Rapid distribution of tryptophol (3-indole ethanol) to the brain and other tissues.

Tryptophol (3-indole ethanol) is a compound which induces sleep, and is formed: (a) in the liver after disulfiram treatment, and (b) by the parasite in trypanosomal sleeping sickness. We prepared, purified, and characterized radiolabeled tryptophol for the purpose of defining its tissue distribution in animals. Tryptophol was found to be highly lipophilic, with an octanol:water partition coefficient of 29.8. Brain extraction, determined after intracarotid injection, was high (brain uptake index = 117 +/- 3.5%), and nonsaturable, suggesting the absence of a carrier system. After intravenous administration, tryptophol distribution to tissues correlated with relative blood flow. More than 85% of the radioactivity remaining in brain 2-5 min after intravenous injection co-migrated with tryptophol standards when analyzed by thin-layer chromatography. Other evidence suggested that tryptophol binds to serum and in vivo may be stripped from serum albumin and taken up by brain in a single capillary transit. Our study suggests that in states such as trypanosomal sleeping sickness or disulfiram treatment, remotely formed tryptophol gains ready access to brain (it is 100% cleared in a single capillary passage), and could thus cause somnolence.

Changes during development in transport processes of the blood-brain barrier.

The permeability of the blood-brain barrier to several classes of compounds was studied in rats between the ages of 15 days and 9 weeks. 14C-labelled test substances were injected simultaneously with two reference isotopes, 3H2O and 113mIn-labelled EDTA, into the common carotid artery followed by decapitation 10 s later. There was evidence that a monocarboxylic acid transport system in 15 to 23 day-old rats had a capacity at least six times greater than that present in adult animals. L-Lactate and acetate showed the highest permeability. At all ages there was a constant ratio between L-lactate and (-)D-3-hyroxybutyrate values. D-Glucose permeability increased with age, while that of several amino acids tested was the same in young and adult rats.

Blood-brain barrier restriction of peptides and the low uptake of enkephalins.

Blood-brain barrier penetration of leucine-enkephalin, methionine-enkephalin, and other peptide-like compounds was measured after intracarotid injection of three isotopes and was found to be non-saturable over the nanomolar range of concentrations tested. No significant differences in brain regional extraction of leucine enkephalin (or morphine or heroin) were observed. In contrast to previous reports, the brain extraction of enkephalins was minimally low (E = 2-3%) and about the same order of magnitude as other putative neurotransmitters. Brain extractions of other peptide-like compounds were similarly small: TRH, E = 1%; glutathione, E = 0.5%; beta-alanyl histidine, E = 1%; and thioacetyl coenzyme A, E = 2%. Extraction of the non-diffusible reference dextran was determined to be 1%, suggesting that the blood brain barrier tends to restrict peptide penetration.

Increased blood--brain barrier transport of protein-bound anticonvulsant drugs in the newborn.

The extraction of heroin, caffeine, diphenylhydantoin, and phenobarbital has been measured in the newborn, suckling, and adult brain. Anticonvulsant drugs such as diphenylhydantoin and phenobarbital are bound by plasma protein, and it is generally believed that only the fraction of drug that is free (dialyzable) in vitro is available for transport through the blood-brain barrier in vivo. In both the adult and neonatal rat or rabbit, lipid-mediated transport of free phenytoin occurs. In addition, a fraction of the drug that enters the capillary bound to plasma protein also gains access to the brain. A greater amount of protein-bound drug permeates the newborn brain, and this is ascribed to a longer capillary transit time in the neonate. With regard to phenobarbital, the total (i.e., both free and protein-bound) plasma drug enters the newborn brain. In contrast, no protein-bound phenobarbital permeates the adult brain, and it is only the free drug fraction that gains access to the brain. Since the blood-brain barrier permeability-surface area product for the two anticonvulsants is unchanged in newborn and older animals, the age-related differences in brain uptake of protein-bound drugs can be attributed to developmental changes in cerebral blood flow and capillary transit time. The increased transport of protein-bound drugs in the newborn may cause increased concentrations (i.e., brain:plasma ratios) of these anticonvulsants in the neonatal brain.

