Quantification of efflux into the blood and brain of intraventricularly perfused [3H]thymidine in the anaesthetized rabbit.

Studies using choroid plexuses incubated in vitro have led to the conclusion that pyrimidine deoxyribonucleosides, such as thymidine, enter the brain predominantly through the blood-cerebrospinal fluid (CSF) barrier across the choroid plexuses. In order to examine this hypothesis, ventriculocisternal perfusions were carried out to determine the magnitude of the passage of [3H]thymidine from the CSF into the brain and blood. These experiments demonstrated that approximately 50% of the [3H]thymidine was eliminated from the CSF perfusate, some 41.6 +/- 5.6% passing into the blood and only 7.6 +/- 0.6% to the brain. Efflux into both the blood and brain was saturable, with a Km of 17.8 microM and a Vmax of 0.46 nM min-1, and partially nitrobenzylthioinosine (NBMPR) sensitive. However, a non-saturable component did exist (Kd, 13.8 microliters min-1). Overall, the rapid removal of [3H]thymidine from the CSF and its low uptake from the CSF into the brain suggests that the choroid plexuses would be an inefficient pathway for the entry of this pyrimidine deoxyribonucleoside into the brain.

Kinetics of circulating vasopressin uptake by choroid plexus.

Uptake of circulating arginine vasopressin (AVP) by choroid plexus was studied by means of the in situ brain perfusion technique in anesthetized guinea pig and by means of single-circulation paired-tracer dilution technique in isolated perfused sheep choroid plexus. Kinetic analysis revealed saturable AVP uptake with Michaelis constant (Km) values of 32 +/- 4 and 31 +/- 5 nM and maximal saturable influx rate (Vmax) of 0.45 +/- 0.06 and 12.1 +/- 0.67 pmol.min-1.g-1 in guinea pig and sheep choroid plexus, respectively. The peptide fragments AVP-(1-8) and [pGlu4,Cyt6]AVP-(4-9), the amino acids L-phenylalanine, L-tyrosine, and 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid, and the aminopeptidase inhibitors Bestatin and bacitracin did not influence hormone kinetics. However, the V1 antagonist [(1-beta-mercapto-beta,beta-cyclo-pentamethylenepropionic acid)-O-methyl-Tyr2]AVP significantly inhibited AVP uptake with inhibitor constant (Ki) values of 0.19 +/- 0.03 (guinea pig) and 0.07 +/- 0.01 microM (sheep). The V2 agonist 1-desamino-8-D-AVP and pressinoic acid produced weak inhibitions only in guinea pig choroid plexus, and Ki/Km ratios indicated 220 and 310 times lower affinities than for AVP, respectively. It is suggested that the membrane mechanism responsible for AVP uptake in choroid plexus has a binding site with properties similar to those of V1 receptor.

Kinetics of arginine-vasopressin uptake at the blood-brain barrier.

Uptake of arginine-vasopressin, VP, at the luminal side of the blood-brain barrier (BBB) was studied by means of an in situ brain perfusion technique in the guinea-pig. Kinetic experiments revealed a saturable peptide influx into the parietal cortex, caudate nucleus and hippocampus with Km between 2.1 and 2.7 microM, and Vmax ranging from 4.9 to 5.6 pmol.min-1.g-1. The non-saturable component, Kd, was not significantly different from zero. Influx of VP into the brain was not altered by the presence of the peptide fragments: VP-(1-8), pressinoic acid and [pGlu4,Cyt6]VP-(4-9) at 4.5 microM, nor yet by the aminopeptidase inhibitor, bestatin (0.5 mM) and the L-amino acid transport system substrates, L-tyrosine and L-phenylalanine at 5 mM. At a perfusate concentration of 4.5 microM, the V1-vasopressinergic receptor antagonist, d(CH2)5[Tyr(Me)2]VP, reduced VP influx; regional Ki values, assuming that the observed inhibitions were purely competitive, ranged between 4.7 and 8.5 microM. It is concluded that there is an apparent cerebrovascular permeability to circulating VP due to the presence of a carrier-mediated transport system for the peptide located at the luminal side. The mechanism for VP BBB uptake exhibits no affinity for peptide fragments and large neutral amino acids, but requires reception of the intact molecule, which may be the same initial step for both the BBB VP transporter and the V1-receptor.

Circulating neuroactive peptides and the blood-brain and blood-cerebrospinal fluid barriers.

