Imaging acute effects of bevacizumab on tumor vascular kinetics in a preclinical orthotopic model of U251 glioma.

The effect of a human vascular endothelial growth factor antibody on the vasculature of human tumor grown in rat brain was studied. Using dynamic contrast-enhanced magnetic resonance imaging, the effects of intravenous bevacizumab (Avastin; 10 mg/kg) were examined before and at postadministration times of 1, 2, 4, 8, 12 and 24 h (N = 26; 4-5 per time point) in a rat model of orthotopic, U251 glioblastoma (GBM). The commonly estimated vascular parameters for an MR contrast agent were: (i) plasma distribution volume (vp ), (ii) forward volumetric transfer constant (Ktrans ) and (iii) reverse transfer constant (kep ). In addition, extracellular distribution volume (VD ) was estimated in the tumor (VD-tumor ), tumor edge (VD-edge ) and the mostly normal tumor periphery (VD-peri ), along with tumor blood flow (TBF), peri-tumoral hydraulic conductivity (K) and interstitial flow (Flux) and tumor interstitial fluid pressure (TIFP). Studied as % changes from baseline, the 2-h post-treatment time point began showing significant decreases in vp , VD-tumor, VD-edge and VD-peri , as well as K, with these changes persisting at 4 and 8 h in vp , K, VD-tumor, -edge and -peri (t-tests; p < 0.05-0.01). Decreases in Ktrans were observed at the 2- and 4-h time points (p < 0.05), while interstitial volume fraction (ve ; = Ktrans /kep ) showed a significant decrease only at the 2-h time point (p < 0.05). Sustained decreases in Flux were observed from 2 to 24 h (p < 0.01) while TBF and TIFP showed delayed responses, increases in the former at 12 and 24 h and a decrease in the latter only at 12 h. These imaging biomarkers of tumor vascular kinetics describe the short-term temporal changes in physical spaces and fluid flows in a model of GBM after Avastin administration.

Adaptation of laser interstitial thermal therapy for tumor ablation under MRI monitoring in a rat orthotopic model of glioblastoma.

BACKGROUND: Laser interstitial thermal therapy (LITT) under magnetic resonance imaging (MRI) monitoring is being increasingly used in cytoreductive surgery of recurrent brain tumors and tumors located in eloquent brain areas. The objective of this study was to adapt this technique to an animal glioma model. METHODS: A rat model of U251 glioblastoma (GBM) was employed. Tumor location and extent were determined by MRI and dynamic contrast-enhanced (DCE) MRI. A day after assessing tumor appearance, tumors were ablated during diffusion-weighted imaging (DWI)-MRI using a Visualase LITT system (n = 5). Brain images were obtained immediately after ablation and again at 24 h post-ablation to confirm the efficacy of tumor cytoablation. Untreated tumors served as controls (n = 3). Rats were injected with fluorescent isothiocyanate (FITC) dextran and Evans blue that circulated for 10 min after post-LITT MRI. The brains were then removed for fluorescence microscopy and histopathology evaluations using hematoxylin and eosin (H&E) and major histocompatibility complex (MHC) staining. RESULTS: All rats showed a space-occupying tumor with T2 and T1 contrast-enhancement at pre-LITT imaging. The rats that underwent the LITT procedure showed a well-demarcated ablation zone with near-complete ablation of tumor tissue and with peri-ablation contrast enhancement at 24 h post-ablation. Tumor cytoreduction by ablation as seen on MRI was confirmed by H&E and MHC staining. CONCLUSIONS: Data showed that tumor cytoablation using MRI-monitored LITT was possible in preclinical glioma models. Real-time MRI monitoring facilitated visualizing and controlling the area of ablation as it is otherwise performed in clinical applications.

Noninvasive measurement of renal blood flow by magnetic resonance imaging in rats.

