David W. McCandless

Cerebral glucose utilization: comparison of [14C]deoxyglucose and [6-14C]glucose quantitative autoradiography.

Abstract: The [14C]deoxyglucose [Sokoloff et al., J. Neurochem.28, 897–916 (1977)] and [6‐14C]glucose [Hawkins et al., Am. J. Physiol.248, C170‐C176 (1985)] quantitative autoradiographic methods were used to measure regional brain glucose utilization in awake rats. The spatial resolution and qualitative appearance of the autoradiograms were similar. In resting animals, there was no significant difference between the two methods among 18 gray and three white matter structures over a fourfold range in glucose utilization rates (coefficient of correlation = 0.97). In rats given increasing frequencies of photoflash visual stimulation, the two methods gave different results for glucose utilization within visual pathways. The linearity of the metabolic response was studied in the superior colliculus using an on‐off checkerboard stimulus between 0 and 33 Hz. The greatest increment in activity occurred between 0 and 4 Hz stimulation with both methods, probably representing recruitment of neuronal elements into activity. Above 4 Hz, there was a progressive increase in labeling with [14C]de‐oxyglucose up to 1.7 times control at 33 Hz. With [6‐14C]‐glucose, there was no further increment in change above a 30% increase seen at 4 Hz. Measurement of tissue glucose revealed no drop in the visually stimulated structures compared to control. We interpret these results to indicate that, with increasing rates of physiological activity, the products of deoxyglucose metabolism accumulate progressively, but the products of glucose metabolism are removed from brain in 10 min.

Effect of Ammonium Chloride on Energy Metabolism of Astrocytes and C6-Glioma Cells In Vitro

Increased ammonia has been considered a key factor in the pathogenesis of hepatic encephalopathy. The high concentration of ammonia interferes with oxidative metabolism in the brain through an inhibitory effect on the tricarboxylic acid cycle (TCA). Inhibition of the TCA cycle may result in depletion of ATP. Due to the involvement of astrocytes in brain detoxification of ammonia, these cells are good candidates for studying ammonias effect on energy stores in the brain. C6-glioma cells, which have altered glycolytic rates, may show greater sensitivity to the toxicity of ammonium chloride than astrocytes. To study the effect of ammonium chlrode on energy storage of both astrocytes and C6-glioma, we observed the acute and chronic effects of NH4Cl (7.5 or 15 mM) on the metabolism of isolated astrocytes and C6-glioma cells. Primary astrocytes were isolated from the cerebral hemispheres of 1–2 day old Sprague-Dawley rats, and C6-glioma cells were purchased from the American Type Culture Collection (ATCC). Following treatment of the cells with ammonia, glucose, lactate, glutamate, ATP, and PCr were assayed. Our data showed that at 15 min following treatment with NH4Cl, there were no significant differences in the concentration of metabolites measured in astrocytes. However, following 15 min of treatment with NH4Cl, the concentration of some metabolites, for example. ATP and lactate, changed significantly in C6-glioma cells. We have shown that 24 h of treatment was sufficient time to see significant biochemical changes but not morphological changes in either cell type. Simultaneous biochemical and morphologicla changes were observed 48 h following treatment in C6-glioma cells and at 9–10 days following treatment in primary astrocytes. In primary astrocytes at 24 h following treatment, glucose utilization increased. This high utilization of glucose was in accordance with the increase in lactate and glutamate production and the decrease in ATP and PCr formation. In C6-glioma cells the utilization of glucose increased but this high utilization of glucose was consistent with a significant decrease in the concentration of lactate, glutamate and ATP.

Responses in primary astrocytes and C6-glioma cells to ammonium chloride and dibutyryl cyclic-AMP

