Search

Purpose To test the hypothesis that a noninvasive dynamic contrast enhanced MRI (DCE-MRI) derived interstitial volume fraction (ve) and/or distribution volume (VD) were correlated with tumor cellularity in cerebral tumor. Methods T1-weighted DCE-MRI studies were performed in 18 athymic rats implanted with U251 xenografts. After DCE-MRI, sectioned brain tissues were stained with Hematoxylin and Eosin for cell counting. Using a Standard Model analysis and Logan graphical plot, DCE-MRI image sets during and after the injection of a gadolinium contrast agent were used to estimate the parameters plasma volume (vp), forward transfer constant (Ktrans), ve, and VD. Results Parameter values in regions where the standard model was selected as the best model were: (mean ± S.D.): vp = (0.81 ± 0.40)%, Ktrans = (2.09 ± 0.65) × 10−2 min−1, ve = (6.65 ± 1.86)%, and VD = (7.21 ± 1.98)%. The Logan-estimated VD was strongly correlated with the standard model's vp + ve (r = 0.91, P < 0.001). The parameters, ve and/or VD, were significantly correlated with tumor cellularity (r ≥ −0.75, P < 0.001 for both). Conclusion These data suggest that tumor cellularity can be estimated noninvasively by DCE-MRI, thus supporting its utility in assessing tumor pathophysiology. Magn Reson Med 71:2206–2214, 2014. © 2013 Wiley Periodicals, Inc.

This paper models the behavior of the longitudinal relaxation rate of the protons of tissue water R1 (R1 = 1/T1), measured in a Look-Locker experiment at 7 Tesla after administration of a paramagnetic contrast agent (CA). It solves the Bloch-McConnell equations for the longitudinal magnetization of the protons of water in a three-site two-exchange (3S2X) model with boundary conditions appropriate to repeated sampling of magnetization. The extent to which equilibrium intercompartmental water exchange kinetics affect monoexponential estimates of R1 after administration of a CA in dynamic contrast enhanced experiment is described. The relation between R1 and tissue CA concentration was calculated for CA restricted to the intravascular, or to the intravascular and extracellular compartments, by varying model parameters to mimic experimental data acquired in a rat model of cerebral tumor. The model described a nearly linear relationship between R1 and tissue concentration of CA, but demonstrated that the apparent longitudinal relaxivity of CA depends upon tissue type. The practical consequence of this finding is that the extended Patlak plot linearizes the ΔR1 data in tissue with leaky microvessels, accurately determines the influx rate of the CA across these microvessels, but underestimates the volume of intravascular blood water. Magn Reson Med, 2011. © 2011 Wiley Periodicals, Inc.

The apparent forward transfer constant, K transa, for albumin was measured in 9L cerebral tumors in 15 rats. An MRI study using gadolinium-labeled bovine serum albumin was followed by terminal quantitative autoradiography (QAR) using radioiodinated serum albumin. Look-Locker MRI estimates of T(1) followed gadolinium-labeled bovine serum albumin blood and tissue concentration. QAR and MRI maps of K transa were coregistered, a region of interest (ROI) that included the tumor and its surround was selected, and the two estimates of K transa from the ROI on QAR and MRI maps were compared by either mean per animal ROI or on pixel-by-pixel data using a generalized estimating equation. An ROI analysis showed a moderate correlation between the two measures (r = 0.57, P = 0.026); pixel-by-pixel generalized estimating equation analysis concurred (r = 0.54, P < 0.0001). The estimates of QAR with MRI of last time points (e.g., 25 min) showed a moderate correlation (ROI r = 0.55, P < 0.035; generalized estimating equation r = 0.58, P < 0.0001). Differences between the QAR and MRI estimates of K transa did not differ from zero, but the MRI 25-min estimate was significantly lower than the QAR estimate. Thus, noninvasive MRI estimates of vascular permeability can serve as a surrogate for QAR measures.

The purpose of this study was to define the cerebrospinal fluid (CSF) clearance kinetics, choroid plexus uptake, and parenchymal penetration of PEPT2 substrates in different regions of the brain after intracerebroventricular administration. To accomplish these objectives, we performed biodistribution studies using [14C]glycylsarcosine (GlySar) and [3H]cefadroxil, along with quantitative autoradiography of [14C]GlySar, in wild-type and Pept2 null mice. We found that PEPT2 deletion markedly reduced the uptake of GlySar and cefadroxil in choroid plexuses at 60 mins by 94% and 82% ( P

The hypothesis that the arterial input function (AIF) of gadolinium-diethylenetriaminepentaacetic acid injected by intravenous bolus and measured by the change in the T1-relaxation rate (ΔR1; R1 = 1/T1) of superior sagittal sinus blood (AIF-I) approximates the AIF of 14C-labeled gadolinium-diethylenetriaminepentaacetic acid measured in arterial blood (reference AIF) was tested in a rat stroke model (n = 13). Contrary to the hypothesis, the initial part of the ΔR1-time curve was underestimated, and the area under the normalized curve for AIF-I was about 15% lower than that for the reference AIF. Hypothetical AIFs for gadolinium-diethylenetriaminepentaacetic acid were derived from the reference AIF values and averaged to obtain a cohort-averaged AIF. Influx rate constants (Ki) and proton distribution volumes at zero time (Vp + Vo) were estimated with Patlak plots of AIF-I, hypothetical AIFs, and cohort-averaged AIFs and tissue ΔR1 data. For the regions of interest, the Kis estimated with AIF-I were slightly but not significantly higher than those obtained with hypothetical AIFs and cohort-averaged AIF. In contrast, Vp + Vo was significantly higher when calculated with AIF-I. Similar estimates of Ki and Vp + Vo were obtained with hypothetical AIFs and cohort-averaged AIF. In summary, AIF-I underestimated the reference AIF; this shortcoming had little effect on the Ki calculated by Patlak plot but produced a significant overestimation of Vp + Vo. Magn Reson Med 63:1502–1509, 2010. © 2010 Wiley-Liss, Inc.

An intravenous step-down infusion procedure that maintained a constant gadolinium-diethylene-triaminepentaacetic acid (Gd-DTPA) blood concentration and magnetic resonance imaging (MRI) were used to localize and quantify the blood-brain barrier (BBB) opening in a rat model of transient cerebral ischemia ( n = 7). Blood-to-brain influx rate constant ( K i) values of Gd-DTPA from such regions were estimated using MRI-Patlak plots and compared with the K, values of Gd-[14C]DTPA, determined minutes later in the same rats with an identical step-down infusion, quantitative autoradiography (QAR), and single-time equation. The normalized plasma concentration-time integrals were identical for Gd-DTPA and Gd-[14C]DTPA, indicating that the MRI protocol yielded reliable estimates of plasma Gd-DTPA levels. In six rats with a BBB opening, 14 spatially similar regions of extravascular Gd-DTPA enhancement and Gd-[14C]DTPA leakage, including one very small area, were observed. The terminal tissue-plasma ratios of Gd-[14C]DTPA tended to be slightly higher than those of Gd-DTPA in these regions, but the differences were not significant. The MRI-derived K i, values for Gd-DTPA closely agreed and correlated well with those obtained for Gd-[14C]DTPA. In summary, MRI estimates of Gd-DTPA concentration in the plasma and brain and the influx rate are quantitatively and spatially accurate with step-down infusions.

