Cerebrospinal fluid production by the choroid plexus and brain.

The production of cerebrospinal fluid and the transport of (24)Na from the blood to the cerebrospinal fluid were studied simultaneously in normal and choroid plexectomized rhesus monkeys. Choroid plexectomy reduced the production of cerebrospinal fluid by an average of 33 to 40 percent and the rate of appearance of (24)Na in the cerebrospinal fluid and its final concentration were proportionately reduced. In both normal and plexectomized animals, (24)Na levels were found to be markedly greater in the gray matter surrounding the ventricles and in the gray matter bordering the subarachnoid space. That sodium exchanges in these two general areas of the brain may be linked to the formation of the cerebrospinal fluid is discussed here.

A choroid plexus papilloma in an elasmobranch (Squalus acanthias).

A choroid papilloma in the choroid plexus of the ala of the fourth ventricle in a mature male elasmobranch, Squalus acanthias, was described. This is apparently the first report of a neoplasm of the central nervous system in a member of the class Chondrichthyes.

Regional [14C]misonidazole distribution in experimental RT-9 brain tumors.

Regional [14C]misonidazole-derived radioactivity (MISO) was measured by quantitative autoradiography in experimental RT-9 brain tumors 0.5, 2, and 4 hr after an i.v. bolus (25 mg) and constant infusion (10 mg/hr). Misonidazole (MISO) concentration in plasma and brain was also measured by high-pressure liquid chromatography; the brain/plasma MISO ratio ranged between 0.5 and 0.7. MISO equivalents were calculated from tissue or plasma 14C radioactivity and [14C]MISO specific activity data. The MISO/MISO equivalents ratio, which represents the nonmetabolized fraction of [14C]MISO, fell gradually in plasma (0.89 at 4 hr) and more rapidly in brain (0.67 at 4 hr) and tumor (0.30 at 4 hr). MISO distributed uniformly throughout the brain at all three time periods. In contrast, MISO distribution in tumor was variable, and tumor concentrations relative to that in brain increased with time. The average tumor/brain MISO ratio was 1.3, 1.7, and 2.6 at 0.5, 2, and 4 hr, respectively, which suggests tumor uptake and binding of MISO or, more likely, MISO-derived 14C-labeled metabolites. In addition, MISO distribution in tumor tissue was strikingly heterogeneous at 4 hr, resulting in an average high/low tumor activity ratio of 4/1 and an average high tumor/brain ratio of 5/1. Tumor regions with high MISO activity correlated in part to viable-appearing cells around necrotic foci.

Regional measurements of [14C]misonidazole distribution and blood flow in subcutaneous RT-9 experimental tumors.

Regional [14C]misonidazole-derived radioactivity (MISO*) was measured by quantitative autoradiography in s.c. RT-9 experimental tumors 0.5, 2, and 4 h after an i.v. bolus (25 mg) and constant infusion (10 mg/h) in rats. Misonidazole (MISO) concentration in plasma, tumor, and other tissues was also measured by high-pressure liquid chromatography. The distribution of MISO* in the tumors always resulted in a characteristic pattern with high peripheral and low central values. The high-activity regions in the tumor rim achieved tissue: plasma MISO* activity ratios of 0.97 and 2.2 by 0.5 and 4 h, respectively; for central tumor regions, this ratio was 0.20 and 0.32 for the same periods, respectively. The limited distribution of MISO* to central tumor regions could be correlated to low values of blood flow (measured with [131I]iodoantipyrine) and to diffusion from peripheral tumor regions. Low blood flow in the central regions of these tumors will significantly limit the distribution of MISO and other drugs to viable-appearing cells in these areas and could account in part for the failures of chemotherapy in certain solid tumors. Pharmacokinetic modeling indicates that 1 to 9 h may be necessary for MISO concentrations in some tumor regions to reach 50% of that in plasma.

In vitro evidence that beta-amyloid peptide 1-40 diffuses across the blood-brain barrier and affects its permeability.

Beta amyloid peptides are major insoluble constituents of amyloid fibrils in senile plaques and cerebrovascular deposits, both characteristic of Alzheimer disease (AD). Low concentrations of soluble forms of amyloid peptides are also present in normal CSF. We previously demonstrated that the 40 amino acid form of soluble beta-amyloid peptide (sAbeta) is rapidly cleared from rat CSF into blood. Herein we hypothesized that a saturable, outwardly directed flux of this peptide occurs at the blood-brain barrier (BBB) and tested whether supraphysiological (possibly pathological) concentrations of sAbeta could alter the permeability of this barrier to a paracellular tracer, polyethylene glycol (PEG). Using an in vitro model of BBB, we showed that influx and efflux of sAbeta were equal, modest (60%-160% greater than that of PEG), and not saturable. These observations suggest that sAbeta moved across the monolayer by a diffusional process, and not via a transporter. PEG flux was doubled immediately after the luminal concentration of cold sAbeta was raised to 5 microM, and was doubled 150 min after the abluminal concentration of sAbeta was increased to 5 microM. Pathological elevations of sAbeta concentration in plasma or brain interstitial fluid may, therefore, alter the permeability of brain capillaries in vivo.

