Embryonic Rat Spinal Cord Research Articles

Achyranthes bidentata polypeptide k enhances the survival, growth and axonal regeneration of spinal cord motor neurons in vitro.

Achyranthes bidentata polypeptide k (ABPPk), a powerful active component from a traditional Chinese medicinal herb-Achyranthes bidentata Bl., has exhibited promising neuroprotective activity due to its multiple-targeting capability. However, the effect of ABPPk on the survival, growth and axonal regeneration of spinal cord motor neurons remains unclear. Here, a modified method, which is more optimized for embryonic cells in ambient carbon dioxide levels, was used for acquisition of rat embryonic spinal cord motor neurons with high survival and purity. ABPPk concentration-dependently enhanced the neuronal viability and promoted the neurite outgrowth. Co-culture of motor neurons and skeletal myocytes model indicated that ABPPk enhanced the neuromuscular junction development and maturation. A microfluidic axotomy model was further established for the axonal disconnection, and ABPPk significantly accelerated the axonal regeneration of motor neurons. Furthermore, we demonstrated that the upregulation of three neurofilament protein subunits in motor neurons might be relevant to the mechanisms of the growth-promoting effect of ABPPk. Our findings provide an experimental and theoretical basis for the development of ABPPk as a potential application in the development of treatment strategy for nerve injury diseases.

CCP1, a Tubulin Deglutamylase, Increases Survival of Rodent Spinal Cord Neurons following Glutamate-Induced Excitotoxicity.

Microtubules (MTs) are cytoskeletal elements that provide structural support and act as roadways for intracellular transport in cells. MTs are also needed for neurons to extend and maintain long axons and dendrites that establish connectivity to transmit information through the nervous system. Therefore, in neurons, the ability to independently regulate cytoskeletal stability and MT-based transport in different cellular compartments is essential. Posttranslational modification of MTs is one mechanism by which neurons regulate the cytoskeleton. The carboxypeptidase CCP1 negatively regulates posttranslational polyglutamylation of MTs. In mammals, loss of CCP1, and the resulting hyperglutamylation of MTs, causes neurodegeneration. It has also long been known that CCP1 expression is activated by neuronal injury; however, whether CCP1 plays a neuroprotective role after injury is unknown. Using shRNA-mediated knock-down of CCP1 in embryonic rat spinal cord cultures, we demonstrate that CCP1 protects spinal cord neurons from excitotoxic death. Unexpectedly, excitotoxic injury reduced CCP1 expression in our system. We previously demonstrated that the CCP1 homolog in Caenorhabditis elegans is important for maintenance of neuronal cilia. Although cilia enhance neuronal survival in some contexts, it is not yet clear whether CCP1 maintains cilia in mammalian spinal cord neurons. We found that knock-down of CCP1 did not result in loss or shortening of cilia in cultured spinal cord neurons, suggesting that its effect on survival of excitotoxicity is independent of cilia. Our results support the idea that enzyme regulators of MT polyglutamylation might be therapeutically targeted to prevent excitotoxic death after spinal cord injuries.

Effect of differentiation, de novo innervation, and electrical pulse stimulation on mRNA and protein expression of Na+,K+-ATPase, FXYD1, and FXYD5 in cultured human skeletal muscle cells.

