Local cerebral glucose metabolism during controlled hypoxemia in rats.

2-Deoxy-[14C]glucose metabolism was examined in brains of hypoxic, normotensive rats by autoradiography, which revealed alternating cortical columns of high and low metabolism. Activity in white matter was increased severalfold over that in adjacent gray matter. The columns were anatomically related to penetrating cortical arteries with areas between arteries demonstrating higher rates of metabolism. The results suggest the presence of interarterial tissue oxygen gradients that influence regional glucose metabolism. The relatively greater sensitivity of white matter metabolism to hypoxia may lead to an understanding of white matter damage in postanoxic leukoencephalopathy.

Alpha-ketoglutaramate: increased concentrations in the cerebrospinal fluid of patients in hepatic coma.

alpha-Ketoglutaramate, a deaminated metabolite of glutamine not previously identified in biological tissues, was measured in the cerebrospinal fluid of human subjects and found to be increased three- to tenfold in patients with hepatic coma. When perfused into the cerebral lateral ventricles of rats, alpha-ketoglutaramate (10 mM) depressed the animals' nocturnal locomotor activity, and at higher doses induced circling behavior and myoclonus. The concentration of alpha-ketoglutaramate in cerebrospinal fluid appears to be a reliable diagnostic indicator of hepatic coma, and its accumulation may contribute to the pathogenesis of this disease.

Effect of acute ammonia intoxication on cerebral metabolism in rats with portacaval shunts.

Rats were made chronically hyperammonemic by portal-systemic shunting and, 8 wk later, were subjected to acute ammonia intoxication by the intraperitoneal injection of 5.2 mmol/kg of ammonium acetate. In free-ranging animals, ammonia treatment induced a brief period of precoma (10-15 min) that progressed into deep, anesthetic coma lasting for several hours and was associated with a high mortality. In paralyzed, artificially ventilated animals that were lightly anesthetized with nitrous oxide, acute ammonia intoxication caused major disturbances of cerebral carbohydrate, amino acid, and energy metabolism that correlated in time with the change in functional state. At 10 min after injection (precoma), the concentrations of most glycolytic intermediates were increased, as was the lactate/pyruvate ratio. Citrate declined, despite a twofold rise in pyruvate, suggesting that the conversion of pyruvate to citrate had been impaired. Concentrations of phosphocreatine, and of the putative neurotransmitters, glutamate and aspartate, declined during precoma, but the concentrations of the adenine nucleotides in the cerebral hemispheres, cerebellum, and brain stem remained within normal limits. At 60 min after injection (coma), ATP declined in all regions of brain; the reduction in total high-energy phosphates was most notable in the brain stem. The findings indicate that cerebral dysfunction in chronic, relapsing ammonia intoxication is not due to primary energy failure. Rather, it is suggested that ammonia-induced depletion of glutamic and aspartic acids, and inhibition of the malate-asparate hydrogen shuttle are the dominant neurochemical lesions.

The dynamics of ammonia metabolism in man. Effects of liver disease and hyperammonemia.

The cyclotron-produced radionuclide, 13N, was used to label ammonia and to study its metabolism in a group of 5 normal subjects and 17 patients with liver disease, including 5 with portacaval shunts and 11 with encephalopathy. Arterial ammonia levels were 52-264 micron. The rate of ammonia clearance from the vascular compartment (metabolism) was a linear function of its arterial concentration: mumol/min = 4.71 [NH3]a + 3.76, r = +0.85, P less than 0.005. Quantitative body scans showed that 7.4 +/- 0.3% of the isotope was metabolized by the brain. The brain ammonia utilization rate, calculated from brain and blood activities, was a function of the arterial ammonia concentration: mumol/min per whole brain = 0.375 [NH3]a - 3.6, r = +0.93, P less than 0.005. Assuming that cerebral blood flow and brain weights were normal, 47 +/- 3% of the ammonia was extracted from arterial blood during a single pass through the normal brains. Ammonia uptake was greatest in gray matter. The ammonia utilization reaction(s) appears to take place in a compartment, perhaps in astrocytes, that includes less than 20% of all brain ammonia. In the 11 nonencephalopathic subjects the [NH3]a was 100 +/- 8 micron and the brain ammonia utilization rate was 32 +/- 3 mumol/min per whole brain; in the 11 encephalopathic subjects these were respectively elevated to 149 +/- 18 micron (P less than 0.01), and 53 +/- 7 mumol/min per whole brain (P less than 0.01). In normal subjects, approximately equal to 50% of the arterial ammonia was metabolized by skeletal muscle. In patients with portal-systemic shunting, muscle may become the most important organ for ammonia detoxification. Muscle atrophy may thereby contribute to the development of hyperammonemic encephalopathy with an associated increase in the brain ammonia utilization rate.