Renal blood flow (RBF) provides important information regarding renal physiology and nephropathies. Arterial spin labeling-magnetic resonance imaging (ASL-MRI) is a noninvasive method of measuring blood flow without exogenous contrast media. However, low signal-to-noise ratio and respiratory motion artifacts are challenges for RBF measurements in small animals. Our objective was to evaluate the feasibility and reproducibility of RBF measurements by ASL-MRI using respiratory-gating and navigator correction methods to reduce motion artifacts. ASL-MRI images were obtained from the kidneys of Sprague-Dawley (SD) rats on a 7-Tesla Varian MRI system with a spin-echo imaging sequence. After 4 days, the study was repeated to evaluate its reproducibility. RBF was also measured in animals under unilateral nephrectomy and in renal artery stenosis (RST) to evaluate the sensitivity in high and low RBF models, respectively. RBF was also evaluated in Dahl salt-sensitive (SS) rats and spontaneous hypertensive rats (SHR). In SD rats, the cortical RBFs (cRBF) were 305 ± 59 and 271.8 ± 39 ml·min-1·100 g tissue-1 in the right and left kidneys, respectively. Retest analysis revealed no differences ( P = 0.2). The test-retest reliability coefficient was 92 ± 5%. The cRBFs before and after the nephrectomy were 296.8 ± 30 and 428.2 ± 45 ml·min-1·100 g tissue-1 ( P = 0.02), respectively. The kidneys with RST exhibited a cRBF decrease compared with sham animals (86 ± 17.6 vs. 198 ± 33.7 ml·min-1·100 g tissue-1; P < 0.01). The cRBFs in SD, Dahl-SS, and SHR rats were not different ( P = 0.35). We conclude that ASL-MRI performed with navigator correction and respiratory gating is a feasible and reliable noninvasive method for measuring RBF in rats.

Optimization of a novel large field of view distortion phantom for MR-only treatment planning.

PURPOSE: MR-only treatment planning requires images of high geometric fidelity, particularly for large fields of view (FOV). However, the availability of large FOV distortion phantoms with analysis software is currently limited. This work sought to optimize a modular distortion phantom to accommodate multiple bore configurations and implement distortion characterization in a widely implementable solution. METHOD AND MATERIALS: To determine candidate materials, 1.0 T MR and CT images were acquired of twelve urethane foam samples of various densities and strengths. Samples were precision-machined to accommodate 6 mm diameter paintballs used as landmarks. Final material candidates were selected by balancing strength, machinability, weight, and cost. Bore sizes and minimum aperture width resulting from couch position were tabulated from the literature (14 systems, 5 vendors). Bore geometry and couch position were simulated using MATLAB to generate machine-specific models to optimize the phantom build. Previously developed software for distortion characterization was modified for several magnet geometries (1.0 T, 1.5 T, 3.0 T), compared against previously published 1.0 T results, and integrated into the 3D Slicer application platform. RESULTS: All foam samples provided sufficient MR image contrast with paintball landmarks. Urethane foam (compressive strength ∼1000 psi, density ~20 lb/ft3 ) was selected for its accurate machinability and weight characteristics. For smaller bores, a phantom version with the following parameters was used: 15 foam plates, 55 × 55 × 37.5 cm3 (L×W×H), 5,082 landmarks, and weight ~30 kg. To accommodate > 70 cm wide bores, an extended build used 20 plates spanning 55 × 55 × 50 cm3 with 7,497 landmarks and weight ~44 kg. Distortion characterization software was implemented as an external module into 3D Slicer's plugin framework and results agreed with the literature. CONCLUSION: The design and implementation of a modular, extendable distortion phantom was optimized for several bore configurations. The phantom and analysis software will be available for multi-institutional collaborations and cross-validation trials to support MR-only planning.

Subcurative radiation significantly increases cell proliferation, invasion, and migration of primary glioblastoma multiforme in vivo.

