Surotomycin versus vancomycin in adults with Clostridium difficile infection: primary clinical outcomes from the second pivotal, randomized, double-blind, Phase 3 trial.

BACKGROUND: The available treatment options for Clostridium difficile infection (CDI) are limited by high recurrence rates. Surotomycin was a novel bactericidal cyclic lipopeptide in development to treat CDI that demonstrated non-inferiority to vancomycin in a Phase 2 trial. OBJECTIVES: To assess surotomycin safety and clinical response (non-inferiority versus vancomycin) at the end of treatment (EOT) of CDI. Additionally, to assess surotomycin response over time and sustained response at 30-40 days post-EOT (superiority versus vancomycin). PATIENTS AND METHODS: Patients with CDI were randomized (1:1) to receive twice-daily oral surotomycin 250 mg alternating with twice-daily placebo or four-times-daily oral vancomycin 125 mg for 10 days in this Phase 3, double-blind, multicentre, international trial. Clinical response over time and sustained clinical response were monitored until the end of the trial, through a follow-up period of 30-40 days. Clinical Trial Registration: NCT01598311. RESULTS: A total of 285 and 292 patients with confirmed CDI were randomized to receive surotomycin and vancomycin, respectively. Surotomycin-associated clinical response at EOT was non-inferior to vancomycin (surotomycin/vancomycin: 83.4%/82.1%; difference 1.4%, 95% CI - 4.9, 7.6). Following treatment with surotomycin, both clinical response over time (stratified log-rank test, P = 0.277) and sustained clinical response (63.3%/59.0%; difference 4.3%, 95% CI - 3.6, 12.2) did not demonstrate superiority versus vancomycin at end of trial. Both treatments were generally well tolerated. CONCLUSIONS: Surotomycin demonstrated non-inferiority to vancomycin for CDI clinical response at EOT. Surotomycin did not demonstrate superiority to vancomycin for clinical response over time or sustained clinical response rate.

Helicase-primase as a target of new therapies for herpes simplex virus infections.

The seminal discovery of acyclovir 40 years ago heralded the modern era of truly selective antiviral therapies and this drug remains the therapy of choice for herpes simplex virus infections. Yet by modern standards, its antiviral activity is modest and new drugs against novel molecular targets such as the helicase-primase have the potential to improve clinical outcome, particularly in high-risk patients. A brief synopsis of current therapies for these infections and clinical need is provided to help provide an initial perspective. The function of the helicase-primase complex is then summarized and the development of new inhibitors of the helicase-primase complex, such as pritelivir and amenamevir, is discussed. We review their mechanism of action, propensity for drug resistance, and pharmacokinetic characteristics and discuss their potential to advance current therapeutic options. Strategies that include combinations of these inhibitors with acyclovir are also considered, as they will likely maximize clinical efficacy.

Cerebral transport and metabolism of 1-11C-D-glucose during stepped hypoglycemia.

Attempts to measure blood-to-brain glucose transport and cerebral glucose metabolism with 11C-glucose have been hampered by methods that require jugular venous sampling or do not adequately account for the efflux of labeled metabolites from the brain. We performed eight positron emission tomography studies with 1-11C-D-glucose in macaques at arterial plasma glucose concentrations of 8.43 to 1.51 mumol ml-1 (152-27 mg dl-1) using a model that includes a fourth rate constant to account for regional egress of all 11C-metabolites. Values for blood-to-brain glucose influx, cerebral glucose metabolism, and brain free glucose concentration agreed closely with values obtained in mammals by other investigators. Values for net extraction fraction corresponded closely to simultaneously measured arteriovenous values. We demonstrated that utilization of a model that includes a fourth rate constant to account for regional egress of all 11C-metabolites with positron emission tomography and 1-11C-D-glucose provides accurate measurements of blood-to-brain glucose transport and cerebral glucose metabolism in vivo without need for jugular venous sampling, even under conditions of severe hypoglycemia.

Tracer-kinetic analysis for measuring regional cerebral blood flow by dynamic nuclear magnetic resonance imaging.

