Transient blood-brain barrier passage of polar compounds at low pH.

The brain uptake index (BUI) of polar 14C-labeled test compounds with molecular weights (MWs) of 79-70,000 was examined using the single-pass carotid injection technique in pentobarbital-anesthetized rats. Compounds were injected in 40 mM malonate, pH 2.5, and 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.4. BUI is defined as (14C-labeled test compound/[3H]H2O in brain)/(14C-labeled test compound/[3H]H2O in injectate) x 100 at a 5-s decapitation time. Increased BUIs of compounds in pH 2.5 buffer were MW dependent with a threshold < 5,000. BUI, extraction, and permeability-surface area product (PS) were significantly greater at pH 2.5 compared with pH 7.4 (P < 0.05). Washouts of [14C]butanol and [3H]H2O at pH 2.5 and pH 7.4 were calculated. Cerebral blood flow and PS increased at low pH. Other buffers, oxalate, glycine, and lactate were used at low pH and also increased BUIs. The duration of the blood-brain barrier (BBB) opening at pH 2.5 was 60 s as estimated by penetrance of the normally excluded dye fluorescein. A plot of BUI or PS at pH 2.5 vs. (MW)1/2 suggests that transient BBB tight junction opening contributed to the passage of polar compounds at low pH.

Terminal endothelial cells of lymph capillaries as active transport structures involved in the formation of lymph in rat skin.

Electron microscopic examination of lymph capillaries of the dermal papillae of rat scalp skin revealed continuous extension of the lymph collection system into 4 to 10 micron diameter lumen capillaries with thin walls, scant basement lamellae (membranes), and blind-endings of 1 to 4 micron lumen diameter within endothelial-type cells. These terminal endothelial cells also displayed intracytoplasmic channels and pinocytotic vesicles, extensive cytoplasmic processes and a high cytoplasmic volume-percent of mitochondria suggesting active transport capabilities of lymphatic endothelia. The mitochondrial cytoplasmic volume-percent (mean 14.5%) exceeded that present in blood capillary endothelial cells of the rat brain (the anatomic substrate of the blood-brain barrier), that have a volume-percent of mitochondria of 10 to 12% (1). Active transport processes centered in such endothelial cells could account for a portion of lymph formation, and explain the continued accumulation of lymphedema distal to blocked lymphatic collection ducts when lymphatic intraluminal pressure is greatly increased. The small lumen diameter capillaries, which correspond spatially to the prelymphatics of other authors (2), typically converge in groups of three to form larger diameter lymph capillaries corresponding to the lymph "initials" previously described (2,3).

Field dependent transverse relaxation rate increase may be a specific measure of tissue iron stores.

The degree to which MRI magnet field strength affects measured transverse relaxation rates (R2) defines a measure termed the field dependent R2 increase (FDRI). We report here the results of in vivo and in vitro experiments that were conducted to evaluate whether FDRI is a potentially useful measure of tissue iron stores. T2 relaxation times were obtained using two clinical MRI instruments operating at 0.5 and 1.5 Tesla, and relaxation rates (R2) were calculated as the reciprocal of T2. The in vivo experiment measured R2 in human brain frontal white matter, caudate nucleus, putamen, and globus pallidus. The FDRI was very highly correlated with published brain iron levels for the four regions examined. The in vitro experiment measured R2 in agarose gel-based phantoms containing physiologic forms and amounts proteins involved in iron storage and transport (ferritin, apoferritin, transferrin, and apotransferrin). Significant field dependence was observed only for the ferritin phantoms. The differences in the R2 values obtained at the two field strengths were striking, and were proportional to the ferritin levels of the phantoms. These studies suggest that FDRI may be a specific measure of tissue ferritin. The quantitative significance of the results to imaging and possible applications to the clinical investigation of pathologic states are discussed.

Blood-brain barrier penetration abolished by N-methyl quaternization of nicotine.

The present study determined the effect of organically quaternizing either of the two tertiary nitrogen sites of nicotine to assess the in vivo effects of the permanently ionized states of the synthesized N-[14C]methylnicotines on brain uptake in rat after intracarotid injection. Male Sprague-Dawley rats were used to measure the brain uptake index (BUI) by single-pass clearance in brain after rapid injection at pH 7.4 into the left common carotid artery (expressed as a percentage) relative to simultaneously injected 3HOH. The BUI of [14C]mannitol, a control for the method background, was measured to be 2.6 +/- 0.6. At physiological pH, in striking contrast to the [pyrrolidine-2-14C]nicotine BUI of 120 +/- 3, the N-[14C]-methylnicotines had a BUI of 3.0 +/- 0.6, which was not significantly different from the method background and which indicated abolition of blood-brain barrier penetration of nicotine with the sensitivity of the BUI method.

Some relationships between addiction and drug delivery to the brain.

