Cultures Of Spinal Cord Explants Research Articles

Antibodies produced by clonally expanded plasma cells in multiple sclerosis cerebrospinal fluid cause demyelination of spinal cord explants.

B cells are implicated in the etiology of multiple sclerosis (MS). Intrathecal IgG synthesis, cerebrospinal fluid (CSF) oligoclonal bands and lesional IgG deposition suggest a role for antibody-mediated pathology. We examined the binding of IgG1 monoclonal recombinant antibodies (rAbs) derived from MS patient CSF expanded B cell clones to central nervous system (CNS) tissue. MS rAbs displaying CNS binding to mouse and human CNS tissue were further tested for their ability to induce complement-mediated tissue injury in ex vivo spinal cord explant cultures. The staining of CNS tissue, primary human astrocytes and human neurons revealed a measurable bias in MS rAb binding to antigens preferentially expressed on astrocytes and neurons. MS rAbs that recognize myelin-enriched antigens were rarely detected. Both myelin-specific and some astrocyte/neuronal-specific MS rAbs caused significant myelin loss and astrocyte activation when applied to spinal cord explant cultures in the presence of complement. Overall, the intrathecal B cell response in multiple sclerosis binds to both glial and neuronal targets and produces demyelination in spinal cord explant cultures implicating intrathecal IgG in MS pathogenesis.

The astroglial cell that guides nerve fibers from growth cone to synapse in organotypic cultures of the fetal mouse spinal cord

We present electron microscopic and autoradiographic studies done using organotypic cultures of spinal cord explants excised from 15 days of gestation mouse embryos. Nerve fibers growing from the spinal cord explant carry at their tips immature mitotic astrocytic cells that lead their growth cones. These glial cells divide only during the active phase of neuronal growth, and correspond ultrastructurally to radial glia. They provide a specific cellular substrate for neuronal growth. Some growth cones form axoglial synapses with smooth membranes of immature glial cells. In contrast, maturing glial cells sprout cytoplasmic processes that tightly wrap individual growth cones and effectively arrest their growth. Next, the processes gather nerve endings into islets and nerve fibers into bundles. After internalizing nerve endings, the glial processes withdraw, bringing the endings into contact with each other. The direct neuronal appositions lead to the transformation of growth cones into presynaptic endings, signaled by their collection of presynaptic vesicles. Clustering of the vesicles at presynaptic axoglial or axodendritic membranes indicates the onset of synaptogenesis-completed by differentiation of spinous and compound synapses. Concomitant with the progress of synaptogenesis, astrocytic investment within the neuropil progressively diminishes. The differentiating astrocytic processes show secretory and tethering activity toward nerve fibers and their endings. Our observations demonstrate that astroglial cells-depending on their developmental stage-first promote and then arrest neuronal growth, and induce synaptogenesis. Thus, at any time, the growing nerve fibers are not only supported but also controlled by the astroglial cells.

1,2-Dioctanoyl-s,n-glycerol-induced activation of protein kinase C results in striking, but reversible growth cone shape changes and an accumulation of f-actin and serine 41-phosphorylated GAP-43 in the axonal process

In spinal cord explant cultures from embryonic chicken (E7) we found that both a long-time downregulation of PKC by phorbol-12,13-dibutyrate (PDBu) and an inhibition of PKC by RO-31-8220 strongly reduce neurite outgrowth. Unlike this, in the presence of a high dose of 1,2-dioctanoyl-s,n-glycerol (diC8, 60 µM), PKCα,β isoforms are not downregulated, but neurite outgrowth appeared reduced up to 37%. A low dose of diC8 (5 µM), however, was found to stimulate neurite outgrowth up to 25%. Using this tissue culture system as well as neuronal cell culture we then studied the effects of diC8 on the shapes and actin-based motility of distal axonal processes and growth cones as well as on the spatial distribution of f-actin and serine 41-phosphorylated GAP-43 (neuromodulin, B50). High-resolution microscopy showed that addition of 30-60 µM diC8 leads within a few minutes to a retraction of filopodia and to an increased protrusion of lamellipodia followed by the formation of club-shaped dense growing tips, axonal varicosities, and a cessation of any actin dynamics. These striking shape changes are completely reversed after replacement of the medium by drug-free medium. Presence of cytochalasins and a panel of different PKC inhibitors prevent or respectively attenuate the diC8 effects. Immuno- and phalloidin-staining confirmed that in control neurons f-actin and serine 41-phosphorylated GAP-43 are confined to and enriched in the growth cones. In parallel with diC8-induced shape changes there is an accretion of f-actin and serine 41-phosphorylated GAP-43 in the entire axonal processes and the rounded growing tips. With respect to the fundamental role of the actin dynamics in growth cone steering and neuronal pathfinding, the data supports the view that in neurons local PKC-regulated phosphorylation of GAP-43 may represent an important mechanism to transduce guiding signals into actin-cytoskeletal responses mediating directed axonal growth.