Denervation reduces the abundance of Na+,K+-ATPase (NKA) in skeletal muscle, while reinnervation increases it. Primary human skeletal muscle cells, the most widely used model to study human skeletal muscle in vitro, are usually cultured as myoblasts or myotubes without neurons and typically do not contract spontaneously, which might affect their ability to express and regulate NKA. We determined how differentiation, de novo innervation, and electrical pulse stimulation affect expression of NKA (α and β) subunits and NKA regulators FXYD1 (phospholemman) and FXYD5 (dysadherin). Differentiation of myoblasts into myotubes under low serum conditions increased expression of myogenic markers CD56 (NCAM1), desmin, myosin heavy chains, dihydropyridine receptor subunit α1S, and SERCA2 as well as NKAα2 and FXYD1, while it decreased expression of FXYD5 mRNA. Myotubes, which were innervated de novo by motor neurons in co-culture with the embryonic rat spinal cord explants, started to contract spontaneously within 7–10 days. A short-term co-culture (10–11 days) promoted mRNA expression of myokines, such as IL-6, IL-7, IL-8, and IL-15, but did not affect mRNA expression of NKA, FXYDs, or myokines, such as musclin, cathepsin B, meteorin-like protein, or SPARC. A long-term co-culture (21 days) increased the protein abundance of NKAα1, NKAα2, FXYD1, and phospho-FXYD1Ser68 without attendant changes in mRNA levels. Suppression of neuromuscular transmission with α-bungarotoxin or tubocurarine for 24 h did not alter NKA or FXYD mRNA expression. Electrical pulse stimulation (48 h) of non-innervated myotubes promoted mRNA expression of NKAβ2, NKAβ3, FXYD1, and FXYD5. In conclusion, low serum concentration promotes NKAα2 and FXYD1 expression, while de novo innervation is not essential for upregulation of NKAα2 and FXYD1 mRNA in cultured myotubes. Finally, although innervation and EPS both stimulate contractions of myotubes, they exert distinct effects on the expression of NKA and FXYDs.

Innervation and electrical pulse stimulation - in vitro effects on human skeletal muscle cells.

Contraction-induced adaptations in skeletal muscles are well characterized in vivo, but the underlying cellular mechanisms are still not completely understood. Cultured human myotubes represent an essential model system for human skeletal muscle that can be modulated ex vivo, but they are quiescent and do not contract unless being stimulated. Stimulation can be achieved by innervation of human myotubes in vitro by co-culturing with embryonic rat spinal cord, or by replacing motor neuron activation by electrical pulse stimulation (EPS). Effects of these two in vitro approaches, innervation and EPS, were characterized with respects to the expression of myosin heavy chains (MyHCs) and metabolism of glucose and oleic acid in cultured human myotubes. Adherent human myotubes were either innervated with rat spinal cord segments or exposed to EPS. The expression pattern of MyHCs was assessed by quantitative polymerase chain reaction, immunoblotting, and immunofluorescence, while the metabolism of glucose and oleic acid were studied using radiolabelled substrates. Innervation and EPS promoted differentiation towards different fiber types in human myotubes. Expression of the slow MyHC-1 isoform was reduced in innervated myotubes, whereas it remained unaltered in EPS-treated cells. Expression of both fast isoforms (MyHC-2A and MyHC-2X) tended to decrease in EPS-treated cells. Both approaches induced a more oxidative phenotype, reflected in increased CO2 production from both glucose and oleic acid. Novelty: Innervation and EPS favour differentiation into different fiber types in human myotubes. Both innervation and EPS promote a metabolically more oxidative phenotype in human myotubes.

Glucocorticoid-Induced Reductions of Myelination and Connexin 43 in Mixed Central Nervous System Cell Cultures Are Prevented by Mifepristone

Repeated stress induces systemic elevations in glucocorticoid levels. Stress is also associated with alterations in central nervous system astrocytes and oligodendrocytes that involve connexins and myelin proteins. Corticosteroid elevation seems a major factor in stress-induced neuropathology. Changes in astrocyte connexins and myelin components may be important mediators for the neurological effects of corticosteroid elevations. Two primary cell culture models, myelination culture from rat embryonic spinal cord (SC) or cerebral cortex (CC) consisting of neurons and glial cells (oligodendrocytes, microglia and astrocytes), and mixed astrocyte-and-oligodendrocyte culture prepared from postnatal rat CC, were used in this study. Cell cultures were treated with either vehicle, corticosterone (CORT) with or without glucocorticoid receptor antagonist mifepristone, or dexamethasone (DEX) during the period of in vitro myelination. Immunoreactivity of astrocyte connexin 43 (Cx43) and oligodendrocyte myelin basic protein (MBP), or the myelination index (co-localization of MBP and phosphorylated neurofilament) was determined by double immunofluorescent labeling. Oligodendrocyte morphology was evaluated by Sholl analysis. Prolonged exposure to CORT or DEX induced dose-dependent reduction of the myelination index, and of immunostaining for MBP and Cx43 in SC and CC myelination cultures, which was prevented by mifepristone. In glial cultures single CORT or DEX exposure caused shrinkage and simplification of/' MBP- or CNPase-positive oligodendrocyte processes. The results support that concurrent effects of glucocorticoids on myelination and astrocyte Cx43 immunoreactivity are mediated by glucocorticoid receptors and may partially account for the involvement of CNS glia in the pathological effects of prolonged stress.

