Nicotine raises the influx of permeable solutes across the rat blood-brain barrier with little or no capillary recruitment.

Nicotine (1.75 mg/kg s.c.) was administered to rats to raise local CBF (lCBF) in various parts of the brain, test the capillary recruitment hypothesis, and determine the effects of this increase in lCBF on local solute uptake by brain. lCBF as well as the local influx rate constants (K1) and permeability-surface area (PS) products of [14C]antipyrine and [14C]-3-O-methyl-D-glucose (3OMG) were estimated by quantitative autoradiography in 44 brain areas. For this testing, the finding of significantly increased PS products supports the capillary recruitment hypothesis. In 17 of 44 areas, nicotine treatment increased lCBF by 30-150%, K1 of antipyrine by 7-40%, K1 of 3OMG by 5-27%, PS product of antipyrine by 0.20% (mean 7%), and PS product of 3OMG by 0-23% (mean 8%). Nicotine had no effect on blood flow or influx in the remaining 27 areas. The increases in lCBF and K1 of antipyrine were significant, whereas those in K1 of 3OMG and in PS for both antipyrine and 3OMG were not statistically significant. The lack of significant changes in PS products implies that in brain areas where nicotine increased blood flow: (a) essentially no additional capillaries were recruited and (b) blood flow within brain capillary beds rises by elevating linear velocity. The K1 results indicate that the flow increase generated by nicotine will greatly raise the influx and washout rates of highly permeable materials, modestly elevate those of moderately permeable substances, and negligibly change those of solutes with extraction fractions of < 0.2, thereby preserving the barrier function of the blood-brain barrier.

Slightly altered permeability-surface area products imply some cerebral capillary recruitment during hypercapnia.

To test the capillary recruitment hypothesis in brain, cerebral blood flow was raised markedly in rats by exposure to 8% CO2 (hypercapnia), and capillary permeability-surface area (PS) products were measured. Local cerebral blood flow (LCBF), volume of radiolabeled blood in parenchymal microvessels (also referred to as the blood space or Vb), plus the local capillary influx rate constants (K1) and PS products of [14C]antipyrine and 3-O-[14C]methyl-D-glucose (3OMG) were estimated in 44 brain areas. Hypercapnia raised PaO2 to 140 mm Hg, elevated LCBF by two- to threefold through out the brain, and increased Vb from 5 to 33% (mean = 22%) in 42 of 44 brain areas; hypercapnia did not, however, alter microvessel hematocrit. With hypercapnia, the influx of antipyrine was increased by 40-65% in all brain areas, and the PS products of antipyrine were elevated from 0-35% (mean = 17%). The PS products of antipyrine plus the parenchymal blood spaces suggest modest (< 30%) capillary recruitment in most brain areas as well as some microvessel dilation, mainly in forebrain gray matter and white matter areas. In contrast, hypercapnia did not appreciably alter K1 nor PS of 3OMG; it slightly but not significantly raised the blood levels of glucose. In view of the blood space and antipyrine evidence for modest capillary recruitment and vasodilation, the lack of change in PS of 3OMG implies that glucose transporter activity was lowered by hypercapnia, an effect similar to that reported for high-dose pentobarbital. Finally, the microvessel hematocrit and 3OMG data suggest that cerebral capillary permeability (P) was not increased by hypercapnia. Overall, hypercapnia seems to increase LCBF mainly by raising the velocity of blood flow; capillary recruitment and dilation appear to play relatively minor roles in this flow increase.

Virtually unaltered permeability-surface area products imply little capillary recruitment in brain with hypoxia.

OBJECTIVE: To test the capillary recruitment hypothesis for the brain with control and hypoxic rats. METHODS: Local cerebral blood flow (LCBF) was sharply raised by respiring 10% O2 (hypoxia). LCBF as well as local influx rate constants (K1) and permeability-surface area (PS) products of 14C-antipyrine and 14C-3-O-methyl-D-glucose (30MG) were estimated for capillary systems in 44 brain areas. RESULTS: With this testing, an increase in PS product would be suggestive of capillary recruitment. In all brain areas, LCBF was increased by 30-90% by hypoxia. Hypoxia modestly raised the influx of antipyrine in brain but did not appreciably alter its PS products. With hypoxia, K1's and PS products of 30MG were significantly lowered (5-25%) throughout the brain, and the blood levels of glucose were sizeably raised. The latter increase would diminish the transfer of 30MG across the blood-brain barrier by the hexose transporter because of increased glucose competition. By applying a glucose-concentration correction to the data, the apparent PS product of 30MC for the hypoxic group became equal to that for the controls, which agrees with the antipyrine PS product results. CONCLUSIONS: Hypoxia, thus, leads to virtually no increase in PS products and no capillary recruitment in brain, and elevates LCBF mainly, perhaps exclusively, by raising the velocity of flow through already perfused capillaries.