Local cerebral glucose metabolism in rats with chronic portacaval shunts.

Local cerebral glucose utilization was measured with the autoradiographic [14C]deoxyglucose method in rats at 2 days and 1, 4, 8, and 12 weeks after the construction of a portacaval shunt, and in weight-matched controls. Local glucose utilization in brain was altered in shunted animals, but the magnitude and direction of the changes differed among 36 neuroanatomical structures, depending upon the length of time that the animals had been shunted. In rats shunted for 4 weeks or less, glucose utilization did not differ from control (30 of 36 structures) or was decreased (6 structures). The largest decreases of glucose utilization, noted at 1 week, occurred in the parietal (-25%) and frontal cortices (-28%) and subcortical white matter (-50%. In rats shunted for 8 weeks, however, glucose utilization was higher than control in many brain structures (13 of 36), and after 12 weeks it was higher than control in most structures (25 of 36). Only the parietal cortex did not follow this trend; it exhibited a decreased rate of glucose utilization in rats shunted for 8 weeks (-21%) that normalized in animals shunted for 12 weeks. Portal-systemic shunting of blood increased arterial blood ammonia concentrations to twice the control value of 85 +/- 3 microM in animals shunted for 1 week, and to approximately 2.5 times control in animals shunted for 4-12 weeks. Kety-Schmidt measurements of cerebral blood flow and arterial-venous (torcular) differences for ammonia across the brains of control rats and rats with portacaval shunts for 8 weeks revealed an increased cerebral uptake of ammonia in the shunted animals. The late-developing morphological changes known to occur in astrocytes and the delayed increases in local glucose consumption in rats with portacaval shunts may be related, and represent an adaptive response to sustained hyperammonemia.

Status epilepticus in immature rats. Protective effects of glucose on survival and brain development.

The role of glucose metabolism in alleviating the complications of status epilepticus (SE) was investigated in developing rats. Pretreatment with glucose reduced mortality from SE by 90% in rats under 1 week of age, 80% in 10-day-old rats, 50% in 15- to 20-day-olds, and not at all in adults. In 4-day-old animals, brain DNA synthesis during seizures, and in survivors, brain weight, DNA, RNA, protein, and cholesterol contents at 7 days of age were reduced less in glucose-treated than in saline-treated littermates. In the saline group, seizures caused a progressive fall in brain glucose level but no fall in blood glucose level, suggesting that glucose transport from blood to brain could not keep pace with glycolytic demands. In glucose-treated rats, blood and brain glucose concentrations remained elevated throughout the convulsive period. There was no reduction of brain adenosine triphosphate levels in either group. Thus, the protection by glucose appears to be related to its roles as a carbon source rather than an energy source. It is concluded that in immature animals, depletion of brain glucose can occur in the absence of hypoglycemia, and may be an important and potentially treatable complication of status epilepticus.

Brain dysfunction in mild to moderate hypoxia.

Hypoxia is commonly invoked to explain alterations in mental function, particularly in patients with cardiac pulmonary failure. The effects of acute graded hypoxia or higher integrative functions are well documented experimentally in man. Hypoxia in experimental animal models demonstrates that the pathophysiology is complex. In mild to moderate hypoxia, in contrast to severe hypoxia and to ischemia, the supply of energy for the brain is not impaired; cerebral levels of adenosine triphosphate (ATP) and adenylate energy charge are normal. In contrast, the turnover of several neurotransmitters is altered by mild hypoxia. For example, acetylcholine synthesis is reduced proportionally to the reduction in carbohydrate oxidation. This relationship holds in vitro and with several in vivo models of hypoxia. Pharmacologic and physiologic studies in man and experimental animals are consistent with acetylcholine having an important role in mediating the cerebral effects of mild hypoxia. These observations raise the possibility that treatments directed to cholinergic or other central neurotransmitter systems may benefit patients with cerebral syndromes secondary to chronic hypoxia.

Comparison of radio-labeled butanol and iodoantipyrine as cerebral blood flow markers.