Vascularization of the Damaged Nerve under the Effect of Experimental Cell Therapy.

Quantitative analysis of blood vessels in the distal segment of rat sciatic nerve after its ligation for 40 sec and subperineurial administration of mesenchymal stem cells or dissociated cells of rat embryonic spinal cord was carried our by immunohistochemical tracing of von Willebrand factor, a marker of endothelial cells of blood vessels. It was found that the number of blood vessels per unit area of the nerve trunk in 21 days after injury and administration of mesenchymal stem cells increased by more than 1.5 times in comparison with the control (damaged nerve). After administration of dissociated cells of the embryonic spinal cord, this effect was not observed. It is assumed that mesenchymal stem cells stimulate the growth of vessels of the damaged nerve via production of angiogenic factors.

Neuron/Oligodendrocyte Myelination Coculture.

Myelination cell culture systems areuseful tools for studying myelin biology and myelin-related disorders. Compared to a number of established protocols for dissociated pure oligodendrocyte (OL) culture, methods for myelination culture are limited. We recently developed a mixed neuron-glia coculture system that generates robust and efficient myelination. By optimizing cell culture conditions, dissociated neural progenitor cells from embryonic rat spinal cords develop into neurons and glial cells including profiles of oligodendrocyte (OL) lineage. Within 4weeks, OL progenitor cells (OPC) proliferate, differentiate into mature OLs, and myelinate axons. The formation of compact myelin sheath is confirmed by electron microscopy. For morphological analysis by light microscopy, cells grown on glass coverslips are fixed and immunostained for various myelin-related proteins, including those embedded within the myelin sheath and those clustered at the node of Ranvier. Myelinated axons can be quantified readily by either manual counting or ImageJ software. The culture system may also be used for electron microscopic analysis by slightly modifying the cell culture procedure.

Brain-derived Neurotrophic Factor Promotes Growth of Neurons and Neural Stem Cells Possibly by Triggering the Phosphoinositide 3-Kinase/ AKT/Glycogen Synthase Kinase-3β/β-catenin Pathway.

Brain-derived neurotrophic factor (BDNF) plays a crucial role in promoting survival and differentiation of neurons and neural stem cells (NSCs), but the downstream regulating mechanisms remain poorly understood. We investigated whether BDNF exerts its effect by triggering the phosphoinositide 3-kinase (PI3K), protein kinase B, PKB (AKT), glycogen synthase kinase-3β (GSK-3β) and β-catenin signaling pathway in cultured neurons and NSCs derived from the rat embryonic spinal cord. Immunocytochemistry was used to detect neuronal and NSCs characteristics. RT-PCR was used to detect PI3K/AKT/GSK3β/β-catenin pathway expression. Neurons and NSCs were successfully separated and cultured from Sprague-Dawley rat embryonic spinal cord and were respectively labeled using immunocytochemistry. Neuron-specific nuclear protein, neuronal class III β-tubulin, and neurofilament expression were detected in neurons; nestin, glial fibrillary acidic protein, microtubule-associated protein 2 and chondroitin sulfate glycosaminoglycan expression were detected in the NSCs. BDNF promoted significant neuronal growth (number, soma size, and average neurite length), as well as NSCs proliferation and differentiation, but BDNF antibody decreased neuronal growth and NSCs proliferation and differentiation. RT-PCR was used to detect changes in BDNF signal pathway components, showing that BDNF upregulated tropomyosin receptor kinase B, phosphoinositide 3-kinase (PI3K), AKT and β-catenin, but downregulated GSK-3β in the neurons and NSCs. BDNF antibody downregulated BDNF, tropomyosin receptor kinase B, PI3K, AKT, β-catenin and cellular-myelocytomatosis viral oncogene, but upregulated GSK- 3β, in the neurons and NSCs. Our findings suggested that BDNF contributed to neuronal growth and proliferation and differentiation of NSCs in vitro by stimulating PI3K/AKT/GSK3β/β-catenin pathways.