Measurement of regional cerebral blood flow in vivo has proved useful in the study of normal and diseased states in the brain. This circumstance has led to a variety of techniques for its quantitative determination and has continued to motivate the search for ever safer and more accurate methods of measurement. Recently, the use of nuclear magnetic resonance (NMR) in medical imaging has stimulated efforts to make it the basis for a non-invasive method of measuring flow in the brain. New advances in fast NMR imaging (MRI) provide data potentially amenable to analysis by tracer-kinetic methods. Such an analysis has not previously been available. In this paper we present theoretical results that may permit measurement of brain blood flow by NMR. The data interpreted by our model are those generated by a novel MRI protocol developed by Perman et al. (1992, Magn. Reson. Med. 28, 74-83; Radiology 185(P), Abstr. 154, 127) that is entirely compatible with existing routine MRI procedures. These data are fast dynamic NMR signals that reflect passage of an intravenously administered paramagnetic contrast agent serving as a plasma tracer. Our equations show how to use such data sequences to determine plasma mean transit time, plasma volume, and plasma and whole-blood flow in arbitrarily selected regions of interest in the brain. The theory accounts rigorously for recirculation of tracer to the imaged regions. Our analysis provides an explanation for the linear relationship observed experimentally by others between regional vascular volumes and time integrals of vascular-tracer residue curves, and shows that this relationship remains valid in the presence of tracer recirculation.

Measurement of regional cerebral blood flow with positron emission tomography: a comparison of [15O]water to [11C]butanol with distributed-parameter and compartmental models.

To further our understanding of the best way to measure regional CBF with positron emission tomography (PET), we directly compared two candidate tracers ([15O]water and [11C]butanol, administered intravenously) and two popular implementations of the one-compartment (1C) model: the autoradiographic implementation representing a single PET measurement of tissue radioactivity over 1 min and a dynamic implementation representing a sequence of measurements of tissue radioactivity over 200 s. We also examined the feasibility of implementing a more realistic, and thus more complex, distributed-parameter (DP) model by assigning fixed values for all of its parameters other than CBF and tracer volume of distribution (Vd), a requirement imposed by the low temporal resolution and statistical quality of PET data. The studies were performed in three normal adult human subjects during paired rest and visual stimulation. In each subject seven regions of interest (ROIs) were selected, one of which was the primary visual cortex. The corresponding ROI were anatomically equivalent in the three subjects. Regional CBF, Vd, tracer arrival delay, and dispersion were estimated for the dynamic data curves. A total of 252 parameter sets were estimated. With [11C]butanol both implementations of the 1C model provided similar results (r = 0.97). Flows estimated using the 1C models were lower (p < 0.01) with [15O]water than with [11C]butanol. In comparison with the 1C model, the constrained version of the DP used in these studies performed inadequately, overestimating high flow and underestimating low flow with both tracers, possibly as the result of the necessity of assigning fixed values for all of its parameters other than CBF and Vd.

A half-Fourier gradient echo technique for dynamic MR imaging.

Recently we developed the simultaneous dual FLASH (SDFLASH) pulse sequence that simultaneously obtains sequential images from the brain and the internal-carotid arteries in the neck with 1-sec temporal resolution using a standard MR scanner. The high temporal resolution (1 sec) of the SDFLASH technique was achieved partly by using a low number of phase-encoding views which thereby limited our in-plane spatial resolution to 6.25 x 3.12 mm pixels. To overcome this limitation we have developed a calibration technique which corrects distortions in signal intensity and object shape when using gradient echo half-Fourier spin warp imaging. Using this calibration technique, the operator can use the 41% decrease in scan time to either double the spatial or temporal resolution. We have successfully used this technique to acquire SDFLASH images of the head and neck with 1.0 sec temporal resolution and 3.12 x 1.6 mm spatial resolution.

Simultaneous MR acquisition of arterial and brain signal-time curves.

Regional cerebral blood flow (rCBF) provides important information about local neuronal functional and cerebrovascular status. Determination of rCBF requires sequential measurements of tracer concentration in arterial blood and brain tissue unless the tracer is trapped in the brain in proportion to rCBF. Since gadopentate dimeglumine is not trapped within brain tissue, we have developed the simultaneous dual FLASH pulse sequence (SDFLASH) which sequentially measures the MR signal change in both the internal carotid artery and brain parenchyma simultaneously during the passage of a bolus of paramagnetic contrast material.

Discordant effects of nisoldipine on coronary vascular resistance and permeability changes during reflow after ischemia in isolated rabbit hearts.

