M. Sensenbrenner

Brain basic fibroblast growth factor stimulates the proliferation of rat neuronal precursor cells in vitro

Neuronal cells from cerebral hemispheres of 13‐day‐old rat embryos were grown in a serum‐free culture medium for 48 h, 4 or 8 days. The neuronal precursor cells proliferate for 5 days. The addition of bovine brain basic fibroblast growth factor stimulates their proliferation as determined by measurement of [121I]‐iododeoxyuridine incorporation and by autoradiographic analysis after [3H]thymidine incorporation. The proliferating responsive cells were characterized as neurons by immunostaining against neurofilament proteins. Five other growth factors tested were without effect on the proliferative activity of these neuronal cells. The present results show that bFGF is mitogenic in vitro for rat neuronal precursor cells of the central nervous system.

Purification of two astroglial growth factors from bovine brain.

Growth factor Cell culture Glial cell

Basic fibroblast growth factor (bFGF) injection activates the glial reaction in the injured adult rat brain

Reactive gliosis is a reaction of glial cells to trauma which is characterized by a phenotypic modification of astrocytes, as well as by a proliferation and a migration of some of these cells to form a glial scar. This scar is currently considered as a physical impediment to neuronal regrowth but it may also be involved in wound healing since the astrocytes beside microglia play a phagocytic role in the clearance of post-traumatic debris. Growth factors are released in the area of the injury and at least some of them could be involved in gliosis. In order to test directly this possibility, we have injected one of them, the basic fibroblast growth factor (bFGF), into several brain areas (cortex, striatum, hippocampus or corpus callosum) of adult 2-month-old rats in the absence of lesion. A glial reaction was observed after 3 days and was maximum after 7 days. It was characterized by an increase in astrocyte proliferation and in glial fibrillary acidic protein (GFAP) expression, resulting in a higher number of GFAP-positive cells per surface unit, and by an increase in the size and branching of the astroglial processes. The GFAP mRNA levels were also strongly increased following the bFGF injection. These effects resemble the reactive gliosis observed after lesion and suggest that bFGF is actually involved in the triggering of glial reactions which follow brain injury. In further experiments, bFGF was injected in the site of electrolytic lesions made in the same various parts of the brain. These injections did not increase significantly the normal reactive gliosis induced by the lesion alone, but it accelerated some of the effects. It also resulted in a higher labeling index and GFAP mRNA levels were strongly enhanced after a 3-day-post-operative delay. This last observation strengthens the idea that one of the main factors driving the astrogliosis is the bFGF normally released in and around the site of the lesion.

Expression of two neuronal markers, growth-associated protein 43 and neuron-specific enolase, in rat glial cells

Abstract Recent studies have revealed that proteins such as growth-associated protein 43 (GAP-43) and neuron-specific enolase (NSE), believed for many years to be expressed exclusively in neurons, are also present in glial cells under some circumstances. Here we present an overview of these observations. GAP-43 is expressed both in vitro and in vivo transiently in immature rat oligodendroglial cells of the central nervous system, in Schwann cell precursors, and in non-myelin-forming Schwann cells of the peripheral nervous system. GAP-43 mRNA is also present in oligodendroglial cells and Schwann cells, indicating that GAP-43 is synthesized in these cells. GAP-43 is also expressed in type 2 astrocytes (stellate-shaped astrocytes) and in some reactive astrocytes but not in type 1 astrocytes (flat protoplasmic astrocytes). These results suggest that GAP-43 plays a more general role in neural plasticity during development of the central and peripheral nervous systems. NSE enzymatic activity and protein and mRNA have been detected in rat cultured oligodendrocytes at levels comparable to those of cultured neurons. NSE expression increases during the differentiation of oligodendrocyte precursors into oligodendrocytes. In vivo, NSE protein is expressed in differentiating oligodendrocytes and is repressed in fully mature adult cells. The upregulation of NSE in differentiating oligodendrocytes coincides with the formation of large amounts of membrane structures and of protoplasmic processes. Similarly, NSE becomes detectable in glial neoplasms and reactive glial cells at the time when these cells undergo morphological changes. The expression of the glycolytic isozyme NSE in these cells, which do not normally contain it, could reflect a response to higher energy demands. This expression may also be related to the neurotrophic and neuroprotective properties demonstrated for this enolase isoform. NSE activity and protein and mRNA have also been found in cultured rat type 1-like astrocytes but at much lower levels than in neurons and oligodendrocytes. Thus GAP-43 and NSE should be used with caution as neuron-specific markers in studies of normal and pathological neural development.

Thrombin is a potent mitogen for rat astroblasts but not for oligodendroblasts and neuroblasts in primary culture

Astroblasts from brain of newborn rat can survive and even proliferate to some extent in a chemically defined medium containing no other growth factor than insulin, providing they are grown first in the presence of fetal calf serum for at least 4 days (Weibel et al., 1984, Int. J. devl Neurosci, 2, 355–366). We found that thrombin is a potent mitogen for these cells, in vitro. The mitogenic activity of thrombin for astroblasts can be compared to that of the astroglial growth factor 2 (AGF2), also known as basic fibroblast growth factor (bFGF), which was, up to now, the most active factor on astroblasts. However, in contrast to the bFGF, thrombin does not modify significantly the morphology of the cells and their synthesis of glutamine synthetase, an astroglial marker in rat brain. Some other proteases are also able to stimulate the proliferation of astroblasts, but to a lesser extent than thrombin. Thrombin does not stimulate the proliferation of oligodendroblasts from newborn rat and of neuroblasts from 13‐day‐old rat embryo. These results suggest that in the central nervous system thrombin might play a role in the induction of astrocyte proliferation after brain injury.