Tumor cell proliferation, infiltration, migration, and neovascularization are known causes of treatment resistance in glioblastoma multiforme (GBM). The purpose of this study was to determine the effect of radiation on the growth characteristics of primary human GBM developed in a nude rat. Primary GBM cells grown from explanted GBM tissues were implanted orthotopically in nude rats. Tumor growth was confirmed by magnetic resonance imaging on day 77 (baseline) after implantation. The rats underwent irradiation to a dose of 50 Gy delivered subcuratively on day 84 postimplantation (n = 8), or underwent no radiation (n = 8). Brain tissues were obtained on day 112 (nonirradiated) or day 133 (irradiated). Immunohistochemistry was performed to determine tumor cell proliferation (Ki-67) and to assess the expression of infiltration marker (matrix metalloproteinase-2, MMP-2) and cell migration marker (CD44). Tumor neovascularization was assessed by microvessel density using von-Willebrand factor (vWF) staining. Magnetic resonance imaging showed well-developed, infiltrative tumors in 11 weeks postimplantation. The proportion of Ki-67-positive cells in tumors undergoing radiation was (71 +/- 15)% compared with (25 +/- 12)% in the nonirradiated group (P = 0.02). The number of MMP-2-positive areas and proportion of CD44-positive cells were also high in tumors receiving radiation, indicating great invasion and infiltration. Microvessel density analysis did not show a significant difference between nonirradiated and irradiated tumors. Taken together, we found that subcurative radiation significantly increased proliferation, invasion, and migration of primary GBM. Our study provides insights into possible mechanisms of treatment resistance following radiation therapy for GBM.

Persistent Peri-Ablation Blood-Brain Barrier Opening After Laser Interstitial Thermal Therapy for Brain Tumors.

Purpose Laser interstitial thermal therapy (LITT) is a minimally invasive, image-guided, cytoreductive procedure to treat recurrent glioblastoma. This study implemented dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) methods and employed a model selection paradigm to localize and quantify post-LITT blood-brain barrier (BBB) permeability in the ablation vicinity. Serum levels of neuron-specific enolase (NSE), a peripheral marker of increased BBB permeability, were measured. Methods Seventeen patients were enrolled in the study. Using an enzyme-linked immunosorbent assay, serum NSE was measured preoperatively, 24 hours postoperatively, and at two, eight, 12, and 16 weeks postoperatively, depending on postoperative adjuvant treatment. Of the 17 patients, four had longitudinal DCE-MRI data available, from which blood-to-brain forward volumetric transfer constant (Ktrans) data were assessed. Imaging was performed preoperatively, 24 hours postoperatively, and between two and eight weeks postoperatively. Results Serum NSE increased at 24 hours following ablation (p=0.04), peaked at two weeks, and returned to baseline by eight weeks postoperatively. Ktrans was found to be elevated in the peri-ablation periphery 24 hours after the procedure. This increase persisted for two weeks. Conclusion Following the LITT procedure, serum NSE levels and peri-ablation Ktrans estimated from DCE-MRI demonstrated increases during the first two weeks after ablation, suggesting transiently increased BBB permeability.

Characterization of the Response of 9L and U-251N Orthotopic Brain Tumors to 3D Conformal Radiation Therapy.

In a study employing MRI-guided stereotactic radiotherapy (SRS) in two orthotopic rodent brain tumor models, the radiation dose yielding 50% survival (the TCD50) was sought. Syngeneic 9L cells, or human U-251N cells, were implanted stereotactically in 136 Fischer 344 rats or 98 RNU athymic rats, respectively. At approximately 7 days after implantation for 9L, and 18 days for U-251N, rats were imaged with contrast-enhanced MRI (CE-MRI) and then irradiated using a Small Animal Radiation Research Platform (SARRP) operating at 220 kV and 13 mA with an effective energy of ∼70 keV and dose rate of ∼2.5 Gy per min. Radiation doses were delivered as single fractions. Cone-beam CT images were acquired before irradiation, and tumor volumes were defined using co-registered CE-MRI images. Treatment planning using MuriPlan software defined four non-coplanar arcs with an identical isocenter, subsequently accomplished by the SARRP. Thus, the treatment workflow emulated that of current clinical practice. The study endpoint was animal survival to 200 days. The TCD50 inferred from Kaplan-Meier survival estimation was approximately 25 Gy for 9L tumors and below 20 Gy, but within the 95% confidence interval in U-251N tumors. Cox proportional-hazards modeling did not suggest an effect of sex, with the caveat of wide confidence intervals. Having identified the radiation dose at which approximately half of a group of animals was cured, the biological parameters that accompany radiation response can be examined.