Interactions of radiolabelled circulating neuroactive peptides: enkephalin-leucine (Enk-Leu), delta sleep inducing peptide (DSIP), thyrotropin-releasing hormone (TRH) and vasopressin-arginine (VP-Arg) with the blood-brain and blood-cerebrospinal fluid barriers were studied by mean of: 1. a vascular perfusion technique in the guinea-pig using multiple-time brain uptake analysis, 2. a vascular perfusion technique of the in situ isolated choroid plexus from sheep using single-circulation paired-tracer dilution or steady-state analysis. It has been demonstrated that Enk-Leu, DSIP and VP-Arg were taken up intact at the luminal side of the blood-brain barrier and blood-tissue interface of the blood-cerebrospinal fluid barrier by a saturable mechanism. On the other hand, a non-saturable mechanism as well as possible enzymatic degradation were shown during TRH interactions with either the blood-brain or blood-cerebrospinal fluid barriers. It is concluded that both, facilitated and simple diffusion, govern circulating neuroactive peptide uptake into the central nervous system.

A saturable mechanism for transport of immunoglobulin G across the blood-brain barrier of the guinea pig.

The existence of an immunological blood-brain barrier to homologous blood-borne immunoglobulin G (IgG) was investigated in the guinea pig using a vascular brain perfusion technique in situ. Cerebrovascular unidirectional transfer constants (Kin) for 125I-labeled IgG (2.5 micrograms/ml) estimated from the multiple-time brain uptake data, ranged from 0.53 to 0.58 ml min-1 g-1 X 10(3) in the parietal cortex, hippocampus, and caudate nucleus, the transfer rate being some 10 times higher than that for [3H]dextran (MW 70,000). In the presence of 4 mg/ml unlabeled IgG, unidirectional blood to brain transfer of 125I-IgG was markedly inhibited. Immunohistochemical analysis of the brain tissue after vascular perfusion with unlabeled IgG revealed a distribution of the blood-borne immunoglobulin in the endothelial cells of microvessels and in the surrounding perivascular tissue. It is concluded that there is a specific transfer mechanism for IgG at the blood-brain barrier in the guinea pig, which is saturated at physiological plasma levels of IgG.

Chronic amphetamine intoxication and the blood-brain barrier permeability to inert polar molecules studied in the vascularly perfused guinea pig brain.

The brain vascular perfusion method, with a multiple-time brain uptake analysis, has been employed to study the effects of chronic amphetamine intoxication on the kinetics of entry of 2 inert polar molecules, D-[14C]mannitol (mol.wt. 180) and [3H]polyethylene glycol (PEG, mol.wt. 4000) into the forebrain of the guinea pig. The unidirectional transfer constants, Kin, determined from graphic analysis 14 and 20 days after chronic amphetamine treatment (5 mg/kg daily, i.p.) showed a marked time-dependent progressive enhancement of transfer for both molecules. The kinetic features of this entry suggest the opening up of pathways through the blood-brain barrier (BBB) which allows mannitol and PEG to pass into the brain at rates which are irrespective of their molecular size and/or lipophilia and these changes cannot be attributed to simple mechanical factors such as hypertension. This opening of the BBB was associated with changes in behaviour (increased locomotor activity, stereotypy, hypervigilance, social withdrawal, and loss of weight) seen in 14- and 20-day amphetamine-treated animals. At 7 and 28 days after the withdrawal of the amphetamine treatment, the behavioural manifestations were absent, and the Kin values for both molecules were not significantly different from those measured in normal control animals which had been treated with placebo injections. The present results suggest a reversible dysfunction of the BBB as a consequence of the chronic amphetamine intoxication which correlates with the behavioural syndrome induced in the guinea pig.

Kinetic analysis of leucine-enkephalin cellular uptake at the luminal side of the blood-brain barrier of an in situ perfused guinea-pig brain.

