In vitro regulation of rat derived microglia.
The cell culture approach to the study of the nervous system attempts to reduce cellular complexity to various extents and to characterize the influences of extrinsic molecules on the cell population under study. To date, the main source of culture model systems to explore CNS function and dysfunction is fetal brain material from experimental animals, typically rodents. We have developed primary microglial cell cultures and focused on the concentration-dependent effects of different amino acids and growth promoting additives on microglial morphology and function. We used Basal Medium Eagle (BME) with 1g/L of glucose instead of Dulbecco's modified Eagle medium (DMEM) as serum-free condition, since BME does not contain L-Glycine (Gly) and L-Serine (Ser), and investigated the effects of these two amino acids on microglial morphology and functions by adding various concentrations of the amino acids to BME and different concentrations of ascorbic acid (10-75 micro g/ ml), hydrocortisone (1-7.5 nM) and DL-alpha-tocopherol (0.01-0.5 micro g/ml) as growth promoters. Under Gly/Ser-free, serum-free condition, and growth promoters-free conditions, the majority of rat microglial cells displayed round morphology, whereas in the presence of 5 micro M Gly and 25 micro M Ser, which correspond to the concentrations of Gly and Ser in the cerebrospinal fluid, they extended multiple branched processes and formed clusters of rough endoplasmic reticulum. Ascorbic acid (25 micro g/ml), 2.5 nM hydrocortisone and 0.05 micro g/ml of DL-alpha-tocopherol elicited the highest level of microglial activation as measured by an increased expression of MHC class-I and MHC class-II antigens. Neuron culture experiments using the conditioned medium obtained from the different microglial culture conditions indicate neurotoxic and neurotrophic effects depending on the concentrations of amino acids as well as on the concentration of the growth promoters. These findings suggest that resting ramified microglial cells with neurotrophic activity can be induced with the combination of BME medium and small amounts of extracellular matrix growth promoters.
Lombardi
Valter R M
VR
Ebiotec Biotechnology Division, La Coruña, Spain. biotecnologia@ebiotec.com
Etcheverría
Ignacio
I
Fernández-Novoa
Lucía
L
Cacabelos
Ramón
R
D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005998:Glycine / Q000008:administration & dosage; D015395:Histocompatibility Antigens Class I / Q000378:metabolism; D000949:Histocompatibility Antigens Class II / Q000378:metabolism; D006854:Hydrocortisone / Q000494:pharmacology; D017628:Microglia / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D009994:Osmolar Concentration; D051381:Rats; D017207:Rats, Sprague-Dawley; D012694:Serine / Q000008:administration & dosage; D024502:alpha-Tocopherol / Q000494:pharmacology
D016895:Culture Media, Serum-Free; D015395:Histocompatibility Antigens Class I; D000949:Histocompatibility Antigens Class II; D012694:Serine; D024502:alpha-Tocopherol; D001205:Ascorbic Acid; D005998:Glycine; D006854:Hydrocortisone