Compartmentalized coculture of rat brain endothelial cells and astrocytes: a syngenic model to study the blood-brain barrier.

The specific structure of the blood-brain barrier (BBB) is based on the partnership of brain endothelial cells and astrocytes. In the last decade, cocultures of these two cell types have been developed as in vitro models. However, these studies did not allow close contacts between both cell types. We report here a syngenic coculture model using rat endothelial cells on one side of a polyethylene terephtalate filter and rat astrocytes on the other. Endothelial cells retain their typical morphology and are factor VIII and OX 26 positive. We optimized the diameter of the membrane pores to establish very close contacts between the cells through the membrane pores without mixing the two cell types. Transmission electron microscopy showed evidence of tight junction formation between the endothelial cells and few pinocytic vesicles. The cocultures reached high electrical resistances up to 1000 Omegacm(2) showing their ability to limit the passage of ions. A 15-fold increase in gamma-glutamyl transpeptidase activity was measured in the endothelial cells in coculture compared to endothelial cell monoculture. Our syngenic coculture represents a useful in vitro model of the rat BBB that may prove to be valuable for studying the passage of substances across the barrier as well as other aspects of the BBB function.

Effects of aluminum exposure on glutamate metabolism: a possible explanation for its toxicity.

The effects of aluminum (Al) exposure on glutamate metabolism were investigated to study the mechanism of Al toxicity in rat brain. In astrocytes, the glutamate-glutamine pathway prevents the accumulation of the excitatory neurotransmitter glutamate, recognized as a neuronal excitotoxin when present in excess in the extracellular space. Changes in the level of l-aspartate, l-glutamate, and its metabolite l-glutamine were investigated in various regions of rat brains following intraperitoneal injection of aluminium gluconate for 2 months. The changes observed were area- and amino-acid-specific. An increase in glutamine, but not in l-glutamate or l-aspartate, was noted in the hippocampus and neocortex of Al-treated rats. This increase in vivo was consistent with observations in vitro. Exposure of cultured astrocytes to Al chloride (200, 400, and 800 microM) specifically increased glutamine synthetase activity for the three concentrations tested. In parallel with this increase, a higher rate of disappearance of glutamate from culture medium was observed during the first 10 min of incubation for the three concentrations tested, as well as an accumulation of glutamine in the cellular extract after 30 min. These observations indicate that the astrocyte population is a potential target for Al toxic action that could mediate the pathogenesis of this metal.

Effects of aluminum exposure on behavioral parameters in the rat.

Adult rats were treated by intraperitoneal injection of aluminum gluconate for 3 months. Rats were submitted to the radial maze test to determine the influence of chronic aluminum intoxication on cognitive and noncognitive behavioral processes. Both learning abilities (working memory and reference memory) and rapidity (time spent to respond and to master a trial) were analyzed. Aluminum concentration was evaluated in the brain, serum, and liver to assess aluminum body burden. While hippocampus and neocortex showed a significant increase in aluminum concentration, aluminum treatment did never affect the animal's performance during cue learning or when the insert cues were removed. The only behavioral difference observed was a decrease in rapidity: both the total time to finish a trial and the latency to make the first choice were lengthened in aluminum-intoxicated rats.

An experimental animal model of aluminium overload.

In order that better therapeutic approaches to disorders in man characterized by aluminium (Al) overload might be developed it is essential to have an appropriate animal model. Chronic oral administration of Al citrate to male Wistar rats leads to an Al overload in a relatively short period of time when compared to previous published animal models. Liver and brain Al levels are increased by 25 and 30-fold respectively compared to control rats after 6 months of loading. Al tissue content was significantly greater when the Al citrate was administered in an iron-free diet. The distribution of Al in brain was similar to that in the Al encephalopathy of patients with chronic renal failure or Alzheimer's disease and is in accord with observations that areas of brain that accumulate greatest amounts of Al have highest concentrations of transferrin receptors. In the brain, the toxic effect of Al at the cellular level was characterized by an extensive cytoplasmic vacuolation in astrocytes (especially) and neurones. These changes are reminiscent of those observed in certain human neurodegenerative diseases.

