Joseph D. Fenstermacher

Rapid distribution of intraventricularly administered sucrose into cerebrospinal fluid cisterns via subarachnoid velae in rat

The intracranial distribution of [14C]sucrose, an extracellular marker infused for 30 s into one lateral ventricle, was determined by autoradiography of frozen-dried brain sections. Within 3.5 min [14C]sucrose appeared in: (i) the third ventricle, including optic, infundibular and mammillary recesses; (ii) the aqueduct of Sylvius; (iii) the velum interpositum, a part of the subarachnoid space that runs along the roof of the third ventricle and contains many blood vessels; (iv) the mesencephalic and fourth ventricles; and (v) the superior medullary velum, a highly vascular extension of the subarachnoid space that terminates at the walls of the mesencephalic and fourth ventricles. Within 5 min, radioactivity was present in the interpeduncular, ambient and quadrigeminal cisterns, which encircle the midbrain. By 10 min, approximately 11% of the radioactivity had passed into the subarachnoid space via a previously undescribed flow pathway that included the velum interpositum and superior medullary velum. At many places along the ventricular system, [14C]sucrose appeared to move from cerebrospinal fluid into the adjacent tissue by simple diffusion, as reported previously (Blasberg R. G. et al. (1974) J. Pharmac. exp. Ther. 195, 73-83; Levin V. A. et al. (1970) Am. J. Physiol. 219, 1528-1533; Patlak C. and Fenstermacher J.D. (1975) Am. J. Physiol. 229, 877-884; Rosenberg G. A. and Kyner W.T. (1980) Brain Res. 193, 56-66; Rosenberg G. A. et al. (1986) Am. J. Physiol 251, F485-F489). Little sucrose was, however, taken up by: (i) circumventricular organs such as the subfornical organ; (ii) medullary and cerebellar tissue next to the lateral recesses; and (iii) the superior and inferior colliculi and cerebral peduncles. For the latter two groups of structures, entry from cerebrospinal fluid was apparently blocked by a thick, multilayered glia limitans. Although [14C]sucrose was virtually absent from the rest of the subarachnoid system after 1 h, it remained in the perivascular spaces and/or walls of pial arteries and arterioles for more than 3 h. Certain transport proteins, protease inhibitors, growth factors and other neurobiologically active materials are present in cerebrospinal fluid, and their distribution to the brain and its blood vessels may be important. The present work shows, in the rat, that the flow of cerebrospinal fluid and the disposition of its constituents is fairly complex and differs among regions. Flow was rapid throughout the ventricular system and into various subarachnoid velae and cisterns, but was surprisingly slow and slight over the cerebral and cerebellar cortices. The cerebrospinal fluid-to-tissue flux of material was relatively free at many interfaces, but was greatly restricted at others, the latter indicating that the old concept of a cerebrospinal fluid-brain barrier may hold at such places. Finally, radiolabeled sucrose was retained longer within the walls and perivascular spaces of pial arteries and arterioles than in other subarachnoid tissues; one function of the cerebrospinal fluid system or third circulation may thus be delivering factors and agents to these pial blood vessels.

Drug “Diffusion” within the Braina

1. The rates and the ways different materials move from ventricular fluid into and through the caudate nucleus vary rather broadly. 2. The major mechanism of solute flow from ventricular CSF into and through the caudate nucleus appears to be simple diffusion. 3. Among solutes that pass across the BBB from brain to blood at very low rates, those that distribute mainly or exclusively in the extracellular space (e.g., sucrose, EDTA, and sodium) diffuse most rapidly into the caudate nucleus. 4. Among solutes which efflux across the BBB at very low rates, the apparent rate of diffusion within the caudate nucleus is the least for those that are readily taken up and accumulated by brain cells (e.g., AIB). 5. Solutes that move moderately to rapidly across the BBB from brain to blood only penetrate distances of several mm or less into the caudate before they are completely cleared from the tissue by the cerebral circulation. 6. In most instances the slopes of the tissue concentration profiles within the caudate nucleus during and after ventriculocisternal perfusion are fairly steep and are complex functions of parameters such as the effective tissue diffusion coefficient (Dt) and the transcapillary efflux rate constant (Ko), each of which are, in turn, dependent on separate sets of variables (e.g., the rate of cellular uptake for Dt and the rate of blood flow for ko). 7. The distribution process and kinetics may be much different in CNS areas adjacent to the subarachnoid space than in the caudate nucleus because of convective flow of subarachnoidal CSF through the perivascular spaces of the penetrating and emerging blood vessels and the arterioles and venules.

Structural and Functional Variations in Capillary Systems within the Braina

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.

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.