Effects of a low dose (5 nM) of nisoldipine on vascular and ventricular function were assessed in isolated rabbit hearts during 2 h of reperfusion after 40 min of global, zero-flow ischemia. External detection of bolus injections of 125I-BSA and pressure data generated during the experiment provided repeated estimates of albumin permeation and vascular hemodynamics (resistance, vascular volume, and fractional rate of intravascular washout of 125I-BSA (k01]. In control hearts perfused continuously for 3.5 h, vascular resistance, vascular volume, LVEDP, and k01 remained constant, while maximum +dP/dt and -dP/dt increased 25% above baseline values, and estimates of albumin permeation increased 1.7 x baseline. Addition of 5 nM nisoldipine to the perfusate after the baseline period produced sustained decreases in vascular resistance (16% vs mean baseline value) without significantly affecting any other parameter. Postischemic perfusion of hearts increased vascular resistance and vascular volume approximately 50% above baseline, decreased k01 by 25% (intravascular washout of 125I-BSA was prolonged), and increased albumin permeation approximately 5 x baseline. While LVEDP remained elevated 3 x baseline, maximum +dP/dt and -dP/dt recovered 100% of baseline values (75-80% of untreated control values at comparable time points). Addition of 5 nM nisoldipine to the perfusate prior to ischemia prevented the increased vascular resistance during reflow, prevented the decrease in k01 and the increase in vascular volume, but did not affect the increased albumin permeation and, in general, did not affect the rate of recovery of left ventricle function. These results indicate that a low dose of nisoldipine preserves postischemic coronary vascular hemodynamics, but has little or no effect on the increased vascular leakage of albumin.

Activity patterns and life changes in people with depression.

The Activity Pattern Indicator (API) (Diller, Fordyce, Jacobs, & Brown, 1978) and the Schedule of Recent Experience (SRE) (Holmes, 1981) were used to determine activity patterns and life changes for 15 depressed patients admitted to an acute care mental health unit. Eight categories on the API were correlated with six categories on the SRE to determine the relationship between activity patterns 1 week and 1 month before hospitalization and life changes for the past year. Two correlations indicated that as the total number of life changes and home and family life changes increase, activity related to personal care decreases. Other correlations showed that as life changes related to health, work, and finance increase, such activities as passive recreation, homemaking, socializing, and personal care also increase. Because activity is the cornerstone of occupational therapy, occupational therapists, in treating patients with depression, might include facilitating close inspection of the patients' activity patterns in relation to the changes that have occurred in their lives.

Myocyte contracture, vascular resistance, and vascular permeability after global ischemia in isolated hearts from alloxan-induced diabetic rabbits.

Coronary vascular hemodynamics, albumin permeation, and myocyte contractility were assessed in isolated hearts from 6-mo alloxan-induced diabetic (ALX-D) rabbits during 3 h of reperfusion after 40 min of global no-flow ischemia. Residue-detection data, generated during the single passage of a bolus of 125I-labeled bovine serum albumin (125I-BSA) through the coronary vasculature, were used to estimate indices of vascular function, including the mean transit time of 125I-BSA, the fractional rate of intravascular clearance of 125I-BSA, and 125I-BSA permeation of coronary vessels. During reflow after ischemia in hearts from control rabbits, vascular resistance increased approximately three times that at baseline, left ventricular end-diastolic pressure (LVEDP) increased 8-10 times, and maximum +dP/dt recovered 0.4 times baseline, whereas the fractional rate of washout of intravascular 125I-BSA decreased to less than one-half of baseline values (was prolonged 2-fold), and albumin permeation and mean-transit time were increased 3 and 5 times baseline, respectively. In hearts from diabetic rabbits, vascular resistance was similar to the control group before ischemia but increased only one-third as much during reflow after ischemia. Increases in LVEDP during reflow were approximately 50% lower than controls, and +dP/dt recovered approximately 2.5 times more than in control hearts. 125I-BSA permeation in diabetics was similar to controls before ischemia, but during reflow increased 6 times (approximately 2 times controls). Washout of intravascular 125I-BSA was prolonged approximately 20% versus baseline during 3 h of reflow in hearts from diabetic rabbits. Thus, ALX-D in the rabbit delayed ischemia-reperfusion injury to myocytes and vascular smooth muscle cells while increasing vascular albumin permeation.

Vascular flow resistance in rabbit hearts: "apparent viscosity" of RBC suspensions.