THE PURPOSE OF THIS STUDY was to develop an MRI technique for evaluating acute drug action (i.e., within one to two hours of administration) upon blood-brain exchange in an experimental cerebral tumor. To accomplish this, a standard cerebral tumor model, the 9L gliosarcoma in Fischer 344 rats (1,2), was employed, and the blood-brain distribution of the macromolecular magnetic resonance contrast agent (MRCA), Gadomer, was assessed quantitatively by MRI both before (test data) and shortly after (retest data) drug administration. Of importance in the selection of this MRCA and the development of this technique, Gadomer is relatively rapidly cleared from the blood (half-life ≈ 30 minutes (3)). To produce an in vivo change in the blood-brain distribution system and test the sensitivity in time of this MRI procedure, dexamethasone was administered to the experimental animals after test data acquisition. Postprocessing analyses yielded sets of blood-brain transfer constants—flux rates and distribution spaces—appropriate to one of three models (4). The test-retest differences in the four transfer constants were calculated and used for statistical analysis of drug effects. As to the choice of drug, the effects of dexamethasone on blood-brain tumor exchange of radioiodinated serum albumin (RISA) has been previously imaged by quantitative autoradiography of a different rat cerebral tumor model, the RG2 glioma (5): The effect of dexamethasone on tumor vascular permeability in another brain tumor model has been previously studied using RISA (5): in that experiment, dexamethasone lowered the transvascular transfer constant and the extravascular distribution space of RISA in the RG2 glioma model, but did not produce a significant change in the tumor plasma volume. Therefore, it was assumed that dexamethasone would have similar effects on the blood-brain distribution of Gadomer in the rat 9L model and this combination of MRCA and tumor would serve as a good proving ground of the test-retest MRI technique for assessing acute drug action.

This study tested the hypothesis that blood-brain barrier (BBB) opening during acute reperfusion permits the passage of smaller macromolecules but not larger ones and that this molecular size restriction disappears over time.Following 3 hours (h) of unilateral middle cerebral artery occlusion and either 3 or 21 h of reperfusion, Wistar rats (n = 42) were injected with Evans blue (EB, a fluorescent dye that binds instantly to plasma albumin yielding EB-tagged albumin, EB-Alb) and with one of three fluorescent dextrans ranging in size from 77- to 2000-kDa. During occlusion and reperfusion, ischemic status of the affected tissue was confirmed by magnetic resonance imaging (MRI). Blood-to-brain transfer of the dextrans relative to that of EB-Alb was examined by fluorescence microscopy within three regions with ischemic damage.Increase in EB-Alb leakage from 3 to 21 h of reperfusion was significant (from 40-60% to 80-90% of fields examined; p0.05). Co-leakage of the largest dextran used 2000-kDa, with EB-Alb was observed in only 40% of the fields at 3+3 h, but nearly in all at 3 + 21 h (p0.01). Parenchymal distribution of the tracers differed among the fields and included considerable cellular uptake of EB-Alb and some of dextrans.Supporting the hypothesis, opening of the BBB was insufficient to allow passage of the largest dextran at 3 + 3 h in about 40% of the microvascular networks viewed. The number of total leaky microvascular beds increased by nearly 50% between 3 + 3 h and 3 + 21 h.

Using an MRI T(1) by multiple readout pulses (TOMROP) image set, an adaptive neural network (ANN) was trained to directly estimate the concentration of a contrast agent (CA), gadolinium-bovine serum albumin (Gd-BSA), in tissue. In nine rats implanted with a 9L cerebral tumor, MRI acquisition of TOMROP inversion-recovery data was followed by quantitative autoradiography (QAR) using radioiodinated serum albumin (RISA). QAR autoradiograms were used as a training set for the ANN. Precontrast and 25 min postcontrast TOMROP image sets were shown to the ANN in the form of a physical feature set related to 24 inversion-recovery images; QAR autoradiograms at 30 min after injection of RISA were taken as the training standard for the network. After training and optimization, the ANN produced a map of Gd-BSA concentration [g-moles/liter]. The prediction by the ANN of CA concentration at 25 min after injection was well correlated (r = 0.82, P < 0.0001) with the corresponding autoradiogram's measure of CA concentration.

To study if the endogenous renin-angiotensin system affects diabetic retinal leukostasis, rats with streptozotocin-induced diabetes were treated with an ACE inhibitor (ramipril), an angiotensin II AT(1) receptor antagonist (losartan) and the Ca channel blocker, (nifedipine). In the diabetic rats, these drug treatments reduced systolic blood pressure by approximately 16 mmHg but did not change blood glucose. After 2 weeks, the rats were examined for retinal leukostasis in vivo with a scanning laser ophthalmoscope (SLO). Retinal leukostasis, which was defined as no movement of arrested leukocytes over 2 min, was markedly higher in diabetic rats than normal controls (P

Arterial spin labeling (ASL) is a potential means of obtaining quantitative images of cerebral blood flow (CBF). However, few validation studies of ASL have been performed in animal models using gold-standard CBF methods. Other methods that use radiolabeled water as a tracer underestimate CBF in high flow states, but this effect has not been evident in ASL studies. In this study the accuracy of ASL measurements of CBF were modeled and experimentally validated, with particular attention paid to high flow rates. The ASL signal as modeled included the contributions from intravascular labeled spins. The modeling demonstrated linearity of the ASL signal with respect to baseline flow, and linearity of ASL signal changes with respect to changes in flow, including high-flow conditions. Validation studies using quantitative autoradiography (QAR) to image flow in a rat model of unilateral cerebral ischemia showed that ASL systematically overestimated CBF by 34%. A similar overestimation was also predicted by modeling. These results indicate that ASL signals are linear with respect to flow (even high flow), but ASL-CBF measurements are systematically overestimated.

Water exchange across capillary walls couples intra- and extravascular (IV-EV) protons and their magnetization. A bolus i.v. injection of an extracellular MRI contrast agent (MRCA) causes a large increase in the spin-lattice relaxation rate, R1, of water protons in the plasma and blood cells within the capillaries and changes the effective relaxation rate R1eff in tissue via IV-EV water exchange. An analysis of the effect of plasma-red cell and IV-EV water exchange on the MRI-measured influx and permeability of capillaries to the MRCA is presented and focused on the brain and the blood-brain barrier. The effect of arrival of a bolus of an MRCA in the capillary on the relaxation rate R1eff in tissue via IV-EV water exchange occurs more rapidly than the MRCA uptake in tissue and can dominate the initial time curve of the R1eff change before the MRCA uptake in tissue becomes significant. This raises the possibility that (tissue dependent) IV-EV rate of exchange of water molecules can affect estimates of MRCA transfer constant. We demonstrate that an approach that considers IV-EV water exchange and uses the theoretical model of blood-brain tracer distribution developed by Patlak et al. (J Cereb Blood Flow Metab 1983;3:1-7) can lead to an accurate estimate of the MRI-determined influx rate constant of the MRCA and to an underestimation of the tissue blood volume.