Two-day starvation does not alter the kinetics of blood--brain barrier transport and phosphorylation of glucose in rat brain.

The blood-brain barrier (BBB) transport and brain phosphorylation of glucose were assessed in conscious rats subjected to 2 days of starvation. Although plasma glucose decreased, no significant changes in brain blood flow, BBB glucose transport, or 2-deoxy-D-glucose phosphorylation were observed. The data suggest that adaptive changes of brain glucose metabolism previously observed in starvation are located beyond the initial steps of brain entry and phosphorylation.

Carotid artery injection technique: bounds for bolus mixing by plasma and by brain.

Estimation of Michaelis-Menten kinetic parameters (Km, Vmax) of blood-brain barrier (BBB) transport processes with the carotid artery single injection technique assumes that mixing of the bolus with unlabeled substrate either from (a) circulating plasma or (b) amino acid efflux from brain, is minimal. The maximum extent to which the bolus could mix by these two sources is quantified in the present studies by measuring 14C-phenylalanine extraction in pentobarbital-anesthetized and conscious rats after the addition of 0-80% rat serum to the arterial injection solution. An upper bound (+/- SE) of bolus mixing due to mixing from both sources, expressed in terms of percentage of rat plasma, is 8.8 +/- 1.9 and 7.0 +/- 2.1% for the anesthetized and conscious rat, respectively. The estimated contribution to bolus mixing due to amino acid efflux from brain is 3.3 and 2.1% for the anesthetized and conscious rat, respectively. Based on these estimates, the upper bound for bolus mixing with circulating rat plasma is only 5.5 and 4.9%, respectively, for the anesthetized and conscious catheterized rat. Thus, any bolus mixing after rapid carotid injection is relatively small and is comparable to the mixing effects observed with the carotid artery infusion technique. Mixing effects on the order of 5% are shown to have no significant effect on the estimation of kinetic parameters of BBB nutrient transport, except for neutral and basic amino acid transport, which are characterized by very low Km values relative to the usual amino acid plasma concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Hippocampal aging in rats: a morphometric study of multiple variables in semithin sections.

Quantitative analyses of pyramidal cells, astrocytes, dark glia (microglia and dark oligodendrocytes), lipofuscin accumulation and astrocyte reactivity were carried out in semithin sections from field CA3 of the hippocampus of rats in 3 age groups (4-7 mo., 13-15 mo., 25-28 mo.). A decrease in neuronal density (approximately 25%) and an increase in dark glia were found in the oldest group. The astrocyte population was stable with age. Lipofuscin increased by 13-15 mo. and increased further by 25-28 mo. Qualitative examples of several kinds of glial reactivity are also described. Our observations indicate that hippocampal changes in aging rats exhibit some similarities to brain changes reported in other mammalian species, and also illustrate the value of semithin sections for examining neuromorphologic correlates of brain aging.

MRI gradient fields increase brain mannitol space.

Following nephrectomy and intravenous injection of tritiated mannitol, adult male rats were exposed to magnetic resonance imaging (MRI) procedures at 1.5 T, 0.5 T, and 0.3 T. Compared to rats similarly handled but not exposed to MRI procedures, brain mannitol concentration, expressed as a percentage of mean body concentration, was significantly increased at 0.3 T and 0.5 T but not at 1.5 T. At 0.3 T, exposure to gradient-field fluctuations used for imaging increased brain mannitol concentration, but exposures to static main field and pulsed radiofrequency energies did not. Increased brain mannitol associated with gradient-field flux may reflect increased blood-brain barrier permeability or blood volume in brain. MRI effects on brain mannitol space are of uncertain clinical significance, but are consistent with prior evidence of an MRI-induced increase of brain capillary endothelial cell transport observed with horseradish peroxidase. Further studies are needed to confirm these findings and to explore the processes underlying changes in mannitol distribution related to MRI.