Isovolumically beating, isolated hearts from male New Zealand rabbits were perfused retrograde via the aorta at a paced rate of 180 to 200 beats per minute. Perfusions were effected at arterial pressures (AP) ranging from 10 to 80 mm Hg. Perfusions (Krebs-Henseleit buffer + 1% BSA + insulin, 100 microliters/liter; same + 40% sheep RBC; same + 40% bovine RBC) were dosed with 15 mM papaverine to induce maximum vasodilation. In each case normalized flow rate (per unit heart mass) varied linearly with AP but all linear regressions extrapolated to a positive, zero-flow value of AP. Normalized flow resistance, defined as the slope of the line fitted to the pressure-drop vs normalized flow (per unit of dry heart weight) data, was not affected by the change from sheep to bovine RBC in the maximally dilated hearts. Data from RBC-free perfusions were inserted in Poiseuille's law to compute an effective geometric factor for the organ vasculature. This is turn was used to calculate apparent viscosities of the RBC suspensions. These were indistinguishable for the different RBC suspensions and fell between 0.5 and 0.6 of the respective high shear-rate values measured in a cone-plate viscometer, thus agreeing with the findings of S.R.F. Whittaker and F.R. Winton (1933, J. Physiol. (London) 78, 339-369) for the dog hindlimb.(ABSTRACT TRUNCATED AT 250 WORDS)

Tracer-kinetic models for measuring cerebral blood flow using externally detected radiotracers.

All tracer-kinetic models currently employed with positron-emission tomography (PET) are based on compartmental assumptions. Our first indication that a compartmental model might suffer from severe limitations in certain circumstances when used with PET occurred when we implemented the Kety tissue-autoradiography technique for measuring CBF and observed that the resulting CBF estimates, rather than remaining constant (to within predictable statistical uncertainty) as expected, fell with increasing scan duration T when T greater than 1 min. After ruling out other explanations, we concluded that a one-compartment model does not possess sufficient realism for adequately describing the movement of labeled water in brain. This article recounts our search for more realistic substitute models. We give our derivations and results for the residue-detection impulse responses for unit capillary-tissue systems of our two candidate distributed-parameter models. In a sequence of trials beginning with the simplest, we tested four progressively more detailed candidate models against data from appropriate residue-detection experiments. In these, we generated high-temporal-resolution counting-rate data reflecting the history of radiolabeled-water uptake and washout in the brains of rhesus monkeys. We describe our treatment of the data to yield model-independent empirical values of CBF and of other parameters. By substituting these into our trial-model functions, we were able to make direct comparisons of the model predictions with the experimental dynamic counting-rate histories, confirming that our reservations concerning the one-compartment model were well founded and obliging us to reject two others. We conclude that a two-barrier distributed-parameter model has the potential of serving as a substitute for the Kety model in PET measurements of CBF in patients, especially when scan durations for T greater than 1 min are desired.

Increased ischemia-reperfusion injury to the heart associated with short-term, diet-induced hypercholesterolemia in rabbits.

The effects of increased dietary cholesterol content on coronary vascular hemodynamics and endothelial cell transport function were assessed in isolated rabbit hearts during 3.5 hours of reperfusion after 30 minutes of global, no-flow ischemia. In control hearts from rabbits fed normal chow, perfusion pressure, left ventricular end-diastolic pressure, maximum +dP/dt, and the rate of intravascular clearance of radiolabelled albumin remained constant during 5 hours of continuous perfusion, while the mean transit time of radiolabelled albumin increased 1.6 X baseline. In ischemic hearts from rabbits fed normal chow, perfusion pressure increased 59% during reperfusion while left ventricular end-diastolic pressure and maximum +dP/dt returned toward control levels. The rate of intravascular clearance of radiolabelled albumin decreased 36%, and the mean transit time of albumin increased approximately 3 X baseline. Ischemia-reperfusion injury to the cardiac vasculature and musculature was markedly increased in hearts of rabbits fed chow supplemented with 2% cholesterol for 2-3 weeks compared to rabbits fed the same diet for a longer duration (5-16 weeks) or rabbits fed normal chow. Prior to ischemia, permeation of the coronary vasculature by albumin was increased twofold in rabbits fed cholesterol for 2-3 weeks while myocyte contractile function was normal relative to chow-fed controls or the group fed cholesterol for 5-16 weeks. These effects of acute cholesterol feeding precede occlusive atherosclerotic coronary artery disease and occur at plasma cholesterol concentrations one third of those in rabbits fed cholesterol for the longer duration.(ABSTRACT TRUNCATED AT 250 WORDS)

Strategies for in vivo measurement of receptor binding using positron emission tomography.