Elevated brain ammonia levels are a major factor in the genesis of hepatic encephalopathy (HE). The mechanism of ammonium chloride (NH4Cl) neurotoxicity involves interruption of oxidative metabolism. This leads to decreased levels of ATP concentration and subsequent glial fibrillary acidic protein (GFAP) degradation of astrocytes and fibrous C6-glioma cells. Our study investigates NH4Cl toxicity by evaluating changes in ATP concentration and mitochondrial function as well as by evaluating alterations in GFAP expression. NH4Cl induced decreases in ATP were detected after 15 minutes in C6-glioma cells and 24 hours in both cell types. Mitochondrial function, assessed by MTT (2–4,5-dimethylthiazol A-yl)-2, 5-diphenyltetrazolium bromide) assay, was impaired in both cell types at 24 hours following NH4Cl treatment. GFAP was also significantly decreased in both cell types. Morphologic and metabolic toxicities were greater in C6-glioma cells. The data clearly indicate that NH4Cl interrupts oxidative metabolism. The greater toxicity seen in C6-glioma cells may be due to their greater dependence on oxidative metabolism. Lastly, the decrease in GFAP is probably a consequence of diminished ATP.

The effect of ammonium chloride on metabolism of primary neurons and neuroblastoma cellsin vitro

Hyperammonemia is a consistent finding in many metabolic disorders. The excess ammonia (NH4Cl) interferes with brain energy metabolism possibly in part by inhibiting the tricarboxylic acid (TCA) cycle. Inhibition of the TCA cycle may result in depletion of ATP in the brain cells. In this study, the acute and chronic effect of NH4Cl (7.5 mM and 15 mM) on the metabolism of isolated neurons and neuroblastoma cells was examined. These cells were treated with NH4Cl for 15 minutes and 24 hours. Morphologic and metabolic toxicity were greater in neuroblastoma cells than in primary neurons. Following 15 minutes treatment, concentration of lactate increased significantly in neuroblastoma cells but, the concentration of other metabolites did not change significantly in neuroblastoma cells and in primary neurons. Following 24 hours treatment, the glucose utilization increased in both cell types. This high utilization of glucose in neuroblastoma cells was in concert with an increase in lactate and decrease in glutamate and ATP. In primary neurons, following 24 hours treatment, the glucose utilization significantly increased, but the concentration of the other metabolites did not change significantly. Neuroblastoma cells consumed more glucose than primary neurons in absence of NH4Cl, but generated the same amount of lactate as neurons.

Auditory Brainstem Response

Auditory brainstem response to auditory stimulation is a non-invasive technique used to test the functional integrity of the brainstem connections and nuclei involved with hearing This method has been used in evaluating potential early brain damage from high serum levels of unconjugated bilirubin. The concept is that small discreet brain regions such as the cochlear (eighth) nucleus is uniquely sensitive to toxic effects of unconjugated bilirubin. Since function of this nucleus and its brainstem connections can be tested without invasive techniques (see Fig. 1), it is an ideal target to determine if unconjugated bilirubin brain damage has occurred in newborn infants.

Valproate-induced limb malformations in mice associated with reduction of intracellular pH.

Valproic acid (VPA) is a commonly used antiepileptic agent that recently has been found useful in the treatment of affective disorders and prophylaxis of migraine. VPA induces congenital malformations, especially spina bifida, in the offspring of women treated with this agent during early pregnancy. The mechanism by which VPA induces abnormal development remains unknown despite many studies in experimental animals in which VPA causes malformations similar to those seen in human infants. Because of its chemical structure as a weak organic acid and its capability to induce postaxial forelimb ectrodactyly in C57BL/6 mice, we postulated that VPA acts to perturb limb morphogenesis by reducing embryonic intracellular pH (pHi). We administered VPA, 200 to 400 mg/kg, to C57BL/6 mice on day 9 of gestation. A dose-dependent incidence of postaxial forelimb ectrodactyly was observed. Forelimb bud pHi was estimated by computer-assisted image analysis from the transplacental distribution of 14C-DMO. At the highest doses, 300 and 400 mg/kg, a decrease of pHi of 0.2 to 0.3 pH units was observed uniformly throughout the limb bud 1 h after VPA treatment. None of these changes were seen after treatment with 2-en VPA, a nonteratogenic analog of VPA. Furthermore, the capability of VPA to induce postaxial forelimb ectrodactyly was greatly enhanced by coadministration of agents that inhibit pHi regulatory processes. These data support the hypothesis that VPA-induced postaxial ectrodactyly in murine fetuses can be attributed to reduction in limb bud pHi.

Effect of 6-aminonicotinamide on metabolism of astrocytes and C6-glioma cells.