In Vitro Innervation as an Experimental Model to Study the Expression and Functions of Acetylcholinesterase and Agrin in Human Skeletal Muscle.

Acetylcholinesterase (AChE) and agrin, a heparan-sulfate proteoglycan, reside in the basal lamina of the neuromuscular junction (NMJ) and play key roles in cholinergic transmission and synaptogenesis. Unlike most NMJ components, AChE and agrin are expressed in skeletal muscle and α-motor neurons. AChE and agrin are also expressed in various other types of cells, where they have important alternative functions that are not related to their classical roles in NMJ. In this review, we first focus on co-cultures of embryonic rat spinal cord explants with human skeletal muscle cells as an experimental model to study functional innervation in vitro. We describe how this heterologous rat-human model, which enables experimentation on highly developed contracting human myotubes, offers unique opportunities for AChE and agrin research. We then highlight innovative approaches that were used to address salient questions regarding expression and alternative functions of AChE and agrin in developing human skeletal muscle. Results obtained in co-cultures are compared with those obtained in other models in the context of general advances in the field of AChE and agrin neurobiology.

Modelling FUS Mislocalisation in an In Vitro Model of Innervated Human Muscle.

Degeneration of distal axons and neuromuscular junctions is an early feature in the pathology of amyotrophic lateral sclerosis (ALS), which culminates in motor neuron loss due to axon retraction and muscle atrophy. The complex interactions in the pathogenesis of ALS between motor neurons, muscle cells and accompanying glia require an appropriate experimental model. Here, we have defined a co-culture model based on human myotubes innervated by neurons from embryonic rat spinal cord explants to investigate the pathology and treatment of ALS. This model was first characterised for endogenous expression and distribution of ALS-related proteins TDP-43 and FUS. Then, wild-type FUS and its mutants were introduced into these co-cultures to determine how FUS defects in nuclear transport modulate the pathological conditions. FUS-bearing plasmids were introduced by classical transfection and electroporation, as novel approaches to deliver plasmids into explants, and their cellular distributions were characterised. Endogenous nuclear expression of TDP-43 and FUS was observed in explants and myoblasts/myotubes. After transfection, wild-type FUS was expressed in nuclei of myoblasts, myotubes and explants, although with low transfection rates. Following successful electrotransfection into explants, the localisation of wild-type FUS was nuclear, and it was detected in neurons, astrocytes, Schwann cells and oligodendrocyte precursors, whereas the FUS∆Y, FUSY526A and FUSY526E mutants were cytoplasmic, and the FUSY526F mutant was nuclear and cytoplasmic. This co-culture model is applicable to the study of neuronal and non-neuronal cell contributions to ALS and other neurodegenerative diseases, and it can be used to investigate drug targets amenable to intervention.

Changes in Oligodendroglial and Microglial Cell Populations in the Embryonic Rat Spinal Cord Following Maternal Immune Activation

