W. David Lust

Neuroprotection in diet-induced ketotic rat brain after focal ischemia.

Neuroprotective properties of ketosis may be related to the upregulation of hypoxia inducible factor (HIF)-1α, a primary constituent associated with hypoxic angiogenesis and a regulator of neuroprotective responses. The rationale that the utilization of ketones by the brain results in elevation of intracellular succinate, a known inhibitor of prolyl hydroxylase (the enzyme responsible for the degradation of HIF-1α) was deemed as a potential mechanism of ketosis on the upregulation of HIF-1α. The neuroprotective effect of diet-induced ketosis (3 weeks of feeding a ketogenic diet), as pretreatment, on infarct volume, after reversible middle cerebral artery occlusion (MCAO), and the upregulation of HIF-1α were investigated. The effect of β-hydroxybutyrate (BHB), as a pretreatment, via intraventricular infusion (4 days of infusion before stroke) was also investigated following MCAO. Levels of HIF-1α and Bcl-2 (anti-apoptotic protein) proteins and succinate content were measured. A 55% or 70% reduction in infarct volume was observed with BHB infusion or diet-induced ketosis, respectively. The levels of HIF-1α and Bcl-2 proteins increased threefold with diet-induced ketosis; BHB infusions also resulted in increases in these proteins. As hypothesized, succinate content increased by 55% with diet-induced ketosis and fourfold with BHB infusion. In conclusion, the biochemical link between ketosis and the stabilization of HIF-1α is through the elevation of succinate, and both HIF-1α stabilization and Bcl-2 upregulation play a role in ketone-induced neuroprotection in the brain.

Substrates of Energy Metabolism Attenuate Methamphetamine‐Induced Neurotoxicity in Striatum

Abstract: High doses of methamphetamine (METH) produce a long‐term depletion in striatal tissue dopamine content. The mechanism mediating this toxicity has been associated with increased concentrations of dopamine and glutamate and altered energy metabolism. In vivo microdialysis was used to assess and alter the metabolic environment of the brain during high doses of METH. METH significantly increased extracellular concentrations of lactate in striatum and prefrontal cortex. This increase was significantly greater in striatum and coincided with the greater vulnerability of this brain region to the toxic effects of METH. To examine the effect of supplementing energy metabolism on METH‐induced dopamine content depletions, the striatum was perfused directly with decylubiquinone or nicotinamide to enhance the energetic capacity of the tissue during or after a neurotoxic dosing regimen of METH. When decylubiquinone or nicotinamide was perfused into striatum during the administration of METH, there was no significant effect on METH‐induced striatal dopamine efflux, glutamate efflux, or the long‐term dopamine depletions measured 7 days later. However, a delayed perfusion with decylubiquinone or nicotinamide for 6 h beginning immediately after the last METH injection attenuated the METH‐induced striatal dopamine depletions measured 1 week later. These results support the hypothesis that the compromised metabolic state produced by METH administration predisposes dopamine terminals to the neurotoxic effects of glutamate, dopamine, and/or free radicals.

Diet-induced ketosis does not cause cerebral acidosis

Ketosis is beneficial for seizure control, possibly through induction of cerebral acidosis. However, cerebral intracellular pH has not previously been measured in ketotic humans and the animal data are sparse. We describe a high‐fat diet, avidly consumed by rats, that induced consistent and moderate ketosis. Adult male rats were fed either the high‐fat ketogenic diet, a high‐carbohydrate diet with the same protein content as the ketogenic diet, or regular laboratory chow. Five to 6 weeks later, the rats were anesthetized, paralyzed, and injected with neutral red; their brains were frozen in situ. Intracellular pH of the cerebral cortex and cerebral glucose, lactate, ATP, phosphocreatine, and y‐aminobutyric acid (GABA) levels were measured. Rats fed the ketogenic diet had > 10–fold increase in their plasma ketones, but we noted no significant differences in cerebral pH or in cerebral metabolites and GABA levels among the three groups. Therefore, the antiepileptic effect of the ketogenic diet probably is not mediated by cerebral acidosis or changes in total cerebral GABA levels.