Measurement of cerebral blood flow by washout of microwave induced heating.

A method is described for measurement of cerebral blood flow utilizing the washout of microwave delivered heating. Using a microwave source attenuated to achieve a brain temperature elevation of 0.5-0.75 degrees C after a 2 second exposure in the rat, cerebral blood flow was calculated from the temperature washout curve monitored by a small thermistor implanted in the brain. The results obtained with this method were comparable to those obtained using the [14C] butanol method. To our knowledge this represents the first description of a method to deliver a blood flow "indicator" atraumatically directly into brain tissue.

Newborn rabbit blood-brain barrier is selectively permeable and differs substantially from the adult.

Examination of blood-brain barrier (BBB) function by the intracarotid injection technique has been utilized in studies of newborn (6-30 h) and adult rabbits. The exclusion of mannitol (mol. wt. 182), dextran (mol. wt. 60,000-90,000), and indium-bound EDTA indicate that the newborn BBB has restrictive properties similar to the adult. At birth, saturable, carrier-mediated transport mechanisms are present, regulating the entry of glucose, amino acids, organic acids, purines, nucleosides, and choline. No difference in brain uptake of glucose was observed between adult and newborn, but considerably higher uptake rates for arginine, choline, and adenine were seen in the newborn. In contrast to suggestions of an immature barrier in young animals, these studies indicate that a sophisticated, selective BBB is operative at birth. Furthermore, the specific selectivity and dramatic increases seen for certain metabolites imply a vital function in the newborn for these carrier systems.

Developmental modulations of blood-brain barrier permeability as an indicator of changing nutritional requirements in the brain.

The intracarotid injection technique has been utilized to examine blood-brain barrier function in studies of newborn (greater than 24 h), 7, 14, 21 and 28 day-old, as well as adult rabbits. The age-related modulations in blood-brain barrier transport of adenine, arginine, choline, lactate and tryptophan were defined and demonstrated to be independent of each other. Lactic acid uptake was unusual in that the brain uptake index (BUI) was found to be greatest at 7 days postpartum. Elevated lactate uptake continues until 14 days and is then reduced. As indicated below, for all of the other metabolites examined, a maximal BUI was observed in the newborn brain and BUIs typically showed some sort of inverse relationship to animal age. The BUI of arginine is apparently halved in the first 7 days postnatally, and continues to decrease, reaching the value seen for the adult rabbit by an age of 21 days. In contrast, the brain uptake of adenine is unusual in that there appears to be a very gradual reduction in brain uptake occurring throughout the suckling period. A 3-fold decrease in the BUI of choline was observed during the first 2 wk postpartum. Tryptophan uptake undergoes a 4-fold reduction in the first 4 wk postnatally . Only minor variations in the uptakes of glucose and butanol (a reference substance which is completely cleared by brain over a wide range of blood flow rates) were observed over the range of ages examined. Therefore modulations in adenine, arginine, choline, lactate and tryptophan permeability are not attributable to blood flow alterations.

Kinetics of transport and phosphorylation of 2-fluoro-2-deoxy-D-glucose in rat brain.