The uptake of enkephalin-(5-L-leucine) (Leu-enkephalin) at the luminal side of the blood-brain barrier was measured by means of an in situ vascular brain perfusion technique in the anaesthetized guinea pig. This method allows measurements of cerebrovascular peptide uptake over periods of up to 20 min, and excludes the solute under study from the general circulation and systemic metabolic influences. A capillary unidirectional transfer constant, Kin, for [tyrosyl-3,5-3H]Leu-enkephalin was estimated graphically from the multiple-time brain uptake data in the presence of different concentrations of unlabelled peptide, and dose-dependent self-inhibition was demonstrated. Analysis of unidirectional influx of blood-borne Leu-enkephalin into the brain revealed Michaelis-Menten saturation kinetics in the parietal cortex, caudate nucleus, and hippocampus, with Vmax between 0.14 and 0.16 nmol min-1 g-1 and Km ranging from 34 to 41 microM, for the saturable component, whereas the estimated diffusion constant, Kd, was not significantly different from zero. Entry of [3H]Leu-enkephalin was not inhibited in the presence of either a 5 mM concentration of unlabelled L-tyrosine, tyrosylglycine, and tyrosylglycylglycine, or aminopeptidase inhibitor, bestatin (0.5 mM), suggesting that the saturable mechanism of the tracer at the luminal side of the blood-brain barrier does not involve uptake of the peptide's N-terminal amino acid and/or its tyrosine-containing fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

Saturable mechanism for delta sleep-inducing peptide (DSIP) at the blood-brain barrier of the vascularly perfused guinea pig brain.

Cellular uptake of [125I] labelled DSIP at the luminal interface of the blood-brain barrier (BBB) was studied in the ipsilateral perfused in situ guinea pig forebrain. Regional unidirectional transfer constants (Kin) calculated from the multiple-time brain uptake analysis were 0.93, 1.33 and 1.66 microliter.min-1 g-1 for the parietal cortex, caudate nucleus and hippocampus, respectively. In the presence of 7 microM unlabelled DSIP the brain uptake of [125I]-DSIP (0.3 nM) was inhibited, the values of Kin being reduced to 0.23-0.38 microliter.min-1 g-1, values that were comparable with the Kin for mannitol. The rapidly equilibrating space of brain, measured from the intercept of the line describing brain uptake versus time on the brain uptake ordinate, Vi, was greater for [125I]-DSIP than for mannitol; in the presence of unlabelled DSIP this was reduced to that of mannitol, and it was suggested that the larger volume for [125I]-DSIP represented binding at specific sites on the brain capillary membrane. L-tryptophan, the N-terminal residue of DSIP, in concentrations of 7 microM and 1 mM, inhibited Kin without affecting Vi. A moderate inhibition of Kin was obtained by vasopressin ([Arg8]-VP), but only at a concentration as high as 0.2 mM. The results suggest the presence of a high affinity saturable mechanism for transport of DSIP across the blood-brain barrier, with subsequent uptake at brain sites that are highly sensitive to L-tryptophan, and may be modulated by [Arg8]-VP.

Blood-brain barrier permeability changes during acute allergic encephalomyelitis induced in the guinea pig.

Blood-brain barrier permeability to homologous serum 125I-IgG and to D-[3H]mannitol was studied by means of the brain vascular perfusion method in guinea pigs with experimental allergic encephalomyelitis (EAE). EAE was induced with homologous myelin basic protein (MBP) after pretreatment with foreign protein and muramyl dipeptide (MDP). The results suggest a significant comparable increase in IgG blood-to-brain clearance in the parietal cortex, hippocampus, and caudate nucleus, during vascular perfusion of the brains of animals, after 7 and 20 days of EAE. On the other hand, unidirectional transfer of mannitol in the same period of EAE was markedly augmented only in the hippocampus, but no significant changes in the parietal cortex or caudate nucleus were observed. Cerebrospinal fluid (CSF)/serum ratios for IgG and albumin were both significantly increased, suggesting an increase in blood-CSF barrier permeability, but more for albumin than for IgG. The results were confirmed by immunohistochemical determination of the IgG deposits in the brains of EAE animals, during vascular perfusion with unlabeled homologous IgG. An important role of the blood-brain barrier for the central nervous system immunoglobulin homeostasis during EAE is suggested.

Effects of sensory-motor cortical lesions on blood-brain permeability in guinea pigs.

Effects of sensory-motor cortical lesions on the function of the blood-brain barrier in distant brain areas are poorly understood. Therefore a brain vascular perfusion method has been used to measure simultaneously the kinetics of entry of two inert polar molecules, D-[14C]mannitol (MW 180) and [3H]polyethylene glycol (PEG; MW 4000), into the parietal cortex, hippocampus, and caudate nucleus in guinea pigs with ipsilateral and contralateral sensory-motor cortical lesions. The graphically determined cerebral capillary unidirectional constant, Kin, indicated a marked increase in blood-to-brain transport of both molecules in all regions studied, the changes being significantly higher after contralateral lesion. The mannitol/PEG cerebrovascular permeability constant ratio, Pman/PPEG, suggested the opening up of channels that permit a flow of fluid carrying substances either with respect to (2 days after ipsilateral lesion) or irrespective of their molecular size, depending on the time after lesion. Amphetamine treatment in the guinea pigs with sensory-motor lesions induced more pronounced blood-brain barrier permeability changes for both molecules in distant brain areas.