Cerebral metabolic profile, selective neuron loss, and survival of acute and chronic hyperglycemic rats following cardiac arrest and resuscitation

Cortical metabolites and regional cerebral intracellular pH (pHi) were measured in normoglycemic (NM), acute hyperglycemic (AH), and chronic hyperglycemic (CH, 2 week duration, streptozotocin-induced) Wistar rat brains during cardiac arrest and resuscitation. During total ischemia in AH and CH rats (plasma glucose approximately 30 mM), cortical ATP, PCr, glucose, and glycogen all fell significantly as expected. Lactate levels increased dramatically in association with a concomitant intracellular acidosis. Although lactate reached higher concentrations in AH and CH than NM, pHi was significantly lower only in the AH group. With 5 min of reperfusion, all groups recovered to near baseline in all variables, though lactate remained elevated. In a separate aspect of the study, animals from each experimental group were allowed to recover for 4 days following resuscitation, with outcome being gauged by mortality rate and hippocampal CA1 neuron counts. NM survival rate was significantly better than AH and CH. In particular, no CH rats survived for 4 days despite rapid initial recovery. After 4 days, the AH group had suffered significantly greater CA1 neuron loss than the NM rats. In summary, our research identified differences in intra-ischemic acid-base status in the two hyperglycemic groups, suggesting that chronic hyperglycemia may alter the brains buffering capacity. These observations may account for differences between acutely and chronically hyperglycemic subjects regarding outcome, and they suggest that factors other than hydrogen ion production during ischemia are responsible for modulating outcome.

Compromised metabolic recovery following spontaneous spreading depression in the penumbra.

Spreading depression (SD) has been demonstrated following focal ischemia, and the additional workload imposed by SD on a tissue already compromised by a marked reduction in blood flow may contribute to the evolution of irreversible damage in the ischemic penumbra. SD was elicited in one group of rats by injecting KCl directly into a frontal craniectomy and the wave of depolarization was recorded in two craniectomies 3 and 6 mm posterior to the first one. In a second group, the middle cerebral artery was occluded using the monofilament technique and a recording electrode was placed 5 mm lateral to the midline and 0.2 mm posterior to bregma. To determine the metabolic response in the penumbral region of the cortex ipsilateral to the occlusion, brains from both groups were frozen in situ when the deflection of the SD was maximal. The spatial metabolic response of SD in the ischemic cortex was compared to that in the non-ischemic cortex. Coronal sections of the brains were lyophilized, pieces of the dorsolateral cortex were dissected and weighed, and analyzed for ATP, P-creatine, inorganic phosphate (Pi), glucose, glycogen and lactate at varying distances anterior and posterior to the recording electrode. ATP and P-creatine levels were significantly decreased at the wavefront in both groups and the levels recovered after passage of the wavefront in the normal brain, but not in the ischemic brain. Glucose and glycogen levels were significantly decreased and lactate levels significantly increased in the tissue after the passage of the wavefront. While the changes in the glucose-related metabolites persisted during recovery even in anterior portions of the cortex in both groups in the aftermath of the SD, the magnitude of the changes was greater in the penumbra than in the normal cortex. SD appears to impose an equivalent increase in energy demands in control and ischemic brain, but the ability of the penumbra to recover from the insult is compromised. Thus, increasing the energy imbalance in the penumbra after multiple SDs may hasten the deterioration of the energy status of the tissue and eventually contribute to terminal depolarization and cell death, particularly in the penumbra.

Activation of caspase-12, an endoplasmic reticulum resident caspase, after permanent focal ischemia in rat.

The endoplasmic reticulum (ER) is emerging as a contributory component of cell death after ischemia. Since caspase-12 has been localized to the ER and is a novel signal for apoptosis, we examined the message levels and protein expression of caspase-12 after cerebral ischemia in vivo. Animals underwent permanent middle cerebral artery occlusion (MCAO) and were sacrificed 24 h after ischemia. Protein analysis revealed a significant increase in caspase-12 and a corresponding up-regulation of caspase-12 mRNA in the ischemia group compared with that in the sham group. Immunohistochemical analysis revealed diffuse positive immunostaining of caspase-12 throughout the striatum and cerebral cortex in animals that underwent ischemia, with more intense caspase-12 immunostaining in the striatum than in the cortex after ischemia. These results demonstrate that cerebral ischemia initiates an ER-based stress response that results in the transcriptional up-regulation and corresponding increased expression of caspase-12 protein, and may provide a new area for therapeutic intervention to ameliorate outcomes following stroke.