Epidemiological studies suggest that maternal immune activation (MIA) may contribute to the development of neurological disorders such as cerebral palsy, schizophrenia, autism, epilepsy and potentially, neurodegenerative diseases. These studies, and those in animal models, have suggested that the effects of MIA on the developing nervous system, and thus the longer term consequences for the offspring, may be spatially and temporally regulated. However, much work remains in order to elucidate fully the CNS regions, cell populations and timing of gestational vulnerability.The in vivo effects of MIA on cellular development in the spinal cord (SC) at embryonic day (E)12, 14 and 16 were investigated in this study. Dams received a single intraperitoneal injection of 50μg/kg lipopolysaccharide (LPS), 100 μg/kg LPS, or saline. Embryos were sacrificed either 5 hours post‐injection or, following their delivery, two weeks postnatally(P). Embryos and pups were fixed and frozen prior to cryosectioning at 15μm. Rostral, intermediate and caudal SC sections were immunofluorescently stained with Olig‐2 (an oligodendrocyte marker), Iba‐1 (a microglial marker), nestin (a radial glial marker) and reelin antisera.Olig2‐positive cells were counted in white matter (WM) and grey matter (GM) regions of the SC. Olig2‐positive cell number in E16 embryos decreased 5 hours following treatment with 100μg/kg LPS but not with treatment with 50μg/kg LPS. At E14 Olig2‐positive cells showed no change at 5 hours in response to 50μg/kg LPS treatment.Iba‐1+ cells decreased in GM and WM of the E16 SC 5Hr post treatment with 100μg/kg LPS.Reelin fluorescence intensity is decreased in the GM of the E16 SC 5 hours after 100μg/kg LPS treatment. Reelin fluorescence intensity is unaltered in E16 spinal cord in response to 50μg/kg LPS. Reelin appears to co‐localise with a specific population of neurons at E16 and P14.In conclusion, MIA appears to have an acute effect on the development of key cell populations at specific time points. Ongoing work is extending these analyses and will seek to identify how such perturbations affect ongoing SC development into the early postnatal period.Support or Funding InformationAnatomical SocietyGraduate Entry Medical School University of Limerick

Effects of electroacupuncture and the retinoid X receptor (RXR) signalling pathway on oligodendrocyte differentiation in the demyelinated spinal cord of rats.

ObjectivesIn spinal cord demyelination, some oligodendrocyte precursor cells (OPCs) remain in the demyelinated region but have a reduced capacity to differentiate into oligodendrocytes. This study investigated whether ‘Governor Vessel’ (GV) electroacupuncture (EA) would promote the differentiation of endogenous OPCs into oligodendrocytes by activating the retinoid X receptor γ (RXR-γ)-mediated signalling pathway.MethodsAdult rats were microinjected with ethidium bromide (EB) into the T10 spinal cord to establish a model of spinal cord demyelination. EB-injected rats remained untreated (EB group, n=26) or received EA treatment (EB+EA group, n=26). A control group (n=26) was also included that underwent dural exposure without EB injection. After euthanasia at 7 days (n=5 per group), 15 days (n=8 per group) or 30 days (n=13 per group), protein expression of RXR-γ in the demyelinated spinal cord was evaluated by immunohistochemistry and Western blotting. In addition, OPCs derived from rat embryonic spinal cord were cultured in vitro, and exogenous 9-cis-RA (retinoic acid) and RXR-γ antagonist HX531 were administered to determine whether RA could activate RXR-γ and promote OPC differentiation.ResultsEA was found to increase the numbers of both OPCs and oligodendrocytes expressing RXR-γ and RALDH2, and promote remyelination in the remyelinated spinal cord. Exogenous 9-cis-RA enhanced the differentiation of OPCs into mature oligodendrocytes by activating RXR-γ.ConclusionsThe results suggest that EA may activate RXR signalling to promote the differentiation of OPCs into oligodendrocytes in spinal cord demyelination.

Transplantation of Embryonic Spinal Cord Derived Cells Helps to Prevent Muscle Atrophy after Peripheral Nerve Injury.

