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 17beta-estradiol (10 microM) 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.

The effect of ammonium chloride on metabolism of primary neurons and neuroblastoma cells in 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.

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.

Effect of alcohol on energy storage of primary astrocytes and C6-glioma cells in vitro.

The present experiments were conducted to investigate the direct effects of ethanol on the energy metabolism of astrocytes and C6-glioma cells. Primary astrocytes were prepared from cerebral cortices of neonatal rats, and C6-glioma cells were purchased from American Type Culture Collection (ATCC). These cells were exposed to different concentrations of alcohol (100 mM, 200 mM, and 300 mM) for 15 minutes and 24 hours. The amount of ATP and PCr was measured by the method of Lowry and Passonneau (1972). Following 15 minutes treatment with different doses of ethanol the amount of ATP and PCr increased, in both cell types. Only the increase of ATP concentration with varying doses of ethanol (100 mM, 200 mM, and 300 mM) was statistically significant. Following 24 hours treatment of astrocytes with different doses of ethanol the concentration of ATP and PCr decreased. The decrease in concentration of ATP was significant with all three doses of ethanol, but the decrease of PCr concentration was only statistically significant with 300 mM ethanol. Following 24 hours treatment of C6-glioma cells to varying doses of ethanol, the concentration of PCr and ATP decreased. The decrease of PCr was statistically significant with all three doses of ethanol and the decrease of ATP concentration was only significant with 300 mM ethanol.

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.

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 ammonia's 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 chloride 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 morphological 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.

Elevated gamma-aminobutyric acid levels attenuate the metabolic response to bilateral ischemia.

Bilateral ischemia has been shown to alter the net brain levels of energy metabolites such as ATP, phosphocreatine, glucose, and glycogen. The amino acid neurotransmitter gamma-aminobutyric acid (GABA) exerts a tonic inhibitory influence on neural activity. The present studies were designed to evaluate the influence of elevated GABA levels on the metabolic sequelae of ischemia. The GABA transaminase inhibitor gamma-vinyl-GABA (GVG; vigabatrin) was administered to Mongolian gerbils before the production of a bilateral ischemic incident. GABA levels were elevated in all regions assayed. Levels of energy metabolites were also increased, an indication of reduced energy utilization. In control animals, in the absence of GVG, 1 min of bilateral ischemia produced decreases in the levels of all metabolites. In animals pretreated with GVG, the effects of 1 min of bilateral ischemia were attenuated. These data suggest that the level of ongoing activity may affect the response to an ischemic insult. Furthermore, GVG may have a clinical indication in reducing the effect of minor ischemic incidents.

Energy metabolism in rat hippocampus during and following seizure activity.

The hippocampus exhibits a post-ictal phenomenon in which it is unresponsive to further stimulation. It has been suggested that this loss of excitability is the basis of post-seizure amnesia. The biochemical events associated with this phenomenon are unclear. In the present study, energy metabolites were measured in the stratum oriens, stratum pyramidale and stratum radiatum in rat hippocampus, and correlated with field potential recordings. Wistar rats were anesthetized and the calvarium removed. Following removal of the cortex by aspiration, the hippocampus was covered with oil, and stimulating and recording electrodes were placed. Stimulation consisted of a train of stimuli at 100 Hz (10-20 m Amps). This stimulation was found to be effective in evoking self-sustaining after-discharges and post-ictal depression. Tissues for metabolite analysis were taken from a series of controls, from animals during active self-sustaining seizures, and from animals which were totally unresponsive to further electrical stimulation. Hippocampal tissue for metabolite analysis was obtained by pouring liquid N2 on the exposed tissue, then removing the frozen tissue. Glucose, ATP, and phosphocreatine were measured in hippocampal layers of CA1 using fluorescence techniques and enzymatic cycling. Results showed that during seizure activity, glucose, ATP, and phosphocreatine were all decreased from 40-80% in the three layers of the hippocampus, whereas from 60 seconds after the onset of hippocampal shutdown, energy metabolites had returned toward normal. Thus, at a time when the hippocampus was unresponsive, energy metabolites were at control levels. These data suggest that the shutdown phenomenon occurs in the presence of adequate energy stores.

Regional depletion of adenosine triphosphate, phosphocreatine, and glucose in ischemic hippocampus.