Dopaminergic ligands labeled with positron-emitting radionuclides have been synthesized for quantitative evaluation of dopaminergic binding in vivo. Two different methods, the explicit method and an operationally simplified ratio method, have been proposed for analysis of these positron emission tomographic (PET) data. The basis for both methods is the same three-compartment model. The two methods differ in the assumptions necessary for practical implementation. We have compared these two approaches using PET data obtained in our laboratory. Sequential scans and serial arterial blood samples from a baboon following intravenous injection of [18F]spiroperidol were collected. Application of the two methods to the same data yielded different values for corresponding parameters. Values calculated by the ratio method for the specific rate constant describing receptor binding varied depending upon the time after tracer injection, thus demonstrating an internal inconsistency in this approach. Tracer metabolism markedly affected the binding measurements calculated with either method and thus cannot be ignored. Our results indicate that the adoption of simplifying assumptions for operational convenience can lead to substantial errors and must be done with caution. Alternatively, we present simple new analytical solutions of the tracer conservation equations describing the complete, unsimplified three-compartment model that vastly reduce the computations necessary to implement the explicit method.

Hyaluronidase does not prevent deterioration of vascular functional integrity during reperfusion after no-flow ischemia in isolated rabbit hearts.

Effects of hyaluronidase on myocardial water content and distribution, and on coronary vascular hemodynamics and endothelial cell transport function were assessed in isolated rabbit hearts during 3.5 hours of reperfusion after 30 minutes of global, no-flow ischemia. In nonischemic control hearts, perfusion pressure, left ventricular end-diastolic pressure, maximum +dP/dt, and intravascular clearance of radiolabeled albumin remained constant during 5 hours of continuous perfusion, while the mean-transit time and vascular into extravascular space clearance of radiolabeled albumin increased 1.5X and 2.5X baseline, respectively. During reperfusion after 30 minutes of no flow, perfusion pressure increased 53% and interstitial fluid volume increased 2-fold, while left ventricular end-diastolic pressure and maximum +dP/dt returned to control levels. The rate of intravascular clearance of radiolabeled albumin decreased 38%, and the mean-transit time and vascular-into-extravascular space clearance of albumin increased approximately 3X and 5X baseline, respectively. Hyaluronidase blocked the ischemia-reperfusion-induced increases in total water content and in interstitial fluid volume and reduced the increases in perfusion pressure and mean-transit time of radiolabeled albumin by 40% and 45%, respectively, but did not prevent the increase in albumin vascular-into-extravascular space clearance and the decrease in albumin clearance from the coronary vasculature. These findings indicate that hyaluronidase does not prevent ischemia-reperfusion-induced increases in albumin permeation of the coronary vasculature, and suggest that its protective effect on ischemic myocardium is mediated, instead, by reducing interstitial edema and vascular resistance.

Coronary vascular hemodynamic and permeability changes during reperfusion after no-flow ischemia in isolated, diltiazem-treated rabbit hearts.

Effects of diltiazem on coronary vascular functional integrity were assessed in isolated rabbit hearts during reperfusion after 30 min of global, no-flow ischemia. External detection of radiolabeled albumin, [125I]bovine serum albumin ([125I]BSA), and compartmental-model analysis were used to estimate the mean transit time of [125I]BSA (tBSA), vascular volume (V1), and vascular into extravascular space clearance (F21) for [125I]BSA. Perfusion pressure, left ventricular (LV) end-diastolic pressure, LV developed pressure, maximum +dP/dt, and V1 remained constant during 5 h of continuous perfusion, while tBSA and F21 gradually increased (1.5 and 2.4 times baseline, respectively). Diltiazem, 4 microM, increased total water content (8.5%) and decreased perfusion pressure (11%), LV developed pressure (22%), and +dP/dt (24%) in nonischemic control experiments, but did not significantly affect estimates of V1, extracellular space, tBSA, or albumin permeation. During reperfusion after 30 min of ischemia, V1 increased 40% and perfusion pressure increased 60%, while tBSA and F21 increased three and eight times baseline, respectively. LV developed pressure and +dP/dt returned to control levels, even though the water content and extracellular space of ischemic hearts were increased significantly. Diltiazem, 4 microM, blocked ischemia-reperfusion-induced increases in water content, extracellular space, vascular resistance, V1, and vascular permeability to [125I]BSA, without reducing LV developed pressure or +dP/dt relative to nonischemic diltiazem controls. These results suggest that protection of ischemic myocardium by diltiazem is mediated, at least in part, by preservation of vascular functional integrity.

