Lee-Hong Chang

Stability of brain intracellular lactate and 31P-metabolite levels at reduced intracellular pH during prolonged hypercapnia in rats.

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 ∼20%, and Pi increased by ∼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 (∼6.5) are well tolerated in rats for periods up to 75 min if adequate oxygenation is maintained. The prolonged stability of metabolite concentrations during hypercapnia makes its use convenient for in vivo animal studies of the relevance of pHi to brain injury.

Tolerance of Low Intracellular pH During Hypercapnia by Rat Cortical Brain Slices: A 31P/1H NMR Study

Abstract: Metabolic tolerance of low intracellular pH (pHi) 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 (sim 3.2 g wet weight) were suspended in an NMR tube, perfused with artificial CSF (ACSF), and studied at 4.7 T with 31P and 1H NMR spectroscopy. Spectra obtained every 5 min monitored relative concentrations of lactate or high‐energy phosphate metabolites, from which pHi and extracellular pH were determined. Unperturbed slice preparations were metabolically stable for > 10 h, with no significant changes occurring in pHi, 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% O2/5% CO2 gas mixture. In each of five separate 1H and 31P experiments, brain slices were recovered within 2 h after termination of exposure to high CO2. The pHi was determined from measurements of the chemical shift difference between phosphoethanolamine and PCr, using a calibration curve obtained for our preparation. During hypercapnia, pHi decreased from 7.18 pmn 0.22 to 6.79 pmn 0.13, 6.60 pmn 0.11, 6.33 pmn 0.40, and 6.37 pmn 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 pmn 0.15 to 1.35 pmn 0.32 (n = 5, p = 0.0024). The administration of 70% CO2 also caused complete depletion of PCr and a 46 pmn 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 pHi attainable in our protocol using 30% O2/70% CO2 in our brain slice preparation was sim 6.3.

Effect of Dichloroacetate on Recovery of Brain Lactate, Phosphorus Energy Metabolites, and Glutamate During Reperfusion After Complete Cerebral Ischemia in Rats

The effects of dichloroacetate (DCA) on brain lactate, intracellular pH (pHi), phosphocreatine (PCr), and ATP during 60 min of complete cerebral ischemia and 2 h of reperfusion were investigated in rats by in vivo 1H and 31P magnetic resonance spectroscopy; brain lactate, water content, cations, and amino acids were measured in vitro after reperfusion. DCA, 100 mg/kg, or saline was infused before or immediately after the ischemic period. Preischemic treatment with DCA did not affect brain lactate or pHi during ischemia, but reduced lactate and increased pHi after 30 min of reperfusion (p < 0.05 vs. controls) and facilitated the recovery of PCr and ATP during reperfusion. Postischemic DCA treatment also reduced brain lactate and increased pHi during reperfusion compared with controls (p < 0.05), but had little effect on PCr, ATP, or Pi during reperfusion. After 30 min of reperfusion, serum lactate was 67% lower in the postischemic DCA group than in controls (p < 0.05). The brain lactate level in vitro was 46% lower in the postischemic DCA group than in controls (p < 0.05). DCA did not affect water content or cation concentrations in either group, but it increased brain glutamate by 40% in the preischemic treatment group (p < 0.05). The potential therapeutic effects of DCA on brain injury after complete ischemia may be mediated by reduced excitotoxin release related to decreased lactic acidosis during reperfusion.

Tolerance of low cerebral intracellular pH in rats during hyperbaric hypercapnia.

Background and Purpose Brain addosis from cerebral ischemia is characterized by average intraceiiular pH levels of approximately 5.8-6.2, which appear in turn to worsen cellular injury. We report that the brain is not injured when hypercapnia is used to reduce intraceiiular pH to about 62 during adequate oxygenation. A hyperbaric chamber is needed to achieve intraceiiular pH values so low because inspired CO2 tensions must be increased to approximately 1 atm. Summary of Report Using in vivo phosphorus-31 and proton nuclear magnetic resonance spectroscopy, we measured brain intraceiiular pH and lactate concentration of rats inside a nonmagnetic polycarbonate chamber at a barometric pressure of 1,500 mm Hg. Intubated rats were ventilated with a 50% O2/50% CO2 gas mixture for specific times. All six rats ventilated for 15 minutes with CO2 tensions of approximately 750 mm Hg woke up without neurological impairment, despite a decrease in intraceiiular pH to about 6.2. Higher CO2 tensions and longer exposures resulted in cardiovascular collapse and sudden death, followed by the postmortem appearance of brain lactate. Conclusions Brain intraceiiular pH values near 62 can be induced briefly in vivo in ventilated rats without injury under hyperbaric hypercapnic conditions. If attempts are made to lower brain pH in vivo even further by increasing PCO2 beyond 750 mm Hg, mean arterial blood pressure and cerebral blood flow decrease to values incompatible with life.

