Dynamic contrast enhanced MRI parameters and tumor cellularity in a rat model of cerebral glioma at 7 T.

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 (K(trans) ), 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)%, 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, 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.

Brain edema and blood-brain barrier opening after photothrombotic ischemia in rat.

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.

Modeling of Look-Locker estimates of the magnetic resonance imaging estimate of longitudinal relaxation rate in tissue after contrast administration.

This paper models the behavior of the longitudinal relaxation rate of the protons of tissue water R(1) (R(1) = 1/T(1) ), 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 R(1) after administration of a CA in dynamic contrast enhanced experiment is described. The relation between R(1) 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 R(1) 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 ΔR(1) 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.

The concordance of MRI and quantitative autoradiography estimates of the transvascular transfer rate constant of albumin in a rat brain tumor model.

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.

MRI and quantitative autoradiographic studies following bolus injections of unlabeled and (14)C-labeled gadolinium-diethylenetriaminepentaacetic acid in a rat model of stroke yield similar distribution volumes and blood-to-brain influx rate constants.

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.

MRI estimation of gadolinium and albumin effects on water proton.

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 < 0.014 and 0.001).

Distribution of glycylsarcosine and cefadroxil among cerebrospinal fluid, choroid plexus, and brain parenchyma after intracerebroventricular injection is markedly different between wild-type and Pept2 null mice.

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 [(14)C]glycylsarcosine (GlySar) and [(3)H]cefadroxil, along with quantitative autoradiography of [(14)C]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<0.001), respectively, and lowered their CSF clearances by about fourfold. Autoradiography showed that GlySar concentrations in the lateral, third, and fourth ventricle choroid plexuses were higher in wild-type as compared with Pept2 null mice (P<0.01). Uptake of GlySar by the ependymal-subependymal layer and septal region was higher in wild-type than in null mice, but the half-distance of penetration into parenchyma was significantly less in wild-type mice. The latter is probably because of the clearance of GlySar from interstitial fluid by brain cells expressing PEPT2, which stops further penetration. These studies show that PEPT2 knockout can significantly modify the spatial distribution of GlySar and cefadroxil (and presumably other peptides/mimetics and peptide-like drugs) in brain.

Multiparametric magnetic resonance imaging and repeated measurements of blood-brain barrier permeability to contrast agents.

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 MRI-measured arterial input function resulting from a bolus injection of Gd-DTPA in a rat model of stroke slightly underestimates that of Gd-[14C]DTPA and marginally overestimates the blood-to-brain influx rate constant determined by Patlak plots.

The hypothesis that the arterial input function (AIF) of gadolinium-diethylenetriaminepentaacetic acid injected by intravenous bolus and measured by the change in the T(1)-relaxation rate (Delta R(1); R(1) = 1/T(1)) of superior sagittal sinus blood (AIF-I) approximates the AIF of (14)C-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 Delta R(1)-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 (K(i)) and proton distribution volumes at zero time (V(p) + V(o)) were estimated with Patlak plots of AIF-I, hypothetical AIFs, and cohort-averaged AIFs and tissue Delta R(1) data. For the regions of interest, the K(i)s estimated with AIF-I were slightly but not significantly higher than those obtained with hypothetical AIFs and cohort-averaged AIF. In contrast, V(p) + V(o) was significantly higher when calculated with AIF-I. Similar estimates of K(i) and V(p) + V(o) were obtained with hypothetical AIFs and cohort-averaged AIF. In summary, AIF-I underestimated the reference AIF; this shortcoming had little effect on the K(i) calculated by Patlak plot but produced a significant overestimation of V(p) + V(o).

Estimating blood and brain concentrations and blood-to-brain influx by magnetic resonance imaging with step-down infusion of Gd-DTPA in focal transient cerebral ischemia and confirmation by quantitative autoradiography with Gd-[(14)C]DTPA.

An intravenous step-down infusion procedure that maintained a constant gadolinium-diethylenetriaminepentaacetic 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(i) values of Gd-[(14)C]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-[(14)C]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-[(14)C]DTPA leakage, including one very small area, were observed. The terminal tissue-plasma ratios of Gd-[(14)C]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-[(14)C]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.