Injuries to peripheral nerves are frequent in serious traumas and spinal cord injuries. In addition to surgical approaches, other interventions, such as cell transplantation, should be considered to keep the muscles in good condition until the axons regenerate. In this study, E14.5 rat embryonic spinal cord fetal cells and cultured neural progenitor cells from different spinal cord segments were injected into transected musculocutaneous nerve of 200–300 g female Sprague Dawley (SD) rats, and atrophy in biceps brachii was assessed. Both kinds of cells were able to survive, extend their axons towards the muscle and form neuromuscular junctions that were functional in electromyographic studies. As a result, muscle endplates were preserved and atrophy was reduced. Furthermore, we observed that the fetal cells had a better effect in reducing the muscle atrophy compared to the pure neural progenitor cells, whereas lumbar cells were more beneficial compared to thoracic and cervical cells. In addition, fetal lumbar cells were used to supplement six weeks delayed surgical repair after the nerve transection. Cell transplantation helped to preserve the muscle endplates, which in turn lead to earlier functional recovery seen in behavioral test and electromyography. In conclusion, we were able to show that embryonic spinal cord derived cells, especially the lumbar fetal cells, are beneficial in the treatment of peripheral nerve injuries due to their ability to prevent the muscle atrophy.

Abstracts from The 12th Symposium of the International Neurotrauma Society February 1-4, 2016 Cape Town, South Africa.

Abstracts from The 12th Symposium of the International Neurotrauma Society February 1-4, 2016 Cape Town, South Africa.

Generation of Functional Inhibitory Synapses Incorporating Defined Combinations of GABA(A) or Glycine Receptor Subunits

Fast inhibitory neurotransmission in the brain is mediated by wide range of GABAA receptor (GABAAR) and glycine receptor (GlyR) isoforms, each with different physiological and pharmacological properties. Because multiple isoforms are expressed simultaneously in most neurons, it is difficult to define the properties of individual isoforms under synaptic stimulation conditions in vivo. Although recombinant expression systems permit the expression of individual isoforms in isolation, they require exogenous agonist application which cannot mimic the dynamic neurotransmitter profile characteristic of native synapses. We describe a neuron-HEK293 cell co-culture technique for generating inhibitory synapses incorporating defined combinations of GABAAR or GlyR subunits. Primary neuronal cultures, prepared from embryonic rat cerebral cortex or spinal cord, are used to provide presynaptic GABAergic and glycinergic terminals, respectively. When the cultures are mature, HEK293 cells expressing the subunits of interest plus neuroligin 2A are plated onto the neurons, which rapidly form synapses onto HEK293 cells. Patch clamp electrophysiology is then used to analyze the physiological and pharmacological properties of the inhibitory postsynaptic currents mediated by the recombinant receptors. The method is suitable for investigating the kinetic properties or the effects of drugs on inhibitory postsynaptic currents mediated by defined GABAAR or GlyR isoforms of interest, the effects of hereditary disease mutations on the formation and function of both types of synapses, and synaptogenesis and synaptic clustering mechanisms. The entire cell preparation procedure takes 2–5 weeks.

Differentiation of Cholinergic Neurons in Rat Spinal Cord Under Conditions of Allotransplantation into a Peripheral Nerve and In Situ Development.

The method of ectopic transplantation of embryonic anlages of CNS allows studying histoblastic potencies of progenitor cells developing under conditions of changed microenvironment. Some progenitor cells in the transplants of rat embryonic spinal cord retained their ability to express choline acetyltransferase after transplantation into the sciatic nerve of adult animals. Comparative analysis of cholinergic neurons in the neurotransplants and neurons formed in rat spinal cord during normal ontogeny showed that choline acetyltransferase-positive cells after transplantation into the nerve reached morphological differentiation of motor neurons at later terms than cells developing in situ. They were scattered one by one and did not form nuclear nerve centers. We did not fi nd structures similar to presynaptic cholinergic buds typical of intact spinal cord near these cells throughout the observation period. Solitary cholinergic neurons survived in the transplants for 19 months.

Rapid transient isoform-specific neuregulin1 transcription in motor neurons is regulated by neurotrophic factors and axon-target interactions.

