Abstract
Abstract: :
Purpose: To determine how glucose or the monocarboxylates, lactate or pyruvate, affect the viability of neurones or glia in mixed rat retinal cultures. Methods: Mixed retinal cultures comprising glia and neurones were prepared from 3–5 day old Wister rat litters. Cultures were grown in Dulbecco’s Modified Eagle Medium (DMEM) containing 5mM glucose and experiments were performed after 6–10 days in vitro. At this time, medium was changed to DMEM lacking glucose and incubations were carried out for up to 72 hours in this medium containing combinations of glucose (100µM–25mM) and/or pyruvate or lactate (100µM–25mM). Glycolysis was inhibited with iodoacetic acid (IOA; 1–100µM) and oxygen–dependent metabolism with the mitochondrial inhibitor, 2–4–dinitrophenol (2,4–DNP; 1–100µM). Glucose and monocarboxylate transport were inhibited with 1–10µM cytochalasin B (CCB) and α–cyano–4–hydroxycinnamate (4–CIN), respectively. Cultures were assessed for overall viability using the 3,(4,5–dimethylthiazol–2–yl)–2,5–diphenyltetrazolium bromide (MTT) reduction assay, immunocytochemistry determined the cell types that were specifically affected and the TUNEL assay was used to detect apoptotic–like cell death. Results: The overall viability of cells in mixed retinal cultures decreased as the glucose concentration was lowered below 2mM for more than 24 hours. Both glia and neurones were affected in this way. Addition of greater than 1mM lactate or pyruvate, concurrently, partially preserved neurones, but had no obvious effect on glial cells. Inhibition of glycolysis led to neurone and glial death that was characteristic of apoptosis, whereas inhibition of oxidative metabolism only affected neurones. Strikingly, inhibition of glucose transport led to death of glial cells alone, whereas blocking monocarboxylate transport clearly caused death of neurones. Conclusions: The results show that both neurones and glia in mixed rat retinal cultures require glucose for optimal survival but that neurones alone have the ability to utilise alternative energy substrates as shown by their partial reliance on monocarboxylates. These data add support to the theory that retinal glial cells release the glycolytic end–product, lactate, for maintenance of neurone functioning in vivo.
Keywords: retinal culture • metabolism • nutritional factors