Regional variations in the apparent diffusion coefficient and the intracellular distribution of water in rat brain during acute focal ischemia.

BACKGROUND AND PURPOSE: The apparent diffusion coefficient of water (ADC) rapidly drops in ischemic tissue after cerebral artery occlusion. This acute drop is thought to be caused by the loss of extracellular fluid and the gain of intracellular fluid. To test the latter possibility, changes in ADC and the size of several cellular compartments were assessed in 3 regions of rat brain at the end of 90 minutes of focal cerebral ischemia. METHODS: One middle cerebral artery was permanently occluded in 8 Sprague-Dawley rats; sham occlusions were performed in 2 other rats. ADC maps were generated 90 minutes later, and the brains were immediately perfusion fixed. Three regions of interest (ROIs) were defined on the basis of ADC range. Various neuronal, astrocytic, and capillary compartments in each ROI were quantified with light and electron microscopy. RESULTS: At the end of 90 minutes of ischemia, mean ADC was normal in the cortex of sham-operated rats and the contralateral cortex of ischemic rats (ROI-a), 25% lower in the ipsilateral frontoparietal cortex (ROI-b), and 45% lower in the ischemic lateral caudoputamen (ROI-c). At this time, the frequency of swollen astrocytic cell bodies and volume of swollen dendrites and astrocytic processes in neuropil were ROI-a

Differences in vulnerability to permanent focal cerebral ischemia among 3 common mouse strains.

BACKGROUND AND PURPOSE: Genetically engineered mice are used to study the role of single genes in cerebral ischemia, but inherent, strain-dependent differences in neuronal vulnerability may affect experimental end points. To examine this possibility, tissue injury resulting from focal ischemia and its relationship to cerebral hemodynamics were determined in 3 common mutant mouse strains. METHODS: Permanent middle cerebral artery ligation was performed in male C57BL/6J, Balb/C, and 129X1/SvJ mice. Mean arterial blood pressure, blood gases, basal and postischemic cortical blood flow ([(14)C]iodoantipyrine autoradiography and laser-Doppler flowmetry), posterior communicating artery patency, and infarct size were determined. RESULTS: Basal cortical blood flow did not differ among strains. Ten minutes after middle cerebral artery ligation, relative red cell flow in the ischemic cortex was 6% to 7% of preischemic flow in every strain. Despite similar hemodynamics, cortical infarcts in Balb/C mice were 3-fold larger than those in 129X1/SvJ and C57BL/6J mice; infarct size in the latter 2 strains was not significantly different. The posterior communicating artery was either poorly developed or absent in >90% of the Balb/C and C57BL/6J but in <50% of the 129X1/SvJ mice. CONCLUSIONS: The extent of ischemic injury differed markedly between the 3 strains. The presence and patency of posterior communicating arteries, although variable among strains, did not affect preischemic or postischemic cortical blood flow or bear any relationship to ischemic injury. Therefore, intrinsic factors, other than hemodynamic variability, may contribute to the differences in ischemic vulnerability among strains. These findings underscore the importance of selecting genetically matched wild-type controls.

Smaller local brain volumes and cerebral atrophy in spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR) have enlarged cerebral ventricles from 8 weeks of age onward and smaller brains than age-matched, normotensive Wistar-Kyoto (WKY) rats (controls). At 6-7 months of age local cerebral glucose utilization is apparently lower in many brain areas of SHR relative to WKY rats. These observations led to the hypothesis that there are morphological differences between these two strains of rats in many, if not all, brain areas. This hypothesis was tested in 6-7-month-old SHR and WKY rats by quantitating 1) the volumes of the ventricular system, whole brain, six gray matter structures, and two white matter areas; 2) the thickness of two regions of the cerebral cortex; and 3) the frequency of neuronal nuclei (neuronal frequency) in nine brain areas. Ventricular volume was twofold greater in SHR than in control rats. The volumes of the entire brain and all six gray matter structures plus the thickness of the two cortical regions were 11-25% less in SHR. Neuronal frequency was, however, similar in the two rat strains. The latter finding coupled with the smaller regional tissue volumes indicates appreciably fewer neurons per brain structure in young adult SHR than in controls. These results indicate significant cerebral structural differences between young adult SHR and WKY rats and suggest that structure as well as metabolism are abnormal in the SHR brain.

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.

Circumventricular organs in chronic serum sickness: a model for cerebral lupus.

