Biocompatibility and proteomic profiling of DMSA-coated iron nanocubes in a human glioblastoma cell line.

Background: Superparamagnetic iron core iron oxide shell nanocubes have previously shown superior performance in magnetic resonance imaging T2 contrast enhancement compared with spherical nanoparticles. Methods: Iron core iron oxide shell nanocubes were synthesized, stabilized with dimercaptosuccinic acid (DMSA-NC) and physicochemically characterized. MRI contrast enhancement and biocompatibility were assessed in vitro. Results: DMSA-NC showed a transverse relaxivity of 122.59 mM-1·s-1 Fe. Treatment with DMSA-NC did not induce cytotoxicity or oxidative stress in U-251 cells, and electron microscopy demonstrated DMSA-NC localization within endosomes and lysosomes in cells following internalization. Global proteomics revealed dysregulation of iron storage, transport, transcription and mRNA processing proteins. Conclusion: DMSA-NC is a promising T2 MRI contrast agent which, in this preliminary investigation, demonstrates favorable biocompatibility with an astrocyte cell model.

MRI is a powerful tool used in the diagnosis of cancer, strokes and other injuries. An MRI scan can be improved with the use of iron oxide nanoparticles, which enhance the contrast of the image. In this study we have developed cube-shaped iron nanoparticles (nanocubes), which have been previously shown to be more effective at inducing contrast. We demonstrated that iron-based nanocubes do not damage or induce stress in cells and work effectively as an MRI contrast agent. We further analyzed how the nanocubes may affect cell functioning by investigating changes to protein levels in the cells. The results of this study are promising steps towards using iron-based nanocubes as a tool to improve the clarity of MRI scans for medical imaging and diagnosis. Future work must determine whether these nanocubes work effectively and safely in an animal model, which is a critical step in progressing to their use in clinical settings.

Evaluation of Dimercaptosuccinic Acid-Coated Iron Nanoparticles Immunotargeted to Amyloid Beta as MRI Contrast Agents for the Diagnosis of Alzheimer's Disease.

Nanoparticle-based magnetic contrast agents have opened the potential for magnetic resonance imaging (MRI) to be used for early non-invasive diagnosis of Alzheimer's disease (AD). Accumulation of amyloid pathology in the brain has shown association with cognitive decline and tauopathy; hence, it is an effective biomarker for the early detection of AD. The aim of this study was to develop a biocompatible magnetic nanoparticle targeted to amyloid beta (Aß) plaques to increase the sensitivity of T2-weighted MRI for imaging of amyloid pathology in AD. We presented novel iron core-iron oxide nanoparticles stabilized with a dimercaptosuccinic acid coating and functionalized with an anti-Aß antibody. Nanoparticle biocompatibility and cellular internalization were evaluated in vitro in U-251 glioblastoma cells using cellular assays, proteomics, and transmission electron microscopy. Iron nanoparticles demonstrated no significant in vitro cytotoxicity, and electron microscopy results showed their movement through the endocytic cycle within the cell over a 24 h period. In addition, immunostaining and bio-layer interferometry confirmed the targeted nanoparticle's binding affinity to amyloid species. The iron nanoparticles demonstrated favourable MRI contrast enhancement; however, the addition of the antibody resulted in a reduction in the relaxivity of the particles. The present work shows promising preliminary results in the development of a targeted non-invasive method of early AD diagnosis using contrast-enhanced MRI.

Site-Specific Antibody Assembly on Nanoparticles via a Versatile Coating Method for Improved Cell Targeting.

Antibody-nanoparticle conjugates are promising candidates for precision medicine. However, developing a controllable method for conjugating antibodies to nanoparticles without compromising the antibody activity represents a critical challenge. Here, a facile and generalizable film-coating method is presented using zeolitic imidazole framework-8 (ZIF-8) to immobilize antibodies on various nanoparticles in a favorable orientation for enhanced cell targeting. Different model and therapeutic antibodies (e.g., Herceptin) are assembled on nanoparticles via a biomineralized film-coating method and exhibited high antibody loading and targeting efficiencies. Importantly, the antibodies selectively bind to ZIF-8 via their Fc regions, which favorably exposes the functional Fab regions to the biological target, thus improving the cell targeting ability of antibody-coated nanoparticles. In combination, molecular dynamics simulations and experimental studies on antibody immobilization, orientation efficiency, and biofunctionality collectively demonstrate that this versatile site-specific antibody conjugation method provides effective control over antibody orientation and leads to improved cell targeting for a variety of nanoparticles.