Differences in Function and Structure of the Capillary Endothelium in Gray Matter, White Matter and a Circumventricular Organ of Rat Brain

Physiological and morphometric studies were conducted on the microvascular endothelium of four individual cerebral structures having different neural activities--the inferior colliculus, sensorimotor cortex (both gray matter regions), genu of the corpus callosum (white matter), and the subfornical organ (a circumventricular organ) of rats. The physiological data, obtained by quantitative autoradiography, produced new findings: the rate of blood-to-tissue flux across capillary endothelial cells of a neutral amino acid, 14C-alpha-aminoisobutyric acid, was 100-400 X more rapid in the subfornical organ than in gray and white matter regions, and the transit time of labeled albumin in the subfornical organ microcirculation was 7-12 X longer than in the blood-brain barrier regions. These quantitative studies suggest that circulating messengers, such as hormones, would have more prolonged receptor contact with capillary endothelial cells and greater rates of transendothelial passage in the subfornical organ than in gray or white matter. Capillary densities, volume fractions, and surface areas were similar for the inferior colliculus (which has the highest rates of tissue glucose metabolism and blood flow among blood-brain barrier regions) and the subfornical organ, but were significantly smaller for sensorimotor cortex and corpus callosum (about 35 and 70% lower, respectively). Electron microscopic morphometry of capillary endothelial cells in the inferior colliculus and corpus callosum indicated the ultrastructural basis of blood-brain barrier mechanisms in these regions--intercellular junctions that appeared tight, few cytoplasmic vesicular profiles, and no fenestrations. Analysis of endothelial cells in the subfornical organ demonstrated 7 X more vesicular profiles and 4 fenestrations per cross section of capillary. These findings represent structural evidence for high rates of solute flux across the capillary endothelium of circumventricular organs.

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.

Topography of Capillary Density, Glucose Metabolism, and Microvascular Function Within the Rat Inferior Colliculus

A midbrain nucleus of the auditory system, the inferior colliculus, was used as a model for analyzing spatial correlations or “coupling” among capillary density, tissue glucose metabolism, and several measures of microvascular function in the rat. The capillary bed of the inferior colliculus was examined with stereological techniques, and physiological measures were obtained with radioactive tracers, quantitative autoradiography, and image processing. Within the colliculus, capillary density, volume fraction, length, and surface area were highest in the central nucleus where the packing densities of neuropil and perikarya are greatest. Rates of glucose metabolism and blood flow correlated closely with capillary density in a 3 × 2 matrix of collicular subregions in the sagittal and coronal planes. The strength of this correlation suggests that estimates of capillary density can be made from measurements of tissue glucose metabolism within this structure under normal conditions. Microvascular blood volume and transcapillary flux of a neutral amino acid, α-aminoisobutyric acid, were homogeneous throughout the colliculus. The studies demonstrate quantitatively in a single brain nucleus a close correspondence between cytoarchitecture, richness of the capillary bed, and complexity of neural activity (inferred from local measures of glucose metabolism and blood flow). Such relationships were suggested by Craigie 67 years ago.

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 (30MG) 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 30MG by 5–27%, PS product of antipyrine by 0–20% (mean 7%), and PS product of 30MG 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 30MG and in PS for both antipyrine and 30MG 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.

Parenchymal microvascular systems and cerebral atrophy in spontaneously hypertensive rats

Spontaneously hypertensive rats (SHR) are hypertensive, hyperactive, and hydrocephalic; furthermore SHR have smaller brain volume and weight than age-matched, normotensive Wistar-Kyoto rats (WKY). At 6-7 months of age, local cerebral glucose is sizably lower in SHR than WKY. The hypothesis that these several abnormalities of SHR lead to variations in cerebral microvascular bed morphology was tested in 6-7-month-old SHR and WKY by quantitating various parameters of small, intermediate, and large parenchymal microvessels (grouped by luminal diameter) in 21 brain areas. Within each rat strain, the microvascular bed properties such as vessel profile frequency (density) varied considerably among the 21 brain areas. In opposition to the hypothesis, mean luminal diameter as well as profile frequency, surface area, and luminal volume of the microvascular beds per unit tissue mass were virtually identical in each brain area of SHR and WKY for the three groups of microvessels. These findings coupled with the reports of less tissue per structure but similar density of neurons throughout the brain of SHR and WKY indicate that there are fewer neurons and less vascular tissue per brain structure in 6-7-month-old SHR than WKY; in addition, they suggest a linkage between the size of parenchymal microvascular beds and the surrounding nervous tissue.

Pharmacology of the Blood-Brain Barrier

All so-called permeability studies of the blood-brain barrier (BBB) actually measure one of several transfer constants (e.g., the extraction fraction) and not a true permeability coefficient. The permeability coefficient or, more simply, the permeability is classically defined as the flux (i.e., the rate of unidirectional solute flow) per unit membrane area divided by the driving forces for the flux, which are the concentration, pressure, and electrical gradients, in most cases. Experimental measurements of permeability involve an assessment of the flux across the membrane of predetermined surface area that separates two solutions of nearly identical composition. For the BBB, which is generally believed to be formed by the capillary endothelium, the classic definition of the permeability coefficient should be retained because it can be used to understand BBB function more clearly. By contrast, the standard methods of measuring the permeability coefficient cannot be employed for the cerebral capillaries, since neither the capillary surface area nor the composition of the blood within the capillaries and the interstitial fluid surrounding them can be tightly controlled or precisely known.