Cerebral energy metabolism and intracellular pH during severe hypoxia and recovery: a study using 1H, 31P, and 1H [13C] nuclear magnetic resonance spectroscopy in the guinea pig cerebral cortex in vitro

1H and 31P nuclear magnetic resonance spectroscopy was used to study intracellular pH (pHi), high-energy phosphates, lactate, and amino acids in cortical brain slices superfused in Krebs-Henseleit bicarbonate buffer during and after severe hypoxia at 0, 10, and 50 mM glucose. An extensive drop in phosphocreatine (PCr) and a rapid build-up of intracellular lactate and H+ were the first signs of hypoxia. Adenosine triphosphate (ATP) was significantly more resistant to hypoxia provided that glucose was present. In the preparations that had been exposed to hypoxia in the presence of glucose, PCr became detectable within 2 min of reoxygenation, and both PCr and ATP concentrations were restored to 72-80% of normoxic levels within 30 min. Lactate was washed out, and pHi returned to normal within 4-8 min. Using 1-[13C]glucose as a tracer, we demonstrated that the rate of lactate production in the immediate posthypoxic period was at the prehypoxic level, indicating that the elevated lactate during this period was due solely to that produced during hypoxia. During reoxygenation of the preparations that were denied glucose during hypoxia, only 30% of total creatine + PCr and 18% of PCr were restored, and ATP was not detectable. The lactate concentration rose twofold in this period, and pHi became significantly more alkaline than before the hypoxic insult. Thus acute metabolic damage was considerably greater if glucose was absent during the insult, suggesting that either anaerobic ATP production or low pH may exert some protective effect against acute cell damage.     

Hypoxia-induced dysfunctions and injury of astrocytes in primary cell cultures

The effects of severe hypoxia were studied in a primary culture of astrocytes prepared from newborn rat cerebral cortex. Hypoxia was created by placing cultures in an airtight chamber that was flushed with 95% N2/5% CO2 for 15 min before being sealed. The hypoxic environment was maintained constant for up to 24 h. During the first 12 h of hypoxia, astrocytes showed no morphological changes by phase-contrast microscopy. After 18 h of hypoxia, some astrocytes in culture became swollen and started to detach from the culture dish. All cells in the culture were destroyed after 24 h of hypoxia. The lactate dehydrogenase level in the culture medium increased more than tenfold between 12 and 24 h of hypoxia. Glutamate uptake was inhibited 80% by similar hypoxic conditions. The cell volume of astrocytes, as measured by 3-O-methyl-[14C]-D-glucose uptake, was increased. These observations suggested cell membrane dysfunction. The malondialdehyde level of hypoxic cultures increased two-fold after 24 h of hypoxia. Verapamil (0.5 mM), furosemide (1 mM), indomethacin (1 mM), MgCl2 (10 mM), and mannitol (10 mM) reduced but never completely abolished the release of lactate dehydrogenase from hypoxic astrocytes. These data suggest multifactorial causes for severe injury in hypoxic astrocytes.     

Aniracetam attenuates apoptosis of astrocytes subjected to simulated ischemia in vitro

The aim of the present study was to establish whether aniracetam is capable of protecting cultured rat astrocytes against ischemic injury. Treatment of the cultures with aniracetam (1, 10 and 100 mM) during 24 h ischemia simulated in vitro significantly decreased the number of apoptotic cells. The antiapoptotic effects of the drug were confirmed by the increase of intracellular ATP and phosphocreatine (PCr) levels and the inhibition of the caspase-3 activity. Aniracetam also attenuated cellular oxidative stress by decreased production of reactive oxygen species (ROS). These effects were associated with the decrease in levels of c-fos and c-jun mRNA in primary astrocyte cultures exposed to 24 h ischemia. When cultured astrocytes were incubated during 24 h simulated ischemia with wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor or PD98059, a mitogen-activated protein (MAP)/extracellular signal regulated kinase (ERK) (MEK) inhibitor the cell apoptosis was accelerated. This effect was antagonized by adding 100 mM aniracetam to the culture medium. These findings suggest that the protective effect of aniracetam is mediated by PI 3-kinase and MEK pathways in the downstream mechanisms.     