Dual-function AAV gene therapy reverses late-stage Canavan disease pathology in mice.

The leukodystrophy Canavan disease is a fatal white matter disorder caused by loss-of-function mutations of the aspartoacylase-encoding ASPA gene. There are no effective treatments available and experimental gene therapy trials have failed to provide sufficient amelioration from Canavan disease symptoms. Preclinical studies suggest that Canavan disease-like pathology can be addressed by either ASPA gene replacement therapy or by lowering the expression of the N-acetyl-L-aspartate synthesizing enzyme NAT8L. Both approaches individually prevent or even reverse pathological aspects in Canavan disease mice. Here, we combined both strategies and assessed whether intracranial adeno-associated virus-mediated gene delivery to a Canavan disease mouse model at 12 weeks allows for reversal of existing pathology. This was enabled by a single vector dual-function approach. In vitro and in vivo biopotency assessment revealed significant knockdown of neuronal Nat8l paired with robust ectopic aspartoacylase expression. Following nomination of the most efficient cassette designs, we performed proof-of-concept studies in post-symptomatic Aspa-null mice. Late-stage gene therapy resulted in a decrease of brain vacuoles and long-term reversal of all pathological hallmarks, including loss of body weight, locomotor impairments, elevated N-acetyl-L-aspartate levels, astrogliosis, and demyelination. These data suggest feasibility of a dual-function vector combination therapy, directed at replacing aspartoacylase with concomitantly suppressing N-acetyl-L-aspartate production, which holds potential to permanently alleviate Canavan disease symptoms and expands the therapeutic window towards a treatment option for adult subjects.

Therapeutic inhibition of MPO stabilizes pre-existing high risk atherosclerotic plaque.

Currently there are no established therapies to treat high-risk patients with unstable atherosclerotic lesions that are prone to rupture and can result in thrombosis, abrupt arterial occlusion, and a precipitous infarction. Rather than being stenotic, rupture-prone non-occlusive plaques are commonly enriched with inflammatory cells and have a thin fibrous cap. We reported previously that inhibition of the pro-inflammatory enzyme myeloperoxidase (MPO) with the suicide inhibitor AZM198 prevents formation of unstable plaque in the Tandem Stenosis (TS) mouse model of plaque instability. However, in our previous study AZM198 was administered to animals before unstable plaque was present and hence it did not test the significant unmet clinical need present in high-risk patients with vulnerable atherosclerosis. In the present study we therefore asked whether pharmacological inhibition of MPO with AZM198 can stabilize pre-existing unstable lesions in an interventional setting using the mouse model of plaque instability. In vivo molecular magnetic resonance imaging of arterial MPO activity using bis-5-hydroxytryptamide-DTPA-Gd and histological analyses revealed that arterial MPO activity was elevated one week after TS surgery, prior to the presence of unstable lesions observed two weeks after TS surgery. Animals with pre-existing unstable plaque were treated with AZM198 for one or five weeks. Both short- and long-term intervention effectively inhibited arterial MPO activity and increased fibrous cap thickness, indicative of a more stable plaque phenotype. Plaque stabilization was observed without AZM198 affecting the arterial content of Ly6B.2+- and CD68+-cells and MPO protein. These findings demonstrate that inhibition of arterial MPO activity converts unstable into stable atherosclerotic lesions in a preclinical model of plaque instability and highlight the potential therapeutic potency of MPO inhibition for the management of high-risk patients and the development of novel protective strategies against cardiovascular diseases.

In Vivo 3D MRI Measurement of Tumour Volume in an Orthotopic Mouse Model of Prostate Cancer.

Prostate cancer (CaP) is the most commonly diagnosed cancer in males in western countries. Orthotopic implantation is considered as an ideal xenograft model for CaP study, and noninvasive measurement of tumor volume changes is important for monitoring responses to anticancer therapies. In this study, the T2-weighted fast spin echo sequence magnetic resonance imaging (MRI) was performed on a CaP orthotopic non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mouse model weekly for 6 weeks post PC-3 CaP cell inoculation, and the fat signal was suppressed using a chemical shift-selective pulse. Subsequently, the MRI data were imported into the image processing software Avizo Standard and stacked into three-dimensional (3D) volumes. Our results demonstrate that MRI, combined with 3D reconstruction, is a feasible and sensitive method to assess tumor growth in a PC-3 orthotopic CaP mouse model and this established monitoring approach is promising for longitudinal observation of CaP xenograft development after anticancer therapy in vivo. Further investigation is needed to validate this protocol in a larger cohort of mice to generate enough statistical power.

