Dusan M. Mitrovic

Measurement of Solute Transport Across the Blood–Brain Barrier in the Perfused Guinea Pig Brain: Method and Application to N‐Methyl‐α‐Aminoisobutyric Acid

Abstract: A technique for the vascular perfusion of the guinea pig head in vivo, suitable for measurements of blood‐to‐brain transport under controlled conditions of arterial inflow, has been developed. With a perfusion pressure ranging between 13 and 18 kPa and Pco2 in the arterial inflow of 5 and 5.5 kPa, cerebral blood flow, measured with [14C]butanol, was about 1 ml min−1 g−1 in the cerebral cortex, hippocampus, and caudate‐putamen of the ipsilateral hemisphere; in the cerebellum and pontine white matter it was considerably less, and much higher perfusion pressures were required to establish equal blood flow throughout the whole brain. Regional water content, Na+/K+ ratio, ATP, energy charge potential, and lactate content of the ipsilateral side of perfused and nonperfused brain were not significantly different after 10 min perfusion. The D‐[3H]mannitol space did not exceed 1% after 30 min of perfusion, indicating the integrity of the barrier. Over this period, EEG, ECG, and respiratory waveform remained normal. When [14C]N‐methyl‐α‐aminoisobutyric acid (MeAIB), and D‐[3H]mannitol were perfused together over periods extending to 30 min progressive uptakes of both solutes by the parietal cortex could be measured, and the unidirectional transfer constants estimated from multiple time‐uptake data. The Kin for MeAIB (0.75 × 10−3 ml min−1 g−1) was some three times that for mannitol. It is concluded that the technique provides a stable, well‐controlled environment in the cerebral microvasculature of the ipsilateral perfused brain hemisphere suitable for examining the transport of slowly penetrating solutes into the brain.

Permeability of the Blood‐Brain Barrier to the Immunosuppressive Cyclic Peptide Cyclosporin A

Abstract: Uptake of the immunosuppressive lipophilic peptide cyclosporin A has been measured by a number of techniques. The brain uptake index (BUI) technique in the rat yields only a small BUI value that is not significantly different from that of sucrose and mannitol and is comparable to other published BUI values for this compound. Brain perfusion studies in the guinea pig produce a unidirectional cerebrovascular permeability constant (Kin) of 1.2 ± 0.28 μg−1 min−1 for the hippocampus. Intravenous bolus injection techniques also in the guinea pig characteristically produce a larger Kin value of 2.53 ± 0.38 μg−1 min−1 for the same brain region, even after a correction for the inulin space of the tissue has been made. Apparent penetration of cyclosporin A into the cerebrospinal fluid (CSF) determined with the intravenous bolus injection technique is small with a Kin of 0.79 ± 0.07 μ g−1 min−1. However it is suggested that the radioactivity present in CSF is largely tritiated water. Studies with cultured cerebral endothelial cells from the rat have also been carried out and show that the cultured cells take up and accumulate cyclosporin A in vitro, achieving a tissue‐to‐medium ratio of 20 after 25 min of incubation. It is suggested that cyclosporin A is primarily taken up from lipoprotein at the blood‐brain interface but, because of tight junctions at the blood‐brain and blood‐CSF barriers, becomes effectively trapped in the cerebral endothelial cells and the choroid plexus.

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.

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.

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.

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 sensorymotor 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.

Penetration of 3H Tiazofurin from Blood into Guinea Pig Brain

Transport of tiazofurin (2-β-D-ribofuranosyl thiazole-4-carboxamide) across the blood-brain barrier (BBB) was studied using brain vascular perfusion method in the guinea pig. The obtained results demonstrate that brain clearance of 3H tiazofurin significantly differs from zero, suggesting that this molecule penetrates from blood into the brain. The values of tiazofurin brain clearance are very close to the values obtained for neuropeptides and other so called “slow penetrating molecules” (regarding the blood brain barrier). Addition of increasing concentrations of unlabelled tiazofurin to the perfusing medium caused a significant decrease in the uptake of [3H] labelled tiazofurin. Therefore, penetration of tiazofurin from blood into brain seems to be a saturable process. Presence of increasing concentrations of unlabelled adenosine has similar effect as the presence of unlabelled tiazofurin in the perfusing medium. However, it did not cause complete inhibition of tiazofurin brain uptake.

Transport of 3H L-Alanine Across the Blood-Brain Barrier of in Situ Perfused Guinea Pig Brain

Transport of 3H L-alanine through the blood-brain barrier (BBB) was studied using brain vascular perfusion method in guinea pig. Our results indicate that L-alanine passes across the luminal side of the BBB. Unidirectional transport constant Kin ranged from 4.871±0.622 μmin−1g−1 in hippocampus to 5.608±0.902 μmin−1g−1 in parietal cortex, which is comparable with the values obtained for other small neutral non-essential amino acids. Addition of unlabelled L-alanine to perfusing medium caused the decrease in L-alanine transport, indicating the importance of saturable component for L-alanine transport. However, presence of high concentrations of unlabelled L-alanine in perfusing medium (up to 12 mmol/l), did not result in complete inhibition of 3H L-alanine transport through the BBB. Therefore, it seems that another mechanism is also involved in 3H L-alanine transport across the endothelial cells’ luminal membrane. Values for Michaelis-Menten constant for L-alanine transport from blood into brain point out that the affinity of this molecule to its carrier(s) is rather small (Km >1 mmol/1). Capacity of 3H L-alanine blood-to-brain transport is very small as well (Vmax <20 nmol/min/g).

Distribution of Small Neutral Amino Acids after Penetrating the Luminal Side of the Guinea Pig Blood-Brain Barrier

Distribution of radio labelled small neutral amino acids between endothelial cells and brain parenchyma after transport across the luminal side of the blood-brain barrier was studied in guinea pig. After in situ brain vascular perfusion, the capillary depletion method was applied (Triguero et al. 1990). Endothelial cells were separated from brain parenchyma, using centrifugation (5400 g) in 13% dextran solution. The concentration ratio between endothelial cells (pellet) and brain parenchyma (supernatant) was determined for each amino acid studied (3H L-serine, 3H L-alanine and 14C L-proline) after different perfusion times (1,3 and 6 minutes).

Opening of the Blood—Brain Barrier to D-Mannitol Induced by Sensorimotor Cortical Lesions in the Anaesthetized Guinea-pig

We applied the brain vascular perfusion method to determine the kinetics of entry to inert polar molecular D-[3H]-mannitol into the parietal cortex, hippocampus and caudate nucleus in guinea-pigs with sensorimotor (SM) and occipital (Oc) cortical lesions. In the control group of animals, with Oc lesions, only a moderate increase of blood-brain (BBB) permeability to D-mannitol was found after the ipsilateral lesion and no permeability changes occurred after the contralateral lesion, which was expressed by graphically determined unidirectional transfer constant, Kin. In a separate series of experiments, in the guinea-pigs with SM cortical lesions, estimated Kin values indicated a marked increase of blood-to-brain transport of D-[3H)-mannitol in all brain regions studied, the changes being significantly higher after the contralateral lesion. Amphetamine pretreatment in the guinea-pigs with SM and Oc cortical lesions induced more pronounced BBB permeability changes for D-[3H]-mannitol in all brain areas studied.