TOLERANCE OF LOW INTRACELLULAR PH DURING HYPERCAPNIA BY RAT CORTICAL BRAIN-SLICES - A P-31/H-1 NMR-STUDY

Metabolic tolerance of low intracellular pH (pH(i)) was studied in well-oxygenated, perfused, neonatal, rat cerebrocortical brain slices (350 mum thick) by inducing severe hypercapnia. In each of 17 separate experiments 80 brain slices (approximately 3.2 g wet weight) were suspended in an NMR tube, perfused with artificial CSF (ACSF), and studied at 4.7 T with P-31 and H-1 NMR spectroscopy. Spectra obtained every 5 min monitored relative concentrations of lactate or high-energy phosphate metabolites, from which pH(i) and extracellular pH were determined. Unperturbed slice preparations were metabolically stable for > 10 h, with no significant changes occurring in pH(i), ATP, phosphocreatine (PCr), inorganic phosphate, or lactate. Different levels of hypercapnia were produced by sequentially perfusing slices with the following different ACSF batches, each having previously been equilibrated with a specific mixture Of CO2 in oxygen: (a) 10% CO2, 15 min of perfusion; (b) 30% CO2, 15 min of perfusion; (c) 50% CO2, 15 min of perfusion; (d) 70% CO2, 30 min of perfusion; (e) 50% CO2, 15 min of perfusion; (f) 30% CO2, 15 min of perfusion; and (g) 10% CO2, 15 min of perfusion. At the completion of this protocol slices were again perfused with fresh ACSF that was equilibrated with a 95% 02/5% CO2 gas mixture. In each of five separate H-1 and P-31 experiments, brain slices were recovered within 2 h after termination of exposure to high CO2. The pH(i) was determined from measurements of the chemical shift difference between phosphoethanolamine and PCr, using a calibration curve obtained for our preparation. During hypercapnia, pH(i) decreased from 7.18 +/- 0.22 to 6.79 +/- 0.13, 6.60 +/- 0.11, 6.33 +/- 0.40, and 6.37 +/- 0.14 when CO2 concentrations were increased to 10, 30, 50, and 70%, respectively. Lactate/N-acetylaspartate ratios were unchanged until the administration of 70% CO2, which caused a relative increase from 0.83 +/- 0.15 to 1.35 +/- 0.32 (n = 5, p = 0.0024). The administration of 70% CO2 also caused complete depletion of PCr and a 46 +/- 22% (n = 5, p = 0.0024) decrease in ATP content. Metabolite and pH values returned to control values after restoration of normocapnia. The lowest tissue pH(i) attainable in our protocol using 30% 02/70% CO2 in our brain slice preparation was approximately 6.3.