Short Diffusion Time Diffusion-Weighted Imaging With Oscillating Gradient Preparation as an Early Magnetic Resonance Imaging Biomarker for Radiation Therapy Response Monitoring in Glioblastoma: A Preclinical Feasibility Study.

PURPOSE: To investigate a novel alternative diffusion-weighted imaging (DWI) approach using oscillating gradients preparation (OGSE) to obtain much shorter effective diffusion times (Δeff) for tumor response monitoring by apparent diffusion coefficient (ADC) mapping in a glioblastoma mouse model. METHODS AND MATERIALS: Twenty-four BALB/c nude mice inoculated with U87 glioblastoma cells were randomized into a control group and an irradiation group, which underwent a 15-day fractioned radiation therapy (RT) course with 2 Gy/d. Therapy response was assessed by mapping of ADCs at 6 time points using an in-house implementation of a cos-OGSE DWI sequence with Δeff = 1.25 ms and compared with a standard pulsed gradient DWI protocol (PGSE) with typical clinical diffusion time Δeff = 18 ms. Longitudinal ADC changes in tumor and contralateral white matter (WM) were statistically assessed using repeated-measures analysis of variance and post hoc (Sidak) testing. RESULTS: On short Δeff OGSE maps tumor ADC was generally 30%-50% higher than in surrounding WM. Areas correlated well with histology. Tumor identification was generally more difficult on PGSE maps owing to nonsignificant WM/tumor contrast. During RT, OGSE maps also showed significant tumor ADC increase (approximately 15%) in response to radiation, consistently seen after 14-Gy RT dose. The clinical reference (PGSE) showed lower sensitivity to radiation changes, and no significant response across the radiation group and time course could be detected. CONCLUSION: Our short Δeff DWI method using OGSE better reflected histologically defined tumor areas and enabled more consistent and earlier detection of microstructural radiation changes than conventional methods. Oscillating gradients preparation offers significant potential as a robust microstructural RT response biomarker, potentially helping to shift important therapy decisions to earlier stages in the RT time course.

Uncoupling N-acetylaspartate from brain pathology: implications for Canavan disease gene therapy.

N-Acetylaspartate (NAA) is the second most abundant organic metabolite in the brain, but its physiological significance remains enigmatic. Toxic NAA accumulation appears to be the key factor for neurological decline in Canavan disease-a fatal neurometabolic disorder caused by deficiency in the NAA-degrading enzyme aspartoacylase. To date clinical outcome of gene replacement therapy for this spongiform leukodystrophy has not met expectations. To identify the target tissue and cells for maximum anticipated treatment benefit, we employed comprehensive phenotyping of novel mouse models to assess cell type-specific consequences of NAA depletion or elevation. We show that NAA-deficiency causes neurological deficits affecting unconscious defensive reactions aimed at protecting the body from external threat. This finding suggests, while NAA reduction is pivotal to treat Canavan disease, abrogating NAA synthesis should be avoided. At the other end of the spectrum, while predicting pathological severity in Canavan disease mice, increased brain NAA levels are not neurotoxic per se. In fact, in transgenic mice overexpressing the NAA synthesising enzyme Nat8l in neurons, supra-physiological NAA levels were uncoupled from neurological deficits. In contrast, elimination of aspartoacylase expression exclusively in oligodendrocytes elicited Canavan disease like pathology. Although conditional aspartoacylase deletion in oligodendrocytes abolished expression in the entire CNS, the remaining aspartoacylase in peripheral organs was sufficient to lower NAA levels, delay disease onset and ameliorate histopathology. However, comparable endpoints of the conditional and complete aspartoacylase knockout indicate that optimal Canavan disease gene replacement therapies should restore aspartoacylase expression in oligodendrocytes. On the basis of these findings we executed an ASPA gene replacement therapy targeting oligodendrocytes in Canavan disease mice resulting in reversal of pre-existing CNS pathology and lasting neurological benefits. This finding signifies the first successful post-symptomatic treatment of a white matter disorder using an adeno-associated virus vector tailored towards oligodendroglial-restricted transgene expression.