Adult rat brain-slice preparation for nuclear magnetic resonance spectroscopy studies of hypoxia

Background When perfused neonatal brain slices are studied ex vivo with nuclear magnetic resonance (NMR) spectroscopy, it is possible to use sup 31 Phosphorus detection to monitor levels of intracellular adenosine triphosphate (ATP), cytosolic pH, and other high‐energy phosphates and1 Hydrogen detection to monitor lactate and glutamate. Adult brain slices of high metabolic integrity are more difficult to obtain for such studies, because the adult cranium is thicker, and postdecapitation revival time is shorter. A common clinical anesthesia phenomenon‐‐loss of temperature regulation during anesthesia, with surface cooling and deep hypothermia, was used to obtain high‐quality adult rat cerebrocortical slices for NMR studies. Methods Spontaneously breathing adult rats (350 g), anesthetized with isoflurane in a chamber, were packed in ice and cooled until rectal temperatures decreased to [nearly equal] 30 degrees C. An intraaortic injection of heparinized saline at 4 degrees C further cooled the brain to [nearly equal] 18 degrees C. Slices were obtained and then recovered at 37 degrees C in oxygenated medium. Interleaved31 Phosphorous/sup 1 Hydrogen NMR spectra were acquired continually before, during, and after 20 min of no‐flow hypoxia (PO2 [nearly equal] 0 mmHg). Histologic (Nissl stain) measurements were made from random slices removed at different times in the protocol. Three types of pretreatment were compared in no‐flow hypoxia studies. The treatments were: (1) hyperoxia; (2) hypercapnia (50% CO2); and (3) hypoxia, which was accomplished by washing the slices with perfusate equilibrated with 100% Nitrogen2 and maintaining a 100% Nitrogen2 gas flow in the air space above the perfusate. Results During hyperoxia,31 Phosphorus NMR metabolite ratios were identical to those seen in vivo in adult brains, except that, in vitro, the Phosphorusi peak was slightly larger than in vivo. A lactate peak was seen in in vitro1 Hydrogen spectra of slices after metabolic recovery from decapitation, although lactate is barely detectable in vivo in healthy brains. The in vitro lactate peak was attributed to a small population of metabolically impaired cells in an injury layer at the cut edge. NMR spectral resolution from the solenoidal coil exceeded that obtained in vivo in surface coil experiments. Phosphocreatine and ATP became undetectable during oxygen deprivation, which also caused a three‐ to sixfold increase in the ratio of lactate to N‐acetyl‐aspartate. Within experimental error, all metabolite concentrations except pHi recovered to control values within 2 h after oxygen restoration. Nissl‐stained sections suggested that pretreatment with hypercapnia protected neurons from cell swelling during the brief period of no‐flow oxygen deprivation. Conclusions Perfused, respiring adult brain slices having intact metabolic function can be obtained for NMR spectroscopy studies. Such studies have higher spectral resolution than can be obtained in vivo. During such NMR experiments, one can deliver drugs or molecular probes to brain cells and obtain brain tissue specimens for histologic and immunochemical measures of injury: Important ex vivo NMR spectroscopy studies that are difficult or impossible to perform in vivo are feasible in this model.

Modulation of Glutamate-Induced Intracellular Energy Failure in Neonatal Cerebral Cortical Slices by Kynurenic Acid, Dizocilpine, and NBQX

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 μm 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 μM) and tetrodotoxin (1 mM) to examine glutamate receptor specificity in this tissue model. Dizocilpine (150 μM) 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%. Additionally, the dizocilpine-induced recovery of ATP levels, complete after 30 min of glutamate exposure, lasted througout 30 additional min of glutamate exposure and 60 additional min of washout with glutamate-free ACSF. Although dizocilpine did not alter the maximum decrease that occurred in PCr (to 36 ± 4% of control), dizocilpine did cause PCr levels to return to within 7 ± 5% of the control after 30 min of glutamate exposure. PCr levels stayed at this value throughout 30 additional min of glutamate exposure. During the washout period PCr immediately rose to a value 5 ± 2% above the control and then remained constant during the rest of the 60-min washout. During the first 20 min of glutamate administration, kynurenic acid (1.0 mM) best improved the high-energy phosphate levels. NBQX (6.0 μM), reported to protect the brain from ischemic injury, decreased PCr depletion during glutamate exposure without affecting the loss of ATP. After 60 min of glutamate washout, PCr levels with kynurenate (84 ± 6% of control) and NBQX (84 ± 2% of control) were significantly higher (p < 0.001) than with glutamate alone (42 ± 6% of control), although ATP levels were not significantly improved by either drug. Acute energy failure in our brain slice model, intended to simulate oxygenated penumbral tissue, probably occurs primarily in neurons. The reason that dizocilpine best preserves high-energy phosphate levels might relate to its mechanism of N-methyl-d-aspartate receptor blockade. Additional energy protection from dizocilpine might also arise from a partial blockade of voltage-dependent Na+ channels, which is possible at the concentration used.