Dynamic contrast enhanced (DCE) MRI estimation of vascular parameters using knowledge-based adaptive models.

We introduce and validate four adaptive models (AMs) to perform a physiologically based Nested-Model-Selection (NMS) estimation of such microvascular parameters as forward volumetric transfer constant, Ktrans, plasma volume fraction, vp, and extravascular, extracellular space, ve, directly from Dynamic Contrast-Enhanced (DCE) MRI raw information without the need for an Arterial-Input Function (AIF). In sixty-six immune-compromised-RNU rats implanted with human U-251 cancer cells, DCE-MRI studies estimated pharmacokinetic (PK) parameters using a group-averaged radiological AIF and an extended Patlak-based NMS paradigm. One-hundred-ninety features extracted from raw DCE-MRI information were used to construct and validate (nested-cross-validation, NCV) four AMs for estimation of model-based regions and their three PK parameters. An NMS-based a priori knowledge was used to fine-tune the AMs to improve their performance. Compared to the conventional analysis, AMs produced stable maps of vascular parameters and nested-model regions less impacted by AIF-dispersion. The performance (Correlation coefficient and Adjusted R-squared for NCV test cohorts) of the AMs were: 0.914/0.834, 0.825/0.720, 0.938/0.880, and 0.890/0.792 for predictions of nested model regions, vp, Ktrans, and ve, respectively. This study demonstrates an application of AMs that quickens and improves DCE-MRI based quantification of microvasculature properties of tumors and normal tissues relative to conventional approaches.

Cerebral myogenic reactivity and blood flow in type 2 diabetic rats: role of peroxynitrite in hypoxia-mediated loss of myogenic tone.

Dysregulation of cerebral vascular function and, ultimately, cerebral blood flow (CBF) may contribute to complications such as stroke and cognitive decline in diabetes. We hypothesized that 1) diabetes-mediated neurovascular and myogenic dysfunction impairs CBF and 2) under hypoxic conditions, cerebral vessels from diabetic rats lose myogenic properties because of peroxynitrite (ONOO(-))-mediated nitration of vascular smooth muscle (VSM) actin. Functional hyperemia, the ability of blood vessels to dilate upon neuronal stimulation, and myogenic tone of isolated middle cerebral arteries (MCAs) were assessed as indices of neurovascular and myogenic function, respectively, in 10- to 12-week control and type 2 diabetic Goto-Kakizaki rats. In addition, myogenic behavior of MCAs, nitrotyrosine (NY) levels, and VSM actin content were measured under normoxic and hypoxic [oxygen glucose deprivation (OGD)] conditions with and without the ONOO(-) decomposition catalyst 5,10,15,20-tetrakis(4-sulfonatophenyl) prophyrinato iron (III), chloride (FeTPPs). The percentage of myogenic tone was higher in diabetes, and forced dilation occurred at higher pressures. Functional hyperemia was impaired. Consistent with these findings, baseline CBF was lower in diabetes. OGD reduced the percentage of myogenic tone in both groups, and FeTPPs restored it only in diabetes. OGD increased VSM NY in both groups, and although FeTPPs restored basal levels, it did not correct the reduced filamentous/globular (F/G) actin ratio. Acute alterations in VSM ONOO(-) levels may contribute to hypoxic myogenic dysfunction, but this cannot be solely explained by the decreased F/G actin ratio due to actin nitration, and mechanisms may differ between control and diabetic animals. Our findings also demonstrate that diabetes alters the ability of cerebral vessels to regulate CBF under basal and hypoxic conditions.

Unaffected ex vivo clotting cascade by experimental hemostatic nanoparticles when introduced in the presence of recombinant tissue plasminogen activator.