Measurement and modeling of diffusion time dependence of apparent diffusion coefficient and fractional anisotropy in prostate tissue ex vivo.

The purpose of this study was to measure and model the diffusion time dependence of apparent diffusion coefficient (ADC) and fractional anisotropy (FA) derived from conventional prostate diffusion-weighted imaging methods as used in recommended multiparametric MRI protocols. Diffusion tensor imaging (DTI) was performed at 9.4 T with three radical prostatectomy specimens, with diffusion times in the range 10-120 ms and b-values 0-3000 s/mm2 . ADC and FA were calculated from DTI measurements at b-values of 800 and 1600 s/mm2 . Independently, a two-component model (restricted isotropic plus Gaussian anisotropic) was used to synthesize DTI data, from which ADC and FA were predicted and compared with the measured values. Measured ADC and FA exhibited a diffusion time dependence, which was closely predicted by the two-component model. ADC decreased by about 0.10-0.15 µm2 /ms as diffusion time increased from 10 to 120 ms. FA increased with diffusion time at b-values of 800 and 1600 s/mm2 but was predicted to be independent of diffusion time at b = 3000 s/mm2 . Both ADC and FA exhibited diffusion time dependence that could be modeled as two unmixed water pools - one having isotropic restricted dynamics, and the other unrestricted anisotropic dynamics. These results highlight the importance of considering and reporting diffusion times in conventional ADC and FA calculations and protocol recommendations, and inform the development of improved diffusion methods for prostate cancer imaging.

Longitudinal measurements of syrinx size in a rat model of posttraumatic syringomyelia.

OBJECTIVE Syringomyelia pathophysiology is commonly studied using rodent models. However, in vivo studies of posttraumatic syringomyelia have been limited by the size of animals and lack of reliable noninvasive evaluation techniques. Imaging the rat spinal cord is particularly challenging because the spinal cord diameter is approximately 1-3 mm, and pathological lesions within the spinal cord parenchyma are even smaller. The standard technique has been histological evaluation, but this has its limitations. The aim of the present study was to determine whether syrinx size could be reliably measured using a preclinical high-field MRI animal system in a rat model of posttraumatic syringomyelia. METHODS The authors used an existing rat model of posttraumatic syringomyelia, which was created using a controlled pneumatic compression device to produce the initial spinal cord injury, followed by a subarachnoid injection of kaolin to produce arachnoiditis. T2-weighted MRI was performed on each animal using a 9.4-T scanner at 7, 10, and 13 weeks after injury. Animals were killed and syrinx sizes were calculated from in vivo MRI and histological studies. RESULTS MRI measurements of syrinx volume and length were closely correlated to histological measurements across all time points (Pearson product moment correlation coefficient r = ± 0.93 and 0.79, respectively). CONCLUSIONS This study demonstrates that high-field T2-weighted MRI can be used to measure syrinx size, and data correlate well with syrinx size measured using histological methods. Preclinical MRI may be a valuable noninvasive technique for tracking syrinx formation and enlargement in animal models of syringomyelia.

Diffusion anisotropy in fresh and fixed prostate tissue ex vivo.

PURPOSE: To investigate diffusion anisotropy in whole human prostate specimens METHODS: Seven whole radical prostatectomy specimens were obtained with informed patient consent and institutional ethics approval. Diffusion tensor imaging was performed at 9.4 Tesla. Diffusion tensors were calculated from the native acquired data and after progressive downsampling RESULTS: Fractional anisotropy (FA) decreased as voxel volume increased, and differed widely between prostates. Fixation decreased mean FA by ∼0.05-0.08 at all voxel volumes but did not alter principle eigenvector orientation. In unfixed tissue high FA (> 0.6) was found only in voxels of volume <0.5 mm(3) , and then only in a small fraction of all voxels. At typical clinical voxel volumes (4-16 mm(3) ) less than 50% of voxels had FA > 0.25. FA decreased at longer diffusion times (Δ = 60 or 80 ms compared with 20 ms), but only by ∼0.02 at typical clinical voxel volume. Peripheral zone FA was significantly lower than transition zone FA in five of the seven prostates CONCLUSION: FA varies widely between prostates. The very small proportion of clinical size voxels with high FA suggests that in clinical DWI studies ADC based on three-direction measurements will be minimally affected by anisotropy. Magn Reson Med 76:626-634, 2016. © 2015 Wiley Periodicals, Inc.