UNLABELLED: The kinetics of transport across the blood-brain barrier and metabolism in brain (hemisphere) of [14C]2-fluoro-2-deoxy-D-glucose (FDG) were compared to that of [3H]2-deoxy-D-glucose (DG) and D-glucose in the pentobarbital-anesthetized adult rat. Saturation kinetics of transport were measured with the brain uptake index (BUI) method. The BUI for FDG was 54.3 +/- 5.6. Nonlinear regression analysis gave a Km of 6.9 +/- 1.2 mM and a Vmax of 1.70 +/- 0.32 mumol/min/g. The Ki for glucose inhibition of FDG transport was 10.7+/-44 mM. The kinetic constants of influx (k1) and efflux (k2) for FDG were calculated from the Km2, Vmax, and glucose concentrations of the hemisphere and plasma (2.3 +/- 0.2 mumol/g and 9.9 +/- 0.4 mM, respectively). The transport coefficient (k1 FDG/k1 glucose)was 1.67 +/- 0.07 and the phosphorylation constant was 0.55 +/- 0.16. The predicted lumped constant for FDG was 0.89, whereas the measured hexose utilization index for FDG was 0.85 +/- 0.16. CONCLUSION: The value for the lumped constant can be predicted on the basis of the known kinetic constants of FDG and glucose transport and metabolism, as well as brain and plasma glucose levels. Knowledge of the lumped constant is crucial in interpreting data obtained from 18FDG analysis of regional glucose utilization in human brain in pathological states. We propose that the lumped constant will rise to a maximum equal to the transport coefficient for FDG under conditions of transport limitation (hypoglycemia) or elevated glycolysis (ischemia, seizures), and will fall to a minimum equal to the phosphorylation coefficient during phosphorylation limitation (extreme hyperglycemia).

Kinetics of regional blood-brain barrier glucose transport and cerebral blood flow determined with the carotid injection technique in conscious rats.

Anesthetics, particularly barbiturates, have depressive effects on cerebral blood flow and metabolism and likely have similar effects on blood-brain barrier (BBB) transport. In previous studies utilizing the carotid injection technique, it was necessary to anesthetize the animals prior to performing the experiment. The carotid injection technique was modified by catheter implantation in the external carotid artery at the bifurcation of the common carotid artery. The technique was used to determine cerebral blood flow, the Km, Vmax, and KD of glucose transport in hippocampus, caudate, cortex, and thalamus-hypothalamus in conscious rats. Blood flow increased two to three times from that seen in the anesthetized rat. The Km in the four regions ranged between 6.5 and 9.2 mM, the Vmax ranged between 1.15 and 2.07 mumol/min/g, and the KD ranged between 0.015 and 0.035 ml/min/g. The Km and KD in the conscious rat did not differ from the values seen in the barbiturate anesthetized rat. The Vmax, on the other hand, increased two- to three-fold from that seen in the anesthetized rat and was nearly proportional to the increase in blood flow seen in the conscious rat. The development of the external carotid catheter technique now allows for determination of BBB substrate transport in conscious animals.

Rapid, transient drop in brain glucose after intravenous phloretin or 3-0-methyl-D-glucose.

Rats were injected intravenously with either phloretin (100 mg/kg) or 3-0-methyl glucose (2 g/kg) to reduce the carrier-mediated flux of glucose into brain. Plasma glucose and brain free glucose (BFG), lactate, and glycogen were measured over a 16 min time course. Injection of these substances caused a rapid drop in BFG to 60% of control at one minute and a minimum (50% of control values) at 4 min., followed by a gradual rise to control levels at 16 min. While plasma glucose fell, and then increased after injection, brain lactate and glycogen content was unaffected. Repeated injections of phloretin eventually caused a drop in brain glycogen; but with either competitor, BFG never fell below 50% of normal values. The i.v. injection of the glucose analog, 3-0-methyl glucose (the less toxic of the two drugs) is proposed as a possible means of cutting off the potentially hazardous supply of blood glucose to the postischemic brain.

Kinetic constants for blood-brain barrier amino acid transport in conscious rats.