Nicotine increases microvascular blood flow and flow velocity in three groups of brain areas.

To examine the mechanism of local cerebral blood flow (LCBF) elevation, nicotine (1.75 mg/kg sc) was administered to rats, and LCBF plus the distribution spaces of radiolabeled albumin (RISA) and red blood cells (RBC) in parenchymal microvessels were measured throughout the brain. Microvascular blood spaces and transit times were calculated from the data. From 1.5 to 3 min after nicotine administration, LCBF was raised by 40-150% in 16 of the brain areas and unaltered in the remaining 28 areas. The affected structures included parts of the visual-auditory, sensorimotor-cortical, and interpeduncular systems. RBC spaces were not changed by nicotine treatment. RISA and blood spaces were increased slightly but not significantly in some of the LCBF-affected areas but nowhere else. Nicotine seemingly elevates LCBF in the affected areas mainly by increasing linear velocity of flow through the microvascular beds. In agreement with this, mean transit time, which is inversely related to velocity, was decreased from 0.3-0.5 to approximately 0.2 s in the microvascular systems of the nicotine-affected areas.

Hypercapnia slightly raises blood volume and sizably elevates flow velocity in brain microvessels.

The increase in local cerebral blood flow (LCBF) caused by hypercapnia may be mainly accomplished by raising the velocity of plasma and/or red blood cell (RBC) flow through the microvessels and not by perfusing more capillaries. This suggestion was tested in awake rats exposed to 8% CO2 and in control rats. LCBF was measured by the 14C-labeled iodoantipyrine method. The volume of blood in small parenchymal microvessels was estimated from the distribution spaces of 125I-labeled serum albumin (RISA) and 55Fe-labeled RBCs. Hypercapnia elevated LCBF 2.0- to 3.5-fold in the 40 brain areas studied, marginally raised the RBC spaces, and significantly increased the RISA and whole blood distribution spaces (approximately 25 and 19%, respectively). These changes in microvessel distribution volumes could be the result of perfusing a slightly larger fraction of capillaries (recruitment), increasing microvessel diameter somewhat, or both. With hypercapnia, the mean transit times fell to approximately 45% of control, which indicated that LCBF was mainly increased by raising the velocity of RBC and plasma flow through already perfused microvessels. Overall, few, if any, capillaries or other microvessels were recruited by hypercapnia.

Hypoxia increases velocity of blood flow through parenchymal microvascular systems in rat brain.

The postulation that hypoxia increases local cerebral blood flow (lCBF) mainly by perfusing more capillaries (the capillary recruitment hypothesis) was tested in awake adult male Sprague-Dawley rats exposed to 10% O2 and control rats. The [14C]iodoantipyrine technique was used to measure lCBF. Local cerebral blood volume was determined by measuring plasma and red cell distribution spaces within the brain parenchyma with 125I-labeled serum albumin (RISA) and 55Fe-labeled red cells (RBC), respectively. Tissue radioactivity in 44 brain areas was estimated by quantitative autoradiography. Hypoxia raised lCBF by 25-90% in all brain areas. In about one-quarter of the brain areas, the rise in blood flow was associated with a small increase in microvascular plasma and blood volumes. This change in blood volume, which could be the result of perfusing more parenchymal microvessels and/or increasing parenchymal microvessel diameter, is not sufficient to account for the observed rise in lCBF. In the remaining areas the RISA, RBC, and blood spaces were either unchanged or only marginally increased by hypoxia. For this hypoxic perturbation, the major mechanism of raising blood flow appears to be increased velocity of microvessel perfusion and not perfusion of more capillaries. These findings provide only limited support for the capillary recruitment hypothesis.

The velocities of red cell and plasma flows through parenchymal microvessels of rat brain are decreased by pentobarbital.

Local cerebral blood flow is lowered in many brain areas of the rat by high-dose pentobarbital (50 mg/kg). In the present study, the mechanism of this flow change was examined by measuring the distribution of radiolabeled red blood cells (RBCs) and albumin (RISA) in small parenchymal microvessels and calculating the microvascular distribution spaces and mean transit times of RBCs, RISA, and blood. In most brain areas, pentobarbital slightly decreased the RISA space, modestly increased the RBC space, and did not alter the blood space. The mean transit times of RBCs, RISA, and blood through the perfused microvessels were considerably greater in treated rats than in controls. These findings indicate that the mechanism by which high-dose pentobarbital diminishes local cerebral blood flow in rat brain is, in the main, a lowered linear velocity of plasma and RBC flow through small parenchymal microvessels and not decreased percentage of perfused capillaries (capillary retirement). This response is probably driven mainly by lowered local metabolism and may well entail a slight increase in the number of small microvessels that are perfused by RBCs.

