RESUMEN
Administration of ethyl pyruvate, which is formed from pyruvate and ethanol, has been found capable of rescuing cells injured by oxidative stress. In one perspective the rescue has been postulated to be metabolic, with the resulting intracellular delivery of pyruvate seen as providing substrate for the TCA Cycle, making it possible to counteract sequela of poly(ADP-ribose)ribosylation, such as depletion of cytosolic NAD(+), glycolytic arrest, and mitochondrial deprivation of pyruvate. The rescue has also been attributed to radical scavenging via the carbonyl groups in ethyl pyruvate and pyruvate. In a previous study we exposed superfused neonatal (P7) brain slices for 60min to 2mM H(2)O(2) and found evidence for both rescue mechanisms. To see if ethyl pyruvate's actions stemmed more from being an antioxidant than from being a nutrient we conducted six new experiments using the same H(2)O(2) protocol, but with two new rescue solutions: [10mM] glucose (glc) plus one of the following: ethyl pyruvate [20mM], or the nonmetabolizable radical scavenger N-tert-butyl-alpha-phenylnitrone (PBN, 1mM). Final ATP values compared to initial, measured in 14.1T (31)P NMR spectra of PCA extracts, were the same: 0.70+/-0.08 for the former (N=3), and 0.64+/-0.08 for the latter (N=3). Quantifications of this study's (1)H NMR metabolites, also measured at 14.1T, exhibited separate clustering when pooled with data from the previous study and compared in a metabolomic multivariate analyses. Because the addition of ethyl pyruvate provided the same ATP protection as the addition of a nonmetabolizable antioxidant, antioxidant protection was its prominent protective mechanism in the chosen, high glucose protocol. Having distinct clusters in the Scores Plot of a Partial Least Squares-Discriminant Analysis suggests the feasibility of constructing statistical models that are predictive.
Asunto(s)
Animales Recién Nacidos/metabolismo , Antioxidantes , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Peróxido de Hidrógeno/toxicidad , Estrés Oxidativo/efectos de los fármacos , Piruvatos/farmacología , Adenosina Trifosfato/metabolismo , Animales , Ciclo del Ácido Cítrico/efectos de los fármacos , Óxidos N-Cíclicos/metabolismo , Técnicas In Vitro , Espectroscopía de Resonancia Magnética , Fosfolípidos/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The effects of hypoxic hypoxia on high-energy phosphate metabolites and intracellular pH (pHi) in the brain of the anesthetized infant rabbit were studied in vivo using 31P nuclear magnetic resonance spectroscopy. Five 10- to 16-day-old rabbits were anesthetized with 1.5% halothane. Ventilation was controlled to maintain normocarbia. Inspired O2 fraction was adjusted to produce three states of arterial oxygenation: hyperoxia (PaO2 greater than 250 mm Hg), normoxia (PaO2 approximately 100 mm Hg), and hypoxia (PaO2 25-30 mm Hg). During hypoxia, blood pressure was kept within 20% of control values with a venous infusion of epinephrine. During hyperoxia, the phosphocreatine-to-ATP ratio was 0.86, a value that is 2-2.5 times less than that reported for adults. During normoxia, ATP decreased by 20% and Pi increased by 90% from hyperoxia values. During 60 min of hypoxia, the concentrations of high-energy phosphate metabolites did not change, but intracellular and arterial blood pH (pHa) decreased significantly. When hyperoxia was reestablished, pHi returned to normal and pHa remained low. These results suggest that during periods of hypoxemia, the normotensive infant rabbit maintains intracellular concentrations of cerebral high-energy phosphates better than has been reported for adult animals.