Slow penetration of thyrotropin-releasing hormone across the blood-brain barrier of an in situ perfused guinea pig brain.

Transport of 3H-labelled thyrotropin-releasing hormone (TRH) across the blood-brain barrier was studied in the ipsilateral perfused in situ guinea pig forebrain. The unidirectional transfer constant (Kin) calculated from the multiple time brain uptake analysis ranged from 1.14 X 10(-3) to 1.22 X 10(-3) ml min-1 g-1, in the parietal cortex, caudate nucleus, and hippocampus. Regional Kin values for [3H]TRH were significantly reduced by 43-48% in the presence of an aminopeptidase and amidase inhibitor, 2 mM bacitracin, suggesting an enzymatic degradation of tripeptide during interaction with the blood-brain barrier. In the presence of unlabelled 1 mM TRH and 2 mM bacitracin together, a reduction of [3H]TRH regional Kin values similar to that obtained with 2 mM bacitracin alone was obtained . L-Prolinamide, the N-terminal residue of tripeptide, at a 10 mM level had no effect on the kinetics of entry of [3H]TRH into the brain. The data indicate an absence of a specific saturable transport mechanism for TRH presented to the luminal side of the blood-brain barrier. It is concluded that intact TRH molecule may slowly penetrate the blood-brain barrier, the rate of transfer being some three times higher than that of D-mannitol.

Passage of delta sleep-inducing peptide (DSIP) across the blood-cerebrospinal fluid barrier.

Unidirectional flux of 125I-labeled DSIP at the blood-tissue interface of the blood-cerebrospinal fluid (CSF) barrier was studied in the perfused in situ choroid plexuses of the lateral ventricles of the sheep. Arterio-venous loss of 125I-radioactivity suggested a low-to-moderate permeability of the choroid epithelium to the intact peptide from the blood side. A saturable mechanism with Michaelis-Menten type kinetics with high affinity and very low capacity (approximate values: Kt = 5.0 +/- 0.4 nM; Vmax = 272 +/- 10 fmol.min-1) was demonstrated at the blood-tissue interface of the choroid plexus. The clearance of DSIP from the ventricles during ventriculo-cisternal perfusion in the rabbit indicated no significant flux of the intact peptide out of the CSF. The results suggest that DSIP crosses the blood-CSF barrier, while the system lacks the specific mechanisms for removal from the CSF found with most, if not all, amino acids and several peptides.

Unidirectional uptake of enkephalins at the blood-tissue interface of the blood-cerebrospinal fluid barrier: a saturable mechanism.

The cellular uptake at the blood-tissue interface of the blood-cerebrospinal fluid (CSF) barrier to tyrosyl-3,5-[3H]enkephalin-[5-L-leucine] (abbreviated to Leu-enkephalin) and of its synthetic analogue D-alanine2-tyrosyl-3,5-[3H]enkephalin-[5-D-leucine] (abbreviated to D-Ala2-D-Leu5-enkephalin) was studied in the isolated perfused choroid plexuses from the lateral ventricles of the sheep, using the rapid (less than 30 s), single circulation, paired-tracer dilution technique, in which D-[14C]-mannitol serves as an extracellular marker. Cellular uptake of peptides was estimated by directly comparing venous dilution profiles of [3H] and [14C] radioactivities in the absence and presence of unlabelled peptide, the N-terminal amino acid (L-tyrosine), the typical L-transport system substrate, 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH) and the inhibitor of aminopeptidase activity, bacitracin. The cellular uptake of both enkephalins was strongly (65-76%) but not completely inhibited by the addition of 5 mM unlabelled peptide to the bolus; the self-inhibition was significantly higher for D-Ala2-D-Leu5-enkephalin than for Leu-enkephalin. The addition to the bolus of L-tyrosine (5 mM), BCH (10 mM) or bacitracin (2 mM) reduced the 3H-radioactivity uptake by the choroid plexus of both enkephalins by 20-40%, the degree of inhibition being greater for [3H]-Leu-enkephalin than for its analogue. It is concluded that during single passage of enkephalins through the choroid plexus circulation, unidirectional uptake at the blood-tissue interface of the blood-CSF barrier consists of two components; a saturable component, which represents uptake of the intact peptide by the choroid epithelium, and a non-saturable component, which reflects enzymatic degradation of peptide in the blood and/or at the barrier, with a liberation of the N-terminal tyrosyl residue. Higher penetration of the blood-CSF barrier by D-Ala2-D-Leu5-enkephalin can be attributed to its greater resistance to hydrolysis.