First Steps Toward Ultrasound-Based Motion Compensation for Imaging and Therapy: Calibration with an Optical System and 4D PET Imaging.

Target motion, particularly in the abdomen, due to respiration or patient movement is still a challenge in many diagnostic and therapeutic processes. Hence, methods to detect and compensate this motion are required. Diagnostic ultrasound (US) represents a non-invasive and dose-free alternative to fluoroscopy, providing more information about internal target motion than respiration belt or optical tracking. The goal of this project is to develop an US-based motion tracking for real-time motion correction in radiation therapy and diagnostic imaging, notably in 4D positron emission tomography (PET). In this work, a workflow is established to enable the transformation of US tracking data to the coordinates of the treatment delivery or imaging system - even if the US probe is moving due to respiration. It is shown that the US tracking signal is equally adequate for 4D PET image reconstruction as the clinically used respiration belt and provides additional opportunities in this concern. Furthermore, it is demonstrated that the US probe being within the PET field of view generally has no relevant influence on the image quality. The accuracy and precision of all the steps in the calibration workflow for US tracking-based 4D PET imaging are found to be in an acceptable range for clinical implementation. Eventually, we show in vitro that an US-based motion tracking in absolute room coordinates with a moving US transducer is feasible.

Information theoretic ranking of four models of diffusion attenuation in fresh and fixed prostate tissue ex vivo.

PURPOSE: To compare the theoretical information content of four popular models of diffusion-weighted signal attenuation. METHOD: Four whole prostates were imaged fresh unfixed and fixed at 9.4T. Biexponential, kurtosis, stretched exponential, and monoexponential models were ranked using Akaike's Information Criterion (AIC) with validation by a leave-one-out test of model prediction error. RESULTS: For unfixed tissue measurements (b-value range: 17-2104 s/mm(2)) the biexponential and kurtosis models had similar information content to each other and this was distinctly higher than for the stretched and monoexponential models. In fixed-tissue measurements (b-value range: 17-8252 s/mm(2)), the biexponential model had much higher information content than the three other models. CONCLUSION: AIC-based model ranking is consistent with an independent prediction accuracy test. Biexponential and kurtosis models consistently perform better than stretched and monoexponential models. The biexponential model has increasing superiority over all three other models as maximum b-value increases above ∼2000 s/mm(2).

3D radial projection technique with ultrashort echo times for sodium MRI: clinical applications in human brain and skeletal muscle.

(23)Na MRI has the potential to noninvasively detect sodium (Na) content changes in vivo. The goal of this study was to implement (23)Na MRI in a clinical setting for neurooncological and muscular imaging. Due to the biexponential T(2) decay of the tissue Na signal with a short component, which ranges between 0.5-8 ms, the measurement of total Na content requires imaging techniques with echo times (TEs) below 0.5 ms. A 3D radial pulse sequence with a TE of 0.2 ms at a spatial resolution of 4 x 4 x 4 mm(3) was developed that allows the acquisition and presentation of Na images on the scanner. This sequence was evaluated in patients with low- and high-grade gliomas, and higher (23)Na MR signals corresponding to an increased Na content were found in the tumor regions. The contrast-to-noise ratio (CNR) between tumor and white matter increased from 0.8 +/- 0.2 to 1.3 +/- 0.3 with tumor grade. In patients with an identified muscular (23)Na channelopathy (Paramyotonia congenita (PC)), induced muscle weakness led to a signal increase of approximately 18% in the (23)Na MR images, which was attributed to intracellular Na(+) accumulation in this region.

[Testing of model assumptions of MR oxygen extraction fraction mapping using a phantom study]. / Validierung von Modellannahmen einer MR-Sauerstoff- extraktionsbildgebung mittels einer Phantomstudie.

Oxygen supply is an important parameter for the evaluation of tissue viability and therefore of high interest in cancer diagnosis and therapy. One promising approach to extract relevant information from imaging data is the determination of oxygen saturation by means of the BOLD-effect. Using a simple model of tissue structure allows to evaluate the susceptibility difference between tissue and venous blood, from which the blood oxygen extraction fraction can be derived indirectly. The present study tested the validity of two model assumptions needed for exact quantification: the independence of the results of both capillary diameter d and the relative blood volume lambda. For this purpose a phantom was built, which allows the evaluation of susceptibility differences depending on d (27 microm-238 microm) and lambda (3%-12%). In agreement with model assumptions, delta chi(lambda) was widely constant and independent of ) lambda. In contradiction to the model, delta chi (d) showed a positive slope (delta chi range: 0.35-0.57 ppm). The present study suggests that the simple model investigated here has shortcomings in the quantification of oxygen extraction due to insufficient model assumptions.