Pathologic changes in brain tissue during and after stroke may lead to injury of the blood-brain barrier (BBB) and subsequent hemorrhagic transformation (HT). In a rat model of HT, the apparent diffusion coefficient of water, cerebral blood flow, relaxation times, T(1) and T(2), and magnetization transfer (MT) related parameters (T(1sat), K(for) and the MT ratio) were repetitively measured during 3 h of focal ischemia and 2 h of reperfusion (n = 8). Areas of BBB opening were identified by sequential assay of the transcapillary influx of Gd-diethylenetriaminepentaacetic acid (Gd-DTPA) by MRI and (14)C-alpha-aminoisobutyric acid (AIB) by quantitative autoradiography. Ischemia-injured regions of interest were identified from the MRI data and divided into those with and without BBB opening. Of the several MRI parameters measured, the T(1sat) in the caudate-putamen and preoptic area during ischemia and the first 2 h of reperfusion correlated best with the regional pattern of BBB opening observed thereafter. These data suggest that an ipsilateral/contralateral T(1sat) ratio > 1.6 demarcates leakage of small molecules such as Gd-DTPA and AIB across the BBB. As to clinical relevance, the quantitation of MT parameters in acute stroke may enable the early detection of areas of BBB opening and potential HT.

This study describes a quantitative method to measure the magnitude and distribution of gene expression following local delivery of an adenoviral vector containing the human sodium iodide symporter (hNIS) reporter gene into the canine prostate. Following systemic administration of Na 99m TcO 4 , autoradiographs of prostate sections depicting hNIS-dependent 99m TcO 4 − uptake were digitized and stacked to produce a three-dimensional reconstruction of gene expression. Frequency histograms reflecting hNIS gene expression magnitude and volume were used to quantify hNIS function. The method demonstrated submillimeter resolution allowing for precise measurements of gene expression magnitude and volume in vivo . The method developed here could be applied to other reporter gene systems in which the readout can be digitized from thin tissue sections.

With the goal of optimizing adenovirus-mediated suicide gene therapy for prostate cancer, we have developed a method based on the human sodium iodide symporter (hNIS) that allows for noninvasive monitoring of adenoviral vectors and quantification of gene expression magnitude and volume within the prostate. A replication-competent adenovirus (Ad5-yCD/ mut TK SR39 rep -hNIS) coexpressing a therapeutic yeast cytosine deaminase (yCD)/mutant herpes simplex virus thymidine kinase ( mut TK SR39 ) fusion gene and the hNIS gene was developed. Ad5-yCD/ mut TK SR39 rep -hNIS and a replication-defective hNIS adenovirus (rAd-CMV-FLhNIS) were injected into contralateral lobes of the dog prostate and hNIS activity was monitored in live animals following administration of Na 99m TcO 4 using gamma camera scintigraphy. Despite the close proximity of the urinary bladder, 99m TcO 4 − uptake was readily detected in the prostate using viral dose levels (10 10 to 10 12 viral particles) that have been safely administered to humans. Due to its rapid clearance and short physical half-life (6 h), it was possible to obtain daily measurements of 99m TcO 4 − uptake in vivo, allowing for dynamic monitoring of reporter gene expression within the prostate as well as biodistribution throughout the body. High-resolution autoradiography of prostate sections coupled with 3D reconstruction of gene expression demonstrated that the magnitude and volume of gene expression could be quantified with submillimeter resolution. Implementation of the GENIS (gene expression of Na/I symporter) technology in the clinic will facilitate optimization of future human gene therapy trials.

The availability of reperfusion therapy for acute ischemic stroke patients has made the causes and significance of hemorrhagic transformation an area of intense interest and controversy. Ninety-two male Wistar rats underwent transient middle cerebral artery occlusion (MCAO) of between 1 and 6 h. Forty animals received 10 mg kg-1 of recombinant tissue plasminogen activator (rtPA), infused over 20 min, starting 5 min before reperfusion. At 18-24 h, the animals were sacrificed. The presence of hemorrhagic transformation (HT) on stained sections was recorded and total ischemic lesion area was quantified using image analysis software. Seventeen animals (11 with HT) were subjected to immunohistochemical analysis for detection of endothelial barrier antigen (EBA), quantified in three sections, in eight different fields per section. Chi-squared analysis and logistic regression were used to assess the contribution of rtPA and duration of occlusion to HT development. Nested, repeated measures analyses of variance were performed to assess the changes in EBA caused by ischemia and associated with HT. Fifty-nine animals developed HT that was significantly associated with occlusion duration (p < 0.0001) and ischemic lesion size (p = 0.0007). The presence of rtPA accelerated HT development. Statistically significant side-to-side differences in the presence of EBA were found in the striatum (core of the infarct) of animals with HT (p < 0.001) and without HT (p < 0.001), but only in animals with durations of occlusion of 2 h or more. Duration of occlusion is an important predictor of HT in transient MCAO in the rat and is closely associated with EBA expression.

The present study determined cerebral blood flow (CBF) in the rat using two different magnetic resonance imaging (MRI) arterial spin-tagging (AST) methods and 14C-iodoantipyrine (IAP)-quantitative autoradiography (QAR), a standard but terminal technique used for imaging and quantitating CBF, and compared the resulting data sets to assess the precision and accuracy of the different techniques. Two hours after cerebral ischemia was produced in eight rats via permanent occlusion of one middle cerebral artery (MCA) with an intraluminal suture, MRI-CBF was measured over a 2.0-mm coronal slice using single-coil AST, and tissue magnetization was assessed by either a spin-echo (SE) or a variable tip-angle gradient-echo (VTA-GE) readout. Subsequently (∼2.5 hours after MCA occlusion), CBF was assayed by QAR with the blood flow indicator 14C-IAP, which produced coronal images of local flow rates every 0.4 mm along the rostral—caudal axis. The IAP-QAR images that spanned the 2-mm MRI slice were selected, and regional flow rates (i.e., local CBF [lCBF]) were measured and averaged across this set of images by both the traditional approach, which involved reader interaction and avoidance of sectioning artifacts, and a whole film-scanning technique, which approximated total radioactivity in the entire MRI slice with minimal user bias. After alignment and coregistration, the concordance of the CBF rates generated by the two QAR approaches and the two AST methods was examined for nine regions of interest in each hemisphere. The QAR-lCBF rates were higher with the traditional method of assaying tissue radioactivity than with the MRI-analog approach; although the two sets of rates were highly correlated, the scatter was broad. The flow rates obtained with the whole film-scanning technique were chosen for subsequent comparisons to MRI-CBF results because of the similarity in tissue “sampling” among these three methods. As predicted by previous modeling, “true” flow rates, assumed to be given by QAR-lCBF, tended to be slightly lower than those measured by SE and were appreciably lower than those assessed by VTA-GE. When both the ischemic and contralateral hemispheres were considered together, SE-CBF and VTA-GE-CBF were both highly correlated with QAR-lCBF ( P < 0.001). If evaluated by flow range, however, SE-CBF estimates were more accurate in high-flow (contralateral) areas (CBF > 80 mL · 100 g−1 · min−1), whereas VTA-GE-CBF values were more accurate in low-flow (ipsilateral) areas (CBF ≤ 60 mL · 100 g−1 · min−1). Accordingly, the concurrent usage of both AST-MRI methods or the VTA-GE technique alone would be preferred for human studies of stroke.