Cerebral glucose utilization and blood flow in adult spontaneously hypertensive rats.

Not only blood pressure but also behavioral activity, brain morphology, and cerebral ventricular size differ between young spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. This suggests that cerebral blood flow and cerebral metabolism may vary between these two rat strains. To test this hypothesis, we measured local cerebral glucose utilization in 31 brain areas of 26-30-week-old rats. Local cerebral blood flow was also assessed in these same areas. Cerebral glucose utilization was measured by the 2-deoxyglucose method; cerebral blood flow was determined by the iodoantipyrene method. In virtually all gray matter structures, the apparent rate of glucose utilization was lower in SHR than in normotensive WKY rats; the interstrain differences varied significantly among structures and were statistically significant (uncorrected t tests) in 14 of 28 gray matter areas. Local cerebral blood flow was fairly similar in the two rat strains. The coupling of blood flow to glucose utilization varied significantly among brain areas in normotensive WKY rats as well as in SHR. In a number of gray matter structures, the coupling of flow to metabolism differed between hypertensive and normotensive animals. These data suggest that for many brain areas, either glucose utilization or glucose partitioning differs between WKY rats and SHR.

Technique-dependent variations in cerebral microvessel blood volumes and hematocrits in the rat.

To quantitate small parenchymal microvessel blood volumes in the brain, the distribution spaces of radiolabeled red blood cells (RBC) and serum albumin (RISA) were assessed in rats by different methods of tissue sampling and radioassay. Three minutes after intravenous administration of 55Fe-RBCs and/or 125I-RISA, the rats were decapitated. The brain was either immediately frozen within the skull and later removed (head-frozen group) or rapidly removed from the skull and then frozen (brain-frozen group). Radioactivity was measured either by liquid scintillation counting of tissue pieces, which contained pial plus large and small parenchymal microvessels, or by quantitative autoradiography (QAR) of tissue sections, which indicated small parenchymal microvessel blood only. In 12 of 15 areas, the RISA, RBC, and blood volumes determined by liquid scintillation counting of head-frozen tissue pieces were equal to or greater than those of brain-frozen tissue; this indicated less than or equal to 25% greater blood retention in pial and parenchymal microvessels with head freezing. At the parenchymal microvessel level (QAR assay), the distribution volumes of RBCs, RISA, and blood were similar with the two freezing techniques; hence with QAR either freezing procedure can be used to assess small parenchymal microvessel blood volumes.

Differences and similarities in albumin and red blood cell flows through cerebral microvessels.

The hypothesis that microvessels in brain parenchyma are continuously perfused by plasma but intermittently perfused by red blood cells (RBCs) was tested in awake Sprague-Dawley rats. The microvascular distribution volumes of radioiodinated serum albumin (RISA) and 51Cr- and 55Fe-labeled RBCs were measured for periods from 15 s to 30 min. Local cerebral blood flow (LCBF) was assessed by the iodoantipyrine technique. The RISA and RBC distribution volumes were constant in the 12 areas studied from 15 s onward. These data fit a model of continuous plasma flow with intermittent RBC flow (and thus support the hypothesis), but they are also consistent with other models, e.g., continuous flow of both plasma and RBCs through all perfused microvessels. In parallel with LCBF, microvascular blood volume varied greater than 10-fold among brain areas. Relative to arterial hematocrit, microvascular hematocrits were low, which indicates that the passage of RBCs through parenchymal microvessels is more rapid than that of RISA. This could be the result of both the Fahraeus effect and intermittent RBC flow.

Pentobarbital produces dissimilar changes in glucose influx and utilization in brain.

The effects of pentobarbital sodium on local cerebral glucose utilization (LCGU) and 3-O-methylglucose (3-MG) influx were measured by quantitative autoradiography in 52 brain areas of control and treated rats. Pentobarbital (50 mg/kg ip) lowered LCGU to a relatively uniform rate (approximately 35 mumol.100 g-1.min-1) in 24 of 25 forebrain areas. Among the 18 hindbrain areas, LCGU was decreased by pentobarbital by 15-55% (range 50-157 and 28-110 mumol.100 g-1.min-1 in control and treated rats, respectively). In contrast, pentobarbital lowered the 3-MG influx rate constant and permeability-surface area product by 20-30% in nearly all brain structures. The 3-MG results fit a model in which both the half-saturation constant and the maximal velocity of the glucose carrier are decreased by pentobarbital. After pentobarbital treatment, the ratio of local cerebral plasma flow (LCPF) to LCGU was the same as in controls for brain areas in which LCGU was less than 35 mumol.100 g-1.min-1 but was higher in brain areas where LCGU exceeded 35 mumol.100 g-1.min-1. Pentobarbital produced dissimilar changes in LCGU, 3-MG influx, and LCPF; these processes may thus not be closely linked during pentobarbital anesthesia.