CONTEXT: Hemostatic nanoparticles (hNPs) have shown efficacy in decreasing intracerebral hemorrhage (ICH) in animal models and are suggested to be of use to counter tissue plasminogen activator (tPA)-induced acute ICH. AIMS: The objective of this study was to test the ability of an hNP preparation to alter the clotting properties of blood exposed to tPA ex vivo. MATERIALS AND METHODS: Fresh blood samples were obtained from normal male Sprague-Dawley rats (~300 g; n = 6) and prepared for coagulation assays by thromboelastography (TEG) methods. Samples were untreated, exposed to tPA, or exposed to tPA and then to hNP. TEG parameters included reaction time (R, time in minutes elapsed from test initiation to initial fibrin formation), coagulation time (K, time in minutes from R until initial clot formation), angle (α, a measure in degrees of the rate of clot formation), maximum amplitude (MA, the point when the clot reaches its MA in mm), lysis at 30 min after MA (LY30, %), and clot strength (G, dynes/cm2), an index of clot strength. STATISTICAL ANALYSIS USED: Kruskal-Wallis test was employed to compare TEG parameters measured for untreated control samples versus those exposed to tPA and to compare tPA-exposed samples to samples treated with tPA + hNPs. Significances were inferred at P ≤ 0.05. RESULTS: Compared to untreated samples, tPA-treated samples showed a trend toward decreased angle and G suggesting potentially clot formation rate and clot strength. The addition of hNP did not affect any of these or other measured indices. CONCLUSIONS: The data demonstrated no hemostatic effects when the hNP was used in the presence of tPA. The lack of change in any of the TEG parameters measured in the present study may indicate limitations of the hNPs to reverse the thrombolytic cascade initiated by tPA.

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.

The impact of initial tumor microenvironment on imaging phenotype.

Models of human cancer, to be useful, must replicate human disease with high fidelity. Our focus in this study is rat xenograft brain tumors as a model of human embedded cerebral tumors. A distinguishing signature of such tumors in humans, that of contrast-enhancement on imaging, is often not present when the human cells grow in rodents, despite the xenografts having nearly identical DNA signatures to the original tumor specimen. Although contrast enhancement was uniformly evident in all the human tumors from which the xenografts' cells were derived, we show that long-term contrast enhancement in the model tumors may be determined conditionally by the tumor microenvironment at the time of cell implantation. We demonstrate this phenomenon in one of two patient-derived orthotopic xenograft (PDOX) models using cancer stem-like cell (CSC)-enriched neurospheres from human tumor resection specimens, transplanted to groups of immune-compromised rats in the presence or absence of a collagen/fibrin scaffolding matrix, Matrigel. The rats were imaged by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and their brains were examined by histopathology. Targeted proteomics of the PDOX tumor specimens grown from CSC implanted with and without Matrigel showed that while the levels of the majority of proteins and post-translational modifications were comparable between contrast-enhancing and non-enhancing tumors, phosphorylation of Fox038 showed a differential expression. The results suggest key proteins determine contrast enhancement and suggest a path toward the development of better animal models of human glioma. Future work is needed to elucidate fully the molecular determinants of contrast-enhancement.

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).

Simvastatin and atorvastatin improve neurological outcome after experimental intracerebral hemorrhage.

BACKGROUND AND PURPOSE: This study investigates the effects of statin treatment on experimental intracerebral hemorrhage (ICH) using behavioral, histological, and MRI measures of recovery. METHODS: Primary ICH was induced in rats. Simvastatin (2 mg/kg), atorvastatin (2 mg/kg), or phosphate-buffered saline (n=6 per group) was given daily for 1 week. MRI studies were performed 2 to 3 days before ICH, and at 1 to 2 hours and 1, 2, 7, 14, and 28 days after ICH. The ICH evolution was assessed via hematoma volume measurements using susceptibility-weighted imaging (SWI) and tissue loss using T2 maps and hematoxylin and eosin (H&E) histology. Neurobehavioral tests were done before ICH and at various time points post-ICH. Additional histological measures were performed with doublecortin neuronal nuclei and bromodeoxyuridine stainings. RESULTS: Initial ICH volumes determined by SWI were similar across all groups. Simvastatin significantly reduced hematoma volume at 4 weeks (P=0.002 versus control with acute volumes as baseline), whereas that for atorvastatin was marginal (P=0.09). MRI estimates of tissue loss (% of contralateral hemisphere) for treated rats were significantly lower (P=0.0003 and 0.001, respectively) than for control at 4 weeks. Similar results were obtained for H&E histology (P=0.0003 and 0.02, respectively). Tissue loss estimates between MRI and histology were well correlated (R2=0.764, P<0.0001). Significant improvement in neurological function was seen 2 to 4 weeks post-ICH with increased neurogenesis observed. CONCLUSIONS: Simvastatin and atorvastatin significantly improved neurological recovery, decreased tissue loss, and increased neurogenesis when administered for 1 week after ICH.