Cerebral blood flow (CBF) techniques based on the principle of indicator fractionation rely upon free diffusibility of the blood flow indicator into brain (i.e. complete cerebral extraction). Extraction of two commonly-used indicators, iodoantipyrine and n-butanol, was evaluated in rats by measuring torcular venous efflux after systemic injection of the indicator under conditions of normal and high CBF. The extraction of n-butanol was found to be virtually complete at all blood flows examined; iodoantipyrine, on the other hand, was completely extracted only at flows under 180 ml/100 g/min, despite the fact that the oil: water partition coefficient for iodoantipyrine exceeds that for n-butanol. Brain uptake indices for the two indicators were also measured: brain uptake of n-butanol was greater than that of iodoantipyrine, and the difference was more marked if the indicator entered brain mixed with blood than if it entered as a bloodless bolus. Blood components may thus interact with iodoantipyrine to retard its movement across the blood-brain barrier and thereby limit extraction of this lipid-soluble substance. Inasmuch as iodoantipyrine is diffusion-limited at blood flows above 180 ml/100 g/min, butanol is a more accurate CBF indicator above the normal flow range in the rat.

Regional energy balance in rat brain after transient forebrain ischemia.

Phosphocreatine, ATP, and glucose were severely depleted, and the lactate levels were increased in the paramedian neocortex, dorsal-lateral striatum, and CA1 zone of hippocampus of rats exposed to 30 min of forebrain ischemia. Upon recirculation of the brain, phosphocreatine, ATP, and lactate concentrations recovered to control values in the paramedian neocortex and CA1 zone of hippocampus and to near-control values in the striatum. The phosphocreatine and ATP concentrations then fell and the lactate levels rose in the striatum after 6-24 h, and in the CA1 zone of hippocampus after 24-72 h. The initial recovery and subsequent delayed changes in the phosphocreatine, ATP, and lactate concentrations in the striatum and hippocampus coincided with the onset and progression of morphological injury in these brain regions. The results suggest that cells in these regions regain normal or near-normal mitochondrial function and are viable, in terms of energy production, for many hours before unknown mechanisms cause irreversible neuronal before unknown mechanisms cause irreversible neuronal injury.

Impaired synthesis of acetylcholine by mild hypoxic hypoxia or nitrous oxide.

The effect of mild hypoxic hypoxia on brain metabolism and acetylcholine synthesis was studied in awake, restrained rats. Since many studies of hypoxia are done with animals anesthetized with nitrous oxide (N2O), the effects of N2O were evaluated. N2O (70%) increased the cerebral cortical blood flow by 33% and the cortical metabolic rate of oxygen by 26%. In addition, the synthesis of acetylcholine in N2O-anesthetized animals, measured with [U-14C]glucose and [1-2H2,2-2H2]choline, decreased by 45 and 53%, respectively. Consequently, mild hypoxia was studied in unanesthetized rats. Control rats breathing 30% O2 (partial pressure of oxygen, PaO2 = 120 mm Hg) were compared with rats exposed to 15% O2 (PaO2 = 57 mm Hg) or 10% O2 (PaO2 = 42 mm Hg). The synthesis of acetylcholine, measured with [U-14C]glucose, was decreased by 35 and 54% with 15% O2 and 10% O2, respectively; acetylcholine synthesis, measured with [1-2H2,2-2H2]choline, was decreased by 50 and 68% with 15% O2 and 10% O2, respectively. Animals breathing either 15% or 10% O2 had normal cerebral metabolic rates of oxygen but had increased brain lactates and increased cortical blood flows compared with animals breathing 30% O2. These results show that even mild hypoxic hypoxia impairs acetylcholine synthesis, which in turn may account for the early symptoms of brain dysfunction associated with hypoxia.

Regulation of local cerebral blood flow in normal and hypoxic newborn dogs.

Local cerebral blood flow (LCBF) was measured autoradiographically in newborn puppies by an indicator fractionation technique using 4-iodo-[14C]antipyrine as the diffusible indicator. Measurements were obtained in unanesthetized, normotensive animals, and the sensitivity of blood flow to hypercapnia and acute hypoxia was determined in 32 brain structures. LCBF in normal and hypoxic puppies was correlated with local cerebral glucose utilization (LCGU) obtained under the same experimental conditions (Duffy et al, 1982). In normocapnic (PaCO2 33 mm Hg) control animals, highest rates of blood flow were found in gray matter nuclei of the brainstem, in the medulla oblongata, and in the posterolateral nucleus of the thalamus (50 to 77 ml/100 gm/min); far lower flows were recorded among white matter structures (5 to 11 ml/100 gm/min). The vasodilatory response to both hypercapnia and hypoxia was greatest among brainstem gray matter structures, intermediate among cortical and diencephalic gray matter structures, and least in white matter. When LCBF was plotted as a function of LCGU for control animals, a positive linear correlation was obtained for all structures (p less than 0.001), implying that in newborns, as in adults, cerebral blood flow and metabolism are physiologically coupled. In hypoxic puppies, no consistent relationship between LCGU and LCBF could be demonstrated; however, there was suggestion that the two measurements correlated inversely, presumably reflecting enhanced anaerobic glycolysis in structures (e.g., hemispheric white matter) that were not adequately protected by compensatory hyperemia. White matter damage, a frequent complication of perinatal hypoxia-asphyxia, may be a consequence in part of the limited capacity of white matter to vasodilate in response to te chemical "signals" of hypercapnia and lactic acidosis.