An improved cryo-jet freezing method.

A new cryo-jet freezing apparatus is described that is easy to use and gives good results using a propane-butene mixture (3:1). Our use of the freezer in the study of mouse spinal cord explant cultures is discussed. At the tissue surface, the quality of tissue preservation from freezing, followed by freeze substitution, rivals that of conventional electron microscopic methods. Certain intracellular structures are better visualized using our methods. There is no evidence of the tissue being distorted by the cryogen jet when the freezer is operated correctly. A new freeze substitution device is also discussed.

Study of platelet-mediated neurotoxicity in rat brain.

The mechanism of ischemic neuronal injury is not fully resolved. The present view is that vascular occlusion per se does not fully account for the extent of neurological dysfunction. We previously hypothesized that platelet secretory products might contribute to neuronal injury in the central nervous system. Our preliminary studies using organotypic rat spinal cord cultures exposed to human platelet and its secretory products revealed that platelet products had neurotoxic properties. Further studies, using the same methods, were conducted, with the addition of several refinements such as use of gel-filtered platelets (as opposed to washed platelets) and adding additional relevant controls including platelet membranes, red blood cells, and washed rat platelets. Exposure of spinal cord explant cultures to platelet secretory products resulted in reduced number of neurons per ventral horn compared with control. Our findings suggest that platelet secretory products have neurotoxic properties. This effect was seen with platelet secretion obtained from physiological platelet concentrations. It appears possible that more abundant release of platelet products at the site of thrombus formation could have pathological significance in vivo.

Gamma-Aminobutyric acid receptors visualized in spinal cord cultures by [3H]muscimol autoradiography.

gamma-Aminobutyric acid (GABA) receptors were visualized in mouse spinal cord explant cultures by [3H]muscimol autoradiography over certain small neurons of the dorsal horn grey matter, in the interneuronal neuropil of dorsal and ventral horns, and (rarely) in small clusters on processes of anterior horn cells. Specificity was indicated in control experiments by inhibition of binding by [3H]muscimol after pretreatment with GABA or a GABA analogue, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, isoxazole 16), or with receptor antagonists bicuculline and picrotoxin. Unlabeled muscimol exchanged effectively with [3H]muscimol and produced autoradiographic label in locations indistinguishable from those found in experiments with [3H]muscimol alone. Pretreatment with the GABA uptake and transport inhibitors (-)-nipecotic acid and guvacine did not affect binding with [3H]muscimol. These experiments indicate that explant culture systems can be used for demonstration of functional receptors.

Neurotrophic effects on the electrical properties of cultured muscle produced by conditioned medium from spinal cord explants

The passive electrical properties of cultured chick skeletal muscle are significantly altered when the muscle is co-cultured with spinal cord explants. A reduced transverse membrane resistance appears to be responsible for the smaller values of input resistance, space constant, and time constant observed in co-cultures relative to those observed in pure muscle cultures. In this report, we show that neuromuscular junctions are not required in order to observe this neurotrophic effect because medium from spinal cord explant cultures is capable of producing the same reduction in transverse membrane resistance as the co-culturing of spinal cord explants with muscle. Control medium from liver explant cultures has no effect on muscle passive electrical properties. These results indicate that a trophic substance which is capable of regulating the electrical properties of excitable cells is released into the culture medium by spinal cord explants.