Ketamine, etomidate, propofol, and pentobarbital were compared for effects on and interactions with the type 1 facilitative glucose transporter (GLUT-1). Fluxes of radiolabeled hexoses were used to determine the effects of anesthetics on GLUT-1 function. Hypotonic hemolysis of human erythrocytes was used to assess perturbations of membrane integrity. Quenching of intrinsic protein fluorescence was used to assess the direct interactions of anesthetics with purified GLUT-1. Pentobarbital, ketamine, etomidate, and propofol inhibited glucose transport in murine fibroblasts with ic 50 values of 0.8, 1.6, 0.1, and 0.4 mM, respectively. Pentobarbital, ketamine, etomidate, and propofol also inhibited sugar transport in human erythrocytes. The ic 50 values for pentobarbital and ketamine exhibited substrate dependence for equilibrium exchange but not unidirectional effluxes. This was not observed for etomidate. Propofol did not inhibit equilibrium exchange but did inhibit unidirectional efflux with little substrate dependence. Pentobarbital protected against hemolysis, whereas etomidate and ketamine promoted hemolysis of erythrocytes. Propofol had no effect on membrane integrity. Pentobarbital, ketamine, and etomidate all interacted directly with GLUT-1, with apparent Kd values of 2.2, 0.8, and 0.5 mM, respectively. Like barbiturates, ketamine, etomidate, and propofol inhibited GLUT-1 at concentrations near to those used pharmacologically. Inhibition of GLUT-1 by these intravenous general anesthetics was complex, exhibiting differential kinetic effects on equilibrium exchange versus unidirectional fluxes and contrasting substrate dependencies. Like barbiturates, ketamine and etomidate bound to GLUT-1 with affinities that paralleled inhibition of glucose transport. As a class, intravenous general anesthetics, in contrast to inhalation anesthetics, inhibit GLUT-1-mediated glucose transport.

This study was designed to characterize the initial and secondary changes of the apparent diffusion coefficient (ADC) of water with high temporal resolution measurements of ADC values and to correlate ADC changes with functional outcomes. Fourteen rats underwent 30 minutes of temporary middle cerebral artery occlusion (MCAO). Diffusion-, perfusion-, and T2-weighted imaging was performed during MCAO and every 30 minutes for a total of 12 hours after reperfusion (n = 6). Neurological outcomes were evaluated during MCAO, every 30 minutes for a total of 6 hours and at 24 hours after reperfusion (n = 8). The decreased cerebral blood flow during MCAO returned to normal after reperfusion and remained unchanged thereafter. The decreased ADC values during occlusion completely recovered at 1 hour after reperfusion. The renormalized ADC values started to decrease secondarily at 2.5 hours, accompanied by a delayed increase in T2 values. The ADC-defined secondary lesion grew over time and was 52% of the ADC-defined initial lesion at 12 hours. Histological evaluation demonstrated neuronal damage in the regions of secondary ADC decline. Complete resolution of neurological deficits was seen in 1 rat at 1 hour and in 6 rats between 2.5 and 6 hours after reperfusion; no secondary neurological deficits were observed at 24 hours. These data suggest that (1) a secondary ADC reduction occurs as early as 2.5 hours after reperfusion, evolves in a slow fashion, and is associated with neuronal injury; and (2) renormalization and secondary decline in ADC are not associated with neurological recovery and worsening, respectively.

Background and Purpose —The early ischemic lesions demonstrated by diffusion-weighted imaging (DWI) are potentially reversible. The purposes of this study were to determine whether resolution of initial DWI lesions is transient or permanent after different brief periods of focal brain ischemia and to evaluate histological outcomes. Methods —Sixteen rats were subjected to 10 minutes (n=7) or 30 minutes (n=7) of temporary middle cerebral artery occlusion or sham operation (n=2). DWI, perfusion-weighted imaging (PWI), and T 2 -weighted imaging (T 2 WI) were performed during occlusion; immediately after reperfusion; and at 0.5, 1.0, 1.5, 12, 24, 48, and 72 hours after reperfusion. After the last MRI study, the brains were fixed, sectioned, stained with hematoxylin and eosin, and evaluated for neuronal necrosis. Results —No MRI or histological abnormalities were observed in the sham-operated rats. In both the 10-minute and 30-minute groups, the perfusion deficits and DWI hyperintensities that occurred during occlusion disappeared shortly after reperfusion. The DWI, PWI, and T 2 WI results remained normal thereafter in the 10-minute group, whereas secondary DWI hyperintensity and T 2 WI abnormalities developed at the 12-hour observation point in the 30-minute group. Histological examinations demonstrated neuronal necrosis in both groups, but the number of necrotic neurons was significantly higher in the 30-minute group (95±4%) than in the 10-minute group (17±10%, P Conclusions —Transient or permanent resolution of initial DWI lesions depends on the duration of ischemia. Transient resolution of DWI lesions is associated with widespread neuronal necrosis; moreover, permanent resolution of DWI lesions does not necessarily indicate complete salvage of brain tissue from ischemic injury.

Laser-scanning confocal microscopy (LSCM) was used to measure at high resolution cerebral plasma volumes (perfusion) using two fluorescent plasma markers in a rat model of embolic stroke. This application of LSCM to study the microvascular circulation in embolic stroke was developed as an alternative to autoradiography to measure cerebral perfusion. An additional benefit of LSCM is that it quantitates with great accuracy the structural relationships of the microcirculation to cells and the pathological alterations of the ischemic brain. Autoradiography allows only a quantitative analysis of cerebral perfusion. For example, in order to study the microcirculation and its relationship to blood brain barrier damage, the volume of perfused cerebral capillaries was measured by administering two fluorescent plasma markers (FITC-dextran and Evans blue) intravenously to a rat. Evans blue was administered before cerebral ischemia and FITC-dextran administered post-ischemia 1 min before sacrifice. Volumes of plasma perfusion were analyzed by means of a system developed for 3D analysis of fixed and stained serial brain histologies. Plasma volumes for the non-ischemic cerebral cortex were 1.00%+/-0.38% while plasma volumes in the caudate/putamen were 0.69%+/-0.17% in good agreement with the previously published values using the autoradiography method. The architecture of the capillaries in the ischemic core showed perfusion of Evans blue but there was no flow of FITC dextran. Our work represents a novel application of this technology to investigation of cerebral vascular disease and identifies its potential to become an important tool for investigation of cerebral pathology.

Acute blood-brain barrier (BBB) opening in cerebral ischemia is an often observed but seldom studied phenomenon. Increased permeability has been implicated with several consequences including exacerbating ischemic injury, leading to hemorrhagic transformation (HT) and also predictive of chronic damage and a way of delivering therapeutics to the diseased parts of brain. Very few studies have investigated the 'size' of such acute openings. Herein the blood-brain distribution of fluorescent isothiocyanate (FITC)- labeled red blood cells (RBCs; approximately 5 tm in diameter) and two different sized plasma flow markers in cerebral microvessels was studied by laser scanning confocal microscopy (LSCM) 6 and 24 hours after the onset of a 3 hour period of focal ischemia. At hour 6, Evans blue-tagged albumin [EB-Alb; molecular weight (MW)= 68 kDa, Stokes-Einstein radius=37 A], a marker of both plasma flow and BBB opening, was seen both inside and around microvessels whereas the RBCs were only intravascular. FITC-labeled dextran (FITC-dextran; MW=2000 kDa, Stokes-Einstein radius = approximately 150 A), another plasma flow tracer, had not leaked across the BBB into the tissue at this time. At hour 24, both RBCs and FITC-dextran were found extravascularly along with EB-Alb. We postulate that smaller sized openings in BBB at hour 6 limited the leaking of the two large tracers (RBCs and FITC-dextran) and that such size-dependency was lost by 24 hours with the progression of the ischemic injury.

