David D. Gilboe

Ischemia-Induced Changes in Cerebral Mitochondrial Free Fatty Acids, Phospholipids, and Respiration in the Rat

Abstract: Changes in the free fatty acid pool size and fatty acyl chain composition of mitochondrial membrane phospholipids and their relation to disruption of mitochondrial function were examined in rat brains after 30 min of cerebral ischemia (Pulsinelli‐Brierley model) and 60 min of normoxic reoxygenation. During ischemia, significant hydrolysis of polyunsaturated molecular species from diacyl phosphatidylcholine, particularly fatty acyl 20:4 (arachidonic acid; 20% decrease) and 22:6 (docosahexaenoic acid; 15% decrease), was observed. Thirty minutes of ischemia caused a 16% loss of 18:2 (linoleic acid) from phosphatidylethanolamine. Recirculation for 60 min did not return the polyunsaturated fatty acid content of phospholipids to normal. Total content of free fatty acids increased during ischemia, particularly 18:2 and 22:6, which exhibited the most dramatic rise. The free fatty acid pool size continued to increase during 60 min of recirculation. The respiratory control ratio decreased significantly during 30 min of ischemia with no apparent recovery following 60 min of reoxygenation. The degree of free radical‐mediated lipid peroxidation in mitochondria was significantly increased during ischemia and reperfusion. It was concluded that (a) 30 min of cerebral ischemia caused differential degradation in each of the phospholipid classes and preferential hydrolysis of the polyunsaturated molecular species and (b) 60 min of normoxic reperfusion failed to promote reacylation of the mitochondrial phospholipids and restoration of normal respiration.

Inhibition of glucose transport into brain by phlorizin, phloretin and glucose analogues

An indicator dilution technique with 22Na+ as the intravascular marker was used to measure unidirectional transport of D-[6-3H]glucose from blood into the isolated, perfused dog brain. 18 compounds which are structurally related to glucose were tested for their ability to inhibit glucose transport. The data suggest that no single hydroxyl group is absolutely required for glucose transport, but rather that glucose binding to the carrier probably occurs through hydrogen bonding at several sites (hydroxyls on carbons 1, 3, 4 and 6). In addition, alpha-D-glucose has higher affinity for the carrier than does beta-D-glucose. A separate series of experiments demonstrated that phlorizin and phloretin are competitive inhibitors of glucose transport into brain; however, phloretin is partially competitive and inhibits at lower concentrations than does phlorizin. Inhibition by phlorizin and phloretin is mutually competitive, indicating that these compounds compete for binding to the glucose carrier. Comparison with the results reported in the literature for similar studies using the human erythrocyte demonstrates a fundamental similarity between glucose transport systems in the blood-brain barrier and erythrocyte.

Effects of Nitrous Oxide on the Cerebrovascular Tone, Oxygen Metabolism, and Electroencephalogram of the Isolated Perfused Canine Brain

Nitrous oxide has been reported to act both as a stimulant and as a depressant of cerebral oxygen metabolism (CMRO2) and blood flow under a variety of experimental conditions in the intact animal. The isolated brain preparation is advantageous because it permits direct measurement of blood flow and allows the study of drug effects without interference from other organ systems or drugs. In this study, six isolated perfused canine brain preparations were used to compare the CMRO2, cerebral vascular resistance (CVR), and the EEG of brains perfused with normocapnic, normoxic blood equilibrated with either 70% N2O or 70% N2. There was no significant change in CMRO2. Cerebral vascular resistance fell [16.4% ± 3.4% SEM (P <0.015)] during exposure to N2O. The EEG pattern was reduced in amplitude, but showed an increase in both low-voltage β activity (14–40 Hz), and 3–5 Hz activity. In the isolated brain, N2O reduces cerebral vascular tone while exhibiting no effect on cerebral oxygen metabolism.