Hemodynamic radioisotope studies of brain blood flow in humans have been correlated with the delivery of some common addictive drugs. Both lipophilic and hydrophilic tracers were used in the hemodynamic studies. Iodoantipyrine is lipophilic and is completely cleared by brain during a single brain circulatory passage, as are cocaine and nicotine. Iodohippurate is hydrophilic, so its brain clearance after IV injection resembles that of morphine. The earlier studies performed in humans have been related here with recent studies of blood brain penetration of drugs of abuse. As presented, these separate studies are consistent with the proposed hypothesis that the interval between drug intake and perceived effect is a significant consideration when explaining severity of addiction. The shorter the time interval between drug intake and its perceived effect, the more severe the addiction appears to be. This relationship may explain differences in severity of addiction to the same drug taken by various routes of administration.

T2 hyperintense foci on magnetic resonance images of schizophrenic patients and controls.

High resolution magnetic resonance imaging (MRI) of the brain was performed on 18 male schizophrenic patients and 15 male normal control subjects using an identical imaging protocol. The number and size of T2 hyperintense foci were clinically quantified by an academic radiologist. Large foci (greater than or equal to 3 mm in diameter) were observed more frequently on patient images (7/18) than on control images (1/15). The imaging protocol detected high rates of focal hyperintensities, but no differences between patients and controls were noted in the total affected brain area (sum of focal areas) or in the presence or absence of foci.

Rat brain free glucose and lactate measurement by a novel method using bisecting decapitation-extrusion and enzyme denaturation at five seconds.

This study investigates a novel method as a means for animal decapitation with rapid brain removal and enzyme denaturation. Briefly, the rat head is simultaneously decapitated and bisected. Either half of the in situ brain is aspirated under -250 mm Hg pressure into a modified small plastic syringe and then extruded through a needle as a fine strand into a relatively large volume of 2 M urea at 95 degrees C. After cooling, sonication, and centrifugation of the brain homogenate, the supernatant is measured enzymatically for brain free glucose and lactate concentration. Enzyme denaturation is effected within 4-6 s. The results are in good agreement with published values for glucose and lactate using other rapid enzyme inactivation techniques.

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.

Trophic changes in the arteries at the base of the rat brain in response to bilateral common carotid ligation.

A rat model was developed to examine changes in the posterior circle of Willis and the basilar and intracranial vertebral arteries after bilateral common carotid ligation. This procedure produced a major redistribution of blood to the head, with increased flow through the vertebral and basilar arteries. Changes in the vertebral, basilar, posterior communicating and proximal segments of the posterior cerebral arteries and neck vessels were assessed by postmortem barium sulfate arteriography and by histology of the middle portion of the basilar arteries serially at four days, and one, two, four and 15 weeks post-ligation. The changes noted were basilar and vertebral artery tortuosity, enlargement, and duplication of the vertebro-basilar junction. By 15 weeks, these intracranial vascular changes had largely regressed toward normal, commensurate with the appearance of multiple collateral vessels which were scattered throughout the soft tissues of the neck and shunted the original ligation sites. A mechanism that could explain these trophic vessel changes in response to increased blood flow is discussed. Some possible correlates of these findings with several brain vascular diseases are presented.

Nuclear magnetic resonance study of obsessive-compulsive disorder.

Magnetic resonance imaging (MRI) of the brains of 32 patients who met the DSM-III criteria for obsessive-compulsive disorder and of 14 normal subjects frequently revealed abnormalities, but none was specific to obsessive-compulsive disorder. Spin-lattice relaxation time (T1) for right frontal white matter was prolonged in the patients compared to the control subjects, and the patients had greater right-minus-left T1 differences for frontal white matter. Right-minus-left T1 differences in the orbital frontal cortex were strongly correlated with symptom severity in the unmedicated patients and in the patients with family histories of obsessive-compulsive disorder.

Simple method for determination of heroin deacetylation in rat and human serum.

This study generally supports previous studies (4,6) of deacetylation rates of heroin by whole blood and serum. The greatly accelerated in vitro deacetylation rates noted in the rat, relative to the human, are consistent with the literature, (6), with the exception that rat serum reaction rates were about 60% faster (Table 1). This in vitro study demonstrates an example of widely differing drug metabolic rates between species. The method employs urea as a denaturing agent, which is also nondamaging to the compounds of interest. Previous studies used a variety of methods, while all of the present methods apply one relatively simple technique which is especially suited to rapid enzymatic reactions in fluid media such as whole blood.

A magnetic resonance imaging study of autism: normal fourth ventricle size and absence of pathology.

Magnetic resonance imaging did not diagnose neuropathology in 15 autistic patients. Measurements of the midsagittal area and volume of the fourth ventricle did not differ between these patients and matched control subjects.

N-acetyl-L-aspartic acid: a literature review of a compound prominent in 1H-NMR spectroscopic studies of brain.