Brain tissue cells have been shown to use two predominant pathways for energy production. The first of these is the pentose phosphate shunt, and the second is glycolysis, followed by the TCA cycle. Inhibition of these pathways can result in a reduction of ATP, and changes in the concentration of various metabolites. In the present study, the acute and chronic effect of 6-aminonicotinamide (6-AN) (0.01, 0.02, and 0.03 mg/ml) was examined on astrocytes and C6-glioma cells. Following this treatment, glucose, lactate, glutamate, ATP, and PCr were assayed according to the procedures of Lowry and Passonneau. Our data indicated that following 15 minutes treatment of astrocytes and C6-glioma with 6AN there was no significant difference in the concentration of metabolites measured. However, following 24 hours treatment there was a significant increase in glucose concentration and significant reduction in the concentration of ATP, PCr, lactate and glutamate in both cell types. Morphological changes appeared later following 48 hours treatment with 6-AN in both cell types. Glucose accumulation can be explained by the fact that it is the precursor to both glycolysis and the pentose phosphate shunt. If these processes are inhibited, glucose will obviously accumulate and products like ATP, PCr, lactate and glutamate will decrease. Additionally, there was significant differences in concentration of glucose and lactate between astrocytes and C6-glioma cells. The significance of these differences has been discussed.

Pentylenetetrazole-Induced Changes in Cerebral Energy Metabolism in Tupaia Glis

Summary: The convulsant pentylenetetrazole was administered to the lower primate, the tree shrew. Shortly after the onset of seizures, the animals were killed using a microwave device at 25 Kw and 915 MHz. The energy metabolites glycogen, glucose, ATP, and phosphocreatine were measured in five layers of the cerebral cortex and three layers of the cerebellum. Results showed that, as compared with controls, seizing animals had decreased energy metabolites selective to certain layers. Glucose was decreased in all cortical layers, but only in the granular layer of the cerebellum. Phosphocreatine was decreased in the outer small pyramidal layer and the polymorphous layer of the cortex but was unchanged in the cerebellum. ATP was decreased only in the outer small polymorphous layer of the cortex. These changes are consistent with the concept that selective changes may occur during seizures and that these changes are localized to layers that contain pyramidal cells. Examination of whole cortex may mask more subtle regional changes

Regional cerebral energy metabolism in bicuculline induced seizures

In order to assess the early regional changes in energy metabolism in bicuculline induced seizures, mice were injected and sacrificed before the onset of overt seizure activity, and shortly after clonic-tonic seizures began. The energy metabolites glucose, ATP, and phosphocreatine were measured in layers of the motor cortex and the cerebellar vermis. Results showed minimal metabolite changes in the cerebellum, whereas changes in energy metabolism in the motor cortex were largely localized to the layers containing pyramidal cells. These results are in agreement with previous studies showing a relative sparing of the cerebellum, and suggest early cortical changes occur in pyramidal cells.

Cytoprotective Effect of Estrogen on Ammonium Chloride–Treated C6-Glioma Cells

The potential cytoprotective effects of estrogen in the brain are of special interest in aging, neurodegenerative diseases, exposure to toxins, and trauma. Estrogen effects on neurons have been widely explored, but less is known about estrogen effects on glia. Glial cells are primary targets of ammonia toxicity, which arises from liver disease or failure (such as from cirrhosis in alcoholics), urea cycle disorders, or inborn errors of metabolism. We examined the ability of estrogen to protect glial cells from ammonium chloride toxicity using an in vitro model system. C6-glioma cells in later passage have many astrocytic characteristics and provided a convenient and well established model system for this work. When C6-glioma cells were exposed to 15 mM ammonium chloride, we observed major cell death (only 32% cell survival relative to control) within 72 h. Pretreatment with 17β-estradiol (10 μM) significantly protected C6-glioma cells from ammonia toxicity (99% cell survival relative to control). In addition to enhancing the viability of C6-glioma cells against ammonia challenge, estrogen pretreatment was also found to protect mitochondrial function as assayed using the MTT reduction assay. Mitochondrial function was reduced to 39% of control levels in ammonia-challenged cultures and was mostly protected by estrogen (72% of control levels). The findings are potentially relevant for the development of therapeutic strategies to protect glial cells against ammonia toxicity resulting from hepatic failure or other causes.