Effect of nitric oxide synthase inhibition during post-hypoxic reoxygenation on Bax and Bcl-2 protein expression and DNA fragmentation in neuronal nuclei of newborn piglets

Previous studies have shown that cerebral tissue hypoxia results in increased generation of oxygen-free radicals including nitric oxide (NO), expression of the proapoptotic protein Bax and fragmentation of nuclear DNA. The present study tests the hypothesis that post-hypoxic reoxygenation for 6 h following hypoxia (FiO2=0.06 for 1 h) results in continued hypoxia-induced, NO-mediated expression of the Bax protein and nuclear DNA fragmentation in the cerebral cortex of newborn piglets. Piglets were divided into normoxic (Nx), hypoxic (Hx, FiO2=0.06 for 1 h), hypoxic with 6 h reoxygenation (Hx+reox) and hypoxic with 6 h reoxygenation injected with 7-nitroindazole sodium salt (7-NINA), a selective nNOS inhibitor, immediately after hypoxia (Hx+7-NINA). Cerebral tissue hypoxia was documented by levels of ATP and phosphocreatine (PCr). Bax and Bcl-2 were analyzed by Western blot and DNA fragmentation was determined by agarose gel electrophoresis. ATP and PCr values in Hx, Hx+reox and Hx+7-NINA were significantly different from Nx (P<0.05 vs. Nx). Bax protein (ODxmm2) was 128.9+/-38.7 in Nx; 223.6+/-45.8 in Hx (P<0.05 vs. Nx); 340.5+/-73.2 in Hx+reox (P<0.05 vs. Nx, Hx and Hx+7-NINA); and 202.2+/-34.8 in Hx+7-NINA (P=NS vs. Hx). Bcl-2 protein (ODxmm2) was 14.9+/-2.7 in Nx, 12.4+/-2.1 in Hx, (P<0.05 vs. Nx), 15.7+/-3.8 in Hx+reox, (P<0.05 vs. Hx) and 13.1+/-2.2 in Hx+7-NINA (P=NS among groups). Nuclear DNA fragmentation (ODxmm2) was 147+/-15 in Nx; 797+/-84 in Hx (P<0.05 vs. Nx); 1134+/-127 in Hx+reox (P<0.05 vs. Nx, Hx and Hx+7-NINA); and 778+/-146 in Hx+7-NINA (P=NS vs. Hx, P<0.05 vs. Hx+reox). The results show that post-hypoxic reoxygenation results in increased expression of Bax protein without affecting Bcl-2 protein and increased fragmentation of nuclear DNA, which are prevented by 7-NINA. We conclude that during post-hypoxic reoxygenation the increase in Bax protein expression and fragmentation of nuclear DNA are mediated by NO derived from nNOS. We propose that in addition to NO-mediated nuclear DNA damage, the hypoxia-induced increased ratio of Bax/Bcl-2 protein will lead to caspase-activated cascade of hypoxic neuronal death during post-hypoxic reoxygenation.     

Exposure of cultured primary rat astrocytes to hypoxia results in intracellular glucose depletion and induction of glycolytic enzymes

Based on the neurotrophic properties of astrocytes in response to ischemia, the current work focuses on the mechanism for cultured astrocytes to adapt to a hypoxic environment. Intracellular glucose levels in primary cultured rat astrocytes exposed to hypoxia fell by 30% within 24 h, in parallel with a decrease in glycogen stores. Glycolytic metabolism was crucial for cell survival during hypoxia, as 2-deoxyglucose resulted in rapid ATP depletion and cell death. The mechanism for maintaining glucose levels under these conditions appeared to be mobilization of glycogen stores, rather than increased extracellular uptake of glucose, as gluconolactone (an inhibitor of beta1-4 amyloglucosidase) induced a rapid fall in cellular ATP in cultures subjected to hypoxia, whereas cytochalasin B was without affect. Addition of cycloheximide diminished the viability of astrocytes in hypoxia, suggesting an obligatory role of de-novo gene expression to respond to hypoxia. Consistently, the results of differential display suggested the induction of glycolytic enzymes, including aldolase A (EC 4.1.2.13), hexokinase II (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1), and triosephosphate isomerase (EC 5.3.1.1) in the hypoxic culture. Marked induction of these glycolytic enzymes in hypoxic astrocytes was confirmed by Northern blot analysis. These data provide a theoretical basis to understand the ability of astrocytes to tolerate ischemic condition.     