We have examined the time course of brain edema and the blood-brain barrier opening in rat after basal ganglia ischemia induced by photothrombotic occlusion of the small vessels within the caudate-putamen. Male SD rats were anesthetized, and Rose Bengal dye was intravenously injected. The left caudo-putamen was exposed to cold white light for 5-10 min via a stereotaxically implanted optic fiber. Ischemic brain edema and the blood-brain barrier, as well as the histological changes, were assessed at various times during the following 6 weeks. Local cerebral blood flow was measured 90 min after photothrombosis by quantitative autoradiography. A round infarct with thrombosed parenchymal vessels surrounded by a layer of selective neuronal death was formed within the caudo-putamen. The ischemic lesion turned into a lacune over a period of 6 weeks. A central zone of markedly reduced blood flow and a surrounding oligemic zone were observed 90 min after light exposure. Early blood-brain barrier opening with edema was observed as early as 4 h after photothrombosis, peaked at day 1, and disappeared at 7 days after photothrombosis. In a model of lacunar infarction, we observed an early and transient brain edema and blood-brain opening after onset of ischemia.

Barbiturates inhibit GLUT-1 mediated hexose transport both in vivo [Gjedde & Rasmussen (1980) J. Neurochem. 35, 1382-1387; Otsuka et al. (1991) Am. J. Physiol. 261, R265-R275] and in vitro [Honkanen et al. (1995) Biochemistry 34, 535-544]. In the present study, the mechanism by which barbiturates inhibit GLUT-1 mediated hexose transport was examined by measuring both unidirectional zero trans and equilibrium exchange fluxes of hexoses in the functionally well-characterized, GLUT-1 rich human erythrocyte system. Unidirectional influx were both inhibited (> 80%) by 10 mM pentobarbital (PB). This symmetrical inhibition of unidirectional flux by PB was virtually independent of cis sugar concentration (2-130 mM) and exhibited an IC50 of approximately 2 mM. In contrast to unidirectional sugar flux, PB inhibition of equilibrium exchange sugar flux is attenuated by increased substrate concentration (e.g., 88% inhibition at 1 mM Glc versus 40% inhibition at 130 mM Glc in the presence of 10 mM PB) and exhibits an IC50 of approximately 10 mM at 100 mM Glc. Other barbiturates were found to inhibit sugar flux in human erythrocytes in this differential manner. These findings, when viewed with kinetic models proposed for GLUT-1 mediated transport [Carruthers (1990) Physiol. Rev. 70, 1135-1176], are consistent with barbiturates being noncompetitive inhibitors of Glc translocation and preferentially inhibiting the unoccupied form of the carrier protein. We propose, therefore, that barbiturates may prevent or alter the conformational changes associated with the reorientation of the carrier protein within the membrane. Overall, these results imply that barbiturates may more strongly inhibit GLUT-1 mediated Glc flux in vivo when the trans Glc is near zero as a result of either metabolism or another transport process.

We have examined the time course of brain edema and the blood–brain barrier opening in rat after basal ganglia ischemia induced by photothrombotic occlusion of the small vessels within the caudate–putamen. Male SD rats were anesthetized, and Rose Bengal dye was intravenously injected. The left caudo-putamen was exposed to cold white light for 5–10 min via a stereotaxically implanted optic fiber. Ischemic brain edema and the blood–brain barrier, as well as the histological changes, were assessed at various times during the following 6 weeks. Local cerebral blood flow was measured 90 min after photothrombosis by quantitative autoradiography. A round infarct with thrombosed parenchymal vessels surrounded by a layer of selective neuronal death was formed within the caudo-putamen. The ischemic lesion turned into a lacune over a period of 6 weeks. A central zone of markedly reduced blood flow and a surrounding oligemic zone were observed 90 min after light exposure. Early blood–brain barrier opening with edema was observed as early as 4 h after photothrombosis, peaked at day 1, and disappeared at 7 days after photothrombosis. In a model of lacunar infarction, we observed an early and transient brain edema and blood–brain opening after onset of ischemia.

To test the hypothesis that a noninvasive dynamic contrast enhanced MRI (DCE-MRI) derived interstitial volume fraction (ve ) and/or distribution volume (VD ) were correlated with tumor cellularity in cerebral tumor.T1 -weighted DCE-MRI studies were performed in 18 athymic rats implanted with U251 xenografts. After DCE-MRI, sectioned brain tissues were stained with Hematoxylin and Eosin for cell counting. Using a Standard Model analysis and Logan graphical plot, DCE-MRI image sets during and after the injection of a gadolinium contrast agent were used to estimate the parameters plasma volume (vp ), forward transfer constant (K(trans) ), ve , and VD .Parameter values in regions where the standard model was selected as the best model were: (mean ± S.D.): vp = (0.81 ± 0.40)%, K(trans) = (2.09 ± 0.65) × 10(-2) min(-1) , ve = (6.65 ± 1.86)%, and VD = (7.21 ± 1.98)%. The Logan-estimated VD was strongly correlated with the standard model's vp + ve (r = 0.91, P0.001). The parameters, ve and/or VD , were significantly correlated with tumor cellularity (r ≥ -0.75, P0.001 for both).These data suggest that tumor cellularity can be estimated noninvasively by DCE-MRI, thus supporting its utility in assessing tumor pathophysiology.

The origin and magnitude of potential errors in whole-blood sampling are prredicted on the basis of a mathematical model. The model describes the kinetics of solute metabolism, breakdown, and interphase distribution (i.e., partitioning and exchange between formed elements and plasma) within a blood sample during sample withdrawal and storage. The model is applied to the determination of the integral over time of solute concentration in the plasma (area-under-the-curve, or AUC) from a sample withdrawn through an arterial or venous catheter. Errors in AUC determination can be substantial and are strongly dependent on the duration of sampling (T), the rate constants for solute degradation processes, the rate constant for solute exchange between the formed elements and the plasma (ke). ,nd the equilibrium ratio for distribution of the solute between formed elements and plasma (R). When the value of the dimensionless group keT/R is small, little solute exchanges between plasma water and formed elements before the two phases of the blood are separated. When keT/R is large, the solute distribution is close to equilibrium at all times. In these two keT/R limits, the contribution of solute redistribution to sampling error is small. Sizable errors resulting from redistribution are associated with intermediate values of keT/R, even in the absence of metabolism and despite rapid separation of the phases at the end of the withdrawal period. Chemical conversion within either of the blood phases introduces additional sampling error under most circumstances.

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.

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.

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.

