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Purification and Culture of Adult Rat Dorsal Root Ganglia Neurons
Delree, P.; Leprince, Pierre; Schoenen, Jean et al.
1989In Journal of Neuroscience Research, 23 (2), p. 198-206
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Abstract :
[en] To study the trophic requirements of adult rat dorsal root ganglia neurons (DRG) in vitro, we developed a purification procedure that yields highly enriched neuronal cultures. Forty to fifty ganglia are dissected from the spinal column of an adult rat. After enzymatic and mechanical dissociation of the ganglia, myelin debris are eliminated by centrifugation on a Percoll gradient. The resulting cell suspension is layered onto a nylon mesh with a pore size of 10 microns. Most of the neurons, the diameter of which ranged from 17 microns to greater than 100 microns, are retained on the upper surface of the sieve; most of the non-neuronal cells with a caliber of less than 10 microns after trypsinization go through it. Recovery of neurons is achieved by reversing the mesh onto a Petri dish containing culture medium. Neurons to non-neurons ratio is 1 to 10 in the initial cell suspension and 1 to 1 after separation. When these purified neurons are seeded at a density of 3,000 neurons/cm2 in 6 mm polyornithine-laminin (PORN-LAM) coated wells, neuronal survival (assessed by the ability to extend neurites), measured after 48 hr of culture, is very low (from 0 to 16%). Addition of nerve growth factor (NGF) does not improve neuronal survival. However, when neurons are cultured in the presence of medium conditioned (CM) by astrocytes or Schwann cells, 60-80% of the seeded, dye-excluding neurons survive. So, purified adult DRG neurons require for their short-term survival and regeneration in culture, a trophic support that is present in conditioned medium from PNS or CNS glia.(ABSTRACT TRUNCATED AT 250 WORDS)
Disciplines :
Neurosciences & behavior
Neurology
Author, co-author :
Delree, P.
Leprince, Pierre ;  Université de Liège - ULiège > GIGA-Neuroscience
Schoenen, Jean  ;  Université de Liège - ULiège > Département des sciences biomédicales et précliniques > Neuro-anatomie
Moonen, Gustave  ;  Université de Liège - ULiège > Département des sciences cliniques > Neurologie
Language :
English
Title :
Purification and Culture of Adult Rat Dorsal Root Ganglia Neurons
Publication date :
June 1989
Journal title :
Journal of Neuroscience Research
ISSN :
0360-4012
eISSN :
1097-4547
Publisher :
John Wiley & Sons, Hoboken, United States - New York
Volume :
23
Issue :
2
Pages :
198-206
Peer reviewed :
Peer Reviewed verified by ORBi
Available on ORBi :
since 30 September 2009

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