Exogenous creatine delays anoxic depolarization and protects from hypoxic damage: dose–effect relationship

Incubation of hippocampal slices with different concentrations of creatine (0.5, 1, 10, 25 mM) results in a dose-dependent increase in intracellular phosphocreatine (PCr). Electrophysiological evidence suggests that this effect can protect neurons from anoxic damage by delaying the depletion of ATP during oxygen deprivation. In this paper we show that incubation of brain slices with varying doses of creatine increases intracellular phosphocreatine and delays anoxic depolarization (AD) in a dose-dependent way. Specifically, addition to the incubation medium of 1 mM creatine significantly increased AD latency during hypoxia and prevented irreversible neuronal damage. Adding 0.5 mM creatine had no significant effect. Higher concentrations of creatine (up to 25 mM) did not provide any better protection. Our data also suggest a linear correlation between intracellular PCr and AD latency. These data report neural protection by exogenous creatine at concentrations lower than those usually reported in the literature.     

p38MAPK在体外培养星形胶质细胞缺氧/复氧损伤中的表达

目的 建立SD大鼠星形胶质细胞缺氧复氧损伤模型,探讨p38MAPK活性变化与星形胶质细胞损伤的关系.方法 体外培养新生SD大鼠星形胶质细胞,实验设正常对照组（N）、SB203580组（SB组,10 μmol/L）、缺氧/复氧组（H/R组）和缺氧/复氧组＋SB203580阻断p38MAPK组（H/R＋SB组）.应用MTT法、WB法、ELISA法检测缺氧4 h、8 h、复氧6 h、12 h、24 h、48 h时细胞存活率,p38MAPK、p-p38（磷酸化p38MAPK）及TNF-α的变化.结果 培养星形胶质细胞GFAP阳性表达率大于97%.缺氧/复氧使星形胶质细胞活力降低,SB203580阻断p38MAPK细胞活力高于H/R组,各组星形胶质细胞总p38MAPK水平无显著变化,缺氧复氧干预后p-p38表达上调,TNF-α水平显著增高.用SB203580阻断p38MAPK通路后,SB＋H/R组较H/R组p-p38、TNF-α水平降低.SB组总p38MAPK、p-p38、TNF-α水平与N组比较无显著变化.结论 p38MAPK信号通路参与了星形胶质细胞缺氧复氧损伤过程.     

Hypoxia induces caspase-9 and caspase-3 activation without neuronal death in gerbil brains

To investigate the in vivo apoptotic machinery in oxygen deprived brain, we examined the expression of caspase-9 and caspase-3 in the hippocampus of Mongolian gerbils subjected to either transient hypoxia (4% O2 for 6 min) or forebrain ischemia (10 min bilateral carotid artery occlusion) followed by 8 h to 7 days of reoxygenation or blood recirculation. Apoptotic death was characterized by isolating hippocampal genomic DNA and analysing DNA fragmentation as well as histological studies including TUNEL assay and toluidine blue staining of brain sections. The results showed that both hypoxic and ischemic gerbil brains exhibited an increase in caspase-9 and caspase-3 gene expression. However, no cell damage was detectable following hypoxia, while marked DNA fragmentation and extensive cell death was observed following ischemia. Moreover, although hypoxia did not lead to cell death, both hypoxia and ischemia were associated with cleavage of procaspase-9 and procaspase-3 and increases in their activities as well as cleavage of poly(ADP-ribose) polymerase-1 (PARP-1), a major caspase-3 substrate. These results indicate that, in vivo, even late apoptotic events such as caspase activation and PARP-1 cleavage in hypoxic brains do not necessarily induce an irreversible commitment to apoptotic neuronal death.     