In previous studies on a rat model of transient cerebral ischemia, the blood and brain concentrations of gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA) following intravenous bolus injection were repeatedly assessed by dynamic contrast-enhanced (DCE)-MRI, and blood-to-brain influx rate constants (K(i)) were calculated from Patlak plots of the data in areas with blood-brain barrier (BBB) opening. For concurrent validation of these findings, after completing the DCE-MRI study, radiolabeled sucrose or α-aminoisobutyric acid was injected intravenously, and the brain disposition and K(i) values were calculated by quantitative autoradiography (QAR) assay employing the single-time equation. To overcome two of the shortcomings of this comparison, the present experiments were carried out with a radiotracer virtually identical to Gd-DTPA, Gd-[(14)C]DTPA, and K(i) was calculated from both sets of data by the single-time equation. The protocol included 3 h of middle cerebral artery occlusion and 2.5 h of reperfusion in male Wistar rats (n = 15) preceding the DCE-MRI Gd-DTPA and QAR Gd-[(14)C]DTPA measurements. In addition to K(i) , the tissue-to-blood concentration ratios, or volumes of distribution (V(R) ), were calculated. The regions of BBB opening were similar on the MRI maps and autoradiograms. Within them, V(R) was nearly identical for Gd-DTPA and Gd-[(14)C]DTPA, and K(i) was slightly, but not significantly, higher for Gd-DTPA than for Gd-[(14)C]DTPA. The K(i) values were well correlated (r = 0.67; p = 0.001). When the arterial concentration-time curve of Gd-DTPA was adjusted to match that of Gd-[(14)C]DTPA, the two sets of K(i) values were equal and statistically comparable with those obtained previously by Patlak plots (the preferred, less model-dependent, approach) of the same data (p = 0.2-0.5). These findings demonstrate that this DCE-MRI technique accurately measures the Gd-DTPA concentration in blood and brain, and that K(i) estimates based on such data are good quantitative indicators of BBB injury.

Breakdown of the blood-brain barrier (BBB) is present in several neurological disorders such as stroke, brain tumors, and multiple sclerosis. Noninvasive evaluation of BBB breakdown is important for monitoring disease progression and evaluating therapeutic efficacy in such disorders. One of the few techniques available for noninvasively and repeatedly localizing and quantifying BBB damage is magnetic resonance imaging (MRI). This usually involves the intravenous administration of a gadolinium-containing MR contrast agent (MRCA) such as Gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA), followed by dynamic contrast-enhanced MR imaging (DCE-MRI) of brain and blood, and analysis of the resultant data to derive indices of blood-to-brain transfer. There are two advantages to this approach. First, measurements can be made repeatedly in the same animal; for instance, they can be made before drug treatment and then again after treatment to assess efficacy. Secondly, MRI studies can be multiparametric. That is, MRI can be used to assess not only a blood-to-brain transfer or influx rate constant (Ki or K1) by DCE-MRI but also complementary parameters such as: (1) cerebral blood flow (CBF), done in our hands by arterial spin-tagging (AST) methods; (2) magnetization transfer (MT) parameters, most notably T1sat, which appear to reflect brain water-protein interactions plus BBB and tissue dysfunction; (3) the apparent diffusion coefficient of water (ADCw) and/or diffusion tensor, which is a function of the size and tortuosity of the extracellular space; and (4) the transverse relaxation time by T2-weighted imaging, which demarcates areas of tissue abnormality in many cases. The accuracy and reliability of two of these multiparametric MRI measures, CBF by AST and DCE-MRI determined influx of Gd-DTPA, have been established by nearly congruent quantitative autoradiographic (QAR) studies with appropriate radiotracers. In addition, some of their linkages to local pathology have been shown via corresponding light microscopy and fluorescence imaging. This chapter describes: (1) multiparametric MRI techniques with emphasis on DCE-MRI and AST-MRI; (2) the measurement of the blood-to-brain influx rate constant and CBF; and (3) the role of each in determining BBB permeability.

The longitudinal relaxivity on the protons of water of a Gd–chelate–albumin compound was measured at 7 T as a function of the macromolecular content of a cross-linked matrix. In agreement with previous works, the results demonstrate that the effect of gadolinium on water proton relaxivity is not constant, rising moderately with increase in the concentration of bovine serum albumin (BSA). About 35% variation in relaxivity was observed over a 0%–25% range of BSA concentrations (ℜ = 3.893 + 0.0502 × BSA [%], SE = 0.0119 and 0.1740, t = 4.215 and 22.383, p

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.

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.

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.

The level of cerebrospinal fluid (CSF) protein is elevated in diseases and disease models that are associated with circulating immune complexes such as serum sickness. Circulatory immune complexes are known to deposit in the basal lamina of fenestrated capillaries and may, as a result, affect both capillary bed and parenchymal function. Since the brain has both fenestrated and unfenestrated capillaries and immune complexes deposit to a varying extent in the fenestrated capillaries in chronic serum sickness, cerebral capillary permeability to protein may be altered in some brain areas and lead to the elevation of CSF proteins. In addition various other cerebrovascular and metabolic functions may also be affected by this condition. In this study either radio-iodinated serum albumin (RISA) or 2-[14C]deoxyglucose (14C-2DG) was intravenously injected into control Wistar rats and Wistar rats with chronic serum sickness; subsequently the tissue levels of radioactivity were measured by quantitative autoradiography in 4 brain areas with fenestrated capillaries and 11 brain areas with unfenestrated capillaries. The 2-min distribution of RISA, which demarcates the volume of circulating plasma in perfused microvessels and is generally proportional to local plasma flow, was the same in control and experimental rats. The passage of RISA from blood into brain over 30 min was negligible in both groups; thus cerebral capillary permeability to albumin was not detectably increased in any of these 15 brain areas by chronic serum sickness. The rate of local cerebral glucose utilization, an indicator of local metabolic and neural activity, was calculated from the 14C-2DG data and was virtually identical in control and experimental rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Background and Purpose— Basal ganglia infarction is typically caused by the occlusion of deep arteries and the formation of relatively small lesions called lacunes. In the present study, a rat model of lacunar infarction was induced by photothrombotic occlusion of the small vessels within the caudate-putamen and subsequently characterized. Methods— Male Sprague-Dawley rats (n=143) were anesthetized, and Rose Bengal dye (20 mg/kg) was intravenously injected. The left caudoputamen was exposed to cold white light for 5 to 10 minutes via a stereotaxically implanted polymethylmethacrylate optic fiber (0.5–0.75 mm diameter). Neurological and morphological changes were assessed at various times during the following 6 weeks. Local cerebral blood flow was measured 90 minutes after photothrombosis by [ 14 C]-N-isopropyl- p -iodoamphetamine quantitative autoradiography. The time course of blood–brain barrier opening and ischemic brain edema as well as the effects of aspirin and tissue plasminogen activator treatment were also determined. Results— A virtually round infarct with thrombosed parenchymal vessels surrounded by a layer of selective neuronal death was formed within the caudoputamen; it turned into a cystic cavity (lacune) over 6 weeks. A central zone of markedly reduced blood flow and surrounding oligemic zone were observed 90 minutes after light exposure. Lesion size was proportional to light exposure, and the severity and duration of neurological deficits paralleled infarct size. Early blood–brain barrier opening with edema peaked at day 1. After tissue plasminogen activator treatment, infarction volume and neurological deficits were reduced. Conclusions— This study describes a new rat model of lacunar infarction by photothrombotic occlusion of the microvessels within the caudoputamen. With this model, infarct size correlates with the severity and duration of the neuropathology and can be varied by altering light exposure.