Asunto(s)
Encéfalo/metabolismo , Espacio Extracelular/metabolismo , Hipoxia/metabolismo , Fosfatos/metabolismo , Adenosina Trifosfato/metabolismo , Anestesia , Animales , Animales Recién Nacidos , Isquemia Encefálica/metabolismo , Metabolismo Energético , Halotano , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Fosfocreatina/metabolismo , Fósforo , ConejosRESUMEN
31P nuclear magnetic resonance (NMR) spectroscopy was used noninvasively to measure in vivo changes in intracellular pH and intracellular phosphate metabolites in the brains of rats during supercarbia (PaCO2 greater than or equal to 400 mm Hg). Five intubated rats were mechanically ventilated with inspired gas mixtures containing 70% CO2 and 30% O2. Supercarbia in the rat was observed to cause a greater reduction in cerebral intracellular pH (pHi) and increase in PCO2 than observed in other experiments with rats after 15 min of global ischemia. Complete neurologic and metabolic recovery was observed in these animals, despite and average decrease in pHi of 0.63 +/- 0.02 pH unit during supercarbia episodes that raised PaCO2 to 490 +/- 80 mm Hg. No change was observed in cerebral intracellular ATP and only a 25% decrease was detected in phosphocreatine. The concentration of free cerebral intracellular ADP, which can be calculated if one assumes that the creatine kinase reaction is in equilibrium, decreased to approximately one-third of its control value. The calculated threefold decrease in the concentration of free ADP and twofold increase in the cytosolic phosphorylation potential suggest that there is increased intracellular oxygenation during supercarbia. Because a more than fourfold increase in intracellular hydrogen ion concentration was tolerated without apparent clinical injury, we conclude that so long as adequate tissue oxygenation and perfusion are maintained, a severe decrease in intracellular pH need not induce or indicate brain injury.
Asunto(s)
Encéfalo/metabolismo , Espacio Extracelular/metabolismo , Hipercapnia/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Análisis de los Gases de la Sangre , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Fosfocreatina/metabolismo , Fósforo , Ratas , Ratas EndogámicasRESUMEN
The tolerance of low intracellular pH (pHi) was examined in vivo in rats by imposing severe, prolonged respiratory acidosis. Rats were intubated and ventilated for 10 min with 20% CO2, for 75 min with 50% CO2, and for 10 min with 20% CO2. The maximum PaCO2 was 320 mm Hg. Cerebral intracellular lactate, pHi, and high-energy phosphate metabolites were monitored in vivo with 31P and 1H nuclear magnetic resonance (NMR) spectroscopy, using a 4.7-T horizontal instrument. Within 6 min after the administration of 50% CO2, pHi fell by 0.57 +/- 0.03 unit, phosphocreatine decreased by approximately 20%, and Pi increased by approximately 100%. These values were stable throughout the remainder of the hypercapnic period. Cerebral intracellular lactate, visible with 1H NMR spectroscopy in the hyperoxic state, decreased during hypercapnia, suggesting either a favorable change in oxygen availability (decreased lactate production) or an increase in lactate clearance or both. All hypercapnic animals awakened and behaved normally after CO2 was discontinued. Histological examination of cortical and hippocampal areas, prepared using a hematoxylin and eosin stain, showed no areas of necrosis and no glial infiltrates. However, isolated, scattered, dark-staining, shrunken neurons were detected both in control animals (no exposure to hypercapnia) and in animals that had been hypercapnic. This subtle histological change could represent an artifact resulting from imperfect perfusion-fixation, or it could represent subtle neurologic injury during the hypercapnia protocol. In summary, extreme hypercapnia and low pHi (approximately 6.5) are well tolerated in rats for periods up to 75 min if adequate oxygenation is maintained.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Acidosis Respiratoria/metabolismo , Encéfalo/metabolismo , Hipercapnia/metabolismo , Lactatos/metabolismo , Fosfatos/metabolismo , Acidosis Respiratoria/etiología , Animales , Hidrógeno , Hipercapnia/complicaciones , Espectroscopía de Resonancia Magnética , Fósforo , Ratas , Ratas EndogámicasRESUMEN
The severity and rapidity of acute, glutamate-induced energy failure were compared in live cerebral cortical slices. In each experiment 80 live cerebral cortical slices (350 microns thick) were obtained from neonatal Sprague-Dawley rats, suspended and perfused in a nuclear magnetic resonance (NMR) tube, and studied at 4.7 T with interleaved 31P/1H NMR spectroscopy. NMR spectra, obtained continually, were determined as 5-min averages. Slices were perfused for 60 min with artificial cerebrospinal fluid (ACSF) containing either glutamate alone or glutamate mixed with one of three glutamate-receptor antagonists: kynurenate, dizocilpine (MK-801), and 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(F)quinoxaline (NBQX). Dose-dependent decreases in high-energy phosphates were studied during glutamate