The pathogenesis of the CNS manifestations of systemic lupus erythematosus (SLE) has been the subject of considerable investigation. The focus of many of these studies has concerned immune complex deposition within the choroid plexus (CP). Involvement of the other brain fenestrated vascular beds, the small, paraventricular circumventricular organs, has not been ascertained. For this purpose, chronic serum sickness, a good immunopathological experimental model of naturally occurring systemic immunological disorders such as SLE, was induced in Wistar rats by prolonged immunization with bovine serum albumin (BSA). The involvement of circumventricular vascular beds by immune deposits was ascertained immunohistochemically. The choroid plexus was found to be the most intensely involved circumventricular structure. Immune complex deposits were also present, in descending order of frequency, in the area postrema, subfornical organ, and pineal gland.

Graphical evaluation of blood-to-brain transfer constants from multiple-time uptake data.

A theoretical model of blood-brain exchange is developed and a procedure is derived that can be used for graphing multiple-time tissue uptake data and determining whether a unidirectional transfer process was dominant during part or all of the experimental period. If the graph indicates unidirectionality of uptake, then an influx constant (Ki) can be calculated. The model is general, assumes linear transfer kinetics, and consists of a blood-plasma compartment, a reversible tissue region with an arbitrary number of compartments, and one or more irreversible tissue regions. The solution of the equations for this model shows that a graph of the ratio of the total tissue solute concentration at the times of sampling to the plasma concentration at the respective times (Cp) versus the ratio of the arterial plasma concentration-time integral to Cp should be drawn. If the data are consistent with this model, then this graph will yield a curve that eventually becomes linear, with a slope of Ki and an ordinate intercept less than or equal to the vascular plus steady-state space of the reversible tissue region.

Transport of alpha-aminoisobutyric acid across brain capillary and cellular membranes.

The transport of alpha-aminoisobutyric acid (AIB), N-methyl-AIB (MeAIB), and diethylenetriaminepentaacetic acid (DTPA) from blood to brain was measured over different experimental periods in eight regions of the rat brain. Unidirectional transfer rate constants were determined from multiple-time/graphical and single-time analysis of the experimental data; values of 0.0018, 0.00057, and 0.000021 ml g-1 min-1, respectively, were obtained for the thalamus by graphical analysis. The initial distribution volume of AIB and MeAIB in brain tissue was several-fold greater than that of DTPA and the tissue plasma volume, and this difference was not accounted for by red blood cell uptake. This discrepancy could be due to rapid transport of AIB and MeAIB into brain endothelial cells in addition to the relatively rapid uptake by choroidal, meningeal, and ependymal associated tissues that was demonstrated by autoradiography. Thus, it may be misleading and erroneous to consider the blood-brain barrier (BBB) to be a simple, single-membrane structure when analyzing the blood-brain transfer data of solutes such as amino acids. The data from the ventriculocisternal perfusion experiments and previously published AIB uptake data in mouse brain slices were used to estimate the transfer rate constants across brain cell membranes. These studies indicated that the transport of AIB into brain cells was approximately 110 to 265 times greater than that across normal brain capillaries per unit mass of brain tissue, and that the BBB limits blood-to-brain cell transport of this amino acid. These observations (low rate of transport across normal brain capillaries and rapid concentrative uptake by brain cells) indicate that AIB is a good marker for measuring moderate to large increases in BBB permeability by experiments that require unidirectional flux of the tracer.

An evaluation of errors in the determination of blood flow by the indicator fractionation and tissue equilibration (Kety) methods.

In this report, the effects of various errors and plasma time courses of indicator concentration on the accurate determination of cerebral blood flow (F) are theoretically analyzed for the tissue equilibration and the indicator fractionation techniques. For the indicator fractionation technique, the impact of sample timing and tissue assaying errors and of indicator backflux were examined; for the tissue equilibration method, errors in the value of the partition coefficient (lambda), sample timing, and tissue assaying were considered. The recommended ways to decrease the effects of errors in the indicator fractionation technique are to administer the indicator by an intravenous bolus and to sample the tissue about 10 s thereafter. Possible errors in the assessment of F by the tissue equilibration technique are diminished by using an indicator infusion schedule which yields a continuous rise in arterial concentration and by selecting a 30-s experiment duration. Surprisingly, the impact of sample timing errors is greater on the determination of F with the tissue equilibration method than with the indicator fractionation technique. For the chosen plasma time courses, there is always a backflux error in an indicator fractionation estimation of F, and this error increases as the flow rate increases. Thus, provided the sample timing and tissue assay errors are small and the value of lambda is known, the tissue equilibration method is the more accurate of the two. If lambda is unknown, then the indicator fractionation technique should be used. In many cases, the indicator fractionation method will provide as accurate an estimate of F as will the tissue equilibration method.