External detection of early microvascular dysfunction after no-flow ischemia followed by reperfusion in isolated rabbit hearts.

To define relationships better between the duration of severe ischemia and microvascular functional integrity with an approach potentially applicable to studies in vivo, the effects of 30 and 60 minutes of global, no-flow ischemia on the coronary vasculature of isolated, perfused rabbit hearts were determined. Residue-detection data, analyzed with a two-compartment model, were used to estimate indices of microvascular function, including the mean-transit time (tBSA) of radiolabeled bovine serum albumin (125I-BSA), vascular into extravascular space clearance, and vascular and extravascular space volumes. It was shown that the Central Volume Principle of tracer kinetics does not hold when transport of label between vascular and extravascular spaces takes place convectively by solvent drag, and a more general expression for tBSA was derived and applied. Left ventricular end-diastolic pressure and left ventricular developed pressure were monitored with an isovolumic balloon. Aortic perfusion pressure, left ventricular end-diastolic pressure, left ventricular developed pressure and vascular space volume remained constant, while mean transit time, vascular into extravascular space clearance and extravascular space volumes increased gradually during 3-hour control perfusions. Perfusion pressure, mean transit time and extravascular space clearance increased significantly with reperfusion after 30 minutes of ischemia even though left ventricular end-diastolic and left ventricular-developed pressures returned to control levels. Vascular space volumes increased minimally, whereas extravascular space volumes increased 5-fold during reperfusion. These changes in 125I-BSA washout and permeation across endothelium with reperfusion after no-flow ischemia indicate that compromised vascular integrity is an early manifestation of ischemia with functional consequences that persist even after ischemia sufficiently brief to permit restoration of left ventricular performance.

An intravenous technique for the measurement of cerebral vascular extraction fraction in the rat.

An intravenous injection method to measure cerebral vascular extraction fractions of highly diffusible substances in the rat is described. The brain extraction fractions of 3H-labeled water (Ew) and ethanol (Ee) were defined as the ratio of either of those tracers to the freely diffusible reference tracer, 14C-butanol, in the brain, divided by the ratio of the tracers available for the extraction during the time between simultaneous intravenous injection of the tracers and decapitation of the rat. Ew and Ee were measured in five regions of brain, including brainstem and cerebellum, under PaCO2 conditions ranging from 15 to 85 mm Hg. The extraction fractions for both test tracers were shown to vary with PaCO2-induced flow changes according to the equation, ln(1 - E) = -PS/F. When PS/F values calculated from regional measurements of Ew and Ee were plotted versus PaCO2, least squares regression equations of the plots could be used to compare permeabilities of both tracers at any given PaCO2 value. Ratios of the permeabilities of water and ethanol varied regionally but were relatively constant in a given region under different flow states. This intravenous injection method allows for accurate measurement of the extraction fractions of even highly diffusible tracers under varied flow conditions in all brain regions regardless of arterial blood supply.

Measurement of cerebral vascular extraction fractions in the rat using intracarotid injection techniques.

A small volume (5 microliters) common carotid arterial injection method is described for the quantitation of cerebral vascular extraction fractions (Et) of diffusion limited tracer molecules in the rat. The method is a modification of a technique introduced by Oldendorf and widely used for the study of blood-brain barrier phenomena. While the Oldendorf technique has proven valuable for estimating the relative permeabilities of substances, it is limited in measuring Et under conditions of physiologically or pharmacologically altered permeability or blood flow. The method described in this paper--using a small volume (5 microliters) common carotid injection, a freely diffusible reference tracer, [14C]butanol, and a 5 sec circulation time--allows for measurements of Et that reflect changes in blood flow and small differences in permeability. The modified method is important for the study of the regulation of cerebral vascular permeability and flow in an inexpensive animal model.