Induction of mRNAs and proteins for Na/K ATPase alpha1 and beta1 subunits following hypoxia/reoxygenation in astrocytes

Characteristics of the cellular response to oxygen deprivation and subsequent reoxygenation (hypoxia/reoxygenation) include redirection of energy metabolism, increased glucose utilization and expression of oxygen-regulated proteins. Inhibition of protein synthesis during early reoxygenation period prevented effective astrocyte adaptation to hypoxia/reoxygenation, resulting in eventual cell death. To elucidate the role of astrocytes in the central nervous system in response to hypoxia/reoxygenation, we analyzed the cDNA library derived from the cultured rat astrocytes subjected to 24 h of hypoxia followed by reoxygenation by differential display, and isolated a cDNA corresponding to Na/K ATPase alpha1 subunit. The expression of Na/K ATPase alpha1 subunit mRNA as well as beta1subunit mRNA was transiently increased after reoxygenation, whereas hypoxia itself did not induce any gene expression change. Na/K ATPase alpha1 subunit protein was transiently increased, whereas the protein expression for Na/K ATPase beta1 subunit showed sustained induction after reoxygenation. Overexpression of beta1 subunit in HEK 293 cells subjected to hypoxia/reoxygenation promoted survival of the cells. These findings suggest that Na/K ATPases may contribute to maintain the cellular environment of astrocytes subjected to hypoxia/reoxygenation.     

Effects of neuroprotective dose of fructose-1,6-bisphosphate on hypoxia-induced expression of c-fos and hsp70 mRNA in neonatal rat cerebrocortical slices

In situ hybridization (ISH) measurements of c-fos and hsp70 expression were made in brain slice studies of hypoxia, with or without fructose-1,6-bisphosphate (FBP) pretreatment. Each experiment used eighty 350 μm thick cerebrocortical slices, obtained from twenty 7-day old rats. Thirty minute periods of hypoxia were followed by 8 h of hyperoxic perfusion. Slices were removed at eight predetermined times, and processed for ISH and immunohistochemistry. In three of six hypoxia experiments, slices were pretreated for 60 min with 2 mM FBP, a condition known to maintain ATP level in brain slices during hypoxia. In three other hypoxia experiments slices received no pretreatment. In two control experiments slices were perfused for 11.5 h without hypoxia. In control experiments, hsp70 mRNA was barely detectable in slices at all times, although moderate c-fos mRNA expression occurred at 1 h after decapitation. Hypoxia produced a modest but statistically significant increase in c-fos mRNA and hsp70 mRNA induction 4 h following reoxygenation. At all times after hypoxia, FBP pretreatment reduced expression of c-fos and hsp70 mRNA. The absence of hsp70 mRNA in control slices suggests that intracellular protein denaturation was minimal in this preparation. In slices made hypoxic, the decrease in c-fos and hsp70 mRNA caused by FBP pretreatment suggests ameliorated progression towards injury. Immunohistochemistry showed no HSP70 protein at any time following hypoxia, with or without FBP pretreatment, presumably due to delayed HSP70 protein synthesis, or to a block in translation, as observed in vivo in other studies.     

Fructose-1,6-bisphosphate reduces ATP loss from hypoxic astrocytes

Hypoxia caused injury and metabolic dysfunction of astrocytes, as indicated by a time-dependent loss of lactate dehydrogenase (LDH) activity and ATP content. The combination of 3.5 mM fructose-1,6-bisphosphate (FBP) and 7.5 mM glucose (GLC) reduced the decrease of ATP and prevented the loss of LDH. These data indicate that the combination of GLC + FBP protects astrocytes from hypoxia. The results also suggest that the maintainance of ATP concentration is the mechanism by which FBP prevents hypoxic injury.     