The effects of aluminum chloride on the rate of secretion of the cerebrospinal fluid.

The claim that AlCl3 could produce 100% inhibition of cerebrospinal fluid secretion was investigated using ventriculocisternal perfusion in the rabbit, and it was shown that a large part of this inhibition was an artefact due to a pH sensitivity of Blue Dextran, used as an indiffusible marker, caused by AlCl3. Thus the true inhibition found by us, using [3H]labeled markers, was about 33% and usually only partly reversible. Penetration of 22Na from blood into the perfused ventricles was partially inhibited by AlCl3. The effects of some other acid buffer systems, namely acetate and phosphate, on rate of secretion were measured; the results were highly variable. When mean arterial pressure was measured, it was found to be unaffected by AlCl3 but elevated with acetate and phosphate buffer mixtures.

Transport of leucine-enkephalin across the blood-brain barrier in the perfused guinea pig brain.

Transport of [tyrosyl-3,5-3H]enkephalin-(5-L-leucine) [( 3H]Leu-enkephalin) across the blood-brain barrier was studied in the adult guinea pig, by means of vascular perfusion of the head in vivo. The unidirectional transfer constant (Kin) estimated from the multiple-time uptake data for [3H]Leu-enkephalin ranged from 3.62 X 10(-3) to 3.63 X 10(-3) ml min-1 g-1 in the parietal cortex, caudate nucleus, and hippocampus. Transport of [3H]Leu-enkephalin was not inhibited by unlabelled L-tyrosine (the N-terminal amino acid) at a concentration as high as 5 mM, or by the inhibitor of aminopeptidase activity bacitracin (2 mM), suggesting that there was no enzymatic degradation of peptide at the blood-brain barrier. By contrast, 2 mM unlabelled Leu-enkephalin strongly inhibited the unidirectional blood-to-brain transport of [3H]Leu-enkephalin by 74-78% in the parietal cortex, caudate nucleus, and hippocampus. The tetrapeptide tyrosyl-glycyl-glycyl-phenylalanine (without the C-terminal leucine of Leu-enkephalin), at a concentration of 5 mM, caused a moderate inhibition ranging from 15 to 29% in the brain regions studied, whereas the tetrapeptide glycyl-glycyl-phenylalanyl-leucine (without the N-terminal tyrosine) at 5 mM was without effect on Leu-enkephalin transport. Unidirectional brain uptake of Leu-enkephalin was not altered in the presence of naloxone at a concentration as high as 3 mM (1 mg/ml), suggesting that there is no binding of Leu-enkephalin to opioid receptors at the blood-brain barrier. It is concluded that there is a specific transport mechanism for Leu-enkephalin at the blood-brain barrier in the guinea pig.(ABSTRACT TRUNCATED AT 250 WORDS)

Steady-state distribution of cycloleucine and alpha-aminoisobutyric acid between plasma and cerebrospinal fluid.

Estimates of the steady-state distribution ratios of two nonmetabolizable amino acids, alpha-aminoisobutyric acid and aminocyclopentane carboxylic acid (cycloleucine), between plasma and cerebrospinal fluid were made with a view to establishing whether or not the low values found with metabolizable amino acids, such as glycine or leucine, could be accounted for by uptake and metabolism by the brain. The estimates, based on the ratios found after i.p. injections either in bolus form or by implantation of "osmotic pumps" containing the labeled amino acids, were comparable with those found for metabolizable amino acids.

Permeability of the blood-cerebrospinal fluid and blood-brain barriers to thyrotropin-releasing hormone.