exposure (0.5 to 10 mM), with and without antagonist protection. Energy recovery after glutamate exposures was measured during a 60-min washout with glutamate-free, antagonist-free ACSF. Reversible and irreversible energy failures were characterized by changes in intracellular pH, and by changes in relative concentrations of ATP, phosphocreatine (PCr), and inorganic phosphate. No changes were observed in intracellular levels of N-acetylaspartate and lactate. Some special studies were also done using R-(-)-2-amino-5-phosphonovaleric acid (100 microM) and tetrodotoxin (1 mM) to examine glutamate receptor specificity in this tissue model. Dizocilpine (150 microM) best ameliorated the energy failure caused by 2.0 mM glutamate. With dizocilpine the maximum ATP decrease was only 6 +/- 5%, instead of 35 +/- 7%.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Corteza Cerebral/metabolismo , Metabolismo Energético , Glutamatos/farmacología , Membranas Intracelulares/metabolismo , Neurotoxinas/farmacología , Adenosina Trifosfato/metabolismo , Animales , Animales Recién Nacidos , Maleato de Dizocilpina/farmacología , Ácido Glutámico , Técnicas In Vitro , Ácido Quinurénico/farmacología , Fosfocreatina/metabolismo , Quinoxalinas/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Qualitatively different responses of ADP levels have previously been observed in the brain during hypercarbia. One investigation has found that cerebral ADP stayed constant during hypercarbia in rats that were anesthetized with halothane, while another observed that ADP decreased during supercarbia in rats that received no supplemental anesthesia. This article reports an in vivo 31P nuclear magnetic resonance study to test the hypothesis that halothane anesthesia accounts for the discrepant observations. Isoflurane anesthesia was also studied in a second group of rats to see if a different general anesthetic agent would cause the same effects that halothane causes. The two groups of five rats underwent dual episodes of hypercarbia that were separated by a 45-min recovery period. General anesthesia, either 0.5% halothane or 1.0% isoflurane, was administered during the first episode but not during the second. Hypercarbia during halothane anesthesia caused the measured phosphocreatine (PCr) to decrease by 40%, while the calculated change in ADP was 10%, in agreement with the former investigation. In contrast, hypercarbia during either isoflurane anesthesia or no anesthesia caused a decrease of only 10% in PCr, which meant that the calculated decrease in ADP was 60%, in agreement with the results of the second investigation. We conclude that during hypercarbia, clinical concentrations of halothane, unlike clinical concentrations of isoflurane, interfere with the regulation of ATP metabolism.
Asunto(s)
Adenosina Difosfato/metabolismo , Encéfalo/metabolismo , Halotano/farmacología , Hipercapnia/metabolismo , Isoflurano/farmacología , Éteres Metílicos/farmacología , Anestesia General , Animales , Encéfalo/efectos de los fármacos , Electroencefalografía , Espectroscopía de Resonancia Magnética , Fosfocreatina/metabolismo , Ratas , Ratas EndogámicasRESUMEN
Clinical studies have shown enhancement of cyclosporine toxicity when co-administered with the immunosuppressant sirolimus. We evaluated the biochemical mechanisms underlying the sirolimus/cyclosporine interaction on rat brain metabolism using magnetic resonance spectroscopy (MRS) and compared the effects of sirolimus with those of the structurally related RAD. Two-week-old rats (25 g) were allocated to the following treatment groups (all n=6): I. control, II. cyclosporine (10 mg kg(-1) d(-1)), III. sirolimus (3 mg kg(-1) d(-1)), IV. RAD (3 mg kg(-1) d(-1)), V. cyclosporine+sirolimus and VI. cyclosporine+RAD. Drugs were administered by oral gavage for 6 days. Twelve hours after the last dose, metabolic changes were assessed in brain tissue extracts using multinuclear MRS. Cyclosporine significantly inhibited mitochondrial glucose metabolism (glutamate: 78+/-6% of control; GABA: 67+/-12%; NAD(+): 76+/-3%; P<0.05), but increased lactate production. Sirolimus and RAD inhibited cytosolic glucose metabolism via lactate production (sirolimus: 81+/-3% of control, RAD: 69+/-2%; P<0.02). Sirolimus enhanced cyclosporine-induced inhibition of mitochondrial glucose metabolism (glutamate: 60+/-4%; GABA: 59+/-8%; NAD(+): 45+/-5%; P<0.02 versus cyclosporine alone). Lactate production was significantly reduced. In contrast, RAD antagonized the effects of cyclosporine (glutamate, GABA, and NAD(+), not significantly different from controls). The results can partially be explained by pharmacokinetic interactions: co-administration increased the distribution of cyclosporine and sirolimus into brain tissue, while co-administration with RAD decreased cyclosporine brain tissue concentrations. In addition RAD, but not sirolimus, distributed into brain mitochondria. The combination of cyclosporine/RAD compares favourably to cyclosporine/sirolimus in regards to their effects on brain high-energy metabolism and tissue distribution in the rat.