N-acetyl aspartic acid (NAA), discovered in 1956 by Tallan, is the major peak seen in water-suppressed NMR proton (hydrogen) spectroscopy. NAA makes up about one thousandth of the wet weight of human brain and appears to be limited solely to neurons. This compound has been shown to be relatively stable for a period of twenty-four hours post-mortem and the concentration of NAA is not changed by insulin-induced hypoglycemia. MAO inhibitors lower its concentration while reserpine and other drugs increase it. NAA has been implicated in many processes of the nervous system: it may be involved in the regulation of neuronal protein synthesis, myelin production, or the metabolism of several neurotransmitters such as aspartate or N-acetyl-aspartyl-glutamate. It is involved in the neurologic disorder Canavan disease and has grown to be a vital component of in vivo 1H-NMR spectroscopic studies.

The effect of pretreatment with pentobarbital on the extent of [14C] incorporation from [U-14C]glucose into various rat brain glycolytic intermediates: relevance to regulation at hexokinase and phosphofructokinase.

In the present investigation we monitored the incorporation of [14C] from [U-14C]glucose into various rat brain glycolytic intermediates of conscious and pentobarbital-anesthetized animals. Labeled glucose was delivered to brain by single bolus intracarotid injection and brain tissue was subsequently prepared at 15, 30, and 45 sec by freeze-blowing. Glycolytic intermediates were then separated by column chromatography. Our results showed a gradual decrease with time of 14C-labeled glucose which gave a calculated rate for glucose metabolism of 0.86 mumol/min/g and 0.56 mumol/min/g in conscious and anesthetized animals, respectively. Compared to the results obtained using conscious animals the administration of pentobarbital not only resulted in a significant attenuation of the rate of glucose metabolism but also caused a similar reduction in the amount of 14C incorporated into several glycolytic intermediates. These intermediates included: glucose 6-phosphate, fructose 6-phosphate, fructose 1,6 diphosphate, dihydroxyacetone phosphate and post glycolytic compounds. In addition, pretreatment with pentobarbital resulted in a 75% increase in the endogenous concentration of glucose, 10% increase in glucose 6-phosphate, no change in fructose 6-phosphate and 42% decrease in lactate compared to levels in brains obtained from conscious animals. These results are discussed in relation to control of glycolysis through coupled regulation at hexokinase-phosphofructokinase.

pH dependence of histidine affinity for blood-brain barrier carrier transport systems for neutral and cationic amino acids.

The effects of pH (3.5-7.5) on the brain uptake of histidine by the blood-brain barrier (BBB) carriers for neutral and cationic amino acids were tested, in competition with unlabeled histidine, arginine, or phenylalanine, with the single-pass carotid injection technique. Cationic amino acid ( [14C]arginine) uptake was increasingly inhibited by unlabeled histidine as the pH of the injection solution decreased. In contrast, the inhibitory effect of unlabeled histidine on neutral amino acid ( [14C]phenylalanine) uptake decreased with decreasing pH. Brain uptake indices with varying histidine concentrations indicated that the neutral form of histidine inhibited phenylalanine uptake whereas the cationic form competed with arginine uptake. Since phenylalanine decreased [14C]histidine uptake at all pH values whereas arginine did not, it was concluded that the cationic form of histidine had an affinity for the cationic carrier, but was not transported by it. We propose that the saturable entry of histidine into brain is, under normal physiological circumstances, mediated solely by the carrier for neutral amino acids.

Diet-induced changes in rabbit serum potassium do not alter size or number of brain capillary endothelial cell mitochondria.

Previous studies have shown brain extracellular (EC) [K+] to remain constant during wide variations of the plasma-brain [K+] gradient. High serum [K+] is hypothesized to increase K+ pumping by the blood-brain barrier (BBB) to maintain normal EC levels. This changing metabolic work could be reflected in endothelial cell mitochondria which have been shown to be three to four times more numerous in the BBB than in other capillary endothelial cells. We measured several morphologic parameters of brain capillary endothelial cells in white male New Zealand rabbits following dietary manipulation of their serum [K+] levels. Four groups, based on serum [K+] levels, were formed: Deficient, Low, Normal, and High. Following the final day's weight and blood samples, rabbits were anesthetized and perfused for transmission electron microscopic planimetry. Variables included number and area of mitochondria, areas of capillary lumen and capillary diameter, area of endothelial cell, and proportion of endothelial cell taken up by mitochondria. With the exception of the area of the endothelial cell (P less than 0.01), which decreased with increasing serum [K+] levels, no differences appeared among the variables in any of the four groups. Lack of anticipated differences in mitochondrial number or proportional area indicates either that pumping K+ occupies a minor fraction of cell energy, or changes in mitochondrial size or number are not sufficiently sensitive indicators of metabolic workload in this model.

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.