Transfer of the inhibitory neurotransmitter gamma-aminobutyric acid across the normal blood-brain barrier is minimal. One prerequisite for gamma-aminobutyric acid in plasma contributing to the neural inhibition of hepatic encephalopathy would be that increased transfer of gamma-aminobutyric acid across the blood-brain barrier occurs in liver failure. The aim of the present study was to determine if brain gamma-aminobutyric acid uptake is increased in rabbits with stage II-III (precoma) hepatic encephalopathy due to galactosamine-induced fulminant hepatic failure. A modification of the Oldendorf intracarotid artery-injection technique was applied. [3H] gamma-aminobutyric acid, [14C] butanol, and 113mIn-labeled serum protein (transferrin) were injected simultaneously 4 s before decapitation. The ipsilateral brain uptake index of gamma-aminobutyric acid was determined from measurements of the 3 isotopes in 5 brain regions. Uncorrected or simple brain uptake indices of [3H] gamma-aminobutyric acid and [113mIn] transferrin were calculated using [14C] butanol as the highly extracted reference compound. The [113mIn] transferrin data were also used to "correct" the brain uptake index of [3H] gamma-aminobutyric acid for intravascular retention of [3H] gamma-aminobutyric acid. The methodology adopted minimized problems attributable to rapid [3H] gamma-aminobutyric acid metabolism, and slow brain washout and recirculation of the radiolabeled tracers. Both the uncorrected and corrected brain uptake indices of gamma-aminobutyric acid as well as the simple brain uptake index of transferrin were significantly increased in both stage II and III hepatic encephalopathy in all brain regions studied. Moreover, these brain uptake indices were significantly greater in stage III hepatic encephalopathy than in stage II hepatic encephalopathy. These findings indicate that transfer of gamma-aminobutyric acid from plasma to brain extracellular fluid is increased in the model of hepatic encephalopathy studied; hence, they provide support for the hypothesis that plasma-derived gamma-aminobutyric acid may contribute to the neural inhibition of hepatic encephalopathy due to fulminant hepatic failure.

The hypothesis that the human sodium-iodide symporter, NIS, can be used to detect NIS expression using standard radiological techniques was tested using adenoviral transduced NIS expression in human tumor xenografts grown in mice and in a naive dog prostate. Nonradioactive iodide was administered systemically to animals that 1-3 days previously had received a local injection of a replication-competent adenovirus expressing NIS under the control of the CMV promoter. The distribution of radiopacity was assessed in mouse tumors using micro-CT and a clinical X-ray machine and in the prostate of an anesthetized dog using a clinical spiral CT. Iodide sequestration and NIS expression were measured using X-ray spectrochemical analysis and fluorescence microscopy, respectively. Radiographic contrast due to NIS gene expression that was observed indicates the technique has potential for use in preclinical rodent tumor studies but probably lacks sensitivity for human use. Copyright # 2007 John Wiley & Sons, Ltd.

Acute vascular- and neuroprotective effects of simvastatin were evaluated in a rat model of transient, focal cerebral ischemia. Male, Wistar rats (n=12) underwent transient middle cerebral artery (MCA) occlusion for 3 hours followed by 3 hours of reperfusion. After 30 minutes of MCA occlusion, four rats each were subcutaneously injected with either 20 or 40 mg/kg of simvastatin. At the end of 3 hours of reperfusion, tissue injury and blood–brain barrier (BBB) opening were quantified by histology and [14C]-alpha-aminoisobutyric acid (AIB)-based quantitative autoradiography (QAR), respectively. Compared with untreated rats, those treated with simvastatin (20 mg/kg) had reduced volumes of AIB leakage, tissue pallor and distribution space for AIB (p

A rat model of transient suture occlusion of one middle cerebral artery (MCA) was used to create a unilateral reperfused cerebral ischemic infarct with blood–brain barrier (BBB) opening. Opening of the BBB was visualized and quantitated by magnetic resonance (MR) contrast enhancement with a Look-Locker T 1 -weighted sequence either following an intravenous bolus injection ( n =7) or during a step-down infusion ( n =7) of gadolinium–diethylenetriaminepentaacetic acid (Gd–DTPA). Blood levels of Gd–DTPA after either input were monitored via changes in sagittal sinus relaxation rate. Blood-to-brain influx constants ( K i ) were calculated by Patlak plots. On the basis of the MRI parameters and lesion size, the ischemic injury was determined to be similar in the two groups. The bolus injection input produced a sharp rise in blood levels of Gd–DTPA that declined quickly, whereas the step-down infusion led to a sharp rise that was maintained relatively constant for the period of imaging. Visual contrast enhancement and signal-to-noise (S/N) ratios were better with the step-down method (S/N=1.8) than with bolus injection (S/N=1.3). The K i values were not significantly different between the two groups ( P >.05) and were around 0.005 ml/(g min). The major reason for the better imaging of BBB opening by the step-down infusion was the higher amounts of Gd–DTPA in plasma and tissue during most of the experimental period. These results suggest that step-down MR contrast agent (MRCA) administration schedule may be more advantageous for detection and delineation of acute BBB injury than the usually used bolus injections.

A macromolecular magnetic resonance contrast agent (MMCA) was prepared by linking bovine serum albumin (BSA) to gadolinium (Gd) via a chelating agent, diethylenetriaminepentaacetic acid (DTPA). Colorimetric testing with 2,7-bis(o-arsenophenylazo)-1,8-dihydroxynaphthalene-3,6-disulfonic acid (arsenazo III) was performed to check for the appearance of free gadolinium during preparation and to quantify the Gd content in the final product. The complex was purified by dialysis, concentrated by lyophilyzation and characterized by magnetic resonance (MR) proton relaxation times. The resultant product had a molecular weight of about 90 kDa, Gd:BSA ratio of 14:1, and T1 and T2 relaxation times of 128.3 and 48.9 ms, respectively, at a field strength of 7Tesla (T) and at 20% concentration. Contrast enhancement of Gadomer-17 (a dendritic MMCA) and Gd-linked to BSA (Gd-BSA) was sequentially evaluated in a rat brain gliosarcoma model (n = 5) by MR imaging (MRI). Following intravenous injection, the blood concentration of Gadomer-17 fell rapidly, whereas that of Gd-BSA was almost constant for the duration of imaging. The areas of enhancement of both MMCAs were comparable. The spatial distribution of Gd-BSA showed good agreement with Evans blue-tagged albumin. Treatment with dexamethasone decreased Gd-BSA enhancement in the tumor. These results suggest that the arsenazo III method is applicable in preparing Gd-BSA to image brain tumors and their response to treatment. This simple method may also be useful for preparing other gadolinium-linked MMCAs.

The present study examined the effects of N-hydroxy-N'-(4-butyl-2 methylphenyl) formamidine (HET0016), a selective inhibitor of the formation of 20-hydroxyeicosatrienoic acid (20-HETE) on the growth of 9L rat gliosarcoma cells in vitro and in vivo. After 48 h of incubation, HET0016 reduced the proliferation of 9L in vitro by 55%, and this was associated with a fall in p42/p44 mitogen-activated protein kinase and stress-activated protein kinase/c-Jun NH(2)-terminal kinase phosphorylation and increased apoptosis. HET0016 inhibited epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)-induced proliferation and diminished phosphorylation of PDGF receptors. A stable 20-HETE analog increased 9L cell proliferation. In vivo, chronic administration of HET0016 (10 mg/kg/day i.p.) for 2 weeks reduced the volume of 9L tumors by 80%. This was accompanied by a 4-fold reduction in the mitotic index, a 3- to 4-fold increase in the apoptotic index, and a approximately 50% decrease in vascularization in the tumor. HET0016 treatment increased mean survival time of the animals from 17 to 22 days. Liquid chromatography/mass spectrometry experiments indicated that neither 9L cells grown in vitro nor 9L tumors removed produce 20-HETE when incubated with arachidonic acid. The normal surrounding brain tissue, however, avidly makes 20-HETE, and this activity is selectively inhibited by HET0016. These results suggest that HET0016 may be the prototype of a class of antigrowth compounds that may be efficacious for treating malignant brain tumors. In vivo, it may act in part by inhibiting the formation of 20-HETE by the surrounding tissue. However, the antiproliferative effects of HET0016 on 9L cells in vitro seem unrelated to its ability to inhibit the formation of 20-HETE.