Asunto(s)
Encéfalo/efectos de los fármacos , Inmunosupresores/farmacología , Mitocondrias/efectos de los fármacos , Animales , Ácido Aspártico/análogos & derivados , Ácido Aspártico/efectos de los fármacos , Ácido Aspártico/metabolismo , Peso Corporal/efectos de los fármacos , Encéfalo/metabolismo , Ciclosporina/sangre , Ciclosporina/farmacología , Sinergismo Farmacológico , Everolimus , Ácido Glutámico/efectos de los fármacos , Ácido Glutámico/metabolismo , Glutamina/efectos de los fármacos , Glutamina/metabolismo , Inmunosupresores/sangre , Espectroscopía de Resonancia Magnética , Mitocondrias/metabolismo , Ácido Oxaloacético/metabolismo , Fosfatos/metabolismo , Ratas , Ratas Wistar , Sirolimus/análogos & derivados , Sirolimus/sangre , Sirolimus/farmacología , Aumento de Peso/efectos de los fármacos , Ácido gamma-Aminobutírico/efectos de los fármacos , Ácido gamma-Aminobutírico/metabolismoRESUMEN
1. SDZ-RAD, 40-O-(2-hydroxyethyl)-rapamycin, is a novel macrolide immunosuppressant. Because of its synergistic interaction, SDZ-RAD is under clinical investigation as immunosuppressant in combination with cyclosporine after organ transplantation. Neurotoxicity is a critical side-effect of cyclosporine. 2. We studied the effect of SDZ-RAD and its combination with cyclosporine on high-energy phosphates, phosphocreatine (PCr) and nucleoside triphosphates (NTP), in brain slices using 31P-magnetic resonance spectroscopy (MRS). 3. Cyclosporine significantly reduced high-energy phosphates after 2 h in a dose-dependent manner (100 micrograms l-1: 93 +/- 3% of control (NTP), 91 +/- 3% (PCr); 500 micrograms l-1: 84 +/- 2% (NTP), 73 +/- 2 (PCr); 5000 micrograms l-1: 68 +/- 3% (NTP), 55 +/- 5% (PCr); n = 6; P < 0.02). 4. In contrast, after perfusion for 2 h, SDZ-RAD (500 micrograms l-1 and 5000 micrograms l-1) significantly increased high-energy phosphate concentrations in the brain slices (P < 0.02). Even at the lowest concentration, SDZ-RAD protected brain energy metabolism against cyclosporine toxicity: 100 micrograms l-1 SDZ-RAD + 5000 micrograms l-1 cyclosporine: 86 +/- 3% (NTP), 83 +/- 7% (PCr), n = 3, P < 0.03 compared to cyclosporine alone. 5. As evaluated using an algorithm based on Loewe isobolograms, the effects of SDZ-RAD/cyclosporine combinations on brain energy reduction were antagonistic. Both drugs were found in mitochondria using h.p.l.c-MS analysis. 6. We conclude that cyclosporine inhibits mitochondrial high-energy phosphate metabolism, which can be antagonized by SDZ-RAD.