Selection of experimental conditions for the accurate determination of blood--brain transfer constants from single-time experiments: a theoretical analysis.

Reliable blood-brain transfer constants can be determined from data obtained in single-time experiments (i.e., a single experimental time for tissue sampling). The accuracy of such measurements depends on factors such as the test molecule used and the experimental time chosen; therefore, the selection of optimal experimental conditions is important. In this presentation, a model of transport across the blood--brain barrier (BBB) was developed and used to determine appropriate experimental protocols for single-time experiments. Transfer numbers derived from published data with alpha-aminoisobutyric acid (AIB; a compound of low BBB permeability that is readily taken up by brain cells) and diethylenetriaminepentaacetic acid (DTPA; a compound of very low BBB permeability that is not taken up by brain cells) were inserted into the model and apparent blood-to-brain transfer constants (K1) were obtained. In addition, the two basic sets of transfer numbers were altered to mimic various experimental and pathological changes in blood--brain transport. The results of this analysis indicate that moderate to large transfer rates across the BBB (0.01-1.0 ml g-1 min-1) are more easily and reliably measured by AIB-like compounds. In contrast, compounds like DTPA are better test-molecules for measuring small changes in the BBB transfer rate (0.0001-0.001 ml g-1 min-1), provided an appropriate experimental time is chosen.

Quantitation of microvascular plasma perfusion and neuronal microtubule-associated protein in ischemic mouse brain by laser-scanning confocal microscopy.

In an exposition of the technique of calculating distribution volumes from laser-scanning confocal microscopic (LSCM) data, three-dimensional images of the distribution of one or two fluorescent markers in mouse brain specimens were generated by LSCM and processed by a system developed for morphometric analysis of fixed and stained serial brain histologic samples. To determine the volume of perfused cerebral capillaries, one of two fluorescent plasma markers, either fluorescein isothiocyanate (FITC)-dextran or Evans blue, was intravenously administered to mice subjected to 1 hour of embolic middle cerebral artery (MCA) occlusion (n = 9) and to mice that were not operated on (n = 3); after 1 minute of circulation, brains were removed, immersion-fixed, and processed for LSCM. In some of these animals (n = 5), the volume of endogenous microtubule-associated protein-2 (MAP2) fluorescence was also determined using immunohistochemical staining. For mice that were not operated on, this methodology yielded highly localized volumes of (1) microvascular plasma, which agree with those determined for rodents by other techniques, and (2) MAP2 expression, which appears physiologically and morphologically reasonable. After 1 hour of MCA occlusion, the MAP2 volumes of distribution were less than 10% of normal in the ipsilateral hemisphere in which plasma perfusion essentially ceased. In conclusion, precise colocalization and quantitation of early ischemic neuronal damage and cerebral plasma perfusion deficit can be done with this three-dimensional, microphysiologic and microanatomic methodology.

A spatial analysis of the blood-brain barrier damage in experimental allergic encephalomyelitis.

Experimental allergic encephalomyelitis was induced in young male Lewis rats. Following the development of neurological signs, the local distribution of perivascular inflammatory cellular infiltrates and the local blood-to-tissue transfer constants (K1) of alpha-aminoisobutyric acid (AIB) were determined, and these results were compared. Perivascular infiltrative lesions were generally found near areas of the CNS that normally lack an effective blood-brain barrier (BBB) such as the choroid plexus and the entry zones of the cranial and spinal nerve roots. This distribution pattern indicates that the entry of the causative agent into CNS tissue may be by way of the permeable microvessels of these structures. In tissue around inflamed veins, the mean transfer constant was slightly but significantly increased (2.8 +/- 1.5 microliter g-1 min-1) compared with uninvolved regions (0.9 +/- 0.2 microliter g-1 min-1) and similar areas in control animals (0.9 +/- 0.3 microliter g-1 min-1). Analysis of the autoradiographic method of determining transfer constants suggested that the AIB influx rate in the lesion areas may actually be manyfold larger than measured, that BBB permeability may be greatly increased at such sites, and that the areas of lymphocytic infiltration and increased K values may be virtually identical.

Rapid amino acid uptake in rat pituitary neural lobe during functional stimulation by chronic dehydration.

We studied the transfer of a small neutral amino acid, alpha-aminoisobutyric acid, from arterial blood into the pituitary neural lobe in normal rats and in rats deprived of water for 5 days. A threefold increase in the neural lobe uptake of the amino acid was found in the chronically dehydrated rats. Possible causes for this effect include enlargement of the capillary and pituicyte membrane surface areas available for solute flux and increased permeability of these membranes. Such functional and structural alterations may be associated with an increase in protein turnover in the neural lobe during dehydration.

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.

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 X 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, alpha-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.