Ultrastructural changes in brain parenchyma during normal aging and in animal models of aging.

During aging, the brain parenchyma of animals and humans share many similarities, both in the gray and the white matter. Unfortunately, until now, neither aged animals nor animal models reproduce the two hallmarks of aging of the human brain: senile plaques and tangles. Therefore, observations performed on animals are limited to some aspects of the involutive process which affects brain parenchyma during aging and their appropriateness to the human situation. One striking aspect concerns the occurrence of vacuolated necrotic cells whose number increases with advancing age. These cells can constitute markers of the brain involutive process and they characterize, both in animal and human, the more vulnerable areas of the brain affected by the neuronal rarefaction. Experimental animal models can be used to study the various conditions which sustain the cell survival and to determine, at the cellular level, the factors leading the brain parenchyma to an irreversible state of degradation.

Regeneration of lesioned cholinergic septal neurons of the adult rat can be promoted by peripheral nerve grafts and a fibrin-fibronectin-containing matrix of peripheral regeneration chambers.

Axonal regeneration of septal cholinergic neurons was examined after lesion of the septohippocampal pathway of the adult rat and implantation of tubes containing peripheral cellular or acellular substrates. After empty tube implantation, no regenerated structures were observed in the conduit. However, after implanting tubes filled with sections of predegenerated sciatic nerves or a fibrin-fibronectin-containing matrix provided by peripheral regeneration chambers, numerous regenerated axons were detected 6 weeks after the operation. At the electron microscopic level, regenerated axons were observed in the grafted sciatic nerves in contact with Schwann cells but also in contact with astrocytes which were able to migrate and send processes into the graft. After fibrin-fibronectin-containing-matrix implantation, the regenerated structure between septum and hippocampus was composed mainly of fibroblasts, astrocytes, and regenerated axons associated to these central glial cells.

Axonal regeneration after peripheral nerve grafting and fibrin-fibronectin-containing matrix implantation on the injured septohippocampal pathway of the adult rat: a light and electron microscopic study.

The damaged septohippocampal pathway was utilized to study the axonal regeneration of injured neurons. Semipermeable tubes, 2-mm long, were placed in the axis of the transected septohippocampal pathway of adult rats. In a first series of experiments, empty tubes were implanted. Even six weeks after the operation, no regenerated axons were observed in the conduit. In a second series of experiments, in order to validate our approach, segments of pre-degenerated sciatic nerves were introduced into the tubes. Under these experimental conditions, acetylcholinesterase (AChE)-containing regenerated axonal processes were detected in the grafted sciatic nerves. Glial fibrillary acidic protein (GFAP)-immunodetection showed that astroglial cells and astrocyte processes were able to progress on and into the peripheral grafts. At the electron microscopic level, axons were observed in close contact with Schwann cells which myelinated some of them. In some other cases, unmyelinated axons were also present at the surface of reactive astroglial cells filled by numerous intermediate filaments. These central glial cells had migrated among the sciatic nerve collagen fibers. No axon was detected without glial cell contact. In a third series of experiments, we implanted semipermeable tubes previously filled with a fibrin-fibronectin-containing matrix provided by peripheral regeneration chambers. One week after the implantation of the tubes containing this peripheral substrate, different cell types were observed migrating into the conduit and replacing the fibrin-fibronectin-containing matrix. Among these cells astrocytes were present as revealed by GFAP-immunocytochemistry and electron microscopic examinations. During the following weeks, axons were detected in contact with the reactive astroglial cells. AChE-histochemistry showed that axons were able to cross the two millimeter distance separating the septal part and the hippocampal part of the lesion site. GABA (γ-aminobutyric acid)-ergic fibers were also detected in the regenerated structure. These experiments show that cellular or acellular substrates provided by the PNS can promote the regeneration of CNS GABAergic and cholinergic neurons. Our observations suggest that astrocytes can take an important part, after their migration or after extending processes, in the axonal regeneration in the adult CNS of the rat, possibly in furnishing a cellular terrain for the progression of growth cones over a distance of two millimeters and in maintaining regenerated axons at least until the sixth week after the operation.