The selective vulnerability of pyramidal neurons in the CA1 hippocampal region in ischemic rat brain may be preceded by regional alterations of energy metabolism during early reperfusion. We measured ATP, phosphocreatine (PCr), and glucose in paramedian and lateral CA1 and in an area showing little postischemic cell loss, CA2. ATP levels in paramedian CA1 were depressed immediately after 30 min of ischemia (P less than or equal to 0.02) and remained abnormal after 2 hr of reperfusion (P less than or equal to 0.05). PCr was reduced substantially in both subdivisions of CA1 immediately after ischemia (P less than or equal to 0.04) but returned to normal levels after 2 hr. Glucose levels were depressed in paramedian CA1 and CA2 after ischemia (P less than or equal to 0.02) but corrected with reperfusion. We determined approximately P, the sum of ATP and PCr, in separate experiments investigating regional differences in consumption of high-energy phosphate metabolites during complete ischemia. The approximately P levels of rats subjected to 30 min of reversible ischemia followed by 2 hr of reperfusion showed a different pattern of regional differences from those seen in sham-ischemic animals (P less than or equal to 0.01), indicating a persistent depression of metabolic rate in CA1 during reperfusion. We conclude that regional depletion of high-energy phosphates and alteration of metabolic rate may contribute to the selective vulnerability of the CA1 region during brain ischemia.

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

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]deoxyglucose 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.

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.

Pentylenetetrazole-induced changes in cerebral energy metabolism in Tupaia glis.

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.

Status epilepticus-induced changes in primate cortical energy metabolism.

The objective of this study was to evaluate changes in cortical energy metabolism in experimentally induced seizures in the primate. Cynamologus fascicularis monkeys were anesthetized, and a craniotomy was performed. Small samples from the motor cortex were removed for measurement of energy metabolites just prior to intravenous bicuculline infusion (0.6 mg/kg), 20 min after the onset of seizures, and 2 h after the second sample. Samples were also taken for electron microscopy. Results showed decreased phosphocreatine values 20 min after the onset of seizures, whereas ATP levels were normal. Two hours after the onset of seizures, phosphocreatine had returned to normal, but ATP levels were below normal. Examination of tissue by electron microscopy showed evidence of cell damage 2 h, 20 min after the onset of seizures. These findings are consistent with the concept that sustained seizures may lead to irreversible cell damage and that alterations in energy metabolism may contribute to the cell death.

Sodium valproate and brainstem energetics.

The effect of the anticonvulsant sodium valproate on cerebral brainstem energy metabolism has been investigated. Stupor and coma were produced in mice by the intraperitoneal injection of sodium valproate at a dose of 600 mg/kg. Glucose, glycogen, ATP, and phosphocreatine were measured in small tissue samples from the ascending reticular activating system. Levels of all metabolites were either normal or elevated in precoma and comatose mice as compared to controls. These data are consistent with the concept that sodium valproate does not have a primary action through depletion of high energy phosphates.

Cerebral acetylcholine and energy metabolism changes in acute ammonia intoxication in the lower primate Tupaia glis.

Ammonia levels are elevated in many patients with hepatic encephalopathy. This observation, coupled with animal studies showing an encephalogenic role for ammonia, has led to the concept that ammonia is an important toxin in the production of neurologic symptoms. Studies in rodents have shown that ammonia alters cerebral energy metabolism in the reticular formation, an area important in the modulation of consciousness. Our study was undertaken to extend these observations to the lower primate Tupaia glis, the tree shrew. The energy metabolites glucose, glycogen, lactate, adenosine triphosphate, and phosphocreatine were measured in the reticular formation by microanalytic techniques and enzymatic cycling. Acetylcholine was measured in brain regions by gas chromatography. Acetylcholine levels were increased significantly only in the medulla-pons and diencephalon in the coma stage. The energy metabolites glucose, glycogen, and phosphocreatine were decreased in reticular formation cells during the coma, whereas lactate was increased. During the precoma, glycogen and phosphocreatine were decreased. It appears, therefore, that the tree shrew has a metabolic response to ammonia similar to that of mice. A lowering of energy metabolism in the area of brain-regulating consciousness may act to place the animal in a coma. This coma in turn acts to decrease overall metabolic demand, which allows the animal an opportunity to conserve its threatened energy reserves.

Octanoic acid-induced coma and reticular formation energy metabolism.