Absence of abundant binding sites for anesthetics in rabbit brain : an in vivo NMR study

Using magnetic resonance spectroscopy, the authors tested whether cerebral concentrations of inhaled anesthetics do not increase proportionately at inspired concentrations exceeding 3% 1) because anesthetics bind to and saturate specific sites in the brain or 2) because anesthetic-induced depression of ventilation limits the increase in alveolar anesthetic partial pressure. New Zealand White rabbits were anesthetized with methohexital, 70% nitrous oxide, and local infiltration of 1% lidocaine. Cerebral concentrations of anesthetic were determined from 19F spectra acquired with nuclear magnetic resonance (NMR). Inspired, end-tidal, and arterial anesthetic concentrations, and end-tidal and arterial partial pressure of carbon dioxide were measured. Blood/gas partition coefficients were determined and used to convert arterial anesthetic concentration to partial pressures. In seven spontaneously breathing animals, halothane (1%; n = 5) or isoflurane (0.8%; n = 2) was administered at a constant inspired concentration for 20 min; NMR spectra were acquired between 10 and 20 min. Thereafter, the inspired concentration was increased and the process repeated until apnea occurred. Two additional rabbits were anesthetized with isoflurane and studied similarly but with higher inspired concentrations during mechanical ventilation. In spontaneously breathing animals, ventilatory depression occurred, documented by marked increases in PaCO2, and cerebral concentrations of anesthetic did not increase proportionately at inspired concentrations exceeding 3%. In contrast to an absence of a correlation of inspired and cerebral concentrations during spontaneous ventilation, arterial and cerebral concentrations correlated linearly during both spontaneous and mechanical ventilation (R2 greater than 0.969). These results are consistent with depression of ventilation, rather than binding to specific cerebral sites as an explanation for the nonlinear relationship between cerebral and inspired anesthetic concentrations.

In situ brain metabolism

As an understanding of cerebral metabolism and circulation may have practical consequences for the treatment of brain injury and for surgery, a considerable body of knowledge has been gathered on the subject over a period of a t least twenty years.’-* Probably the most striking aspect of the subject is its complexity. The interplay of biochemical and physiological events when cerebral ischemia and hypoxia occur has still not been elucidated. Ischemia, i.e., either partial or total restriction of cerebral blood flow, presents the major medical problem of stroke. Hypoxia (low oxygen levels with normal cerebral blood flow) poses a concern during pulmonary failure, anesthetic malfeasance, and in high altitudes. The effects of ischemia and hypoxia are not identical. These cerebral insults exert various interrelated effects on morphological structure, function, and chemistry that are not simply reversed with reperfusion or restoration of oxygen. Since N M R spectroscopy has the potential for following some metabolic processes noninvasively, there has been some effort made to develop N M R as a technology to examine cerebral metabolism. Much is known about the mechanism of injury associated with cerebral ischemia. Gross physiological problems include brain edema, increased intracranial pressure, microcirculatory compromise, and post-ischemic recirculation problems, such as the “no-reflow” phenomenon and “loss of reperfusion” syndrome. Biochemical and in tracellular physiological aspects include low intracellular pH; the calcium-induced arachidonic acid cascade, excitotoxins, preischemic glucose excess, and oxygenderived free radical toxicity. Although much is known, optimum clinical stratagems for “brain protection” and “brain resuscitation” remain to be developed. It is suspected that much of the injury secondary to ischemia occurs during reperfusion and that an optimum regimen for reperfusion has yet to be developed. Many patients must also tolerate unavoidable periods of cerebral ischemia during surgery. Regulation of cellular energy metabolism and the consequences of lack of regulation are central to the problems of ischemia and hypoxia. The important factors fur regulation have been r e ~ i e w e d . ~ ATP is the bridge between the metabolic reactions that produce energy (glycolysis in the cytosol and oxidative phosphorylation in the mitochondria) and the energy-requiring functions of the cell including biosynthesis (gluconeogenesis, lipogenesis, protein synthesis, and nucleic acid synthesis), muscle contraction, and ion transport (to maintain ion gradients across cell membranes, transepithelial transport, and nerve conduction). The vast majority of ATP is produced

19F NMR calcium changes, edema and histology in neonatal rat brain slices during glutamate toxicity

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.

Modulation of Edema by Dizocilpine, Kynurenate, and NBQX in Respiring Brain Slices After Exposure to Glutamate

Brain edema caused by glutamate excitotoxicity was studied in well oxygenated neonatal cerebrocortical brain slices (350 mu thick). Slices exposed to 60 minutes of 2 mM glutamate, with or without glutamate antagonists (dizocilpine, kynurenate, or NBQX), were allowed to recover for 60 minutes. The protocol was identical to that in noninvasive multinuclear NMR spectroscopy studies (31P/1H/19F) of live slices. Percent water and swelling were determined invasively in isolated slices by wet and dry weight measurements before and after glutamate exposure. Edema was detectable within minutes in all experiments with glutamate exposures, but not in untreated control slices. Dizocilpine, kynurenate, and NBQX differently affected swelling, which correlated with PCr and ATP loss in separate NMR studies. Synaptic glutamate receptor activation appears to initiate events causing both edema and energy failure. Multiple glutamate receptor types seem to be involved. No glutamate antagonist provided greater protection against both edema and energy loss than dizocilpine. Dizocilpine might also block voltage-dependent Na+ channels, and provide protection via mechanisms other than NMDA-receptor dependent channel antagonism.