Asunto(s)
Química Encefálica/efectos de los fármacos , Ciclosporina/antagonistas & inhibidores , Inmunosupresores/farmacología , Fosfatos/metabolismo , Sirolimus/análogos & derivados , Adenosina Trifosfato/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Ciclosporina/farmacología , Relación Dosis-Respuesta a Droga , Metabolismo Energético/efectos de los fármacos , Everolimus , Técnicas In Vitro , Espectrometría de Masas , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Perfusión , Fosfocreatina/metabolismo , Ratas , Ratas Wistar , Sirolimus/farmacologíaRESUMEN
Acetazolamide (AZ), a potent carbonic anhydrase inhibitor in human and animal tissues, increases cerebral blood flow (CBF) by acidifying cerebral extracellular fluids. To demonstrate the relationship of increased CBF to brain O2 availability after AZ administration, a compensated fluorometer was used to study changes in the cerebrocortical redox balance in rabbits. Seven rabbits were anesthetized with pentobarbital sodium. Excitation light (366 nm) was conducted to the cerebrocortical surface of each animal by a 4-mm-diam fiberoptic light guide. Fluorescence emissions from cerebrocortical NADH (450 nm) were compared at different inspired O2 (FIO2) tensions. Reflected light (366 nm), which was used to determine a correction to the fluorescence signal, was separately quantitated and interpreted as an index of cerebrocortical blood volume. Reductions in FIO2 from 1.0 to 0.21, 0.14, 0.10, and 0.07 resulted in increases in both tissue blood volume and [NADH]. Intravenous AZ (25 mg/kg) increased cerebrocortical blood volume and reduced the [NADH], even during ventilation with 100% O2. The changes in brain redox balance caused by vasodilation with AZ were compared with those caused by vasodilatation with CO2. The NAD+/NADH redox state was a continuous function of FIO2 at all levels of arterial PCO2 (PaCO2), both before and after AZ administration. The improvement in cerebral O2 delivery caused by AZ-induced vasodilation was comparable to that caused by the vasodilatation that results from a PaCO2 elevation approximately equal to 12-15 Torr above normal. The slope of the relationship between [NADH] and FIO2 was similar at normal, low, and high levels of PaCO2. We conclude that AZ administration and PaCO2 elevation improve cerebral oxygenation by similar mechanisms.
Asunto(s)
Acetazolamida/farmacología , Volumen Sanguíneo/efectos de los fármacos , Corteza Cerebral/efectos de los fármacos , NAD/metabolismo , Animales , Dióxido de Carbono/análisis , Ácido Carbónico/análisis , Corteza Cerebral/metabolismo , Fluorometría , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Oxígeno/farmacología , ConejosRESUMEN
Acetazolamide (AZ) inhibition of brain and blood carbonic anhydrase increases cerebral blood flow by acidifying cerebral extracellular fluid (ECF). This ECF acidosis was studied to determine whether it results from high PCO2, carbonic acidosis (accumulation of H2CO3), or lactic acidosis. Twenty rabbits were anesthetized with pentobarbital sodium, paralyzed, and mechanically ventilated with 100% O2. The cerebral cortex was exposed and fitted with thermostatted flat-surfaced pH and PCO2 electrodes. Control values (n = 14) for cortex ECF were pH 7.10 +/- 0.11 (SD), PCO2 42.2 +/- 4.1 Torr, PO2 107 +/- 17 Torr, HCO3- 13.8 +/- 3.0 mM. Control values (n = 14) for arterial blood were arterial pH (pHa) 7.46 +/- 0.03 (SD), arterial PCO2 (PaCO2) 32.0 +/- 4.1 Torr, arterial PO2 (PaO2) 425 +/- 6 Torr, HCO3- 21.0 +/- 2.0 mM. After intravenous infusion of AZ (25 mg/kg), end-tidal PCO2 and brain ECF pH immediately fell and cortex PCO2 rose. Ventilation was increased in nine rabbits to bring ECF PCO2 back to control. The changes in ECF PCO2 then were as follows: pHa + 0.04 +/- 0.09, PaCO2 -8.0 +/- 5.9 Torr, HCO3(-)-2.7 +/- 2.3 mM, PaO2 +49 +/- 62 Torr, and changes in cortex ECF were as follows: pH -0.08 +/- 0.04, PCO2 -0.2 +/- 1.6 Torr, HCO3(-)-1.7 +/- 1.3 mM, PO2 +9 +/- 4 Torr. Thus excess acidity remained in ECF after ECF PCO2 was returned to control values. The response of intracellular pH, high-energy phosphate compounds, and lactic acid to AZ administration was followed in vivo in five other rabbits with 31P and 1H nuclear magnetic resonance spectroscopy.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Acetazolamida/farmacología , Equilibrio Ácido-Base/efectos de los fármacos , Encéfalo/efectos de los fármacos , Animales , Dióxido de Carbono/análisis , Ácido Carbónico/análisis , Espacio Extracelular/análisis , Femenino , Lactatos/análisis , Ácido Láctico , Espectroscopía de Resonancia Magnética , Masculino , ConejosRESUMEN
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 microns 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.