Caspase-9 Inhibition after Focal Cerebral Ischemia Improves Outcome following Reversible Focal Ischemia

Cerebral ischemia initiates a program of cell death known as apoptosis. Early steps in these death promoting events are the release of cytochrome c from the mitochondria and activation of caspase-9. The purpose of this report is to determine if the administration of a specific caspase-9 inhibitor, Z-Leu-Glu(Ome)-His-Asp(Ome)-FMK·TFA (Z-LEHD-FMK) would attenuate apoptosis and the resultant brain injury after ischemia. Adult Wistar rats underwent 3 h of temporary middle cerebral artery occlusion (MCAO) followed by 24 h of reperfusion. An intraventricular injection of 4.8 μg of Z-LEHD-FMK was given 15-min postreperfusion. Administration of the caspase-9 inhibitor, Z-LEHD-FMK, to the experimental group (n = 12) reduced total infarction volume by 49% (p < 0.05) and improved neurological outcome by 63% (p < 0.01) as compared to the control group (n = 12). Western blot analysis of animals that underwent ischemia-reperfusion showed the appearance of the active form of caspase-9. Inhibition of caspase-9, the apical caspase in cytochrome-c-dependent apoptosis, is an effective intervention to attenuate neurological injury after focal ischemia.

Formation of free choline in brain tissue during in vitro energy deprivation

Free choline and ATP contents were measured in Mongolian gerbil hippocampal slices (tissue) and incubation media (media) during exposure to 30 min of aglycemia, high potassium, anoxia, or ischemia. Changes in choline levels reflected the degree of energy reduction, lower ATP levels being associated with high choline (4-fold increase during exposure to high potassium and anoxia, and 11-fold increase during ischemia). Media (extracellular) choline was particularly affected and increased about twofold during relatively mild energy depletion (e.g., aglycemia), but tissue choline content was less sensitive to energy reduction. A plot of choline vs. ATP levels indicated a nonlinear correlation, and the sharp increase in choline occurred when ATP values fell to about 2.5 nmol/mg of protein. Inhibition of acetylcholine sterase by 10 μM physostigmine during ischemia did not prevent an increase in choline contents but rather enhanced them, indicating that acetylcholine hydrolysis was not the source of free choline. Formation of free choline was Ca2+ independent. These findings suggest the involvement of phospholipase D and phosphatidylcholine hydrolysis in free choline formation during energy stress. The extent of choline formation may be an indicator of the degree of membranal damage, which in turn reflects damage to the metabolic machinery of the cell.

Metabolism in the hamster brain during hibernation and arousal

Hibernation was induced in hamsters by placing them in a cold room for an extended period of time, after which the hibernating state was confirmed by marked reductions in heart rate, body temperature, and the respiratory rate. The animals were either frozen intact in liquid nitrogen, or aroused and then frozen when body temperature reached 8, 12, 16, 20, 24 or 32 degrees C. A metabolite profile, including glucose-related metabolites, high-energy phosphates, gamma-aminobutyric acid (GABA) and cyclic nucleotides, was determined for both the cerebral cortex and cerebellum. In general, the metabolite changes in the two regions elicited by hypothermia were alike, although some differences were evident. The brains of hibernators were biochemically characterized by (1) a high concentration of energy reserves including glycogen, glucose, adenosine triphosphate, and P-creatine, (2) significantly elevated levels of lactate and GABA, and (3) near depletion of cyclic guanosine monophosphate with only a moderate depression of cyclic adenosine monophosphate. During arousal, the metabolites were restored to near normal values and there was little or no indication that the brain energy metabolism was compromised by the arousal process. The study provides certain insights into the metabolic adaptation of the brain to prolonged periods of profound hypothermia in a hibernating species.

Comparison of Glucose Influx and Blood Flow in Retina and Brain of Diabetic Rats

Diabetes is associated with extensive microvascular pathology and decreased expression of the glucose transporter (GLUT-1) in retina, but not brain. To explore the basis of these differences, the authors simultaneously measured glucose influx (μmol · g−1 · min−1) and blood flow (mL · g−1 · min−1) in retina and brain cortex of nondiabetic control rats (normoglycemic and acute-hyperglycemic) and in rats with streptozotocin-induced diabetes (with or without aminoguanidine (AMG) treatment) using a single-pass, dual-label indicator method. In addition, tissue glucose and adenosine triphosphate (nmol/mg dry weight) levels were measured. Glucose influx in retina exceeded that of cortex by about threefold for both the nondiabetic and diabetic groups. In contrast, blood flow in retina was significantly lower than in cortex by about threefold for each group. Retinal and cortical glucose influx in the diabetic rats was lower than in the nondiabetic acute-hyperglycemic group, but not in the normoglycemic group. Blood flow in these tissues remained relatively unchanged with glycemic conditions. The glucose levels in the diabetic retina (aminoguanidine untreated and aminoguanidine treated) were fourfold to sixfold greater than the nondiabetic retina. The cortical glucose levels remained unchanged in all groups. These data suggest that the accumulation of glucose in the diabetic retina cannot be explained by increased endothelial-glucose uptake or increased vascular membrane permeability.