Variation in local cerebral blood flow response to high-dose pentobarbital sodium in the rat.

Microvascular bed structure and functions are known to vary throughout the brain. Microvascular responses to high doses of pentobarbital sodium might therefore differ among brain areas. This possibility was examined by measuring local cerebral blood flow (LCBF) with [14C]iodoantipyrine in 52 brain areas at 5, 10, 25, and 60 min after intraperitoneal administration of pentobarbital (50 mg/kg). From 5 to 60 min, LCBF was significantly lowered in 17 of 25 forebrain gray matter areas but in only 1 of 18 hindbrain gray matter structures, the pontine nuclei. Smaller, shorter duration lowering of LCBF was also observed in ten other brain areas. In both control and treated rats, LCBF was found to vary within individual brain structures. The pattern of these LCBF variations was columnar in the cerebral cortex and the hippocampus but was patchy in the caudate-putamen, thalamus, and inferior colliculus. These results indicate that pentobarbital anesthesia more strongly alters LCBF in the forebrain than in the hindbrain and produces different patterns of changes in LCBF than in local cerebral glucose utilization, which was measured with 2-deoxyglucose in a companion study.

Functional variations in parenchymal microvascular systems within the brain.

Variations in microvascular system functions were observed among a number of brain areas. The rates of local blood flow varied 18-fold among areas and were extremely high in neuroendocrine structures. Marked differences in blood flow were also found within some brain structures. The volume of radiolabeled blood in perfused parenchymal microvessels ranged from 5 to 70 microliters/g and correlated closely with local cerebral blood flow. The hematocrits within parenchymal microvessels were 45-75% of the arterial hematocrit, which indicates that red cells more rapidly traverse brain microvessels than do plasma proteins. The mean transit times of blood through parenchymal microvessels were extremely short and ranged from 0.3 to 0.6 s.

Quantitative autoradiographic assessment of 55Fe-RBC distribution in rat brain.

A simple in vivo technique of labeling erythrocytes (RBCs) with 55Fe was developed for quantitative autoradiography (QAR). This procedure involved injecting 5-6 ml of [55Fe]ferrous citrate solution (1 mCi/ml) intraperitoneally into donor rats. The number of labeled RBCs reached a maximum at around 7 days and declined very slowly thereafter. Labeled RBCs were harvested from donor rats and used for RBC volume measurement in awake rats. Brain radioactivity was assayed by QAR, which yielded spatial resolution of greater than 50 microns. Tight nearly irreversible binding of 55Fe to RBCs was found in vivo and in vitro. More than 99.5% of the 55Fe in the blood of donor rats was bound to RBCs. Because of this, labeled blood can be taken from donors and injected into recipients without further preparation. The tissue absorption of 55Fe emissions was the same in gray and white matter. Microvascular RBC volumes measured with 55Fe-labeled RBCs agreed with those assayed with 51Cr-labeled RBCs for many, but not all, brain areas. In conclusion, 55Fe-RBCs can be readily prepared by this technique and accurately quantitated in brain tissue by QAR.

Structural and functional variations in capillary systems within the brain.

The major hypothesis of this study is that there are differences among brain areas in capillary bed structure and function. Three general differences between circumventricular organ and non-CVO capillary beds were found. First, the PS products for AIB were about 300 times greater in CVO capillaries than in non-CVO (blood-brain barrier) capillaries. Second, the frequency of endothelial cell fenestrations was much greater in CVO capillaries than in non-CVO capillaries and the fenestrae may be structural modifications of endothelial cells that permit ready passage of solutes such as AIB. Third, the frequency of mitochondria was greater in BBB capillaries than in CVO capillaries; this high metabolic potential of BBB capillaries may be associated, in part, with "carrier-mediated" transport of various solutes between plasma and cerebral interstitial fluid. Capillary bed differences among all (i.e., both CVO and non-CVO) brain structures were also observed. Among these differences are: rate of blood flow, mean transit time of albumin, capillary volume and surface area, perfused microvessel blood volume, apparent percentage of perfused capillaries, PS products for AIB, and frequency within the endothelium of vesicular profiles.