Asunto(s)
Corteza Cerebral/metabolismo , Fructosadifosfatos/farmacología , Proteínas HSP70 de Choque Térmico/biosíntesis , Hipoxia Encefálica/metabolismo , Fármacos Neuroprotectores , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Transcripción Genética/efectos de los fármacos , Animales , Animales Recién Nacidos , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/patología , Hipoxia , Hipoxia Encefálica/patología , Inmunohistoquímica , Hibridación in Situ , Técnicas In Vitro , Cinética , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Respiring neonatal rat cerebrocortical slices were exposed for 30 min to toxic concentrations of N-methyl-D-aspartate (NMDA; 100 microM, 500 microM and 1000 microM). In situ hybridization was used to study c-fos and hsp70 mRNA before, during, and for 8 h after NMDA exposure. Cell swelling and nuclear morphology were assessed using Cresyl violet (Nissl) staining. Possible evidence for apoptosis was examined using in situ terminal transferase d-UTP nick-end labeling (TUNEL) staining and agarose-gel electrophoresis of extracted slice DNA. After NMDA administration c-fos and hsp70 mRNA expression increased, with maxima occurring, respectively, at 1 h and 4 h after NMDA exposure. When treatment with dizocilpine (MK-801; 10 microM), a non-competitive NMDA antagonist, was started before NMDA exposures, expression of both c-fos and hsp70 mRNA was decreased to values near control, indicating that activation of NMDA receptors induces both genes. Only a minority of induced cells expressed FOS protein and no HSP70 protein expression was seen. These apparent failures of translation might be related to the stress response. Histologically, 1000 microM NMDA produced substantial necrosis, with no evidence of apoptosis. Evidence for apoptosis was found at the two lower NMDA concentrations, which produced TUNEL-positive fragmented nuclei and faint ladder patterns in DNA electrophoresis. Dizocilpine pre-treatment blocked NMDA-induced necrosis and attenuated TUNEL-positive staining in slice parenchyma. TUNEL-positive staining with a different morphology was found in the injury layer, a region 50-micron thick where mechanical trauma was inflicted when slices were cut from brain. When slices received dizocilpine immediately after decapitation, TUNEL-positive staining no longer occurred in the injury layer, in agreement with previous cell culture studies that implicated NMDA receptor activation after mechanical trauma to neurons. We conclude that at the toxic doses studied, NMDA receptor activation results primarily in necrosis. However, data at low NMDA concentrations are consistent with a small amount of apoptosis.
Asunto(s)
Apoptosis/fisiología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/fisiología , Proteínas HSP70 de Choque Térmico/biosíntesis , N-Metilaspartato/farmacología , Neuronas/fisiología , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Transcripción Genética/efectos de los fármacos , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Corteza Cerebral/citología , Fragmentación del ADN , Maleato de Dizocilpina/farmacología , Genes fos , Técnicas In Vitro , Necrosis , Neuronas/citología , Neuronas/efectos de los fármacos , Perfusión , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Respiring neonatal cerebrocortical slices (350 microns thick), loaded with the free calcium indicator 5F-BAPTA, were perfused in a 20-mm-diameter glass NMR tube with oxygenated artificial CSF, exposed to extracellular glutamate and studied at 4.7 Tesla with 19F NMR spectroscopy. 31P/1H NMR spectra, obtained concurrently, were used to assess slice integrity from determinations of intracellular pH, ATP, PCr, lactate and N-acetylaspartate. 60-min periods were induced of recoverable and nonrecoverable glutamate toxicity-defined from changes in NMR metabolites. In other NMR studies, where 5F-BAPTA was not used, metabolic toxicity was modulated by three glutamate receptor antagonists: dizocilpine, NBQX and kynurenic acid. Outcome measurements were made of edema, determined invasively in isolated slices from % swelling and water content and from histological changes in Nissl stains of slice sections. Edema was (1) detectable in all slices within minutes after onset of glutamate exposure, though never in untreated control slices, and (2) modulated differently by dizocilpine, NBQX and kynurenate. Correlations were observed between edema and NMR decreases in PCr and ATP. Nissl stains of sections from slices treated with the most protective agent, dizocilpine, showed preservation of neuronal processes. As was expected in 7-day-old rats with immature NMDA receptors, 19F NMR spectroscopy revealed only small increases in free intracellular calcium ([Ca2+]i). These occurred late during glutamate exposure and reversed early during glutamate washout. The studies demonstrate that it is possible to study correlations between repeated noninvasive NMR spectra in ensembles of brain slices and invasive measures of early cellular responses.