The ability of gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA) enhanced MRI to localize and quantitate blood–brain barrier (BBB) opening was evaluated against quantitative autoradiographic (QAR) imaging of 14C-α-aminoisobutyric acid (AIB) distribution. The blood-to-brain transfer constant (Ki) for Gd-DTPA was determined by MRI in rats after 3 h of focal cerebral ischemia plus 2.5 h of reperfusion (n = 9), and that of AIB was determined by QAR shortly thereafter. Tissue regions of interest (ROIs) for Gd-DTPA leakage were identified by ISODATA segmentation of pre- and post-Gd-DTPA Look–Locker (L-L) T1 maps. Patlak plots were constructed using time course of blood and tissue T1 changes induced by Gd for estimating Ki. Among the nine rats, 14 sizable regions of AIB uptake were found; 13 were also identified by ISODATA segmentation. Although the 13 MRI-ROIs spatially approximated those of AIB uptake, the segmentation sometimes missed small areas of lesser AIB uptake that did not extend through more than 60% of the 2.0-mm-thick slice. Mean Ki's of AIB were highly correlated with those of Gd-DTPA across the 13 regions; the group means (±SD) were similar for the two tracers (7.1 ± 3.3 × 10−3 and 6.8 ± 3.5 × 10−3 ml.g−1 · min−1, respectively). In most instances, Gd-DTPA MRI accurately localized areas of BBB opening. Magn Reson Med, 2005. © 2005 Wiley-Liss, Inc.

Vasculature in and around the cerebral tumor exhibits a wide range of permeabilities, from normal capillaries with essentially no blood-brain barrier (BBB) leakage to a tumor vasculature that freely passes even such large molecules as albumin. In measuring BBB permeability by magnetic resonance imaging (MRI), various contrast agents, sampling intervals, and contrast distribution models can be selected, each with its effect on the measurement's outcome. Using Gadomer, a large paramagnetic contrast agent, and MRI measures of T1, over a 25-min period, BBB permeability was estimated in 15 Fischer rats with day-16 9L cerebral gliomas. Three vascular models were developed: (1) impermeable (normal BBB); (2) moderate influx (leakage without efflux); and (3) fast leakage with bidirectional exchange. For data analysis, these form nested models. Model 1 estimates only vascular plasma volume, vD, Model 2 (the Patlak graphical approach) vD and the influx transfer constant K1- Model 3 estimates vD, Ki and the reverse transfer constant, kb, through which the extravascular distribution space, ve, is calculated. For this contrast agent and experimental duration, Model 3 proved the best model, yielding the following central tumor means (±s.d.; n = 15): vD = 0.07±0.03 for Ki = 0.0105±0.005 min −1 and ve = 0.10±0.04. Model 2 Ki estimates were approximately 30% of Model 3, but highly correlated ( r 0.80, PD, Ki and kb appeared within each tumor. We conclude that employing nested models enables accurate assessment of transfer constants among areas where BBB permeability, contrast agent distribution volumes, and signal-to-noise vary.

Background Putatively active drugs are often intraventricularly administered to gain direct access to brain and circumvent the blood-brain barrier. A few studies on the normal central nervous system (CNS) have shown, however, that the distribution of materials after intraventricular injections is much more limited than presumed and their exit from cerebrospinal fluid (CSF) is more rapid than generally believed. In this study, we report the intracranial distribution and the clearance from CSF and adjacent CNS tissue of radiolabeled insulin-like growth factor-1 after injection into one lateral ventricle of the normal rat brain. Methods Under barbiturate anesthesia, 125I-labeled insulin-like growth factor-1 (IGF-1) was injected into one lateral ventricle of normal Sprague-Dawley rats. The subsequent distribution of IGF-1 through the cerebrospinal fluid (CSF) system and into brain, cerebral blood vessels, and systemic blood was measured over time by gamma counting and quantitative autoradiography (QAR). Results Within 5 min of infusion, IGF-1 had spread from the infused lateral ventricle into and through the third and fourth ventricles. At this time, 25% of the infused IGF-1 had disappeared from the CSF-brain-meningeal system; the half time of this loss was 12 min. The plasma concentration of cleared IGF-1 was, however, very low from 2 to 9 min and only began to rise markedly after 20 min. This delay between loss and gain plus the lack of radiotracer in the cortical subarachnoid space suggested that much of the IGF-1 was cleared into blood via the cranial and/or spinal nerve roots and their associated lymphatic systems rather than periventricular tissue and arachnoid villi. Less than 10% of the injected radioactivity remained in the CSF-brain system after 180 min. The CSF and arteries and arterioles within the subarachnoid cisterns were labeled with IGF-1 within 10 min. Between 60 and 180 min, most of the radioactivity within the cranium was retained within and around these blood vessels and by periaqueductal gray matter. Tissue profiles at two sites next to ventricular CSF showed that IGF-1 penetrated less than 1.25 mm into brain tissue and appreciable 125I-activity remained at the tissue-ventricular CSF interface after 180 min. Conclusion Our findings suggest that entry of IGF-1 into normal brain parenchyma after lateral ventricle administration is limited by rapid clearance from CSF and brain and slow movement, apparently by diffusion, into the periventricular tissue. Various growth factors and other neuroactive agents have been reported to be neuroprotective within the injured brain after intraventricular administration. It is postulated that the delivery of such factors to neurons and glia in the injured brain may be facilitated by abnormal CSF flow. These several observations suggest that the flow of CSF and entrained solutes may differ considerably between normal and abnormal brain and even among various neuropathologies.

The preferential accumulation of sickle blood cells in tumor vasculature is demonstrated noninvasively using MRI and sickle red blood cells loaded with Gd-DTPA and invasively by two other techniques. The distribution of red blood cells in rat brain tumors relative to normal brains were measured using three separate techniques: MRI of Gd-DTPA loaded cells, fluorescent microscopy detection of Oregon Green 488 fluorescence conjugated to a streptavidin-biotin complex that binds to red blood cell surface proteins, and autoradiography using a technetium (99m)Tc-labeling kit. Labeled red cells were infused intravenously in rats with brain tumors. Sickle cells preferentially accumulated in tumor relative to normal brain, with highest concentrations near the tumor / normal tissue boundary, whereas control normal red cells did not preferentially aggregate at the tumor periphery. This demonstrates the potential of sickle red blood cells to accumulate in the abnormal tumor vessel network, and the ability to detect their aggregation noninvasively and at high spatial resolution using MRI. The application of the noninvasive measurement of sickle cells for imaging tumor neovasculature, or as a delivery tool for therapy, requires further study.