Molecular magnetic resonance imaging of myeloperoxidase activity identifies culprit lesions and predicts future atherothrombosis.

Aims: Unstable atherosclerotic plaques have increased activity of myeloperoxidase (MPO). We examined whether molecular magnetic resonance imaging (MRI) of intraplaque MPO activity predicts future atherothrombosis in rabbits and correlates with ruptured human atheroma. Methods and results: Plaque MPO activity was assessed in vivo in rabbits (n = 12) using the MPO-gadolinium (Gd) probe at 8 and 12 weeks after induction of atherosclerosis and before pharmacological triggering of atherothrombosis. Excised plaques were used to confirm MPO activity by liquid chromatography-tandem mass spectrometry (LC-MSMS) and to determine MPO distribution by histology. MPO activity was higher in plaques that caused post-trigger atherothrombosis than plaques that did not. Among the in vivo MRI metrics, the plaques' R1 relaxation rate after administration of MPO-Gd was the best predictor of atherothrombosis. MPO activity measured in human carotid endarterectomy specimens (n = 30) by MPO-Gd-enhanced MRI was correlated with in vivo patient MRI and histological plaque phenotyping, as well as LC-MSMS. MPO-Gd retention measured as the change in R1 relaxation from baseline was significantly greater in histologic and MRI-graded American Heart Association (AHA) type VI than type III-V plaques. This association was confirmed by comparing AHA grade to MPO activity determined by LC-MSMS. Conclusion: We show that elevated intraplaque MPO activity detected by molecular MRI employing MPO-Gd predicts future atherothrombosis in a rabbit model and detects ruptured human atheroma, strengthening the translational potential of this approach to prospectively detect high-risk atherosclerosis.

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.

The combined use of DTI and MR elastography for monitoring microstructural changes in the developing brain of a neurodevelopmental disorder model: Poly (I:C)-induced maternal immune-activated rats.

Early neuropathology mechanisms in neurodevelopmental disorders are partially understood because routine anatomical magnetic resonance imaging (MRI) cannot detect subtle brain microstructural changes in vivo during postnatal development. Therefore, we investigated the potential value of magnetic resonance elastography (MRE) and diffusion tensor imaging (DTI) in a rat model of neurodevelopmental disorder induced by maternal immune activation. We studied 12 offspring of mothers injected with polyriboinosinic-polyribocytidylic acid (poly (I:C), 4 mg/kg) on gestational day 15, plus 8 controls. T2-weighted anatomical MR images, MRE (800 Hz) and DTI (30 gradient directions, b = 765.8 s/mm2, 5 images, b = 0 s/mm2) were collected when the rats were 4 and 10 weeks old, and results were compared with histological analysis performed at week 10. Ventricles were ~1.4 fold larger from week 4 in poly (I:C) rats than in controls. No other morphological abnormalities were detected in poly(I:C) rats. At week 4, larger ventricles were correlated with lower external capsule fractional anisotropy and internal capsule radial diffusion (Pearson, r = -0.53, 95% confidence intervals (CI) [-0.79 to -0.12], and r = -0.45, 95% CI [-0.74 to -0.01], respectively). The mean and radial diffusion of the corpus callosum, the mean and axial diffusion of the internal capsule and the radial diffusion properties in the external capsule increased with age for poly (I:C) rats only (Sidak's comparison, P<0.05). Cortical stiffness did not increase with age in poly (I:C) rats, in contrast with controls (Sidak's comparison, P = 0.005). These temporal variations probably reflected abnormal myelin content, decreased cell density and microglia activation observed at week 10 after histological assessment. To conclude, MRE and DTI allow monitoring of abnormal brain microstructural changes in poly (I:C) rats from week 4 after birth. This suggests that both imaging techniques have the potential to be used as complementary imaging tools to routine anatomical imaging to assist with the early diagnosis of neurodevelopmental disorders and provide new insights into neuropathology.

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.

Magnetic resonance imaging and diffusion tensor imaging reconstruction of connectomes in a macropod, the quokka (Setonix brachyurus).