Carbohydrate metabolism in fetal and neonatal rat brain during anoxia and recovery.

Systemic and cerebral metabolic responses to acute anoxia were studied in term-fetal and neonatal rats in order to account for the greater anoxic tolerance of fetuses. Measurements of blood acid-base balance were correlated with changes in the concentrations of adenine nucleotides, creatine, phosphocreatine, and glycogen in brain, and of glucose, pyruvate, and lactate in brain, blood, and cerebrospinal fluid during 1) exposure (20-40 min) to 100% nitrogen at 37 degrees C, and 2) subsequent recovery in air. Blood PCO2 was higher initially in fetuses and increased more rapidly during anoxia in fetuses than in neonates, exceeding 150 mmHg after 20 min. Brain glycogen, phosphocreatine, and total adenine nucleotides declined more slowly in fetuses than in neonates during anoxia, whereas brain glucose levels declined at similar rates in the two groups. From the changes in these preformed and potential energy stores, it was estimated that total cerebral energy consumption during anoxia was significantly lower in fetuses. The data suggest that the more severe hypercapnia superimposed on anoxia in fetuses decreased cerebral metabolic demands, and thus prolonged survival. An incidental finding was that L-lactate readily enters the immature brain from the blood during anoxia, and in the early recovery phase may constitute the preferred substrate for cerebral oxidative metabolism, sparing glucose.

Use of beta-methylene-D,L-aspartate to assess the role of aspartate aminotransferase in cerebral oxidative metabolism.

Several inhibitors of aspartate aminotransferase, a key enzyme of the malate-aspartate shuttle, were investigated for their effects on cerebral oxidative metabolism in vitro. beta-Methylene-D,L-aspartate (2 mM), aminooxyacetate (0.1 mM), and D,L-vinylglycine (20 mM) all significantly reduced the activity of aspartate aminotransferase and the rate of oxygen consumption of rat cerebral cortex slices respiring on glucose. In the presence of beta-methyleneaspartate, a one-to-one correlation was found between the degree of inhibition of tissue respiration and the degree of inhibition of transaminase activity. Slices of rat liver incubated in the presence of glucose and beta-methyleneaspartate showed a similar one-to-one relationship between inhibition of oxygen comsumption and inhibition of aspartate aminotransferase activity, whereas with rat kidney cortex slices, the inhibition of aspartate aminotransferase activity was greater than the inhibition of oxygen consumption. Structural analogs of beta-methyleneaspartate (D,L-beta-methyl-D,L-aspartate, gamma-methyl-D,L-glutamate, and alpha-methyl-D,L-didehydroglutamate) that did not inhibit the activity of aspartate aminotransferase similarly did not inhibit the rate of oxygen consumption by cerebral cortex slices. In the presence of beta-methyleneaspartate, pyruvate oxidation by cerebral cortex slices was inhibited to almost the same extent as was glucose oxidation, and the oxidation of succinate was decreased by approximately 20%. The artificial electron acceptor phenazine methosulfate (0.1 mM) only partially overcame the beta-methyleneaspartate-mediated inhibition of respiration with glucose as substrate. The content of ATP and phosphocreatine declined steadily in slices incubated with glucose and beta-methyleneaspartate. At 1 h the concentration of lactate and the lactate/pyruvate ratio, an indicator of the cytoplasmic redox state, increased threefold, whereas the concentrations of malate, citrate, and aspartate decreased. The findings are interpreted in the context of the hypothesis that enzymes common to the malate-aspartate shuttle and the tricarboxylic acid cycle are physically complexed in brain, so that inhibition of aspartate aminotransferase, a component of the complex, impedes the flow of carbon through both metabolic pathways.(ABSTRACT TRUNCATED AT 400 WORDS)

A mask for delivery of inhalation gases to small laboratory animals.

A mask was developed for the administration of volatile anesthetics and other gases to small, spontaneously breathing laboratory animals. A vacuum-powered venting system surrounding the inspiratory gas supply prevented potentially hazardous gases from escaping into the environment. This system was used to deliver nitrous oxide, halothane, and methoxyflurane to rats, gerbils, and newborn dogs. It was used to vary the oxygen and carbon dioxide concentrations inspired by spontaneously breathing animals undergoing physiological experiments.