Delivery of human interferon-alpha to brain by transient osmotic blood-brain barrier modification in the rat.

Pharmacokinetic parameters of human lymphoblastoid interferon (IFN-alpha) delivery to normal rat brain were examined. IFN-alpha concentrations in brain parenchyma could only be detected 120 min after its intravascular administration, and were 0.003% per gram of the administered dose. The mean cerebrovascular permeability-surface area (permeability x surface area) product to IFN, 120 min after infusion, was 0.35 x 10(-6) sec-1, which is not significantly different from zero. Neither i.v. nor intracarotid IFN-alpha administration significantly affected delivery to brain. Intrathecal administration of IFN-alpha, via the cisterna magna, resulted in undetectable concentrations in brain tissue and plasma at 30 and 60 min. However, osmotic blood-brain barrier opening significantly increased IFN-alpha delivery to brain after its carotid administration. A maximum concentration of 0.18% per gram of the total administered dose was achieved at 120 min, and the cerebrovascular permeability-surface area was increased to 30.8 x 10(-6) sec-1. Intracerebral IFN-alpha concentrations did not decline significantly during the 240 min study. Osmotic blood-brain barrier opening increased the area under the brain concentration vs. time curve, measured between 30 and 240 min, from 0.012 x 10(6) U.min/g, in controls, to 1.24 x 10(6) U.min/g, at least 100-fold. This study indicates that osmotic blood-brain barrier opening significantly increases the delivery of IFN-alpha into brain, and that delivered remains within the brain for many hours.

Reversible osmotic opening of the blood-brain barrier in mice.

Unilateral reversible osmotic opening of the blood-brain barrier can be produced in mice. Infusion of 1.8 molal arabinose in water at a rate of 0.64 ml/min for 30 seconds into the internal carotid artery consistently results in ipsilateral brain staining by intravascular Evans blue dye. Osmotic opening is concentration-dependent (threshold, 1.6 molal arabinose) and reversible within 4 hours. No long-term neurologic deficit occurs. These results suggest that reversible osmotic blood-brain barrier opening can be applied to disease models in mice.

Regional cerebral blood flow in awake beagles of different ages.

Regional cerebral blood flow (rCBF) was measured in 35 brain regions of awake beagles of different ages, by infusing 14C-iodoantipyrine intravenously. Between 1-3 and 6-12 yrs of age, rCBF was reduced significantly (P less than 0.01) in only 6 of the 35 regions, by 16 to 25%, and mean weighted CBF was not significantly altered. At 14-15 yrs, rCBF was reduced significantly by 25 to 50% in 13 of 28 grey matter regions examined, but in no white matter region, as compared with rCBF at 1-3 yrs of age, and mean weighted CBF was reduced by 30% (P less than 0.01). The course of rCBF during ageing of the beagle corresponded to the reported course of the regional cerebral metabolic rate for glucose. The results demonstrate that cerebral functional activity in most brain regions is constant during much of the adult life of the beagle, and that coupling between blood flow and metabolism also is constant. Compensatory morphological and neurochemical mechanisms may account for this homeostasis of CBF. In extreme senescence, generalized reductions in rCBF are found, and probably reflect reduced sensory input to the brain as well as systemic disease.

The lumped constant in the deoxyglucose procedure declines with age in Fischer-344 rats.

Concentrations of [14C]2-deoxy-D-glucose ([14C]DG) and of glucose were measured in plasma of arterial and sagittal sinus venous blood from awake Fischer-344 rats at 3, 12, and 24 months of age, during continuous intravenous infusion of [14C]DG and after a steady-state arterial plasma concentration of [14C]DG was reached. Brain extraction, i.e., the difference between arterial and venous plasma concentrations divided by the arterial plasma concentration, was calculated for both [14C]DG and glucose. Because exchange of both substances between rat plasma and erythrocytes is slow, the ratio of the brain extraction of [14C]DG to that of glucose is identical to the lumped constant in the deoxyglucose procedure of Sokoloff et al. [J. Neurochem. 28, 897-916. (1977)]. This ratio equaled 0.502 +/- 0.015 (SEM) at 3 months, 0.456 +/- 0.007 at 12 months, and 0.418 +/- 0.006 at 24 months of age (n = 15); the means differed significantly from each other (p less than 0.05). The results indicate that the lumped constant declines between 3 and 24 months of age in awake rats, and suggest that many reported age reductions in regional cerebral glucose utilization, of 15-25%, are artifactual.