The diversity of the diprotodontids provides an excellent opportunity to study how a basic marsupial cortical plan has been modified for the needs of the mammals living in different habitats. Very little is known about the connections of the cerebral cortex with the deep brain structures (basal ganglia and thalamus) in this evolutionarily significant group of mammals. In this study, we performed mapping of brain regions and connections in a diprotodontid marsupial from data obtained from an excised brain scanned in high-field (9.4 T) microstructural magnetic resonance imaging (MRI) instrument. The analysis was based on two MRI methodologies. First, high-resolution structural scans were used to map MRI visible brain regions from T1w and T2w images. Second, extensive diffusion tensor imaging (DTI) data were obtained to elucidate connectivity between brain areas using deterministic diffusion tracking of neuronal brain fibers. From the data, we were able to identify corticostriate connections between the frontal association and dorsomedial isocortex and the head of the caudate, and between the lateral somatosensory cortex and the putamen. We were also able to follow the olfactory and limbic connections by tracing fibers in the fornix, cingulum, intrabulbar part of the anterior commissure, and lateral olfactory tract. There was segregation of fibers in the anterior commissure such that olfactory connections passed through the rostroventral part and successively more dorsal cortical areas connected through more dorsal parts of the commissure. Our findings confirm a common pattern of cortical connectivity in therian mammals, even where brain expansion has occurred independently in diverse groups.

Developmental delay and late onset HBSL pathology in hypomorphic Dars1M256L mice.

The leukodystrophy Hypomyelination with Brainstem and Spinal cord involvement and Leg spasticity (HBSL) is caused by recessive mutations of the DARS1 gene, which encodes the cytoplasmic aspartyl-tRNA synthetase. HBSL is a spectrum disorder with disease onset usually during early childhood and no available treatment options. Patients display regression of previously acquired motor milestones, spasticity, ataxia, seizures, nystagmus, and intellectual disabilities. Gene-function studies in mice revealed that homozygous Dars1 deletion is embryonically lethal, suggesting that successful modelling of HBSL requires the generation of disease-causing genocopies in mice. In this study, we introduced the pathogenic DARS1 M256L mutation located on exon nine of the murine Dars1 locus. Despite causing severe illness in humans, homozygous Dars1 M256L mice were only mildly affected. To exacerbate HBSL symptoms, we bred Dars1 M256L mice with Dars1-null 'enhancer' mice. The Dars1 M256L/- offspring displayed increased embryonic lethality, severe developmental delay, reduced body weight and size, hydrocephalus, anophthalmia, and vacuolization of the white matter. Remarkably, the Dars1 M256L/- genotype affected energy metabolism and peripheral organs more profoundly than the nervous system and resulted in reduced body fat, increased respiratory exchange ratio, reduced liver steatosis, and reduced hypocellularity of the bone marrow. In summary, homozygous Dars1 M256L and compound heterozygous Dars1 M256L/- mutation genotypes recapitulate some aspects of HBSL and primarily manifest in developmental delay as well as metabolic and peripheral changes. These aspects of the disease might have been overlooked in HBSL patients with severe neurological deficits but could be included in the differential diagnosis of HBSL in the future.

Applications of magnetic particle imaging in the dementias.

PURPOSE OF REVIEW: This review discusses recent developments in the application of magnetic particle imaging (MPI) to dementia research. RECENT FINDINGS: MPI is a tracer method that is currently in the preclinical development stage. It provides high sensitivity for the detection and localization of magnetic nanoparticles with very high spatial and temporal resolution and a similar application spectrum as PET. Unlike MRI, the MPI signal is not contaminated by background signal from tissues and is highly quantifiable in terms of local tracer concentrations. These properties make the technology ideally suited for localization of specific targets or quantification of vascular parameters. MPI uses magnetic nanoparticles which can be modified by various coatings, and by adding ligands (i.e. peptides or antibodies) for specific targeting. This makes MPI an attractive tool for the potential detection of abnormal protein deposits, such as Aß plaques, with greater specificity than MRI. Neural stem cells can also be labelled with these nanoparticles ex vivo to monitor their migration in vivo. SUMMARY: The capabilities of MPI opens the potential for several applications of MPI in neurocognitive disorders, including vascular imaging, detection of amyloid plaques and potentially other pathological hallmarks of Alzheimer's disease and stem-cell tracking.