The kinetic constants for large neutral amino acid (LNAA) transport across the blood-brain barrier (BBB) of conscious rats were determined in four brain regions: cortex, caudate-putamen, hippocampus, and thalamus-hypothalamus. Indwelling external carotid artery catheters allowed for single-bolus (200 microliters) injections directly into the arterial system of unanesthetized and lightly restrained animals. Our results showed lower brain uptake index values for conscious rats compared to previous reports for anesthetized animals which are consistent with higher rates of cerebral blood flow in the conscious animals. Km values were lower in the conscious animals and ranged from 29% to 87% of the Km values in pentobarbital-anesthetized animals whereas the KD values were about twofold higher in the conscious animals. No apparent regional differences were observed. Influx rates were determined which take into consideration flow rates and plasma amino acid concentrations. Our results showed an average amino acid influx value of 5.2 nmol/min/g, which is 53% higher than the average influx in pentobarbital-anesthetized animals. The present results in conscious animals regarding the low Km of LNAA transport across the BBB lend further support to the importance of fluctuations in plasma amino acid concentrations and LNAA transport competitive effects on brain amino acid availability.

Effect of pharmacological doses of 3-0-methyl-D-glucose and 2-deoxy-D-glucose on rat brain glucose and lactate.

The present investigation examined the effects of two glucose analogues, 3-0-methyl-D-glucose (30MG) and 2-deoxy-D-glucose (2DOG) on basal levels of rat brain glucose and lactate. The results showed that pretreatment (iv) with 30MG up to 2 g/kg caused a transient drop in brain glucose levels to 42% of control value within 2.5 min and a drop in lactate levels to 75% of control value by 5 min. 2DOG administration (2 g/kg) affected glucose in a biphasic response with an initial drop to 46% of control value seen by 2.5 min, followed by a progressive increase to 290% of the control value by 40 min. This elevated level of glucose was sustained for approximately 40 min. Lactate levels responded to 2DOG administration by a decrease to 37% of control value within 10 min post-injection and returned to near basal levels by 160 min. A dose response was also examined for both compounds. Behaviorally 30MG had no apparent effects. However, the response to 2DOG was a reduction in voluntary movements, piloerection, irregular clonic jerks, splayed limbs and fits of wild running. These experiments were designed to evaluate the potential of 30MG or 2DOG for attenuating the well documented rise in brain lactate levels following an ischemic insult. Our results suggest that under certain experimental conditions either 30MG or 2DOG could prevent brain lactate rise and might have beneficial effects in minimizing the neuropathological consequences of ischemic damage that could be related to increases in brain lactate.

Measurement of cerebral glucose utilization using washout after carotid injection in the rat.

The carotid injection technique, used previously to quantitate the kinetics of blood-brain barrier transport of metabolic substrates, may be modified to analyze the rate of cerebral glucose utilization. A 0.2-ml solution of [14C]glucose (GF) and [3H]methylglucose (M), an internal reference, is rapidly injected into the carotid artery, followed by microwave fixation of brain at various times up to 4 min after injection. The brain radioactivity is separated into a fraction containing neutral hexoses (GF and M) and a fraction containing metabolites of glucose. The GF/M ratio is related to the rate constant (k3) of brain glucose utilization by the simple, linear equation: 1n(GF/M) = ln(GF0/0)--k3t, where GF0/M0 = the brain uptake index of glucose, relative to methylglucose, at 5--15 s after injection, and t = the time after carotid injection, e.g., 1--4 min. It is assumed that (a) the rate of influx due to recirculation of label is minimal during the 4-min circulation period; and (b) the rate constants of glucose efflux (k2) and methylglucose efflux (k2*) are identical. Independent estimates of k2 and k2* showed these parameters to be identical: k2 = 0.14 +/- 0.08 min-1; k2* = 0.14 +/- 0.02 min-1. A logarithmic plot of GF/M ratios versus time was linear (r = 0.99), and was described by the slope k3 = 0.21 +/- 0.02 min-1. Assuming glucose is uniformly distributed in brain, then the glycolytic rate = k3 x brain glucose = (0.21 min-1) (2.6 mumol g-1) = 0.55 mumol min-1 g-1 for the cortex of the barbiturate-anesthetized rat. These studies provide the basis for a simple method of measurement of regional brain glycolysis that does not require either the use of correction factors, e.g., the lumped constant, or the use of differentially labeled glucose.