The neuregulins (NRGs) are a family of alternatively spliced factors that play important roles in nervous system development and disease. In motor neurons, NRG1 expression is regulated by activity and neurotrophic factors, however, little is known about what controls isoform-specific transcription. Here we show that NRG1 expression in the chick embryo increases in motor neurons that have extended their axons and that limb bud ablation before motor axon outgrowth prevents this induction, suggesting a trophic role from the developing limb. Consistently, NRG1 induction after limb bud ablation can be rescued by adding back the neurotrophic factors BDNF and GDNF. Mechanistically, BDNF induces a rapid and transient increase in type I and type III NRG1 mRNAs that peak at 4h in rat embryonic ventral spinal cord cultures. Blocking MAPK or PI3K signaling or blocking transcription with Actinomycin D blocks BDNF induced NRG1 gene induction. BDNF had no effect on mRNA degradation, suggesting that transcriptional activation rather than message stability is important. Furthermore, BDNF activates a reporter construct that includes 700bp upstream of the type I NRG1 start site. Protein synthesis is also required for type I NRG1 mRNA transcription as cycloheximide produced a super-induction of type I, but not type III NRG1 mRNA, possibly through a mechanism involving sustained activation of MAPK and PI3K. These results reveal the existence of highly responsive, transient transcriptional regulatory mechanisms that differentially modulate NRG1 isoform expression as a function of extracellular and intracellular signaling cascades and mediated by neurotrophic factors and axon-target interactions.

Neuronal regulation of expression of hydrogen sulfide-producing enzyme cystathionine β-synthase in rat spinal cord astrocytes

Cystathionine β-synthase (CBS), expressed in astrocytes, generates a gaseous neuromodulator, hydrogen sulfide (H2S) in the central nervous system (CNS). However, little is known about the regulatory mechanisms of astrocytic CBS expression and activity. This study evaluated the influence of neurons on astrocytic CBS expression by employing multiple culture systems. Substantial CBS expression was observed in the intact neonatal rat spinal cord, while CBS content was markedly reduced in an astrocyte-enriched culture prepared from the neonatal spinal cord. Immunofluorescence analysis confirmed the localization of spinal cord CBS in astrocytes, but not in neurons. Although CBS expression was weak in the embryonic rat spinal cord, enzyme levels were time-dependently increased in a neuron/astrocyte mixed culture originating from embryonic spinal cord. The reduced CBS expression in isolated neonatal astrocytes was restored by co-culture with embryonic neurons. Together with the observed CBS expression levels, H2S production was relatively low in astrocytes cultured alone, but was considerably higher in astrocytes cultured with neurons. These results indicate that neurons are essential for maintaining the expression and H2S-producing activity of astrocytic CBS in the rat spinal cord.

Effect of allotransplants containing dissociated cells of rat embryonic spinal cord on nerve fiber regeneration in a recipient.

Regeneration of nerve fibers in rat sciatic nerve was quantitatively assessed after injury (ligation) and injection of dissociated cells derived from embryonic spinal cord. A suspension of dissociated spinal cord cells from rat embryos was transplanted under the perineurium of a nerve trunk. After transplantation, bromodeoxyuridine-labeled precursor cells survived and retained the label for more than 2 months; some of these cells differentiated into NeuNpositive neurons. Analysis of semithin sections of the distal nerve segment from the recipient taken at a distance of 0.5 cm from the site of injury showed that transplantation of dissociated cells of embryonic spinal cord led to an increase in the number of myelinated nerve fibers in the recipient nerve.

A phenotypic culture system for the molecular analysis of CNS myelination in the spinal cord

Studies of central nervous system myelination lack defined in vitro models which would effectively dissect molecular mechanisms of myelination that contain cells of the correct phenotype. Here we describe a co-culture of purified motoneurons and oligodendrocyte progenitor cells, isolated from rat embryonic spinal cord using a combination of immunopanning techniques. This model illustrates differentiation of oligodendrocyte progenitors into fully functional mature oligodendrocytes that myelinate axons. It also illustrates a contribution of axons to the rate of oligodendrocyte maturation and myelin gene expression. The defined conditions used allow molecular analysis of distinct stages of myelination and precise manipulation of inductive cues affecting axonal–oligodendrocyte interactions. This phenotypic in vitro myelination model can provide valuable insight into our understanding of demyelinating disorders, such as multiple sclerosis and traumatic diseases such as spinal cord injury where demyelination represents a contributing factor to the pathology of the disorder.