Effects of anoxia on net uptake and unidirectional transport of glucose into the isolated dog brain

Unidirectional glucose transport and diffusion and net glucose uptake were measured before, during and after perfusion of the isolated canine brain with anoxic blood. An indicator dilution technique with 22Na as the intravascular marker was used to measure unidirectional transport of d-[6-3H]glucose from blood into brain while the rate of unidirectional diffusion was estimated from similar studies using 22Na and d-[6-3H]fructose. Net glucose uptake was calculated by multiplying the arteriovenous concentration difference by the plasma flow rate. During the first 2 min of anoxia, the rate of net glucose uptake more than doubled, but by 10 min of anoxia it had declined to near the pre-anoxic level. Net uptake did not change significantly between 10 and 30 min of anoxia nor during the first hour of post-anoxic recovery. The kinetics of unidirectional glucose transport were unchanged after 1 min of anoxia; however, after 10 min, unidirectional transport was decreased to an average of 39% of the control rate. After 1 h of recovery from 30 min of anoxia, transport was 59% of the control rate. The rate of simple diffusion of glucose was unchanged during and after anoxia. It was concluded that the initial increase in net glucose uptake is due to a decrease in the rate of unidirectional efflux of glucose from the brain as a result of reduced brain glucose levels. The subsequent decrease in net uptake is caused by an impairment of unidirectional glucose transport. These data indicate that transport of glucose from the blood to the brain may be an energy dependent process.

Evaluation of the Sakaguchi Reaction for Quantitative Determination of Arginine.

Summary The Sakaguchi reaction has been studied to obtain a reliable quantitative method for the colorimetric estimation of arginine. The best conditions for this method have been investigated and reported. The method has been found to be entirely satisfactory for determining arginine over the range of 0-30 γ. Duplicability of the method is ± 0.5%, (Standard deviation).

The Characteristics of Glucose Transport Across the Blood Brain Barrier and its Relation to Cerebral Glucose Metabolism

The evidence suggests that glucose transport across the blood brain barrier (BBB) in the dog is normally not a rate-limiting step in cerebral metabolism; however, transport may become rate-limiting under conditions of extreme hypoglycemia or anoxia. Studies on the mechanism of glucose transport from blood to brain do not at this time permit us to distinguish between active transport and facilitated diffusion; however, a decrease in the rate of unidirectional transport during anoxia suggests that an energy-dependent process may be involved. In spite of this evidence, glucose transport across the BBB is similar to the facilitated diffusion of glucose into the red cell in terms of the structural requirements of the glucose molecule, the pattern of inhibition by phlorizin, phloretin and cytochalasin B, and the lack of sensitivity to Na+ or insulin.

Demonstration of Alpha and Beta Adrenergic Receptors in Canine Cerebral Vasculature

The cerebral vascular effects of various adrenomimetic agents were examined in 12 isolated canine brains perfused with blood at constant flow. Changes in cerebral vascular resistance (CVR) and the magnitude of pressor or depressor responses observed following drug injection were examined. The presence of alpha adrenergic receptors in this vascular bed is indicated by the increased CVR observed when phenylephrine, norepinephrine, and epinephrine were administered and by the reduced or reversed pressor actions of these compounds following treatment with the alpha adrenergic blocking drug phenoxybenzamine HCl. The presence of beta adrenergic receptors in this vascular bed is indicated by the decreased CVR noted when isoproterenol was given and by the reduced depressor actions of this compound following treatment with the beta adrenergic blocking drug propranolol. Further evidence for the presence of beta adrenergic receptors is demonstrated by the vasomotor reversal to epinephrine which was observed following alpha suppression with phenoxybenzamine. As catecholamine blood levels in intact dogs are low in comparison to those achieved in these studies, it appears doubtful that circulating catecholamines play an important physiological role in the regulation of CVR. Possible explanations are considered for the lower response of the cerebral vasculature to catecholamines when this response is compared to that observed in other vascular beds.