The permeability of the blood-cerebrospinal fluid (CSF) barrier to 3H-labelled thyrotropin-releasing hormone (TRH), was studied at the blood-tissue interface of the isolated perfused choroid plexus of the sheep, using a rapid (less than 30 s), single circulation paired-tracer dilution technique, in which D-[14C]mannitol serves as an extracellular marker. Arterio-venous loss of 14C radioactivity reflects the percentage of the D-mannitol dose that crosses the blood-CSF barrier using a non-specific pathway. This loss suggests that the choroidal epithelium is moderately leaky. Cellular uptake of TRH, estimated by directly comparing venous dilution profiles of [3H]TRH and D-[14C]mannitol was independent of this leakiness. The unidirectional transport of TRH could not be saturated with unlabelled TRH at a concentration as high as 10 mM, but was markedly reduced by 10 mM proline and by the inhibitor of amidase and aminopeptidase activity, bacitracin (2 mM). Permeability of the blood-brain barrier to [3H]TRH was studied in the adult rat, employing the intracarotid injection technique of Oldendorf in which [14C]butanol served as an 'internal standard'. Brain-uptake of 3H radioactivity corrected for residual vascular space indicated a low extraction from the blood of TRH during a 15 s period of exposure to the peptide. Self-inhibition of [3H]TRH uptake by unlabelled TRH (10 mM) could not be demonstrated, but L-proline (10 mM) and bacitracin (2 mM) strongly inhibited this uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Ventriculo-cisternal perfusion of twelve amino acids in the rabbit.

The clearances of twelve amino acids from the ventricles during ventriculo-cisternal perfusion in the rabbit have been measured; uptake by the brain was also measured and this permitted the separate computation of loss to brain and loss to blood during the perfusion. Clearance under carrier-free conditions was greater than when a concentration of 5mM unlabeled amino acid was present in the perfusion fluid. Brain uptake was also usually reduced by the presence of unlabeled amino acid due presumably to suppression of accumulation by brain cells. Reduction of transport across the blood-brain barrier would tend to increase brain uptake, and there was some evidence for a balance between the two opposing tendencies. Inhibition of clearance of a given labeled amino acid could be brought about by unlabeled amino acids of different molecular species. In general, the amino acids fell into three categories: neutral, acidic, and basic, and there was some overlap between them; of the neutral amino acids the A- and L-classification of Christensen was valid, although once again there was some overlap. If, during ventriculo-cisternal perfusion of a labeled amino acid, the activity of this labeled amino acid in the blood was raised well above that in the inflowing perfusion fluid, the labeled amino acid continued to be cleared from the perfusion fluid, suggesting uphill transport. On this basis it was suggested that the normally low concentrations of amino acids in the cerebrospinal fluid (CSF), by comparison with those in plasma, were due to an active transport from the CSF to the blood. Substrate-facilitated transport, whereby the penetration of labeled amino acid into the perfusion fluid from blood could be accelerated by adding unlabeled amino acid to the perfusion fluid, or vice versa, was demonstrated.

Cerebrospinal fluid drainage as influenced by ventricular pressure in the rabbit.

Artificial cerebrospinal fluid (CSF) containing radioisotope iodinated (125I) serum albumin (RISA) and either blue dextran or indigo carmine was given to white New Zealand rabbits over 4 hours. In one group it was given by ventriculocisternal perfusion, in one by ventricular infusion, and in one by cisterna magna infusion. Blood was sampled continuously from the superior sagittal sinus (SSS) or intermittently from the systemic arterial circulation. Removal of CSF from the cisterna magna during the ventriculocisternal perfusion kept the intracranial pressure (ICP) at 0 to 5 torr, whereas ventricular or cisterna magna infusion raised the ICP to 20 to 30 torr and 15 to 20 torr, respectively. In the two groups with raised ICP, an increased concentration of RISA was present in the optic nerves, olfactory bulbs, episcleral tissue, and deep cervical lymph nodes; but this was not found in the group with normal ICP. In all three groups, the concentration of RISA in the SSS blood was the same as in the systemic arterial blood. The concentration gradient of RISA across the cerebral cortex was similar in both the ventriculocisternal perfusion and the ventricular infusion groups. With cisterna magna infusion, the concentration of RISA was the same on the cortical surface and less in the ventricles compared with the ventricular infusion. It is concluded that, with elevated ICP, CSF drained via pathways that are less evident under normal pressure. Drainage of CSF was similar irrespective of whether the infusion site was the ventricles or cisterna magna. It did not appear that acute dilatation of the ventricles during ventricular infusion compromised the subarachnoid space over the surface of the hemisphere, as the concentration of RISA on the convexities and in the SSS blood did not significantly differ between the groups. Transcortical bulk transfer of CSF was not evident with raised ICP.