The medium chain fatty acid octanoic acid was injected i.p. into 20-22 g Swiss-Albino mice at a dose of 15 mumol/g. This dose produced a reproducible response consisting of a 3-4 min period of drowsiness, followed by coma. These mice as well as suitable controls were sacrificed by rapid submersion in liquid N2, or by microwave irradiation in a 7.3 kW microwave oven. Tissue from the reticular formation and the inferior colliculus was prepared for microanalysis of the energy metabolites glucose, glycogen, ATP and phosphocreatine. Results from this study showed a selective effect on energy metabolism in cells of the reticular formation. Both glucose and glycogen were elevated in the coma and precoma state. In addition, ATP and phosphocreatine were decreased in the reticular formation during coma. These results show a selective effect of octanoic acid on energy metabolism in the reticular formation both in the precoma stage, and during overt coma. The selective vulnerability of the reticular formation to metabolic insult may act in a beneficial manner to the animal by inducing coma. This lowers the overall demand for energy, thereby placing the animal in a milieu in which there is an increased chance for correction of the perturbation.

Disruption of the blood-brain barrier in hyperammonemic coma and the pharmacologic effects of dexamethasone and difluoromethyl ornithine.

Both hyperammonemia and blood-brain barrier (BBB) breakdown have been implicated in the evolution of hepatic encephalopathy. To define a possible relationship, Swiss Albino mice were subjected to sublethal encephalopathic doses of ammonium acetate; the integrity of the BBB was determined grossly with Evans blue and quantitatively with [14C]-alpha-aminoisobutyrate (AIB). Some animals were injected with a dose of ammonium acetate sufficient to maintain coma for 1 hr (AC group). One group, termed stuporous (AS), received only enough ammonium acetate to interfere with grooming and exploratory activity; this dosage was insufficient to completely block the righting response, which was absent in the AC group. When compared to that of controls (CON) receiving normal saline instead of ammonium acetate, cerebral tissue from the AC group was stained blue and contained nearly double the amount of AIB; AS group brain tissue was unstained and the AIB content did not differ significantly from normal. Some of the AC group were pretreated with drugs known to retard BBB breakdown; one set received dexamethasone (AC-DXMN), another the ornithine decarboxylase inhibitor difluoromethyl ornithine (AC-DFMO), and a third L-ornithine (AC-ORN). Brain tissue from the AC-ORN group stained blue and AIB content did not differ significantly from that of the untreated AC group. Cerebral tissue of the AC-DXMN pretreatment group stained light blue; AIB content was significantly lower than in the AC group and greater than the CON group. The AC-DFMO brains were unstained and AIB content was significantly lower than in the AC group but did not differ significantly from CON. These results indicate that hyperammonemia may induce BBB breakdown but that the disruption of barrier integrity is not antecedent to the development of coma, although it seems to coincide with coma in time.(ABSTRACT TRUNCATED AT 250 WORDS)

Maintenance of regional chemical integrity for energy metabolites in microwave heat inactivated mouse brain.

A method is described in which brain tissue from microwave heat inactivated mouse brain is prepared for microregional energy metabolite analysis. This method permits the sectioning of the tissue into sections 40 microns thick in which layers can be visualized and dissected. Results show no movement or leaching of metabolites from one microregion to another. This method therefore, permits the combined use of the two powerful neurochemical techniques of microwave irradiation for sacrifice and microregional analysis for energy metabolite determination.

Audiogenic seizure-induced changes in energy metabolites in cerebral cortical and cerebellar layers.

Audiogenic seizure-prone mice (DBA/2J) were exposed to a broad band noise source. A reproducible response consisting of wild run, clonus, and tonic stages resulted in all mice. Layers 1 and pyramidal from the parietal cortex and the molecular and Purkinje cell-rich layers from the cerebellar vermis were separately analyzed for glucose, glycogen, ATP, and phosphocreatine. Results showed a biphasic cerebellar response, with decreases in high energy phosphates occurring during the wild run and tonic stage. In the cortex, similar changes occurred in the pyramidal cell layer, but the decreases were not as pronounced as those in the cerebellum. Cells from layer 1 of the parietal cortex were not affected as much as those of the pyramidal layer, suggesting a differential effect between neuronal and nonneuronal cell populations. The greater response of the cerebellum could indicate an attempt to reduce the severity of both the wild run and the tonic extension seizure.