AC133+ progenitor cells as gene delivery vehicle and cellular probe in subcutaneous tumor models: a preliminary study.

BACKGROUND: Despite enormous progress in gene therapy for breast cancer, an optimal systemic vehicle for delivering gene products to the target tissue is still lacking. The purpose of this study was to determine whether AC133+ progenitor cells (APC) can be used as both gene delivery vehicles and cellular probes for magnetic resonance imaging (MRI). In this study, we used superparamagentic iron oxide (SPIO)-labeled APCs to carry the human sodium iodide symporter (hNIS) gene to the sites of implanted breast cancer in mouse model. In vivo real time tracking of these cells was performed by MRI and expression of hNIS was determined by Tc-99m pertechnetate (Tc-99m) scan. RESULTS: Three million human breast cancer (MDA-MB-231) cells were subcutaneously implanted in the right flank of nude mice. APCs, isolated from fresh human cord blood, were genetically transformed to carry the hNIS gene using adenoviral vectors and magnetically labeled with ferumoxides-protamine sulfate (FePro) complexes. Magnetically labeled genetically transformed cells were administered intravenously in tumor bearing mice when tumors reached 0.5 cm in the largest dimension. MRI and single photon emission computed tomography (SPECT) images were acquired 3 and 7 days after cell injection, with a 7 Tesla animal MRI system and a custom built micro-SPECT using Tc-99m, respectively. Expression of hNIS in accumulated cells was determined by staining with anti-hNIS antibody. APCs were efficiently labeled with ferumoxide-protamine sulfate (FePro) complexes and transduced with hNIS gene. Our study showed not only the accumulation of intravenously administered genetically transformed, magnetically labeled APCs in the implanted breast cancer, but also the expression of hNIS gene at the tumor site. Tc-99m activity ratio (tumor/non-tumor) was significantly different between animals that received non-transduced and transduced cells (P < 0.001). CONCLUSION: This study indicates that genetically transformed, magnetically labeled APCs can be used both as delivery vehicles and cellular probes for detecting in vivo migration and homing of cells. Furthermore, they can potentially be used as a gene carrier system for the treatment of tumor or other diseases.

Detection of migration of locally implanted AC133+ stem cells by cellular magnetic resonance imaging with histological findings.

This study investigated the factors responsible for migration and homing of magnetically labeled AC133(+) cells at the sites of active angiogenesis in tumor. AC133(+) cells labeled with ferumoxide-protamine sulfate were mixed with either rat glioma or human melanoma cells and implanted in flank of nude mice. An MRI of the tumors including surrounding tissues was performed. Tumor sections were stained for Prussian blue (PB), platelet-derived growth factor (PDGF), hypoxia-inducible factor-1alpha (HIF-1alpha), stromal cell derived factor-1 (SDF-1), matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor (VEGF), and endothelial markers. Fresh snap-frozen strips from the central and peripheral parts of the tumor were collected for Western blotting. MRIs demonstrated hypointense regions at the periphery of the tumors where the PB(+)/AC133(+) cells were positive for endothelial cells markers. At the sites of PB(+)/AC133(+) cells, both HIF-1alpha and SDF-1 were strongly positive and PDGF and MMP-2 showed generalized expression in the tumor and surrounding tissues. There was no significant association of PB(+)/AC133(+) cell localization and VEGF expression in tumor cells. Western blot demonstrated strong expression of the SDF-1, MMP-2, and PDGF at the peripheral parts of the tumors. HIF-1alpha was expressed at both the periphery and central parts of the tumor. This work demonstrates that magnetically labeled cells can be used as probes for MRI and histological identification of administered cells.

