• RouxPeterD Le and Susana Esquenazi.
• Department of Neurosurgery, University of Pennsylvania, Philadelphia 19107, USA. leroux@uphs.upenn.edu
• Neurol. Res. 2002 Jan 1; 24 (1): 81-92.

AbstractAstrocytes occupy a central role in central nervous system (CNS) function. In particular astyrocytes can support neurite growth, in part, by release of diffusable factors. We therefore performed biochemical analysis of astrocyte conditioned medium to examine possible mechanisms of astrocyte mediated axon and dendrite growth in the mammalian CNS. Culture medium was conditioned on purified astrocyte monolayers derived from P3 rat cerebral cortex or on fibroblasts. Conditioned medium (CM) was subject to protein denaturation, molecular weight fractionation, and heparin affinity chromatography. E18 mouse cerebral cortical neurons were then cultured in the various media or directly on astrocyte monolayers and axon and dendrite growth from 50 neurons in each condition quantified after 3 DIV using double-labeled immunohistochemical techniques. Axon and dendrite growth was supported by astrocyte CM and both were significantly greater than process growth from neurons incubated in fibroblast CM. Protein denaturation significantly reduced astrocyte CM support of axon and dendrite growth. Following ultrafiltration and dialysis dendrite and axon growth was observed in the molecular weight fraction between 10 and 100 kDa. Axon growth also was observed in the CM molecular weight fraction greater than 100 kDa. Conditioned medium was eluted on a heparin column; when the bound fragment was reconstituted in chemically defined medium extensive dendrite and axon growth was observed. Since fibroblast growth factor (FGF) has these biochemical characteristics we added anti-bFGF neutralizing antibodies to astrocyte monolayers or CM; this significantly reduced astrocyte support of process growth. By contrast, the addition of heparin, which helps activate FGF receptors, to astrocyte CM further enhanced process growth. Western blot analysis confirmed that bFGF was present in astrocyte CM. We then examined axon and dendrite growth from cortical neurons after the addition of various growth factors to chemically defined medium. Axon and dendrite growth, similar to that found in astrocyte CM was observed after the addition of bFGF or aFGF. Astrocyte support of cerebral cortical neuron axon and dendrite growth in vitro may be explained, in part, by FGF release.

39 keywords...

hide…