Asunto(s)
Edema Encefálico/inducido químicamente , Encéfalo/efectos de los fármacos , Calcio/metabolismo , Glutamatos/toxicidad , Animales , Animales Recién Nacidos , Encéfalo/patología , Edema Encefálico/patología , Ácido Egtácico/análogos & derivados , Flúor , Ácido Glutámico , Técnicas In Vitro , Espectroscopía de Resonancia Magnética , Ratas , Ratas Sprague-DawleyRESUMEN
Spin-echo 19F magnetic resonance imaging was performed at 2.0 T to explore the in vivo spatial distribution of halothane in the rabbit head. Because the halothane concentration is low in vivo, and because the measured relaxation times of the 19F resonance peak for halothane were T1 approximately equal to 1.0 sec and T2 approximately equal to 3.5-65 msec, 1-3-h imaging times were required (TR = 1 sec, TE = 9 msec) in order to obtain adequate images with a 64 X 256 raw data matrix and a 20-mm slice thickness. With this technique, halothane was primarily detected in lipophilic regions of the rabbit head, but little or no halothane was observed in brain tissue. Because T2 was shorter in brain tissue than in surrounding fat, a shorter TE than we could obtain is needed for optimal spin-echo imaging of brain halothane.
Asunto(s)
Química Encefálica , Halotano/análisis , Espectroscopía de Resonancia Magnética , Animales , Médula Ósea/análisis , Lípidos/análisis , ConejosRESUMEN
Although mechanisms of hypothermic neuroprotection during oxygen deprivation have long been investigated, further characterizations of various molecular mechanisms are appropriate. Anticipating future studies of hypothermia and hypoxia/ischemia, we investigated the extent to which our ex vivo, NMR-based, superfused brain slice model might be helpful. (Slices are approximately 350 microm thick, with 18 slices per 8 mm NMR tube.) 31P NMR spectroscopic measurements were made of hypothermia-induced changes in high energy phosphates, while simultaneously monitoring and controlling tissue temperature, using 1H NMR, the high spectroscopic resolution available at 14.1 Tesla (600 MHz for protons), and a recently published protocol. NTP and PCr concentrations in healthy, well-oxygenated slices decreased to (55 +/- 15)% and (66 +/- 30)% of their respective values at 28.0 degrees C when warmed to 38.0 degrees C, in approximate agreement with earlier in vivo studies by others. During 30 min hypoxia NTP and PCr decreased to non-observable values, regardless of temperature. After reoxygenation, NTP and PCr recoveries as percentages of respective prehypoxia values were (63% +/- 16%; 70%) +/- 5%) for hypothermic slices (28.0 degrees C), and (46% +/- 13%; 41% +/- hypothermic neuroprotection during oxygen deprivation in this model, which appears suitable for use in further studies.
Asunto(s)
Corteza Cerebral/metabolismo , Frío , Hipoxia/metabolismo , Nucleótidos/metabolismo , Consumo de Oxígeno , Fosfatos/metabolismo , Fosfocreatina/metabolismo , Animales , Animales Recién Nacidos , Técnicas In Vitro , Espectroscopía de Resonancia Magnética , Perfusión , Protones , Ratas , Ratas Sprague-DawleyRESUMEN
Phosphorylation of Akt before hypoxia (30 min) and during reoxygenation (4 h) was evaluated in superperfused neonatal rat cerebrocortical slices (350 microm, P7, Sprague-Dawley). Cytosolic cytochrome c intensities in Western blots, which were increased at the end of hypoxia. were decreased during reoxygenation. Western blot intensities of phosphorylated Akt (phospho-Akt), nearly undetectable at the end of hypoxia, recovered quickly during reoxygenation, in a trend opposite that for cytochrome c. At 1.5 h and 4 h after hypoxia they became larger or the same as before hypoxia. Total Akt was unchanged by hypoxia and reoxygenation. Phosphocreatine (PCr) and nucleotide triphosphates (NTP) were measured in parallel 14.1 Tesla ex vivo 31P NMR superfused brain slice studies. PCr and alpha-NTP were nearly undetectable at the end of hypoxia. Although they recovered quickly after hypoxia, they were lower than before hypoxia. Reductions in phospho-Akt during hypoxia were consistent with the general unavailability of basic high energy phosphates. Preferential phosphorylation of Akt after hypoxia suggested that substantial reductions in intracellular energy, as indicated by PCr and NTP, might be tolerated by processes important for generating phospho-Akt. Additionally, the post-hypoxia increase in phospho-Akt might have contributed to concomitant reductions in cytosolic cytochrome c.