Identification of variations in blood-brain barrier opening after cerebral ischemia by dual contrast-enhanced magnetic resonance imaging and T 1sat measurements.

BACKGROUND AND PURPOSE: Variations in blood-brain barrier (BBB) opening after ischemia have been suggested by some tracer and magnetization transfer studies, although direct in vivo proof is still lacking. Contrast-enhanced magnetic resonance imaging (MRI) is also often used to visualize BBB damage in stroke. We hypothesized that MR contrast agents of different sizes enhance differently when BBB openings vary in size and that magnetization transfer alterations, measured by T(1) in the presence of off-resonance radiofrequency saturation (T(1sat)), in these regions reflect such differences. METHODS: Male Wistar rats ( approximately 300 g, n=7) were subjected to 3 hours of suture occlusion of the middle cerebral artery followed by reperfusion. Status of the BBB at 24 hours after the ictus was assessed first by Gd-DTPA (554 Da) MRI and then by Gd-bovine serum albumin linked to Evans blue (Gd-BSA-EB; approximately 68 kDa) MRI for contrast enhancement; T(1sat) changes, cerebral blood flow, and blood-to-brain transfer constants (K(i)s) for the 2 contrast agents were measured. After MRI, rats were injected with fluorescent dextran and brains were studied by fluorescence microscopy. RESULTS: The Gd-BSA-EB-enhancing areas were always smaller (147+/-80 pixels) than those for Gd-DTPA (308+/-204 pixels) and were contained within the latter. The difference between the 2 areas was significant (P=0.024). Changes in T(1sat) were larger in Gd-BSA-EB-enhancing areas (ipsilateral to contralateral [I/C]=1.53+/-0.20) than in Gd-DTPA-enhancing areas (I/C=1.40+/-0.24, P=0.005). The differences in cerebral blood flow values between the 2 regions were not significant (P=0.62), but those for the K(i) values of the 2 tracers were different (P=0.01 to 0.02). Excellent agreement between regions of Gd-BSA-EB enhancement and EB fluorescence was also observed. CONCLUSIONS: These results substantiate earlier reports of regional differences in BBB opening after stroke and provide the first in vivo evidence for this phenomenon. They also support the possible use of T(1sat) in quantifying stroke-induced graded BBB damage in the absence of contrast-enhanced MRI.

Temporal MRI assessment of intracerebral hemorrhage in rats.

BACKGROUND AND PURPOSE: MRI was used to evaluate the effects of experimental intracerebral hemorrhage (ICH) on brain tissue injury and recovery. METHODS: Primary ICH was induced in rats (n=6) by direct infusion of autologous blood into the striatum. The evolution of ICH damage was assessed by MRI estimates of T(2) and T(1sat) relaxation times, cerebral blood flow, vascular permeability, and susceptibility-weighted imaging before surgery (baseline) and at 2 hours and 1, 7, and 14 days post-ICH. Behavioral testing was done before and at 1, 7, and 14 days post-ICH. Animals were euthanized for histology at 14 days. RESULTS: The MRI appearance of the hemorrhage and surrounding regions changed in a consistent manner over time. Two primary regions of interest were identified based on T(2) values. These included a core, corresponding to the bulk of the hemorrhage, and an adjacent rim; both varied with time. The core was associated with significantly lower cerebral blood flow values at all post-ICH time points, whereas cerebral blood flow varied in the rim. Increases in vascular permeability were noted at 1, 7, and 14 days. Changes in T(1sat) were similar to those of T(2). MRI and histological estimates of tissue loss were well correlated and showed approximately 9% hemispheric tissue loss. CONCLUSIONS: Although the cerebral blood flow changes observed with this ICH model may not exactly mimic the clinical situation, our results suggest that the evolution of ICH injury can be accurately characterized with MRI. These methods may be useful to evaluate therapeutic interventions after experimental ICH and eventually in humans.

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.