RESUMEN
Neonatal rat brain astrocyte secondary cultures were established on nitrocellulose membrane filters (13-mm diameter Millipore disk) and on plastic coverslips in serum-supplemented medium. On these substrata, cultured astrocytes changed their shape from flat and polygonal to stellate in the absence of hormones or growth factor supplements. Cultures became confluent after 4 days, and astrocytes on nitrocellulose filters continued to differentiate morphologically and biochemically, as evidenced by extensive cytoplasmic process formation and glial fibrillary acid protein (GFAP) accumulation. Cultures were immunostained for GFAP and vimentin. mRNAs to GFAP, vimentin, alpha and beta tubulin, and actin also were detected by in situ hybridization with biotinylated cDNA probes. The astrocyte culture method on nitrocellulose provides a simple, versatile means of comparing undifferentiated, morphologically mature, reactive, and neoplastic astrocytes in vitro.
Asunto(s)
Astrocitos/análisis , Proteínas del Citoesqueleto/análisis , Hibridación de Ácido Nucleico , ARN Mensajero/análisis , Animales , Células Cultivadas , Corteza Cerebral/análisis , Corteza Cerebral/citología , Colodión , ADN , Ensayo de Inmunoadsorción Enzimática , Proteína Ácida Fibrilar de la Glía/análisis , Proteína Ácida Fibrilar de la Glía/metabolismo , Membranas Artificiales , Ratas , Ratas Endogámicas , Coloración y Etiquetado , Vimentina/análisis , Vimentina/metabolismoRESUMEN
The rate of clearance of porcine myelin basic protein (MBP) from plasma of rabbits was determined following intravenous injection of 20 mg MBP. The MBP level in the plasma was measured by a 2-site immunoradiometric assay with specific antibody to guinea pig MBP produced in rabbits. Plasma MBP-antibody levels were determined by competitive binding radioimmune assay (RIA). Unsensitized and those sensitized with complete Freund's adjuvant (CFA), with porcine MBP in CFA, and with whole porcine spinal cord in CFA were studied. Unsensitized and CFA sensitized rabbits exhibited maximum MBP levels in the plasma within two minutes after injection with rapid decrease to undetectable levels in one hour. Thirty-nine of the unsensitized (control) rabbits exhibited normal, rapid clearance and no subsequent physical signs of EAE while one of the control rabbits exhibited a slightly retarded clearance rate. Histologic examination of autopsy tissues from the control group revealed that five rabbits showed lesions which could be attributed to Encephalitozoan cuniculi or Toxoplasma and one rabbit autopsied 65 days after clearance had minimal EAE lesions. Rabbits sensitized with MBP exhibited a retarded rate of clearance at the acute stage of EAE and following recovery. Rabbits sensitized with whole spinal cord in CFA also exhibited a retarded rate of MBP clearance. Anti (MBP) antibodies were detected in the plasma of all rabbits which exhibited a retarded rate of MBP clearance. Significant rates of retardation were not detected until approximately three weeks after sensitization with CFA-MBP or CFA-spinal cord. While MBP antibody levels in most animals were not detected by the immunodiffusion technique, antibodies were demonstrated by RIA. The 20 mg MBP given intravenously is probably in great antigen excess and conducive to the formation of soluble MBP-anti (MBP) complexes in the blood.