Behaviour of neuroblasts in the presence of glial cells, fibroblasts and meningeal cells in culture☆

Abstract Dissociated nerve cells from 7-day-old chick embryo cerebral hemispheres were cultivated on plastic surfaces, astroblast layers, fibroblast layers and meningeal cell layers. The cell suspensions obtained by mechanical dissociation and plated on these layers contained primarily neuroblasts. The neuroblasts cultured on astroblast layers behaved differently from those cultured on fibroblast or on meningeal cell layers. They adhered within 2 h to the preformed astroblast monolayers and remained scattered over it. In contrast, in the two other cases, the neuroblasts formed floating aggregates which adhered to the layers only after 24 h. Neuroblasts behaved on monolayers made of fibroblasts or meningeal cells as on plastic surfaces. The neuronal cells grown on astroblast layers were much more differentiated than those plated on plastic or on the two other layers studied. After 2–3 weeks of culture the neurons were large and the fibres were longer, thicker and more ramified. However, the fibroblast and the meningeal cells enhanced slightly the growth of the neuroblasts relative to plastic surfaces. These results support the possibility of specific interactions between astroblasts and neuroblasts.

Rat brain glial cells in culture: Effects of brain extracts on the development of oligodendroglia-like cells

Abstract Cells dissociated from brains of newborn rats and grown on plastic surfaces develop into a glial culture, composed of at least three morphologically different cell types. The predominating cell type consists of astroglial cells, which form a monolayer. The second cell type, rarely observed, consists of ependymal cells. The third type consists of small cells scattered upon the astroglial layer. After 3 weeks very few of these small cells remain and the glial culture develops into a more homogenous appearance, mainly composed of astroglial cells. The effects of various brain extracts on the development of the small cell type was investigated. The treatment by either rat or chick brain extracts caused an increase in the number of these cells, which were seen to form clusters. Brain extracts from older animals have a stronger effect than brain extracts from younger animals. These data suggest that factors contained in the brain during and after the myelination period influence the development of this cell type in dissociated cultures. The small cells were tentatively identified as oligodendroglial cells by ultrastructural and histochemical criteria. They did not contain acetylcholinesterase (AChE) and did not bind tetanus toxin. Furthermore, they did not contain glial fibrillary acidic (GFA) protein. But carbonic anhydrase II (CAII) was found in them at light and electron-microscopical level. CAII was found to be localized essentially on the plasmic membrane and on the endoplasmic reticulum of these cultured oligodendroglial cells.

Isolation of a glial maturation factor from beef brain

Soluble brain extracts from chick embryos and from newborn rats stimulate the morphological maturation of chick neuronal and glial cells in mixed cultures [ 1,2]. Chick brain extracts induce morphological changes in pure astroglial cells in culture derived from brains of newborn rats, with an increase of the level of the glial-specific protein SlOO [3]. In these rat glial structures, brain extracts prepared from postnatal rats and adult beef elicit the same morphological and biochemical stimulatory effects [4] as well as enhance glial cell proliferation. Similar changes induced by brain extracts from rats and pigs in different astroglial cultures have been reported [5-71. It is not known whether the different effects, i.e., morphological changes, enhanced proliferation and biochemical maturation are induced by a single active factor or by several different factors. Therefore, we purified a crude fraction from beef brain extract and could demonstrate that the isolated factor induces the three events simultaneously.

Influence of meningeal cells on the proliferation and maturation of rat neuroblasts in culture

SummaryNeuronal cells were obtained by dissociating cells from the cerebral hemispheres of rat embryos (10 to 17-day-old), either cleaned entirely or only partially of their meningeal membranes. These cells were seeded on poly-lysine-coated Petri dishes in serum-containing medium. The cultures most enriched in neuronal cells were obtained from brains of 13- to 15-day-old embryos and after 2 h, the culture medium was switched to Dulbeccos modified Eagles medium, without serum, supplemented with the N1 supplements as described by Bottenstein et al. (1980). The proliferation of neuroblasts from 13-day-old embryos in the presence or absence of meningeal cells was studied by using a combination of tritiated thymidine autoradiography and immuno-staining against neurofilament proteins. The neuroblasts seem to proliferate during the first 3 days. The proliferative activity was further enhanced in the presence of meningeal cells. The glioblasts multiply only after a period of one week in culture conditions as observed here. The subsequent development of the neuroblasts was followed over a period of 4 weeks and the ultrastructural appearance of these cells was investigated at 2 and 3 weeks. In the presence of meningeal cells, many neurons, intensely stained for neurofilament proteins, survived for 21 days, while in control cultures they underwent massive degeneration after 2 weeks. Synapses with numerous clear vesicles were abundant in cultures grown under the influence of meningeal cells; they were rare and possessed few vesicles in control cultures. The data indicate that meningeal cells affect the proliferation and maturation of rat neuroblasts in culture.

RESPIRATION BY CULTIVATED ASTROCYTES AND NEURONS FROM THE CEREBRAL HEMISPHERES

‐Rates of oxygen uptake were measured in chick and/or rat astrocytes and neuronal cells cultivated for 2–4 weeks in Falcon flasks or Rose chambers. All the preparations were found to have respiratory rates between 0.4 and 0.8 × 10−5μl/h O2 per cell. Based upon measurements of cell diameters these values were recalculated to about 570 μmol/g wet wt. for the neuronal cells and 130 μmol/g wet wt. for the glial cells. The results are compared with previous data of oxygen uptake by neurons and glial cells separated by other procedures.