31P-MRS-Based Determination of Brain Intracellular and Interstitial pH: Its Application to In Vivo H+ Compartmentation and Cellular Regulation during Hypoxic/Ischemic Conditions

In the last decade, significant progress has been made in the characterization of pH regulation in nervous tissue in vitro. However, little work has been directed at understanding how pH regulatory mechanisms function in vivo. We are interested in how ischemic acidosis can effect pH regulation and modulate the extent of post-ischemic brain damage. We used 31P-MRS to determine normal in vivo pHi and pHe simultaneously in both the isolated canine brain and the intact rat brain. We observed that the 31Pi peak in the 31P-MRS spectrum is heterogeneous and can be deconvoluted into a number of discrete constituent peaks. In a series of experiments, we identified these peaks as arising from either extracellular or intracellular sources. In particular, we identified the peak representing the neurons and astrocytes and showed that they maintain different basal pH (6.95 and 7.05, respectively) and behave differently during hypoxic/ischemic episodes.

Effect of the Platelet‐Activating Factor Antagonist BN 50739 and Its Diluents on Mitochondrial Respiration and Membrane Lipids During and Following Cerebral Ischemia

Abstract: Recent evidence suggests that platelet‐activating factor plays a role in ischemia‐induced neural injury. The Pulsinelli‐Brierley four‐vessel occlusion model was used to study the effect of a synthetic platelet‐activating factor antagonist, BN 50739, and its solvents, either dimethyl sulfoxide or hydroxypropyl‐β‐cyclodextrin, on cerebral ischemia‐reperfusion. Rats were subjected to either 30 min of ischemia or 30 min of ischemia followed by 60 min of recirculation. Changes in the brain mitochondrial free fatty acid pool size, fatty acyl composition of phospholipids, and respiratory function were monitored. When the BN 50739 (2 mg of BN 50739/kg of body weight i.v.) was administered at the onset of recirculation, it significantly reversed the ischemia‐induced accumulation of mitochondrial free fatty acids and loss of polyunsaturated fatty acyl chains from phosphatidylcholine and phosphatidylethanolamine while simultaneously improving mitochondrial respiration. Dimethyl sulfoxide alone decreased the mitochondrial level of malonyldialdehyde and total free fatty acid pool size, but there was no improvement in mitochondrial respiration. Hydroxypropyl‐β‐cyclodextrin was reported to be pharmacologically inactive and capable of dissolving BN 50739. However, hydroxypropyl‐β‐cyclodextrin alone also caused a significant increase in content of cerebral mitochondrial membrane free fatty acids and hydrolysis of phosphatidylcholine in normoxic control animals. The overall effect of BN 50739 on mitochondrial structure and energy metabolism supports the hypothesis that platelet‐activating factor may play a key role in ischemia‐induced cerebral injury.

Accelerative exchange diffusion kinetics of glucose between blood and brain and its relation to transport during anoxia

An earlier study showed that unidirectional glucose transport from blood to brain decreases during perfusion with anoxic blood (Betz, A. L., Gilboe, D. D. and Drewes, L. R. (1974) Brain Res. 67, 307-316). Brain glucose levels also decrease during anoxia. Therefore, the present study was designed to investigate whether the decreased transport might be the result of decreased accelerative exchange diffusion when brain glucose levels are low. The rate of undirectional transport into brain (v) of D-[6-3H]glucose was studied in 22 isolated, perfused dog brains by means of an indicator dilution technique using 22Na as the intravascular reference. The kinetics of transport were determined over a range of blood glucose concentrations (S1) at each of five different brain glucose levels (S2). The existence of accelerative exchange diffusion for glucose was indicated by a decrease in the intercept (increase of apparent V) of a double reciprocal plot (1/v versus 1/S1) as S2 increased. This phenomenon is consistent with a model for facilitated diffusion in which the mobility of the loaded carrier is greater than that of the unloaded carrier. Although the data predict a decrease in glucose transport during anoxia, the predicted decrease (5%) is less than the observed decrease (35%). It is concluded that the simple mobile-carrier model for facilitated diffusion cannot, by itself, describe all properties of blood-brain glucose transport.