A Biomimetic Approach toward Enhancing Angiogenesis: Recombinantly Expressed Domain V of Human Perlecan Is a Bioactive Molecule That Promotes Angiogenesis and Vascularization of Implanted Biomaterials.

Angiogenic therapy involving delivery of pro-angiogenic growth factors to stimulate new blood vessel formation in ischemic disease is promising but has seen limited clinical success due to issues associated with the need to deliver supra-physiological growth factor concentrations. Bio-inspired growth factor delivery utilizing the native growth factor signaling roles of the extracellular matrix proteoglycans has the potential to overcome many of the drawbacks of angiogenic therapy. In this study, the potential of the recombinantly expressed domain V (rDV) of human perlecan is investigated as a means of promoting growth factor signaling toward enhanced angiogenesis and vascularization of implanted biomaterials. rDV is found to promote angiogenesis in established in vitro and in vivo angiogenesis assays by potentiating endogenous growth factor signaling via its glycosaminoglycan chains. Further, rDV is found to potentiate fibroblast growth factor 2 (FGF2) signaling at low concentrations that in the absence of rDV are not biologically active. Finally, rDV immobilized on 3D porous silk fibroin biomaterials promotes enhanced vascular ingrowth and integration of the implanted scaffolds with the surrounding tissue. Together, these studies demonstrate the important role of this biologically active perlecan fragment and its potential in the treatment of ischemia in both native and bioengineered tissues.

Nanoparticles as contrast agents for the diagnosis of Alzheimer's disease: a systematic review.

Nanoparticle (NP)-based magnetic contrast agents have opened the potential for MRI to be used for early diagnosis of Alzheimer's disease (AD). This article aims to review the current progress of research in this field. A comprehensive literature search was performed based on PubMed, Medline, EMBASE, PsychINFO and Scopus databases using the following terms: 'Alzheimer's disease' AND 'nanoparticles' AND 'Magnetic Resonance Imaging.' 33 studies were included that described the development and utility of various NPs for AD imaging, including their coating, functionalization, MRI relaxivity, toxicity and bioavailability. NPs show immense promise for neuroimaging, due to superior relaxivity and biocompatibility compared with currently available imaging agents. Consistent reporting is imperative for further progress in this field.

A Hypomorphic Dars1 D367Y Model Recapitulates Key Aspects of the Leukodystrophy HBSL.

Hypomyelination with brain stem and spinal cord involvement and leg spasticity (HBSL) is a leukodystrophy caused by missense mutations of the aspartyl-tRNA synthetase-encoding gene DARS1. The clinical picture includes the regression of acquired motor milestones, spasticity, ataxia, seizures, nystagmus, and intellectual disabilities. Morphologically, HBSL is characterized by a distinct pattern of hypomyelination in the central nervous system including the anterior brainstem, the cerebellar peduncles and the supratentorial white matter as well as the dorsal columns and the lateral corticospinal tracts of the spinal cord. Adequate HBSL animal models are lacking. Dars1 knockout mice are embryonic lethal precluding examination of the etiology. To address this, we introduced the HBSL-causing Dars1 D367Y point mutation into the mouse genome. Surprisingly, mice carrying this mutation homozygously were phenotypically normal. As hypomorphic mutations are more severe in trans to a deletion, we crossed Dars1 D367Y/D367Y mice with Dars1-null carriers. The resulting Dars1 D367Y/- offspring displayed a strong developmental delay compared to control Dars1 D367Y/+ littermates, starting during embryogenesis. Only a small fraction of Dars1 D367Y/- mice were born, and half of these mice died with hydrocephalus during the first 3 weeks of life. Of the few Dars1 D367Y/- mice that were born at term, 25% displayed microphthalmia. Throughout postnatal life, Dars1 D367Y/- mice remained smaller and lighter than their Dars1 D367Y/+ littermates. Despite this early developmental deficit, once they made it through early adolescence Dars1 D367Y/- mice were phenotypically inconspicuous for most of their adult life, until they developed late onset motor deficits as well as vacuolization and demyelination of the spinal cord white matter. Expression levels of the major myelin proteins were reduced in Dars1 D367Y/- mice compared to controls. Taken together, Dars1 D367Y/- mice model aspects of the clinical picture of the corresponding missense mutation in HBSL. This model will enable studies of late onset deficits, which is precluded in Dars1 knockout mice, and can be leveraged to test potential HBSL therapeutics including DARS1 gene replacement therapy.