Effects of hypoxia on rat brain metabolism: unilateral in vivo carotid infusion.

An in vivo brain perfusion technique was used to examine effects of hypoxia on cerebral cortical metabolism in barbiturate-anesthetized rats. Dulbecco's phosphate-buffered solution (PBS), or Dulbecco's PBS + 6 mM glucose, was infused into the right carotid circulation for 0 to 3 min, at a rate that reduced regional cerebral blood flow to the ipsilateral parietal lobe by more than 40% and O2 delivery by about 50%. The duration of infusion of either solution was correlated negatively with the ipsilateral parietal lobe concentrations of glucose, ATP, and phosphocreatine (PCr), and positively with parietal concentrations of lactate and cAMP. cGMP increased in relation to infusion duration of Dulbecco's PBS. Statistically significant elevations of brain lactate occurred after 1 min of infusion of Dulbecco's PBS; lactate was elevated and glucose was reduced after 2 min of infusion of either solution. Brain ATP, PCr, and glycogen concentrations decreased in relation to the elevation in brain lactate, and the [PCr]:[ATP] ratio declined. The results demonstrated that limited hypoxia stimulated cerebral glycolysis and produced a concurrent decrease in brain ATP and PCr. However, ATP was spared to a degree, at the expense of PCr.

Clearance of bilirubin from rat brain after reversible osmotic opening of the blood-brain barrier.

Utilizing multicomponent spectrophotometry, we assayed the bilirubin content of rat cerebral hemispheres. With this assay, we determined the clearance of bilirubin from the rat brain following reversible, osmotic opening of the blood-brain barrier. Clearance was rapid, with a half-time of 1.7 h. This half-time was the same as that for clearance of bilirubin from the serum, suggesting that brain bilirubin was removed by transport or diffusion back into the general circulation. Osmotic opening does not damage brain tissue. Thus, in the undamaged rat brain, bilirubin is rapidly cleared, in contrast to its persistence in autopsy-proven human kernicterus. The potential for clearance of bilirubin from human neonatal brain should be considered, especially in the absence of underlying tissue damage.

[3H]Methotrexate loss from the rat brain following enhanced uptake by osmotic opening of the blood-brain barrier.

Right brain regions of anesthetized rats were loaded with [3,5,7-3H]methotrexate ([3H]MTX) or with [14C]sucrose by infusing the tracers into the right carotid artery, after the blood-brain barrier had been opened by right carotid infusion of a hypertonic arabinose solution. During the 6 hr following the procedure, the [3H]MTX concentration in 7 right-sided brain regions, when normalized to the plasma concentration integral during tracer infusion, fell, with an average half-time of 4.8 hr as compared to less than 20 min for the initial rate of loss [14C]sucrose. Right-left brain concentration differences 3 hr after treatment were statistically significant (p less than 0.05) for [3H]MTX but not for [14C]sucrose. The results indicate that intracerebral [3H]MTX is lost more slowly than is intracerebral [14C]sucrose, possibly because [3H]MTX enters brain cells, whereas [14C]sucrose remains largely extracellular.

Cerebral blood flow and oxidative metabolism in conscious Fischer-344 rats of different ages.

The cerebral metabolic rates for O2 and for glucose were measured in conscious, fasted male Fischer-344 rats at the ages of 3, 12, and 24 months, and cerebral blood flow was determined with 14C-iodoantipyrine. The metabolic rates for oxygen and glucose were obtained by multiplying blood flow by the O2 and glucose concentration differences, respectively, between blood in the femoral artery and in the superior sagittal sinus. Mean cerebral blood flow and the metabolic rates for oxygen and glucose did not differ significantly (p greater than 0.05) between 3 and 12 or between 12 and 24 months. Nor did the arteriovenous differences for O2 and for glucose change significantly with age. Because the superior sagittal sinus drains blood mainly from the cerebral cortex, the results indicate that average cerebral cortical oxidative metabolism, and the coupling ratios between the cerebral metabolic rate for oxygen and cerebral blood flow and between the cerebral metabolic rate for glucose and cerebral blood flow, do not change significantly with age in the Fischer-344 rat.