凋亡敏感基因在短暂性缺氧再复氧复注血清后的大鼠星形胶质细胞中的表达及其意义

目的研究一种新发现的抗氧化蛋白质--凋亡敏感基因(SAG)在短暂性缺氧再复氧复注血清诱导的细胞坏死和凋亡中的作用.方法用短暂性缺氧再复氧复注血清来诱导原代培养的大鼠大脑皮质星形胶质细胞损伤,用免疫细胞化学方法检测凋亡敏感基因的表达,并作图像分析;用流式细胞仪检测胶质细胞在短暂缺氧再复氧复注血清后不同时间点的凋亡率.结果凋亡敏感基因在缺氧15 min再复氧复注血清5 h后表达最高,在复氧复注血清16 h后恢复至对照组水平;细胞凋亡率在缺氧15 min再复氧复注血清1h时达到最高,而在再复氧复注血清5 h后降至对照组水平.结论凋亡敏感基因具有抗凋亡的作用,可减轻星形胶质细胞的缺血再灌注损伤.     

Molecular mechanisms of glutamate neurotoxicity in mixed cultures of NT2-derived neurons and astrocytes: protective effects of coenzyme Q10

Although glutamate excitotoxicity has long been implicated in neuronal cell death associated with a variety of neurological disorders, the molecular mechanisms underlying this process are not yet fully understood. In part, this is due to the lack of relevant experimental cell systems recapitulating the in vivo neuronal environment, mainly neuronal-glial interactions. To explore these mechanisms, we have analyzed the cytotoxic effects of glutamate on mixed cultures of NT2/N neurons and NT2/A astrocytes derived from human NT2/D1 cells. In these cultures, the neurons were resistant to glutamate alone (up to 2 mM for 24-48 hr), but they responded to a simultaneous exposure to 0.5 mM glutamate and 6 hr of hypoxia. Neuronal cell death occurred during subsequent periods of reoxygenation (>30% within 24 hr). This was associated with a marked decrease of intracellular ATP, a significant increase in reactive oxygen species (ROS) and downregulation of glutamate uptake by astrocytes. Thus, under energy failure and high levels of ROS production, only the neurons from these mixed cultures succumbed to glutamate neurotoxicity; the astrocytic cells remained unaffected by the treatment. Taken together, our data suggested that glutamate excitotoxicity might be due to the energy failure and oxidative stress affecting the properties of the NMDA glutamate receptors and causing impairment of glutamate transporters. Cells pretreated for 72 hr with 10 microg/ml of coenzyme Q(10) (functions both as a ROS scavenger and co-factor of mitochondrial electron transport), were protected, suggesting a useful role for coenzyme Q(10) in treatments of neurological diseases associated with glutamate excitotoxicity. A model of the complex interactions between neurons and astrocytes in regulating glutamate metabolism is presented.     

缺氧/复氧后星形胶质细胞AQP5表达的变化及亚低温的干预效应

目的观察缺氧/复氧条件下大脑星形胶质细胞水通道蛋白5(AQP5)的表达变化以及亚低温对其表达的影响。方法利用新生24 h内的SD大鼠,进行原代、传代培养,将星形胶质细胞分为对照组、常温组及亚低温组。用台盼蓝染色法测定37℃及32℃时,缺氧/复氧不同时间点星形胶质细胞的存活率,作为细胞受损指标,用倒置相差显微镜对细胞进行形态学观察,应用细胞免疫化学技术检测星形胶质细胞缺氧/复氧各个时间点AQP5的表达变化及亚低温的干预效果。结果 (1)缺氧4、8 h细胞形态变化不明显,随着复氧时间的延长,可见活化逐渐明显,而亚低温干预的细胞形态及细胞存活力变化均较相应的常温组明显减轻;(2)缺氧及复氧早期AQP5的表达水平降低,复氧后6 h随着时间延长AQP5表达明显增多,在复氧≤8 h常温组及亚低温组的表达水平均低于对照组(P<0.05或0.01),而复氧后10、12 h AQP5蛋白表达水平均明显高于对照组(P<0.05或0.01);(3)在复氧后各时间点亚低温组AQP5的表达水平均明显低于常温组(P<0.05或0.01)。结论亚低温可以减轻缺氧/复氧后星形胶质细胞的损伤,通过降低AQP5的表达水平,可能是亚低温减轻缺血性脑水肿的作用机制之一。     