Assessment of irradiated brain metastases by means of arterial spin-labeling and dynamic susceptibility-weighted contrast-enhanced perfusion MRI: initial results.

RATIONALE AND OBJECTIVES: To assess if preradiation and early follow-up measurements of relative regional cerebral blood flow (rrCBF) can predict treatment outcome in patients with cerebral metastases and to evaluate rrCBF changes in tumor and normal tissue after stereotactic radiosurgery using arterial spin-labeling (ASL) and first-pass dynamic susceptibility-weighted contrast-enhanced (DSC) perfusion MRI. METHODS: In 25 patients with a total of 28 brain metastases, DSC MRI and ASL perfusion MRI using the Q2TIPS sequence were performed with a 1.5-T unit. Measurements were performed prior to and at 6 weeks, 12 weeks, and 24 weeks after stereotactic radiosurgery. Follow-up examinations were completely available in 25 patients for Q2TIPS and 17 patients with 18 metastases for DSC MRI. The rrCBF of the metastases and the normal brain tissue was determined by a region-of-interest analysis. rrCBF values were correlated with the treatment outcome that was classified according to tumor volume changes at 6 months. RESULTS: The alteration of the rrCBF at the 6-week follow-up was highly predictive for treatment outcome. A decrease of the rrCBF value predicted tumor response correctly in all metastases for Q2TIPS and in 13 of 16 metastases for DSC MRI. The pretherapeutic rrCBF was not able to predict treatment outcome. The rrCBF values in normal brain tissue affected by radiation doses less than 0.5 Gy remained unchanged after therapy. CONCLUSION: These preliminary results suggest that ASL and DSC MRI techniques determining rrCBF changes in brain metastases after stereotactic radiosurgery allow the prediction of treatment outcome.

Small-angle X-ray scattering-based three-dimensional reconstruction of the immunogen KLH1 reveals different oxygen-dependent conformations.

For decades the respiratory protein keyhole limpet hemocyanin (KLH1) from the marine gastropod Megathura crenulata has been used widely as a potent immunostimulant, useful hapten carrier, and valuable agent in the treatment of bladder carcinoma. Although much information on the immunological properties of KLH1 is available, biochemical and structural data are still incomplete. Small-angle x-ray scattering revealed the existence of two conformations, an oxy state being slightly more compact than the deoxy state. Based on small-angle scattering curves, a newly developed Monte Carlo algorithm delivered a surface representation of proteins. The massive changes of the surfaces of reconstructed didecameric KLH1 molecules are explained as a twist of the two non-covalently associated decameric half-molecules. Upon oxygenation, the KLH1 molecule becomes longer and skinnier. This study provides the first real evidence how a molluscan hemocyanin changes conformation during an allosteric transition.

Comparison of arterial spin-labeling techniques and dynamic susceptibility-weighted contrast-enhanced MRI in perfusion imaging of normal brain tissue.

OBJECTIVES: To evaluate relative cerebral blood flow (rCBF) in normal brain tissue using arterial spin-labeling (ASL) methods and first-pass dynamic susceptibility-weighted contrast-enhanced (DSC) magnetic resonance imaging (MRI). METHODS: Sixty-two patients with brain metastases were examined on a 1.5 T-system up to 6 times during routine follow-up after stereotactic radiosurgery. Perfusion values in normal gray and white matter were measured using the ASL techniques ITS-FAIR in 38 patients, Q2TIPS in 62 patients, and the first-pass DSC echo-planar (EPI) MRI after bolus administration of gadopentetate dimeglumine in 42 patients. Precision of the ASL sequences was tested in follow-up examinations in 10 healthy volunteers. RESULTS: Perfusion values in normal brain tissue obtained by all sequences correlated well by calculating Pearson's correlation coefficients (P < 0.0001) and remained unchanged after stereotactic radiosurgery as shown by analysis of variance (P > 0.05). CONCLUSION: Both ASL and DSC EPI MRI yield highly comparable perfusion values in normal brain tissue.