Effects of immobilization stress on cerebral blood flow and cerebrovascular permeability in spontaneously hypertensive rats.

Immobilization of unanesthetized, freely breathing, 10-12 month-old, spontaneously hypertensive rats (SHR) did not significantly alter regional cerebral blood flow (rCBF) in 13 of 14 brain regions assayed. After 5 or 15 min of immobilization, rCBF was unchanged except at the frontal lobe, where it rose significantly by 21%. Furthermore, immobilization did not increase the cerebrovascular permeability-area product for 14C-sucrose, except at three brain regions. The results indicate that immobilization of SHR does not significantly affect rCBF or blood-brain barrier permeability in most regions of the brain, and suggest that adequate autoregulation of rCBF is maintained under stress.

Regional cerebral blood flow during development and ageing of the rat brain.

Regional cerebral blood flow (rCBF) in the conscious Fischer-344 rat was measured in 14 brain regions at 5 different ages. rCBF increased significantly (P less than 0.05) in most anterior brain regions between 1 and 3 months of age, but not in phylogenetically more primitive regions from the mid- and hindbrain that may have matured prior to 1 month of age. rCBF tended to increase or remain constant between 3 and 12 months, and rose significantly in the frontal lobe. Between 12 and 24 months of age, rCBF declined by an average of 17 per cent and fell significantly in 5 brain regions, mainly from the posterior brain, and in some, possibly in relation to partial functional deafferentation. There were no statistically significant changes in rCBF between 24 and 34 months of age. rCBF and local cerebral glucose utilization (LCGU) do not follow identical time courses during development and maturation of the rat brain. A fall in LCGU between 3 and 12 months of age, when rCBF remains constant or tends to rise, may reflect increased sensitivity of the cerebrovascular bed to metabolic factors which regulate cerebral blood flow.

Effects of immobilization stress on regional cerebral blood flow in the conscious rat.

Immobilization stress of conscious, normotensive, freely breathing 10-month-old Wistar-Kyoto rats produced an overall decline in regional cerebral blood flow (rCBF), as measured with [14C]iodoantipyrine, except at the frontal lobe. In 14 brain regions, rCBF fell by an average of 14.3% after 5 min of immobilization and by 11.9% after 15 min. Immobilization stress also stimulated hyperventilation and thereby reduced PaCO2. The slope relating rCBF to PaCO2 averaged 1.5 ml 100 g-1 min-1 mm Hg-1 in 9 significantly affected regions. The findings suggest that rCBF declines during immobilization stress because of cerebrovascular constriction caused by a reduction in PaCO2. Comparison of the average slope with published values in indicates furthermore that were PaCO2 to remain unchanged during immobilization, rCBF would increase by at most 20%.

Quantitative aspects of reversible osmotic opening of the blood-brain barrier.

Retrograde infusion of a hypertonic arabinose solution into the right external carotid artery of rats reversibly increases cerebrovascular permeability to [14C]sucrose in the right cerebral hemisphere. PA ([14C]sucrose permeability x capillary surface area) rises from a control mean of 11 x 10(-6) S-1 to above 200 x 10(-6) S-1. The rise correlates with an increased staining of the brain by intravascular Evans blue, and is followed by a transient, 1-1.5% increase in brain water content. At least 20 s of infusion is required for 1.6 M arabinose solution to effectively open the blood-brain barrier. The increase in cerebrovascular permeability is temporary, however, because PA remains slightly elevated 1-2 h after infusion and is normal 6 h after infusion. It is suggested that osmotic barrier opening is mediated by cerebrovascular dilatation as well as by shrinkage of the vascular endothelium. By quantitatively defining thresholds of infusate concentration and infusion time for osmotic barrier opening, and by characterizing the time course of increased PA, the experiments establish criteria for applying the osmotic method to experimental pharmacology of the central nervous system.

Osmotic opening of the blood-brain barrier to methotrexate in the rat.

