. MAIN OUTCOME MEASURES: At 1 hour, as well as 1, 3, 5, 7, and 14 days after spinal cord injury, iNOS- and Bcl-2/Bax-positive cells were quantified with immunohistochemistry. Pathological changes were detected using hematoxylin-eosin staining under an optical microscope. RESULTS: Spinal cord injury in the ginkgo biloba leaf extract and hormone groups was milder compared with the trauma group. Demyelination was significantly ameliorated and the necrotic cavity was obviously reduced in the injured spinal cord of rats in the ginkgo biloba leaf extract and hormone groups at each time point. iNOS expression was increased in the injured spinal cord, and reached a peak at 5 days. The number of iNOS-positive cells was lower in the ginkgo biloba leaf extract and hormone groups compared with the trauma group (P < 0.05-0.01). The number of iNOS-positive cells was lower in the ginkgo biloba leaf extract group compared with the hormone group at 7 and 14 days after spinal cord injury (P < 0.05). Bcl-2 expression reached a peak at 3 days, and Bax expression reached a peak at 5 days following rat spinal cord injury. Bcl-2 expression was increased, but Bax expression was decreased in the ginkgo biloba leaf extract and hormone groups compared with the trauma group (P < 0.05-0.01). Bcl-2 expression was greater, but Bax expression was reduced in the ginkgo biloba leaf extract group compared with the hormone group at 7 and 14 days after spinal cord injury (P < 0.05). CONCLUSION: Ginkgo biloba leaf extract exhibits neuroprotective effects by upregulating Bcl-2 expression, downregulating Bax expression, and significantly inhibiting high expressions of iNOS in the injured spinal cord. The neuroprotective effects of ginkgo biloba leaf extract are greater compared with methylprednisolone at 1 week after spinal cord injury. Key Words: apoptosis; Bcl-2/Bax; ginkgo biloba leaf extract; inducible nitric oxide synthase; methylprednisolone; neuroprotection; spinal cord injury     

Schwann cells transplanted into normal and X-irradiated adult white matter do not migrate extensively and show poor long-term survival

Although Schwann cells are able to enter the central nervous system (CNS) when the integrity of the glia limitans is disrupted, their ability to migrate through intact CNS remains unclear. We have addressed this issue by transplanting lacZ-labeled Schwann cells into normal adult spinal cord white matter, and into X-irradiated spinal cord (an environment that, unlike normal spinal cord, permits the migration of transplanted oligodendrocyte progenitors). Schwann cell cultures, obtained from neonatal rat sciatic nerve and expanded using bovine pituitary extract and forskolin, were transfected by repeated exposure to retroviral vectors encoding the Escherichia coli lacZ gene. The normal behavior of the transduced cells was confirmed by transplantation into a nonrepairing area of demyelination in the spinal cord, where they formed myelin sheaths around demyelinated axons. A single microliter containing 4 x 10(4) cells was then transplanted into unlesioned normal and X-irradiated white matter of the spinal cord of adult syngeneic rats. One hour after injection, blue cells were observed as a discrete mass within the dorsal funiculus with a longitudinal distribution of 2-3 mm, indicating the extent of passive spread of the injected cells. At subsequent survival times (1, 2, and 4 weeks posttransplantation) blue cells had a distribution that was no more extensive than that seen 1 h after transplantation. However, the number of Schwann cells declined with time following transplantation such that at 4 weeks there were few surviving Schwann cells in both X-irradiated and nonirradiated spinal cord. These results indicate that transplanted Schwann cells do not migrate extensively and show poor long-term survival when introduced into a normal CNS environment.