Microchannels Are an Architectural Cue That Promotes Integration and Vascularization of Silk Biomaterials in Vivo.

Functional integration of implanted biomaterials and bioengineered tissues in vivo requires effective and timely vascular ingrowth. While many vascularization strategies rely on delivery of angiogenic growth factors or endothelial cells to promote vascular ingrowth, the effect of physical and architectural features of biomaterials on the vascularization process is less well understood. Microchannels are a simple, accessible architectural feature frequently engineered into 3D biomaterials to promote mass transfer. In this study, the effect of microchannels on the integration and vascularization of 3D porous silk scaffolds was explored over a 14 week period. An array of 508 µm diameter microchannels spanning the length of critically sized, porous silk scaffolds significantly improved tissue ingrowth into the constructs. At week 6, all silk scaffolds (n = 8) with microchannels showed complete tissue infiltration throughout the construct, while only one of eight (12.5%) did so in the absence of microchannels. The presence of microchannels improved silk scaffold vascularization with significantly more vessels per unit area in the presence of microchannels. The vessel size distribution was similar in both scaffold types, but a shift in distribution toward smaller vessels was observed in the presence of microchannels. The blood vessels in silk scaffolds were perfused, functional and connected to the animal's cardiovascular system, as demonstrated by the presence of red blood cells in the vessel lumens, and effective delivery of a contrast agent the vessels inside the scaffold. This study demonstrates the utility of microchannels as a simple architectural feature that significantly improves vascularization and integration of implanted biomaterials.

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.

Myeloperoxidase is a potential molecular imaging and therapeutic target for the identification and stabilization of high-risk atherosclerotic plaque.

Aims: As the inflammatory enzyme myeloperoxidase (MPO) is abundant in ruptured human atherosclerotic plaques, we aimed to investigate the role of MPO as a potential diagnostic and therapeutic target for high-risk plaque. Methods and results: We employed the tandem stenosis model of atherosclerotic plaque instability in apolipoprotein E gene knockout (Apoe-/-) mice. To test the role of MPO, we used Mpo-/-Apoe-/- mice and the 2-thioxanthine MPO inhibitor AZM198. In vivo MPO activity was assessed by liquid chromatography-tandem mass spectrometry detection of 2-chloroethidium generation from hydroethidine and by bis-5HT-DTPA-Gd (MPO-Gd) molecular magnetic resonance imaging (MRI), while plaque phenotype was verified histologically. Myeloperoxidase activity was two-fold greater in plaque with unstable compared with stable phenotype. Genetic deletion of MPO significantly increased fibrous cap thickness, and decreased plaque fibrin and haemosiderin content in plaque with unstable phenotype. AZM198 inhibited MPO activity and it also increased fibrous cap thickness and decreased fibrin and haemosiderin in plaque with unstable phenotype, without affecting lesion monocytes and red blood cell markers or circulating leukocytes and lipids. MPO-Gd MRI demonstrated sustained enhancement of plaque with unstable phenotype on T1-weighted imaging that was two-fold greater than stable plaque and was significantly attenuated by both AZM198 treatment and deletion of the Mpo gene. Conclusion: Our data implicate MPO in atherosclerotic plaque instability and suggest that non-invasive imaging and pharmacological inhibition of plaque MPO activity hold promise for clinical translation in the management of high-risk coronary artery disease.

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.