Osmotic opening of the blood-brain barrier in the rat, by intracarotid infusion of 1.6 molal (m) arabinose solution, increases cerebrovascular permeability to 3H-methotrexate by a factor of about seven. PA (product of permeability and capillary surface area) increases from a mean of 3.3 X 10(-5) sec-1 in brains of control rats to as much as 28 X 10(-5) sec-1 at the cerebral hemisphere ipsilateral to carotid infusion. If tracer is introduced into the carotid artery after osmotic treatment, brain uptake is increased by a net factor of 50 (a factor of 70 due to elevation of PA, multiplied by 7 due to infusion by the carotid route) as compared to uptake by normal, untreated brain with infusion into a peripheral vein. Osmotic barrier opening followed by carotid drug administration may be of use in experimental chemotherapy of nervous system disease.

Modification of the blood-brain barrier: increased concentration and fate of enzymes entering the brain.

The blood-brain barrier of rats was opened reversibly by infusing a hyperosmotic solution of arabinose into the external carotid artery. Permeability was increased maximally in the first 15 min and remained slightly elevated at 1 hr. Osmotic barrier opening significantly increased brain uptake of intravenously injected alpha-mannosidase (alpha-D-mannoside mannohydrolase, EC 3.2.1.2.4) (derived from human placenta) and horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7). By injection of 4 X 10(5) units of alpha-mannosidase into an animal, brain activity rose to about twice the normal control activity of the enzyme. After 30 min, activity of administered enzyme in the extracellular space of the brain was calculated to be 30% of the serum concentration. Biochemical and histological studies with horseradish peroxidase showed that exogenously administered enzyme entered the brain extracellular space immediately after barrier opening and was incorporated within neuronal lysosomal packets during the next 24 hr. Measurable peroxidase activity was found in brain as much as 72 hr after osmotic treatment. The results demonstrate that the blood-brain barrier can be reversibly opened to enzymes, that a glycoprotein enzyme is incorporated into neuronal lysosomes, and that the brain may now be considered a potential target for enzyme replacement therapy in heritable metabolic disorders.

Entry of [3H]norepinephrine, [125I]albumin and Evans blue from blood into brain following unilateral osmotic opening of the blood-brain barrier.

The blood-brain barrier in adult rats was opened unilaterally by infusing 1.58 M L (+)-arabinose in 0.9% NaCl solution into the internal carotid artery, via a catheter in the external carotid. The common carotid remained patent during the procedure. Osmotic barrier opening allowed entry into the brain of three intravascularly administered tracers--a visual tracer Evans blue (pulsely injected) and radioactive tracers [3H]norepinephrine (continuously infused) and [125I]albumin (pulsely injected). In osmotically perfused brain tissue, uptake of both 3H and 125I from blood was increased 2-5-fold above control, with maximal increases observed in the caudate nucleus, hippocampus and thalamus. In control brain regions, Evans blue and albumin remained intravascular, whereas norepinephrine was taken up, possibly by sympathetic nerve endings in cerebral vessels, as a function of blood plasma concentration and duration of exposure. The barrier closed within 4 h after intracarotid arabinose infusion, and barrier opening was not associated with edema as measured two days after infusion.

Isolation and antigenic characteristics of axial filaments from the Reiter Treponeme.

Axial filaments were isolated and purified from Reiter treponemes after detergent solubilization of the cells" outer envelope. The axial filaments were separated from the spirochetal cells by shearing, purified by density gradient centrifugation, and fragmented by ultrasonication. Acrylamide gel electrophoresis of dissociated filaments revealed two major protein bands. Gel diffusion precipitin tests and immunoelectrophoresis between a purified axial filament suspension and anti-Reiter treponeme serum gave a single precipitin line. Checkerboard complement fixation tests also gave results consistent with a single antigen-antibody system. Tests with immune sera to other cultivable spirochetes were positive with some and negative with others. In addition, strongly positive reactions were obtained in complement fixation and precipitin tests with sera from rabbits and humans with syphilis and other treponematoses. However, both serological tests gave reactions of partial identity between the antigen(s) of Reiter treponeme axial filaments and those of the pathogenic treponemes. It was concluded from these studies that the axial filaments were probably the cellular locus of the so-called "Reiter protein" antigen of syphilis serology.