Neuroprotective effects of allopregnenolone on hippocampal irreversible neurotoxicity in vitro

1. The effects of allopregnenolone, a neurosteroid, endowed with GABAmimetic properties, were tested towards two models of irreversible hippocampal neurotoxicity: i) the irreversible depression produced by hypoxia on the CA1 evoked field potentials in rat hippocampal slices, and ii) glutamate-induced irreversible changes in intracellular calcium concentration in primary hippocampal cell coltures. 2. In control conditions during the reoxygenation period after the application of 15 min of hypoxia, the CA1 evoked field potentials were irreversibly suppressed in almost the 50% of the experiments. In the remaining experiments there were a significative (p<0.01) irreversible reduction of the magnitude of the CA1 population spike with respect with the pre-hypoxia values. Allopregnenolone (50-75 microM) perfused 30 min before, during and 30 min after hypoxia produced a significative (p<0.05) decrease both in the hypoxia-induced irreversible suppression of the CA1 PS and both in the irreversible decrease of the CA1 PS at the end of reoxygenation. 3. The exposition of the primary hippocampal cultured cells to glutamate 0.5 mM for 10 min was followed by a sustained elevation of [Ca2+]i, that persisted at 70-80% of maximal increase for the rest of the experiment (60 min). When a pretreatment with 10-50 microM allopregnanolone preceded Glu 0.5 mM application, [Ca2+]i increased to a maximal value during the glutamate application, after which a fast decrease to 50% was observed, followed by a slow recovery within about 30 min. 4. The results showed that the neurosteroid allopregnenolone, endowed with GABAmimetic properties, ameliorated the functional correlates of irreversible hippocampal neurotoxicity.     

Neuroprotection by nicotine against hypoxia-induced apoptosis in cortical cultures involves activation of multiple nicotinic acetylcholine receptor subtypes

Activation of neuronal nicotinic acetylcholine receptors (nAChR) by nicotine has been suggested to protect neurons against a hypoxic insult. The objective of this study was to examine the nature of cell death induced by acute hypoxia in rat primary cortical cultures and the neuroprotective potential of nicotine in ameliorating these processes. Neuronal cell death induced by a 4-h exposure to hypoxia (0.1% O(2)) was apoptotic, as shown by TUNEL staining and assays monitoring DNA strand breaks and caspase-3/7 activity. The presence of nicotine (10 microM) during the hypoxic insult protected a subpopulation of susceptible neurones against DNA damage and apoptosis induced by oxygen deprivation. This protective effect of nicotine was prevented by a 30-min pre-incubation with either 100 nM alpha-bungarotoxin or 1 microM dihydro-beta-erythroidine, but not 1 microM atropine, suggesting that activation of at least two subtypes of nAChR, alpha7 and beta2* nAChR, is involved in mediating nicotine neuroprotection.     

P~(38)激酶在PC12细胞缺氧/再给氧损伤中的作用

目的:探讨PC12细胞缺氧/再给氧损伤的信号转导机理。方法:培养的PC12细胞先缺氧(95％N2/5％CO2)6h,然后重新给氧,观测不同时间点细胞的存活率和caspase-3的活性;用MTT法测存活率,caspase-3检测试剂盒测caspase-3活性。用p38拮抗剂SB203580孵育细胞2h,之后缺氧/再给氧,观察SB203580对细胞存活率和caspase-3活性的影响。结果:PC12细胞缺氧/再给氧后caspase-3活性明显增加并使细胞存活率下降,SB203580明显降低缺氧/复氧后caspase-3的活性并使细胞死亡减少。结论:PC12细胞缺氧/再给氧后至少可以通过激活p38、caspase-3信号分子诱导PC12细胞死亡。