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.

ABSTRACT. 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. Hyperbilirubinemia is likely the most common medical diagnosis in the newborn. Physicians treat neonatal jaundice in order to prevent the neurotoxicity associated with brain uptake of bilirubin (kernicterus). Research on the cerebral transport and toxicity of bilirubin continues, but the results remain unclear (1). Uncertainty extends to clinical practice as well. For example, the preceding edition of Hospital Care of Newborn Infants, published by the American Academy of Pediatrics, included guidelines for exchange transfusion (2). The current edition omits guidelines, noting a diversity of approaches among physicians (3). In part, this diversity results from uncertainty about how bilirubin enters the brain and the anatomic and biochemical loci of bilirubin toxicity.The blood-brain bamer normally restricts exchange of watersoluble substances and proteins between blood and brain, but is damaged in diseases such as hypertension, by ischemia or trauma. Experimentally, the blood-brain bamer can be unilaterally and reversibly opened without causing brain damage by infusing a hypertonic solution of arabinose into one of the carotid arteries (4). Permeability returns to normal within about I h after arabinose infusion (5). We have previously shown that circulating albumin-bound bilirubin enters the side of the rat brain receiving the arabinose (4, 6). Furthermore, the regional cerebral distribution of albumin mimicks that seen in human kernicterus, providing a potential model for the human disease.We designed this study to determine the time-course of bilirubin entry into and removal from the brain after osmotic opening of the blood-brain bamer. For the study, we also developed rapid, simple techniques for the extraction and quantitation of bilirubin in brain tissue.

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In a review of cerebral metabolism and function in man, Kety (1950) reported that one subject, who demonstrated marked apprehension during a test situation, had an unusually high cerebral metabolic rate for oxygen (CMR02), 5.0 ml O2 100 g-l min-I, as compared to his normal range of 3.2-4.2 ml O2 100 g-I min-I. Kety suggested that anxiety, or psychic stress, can increase brain metabolism. A recent study in man also indicates that pain-induced stress significantly increases blood flow in gray matter by about 10%, particularly in the frontal lobes (lngvar et aI., 1976).These clinical observations appear to be sup ported by recent findings in rats. Immobilization for 5 -30 min (conscious rats were artificially ventilated while paralyzed by a muscle relaxant) increases both CMR02 and cerebral blood flow (CBF) up to twofold (Carlsson et aI., 1975(Carlsson et aI., , 1977. These cerebral effects in rats are ascribed to the f3-adrenergic re ceptor action of catecholamines, particularly epi-

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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.

The blood-brain barrier of rats was opened reversibly by infusing a hyperosmotic solution of

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. 12 to augment brain entry of intravenous water-soluble drugs and proteins normally excluded from the central nervous system. Central nervous system chemotherapy in human patients also has been conducted with this technique. l314 The method has been shown to increase delivery of substances to the brain without causing long-term cerebral damage.2 7 1 2 " However, a number of disease models in which it would be useful to study the central nervous system effects of drugs, enzymes, and antibodies administered systemically have been developed largely in mice. These models include viral and fungal infections, immune deficiencies, experimental tumors, and genetic abnormalities.16 Therefore, we thought it of interest to extend the osmotic method to unilaterally and reversibly open the BBB in mice. An abstract of this work has been published. 17Materials and Methods BALB/c female mice (Charles River Breeding Laboratories, Wilmington, Massachusetts) weighing 15-22 g, 6-10 weeks of age, were anesthetized with i.p. 50 mg/kg sodium pentobarbital (Somnifer, diluted 1:4 vol: vol with water; Richmond Veterinary Supply, Richmond, Virginia). A PE-50 polyethylene catheter with the end tapered to 0.4 mm diameter with heat was inserted retrograde in the right external carotid artery and secured with 6-0 surgical silk. The catheter tip was placed just above the bifurcation with the internal carotid artery. During surgery, the superior thyroid and occipital arteries were cauterized (Week, Research Triangle Park, North Carolina), but carotid circulation to the brain never was interrupted. The catheter was filled with 0.04 ml 100 units heparin/ml 0.9% (wt: vol) NaCl, filtered with a 0.22 /Am-diameter filter (Millipore, Bedford, Massachusetts). Evans blue dye (Chroma-Gesellschaft, Stuttgart, F.R.G., 2 ml/kg of a 2% [wt: vol] into a saphenous vein. The dye forms a complex with plasma albumin and is used as a marker of BBB integrity. 5Five minutes after Evans blue injection, 41 mice were infused for 30 seconds with a warm (37° C) solution of hypertonic L-( + )-arabinose (Sigma, St. Louis, Missouri) in distilled water filtered with a 0.2-/um filter (Nalge, Rochester, New York). Five control mice were infused with filtered 0.9% (wt: vol) NaCl (isotonic saline). Infusions at a constant rate of 0.64 ml/min were delivered with an infusion pump (Harvard 944, Millis, Massachusetts). The interface between common carotid blood and infusate was observed during infusion. If infusate was seen to pass into the...