Development of a rat model of photothrombotic ischemia and infarction within the caudoputamen.

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.

MRI measurement of change in vascular parameters in the 9L rat cerebral tumor after dexamethasone administration.

PURPOSE: To demonstrate in the rat 9L cerebral tumor model that repeated MRI measurements can quantitate acute changes in the blood-brain distribution of Gadomer after dexamethasone administration. MATERIALS AND METHODS: A total of 16 Fischer 344 rats were studied at 7T, 15 days after cerebral implantation of a 9L tumor. MRI procedures employed a T-One by Multiple Read Out Pulses (TOMROP) sequence to estimate R(1) (R(1) = 1/T(1)) at 145-second intervals before and after administration of Gadomer (Bayer), a macromolecular contrast agent (CA). Two baseline studies preceded Gadomer administration and 10 subsequent R(1) maps tracked CA concentration in blood and brain for 25 minutes. Thereafter, either dexamethasone (N = 10) or normal saline (N = 6) was administered intravenously. A total of 90 minutes later a second series of 12 TOMROP measurements of Gadomer distribution was performed. The influx constant, K(1), plasma distribution volume, v(D), backflux constant, k(b), and interstitial space, v(e), were determined, and the test-retest differences of each of four vascular parameters were calculated. RESULTS: Dexamethasone decreased K(1) approximately 60% (P = 0.02), lowered k(b) and v(D) (P = 0.03 and P < 0.01, respectively), and marginally but insignificantly decreased v(e). CONCLUSION: This noninvasive MRI technique can detect drug effects on blood-brain transfer constants of CAs within two hours of administration.

Acute leakage patterns of fluorescent plasma flow markers after transient focal cerebral ischemia suggest large openings in blood-brain barrier.

OBJECTIVE: 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. METHODS: 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. RESULTS: Increase in EB-Alb leakage from 3 to 21 h of reperfusion was significant (from 40-60% to 80-90% of fields examined; p < 0.05). 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 (p < 0.01). Parenchymal distribution of the tracers differed among the fields and included considerable cellular uptake of EB-Alb and some of dextrans. CONCLUSIONS: 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.

Reporter gene imaging using radiographic contrast from nonradioactive iodide sequestered by the sodium-iodide symporter.

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.

MRI estimation of contrast agent concentration in tissue using a neural network approach.

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.

Relative distribution of plasma flow markers and red blood cells across BBB openings in acute cerebral ischemia.

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.

Step-down infusions of Gd-DTPA yield greater contrast-enhanced magnetic resonance images of BBB damage in acute stroke than bolus injections.

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.

Role of angiotensin II in retinal leukostasis in the diabetic rat.

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<0.01). Leukostasis was significantly decreased by ramipril and losartan (P<0.01 vs. untreated diabetic rats) but was still higher than normal. Retinal leukostasis after nifedipine treatment was not significantly different than in untreated diabetic rats. The same trend was observed when leukostasis was analyzed on retinal flat mounts with concanavalin A and CD45 immunofluorescence; ramipril and losartan treatment, however, decreased leukostasis to values no different than controls. Retinal leukostasis was lowered by nifedipine (P<0.05, untreated diabetes vs. nifedipine-treated) but was still higher than in normal, ramipril-, or losartan-treated rats. Assays of gene expression of retinal intercellular adhesion molecule (ICAM-1) by semi-quantitative RT-PCR indicated that ICAM-1 mRNA was increased in diabetic rats but was decreased markedly by treatment with losartan or ramipril, and modestly by nifedipine. In summary, suppressing the activity of the endogenous renin-angiotensin system markedly decreases, perhaps even normalizes, the retinal leukostasis that accompanies type I diabetes in rats. These effects seem to be partly independent of blood pressure and to be associated with a decrease in ICAM-1 gene expression. Angiotensin II may, thus, mediate retinal leukostasis in early diabetes.

Application of arsenazo III in the preparation and characterization of an albumin-linked, gadolinium-based macromolecular magnetic resonance contrast agent.

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.

9L gliosarcoma cell proliferation and tumor growth in rats are suppressed by N-hydroxy-N'-(4-butyl-2-methylphenol) formamidine (HET0016), a selective inhibitor of CYP4A.

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.