Acidosis has opposite effects on neuronal survival during hypoxia and reoxygenation Runar Almaas,* Morten Pytte,* Julie K. Lindstad,* Marianne Wright,* Ola Didrik Saugstad,* David Pleasure and Terje Rootwelt* *Department of Pediatric Research, Rikshospitalet, Oslo, Norway  Neurology Research, Children’s Hospital of Philadelphia, Philadelphia, USA Abstract To study the effect of extracellular acidosis on apoptosis and necrosis during ischemia and reoxygenation, we exposed human post-mitotic NT2-N neurones to oxygen and glucose deprivation (OGD) followed by reoxygenation. In some experiments, pH of the cell medium was lowered to 5.9 during either OGD or reoxygenation or both. Staurosporine, used as a positive control for apoptosis, caused Poly(ADP-ribose)- polymerase (PARP) cleavage and nuclear fragmentation, but no PARP cleavage and little fragmentation were seen after OGD. Low molecular weight DNA fragments were found after staurosporine treatment, but not after OGD. No protective effect of caspase inhibitors was seen after 3 h of OGD and 21 h of reoxygenation, but after 45 h of reoxygenation caspase inhibition induced a modest improvement in 3-(4,5- dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) cleavage. While acidosis during OGD accompanied by neutral medium during reoxygenation protected the neurones (MTT: 228 ± 117% of neutral medium, p < 0.001), acidosis during reoxygenation only was detrimental (MTT: 38 ± 25%, p < 0.01). We conclude that apoptotic mechanisms play a minor role after OGD in NT2-N neurones. The effect of acidosis on neuronal survival depends on the timing of acidosis, as acidosis was protective during OGD and detri- mental during reoxygenation. Keywords: acidosis, apoptosis, human, ischemia, neurone, reoxygenation. J. Neurochem. (2003) 84, 1018–1027. Traditionally, hypoxic/ischemic neuronal death has been thought to occur through necrotic mechanisms, but recent studies have also reported apoptotic features in infants who died after severe asphyxia (Edwards et al. 1997). However, electron microscopic findings in gerbils after global ischemia were not consistent with apoptosis (Colbourne et al. 1999), and protective effects of caspase inhibitors have been observed in focal, but not in global ischemia (Li et al. 2000). Exposure of neurones to low concentrations of oxidants or excitotoxins causes apoptosis, while more severe insults cause necrosis (Bonfoco et al. 1995). However, Colbourne et al. did not find apoptotic morphology even when the ischemic insult was reduced (Colbourne et al. 1999). The intensity of the hypoxic/ischemic insult may not, therefore, explain all the differences between experimental models with and without evidence of apoptosis. The effect of pH on hypoxic/ischemic damage has been evaluated in experiments with hyperglycemia, which increa- ses the formation of lactate and aggravates acidosis. Most authors have found that hyperglycemia worsens hypoxic/ ischemic damage in adult models, but there are also some reports of beneficial effects of hyperglycemia (Wass and Lanier 1996). The results from neonatal animal models have given conflicting results, as hyperglycemia is protective in some models with rodents (Holowach-Thurston et al. 1974; Voorhies et al. 1986) and detrimental in models with pigs (LeBlanc et al. 1993) and dogs (Vannucci et al. 1996). Acidosis reduces ischemic damage in cell cultures (Giffard et al.1990;TombaughandSapolsky1990).Thismaybedue to the inhibitory effect of acidosis on NMDA- (Giffard et al. 1990; Tang et al. 1990) and kainate-receptors (Giffard et al. Received June 24, 2002; revised manuscript received October 30, 2002; accepted November 11, 2002. Address correspondence and reprint requests to Runar Almaas, Department of Pediatric Research, Rikshospitalet, N-0027 Oslo, Norway. E-mail: runaralm@start.no Abbreviations used: DMEM, Dulbecco’s modified Eagle’s medium; LDH, lactate dehydrogenase; MTT, 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide; OGD, oxygen and glucose deprivation; PARP, poly(ADP-ribose)-polymerase; PBS, phosphate-buffered saline. Journal of Neurochemistry , 2003, 84, 1018–1027 doi:10.1046/j.1471-4159.2003.01593.x 1018 Ó 2003 International Society for Neurochemistry, J. Neurochem. (2003) 84, 1018–1027